David 

J. Beale
Konstantinos A. Kouremenos
Enzo A. Palombo Editors

Microbial
Metabolomics
Applications in Clinical, Environmental,
and Industrial Microbiology
Microbial Metabolomics
David J. Beale Konstantinos A. Kouremenos
•

Enzo A. Palombo
Editors

Microbial Metabolomics
Applications in Clinical, Environmental,
and Industrial Microbiology

123
Editors
David J. Beale Enzo A. Palombo
Land and Water Swinburne University of Technology
CSIRO Hawthorn, VIC
Dutton Park, QLD Australia
Australia

Konstantinos A. Kouremenos
Metabolomics Australia, Bio21 Molecular
Science & Biotechnology Institute
The University of Melbourne
Parkville, VIC
Australia

ISBN 978-3-319-46324-7 ISBN 978-3-319-46326-1 (eBook)

DOI 10.1007/978-3-319-46326-1
Library of Congress Control Number: 2016950891

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Preface

Systems biology has greatly increased our understanding of many cellular functions
and cell–cell interactions. Our awareness of the enormous range of activities per-
formed by microorganisms has been greatly enhanced by major developments in
the fields of genomics, proteomics and transcriptomics. However, understanding
these functions does not fully describe the global metabolic features of single cells
or cell populations (mono-specific or multi-species). Characterization of the
metabolome brings us closer to the phenotype of the cell and more closely reflects
the activities of microbes in response to environmental stimuli.
Microbial metabolomics is an emerging field that has developed rapidly in recent
years. This development has been paralleled and supported by important advances
in analytical instrumentation and technologies, in particular chromatographic and
mass spectrometric methods, coupled with new and more powerful computational
tools.
This book brings together contributions from global experts from diverse areas
that have facilitated the exciting advances in microbial metabolomics, with special
attention given to the development of relevant hardware and software platforms.
Thus, the principles of these technologies will be a major focus of the book.
The main application of metabolomics is likely to be in the field of clinical and
veterinary microbiology with a focus on disease-causing microorganisms.
However, there is a great potential to apply metabolomics to help better understand
complex biological systems that are dominated by multi-species microbial popu-
lations exposed to changing growth and nutritional conditions. In particular,
environmental (e.g. water and soil), food (e.g. microbial spoilage and food patho-
gens), agriculture and industrial applications are seen as developing. As such, this
book looks at the application metabolomics from clinical, environmental and
industrial perspectives.

Dutton Park, QLD, Australia David J. Beale

Parkville, VIC, Australia Konstantinos A. Kouremenos
Hawthorn, VIC, Australia Enzo A. Palombo

v
Contents

1 Introduction to Microbial Metabolomics . . . . . . . . . . . . . . . . . . . . . . 1

Silas G. Villas-Boas
2 Microbes, Metabolites and Health . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
Trevor J. Lockett, Anthony R. Bird, Claus Christophersen,
Julie M. Clarke, Michael A. Conlon and David L. Topping
3 Exploring the Bioactive Landscape of the Gut Microbiota
to Identify Metabolites Underpinning Human Health. . . . . . . . . . . . 49
Páraic Ó Cuív, Sriti Burman, Sian Pottenger and Mark Morrison
4 Using Metabolomic Approaches to Characterize the Human
Pathogen Leishmania in Macrophages . . . . . . . . . . . . . . . . . . . . . . . . 83
Joachim Kloehn, Eleanor C. Saunders and Malcolm J. McConville
5 Exometabolomics for Linking Soil Carbon Dynamics to
Microbial Communities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119
Andrea Lubbe and Trent Northen
6 Soil Microbial Metabolomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Michael W. Heaven and Devin Benheim
7 Community Metabolomics in Environmental Microbiology . . . . . . . 199
Oliver A.H. Jones, Gavin Lear, Aalim M. Welji, Gavin Collins
and Christopher Quince
8 Metabolomics: Applications to Food Safety
and Quality Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
Farhana R. Pinu
9 Microbial Metabolomics in Biomass Waste Management . . . . . . . . . 261
Avinash V. Karpe, David J. Beale, Ian H. Harding
and Enzo A. Palombo
10 Beyond Metabolomics: A Review of Multi-Omics-Based
Approaches . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 289
David J. Beale, Avinash V. Karpe and Warish Ahmed
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 313

vii
Chapter 1
Introduction to Microbial Metabolomics

Silas G. Villas-Boas

1 Introduction

The ultimate goal of metabolomics is the unbiased and nontargeted analysis of cell
metabolites combined with the detection, identification, and sometimes also the
absolute quantification of a multitude of compounds in biological samples. Despite
this being a very ambitious goal due to all the analytical challenges associated with
the analysis of metabolomes (and our inability to design completely unbiased
analytical methods), there has been an enormous development in the field of
metabolomics in the past 16 years that has placed metabolomics in the spotlight of
cutting-edge technologies for life sciences in the twenty-first century (Reeves and
Rabinowtiz 2011; Nguyen et al. 2012; Russell and Duncan 2013; Zhang et al.
2014).
Microbiology greatly benefited from developments in metabolomics since its
conception (Allen et al. 2003; Castrillo et al. 2003; Villas-Bôas et al. 2005a, b,
2006; Wang et al. 2006; van der Werf et al. 2007). Microbial systems are usually
simpler and highly controllable compared to higher (multicellular) organisms,
which make them the ideal platform for developing, applying, and validating
metabolomics tools. Moreover, the development of genome-scale metabolic models
for a wide range of microbial species—based on genome sequence data—has
offered the best scaffold for interpretation of metabolomics data and, more
importantly, for integration of metabolomics data with other omics (Lee et al.
2005).
However, microbial metabolomics is not free of its challenges. The microbial
metabolome is sparsely distributed into three very distinct matrices: (i) inside the
cell (intracellular); (ii) outside the cell, in the medium (extracellular); and (iii) in the

S.G. Villas-Boas (&)

School of Biological Sciences, University of Auckland, 3A Symonds
Street, Auckland 1142, New Zealand
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 1

D.J. Beale et al. (eds.), Microbial Metabolomics,
DOI 10.1007/978-3-319-46326-1_1
2 S.G. Villas-Boas

culture headspace (volatile compounds). Although this feature is not unique to

microbial systems, for microorganisms the metabolite profile of each matrix can be
equally important and it is not always easy to assess them separately; and most
importantly, we know very little about the metabolic mechanism driving their
distribution (Granucci et al. 2015). These challenges combined with the great
diversity of microbial cell types provide a glimpse to the main technical issues that
microbial metabolomics still faces today.
In this chapter, the state-of-the-art of microbial metabolomics field will be briefly
reviewed, including the main applications of metabolomics in microbiology, current
challenges, major achievements, and a future perspective for this field.

2 Microbial Metabolomics: From Early Metabolite

Profiles to Metabolome Databases

A summarized timeline of main developments in microbial metabolomics is pro-

vided in Table 1. The first peer-reviewed articles that one finds when searching
scientific literature databases using the keyword “metabolome” is focused on the
two best known microorganisms: Escherichia coli (Tweeddale et al. 1998) and
Saccharomyces cerevisiae (Oliver 1997; Oliver et al. 1998). Therefore, we could
say that the concept of metabolomics started with microorganisms. Nonetheless,
microbial metabolite profiling precedes the concept of metabolomics (Collar 1991;
Montel et al. 1991; Andersen et al. 1995; Smedsgaard and Frisvad 1996). Since the
early 1990s, metabolite profiling has been applied to microbiology mainly for
phenotypic characterization of closely related microbial strains or species (Collar
1991; Montel et al. 1991; Andersen et al. 1995; Smedsgaard and Frisvad 1996) as
well as a tool for microbial identification (Montel et al. 1991).
The first two publications which appear in metabolomics originate from the
group of Professor Thomas Ferenci at the University of Sydney in Australia,
describing the metabolic response of E. coli to different environmental perturbations
(Tweeddale et al. 1998, 1999). The authors assessed the metabolic response of
E. coli to different growth rates when grown on glucose and minimal medium
(Tweeddale et al. 1998) and to oxidative stress (Tweeddale et al. 1999). The global
metabolite pool (metabolome) of E. coli was analyzed through two-dimensional
thin-layer chromatography of all 14C-glucose labeled compounds extracted from
bacterial cells. Since then, the microbial metabolomics field has progressed sig-
nificantly, mostly taking advantage of developments in mass spectrometry tech-
nology and major advances in plant metabolomics.
Although the application of metabolomics apparently started around microbial
systems, plant metabolomics was the main area that initially launched metabolomics
to the world and drove the main developments around mass spectrometry-based
metabolomics. The pioneering works on mass spectrometry-based plant metabo-
lomics came from the Max Planck Institute in Golm, Germany (Fiehn et al. 2000;
1 Introduction to Microbial Metabolomics 3

Table 1 Timeline of important developments in microbial metabolomics field

Year Development References
Era pre-metabolome
1991 Metabolite profiling applied to metabolism assessment and Collar (1991)
systematics of microorganisms Montel et al. (1991)
1995 Metabolite profiling to phenotype microbial cultures Andersen et al.
(1995)
1996 Direct electrospray mass spectrometry for fungal taxonomy Smedsgaard and
and secondary metabolite profiling Frisvad (1996)
Metabolome era
1997 The word metabolome first proposed Oliver 1997
1998 Metabolome as a functional genomic tool and first paper Oliver et al. (1998)
traceable using the keyword “metabolome” Tweeddale et al.
(1998)
1999 Soga and Ross
(1999)
Tweeddale et al.
(1999)
2000 Fiehn et al. (2000)
Roessner et al.
(2000)
Soga and Heiger
(2000)
2001 Fiehn (2001)
Soga and Imaizumi
(2001)
2002 Fiehn (2002)
Roessner et al.
(2001)
Soga et al. (2002a,
b
2003 Allen et al. (2003)
Castrillo et al.
(2003)
Maharjan and
Ferenci (2003)
Villas-Bôas et al.
(2003)
2004 Mashego et al.
(2003)
Wittmann et al.
(2004)
2005 Villas-Bôas et al.
(2005a, b)
2012 Publication of the yeast metabolome database Jewison et al.
(2012)
2013 Publication of the E. coli metabolome database Guo et al. (2013)
4 S.G. Villas-Boas

Fiehn 2001, 2002; Roessner et al. 2000, 2001) and paved the way for modern
metabolomics. However, the microbial metabolomics field was also particularly
influenced by proposed approaches and methodologies developed by the group of
Professor Stephen Oliver at that time at the University of Manchester, UK (Allen
et al. 2003; Castrillo et al. 2003). Oliver’s group was not only the first group to
propose the concept of metabolic fingerprinting using direct infusion mass spec-
trometry and S. cerevisiae as the model organism (Castrillo et al. 2003); they also
demonstrated the importance of extracellular metabolites found in the culture media
for phenotyping purposes, referring to it as the metabolic footprint (Allen et al.
2003). Other important contributions to early developments in microbial metabo-
lomics include the global metabolite profiling of intra- and extracellular metabolites
of microbial cells and cultures by methyl chloroformate (MCF) derivatization and
gas chromatography–mass spectrometry (GC-MS) analysis (Villas-Bôas et al. 2003,
2005a, b); assessment of sampling and sample preparation for microbial metabo-
lomics (Maharjan and Ferenci 2003; Wittmann et al. 2004; Villas-Bôas et al. 2005a,
b); quantitative microbial metabolomics using capillary electrophoresis–mass
spectrometry (CE-MS) (Soga and Ross 1999; Soga and Heiger 2000; Soga and
Imaizumi 2001; Soga et al. 2002a, b); and the application of stable isotope dilution
theory in metabolome characterization of S. cerevisiae cultures, where each
metabolite concentration is quantified relative to the concentration of its
U-13C-labeled equivalent (Mashego et al. 2003).
In the past 14 years, metabolomics has been successfully applied to a wide range
of microbiological applications, from microbial functional genomics (Allen et al.
2003; Castrillo et al. 2003; Moxley et al. 2009), phenotyping (Bundy et al. 2005;
Villas-Bôas et al. 2008), to the detection of microbial contaminations in foods
(Jahangir et al. 2008; Cevallos-Cevallos et al. 2011), fermentation broth (Sue et al.
2011); and to characterize microbial symbiotic associations with different hosts
(Martin et al. 2007; Russell and Duncan 2013). In addition, the microbial meta-
bolomics field includes two important microbial metabolome databases developed
at the University of Alberta (Canada): The Yeast Metabolome Database (YMDB)
(http://www.ymdb.ca/) and the E. coli Metabolome Database (ECMDB) (http://
ecmdb.ca/) (Jewison et al. 2012 and Guo et al. 2013). These are databases of
metabolites found in cultures or cells of S. cerevisiae and E. coli, respectively.
The YMDB contains 2027 small molecules with 980 associated enzymes and 138
associated transporters and the ECMDB contains 3755 small molecules with 1402
associated enzymes and 387 associated transporters. For each metabolite there is a
description of the compound, its chemical characteristics, synonyms and links to
spectral and chemical databases. Therefore, these two databases represent a land-
mark for microbial metabolomics, offering powerful Web-based tools to aid in
microbial metabolomics data analysis and interpretation.
1 Introduction to Microbial Metabolomics 5

3 Challenges

As mentioned above, microbial metabolomics still faces some important challenges,

with the efficient quenching of microbial cell metabolism during sampling com-
bined with separation of intra- and extracellular metabolites being the most
important. Microbial cells are usually diffused and diluted in a large volume of
culture medium containing high levels of unutilized substrates and metabolic waste
products. Adding to that is the fact that the ratio between microbial cell biomass and
extracellular medium is often extremely low, making it often difficult to obtain a
reasonable amount of intact cells for intracellular metabolite analysis per sample
unit. Therefore, sampling and sample preparation in microbial metabolomics have
to be carefully considered and tested before any study begins. To date, there are no
standard protocols that can be widely applied to different microbial cell types and
experimental conditions.
Ideally, microbial metabolomics needs robust sampling methods that quench the
metabolic activity of microbial cells simultaneously with sampling and allow the
separation of microbial biomass from the culture medium without affecting the
metabolic state and integrity of the cells. Unfortunately, to a large degree, this has
not been satisfactorily achieved as yet. There have been numerous studies com-
paring different sampling (quenching) methods for different microbial cells
(Villas-Bôas et al. 2005b; Bolten et al. 2007) and proposing new methods
(Villas-Bôas and Bruheim 2007; Canelas et al. 2008). However, many of those
studies show controversial results (mainly regarding the popular cold methanol
water solution for sampling microbial cells) and most protocols seem to be suitable
only to specific cell types (Faijes et al. 2007; Tredwel et al. 2011).
Another challenge that metabolomics still faces specific to microbial metabo-
lomics is the metabolome coverage by different analytical methods. It is common
knowledge in metabolomics today that there is no single analytical method capable
of detecting and identifying the whole metabolome of a cell. Therefore, metabo-
lomics is achieved through the combination of different analytical tools in order to
get as much information as possible about the metabolite profile of a population of
cells or organism. Considering that microbial samples are usually short in biomass
concentration, obtaining enough samples from a single experiment to be analyzed
by different instruments or methods can be an issue. Moreover, identification of
microbial secondary metabolites (and characterization of novel ones) is still a rel-
atively low throughput process, similar to plant secondary metabolites.
Finally, microbial metabolomics suffers from a huge knowledge gap regarding
the metabolic relationship between intra- and extracellular metabolites (Granucci
et al. 2015). The transport of substrates to the intracellular medium is an essential
function to any live cell and it has been extensively studied (Aguilar-Barajas et al.
2011; Jojima et al. 2010; Hofman-Bang 1999). However, the transport of chemical
molecules through the plasmatic membrane is not exclusively to provide nutrients
6 S.G. Villas-Boas

and other substrates to the cells, but to also remove metabolic waste products in
order to maintain intracellular homeostasis. Therefore, the secretion of metabolites
is also an essential biochemical function to all living cells, but it is very little studied
if compared to the process of nutrient uptake.
The secretion of macromolecules such as proteins (enzymes) and nucleic acids
have been widely studied in microorganisms (Alvarez-Martinez and Christie 2009;
Chapon-Hervé et al. 1997; Genin and Boucher 1994). The best known mechanisms
of macromolecule secretion are the xcp secretion pathway (Chapon-Hervé et al.
1997), the transmembrane protein family PulD (Genin and Boucher 1994), and the
type IV secretion systems (T4SS) (Alvarez-Martinez and Christie 2009). On the
other hand, recent studies have shown that ABC transmembrane proteins
(ATP-binding cassette) and MFS (Major Facilitator Superfamily) play a very
important role in the secretion of secondary/toxic metabolites in microbial systems
such as endogenous and exogenous antibiotics (Stergiopoulos et al. 2002; Martín
et al. 2005). It is believed that small metabolites, mainly those end products of
microbial fermentation (e.g., acetate, lactate, butyrate, ethanol, butanol, acetone,
etc.) are excreted passively through the plasma membrane, or secreted by spe-
cialized mechanisms such as response reaction to hypo-osmotic stress (Krämer
1994) and uniport, synport, and antiport transport systems (Krämer 1994).
Although there is little experimental data to validate these mechanisms as being
responsible for the metabolic efflux, all of them are based on the concept of
metabolic overflow, which says that under specific metabolic conditions, a massive
excretion of some metabolic intermediates is observed due to intracellular accu-
mulation of one or more intermediates from one or more metabolic pathways
(Krämer 1994). Although this concept seems to be appropriate to explain the
secretion of some metabolic intermediates, it does not apply to many cases studied
during continuous culture (chemostats) (Krämer 1994). Microbial metabolomics
data obtained during continuous culture in our laboratory challenge the concept of
metabolic overflow (Granucci et al. 2015). For instance, in several studies in which
microbial samples were collected before, during and after an environmental per-
turbation (during continuous culture), we observed that some intracellular
metabolites are actively secreted to the extracellular medium in response to an
environmental stimulus, demonstrating that the secretion of a given metabolite to
the extracellular medium does not take place exclusively as a result of its accu-
mulation in the intracellular medium. Our results suggest that microbial cells very
often remove some intracellular metabolites, even though these metabolites could
be key intermediates of central carbon metabolism such as pyruvate, phospho-
enolpyruvate, and several amino acids. Therefore, a better understanding about the
relationship between intra- and extracellular metabolites is essential if we want to
make better use of extracellular metabolomics data.
1 Introduction to Microbial Metabolomics 7

4 Cutting-Edge Applications

4.1 Assessing the Metabolic State of a Microbial Community

It is evident today that the metabolite profile of a pure microbial culture is very
distinct from that of a mixed culture and the metabolite profile of a mixed culture is
not a simple sum up of the metabolite profiles of the different species composing the
community (Sue et al. 2011; Kimes et al. 2013; Lv 2013; Ponnusamy et al. 2013).
Microbial cells sense the presence of a different microbial species in their envi-
ronment and immediately respond to the foreign presence by changing their
metabolic state. This change in metabolic state can be easily detected through
metabolomics (e.g., metabolic footprint analysis) (Sue et al. 2011). Therefore,
metabolomics has become an important tool for assessing the metabolic state of
microbial communities both in vitro (Sue et al. 2011; Ponnusamy et al. 2013) as
well as in nature (Badri et al. 2013; Kimes et al. 2013). Nevertheless, it is essential
to any metabolomic study involving microbial communities to keep track of the
microbial community dynamics in terms of its composition both qualitative and
quantitatively.

4.2 Host–Microbe Interactions

The relationship of metabolite with the microbial population profiles derived

from culture-independent molecular techniques can help to unravel the inherent
and intimate host–microbe relationships. This approach has been increasingly
applied to study the molecular mechanisms behind host–microbe interactions,
with demonstrated potential to elucidate the causes of various pathologies
resulting from host–microbial associations (Ming et al. 2012; Xie et al. 2012;
Marcobal et al. 2013; Storr et al. 2013), including human enteric infections (Han
et al. 2010). Gut microorganisms and their metabolites affect the luminal
environment and alter development, motility, and homeostasis of the gut (Han
et al. 2010). Therefore, insights regarding the metabolic principles coordinating
gut microbiota will guide future manipulation of the gut microbiota to promote
human health.

4.3 New Diagnosis of Microbial Infection Through

Metabolomics

The identification of the cause of microbial infection is not always simple and
usually requires days of culturing and sometimes evasive sampling procedures. The
discovery of metabolic biomarkers specific to different infective agents through
8 S.G. Villas-Boas

metabolomics can pave the way for development of fast, sensitive, and accurate
methods of diagnosis through metabolite profiles. Metabolomics has been suc-
cessfully applied to distinguish different causative agents of bacterial pneumonia in
animal models (Slupsky 2010; Hoerr et al. 2012) as well as for early diagnosis of
pediatric septic shock (Mickiewicz et al. 2013). In addition, the opportunity to use
noninvasive sampling procedures such as the use of exhaled breath for diagnostic of
infectious diseases through metabolite profiling of volatile metabolites promises to
be the future of clinical diagnosis (Boots et al. 2012).

5 Future Perspective

The future of microbial metabolomics certainly lies in further advances in analytical

techniques showing ultrasensitivity and specificity to allow the detection and
identification of intracellular metabolites using very small biomass samples. Mass
spectrometry seems to be the technology with best potential to achieve this goal.
Important recent steps have been taken towards this goal, such as the development
of an ionization method called laser ablation electrospray ionization (LAESI)
capable of in situ mass spectrometric analysis of individual cells at atmospheric
pressure (Zenobi 2013). Mass spectrometry imaging is another novel technique
which makes use of matrix-assisted laser desorption/ionization (MALDI) and
secondary ion mass spectrometry (SIMS) imaging to visualize metabolites within a
spatial resolution of <1 μm, at ambient pressures, and with very high mass accuracy
and mass resolution (Zenobi 2013).
Consequently, single-cell metabolomics should also soon become a reality for
microbial systems. Impressive advances in single-cell metabolomics have been
achieved recently, but they have so far failed to generate meaningful insights into the
phenotypic diversity of single cells within a population (Zenobi 2013). The main
reason for that is probably the lack of sensitivity of the analytical methods available
to measure low-concentrated metabolites within a single cell. Therefore, more
extensive coverage of microbial metabolomes is the main requirement for future
advances in microbial metabolomics, which is intrinsically linked to the develop-
ment of ultrasensitive analytical methods. The size of the model yeast S. cerevisiae
metabolome is around 1168 metabolites and the yeast metabolome database (http://
ymdb.ca) lists over 2000 molecules. Still, most current metabolomics methods are
capable of detecting and identifying less than 10 % of the full yeast metabolome
(Zenobi 2013). Therefore, ultrasensitive analytical methods, combined with faster
identification of metabolites from high-throughput measurements using small
sample sizes (e.g., single cell metabolomics) will pave way for the future of
microbial metabolomics.
1 Introduction to Microbial Metabolomics 9

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Chapter 2
Microbes, Metabolites and Health

Trevor J. Lockett, Anthony R. Bird, Claus Christophersen,

Julie M. Clarke, Michael A. Conlon and David L. Topping

1 Introduction

High throughput DNA sequencing has revolutionised the analysis of complex

communities of microorganisms (microbiota). In humans, this paradigm shift,
allowing analysis of the membership of microbial communities without the need to
culture individual organisms, has spawned an avalanche of research and a number
of important collaborative programs characterising not only the catalogue of species
comprising the microbiota associated with different ecological niches in the body in
states of both health and disease (Group et al. 2009) but also their genetic potential
(Qin et al. 2010). Nowhere in the human body are the microbes more numerous or
the microbiota more complex than in the colon of the gastrointestinal tract (Savage
1977). With its trillions of bacteria, comprising over one thousand individual
microbial species present in varying abundances, the microbiota of the human gut
and the genes they carry (collectively the microbiome) provides the host with a vast
metabolic potential over and above that afforded by the 20,000–25,000 protein
coding genes of the human genome.
The composition of the human colonic microbiota is remarkably diverse between
individuals with each person’s microbiota being unique (Qin et al. 2010;
Yatsunenko et al. 2012). Despite this diversity at the taxonomic level, when
analysed for functional potential by whole genome analysis at the gene level,
conservation of bacterial genes between individuals was high suggesting that

T.J. Lockett (&)

CSIRO Health and Biosecurity, North Ryde, NSW, Australia
e-mail: [email protected]
A.R. Bird
J.M. Clarke
M.A. Conlon
D.L. Topping
CSIRO Health and Biosecurity, Kintore Ave, Adelaide, SA, Australia
C. Christophersen
Edith Cowan University, Joondalup, WA, Australia

© Springer International Publishing Switzerland 2016 13

D.J. Beale et al. (eds.), Microbial Metabolomics,
DOI 10.1007/978-3-319-46326-1_2
14 T.J. Lockett et al.

diverse microbial populations could deliver very similar functional outcomes for
both the microbial community and the host (Huttenhower et al. 2012).
There is growing evidence that the gut microbiota impacts significantly on the
health of the host. Animal studies have shown that the gut microbiota plays a key role
in the post-natal maturation of both the gut itself and the gut immune system (Smith
et al. 2007; Chung et al. 2012). In humans, changes in gut microbiota structure have
been associated with gut-associated disorders including: inflammatory bowel dis-
eases (Gevers et al. 2014; Frank et al. 2007; De Cruz et al. 2015) and colorectal
cancer (Garrett 2015); metabolism-related disorders such as obesity (Ley et al. 2006),
metabolic syndrome (Le Chatelier et al. 2013; Haro et al. 2016) and diabetes (Qin
et al. 2012) but also with systemic disorders including cardiovascular disease, neu-
rodegenerative diseases including Alzheimer’s (Alam et al. 2014; Naseer et al. 2014)
and Parkinson’s (Goldman et al. 2014; Scheperjans et al. 2015) diseases and
neuro-developmental and psychiatric conditions including autism spectrum disorder
(Louis 2012; Mulle et al. 2013) and schizophrenia (Nemani et al. 2015).
Of these disorders, the role of the microbiota has probably been studied most
extensively in obesity which is also a well-recognized risk factor for many of the
other chronic diseases mentioned above. Obese humans, when compared to normal
weight individuals, displayed a reduced ratio of microbes from the Bacteroidetes
phylum relative to those of the Firmicutes phylum in their faeces and this ratio
increased in obese individuals who lost weight (Ley et al. 2006). Important insights
into the functional significance of these associations have come from the use germ
free, gnotobiotic and gene mutant animal models. This same relationship of
Bacteroidetes to Firmicutes was also observed in obese mice carrying two copies of
the ob mutation in the leptin gene (ob/ob mice) relative to their ob/+ and +/+ litter
mates. In this animal model, biochemical analyses revealed that these different
microbiota varied in the efficiency of energy harvest from the diet and that both the
obese-prone and lean phenotypes was transferrable to germ free mice by colo-
nization with the microbiota from obese and lean donors respectively (Turnbaugh
et al. 2006). This same result was observed when human faecal microbiota from
female twins discordant for obesity were used to colonize germ free mice (Ridaura
et al. 2013). Mice practise coprophagy. Co-housing of mice, conventionalised with
the obese twin’s microbiota, with other mice conventionalised with the lean twin’s
microbiota prevented acquisition of the obese phenotype. Microbial community
analysis revealed that this prevention of obesity was associated with an invasion of
Bacteroidetes from the lean microbiota into the microbiota of the
obese-conventionalised animals (Ridaura et al. 2013). Interestingly this effect was
diet-dependent and was most marked when the animals were fed a diet high in fruit
and vegetables. When the animals were fed a diet high in saturated fat and low in
fruit and vegetables, levels of Bacteroidetes invasion from the lean convention-
alised animals was very low (Ridaura et al. 2013) highlighting the importance of
diet as a selective force in gut microbiota structure/function determination. While
microbial population changes associated with diseases have been extensively
studied, the mechanisms by which these changes elicit their physiological effects on
the host are just beginning to be unraveled.
2 Microbes, Metabolites and Health 15

Metabolites and other products made by the gut microbiota play a key role in the
dialogue between the gut microbiota and the host. Contributions of the gut micro-
biota to the nutritional wellbeing of the host are well known and include the provision
of vitamin K, B group vitamins, B12, biotin, riboflavin, thiamine and folate (Koenig
et al. 2011; Said 2011) and amino acids (Yatsunenko et al. 2012). However, gut
microbes also make a vast array of proteins, peptides and low molecular weight
metabolites and chemically transform others of host or bacterial origin, all of which
impact on the structure and functionality of the microbiota and the health of the host
(Nicholson et al. 2012). Some bacteria help shape their local community by pro-
ducing bacteriophage or bacteriocins to kill off specific competitors. The production
of bacteriocins by commensal Escherichia coli can, for example, inhibit the invasion
of their niche by pathogenic E. coli O157:H7 (Schamberger and Diez-Gonzalez
2002). Others form feeding chains where one species performs an initial break down
of a starting nutrient source to release intermediate breakdown products that can
themselves provide substrates for other types of bacteria as exemplified using in vitro
fermentation studies to examine the degradation of dietary glycans (Leitch et al.
2007a, b) and host-derived mucins (Png et al. 2010). Others can induce the host to
create local environments that provide a colonisation and proliferation advantage for
themselves and potentially other bacteria (Collier et al. 2008), while others can have
direct effects on host functions e.g. the production of gamma amino butyric acid
(GABA) (Barrett et al. 2012), the major inhibitory neuro transmitter in the mam-
malian central nervous system (CNS) with a major role in the regulation of muscle
tone in humans. Although GABA produced peripherally does not cross the blood
brain barrier and hence is unlikely to impact on CNS functions, it may modify gut
motility and blood pressure (Inoue et al. 2003).
Likewise the host helps shape the gut microbiota through the secretion of
bioactive molecules into the lumen of the gut. Defensins, (Gallo and Hooper 2012)
secreted from the Paneth cells and most enterocytes of the gut mucosa, and IgA
(Cerutti 2008), secreted from plasma cells in the lamina propria and through the gut
epithelium, provide protection against overgrowth of many pathogenic microbial
species. Further, using a germ free/gnotobiotic maternal separation mouse model of
stress it has been demonstrated recently that stress can alter the colonic environment
leading to an altered colonic microbiota. While this altered microbiota was insuf-
ficient to recapitulate the stress-associated changes in host physiology upon
microbial transplantation to non-stressed germ free animals, transplantation was
associated with a transfer of anxiety-like behavior and behavioral despair but the
molecular effectors are yet to be defined.
So what are the key influencers of gut microbiota structure and function and how
might they elicit their impacts on health? While the gut of the near term foetus may
have some microbes present, the numbers are very low (Jimenez et al. 2008). The
mode of delivery (vaginal vs. caesarean section) impacts significantly on the
structure of the gut microbiota of the neonate (Dominguez-Bello et al. 2010) with
vaginally delivered infants developing bacterial communities resembling those of
their mother’s vaginal microbiota, while the gut microbiota of caesarean-delivered
infants’ reflected more closely those of their mother’s skin. Prior to weaning the gut
16 T.J. Lockett et al.

microbiota is relatively simple, dominated by Bifidobacteria and lactic acid bac-

teria. This changes significantly with the introduction of solid food, acquiring
something close to its mature composition by the time of their first birthday
(Backhed et al. 2015; Favier et al. 2002; Koenig et al. 2011). The base microbial
population structure of a human’s colon is relatively stable through childhood and
much of adult life but begins undergoing change and simplification with aging and
associated changes in health status, diet and lifestyle (Claesson et al. 2012). Despite
this stability of an individual’s core microbiota, components still show a dynamic
response, both in composition and biological functionality, to changing dietary and
environmental conditions (Clemente et al. 2012). Diet composition, in particular the
amounts and nature of non-digestible components of the diet, can modify the of gut
microbiota markedly (Gibson and Roberfroid 1995; Flint et al. 2012). Dietary fibre
is the best understood of these. Epidemiological studies have associated diets high
in dietary fibre with a reduced risk of a number of disorders including colorectal
cancer and inflammatory bowel diseases (Hou et al. 2011). Here we discuss what is
known about dietary fibre, the early research implicating it in promotion of gas-
trointestinal health, its interaction with the gut microbiota to produce a range of
bioactive metabolites and our growing understanding of how these metabolites
elicit their associated health benefits in the host.

2 Dietary Fibre and Gut Health: A Brief History

The high community awareness of the health potential of dietary fibre is relatively
new. This interest was triggered by early work of a number of British physicians in
South and East Africa. They saw that native Africans ate a diet high in whole grain
foods and were essentially free of the non-infectious diseases which affected
Europeans living in the same geographical area who ate highly refined foods
(Burkitt 1973). Initially, they focused on large bowel disorders including consti-
pation, diverticular disease, appendicitis and related problems. They linked these
differences to their dietary fibre intakes and suggested that the disease profile of the
Europeans reflected a simple dietary fibre deficiency. These early studies relied on
observation (not measurement) and were quite limited in scope. They were limited
also by the early definition of dietary fibre as plant cell wall material resistant to
human small intestinal enzymes i.e. indigestible and largely insoluble plant mate-
rial. The total indigestibility of these non-starch polysaccharides (NSP) explained
their faecal bulking and laxating effects and protection against large bowel diseases
very neatly (Cummings et al. 1992). However, fibre (as NSP) has proved very
disappointing for protecting against other diseases of affluence, especially colorectal
cancer (CRC), where it was expected to lower risk substantially. While some
studies (e.g. the European Prospective Investigation into Cancer and Nutrition,
EPIC) have shown a dose-dependent reduction in CRC risk (Murphy et al. 2012),
others have not (Park et al. 2005; Lanza et al. 2007). The Australian paradox also
fits with these inconsistencies. Australia seems to be one of the few countries where
2 Microbes, Metabolites and Health 17

fibre consumption has increased substantially over time with a reported population
average of 28 g/person/day (Baghurst et al. 1996) but CRC morbidity and mortality
rates remain high (AIHW 2012). This paradox is leading to a revision of the relative
importance of the different components which contribute to fibre intake in low risk
populations and also the way in which dietary fibre actually works in the large
bowel.
Much of the credit for the popularization of the dietary fibre hypothesis is due to
Dennis Burkitt (Trowell and Burkitt 1987) but it seems that the focus on fibre as an
indigestible bulking agent may be misplaced. It is now impossible to quantify actual
food consumption rates in the original African population. Changes due to
urbanization and higher incomes have altered the traditional rural lifestyle patterns
of native South Africans so that their activity levels and total dietary fibre
(TDF) consumption have fallen. TDF intakes have declined from an estimated 25–
35 g/person/day (2 generations ago) to 15–20 g/person/day (now) (Segal et al.
2000). One would have expected this to lead to an increase in CRC rates but this
does not seem to have happened. Comparison of the diet of indigenous Africans in
South Africa and African Americans in the United States shows that they are quite
similar in their low TDF content (O’Keefe et al. 2007) but the latter have one of the
highest global rates of CRC of any ethnic group. One suggestion is that the high
content of animal products in the African American diet is responsible. It is true that
CRC risk has been linked to greater consumption of red and processed meat (Norat
et al. 2005) but the work of Walker and others suggest very strongly that the type of
fibre consumed by Africans may be equally important (Walker et al. 1986). The
original concept underpinning the differential in disease risks was the high whole
grain consumption of the Africans. Put simply, the notion of “fibre” came later,
essentially as roughage (Topping and Illman 1986). However, it has emerged that
the culinary habits of the Africans are an important factor. Many native South
Africans consume reheated or cold maize porridge on a daily basis, particularly in
rural areas. Cooking gelatinises the starch, increasing its small intestinal
digestibility but cooling leads to self-association of the chains. This process (ret-
rogradation) leads to resistance to amylolysis i.e., the formation of resistant starch
(RS). Resistance to amylase is a key feature of TDF meaning that RS is also a fibre
component. It has been shown that hot traditionally cooked maize meal contains
18 g of RS/100 g while cooked and cooled maize contains a higher RS content than
that of a hot maize meal (Heneker et al. 1998). Cooking is not the only factor. The
amylose content of South African maize is 37–40 % of total starch which is rela-
tively high (37.1–39.9 %) (van der Merwe et al. 2001). Amylose is a relatively
compact starch polymer which is slower to gelatinize than amylopectin and also
quicker to retrograde meaning that it is digested more slowly than low amylose
starches and also yields more RS in food products.
One of the most important observations driving the reappraisal of the relative
importance of fibre (i.e., NSP and RS) was the lack of relationship between TDF
and bowel habit in native South African children. This has led to the current focus
on the role of the microbiota in human health, specifically through the production of
short chain fatty acids (SCFA). Knowledge of the existence of RS is not new nor is
18 T.J. Lockett et al.

the presence of SCFA in the large bowel of omnivores (Elsden et al. 1946). It was
known that TDF polysaccharides are lost during whole of gut transit (Schneeman
1986). For NSP, the extent of loss varies by source and ranges from *0 (for
cellulose) to >90 % for pectin. In contrast very little starch appears in normal
human faeces which, coupled with its susceptibility to amylolysis, gave the obvious
conclusion that small intestinal starch digestion was complete. The existence of
potentially significant levels of indigestible starch (i.e., amylase resistant starch—
RS) in foods was shown by Englyst and colleagues (Englyst et al. 1982). There was
earlier (indirect) evidence from Levitt’s laboratory (Anderson et al. 1981) in intact
humans showing that, following consumption of a low-fibre convenience cereal
product, breath H2 evolution rose significantly. Breath H2 is a marker for large
bowel fermentation and their observations can now be interpreted as showing the
presence of RS and also that fermentation was occurring. Both are now established
as significant factors in human gut physiology (Topping and Clifton 2001). RS is
defined in terms of starch which escapes into the large bowel and the products of its
fermentation. RS is today recognized as a component of fibre and not a separate
entity.
There are striking similarities between large bowel fermentation by the micro-
biota of humans and other omnivores and that of obligate herbivores (Topping and
Clifton 2001). The end products are similar i.e., SCFA (principally acetate, pro-
pionate and butyrate), gases (CH4, H2 and CO2) and the energy to fuel bacterial
growth. In humans and relevant model species (most notably pigs), SCFA levels are
highest in the proximal large bowel and decline with passage of the digesta stream.
This is consistent with the high production rates in the caecum and proximal colon
i.e., where substrate availability is greatest. The fall in SCFA levels along the colon
reflects substrate depletion but also their uptake by the large bowel so that <10 % of
total production is excreted in faeces. This distributional profile has very important
implications for gut health and is believed to relate to the higher prevalence of
non-infectious disease in the distal colon and rectum (Topping and Clifton 2001;
Cats et al. 1996).
SCFA have a number of general effects which contribute to large bowel health
(Topping and Clifton 2001; Brouns et al. 2002; Wong et al. 2006). Their production
acidifies the digesta which inhibits the overgrowth of potential pathogens and limits
the absorption of potentially toxic compounds e.g. NH3. The latter cannot enter
colonocytes as NH4+ so lower pH values limit their exposure to this and other
cytotoxic and genotoxic agents (Topping 2007). A study in normal humans con-
suming their usual diets with supplements of NSP or NSP + RS showed an increase
in faecal NH3 excretion which correlated closely with greater butyrate excretion
(McOrist et al. 2011). This is consistent with diminished absorption by (and less
exposure to) colonocytes. SCFA enhance visceral blood flow through dilation of
resistance vessels in the colon which facilitates O2 supply (Topping and Clifton
2001). The history of research in dietary fibre is littered with misconceptions,
revisions and reappraisals. The current focus on the microbiome is another case in
point. The emergence of modern technologies has given us an opportunity to
identify the species which are present and how they are altered by diet. Previously,
2 Microbes, Metabolites and Health 19

this was almost impossible using the techniques of traditional microbiology.

However, it should be noted that it is the products of bacterial action in the large
bowel which influence human health. While they have these general effects, the
individual acids have specific actions which benefit visceral function, health and
wellbeing.

3 Dietary Fibre: a Complex Mixture

Dietary fibre is not a single entity but an umbrella term for a large, diverse group of
substances, predominately but not exclusively carbohydrate polymers of plant
origin, that pass through the small intestine and undergo partial or complete fer-
mentation in the colon.
The global consensus definition of dietary fibre (Codex 2010), has broadened
from a botanical and structural focus (plant cell wall polymers) and now captures
other salient properties of fibre, notably compositional diversity and physiological
functionality.
The major classes of dietary fibre are NSP, RS and non-digestible oligosac-
charides although inclusion of the latter can vary with jurisdiction (Codex 2010).
Fibre also includes analogous compounds, non-digestible carbohydrate polymers,
of animal origin, and plant secondary metabolites that are intimately associated with
plant cell wall polysaccharides, notably lignin.
Fibres, both natural and synthetic, differ markedly in their physiological prop-
erties and capacity to promote human health (Dahl and Stewart 2015; Bird 2007;
Cummings and Stephen 2007; Buttriss and Stokes 2008; Brownlee 2014; Edwards
et al. 2015). While physiological functionality is included in the definition of fibre,
it is applied only to foods containing carbohydrate polymers sourced from either
raw food materials or chemically synthesized. A demonstrable beneficial physio-
logical effect in humans is a requirement for foods to be advertised as containing
added/functional fibre.
A prerequisite property and defining feature of fibre is that it is potentially
fermentable by colonic microbiota. In recent times there has been growing scientific
and commercial interest in highly fermentable fibres, notably non-digestible
oligosaccharides and resistant starches, owing to their favourable effects on gut
microbiota composition and activity, and host health (Bird 2007; Conlon and Bird
2015; Bird and Topping 2001; Broekaert et al. 2011, de Menezes et al. 2013).
NSP, mainly cellulose and hemicelluloses, and lignin, are the fundamental
structural elements of plant cell walls and have been integral to all descriptions of
dietary fibre (Miller Jones 2014). Cellulose and hemicelluloses are widely dis-
tributed in the plant kingdom and are ubiquitous in the plant foods that humans eat.
NSP typically comprise long chains of monosaccharides, mostly glucose moieties,
joined by b-1,4 linkages. Cellulose is a high molecular weight homopolymer
consisting of linear b-1,4 linked-chain of glucose units. The chains are closely
packed and the hydrogen bonds that form between adjacent chains ensure that this
20 T.J. Lockett et al.

structure is highly resistant to digestion by human hydrolases, which specifically

attack monosaccharide units linked by a-1,4 and a-1,6 bonds (Tungland and Meyer
2002; Englyst et al. 2013). Hemicelluloses, in contrast, are smaller, branched,
heteropolymers comprising a variety of monomeric units, commonly xylose and
arabinose, and mannose, glucose, rhamnose and glucuronic acid, joined by various
types of chemical bonds. Hemicelluloses comprise a large variety of glycans, both
water insoluble and soluble (Tungland and Meyer 2002; Englyst et al. 2013).
NSP generally conform to the notion of “insoluble fibre” and are concentrated in
highly fibrous foods such as cereal brans (Bird 2007). Fibres such as b-glucans and
arabinoxylans tend to be water soluble (water extractable). Other examples of
soluble non-starch polysaccharides include gum arabic (acacia gum), guar gum,
psyllium, pectins and alginates (Tungland and Meyer 2002; Englyst et al. 2013).
However, solubility is not strictly a fixed property. Mixed-linkage glucans, such as
those found in oats and barley, exhibit varying degrees of solubility depending on
the source, analytical methodology and grain processing (Tosh et al. 2008;
Agbenorhevi et al. 2011).
RSs are starches which escape small intestinal digestion. Like highly digestible
starches, RS is a polymer of glucose monomers joined a-1,4 and a-1,6 glycosidic
linkages at branch points and hence are not intrinsically resistant to digestion by
enteric enzymes of humans. Rather they are rendered less accessible to the amylases
of the upper gastrointestinal tract by some feature of the starch itself, e.g. its
physicochemical state and/or the physical complexity of the food matrix in which
the starch is presented e.g. whole grains or some feature of the host e.g. compro-
mised dentition or a rapid food transit through the upper GI tract (mouth, stomach,
small intestine) (Bird et al. 2000). In particular, the duration of exposure of ingested
starch to hydrolytic processes in the upper gut, which is governed by gut motility
and digesta transit rate, is a major determinant of the amount of dietary starch
reaching the colonic microbiota. RS therefore represents the sum of the undigested
starch and intermediates of starch digestion that are not absorbed in the small
intestine and hence enter the large bowel of healthy humans (Bird et al. 2010,
2012). While most fibres are reasonably stable to culinary practices, RS can be
formed or easily depleted by cooking and other processes involved in food pro-
duction and preparation (Bird et al. 2012).
Non-digestible oligosaccharides are important contributors to the total fibre
content of many plant foods. They are low molecular weight fibres that have a
degree of polymerization (chain length; DP) between 3 and 9 (de Menezes et al.
2013; Englyst et al. 2013). Fructans are polyfructoses consisting of 3–20 units with
a terminal glucose unit (Gibson et al. 2004; Roberfroid 2005). Food sources rich in
fructans include Jerusalem artichoke, onions, garlic and bananas. Legumes, such as
soybeans, contain galactooligosaccharides (raffinose, stachyose and verbascose)
(Muir et al. 2009). Cereal fructans consist of straight and branched chain fructose
polymers (Jenkins et al. 2011). Commercial sources of dietary fructans, commonly
derived from chicory, inulin and fructooligosaccharides (FOS), are extensively used
as food ingredients (Roberfroid 2007). Inulin is a large molecule with a DP of
2 Microbes, Metabolites and Health 21

between 2 and 60 fructose residues whereas FOS comprises smaller molecules with
a DP between 2 and 8. ‘Oligofructose’ is a partial enzymatic hydrolysate of inulin.
Fibre also encompasses a range of other plant substances, including lignin, cutin,
waxes, suberin, tannins, saponins and phytosterols, that are naturally associated
with botanical cellular structures (Bird 2007; Englyst et al. 2007; Miller Jones
2014). Lignin is a heterogenous polyphenolic ether, not a carbohydrate, that is
covalently bonded to cellulose (Tungland and Meyer 2002). Most of these sub-
stances are quantitatively minor constituents of human diets and are not metabo-
lized extensively by the gut microbiota (Topping and Clifton 2001). However, they
may impair fermentation of plant structural and storage polysaccharides (Stephen
1994; Edwards et al. 2012).
In certain disease settings, dietary carbohydrates that are normally digestible in
the upper gut (‘available carbohydrate’) can also make their way to the colon and
essentially function as dietary fibre. For instance, lactose can be an important
substrate for the microbiota in those individuals with lactase deficiency (Deng et al.
2015; Lukito et al. 2015; Wahlqvist 2015; Windey et al. 2015). The contribution of
this carbohydrate is dependent on the degree of lactase deficiency. Other bowel
diseases and conditions that compromise the functional capacity of the upper gut,
such as celiac and Crohn’s diseases, will also increase the amount of malabsorbed
carbohydrate, and other dietary constituents, reaching the colonic microbiota
(Conlon and Bird 2015).
Physicochemical and functional properties of fibre, including solubility in water,
viscosity and gel formation, and water holding capacity, have formed the basis of
various classification schemes. Soluble/insoluble fibre classification has proven
popular, however it is inconsistent with the accepted definitions for fibre and is an
unreliable predictor of physiological function. ‘Solubility’ relates to an in vitro
environment, not the gut lumen. The proportion of fibre that is solubilised is often a
function of the assay conditions used and in many instances the results are method
specific (Cummings and Stephen 2007; Englyst et al. 2007). The proportion of
soluble fibre can also change during food processing and passage of the fibre
through the upper gut (Arrigoni 2001; Comino et al. 2016).
Dietary fibres can be classified on their fermentability (Dahl and Stewart 2015;
Slavin 2001). But, gauging the rate and extent of fibre fermentation in vivo is
challenging, which is why knowledge of fermentation of fibre-rich substrates is
based largely on studies deploying in vitro techniques. These commonly use human
faecal inocula in static and flow-through incubation systems to simulate conditions
and events in the complex, anaerobic and highly dynamic micro-ecosystem of the
large bowel. As such, they provide limited indicative data on fibre degradation by
the colonic microbiota.
Generally, about 70 % of the fibre that is consumed by people on mixed diets is
considered to be fermented to completion in the colon (Slavin 2001; Topping and
Clifton 2001). However, individual fibres vary markedly in their fermentability
although most are partially fermentable. Water soluble fibres, such as those from
vegetables & fruits, are usually more easily fermented compared to insoluble fibres,
such as cellulose, which is quite resistant to fermentation. For instance, only about
22 T.J. Lockett et al.

30 % of the fibre contained in wheat bran is fermented completely in humans

(Topping and Clifton 2001). Not surprisingly, degradation of the smaller molecular
weight carbohydrates and soluble NSPs occurs in the more proximal regions,
whereas the breakdown of fibre that is more difficult to ferment occurs further along
the large bowel. In general, the colonic microbiota metabolises individual fibres in
the following order: mono- and oligosaccharides are preferentially fermented fol-
lowed by starches, soluble NSPs (soluble b-glucans, arabinoxylans) and then
insoluble NSPs (Bach Knudsen 2015). However, there are many exceptions to the
rule.
There are myriad factors that influence the rate and extent of fermentability of
carbohydrates aside from the colonic microbiota per se. Physicochemical attributes
are the primary intrinsic determinants, notably chemical structure (monosaccharide
composition, linkage type) and molecular size (chain length) but diet, food form
(matrix and particle size) and physiological factors that alter the gut microbial
habitat, such as digestive and absorptive capacity and digesta transit time, are also
important, as is the amount (dose/concentration) and composition of
fibre-containing foods in the diet.
The fibre content of most western diets is exceedingly low and consequently, so
too are fibre intakes. Many populations consume inadequate amounts of fibre,
falling well short of intake targets recommended by health authorities (i.e., *14 g
total fibre/1000 kcal or *30 g dietary fibre/day for an adult) (Dahl and Stewart
2015; Baghurst et al. 1996). Furthermore, there is a limited diversity of fibre
components in most modern diets. Insoluble NSP predominate (Baghurst et al.
1996; Conlon and Bird 2015). Oligosaccharides are consumed in much smaller
amounts, whereas resistant starch is essentially nonexistent in most modern diets
simply because modern processed and staple foods contain only small amounts
(Bird et al. 2012).
Ancestral hunter-gatherer diets provided five times more fibre than do contem-
porary diets (Eaton 2006, Leach 2007). The range of fibre sources would also have
been considerably greater than that consumed by modern populations, which
suggests that our early forebears harboured a much larger, richer and more diverse
gut microbiota.
Cereals and cereal products are the major source of fibre for most human pop-
ulations, but the variety of cereal sources is limited (Baghurst et al. 1996). Wheat
predominates and its increasing worldwide popularity explains why insoluble NSP
such as cellulose and arabinoxylans, are the major type of dietary fibre for most
people. Fruits and vegetables are the next most important source of fibre but they
too tend to supply mainly NSP (Baghurst et al. 1996). There is also an increasing
range of synthetic fibres (e.g. chemically modified resistant starches such as
cross-bonded starches) in processed foods. Most are derivatives of plant polymers.
NSP account for the major portion of dry matter (20–45 %; equivalent to 10–
25 g/day) supplied to the colon of individuals consuming western diets (Conlon and
Bird 2015). Monosaccharides and oligosaccharides account for a further 10 %, and
RS, which includes partial hydrolysis products, is unlikely to be more than 10 %
(Conlon and Bird 2015); Small quantities of sugar alcohols also serve as
2 Microbes, Metabolites and Health 23

fermentable substrates but the amount and type is very much diet dependent
(Grembecka 2015; Halmos et al. 2015; Muir et al. 2009; Tennant 2014).
Interactions between different fibre components in a food as well as its physical
form (matrix), in particular the structural integrity of plant cell walls, will influence
the rate and extent of fermentation of the component fibres. Milling, processing,
preparation and storage conditions of foods can alter their fibre content and func-
tionality and hence their impact on the gut microbiota (Tuohy et al. 2012). For
instance, soluble fibre may be lost through leaching during the cooking process.
Heat treatment may solubilise certain fibres, thereby increasing the ratio of soluble
to insoluble fibre. Short chain fructans can be degraded by yeast enzymes and also
exposure to acidic conditions. A sufficiently large particle size is needed to ensure
an intact cell wall structure resists extensive fermentation. Coarse bran loses both its
water holding capacity and some of its laxative effects when it is finely ground
simply because it is more extensively fermented in the large bowel (Topping and
Bird 1999).
Another important consideration is functional capacity of the upper gut. Small
intestinal digesta transit rate and nutrient assimilation can differ markedly among
people. For instance, some individuals are especially efficient in digesting and
fermenting starch (Thornton et al. 1987). Furthermore, some fibres may undergo
degradation in the upper digestive tract owing to the action of bacterial glycanases
in that region of the gut (Bach Knudsen 2015). However, the loss of fermentable
substrate is probably small.
Poorly fermented carbohydrates may alter the metabolic capacity of the gut
microbiota through various mechanisms. For instance, by acting as a physical
scaffold for colonic microbes, resident and transitory, it may facilitate the fer-
mentation process. Fibres in general also influence fermentation events by aug-
menting intestinal motility and flow of the faecal stream (Topping and Bird 1999;
James et al. 2015).

4 Dietary Fibre and Its Interaction with the Gut

Microbiota

The dietary fibres described in the previous section have distinct effects on the gut
microbiome. This can be because of their solubility, structure or where in the gut
the fibre is fermented. This differential effect will lead to selection of certain groups
of bacteria, specific fermentation outputs, like SCFA and gas production, and likely
inhibition of pathogenic bacteria.
Some of the most widely studied prebiotic fibres are inulin, fructooligosaccharies
(FOS) and galactooligosaccharides (GOS) with the latter two encompassing
numerous different types of fibres. All of these fibres are soluble and highly fer-
mentable which means they are fermented in the terminal ileum or the proximal
colon (Eswaran et al. 2013). FOS and GOS were both found to have a bifidogenic
24 T.J. Lockett et al.

effect and increased levels of Lactobacillus in both animals and humans (Davis
et al. 2011; Djouzi and Andiueux 1997; Kleessen et al. 2007a). These effects were
accompanied with higher levels of SCFA and a lower pH in the large bowel,
however FOS were found to favour an increase in butyrate whereas GOS advocated
for higher propionate levels (Djouzi and Andiueux 1997). FOS and GOS also had
differential effects on gas production in rats, with FOS feeding increasing both
hydrogen and methane excretion, but GOS only induced higher methane excretions
(Djouzi and Andiueux 1997). Inulin seems to have differential effects in animals
and humans. This difference was found albeit using a human microbiota-associated
rat model. In the rat model, no difference in Bifidobacterium spp. was observed after
inulin intake, but higher levels of fibre degrading bacteria Roseburia spp. and
Eubacterium rectale were observed and a reduction in potentially pathogenic
bacteria (Clostridium spp.) (Kleessen et al. 2007a; Van den Abbeele et al. 2011).
SCFA concentrations increased and pH levels reduced with inulin intake in ani-
mals. Human intervention trials have not been able to replicate these changes. In
these studies, higher levels of Bifidobacterium, Faecalibacterium prasnitzii,
reduced numbers of Bacteroides-Prevotella and potentially pathogenic bacteria
were observed (Kleessen et al. 2007b; Ramirez-Farias et al. 2009; Costabile et al.
2010). In a later study, Ramirez-Farias et al. (2009) showed that it was certain
species of Bifidobacterium; B. adolescentis, B. longum and B. bifidum that changed
with inulin intake. Another significant difference between the animals and human
studies was the absences of changes observed in faecal SCFA levels in humans
given inulin (Kleessen et al. 2007b; Costabile et al. 2010). Another way to evaluate
the effect of certain fibres is to remove them from the diet. This was done by
Halmos et al. (2015) in a randomised control crossover trial of a low Fermentable
Oligo- Di- and Monosaccharides And Polyols (FODMAP) diet (no inulin) and a
diet containing a typical level of FODMAP’s found in an Australian diet. This study
showed that by avoiding FODMAP the overall bacterial abundance, and the relative
abundance of Clostridium cluster XIVa (butyrate-producing bacteria) and the
mucus-associated bacterium Akkermansia muciniphila was reduced. However,
these changes did not result in differences in faecal SCFA levels, although pH was
higher on the low FODMAP diet.
A possible reason for not noticing any differences in faecal SCFA levels may
well be because >95 % of the SCFAs produced in the human colon are taken up by
the gut epithelia (Kleessen et al. 2007b; Costabile et al. 2010). This does not
exclude that there might be a difference in SCFA levels in the proximal colon
however, when SCFAs are measured in faeces, levels are diminished due to uptake
along the colon.
RS, can also change the gut microbiome and the metabolic components pro-
duced, however the RS type plays an important role. All of the RS types are
generally digested in the proximal and the transverse colon, but some are digested
throughout the colon like RS type 1 and 4 (Clarke et al. 2011b; Eswaran et al.
2013).
Animal studies have revealed great benefit and potential for RS as a prebiotic
fibre. A limited number of studies have been conducted using RS type 1; in animals
2 Microbes, Metabolites and Health 25

(rats), this type of RS did promote higher abundances of Bifidobacterium spp. and
increased SCFA levels at month five compared to control (Kleessen et al. 1997).
The RS type 2 fibre is often used in animal studies and seems to have a broader effect
on the gut microbiota. It was shown in rat and pig studies that RS type 2 can increase
the abundances of Bifidobacterium spp., Lactobacillus spp., Enterobacteriaceae and
Ruminococcus bromii compared to control animals (Conlon et al. 2012; Abell et al.
2011; Kleessen et al. 1997; Le Blay et al. 1999; Martin et al. 1998). These microbial
changes were accompanied by higher SCFA levels due to RS type 2 feeding with
certain studies observing specific increases in propionate (Conlon et al. 2012; Abell
et al. 2011) and butyrate (Conlon et al. 2012; Abell et al. 2011; Le Blay et al. 1999;
Martin et al. 1998) excretion. RS type 3 have also been shown in rat and pig studies
to select for Bifidobacterium spp., Lactobacillus spp. and Faecalibacterium
prausnitzii, but lower the abundances of gamma-Proteobacteria (Conlon et al. 2012;
Haenen et al. 2013). Again, these microbial changes were in conjuction with
increased SCFA levels, especially butyrate and propionate. The gut microbiota can
also be modified with RS type 4 with a rat study conducted with chemically modified
RS showing increases in Lactobacillus spp. and Parabacteroides distasonis com-
pared to control (Abell et al. 2011). This study also recorded increases in SCFA
levels.
Human studies have confirmed many of the changes observed in the animal gut,
however with a high inter-individual variation for both microbial and biochemical
composition. Studies in humans are generally conducted using RS type 2, 3 and 4.
RS type 2 and 3 have very similar effects in the human gut promoting higher levels
of R. bromii, F. prausnitzii and E. rectale compared to a Non-Starch Polysaccharide
(NSP) control (Abell et al. 2008; Martinez et al. 2010; Walker et al. 2011). For two
of these studies, SCFA were also measured and the RS consumption was associated
with higher levels of faecal SCFA levels, especially butyrate (Abell et al. 2008;
Walker et al. 2011; McOrist et al. 2011). This increase in faecal butyrate levels
successfully correlated with the increasing faecal abundance of F. prausnitzii and E.
rectale (Abell et al. 2008; Walker et al. 2011). Human research studies have been
conducted with RS type 4 showing beneficial effects on the gut microbiota with
higher abundances of Actinobacteria, Bacteroidetes and a reduction in Firmicutes
(Martinez et al. 2010), more specifically Clostridium cluster XIVa and IV,
Lactobacillus, B. adolescentis, P. distasonis and R. bromii (Clarke et al. 2011b,
Martinez et al. 2010; West et al. 2013; Le Leu et al. 2015). Reductions were also
observed in bacteria, Ruminococcus gnavis, R. torques and E. coli, previously
associated with gut diseases (Le Leu et al. 2015). RS type 4 consumption also
resulted in higher SCFA level in the faeces of volunteers consuming these fibres.
These substantial shifts in bacterial composition imply that the different RS types
have the ability to select for certain groups of bacteria, despite a relative high
inter-individual variation. Combined, these studies strongly suggest a key role for
R. bromii in the degradation of RS and these bacteria have also been found to be the
predominant ones to colonise RS (Leitch et al. 2007a; Ze et al. 2012). It is also
worthwhile keeping in mind that these prebiotic effects are not universal, as some
volunteers are non-responders. The response in any volunteer will always depend
26 T.J. Lockett et al.

on the initial composition of the gut microbiota which helps to explain the often
large inter-individual variation seen in human intervention studies examining the
gut microbiome.
Fibres, like NSP, with a very limited prebiotic effect because up to 75 % of the
ingested fibre is not fermented in the gut (Backman 2009; Roberts et al. 2010) and
no selective promotion of specific bacterial populations (Abell et al. 2008; Walker
et al. 2011) can still affect the gut microbiota. It is suggested that NSP can interact
with the intestinal bacteria via contrabiotic effects, whereby they prevent potentially
harmful interactions between bacteria and the gut epithelium that occur upon
dysbiosis (Backman 2009; Parsons et al. 2014; Roberts et al. 2010, 2013). The
contrabiotic effect has been tested for a range of soluble NSP’s and they are not all
equally effective. NSP from plantain bananas and broccoli seems to have greater
effect than NSP from apple and leek (Roberts et al. 2010). These tests have mainly
been conducted in vitro testing the ability of the fibres to block the attachment of
adherent, invasive E. coli (AIEC). Soluble plantain NSP’s have not only been able
to block AIEC but also adherent enteric pathogens, like Shigella spp., Clostridium
difficile, enterotoxigenic E. coli and Salmonella spp. (Parsons et al. 2014; Roberts
et al. 2013). Parsons et al. (2014) also demonstrated that NSP from plantain bananas
can block Salmonellosis in chicken when added to their feed and translocation of
Salmonella Typhimurium across isolated follicle-associated epithelium from human
ileum. It is believed that microfold (M)-cells overlying Peyer’s patches in the
human ileum and lymphoid follicles in the colon are potential points of entry for
AIEC (Roberts et al. 2010). It is suggested that the contrabiotic effect is mediated
via an interaction between the soluble NSP and the epithelial cell causing an
electrogenic chloride secretion preventing the adhesion of the pathogens (Parsons
et al. 2014). Another and more simple explanation is the direct interaction between
plantain NSP and E. coli, C. difficile and Salmonellae as they can all utilise plantain
NSP as an energy source (Roberts et al. 2010, 2013).

5 Dietary Fibre, Short Chain Fatty Acids and Their

Impact on Gut Health

Collectively, the microbes present within the GI tract can produce a vast array of
bioactive compounds, many of which can influence the functions of host tissues.
A comprehensive review of the many known gut microbe-derived metabolites and
their functions can be found elsewhere (Nicholson et al. 2012). We will focus our
discussion on the impacts of some of the primary fermentation end-products, par-
ticularly the SCFA, which are considered beneficial and largely a result of the
breakdown of dietary polysaccharides, and some toxic products such as ammonia,
phenols and hydrogen sulphide which are largely derived from fermentation of
dietary protein.
2 Microbes, Metabolites and Health 27

The wide degree of variation in microbiota composition between individuals

would be expected to be reflected in different gut microbial metabolite profiles and
susceptibility to diseases. For example, levels of SCFA in the large bowel of
humans vary by ten-fold, and although dietary and environmental factors contribute
to the variation, microbial differences appear to play a major part (McOrist et al.
2011). Our understanding of what represents a normal or healthy complement of
gut microbial taxonomic units is poor, as is our understanding of the factors which
can disturb the commensal microbial populations and potentially impact health.
Studies examining the cause(s) and potential treatments of inflammatory bowel
disease (IBD) have provided our greatest insights into this area.
Gut microbes play a central role in the causation of IBD. For example, it is not
possible to induce ulcerative colitis (UC) in germ-free animals (Sellon et al. 1998).
Also, antibiotics can improve symptoms of IBD (Khan et al. 2011), as can diversion
of faeces away from the large bowel of individuals with UC by creation of a tem-
porary ileostomy. Conversely, it is also possible to induce diversion colitis in
individuals with an ileostomy (Harig et al. 1989), which suggests removal of healthy
microbes from the colon also has a detrimental effect. It is also now well documented
that the gut microbiota populations of individuals with IBD are significantly altered
compared to community controls (Manichanh et al. 2006; De Cruz et al. 2012;
Rajilic-Stojanovic et al. 2013), adding further support for a role in causation. A key
change is a reduced diversity, largely a result of a decrease in bacteria of the gram
positive Firmicutes phylum, especially Clostridium clusters IV and XIV, whereas
some bacteria belonging to the Proteobacteriaceae, particularly E. coli and
Enterobacteriaceae, are increased in relative abundance. Other pathogens such as
Fusobacterium sp., Peptostreptococcus sp., Helicobacter sp., Campylobacter
sp. and C. difficile also tend to increase in number (Rajilic-Stojanovic et al. 2013).
Much of the diversity of the human gut microbiota seems to be a consequence
of the need to use their enzymatic and metabolic machinery to breakdown and
utilise the wide range of polysaccharides available to us as food. In the large
bowel, the cells of the gut mucosa are reliant on fermentation by the resident
bacteria to generate SCFA in order to maintain their integrity and function, and
polysaccharides derived from the diet are the principal substrates for this. Of
particular interest then, are the observations suggesting an altered microbial
capacity to generate SCFA occurs in IBD (Galecka et al. 2013; Machiels et al.
2014). For example, significant reductions can be seen for the key butyrate
producers E. rectale, Roseburia spp. (Rajilic-Stojanovic et al. 2013) and F.
prausnitzii, the latter of which also has some other anti-inflammatory properties
(Sokol et al. 2008). R. bromii plays an important role in the initial breakdown of
dietary resistant starch (Abell et al. 2008), a substrate that is readily converted to
SCFA, and reduced abundance of this species is also associated with UC
(Rajilic-Stojanovic et al. 2013). Added to this, there are now a number of studies
which have shown that treatments which increase butyrate in the large bowel can
improve colitis symptoms using experimental animal models (Komiyama et al.
2011; Vieira et al. 2012; Le Leu et al. 2013). In humans, rectal administration of
28 T.J. Lockett et al.

butyrate has been shown to improve diversion colitis symptoms (Harig et al.
1989), helping to prevent atrophy of colonic tissues (Luceri et al. 2016).
Enteropathogenic microbes such as certain strains of E. coli and Enterococcus
may contribute significantly to IBD and other gut conditions. In addition to the
evidence of raised numbers in affected individuals there is experimental evidence to
support such activities. To add weight to an important role for SCFA production,
and the microbes which mediate this, in helping to protect against effects of these
bacteria, the toxic effects of enterohaemorrhagic E. coli O157:H7 can be inhibited
in vivo through increased production of acetate (Fukuda et al. 2011b). The acetate
derived from bacteria of the genus Bifidobacterium was able to mediate this pro-
tection, suggesting potential for the use of appropriate strains as probiotics to treat
conditions where E. coli or other similar pathogens contribute to disease. It is
important to note that some strains of E. coli may benefit health e.g. by maintaining
insulin-like growth factor signalling to muscle and preventing wasting associated
with intestinal infections by certain pathogenic microbes (Schieber et al. 2015).
Nevertheless, increasing numbers of gram negative bacteria such as E. coli can
increase the risk of inflammation as components of their cell well, lipopolysac-
charide, promote inflammatory responses in tissues.
Despite numerous microbial species having been linked to IBD, it has not been
possible to consistently pinpoint any particular microbes as a cause. It is quite likely
that many gut microbes have the potential to trigger IBD under the right conditions,
especially those where host defences, particular the mucus gut barrier, are weakened
(Swidsinski et al. 2009). A large proportion of host immune defences reside within the
gut to deal with the threat of luminal microbes invading the tissues. One of the first lines
of defence is the mucus barrier which lines the gastrointestinal tract. Microbes are
present within the outer layer of this mucus, and many use it as a nutritional substrate,
but they are not normally present within the innermost layer. A breakdown of mucus
barrier integrity is implicated in IBD, in part because the numbers of bacteria that
contribute to large bowel mucus turnover have been shown to be altered in those with
the disease, i.e. A. muciniphila, Ruminococcus torques and Ruminococcus gnavus (Png
et al. 2010). Interestingly, some of these bacteria have also been found to be altered in
the stool of children with autism spectrum disorder, who also often report a significant
level of GI discomfort (Wang et al. 2011).
The maintenance of tight junctions between cells of the GI tract is also a critical
line of defence by helping to prevent passage of unwanted microbes or molecules
into host tissues. SCFA, but particularly butyrate, appear to play an important role
in the maintenance of tight junctions (Fukuda et al. 2011b; Suzuki et al. 2008).
A breakdown of this defence, a so-called ‘leaky gut’, may be a significant con-
tributor to inflammation in IBD and is also linked to a growing number of other
conditions (Michielan and D’Inca 2015). The recognition that a ‘dysbiosis’ of gut
microbe populations is a likely contributor to IBD has recently led to the testing of a
seemingly radical new treatment, faecal microbial transplantation (FMT), in which
an individual receives stool donated by healthy individuals. It is hypothesised that
the healthy complement of microbes will become established in the large bowel,
promote re-establishment of tight junctions, a protective mucus lining, repopulate
2 Microbes, Metabolites and Health 29

the mucus layer with commensal bacteria and thereby curtail microbe-mediated
inflammation. Although relatively unproven for treatment of IBD and other con-
ditions where microbes contribute to the etiology, FMT is now firmly established as
a successful means of treating C. difficile infections (which often resist all other
known treatments) through rectal administration and through ingestion of capsules
containing stool (Borody and Khoruts 2012; Mattila et al. 2012).
Gut microbes and their products also appear to play a role in the aetiology of
colorectal cancer (CRC). Individuals with IBD have an elevated risk of CRC,
suggesting that microbe-associated inflammation can contribute to oncogenesis. In
otherwise healthy individuals there is a significantly greater risk of CRC when diets
high in red or processed meat and low in fibre, which are typical of western style
diets, are consumed (Norat et al. 2005; Murphy et al. 2012). A range of mechanisms
has been proposed to explain this. Dietary protein can escape digestion and undergo
fermentation in the large bowel, generating toxic by-products which can include
ammonia, phenols, cresols, amines, and hydrogen sulphide (Windey et al. 2012).
N-nitroso compounds (NOC) are alkylating agents which can also be produced
from meat proteins by the action of gut microbes and have also been suggested as
contributing to CRC through their ability to form DNA adducts in human colonic
cells (Bingham et al. 1996). There is growing experimental evidence in animals and
humans that consuming high levels of dietary protein, particularly as red meat,
increases the level of DNA damage in the colon (which is regarded as a required
step in oncogenesis) (Toden et al. 2007; Winter et al. 2011; Conlon et al. 2012;
O’Callaghan et al. 2012; Le Leu et al. 2015). These studies also show that addition
of fermentable dietary fibre to the diet in the form of resistant starch protects against
this damage through mechanisms that appear to be at least partly mediated by the
actions of SCFA produced by the microbiota. Gut microbes, including members of
Lactobacillus, Bifidobacterium and Bacteroides genera, mediate secondary bile
acid formation and turnover (Nicholson et al. 2012). These acids, particularly
deoxycholic acid, are potentially carcinogenic and there is evidence that their levels
are higher when SCFA levels are lower (Ou et al. 2012). Numerous species of gut
microbes have been implicated in CRC, and these may act through a variety of
mechanisms, such as the production of compounds like toxins or forms of reactive
oxygen which damage tissues, or through exclusion of beneficial bacteria (Yu and
Fang 2015). Some of the bacteria implicated include Fusobacterium nucleatum,
invasive bacteria found to be strongly associated with the neoplastic tissues of CRC
patients (Castellarin et al. 2012), Bacteroides fragilis, Enterococcus faecalis,
Helicobacter pylori and Strepococcus bovis (Yu and Fang 2015).

6 Short Chain Fatty Acids, Inflammation and Allergy

The SCFA products of dietary fibre fermentation by the gut microbiota are pleio-
tropic metabolites that elicit their biological effects via both intra- and extra-cellular
mechanisms. While un-ionized SCFA can cross cellular membranes by simple
30 T.J. Lockett et al.

diffusion, with pKa values of around 4.8, the majority of these acids exist in the
ionised state at normal colonic pH. Therefore, most uptake of SCFA by the tissues
of the colonic mucosa is facilitated by membrane bound transporter proteins. These
include the low affinity/ high capacity transporters SLC16A1 (proton-coupled
monocarboxylate transporter-1, MCT-1), SLC16A3 (MCT-3) and SLC16A7
(MCT-16) which are important when SCFA concentrations are relatively high and
high affinity/ low capacity sodium-coupled monocarboxylate transporter-1
(SMCT-1; or SLC5A8) which are important when digesta and tissue SCFA con-
centrations are low. Low affinity receptors such as MCT-1 are distributed
throughout the gut with expression highest in the large intestine of most mammals
and reside on the apical (luminal) surface of the cell. In contrast, the high affinity
transporter SMCT-1, which also promotes water absorption, is expressed on the
apical epithelial membrane of the distal ileum and large intestine of mice, with
expression highest in the colo-rectum (Iwanaga and Kishimoto 2015). SMCT-1 is
also found on cells within the lamina propria (the tissue immediately below the
mucosal layers) where SCFA, particularly butyrate, influences immune cells and
enteric neurons (Vadivel et al. 2014). The relative affinity of SMCT-1 for its dif-
ferent SCFA substrates is: butyrate > propionate > lactate > acetate, which may
contribute to its proposed tumour suppressor role (Ganapathy et al. 2005).
Past research has focused on the ability of SCFA to modulate biological
responses by directly inhibiting histone deacetylases (HDACs) which results in
increased histone acetylation and altered gene expression. The potent HDAC
inhibitory effects of butyrate on the expression of genes that regulate proteins
involved in cellular apoptosis, cell cycle regulation and DNA repair that may
protect from colorectal oncogenesis (Fung et al. 2012) has received considerable
attention, although HDAC inhibition may be SCFA and tissue specific. Acetate has
recently been found to increase brain histone acetylation-state in rodents (Soliman
and Rosenberger 2011). The effects on the immune response of SCFA on HDAC
inhibition are largely anti-inflammatory (Tan et al. 2014) affecting not only cells of
the innate immune system (Lin et al. 2015) but also increasing Foxp3 gene
expression and the numbers and immunosuppressive function of T regulatory cells
in mice (Tao et al. 2007).
SCFA also modulate biological responses by activating G-protein-coupled
receptors (GPCRs), mainly GPR41 (FFAR3), GPR43 (FFAR2) and GPR109A,
which are expressed on the apical membrane of colonic epithelial and enteroen-
docrine cells (see reviews by Vadivel et al. 2014; Tan et al. 2014). GPR41 and
GPR43 are activated by all three SCFA with propionate the most potent agonist for
both GPR41 and GPR43, and with acetate more selective for GPR43 (Le Poul et al.
2003a). GPR109A is only activated by butyrate (Thangaraju et al. 2009) and is
expressed in adipocytes, intestinal epithelial cells and some immune cells (see
review by Singh et al. 2014). GPR43 was first described in neutrophils (Le Poul
et al. 2003a) and has since been described in other immune cells, the gastroin-
testinal tract including endocrine L-cells of the ileum and colon producing intestinal
peptide YY (PYY) and glucagon-like peptide 1 (GLP-1) (Tolhurst et al. 2012).
GPR41 is more broadly expressed in a number of tissues (Le Poul et al. 2003b).
2 Microbes, Metabolites and Health 31

The gut intestinal barrier consists of a layer of epithelial cells and products
including mucus, immunoglobulins and other antimicrobial agents and microbes
that interact to provide a selective barrier to protect the host. The barrier enables the
uptake of water, electrolytes, nutrients and other molecules while preventing the
entry of antigens and microorganisms (Camilleri et al. 2012). The epithelium forms
the main physical barrier between the gut lumen and the mucosal tissues with
absorption occurring either transcellularly by active transport or passive diffusion,
or through the paracellular spaces between the cells by passive movement. The
selective paracellular barrier is maintained by three adhesive protein complexes:
desmosomes, adherens junctions, and tight junctions (Groschwitz and Hogan
2009). The tight junctions seal the intercellular space and involve claudins and
occludins that form the paracellular pore, and scaffold and other cytoplasmic pro-
teins including zonula occludens (ZO-1, 2 and 3).
Intestinal barrier dysfunction enabling gut translocation of bacteria causing
inflammation and autoimmune disease has also been suggested as a causative factor
in a range of diseases including irritable bowel syndrome (Piche 2014;
Vivinus-Nebot et al. 2014), inflammatory bowel disease (Michielan and D’Inca
2015; Ploger et al. 2012) (IBD), food allergies (Perrier and Corthesy 2011), celiac
disease, asthma (Hijazi et al. 2004) and types 1 (T1D) and 2 (T2D) diabetes (de
Kort et al. 2011). Butyrate, but not acetate, decreased bacterial translocation in cell
models (Lewis et al. 2010) and enhanced intestinal barrier function in vitro by
modifying the expression of the tight junction protein Claudin-1 (Wang et al. 2012).
Elsewhere, acetate has been shown to enhance gut barrier function and protect mice
treated with E. coli O157:H7 from translocation of Shiga toxin from the gut lumen
(Fukuda et al. 2011a).
Impaired tight junction function has been found in patients with Crohn’s disease
(Zeissig et al. 2007) (CD) and ulcerative colitis (Heller et al. 2005) (UC) and first
degree relatives of patients with Crohn’s disease have increased intestinal perme-
ability (Peeters et al. 1997). Paracellular permeability is increased in patients with
quiescent IBD and IBS-like symptoms associated with persistent subclinical
intestinal inflammation (Vivinus-Nebot et al. 2014). Butyrate-producing bacteria
are reduced in the faeces of CD (Takahashi et al. 2016) and UC (Machiels et al.
2013) patients compared to healthy controls. The physical and chemical protective
mucus layer is thinner and more variable in thickness in UC disease patients (Pullan
et al. 1994); in rodents, diets containing resistant starch delivering high concen-
trations of butyrate to the large bowel increased mucus thickness (Toden et al.
2014). However, the use of butyrate enemas to control or maintain relapse in IBD
patients has had mixed results (Breuer et al. 1997; Scheppach 1996; Steinhart et al.
1996; Hamer et al. 2010) which may be due to difficulty maintaining prolonged
mucosal contact. Butyrate metabolism is impaired in the inflamed mucosa of IBD
patients, which may be due to a reduction in butyrate uptake due to a downregu-
lation of the MCT-1 butyrate transporter in IBD (Thibault et al. 2007), leading to
the suggestion that butyrate deficiency is a causative factor for the inflammation
(Thibault et al. 2010). Butyrate delivered to the colon by butyrylated starch
32 T.J. Lockett et al.

ameliorated colitis in an adoptive transfer mouse model whereas starches delivering

acetate and propionate had no protective effect (Furusawa et al. 2013).
Increased gastric and small intestinal permeability precedes the development of
diabetes in the BB rat (Meddings et al. 1999) and increased permeability resulting
in autoimmune destruction of the pancreatic beta cells may play a role in the
development of diabetes. Furthermore, glucagon-like peptide-2 (GLP-2), an
enteroendocrine peptide involved with satiety control released in response to the
ingestion of a prebiotic, improves barrier function and reduces LPS concentrations
in mice (Cani et al. 2009). Dietary resistant starch (RS) provides substrate for
colonic fermentation and the production of SCFA. These diets are associated with
improved bowel health, reduced abdominal fat and improved insulin sensitivity and
increased serum GLP-1 is likely to play a role in mediating these effects (Keenan
et al. 2015). Diets providing high levels of colonic SCFA may have potential to
improve metabolic control in type 2 diabetes (Puddu et al. 2014).
SCFA have been shown to affect the immune system through multiple mechanisms
that contribute to colonic health and homeostasis (see reviews by (Tan et al. 2014; Fung
et al. 2014; Vinolo et al. 2011b). These mechanisms include modulating chemotaxis
and the activity of macrophages and neutrophils (Maslowski et al. 2009; Vinolo et al.
2011a); the production of reactive oxygen species by neutrophils (Maslowski et al.
2009); suppressing pro-inflammatory and inhibiting anti-inflammatory cytokine pro-
duction by macrophages and neutrophils (TNF-a, IL-2, IL-6 and IL-10), the interaction
of neutrophils with endothelial epithelium (Vinolo et al. 2009); and modulating NF-KB
activity by inhibition of HDAC activity (Inan et al. 2000).
SCFA also induce Treg cells from naive CD4+ T-cell precursors in the colonic
lamina propria by inhibiting histone deacetylases and facilitating Foxp3 expression
(Furusawa et al. 2013, Arpaia et al. 2013). SCFA have been shown to expand the
existing Treg cell pool via a GPR43-dependent mechanism (Smith et al. 2013). Treg
cells have an important role moderating allergic and inflammatory responses
(Sakaguchi et al. 2008) and may also be induced through the production of TGFb1
by Clostridia species from clusters IV, XIVa, and XVIII (Atarashi et al. 2013). In
addition, butyrate promotes anti-inflammatory properties in macrophages and
dendritic cells in the colonic lamina propria and enables them to induce differen-
tiation of IL-10-producing Treg cells via GPR109A signaling (Singh et al. 2014).
The route of administration may determine the effects of SCFA. When indi-
vidual SCFA are administered by drinking water they are largely absorbed in the
small intestine and acetate and propionate delivered by this route are more effective
at accumulating colonic Tregs than butyrate, suggesting acid absorbed in the small
intestine may be important for migration of Treg cells into the colon from lymphoid
tissues (Smith et al. 2013). Butyrate delivered in the drinking water increased
peripheral but not thymic or colonic Treg cells, whereas butyrate delivered by
enema increased Treg numbers in the colonic lamina propria (Arpaia et al. 2013).
Likewise, butyrate delivered luminally to the colon via butyrylated starch induced
colonic Treg differentiation but propionate delivered from propionylated starch was
less effective and acetate from acetylated starch had no effect (Furusawa et al.
2013).
2 Microbes, Metabolites and Health 33

The anti-inflammatory effects of acetate administered in drinking water have

been demonstrated in animal models of colitis, arthritis and asthma. Acetate ame-
liorated disease symptoms implying a major role for the acetate-GPR43 axis
(Maslowski et al. 2009). Rodent studies suggest a maternal diet high in acetate may
reduce the incidence of asthma in offspring by influencing the expression of certain
genes in the fetal lung (Thorburn et al. 2015).
Early studies showed that SCFA are rapidly taken up in the large bowel with the
absorption of sodium and chloride (Rajendran and Binder 1994) and with the
secretion of bicarbonate (Fleming et al. 1991). SCFA have since been shown to
induce the activity of sodium-hydrogen exchange transporters (NHE) in intestinal
cells that promote the absorption of fluid and Na-H, SCFA-HCO3− and Cl-SCFA
exchanges (see review Binder 2010). Multiple isoforms of NHEs have been
identified in intestinal epithelial cells but the activity of NHE3 (Musch et al. 2001)
is increased by SCFA and with NHE2 is found in the small and large intestine of
humans (Hoogerwerf et al. 1996). Butyrate also stimulates intestinal NHE8
expression enhancing sodium uptake in vitro (Xu et al. 2015). The transport protein
(s) responsible for SCFA-HCO3− exchange have not been described (Binder 2010)
although a low-affinity, high capacity butyrate-HCO3− process and a high affinity,
low-capacity proton-monocarboxylate co-transporter have been described (Lecona
et al. 2008).
Unlike several other intestinal transport processes, SCFA-stimulated absorption
of fluid and electrolytes is not inhibited by mucosal cAMP (Ramakrishna et al.
1990). Mucosal cAMP is produced in response to enterotoxins secreted by many
bacterial pathogens responsible for acute infectious diarrhoea, including Vibrio
cholera (Kimberg et al. 1971), S. typhimurium, Shigella dysenteriae type 1 toxin,
Campylobacter jejuni (see review by Binder 2009). Butyrate has been found to
restore the activities of NHE2 and 3 isoforms inhibited by cAMP in vitro
(Subramanya et al. 2007) and also decrease by 40 % the cholera-toxin induced
increase in cAMP (Subramanya et al. 2007). Hence, delivering SCFA to the colon
has potential to reduce fluid and electrolyte loss and improve the clinical response
to oral rehydration solution (ORS) in patients with acute infectious diarrhoea.
SCFA can be produced in the colon by the fermentation of ingested RS, and RS
added to ORS promotes fluid and electrolyte uptake in a perfused-gut rat model of
cholera-toxin (CT) diarrhoea (Subramanya et al. 2006). It also reduces fluid loss
and recovery time in human adults (Ramakrishna et al. 2000) and duration of
diarrhoea in children (Raghupathy et al. 2006). Acylated starches are largely
resistant to small intestinal digestion and deliver specific SCFA rapidly to the large
bowel (Clarke et al. 2008, 2011c). Acetylated starch was more effective at pro-
moting fluid and electrolyte uptake than RS or acylated starch delivering propionate
or butyrate in the perfused-gut rat CT model (Clarke et al. 2011a). This may be the
result of more rapid and complete release of bound acetate resulting from the more
disrupted starch structure compared to starches acylated with propionate and
butyrate. The acetylated starch shortened the duration of diarrhoea in patients with
acute infectious diarrhoea (Pal et al. 2013) but not faecal volume.
34 T.J. Lockett et al.

7 Future Prospects

Modern techniques in metagenomics analysis continue to dramatically enhance our

understanding of how microbial taxonomy and the genetic potential of the human
microbiota change in health and disease and how they respond to changing envi-
ronmental conditions such as diet. However, this is only part of the equation.
Understanding how the function of the microbiota changes, how this impacts on the
health and wellbeing of the host and what levers can be pulled to efficiently shape
the microbiome and its function for improved health will be important areas for
future research.
Dietary fibre provides a glimpse into the multifaceted impacts that food and
xenobiotics can have on the microbiota and the host, and highlights the importance
of small molecule metabolites as mediators of change. As discussed above, pro-
vision of a fermentable substrate alone provides a growth advantage to bacteria in
the gut ecosystem capable of metabolizing these substrates. Selective pressure
within the gut microbial population is further modified by changes in the chemical
environment caused by the accumulation of the metabolic end products of fer-
mentation. Dropping digesta pH resulting from the accumulation of SCFA further
advantages some microbes over others while the SCFA themselves, pleiotropic
bioactive agents in their own right, provide vehicles to the host for energy salvage,
immunological signaling, promotion of gut homeostasis and repair, motility mod-
ification, cellular signaling (both via GPCRs and histone deacetylase inhibition) and
satiety promotion. But fibre is just one dietary substrate capable of modifying the
gut microbial structure and function. Other macronutrients entering the colon can
also promote the growth of other bacterial populations leading to the production of
other metabolites with different outcomes for other microbes and the host. Yet other
dietary molecules or phytonutrients may alternatively act by modifying the effi-
ciency of other microbial processes e.g. the inhibition of RS fermentation efficiency
by cutins and waxes (Edwards et al. 2012).
The human metabolome is very complex. Lipids alone have been estimated to
have the potential to generate 9000–10,000 different molecular species (Yetukuri
et al. 2008). With the gut microbiome carrying around 40 times the number of
protein coding genes as the human genome, it is likely that the metabolome pro-
duced by the gut microflora will be substantially more complex. While SCFA are
undoubtedly interesting and important metabolites, there are many other gut
metabolites of microbe, host or dietary origin, both known and yet to be identified,
that are capable of eliciting potent physiological effects. These include quorum
sensing and virulence factors that regulate the composition of the surrounding
microbiota as well as metabolites modulating host functions including the gut
immune system (Nicholson et al. 2005, 2012).
The challenge will be to bring together our growing understanding of factors
affecting the gut microbiome and the extent and variation in the gut metabolome
with knowledge of the associated impacts on host physiology to develop predictive
personalized approaches to health management and chronic disease prevention. But
2 Microbes, Metabolites and Health 35

there remains much to be done. Expanding our knowledge of the gut microbiome
and its metabolic potential will be important. Only some 10 % of the currently
sequenced open reading frames in the microbiome are recognized as encoding
proteins with an assignable or predicted structural or enzymatic function. Further
our understanding of the metabolome is in its infancy while attribution of the
physiological impacts of metabolites continues to be assessed on a candidate by
candidate basis (Martin et al. 2007). While there is much important work to be done
in the expansion of these individual catalogues (genes, their function; metabolites
and their impact on physiology) there is now a real need to bring these areas closer
together. With the increasing use of high resolution spectroscopy methods to
analyse metabolite profiles of microbial origin, opportunities now exist to statisti-
cally integrate large and complex metabolic and metagenomic data sets, using
informatics to reveal pathways and putative functional relationships previously
inaccessible. However, validation of these proposed relationships will be crucial. In
this regard, the development of improved in vitro fermentation systems that more
accurately represent the processes occurring in the gut digesta and the availability of
gut organoid culture systems that accurately recapitulate biological responses of the
gut mucosa to bioactive metabolites are providing an exciting prospect of a medium
throughput screening of these in silico predictions en route to more classical animal
and human substantiation of the strongest candidates. While we are only in the early
stages of this new, data-driven revolution, if it lives up to its promise then the path
to a more detailed understanding of the gut, its metabolites and their impact on
human health becomes clearer and the promise of translating this knowledge to help
inform the personalization of health care, a tangible and exciting prospect.

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Chapter 3
Exploring the Bioactive Landscape
of the Gut Microbiota to Identify
Metabolites Underpinning Human Health

Páraic Ó Cuív, Sriti Burman, Sian Pottenger and Mark Morrison

1 Introduction

North America, Europe and Australasia have amongst the highest incidences of
chronic gastrointestinal and metabolic diseases including inflammatory bowel dis-
eases (IBD), colorectal cancer (CRC) and obesity (Molodecky et al. 2012; Stevens
et al. 2012; Bray et al. 2013). Although once considered rare in large parts of the
world the incidences of IBD and obesity in particular have also been steadily
increasing in Asia, South America and the Middle East (Ng et al. 2014; Kaplan
2015). These diseases are associated with considerable socioeconomic costs; for
example, the estimated costs to the global economy from obesity approaches US$2
trillion per annum, which equates to 2.8 % of global gross domestic product (Dobbs
et al. 2014). Thus, there is an urgent need to develop more effective preventative
and therapeutic strategies to ameliorate the impacts of these diseases.
Genomic studies have revealed that IBD, CRC and obesity are underpinned by
specific host genetic susceptibilities that are considered to be necessary but often
not sufficient for disease to develop (Jostins et al. 2012; Peters et al. 2015; Locke
et al. 2015), and it is now recognised that environmental factors and lifestyle
choices also affect disease risk. Epidemiological studies also suggest that host
genetic, environmental factors and lifestyle choices either alone or in combination
does not fully explain disease risk implying that other risk factors remain to be
identified. With that context, the human gastrointestinal tract harbours a diverse
microbial community (gut microbiota) that provides a range of ecological and
metabolic functions relevant to host health and well-being (reviewed by Backhed
et al. 2005). Human- and animal-based studies have now also identified the gut
microbiota as an important risk factor in the aetiology of chronic gut diseases. First,

P. Ó Cuív (&)
S. Burman
S. Pottenger
M. Morrison

The University of Queensland Diamantina Institute, The University of Queensland,
Translational Research Institute, Woolloongabba QLD 4102, Australia
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 49

D.J. Beale et al. (eds.), Microbial Metabolomics,
DOI 10.1007/978-3-319-46326-1_3
50 P. Ó Cuív et al.

human studies have revealed that the microbiota varies between healthy and dis-
eased individuals (e.g. Turnbaugh et al. 2009; Qin et al. 2010; Nakatsu et al. 2015)
and these variations are associated with changes in the disease state (Cotillard et al.
2013; Nakatsu et al. 2015; De Cruz et al. 2015). Second, germ-free animals are
protected from disease but become susceptible following microbiota transfer
(Turnbaugh et al. 2006; Zackular et al. 2013; Schaubeck et al. 2016). Third, both
human- and animal-based studies have revealed these diseases are responsive to
interventions that modulate the activity of the gut microbiota including antibiotics
(Zackular et al. 2013; Murphy et al. 2013; Schaubeck et al. 2016), diet (Donohoe
et al. 2014; Quince et al. 2015), probiotics (Kadooka et al. 2010; Bassaganya-Riera
et al. 2012) and faecal microbiota transfers (Suskind et al. 2015).
Even though IBD, CRC and obesity are a heterogeneous group of diseases, they
are all characterised by an activated inflammatory response. Nuclear factor-kappa
B (NF-κB) is a master regulator of gut epithelial integrity and inflammation, and
activation of the NF-κB signalling pathway plays a key role in driving the
inflammatory response during the onset and progression of these diseases.
Consistent with this, the NF-κB signalling pathway is a validated therapeutic target
for the treatment of IBD (Atreya et al. 2008) (Fig. 1), and it is also a recognised
therapeutic target for CRC (Sakamoto and Maeda 2010), and for obesity and its
co-morbidities (Donath 2014; Esser et al. 2015). The NF-κB pathway is particularly
well recognised as a therapeutic target for IBD, however, many of the current
therapeutics are only partially effective and/or have significant side effects. For
instance, glucocorticosteroids can affect linear growth and bone health in paediatric
subjects; methotrexate can cause hepatotoxicity, and as a teratogen, the treatment of
female subjects is complicated; salicylates are associated with an increased risk of
bleeding. Similarly, the newer biologics (e.g. anti-TNFα factors) are expensive,
increase the risk of infection and suffer from a loss of response. Interestingly, the
gut microbiota plays a central role in modulating the host immune response and
specific gut microbes have been shown to possess potent NF-κB suppressive
capabilities that can ameliorate the inflammatory response (Ménard et al. 2004;
Sokol et al. 2008; Heuvelin et al. 2009; Petrof et al. 2009; Eeckhaut et al. 2012;
Khokhlova et al. 2012; Kaci et al. 2013). This suggests that exploiting gut
microbe-derived NF-κB suppressive bioactives may provide new opportunities to
maintain host health. In this Chapter, we examine our current understanding of the
host-microbiota interaction and outline strategies to identify and characterise the
NF-κB suppressive capabilities of the gut microbiota. In particular, we propose that
an integrated approach combining culture-dependent and independent approaches
with a more mechanistic dissection of the microbiota provided by improved cul-
tivation techniques, high-throughput functional screens and metabolomic and
genetic dissections is necessary to transform our understanding of gut health and
support the development of new preventative and therapeutic strategies.
3 Exploring the Bioactive Landscape of the Gut Microbiota … 51

Fig. 1 The NF-κB pathway as a validated drug target for the treatment of chronic gut diseases.
The NF-κB pathway can be activated by several mechanisms including microbe-associated
molecular patterns (MAMP; e.g. via lipopolysaccharide, flagellin from the gut microbiota),
damage associated molecular patterns (DAMP; e.g. via extracellular detection of normally
intracellular proteins) or cytokines. Targeting of the NF-κB pathway for the treatment of chronic
gut diseases is best recognised for IBD with glucocorticosteroids (corticosteroids), methotrexate,
salicylates (e.g. mesalazine, sulfasalazine) and anti-TNFα biologics interfering with pathway
signalling. However, this pathway is also increasingly targeted for CRC, and obesity and its
co-morbidities

2 The Human Holobiont: An Emergent Paradigm

of Human Health

The publication of the human genome sequence was a seminal milestone in our
history. Published with much excitement in 2001, it promised new insights and
understanding of what it means to be human (Venter et al. 2001; Lander et al.
2001). Initial estimates of the number of protein-coding genes deemed necessary to
explain the biological and phenotypic complexity characteristic of humans varied
widely, however, there was considerable surprise when it was revealed that the
human genome is comprised of as few as 25,000 genes (International Human
Genome Sequencing Consortium 2004). Humans are not autonomous and fol-
lowing a period of introspection it was increasingly recognised that our associated
microbiota provides a range of functions relevant to health and disease. Thus, in its
aftermath, there was an increasing call to sequence our second genome—that of the
human microbiota (Davies 2001; Relman and Falkow 2001). This international
effort to sequence the human microbiome has principally been led by the Human
Microbiome Project (HMP) funded by the US National Institutes of Health
(Peterson et al. 2009) and the MetaHIT Project (Ehrlich 2010) funded by the
European Union, with additional coordination of other global efforts mediated
through the International Human Microbiome Consortium.
Humans and their associated microbiota co-exist as a symbiotic multispecies
assemblage termed a “holobiont” that is defined as a physical association between a
52 P. Ó Cuív et al.

host and its associated microbiota for signifıcant portions of their life history
(Bordenstein and Theis 2015). The emergence of the holobiont concept has dra-
matically altered our perception of human health—where the role of microbes was
traditionally viewed from the perspective of infectious diseases—to one where the
microbiota is viewed as an integral component that contributes essential function-
alities relevant to the fitness of the holobiont. The assembly of a holobiont is a
dynamic process that impacts both the host and microbiota (Gilbert 2014). For
instance, the human gut provides a wide variety of ecological niches that are
characterised by a constant temperature, oxygen tension, humidity and nutrient
supply. This supports colonisation by a numerically abundant and diverse micro-
biota that in return helps prevent colonisation by potential pathogens, detoxifies
harmful compounds, produces essential nutrients and catalyses the biotransforma-
tion of dietary substrate so they can be utilised by the host (Fig. 2). The “holo-
genome” then is comprised of the genetic potential encoded by the host’s genome
and their associated microbiota (microbiome) and can thus be considered as an
extension of the host genotype itself. Notably, the hologenome is dynamic in terms
of its composition with the potential to change more rapidly than the host genome
alone via gene acquisition or loss which also confers a greater adaptive potential to
the holobiont (Quercia et al. 2014).
The holobiont concept provides a new paradigm for a more holistic under-
standing of the aetiology of chronic gut diseases. For instance, some of the genetic

Fig. 2 The emergent holobiont model of human health. The holobiont assembly impacts the
fitness of the host and its associated microbiota and is characterised by specific host–microbiota
interdependencies. The hologenome is comprised of the genetic capacity of both the human
genome and microbiome. The holobiont provides a new model to examine the impact of
environmental factors and lifestyle choices on host health and disease risk
3 Exploring the Bioactive Landscape of the Gut Microbiota … 53

susceptibility loci for these diseases also affect the ability of specific microbes to
colonise the gut (reviewed by Spor et al. 2011) suggesting that the contribution of
genetic susceptibility and microbiota composition to disease risk may be intrinsi-
cally linked. In addition, the holobiont has provided a framework on which the
impact of environmental factors and lifestyle choices on health and disease risk can
be dissected and this has informed the development of new strategies to rationally
modulate the holobiont phenotype to improve host health (Zeevi et al. 2015). The
holobiont may also represent an optimum biological system to bioprospect for
novel NF-κB suppressive bioactives as the gut microbiota has co-evolved with the
development of the host mucosal immune system. In particular, we hypothesise that
select microbes produce bioactives that actively suppress the NF-κB-mediated
immune response perhaps as an essential capability to allow for successful
colonisation and persistence. These NF-κB suppressive bioactives may have
specific attributes that are relevant to the development of new therapeutics including
high bioactivity, bioavailability and target site specificity, as well as stability in the
gut environment. Thus, these bioactives could potentially be used directly or serve
as lead molecules for the development of novel NF-κB suppressive therapeutics.
Alternatively, determining the mechanism by which they exert their suppressive
effects could help to identify new cellular targets that could be drugged by existing
or new therapeutics. Taken together the identification and characterisation of these
bioactives may help realise new opportunities to prevent or treat chronic gut
diseases.

3 Insights into the Structure: Function Capacity

of the Human Gut Microbiota

Our understanding and appreciation of the diversity and functional capacity of the
gut microbiota are largely based on distinct yet complementary culture-dependent
and culture-independent analyses of the gut environment (Fig. 3). Many of the
current reference strains used in gut microbiota research were first isolated in the
mid-twentieth century following the advent of techniques in anaerobic microbiol-
ogy (for a historical perspective see Rajilić-Stojanović and de Vos 2014). However,
it has long been recognised that the vast majority of gut microbes are resistant to
cultivation as revealed by the discordance between microscopic counts of microbial
cells and those recovered using traditional laboratory based cultivation. This phe-
nomenon was first described in aquatic environments and termed “the great plate
count anomaly” (reviewed by Staley and Konopka 1985). Instead, advances in
molecular biology and DNA sequencing technology culminated in the establish-
ment of culture-independent approaches to study the microbiota, based largely on
16S rRNA gene community profiling and metagenomics. A crucial discovery was
that the microbial 16S rRNA gene could be used as a molecular clock to infer
phylogeny and provide an estimate of microbial diversity (Woese and Fox 1977).
54 P. Ó Cuív et al.

Fig. 3 The analysis of the gut microbiota by culture-dependent and culture-independent

approaches. For culture-dependent approaches gut microbes are ideally recovered from gut
samples (e.g. faeces, biopsy tissue) as axenic cultures. The phylogeny and functional potential of
the isolates can then be assessed by 16S rRNA gene and/or genome sequencing. The functional
characteristics of the isolates can be assessed by phenotypic profiling. For culture-independent
approaches bulk DNA is typically recovered directly from gut samples and the DNA is then used
for 16S rRNA gene profiling and/or metagenomic sequencing. The diversity and functional
capacity of the microbiota can be assessed from the resultant sequence data

The 16S rRNA gene is approximately 1550 bp in length and has a divergence rate
of 1–2 % per 50 million years (Ochman et al. 1999). The gene is comprised of
conserved and (hyper)variable regions and this architecture has been exploited in
culture-independent studies to assess microbial diversity. Here, near full-length or
subsections of the 16S rRNA gene are amplified by polymerase chain reaction
(PCR) using primers targeting the conserved regions and the intervening variable
regions are used to infer phylogeny (Klindworth et al. 2013). The length of the 16S
rRNA gene sequence can affect phylogenetic assignment (Kim et al. 2011; Franzén
et al. 2015) and it does not provide any information on the functional potential of
the taxa identified. However, the development of metagenomic approaches, facil-
itating the sequencing of bulk DNA recovered from microbial communities has
now provided new opportunities to both assess microbial diversity through
sequencing of defined phylogenetic marker genes (Sunagawa et al. 2013) and the
functional capacity of the microbiome (Qin et al. 2010; Li et al. 2014a). In a
landmark study Qin et al. (2010) examined the microbiome of 124 subjects and
determined that it is comprised of a genetic pool of up to 3.3 million non-redundant
genes that is as much as 150× that of the human genome. In practical terms the
3 Exploring the Bioactive Landscape of the Gut Microbiota … 55

functional activity of an individual’s gut microbiome is supported by *500,000

non-redundant genes. In line with an earlier estimate (Yang et al. 2009), a subse-
quent study by Li et al. (2014a) identified over 9.8 million non-redundant genes in
the human gut microbiome. It is likely that the number of non-redundant genes
remains underestimated, however, metagenomics may have reached a point of
diminishing returns where a greater effort has to be expended to provide an even
deeper insight into the gene repertoire of the gut microbiota. Together, both 16S
rRNA-based profiling and metagenomic sequencing have provided a unique insight
into the gut microbiome and revealed that the vast majority of gut microbes remain
uncultured (reviewed by Rajilic-Stojanovic et al. 2007; Rajilić-Stojanović and de
Vos 2014).
The adult human gut microbiota is comprised of viruses, bacteria, archaea and
eukaryotes with the number of microbial cells inhabiting the adult human gut
outnumbering host cells by an order of magnitude (Savage 1977). The gut envi-
ronment is characterised by a host driven top-down pressure on the microbiota that
selects for a community of distantly related microbes with similar functional
capabilities ensuring redundancy of microbial processes essential for the host. In
contrast, intra-microbiota competition results in a bottom-up pressure that selects
for functional specialisation. Consequently, the structure of the gut microbiota is
characterised by distinct inter-subject variability although the core functional
capabilities of the microbiota (e.g. short chain fatty acid (SCFA) production,
vitamin biosynthesis) are largely conserved (Turnbaugh et al. 2009; Lozupone
et al.2012). The diversity and functional attributes of the bacterial and archaeal
communities in the human gut is best understood. The human gut microbiota is
dominated by bacteria affiliated with the phyla Firmicutes and Bacteroidetes with
smaller numbers of other phyla including Actinobacteria, Fusobacteria,
Proteobacteria and Verrucomicrobia also present (Rajilic-Stojanovic et al. 2007;
Lozupone et al. 2012). The diversity of the microbiota becomes increasingly
complex at deeper phylogenetic levels and the human gut can harbour up to several
hundred individual strains that vary substantially between individuals (Greenblum
et al. 2015; Yassour et al. 2016). The gut archaea have a low abundance and are
comprised of methanogenic and non-methanogenic archaea (Rieu-Lesme et al.
2005; Gill et al. 2006; Nam et al. 2008; Oxley et al. 2010; Ó Cuív et al. 2011a). The
methanogenic gut archaea are dominated by strains affiliated with
Methanobrevibacter and Methanosphaera spp. although the diversity of human gut
methanogenic archaea may be underestimated (Gill et al. 2006; Nam et al. 2008;
Scanlan et al. 2008; Mihajlovski et al. 2008; Ó Cuív et al. 2011a). Despite the
substantial inter-subject variability, the healthy gut microbiota has been shown to be
comprised of a core microbiota that is widely shared between individuals and that
includes some of the most abundant members of the microbiota (Tap et al. 2009;
Qin et al. 2010; Jalanka-Tuovinen et al. 2011; Sekelja et al. 2011;
Rajilic-Stojanovic et al. 2012; Martínez et al. 2013; Li et al. 2013), and an accessory
microbiota that is less widely shared and typically comprised of low abundance taxa
that are nonetheless metabolically active (Peris-Bondia et al. 2011).
56 P. Ó Cuív et al.

Both culture-dependent and culture-independent approaches have helped to

identify important differences to the structure-function activity between the gut
microbiota of healthy individuals and those with chronic gut diseases. For instance,
perturbations of signature bacterial species from the core microbiota have been
associated with chronic gut diseases (Qin et al. 2010; Nakatsu et al. 2015). In CD,
the abundance of specific core bacteria differs from the healthy gut (Kang et al.
2010; Mondot et al. 2011; Prideaux et al. 2013; De Cruz et al. 2015), these dif-
ferences are coincident with the onset of active disease (De Cruz et al. 2015) and a
restoration of their abundance may support remission (Dey et al. 2013; De Cruz
et al. 2015). Furthermore, in vivo and in vitro-based experiments have demon-
strated that structure-function differences between the healthy and diseased gut
microbiota are associated with variations in biological activities that are relevant to
gut health. While the immunoregulatory capacity of the microbiota remains largely
unknown several representative isolates from the core microbiota produce bioactive
factors that can suppress NF-κB activation (Ménard et al. 2004; Sokol et al. 2008;
Heuvelin et al. 2009; Khokhlova et al. 2012; Quevrain et al. 2016), modulate the
balance and/or activity of regulatory and effector T-cell populations (Atarashi et al.
2011; Atarashi et al. 2013; Qiu et al. 2013; Li et al. 2014b) and restore barrier
function (Martin et al. 2015), thus attenuating the host inflammatory response and
helping to maintain gut homeostasis. Based on these observations, members of the
core gut microbiota have been proposed as “next-generation” probiotics for the
treatment of chronic gut diseases (Neef and Sanz 2013). There continues to be a
growing appreciation of the NF-κB suppressive capabilities of individual members
of the core microbiota and other gut bacteria. However, while some of the NF-κB
suppressive factors produced by gut bacteria have been identified, in many
instances they remain to be determined (Kelly et al. 2004; Lakhdari et al. 2011;
Kaci et al. 2011; Santos Rocha et al. 2012; Kaci et al. 2013).
Advancements in DNA sequencing technologies continue apace and the cost and
speed at which sequence data can be produced and annotated continues to dra-
matically improve (Loman et al. 2012; Land et al. 2015). However, despite the
wealth of gut microbiome associated sequence data now available in the public
databases, the overwhelming majority of gene products have not been functionally
characterised. Indeed, it is estimated that up to 75 % of protein families are assigned
to uncharacterised orthologous groups and novel families (Qin et al. 2010; Ellrott
et al. 2010; The Human Microbiome Project Consortium 2012). This challenge is
further compounded by the fact that DNA sequence data are typically annotated
using automated pipelines with little manual curation resulting in the introduction
and propagation of annotation errors, and ultimately spurious function prediction
(Schnoes et al. 2009; Promponas et al. 2015). It is widely acknowledged that the
ability to functionally dissect the gut microbiome has not kept pace with DNA
sequencing technology and it is notable that the functions of over one-third of the
gene complement of the model organism and best characterised gut bacterium
Escherichia coli (E. coli) K-12 remain undetermined (Hu et al. 2009). This
shortcoming is increasingly being addressed (Nichols et al. 2011; Meng et al. 2012;
Paradis-Bleau et al. 2014; Rajagopala et al. 2014) supported largely by E. coli’s
3 Exploring the Bioactive Landscape of the Gut Microbiota … 57

ease of propagation and its amenability to genetic dissection. In contrast, the vast
majority of gut microbes are fastidious anaerobes that are not known to be
amenable to genetic dissection and hence their genetic potential remains cryptic.
This has led to suggestions that an increased effort must be expended to functionally
characterise existing gene sets as this will provide new insights into the microbial
factors supporting gut health or driving disease (Roberts 2004; Galperin and
Koonin 2010; Anton et al. 2014; Joice et al. 2014).
Based on these collective observations, we contend that new advances in
microbial isolation coupled with parallel developments in functional characterisa-
tion and dissection approaches will provide the best opportunities to develop
streamlined strategies to identify NF-κB suppressive and other types of bioactives
produced by gut microbes. In particular, considering the complexity of the gut
microbiota these strategies must be cost-effective, scalable and amenable to
automation, and the following sections provide an overview of each of these
aspects.

4 Bringing the Microbiome to Life: Culture-Dependent

Analysis of the Gut Microbiota

It is indisputable that the development of new approaches to isolate and propagate

fastidious gut microbes has not kept pace with those of culture-independent
approaches. In particular, microbial culturing is widely perceived to be a time and
labour intensive process and much information can now be provided without
having to isolate individual microbes (Table 1). Nonetheless, both approaches are
complementary and in some instances culture-dependent approaches provide the
best opportunity to dissect the functional capacity of the microbiota. For instance,
culture-dependent approaches allow specific axenic isolates to be directly linked
with NF-κB suppressive capabilities and, moreover, they provide a valuable
resource to test experimental hypotheses (e.g. Koch’s postulates).
The vast majority of gut microbes are strict anaerobes and require an environ-
ment with a low redox potential in which to grow. The history of isolating and
cultivating fastidious gut microbes extends from the late-nineteenth century
(Rajilić-Stojanović and de Vos 2014). Many of the techniques used in contempo-
rary laboratories were developed and adapted by Hungate (1969) and colleagues
(Eller et al. 1971; Macy et al. 1972; Bryant 1972; Balch et al. 1979) and have been
used to isolate and propagate facultative anaerobic microbes (e.g. E. coli), micro-
aerophilic microbes (e.g. Lactobacillus spp.), aerotolerant anaerobic microbes (e.g.
Bacteroides spp.) and obligate anaerobic microbes (e.g. Clostridium spp.) (Virginia
Polytechnic Institute and State University Anaerobe Laboratory 1975; Dowell et al.
1981). These techniques can be readily established and remain relevant today
although the ability to isolate and propagate fastidious anaerobic microbes has been
advanced by the development of anaerobic chambers for microbiological culturing
58 P. Ó Cuív et al.

Table 1 Culture-dependent analysis of the gut microbiota—opportunities and challenges

Culture-dependent Advantage Disadvantage
approach
Microbial isolation • Enables experimental hypotheses • Time-consuming and labour
to be evaluated (e.g. Koch’s intensive
postulates)
• Provides a resource for further
experimentation
Genomic • Enables the functional potential • Genomic data can be provided
characterisation of an isolate to be assessed by culture-independent means
• The 16S rRNA gene sequence • Genome annotations can result
can be associated with specific in a high number of genes of
functional genes unknown function
• Intraspecies genetic variability
can be assessed where multiple
isolates are available
Functional • Facilitates phenotypic profiling • Limited ability to genetically
characterisation (e.g. metabolic, physiological dissect microbial isolates
characteristics)
• Functional attributes can be
linked with the 16S rRNA gene
and/or genomic content

which further reduce the risk of inadvertent oxygen contamination and allow many
standard techniques (e.g. spread plates, streak plates) to be used to isolate target
microbes.
The distinct ecological niches present along the human gut can be challenging to
replicate in a laboratory environment particularly as the nutritional requirements of
many target microbes are unknown. The use of “habitat simulating” media has been
widely used to circumvent this challenge and typically includes sterile aqueous
extracts of faecal or rumen digesta, in addition to sources of amino acids, carbo-
hydrates and other nutrients (Eller et al. 1971; Barcenilla et al. 2000; McSweeney
et al. 2005; Lagier et al. 2012), leading to the isolation of phylogenetically diverse
gut microbes including bacteria that have specific host dependencies such as
Akkermansia muciniphila (Derrien et al. 2004) and the obligate symbiont seg-
mented filamentous bacterium (Schnupf et al. 2015). Although habitat simulating
media often support the growth of subdominant populations, their enrichment and
isolation is often complicated because of rapid overgrowth by fast growing,
numerically abundant microbes. More selective media have been developed for the
isolation of specific gut taxa including Bacteroides spp. (Livingston et al. 1978),
Bifidobacterium spp. (Ferraris et al. 2010) and Enterococcus spp. (Isenberg et al.
1970) by identifying specific nutritional dependencies, and promoters/inhibitors of
growth (e.g. antibiotics, bile salts, sodium azide). Alternatively, subdominant
populations can be enriched by selecting for a specific phenotype (e.g. spore for-
mation) and this has enabled taxonomically novel microbes to be directly recovered
on nutrient-rich habitat simulating media (Atarashi et al. 2013; Browne et al. 2016).
3 Exploring the Bioactive Landscape of the Gut Microbiota … 59

Accordingly, based on these, our own (Ó Cuív et al. 2011b, 2015) and other
(Rettedal et al. 2014; Ma et al. 2014) observations, many “uncultured” microbes
grow reproducibly well in vitro when isolated as axenic cultures. Thus, many more
novel gut microbes could be recovered if the practical considerations involved with
screening large numbers of microbial isolates under strict anaerobic conditions
could be overcome.
To improve the throughput of microbial isolation, Stevenson et al. (2004)
developed an approach called “Plate wash PCR” to recover axenic isolates of
previously uncultured bacteria from agricultural soil and the guts of wood-feeding
termites. Briefly, an inoculum is plated in duplicate on solid medium and following
growth the colonies are re-suspended en masse from one of the replicate plates and
the sample extracted DNA is screened using specific PCR primers. By this
approach, a broad range of growth parameters can be rapidly screened to determine
conditions supporting the growth of target taxa. Once identified, colonies from the
matching replica plate are grown in multiwell plates and screened with specific
primers to identify the target isolate. Plate wash PCR was successfully used to
isolate a Lachnospiraceae affiliated bacterium that inhibits colonisation of the
murine gut by Clostridium difficile VPI 10463 (Reeves et al. 2012), and it has been
adapted to support the isolation of human gut bacteria affiliated with the HMP’s
most-wanted taxa using a microfluidic platform (Ma et al. 2014). Goodman et al.
(2011) described a similar approach but determined the diversity of microbial
isolates recovered on the replica culture plate by 16S rRNA-based microbial pro-
filing. In addition, to further improve the throughput of the isolation process, a most
probable number (MPN) approach was used to create, in 384 well plates, person-
alised archived culture collections of axenic isolates directly from faecal samples
without picking individual colonies. The MPN approach is based on extinction
culturing, whereby diluting microbial cells so that ≤1 culturable cell is used as an
inoculum supports the production of axenic cultures (Button et al. 1993). This
favours the isolation of the most abundant rather than the fastest growing or most
culturable microbes and the MPN method has also been used to produce axenic
cultures of previously uncultured rumen bacteria (Kenters et al. 2011). Rettedal
et al. (2014) also used 16S rRNA profiling to profile gut bacteria recovered on a
broad range of solid growth media. Then, by a process termed cultivation-based
multiplex phenotyping, they combined growth on solid medium with antibiotic
selection and 16S rRNA profiling to selectively target and recover target bacteria
including members of the HMP’s most-wanted taxa (Fodor et al. 2012). Recently,
Browne et al. (2016) applied a similar approach to isolate spore forming bacteria
from the human gut.
Separately, Raoult and colleagues (Lagier et al. 2012) coined the term “cultur-
omics” and demonstrated that increasing the throughput of microbial isolation
greatly extended the number of cultured isolates from the human gut. By this
approach, 32,500 colonies representing 340 bacterial species and including 31
previously unidentified species were obtained using 212 culture conditions and
60 P. Ó Cuív et al.

three human faecal samples. Culturomics was also shown to be superior to

culture-independent approaches in its ability to detect bacteria that were below the
detection threshold of 16S rRNA profiling approaches (Lagier et al. 2012; Dubourg
et al. 2013). It is notable that these studies were performed using methodologies that
could be readily established in a standard microbiological laboratory (e.g. the use of
anaerobic jars to produce microaerobic or anaerobic conditions). It is likely that the
use of an anaerobic chamber could have further increased the recovery of fastidious
obligate anaerobes, however, manipulating large numbers of isolates in multiwell
plates and a confined environment is challenging. Interestingly, Raoult and col-
leagues (La Scola et al. 2014; Dione et al. 2015) discovered that the addition of
antioxidants to the growth medium permitted the growth of strict anaerobic bacteria
under atmospheric conditions. This observation could revolutionise our ability to
isolate fastidious gut bacteria particularly if it can be verified that their growth and
metabolic activity is similar under aerobic and anaerobic conditions, and it com-
plements advancements in automated colony picking robotic platforms that
are capable of operating in an anaerobic chamber.
The wealth of sequence data now available for gut microbes has also helped to
direct the isolation of gut microbes. For instance, Pope et al. (2011) described the
successful isolation of an uncultured bacterium affiliated with the
Succinivibrionaceae from foregut digesta samples collected from Tammar walla-
bies. Here, metagenomic data were used to partially reconstruct and model the
bacterium’s metabolism and physiological features, and then tailored culture con-
ditions were developed to direct the axenic cultivation of the bacterium by a process
termed metagenome directed isolation. Bomar et al. (2011) similarly used meta-
transcriptomic data to direct the isolation of an abundant Rikenella-like bacterium
from the gut of a medicinal leech. Recently, Oberhardt et al. (2015) developed a
web-based platform that uses a database of microbe-medium combinations to
predict media for microbes based on their 16S rRNA sequence. The exploitation of
sequence data to help bring the microbiome to life is a vital development as much
of these data languishes mostly unused in online databases.
The throughput of microbe identification has also been expedited by develop-
ments in matrix-assisted laser desorption/ionisation-time of flight mass spectrom-
etry (MALDI-TOF-MS) based analyses. The early classification of microbes was
primarily based on physiological and morphological characteristics (Virginia
Polytechnic Institute and State University Anaerobe Laboratory 1975), however,
the development of 16S rRNA-based phylogenetics allowed the genetic relatedness
of these isolates to be determined. The gold standard of 16S rRNA-based phy-
logeny taxonomy is based on the production of near full-length gene sequences that
are used to infer relatedness (Kim et al. 2011; Franzén et al.2015). The identifi-
cation of microbial isolates is typically achieved by low-throughput Sanger
sequencing of the 16S rRNA gene, however, due to its low rate of divergence, it is
widely recognised that the 16S rRNA gene is limited in its ability to provide
3 Exploring the Bioactive Landscape of the Gut Microbiota … 61

phylogenetic resolution of the microbiota at lower phylogenetic levels. Other genes

can also be used as phylogenetic markers [e.g. gyrB, rpoB (Sunagawa et al. 2013;
Fish et al. 2013)] but these are less well established and not routinely used. Instead,
MALDI-TOF-MS-based analyses now provide an alternative and in many respects
a superior means to identify microbial isolates. The ability to identify specific
isolates is typically based on the mass patterns of ribosomal or other abundant
housekeeping proteins and is determined by reference to a database of spectra
produced using representative isolates. This approach is particularly valuable in
providing a cost-effective rapid and sensitive assessment of intraspecies variability
without any prior knowledge of the strains being tested, although the ability to
distinguish between very closely related strains can be challenging (Sandrin et al.
2013). We anticipate that MALDI-TOF-MS-based identification of microbial iso-
lates will increasingly supplant 16S rRNA gene-based identification as the reference
databases become more comprehensive and the technology more robust and
affordable.
In summation, meaningful progress has been made to increase the efficiency and
throughput of microbial isolation and these have increased the diversity of gut
microbes that are available in international biorepositories (e.g. Biodefense and
Emerging Infections Research Resources Repository (BEI Resources), Deutsche
Sammlung von Mikroorganismen und Zellkulturen (DSMZ) GmbH) or that are
held in private laboratory culture collections. Continued advances in automated
microbial isolation and identification will further expedite these efforts and support
a more mechanistic dissection of the gut microbiota in the maintenance of gut
homeostasis and the prevention of chronic gut diseases, although important chal-
lenges remain. Much of our understanding of the diversity and functional capability
of the gut microbiota is based on analyses of faecal associated microbiota, which
can be collected in a non-invasive manner and up to 54 % of the faecal mass is
comprised of microbial biomass (Stephen and Cummings 1980; Rose et al. 2015)
thus providing copious material for experimental interrogation. However, it has
been long recognised that the faecal and mucosa associated microbiota differ
(Zoetendal et al. 2002; Ott et al. 2004; Lepage et al. 2005; Eckburg et al. 2005) and
it is now also recognised that the mucosa associated microbiota is also characterised
by a distinct biogeography (Obata et al. 2010; Aguirre de Carcer et al. 2011; Pedron
et al. 2012; Sonnenberg et al. 2012; Zhang et al. 2014) that likely reflects different
ecological niches driven by variations in nutrient availability, oxygen tension, pH
and immune activation (reviewed by Donaldson et al. 2015). The aetiology of
several chronic gut diseases is characterised by site-specific differences with CD
predominantly affecting the ileum and proximal colon, and UC and CRC pre-
dominantly affecting the distal colon. Thus, the spatial distribution of the gut
microbiota may have implications for our understanding of host–microbe interac-
tions and their relationship to health and disease, and future efforts should seek to
preferentially culture gut microbes from sites relevant to disease.
62 P. Ó Cuív et al.

5 Dissecting the Functional Potential of the Gut

Microbiota: Advances in In Vitro Approaches
to Identify NF-κB Suppressive Gut Microbes

In 2014 it was reported that over 1000 cultured gut microbial species had been
described in the scientific literature and this number continues to increase rapidly
due to the new advances in microbial cultivation techniques (Rajilić-Stojanović and
de Vos 2014). The NF-κB suppressive activities of the vast majority of existing
isolates have not been assessed but taken together with the increasing rate of
microbial isolation there is a need for improved functional screening strategies to
effectively identify these strains. Strategies to identify immunomodulatory microbes
should address three key criteria. First, the assays should be biologically relevant,
sensitive and specific, facilitating the identification of virulent or cytotoxic microbes
at an early point in the screening process. Second, the assays should allow the
extent of immunomodulatory activity to be quantified and the host pathways
affected to be readily identified and dissected to determine the target of the
bioactive. Third, the assays should be cost-effective and robust, easy to perform and
amenable to scaling to an automated high-throughput format. Historically, the use
of well-established cell lines to identify NF-κB suppressive microbial isolates
broadly fulfils these criteria.
The NF-κB pathway has been extensively characterised and transcription can be
activated via two alternate pathways, called the canonical and non-canonical path-
ways. These pathways can be activated either independently (e.g. TNFα/IL-1β
activates the canonical pathway, B-cell activating factor activates the non-canonical
pathway) or in tandem (e.g. CD40L/Lipopolysaccharide activate both pathways).
Many gut bacteria are considered to be pathobionts—symbionts that are capable of
acting as pathogens under certain environmental conditions—and are capable of
stimulating an immune response. Consequently, the ability of gut bacteria to supress
NF-κB activation is often initially assessed using peripheral blood mononuclear cells
(PBMC) as several studies have reported that peripheral blood cells predict the
in vivo immunomodulatory potential of different bacteria (Foligne et al. 2007; Sokol
et al. 2008). Alternatively, NF-κB suppressive capability can be assessed using
peripheral blood derived cell lines (e.g. human monocyte-like THP-1 cell line,
murine RAW macrophage cell line) stimulated with a specific NF-κB pathway
agonist. These cell lines have rapid and reproducible growth characteristics and they
express a broad range of Toll-like receptors (TLR) [e.g. THP-1 cells expresses all
TLRs including the surface TLRs (i.e. TLR1/2, TLR2, TLR4, TLR5 and TLR6/2)].
These characteristics can be used to identify microbes that modulate NF-κB activity,
or that express virulence or cytotoxic factors, in a high-throughput manner.
Despite the usefulness of immune cell lines the ability of gut microbes to sup-
press NF-κB activity is typically assessed using intestinal epithelial cell culture
lines. Numerous epithelial cell lines are widely used by researchers, however, the
HT-29, Caco-2 and T84 cell lines and their derivatives are amongst the most widely
used to assess immunomodulatory activity. Gut epithelial cells are constantly
3 Exploring the Bioactive Landscape of the Gut Microbiota … 63

exposed to microbial factors and are thus broadly unresponsive to stimulation by

the healthy gut microbiota. Consistent with this, HT-29, Caco-2 and T84 cells
express a subset of functional TLRs (e.g. TLR2, TLR3, TLR4 and TLR5) (Cario
et al. 2000; Melmed et al. 2003; Lakhdari et al. 2010), and the cell surface receptors
are predominantly expressed basolaterally. Several gut epithelial cell lines carrying
NF-κB reporter genes including secreted embryonic alkaline phosphatase (Lakhdari
et al. 2010), luciferase (Kaci et al. 2011) and green fluorescent protein
(Mastropietro et al. 2015) have been described. Using reporter cell lines, Blottière
and colleagues at the Institute National de la Recherche Agronomique
(France) have led efforts to identify gut bacteria and metagenomic clones capable of
modulating NF-κB expression using high-throughput screening approaches (e.g.
Lakhdari et al. 2010, 2011; Santos Rocha et al. 2012). In the most exhaustive study
to date, Lakhdari et al. (2011) used a series of immune and intestinal epithelial
reporter cells to determine the NF-κB suppressive capabilities of 49 strains of
well-described gut bacteria. Interestingly, thirteen NF-κB suppressive strains were
identified although their activity was cell line-dependent (one isolate suppressed
NF-κB activation in HT-29 cells whereas the other twelve isolates suppressed
activation in Caco-2 cells) suggesting that the responsiveness may be affected by
the genotype of the cell lines.
While cancer derived intestinal epithelial cell lines may provide biological
insights relevant to CRC, a major criticism is that they typically lack the genetic
susceptibilities relevant to IBD and obesity. For instance, IBD is associated with
over 160 genetic susceptibility loci (McGovern et al. 2010; Jostins et al. 2012) and
is characterised by disease heterogeneity with differences in location, severity and
extent that may change over time. Host genetics can also influence therapeutic
responsiveness and CD carriers of the nod2 mutation are more likely to be
refractory to glucocorticosteroid treatment although they can be effectively treated
by TNFα biologics (Niess et al. 2012). While primary cells can be used as an
alternative to immortal cell lines, they have a finite life span which typically pre-
cludes long-term study. Also, the diversity in cell lineages found in the gut
epithelium (e.g. epithelial, goblet, enteroendocrine, Paneth cells) is not reflected in
homogenous primary cells or immortal cell lines. This issue has been addressed by
recent advances in gut epithelial culture methods from human and laboratory ani-
mals which have resulted in the generation of “mini-guts” from intestinal samples
containing adult, human embryonic or inducible stem cells that retain the phenotype
of the tissue of origin. Mini-guts produced from embryonic or induced pluripotent
stem cells are termed induced intestinal organoids while those produced from adult
stem cells are termed enteroids (small intestinal) or colonoids (colonic) (Stelzner
et al. 2012). Induced intestinal organoid cultures take longer to establish and retain
a foetal phenotype and consequently enteroids/colonoids are considered to be a
more representative model for human disease. Enteroids/colonoids are derived from
intestinal samples containing adult stem cells following cultivation in the presence
of growth factors and ultimately form three-dimensional cultures containing dif-
ferentiated epithelial cells (Sato and Clevers 2013; VanDussen et al. 2015; Mahe
et al. 2015). The cultured cells can be grown as spheroids with the apical membrane
64 P. Ó Cuív et al.

facing a single internal lumen compartment or, alternatively, they can be grown as
monolayers in a transwell system. These cell cultures can also be stably maintained
through repeated rounds of propagation and freezing thus recapitulating the main
elements of cancer cell culture lines and providing a superior in vitro model to
assess NF-κB suppressive capabilities. For instance, the impact of NF-κB sup-
pressive bioactives on individual epithelial cell subtypes could be assessed by
fluorescence-activated cell sorting using antibodies targeting the NF-κB complex
and lineage specific markers. Enteroids/colonoids can be generated from animals
carrying reporter genes or, alternatively, Schwank et al. (2013) reported that the
CRISPR/Cas9 system could be used to edit organoid genome sequences. Along
with new developments in CRISPR/Cas9-based large fragment deletions and
insertions (Wang et al. 2015; Zhang et al. 2015a), this may provide new oppor-
tunities to produce patient-specific reporter cell lines. Together, these developments
offer new opportunities to identify and dissect disease-specific pathways as well as
assess their responsiveness to different therapeutics.

6 Metabolomic-Based Strategies to Identify NF-κB

Suppressive Bioactives

The healthy gut microbiota produces a diverse array of factors including proteins
(Rieu et al. 2014), peptides (Kaci et al. 2011; Quevrain et al. 2016),
polysaccharide-peptidoglycans (Matsumoto et al. 2009) and secondary metabolites
(Bansal et al. 2010; Gonzalez-Sarrias et al. 2010; Lim et al. 2015; Lee et al. 2015)
that are capable of suppressing NF-κB, revealing this capability is characterised by
a high degree of functional redundancy. Metabolomic approaches have played a
central role in the identification of these factors although they have been challenged
by the sheer diversity of metabolites produced by gut microbes. In addition, many
of these metabolites are produced at low concentrations and include novel
metabolites that are not represented in existing databases, further hindering iden-
tification efforts. Nonetheless, effective bioassay guided fractionation strategies that
typically involve successive fractionation coupled with functional assays to track
the fraction(s) retaining suppressive activity can be devised to identify NF-κB
suppressive bioactive factors (Fig. 4).
Microbes in the healthy gut environment are physically separated from epithelial
cells by a mucus layer and bioactive factors must be capable of traversing this
barrier to reach their cellular target. Many NF-κB bioactives are secreted into the
extracellular milieu and the first stage of the screening process involves the
preparation of a cell-free supernatant fraction of spent medium that can be assessed
for suppressive activity. The supernatant fraction of most fastidious gut microbes is
likely to contain SCFA which are produced by anaerobes as an end product of
fermentation, and are amongst the most abundant metabolites produced. Acetate,
propionate and butyrate are produced at the highest concentrations with other SCFA
3 Exploring the Bioactive Landscape of the Gut Microbiota … 65

Fig. 4 Bioassay guided fractionation strategy to enrich and purify NF-κB suppressive bioactives.
The bioactive fractions are successively fractionated and the NF-κB suppressive activity in the
fractions is assessed after each treatment. Fractions enriched in primary and secondary metabolites
(1° and 2° respectively), and peptides are typically produced by size fractionation.
Fractions >3 kDa are typically not considered to be suitable for drug development but may help
to identify new drugable targets. Metabolites and peptide bioactives can be further fractionated
using biochemical treatments (e.g. protease, denaturant, thermal treatment). Peptide bioactives can
be further fractionated and used to identify new drugable targets or as lead molecules for drug
development. The 1° and 2° metabolites can be further fractionated (e.g. solid phase extractions,
HPLC based fractionation) to identify the bioactive factor. Secondary metabolites can also be used
to identify new drugable targets or as lead molecules for drug development

produced at lower concentrations. SCFA bind to G-protein coupled receptors

including GPR41 and GPR43 (acetate, butyrate, propionate), GPR109A (butyrate)
and OLFR78 (lactate, propionate). These receptors are found on a range of cells
including immune and epithelial cells (Karaki et al. 2008; Pluznick et al. 2013;
Tazoe et al. 2009; Thangaraju et al. 2009; Vinolo et al. 2011). SCFA are amongst
the most bioactive metabolites produced by the microbiota and they affect a variety
of cellular process including NF-κB activity (Inan et al. 2000; Tedelind et al. 2007;
Lakhdari et al. 2011). To identify culture supernatants possessing non-SCFA
suppressors of NF-κB activity, the SCFA concentrations in the spent culture
supernatant are typically first determined and the ability of similar concentrations of
SCFA to suppress NF-κB activation is then assessed (Lakhdari et al. 2011).
A variety of fractionation strategies have been described to differentiate between
bioactives with specific biochemical characteristics. For instance, small peptides
and secondary metabolites are considered to be more conducive to drug develop-
ment as they are more likely to possess desirable characteristics and be less costly to
produce (Uhlig et al. 2014; Fosgerau and Hoffmann 2015; Harvey et al. 2015).
These bioactives can often be readily separated from larger macromolecules on the
66 P. Ó Cuív et al.

basis of size and a simple 3 kDa molecular weight cut-off filter allows peptides up
to 27 amino acids long (assuming an average amino acid size of 110 Da) to be
easily separated from larger molecules. Bioactive secondary metabolites and pep-
tides can subsequently be distinguished using routine (e.g. protease, denaturing,
thermal) treatments and the sample can subsequently be further fractionated (e.g.
solid phase extractions, HPLC based fractionation) to further reduce the complexity
of the samples. This approach has been used effectively to identify
NF-κB suppressive peptides produced by Faecalibacterium prausnitzii (Quevrain
et al. 2016). However, the identification of secondary metabolites can be more
challenging and it can be necessary to fractionate large sample volumes to identify
the metabolites of interest, although this process can be expedited if isogenic mutant
or non-suppressive strains can be processed in parallel (Donia et al. 2014). Once
sufficiently enriched and concentrated the bioactives can be identified using spe-
cialist metabolomic methodologies and equipment.

7 Genetic-Based Strategies to Identify NF-κB

Suppressive Bioactives

Much of our understanding of the functional capacity of the microbial world has
been provided by the genetic dissection of clinically and agriculturally relevant
bacteria that are only distantly related to the microbes that typically inhabit the
human gut. The ability to conclusively link genes and function is a central challenge
in elucidating the functional potential of the microbiota. However, with limited
exceptions (Rey et al. 2013; Ichimura et al. 2013), few molecular tools have been
described for the characterisation of gut microbes. The vast majority of the currently
available microbial isolates are not known to be amenable to genetic transformation
although (meta)genomics has revealed evidence of extensive lateral gene transfer
within the gut microbiome. To address this challenge, we recently developed an
innovative approach termed metaparental mating that expedites the directed isola-
tion of genetically tractable gut bacteria from mixed microbial communities (Ó
Cuív et al. 2015). The metaparental mating approach is based on the
well-established biparental mating approach (Simon et al. 1983; Simon et al. 1986)
and uses RP4 (RK2)-mediated bacterial conjugation and a broad host range
mobilisable shuttle vector. Metaparental mating has several advantages over
alternative natural (i.e. transduction, transformation) or contrived (e.g. electropo-
ration, sonoporation) genetic transformation approaches. First, RP4-based conju-
gation is very promiscuous and has been shown to mediate the transfer of DNA to a
diverse range of bacteria (Whitehead and Hespell 1990; Picardeau 2008; Tolonen
et al. 2009; Dominguez and O’Sullivan 2013) and also to archaea (Dodsworth et al.
2010), fungi (Nishikawa et al. 1990) and animal cells (Waters 2001). Second, the
metaparental mating can be performed under anaerobic conditions and stably
transformed recipients can be recovered by selection of a vector encoded marker. In
3 Exploring the Bioactive Landscape of the Gut Microbiota … 67

addition, as the antibiotic resistance phenotype of the recipients may not be known,
the laboratory E. coli ST18 donor strain can be efficiently counter selected without
antibiotics by nutritional auxotrophy (i.e. the omission of δ-aminolevulinic acid
from the selection medium). Third, the RP4-based conjugation can be readily
scaled and automated (Clarke et al. 2005) to increase the throughput of the meta-
parental mating mediated isolation process.
We used the metaparental mating approach to specifically target bacteria affili-
ated with the Firmicutes as these comprise the majority of the human gut microbial
core although they are underrepresented in microbial culture collections.
Furthermore, few of these bacteria, and in particular those affiliated with the
Clostridia, have been genetically characterised although many strains are capable of
modulating the host immune response (Sokol et al. 2008; Ivanov et al. 2009;
Atarashi et al. 2011, 2013; Li et al. 2014b; Quevrain et al. 2016). In support of this
effort we developed a series of modular vectors termed pEHR5 that can be con-
jugated from an E. coli host to a pool of potential recipients. As the efficiency of
conjugation can be affected by the size of the vectors, their modular architecture
helps minimise their overall size. In addition, it allows individual modules to be
easily exchanged ensuring that the base vectors are flexible and can be readily
re-purposed. Similar modular vectors have been used in a broad range of non-
E. coli hosts to support protein expression and the construction of fluorescently
labelled bacterial strains (Herrero et al. 1990; Charpentier et al. 2004; Fodor et al.
2004; Heap et al. 2009; Dammeyer et al. 2013; Wright et al. 2015). By this
approach, we recovered a broad suite of axenic fastidious gut bacteria affiliated with
the Firmicutes that were stably transformed with pEHR5-based vectors. In addition,
we demonstrated that the metaparental mating approach and the pEHR vectors can
be used for heterologous protein expression by constructing fluorescently labelled
gut bacteria (Ó Cuív et al. 2015).
The pEHR5 vector system is freely available to the research community without
the need for a restrictive material transfer agreement and it offers a basis for the
development of a uniform and streamlined set of molecular tools for the isolation
and functional genetic characterisation of fastidious microbes. Nonetheless, the
metaparental mating approach can plausibly be applied with any RP4 mobilisable
vector bearing an appropriate resistance marker(s) and origin(s) of replication, thus
allowing genetically tractable bacteria to be recovered from complex microbial
communities. We confirmed this hypothesis by using the narrow host range vector
pJQ200sk(+) (Quandt and Hynes 1993) to demonstrate that E. coli transconjugants
bearing pJQ200sk(+) could be selectively recovered from an anaerobic enrichment
from human faeces by metaparental mating (Fig. 5). In addition, we used the vector
pGusAmob [(Girbal et al. 2003), pGusA modified to carry an oriT] to target the
recovery of Firmicutes affiliated bacteria and isolated transconjugants affiliated with
Blautia hathewayi, Streptococcus pleomorphus and Anaerococcus vaginalis on
M10-based medium. We have now also demonstrated that the pEHR vectors can be
cured using standard molecular techniques to yield naïve strains (Pottenger and Ó
Cuív, Unpublished data).
68 P. Ó Cuív et al.

Fig. 5 a Identification of E. coli transconjugants carrying pJQ200sk(+) (5.4 kb) recovered by

metaparental mating. The E. coli transconjugants were recovered on LB medium and replica plated
onto MacConkey Agar supplemented with 30 μg.ml−1 gentamicin sulphate to differentiate between
the different laboratory and commensal strains. The laboratory strain E. coli JM109 carrying
pJQ200sk(+) was characterised by a clear zone around the patched culture consistent with its
inability to ferment lactose. In contrast, the recent human gut isolate E. coli PC1101 was capable of
fermenting lactose and the 13 transconjugants recovered exhibited a similar phenotype. As
expected, E. coli ST18 did not grow on MacConkey agar due to its nutritional requirement for
δ-aminolevulinic acid. b The identity of the transconjugants was confirmed by PCR using E. coli
specific primers (Sabat et al. 2000). Successful confirmation is indicated by a 545 bp product and,
as expected, E. coli ST18 carrying pJQ200sk(+) (Lane 2) and all of the transconjugants (Lanes 4–
16) produced a product of the correct size. In contrast, no products were observed for PCR lacking
DNA template (Lane 1) or containing Campylobacter jejuni DNA template (Lane 3). c The
presence and integrity of the plasmid vector was assessed by agarose gel electrophoresis. Plasmid
vector prepared from each of the 13 transconjugants (Lanes 3–15) exhibited similar mobility to
plasmid DNA prepared from E. coli JM109 (Lane 2) confirming that they were stably transformed

The metaparental mating approach and the pEHR vector series are significant
developments for the genetic dissection of the gut microbiota by forward and
reverse genetic approaches. For instance, it is now known that many gut bacteria
carry putative biosynthetic gene clusters for secondary metabolites (Letzel et al.
2013; Donia et al. 2014; Cimermancic et al. 2014; Donia and Fischbach 2015;
Hadjithomas et al. 2015), some of which may encode for NF-κB suppressive
bioactives. The specific gene clusters underpinning the production of NF-κB sup-
pressive bioactives could potentially be identified by comparative genomics of
suppressive and non-suppressive strains, however, reverse genetic approaches can
now also plausibly be applied to specifically disrupt target genes and conclusively
confirm their role in the production of specific bioactives (Donia et al. 2014).
Consistent with this, mutagenesis strategies based on homologous recombination
(Al-Hinai et al. 2012; Heap et al. 2012; Faulds-Pain and Wren 2013) and the Ll.
ltrB group II intron (Chen et al. 2005; Heap et al. 2007; Tolonen et al. 2009) have
been described for a diverse range bacteria affiliated with the Firmicutes. While the
specific factors underpinning NF-κB suppressive activity have been identified in
some cases (e.g. Rieu et al. 2014; Quevrain et al. 2016) in most instances, they
3 Exploring the Bioactive Landscape of the Gut Microbiota … 69

remain cryptic and forward genetic approaches including transposon mutagenesis

(Liu et al. 2013; Ichimura et al. 2013; Zhang et al. 2015b) and in vivo transpo-
somics (Vidal et al. 2009; Veeranagouda et al. 2012) have been successfully
developed for fastidious bacteria. While forward genetic approaches can be applied
with fastidious gut microbes they are constrained by the number of mutant clones
that have to be screened to achieve good coverage of the genome. For example,
assuming a genome size of 4 Mb and an average gene size of 753 bp (Li et al.
2014a), over 12,000 mutants would have to be screened to achieve 90 % coverage
of the genome (Clarke and Carbon 1976).
An alternative approach involves the construction of medium/large insert gene
libraries (e.g. plasmid, cosmid, fosmid, BAC libraries) that are screened for NF-κB
suppressive activity in a suitable microbial host. Using this approach, approxi-
mately 230 clones would have to be screened to achieve 90 % coverage of the
genome assuming a genome size of 4 Mb and an average insert size of 40 kb. Gene
libraries generally assume that all of the genetic elements supporting the
immunomodulatory activity are linked, expressed and functional in the microbial
host. E. coli has relaxed requirements for promoter recognition and this approach
has been exploited to identify metagenomic fosmids derived from human gut
microbiota that are capable of suppressing/activating NF-κB (Lakhdari et al. 2010;
Cohen et al. 2015). Nonetheless, as few as 40 % of heterologous genes are
expressed in E. coli (Aakvik et al. 2011) and new cloning vectors have been
developed that have extended the host range of large insert vectors (e.g. Aakvik
et al. 2009; Kakirde et al. 2011). Currently, the replication range of these vectors is
mostly limited to proteobacteria and examples of vectors for more distantly related
phyla, especially the Firmicutes, are limited (Hain et al. 2008; Liu et al. 2009).

8 Bioprospecting for NF-κB Suppressive Bioactives:

Faecalibacterium prausnitzii as a Case Study

The butyrate producing gut bacterium, F. prausnitzii, comprises part of the core
microbiota in healthy adult humans and is ubiquitously found in the gut of mam-
mals and insects (Foglesong et al. 1984; Bjerrum et al. 2006; Castillo et al. 2007;
Qin et al. 2010; Nava and Stappenbeck 2011; Miquel et al. 2013; Oikonomou et al.
2013). This suggests that F. prausnitzii plays a critical role in host metabolism and
physiology and consequently it is widely considered to be a model gut bacterium
with relevance to health and disease. In that context, much progress has been made
in identifying the true metabolic potential of F. prasunitzii and its contribution to
health and well being (Sokol et al. 2008; Quevrain et al. 2016). Swidsinski et al.
(2008) first reported a reduced population of F. prausnitzii in CD subjects and
Sokol et al. (2008) subsequently demonstrated a low abundance of F. prausnitzii in
ileal biopsies from CD subjects at the time of surgery was associated with recur-
rence six months postoperatively, and that the abundance at six months was
70 P. Ó Cuív et al.

consistently lower in subjects with recurrent disease in comparison to those in

remission. In support of this observation a longitudinal study with an Australian CD
cohort examined the mucosa associated microbial communities in subjects under-
going ileal resection and determined that patients who were in remission 6 months
postoperatively had a higher population of F. prausnitzii and other members of the
Firmicutes at surgery (De Cruz et al. 2015). Notably, changes in the abundance of
F. prausnitzii in CD subjects have been reported in different ethnic populations
(Prideaux et al. 2013) and perturbations have also been reported in other inflam-
matory and metabolic disorders like ulcerative colitis, coeliac disease, juvenile
spondyloarthritis and type 2 diabetes (Sokol et al. 2009; De Palma et al. 2010;
Remely et al. 2014; Gill et al. 2015) suggesting that it plays an important role in
maintaining gut homeostasis.
The immunomodulatory potential of F. prausnitzii A2-165 was first identified by
Sokol et al. (2008) who demonstrated that the bacterium exerted an
anti-inflammatory effect in PBMCs by inducing IL-10 and suppressing IL-12 and
INFγ secretion. They also showed that spent culture supernatant but not sterile
medium, UV-killed F. prausnitzii or cellular fractions were able to block the
activation of NF-κB and reduce IL-8 secretion in Caco-2 cells. Butyrate exerts
physiological and anti-inflammatory effects in the gut (Canani et al. 2011; Ploger
et al. 2012), however, the presence of butyrate in the spent culture supernatants did
not suppress NF-κB activation in Caco-2 cells suggesting that other bioactive
factors were responsible for the anti-inflammatory effects (Sokol et al. 2008).
Critically, F. prausnitzii whole cells as well as filter-sterilised culture supernatant
could attenuate the overall severity of trinitrobenzene sulphonic acid induced colitis
in BALB/c mice by both a gut-dependent and gut-independent route. Separate
studies have also supported these observations and revealed that F. prausnitzii
and/or its supernatant can induce Treg proliferation (Qiu et al. 2013; Martin et al.
2014), modulate T-cell responses (Rossi et al. 2016) and improve gut barrier
function (Carlsson et al. 2013; Martin et al. 2015; Laval et al. 2015), thus also
contributing to the suppression of inflammation. Together, these observations
indicated that the anti-inflammatory activity could be largely attributed to a secreted
bioactive.
In addition to butyrate, it is now known that F. prausnitzii produces a range of
distinct immunomodulatory bioactives relevant to host health including peptides
and secondary metabolites. Using a peptidomic approach, Quevrain et al. (2016)
identified 7 peptides derived from a 15 kDa protein termed MAM (Microbial
Anti-inflammatory Molecule) that is phylogenetically narrowly distributed.
Intracellular expression of the MAM protein in human epithelial cells suppressed
NF-κB activation in a specific and dose-dependent manner possibly by affecting
IκκB function. Furthermore, Lactococcus lactis expressing MAM was capable of
ameliorating dinitrobenzene sulphonic acid induced colitis in BALB/c mice. The
mechanism of action of the MAM protein remains to be determined including
whether its NF-κB suppressive activity is mediated by the intact protein and/or its
derived peptides, and how these are delivered to the cell. In addition to MAM, F.
prausnitzii produces a range of (precursor) anti-inflammatory secondary
3 Exploring the Bioactive Landscape of the Gut Microbiota … 71

metabolites. Using a gnotobiotic mouse model, Miquel et al. (2015) revealed that
the protective effect of F. prausnitzii following colonisation was associated with the
presence of salicylic acid and shikimic acid in gut and serum metabolomic profiles.
Salicylic acid is a precursor of 5-aminosalycilic acid and is capable of suppressing
IL-8 secretion from TNFα stimulated HT-29 cells. In contrast, shikimic acid is not
capable of suppressing IL-8 secretion from TNFα stimulated HT-29 cells, however,
this molecule is a precursor of anti-inflammatory aromatic compounds including
salicylic acid and 3,4-oxo-eisopropylideneshikimic acid (Xing et al. 2013).
These observations underline the role played by F. prausnitzii in the mainte-
nance of gut homeostasis and reveal the evolution of a variety of strategies to affect
specific aspects of gut function and the immune response. Consistent with this, F.
prausnitzii has been suggested as a candidate next generation probiotic for the
treatment of gut inflammatory diseases (Sokol et al. 2008; Neef and Sanz 2013).
Critically, the characterisation of F. prausnitzii has provided a template by which
the contribution of other microbes to gut health can be examined. It should be noted
that although F. prausnitzii A2-165 has been known to suppress NF-κB since 2008
and its genome was sequenced in 2009, the specific bioactives supporting this
activity remained unidentified until 2015, highlighting the limited capacity of -omic
approaches to identify novel functional capabilities. We have now used meta-
parental mating to isolate genetically tractable strains of F. prausnitzii (Ó Cuív et al.
2015), and we anticipate that these will further expedite the functional dissection of
this important gut bacterium.

9 Concluding Remarks and Future Perspective

The healthy gut microbiota plays a vital role in helping to maintain gut homeostasis
and preventing the onset of chronic gut disease. Surprisingly, little is known about
the essential functionalities that underlie this capability and how they might be
exploited to develop more effective therapeutic interventions. The rapid advances in
DNA sequencing technologies continue to provide an unprecedented insight into
structure-functional activity of the gut microbiome. In contrast, the development of
complementary approaches including microbial culturing, functional assays,
metabolomics and genetic technologies have not kept pace with these develop-
ments. This has hindered efforts to realise the functional potential of the microbiota,
however, the successful metabolomic dissection of F. prausnitzii will encourage
and inform the development of improved methodologies for other gut bacteria.
Similarly, new advances in microbial culturing and genetic techniques will provide
new opportunities to support a more mechanistic dissection of these functionalities.
We anticipate that the effective integration of these disparate yet complementary
approaches will afford the best opportunity to effectively bioprospect the gut
microbiota and support the discovery of novel bioactives, and the development of
new therapeutics.
72 P. Ó Cuív et al.

Acknowledgements We gratefully acknowledge the financial support of the University of

Queensland Diamantina Institute to PÓC, SP and MM. SB is the recipient of an Australian
Postgraduate Award. The Translational Research Institute is supported by a grant from the
Australian Government.
Competing interest
The authors declare no competing interest.

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Chapter 4
Using Metabolomic Approaches
to Characterize the Human Pathogen
Leishmania in Macrophages

Joachim Kloehn, Eleanor C. Saunders and Malcolm J. McConville

1 Leishmania and Leishmaniasis

Leishmania spp., are protozoan parasites that belong to the order Kinetoplastida
(Phylum Euglenozoa). The Kinetoplastida encompass a large group of flagellated
protists including the uniflagellated Trypanosomatidae, which all have parasitic life
styles, and the biflagellated Bodonidae which are typically free living. The family
Trypanosomatidae comprises the medically important genera Trypanosoma, which
includes Trypanosoma brucei and Trypanosoma cruzi, the causative agent of
human African trypanosomiasis and Chagas disease, respectively, as well as
Leishmania spp., which are the cause of a spectrum of diseases collectively termed
the leishmaniases (Stuart et al. 2008). T. brucei, T. cruzi, and Leishmania spp., all
rely on an insect vector as well as a mammalian host to complete their complex
lifecycle. While T. brucei and T. cruzi are transmitted by the tsetse fly and tri-
atomine bugs, respectively, Leishmania spp., are transmitted by phlebotomine
sandflies.
The pathology of leishmanial diseases range from localized self-healing cutaneous
or diffuse cutaneous infections (CL and DCL, respectively) to mucocutaneous
leishmaniasis (MCL) and visceral leishmaniasis (VL, also called kala-azar) (Pearson
and Sousa 1996). The pathology can be linked to different species—e.g., Leishmania
donovani, Leishmania infantum, and Leishmania chagasi cause VL, while
Leishmania major, Leishmania mexicana, Leishmania amazonensis, and Leishmania
aethiopica cause CL or DCL, and Leishmania braziliensis, Leishmania peruviana,

J. Kloehn
E.C. Saunders
M.J. McConville (&)

The Department of Biochemistry and Molecular Biology, Bio21 Molecular Science
and Biotechnology Institute, University of Melbourne, Parkville, VIC 3010, Australia
e-mail: [email protected]
M.J. McConville
Metabolomics Australia, Bio21 Molecular Science and Biotechnology Institute,
University of Melbourne, Parkville, VIC 3010, Australia

© Springer International Publishing Switzerland 2016 83

D.J. Beale et al. (eds.), Microbial Metabolomics,
DOI 10.1007/978-3-319-46326-1_4
84 J. Kloehn et al.

and Leishmania guyanensis cause MCL. CL is characterized by open sores around the
site of the sandfly bite, which can take years to heal and leave disfiguring scars.
Furthermore, CL can recur years after patients have seemingly healed from the initial
infection (Marovich et al. 2001; Gangneux et al. 2007). DCL is a more severe form of
CL that results in the formation of several hundred nodules or ulcers. The marring
lesions and disfiguring scars caused by CL and DCL can result in stigmatization and
lead to social exclusion and economic disadvantage (Kassi et al. 2008). MCL results
from the dissemination of parasites to mucosal membranes around the mouth and nose
and, in some cases, to the genital or optical mucosa (Huna-Baron et al. 2000). This
severe form of leishmaniasis can result in devastating destruction and deformation of
the face with high risk of secondary infections. VL, or kala-azar, occurs when para-
sites disseminate to the bone marrow, liver, and spleen, resulting in anemia, fever,
weightloss, and enlargement of liver and spleen. If left untreated, VL leads to death in
nearly 100 % of cases within a two-year period (WHO.int).
Leishmaniasis is endemic in 88 countries throughout the tropics, subtropics, and
the Mediterranean Basin with over 350 million people at risk of infection (WHO.int).
Disease prevalence is estimated at 12 million people with more than 2 million new
infections occurring annually (WHO.int). Mortalities from VL are increasing
worldwide and currently stand at >20,000 deaths annually, making it the second
deadliest parasitic disease after malaria (WHO.int; Desjeux 2004; Reithinger 2008;
Alvar et al. 2012). The current war in Syria and instability in the Middle East have led
to increased incidence and spread of leishmaniasis in the area due to a number of
factors including greater refugee migration and an inability to access staff and
facilities for diagnosis and treatment (Alawieh et al. 2014).

2 Treatment of Leishmaniasis

Current antileishmanial treatments suffer from one or more major limitations

including high toxicity/severe side effects (amphotericin, pentamidine, paro-
momycin, miltefosine and sitamaquine), the requirement for long-term/parenteral
administration (pentavalent antimonials, amphotericin B and paromomycin), high
cost (liposomal amphotericin B), variable efficacy (e.g. species specificity, pen-
tavalent antimonials, imidazole and pentamidine), and resistance or likely devel-
opment of resistance (pentavalent antimonials and miltefosine) (Bouchard et al.
1982; Rangel et al. 1996; Seifert et al. 2003, 2007; Ouellette et al. 2004; Olliaro
et al. 2005; Croft et al. 2006; Sindermann and Engel 2006; Bhattacharya et al. 2008;
Sundar et al. 2007; Davidson et al. 2009; Moore and Lockwood 2010; Chakravarty
and Sundar 2010; Seifert 2011; Freitas-Junior et al. 2012). While combination
treatments are being used to reduce the emergence of resistant strains (van
Griensven et al. 2010), new drugs which overcome the limitations of the current
drugs are needed urgently (Freitas-Junior et al. 2012). Furthermore, despite the fact
that humans can generate strong protective immunity against Leishmania
4 Using Metabolomic Approaches to Characterize the Human Pathogen … 85

infection/reinfection (Evans and Kedzierski 2012), no efficacious defined vaccine

for preventing human leishmaniasis has been developed to date (Handman 2001; de
Oliveira et al. 2009; Kedzierski 2010; McCall et al. 2013; Joshi et al. 2014; Kumar
and Engwerda 2014).

3 The Lifecycle of Leishmania

Leishmania differentiate through several morphologically and physiologically dis-

tinct stages during their complex digenetic lifecycle in their insect vector and
animal hosts (Fig. 1). The major developmental stage in the sandfly vector
(Phlebotomus and Lutzomyia) is the motile promastigote, which possesses a single
long flagellum that emerges from the anterior flagellar pocket. Sandflies become a
vector following the uptake of infected cells or free Leishmania parasites (typically
low, usually 10–100 parasites) when feeding on a mammalian host (Anjili et al.
2006). Ingested parasites initially differentiate to promastigotes, which undergo a
period of rapid proliferation, exploiting the nutrient-rich milieu of the blood meal as
it is progressively digested by the sandfly’s hydrolases (Pimenta et al. 1997).
Following the breakdown of the peritrophic membrane that encapsulates the initial
blood meal, the promastigote differentiates through several developmental stages
which are distinguished by markedly different replication rates (Gossage et al.
2003; Dostalova and Volf 2012) including the nondividing metacyclic stage that
accumulate in the sandfly foregut. This stage exhibits a similar physiology to
nondividing (stationary phase) promastigotes (Prostat) in in vitro culture and appears
to be highly virulent and preadapted for life in the mammalian host (Sacks and
Perkins 1984, 1985; da Silva and Sacks 1987; Sacks and da Silva 1987; Sacks
1989). The accumulation of large clusters or aggregates of these metacyclic pro-
mastigotes at the sandfly mouthparts (specifically the stomodeal valve) causes an
alteration in sandfly feeding behavior (e.g. repeated probing of the skin), damage to
the stomodeal valve, and regurgitation of the parasite bolus, which may enhance
transmission of the parasite to the host (Killick-Kendrick et al. 1977; Beach et al.
1984, 1985; Bates 2007; Rogers and Bates 2007, Schlein et al. 1992; Volf et al.
2004).
The number of Leishmania transmitted during a blood meal is typically low
(<600 parasites) but can, in some cases, be as high as 100,000 cells (Kimblin et al.
2008). Infectious metacyclics are transmitted along with sandfly saliva and a highly
immunogenic polysaccharide gel secreted by the promastigote (Titus and Ribeiro
1988; Bates 2007). Injected promastigotes are initially phagocytosed by polymor-
phonuclear leucocytes (PMNs) that are rapidly recruited to the sandfly bite site (van
Zandbergen et al. 2004). These PMNs undergo apoptosis within a few days and the
cellular debris (including released parasites) and/or intact PMNs containing para-
sites, are phagocytosed by a wave of macrophages that are recruited to the damaged
tissue. Macrophages are the primary host cells of Leishmania (Handman and Bullen
2002) and it has been proposed that the initial infection of PMNs (in which the
86 J. Kloehn et al.

Fig. 1 Leishmania undergo a complex digenetic lifecycle developing within the sandfly vector
and the mammalian host. Transmission occurs when an infected sandfly takes a blood meal.
Amastigotes/infected macrophages are taken up by the sandfly during a blood meal (1) (Anjili
et al. 2006) and are initially enclosed within a digestive peritrophic sac in the sandfly gut (red)
(Pimenta et al. 1997). Amastigotes differentiate to flagellated procyclic promastigotes (2) which
undergo multiple rounds of division before slowing their replication and differentiating into highly
motile, but nondividing, nectomonads which escape the peritrophic sac (Gossage et al. 2003;
Dostalova and Volf 2012). Nectomonads adhere to the mid-gut wall (3) and develop into
leptomonads in the thoracic mid-gut (4). Leptomonads undergo further rounds of rapid replication
and are immobilized in a gel-like matrix which is secreted by this life-cycle stage. Leptomonads
differentiate into either nondividing haptomonads, which form a plug by attaching to each other
and the stomodeal valve (5), or nondividing metacyclics, which are highly infectious and are
transmitted during a blood meal (6) (Sacks and Perkins 1984, 1985; da Silva and Sacks 1987;
Sacks and da Silva 1987; Sacks 1989). In the mammalian host, metacyclic promastigotes are
phagocytosed by polymorphic neutrophils (PMN), which are first to reach the site of inflammation
following the sandfly bite (7, 8) (Laskay et al. 2003; van Zandbergen et al. 2004). PMNs have a
short life span and macrophages phagocytose released parasites and/or infected apoptotic PMNs
(8, 9) (Laskay et al. 2003; van Zandbergen et al. 2004; Ritter et al. 2009). Within the
phagolysosome of macrophages, Leishmania differentiate into the amastigote stage, adapt to their
new milieu (10) and start replicating (11) (Kaye and Scott 2011). Amastigotes are released when
heavily infected macrophages rupture (12) allowing infection of other cells (Noronha et al. 2000).
The lifecycle begins again when sandflies ingest free amastigotes or infected cells during a blood
meal (1)

parasites do not replicate) represents a ‘Trojan Horse’ strategy for targeting mac-
rophages, the preferred host cell for these pathogens (Laskay et al. 2003; van
Zandbergen et al. 2004, 2007). Alternatively, extracellular Leishmania may be taken
up incidentally during the phagocytosis of necrotic PMNs (the ‘Trojan rabbit’
model) (Ritter et al. 2009). Following uptake by macrophages and the maturation of
the vacuole into a phagolysosomal compartment, promastigotes differentiate to
4 Using Metabolomic Approaches to Characterize the Human Pathogen … 87

small, aflagellate amastigotes which re-enter a proliferative state that is associated

with progression of the disease (Kaye and Scott 2011). Further recruitment of naïve
macrophages to the site of infection leads to the development of granulomatous
lesions that are predominantly composed of infected and uninfected macrophages, as
well as other populations of monocytes, PMNs, dendritic cells, and lymphocytes
(Amaral et al. 2000; Souza-Lemos et al. 2011). Infected macrophages, or even
extracellular amastigotes, can also disseminate to other tissues and establish infec-
tion in lymph nodes, mucosal membranes (MCL), or the spleen and liver
(VL) (Ridley et al. 1989; McElrath et al. 1988). Subpopulations of Leishmania-
infected macrophages in lymph nodes are thought to be responsible for long-term
persistence of the pathogen, but are inadequately characterized (Dereure et al. 2003).
Macrophages are specialized phagocytic cells that are a vital part of the innate
and adaptive immune system. These cells are responsible for internalizing and then
killing microbes within the phagolysosome which has a low pH, a complex array of
hydrolytic enzymes, and is the target of key microbicidal processes such as the
oxidative burst and nitric oxide (NO) synthesis (Russell et al. 2009). However, a
number of bacterial (Coxiella brunetti, Listeria monocytogenes, Shigella spp.,
Mycobacterium tuberculosis), fungal (Cryptococcus neoformans), and protozoan
pathogens (Leishmania spp., T. cruzi and Toxoplasma gondii) actively target
macrophages (Dermine and Desjardins 1999). While most of these organisms
(including Leishmania promastigotes) exhibit mechanisms to escape the
phagolysosome or prevent its maturation (Desjardins and Descoteaux 1997;
Dermine et al. 2000, 2005; Moradin and Descoteaux 2012), Leishmania amastig-
otes and Coxiella burnetti have evolved to withstand the harsh conditions
encountered within the mature phagolysosome (Dermine and Desjardins 1999; Thi
et al. 2012). In order to survive the acidic pH within the phagolysosome,
Leishmania amastigotes express proton pumps to maintain a near-neutral intracel-
lular pH (Glaser et al. 1988; Zilberstein et al. 1989; Grigore and Meade 2006). The
mechanisms by which Leishmania amastigotes withstand the high concentration of
proteases within the phagolysosome are poorly understood (Prina et al. 1990).
Possibly, Leishmania cell surface glycoconjugates play a role in acid resistance
and/or Leishmania membrane proteins are resistant to proteolysis (Chang and Fong
1983; Prina et al. 1990). Prina et al. (1990) proposed that the enlarged
phagolysosomes observed in macrophages infected with L. mexicana and related
species are beneficial to the parasite as they result in dilution of the lysosomal
proteases. Due to the difficulty in studying intracellular amastigotes, most studies
rely on the axenic differentiation of promastigotes to amastigotes (Amaaxenic) which
can be achieved by lowering the pH of the culture medium and increasing the
temperature or through depletion of iron in the culture medium to mimic the
conditions of the host macrophage (Saar et al. 1998; Mittra et al. 2013).
88 J. Kloehn et al.

4 Characterizing Leishmania Metabolism: Insights

Offered by ‘-Omics’Approaches

The paucity of good treatment options and no vaccine means that there is an urgent
need to identify new therapeutic targets in Leishmania. Many of these targets will
ideally be specific to parasite metabolism, targeting pathways critical for the sur-
vival and proliferation of the intracellular amastigote stage. Understanding the
interplay of host and parasite metabolism is critical in identifying targets that will
not affect the host metabolism. Finally, by mapping parasite metabolism it is
anticipated that the mode of action (MOA) of current chemotherapies will be
revealed and new insights into drug resistance mechanisms gained. The metabolism
of Leishmania has been studied for several decades using a range of approaches and
techniques. Here we discuss these techniques, the insights they have provided into
Leishmania metabolism as well as their limitations.

5 Genome Studies and Leishmania Metabolism Mapping

Genome sequencing has revealed many new potential drug targets in

Leishmania. To date, the genomes of several Leishmania species have been
sequenced (Ivens et al. 2005; Peacock et al. 2007; Downing et al. 2011; Real et al.
2013) providing important new insights into the biology of these parasites,
including the identification of novel surface proteins, protein kinases, and phos-
phatases that could be involved in signaling pathways, differentiation and parasite
stress responses, as well as many metabolic pathways (Myler et al. 2000; Worthey
et al. 2003; Ivens et al. 2005). Genome-wide annotations have led to the devel-
opment of curated biochemical pathways databases, such as LeishCyc and Kyoto
Encyclopedia of Genes and Genomes (KEGG) pathways (Doyle et al. 2009;
Saunders et al. 2012). Furthermore, genome-wide flux balance models of
Leishmania metabolism give unique insight into crucial reactions, predicted aux-
otrophies and minimal culture media components, potentially lethal or
growth-reducing gene deletions, and novel drug target identification (Chavali et al.
2008, 2012; Silva et al. 2015; Subramanian et al. 2015). These tools are particularly
useful in providing a means to contextualize and map metabolomics data and are
also valuable in hypothesis-driven metabolomic analyses.
However, as is the case with other protozoan genomes, more than half of the
genes in the Leishmania genomes have not yet been assigned a function (Pawar
et al. 2014), highlighting the importance of pairing genomic data with functional
gene analysis tools such as metabolomics. Another striking feature to emerge from
these analyses is the extraordinarily high level of gene synteny identified by the
comparative analysis of T. brucei, T. cruzi, and L. major genomes (El-Sayed et al.
2005). About 6150 genes were found to be shared between these species, while the
authors identified about 900 Leishmania-specific genes. Furthermore, a comparison
4 Using Metabolomic Approaches to Characterize the Human Pathogen … 89

of L. major, L. braziliensis, and L. infantum genomes, identified only about 80

genes that are restricted to an individual species despite the early divergence of
these species and the distinct clinical manifestation observed during infection with
these species (Peacock et al. 2007). This indicates that there are only few
species-specific genes which contribute to the different biology of each species,
indicating that species and strain-specific differences may result from gene dosage
(copy number) (Rogers et al. 2011). Additionally, it should be noted that the
genome provides insights into the overall metabolic capacity of Leishmania, but
allows no conclusion about the significance of metabolic pathways in specific
developmental stages and given conditions (e.g. nutrient restriction, drug treatment
etc.). For example, enzymes/pathways which may be crucial for survival in the
sandfly vector might be dispensable during mammalian host infection and vice
versa.
Finally, models of Leishmania metabolism may not adequately account for
niche/species/stage-specific changes to enzyme compartmentalization which in turn
will affect metabolic flux. For example, Leishmania and other kinetoplastids
maintain an unusual peroxisome-like organelle termed the glycosome (Haanstra
et al. 2016). This organelle contains the enzymes of several metabolic pathways
including glycolysis, gluconeogenesis, the pentose phosphate pathway (PPP), fatty
acid β-oxidation, purine salvage, and pyrimidine synthesis as well as hypothetical
proteins with unknown function (Michels et al. 2006; Jamdhade et al. 2015). The
localization of enzymes within the glycosome has repercussions on metabolic flux
and is thought to provide a means of regulation in the absence of traditional feedback
loops. For example, Leishmania hexokinase and phosphofructokinase lack con-
ventional activity regulation which could cause the toxic accumulation of hexose
phosphates (Bakker et al. 2000; Michels et al. 2006). Indeed, the relocalization of
glycolytic enzymes, such as the phosphoglucokinase and the triose phosphate iso-
merase, to the cytoplasm proved to be fatal for T. brucei (Blattner et al. 1998; Helfert
et al. 2001). Furthermore, the number and enzymic content of the glycosome
changes during the promastigote to amastigote differentiation thereby affecting the
metabolic regulation and capacity of the parasite (Cull et al. 2014).

6 Transcriptomic and Proteomic Studies

Unlike the situation in other eukaryotes, protein-encoding genes in Leishmania lack

introns and are organized and constitutively transcribed as large polycistronic
clusters (Myler et al. 2000; Worthey et al. 2003; Ivens et al. 2005). Transcription is
not regulated by transcription factors and the polycistronic mRNA is subsequently
processed into individual mRNAs by transsplicing and polyadenylation (Ivens et al.
2005). As a result of this unusual mode of gene expression, relative levels of mRNA
expression remain remarkably constant across different life-cycle stages under dif-
ferent growth conditions (Leifso et al. 2007). Consequently, transcriptomic studies
have not been particularly fruitful in identifying enzymes or metabolic pathways that
90 J. Kloehn et al.

are activated (or repressed) in amastigotes following their uptake by macrophages, as

has been the case in some other bacterial or fungal pathogens (Haile and
Papadopoulou 2007). However, there are a limited number of examples where
stage-specific changes in protein expression are regulated at the level of mRNA
stability. Examples include some of the lysosomal cysteine proteases, as well as
some enzymes involved in metabolism and protein synthesis (Saxena et al. 2007).
A recent analysis of the L. major transcriptome reported that the polycistronic
transcripts can be highly heterogeneous in length which may lead to differences in
stability, further complicating the interpretation of transcriptomic data (Rastrojo
et al. 2013). Lastly, a general limitation of transcriptome analyses is the potentially
poor correlation between mRNA and protein levels or between transcript levels and
enzymatic activity as observed in other organisms (Miyamoto et al. 2001; Maier
et al. 2009; Hoppe 2012; Vogel and Marcotte 2012).
Proteomic approaches have been used to identify differentially expressed pro-
teins in promastigote and amastigote stages, and have given some crucial insights
into the metabolism of the two life-cycle stages (Handman et al. 1995; Thiel and
Bruchhaus 2001; El Fakhry et al. 2002; Bente et al. 2003; Nugent et al. 2004;
Walker et al. 2006; Rosenzweig et al. 2008a, b). Promastigote to amastigote dif-
ferentiation is associated with a global reduction in protein synthesis but increased
expression of some amastigote specific proteins such as A2, an ATP-dependent
RNA helicase, the amastin family of proteins, lysosomal cysteine proteinases, some
protein chaperones and histones (Handman et al. 1995; Carvalho et al. 2002;
Barhoumi et al. 2006; Nasereddin et al. 2010). Comparing the proteome of cultured
promastigotes and Amaaxenic, Rosenzweig et al. (2008a) reported significant dif-
ferences in the levels of some metabolic enzymes including increases in enzymes
involved in gluconeogenesis, β-oxidation, amino acid catabolism, the tricarboxylic
acid (TCA) cycle, and the mitochondrial respiratory chain, suggesting stage-specific
remodeling of amastigote metabolism. A number of other studies have identified
proteins that are differentially expressed in the distinct life-cycle stages (El Fakhry
et al. 2002; Bente et al. 2003; Nugent et al. 2004; Walker et al. 2006). However, the
identified differences were surprisingly small and interpretation of these analyses
are complicated by the fact that many changes in protein expression are inconsistent
across different species and/or the possibility that at some of these changes may be
attributed to stage-specific changes in the size or complement of mitochondria and
glycosomes in the different developmental stages.
More broadly, while protein levels are commonly considered to provide a
measure for the activity of an enzyme/pathway, this correlation is not always
observed (Miyamoto et al. 2001). Furthermore, relatively few Leishmania proteins
have been functionally characterized and there is an increasing number of reports of
parasite proteins that have divergent or repurposed roles from those inferred from
homology alignments (Oppenheim et al. 2014). For example, Leishmania hexoki-
nase functions as a metabolic enzyme in glycosomal glycolysis, as well as heme
receptor in the flagellar pocket and mitochondrion, while enolase ‘moonlights’ as a
microtubule-binding protein (Krishnamurthy et al. 2005; Quinones et al. 2007;
4 Using Metabolomic Approaches to Characterize the Human Pathogen … 91

Vanegas et al. 2007; Collingridge et al. 2010; Tonkin et al. 2015), complicating the
interpretation of proteomics analyses.
These studies suggest that Leishmania may be more dependent on posttransla-
tional regulatory mechanisms during differentiation or adaptation to changing
environmental circumstance than other eukaryotes. Indeed gene families for pro-
teins involved in protein posttranslational modifications (kinases, phosphatases,
etc.) are commonly amplified in these parasites. In particular, phosphoproteomic
studies have revealed major changes in protein phosphorylation during pro-
mastigote to amastigote differentiation (Rosenzweig et al. 2008b; Morales et al.
2010a; Tsigankov et al. 2013, 2014). Interestingly, some of the most abundant
proteins to be phosphorylated during differentiation were heat-shock proteins which
are otherwise constitutively expressed (Morales et al. 2010a). Rosenzweig et al.
(2008b) utilized isobaric tags for relative and absolute quantification/liquid
chromatography-mass spectrometry/mass spectrometry (iTRAQ/LC-MS/MS) to
characterize a number of posttranslational modifications in Leishmania pro-
mastigotes and Amaaxenic. The Leishmania genomes also encode large numbers of
protein kinases and phosphatases, several of which have been shown to be essential
for differentiation and/or amastigote survival (Ivens et al. 2005; Morales et al.
2010b; Cayla et al. 2014). To date, however, no complete signaling pathways in
Leishmania (from surface receptor/sensor to downstream effector(s)) have been
delineated and the function of most of these posttranslational modifications remains
poorly defined. Nonetheless, studies on proteins involved in regulating mitochon-
drial proteins through the covalent attachment of ubiquitin-like proteins
(Gannavaram et al. 2011, 2012) suggest that this will be a rewarding endeavor. In a
recent study, Goldman-Pinkovich et al. (2016) described a coordinated arginine
deprivation response (ADR) that is activated in response to low arginine levels
which are expected to occur in the macrophage phagolysosome. L. donovani
responded to low external arginine levels by rapidly upregulating the arginine
transporter, LdAAP3. This response was dependent on expression of the mitogen
activated protein kinase 2 (MAPK2)-dependent signaling pathway
(Goldman-Pinkovich et al. 2016). Similarly, stage-specific regulation of expression
of the major plasma membrane glucose transporters in L. mexicana was found to be
regulated by ubiquitination of the cytoplasmic tails of the transporter proteins and
their internalization and degradation in the lysosome (Vince et al. 2011).

7 Insights into Leishmania Metabolism using

Metabolomics

Further advances in our understanding of Leishmania adaptive responses in both

the sandfly and mammalian hosts will be dependent on the use of complementary
approaches, such as metabolomics. Metabolomics is increasingly being used alone
or in combination with other ‘-omics’ approaches to identify new or unanticipated
92 J. Kloehn et al.

metabolic pathways and to characterize metabolic networks in microbial pathogens

(Holmes 2010; Creek and Barrett 2014; McConville 2014; McConville et al. 2015;
Saunders et al. 2015; Lau et al. 2015; Kim and Creek 2015; Kloehn et al. 2016).
The metabolome of a cell/tissue/organism can be considered the major downstream
phenotype of changes in the transcriptome and proteome, or the most upstream
input into cellular processes from the environment. The later point is particularly
relevant when considering pathogens which are highly responsive to changes in the
nutrient levels within their specific host niches. Trindade et al. (2016) demonstrated
this in a recent study as they identified a major reservoir of T. brucei cells in adipose
tissue. In comparison with parasites in the bloodstream and central nervous system,
the adipose tissue form (ATF) exhibited a distinct metabolism, as it is adapted to its
niche by utilizing myristate as a major carbon source through β-oxidation.
Furthermore, metabolomics has proven to be particularly useful in drug target
discovery research and is expected to become increasingly valuable due to the rapid
refinement of existing approaches and the development of new analytical tech-
niques (Rabinowitz et al. 2011). To date in Leishmania spp., metabolomics
approaches have been employed to characterize mutants, elucidate the MOA of
drugs and mechanisms of resistance, and to describe the nutritional requirements
and central carbon metabolism of different parasite developmental stages as well as
different species (Naderer et al. 2006; De Souza et al. 2006; Creek and Barrett 2014;
Vincent et al. 2014; Rojo et al. 2015; Saunders et al. 2015; Arjmand et al. 2016;
Westrop et al. 2015). Though beyond the scope of this review, metabolomics has
also been used to investigate changes to the host cell upon infection (Lamour et al.
2012; Moreira et al. 2015).

8 Characterization of Genetic Knockouts using Metabolite

Profiling

Notwithstanding the technical difficulties of deleting genes in a diploid organism

such as Leishmania, a significant number of genetically defined mutants have been
generated, which have provided new insights into the metabolic requirements of
intracellular amastigotes (McConville et al. 2007). To date, metabolomic profiling
in Leishmania spp., has predominantly been used in a highly targeted fashion to
validate the deletion of the desired target gene, complementing detailed molecular
biology, biochemistry, and virulence data (Naderer et al. 2006; Saunders et al.
2012; Naderer et al., 2015). For example, Naderer et al. (2006) deleted the gene
encoding the gluconeogenic enzyme, fructose 1,6-bisphosphatase (FBPase) in L.
major. In contrast to wild-type cells, these parasites were unable to utilize gluco-
neogenic carbon sources (glycerol) to synthesize hexose sugars and mannogen, a
unique storage polysaccharide composed of β1,2-linked mannose residues. While
the L. major ΔFBPase promastigotes grew normally in rich culture medium, they
were unable to replicate in macrophages and displayed highly attenuated virulence
4 Using Metabolomic Approaches to Characterize the Human Pathogen … 93

in mice suggesting that gluconeogenesis is essential for Leishmania survival in the

macrophage, possibly due to low levels of hexoses in the phagolysosome.
Paradoxically, other genetic studies have shown that Leishmania spp., are also
dependent on the uptake of sugars (Burchmore et al. 2003; Naderer et al. 2010). An
abundant source of sugars in the macrophages phagolysosome are likely to be
aminosugars (N-acetyl-glucosamine and glucosamine) derived from the breakdown
of glycosaminoglycans which are internalized by macrophages (Naderer et al. 2010,
2015) which would provide amastigotes with a source of both carbon skeletons and
nitrogen groups. The significance of aminosugar uptake and utilization in
amastigote metabolism was supported by the finding that deletion of the gene
encoding the enzyme glucosamine 6-phosphate deaminase (GND), which converts
glucosamine (GlcN) to fructose 6-phosphate, resulted in severe loss of virulence in
mice and the capacity to grow in macrophages, while loss of the enzyme
N-acetylglucosamine acetyltransferase (GNAT), which converts these parasites to
GlcN auxotrophs had no effect on intracellular growth or virulence (Naderer et al.
2015).
In the future, it is anticipated that metabolite profiling will be increasingly used
to analyze entire pathways (or nodes) as, for example, demonstrated in its recent
application to analysis of the polyamine pathway in L. amazonensis
(Castilho-Martins et al. 2015). An untargeted (or at least more extensive) metabolite
profiling of gene knock-out lines and wild-type cells is particularly recommended in
Leishmania spp., in order to prevent overlooking unforeseen (side-) effects of the
gene depletion. Due to the complexity and interconnectivity of the metabolic net-
work, as well as our still limited understanding of the metabolic network and
metabolite-protein interactions, unexpected effects of gene depletions are likely to
be relatively common (Hellerstein 2003).
Untargeted metabolomics may also be combined with functional genomics to
assign identities to a large number of unknown/hypothetical Leishmania genes.
Although this approach has not been systematically applied to Leishmania spp.,
such approaches have been useful in identifying pathways and annotating genes in
other, more intensely studied organisms such as Saccharomyces cerevisiae
(Raamsdonk et al. 2001) and Arabidopsis thaliana (Fukushima et al. 2014). Indeed,
the development of a robust and high-throughput means to characterize mutants
will become increasingly important as new technologies, such as the CRISPR
(clustered regularly interspaced short palindromic repeat)-Cas9 system (Sollelis
et al. 2015; Zhang and Matlashewski 2015), are applied to Leishmania making
gene disruption easier and feasible in large-scale assays. Extensive annotation of
metabolic phenotypes and perturbations will help to refine existing in silico gen-
erated metabolic networks and the usefulness of these models for predicting
lethality, auxotrophy, and drug susceptibility. When combined with traditional
phenotypic data of a given mutant (e.g. growth rate, virulence, viability of each life
cycle stage) metabolomics is a powerful way to characterize a mutant and poten-
tially offers a means to ascribe gene function in a high-throughput manner.
94 J. Kloehn et al.

9 Studying a Drug’s Mode of Action and the Development

of Resistance in Leishmania

Metabolomics represents a powerful tool to understand both the MOA of drugs as

well as how drug resistance occurs (Vincent et al. 2014). In the case of anti-
Leishmania chemotherapies, this is especially important as the MOA of several
frontline drugs remains poorly defined (Croft and Coombs 2003) and the drug
efficacy is highly variable dependent on the Leishmania species and host genetics
(Rangel et al. 1996; Schriefer et al. 2008).
Pentavalent and trivalent antimonials (SbV, SbIII) have been used to treat VL as
well as CL, since the 1960s. Pentavalent antimonials remain effective in 95 % of
patients in many areas (Seifert 2011), however, the widespread use of the drugs for
several decades has given rise to resistant strains in hyperendemic areas in India
(Croft et al. 2006; Ashutosh et al. 2007). Aside from increasing resistance, a major
drawback of pentavalent antimonials is their severe cardiotoxicity, which is
observed in as many as 10 % of patients (Sundar et al. 1998; Sundar and
Chakravarty 2010), and the requirement of parenteral administration as no oral
preparation is available. Despite the use of antimonials to treat leishmaniasis (VL
and CL) for about 60 years, the compounds’ MOA remains unclear (Haldar et al.
2011). Several studies have employed metabolomics in order to determine the effect
of drugs on metabolism and understand how resistance occurs (Canuto et al. 2012;
t’Kindt et al. 2010; Berg et al. 2013). Most of these studies have employed an
untargeted approach to quantify as many metabolites as possible, although the
technology employed (capillary electrophoresis-electron spray ionization-time of
flight-mass spectrometry (CE-ESI-TOF-MS), LC-MS (Orbitrap)), as well as the
Leishmania species (L. infantum, L. donovani), and isolates differ between the
publications. t’Kindt et al. (2010) profiled (untreated) antimonial susceptible and
resistant clones of L. donovani, identifying major differences in phospholipid and
sphingolipid metabolism and pools of amino acid/amino acid derivatives. Berg
et al. (2013) conducted a more comprehensive analysis, profiling the metabolism of
three L. donovani clinical isolates that differed in their susceptibility to antimony.
Rapidly dividing and nondividing promastigotes (Prolog and Prostat respectively)
were analyzed revealing significant differences in several metabolic pathways
including arginine metabolism, cysteine transsulfuration pathway, acylglycines,
indole acrylate, glycerophospholipids, and amino acid levels. From these studies it
was inferred that drug (SbIII) resistance may be linked to (1) increased protection
from drug-induced oxidative stress via greater thiol production and tryptophan
degradation, (2) changes to mitochondrial metabolism, (3) increased membrane
fluidity (protecting the cell from host-derived oxidants and/or affect drug
uptake/export), and (4) accumulation of nonessential amino acids as alternative
carbon source for use upon host cell invasion. More recently, Rojo et al. (2015)
used a multiplatform approach (LC-MS, CE-MS and gas chromatography (GC)-
MS) to increase the diversity of metabolites detected in antimony susceptible and
resistant L. infantum. Consistent with other metabolomics experiments, a depletion
4 Using Metabolomic Approaches to Characterize the Human Pathogen … 95

of urea cycle and polyamine biosynthetic pathway intermediates was observed

when sensitive promastigotes were treated with SbIII which was not apparent (or
reversed) in resistant parasites. Treated parasites also appeared to have a disturbed
TCA cycle, with several important intermediates (malate and anaplerotic amino
acids, aspartate and glutamate) significantly reduced, as well as changes to parasite
membrane composition.
The alkylphosphocoline, miltefosine, was the first oral drug to be licensed for
use against leishmaniasis. It is used to treat VL on the Indian subcontinent and CL
in South America. Miltefosine was initially developed as an anticancer drug in the
1980s (Eibl and Unger 1990) and was later repurposed as an antileishmanial drug.
Drug repurposing/repositioning has been suggested to be an important cost- and
time-saving strategy for the identification of new antiparasitic drugs (Andrews et al.
2014). Given that miltefosine is thought to affect lipid metabolism and membrane
composition, Vincent et al. (2014) compared miltefosine treated/untreated L.
infantum promastigotes using a lipidomics approach. Changes to intracellular lipid
metabolism were observed as an increase in the abundance of short alkanes,
although no change in membrane lipids was detected. These finding contradict
earlier studies, which detailed changes to fatty acid metabolism, sterol pathways,
and phospholipid levels (Rakotomanga et al. 2004, 2007) although comparison
between the two studies is complicated by differences in the experimental design
(Vincent et al. 2014). Vincent et al. (2014) also observed an increase in the
abundance of selected sugars and DNA damage (released nucleotide fragments).
Canuto et al. (2014) performed a more extensive analysis comparing
miltefosine-treated/untreated and resistant lines of L. donovani while also broad-
ening the metabolite base by employing three different platform technologies
(LC-MS, GC-MS and CE-MS). Upon miltefosine treatment, a decrease in the
abundance of several key intermediates in the arginine/polyamine pathway (argi-
nine, ornithine and citrulline) was observed in susceptible lines. This suggests that
the parasites, due to a decreased trypanothione availability, are particularly sus-
ceptible to oxidative stress (potentially induced by miltefosine). In resistant para-
sites, the abundance of these metabolites, as well as spermidine and intermediates of
trypanothione biosynthesis, was increased. The amino acid profile also differed in
susceptible and resistant lines. In the resistant line, the abundance of most amino
acids was increased, while in the susceptible line many amino acids showed a
moderate to strong decrease in abundance upon exposure to the drug suggesting
some degree of amino acid starvation. The importance of amino acids as a potential
carbon source is detailed below.
Metabolomics has also been used to explore changes in the metabolism of single
and combined therapy-resistant (CTR) L. donovani lines (Berg et al. 2015).
Combination therapies are commonly used in the field with the aim of shortening
treatment time, improving efficacy, and delaying the emergence of resistance (Croft
and Olliaro 2011). Researchers compared the metabolite profiles, acquired by
LC-MS, of lines resistant to amphotericin B-, miltefosine-, antimonial (SBIII)-,
paromomycin-, and combinations thereof, in order to identify unique and common
metabolic features. Comparing CTR lines with their singly resistant counterparts
96 J. Kloehn et al.

revealed that changes in the metabolome were highly varied. For some combina-
tions, the changes were not additive (amphotericin-B/paromomycin, amphotericin/
miltefosine and amphotericin-B/antimonial), while in other cases changes to the
metabolome were greater than what would be predicted from singly resistant lines
(miltefosine/paromomycin and antimonial/paromomycin). Quantitative analysis of
amphotericin B/antimonial (AS) and antimonial/paromomycin (SP) resistant lines
identified several shared changes in metabolites of proline biosynthesis and the
transsulfuration pathway which the researchers suggest is indicative of increased
protection against oxidative stress in these CTR lines (proline as a general
stress-response metabolite and free radical scavenger, products of transsulfuration
pathway feeding into trypanothione biosynthesis). Changes in the abundance of
lipid and sterol pathway intermediates were also observed in the tested CTR lines
suggesting alterations in membrane composition. Importantly, the researchers
sought to validate these conclusions using functional assays (susceptibility to
drug-induced and extracellular reactive oxygen stress, genomic DNA damage and
membrane fluidity).
As with many other metabolomics studies in Leishmania, the bulk of
MOA/resistance experiments have been undertaken only in the promastigote stage
(Vincent and Barrett 2015). Ultimately, however, any conclusions made about a
given drug’s MOA or proposed mechanism of resistance need to be verified in the
disease causing amastigote stage, such as those generated in vitro (Amaaxenic) or
isolated from in vitro infected macrophages (AmaMΦ) or from murine lesions
(Amalesion) (Vincent and Barrett 2015). As detailed in the following sections, each
Leishmania developmental stage is characterized by unique metabolic features and
growth rate which may result in significantly different drug efficacy, MOA, and
mechanisms of resistance. Furthermore, the host environment may additionally alter
a drug’s effectiveness by altering the parasite’s environment (e.g. nutrient restriction
and stress induced by host microbicidal responses), modulate a drug’s availability
(e.g. membrane permeability and prodrug catabolism), or indirectly kill the
amastigote by inhibiting targets in the macrophage host (e.g. immunomodulatoy
effects). Indeed, compound screening experiments have revealed significant dif-
ferences in the sensitivity of promastigote and amastigote stages to many drugs (De
Muylder et al. 2011; De Rycker et al. 2013). These findings indicate that under-
standing the nutritional environment and metabolism of the host cell is also criti-
cally important when delineating a drug’s MOA and the parasite’s development of
resistance. For example, it has been suggested that the accumulation of amino acids
in drug-resistant lines preserves the parasite’s fitness for the invasion and prolif-
eration in the nutritionally limited environment of the host macrophage by pro-
viding an alternative carbon source (Vermeersch et al. 2009; Berg et al. 2013;
Canuto et al. 2014). Finally, care needs to be taken when studying field isolates so
that detected genetic differences can be aligned with drug susceptibility/resistance
measures, virulence, and metabolomics data.
4 Using Metabolomic Approaches to Characterize the Human Pathogen … 97

10 Metabolomics Reveals Key Differences Between

Leishmania Developmental Stages

10.1 Footprinting Approaches

As discussed above, Leishmania parasites progress through several different

developmental stages during their lifecycle. Many of these developmental stages
can be generated in vitro by manipulating temperature and culture conditions
allowing detailed studies on parasite metabolism in the absence of the confounding
influence of host metabolism (with the caveat mentioned above that in vitro con-
ditions may be an inadequate model for the in vivo environment). A number of
studies have measured changes in the culture supernatant (footprinting (Kell et al.
2005)) of these different axenic parasite stages to infer the operation of specific
pathways in central carbon metabolism and overall metabolic fluxes (Hart and
Coombs 1982; Rainey and MacKenzie 1991; Castilla et al. 1995). Hart and
Coombs measured changes in the sugars, amino acids, and fatty acids in the
medium of axenically-derived L. mexicana promastigotes and amastigotes using a
variety of analytical approaches, including enzyme assays, gas chromatography,
and radioassays (Hart and Coombs 1982). Their results suggested that promastig-
otes have a highly glycolytic metabolism with significant amino acid uptake, while
Amaaxenic exhibit a shift toward greater reliance on fatty acid utilization via
β-oxidation (with a concordant decrease in glucose uptake). These conclusions were
supported by the increased sensitivity of Amaaxenic to the β-oxidation inhibitors,
4-pentenoate, and 2-mercaptoacetate (Hart and Coombs 1982). The researchers also
measured several metabolic end products—succinate, acetate, alanine, and CO2—
with the amount secreted varying depending on the developmental stage assayed. In
a complementary approach, Rainey and MacKenzie (1991) used 13C nuclear
magnetic resonance (NMR) spectroscopy to analyze the end products of
Leishmania pifanio metabolism. Analysis of the positional isotopologs provided
direct evidence for operation of the glycosomal succinate fermentation
(GSF) pathway in Leishmania in which glycolytic phosphoenolpyruvate is
imported back into the glycosomes and reduced to succinate via a series of reactions
that replicate those in the TCA cycle. These studies supported a model for pro-
mastigote metabolism in which glucose is catabolized via glycolysis to generate
phosphoenolpyruvate (PEP) and pyruvate, which are either converted to succinate
(via the GSF) or alanine (via alanine transaminase) and secreted or further catab-
olized in the TCA cycle to generate CO2, and reducing equivalents for oxidative
phosphorylation (Fig. 2). While amastigotes also utilize glucose, they do so at
a decreased rate, which coincided with increased use of fatty acids, although these
studies did not quantitate the relative contribution of these different carbon sources.
Notably, Leishmania lack a functional glyoxylate cycle and are therefore unable to
generate C4 precursors for sugar hexose synthesis, suggesting that any increase in
fatty acid β-oxidation would primarily be used to sustain ATP synthesis or to top-up
(anaplerosis) TCA cycle intermediates.
98 J. Kloehn et al.

Fig. 2 Central carbon metabolism in Leishmania. The schematic shows the acquisition of carbon
sources and key metabolic pathways. Glycolysis and gluconeogenesis as well as the GSF pathway occur
in specialized peroxisomes termed glycosomes, while the TCA cycle and β-oxidation occur in
mitochondria (enzymes for β-oxidation also localize to the glycosomes but mitochondria are likely the
main site of β-oxidation). The major carbohydrate reserve material of Leishmania is comprised of short
oligosaccharides (4–40 mannose residues long), termed mannogen which accumulates in the cytosol and
may play a major role in regulating substrate availability for gluconeogenesis and glycolysis. The role of
several enzymes/pathways in Leishmania metabolism was assessed by generating gene knock-outs or
using metabolic inhibitors. Essential pathways are indicated by red arrows. In many cases, the effects of
gene depletions or metabolic inhibitors on Leishmania viability differ between the promastigote and
amastigote stage (see text), highlighting the distinct metabolism of the two life-cycle stages. For example
parasites lacking the gluconeogenic enzyme FBPase grow normally as promastigotes in rich media but
display attenuated virulence in macrophages and mice. Other pathways appear to be essential in both
life-cycle stages—e.g., Luque-Ortega et al. (2008) showed that inhibition of the ATP synthase in
Leishmania through histatin 5 is lethal for promastigotes and amastigotes. The salvage of some essential
metabolites such as purine and vitamins are discussed elsewhere (e.g. McConville and Naderer 2011).
AcCoA Acetylcoenzyme A; C Cytochrome C; ETC Electron transport chain; Fru6P
Fructose-6-phosphate; Fru1,6P2, Fructose-1,6-bisphosphate; GDP-Man Guanosine diphosphate man-
nose; Glc6P Glucose-6-phosphate; GlcN6P Glucosamine-6-phosphate; Hst5 Histatin 5; Man6P
Mannose-6-phosphate; Man1P Mannose-1-phosphate; NaFAc Sodium fluoroacetate; KG Ketoglutarate;
Mann Mannogen oligosaccharides; NAD Nicotinamide dinucleotide; PM Plasma membrane; PPP
Pentose phosphate pathway; Rib5P Ribose-5-phosphate; TCA Tricarboxylic acid; Triose-P Triose
phosphate; UQ Ubiquinone; I Complex I (NADH dehydrogenase); II Complex II (fumarate reductase);
III Complex III (cytochrome bc1 complex); IV Complex IV (cytochrome c oxidase); V Complex V (ATP
synthase)

10.2 Intracellular Metabolite Levels

While metabolic footprinting has provided important insights into the distinct
metabolism of the Leishmania life-cycle stages, the information gained from ana-
lyzing extracellular metabolites is limited. An alternative approach is to measure
4 Using Metabolomic Approaches to Characterize the Human Pathogen … 99

changes in intracellular metabolite pools. Several studies on Leishmania pro-

mastigotes and amastigotes using NMR spectroscopy (Rainey et al. 1991; Gupta
et al. 1999; Arjmand et al. 2016) identified a range of amino acids (Ala, Arg, Glu,
Gln, Gly, Ser, Val, Iso/Leu), sugars (mannose), organic acids (lactate, succinate),
and other metabolites (acetate, creatinine, β-hydroxybutyrate, glycerol 3-phosphate,
glycerol, a-glycerophosphoryl choline, acetoacetate) that differed between the two
developmental stages, reinforcing the importance of glucose catabolism in the
promastigote stage and a shift to fatty acid/lipid catabolism in the amasitogote
stage. In fact, Berg et al. (2015) proposed that the switch from a promastigote-like
metabolism (highly glycolytic) to the amastigote-like metabolism (increased reli-
ance on the TCA cycle and β-oxidation for energy generation) occurs gradually, as
Prostat exhibit decreased levels of amino acids and sugar phosphates, which more
closely resembles the metabolite profile of Amaaxenic than Prolog. Gupta et al. (2001)
compared the profiles of Amaaxenic with that of Amalesion and found some striking
differences suggesting that Amaaxenic may represent an intermediate stage between
Prostat and Amalesion. While many studies have suggested that Amaaxenic closely
resemble Amalesion with regards to morphology, protein expression, infectivity, etc.,
differences between Amaaxenic and Amalesion potentially reflect inconsistencies in
the nutritional conditions of the culture medium and lesion environments. These
findings underscore the importance of undertaking future metabolomics work (and
proteomics/transcriptomics work) in vivo where possible (see below), alternatively
using purified extracted Amalesion or improving culture conditions to more closely
resemble the in vivo milieu.
While these approaches point to the operations of specific pathways, they provide
little information on overall flux or relative partitioning between different arms of
central carbon metabolism. In particular, they do not allow distinction between the
possibilities that changes in metabolite levels can be driven by changes in
synthesis/degradation, and uptake/secretion. Even when uptake data are considered,
it can be unclear whether a substrate is used for energy generation or metabolite
biosynthesis. Given the dynamic nature of metabolite pools, stable isotope (e.g. 13C,
2
H, 15N) labeling approaches, provide a powerful means to measure metabolic flux.

10.3 Flux Analyses

A detailed analysis of metabolic networks of L. mexicana promastigotes and

amastigotes was recently undertaken using comprehensive 13C-stable isotope
labeling (Saunders et al. 2014). All major developmental stages (Prolog, Prostat,
Amaaxenic isolated Amalesion) were labeled in defined medium with 13C-labeled
glucose, 13C-amino acids, and 13C-fatty acids and the incorporation of tracer car-
bons into a targeted list of 30 intracellular metabolites was quantitated by GC-MS.
Both Amaaxenic and Amalesion were found to exhibit a distinct stringent metabolic
response that was characterized by decreased glucose and amino acid uptake and
more efficient utilization of these carbon sources (Saunders et al. 2014) (Fig. 3).
100 J. Kloehn et al.

This glucose-sparing metabolism of amastigotes stands in stark contrast to the

glucose-wasting metabolism observed in rapidly dividing Prolog as well as nondi-
viding Prostat, which were characterized by high rates of glucose uptake and
secretion of partly oxidized end products (succinate, alanine, acetate) (Saunders
et al. 2014). Interestingly, both nondividing (Prostat) as well as replicating pro-
mastigotes (Prolog) exhibited a glucose-wasting metabolism, indicating that the
 4 Using Metabolomic Approaches to Characterize the Human Pathogen … 101

b Fig. 3 Differences in the central carbon metabolism of Leishmania life-cycle stages.

a 13C-labeling experiments were carried out to determine differences in the central carbon
metabolism and carbon source utilization of the Leishmania insect stage (Prolog) and the disease
causing amastigote stage (Amaaxenic) (Saunders et al. 2014). Following incubation of Prolog and
Amaaxenic in media containing U-13C-glucose (Glc), U-13C-amino acid mix (AA), U-13C-alanine
(Ala), U-13C-asparate (Asp), or U-13C glutamate (Glu), the 13C-enrichment was quantified in
numerous metabolites using GC-MS. b The study revealed marked differences between the two
life-cycle stages—promastigotes exhibit a glucose-wasting metabolism which is characterized by
rapid uptake and utilization of glucose as well as secretion of organic acids and alanine (green
arrows indicate uptake of metabolites, red arrows indicate secretion of end products). In contrast,
amastigotes display a glucose sparing metabolism and show drastically reduced rates of glucose as
well as amino acid uptake and utilization together. Both stages scavenge fatty acids, but
promastigotes primarily use these for membrane synthesis while amastigotes oxidize acquired fatty
acids. G6P Glucose 6-phosphate; F6P Fructose 6-phosphate; S7P Seduheptulose 7-phosphate;
Ru5P Ribulose 5-phosphate; 3PG 3-phosphoglycerate; 2PG 2-phosphoglycerate; PEP
Phosphoenolpyruvate; Suc Succinate; Mal Malate; Fum Fumarate; Cit Citrate; Ala Alanine; Asp
Aspartate; Glu Glutamate; Gly Glycine; Ser Serine; Thr Threonine; Pro Proline; Ile Isoleucine; Leu
Leucine; Lys Lysine; Phe Phenylalanine; Val Valine; Put Putrescine; Orn Ornithine; MTA
5-methylthioadenosine; Ura Uracil; CHO1 Mannogen; I3P Inositol 3-phosphate; MI Myo-inositol;
G3P Glycerol 3-phosphate; Sucr Sucrose; Glc Glucose; FA Fatty acids; AcO− Acetate; GlcN
Glucosamine; Man Mannose; ATP Adenosine triphosphate; NAD Nicotinamide adenine
dinucleotide. The figures are adapted from Saunders et al. (2014) and McConville et al. (2015)

stringent response was not just a consequence of reduced replication but rather a
hard-wired response that is induced during amastigote differentiation. Amastigotes
also exhibited increased fatty acid β-oxidation and increased reliance on the TCA
cycle metabolism (Saunders et al. 2014), consistent with results from earlier pro-
teomic analyses and metabolomics analysis (see above).
These labeling studies were used to predict pathways essential for amastigote
survival in vivo. In particular, the finding that amastigotes stages exhibited negli-
gible rates of glutamate uptake (compared to promastigotes) while the carbon
backbones derived from 13C-glucose and 13C-fatty acids were incorporated into
glutamate/glutamine, suggested that the TCA cycle may have an important role in
generating precursors, such as α-ketoglutarate for glutamate/glutamine synthesis.
These amino acids serve as essential amino donors for a number of other essential
pathways including aminosugar synthesis, pyrimidine synthesis, and
glutathione/trypanothione synthesis. Consistent with the proposal, treatment of
infected macrophages with either sodium fluoroacetate (NaFAc, an inhibitor of
TCA cycle aconitase enzyme) or methionine sulfoxime (MSO, an inhibitors of
glutamine synthetase) resulted in death of intracellular L. mexicana amastigotes
(Saunders et al. 2014). While all developmental stages exhibited some growth
sensitivity to these inhibitors in axenic cultures, particularly when exogenous
glutamate or glutamine was absent, Amaaxenic were much more sensitive to these
inhibitors than Prolog. While Prolog ceased growth during treatment with 5 mM
NaFAc, Amaaxenic lost viability at 50–100-times lower concentrations. The effect of
NaFAc on Amaaxenic was only partially rescued by addition of very high concen-
trations of glutamate (5 mM). Similarly, MSO treatment led to a growth arrest in
Prolog which was rescued by supplementation with glutamine. Interestingly, growth
102 J. Kloehn et al.

of intracellular AmaMΦ could not be rescued by addition of exogenous glutamine to

cultures suggesting that uptake of this amino acid into the phagolysosome by bulk
vesicle flow is inefficient and/or that expression of glutamate amino acid trans-
porters on intracellular amastigote stages is highly repressed. Taken together, these
results suggest that amastigotes rely heavily on a functional TCA cycle to produce
glutamate and glutamine and appear to downregulate the uptake of these amino
acids. This is particularly surprising, as the phagolysosome is the major site of
protein degradation and is expected to be rich in amino acids (Saunders et al. 2014).
These 13C-labeling studies have also been used to define the objective function of
a constraint-based model of L. infantum energy metabolism (i.e. not genome-wide)
(Subramanian et al. 2015). The resulting model, consisting of over 230 reactions and
5 cellular compartments, was able to accurately predict in silico the growth phe-
notypes of previously experimentally generated knock-out mutants (for example, the
L. major ΔFBPase and L. mexicana Δphosphomannomutase mutants, Naderer et al.
2006; Garami et al. 2001). Using the model, 61 single reaction combinations and
10,884 double reaction combinations were predicted that, when knocked-out, are
anticipated to be lethal in L. infantum and therefore may prove useful drug targets.
The model was also used to investigate changes to metabolism under different
nutritional environments, for example the researchers observed that when optimum
oxygen uptake is achieved, glucose is completely catabolized with succinate from
the GSF entering the TCA cycle rather than being secreted. As oxygen uptake
decreases, partially oxidized end products (acetate and succinate) are secreted from
the cell. As described in the profiling and labeling experiments described above,
glucose catabolism is critical even when other alternative carbon sources are
abundant (the model predicts co-utilization of nonessential amino acids alters fluxes
through glycolysis, glutamate biosynthesis and glycine/serine biosynthesis). In
support of this, when glucose availability is restricted, the model predicts no parasite
proliferation despite the availability of amino acids. Nonetheless, nonessential amino
acids were still important when catabolized in conjunction with glucose, increasing
biomass 1–1.5-fold over glucose alone (indeed Subramanian et al. highlight the
importance of glutamate in ATP generation). Finally, the researchers created pro-
mastigote and amastigote scenarios in their model and were able to predict the large
reduction in glycolysis, TCA cycle, ATP synthesis, and amino acid metabolism in
the amastigote stage (reduced, but essential, glucose uptake, reduced glutamate
uptake and reduced overflow metabolite excretion).

11 Studying Leishmania Metabolism in Vivo: Amastigote

Proliferation and Macromolecule Turnover

All of the studies described above rely on the analysis of axenically cultured
amastigotes or isolated Amalesion that have been incubated in rich media which may
not reflect the nutrient levels in vivo (Saunders et al. 2014). Indeed, the nutrients
4 Using Metabolomic Approaches to Characterize the Human Pathogen … 103

present in the phagolysosome as well as their concentrations are largely undefined

(Lorenz and Fink 2002; McConville and Naderer 2011), making it difficult to
establish culture media which replicates the conditions in vivo. An alternative
approach is to use stable isotope labeling in vivo. A number of studies have infused
animals with 13C-labeled precursors (glucose or amino acids) in order to measure
metabolic dynamics in vivo utilizing NMR spectroscopy or MS (Neurohr et al.
1983; Stromski et al. 1986; Shalwitz et al. 1989; Magkos and Mittendorfer 2009;
Maher et al. 2012; Kowalski et al. 2015). However, these experiments are tech-
nically complex and limited by several factors. First, 13C-labeled tracers are costly
and in vivo analyses require high quantities of the tracer to achieve detectable levels
in the analyzed tissue. While short-term labeling with these tracers can be easily
achieved through an oral bolus (gavage) of the tracer, the long-term administration
of 13C-tracers requires continuous infusion, which is costly and stressful to the
animal. Maintaining constant levels of a tracer within the analyzed tissue can also
be challenging due to rapid clearance, catabolism, or zonation effects. Additionally,
interpreting in vivo 13C tracer data is expected to be particularly challenging when
analyzing intracellular parasites, as the tracer is rapidly metabolized by the host
with the pathogens potentially taking up a variety of labeled tracer-derived
metabolites and intermediates. Hence, long-term 13C-labeling approaches are lar-
gely restricted to in vitro applications and are not well suited for the study of
intracellular pathogens.
An alternative approach to using 13C-labeled tracers is the use of 2H2O (also
known as heavy water, deuterium oxide and D2O) as the tracer (Hellerstein 2004).
2
H2O differs from natural abundance water (H2O) in that it contains two deuterium
atoms (2H) instead of hydrogen (typically available as >99.9 % 2H2O, v/v). 2H2O is
utilized by many enzymes that include water in their catalytic mechanisms,
resulting in the incorporation of 2H into stable C–H/2H bonds in a wide variety of
metabolites. This enzymatic 2H2O-labeling differs from 2H-exchange that occurs
across labile N–H or O–H bonds which is commonly used to identify exposed
residues in protein folding studies (Englander et al. 1997). While high concentra-
tions of 2H2O (>20 %, v/v) can be toxic to some organisms because of a solvent
isotope effect (where 2H2O/2H can interfere with the catalytic efficiency of some
enzymes) (Reuter et al. 1985; Takeda et al. 1998), few or no adverse effects are
observed when cells/animals are exposed to low (≤15 %, v/v) concentrations
(Thomson and Klipfel 1960; Lester et al. 1960; Kushner et al. 1999; Busch et al.
2007; Berry et al. 2015). The incorporation of 2H into different metabolite pools
(sugars, amino acids and fatty acids) and downstream macromolecules (DNA,
RNA, proteins, lipids and polysaccharides) can be readily quantitated using MS or
2
H-NMR spectroscopy directly or after depolymerization of macromolecules of
interest (Dufner and Previs 2003). Importantly, 2H2O rapidly equilibrates across all
tissues and cells and can be administered to cell cultures or animals safely and
easily for weeks or months, making it particularly suitable for measuring processes
that have turnover times on the scale of days or longer (Dufner and Previs 2003;
Busch et al. 2007). Administration of 2H2O also results in no perturbation to
external or intracellular metabolite levels in biological systems, such as occurs
104 J. Kloehn et al.

when a bolus of 13C-labeled metabolites is introduced into cultures or animals

(Berry et al. 2015). As a result of these features, 2H2O-labeling is increasingly being
deployed to measure multiple cellular processes in physiology and nutrition
including human studies (Landau et al. 1995; Busch et al. 2006, 2007; Murphy
2006). 2H2O cannot replace but may complement 13C tracers as the processes which
can be measured using 2H2O are limited. However, several approaches have been
developed which allow the quantification of the following processes in vivo using
2
H2O: Replication/DNA turnover (Neese et al. 2002; Hsieh et al. 2004; Busch et al.
2007; Pouteau et al. 2009), protein synthesis (Busch et al. 2006; Gasier et al. 2010),
lipogenesis and cholesterol synthesis (Murphy 2006; Pouteau et al. 2009; Previs
et al. 2011), as well as gluconeogenesis (Landau et al. 1995; Antoniewicz et al.
2011).
Kloehn et al. (2015) have recently shown that heavy water (2H2O–) labeling
approaches can be employed to measure multiple physiological and metabolic
processes in both cultured and tissue stages of Leishmania (Fig. 4). The labeling of
the major life-cycle stages of L. mexicana with 5 % (v/v) 2H2O in culture can be
used to accurately determine replication rates by measuring the incorporation of
deuterium into the deoxyribose moiety of DNA using GC-MS. This approach was
subsequently used to measure the growth rate of L. mexicana amastigotes in
inflammatory lesions in infected BALB/c mice following enrichment of the host’s
body water with 2H2O. Previously, the in vivo growth rate of pathogens was typ-
ically inferred from changes in the microbial burden in the relevant tissues as
determined by direct enumeration of pathogen levels in tissue biopsies following
microtitration and limiting cell dilution assays (Titus et al. 1985; Cotterell et al.
2000) or from measurements of pathogens that have been genetically manipulated
to express bioluminescent or fluorescent proteins (Lang et al. 2005; Thalhofer et al.
2010; Millington et al. 2010; Michel et al. 2011). However, these methods only
provide a measure of the net changes in microbial burden that reflect multiple
parameters in addition to replication rate, such as death rate and pathogen dis-
semination to other tissues. In contrast, 2H2O-labelling allows the measurement of
cell turnover in vivo (Busch et al. 2007). Lesion amastigotes were found to have a
doubling time of nearly 12 days, consistent with the 13C-labeling studies showing
that these stages enter a metabolically quiescent state. Although slow, this rate of
doubling can still account for the observed increase in parasite burden in lesion
granulomas, assuming parasite death is minimal (Kloehn et al. 2015). Taken
together, these analyses indicate that activation of the stringent response may allow
Leishmania amastigotes to sustain a very slow rate of replication and persist within
long-lived macrophages. An intriguing implication of the finding that both parasite
and macrophage populations are both long lived is that expansion of lesions and
parasite numbers may occur via the slow replication of macrophages and parti-
tioning of amastigote-containing phagolysosomes to each of the daughter cells.
Such a mechanism would be consistent with microscopy observations that rarely
detect extracellular parasites in granuloma tissues. Collectively, these findings
indicate that murine inflammatory lesions constrain Leishmania growth but
4 Using Metabolomic Approaches to Characterize the Human Pathogen … 105

Fig. 4 The physiological state of Leishmania life-cycle stages. Differences in the replication rate
as well as protein, RNA, and lipid turnover were measured using 2H2O-labeling in vitro and
in vivo (Kloehn et al. 2015). Leishmania Prolog show the fastest turnover rate for all
macromolecules indicating rapid replication, protein and RNA, and lipid synthesis. Strikingly,
the protein, RNA, and lipid turnover rates of Amalesion are reduced about 15–30-fold compared to
Prolog and are markedly reduced even compared to nondividing Prostat. These results indicate that
Leishmania enter a semiquiescent state in the lesion environment which is characterized by slow
growth and low activity of other energy intensive cellular processes such as protein synthesis

otherwise provide a highly permissive niche, which allows continuous expansion of

the parasite population (Kloehn et al. 2015). This strategy differs from that of other
granuloma-inducing pathogens such as M. tuberculosis (Munoz-Elias et al. 2005;
Gill et al. 2009).
The 2H2O-labeling approach was further extended to measure global changes in
RNA and protein turnover by measuring 2H-incorporation into RNA-ribose and
protein derived amino acids (Kloehn et al. 2015). As transcription and protein
translation represent the most energy consuming processes in cells, these parame-
ters are excellent indicators of the bioenergetic state of a cell. Significant differences
were observed in RNA and protein turnover between major life-cycle stages. In
particular, Amalesion were found to have much lower rates of RNA/protein turnover
than other stages, including nondividing promastigotes (Kloehn et al. 2015),
106 J. Kloehn et al.

providing further support for the notion that amastigotes enter into a distinct state of
slow growth and metabolic quiescence.
Several studies on Amaaxenic have suggested that global rates of protein synthesis
are downregulated, based on measurements of rates of 35S-methionine incorpora-
tion and polysome profile analysis (Lahav et al. 2011; Cloutier et al. 2012). Another
interesting approach for estimating protein turnover/metabolic activity in Amalesion
in vivo has recently been developed using L. major that express a photoconvertible
fluorescent protein (Muller et al. 2013). The authors suggest that protein synthesis
in situ is reduced due to the suppression of metabolism and cell division by sub-
lethal levels of NO. However, a broader application of this approach is limited
given that the fluorescence readout is difficult to calibrate to overall protein turnover
rates and also requires the generation of transgenic parasites lines with possible
associated virulence reduction (da Silva and Sacks 1987; Moreira et al. 2012; Ali
et al. 2013).
Additionally, 2H2O-labeling was shown to delineate specific metabolic pathways
in culture and in vivo. Analysis of 2H-incorporation into total cellular fatty acid
pools in Amalesion showed that this stage largely relies on the scavenging of fatty
acids, in contrast to the situation in promastigotes. However, Amalesion were still
dependent on the synthesis of linoleic acid (C18:2), as shown by the labeling of this
fatty acid in parasite extracts but not host cell serum and tissue (Kloehn et al. 2015).
Linoleic acid is the major polyunsaturated fatty acid of these stages and is syn-
thesized by desaturation of oleic acid. The enzyme oleate desaturase is absent in
mammals, and may therefore be a promising drug target.

12 Conclusion and Outlook

Metabolomic approaches have provided important new insights into the biology of
Leishmania and will continue to be an essential tool for investigating the meta-
bolism of these parasites given their dependence on posttranslational regulatory
processes. In particular, metabolomics has been used to map metabolic networks in
different developmental stages and parasite mutant lines, as well as to define the
mode of action of drugs and understand resistance mechanisms. Importantly, var-
ious 13C/2H labeling strategies have now been used to map parasite metabolism and
physiology in vitro and in infected tissues. We suggest that metabolomic mea-
surements using noninvasive stable isotope tracers will be increasingly important to
understand Leishmania metabolism in the mammalian host. Combining metabo-
lomic approaches with other ‘-omics’ approaches as well as molecular biology (e.g.
gene depletion), biochemistry (e.g. metabolic inhibitors), and biological data (e.g.
virulence) are expected to advance our understanding of Leishmania metabolism.
Last, phenotypically heterogeneous cells and the mechanism underlying cell-to-cell
variability have been identified and studied in a number of bacterial infections
(Lewis 2010; Helaine and Holden 2013; Helaine et al. 2014; Bumann 2015; Kopf
et al. 2016) but are poorly understood in protozoan infections, despite their crucial
4 Using Metabolomic Approaches to Characterize the Human Pathogen … 107

role in development (e.g. Plasmodium gametocytes and hypnozoites; Toxoplasma

bradyzoites, Leishmania metacyclics), persistence, and the emergence of drug
resistance (Seco-Hidalgo et al. 2015). However, the majority of studies described in
this chapter rely on bulk measurement, which provides an average of an entire
population of typically >107 cells masking any variability within the population.
Hence, novel single cell metabolomics approaches will be invaluable to identify
cell-to-cell heterogeneity in protozoan parasite populations (Zenobi 2013;
Seco-Hidalgo et al. 2015).

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Chapter 5
Exometabolomics for Linking Soil Carbon
Dynamics to Microbial Communities

Andrea Lubbe and Trent Northen

1 Introduction

Microbial metabolism has helped shape the world into what it is today, and con-
tinues to drive biogeochemical cycles (Falkowski et al. 2008) including the carbon
cycle. Soil microorganisms play a central role in the global carbon cycle, with an
estimated soil carbon pool of 2500 Gt, over three times the size of the atmospheric
carbon pool (Lal 2004). Inputs of organic carbon into soil is largely plant and
microbial biomass-derived, and carbon is released from soil into the atmosphere
mainly as CO2, the product of plant root, and microbial respiration (Johnston et al.
2004). While we are able to measure emergent properties such as the total release of
CO2 from soil and total organic carbon in soil at a particular time, the underlying
processes that occur between input and release are not well defined. This limits our
ability to understand how human activities are altering the balance of the global
carbon cycle, and how this will affect soil carbon dynamics (Lal 2004) that are
mediated by soil microbial community metabolism.
The bulk of microbial community studies have been based on metagenomics
approaches, where total genomic DNA from soil is sequenced (Delmont et al. 2011;
Fierer et al. 2012; Roesch et al. 2007). This culture-independent approach yields
insights into the microbial community structure (phylogenetic makeup), and has
become a very active field of research due to advances in sequencing technologies
(Franzosa et al. 2015). Besides community structure, metagenomes also reveal the
complement of genes present in soil microbial communities, reflecting their
potential metabolic functions which are inferences based on often poorly annotated

A. Lubbe
T. Northen (&)

Lawrence Berkeley National Laboratory, 1 Cyclotron Road, Berkeley, CA, USA
e-mail: [email protected]
A. Lubbe
e-mail: [email protected]

© Springer International Publishing Switzerland 2016 119

D.J. Beale et al. (eds.), Microbial Metabolomics,
DOI 10.1007/978-3-319-46326-1_5
120 A. Lubbe and T. Northen

genomes. These inferences of in situ metabolic processes can be strengthened

through metatranscriptomics and metaproteomic studies of environmental samples,
since these analyses enable correlation of genes actively transcribed and translated
(respectively) in the community with environmental variables and stresses (Morales
and Holben 2011).
Metabolomics is emerging as a very promising complement to soil metage-
nomics approaches as it can provide direct insights into the functioning of soil
microbial communities in their environment. Exometabolomics, the study of how
cells transform their extracellular small molecule environment (Silva and Northen
2015), is particularly relevant for studying soil metabolic processes and provides an
experimental approach to link organic carbon in the soil to the metabolism of
particular microorganisms or taxonomic group. Soils are complex mixtures of
organic and inorganic components, and are estimated to contain more than
two-thirds of the carbon in the terrestrial biosphere (Lal 2004). The organic com-
ponents, known as soil organic matter (SOM), make up most of this pool, and
thereby comprise the largest reservoir of carbon on Earth. While total pools of
organic carbon in soil can be estimated, the form it takes has been contested
(Lehmann and Kleber 2015). Most organic matter inputs to soil decompose within a
year (Jenkinson and Rayner 1977). Initial degradation is performed by exoenzymes
released by fungi and bacteria that break down organic matter into pieces small
enough to be assimilated by microbial cells (Baldock and Nelson 2000; Weiss et al.
1991). A longstanding view was that some of the degraded organic carbon was
assimilated into microbial biomass, and the rest was converted to large stable
polymeric compounds called humic substances (Stevenson 1994). Their stability
was thought to account for the large belowground pool of organic carbon. However,
advances in analytical techniques in the last few decades revealed a lack of evi-
dence for polymeric humic substances in soil (Piccolo 2002). Recent evidence
suggests that soil organic matter is rather a continuum of progressively decom-
posing organic compounds (Fig. 1) (Lehmann and Kleber 2015). The new view
suggests that much of the SOM exists in the form of lower molecular weight
molecules (below 600 Da). Their persistence in soils is not due to any inherent
recalcitrance of these molecules, but rather to factors related to the environment,
such as absence of degraders or consumers in the immediate environment, sorption
onto mineral surfaces, formation of noncovalently bonded aggregates, water
availability, pH, and redox state (Schmidt et al. 2011).

2 Exometabolomics for Analysis of Soil Organic Matter

Metabolomics involves the study of the metabolome, defined as the low

molecular-weight metabolites (typically less than 2000 Da) present in a cell or
living organism under a given set of physiological conditions (Harrigan and
Goodacre 2003; Oliver et al. 1998). By contrast, exometabolomics aims to char-
acterize extracellular small metabolites (Silva and Northen 2015). By studying
5 Exometabolomics for Linking Soil Carbon Dynamics … 121

Fig. 1 Schematic representation of three competing models for the fate of organic inputs to soil
(top), and the recently proposed soil continuum model (below). Selective preservation assumes that
some organic materials are preferentially mineralized, leaving intrinsically ‘stable’ decomposition
products behind. Progressive decomposition reflects the concept of microbial processing of large
plant biopolymers to smaller molecules. In the proposed SCM, a continuum of organic fragments
is continuously processed by the decomposer community from large plant and animal residues
toward smaller molecular size. At the same time, greater oxidation of the organic materials
increases solubility in water as well as the opportunity for protection against further decomposition
through greater reactivity toward mineral surfaces and incorporation into aggregates. Dashed
arrow lines denote mainly abiotic transfer; solid lines denote mainly biotic transfer; thicker lines
indicate more rapid rates; larger boxes and ends of wedges illustrate greater pool sizes; all
differences are illustrative. All arrows represent processes that are a function of temperature,
moisture, and the biota present. Reprinted from Lehmann and Kleber (2015), with permission
122 A. Lubbe and T. Northen

metabolites consumed from or secreted into the extracellular environment, insights

can be gained into the metabolic activity of the cell (Kell et al. 2005). This approach
(also known as metabolic footprinting) has been applied to characterize yeast
mutant metabolism and phenotypes (Allen et al. 2003; Castrillo et al. 2007; Mas
et al. 2007). Exometabolomics has also been applied in industrial settings, where
analysis of extracellular fermentation media are part of the process of optimizing
yeast fermentation conditions (Devantier et al. 2005; Fu et al. 2014), for monitoring
various industrially important bacterial and yeast strains in bioreactor cultures
(Paczia et al. 2012), and to study the breakdown of polysaccharides by anaerobic
bacterial strains (Villas-Boas et al. 2006). Apart from some recent studies (Swenson
et al. 2015b; Warren 2014), few examples of the application of exometabolomics to
characterize soil microbial communities have been reported, though dissolved
organic matter has been characterized in sea and river water with an exometabo-
lomics approach (Kido Soule et al. 2015; Morales-Cid et al. 2009).
A major reason for the paucity of soil exometabolomics studies is the complexity
of soil, and the associated challenges of extraction and sample preparation. The
extraction method used in any metabolomics experiment is critical to the quality of
the data obtained. The choice of extraction method should allow effective extraction
of metabolites from the system under study, without artifact formation or compound
degradation. In our current understanding of the nature of SOM (Lehmann and
Kleber 2015), the organic compounds that make up SOM exist in different com-
partments with different degrees of biological accessibility. The soluble component
of SOM is the most accessible to processing by soil microbes, and is referred to as
dissolved organic matter (DOM). DOM is often defined as dissolved metabolites
able to pass through a 0.45 lm filter (Gregorich et al. 2000), to differentiate it from
particulate organic matter. In order to characterize DOM in traditional soil science,
various methodological approaches involving extraction from soils have been
developed (Zsolnay 2003). These often involve extraction of soil under relatively
gentle conditions (e.g. aqueous salt solutions) to yield a fraction referred to as water
extractable organic matter (WEOM). This fraction conceptually consists of the
mobile and available portion of the total DOM pool (Corvasce et al. 2006). An
example of such an extraction procedure involves extraction of soil with concen-
trated salt solutions (e.g. up to 500 mM K2SO4) for a few hours, followed by
filtration or centrifugation, and analysis for total organic carbon (Jones and Willett
2006). The high salt concentration in the extraction buffer helps extract mineral
sorbed metabolites, but can cause issues with downstream sample preparation and
metabolite analysis in metabolomics methods used to characterize individual
components of DOM (e.g. formation of salt crystals in samples, ion suppression in
mass spectrometry, and decrease in sensitivity in NMR) (Annesley 2003; Kelly
et al. 2002). Therefore, in recent metabolomics studies, water-based extraction
methods were developed to extract organic matter from soils. Warren (2013a, b)
extracted field-moist soils in water by shaking for 10 min, followed by centrifu-
gation and filtration. The relatively short extraction time addressed concerns over
continued metabolism during extraction, which could give rise to altered metabolite
profiles (Rousk and Jones 2010). Swenson et al. (2015b) followed a similar process
5 Exometabolomics for Linking Soil Carbon Dynamics … 123

but performed aqueous extraction of soils for one h at 4 °C to slow any metabolic
activity present. Another concern with aqueous extraction is enrichment of the
metabolite profile by intracellular metabolites. Osmotic shock can potentially lyze
microbial cells and cause leakage of metabolites (Gregorich et al. 2000). Swenson
et al. (2015b) compared aqueous soil extracts to samples extracted in 10 mM
K2SO4 and 10 mM NH4HCO3, and found no significant qualitative differences in
metabolites detected. It was concluded that water is a suitable extractant for soil
exometabolomics of DOM and that these extracts would be most representative of
the types of resources available for soil microbes.
In some cases, an experiment may require analysis of soil intracellular and
extracellular metabolites. In this case, cell lysis is an important and desirable step in
sample preparation. To access intracellular metabolites, a traditional approach used
in soil science involves chloroform fumigation of soil to lyze microbial cells,
followed by extraction (Brookes et al. 1985; Vance et al. 1987). Swenson et al.
(2015b) compared metabolite profiles of water extracts of fumigated and unfumi-
gated soil samples (Fig. 2). A significant increase in the number and abundance of
metabolites was observed, however, the fumigation technique requires long times
of exposure to chloroform vapors, which raise concerns about continued metabolic
activity or increased enzymatic degradation of metabolites (Warren 2013a, b). The
use of organic solvents which are able to lyze microbial cells is another way to
obtain soil extracts containing intracellular and extracellular metabolites. This was
demonstrated by Swenson et al. (2015b) who used hierarchical cluster analysis to
show similarity in metabolite patterns between fumigated soil extracts and organic
solvent extracts of unfumigated soil. Soil was also directly extracted with chloro-
form and K2SO4 solution (1:4, v/v) to obtain extracts containing intracellular
metabolites (Kakumanu et al. 2013). Rochfort et al. (2015) extracted freeze-dried,
finely ground soil with an 8:2 methanol-water solution by sonication for 10 min.
Since this method breaks up soil aggregates in addition to using organic solvents, it
is not surprising that it provided extracts with a wide coverage of metabolite classes,
derived from the intracellular and extracellular soil metabolite pools.
The major analytical methods used in the field of metabolomics are based on
Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR) (Dettmer et al.
2007; Dunn et al. 2005; Forseth and Schroeder 2011). Each method has advantages
and disadvantages related to sensitivity, structural information, ease of quantitation,
breadth of metabolite coverage, and availability of structural databases for identi-
fication (Lenz and Wilson 2007). Although studies on the soil microbial exome-
tabolome are limited, a few recent examples demonstrate the utility of these
methods to this field.
Gas Chromatography coupled with Mass Spectrometry (GC-MS) has previously
been applied to targeted analyses of particular chemical classes of small soil
metabolites such as sugars or amino acids (Kakumanu et al. 2013). GC-MS is also
well suited for measuring a broad range of small metabolite classes, and has been
widely used in untargeted metabolomics studies in plants (Jenkins et al. 2004),
human biofluids (Garcia and Barbas 2011), and microbial biomass (Koek et al.
2006). Analysis of hydrophilic/polar metabolites by GC-MS requires derivatization
124 A. Lubbe and T. Northen

Fig. 2 a Workflow for soil DOM extraction: A Soil is sieved through 2 mm and fumigated with
chloroform for 24 h to access intracellular metabolites or left unfumigated for extracellular
metabolites. B Soil is extracted with the appropriate solution in a 1:4 ratio (2 g soil: 8 mL
extractant) on an orbital shaker for 1 h at 4 °C. C Extract is centrifuged to pellet soil and the
supernatant filtered through 0.45 lm filter discs. D Dried down and derivatized for GC-MS.
E Data are analyzed for metabolite identification. b Relative intensity of metabolites in extracts of
unfumigated and fumigated soil prepared with different extractants, and analyzed by GC-MS.
Reprinted from (Swenson et al. 2015b), with permission
5 Exometabolomics for Linking Soil Carbon Dynamics … 125

to increase the volatility of compounds. Swenson et al. (2015) characterized soil

extracts using GC-MS. After extraction with different solvents, samples were
derivatized by methoxyamination and trimethylsilylation. Hundreds of unique
features were detected, of these 55 were confidently annotated using the Fiehn
spectral metabolite database (Kind et al. 2009) and comparison with authentic
standards. Metabolites detected in all samples included sugars, sugar alcohols,
amino acids and amino acid metabolites, nucleobases, carboxylic acids, and sterols
(Swenson et al. 2015b).
Liquid Chromatography coupled with Mass Spectrometry (LC-MS) has become
an important analytical tool in metabolomics, and has also been applied in studies
on many biological systems (Theodoridis et al. 2008; Zhou et al. 2012). Separation
of metabolites is achieved by LC using various stationary phases depending on the
polarity of the target metabolites. There are various options available for ion
sources and mass analyzers in LC-MS systems (reviewed by Zhou et al. 2012). Due
to the high structural diversity of metabolites, a particular sample typically needs to
be analyzed in positive and negative ionization mode to obtain a good coverage of
the metabolome. DOM is by definition composed of small metabolites dissolved in
water in situ (Leenheer and Croué 2003). This fraction of SOM is therefore
amenable to separation by hydrophilic interaction liquid chromatography (HILIC),
a variant of normal phase chromatography (Alpert 1990). Baran et al. (2015)
analyzed extracellular soil water, as well as intracellular metabolites of isolates from
biological soil crust, with LC-MS using zwitterionic HILIC chromatography and
electrospray ionization (ESI). Out of nearly 500 molecular features detected in this
study, 79 metabolites were identified based on MS/MS data and comparison with
authentic reference standards. A similar method was used by Swenson et al. (2015a)
to study sorption of microbially derived metabolites onto mineral surfaces.
Capillary electrophoresis mass spectrometry (CE-MS) is suitable for the analysis
of charged metabolites and has found applications in metabolomics studies sum-
marized in a series of reviews (Ramautar et al. 2009, 2011, 2013). In a study
focusing on the pool of nitrogen metabolites in soil (dissolved organic nitrogen:
DON), Warren (2013a) employed a CE-MS procedure. This method allowed
detection of small metabolites ionizable by electrospray that are cationic at low pH.
Approximately 100 nitrogen-containing metabolites with a wide range of polarities
were detected, of which 57 were identified (Warren 2013a).
Fourier transform-ion cyclotron resonance-mass spectrometry (FT-ICR-MS) is
an established method for analyzing natural organic matter, and has been widely
used to characterize complex organic mixtures in environmental samples
(Kujawinski 2002). This method allows the detection of ions with excellent mass
accuracy and resolving power, so that unique empirical formulas can be assigned to
most of the thousands of signals detected (Hockaday et al. 2006). Based on atomic
ratios (e.g. H:C, O:C) these formulas can be assigned to chemical classes such as
carbohydrates, lipids, lignins, tannins, and proteins (Ohno et al. 2014). While
individual features are not unambiguously identified using this technique,
FT-ICR-MS is very useful for obtaining overviews of patterns in soil DOM
dynamics (Hockaday et al. 2006, Kujawinski et al. 2004; Ohno et al. 2014).
126 A. Lubbe and T. Northen

NMR is an established analytical platform in the field of metabolomics. It has

been applied in analysis of human biofluids (Nicholson and Lindon 2008), plants
(Kim et al. 2010), and microbiological samples (Grivet et al. 2003). NMR exo-
metabolomics has been extensively applied to study microbial cell culture systems
(Behrends et al. 2014; Resmer and White 2011; Szeto et al. 2010). While solid-state
NMR techniques have been employed to analyze macromolecules and structural
aspects in soils (Baldock et al. 1992; Kögel-Knabner 1997), there are few examples
of NMR used for the characterization of the small metabolite complement (mi-
crobial- or plant-derived) of SOM. Jones et al. (2014) analyzed extracts of soils
from former mine sites by NMR. The aim was to obtain a survey of the naturally
occurring products of soil community metabolism (including intracellular
metabolites). NMR spectra were dominated by sugars, and a range of other
metabolites such as amino acids and nucleosides were detected. A recent study also
characterized soil extracts by NMR for a comparison of native versus agricultural
soils (Rochfort et al. 2015). Complex spectra were obtained that were dominated by
sugar resonances. Lipophilic compounds (terpenes, lipids) were also detected due to
the extraction solvents having a higher organic solvent composition than that used
by Jones et al. (2014).

3 Exometabolomics for Analysis of Whole Microbial

Communities

The exometabolome of a complex soil microbial community comprises the sum of

small metabolites being produced or released, and consumed by all the metabolic
activity in the soil. The exometabolome is thus a reflection of the net metabolic state
of the community. Studying differences in the microbial community exometabo-
lome under different conditions can lead to insights into the response of commu-
nities as a whole. In one of the first exometabolomics studies on complex microbial
communities, Henriques et al. (2007) applied an LC-MS based approach to analyze
soluble metabolites in wastewater treatment plant communities. Activated sludge
cultures from four different wastewater treatment plants were exposed to four dif-
ferent chemical stressors known to affect the processing ability of such commu-
nities. Comparisons of metabolite profiles between untreated and treated samples
using multivariate statistical methods revealed clear patterns between the different
toxin-stressed cultures. A limited number of variables were able to discriminate
samples based on chemical treatment, which was community-independent. It was
concluded that the discriminant metabolites may be universal biomarkers for these
stress conditions, and that these may be used in developing early warning tools for
toxins in these systems (Henriques et al. 2007). Exometabolomics has also been
applied to analyze uptake and release of extracellular metabolites from microbial
biofilm consortia occurring in water pipes (Beale et al. 2010). Small metabolite
profiles, obtained by GC-MS, of water flowing through copper pipe systems
5 Exometabolomics for Linking Soil Carbon Dynamics … 127

differentiated samples exposed to copper corroding microbial biofilms from those

that were not (Beale et al. 2012). In a pilot study this approach was applied to a
water supply network, where it provided information on biofilm activity in the
system. This approach showed potential for elucidating the relationship between
specific metabolites in water supply networks and issues related to water quality,
caused by microbial biofilms (Beale et al. 2013).
The effect of human activities on soil systems have been the topic of metabo-
lomics field studies. In a report on soils from former mine sites in the UK, Jones
et al. (2014) employed NMR and principal component analysis (PCA) to compare
metabolite profiles of soil extracts. Soil sites under study were geographically
dispersed and had a range of physicochemical properties. The PCA grouped some
sites together based on similarity of their overall profiles. The authors concluded
that the observed patterns are likely due to the similar pollution patterns at the sites,
but did not do further in-depth analysis of the factors potentially underlying the
observed patterns. Another NMR-based study compared soil extracts (intra- and
extracellular metabolites) of geochemically matched remnant (native) and agricul-
tural (managed) soils (Rochfort et al. 2015). When subjected to multivariate data
analysis, samples were grouped together based on land use. NMR resonances
responsible for the observed groupings were assigned to characteristic terpene and
aromatic compound signals in the remnant soils, and sugar and lipid signals in the
agricultural soils. Soil samples were analyzed in parallel by mid-infrared
(MIR) spectroscopy, a technique that employs absorption and transmission of
photons in the infrared energy range (about 2500–25,000 nm in the electromagnetic
spectrum), to characterize molecules based on their constituent bonds.
(Bellon-Maurel and McBratney 2011). Multivariate data analysis of these data
resulted in samples clustering together based on location, irrespective of land use.
Soil extracts were also tested for antibacterial activity, and the most active extracts
were from native soil samples that clustered together by PCA analysis. This study
established that the two analytical methods captured different aspects of the soil,
namely soil biochemistry (NMR) and soil physicochemistry (MIR). It also
demonstrated how biochemical characteristics as measured in this metabolomics
study can be related to functional aspects of soil communities as a whole.
The above studies followed an untargeted metabolomics approach, where
metabolite profiles were measured and compared between samples without iden-
tifying the compounds responsible for discriminating groups. Rochfort et al. (2015)
were able to assign important discriminating NMR signals to compound classes
(e.g. terpenes and aromatics), but noted that further characterization would be
needed to confidently identify individual metabolites. This untargeted approach is
widely used in other fields employing metabolomics (Sévin et al. 2015), but also
points to a larger issue in soil exometabolomics, i.e., very little data on the com-
position of soil metabolites. The studies mentioned in the analytical methods sec-
tion comprise the few that have contributed to the broad qualitative profiling of
multiple compound classes in soil (as opposed to targeted methods for one com-
pound class at a time). Even fewer have attempted to characterize soil metabolites
in a quantitative manner. One exception is Warren (2013b) who performed a broad
128 A. Lubbe and T. Northen

analysis of small nitrogen-containing metabolites in different soil types dominated

by different vegetation. The relative proportions of the different compound classes
in this pool of small metabolites were determined. The relative abundance of the top
ten small nitrogen-containing metabolites in each soil type was also analyzed. Even
though the study of Warren (2013b) focused on a particular subset of metabolites,
such a detailed quantitative analysis lays an important foundation for understanding
what is in the soil exometabolome. Similar characterizations are needed that include
a broader range of metabolite classes, and relate these to differences in factors such
as vegetation type and physicochemical factors.
Warren (2013b) pointed out that soil water extracts may not accurately represent
what is biologically available, since differential adsorption to the soil stationary
phase may occur. The mineral content and surface area of soils are known to affect
the solid-state partitioning of and thereby the accessibility of DOM components to
microorganisms (Kalbitz et al. 2000). However, these processes are not understood
down to a metabolite-specific level. Recently, Swenson et al. (2015a) investigated
the sorption of small metabolites from a soil bacterial lysate on an iron oxide
mineral, ferrihydrite. Different metabolite classes were adsorbed to different
degrees, with phosphate-containing metabolites, for example, showing the highest
sorption (Fig. 3), while other metabolites were not adsorbed, suggesting their
higher degree of bioavailability in iron-rich soils. Since high-sorbing metabolites
were able to displace sorbed phosphate from the ferrihydrite, the authors hypoth-
esized that the release of such metabolites by soil microbes may be a strategy to
access phosphate in soils where it is limiting. More studies of the effects of minerals
on the bioavailability of small metabolites will help elucidate the role abiotic factors
play in SOM dynamics of different environments.

4 Who Does What in Soil Community: Characterizing

Metabolism of Individual Members

One approach to understanding the dynamics of a microbial community is to

characterize the individual members of the community in isolation. Studying the
uptake and release of metabolites through a particular microbial isolate in the lab-
oratory, insights can be gained into its metabolic interactions with the environment.
Baran et al. (2011) used an untargeted metabolite footprinting approach to charac-
terize the marine cyanobacterium Synechococcus sp. PCC 7002 cultured in different
growth media (Fig. 4). A wide variety of metabolites were found to be taken up by
this strain, and analysis of intracellular metabolites also provided insights into which
metabolites were actively turned over and which were maintained in cells in their
native states. A study on acid mine drainage used an exometabolomics approach to
study the role of the primary producer Euglena mutabilis in these oligotrophic
environments (Halter et al. 2012). The exo- and endo-metabolome of E. mutabilis
was profiled in situ and also for laboratory grown cultures. A number of metabolites
5 Exometabolomics for Linking Soil Carbon Dynamics … 129

Fig. 3 Sorption of small metabolites from a soil bacterial lysate on an iron oxide mineral,
ferrihydrite. For each metabolite, the percent sorption (relative to the non-mineral control) is
displayed as mineral concentration increased from 0.5–32 mg. Metabolites were analyzed by
LC-MS. Putatively identified metabolites are indicated by parentheses. Reprinted from Swenson
et al. (2015a), with permission

in the acid mine drainage exometabolome were found to be secreted by the cells in
laboratory cultures. This suggested an important role in organic matter production by
E. mutabilis for consumption by other microbial strains in this ecosystem.
Baran et al. (2015) extended this approach by characterizing multiple isolates
from a biological soil crust (biocrust) community. The primary producer in this
community, the filamentous cyanobacterium Microcoleus vaginatus, was cultured
in the laboratory. Exo- and endo-metabolite profiling revealed that many metabo-
lites were released into the culture medium by this strain. Seven bacterial isolates,
representing diverse phyla from the biocrust environment, were cultured individ-
ually in different-rich media to characterize their substrate preferences. Only a small
proportion of metabolites detected in the media were taken up by any given strain,
and there was little overlap between the strains’ preferred substrates. Metabolite
130 A. Lubbe and T. Northen

Fig. 4 Comparison of levels of selected metabolites in the growth media following growth of
Synechococcus (full bars) against their levels in control media (open bars, n = 4), as determined
by LC-MS. The peak areas axis was scaled with a square root to improve the visualization of
smaller peaks. Statistically significant differences are indicated as “*” (p < 0.05), “**” (p < 0.01),
or “***” (p < 0.001). An arrow is shown next to the name of a metabolite if it was found to be
significantly consumed (←), released (!), or both consumed and released ($). Reprinted from
Baran et al. (2011), with permission

profiling of the biocrust soil water was also performed to link the observed patterns
from the isolates to the intact microbial community. Changes in metabolite profiles
at different times following wet up of desiccated biocrust showed patterns similar to
those observed in the individual isolate experiments. This study revealed the par-
ticular substrate preferences of sympatric isolates from a soil community, which
suggest that exometabolite niche partitioning may be an important driver in
5 Exometabolomics for Linking Soil Carbon Dynamics … 131

maintaining soil microbial diversity. Conversely, if different microbial phyla have

different roles in processing soil organic matter, it follows that changes in soil
microbial diversity may affect carbon cycling in soils.
Integrating exometabolomics data from various soil isolates would be a useful
way to form hypotheses about the relationships between different strains in a par-
ticular environment. An online data repository, Web of Microbes, has been
developed for such exometabolomics data (webofmicrobes.org). This tool allows
rapid visualizations of large exometabolomics datasets of individual isolates that
enables predictions to be made about how they behave in a community. This
includes interactions such as potential resource competition and cross-feeding
between strains, and how these relationships would be affected by changes in the
chemical environment. Characterizing individual isolates from a soil community
can shed light on how they behave in relation to other soil community members.
However, this approach is limited to strains that can be cultured outside of their
native habitat, thereby excluding the vast majority of the soil microbial diversity
(Schloss and Handelsman 2003). Hence, there is a need for methods that enable the
study of soil microbial communities in situ, to link specific functions to particular
community members, and to elucidate the metabolic interactions between them.

5 Stable Isotope Probing: Tracking Flow of Substrates

Through Communities

Stable isotope probing (SIP) techniques involves addition of a stable isotope enri-
ched substrate, and tracking its fate as it is transformed by the metabolism of
community members into labeled molecules/biomarkers (Dumont and Murrell
2005). Variations of SIP target different biomarkers that become labeled as a result
of growth on the labeled substrate. One approach targets microbial phospholipid
fatty acids (PLFAs). Since different microbial classes possess characteristic fatty
acids as part of their cell membranes, selective extraction and analysis of PLFA
patterns is an established approach for determining the composition of microbial
communities (Zelles 1999). In PLFA-SIP, tracking which is the characteristic
phospholipid fatty acids become labeled with the stable isotope yields information
about which groups of microbes were responsible for metabolizing the labeled
substrate. This approach has been used to identify groups of microorganisms per-
forming particular functions in soils based on labeling with substrates such as 13CH4
(Bull et al. 2000), 13C-acetate (Boschker et al. 1998), 13C- glucose, and -ribose
(Apostel et al. 2015). Uniformly labeled 13C-cellulose and 13C-lignin were substrates
in a PLFA-SIP study on the role of diverse microbial groups in plant polymer
degradation (Torres et al. 2014). Treonis et al. (2004) combined a 13CO2-labeling
experiment with PLFA analysis to identify microbes assimilating plant root
132 A. Lubbe and T. Northen

exudates. The disadvantage of this approach is based on PLFA biomarkers

determined from cultivated microbes limiting application to uncultivable
microorganisms.
Another approach relies on combining SIP with nucleic acid analysis. This relies
on the incorporation of the isotopic label into DNA or RNA, so that subsequent
separation and sequencing of the labeled fraction identifies community members
actively incorporating the labeled substrate (Dumont and Murrell 2005). DNA-SIP
has mostly been used with 13C-labeled substrates, such as 13CH3OH to study soil
methylotrophs (Radajewski et al. 2000), and 13C-naphthalene and other organic
compounds to characterize pollutant biodegraders (Padmanabhan et al. 2003).
Using 13C-cellulose as a substrate, El Zahar Haicher et al. (2007) identified cel-
lulose degraders in a soil community using a DNA-SIP approach. These included
bacteria not previously known to have this ability, as well as a number of uncul-
tured strains. A disadvantage of DNA-SIP is that a relatively large amount of
labeled substrate is needed, together with long incubation times to allow production
of highly labeled 13C genomic DNA. Artificially high substrate concentrations are
thus often applied to soils, which can cause biases in how the community behaves
(Dumont and Murrell 2005). The incorporation of 13C into RNA occurs earlier than
DNA (Manefield et al. 2002), therefore targeting RNA as the labeled biomarker
molecules in RNA-SIP allows the use of shorter incubation times. RNA-SIP also
allows detection of cells which are metabolically active, even though they are not
dividing or growing (El Zahar Haichar et al. 2007). A combination of DNA- and
RNA-SIP can potentially be a very powerful approach. Recently, H18 2 O was applied
to a soil bacterial community as a universal substrate, and was found to be an
effective label for DNA- and RNA-based SIP approaches for studying active
members of the community (Rettedal and Brözel 2015).
SIP approaches monitoring the changes in labeling patterns over time can yield
valuable information on how nutrients flow through a microbial community.
Labeled substrates will become incorporated into cells (e.g. as part of PLFA, DNA,
RNA) but a proportion will be transformed and transported out of the cell, where it
may be consumed by other members of the community food web (DeRito et al.
2005). Extending the analysis of labeled biomolecules to the community
exometabolite pool is thus a potentially powerful approach for elucidating how
these trophic interactions occur. Date et al. (2010) combined DNA-SIP with NMR
exometabolomics to study microbial variability and metabolic dynamics in a mouse
gut microbial community. Using labeled glucose as a sole carbon source,
metabolic-microbial correlation analysis was performed, allowing identification of
glucose-utilizing gut microbes and their extracellular metabolites. Microbial strains
consuming the metabolites produced by the glucose utilizers were also identified,
together with their extracellular metabolites. The study demonstrated that the fea-
sibility of this approach for tracking carbon flux within a microbial community by
identifying members of the community involved at different steps, as well as the
metabolites that mediate their interactions. This approach clearly has great potential
to address questions about carbon flux in the context of soil microbial communities.
5 Exometabolomics for Linking Soil Carbon Dynamics … 133

Labeling experiments with stable isotopes can also aid analysis of the highly
complex soil exometabolome. For example, in NMR-based studies, the low natural
abundance of the magnetic isotope of carbon (13C) results in low sensitivity of
detection in unlabeled systems. Signals are dramatically enhanced as metabolites
become labeled with 13C isotopes, thereby facilitating identification of metabolites
downstream of the labeled substrate (Schneider et al. 2003). In mass spectrometry
based methods coupled to chromatographic separations, labeled metabolites can be
detected at the same retention time as their unlabeled counterparts, with charac-
teristic shifts in mass spectral features corresponding to the number of incorporated
labeled isotopes (Rodgers et al. 2000). Computational methods have been devel-
oped for the quantitative detection of features derived from a particular labeled
compound, even when not all metabolites are identified (Hiller et al. 2010).
However, stable isotope labeling can greatly facilitate unambiguous assignment of
chemical formulas, and thereby identification of unknown features (Baran et al.
2010). Thus, SIP methods show great promise for reducing noise by highlighting
relevant metabolites and pathways, and identifying unknown metabolites in com-
plex datasets such as those generated in soil exometabolomics experiments.

6 Metabolite Imaging: Microbial Interactions Through

Space

Soil is a very heterogeneous matrix, in which biotic and abiotic factors combine to
create diverse microclimates. Studies on soil microbial communities are often
conducted on homogenized bulk soil samples, in which the spatial structure of soil
and soil microorganisms are disrupted (Becker et al. 2006). Yet, observations of
microbial communities at the micron scale have revealed defined spatial organi-
zation. For example, in dental plaque, a nine taxon microbial consortium was
observed to be radially arranged around cells of filamentous bacteria (Welch et al.
2016). Obligate aerobes were arranged around the periphery of the consortium,
anaerobes were found in the interior, and others were localized in ways suggestive
of their functional roles in the consortium. Such structured assemblages have been
observed in biofilms and consortia occurring in aquatic systems, on leaf and root
surfaces, and in pathogenic or commensal associations with humans (Almstrand
et al. 2013; Wessel et al. 2013). It is believed that the spatial arrangement of soil
microbial communities is a very important driver of microbial diversity in soil,
thereby also of microbial community functioning (Ettema and Wardle 2002).
Therefore, the next level of detail required to understand microbial soil commu-
nities is characterizing their functioning in space. To achieve this, experimental
methods are needed to observe specific community members and their activities in
relation to other community members in their native spatial arrangement.
134 A. Lubbe and T. Northen

Experimental approaches utilizing labeled substrates have been successfully

used to visualize and identify labeled microbial cells taking up a specific com-
pound. FISH-microautoradiography or fluorescence are approaches for detecting
bacterial cells that have consumed and metabolized a specific radioactive substrate,
and identification of these cells using an oligonucleotide probe (Adamczyk et al.
2003; Lee et al. 1999). The use of radioactive labels are less desirable, and recent
technological advances have yielded promising alternative approaches for analyz-
ing interactions between microorganisms and their chemical environment (Wessel
et al. 2013). Orphan et al. (2001) used FISH in combination with Secondary Ion
Mass Spectrometry (SIMS) to detect and visualize 13C profiles in microbial con-
sortia composed of archaea and sulfate-reducing bacteria. Lower 13C/12C ratios in
both the archaea and associated bacteria provided evidence that methane was
consumed by the former, and that methane-derived carbon was transferred to the
latter consortium members. SIMS is ideally suited to detect isotopes at very fine
resolution, for example, nanoSIMS can image with some 50 nm resolution.
Therefore, the combination of SIMS with stable isotope probing (SIP) shows great
promise for spatially resolved analysis of single microbial cells and their utilization
of particular substrates (Behrens et al. 2008; Chandra et al. 2008; Cliff et al. 2002).
The above-mentioned methods allowed tracking of substrates into identifiable
microbial cells. Ideally, the metabolites released into the environment and
exchanged between community members should also be characterized.
Besides SIMS, other Mass Spectrometry Imaging techniques are potentially well
suited to do this, since a broad range of metabolites can be detected without the
need for labeling with a radioactive or stable isotope (reviewed by Watrous and
Dorrestein 2011). In Mass Spectrometry Imaging techniques, ionization probes
generate ions on the sample surface, and the sample stage is moved in the x–y plane
so that this is done across a defined sample area. Mass analyzers detect the gen-
erated ions, resulting in a grid of data points each with its own mass spectrum. An
ion map can be made from these data showing the location and intensity of detected
ions across the measured sample surface.
In specific Mass Spectrometry Imaging techniques (e.g. nanoDESI-IMS), sam-
ples are detected at atmospheric pressure, and samples can be wet (e.g. fresh
samples of bacteria on an agar plate can be analyzed directly), while for others (e.g.
MALDI-IMS), dry samples are covered in matrix and ionization and detection
occurs under high vacuum (Wessel et al. 2013). In nanostructure-initiator mass
spectrometry (NIMS), microbial agar cultures cannot be analyzed directly since
desorption/ionization occurs at the bottom of the sample (Woo et al. 2008). For this
technique, “replica extraction transfer” is used to transfer metabolites from the
culture surface to the NIMS surface, allowing spatial arrangement of metabolites to
be retained (Louie et al. 2013). These approaches have been used to characterize
metabolites produced and released by microorganisms on solid culture media
(Fig. 5). For example, Traxler et al. (2013) detected a suite of secondary metabo-
lites released by Streptomyces coelicolor in response to interactions with various
Actinomycetes. Watrous et al. (2013) used nanoDESI to profile metabolites in
single colonies of Schewanella oneidensis MR-1 and Bacillus subtilis 3610, as well
5 Exometabolomics for Linking Soil Carbon Dynamics … 135

(a)

P. stutzeri
RCH2

S. oneidensis
MR1

(b)

2.5 mm
m/z 575.5 576.5 523.5

Fig. 5 Mass spectrometry imaging of P. stutzeri RCH2 and S. oneidensis MR1 coculture.
a Optical image of coculture on solid medium. b Tricolor mass spectrometry image of coculture
with m/z corresponding to species-specific lipids (Katherine Louie, unpublished)
136 A. Lubbe and T. Northen

as a mixed biofilm of these strains. A range of peptides, lipids, and small molecules
were detected (Watrous et al. 2013). A REX-NIMS approach was used by Louie
et al. (2013) to identify ions localized to regions within and between bacterial
colonies cultured individually and in coculture on agar media. In a study on
methanotrophic microbial mats from sea shelf methane seeps, Time-of-Flight
Secondary Ion Mass Spectrometry (TOF-SIMS) was used to characterize microbial
lipid biomarkers (Thiel et al. 2007). Characteristic lipid classes were detected in
distinguished areas, which matched the presence of different microbial colonies as
determined by conventional microscopic techniques.
Mass Spectrometry Imaging techniques show great promise for characterization
of microbial communities in space, however, technical challenges limit their
application to complex microbial communities in soil environments. Benefits and
challenges of different imaging mass spectrometry techniques were comprehen-
sively reviewed by Watrous and Dorrestein (2011). While able to detect metabolites
with a wide mass range, the resolution of methods such as MALDI-IMS and
nanoDESI currently does not allow imaging down to the single-cell level.
Dynamic SIMS offers the best spatial resolution (sub-µm scale) but does not pro-
vide molecular information beyond elements and small atomic clusters. Another
challenge of imaging experiments targeting small metabolites lies in the sample
preparation. Methods for preparing thin soil sections as used in soil sciences typ-
ically involve fixing in formaldehyde, washing and impregnation in resin (Nunan
et al. 2001), which would not be suitable for IMS. Tissue samples (e.g. mammalian,
plant) are usually embedded in a substance such as OTC polymer, gelatin, or
agarose gel for stabilization during cryosectioning (Cornett et al. 2007; Lee et al.
2012). Even if such treatment would not cause delocalization of metabolites in soil
samples, the heterogeneous physical structure of soil may hamper cutting thin
sections for imaging. Most IMS experiments to date involve laboratory cultured
agar samples which can be analyzed fresh or mounted and dehydrated in prepa-
ration for imaging experiments (Traxler et al. 2013; Yang et al. 2012). Studies
where microbial consortia from a natural environment were used either involved
smears of soil on the sample target (Orphan et al. 2001), or cryosectioning of
well-structured communities such as microbial mats (Thiel et al. 2007) or sym-
bionts associated with other organisms (Lechene et al. 2007; Schoenian et al. 2011).
Given the great potential of these approaches, there is an urgent need for improved
sample preparation methods that will enable small metabolite imaging of soil
microbial communities in their natural spatial arrangement.

7 Conclusions and Future Outlook

Recent technological and methodological advances have led to great progress in

understanding the linkages between microbial diversity and ecosystem functioning
(Bardgett et al. 2008). Metagenomics approaches have enabled characterization of
the members of the soil community, including uncultivable microorganisms. Other
5 Exometabolomics for Linking Soil Carbon Dynamics … 137

molecular methods and SIP approaches have improved understanding of the par-
ticular members of the soil community’s metabolic capabilities. What members of a
particular soil microbial community actually do, will depend on the substrates that
are available in their environment. Therefore, exometabolomics is a very promising
approach in that it provides direct evidence of the soil metabolites available to soil
microorganisms and how the available substrates are transformed by microbial
community metabolism.
This chapter reviewed the handful of reports where an exometabolomics
approach was applied to the study of intact soil microbial communities, or to
laboratory experiments focusing on a particular aspect of such complex systems.
Soil microbial communities are very complex, and soils are extremely heteroge-
neous matrices, so it is not surprising that there are many technical challenges that
remain to be resolved in this field. Care should be taken to use sample preparation
methods appropriate for the specific question being asked and analytical method
being used. There is no single analytical method that can detect the massive
diversity of metabolites across large dynamic ranges in an unbiased way. The
choice of analytical method will depend on the focus and needs of the study, and
combinations of complementary techniques may offer a more comprehensive
coverage of diverse chemical classes (Simpson et al. 2004; Werf et al. 2007). It is
important to keep in mind that the mineral composition and other factors may
confound analysis by preferentially sorbing certain metabolites (e.g. ferrihydrite
sorbing phosphate containing metabolites) making it challenging to compare soil
types.
As with any metabolomics workflow, soil community exometabolomics
experiments generate large datasets. Untargeted metabolomics results usually
include many detected features that remain unidentified. There are several
well-established mass spectrometry and NMR databases that can aid in identifica-
tion of such unknowns (Kind et al. 2009; Smith et al. 2005; Wishart 2007). Many of
these target intracellular metabolism of organisms such as yeast or plants (Bais et al.
2010; Hummel et al. 2007; Jewison et al. 2011). Since much soil organic matter is
derived from plant and microbial biomass, these are useful to soil organic matter
characterizations. There is currently great interest in secondary metabolites from
soil microorganisms, and increasing the number of entries in databases of such
compounds will also be very helpful in the context of soil exometabolomics
(Hadjithomas et al. 2015). Many workflows have been developed for the analysis of
large metabolomics datasets, which are also applicable to exometabolomics data
analysis (Bowen and Northen 2010; Rübel et al. 2013; Tautenhahn et al. 2012; Xia
et al. 2012). Data analysis tools for interpreting data in SIP experiments will be of
particular value (Hiller et al. 2010; Huang et al. 2014). Any experimental setup and
data reported should meet the quality and reporting standards as set by the larger
metabolomics community (Goodacre et al. 2007).
The full potential of soil microbial community exometabolomics will be realized
when it can be integrated with other approaches such as metagenomics, meta-
transcriptomics, and metaproteomics. A recent review describes examples where
such multiomics approaches were applied to understanding microbial communities
138 A. Lubbe and T. Northen

(Franzosa et al. 2015). Careful planning of experimental design and data integration
strategies are needed to derive the most value out of such combined approaches
(Muller et al. 2013). Such data integration should result in improved mechanistic
models of the structure and functioning of soil microbial communities that can be
tested in combinations of laboratory and field experiments (Franzosa et al. 2015).
This will enable better predictions of the effects of environmental perturbations on
soil carbon cycling by soil microorganisms.

Acknowledgments T.R.N. gratefully acknowledges support from ENIGMA—Ecosystems and

Networks Integrated with Genes and Molecular Assemblies (http://enigma.lbl.gov), a Scientific
Focus Area Program at Lawrence Berkeley National Laboratory is based upon work supported by
the US Department of Energy, Office of Science, Office of Biological & Environmental Research
under contract number DE-AC02-05CH11231. A.L. was supported by the Office of Science,
Office of Biological and Environmental Research, of the US Department of Energy, Award No.
DE-SC0012627. We thank Katherine Louie for providing the mass spectrometry image of a
microbial coculture.

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Chapter 6
Soil Microbial Metabolomics

Michael W. Heaven and Devin Benheim

1 Soil Complexity

The world demand for food is currently expected to increase by two- to fivefold by
2030 (Ewert et al. 2005; Food and Agriculture Organization (FAO) 2002). This
projection requires food production to increase from 60 to 70 % (Clair and Lynch
2010), although there is some confusion due to the types of food stuffs the global
population will be consuming at that time (Alexandratos and Bruinsma 2013) and
the exclusion from earlier studies of important food types (e.g. fruit and vegetables).
Agricultural practices over the last century have succeeded in significantly
increasing crop yields. For instance, global cereal production doubled between
1960 and 2000 (Tilman et al. 2002). However, the yield increases were driven
largely by intensification in the use of non-renewable synthetic fertilisers (Lynch
2007). They were seminal in improving western lifestyles but provided limited
relief in many regions of the world such as Africa, Asia and South America.
Moreover, the technologies were double edged, with gains in agricultural produc-
tion coinciding with increased soil erosion (Matson et al. 1997), industrial agri-
cultural pollution (Horrigan et al. 2002), declines in water quality (Foley et al.
2005) and, possibly most importantly, loss of biodiversity (including genetic ero-
sion) (Aguilar et al. 2008; Balestrini et al. 2015). Arguably, a sustainable way
forward is ‘ecological intensification’. This paradigm expands agricultural inten-
sification that promotes the development of food systems with enhanced nutrient
uptake and water use efficiency (Cassman 1999), reductions in pest and disease

M.W. Heaven (&)

Department of Economic Development, Jobs, Transport and Resources,
1301 Hazeldean Road, Ellinbank, Victoria, Australia
e-mail: [email protected]
D. Benheim
Department of Physiology, Anatomy and Microbiology,
School of Life Sciences, La Trobe University, Victoria, Australia

© Springer International Publishing Switzerland 2016 147

D.J. Beale et al. (eds.), Microbial Metabolomics,
DOI 10.1007/978-3-319-46326-1_6
148 M.W. Heaven and D. Benheim

control (Bommarco et al. 2013) and a restoration in soil fertility (Balestrini et al.
2015; Matson et al. 1997; Tittonell 2014). Many of these goals are only achievable
today due to the advances in analytical technologies and scientific knowledge that
underpins methodologies like metabolomics. As useful as the broad brush of tra-
ditional physicochemical analyses has been for agricultural systems (Sinsabaugh
et al. 2016) (to understand how to manipulate plant growth), it would be advan-
tageous to determine biogeochemical processes that occur in soil (Rockström et al.
2009; Sardans et al. 2011).
The soil matrix is one of the most diverse ecological systems on the planet
(Torsvik and Øvreås 2002). Home to a plethora of organisms, from the largest
redwood to the smallest microbe, there are numerous chemical (McBride 1994),
physical (Marshall and Holmes 1980) and biological (Barea et al. 2005; Lorenz and
Wackernagel 1994) interactions occurring in soil. Until recently, quantifying these
biogeochemical processes as a metabolome of “soil” was not considered feasible,
with only narrow components of the soil matrix, such as plants and animals, being
studied (Maron et al. 2011; Mendes et al. 2013; Mosier et al. 2013). However, a
holistic approach to understanding the soil community is something of increasing
interest with examples in organic nutrient pools [(Warren 2013, 2014), Table 1],
pollution assessment (Jones et al. 2014; Tremaroli et al. 2009), effects of climate
change [(Pang et al. 2016), Table 2], plant [(Badri et al. 2013a; van Dam and
Bouwmeester 2016), Table 3] and/or microbial metabolism (Ponomarova and Patil
2015; Warren 2015). Recent progress in performing untargeted metabolomics to
identify soil organic matter (SOM) metabolites (e.g. lipids and organic acids),
which can be linked to nutrient uptake [(Swenson et al. 2015b), Table 3], has
shown that “soil metabolomics” is now beyond theory. Still, the ability to study soil
holistically is at an early stage of development. The potential for using metabo-
lomics to advance study into how the soil matrix operates will increase as the
technologies underpinning the methodology (i.e. gas and liquid chromatography–
mass spectrometry (GC–MS and LC–MS), and nuclear magnetic resonance
(NMR)) improve (Singh 2006). Likewise, metabolomics can provide a holistic
understanding of the impact of the increasing resource demands has on soil
(Rockström et al. 2009). Through understanding of the important metabolomic
pools and fluxes in soil, so as to understand and monitor soil health as it is managed
by farmers, foresters and the community, is likely to improve productivity and the
environment while reducing costs (Abhilash et al. 2012; Desai et al. 2010).
Metabolomics of the soil would identify the largest component of metabolites
coming from the most varied and numerous collection of microbes known (Barea
et al. 2005; Huang et al. 2014; Mendes et al. 2013), with estimates of between 1 and
40 million species per gram of soil (Burns et al. 2013; Desai et al. 2010; Řezanka
and Sigler 2009). Despite the importance of microbes to the agricultural and
environmental communities, knowledge of the composition of the microbial bio-
mass is limited. For instance, estimates of fungi range from 700 000 to 1.5 million
(Lumbsch and Leavitt 2011; Ponomarova and Patil 2015; Rastogi and Sani 2011).
However, only 100,000 have been detailed. A large part of this struggle to
understand the composition of the soil microbial community is that <1 % has been
6 Soil Microbial Metabolomics 149

Table 1 Typical metabolites found in soil-related samples using other techniques

Metabolite Technique Spectroscopic Why? Publication
class (Column for range (min)
GC or LC)
Amino acids, N CE–MS (bare 10.99–35.32 Identification of Warren
containing fused silica organic N (2013)
compounds, capillary) molecules in soil
dipeptides water
FAME GC-FAME 8.23–11.71 Influence of Shah and
nanoparticles on Belozerova
soil microbes (2009),
Sasser
(2006)
Essential oils GC-FID 10.70–25.20 Rhizobacteria Cappellari
(Supelcowax inoculation et al. (2013)
10)
PLFA GC-IRMS (HP 22.30–40.79 Microbial PLFA Watzinger
5-MS fused biomarkers using (2015)
silica capillary stable isotope
column) methods
Carbohydrates, HPLC 8.07–18.62 Effects of VAM Azaizeh
amino acids, fungi on maize et al. (1995)
phenolics
Flavonoids, RP-HPLC 15.34–69.73 Secondary Chamam
organic acids, (DAD) and metabolite profiling et al. (2013)
resorcinols LC–MS to identify
(Nucleodur bacterial–rice
Sphinx RP) associations

able to be cultured (Kirk et al. 2004; van den Berg et al. 2015). Metabolomics,
being a microbe-independent technique, could shed light on this rich network via
metabolite fluxes and identifying previously unknown metabolites and biogeo-
chemical processes, with genomic studies identifying numerous other microbial
clades that have been identified only by their molecular sequences (Cesco et al.
2012; Cheynier et al. 2013; Swenson et al. 2015b). There is potential to extract
useful information using metabolomics (Alivisatos et al. 2015); although it is
acknowledged that to date, studies have been limited due to the complexity of the
soil matrix (Ponomarova and Patil 2015).
This chapter uses the lens of metabolomics to discuss connections between soil
microbes and the complex interactions they undergo with plants, animals and
human intervention. Although studies of soil microbes using metabolomic tech-
niques are scant, associated studies that can shed light on the complex biogeo-
chemical interactions have been undertaken and provide direction. Due to the drive
to feed the planet and increase agricultural productivity, the rhizosphere of plants
and how to manipulate plant–microbe interactions has probably been of the greatest
research interest in this area (Bertin et al. 2003; De-la-Pena and Loyola-Vergas
Table 2 Typical metabolites found in soil-related samples using 1H-NMR
150

Sample; treatments Metabolite class Techniques used Metabolites Spectroscopic Why? Publication
range (ppm)
(a) Grape skin; soil, Amino acids 1H-NMR (D2O, Isoleucine, leucine, 0.90–1.10 Biomarkers (a) Pereira et al.
weather, cultivar. TSP), 1H-NMR valine (2006). (b) Liebeke
(b) Soil; bacterial (D2O, TSP or et al. (2009).
strain, location. CDCl3) (c) Rochfort et al.
(c) Worms; soil type, (2009)
organic versus
chemical fertiliser
(a) Plant cuts; time Amino acids 1H-NMR TOCSY, Threonine 1.30, 1.45 Biomarkers, (a) Bertram et al.
treatments. NOESY, HSCQ; Salinity (2010). (b) Pang
(b) Seedlings and (D2O with TSP), et al. (2016).
plants; NaCl and 1H-NMR NOESY (c) Rochfort et al.
NaHCO3. (D2O, Na2HPO4, (2009)
(c) Worms; soil type, NaH2PO4, TSP),
organic versus 1H-NMR (D2O, TSP
chemical fertiliser or CDCl3)
Worms; soil type, a-Hydroxy acids 1H-NMR (D2O, TSP Lactate 1.30 Biomarkers Rochfort et al.
organic vs. chemical or CDCl3) (2009)
fertiliser
Wine; bacterial strain Alcohols 1H-NMR 2,3-Butanediol 1.37 Vintage Lee et al. (2009)
NOESYPREAST
(D2O, oxalate, DSS)
(a) Grape skin; Soil, Amino acids 1H-NMR (D2O, Alanine 1.46–1.70 Biomarkers, (a) Pereira et al.
weather, cultivar. TSP), 1H-NMR salinity, (2006). (b) Bertram
(b) Plant cuts; time TOCSY, NOESY, vintage et al. (2010).
treatments. (c) Soil; HSCQ; (D2O with (c) Liebeke et al.
bacterial strain, TSP), 1H-NMR (2009). (d) Pang
location. NOESY (D2O, et al. (2016).
(continued)
M.W. Heaven and D. Benheim
Table 2 (continued)
Sample; treatments Metabolite class Techniques used Metabolites Spectroscopic Why? Publication
range (ppm)
(d) Seedlings and Na2HPO4, (e) Rochfort et al.
plants; NaCl and NaH2PO4, TSP), (2009). (f) Lee et al.
NaHCO3. 1H-NMR (D2O, TSP (2009)
(e) Worms; soil type, or CDCl3), 1H-NMR
organic versus NOESYPREAST
chemical fertiliser. (D2O, oxalate, DSS)
(f) Wine; bacterial
6 Soil Microbial Metabolomics

strain
Grape skin; soil, Amino acids 1H-NMR (D2O, Arginine 1.70 Biomarkers Pereira et al. (2006)
weather, cultivar TSP)
(a) Soil; bacterial Organic acids 1H-NMR (D2O, Acetic acid 1.92 Biomarkers, (a) Liebeke et al.
strain, location, TMSP), 1H-NMR ecotoxicity (2009). (b) Simpson
(b) Earthworms; (Various) and McKelvie
various (2009)
Grape skin; soil, Amino acids 1H-NMR (D2O, GABA + proline 1.94 Biomarkers Pereira et al. (2006)
weather, cultivar TSP)
Grape skin; soil, Amino acids 1H-NMR (D2O, Proline 1.98 Biomarkers Pereira et al. (2006)
weather, cultivar TSP)
Soil; bacterial strain, Amino acids 1H-NMR (D2O, Glutamic acid 2.06 Biomarkers Liebeke et al.
location TMSP) (2009)
Seedlings and plants; Amino acids 1H-NMR NOESY Glutamate 2.10 Salinity Pang et al. (2016)
NaCl and NaHCO3 (D2O, Na2HPO4,
NaH2PO4, TSP)
Seedlings and plants; Amino acids 1H-NMR NOESY Glutamine 2.20 Salinity Pang et al. (2016)
NaCl and NaHCO3 (D2O, Na2HPO4,
NaH2PO4, TSP)
(continued)
151
Table 2 (continued)
152

Sample; treatments Metabolite class Techniques used Metabolites Spectroscopic Why? Publication
range (ppm)
Grape skin; soil, Organic acid 1H-NMR (D2O, Shikimic acid 2.22 Biomarkers Pereira et al. (2006)
weather, cultivar TSP)
Earthworms; various Organic acids 1H-NMR (Various) Acetoacetate 2.23 Ecotoxicity Simpson and
McKelvie (2009)
Soil; bacterial strain, Organic acids 1H-NMR (D2O, Pyruvic acid 2.27 Biomarkers Liebeke et al.
location TMSP) (2009)
Wine; bacterial strain Organic acids 1H-NMR Acetate 2.28 Vintage Lee et al. (2009)
NOESYPREAST
(D2O, oxalate, DSS)
(a) Seedlings and Organic acid 1H-NMR NOESY Succinate 2.40, 2.42, Salinity, (a) Pang et al.
plants; NaCl and (D2O, Na2HPO4, 2.82 biomarkers, (2016), (b) Liebeke
NaHCO3. (b) Soil; NaH2PO4, TSP), ecotoxicity, et al. (2009).
bacterial strain, 1H-NMR (D2O, vintage (c) Simpson and
location. TMSP), 1H-NMR McKelvie (2009).
(c) Earthworms; NOESYPREAST (d) Lee et al. (2009)
various. (d) Wine; (D2O, oxalate, DSS)
bacterial strain
Grape skin; soil, Amino acids 1H-NMR (D2O, GABA + glutamine 2.46 Biomarkers Pereira et al. (2006)
weather, cultivar TSP)
(a) Earthworms; Amine 1H-NMR (Various), Dimethylamine 2.75, 2.80 Ecotoxicity, (a) Simpson and
various. 1H-NMR NOESY salinity McKelvie (2009).
(b) Seedlings and (D2O, Na2HPO4, (b) Pang et al.
plants; NaCl and NaH2PO4, TSP) (2016)
NaHCO3
Soil; bacterial strain, Amino acids 1H-NMR (D2O, Aspartic acid 2.79 Biomarkers Liebeke et al.
location TMSP) (2009)
(continued)
M.W. Heaven and D. Benheim
Table 2 (continued)
Sample; treatments Metabolite class Techniques used Metabolites Spectroscopic Why? Publication
range (ppm)
Seedlings and plants; Organic acid 1H-NMR NOESY 2-Oxoglutarate 3.00 Salinity Pang et al. (2016)
NaCl and NaHCO3 (D2O, Na2HPO4,
NaH2PO4, TSP)
(a) Grape skin; soil, Neurotransmitter 1H-NMR (D2O, GABA 1.85, 2.17, Biomarkers, (a) Pereira et al.
weather, cultivar. (Amino acid) TSP) 2.25, 2.84, vintage (2006). (b) Bertram
(b) Plant cuts; time 3.02, 3.06, et al. (2010). (c) Lee
treatments. (c) Plant 3.10, 3.28 et al. (2009)
6 Soil Microbial Metabolomics

cuts; time treatments

(a) Seedlings and Amino acids 1H-NMR NOESY Choline 3.20, 3.27 Salinity, (a) Pang et al.
plants; NaCl and (D2O, Na2HPO4, biomarker (2016). (b) Bertram
NaHCO3. (b) Plant NaH2PO4, TSP), et al. (2010)
cuts; time treatments 1H-NMR TOCSY,
NOESY, HSCQ;
(D2O with TSP)
in situ; Cytochrome Xenobiotics 1H-NMR Thiodiglycolic acid 3.25 Degradation Delort and
P450 inhibitor; (D2O + TSP), of Combourieu (2001)
xenobiotic ISP-MS xenobiotics
Seedlings and plants; Amino acids 1H-NMR NOESY Betaine 3.30 Salinity Pang et al. (2016)
NaCl and NaHCO3 (D2O, Na2HPO4,
NaH2PO4, TSP)
(a) Grape skin; soil, Sugars 1H-NMR (D2O, Glucose, fructose, 3.42–4.22 Biomarkers (a) Pereira et al.
weather, cultivar. TSP), 1H-NMR sucrose (2006). (b) Bertram
(b) Plant cuts; time TOCSY, NOESY, et al. (2010)
treatments HSCQ; (D2O with
TSP)
(continued)
153
Table 2 (continued)
154

Sample; treatments Metabolite class Techniques used Metabolites Spectroscopic Why? Publication
range (ppm)
(a) Soil; bacterial Amino acids 1H-NMR (D2O, Glycine 3.56–3.60 Biomarkers, (a) Liebeke et al.
strain, location. TMSP), 1H-NMR ecotoxicity, (2009). (b) Simpson
(b) Earthworms; (Various), 1H-NMR salinity and McKelvie
various. NOESY (D2O, (2009). (c) Pang
(c) Seedlings and Na2HPO4, et al. (2016)
plants; NaCl and NaH2PO4, TSP)
NaHCO3
in situ; Cytochrome Xenobiotics 1H-NMR Glycolate 3.95 Degradation Delort and
P450 inhibitor; (D2O + TSP), of Combourieu (2001)
xenobiotic ISP-MS xenobiotics
Seedlings and plants; Amino acids 1H-NMR NOESY Malate 4.40 Salinity Pang et al. (2016)
NaCl and NaHCO3 (D2O, Na2HPO4,
NaH2PO4, TSP)
(a) Grape skin; soil, Sugars 1H-NMR (D2O, Glucose, maltose, 4.62–5.42 Biomarkers (a) Pereira et al.
weather, cultivar. TSP), 1H-NMR sucrose (2006). (b) Liebeke
(b) Soil; bacterial (D2O, TMSP), et al. (2009).
strain, location. 1H-NMR TOCSY, (c) Bertram et al.
(c) Plant cuts; time NOESY, HSCQ; (2010). (d) Rochfort
treatments. (D2O with TSP), et al. (2009)
(d) Worms; soil type, 1H-NMR (D2O, TSP
organic versus or CDCl3)
chemical fertiliser
Wine; bacterial strain Organic acids 1H-NMR Tartrate 4.63 Biomarkers Lee et al. (2009)
NOESYPREAST
(D2O, oxalate, DSS)
Soil; bacterial strain, Sugars 1H-NMR (D2O, Trehalose 5.19 Biomarkers Liebeke et al.
location TMSP) (2009)
(continued)
M.W. Heaven and D. Benheim
Table 2 (continued)
Sample; treatments Metabolite class Techniques used Metabolites Spectroscopic Why? Publication
range (ppm)
Soil; bacterial strain, Organic acids 1H-NMR (D2O, Malic acid 6.03 Biomarkers Liebeke et al.
location TMSP) (2009)
(a) Worms; soil type, Organic acids 1H-NMR (D2O, TSP Fumarate 6.20–6.52 Biomarkers, (a) Rochfort et al.
organic versus or CDCl3), 1H-NMR ecotoxicity (2009). (b) Pang
chemical fertiliser. NOESY (D2O, et al. (2016).
(b) Seedlings and Na2HPO4, (c) Liebeke et al.
plants; NaCl and NaH2PO4, TSP), (2009). (d) Simpson
6 Soil Microbial Metabolomics

NaHCO3. (c) Soil; 1H-NMR (D2O, and McKelvie

bacterial strain, TMSP), 1H-NMR (2009)
location. (Various)
(d) Earthworms;
various
Earthworms; various Organic Acids 1H-NMR (Various) Orotic acid 6.20 Ecotoxicity Simpson and
McKelvie (2009
(a) Soil; bacterial Amino acids 1H-NMR (D2O, Tyrosine 6.92–7.00 Biomarkers (a) Liebeke et al.
strain, location. TMSP), 1H-NMR (2009). (b) Rochfort
(b) Worms; soil type, (D2O, TSP or et al. (2009)
organic versus CDCl3)
chemical fertiliser
Worms; soil type, Amino acids, 1H-NMR (D2O, TSP Phenylalanine 7.40 Biomarkers Rochfort et al.
organic versus Sulfonate, Lipids, or CDCl3) (2009)
chemical fertiliser Triacylglycerides
(TAG)
(a) Worms; soil type, Organic acids 1H-NMR (D2O, TSP Formate 8.35 Biomarkers, (a) Rochfort et al.
organic versus or CDCl3), 1H-NMR ecotoxicity (2009). (b) Liebeke
chemical fertiliser. (D2O, TMSP), et al. (2009).
(b) Soil; bacterial 1H-NMR (Various) (c) Simpson and
strain, location. McKelvie (2009)
155

(c) Earthworms;
various
 156

Table 3 Details of selected GC–MS analyses reported in this review

Sample; Metabolite class Technique Spectroscopic Why? Publication
treatments (Column) range (min)
(a) Soil: time, PLFA (a) GC–MS 1.50–48.83 (a) Transformations of soil (a) Apostel
fumigation, 13C (DB1-MS column). monosaccharides (b) Incorporation et al.
incorporation. (b) GC–MS (HP-1 of organic compounds into microbes (2015).
(b) Soil columns methylpolysiloxane) (b) Gunina
on farm; time et al.
(2014)
Root exudates; Sugars, Phenolics, Amino acids, GC–MS 3.57–17.14 Identifying root exudates that affect Badri et al.
soil; solvent Sugar alcohols (rtx5Sil-MS soil microbiome (2013a)
extraction column)
fraction
Roots; microbe Nitrogenous compounds, GC–MS (Rtx-5MS 3.98–45.54 Root exudates after inoculation with Lima et al.
inoculation, Carbohydrates, Fatty acids, WCOT capillary microbes and humic acids (2014)
humic acids, time Organic acids, Aromatic and column)
phenol derivatives, Terpenoids
and steroids, Alcohols
Plant rosettes; Amino acids, Organic acids, GC-QQQ-MS 10.68–47.99 How Arabidopsis and Thellungiella Lugan et al.
NaCl loading, Alcohols, Carbohydrates, (Rtx-5Sil MS react to salt and/or water stress (2010)
plant species, Amines capillary column)
water and GC-FID (J&W
concentration DB5)
Untargeted soil Amino acids, Organic acids, GC–MS 8.42–24.32 Development of a simple soil Swenson
profiling, Sugar alcohols, Carbohydrate, (Rtx5Sil-MS metabolomics workflow and a novel et al.
fumigation, Lipids, Nucleosides, column) spike recovery approach using 13C (2015b)
labelled (13C) Nucleobases, Sugars, Sterols bacterial lysates to assess the types
and others of metabolites remaining in the
water extractable organic fraction
M.W. Heaven and D. Benheim
6 Soil Microbial Metabolomics 157

2014; van Dam and Bouwmeester 2016; Zahir et al. 2004). Other areas of dis-
cussion beyond the plant–microbe interactions include specific foci on how soil
microbes are affected by pollution, diseases, pests and potential climate change.
A theme of the chapter will be how metabolomics may be used to improve soil
management (Rochfort et al. 2015; Zhang et al. 2015), both for increasing pro-
ductivity of the soil and mitigating environmental effects.

2 Soil Metabolomics Is a Nascent Field

Soil is often a secondary topic for metabolomics analyses. The focus of the research
is often on how microbes interact with plants such as grasses (e.g. Trifolium: clover)
or legumes [(Bertram et al. 2010), Table 2], weeds such as Arabidopsis [(Gamir
et al. 2014), Table 4], trees such as Aspen (Wallenstein et al. 2010) or invertebrates
that inhabit the soil such as earthworms Aporrectodea caliginosa [(Rochfort et al.
2009), Table 2] or Eisenia fetida (Simpson and McKelvie 2009). Recent reviews
have discussed soil microbial metabolites within the concept of environmental
metabolomics. For instance, a review on environmental metabolomics suggests the
field is fragmented, as this new “holistic” methodology was mainly being used to
study single species of the researchers’ interest, including those involving soil
microbes (Bundy et al. 2009). The authors reiterated that this misses one of envi-
ronmental metabolomics assets in gathering an understanding of the interactions
between species and their environment. Another review focused on using meta-
bolomics to assess soil contamination (Hernandez-Soriano and Jimenez-Lopez
2014). Earthworms, usually of the genus Eisenia, were found to be typical subjects
for metabolomic analyses. Studies related to microbial metabolomics were small,
with the only reference to work on the response to tellurite by Pseudomonas
pseudoalcaligenes (Tremaroli et al. 2009). Earthworms were also considered the
main target for soil metabolomics of another review on environmental sciences and
metabolomics (Lin et al. 2006). This review detailed the various effects of con-
taminants on the metabolic profiles of a variety of earthworms, particularly halo-
genated compounds and metal-contaminated soils. Another review discussed
‘eco-metabolomics’, the use of metabolomics and how it relates to ecology, with
regard to interactions between organisms, including those in the soil (Sardans et al.
2011). While mainly related to how plants and worms react to changes in envi-
ronment, some discussion was made on how fungi Magnaporthe grisea and
Sclerontinia sclerotiorum release metabolites to suppress plant defences.
Various studies that have examined microbes and/or the metabolites associated
with them. However, only in 2015 has there been research that specifically men-
tions the use of “soil metabolomics” to identify biogeochemical processes occurring
in soils [(Swenson et al. 2015a; Swenson et al. 2015b), Table 4]. The research
sought to understand the fluxes of microbial metabolites in SOM that might occur
due to climate change. Comparing fumigated and unfumigated soil samples using
 158

Table 4 Details of selected LC–MS analyses reported in this review

Sample; treatments Metabolite class Technique (Column) Spectroscopic Why? Publication
range (min)
Plant roots; arbuscular Amino acids, Organic acids, LC-ESI Q-TOF-MS 0.50–13.40 Mycorrhizal Rivero
mycorrhizal fungi Phosphate compounds, Acetates, (SunFire C18 analytical association of AMF et al.
(AMF) strains, time Nucleosides, Vitamins, Indoles, column) with tomato plants (2015)
Purines
Roots; AMF Lipids (PE, PC, LPE, Carnitine) LC-HILIC-Q-TOF-MS 2.60–8.82 AMF effects on Saia et al.
and/or plant (Acuity 1.7 lm wheat in N limited, (2015b)
growth-promoting BEH HILIC) or P-rich environment
rhizobacteria (PGPR) GC-TOF-MS
(Rtx-5SilMS column)
Arabidopsis cells; Phytochelatin (PC) LC–MS (X-Terra MS 9.70–25.00 Plant response to Sarry et al.
Nutrients C18 column) cadmium stress (2006)
Ferrihydrite: Organic Phosphate, Dicarboxylate, Aromatic, LC–MS (ZIC-pHILIC) 1.70–23.6 Microbial Swenson
Carbon, temperature N-carboxylate, other N organic metabolites on et al.
compounds ferrihydrite (2015a)
Leaves; pre- and Amino acids, Organic acids, LC-QQQ-MS (Pack 0.53–13.40 Understanding Gamir
post-fungal treatments Aldehydes, Indoles ODS-A reversed-phase priming of plants et al.
C18) against microbes (2014)
M.W. Heaven and D. Benheim
6 Soil Microbial Metabolomics 159

water extractable SOM, it was possible to identify metabolites associated with

microbial species. Initial studies using GC–MS identified 55 metabolites via
comparison with accurate standards or carbon-labelled samples (Swenson et al.
2015b). Up to 300 molecular features (after extensive sample preparation using
GC-friendly water soluble solvents) were identified in soil samples following
optimisation of the extraction media water, aqueous potassium sulphate or
ammonium carbonate, isopropanol and methanol. Stable isotope labelling studies,
using 13C-acetate as a growth medium, allowed the differentiation of microbial
metabolites from other compounds in the soil. This method identified sugars and
amino acids as most likely to be co-extracted with other metabolites that contained
hydrogen bonding functional groups [e.g. fatty acids (FAs) and sterols]. A followup
study using LC–MS identified a further 55 metabolites that interacted with iron
oxides in soil, reducing access to these compounds by microbes (Swenson et al.
2015a). Metabolites from this study could be grouped into phosphate containing
(e.g. AMP), dicarboxylates (e.g. fumarate), aromatic and nitrogen containing (e.g.
phenylalanine), carboxylate and nitrogen containing (e.g. creatinine) and others
such as thymine. Future research that draws from the work described below should
allow for a more holistic approach to understanding how microbes in soil can affect
soil.

3 The Rhizosphere

3.1 Map of the Rhizosphere

The rhizosphere, comprising the endorhizosphere, the rhizoplane and the ectorhi-
zosphere (Fig. 1), defines the narrow region between roots and soil directly influ-
enced by both root exudates and exfoliates, and associated microorganisms (Jones
1998). At the heart of the rhizosphere is the root of a plant that is undergoing
symbiosis. The root surface (epidermis and outer cortex) and its adhering soil are
collectively termed the rhizoplane: the interface where both microbial population
and biochemical plant–microbe interactions are at their maximum. The root systems
of plants serve critical roles in the provision of anchorage, water and mineral
absorption and conduction, lateral movement, reproduction, metabolite synthesis
and food storage centre (Kenrick 2013; Kramer and Boyer 1995; Selosse and
Strullu-Derrien 2015). Roots are linear units composed of multiple regions along
the root growth axis. The units—the root cap, root tip, elongation zone, root-hair
zone and mature zone—are uniquely differentiable and perform distinct functions
(Minz et al. 2013). Each of these units uses different libraries of metabolites for
communication to other units while releasing a multitude of metabolites that
modulate plant–microbe interactions in the rhizosphere (Huang et al. 2014). Root
exudates, used as both substrates and signalling molecules by soil microbes,
comprise of both low (e.g. amino acids, organic acids, carbohydrates, phenolics and
160 M.W. Heaven and D. Benheim

Fig. 1 Schematic representation of the rhizosphere showing commonly associated microbial

associations with plant root systems

other secondary metabolites) and high molecular weight compounds (e.g. mucilage
and proteins) (Bais et al. 2008; Walker et al. 2003; Ziegler et al. 2013). The nature
of root exudates is observed to vary significantly between plant species.
Consequently, the rhizosphere microbiome differs with both plant species and soil
type (Tate 2000; Wieland et al. 2001). The influence of the rhizosphere can even
extend beyond the immediate area of the plant, as discovered with metabolomic
analyses of leaf litter around different tree species (Wallenstein et al. 2013;
Wallenstein et al. 2010).
Apart from the root, the rhizosphere’s main constituents are eukaryotic and
prokaryotic microbial species, with a diversity of microbes that can range from
thousands (Berendsen et al. 2012) to millions (Nihorimbere et al. 2011). The
dynamic microbiome which surrounds the roots is of critical importance to
6 Soil Microbial Metabolomics 161

ecosystem function (Bertin et al. 2003; Sardans et al. 2011), below-ground carbon
(Mendes et al. 2011) and nutrient cycling (Swenson et al. 2015b; Van Der Heijden
et al. 2008), affecting overall plant fitness and soil quality (Barea et al. 2005; Minz
et al. 2013). Plants spend up to half of their energy producing exudates into the
rhizosphere (van Dam and Bouwmeester 2016), though it has been argued that the
source of metabolites (e.g. plant vs. microbe) in the rhizosphere is still to be
elucidated convincingly (Dennis et al. 2010). A major role of exudates from roots is
the communication to soil-borne microbes, the most dominant class of soil biota
(Van Der Heijden et al. 2008). These microbes are phylogenetically diverse (e.g.
bacteria, archaea and fungi), and comprise symbionts, pathogens and saprotrophs
(Balestrini et al. 2015; Reynolds et al. 2003).
Saprotrophic microbes are crucial to nutrient cycling in terrestrial ecosystems,
generating the majority of nutrients required by terrestrial vegetation (Crowther
et al. 2012; Schlesinger 1991). The soil microbial community is able to do this
primarily via photosynthetically fixed carbon introduced to the soil ecosystem in the
form of plant biomass and root exudates (Badri et al. 2013a; Dennis et al. 2010;
Tate 2000). While there is competition between plants and microbes for available
nutrients (Kaye and Hart 1997; Kuzyakov and Xu 2013), two key plant–microbe
symbiotic associations, arbuscular mycorrhizal fungi (AMF) (Van Der Heijden
et al. 1998) and root nodule symbiosis (RNS) (Li et al. 2013), impart significant
benefits to both microbes and plants. AMF improve the supply of water and
nutrients (such as phosphate and nitrogen) to the plant via extraradical hyphae
(Kawaguchi and Minamisawa 2010; Parniske 2008). For RNS, nitrogen-fixing
bacteria enable enzymatic conversion of atmospheric nitrogen into bioavailable
ammonia for plant growth (Brewin 2010). Ecological benefits of symbioses are also
contributed as is the case of AMF, which increases host resilience toward drought
(Baum et al. 2015), decreased susceptibility to diseases (Saia et al. 2015b),
improved heavy metal (Schützendübel and Polle 2002), excess salinity tolerance
(Luo et al. 2009) and other abiotic factors (Habib et al. 2013).

3.2 Rhizosphere Metabolomics

The rhizosphere has undergone scrutiny using various omic techniques including
genomics, metagenomics, proteomics, transcriptomics and more recently metabo-
lomics (van Dam and Bouwmeester 2016). The principle metabolomic tools are
NMR spectroscopy [Table 2, (Sardans et al. 2011)], LC–MS [Table 4, (Allwood and
Goodacre 2010)], GC–MS [Table 3, (Kusano et al. 2011)], although other tech-
niques such as capillary electrophoresis–time of flight–MS (CE–TOF–MS, Table 1)
(Abhilash et al. 2012) have been utilised. For the symbiotic process to occur between
microbes and plants, there must be a series of metabolites that are transferred
between the two moieties (Rasmussen et al. 2012). Major bioactive metabolites
found in the rhizosphere include flavonoids (Cesco et al. 2012; Cheynier et al. 2013;
Narasimhan et al. 2003), phenolic compounds (Badri et al. 2013a;
162 M.W. Heaven and D. Benheim

Kakumanu et al. 2013; Rawat et al. 2011), exopolysaccharides (Workentine et al.

2010), antibiotics (Frisvad et al. 2004) and those participating in quorum sensing
(QS) signals (De-la-Pena and Loyola-Vergas 2014; Jia et al. 2016). QS describes the
process by which bacterial population density, biofilm formation and gene expres-
sion (modulating niche persistence and root colonisation) are controlled within the
population through production of low-mass signalling molecules [(Braeken et al.
2008; Lima et al. 2014), Table 3]. Bacterial pathogens and symbionts are largely
dependent on QS to colonise and infect their respective hosts, as in the case of
Pseudomonas aeruginosa, which has been shown to have up to 20 % of its genes
and proteins regulated via QS (Bauer and Mathesius 2004; Das and Mukherjee
2007). Other abiotic soil factors including nutrient availability (Saia et al. 2015b),
soil pH (Dakora and Phillips 2002), salinity (Oikawa et al. 2011) and other envi-
ronmental stressors are also known to correlate with changes in plant metabolite
composition. The physiological impact of the soil microbiome on plant metabolism
is receiving increasingly more attention due to changes to the climate and the need to
feed an increasing population (Park et al. 2014; Sanchez et al. 2008). While
assessment and identification of the full complement of rhizosphere microbes pre-
sents significant challenges, rhizosphere microbes have demonstrated a capacity to
alter plant morphology, enhance plant growth and increase mineral content
(Berendsen et al. 2012; Lakshmanan et al. 2014; Mendes et al. 2011). The following
sections demonstrate several notable examples highlighting the use of rhizosphere
metabolomics in understanding the plant–microbe interactions that underpin the
practical aspects of enhancing plant performance.

4 Exudates

Plant communication to soil microbes is almost solely conducted using metabolites

called exudates (Bais et al. 2004; Bronick and Lal 2005). Exudates directly influ-
ence the structure and function of the soil microbiome and are strong mediating
factors in preferential microbiome selection (Coats and Rumpho 2014). The exu-
dates are commonly amino acids and sugars (Broeckling et al. 2008). Secondary
metabolites that may be active in exudates include flavones (Redmond et al. 1986)
and terpenes (Hartmann et al. 2008). The secondary metabolites mediate processes
to improve nutrient uptake and microbial resistance. To date, most studies have
focussed on how a single microbial species reacts to plant exudates, with only
recent research taking into account the multitude of microbial species in soil
(Swenson et al. 2015a). The manner in which exudates are utilised by and affect
microbes varies considerably and has been extensively reviewed (Bais et al. 2006;
Bertin et al. 2003; Boyce 2005; Brewin 2010; Huang et al. 2014).
A variety of positive and negative interactions with microbes have been
observed occurring directed by exudates from plants (Bais et al. 2006). Positive
microbial interactions include the following:
6 Soil Microbial Metabolomics 163

• facilitating plant growth-promoting rhizobacteria (PGPRs) and symbiosis with,

among others, acids, sugars and vitamin metabolites (Ahemad and Kibret 2014);
• biocontrol of nutrient fluxes including macronutrients such as P and micronu-
trients including Fe (Carvalhais et al. 2011, 2013; Dakora and Phillips 2002;
Valentinuzzi et al. 2015);
• isoflavanoids (Morandi et al. 1984) and phenolic acids [(Azaizeh et al. 1995;
Mandal et al. 2010), Table 1] involved with vesicular-AMF symbiosis;
• alkaloids and nitrogen containing metabolites produced by endophyte symbio-
sis, often to counteract insect predation of plants (Rasmussen et al. 2012).
Interactions that involve defence or attack against invaders of the rhizosphere
include the following:
• QS metabolites such as bradyoxetin from the soybean symbiont, bradyrhizo-
bium japonicum (Loh et al. 2002), that appears to help the bacterium fight off
invading microbes;
• the use of lactones such as N-(3-oxohexanoyl)homoserine lactone in the rhi-
zosphere of ginger (Zingiber officinale) to defend against plant viruses using
bacteria such as Acinetobacter and Burkholderia (Braeken et al. 2008; Chan
et al. 2011; Cooper 2007);
• phytotoxin use against invasive plants, including metabolites that include phe-
nolics, coumarins and quinines (Khanh et al. 2005), and antimicrobial agents
such as rosmarinic acid (Bais et al. 2008; Haichar et al. 2012; Hartmann et al.
2008).
The research listed identifies that there are still many unknown aspects of
exudate processes that have only been hinted at with current research efforts
(Bonanomi et al. 2009; Rabie 1998). Further understanding of rhizosphere
microbe–exudate interactions would increase our capacity to engineer the rhizo-
sphere to suit particular applications, as for example the use of AMF as biofer-
tilisers (Bonfante and Genre 2010). This strategy entails engineering ideal
rhizospheric growth conditions which target particular microbes for their ability to
metabolise distinct nutrients.

4.1 Rhizoengineering

Rhizoengineering entails controlling the plant’s rhizosphere, a considerable chal-

lenge considering the number of different entities involved. This could be to
improve plant yield [e.g. wheat (Saia et al. 2015a)] or output (with Arabidopsis
being a plant of focus in this research) (Kabouw et al. 2012), or to use the plants to
improve the surrounding environment (as was found with grasses (Hyparrhenia
hirta) and beans (Zygophyllum fabago) taking up heavy metals from mine tailings
164 M.W. Heaven and D. Benheim

in Spain) (Padmavathiamma and Li 2007). A notable example of rhizoengineering

used a metabolomics-driven approach to enhance plant–microbe interactions for
bioremediation of soil contaminated by polychlorinated biphenyls (PCBs)
(Narasimhan et al. 2003). In the study, an Arabidopsis–Pseudomonas rhizosphere
model was established in which 76 % were phenylpropanoids [e.g. ampelopsin
(dihydromyricetin)] of 125 identified compounds (identified by metabolic profiling
of Arabidopsis Thaliana). The root exudates identified by LC–MS were found to
create a nutritional bias for efficient rhizocolonising strain of Pseudomonas putida
PLM2. The strain was chosen both for its ability to utilise a diverse range of
phenylpropanoid compounds, and its PCB-degrading capabilities. Using a gnoto-
biotic system, the study showed a 90 % reduction in PCBs in flavonoid-producing
Arabidopsis thaliana strains.
Rhizoengineering need not only be limited to in planta studies. Innovative
efforts have been made to examine microbial community dynamics through the
development of artificial root models using agarose-covered slides amended with
various carbon-rich compounds (i.e. glucose, malic acid and serine) simulating root
exudate composition (Ziegler et al. 2013). Such novel approaches could theoreti-
cally be modified to provide convenient models for the simulation of root–microbe
metabolism. Similar research into artificial roots has utilised mucilage (Ahmed et al.
2014), a polymeric gel exuded by plants that includes metabolites xylose, glucose
and uronic acids. The authors acquired mucilage from chia seeds (Salvia hispanica
L.) to emulate maize (Zea mays L.) root exudate. Ostensibly, this was to identify
how soil in the rhizosphere is often wetter than the roots of the plant producing the
exudate. The artificial roots in this system were simplified with the assumption that
chia mucilage is similar to maize. The use of artificial roots highlights the diffi-
culties in accurately measuring metabolomics fluxes in the rhizosphere.
Another effort to undertake rhizoengineering was to accentuate microbial con-
sumption of polychlorinated biphenyls (PCB) (Narasimhan et al. 2003).
Metabolites in soil were identified and delineated between microbial and those of
Arabidopsis. A number of metabolites (e.g. flavonoids, lignins, indoles, antho-
cyanins) identified in the plant and soil led to the realisation that phenylpropanoids
could be target metabolites for rhizoengineering of the soil. This was based on the
criteria that phenylpropanoid metabolites are complicated enough to be resistant to
microbial degradation, thus allowing them to act as a nutrient source for bacteria
that will selectively degrade PCBs.
Rhizoengineering could be an exciting new area of research. As promoted by
these papers, it is expected that a mixture of different species of plants and microbes
will be required to exude metabolites to the required composition to improve soil
productivity. Metabolomics could be a useful approach to characterise and optimise
the processes to successfully engineer the rhizosphere to suit society’s needs
(Abhilash et al. 2012; Bonfante and Genre 2010).
6 Soil Microbial Metabolomics 165

5 Metabolite Coverage Over the Lifespan of Plants

Another aspect yet to be fully elucidated by research and conducive to metabo-

lomics analyses is studying temporal variabilities to metabolites as a plant matures
and is affected by external stimuli such as climate change, or is situated in different
soil types (Bais et al. 2008; Borisjuk et al. 2012). For instance, the nature of
metabolite secretions from the roots of Arabidopsis thaliana has been found to
differ over the plant lifespan which, by extension, differentially affect root microbes
(Chaparro et al. 2013). Analysis of the root exudates by GC–MS identified 57
metabolites from 107 possible compounds. As the plant developed over 31 days,
the metabolites showed a comparative decrease in the cumulative secretions of
sugars (e.g. fructose, glucose) and sugar alcohols (e.g. glycerol) and an increase in
secretion levels of amino acids (e.g. glycine, alanine) and other metabolites (i.e.
organic acids, carboxylic acids, FAs and other phenolic compounds). This was
noted as being suggestive of a genetically programmed developmental pattern of
varied phytochemical root exudation. Rhizosphere mRNA pyrosequencing showed
strong correlations between microbial functional genes involved in carbohydrate,
amino acid and secondary metabolite metabolism, and metabolites secreted by
Arabidopsis thaliana at specified developmental stages. Another metabolomic
study of interactions over time between potential soil-borne pathogens
Phytophthora infestans on potatoes showed a similar pattern of amino and organic
acid metabolites concentration increase and sugar concentration decrease in
response to microbial inoculation (Abu-Nada et al. 2007). Similar results have also
been seen due to fungal infection of soybean (Scandiani et al. 2015) and straw-
berries (Valentinuzzi et al. 2015). One interesting study showed how maize uses the
soil fungus Fusarium verticillioides to attack another fungus, Ustilago maydis, over
a 7-day period (Jonkers et al. 2012). As time progressed, the battle between the two
fungi could be monitored through the changes in metabolite concentrations of
compounds such as fusaric acid and a mannosylerythritol lipid (Arutchelvi et al.
2008). Generally, however, the manner in which rhizosphere microbiome function
is affected by temporally varied root exudates over the course of plant development
remains largely unknown. Advances in analytical technologies that allow for
real-time monitoring [e.g. portable MS (Yang et al. 2008)] may allow for a greater
interest in using metabolomics to study how metabolites change over time.

6 Microbial Soil Inoculants

The interactions of beneficial rhizosphere soil microbes with root systems have
pivotal roles in the growth, development and ecological fitness of their plant hosts.
The prevalences of intensive farming practices that are high yield and/or quality
centric are traditionally predicated on extensive use of environmentally harmful and
costly chemical fertilisers (Riding et al. 2015; Wissuwa et al. 2008). Subsequently,
166 M.W. Heaven and D. Benheim

this has led to increased industry interest in the use of sustainable and environ-
mentally ethical farming practices (Nihorimbere et al. 2011; Zahir et al. 2004). The
use of soil inoculants or ‘biofertilizers’ comprising beneficial soil microbes has
been observed to strongly fulfil this niche through enhancement of plant growth,
biological control of plant pathogens, nutrient supply and promotion of soil pro-
ductivity [(Cappellari et al. 2013), Table 1]. Examples of soil inoculants are
mycorrhizal fungi, the filamentous fungi Trichoderma spp. and plant
growth-promoting rhizobacteria (PGPR) (including, but not limited to, genera
Acetobacter, Azospirillum, Bacillus, Burkholderia, Herbaspirillum, Paenibacillus,
Phyllobacterium and Pseudomonas) [(Chamam et al. 2013; Saharan and Nehra
2011), Table 1].
For example, the effect of soil inoculation with PGPRs in the commercially
valuable wild marigold (Tagetes minuta) has been assessed (Cappellari et al. 2013).
Wild marigold produces an essential oil (EO) known as “Tagetes oil” sought for the
preparation of high-grade perfumes. The effect of single and co-inoculation of
Tagetes minuta with Pseudomonas fluorescens and Azospirillum brasilense on
plant growth parameters and essential oil production was assessed under glasshouse
conditions. Azospirillum has been used for growth and yield promotion in cereal
plants as rice, maize and wheat (Chamam et al. 2013). Both single and
co-inoculations showed an increase in shoot fresh weight by approximately 50 %.
Total phenolic content of shoots was upregulated by up to twofold with total EO
yield increased by 70 % in single and co-inoculated plants. Major components of
the EO that significantly increased (p < 0.05) included nine types of terpenoids,
such as tagetone and ocimenone, identified by GC–MS. While individual inocu-
lation with either Azospirillum brasilense or Pseudomonas fluorescens increased
plant growth and EO production, significant enhancement to both metrics was
observed in co-inoculated plants suggesting synergy between the two bacterial
genera.
PGPRs demonstrate significant capacity for plant enhancement. In some cases,
the magnitude of enhancement may be both bacterial strain and plant
cultivar-dependant (Chamam et al. 2013). This study showed a strain-dependent
effect in the association between two types of nitrogen-fixing bacterium
Azospirillum and Asian rice (Oryza sativa). The bacteria Azospirillum lipoferum 4B
(rhizosphere-colonising strain isolated from Oryza sativa cultivar Cigalon) and
Azospirillum sp. B510 (an endophytic strain isolated from Oryza sativa cultivar
Nipponbare) were observed to significantly increase (p < 0.05) growth of the
cultivar by up to 1.5 mg/plant over 10 days if they were used to inoculate the rice
strain from which they were isolated. Metabolic profiling data from reverse-phase
LC–MS demonstrated significant modification in rice secondary metabolites in
PGPR-inoculated plants, with 17 flavonoids, 10 hydroxycinnamic acid derivatives
and four alkylresorcinols the most affected metabolite classes. Moreover, the
metabolites were unique to the cultivar with only a few compounds, such as
tryptophan, found common to all cultivars. This research stands as a strong example
of how metabolomics techniques can be used to directly assess the nature of host–
PGPR symbiotic interactions, here being able to distinguish the physiological
6 Soil Microbial Metabolomics 167

responses of both rice cultivars to specific Azospirillum strains with opposing root
colonisation strategies. In another case, similar changes in plant secondary meta-
bolism were observed in a study investigating maize-Azospirillum interactions
(Walker et al. 2011). Major secondary metabolic changes were exclusively
observed in the roots of the Cigalon-4B pairing, predominantly for benzoxazinones
and benzoxazolinones, while the endophytic B510 strain elicited a systemic
response inducing metabolic shifts in both shoots and roots of both rice cultivars
tested.
Compared to bacterial soil inoculants, fungal soil inoculants including
Trichoderma spp., soil fungi that are often associated with plant root ecosystems
(Vinale et al. 2008), and AMF can impart significant benefit to host plants
(Contreras-Cornejo et al. 2011). Considered beneficial to plants, they protect them
from disease by attacking phytopathogenic fungi. Metabolomic studies have
identified that Trichoderma detect fungi due to the exudation of metabolites from
cell wall degrading enzymes and sensing the sugars released by degradation. More
importantly, Trichoderma releases secondary metabolites that are antifungal,
including antibiotics, water soluble acids and peptaibols. The non-polar nature of
the antibiotics (e.g. 6-pentyl-a-pyrone) suggests that these are used as a long-range
defence, while more polar metabolites (i.e. peptaibols) are used to attack fungi at
‘close quarters’. The positive relationship between Trichoderma and plants, which
includes increasing crop productivity by up to 300 %, along with activation of the
plant’s defences (Woo et al. 2006), has led to the use of this fungi as a natural
biocontrol agent.
Other research that has identified microbially related metabolites involved in
systemic acquired resistance and pathogen protection include the following:
• salicylic (SA) and jasmonic acid (JA) (Segarra et al. 2007) in cucumbers;
• SA, JA and indole-3-carboxaldehyde (Contreras-Cornejo et al. 2011), sugars,
amino acids, ethanolamine, tagatose, oxoproline, GABA and urea (Chaparro
et al. 2013) in Arabidopsis thaliana;
• the increased production of phytoalexins (e.g. flavonoids, terpenoids, indoles)
after inoculation by Trichoderma that showed increased root development and
biomass of Arabidopsis thaliana over 8 days (Contreras-Cornejo et al. 2011);
• 2,4-diacetylphloroglucinol, from AMF (Wehner et al. 2010);
Another reason for inoculation has been to counteract stressors on plants such as
drought or salinity. Abiotic stress adaptation has been shown to involve a range of
metabolites including the following:
• rhizospheric fungi Trichoderma harzianum Rifai strain T-22 using lipid per-
oxides in tomatoes as stress biomarkers (Mastouri et al. 2010);
• drought protection of plant through the increased production of hormones such
as indole-3-acetic acid, JA, SA, ethylene, auxins, cytokinins and gibberellins
and amino acids like proline (Azcón et al. 2013);
• reduced concentrations of malondialdehyde and increased concentrations in
proline and phenol metabolites in wheat due to salinity (Rawat et al. 2011); and,
168 M.W. Heaven and D. Benheim

• in the case of Trichoderma spp., the regulation of root system architecture that
was made possible with reactive oxygen species that convert into hydrogen
peroxide (Contreras-Cornejo et al. 2013; Mastouri et al. 2010; Samolski et al.
2012).
Metabolomics can also be used as a tool to find soil microbial metabolites that
are a signal of diseases occurring within the soil (Bundy et al. 2009). For example,
the fungi Trichoderma spp. (Lorito et al. 2010; Vinale et al. 2008) have been found
to improve plant defences through secondary metabolite communication.
A pathogen attacking a plant results in Trichoderma expressing amino acids to
create small proteins called hydrophobins that act to coat the plant as a barrier.
Other metabolites used for defence included heptelidic (koningic) acid (Itoh et al.
1980), along with isocyanide derivatives, proteins with a-aminoisobutyric acid and
a range of FAs (e.g. C8, C10, C10:1).

7 Humus

Another major component within the rhizosphere is humus. Humus is a complex,

amorphous and colloidal substance of natural organic matter, a condensation of
phenolics and nitrogen containing compounds derived from plant and animal tissue
decay (Paul 2016; Sutton and Sposito 2005). Humic substances, which until
recently (Lehmann and Kleber 2015) have been considered largely the products of
microbial metabolism (Manlay et al. 2007), are recognised as promoting both
microbial and plant growth (Coûteaux et al. 1995; Ponge 2013). A recent study
examined the changes in the metabolic profile of maize seedling root exudates by
1
H NMR and GC–MS following co-inoculation with the PGPR diazotrophic
b-proteobacterium Herbaspirillum seropedicae and humic acid (Lima et al. 2014).
The classes of compounds detected in maize exudates included nitrogenous com-
pounds, FAs, organic acids, steroids and terpenoid derivatives. Substantial changes
in the exudation patterns 14 days post-inoculation were observed. For instance, root
exudates from seedlings exclusively treated with humic acids demonstrated dif-
fering quantities of FAs, phenols and organic acids from that of the controls. Those
seedlings treated singularly with Herbaspirillum seropedicae or in combination
with humic acids exclusively exuded a diversity of heterocyclic, nitrogenous ben-
zilamines and polyamines. The study showed that enhanced root colonisation of
Herbaspirillum seropedicae in the presence of humic acids could be explained by
the interplay between increased endophytic colonisation of Herbaspirillum sero-
pedicae and sorption of humic acids to plant cell wall surfaces (Canellas et al.
2013). Interestingly, compounds identified as possible QS-inducing agents (e.g.
substituted c-butyrolactones and 3-hydroxypalmitic acid methyl ester) were also
detected.
6 Soil Microbial Metabolomics 169

There is some controversy with the metabolomic characterisation of humus, with

some researchers claiming that extraction methods are creating the larger “bio-
molecules” from smaller soil-sourced metabolites (Schmidt et al. 2011). For
example, they argue that the well-known but difficult to characterise humics are
actually synthesised from smaller soil metabolites such as carboxylates that con-
dense due to the extraction process, as evidenced by the use of non-destructive
analytical techniques, and in situ observations (e.g. near-edge X-ray fine structure
spectroscopy combined with scanning transmission X-ray microscopy).

8 Terroir

In the viticulture industry, the word ‘terroir’ describes regional influences associ-
ated with climate, soil factors and plant genotype which strongly affect varietal
flavours and aromas (Styger et al. 2011). Biochemically, the same term relates to
modifications to the metabolome of a plant that impart a set of discrete desired
qualities, such as increased plant growth or reduced feeding by larvae (e.g. cabbage
“worm” caterpillar Trichoplusia ni) on plants (Badri et al. 2013b). The ‘terroir
effect’ is shown to manifest itself in cultivated grape varieties that have been
resident in the same location for extended time periods (van Leeuwen and Seguin
2006), and this concept has begun to be used to describe ‘microbial terroir’ (del
Mónaco et al. 2016). It has been suggested that this effect could likely be attributed
to soil microbes or the overall soil microbiome actively adjusting metabolite fluxes
in response to signals from plants (Badri et al. 2013b). In one study, GC–MS was
used to assess the effect of diverse soil microbiomes applied to the roots of
Arabidopsis thaliana on the leaf metabolome and, by extension, whether the
changes in leaf metabolome influenced the feeding behaviour of Trichoplusia ni
larvae. The study demonstrated that variations in soil microbiome composition
(primarily comprised >60 % actinobacteria and proteobacteria) produced a differ-
ential response to canopy and root biomass accumulation in A. thaliana plants. In
comparison to control soil slurries (absence of soil microbiome), all A. thaliana
plants showed an upregulation of amino acids, phenolics, sugars and sugar alcohols
in leaf material. In addition, presented evidence suggested a strong capacity of soil
microbial communities to either modulate above-ground feeding behaviour of
Trichoplusia ni or to enhance the herbivory resistance of 4-week-old A. thaliana
plants. It is surprising that in examining what makes a quality grape for wines that
soil and the microbial life has until recently been largely ignored (Burns et al.
2015). The reason behind this is that soil microbial effects are subtle versus climate
[(Pereira et al. 2006), Table 2], rainfall [(Lee et al. 2009), Table 2] and soil texture
(Pereira et al. 2006) which have been found to be larger drivers of terroir.
170 M.W. Heaven and D. Benheim

9 Nutrients in the Rhizosphere

Nutrient availability is at the heart of producing a healthy crop or pasture, and

changes to the macro- (e.g. N, P and K) and micronutrients (e.g. S, Mg, Ca) affect
plants and soil, with soil microbes adjusting accordingly. As the soil environment
changes due to season, moisture content, pH, soil texture, etc., nutrient fluxes in soil
result in changes in metabolite concentrations as microbes adapt or suffer from
these changes (Lorenz and Wackernagel 1994).
Phosphorus, due to its low solubility (  10 lM), remains one of the most
challenging soil nutrients for plants to acquire (Smith et al. 2011). For instance,
organic P is thought to be made up of predominantly inositols, DNA, RNA and
phospholipids (Nash et al. 2015). Often complexed to the minerals that make up the
soil (e.g. iron and aluminium), they can be difficult for soil microbes to access
(Jones 1998). One study examining organophosphorus metabolites used
glucose-6-phosphate with labelled 33P or 14C atoms to identify how microbes are
affected by deficiencies in nutrients (Heuck et al. 2015). The authors determined
that soil microbes prefer utilisation of this metabolite as a C rather than P source
with even complete uptake of the sugar resulting in excretion of P. The addition of
the additional organophosphorus compound revealed an increase in microbial
activity, which seemed to level off after 66 h, although this was not further
examined to see if this was due to other nutrient deficiencies (i.e. N) or specific to
fungi or bacteria. A metabolomic analyses might have also revealed the type of C
containing compounds that were being taken up. Increase in P availability to the
host plant is one reported benefit of mycorrhizal root colonisation, which plants do
through the expenditure of energy and organic metabolites to organisms such as
AMF (Smith and Smith 2015). The trade off to acquire P has been observed in
tomato roots from AMF Funneliformis mosseae and Rhizophagus irregularis
colonisation experiments [(Rivero et al. 2015), Table 4]. The authors observed that
in acquiring P, the use of AMF led to significantly greater N in mycorrhizal roots
(p < 0.05). However, shoot and root biomass, root/shoot ratio and total C were not
significantly altered. The cost to the plant appeared to be the reduction in phenyl
alcohols and vitamins, along with some amino acids (i.e. tryptophan, tyrosine,
phenylalanine, alanine and leucine). However, increases in root concentration of
intermediaries to amino acid (i.e. phenylalanine and tyrosine), sugar, carboxylic
acids and fatty acid metabolites revealed the benefit of the interaction between plant
and fungi, from the increased uptake of nutrients to the improved stress response of
the colonised plants.
Nitrogen has been the major driver of increases in pasture and crop production
since the 1950s. Due to the success of inorganic N used to increase plant biomass,
our knowledge of organic N is surprisingly limited (Warren 2013, 2014).
Characterising organic N metabolites that are produced by microbes is also a
nascent field, and soil metabolomic data would certainly advance the knowledge
base of this important nutrient. To date, characterisation of amino acids in soil has
been the most studied group of metabolites that are associated with microbes, with
6 Soil Microbial Metabolomics 171

fumigation of samples often used to determine microbial metabolites (Heuck et al.

2015; Swenson et al. 2015b; Warren 2015). However, there has been progress in
identifying key metabolites that microbes use in the soil.
AMF are also important soil microbes that have been studied for understanding
the movement of organic N. However, the study of N fluxes between plants and
AMF has been limited compared to the current focus of understanding the transfer
of C from plants (Hodge and Fitter 2010). Until recently, it was thought that AMF
received most N from its host plant, with transport generally occurring through the
metabolite arginine. However, it has been shown that AMF also seek out and
promote decomposition of organic compounds for the acquisition of N, with a
substantial concentration retained within arbuscular mycorrhizae structures for their
own growth. As N limitation is reported to reduce the benefits of AMF symbioses
(particularly with excess P) (Huang et al. 2014), a comparable study examined the
effect of AMF field inoculation (either single or co-inoculated with PGPRs) on the
root metabolome of durum wheat (Triticum durum Desf.) under N-limited, P-rich
conditions [(Saia et al. 2015b), Table 4]. Metabolomics was used to determine how
AMF or plant growth-promoting rhizobacteria (PGPR) affect wheat growth. Despite
low N and high P soil conditions, plant growth doubled when adding AMF at the
expense of amino acids and saturated fatty acid concentrations in roots. However,
with the addition of PGPR, the acids were retained. Overall, 118 metabolites were
identified and using the Kegg database (http://www.genome.jp/kegg/) metabolic
pathways were delineated from 83 identified compounds. Multivariate analyses
showed separation by treatment effects, with amines and unsaturated FA conserved
in the AMF treatments. Organic acids correlated with AMF + PGPR treatments
versus the control samples, which were also correlated with P-containing com-
pounds, saturated FA, carbohydrates and amino acids. An interesting observation
was the increase in xylitol, indicating a strong interaction between AMF and wheat.
A recent paper has shown how a multi-omics approach can lead to an improved
understanding of fungi and plant interactions (Larsen et al. 2016). The experiments
were designed to identify the signalling metabolites between aspen trees (Populus
tremuloides) and the mycorrhizal fungi Laccaria bicolour. Combining transcrip-
tomics, metabolomics and genomics, the authors were able to identify a number of
biochemical processes related to communication between the plants and fungi.
Metabolites identified from the fungi and correlated to plant gene regulation
included amino acids and phosphor sugars related to biochemical pathways for the
biosynthesis of aromatic compounds, plant hormones, plant metabolites including
quinines and their precursor, and metabolites related to plant terpene biosynthesis.
These metabolites were identified as being involved with fungal communication
with the aspen to modulate cell adhesion, defence response and cell wall modifi-
cation, presumably to facilitate the symbiosis between the two. A similar
multi-omics approach has also been reported for novel compounds such as
nanoparticles that can potentially exhibit unknown and potentially hazardous effects
(MacCormack and Goss 2008).
Other examples of enhancement of nutrient availability and the metabolites
involved include the following:
172 M.W. Heaven and D. Benheim

• the suppression of soil pathogens and production of auxins, peptides, ketones

and terpenes (López-Bucio et al. 2015) by Trichoderma spp. protecting chick-
peas (Rudresh et al. 2005) so as to increase P uptake; and,
• interactions between fungi and plants that express lipids and trehalose that result
in microbe (e.g. Glomus versiforme) mediated exchange of nitrates, phosphates
and amino acids [particularly arginine (Smith and Smith 2015)] from soil to
plant (Bonfante and Genre 2010). This two-way interaction includes the release
of metabolites such as strigolactones from plants into the rhizosphere.
Tree litter is another important source of nutrients on which soil microbes can
feed. Research has shown that microbial communities adapt to the tree they are
under (Ayres et al. 2009). A study of leaf litter revealed that different tree species
litter, monotypic stands of trembling aspen (Populus tremuloides), lodgepole pine
(Pinus contorta), and Engelmann spruce (Picea engelmannii), resulted in different
populations of microbes (Wallenstein et al. 2010). A number of metabolites were
identified that varied significantly between the tree species. Although the metabo-
lites were only identified by retention time and mass, it was suspected with com-
parison to other studies that the metabolites were products of soil microbes or fungi.
A followup study using pyrolysis molecular beam MS (py-MBMS) was able to
determine that the metabolites were a mixture of lower mass (<137 m/z) carbo-
hydrates, phenols and lignin monomers combined with higher mass (m/z = 252–
706), lipids, alkanes, alkenes and FA (Wallenstein et al. 2013).
This research was expanded to analyses of typical oak, beech and grassland
soils, and likewise identified that soil microbes were adapting to tree species dif-
ferences, expressed through changes to a range of soil metabolites [(Liebeke et al.
2009), Table 2]. Grassland and beech forest soils were found to be less diverse than
oak forest. This research contrasted that with improvements to soil quality when
using soil that the microbes were collected from compared with artificial substrates,
due to the variety of metabolites were available for each of the three groups of soil.
Glutamic acid was the predominant amino acid (approximately 70 µM in con-
centration) in oak and in the top three for the other two soil types (although at lower
concentration). Leucine and valine were also common to all three, and of the other
amino acids, only the oak soil sample had sulphur containing methionine. Amino
acids were attributed to degradation of proteins in the soil from microbial
decomposition. The most concentrated sugar was trehalose, again being an order of
magnitude greater in concentration in oak compared to the other soil samples. Other
sugars and organic acids were identified with the total mass of these compounds
revealing the richness of the oak soil metabolites (136 µM vs. 7 µM for grassland
and beech), a similar ratio as found for the physicochemical analyses (e.g. soil
organic carbon). Gram-positive and Gram-negative bacteria grew exponentially in
the oak soils, and the types of soil metabolites seemed to indicate what biochemical
processes were being used by the bacteria that were found.
6 Soil Microbial Metabolomics 173

10 Extracellular Enzymes

As the previous sections attest, there is no doubt that it can be difficult to identify
microbial metabolites from the myriad of chemical compounds in soil (Ponomarova
and Patil 2015). This becomes more problematic when sampling away from the
microbial powerhouse of the rhizosphere. One example is extracellular enzymes
(EE), enzymes released by microbes or plants into the soil to facilitate biochemical
processes (Wallenstein and Weintraub 2008). These processes include degrading
recalcitrant fractions of SOM for uptake by the microbes, secretion of metabolites to
“sense” what predators or prey are in the immediate environment, and release of
antibiotics to attack other microbes (Burns et al. 2013; González-Fernández et al.
2015; Wallenstein and Weintraub 2008). For example, EE from fungi produce
metabolites such as the lactone-based botcinolides and the terpene-based botrydial
compounds (González-Fernández et al. 2015). These metabolites are thought to be
excreted to attack plants through decomposition of plant cell walls followed by
nutrient acquisition from the plants. EEs from soil bacteria and fungi are known to
consume carbon-rich biomolecules such as chitin (Roberts and Selitrennikoff 1988),
lignin (Burns et al. 2013), tannins (Joanisse et al. 2007) and pectins
(González-Fernández et al. 2015; Tepper and Anderson 1990). Sugars and amino
acids from glycoproteins are also found on microbial adhesives (Wang et al. 2014)
from EE-producing microorganisms after consumption of the plant and microbial
debris. Plant litter is also a rich source of nutrients for EE-producing microorgan-
isms. Monitoring of lignin decomposition has revealed the production of metabo-
lites such as quinines and radical lipids that may potentially form humic compounds
in soils with access to phenols, peptides and carbohydrates (Schmidt et al. 2011).
Quantitatively and qualitatively, there is still ambiguity in the sources and
consumption of metabolites in soil. Whether the source is via a combination of EE
excretion, plant decomposition or experimental error, metabolomics techniques
may be amenable to characterising the macromolecules produced by EEs (e.g.
polysomes). A potential solution described in the literature involves attaching
coloured marker molecules to EEs in a dilute soil slurry, as the enzymes tend to stay
fixed to the soil and unavailable for analyses through typical extraction techniques
(Burns et al. 2013). Limitations of the current methodology include no knowledge
of enzyme turnover rates, limited number of fluorescent markers available to attach
to a limited number of functional groups and selection of only those enzymes
capable of being stabilised in the slurry, versus in situ soil samples.
Metabolomics has been suggested as a way of removing these limitations by
detecting the entire metabolome of a soil sample. Both pyrolysis-GC-IRMS and
LC–MS analyses have provided examples of how this might work. For instance, a
general survey of EE metabolites involved soil from the USA and Germany
(Liebeke et al. 2009). Analyses of soil involved both untargeted metabolomics of
GC–MS and 1H-NMR of SOM, while targeting metabolites of microbe Bacillus
licheniformis for comparison. A range of FAs, sugars, amines, amino acids and
organic acids were identified. Most metabolites were species dependent though
174 M.W. Heaven and D. Benheim

metabolites acetic, fumaric, aspartic and glutamic acid, glycine, proline, serine,
glucose and sucrose were found ubiquitously across the varied agricultural types.

11 Anthropogenic Effects on Soil Microbes

It is argued that we are in the Anthropocene age (Bundy et al. 2009; Desai et al.
2010; Rockström et al. 2009), so it is not surprising that soil microbes have had to
adapt to human endeavours, some of which can be deleterious to the environment.
Estimates made in 1998 include up to 100 000 chemicals which were available for
purchase (Rockström et al. 2009), many of which will end up in the environment.
Since then, material synthesis and engineering has advanced greatly, and so it
should be no surprise that many of these compounds end up affecting the soil
microbial food chain [(Simpson and McKelvie 2009), Table 2]. Indictor animals
such as earthworms are often used to determine soil health (Rochfort et al. 2009;
Whitfield et al. 2013), but there is an increasing amount of research into the effects
of synthetic chemicals on soil microbes (Hernandez-Soriano and Jimenez-Lopez
2014). The use of microbes to monitor or remediate contaminated sites is one of
particular interest (Desai et al. 2010; Jones et al. 2014). A soil metabolome would
allow for the ability to look across a range of biogeochemical factors that may affect
soil health. The discussion here will focus solely on metabolomics research as it
relates to microbes.
Agricultural changes of soil from pristine forest to farm land has been a major
change to the environment that has been occurring for thousands of years (Foley
et al. 2005). Changes occurring in soil management have been described through
the lens of metabolomics (Singh 2006). For instance, land use provided an
opportunity to use NMR metabolomics combined with mid-infrared spectroscopy
(MIR) to identify the effect of land management across four different regions of
Victoria, Australia (Rochfort et al. 2015). Comparing soil samples from relatively
untouched native land and adjacent farm land of oats or wheat, 1H-NMR was able
to identify a series of signals from lipid, terpene and sugar. The study identified that
these metabolites could be differentiated by NMR due to different concentrations
depending on land use, whereas soil location was differentiated using MIR. This is
similar to a 1H-NMR study of various mine sites across England (Jones et al. 2014)
which identified a similar series of metabolites, with differences between com-
pounds thought to have occurred due to a different solvent extraction system
(methanol for this study versus deuterium oxide). In both cases, the assumed
microbial source of these metabolites was explored, using a target microbe, Bacillus
subtilis, for the Australian study and the identification that most soil metabolites
were microbially based for the English analysis. Labelling techniques may give
enhanced information, as was shown in the effects of microbes feeding on mine
waste. Metabolite confirmation of mine waste and its effects on microbes was
conducted using stable isotope labelling (Mosier et al. 2013). Using this method
with 15N labelling allowed for the identification of 80 metabolites from 3500
6 Soil Microbial Metabolomics 175

metabolite features that included artefacts, non-biological metabolites, adducts, etc.,

the latter of which were considered predominantly microbial.
How soil microbes are able to adapt to new environments and unusual
metabolites has found use in regions away from their original habitat, as for
example in recovering petroleum oil (Arora et al. 2014). For example, the use of
mutant bacteria known to be resistant to metalloids, Pseudomonas pseudoalcali-
gene, was used to remove polychlorinated biphenyls (Tremaroli et al. 2009). To
identify the mode of action, metabolomic studies identified that thiols were oxidised
when the microbe reacted to the metal. 1H-NMR combined with multivariate
analyses showed that wild-type and mutant bacteria resulted in changes to con-
centrations of amino acids (i.e. glutamate, aspartate, glycine, histidine, tryptophan
and tyrosine), betaine and NAD+. Pseudomonas pseudoalcaligene was found to be
resistant to other toxic compounds, including caffeine, sulphates, streptomycin and
chlorinated compounds.

11.1 Engineered Nanomaterials (ENM)

Novel compounds, often of the size or smaller than the microbes themselves (e.g.
nanometre), are increasingly being developed, used and disposed of, on soils. The
design and use of engineered nanomaterials (ENM) is in part because they have
unusual and useful properties (e.g. strength and conductivity) that are different from
the bulk compound found in nature (Dinesh et al. 2012). ENM, so called to dis-
tinguish them from natural soil nanoparticles (e.g. colloids), are receiving
increasing attention in agricultural and environmental literature. As production of
ENM increases to meet the demand of high-tech materials, these products are more
likely to end up in soil. Products with ENM include sunscreens, cleaning products
and therapeutic goods. Some ENM, such as zero-valent Fe, are widely used in parts
of the world for cleaning up toxic chemicals (Lee et al. 2008). Until recently, the
effect of ENM on the environment had not been systematically studied, in part due
to the cost of manufacture, the similarity in size and composition of natural colloids
(Klaine et al. 2008), as well as the physicochemical aspects of the soil
(MacCormack and Goss 2008). Various studies have shown that ENM are poten-
tially hazardous in a laboratory setting, but there are few field studies (Dinesh et al.
2012; Johansen et al. 2008). To date, ENM have been predicted to be in the
environment (i.e. surface waters) in concentrations of 0.8 ng/L for carbon-based
ENM up to 10 µg/L for Ag-, Ti- and Zn-based compounds (Maurer-Jones et al.
2013). No such study has been conducted for concentrations of ENM in soil.
Adding ENM to soil to see how they affect the biosphere has been the main
method of determining their effects [(Jin et al. 2014; Johansen et al. 2008; Shah and
Belozerova 2009), Table 1]. This has enabled studies of how the dosage of ENM
affects the soil environment. As ENM are in a solid matrix, it can be difficult to
ascertain the dose actually received by microbes, and so studies may overestimate
their soil concentration. Although it is well recognised that they have the potential
176 M.W. Heaven and D. Benheim

to cause pollution in soil through accumulation, ENM have particular properties

that can make it difficult to determine their interactions with denizens of the soil
matrix, including microbes. This includes aggregation into larger particles and
adsorption to minerals within the soil. It is inevitable that nanoparticles are found in
soil due to accidental release as they have already been detected in marine and
airborne environments.
This lack of knowledge means that these compounds are an unknown threat to
the soil and its microbial community. A recent review of 10,000 papers on ENM
found that despite the explosion of research on human health, the consensus on their
toxicity is at best, weak and often misleading (Krug 2014). As to be expected,
research on ENM and their effects on soil and its inhabitants are even less clear.
Beyond health and environmental aspects, ENM are also being explored as inter-
mediaries and markers in microbe communication to help researchers identify
disease rates in soil (MacCormack and Goss 2008).
Carbon fullerenes and nanotubes are arguably the most well-known ENM. They
are also increasingly finding their way into the environment, and into soil (Berry
et al. 2016). However, studies to date find limited interactions with soil microbes
(Pettibone and Louie 2015). Two papers by researchers in South Korea highlighted
how microbes are affected by carbon nanotubes in their environment (Jin et al.
2014; Jin et al. 2013). The metabolic profile of the microbes, revealed through
phospholipid fatty acid analyses (PLFA), showed that microbial FA typically
changed abundance depending on whether the soil were treated with powdered
SWCNT (single-walled carbon nanotubes) or solvent suspended SWCNT.
Generally, as the concentration of SWCNT increased, the biomarker fatty acid (i.e.
odd chained and/or hydroxyl grouped and/or cyclic unsaturated) metabolite con-
centration for Gram-positive bacteria, Gram-negative bacteria and fungi signifi-
cantly decreased. This effect on microbes occurred for at least 25 days. Increases in
other types of FA (e.g. iso-branched) led the authors to believe that microbes
change their lipid composition to defend against SWCNT. Another study of bac-
teria and protozoa and how they are affected by C60 fullerene (Johansen et al. 2008)
noted that systematic biases may be an issue leading to difficulty in comparing
results with other research. In particular, using pure solvents versus soil samples
meant that other chemical characteristics of fullerenes are not taken into account
when the effects on microbes are analysed. However, as was found in studies with
cleaner conditions, fast-growing microbes suffered significantly upon addition of
C60 to soil (p = 0.004–0.033). The bacteria eventually recovered from fullerene
exposure. Suggested reasons for this included absorption of C60 to soil or other
particles that coated the ENM and minimised contact with microbes.
Metal ENM have a longer history than carbon ENM, and have been used,
sometimes unknowingly, since ancient times [i.e. gold nanoparticles for decoration
of ancient Roman sculpture (http://phys.org/news/2013-08-goblet-ancient-romans-
nanotechnology.html)]. Despite their more extensive history, research into their
interactions with microbes in the soil is limited. Like other ENM, metal ENM
analyses are complicated by the number of naturally occurring nanoparticles or
similar minerals already present in the soil. It has been suggested that labelling
6 Soil Microbial Metabolomics 177

metal ENM with unusual isotopes could improve the analyses of these particles
(Klaine et al. 2008). Most research of metal ENM has shown that they act as
bactericides (Lee et al. 2008). As these compounds have been used for water
treatment and fabric formation, some of the pollution may end up in soil (Stefaniuk
et al. 2016). Measuring microbial responses to metal ENM have usually focussed
on their toxicity through the use of fatty acid methyl esters (FAMEs) analysis. For
instance, a study on microbial response to Pd, Cu, Si and Au ENM used FAMEs
typical to microbes to determine how microbes survived in their presence [(Sasser
2006; Shah and Belozerova 2009), Table 1]. Despite using two comparative con-
centrations of metal ENM in soil over 15 days (0.013 and 0.066 % w/w), no
significant change in these microbial biomarkers metabolites was seen. However, it
was speculated that ENM may exhibit an indirect effect through interaction of
compounds required by microbes. This was shown in a concurrent experiment
where the growth of lettuce seeds was reduced in the presence of Pd and Au ENMs.
One positive review discussed how microbes could be used to create metal ENM
for commercial use, based on microbes being able to create metal organic com-
plexes such as iron oxide magnets, metal phosphate medicines and transition metal
catalysts (Lloyd et al. 2008).

11.2 Heavy Metal Contamination

Microbes such as AMF can be affected by heavy metal contamination (Karimi et al.
2011), and it has been shown that the metals reduce microbial biomass. Metal
wastes reaching soils are another environmental issue (Maurer-Jones et al. 2013;
Simpson and McKelvie 2009). Similarity in atomic size to nutrients, some heavy
metals can access microbes through channels designed to diffuse and transport
cations (Singh et al. 2016). This similarity has been put to use with Cu used as a
fungicide that has been shown to improve crop yield (Dhawi et al. 2015). As metals
cannot be degraded, microbial action tends to involve immobilising the contami-
nation so that its toxic effects are mitigated (Azcón et al. 2013). The success of
microbes and plants in resisting radiation effects (Stone 2009) and reducing con-
centrations of toxic metals has been identified in places of recent disasters such as
the Chernobyl and Fukishima nuclear accidents (Aung et al. 2015; Geras’kin et al.
2008). It has been noted that continuous exposure to heavy metals can result in
tolerance forming in microbes as they employ metabolic strategies to reduce metal
toxicity though reduction of the metal’s oxidation state or coordination of
metabolites to soluble metals ions (Jones 1998; Karimi et al. 2011). For instance,
the microbe Klebsiella mobilis CIAM 880 was able to release metabolites that bind
with Cd to promote plant growth in soil with high cadmium concentrations, pre-
cipitating the metal (Nies and Silver 1995; Pishchik et al. 2002). This was seen in
the plant having a larger root system and increased exudate concentrations released
into the soil.
178 M.W. Heaven and D. Benheim

The compound that probably typifies pollution since the industrial era is lead
(Nriagu and Pacyna 1988). One metabolomics study identified metabolites that
changed in concentration due to mycorrhizal microbe influence attenuating the
plants response to Pb contamination (Souza et al. 2014). The analyses identified a
series of amino acids that may complex heavy metal and remove the metal’s ability
to affect the plants. The amino acids include asparagine, histidine, proline and
glutamine. Using the AMF Glomus etunicatum, it was possible to identify potential
microbial interactions with plants (e.g. N and carbohydrate metabolism) that offer
protection from Pb. Pb was also identified as a factor in metabolomics analyses of a
series of mines in the UK [see the “Community Metabolomics” chapter by Jones
et al. (2014) this book]. Using 1H-NMR, metabolomics was used across 11 sites
that mined Pb and Zn (Jones et al. 2014). A simple methodology with minimal
extraction meant that, along with the microbial community, soil and invertebrates
were also sampled. The authors examined specific groups of metabolites (nu-
cleotides, sugars, lactate and amino acids) using multivariate statistics. They were
able to show that there were sites that had similar metabolic profiles and lower Fe
concentrations even though the mines were otherwise different in geochemical
makeup. The authors proposed that monitoring metabolomics could act as an early
warning to hazardous pollutant levels before any visible effects were seen. PLFA
was used in the Czech Republic to determine how mining waste materials fly ash
and mine digestate that contained high Pb, Si and Zn concentrations affected soil
microbes (Garcia-Sánchez et al. 2015). Organic compounds’ concentrations,
including phenolics, were identified as increasing under both contaminants, with the
researchers identifying this via increased microbial activity, particularly fungi,
under the digestate treatment (9.3 ± 1.4 µg/kg after 60 days, p  0.05). Other
compounds found due to contamination included carbohydrates, carboxylic and
amino acids, amines and polymers, although the researchers cautioned that the
metabolomics and genomics analyses could not differentiate between active and
passive microbes in the soil. A timed study showed that after two months, amine
compounds had become preferentially consumed. Digestate contaminated soil was
shown to result in a preference for microbes to consume carboxylic acids taken
from the SOM. The PLFA analyses found that metabolites associated with fungi
were most attenuated by the digestate and fly ash. Metabolites associated with
Gram-negative bacteria were also affected. The digestate increased the concentra-
tion of Gram-positive bacteria after two months. Fly ash was found to be beneficial
to soil community structure, with the metabolites associated with most types of
microbes increasing. Biofilms in mines have also proven to be a rich source of
metabolomic data on how microbes are affected by various metal wastes that
included sulphate, iron, zinc, copper and arsenic (Mosier et al. 2013).
Cadmium contamination is also a concern in soil as it affects the life cycles of
most species [(Sarry et al. 2006), Table 2]. Cd has been found to affect people
through kidney damage after consumption of contaminated food, with the suspicion
that the metal’s affinity to thiols is deleterious to organisms. This includes yeasts
and fungi that are found in a variety of forms in agriculture (Sláviková and
Vadkertiová 2003). Yeast under the effects of cadmium was studied using
6 Soil Microbial Metabolomics 179

metabolomic and proteomic techniques (Lafaye et al. 2005). The research identified
that the typical sulphur pathway in yeast for producing glutathione increased in rate
and the sulphur amino acid concentration reduced by 30 % as the microbe
attempted to remove cadmium. Proteome and metabolomic results were correlated
when Cd was used, but other treatments (e.g. sulphur starvation) that also result in
increased glutathione production were not, indicating an independent pathway is
initiated when Cd contamination is present.
A controlled study of increasing Fe concentrations was used to determine if the
microbe Pseudomonas stutzeri RCH2 was affected by metal-poor or metal-rich soils
(Swenson et al. 2015a). The idea was that Fe will affect the microbe’s metabolite
output as the competition for sites on either the metal or carbon of SOM in soil is
changed. As expected, increasing the Fe concentration increased the sorption of all
metabolites including those with phosphate, N-containing and carboxylate func-
tional groups. The research identified that concentration change in metabolites was
correlated to the charge of the anion (e.g. Phosphate− − Fe+) when it came to
sorption to Fe. The authors felt that it was important to conduct metabolomic
analyses to ascertain the rates of sorption due to a typical mix of microbial
metabolites, which may differ due to competing interactions compared to when
separate metabolites being tested. While the analyses did not bring many surprising
results (i.e. phosphates and dicarboxylates adsorb strongest to Fe), this paper is a
rare example of the application of metabolomics to understand the holistic system
of soil–microbe interactions.

11.3 Organic Contaminants

Research on organic compound contamination has been presented that offers not
just problems but solutions using metabolomics of soil microbes. For instance,
metabolomics was used to determine how microbes might be able to recover pet-
roleum from reservoirs that are not cost effective by traditional extraction tech-
niques (Arora et al. 2014). This study used microbial enhanced oil recovery of an
oil well on soil samples from India to determine how efficient the microbes were
and what decomposition products they produced during the extraction. Using
indigenous hyperthermophilic Clostridium sp., they tested how these microbes may
extract oil at high temperature sites (>91 °C). The use of water from the oil well site
ensured no contamination of microbes from elsewhere in the soil. Metabolites
collected included biosurfactants, organic acids, solvents, exopolysaccharides and
volatile FAs. Following a targeted analysis of the metabolite mix from different
groups of bacteria, the researchers were able to optimise conditions to increase the
concentration of metabolites that would be suitable for extraction, including sugars
(particularly sucrose), nitrates and ammonium metabolites (particularly urea).
Metabolomics has also been used to better understand the bioremediation of soils or
soil models (Singh 2006). NMR studies have generally used soil samples rather
than solutions, although there are metabolomic studies using liquid-state NMR to
180 M.W. Heaven and D. Benheim

monitor microbial degradation of xenobiotics. For instance, in one study on how

Mycobacterium biodegrades morpholine, piperidine and thiomorpholine, scientists
were able to use enriched carbon and nitrogen compounds to identify the
metabolites formed as the bacterium consumed the antibiotics [(Delort and
Combourieu 2001), Table 2].
Persistent organic pollutants (POP) are another source of contamination that can
affect all species, including soil microbes (Wania and Mackay 1996). These are
small compounds with aromatic rings and potentially halogenated functional
groups. While it has been thought that condensation of these compounds into soil is
a better alternative to POP being airborne, the effect on organisms is still a problem.
Polycyclic aromatic hydrocarbons (PAH) are one target of active research, partic-
ularly on contaminated sites such as oil fields and fires (Seo et al. 2009). One study
identified a series of metabolites isolated from the microbe Sinorhizobium sp. C4
that was extracted from soils contaminated with another PAH, phenanthrene (Keum
et al. 2008). After determining that the mode of degradation was ring opening, the
authors used untargeted analyses to monitor polar metabolites such as FAs and
polyhydroxyalkanoates as the bacteria were fed phenanthrene. This was detrimental
to the microbe, with more than 70 % of these metabolites decreasing in concen-
tration after being fed phenanthrene. POP eradication via soil microbes is another
area of active research. Polychlorinated biphenyls (PCBs) have had a 90 % removal
rate over one month when placed in the rhizosphere of Arabidopsis (Narasimhan
et al. 2003). The expression of phenylpropanoids increased by over 100-fold
compared to control samples which the authors linked to the breakdown of PCBs. It
was proposed that by adding to the soil, 10–100 fold the current concentration of
the microbe Pseudomonas spp., the current estimates of PCBs in soil [328 000
tonnes (est. 1988)] could be significantly removed. A consensus is that soil
microbes in the rhizosphere have adapted to using PCBs as a source of carbon and
energy, utilising exudates to degrade them, through the use of metabolites such as
biotin, thiamine, amino acids and isoflavanoids (Jha et al. 2015). The two major
metabolic processes identified from this research were anaerobic dechlorination and
aerobic biodegradation.
Sometimes, as in the case of pesticides and antibiotics, chemicals are deliber-
ately applied to soil. How soil microbes are affected is often overlooked. Pesticides
have a long history in agriculture and so their effects on non-target organisms have
been overshadowed by the benefit to plant yield and productivity (Imfeld and
Vuilleumier 2012). Antibiotics in agriculture, either by accident or design, are
becoming a real concern due the resistance of many bacteria that infect humans (Di
Marco et al. 2014), even when their primary use is intended to promote animal
growth (Horrigan et al. 2002).
Pesticide reduction through improved efficiency of soil microbes such as AMF
has been proposed (Baum et al. 2015) with Trichoderma spp. providing an
excellent example of how this can be achieved (Vinale et al. 2008). It should be
noted that studies have shown that the replacement of conventional pesticides with
“biopesticides” must be a gradual process as mycorrhizae concentrations have
decreased while synthetic fertilisers were applied (Imfeld and Vuilleumier 2012;
6 Soil Microbial Metabolomics 181

Ruzicka et al. 2012). Metabolomics can monitor how biopesticides function by

observing concentrations of carbohydrates, lipids and n-acetylglucosamine, among
others (Baum et al. 2015). Other secondary metabolites from plants that also reduce
the need for pesticides include phenolics, alkaloids and coumarins. For instance,
phenolics have been shown to reduce weed growth (Khanh et al. 2005). Another
example is the effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on Escherichia coli
(Bhat et al. 2015). Previous conflicting research identified that there were significant
disturbances to soil microbe communities. Using GC–MS for the analysis [com-
bined with analyses using scanning electron microscopy (SEM) and atomic force
microscopy (AFM)] the authors were able to determine a specific pathway attrib-
uted to 2,4-D exposure. This involved a combination of attenuation of oxidative
phosphorylation, ABC transporters, peptidoglycan biosynthesis, glutathione meta-
bolism and purine/pyrimidine metabolism, and an increase in amino acid, protein,
sugar and starch metabolism. There was also a significant reduction in the con-
centration of metabolites associated with membranes and cell walls.
In regards to antibiotics, the decreasing effectiveness of current medicines can be
balanced with the discovery of new antibiotics in soil created by microbial action,
as in the example of teixobactin (Ling et al. 2015). For instance, one compound that
is rapidly losing effectiveness as a medicine, tetracycline, has been examined and
found to affect the rhizosphere leading to a loss of exuded metabolites (e.g. phenols,
flavonoids) by up to 48 % (Di Marco et al. 2014). Similar research looking at maize
and its interactions with AMF found up to sixfold increases in carbohydrates, amino
acids and phenolics in the soil when the antibiotics Cefotaxime and Trimethoprim
were added, showing how deleterious antibiotics can be (Azaizeh et al. 1995).
Research detailing how microbes and plants can themselves act as antibiotics is
demonstrated by a recent study showing that Bacillus subtilis was inhibited when
placed in soil samples from wheat farms and untouched forest (Rochfort et al.
2015). Correlating data between the lipids, terpenes and sugars in the soil was able
to be matched with the antibiotic effects against the microbe. Metabolites such as
fulvic acids, isochromantoxins, organic acids and xanthocillins have been identified
in a wide ranging study of microbes including Aspergilloides, Furcatum and
Penicillium (Frisvad et al. 2004). It should be noted that due to technological
limitations, the metabolites of these well-known microbes may be misidentified.

11.4 Climatic Change Effects

Potentially, the greatest anthropogenic effect of the current century is how the
planet responds to an increasingly variable climatic pattern of weather (Edenhofer
et al. 2014). Metabolomics has shown promise as one way to quickly quantitate
metabolites that microbes exude in response to climate stressors (Simpson et al.
2012). Expected changes in weather and climate have led to the call and use of
metabolomics techniques to understand how the denizens of the land will adapt
(Ahuja et al. 2010). Climatic effects on soil microbes are expected as changes in
182 M.W. Heaven and D. Benheim

temperature along with water and nutrient concentrations become more variable
(Rennenberg et al. 2009).
Salinity is one climate change issue that is already common. Salinity is
increasing and the replenishment of water tables is difficult. Exacerbating this
problem is research showing that replenishment of water tables is more difficult as
temperature rises (Lozupone and Knight 2007). Metabolomics approaches have
predominantly examined plants, but microbial interactions with plants due to
salinity have also been examined. Studies of a variety of plants (i.e. Arabidopsis
thaliana, Lotus japonicus and Oryza sativa) seem to suggest metabolites used to
communicate between microbes and plants in the rhizosphere will be significantly
affected (Sanchez et al. 2008). These metabolites include organic and amino acids,
along with sugars. This may be mitigated depending on the plant species, as was
found in the case of the highly salt-tolerant Brassicaceae, Thellungiella salsuginea,
where there appeared to be little change regardless of salinity levels [(Lugan et al.
2010), Table 3].
One issue with salinity is that the high salt concentration in these soils can
interfere with the sample, leading to loss of data through ion suppression (Oikawa
et al. 2011). To address this issue, the authors used capillary electrophoresis–mass
spectrometry (CE–MS) in combination with solid-phase extraction (SPE) to
selectively remove up to 17 cations from soil solution. They were also able to
differentiate between plant and microbial cations. Optimising the method with a
model soil solution of 78 organic and 12 inorganic compounds, the method was
then used on soil solutions from rice farms. Significant differences between soil
with and without rice were obtained, with microbial metabolites such as histamine
and tyrosine present only in the absence of plants, and leucine, isoleucine,
phenylalanine and serine significantly more concentrated without plants.
Climate change will probably result in increased periods of drought, and irri-
gation is one method to ensure crop survivability. This results in a dry–wet event
that can lead to various stresses to both plant and microbial life (Kakumanu et al.
2013). In particular, a change in osmotic potential between the soil and intracellular
contents of microorganism can result in reduced life expectancy of the microbial
biomass. Besides K+, which acts as a regulator of ionic strength in cells, amino
acids, carbohydrates, quaternary amines and tetrahydropyrimidine are regulated and
maintained within microbial cells when the amount of water available is reduced.
Fungi have been found to preferentially accumulate polyols while bacteria have a
preference for amino acids and sugars. Other factors that may improve drought
resistance are a robust AMF that will increase N uptake to plants (Baum et al.
2015),
At the end of drought when rains have come, the sudden influx of fresh water
generally results in a flush of microbial activity (Kakumanu et al. 2013). It has still
not been determined whether this is due to the lysis of microbes, a change in
equilibrium of microbes as the concentration gradient changes, or other reasons.
The authors reasoned that they could quantify metabolites that are released when
the concentration gradient changes. It was found that the accumulation of
6 Soil Microbial Metabolomics 183

metabolites appeared more related to keeping energy-rich compounds at hand than

to regulate osmotic pressure. Metabolite composition of sugars and polyols in
drought and non-drought prone areas revealed that microbial lysis was unlikely to
have occurred when soils were dry. The authors speculated that metabolite accu-
mulation during droughts had more to do with energy and nutrient conservation,
and production of survival metabolites, such as exopolysaccharides.

12 Tools for Microbial Soil Metabolomics

When looking for microbial metabolites, it is often necessary to utilise as many

methods as possible to identify the source of metabolites. Attempts at a broad
understanding of microbes in soil have also been elucidated through model com-
munities (Ahmed et al. 2014; Ziegler et al. 2013) or plants (Ahmed et al. 2014).
However, extrapolating laboratory results to the field can be risky and increasing
the types of data collected can be useful. Correlations with other parameters such as
genomic data or physicochemical parameters can be helpful in understanding why
metabolites are present (Larsen et al. 2016; Rochfort et al. 2015). Recent combined
analyses have shown that a single analytical technique can give an incomplete
picture of microbial fluxes (Larsen et al. 2016).
One common enabling technology compatible with metabolomics methods is
stable isotope labelling, which has been shown to identify a variety of soil functions
of soil microbes, from rhizosphere signalling molecules to nutrient-associated
metabolites [(Gunina et al. 2014; Haichar et al. 2012; Watzinger 2015), Tables 3
and 4]. Probably, the most common method to identify metabolites of microbial
origin is to feed labelled substrates which produce digested products that are easy to
identify (Heuck et al. 2015). Sugars are an important energy source for microbes,
and this has led to metabolomic studies to determine how sugars are taken up by
soil microbes [(Apostel et al. 2015), Table 3]. Using 13C-labelled glucose and
ribose with PLFA to identify microbe type, a loamy soil in Germany was dosed to
determine sugar uptake by the soil. Initial decomposition of the sugars occurred
within 3 days. Glucose concentration then decreased by 50 % between 3 and
10 days, while ribose remained relatively constant. The position of the carbon on
the sugars was important, with the majority of carbon being incorporated from
glucose C-2 (approximately 90 %) and ribose C-5 (approximately 70 %). PLFA
identified the majority of detected microbes taking up sugars were Gram-negative
bacteria while Gram-positive bacteria and actinomycetes incorporated the greatest
concentration of labelled sugars into their PLFA (0.2–0.4 %). Other microbes found
to take up labelled sugars included protozoa, VA-mycorrhiza, anaerobes and fungi.
Gram-negative bacteria were also found to take up the greatest concentrations of
labelled carbon, while Gram-positive bacteria appeared to prefer sugars from older
SOM. A similar study sought to measure the use of plant versus soil organic carbon
by microbes in maize, wheat and rye farms, also in Germany (Kramer and Gleixner
2006). Following the rate and mechanism of these metabolites allowed the
184 M.W. Heaven and D. Benheim

researchers to determine that glycolysis and the pentose phosphate pathway were
parallel processes and characteristic to the soils studied. Initially, both glucose and
ribose were consumed at similar rates. After 10 days, concentration decreases in the
C-5 labelled ribose showing the preference by microbes to incorporate this
metabolite over glucose, presumably due to the formation of RNA and DNA. Data
for other labelled sugars showed a complex series of reactions occurring in the soil
that include glycolysis, pentose phosphate and gluconeogenesis metabolic path-
ways. PLFA data revealed a preference for the 13C substrates to be taken up by
Gram-negative bacteria, known to be the dominant species around the rhizosphere,
while methanotrophs were found to consume non-plant material, despite its
proximity.
The flux of a series of amino acids was determined using labelled compounds
(Gunina et al. 2014). Compared to fungi, sugars were more efficiently taken up by
bacteria, especially glucose and sucrose, if the concentrations of these metabolites
were low in the soil (Gunina et al. 2014). Ribose uptake was similar to glucose for
Gram-negative bacteria. The pentose phosphate pathway for these bacteria was also
characterised by the uptake of xylose. In contrast, fungi appeared to have a pref-
erence for larger, more complex sugars along with decomposition of glucose to
form triacylglycerols. Acetate, a common metabolite in soils due to it being sourced
in plant litter and cattle slurry, was found to be incorporated preferentially into
Gram-negative bacteria.
Using these labelled metabolites helped the authors to identify that
Gram-negative bacteria were the most efficient at using low molecular weight
metabolites, due to the preference of this type of bacteria to use the anabolic pentose
phosphate pathway. Fungi and filamentous microorganisms were found to be better
utilisers of acidic and complex organic compounds like palmitate and
double-bonded FA. Carbon-13 labelling allowed for comparison between microbial
and soil metabolites in a series of experiments investigating SOM accessibility of
nutrients (Swenson et al. 2015b). Comparing samples of soil that had been fumi-
gated versus untouched soil samples, it was possible to identify the labelled
metabolites of the microbe Pseudomonas stutzeri RCH2. Extracellular metabolites
were found to be more likely to be detected than intracellular ones, indicating that
additional steps such as adding salts to reduce osmosis may not be required. As
minimal processing was a goal of this research, water was used as an extractant, a
solution the authors point out is not ideal for the study of metabolites such as FA or
sterols. Despite this, the labelled metabolites identified included amino acids and
analogues, nucleobases such as uracil, and a series of organic acids.

13 Conclusion

Soil is a rich, complex ground for metabolomics research. The majority of microbes
within soil are still a mystery, but metabolomics is beginning to reveal their secrets.
Even with the advances in understanding how soil microbes interact with plants
6 Soil Microbial Metabolomics 185

(Mendes et al. 2011), significant challenges remain in characterising the spatial

separation and metabolic compartmentalization of differing metabolic pathways and
metabolites to their respective source—plant, microbe or systemic metabolic
response. Metabolomics techniques will help to holistically understanding how
humans have changed the soil environment; hopefully we will learn how to sustain
not just the soil but the environment that microbes interact with, whether it be in the
ground (Holland 2004), waterways (Lozupone and Knight 2007) or sky (Conrad
1996). As advances in research occur, many of the mysterious interactions between
biology, geology and chemistry will become apparent.
The knowledge acquired from the interactions between soil microbes will then
allow for specific manipulation of soil from improving crop yield to soil remedi-
ation (Mosa et al. 2016). Soil microbes are potential vectors that could be
manipulated and eventually synthesised to help with soil management. As humanity
comes to master concepts like rhizoengineering, metabolomic techniques may be
needed to monitor the efficiency of designed microbial consortiums, as they are
used to inoculate soil for improved soil health and productivity (Jia et al. 2016;
Zhang et al. 2015). While by no means the only technique to understand microbial
life (Desai et al. 2010), metabolomics has the advantage of being able to cast a wide
net over the biological, chemical and geological interactions occurring in soil.

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Chapter 7
Community Metabolomics
in Environmental Microbiology

Oliver A.H. Jones, Gavin Lear, Aalim M. Welji, Gavin Collins

and Christopher Quince

1 Introduction

Metabolomics may be defined as the analysis of thousands of naturally occurring

small biomolecules (metabolites) that are the substrates and products of both pri-
mary and secondary cellular metabolism (Jones and Cheung 2007). Such molecules
include sugars, organic acids, amino acids, flavonoids, lipids and nucleotides
amongst many others. An organism’s “metabolome” is its full complement of
metabolites, in the same way that its genome comprises its complete genetic content

O.A.H. Jones (&)

Australian Centre for Research on Separation Science (ACROSS),
School of Science, RMIT University, GPO Box 2476, Melbourne, VIC 3001, Australia
e-mail: [email protected]
G. Lear
School of Biological Sciences, University of Auckland, Private Bag 92019,
Auckland 1010, New Zealand
e-mail: [email protected]
A.M. Welji
10-113 Smilow Centre for Translational Research, University of Pennsylvania,
3400 Civic Centre Blvd, Bldg. 421, Philadelphia, PA 19104, USA
e-mail: [email protected]
G. Collins
C. Quince
Department of Civil Engineering (Rankine Building), Glasgow University,
76 Oakfield Avenue, Glasgow G12 8LT, UK
e-mail: [email protected]
C. Quince
e-mail: [email protected]
G. Collins
Microbial Ecology Laboratory, Department of Microbiology, School of Natural
Sciences, National University of Ireland, University Road, Galway, Ireland

© Springer International Publishing Switzerland 2016 199

D.J. Beale et al. (eds.), Microbial Metabolomics,
DOI 10.1007/978-3-319-46326-1_7
200 O.A.H. Jones et al.

Fig. 1 Simplified overview of a metabolomics experiment

and a proteome entails the complete set of proteins expressed and modified fol-
lowing their expression by the genome (Wilkins 2009). A very basic outline of a
metabolomics experimental setup is shown in Fig. 1.
The metabolome can be used to study the underlying biochemical responses of
an organism/population/community to an environmental stimulus or combination of
stimuli. However, because both populations and communities of microorganisms
are often difficult to culture, metabolomic analysis of such assemblages must take a
different approach from that taken by studies using tissue or biofluid(s) from larger
organisms such as humans. A new and effective way to do this is to borrow
techniques and approaches from the field of metagenomics.
Metagenomics is a rapidly growing area of the genome sciences founded by
Venter et al. (2004) who hit upon the idea of undertaking the analysis of the total
complement of microbial DNA extracted directly from entire microbial commu-
nities in their native habitats without worrying about which species it came from.
This approach allowed them to sequence such communities in their entirety,
regardless of the ability of individual component members to be cultured in the
laboratory (Handelsman 2004). Early studies had great success. For instance, when
the technique was applied to the analysis of samples from the Sargasso sea over 1.2
million previously unknown genes were identified (Venter et al. 2004).
Metagenomics allows one to see, in ever-increasing detail, the vast microbial and
metabolic diversity that exists in the biosphere. It has been utilised in several
high-profile publications in studies ranging from the aforementioned study of the
genetic diversity of microbes in seawater (Venter et al. 2004) to phosphorus and
carbon management in sewage sludge (Garcia et al. 2006; Sales and Lee 2015), to
the effects of warming on carbon sequestration and lignin oxidation in soil (Feng
et al. 2008).
It is of note, however, that the other omic sciences have yet to replicate this
success. The first detailed study on the NMR-based metabolomics of microbial
communities in environmental samples was published by Jones et al. (2014) who
looked at the effects of pollutants on soil metabolic profiles in old mine sites in the
UK. There have since been a few similar studies including Swenson et al. (2015)
and Rochfort et al. (2015) who both looked at metabolites in the extractable organic
matter in soil, and Llewellyn et al. (2015) who used community metabolomics as a
7 Community Metabolomics in Environmental Microbiology 201

new approach to discriminate marine microbial particulate organic matter in sedi-

ments from various locations in the English Channel. Plant-associated fungal
communities assessed by meta-omics techniques were also the subject of a recent
review by Peršoh (2015).
Much remains to be learnt from this area of research through what might be
termed “Meta-Metabolomics” or, as proposed by Jones et al. (2014), “Community
Metabolomics”. Under this definition, in the same way as metagenomics indicates
the analyses of all the DNA from a given sample, community metabolomic analysis
uses all of the thousands of naturally occurring metabolites from the meta-
population of a sample of a given environment such as soil or water, and perhaps
even air (Castillo et al. 2012), as opposed to those from simple, laboratory based
monoclonal cultures (Eisen 2007) or clinical isolates (Bundy et al. 2005).
Community metabolomics has the potential to influence a range of “bio” related
disciplines, including medicine. For instance, Bundy et al. (2005) showed that one
can use metabolomics to classify pathogens on the basis of their metabolic profile,
even when it is not possible to infer a direct link to known virulence factors. This
could also have a wide range of possible applications in both general microbiology
and microbial ecology for distinguishing and identifying different functional/
physiological ecotypes of bacterial strains or species. In a similar vein, pioneering
work by Nicholson et al. (2005) at Imperial College London proposed that the
appropriate consideration of individual human gut microbial activities is a neces-
sary part of future personalised healthcare. This is because the gut microbiota of
most species interacts extensively with the host through metabolic exchanges and
cometabolism of substrates. Such interactions are presently poorly understood but
are highly likely to be involved in the aetiology of many human diseases as well as
the fate and toxicity of drugs taken to alleviate said diseases (Nicholson et al. 2005).
This topic is expanded upon elsewhere in this book, so will not be explored further
here.

2 Soil Science

Environmental science is another area where microbial metabolomics has great

potential and it is this area on which this chapter will focus. Whilst most work
carried out on this topic has been, thus far, relatively simple, it illustrates the
potential of the approach. For instance, Fourier Transform-Infrared Spectroscopy
(FT-IR) has been demonstrated to allow the chemically based discrimination of
microbial genotypes, and to produce biochemical fingerprints which are both
reproducible and distinct for different bacteria and fungi (Timmins et al. 1998).
Scullion et al. (2003) also tested the potential of FT-IR spectroscopy for investi-
gating microbial communities and their activities in soil. A range of samples
including laboratory cultures of similar soil bacteria, plant materials and earthworm
casts from worms of various ages and feeding regimes, were analysed using cluster
analyses and this proved capable of differentiating between different bacterial, litter
202 O.A.H. Jones et al.

and cast samples. However, this work has not been followed up in detail and the
amount of diagnostic information that can be obtained from FT-IR is limited.
A more complicated study was that mentioned above by Jones et al. (2014),
which utilised 1H-nuclear magnetic resonance spectroscopy (NMR)-based com-
munity metabolic profiling to assess the changes in biochemical profiles of com-
munities living in contaminated soils from various sites in the UK, each with very
different physicochemical characteristics (levels of metal contamination, underlying
geology and soil type). Each site could be clearly distinguished on the basis of the
metabolic profile of its microbial community. While some of these site differences
may also have been caused by additional abiotic factors (such as soil type or pH),
pattern recognition analysis of the data showed that both site- and
contaminant-specific effects on the metabolic profiles could be discerned. The study
therefore acts as a proof of principle for the use of community metabolomics of
microbial populations from whole soil samples (rather than single isolates) as a
diagnostic tool for pollution assessment. Assigning peaks in NMR spectra of soil
extracts is difficult but software tools such as Chenomx NMR Suite that overlay
library spectra of known compounds over the sample spectra can help (an example
is shown in Fig. 2). Recently developments in NMR software such as Bayesil,
(http://bayesil.ca/) a web based system that automatically identifies and quantifies
metabolites developed at the University of Alberta (Canada) may also be of help in
future.
Rochfort et al. (2015) also used NMR to assess metabolites in soils. They found
similar results to Jones et al. (2014), although in the Rochfort study there were more

Fig. 2 Section of a 1H NMR of soil extract (black) overlain with NMR spectra of lactate (red) and
valine (blue)
7 Community Metabolomics in Environmental Microbiology 203

resonances as a result of lipids and terpenes in the NMR spectra. The authors put
this down to differences between the extraction methodologies employed in each
study, although it seems equally possible that soil type, condition or land use could
have been responsible. Both the Jones and Rochfort studies demonstrated that soil
extracts could be measured by NMR, and there were no obvious interference or line
broadening as a result of paramagnetic materials that might have been present in the
samples. It is interesting to note that Rochfort et al. also used mid-infrared spec-
troscopy (MIR) to analyse their soil samples and found some differences. Whilst the
NMR metabolomic data were more associated with land use rather than location,
the MIR data were correlated more to location and inorganic chemical analysis. The
results may reflect the differences between NMR and MIR data (the latter technique
is far less sensitive) but may also indicate that it may be beneficial to combine two
or more analytical methods in such studies to ensure a comprehensive soil analysis.
It should also be noted that some microbial metabolites rapidly adsorb to soil and
thus one should also keep in mind that soil physical properties such as mineral
composition, surface area, shape and porosity present a notably different set of
challenges in soil analysis compared to the cell and tissue extractions more common
in metabolomics. Swenson et al. (2015) showed that even after a short incubation,
with water, many metabolites, for example amines and carboxylic acids, could be
adsorbed to soil and/or chelated by metals or other ions in the soil or extraction
medium. Cationic and anionic metabolites seem to be most affected, whilst the
majority of neutral metabolites were recovered. This is an important finding since
metabolites not recovered in the extraction procedure could be overlooked in the
subsequent analysis. Other factors that might affect such binding include soil type,
metabolite-soil contact time, temperature, moisture levels, and which extraction
solvents are used. Swenson et al. (2015) point out that there is, however, a silver
lining to this problem as understanding how specific metabolites bind to soil par-
ticles under differing environmental conditions may also increase our understanding
of how such compounds will behave in response to future climatic changes (e.g.
heat or precipitation) and thereby affect substrate availability and the structure of
soil microbial communities in the future.
Analysing small molecule metabolites, in addition to more abundant macro-
molecules, could also help to increase knowledge of biogeochemical cycles. For
example, 13C-labelled carbon sources could be fed to plants or pumped into/through
the soil and the bacterial metabolome studied to assess where the carbon is trans-
ported or to detect subtle shifts in microbial populations interacting with plant litter.
Indeed, the use of NMR techniques has already provided valuable data supporting
the occurrence, diversity and extent of carbon cycling in the carbohydrate meta-
bolism of microorganisms; for a review see Portais and Delort (2002).
Targeted studies have also been undertaken in order to measure flux rates of
specific metabolic pathways, for example through the use of isotope labelling
studies. Scullion et al. (2003) used FT-IR-based metabolic analysis to assess several
potential foods (oat grain, and fresh and aged tobacco) for the earthworm species
Lubricus terrestris and L. rubellus. As casts aged, there was a predictable microbial
succession (from bacteria to fungi) in both earthworm species. Each species was
204 O.A.H. Jones et al.

also found to differ in terms of their cast microbiology in response to food type.
Analysis of ageing casts by FT-IR spectroscopy indicated greater chemical changes
in casts of L. terrestris than L. rubellus irrespective of food type.
Community metabolomics of soils is potentially very useful since the identifi-
cation of biomarkers indicative of a defined response to a pollutant or pollutants,
before major outward changes become apparent, would be very useful in preventing
damage to a variety of sensitive systems (e.g. eutrophication). This could poten-
tially have applications in testing of contaminated land prior to redevelopment (e.g.
house-building on old industrial sites). It could also be useful for the water industry
since the discharge of toxic substances to the sewage network can have negative
effects on wastewater treatment plants, which rely heavily on bacteria and other
microorganisms to function effectively. Similar research could also identify
microorganisms with favourable metabolisms for bioremediation work.
Community metabolomics is not limited to the terrestrial environment.
Llewellyn et al. (2015) used ‘non-targeted’ community metabolomics to determine
patterns in metabolite profiles associated with particulate organic matter (POM) at
four locations from two long-term monitoring stations in the western English
Channel. They identified a range of compounds including amino-acid derivatives,
diacylglyceryltrimethylhomoserine (DGTS) lipids, oxidised fatty acids (oxylipins),
various glycosylated compounds, oligohexoses, phospholipids, triacylglycerides
(TAGs) and oxidised TAGs. Metabolic profiles varied significantly across the four
locations with the largest differences for both the polar and lipid fractions again
being due to geographic location (and/or time). Smaller differences were also
associated with depth.
The work demonstrated that community metabolomics is not only applicable to
the aquatic, as well as the terrestrial, environment but that it has the potential to
contribute significantly to the comprehensive and unbiased characterisation of
marine microbial populations; particularly if linked to metagenomic data. The
authors also speculated that the oxylipins could have a possible link to the for-
mation of oxygenated volatile organic compounds produced by microbial species
that are important in atmospheric chemistry.

3 Case Studies in Community Metabolomics

3.1 Bacteria and Metal Toxicity

As microorganisms form an extremely important, sessile component of ecosystems,

it is little surprise that they have evolved numerous strategies to deal with envi-
ronmental stressors. Laboratory investigations of individual bacterial strains pro-
vide well-controlled conditions from which to study mechanistic behaviour in
response to stress. The ability of microbial strains to survive in the presence of
relatively high levels of metal ions is an important environmental adaptation to
7 Community Metabolomics in Environmental Microbiology 205

toxicological stress. Recently, two specific approaches to microbial adaptation have

been examined using metabolomics methods: (i) an innate metabolic ‘priming’
which confers adaptive advantages to bacteria via specific pathways, and (ii) mor-
phological adaptation into biofilm communities which completely alter the bacterial
metabolism and consequent response to metal toxicity.
The negative environmental consequences of heavy metal toxicity have long
been known, and recent political and regulatory awareness has sparked a renewed
vigour in attacking this problem scientifically. In living systems, such as microbial
communities, heavy metal toxicity manifests itself through numerous mechanisms
(reviewed in Harrison et al. 2007 amongst others). Briefly, toxic effects include:
substitution for other essential inorganic elements, interaction with protein and
metabolite thiol groups, production of reactive oxygen species through Fenton-type
reactions, competitive inhibition of the membrane transport process and siphoning
electrons directly from respiratory pathways.

3.1.1 Tellurite Resistance

Incorporation of Tellurite (tellurium dioxide—TeO2) into electronics and industrial

applications has led to an increased interest in the study of the effects of environ-
mental exposure (Chasteen and Bentley 2002). A picture of pure Tellurium can be
seen in Fig. 3 but this element is not often found in its native form. A common,
soluble oxyanion form of this metal, however, is tellurium oxide (TeO3−), which is
highly toxic to most organisms even at low concentrations. Pseudomonas pseu-
doalcaligenes KF707 is a naturally Te-resistant (TeR) bacterial strain. The exact
mechanisms by which KF707 is resistant to Te are poorly understood, although
there are known interactions with thiol groups that mediate the bacterial interaction
with Te (Zannoni et al. 2008).

Fig. 3 Pure tellurium

206 O.A.H. Jones et al.

In a metabolomics study aimed at understanding the metabolic shifts associated

with Te exposure, a hyper-resistant KF707 strain (T5) was isolated and studied
using an NMR-based approach (Tremaroli et al. 2009). In an initial experiment
using an established biochemical assay (Ellman’s reagent, 5,5′-dithiobis-
2-nitrobenzoic acid) to measure cellular thiol content (RSH), it was found that
exposure to 25 µg/ml K2TeO3 for 30 min resulted in an equivalent depletion of
RSH content in both the wild-type KF707 and hyper-resistant T5 strains.
Intriguingly, the viability of T5 cells did not decrease further with extended
exposure, and infact, the cells were able to replenish RSH. Similarly, T5 cells
demonstrated resistance to perturbations of the membrane potential induced by
metal exposure.
In order to better understand the metabolic adaptations of the T5 strain to
counteract the effects of Te, an untargeted metabolite analysis was performed on
both the wild-type and T5 strains ±TeO3− (Tremaroli et al. 2009). This approach
consisted of quantitatively profiling 28 metabolites using the targeted approach
which accounted for the majority of peaks in NMR spectra of the samples.
Orthogonal partial least square-discriminant analysis (OPLS-DA) comparing two
sample types at a time revealed that there were unique metabolite profiles for
baseline KF707 versus. (a) T5 cells not exposed to TeO3−, (b) KF707 cells ±TeO3−
and (c) T5 cells ±TeO3−. Notably, there was no significant difference between
KF707 and T5 cells exposed to TeO3−, indicating that the post-exposure profile of
the two strains were similar, and that it is the pre-exposure metabolism effects
which confers the selective advantage to the T5 strain. One of the compounds
elevated in the baseline T5 profile compared to wild-type was glutathione, which is
important in responding to cellular oxidative stress. Levels of betaine, which plays a
role in response to hyperosmolar stress (Lisa et al. 1994), were also found to be
elevated .
Collectively, the metabolomics experiments described above lead to the con-
clusion that the hyper-resistance of the T5 cells could be attributed to a series of
basal adaptations that ‘prime’ the cells for exposure. This conclusion was supported
by further experimental evidence using phenotype microarrays which provide a
series of chemical stressors to evaluate the response of exposure to a host of toxic
chemicals (Bochner et al. 2001). In addition to tellurite, T5 cells exhibited an
increased tolerance to a number of other ions, including selenite, chromium, alu-
minium and caesium, while there was no altered response to other metals. Also
noteworthy was the fact that T5 demonstrated increased resistance to compounds
which interact with glutathione, glutathione-related proteins or RSH groups
(namely diamide, 1-chloro-2,4-dinitrobenzene and phenylarsine oxide). This result
provides a demonstrable link between the higher basal levels of glutathione in T5
observed in the metabolomics experiment and the phenotypic response of the
bacteria to toxicants. This is a useful demonstration of the power of community
metabolomics and the work could easily be extended to a range of other metals and
metalloids.
7 Community Metabolomics in Environmental Microbiology 207

3.1.2 Investigations of Biofilms and Morphological Variants

Biofilms are increasingly recognised as potentially important morphological com-

munities by which bacteria are able to survive under conditions that would be
otherwise detrimental. Recent evidence suggests that this transformation is
accompanied by specialisation, analogous to differentiation in higher organisms
(Nadell et al. 2009). Such adaptation into synergistically functional communities
confers a wide range of physical and chemical characteristics unique to the biofilms
compared to planktonic cultures of the same strain.
One of the interesting features of biofilms in both laboratory and ‘real-world’
settings is heterogeneity in colony morphologies when the bacteria are grown on
solid media (Boles et al. 2004). Within Pseudomonas, sp., two of these so-called
phenotypic variants have been well characterised, namely small-colony variants
(SCVs) and rugose small-colony variants [RSCVs, or wrinkly spreaders (WS)].
These morphological variants exhibit increased resistance to anti-microbial com-
pounds and, under laboratory settings, they are selected by exposure to environ-
mental stress agents such as oxidative agents, anti-microbials and metals.
One of the key genetic pathways associated with phenotypic morphological
variation is the global activator of cyanide biosynthesis/regulator of the secondary
metabolism (gac/rsm) signal transduction system (Petrova and Sauer 2009). In
experiments examining the morphological response to metal ion exposure of
P. fluorescens, four distinct populations were observed: (1) the parental wild-type
(CHA0), which did not give rise to any morphological variants; (2) a gacS
(CHA19) strain which gave rise to (3) SCV and (4) WS morphological variants
upon exposure to toxicants. Using an NMR metabolomics approach, the metabolic
difference between these four population types was characterised (Workentine et al.
2010).
Metabolites were extracted from the four groups from cultures grown to mid-log
phase and subjected to 1D 1H-NMR analysis and the results analysed in detail by
both principle component analysis (PCA) and partial least squares discriminant
analysis (PLS-DA). In this case 32 metabolites were identified and quantified,
revealed significant differences between all four populations, both in a combined
PLS-DA approach, and via comparisons of the various strains/morphological types.
Clear metabolic differences were observed between the SCV and WS morpho-
logical variants compared to both the wild-type and CHA19 strains (SCV and WS
were different to both wild-type and CHA19 strains). For example, valine,
phenylalanine and glycine were uniquely found to be important in the WS models,
whereas acetate, pyruvate, aspartate, proline and glutamate were important to
explaining the SCV metabolic differences. Other metabolites, such as tryptophan,
were also found to be important in both variants.
It is not entirely clear that what types of advantages or disadvantages are con-
ferred to morphological variants compared to the parental strains. Metal suscepti-
bility (AgNO3, CuSO4 and NiSO4) was used to evaluate the quantitative survival of
the bacteria in response to exposure. The gacS clone was susceptible to all three
metals, while the SCV and WS variants were both tolerant to NiSO4; however, SCV
208 O.A.H. Jones et al.

was uniquely tolerant to CuSO4 and WS was uniquely tolerant to AgNO3. These
results suggest that the morphological variants are imbued with unique properties.
To further explore the link between the metabolite profiles and metal tolerance,
another set of PLS-DA models were built which related the specific metabolites
responsible to metal tolerance across all three strains (in other words, the basal
metabolite states across all strains was considered together). In this analysis,
tryptophan, glutathione, methionine, adenosine and glucose were elevated in sen-
sitivity for both copper and silver, whereas proline was strongly correlated to
sensitivity for copper only and lactate and NAD+ increased with sensitivity to
silver.
Overall, the results from this study indicate that the biofilms under study had
distinct metabolic states that conferred some advantages to stress, and that mor-
phological variants further allow the bacteria to develop unique profiles. These
laboratory results are particularly important to understanding the environmental
survival and viability of bacterial strains.

3.1.3 Biofilm Versus Planktonic Response to Copper

One of the proposed mechanisms by which biofilms are known to differ from
planktonic cultures is in sugar metabolism. In particular, exo-polysaccharide
matrices are thought to provide structure for formation of the biofilms (Harrison
et al. 2007). The NMR approach commonly employed for metabolomics analysis is
advantageous in that it is robust, quantitative and highly reproducible. One disad-
vantage, however, is the relatively small number of metabolites (*30) that can be
characterised from extracts, with specific metabolites related to sugar metabolism
being poorly characterised. For this reason, in a study of P. fluorescens response to
copper (Booth et al. 2011) a more sensitive gas chromatography-mass spectrometry
(GC-MS) method was employed in addition to NMR. The two analytical platforms
provided identification of 79 unique metabolites, and hence a much larger coverage
of metabolic space. Pairwise multivariate comparisons indicated that while copper
exposure induces a significant metabolic response in both planktonic and biofilm
cultures, the nature of this response is highly different. Only three metabolites
(NAD+, phosphoric acid and glutathione) were common to both responses.
Overall, the planktonic cultures were found to be far more ‘reactive’ to exposure,
characterised by an oxidative stress response with changes to the tricarboxylic acid
(TCA) cycle, glycolysis, pyruvate and nicotinate and niacinamide metabolism. On
the other hand, the biofilm response was dominated by shifts in exo-polysaccharide
metabolism, suggesting a ‘protective’ response. While alterations in levels of glu-
tathione indicate that there was still oxidative stress in the biofilms, the lack of
involvement of energy pathways suggests that the biofilms have alternate methods
for enhancing protection, which is consistent with previous observations (González
et al. 2010).
7 Community Metabolomics in Environmental Microbiology 209

3.1.4 Methodological Considerations and Conclusions

Given the obvious advantages of being able to tightly control growth and exposure
conditions in a laboratory setting, it is no surprise that this is one of the most
popular methods used to study the effect of exposure to environmental toxicants.
Studies of bacterial metabolism are exquisitely sensitive to a multitude of factors,
including sampling conditions, sample processing, and in the context of metabo-
lomics, data acquisition and analysis. Numerous studies have been conducted on
appropriate methods to quench bacterial metabolism, which has recently been
assessed in the context of bacterial metabolomics (van Gulik 2010). The studies
described above were accomplished using cold methanol quenching, which is the
most common technique, but may suffer from leakage of metabolites during the
process. As the metabolomic scientists interest is often not in absolute quantifica-
tion of metabolites in the cells, but relative quantification between different popu-
lations, an inherent assumption in these experiments is that the quenching and
metabolite extraction processes impact the different populations in the same way.
In spite of these limitations, our understanding of the highly unique and specific
alterations in microbial metabolism in response to metals has been greatly enhanced
by metabolomics methods. The studies described here provide some clues as to how
bacteria can adapt, or exist in a uniquely ‘primed’ state for exposure to toxicants.
There remains much to do in the laboratory, including further testing of mixed
microbial communities, testing of complex toxicity profiles consisting of multiple
toxicants more closely reflective of real-world situations under chronic exposure
conditions. From a technical perspective, further expansion of the metabolome
coverage would also be advantageous, for example using liquid chromatography-
mass spectrometry (LC-MS) methods. In summary, metabolomics experiments in
the laboratory have contributed significantly to our understanding of the mecha-
nisms by which microbial populations are metabolically differentiated and, in fact,
these types of studies could have important industrial applications.

4 Potential Industrial Uses of Community Metabolomics

4.1 Using Community Metabolomics to Elucidate Metabolic

Pathways in Microbial Fuel Cells and Anaerobic Waste
Conversion Applications

Effective waste treatment is critical to maintaining ecosystems and human health.

Furthermore, in the context of increasing energy costs and the mitigation of
greenhouse gas emissions, the development of efficient waste conversion systems is
critical. Two related technologies, which promise to help address the challenge of
efficient treatment of wastes and wastewaters, are anaerobic digestion (AD) and
microbial fuel cells (MFCs).
210 O.A.H. Jones et al.

Digestion (both anaerobic and aerobic) is an established waste treatment tech-

nology for residues from various sources, including industrial processes and agri-
culture (Lester and Birkett 1999). Anaerobic digestion can be defined as the
biological mineralisation of organic material to biogas, in the absence of oxygen, by
the sequential activity of several microbial groups (Lester and Birkett 1999). It has
several advantages over aerobic biological treatment, including lower operating
costs and, notably, biogas production (Lettinga 1995). Biogas is similar to natural
gas and consists of a mixture of 50–85 % methane and 15–50 % carbon dioxide
with trace amounts of other gases. It is a renewable energy source and is used for
the generation of heat and electricity, or as vehicle fuel. A very interesting study by
Beale et al. (2016), combined metagenomics and community metabolomics to
characterise the microbiota in AD digesters, and investigate the resilience of said
microbial populations when exposed to operational shocks in the form of temper-
ature and the addition of additional substrates in the form of fats oils and grease.
The results provided a good deal of useful information of the structure and function
of microbe communities in AD units. It was also found that AD performance was
not greatly affected by temperature shocks, but that the addition of fats oils and
grease led to significantly promote biogas production.
The anaerobic reactors used for full-scale wastewater treatment and biogas
production are high-rate systems based on self-immobilised, or granular, biofilms,
e.g. the upflow anaerobic sludge blanket (UASB) reactor (Lettinga et al. 1980;
Lettinga 1995). Biofilms are a potentially multi-species community of microbes
embedded in a self-generated matrix of extracellular polymeric substance (EPS).
They are often found on surfaces but sometimes exist in granules comprising solely
EPS and microbes. In the case of the AD applications, each granule theoretically
contains all of the trophic groups required for the waste conversion. Increased
application of AD could provide benefits, both economically—by the reduction of
energy and operational costs—and environmentally—by preserving fossil fuels and
reducing emissions for CO2, particulate matter and other pollutants.
In the recent years AD research has moved towards: (i) energy-efficient,
low-temperature (psychrophilic) waste treatment applications (McHugh et al.
2003), (ii) biorefinery applications for the conversion of a range of non-food
feedstocks—such as grass and solid wastes—to valuable products, including
organic acids with industrial value, bioplastics and alcohols and (iii) anaerobic
fermentation processes for biohydrogen production (Oh and Logan 2005) and
electricity generation in MFCs (Chaudhuri and Lovley 2003).
MFCs are bioreactors, divided into two compartments separated by means of a
membrane that is permeable to cations but is impermeable to oxygen; electrodes are
inserted into the compartments and connected through an electrical circuit (Min
et al. 2005). Typically, anaerobic microbial consortia oxidise an organic substrate
(e.g. glucose or secondary wastewater) in the anodic compartment; electrons gen-
erated in the reaction are transferred to the anode and delivered to the cathodic
compartment in which they reduce an oxidised substrate (e.g. oxygen or oxidised
metal). Thus, the MFC converts chemical energy directly into electrical energy.
The energy potentially available in MFCs is significant, but their efficiency is
limited by several factors. These include the presence of electron acceptors in the
7 Community Metabolomics in Environmental Microbiology 211

medium, the incomplete oxidation of the substrate and the kinetic limitations in
electron transfer from microorganisms to the anode. This latter process was pre-
viously thought to occur strictly through redox mediators that shuttle the electrons.
However, only a decade ago, microbes capable of direct electron transfer have been
discovered (Min et al. 2005). Although not currently capable of large-scale elec-
tricity generation, MFCs present an attractive technology for self-sufficient
wastewater treatment with modest electricity generation.
Complex microbial communities underpin AD and MFC applications but the
metabolic roles of the individual microbes are still largely undetermined. Moreover,
the species are strongly connected through syntrophies where the waste products of
one provide resources for another. Consequently, to understand, exploit and extend
the application of these systems the ‘ecophysiological’ roles of the individual
populations and species must be determined. Questions, such as how do the
resulting syntrophic interactions structure the system-level behaviour? can then be
addressed to support full-scale engineering and operation. We propose that a uni-
fication of metabolomics and other ‘omics approaches—particularly metagenomics
—could provide a high-throughput solution to link taxonomy with function. This
approach could also enable the construction and validation of ‘ecosystems biology’
models operating at the level of the whole community.

4.2 State of the Science

The vast majority of organisms in anaerobic reactor biofilms and MFCs have not
yet been cultured. To study these organisms requires the application of techniques
from the ‘molecular toolbox’ to characterize the complex, mixed microbial con-
sortia present. Much prior research has focused on extracting DNA from these
communities and polymerase chain reaction (PCR)-amplifying 16S rRNA genes.
This gene is present in all prokaryotes and can be used as a marker for the taxa
present (Clarridge 2004). To date, most 16S rRNA studies have used either
gel-based, fingerprinting techniques, such as DGGE, which provides a ‘barcode’ of
community diversity but without direct sequence information, or clone libraries,
which do provide direct sequence reads but through cost and time constraints are
limited in size (Phung et al. 2004). Both methods will fail to resolve rare taxa.
Quantitative PCR assays can be used to obtain absolute abundances of even rare
organisms but only for a predetermined target.
Until recently there was no economical method capable of resolving the com-
plete community structure. Next-generation sequencing technologies have trans-
formed this situation by providing orders-of-magnitude more sequence data at the
same cost (Margulies et al. 2005). This is driving the ‘omics revolution’, allowing
the sequencing of complete genomes from isolated organisms and environmental
DNA.
Application to MFCs and AD systems is still in its infancy. The genomes of
potentially important species have, however, been sequenced; for example,
212 O.A.H. Jones et al.

Geobacter sulfurreducens (Methe et al. 2003) and Shewanella oneidensis

(Heidelberg et al. 2002), both of which appear capable of direct mediator-less
electron transfer in MFCs. These two genomes have been extensively studied and
their genes annotated to functions which have been confirmed by experiments and
regulatory networks inferred from transcription profiles. This information has also
been integrated into metabolic pathway models. However, even for these two
highly studied organisms, a complete systems level understanding is still some way
off (Fredrickson et al. 2008). Indeed, to extend this highly reductionist approach to
all organisms that could be present in a system that is open to the environment—
although potentially desirable—is impossible.
Metagenomics can be considered as the extension of genome sequencing to a
community. In shotgun metagenomics, short reads are taken indiscriminately from
all organisms and then assembled into longer individual genome fragments or
contigs (Handelsman 2004). When only a few genomes are present, it is possible to
obtain long contigs, and even complete genomes, from which the same inferences
regarding gene function and putative metabolic pathways as for a single genome
can be made. This technique can prove very effective for simple communities such
as those associated with the breakdown of a particular compound in anaerobic
bioreactors (Lykidis et al. 2011). Gene annotation is, however, only the suggestion
of potential function; the methods are far from perfect, and that gene may either not
be actively expressed or its associated protein could be rendered inactive by
post-translational processes. For more complex communities, metagenomics will
fail to recover substantially sized contigs unless the sequencing depth is very high.
This motivates the amplification of focal genes, most often marker genes such as the
16S rRNA. These amplified gene fragments can then be sequenced to very high
depth without dissipating effort through the whole meta-genome and sequences can
be recovered from a substantial portion of the community. Unfortunately, the
diversity of information associated with the genome fragments of metagenomics is
lost and the connection of taxonomy to function is even more tenuous.
Using a careful combination of molecular and analytical techniques, it is pos-
sible to link taxa to function in these systems. This can be illustrated with a case
study: the detection of Crenarchaeota in sludge granules from AD bioreactors
(Amann et al. 1990). These organisms were first observed in bioreactors using 16S
rRNA gene cloning. Fluorescence in situ hybridisation (FISH), a microscopy
technique based on probing mixed-species specimens with fluorescently tagged
oligonucleotides, was used to determine the spatial location of Crenarchaeota cells
which were observed in close association with acetate-consuming methanogens
(Collins et al. 2005). Based on this, it was hypothesised that the Crenarchaeota in
AD bioreactors are H2-oxidising autotrophs that generate acetate for syntrophic,
methanogenic partners. Incubations of granules with radiolabelled acetate
(14C-sodium acetate), followed by biofilm cross-sectioning and beta-microimaging
to detect isotope label uptake, revealed that the acetate was confined to zones
dominated by the co-located Crenarchaeota and acetoclastic methanogens (Collins
et al. 2005). Stable isotope probing (DNA-SIP) experiments using 13C-bicarbonate
and sludge granules, with separate PCR-amplification of 16S rRNA genes from
7 Community Metabolomics in Environmental Microbiology 213

‘heavy’ and ‘light’ DNA fractions, indicated the autotrophic—or at least, mixo-
trophic—potential of crenarchaeal clones. This is a fascinating example of how
culture-independent techniques, and disparate datasets, can together reveal the
metabolic function of target groups, but it is not a high-throughput and generically
applicable approach while metagenomics is a high-throughput technique, the
connection to function is not direct.

4.3 An Integrated Approach for ‘Ecosystems Biology’

An integration of ‘omics techniques could provide the high-throughput approach

required to link taxonomy with function, and to deconstruct the complex microbial
communities found in AD bioreactors and MFCs. Next-generation sequencing of
marker genes will provide a high-resolution picture of community structure gen-
erating relative frequencies of even low-abundance taxa. It is important to sample
the minor community constituents, as it has been hypothesised that this so-called
‘rare biosphere’ may be important in ecosystem functioning (Sogin et al. 2006).
This can be coupled to techniques capable of resolving the absolute abundance of
higher level taxonomic groups, for example microfluidics cell-counting coupled to
quantitative PCR or fluorescence labelling of marker genes. This approach can
determine ‘who’ is there—and in what numbers; metagenomics then allows
determination of the functional genes they possess. Annotating those genes can
provide a framework of potential metabolic pathways on which further information
can be attached. Linking the same marker genes that have been amplified through
co-occurrence on contigs to the functional genes could allow the compartmentali-
sation of metabolic functions to particular species and, consequently, enable a
connection to taxa abundance.
The next step is to resolve from these potential pathways the true metabolic
processes occurring in the community; central to this is metabolomics. Using
methods such as NMR and GC-MS, it is possible to determine the concentrations of
small metabolite intermediates along the active pathways. Even more powerful is to
couple this with experiments pulsing stable isotope-labelled substrates through the
community—through the application of flux balance analysis—to calculate the rates
of reaction along targeted pathways. At the same time, mass balance measurements
based on resolving the concentrations of input substrates and ions in MFCs and
bioreactors, and reaction endpoints, provide a useful corollary. Less obviously,
further ‘omics information’ can be incorporated to resolve ambiguities, or to pro-
vide verification of the predictions. For example, transcriptomics can be used to
determine which functional genes are expressed and proteomics can be utilised to
search for enzymes. There is also a role for more targeted techniques, such as FISH
to identify the spatial location of particular groups of organisms.
In summary, metagenomics can be married to community metabolomics to
directly elucidate the functional role of whole (but exceedingly diverse) microbial
communities. This is an experimental strategy for obtaining the information
214 O.A.H. Jones et al.

necessary to resolve the pathways in individual samples and to link the components
of those pathways to species. Its success, however, is dependent on coupling it to a
computational pipeline capable of extracting the information and an experimental
strategy that maximises the statistical power of the complete data set to give
meaningful and valid results. The individual components of such a pipeline exist; it
is possible to identify microbial taxa from marker genes using resources such as the
Ribosomal Database Project (Cole et al. 2009); metagenomics reads can be anno-
tated to functional genes using MG-RAST (Meyer et al. 2008); and potential
metabolic pathways can be constructed from the KEGG database (Kanehisa et al.
2004). Similarly, tools exist for converting GC-MS peaks into predictions of
molecule identity (Horai et al. 2010; Atherton et al. 2006). The key will be inte-
grating the information in a single pipeline within a single statistical framework that
allows for errors, mislabelling and potentially contradictory information.
Deriving the optimal or most likely set of possible pathways and their divisions
amongst species will be a highly complex optimisation problem. A Bayesian
probabilistic approach will most likely provide the best method to solve this. This
will have the advantage of predicting not a single solution but a complete distri-
bution of possible results. Finally, any statistical technique is limited by the quality
of the data set. It is vital to link these tools to experiments that maximise the
information available. This should be achieved by running replicate systems to
determine the variability of the responses and, conversely, by using carefully
planned perturbations to expand the range of system states explored. These per-
turbations could be through changing substrate concentrations or operating condi-
tions, such as temperature. These requirements underpin the importance of the
high-throughput nature of ‘omics techniques to obtain, at reasonable cost, multiple
datasets, each containing huge amounts of information.
The final step of an ecosystems biology approach is integrating this information
into mathematical models capable of predicting both community composition and
bioreactor function. These will take the metabolic-taxonomic framework elucidated
above and add dynamics. The actual mathematical structures to do this are well
established: in a well-mixed bioreactor, a system of ordinary differential equations
would be appropriate, possibly with the addition of environmental noise; more
complex models will be necessary if spatial localisation of groups of organisms in
anaerobic granules or on the anodes of MFCs are deemed important. The greater
challenge than the model structure will be parameterisation. Once again a Bayesian
approach will be vital to propagate that uncertainty into predictions that formally
account for it.

4.4 Outlook for the Future

The holistic, polyphasic approach outlined above can be viewed as systems biology
at the community level. It is not desirable to reconstruct and model every reaction
and process in every microbial cell, but it is desirable to identify those pathways,
7 Community Metabolomics in Environmental Microbiology 215

syntrophies and ecological interactions that are critical for controlling the com-
munity—and by extension, process—behaviour. The integration of ‘omics’
approaches coupled to process monitoring, and advanced microscopy, can generate
comprehensive, integrated datasets at microorganism, biofilm and bioreactor level.
This will enable the link between processes occurring at microorganism level (scale
c. 1 µm–1 mm) and the processes occurring within bioreactors (scale > 1 m). This
will, in turn, lead to improved reactor monitoring capability, reactor design, per-
formance and operational stability.

5 Upcoming Challenges: What Can Community

Metabolomics Learn from Metagenomics?

5.1 The Challenges Ahead

The past two decades has seen the rapid expansion of ‘omics’ oriented research,
examining the abundance of the vast array of genes (metagenomics), RNA mole-
cules (meta-transcriptomics), proteins (meta-proteomics) and small molecule
metabolites (meta/community metabolomics) present in a wide range of different
environments. To date, it is genomics that has been the poster child of the omics
era, with the global media avidly following the progress of high-profile research
projects that have enabled us to map the human genome (Human Genome
Sequencing 2004), the genomes of various economically important plants (Goff
et al. 2002), animals (Hillier et al. 2004) and disease causing organisms (Heidelberg
et al. 2000). More recently, we have begun to describe the vast content of
‘metagenomic’ DNA, seemingly aiming to catalogue the entirety of life around us
using the DNA barcode.
As our analysis of the global meta-genome continues to benefit from recent
advances in robotics, DNA sequencing chemistries, analytical and computational
procedures, the costs associated with sequencing such large volumes of DNA
continue to tumble. For example, the consumable costs associated with sequencing
the human genome have decreased from tens of millions of (US) dollars in 2007, to
just a few thousand (US) dollars or under today (Drmanac et al. 2010). With so
much data now being generated, the major challenges associated with most
large-scale metagenomic studies concern data storage and how to make sense of the
vast amounts of DNA sequence data generated.
There are several terms in use for the application of metabolomics in
ecological/environmental studies. For instance, the term “Ecotoxicogenomics” was
proposed by Snape et al. (2004) to describe the integration of genomic-based
science into the field of ecotoxicology. “EcoGenomics” (or ecological genomics)
was used by Chapman (2001) to describe the application of genomics based
techniques to ecology. In both cases the term genomics was taken to encompass all
the ‘omic sciences’ namely genomics (genome sequencing and the annotation of
216 O.A.H. Jones et al.

function to genes), transcriptomics (gene expression at the transcription level),

proteomics (protein abundance) and metabolomics (metabolite/small molecule
levels). In addition, the phrase “Environmental metabolomics” was defined by
Viant (2007) as the “application of metabolomics to characterize the metabolism of
free-living organisms obtained from a natural environment, and of organisms reared
under laboratory conditions, where those conditions serve to mimic scenarios
encountered in the natural environment”.
Below, we describe how researchers in the field of metagenomics are meeting
the new challenges provided by their new ‘mega-data sets’ and how researchers of
community metabolomics might benefit from the many lessons learnt by its ‘bigger
brother’, metagenomics.

5.2 The Need for Better Reference Samples

To date, most attempts to ‘sequence’ the meta-genome of varied environments such

as the soil, marine waters or the human intestinal tract have been largely superficial,
only exploring as much of the meta-genome as is necessary to enable some
descriptions of the diversity and function of the community to be made, and with
the volume of data analysed normally dictated by financial or technical constraints.
There is therefore an urgent need for better reference data, comprising the complete
metagenomic data of various sample media. Several years ago, an international Soil
Meta-genome Consortium, ‘Terragenome’, (http://www.terragenome.org/) was
established (Vogel et al. 2009) with the aim of generating the first complete
meta-genome sequence of a complex environmental system (soil from Park Grass,
Rothamsted, UK). This study will provide reference data, to which data collected
from all other soils around the world can be compared. It is only with the provision
of these ‘full’ datasets that we will be able to answer key questions including: What
is the extent of microbial diversity and what is the diversity of functional genes
within the sample media? What is the abundance and functional significance of
each species within the soil, across different domains of life (e.g., bacteria, fungi,
protozoa, etc.)? How does the composition and function of the community differ
across spatial and temporal scales? And, what ‘core’ genes are present in most
soils?
While it may take many years of data collection and analysis before the com-
plexity of the soil meta-genome is fully understood, no serious efforts are currently
underway to catalogue the entire community metabolome of similarly complex
environmental samples. The generation of entire reference sets of community
metabolomic data from complex communities is essential if we are able to better
understand the diversity of functional activities undertaken within them and to
prevent community metabolomics being out-shadowed by its metagenomics for
years to come.
7 Community Metabolomics in Environmental Microbiology 217

5.3 The Importance of Meta-Data

Meta-data (or data about data) provides an essential environmental context to

metagenomic data, detailing key factors including site and sample descriptions,
chemical and physical properties and methods of analysis. To ensure that valid
comparisons can be made between the metagenomic data collected by different
researchers at different times and locations, and to reduce any unnecessary exper-
imental duplication, it is prudent that researchers should adhere to a standard set of
reporting requirements. Possibly inspired by the broadly accepted MIAME
(Minimal Information about a Microarray Experiment) standard (Brazma et al.
2001), the Genomics Standard Consortium (GSC, http://gensc.org) has driven the
development of MIGS (Minimal Information about a Genome Sequence), MIMS
(Minimal Information about a Metagenome Sequence) and MIENS (Minimal
Information about an Environmental Sequence) standards. Similar initiatives have
also been undertaken to provide comprehensive reporting standards for environ-
mentally derived metabolomics data (Morrison et al. 2007).
However, despite the efforts of such groups, most current studies of the
meta-genome (and community metabolome) do not attempt to adhere to such
standards. If the vast increases in data generated by metagenomics and community
metabolomics are to be used to their greatest potential, it is a matter of urgency that
standards for the provision of metadata will be adopted and required for major
scientific journals, as they are already required for microarray experiments (e.g., see
instructions for authors in the ISME Journal). Such a universal approach would help
to avoid unnecessary duplications of effort, whilst also benefiting various fields of
ecosystems biology, by helping to improve our understanding of the ‘connectivity’
between the parts lists generated by the ever growing number of omics approaches
(including metagenomics, meta-transcriptomics, meta-proteomics, and community
metabolomics).

5.4 Data Storage and Sharing

Modern molecular methods have provided tools for the global scientific community
to produce a continual deluge of DNA sequence data. These data, which are gen-
erated at an exponential rate, perhaps doubling in volume every 18 months (Lathe
et al. 2008), now requires terabyte-scale computation and ever expanding storage
facilities to reduce the widening gap between rates of data collection and inter-
pretation. Whilst it is the aim of researchers to increase the volume of useful DNA
data stored in their own databases and in online repositories such as NCBI’s
GenBank (www.ncbi.nlm.nih.gov/genbank/), much of the data being stored by
researchers are of limited value. Increasingly, it is being recognised that much, or
even most, of the storage space is used to house data that could be thrown away.
For this reason, it is now common practice to delete raw data files (e.g.
218 O.A.H. Jones et al.

electropherogram data) immediately after the data have been processed and con-
densed into far smaller text files containing the DNA sequence data. Due to recent
technical advances, the amount of data produced by metabolomic studies is
beginning to pose similar challenges and new standards of data storage and inter-
pretation are now required to minimise the cataloguing of redundant information.

5.5 Knowledge Transfer to Applied Research Outcomes

Even once all of our data have been collected, adequately stored and compared to
appropriate reference and metadata there remain many challenges to maximise the
value of the large datasets characteristic of meta-omic studies. To date, many
large-scale metagenomic and community metabolomic studies have sought only to
address primarily pure, or fundamental, research aims, cataloguing the complexity
of various biological systems and to provide new insights into the functional
capabilities of many different microbial communities. Whilst these are worthy
research aims, it remains crucial that our new understanding of microbial diversity
and function be exploited to its full potential, addressing much broader, applied
research questions of benefit to key industrial partners, healthcare providers and
environmental protection agencies.
The development and application of new, more sensitive, biological indicators of
environmental health will be a key growth area of metagenomics and metabolomics
led research. For example, there is an increasing interest in the monitoring and
restoration of freshwater systems, aimed at maximising their value for ecological,
recreation and economic purposes. Macroinvertebrate communities have been used
around the globe as biological indicators of stream health for decades. Extensive
research has permitted classification of a wide range of macroinvertebrate taxa into
pollution tolerant, sensitive and facultative categories which are used to provide
indices of water quality (e.g., Hilsenhoff 1987) and a well-researched body of
information now supports their use (Feld and Hering 2007; Trigal et al. 2007).
This macroinvertebrate community data provide little or no information as to the
potential causes of any observed declines in freshwater ecological health. However,
recent advances in meta-omics centred research mean that it is now possible to
rapidly characterise (i) the abundance of ‘functional groups’ of bacteria, such as
those conferring resistance to any heavy metal (e.g., cobalt, zinc and cadmium) or
the resistance and ability to degrade organic pollutants (e.g., PCBs), (ii) the
abundance and activity of genes encoding for these ‘key’ functions and (iii) the
presence and abundance of functionally related metabolites. As such, the era of
meta-omics research promises to revolutionise the application of biological indi-
cators such that the rapid analysis of microbial communities may not only be used
to provide an indication of general ecological stress, but also to identify specific
drivers of environmental degradation within any site. This is of particular impor-
tance for the restoration of urban ecosystems in which the primarily drivers of
environmental changes are frequently hard to distinguish amongst the large
7 Community Metabolomics in Environmental Microbiology 219

background of other potentially harmful anthropogenic influences using traditional

chemical analysis.
In contrast, the holistic approach adopted by metagenomic and metabolomic
studies aid the identification of key drivers of microbial community function, akin to
analysing the total chemical profile within a sample. This information can be used to
design highly targeted restoration strategies on a site-specific basis, with no prior
knowledge of the site’s history required. The field of biomedical sciences similarly
stands to benefit from predicted increases in metagenomic and community meta-
bolomic derived data. For example, a number of studies have already sought to
catalogue the presence and abundance of microbial genes within the human gut, also
identifying key differences in the gut microflora of healthy individuals and patients
with inflammatory bowel disease (Qin et al. 2010). By improving our understanding
of the links between the diversity and function of microbial communities, and their
metabolites, on and in the human body, it is expected that we may devise better
treatments and preventative care for a wide range of microbial diseases. For
example, could changes in the abundance of certain bacterial genes or metabolites in
the human gut be used as an early warning signal to detect the onset of inflammatory
bowel disease? It is already evident that the primary legacy of the new ‘omics’ era of
microbial research will not be the catalogue of parts that list diversity of microbial
life on earth, but it is possible by applying our new knowledge of the complex
relationships between the functional capability and versatility of microbial com-
munities and the complex environments in which they live.

Acknowledgments OAHJ thanks colleagues from the Australia and New Zealand Metabolomics
Network (ANZMN), Proteomics and Metabolomics Victoria (PMV), RMIT University (Australia),
the University of Cambridge (UK) and the Centre for Ecology and Hydrology Wallingford
(UK) for help and support in metabolomics research over the years.

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Chapter 8
Metabolomics: Applications to Food Safety
and Quality Research

Farhana R. Pinu

1 Introduction

Food safety is a major concern worldwide, which has gained enormous attention in
the last two decades mainly because of the emergence of new food-borne pathogens
and other chemical hazards. Additionally, there has been an extreme increase in the
food-borne illness incidences along with large-scale outbreaks. Many factors con-
tribute to these growing concerns, including the industrialisation and mass pro-
duction of agricultural products, the increasing number of imported food products,
and changes in food consumption patterns due to consumer lifestyle changes
(Motarjemi et al. 2008). Ready-to-eat and fast foods, especially, have gained great
popularity, and thus also resulted in many food-borne illness incidences in recent
years. Moreover, the tremendous upsurge in global population is also almost
forcing producers towards mass production of agricultural products without giving
much attention to quality and safety issues (Motarjemi and Lelieveld 2014). The
world has already seen an introduction of a massive amount of genetically modified
(GM) crops in the last 40 years, in response to the need to feed over 6.5 billion
people. Consumers are doubtful about the safety of consuming GM food products;
therefore, scientific attention is required to unravel the facts about the safety of
eating such foods (Pinu 2015).
In many third-world countries, food quality and safety are often overlooked. In
addition, many food vendors deliberately contaminate food products with unwanted
materials (e.g. melamine in powdered milk) to increase profits, and others do so

F.R. Pinu (&)

The New Zealand Institute for Plant and Food Research Limited,
Private Bag, 92169, Auckland 1142, New Zealand
e-mail: [email protected]
F.R. Pinu
The Liggins Institute, University of Auckland, Private Bag, 92019, Auckland, New Zealand

© Springer International Publishing Switzerland 2016 225

D.J. Beale et al. (eds.), Microbial Metabolomics,
DOI 10.1007/978-3-319-46326-1_8
226 F.R. Pinu

unintentionally because of their lack of knowledge of food hygiene and safety

(Unnevehr 2015). Therefore, insecurity about raw foods and other food products is
on the rise because of the deterioration of quality in different food products that are
produced in large amounts and the lack of adequate information about their
nutritional properties. However, the good news is that consumers around the world
are now more aware about the risks and safety of different food products because of
the availability of information via the internet and social media (Pinu 2015).
Therefore, there is growing interest and a need from both food producers and
consumers in implementing proper management of food safety and quality.
Quality control is a routine activity in different food industries, and can ensure
the adequate safety of food, helping to maintain the trust of consumers. However,
there is a strong demand for the development of techniques that might help to
determine the key chemical, microbiological and nutritional features of a food
product. Although many technologies are already available for the routine checking
of many food products, these can be inadequate and often cannot determine
emerging chemical and microbiological hazards present in foods. There is indeed
scope for improvement and this is where metabolomics has a potential in food
safety and quality research—mainly by introducing, developing and improving
techniques to detect contaminants (both microbiological and chemical) present in
food products. In this chapter, the ways in which metabolomics can be used (both
targeted and non-targeted approaches) to ensure the safety and quality of raw,
processed and GM foods and food products will be discussed. The importance of
improvement on different analytical approaches that may lead to the management of
toxins and food-borne illnesses caused by different microorganisms will also be
highlighted.

2 The Concept of Food Safety and Current Practices

Food safety issues around the world are mainly monitored by the World Health
Organisation (WHO) and The Food and Agriculture Organisation (FAO) of the
United Nations. The Codex Alimentarius Commission (CAC) is an intergovern-
mental body that is operated under WHO and FAO. In 1997, CAC defined food
safety as the assurance that a food or food product will not cause any harm to the
consumers when it is prepared and/or eaten according to its intended use. Therefore,
a food will only be considered as safe when it will not cause any long- or short-term
illness to the consumer. It is the responsibility of food safety management to make
sure that a food is safe before it is sold to its intended consumers. The maintenance
of food hygiene is another important parameter during industrial food production
that can ensure the safety and quality of food products. However, consumers are
also responsible for following the instructions provided on the packaging to ensure
the post-sale safety of food. Therefore, the key responsibilities that allow the proper
maintenance of food safety follow a chain of action from regulators, to producers
and then to consumers (Fig. 1).
8 Metabolomics: Applications to Food Safety and Quality Research 227

Fig. 1 The food safety regulatory model

The role of regulatory organisations and bodies is of foremost importance

(Fig. 1), and relies on the cooperation of different sectors, including government,
industry, consumers, and even academia (Motarjemi and Mortimore 2014). While
the main task of a government in this area is to foresee all infrastructure and public
health services needed for food safety management and to implement and enforce
the laws and regulations, the industry is the one that actually ensures the safety of a
food product by following all the regulatory protocols. The codes of good practices
(CGP) are the first line of defence for any food industry and are a set of principles
and measures that have been previously identified, based on past incidences.
Generally, CGPs are applicable to most food industries; however, these may vary
slightly depending on the type of industries. Hazard Analysis Critical Control Point
(HACCP) is the second line of defence, which is also widely implemented in
different food industries. There are seven principles under HACCP (Fig. 2) that
include identifying the potential hazards or problems, taking measurements and
then appropriate precautions to control the situation. Although HACCP is very
beneficial in an industrial setting to ensure the safety of the product, industry staff
may feel burdened by administrative requirements (Motarjemi 2014). However, the
implementation of HACCP rules allows setting up critical control points (CCPs)
that can be monitored, and corrective actions that can be undertaken if necessary to
avoid any further loss of industrial food production (Fig. 2). Verifications are
228 F.R. Pinu

Fig. 2 The seven principles of Hazard Analysis Critical Control Points (HACCP)

carried out to determine the food quality by testing the raw material and the end
product, by monitoring the environment before releasing the products. Auditing and
consumer complaints handling are also associated with HACCP implementation in
an industry.
In addition to regulatory bodies and industries, consumers also play a vital role
in maintaining food hygiene and safety. Many consumers prefer to buy inexpensive
food materials that do not have appropriate labels and safety information, and
consumptions of these foods might pose a health risk (Motarjemi et al. 2001).
Moreover, some ignore the instructions provided within the packaging of the food
products and some never report defective foods that may cause public health
concerns. Therefore, the liability of consumers cannot be overlooked and raising
awareness might help to improve current food safety issues. Food safety-related
campaigns organised by government and non-governmental organisations and
social media can help in raising awareness in consumers.
The role of the scientific community is also important in maintaining food safety
and quality. Ongoing research related to food products is always beneficial,
8 Metabolomics: Applications to Food Safety and Quality Research 229

bringing new insight into different food pathogens and chemicals that contaminate
food and food products. Moreover, toxicological and ecological knowledge of
microbial and chemical spoilage provided by scientists allows us to manage the
food safety situation in a better way by undertaking control measurements
(Motarjemi and Lelieveld 2014).

3 Food Safety and Quality Research: The Main Problems

The current mass production and industrialisation of different foods and food
products have been initiated in order to feed the increasing world population. The
‘green revolution’ has also taken place in the last decades, enabling production of
vast amounts of crops mainly using genetically modified (GM) plants. There is still
an insecurity about GM food products, as many believe that long-term consumption
of these food materials may cause some deleterious effects on human health
including allergies and other immunological disorders (Maghari and Ardekani
2011; Krimsky 2015). However, this is still under question as at least two different
groups of scientists exist who either consider GM food as safe or harmful (Krimsky
2015). Due to the lack of long-term and consistent research on the effect of GM
food on human health, it is still not possible to come to a conclusion and this is why
consumers are even more doubtful about the safety of GM products. In addition,
climate change is another key factor that has prompted development of new plants
or crop varieties that will withstand the gradual changes in environmental condi-
tions. For instance, many efforts have already been undertaken to develop new rice
varieties that can withstand considerably unfavourable conditions including heat
stress, drought and high salinity (Nokkoul and Wichitparp 2014; Van Oort et al.
2015; Nguyen et al. 2016). Barley is another crop variety that has been used widely
as a model to study and develop climate-resilient crops (Dawson et al. 2015).
Therefore, the world has observed huge changes in the quality and type of foods
and raw materials, which pose both a significant advantage and sometimes a new
threat to food safety and quality. Chemical contaminants and xenobiotic molecules,
including pesticide residues and organic halogenated compounds, also pose sig-
nificant potential impacts on both human health and the environment, as these
molecules can take a very long time to break down (over 50 years) in the envi-
ronment. The effects of pesticides and other xenobiotics on aquatic and other
environments (e.g. infertility of sea birds) is well documented, and attempts have
already been undertaken to reduce the rate of contamination by banning many
known chemical contaminants (Walker 1990; Falkowska and Reindl 2015;
Gustafson et al. 2015; Pérez et al. 2015). In addition to chemical hazards, food
safety is also threatened by microbial exposure. For instance, overall changes in
lifestyle have steadily forced us to adapt to comparatively different food habits, and
introduced new types of foods (e.g. raw food). As a result, many new food-borne
pathogens have emerged or other pathogens re-emerged because of these newly
found transmission vehicles. Thus, many food-borne outbreaks which occurred in
230 F.R. Pinu

the last 20 years were caused by bacteria, viruses, and protozoa, and many more
pathogens are being introduced via food contamination every year. Therefore, there
is an ongoing need for a proper risk assessment and management system to control
outbreaks or even a pandemic related to any food-borne pathogen.
Major current challenges can be identified as follows:
I. Emergence of new food pathogens and control of outbreaks
II. Potential effects from genetically modified foods
III. Emerging chemical contaminants and xenobiotics
IV. Adulterations of food materials.

4 Current State of the Art of Food Safety and Quality

Research

Food safety is not only a public health issue, but also has serious social and
economic consequences. Food-borne illness can cause havoc in any country and
sometimes worldwide when it is an epidemic that cause loss of many lives. For
instance, food- and water-borne diseases are the main reasons of death of over 2.2
million people annually (WHO 2006), children, immunocompromised and elderly
populations are often the most affected. This is not only a burden for many poor
countries, but also endangers the international developmental efforts that have been
undertaken to combat poverty (Kuchenmüller et al. 2009). These food-borne ill-
nesses can be caused by various agents including pathogenic microbes, heavy
metals, chemical contaminants and other toxic materials found in different food
sources (e.g. toxins in wild mushrooms) (Anater et al. 2016; Signes-Pastor et al.
2016). Among pathogenic microbes, different strains of Escherichia coli, Listeria
monocytenes, Salmonella spp. and even some food-borne viruses including nor-
ovirus and Hepatitis C have gained particular attention because of outbreaks related
to contaminated food consumption in many countries (Marušic 2011). Moreover,
many incidents were also reported of contamination of foods due to the presence of
heavy metals and chemicals. In particular, the presence of arsenic in rice and other
crops in many developing countries and melamine in milk products have gained
public attention in recent years (Signes-Pastor et al. 2016). Most previous research
on food quality and safety have mainly focused on determining the food compo-
sition and stress response of food pathogens, and have helped to add knowledge on
what should be present in a particular food and how the growth of pathogenic
microbes can be controlled using different techniques.
8 Metabolomics: Applications to Food Safety and Quality Research 231

4.1 Study of Food Composition and Its Importance in Food

Quality and Safety Research

To know more about the quality of food and food products, it is important to
determine the composition, which also allows us to decide whether a given food is
safe to consume, by providing information about any potential hazard. Much
research has already been undertaken by focusing mainly on food composition of
different crops, fresh food materials, and raw ingredients for other food products.
Determination of food composition is not only focused on differentiating between
different food products to ensure their safety, but is also intended to gain insight
about the originality of some speciality foods and fermented food products, e.g.
fruit juices, balsamic vinegars, and wines. One of the main reasons for determining
originality is to control the adulteration of food material with unwanted ingredients
that should not be present in that specific food. For instance, nuclear magnetic
resonance (NMR) profiling of fruit juices has been carried out to determine the
country of origin and also to discover if those fruit juices were produced using real
juice or concentrate (Spraul et al. 2009a, b; Tomita et al. 2015). In addition, volatile
compound analysis of balsamic vinegars through the use of gas chromatography
and mass spectrometry (GC-MS) allowed determination of the effects of ageing
materials or woods on vinegar quality (Chinnici et al. 2009; Callejón et al. 2010).
Many studies have also focused on regional or country-specific food products
including fresh vegetables, seafood, fish and ready-to-eat foods. This is mainly true
for those countries either with low average incomes or where environmental pol-
lution is a considerably bigger problem than in other developed countries. In
countries that lack proper implementation of laws, regulations and auditing systems
required for maintaining food safety, food stuffs can become deliberately contam-
inated with unwanted materials by vendors in order to gain more profit (Pinu 2015).
In this situation, food composition studies that help to determine the type and
diversity of contaminants are very useful. However, there is still a huge scope for
improvements to technologies for rapid detection and identification of those food
contaminants. Using these technologies, food auditors will be able to manage sit-
uations better by analysing food samples in real time while examining a food
processing unit. Such technologies are still very limited currently, and research
should focus on developing more user-friendly techniques and detectors using
cutting-edge technologies.

4.2 Food Safety: Pathogenic Microbes and Their Stress

Responses

One of the most demanding areas of research in food safety is the study of
food-borne pathogens, which is mainly because of the many outbreaks related to the
consumption of food products contaminated with pathogenic microbes. Food is
232 F.R. Pinu

mostly considered as an ideal growth medium for a wide range of microorganisms,

and thus also an ideal vehicle for transmission of food-borne illnesses. Although
vast numbers of people from most low-income countries where food hygiene is not
maintained properly suffer from food-borne illnesses frequently, few data are
available about those illnesses or outbreaks, mainly because of the lack of appro-
priate reporting systems. Therefore, most of the available data about food-borne
outbreaks related to the consumption of contaminated food materials from restau-
rants to homes are from developed countries, where these cases are well docu-
mented. In recent years (2009–2015), a few developed countries, including
Germany and the United States, have seen food-borne outbreaks associated with
pathogenic Escherichia coli strains (O104:H4, O157:H7, O27 and O121) from the
consumption of fresh vegetables, sprouted foods, ground beef, chicken salad and
chipotle Mexican grill (Raupp 2014; Gelting et al. 2015; Ison et al. 2015;
Radosavljevic et al. 2015). Different species of Salmonella have also caused
food-borne outbreaks in developed countries including the USA and Australia from
the consumption of chicken, cucumber, nut butter and raw cashew cheese
(OzFoodNet Working 2014; Herman et al. 2015; Laufer et al. 2015; McWhorter
et al. 2015). In addition to bacteria, food-borne illnesses associated with viruses,
such as norovirus and rotavirus, were also quite common, and caused severe gas-
troenteritis disorders in many countries (Trivedi et al. 2012; Pacilli et al. 2015).
Food pathogens are very efficient in responding to the environmental changes
during the different steps of food processing and they evolve to adapt to these harsh
environments, mainly by changing their gene expression or by producing quorum
sensing molecules, thus showing potential for causing disease in vulnerable hosts
(Humphrey 2004; Begley and Hill 2015; Rul and Monnet 2015). Therefore, many
studies have been undertaken to find out the mechanisms of adaptation of patho-
genic microbes under various stresses including heat, acid, salt and oxidative stress
(Samelis et al. 2001; Humphrey 2004; Koutsoumanis and Sofos 2004; Tiganitas
et al. 2009; Shen and Fang 2012; Stackhouse et al. 2012; Alvarez-Ordóñez et al.
2015; O’Leary et al. 2015). For instance, biofilm formation is one of the key
adaptation mechanisms that has been observed in many food pathogens, including
Listeria monocytogenes and Salmonella enterica (Rodrigues et al. 2011; O’Leary
et al. 2015). Preventive measures include the use of either chemical or physical
forces and using bacteriophages that do not allow these food pathogens to form the
biofilms, thus diminishing their chances of growing in the food products (Soni and
Nannapaneni 2010; Van Houdt and Michiels 2010; Da Silva and De Martinis
2013). Therefore, studies of stress adaptation of food pathogens are very useful in
the area of food safety, and generate in-depth knowledge about the molecular
mechanisms behind this process. Control measures can be undertaken or developed
based on what causes food pathogens to adapt to these adverse situations.
Moreover, these may also lead to the development of techniques to detect the
microbial contamination in the early stages of growth, thus preventing the infection
from being transmitted widely by the consumption of food.
8 Metabolomics: Applications to Food Safety and Quality Research 233

5 Metabolomics and Food Safety and Quality Research

5.1 Food Metabolomics

Metabolomics is one of the most recently introduced ‘omics’ that aims to analyse
small molecules (metabolites) in a given biological sample. Although metabolomics
was initially defined as the technical area by which it would be possible to detect
and identify all the metabolites produced by a cell or an organism (Fiehn 2002;
Bino et al. 2004), this has not been realised, mainly due to the diversity of
metabolites. However, metabolomics is already established as a powerful tool for
studying the metabolism and physiology of many living organisms, and thus this
approach has been applied to a diverse range of research areas, including biomarker
and drug discovery, agriculture, nutrition, bioremediation, plant biotechnology and
also food science (Anizan et al. 2012; Badilita et al. 2014; Hall and de Maagd 2014;
Li et al. 2014; Lima et al. 2014; Booth et al. 2015; El Amrani et al. 2015; Melnik
2015). Metabolomics has been used to study food systems including food ingre-
dients, food processing and food pathogens; it has gained popularity in the last
10 years and numerous studies have already been carried out (Vikram et al. 2004;
Badilita et al. 2014; Kusano et al. 2014; Inoue et al. 2015; Le Boucher et al. 2015;
Ragone et al. 2015). Therefore, a distinct research area entitled ‘food metabolomics’
is well established that refers to the application of metabolomics in food system
processes, from farm to consumers (Kim et al. 2016). Recently a term ‘foodomics’
has been introduced within the scientific community that refers to the application of
‘omics approaches including genomics, proteomics, transcriptomics and metabo-
lomics in food science (Herrero et al. 2012; Cifuentes and Rutledge 2013;
D’Alessandro and Zolla 2013; Ibáñez and Cifuentes 2014; Laghi et al. 2014; Inoue
and Toyo’oka 2015). However, food metabolomics deals only with the most
downstream product of cell metabolism, metabolites, present in a given food or
food system.
The food metabolome is very complex in nature, and also widely variable,
depending on the type of food and the raw materials. Therefore, many thousands of
metabolites are present in food are highly variable in terms of polarity and
molecular weight (Shulaev 2006). The differing concentrations of metabolites in
food items pose a major challenge in the development of analytical tools to detect as
many metabolites as possible within a single analysis (Kueger et al. 2012). So far,
there is no such analytical instrument available; therefore, for the better under-
standing of the metabolome by analysing as many metabolites as possible, the use
of multiple analytical technologies is suggested by many scientists (Hall et al. 2002;
Dunn and Ellis 2005; Villas-Bôas et al. 2007; Sumner 2010; Hall and Hardy 2012;
Pinu et al. 2014). In addition, one of the major challenges that a food scientist needs
to overcome while analysing food is the complex sample matrix. The matrix effect
(ME) can create challenges in the detection and quantification of compounds that
are present at very low concentrations in different food. ME is also responsible for
poor and unreliable data that can affect the reproducibility, repeatability, linearity
234 F.R. Pinu

and accuracy of the methods used by various analytical instruments (Trufelli et al.
2011). To avoid and reduce the ME, a sample clean-up step using Solid Phase
Extraction (SPE) or Solid Phase Microextraction (SPME) or liquid extraction is
usually necessary before analysis by other methods (Jiang et al. 2012). More effi-
cient chromatographic separation is also suggested by Trufelli et al. (2011), in
which two-dimensional separation techniques (both GC and LC) can also be
applied (Marriott et al. 2012; Mondello et al. 2012). However, pre-analytical steps
can be time-consuming, arduous and often can cause loss of analytes, which is not
appropriate for an unbiased profiling approach (Villas-Bôas et al. 2007; Cappiello
et al. 2010; Trufelli et al. 2011). Although metabolomics was initially introduced
mainly as an unbiased and non-targeted approach, both targeted and non-targeted
analyses are performed for any biological samples to better answer the research
questions. Therefore, both targeted and non-targeted metabolomics are gaining
popularity for the analysis food products.
A significant improvement has also been achieved in metabolomics workflow,
including sample preparation, quenching, metabolite extraction and acquisition of
data (Fig. 3). In recent years, many sample preparation protocols have been pub-
lished that allow better detection of metabolites with a wide range of chemical
properties (Anizan et al. 2010; Biais et al. 2012; Teo et al. 2013; Brennan 2014;
Chan et al. 2014; Le Gall 2015; Rejczak and Tuzimski 2015). In short, an appro-
priate quenching method is used to stop the ongoing enzymatic activities after the
collection of a food or microbial samples. After quenching, metabolites are
extracted using a suitable extraction solvent (e.g. chloroform/methanol/water).
However, it is noteworthy that metabolite profiles may vary depending on the
metabolite extraction protocols; therefore, it is better to use at least a few extraction
protocols to obtain a global metabolite profile of any biological sample (Duportet
et al. 2012, Jäpelt et al. 2015). Once metabolites are extracted from the sample, they
are ready for analysis by an instrument of choice. The acquired data then needs to
be explored using different statistical and chemometric approaches (Aggio et al.
2011, 2014; Gowda et al. 2014; Pluskal et al. 2010; Robotti and Marengo 2016;
Smith et al. 2006; Xia et al. 2012): including feature detection, alignment,
biomarker identification and chemical structure elucidation (Fig. 3).

5.2 Recent Advancements of Analytical Instruments

in Metabolomics

Due to advancements in different analytical instruments in the last decade, it is now

possible to analyse thousands of metabolites from a food sample in a single anal-
ysis. The sample preparation and data handling processes have also been improved
tremendously, making it easier for scientists to analyse samples in a short time.
Moreover, there are many commercial and in-house metabolite databases available,
which is also beneficial for the identification and sometime structural elucidation of
8 Metabolomics: Applications to Food Safety and Quality Research 235

Fig. 3 Workflow for the analysis of the food metabolome. Here, MS Mass spectrometry and NMR
Nuclear Magnetic Resonance spectroscopy

unknown metabolites present in a food. It is well known that powerful detectors are
the main factors for the analysis of metabolites. Two technologies, Nuclear
Magnetic Resonance (NMR) and Mass Spectrometry (MS) have been employed
widely in metabolomics (Shulaev 2006; Villas-Bôas et al. 2007; Dieterle et al.
2011; Herrero et al. 2012; Kueger et al. 2012; Zhang et al. 2012; Balan et al. 2013;
Ibáñez et al. 2013; Badilita et al. 2014; Senyuva et al. 2015). However, there are
many other instrumental techniques, including Fourier transform infra-red spec-
troscopy (FTIR), which are available for metabolite profiling of food samples.
NMR has been broadly used for untargeted metabolite profiling of complex
mixtures (i.e. fruit juices, wines, spirits, urine and blood) (Ogrinc et al. 2003). NMR
spectroscopy is increasingly renowned for its efficacy, non-invasiveness
(non-destructive), throughput and linearity (Laghi et al. 2014). Moreover, NMR
spectroscopy also provides structural, chemical-kinetics and other information in
multidimensional applications (Dieterle et al. 2011). Thus, high resolution NMR
spectroscopy along with multivariate data analysis has been used for direct char-
acterisation of fruit juices (Cuny et al. 2008), wine (Lee et al. 2009; Pinu et al.
2014), grape berries (Pereira et al. 2006; Mulas et al. 2011), olive oil (Del Coco
et al. 2012; Piccinonna et al. 2016) and beer (Almeida et al. 2006; Rodrigues et al.
2011). To obtain a global metabolite profile of a complex sample, NMR needs to be
coupled with another non-targeted analytical approach (e.g. MS). But there are
some serious drawbacks to using NMR for metabolome analysis. Different
parameters of the sample, e.g. salinity, pH and concentrations of metal ions, can
affect the sensitivity of NMR spectrometers and can cause difficulty in
bioinformatics-based resonance assignments (Lewis et al. 2012). To avoid these
236 F.R. Pinu

problems, consistency in sample preparation is required. Moreover, an NMR

spectrometer cannot detect metabolites with low concentrations; samples may need
to be concentrated before analysing, as well as require larger sample volumes. To
dissolve the dried samples, high concentration of deuterated solvents are required
and these solvents also cause bias in sample preparation and data analysis (Lewis
et al. 2012).
On the other hand, the MS technique has gone through remarkable developments
and it is now an important instrument for many researchers (El-Aneed et al. 2009).
MS is the most extensively used instrumental approach in metabolomics
(Villas-Bôas et al. 2005; Dunn 2011; Vestal 2011; Kueger et al. 2012). It is also
considered the technique of choice in metabolite profiling mainly because of its
high sensitivity and also its ability to profile a wide range of metabolites in a
mixture and within a single analysis (Glinski and Weckwerth 2006). Significant
improvements have been achieved in terms of mass analysers, including quadrupole
(Q), quadrupole ion-trap (QIT), time of flight (ToF), orbitrap, ion-mobility spec-
trometry (IMS) and Fourier transform ion cyclotron resonance (FTICR).
Quadrupole mass analysers are very robust, low cost and simple to use, but they
provide lower mass resolution and accuracy than other mass analysers (Villas-Bôas
et al. 2005). ToF, FTICR and orbitrap are considered excellent instruments that
offer the highest mass resolution of all mass analysers.
MS is mostly used in combination with a few powerful separation techniques to
enhance its identification power. Gas chromatography (GC), liquid chromatography
(LC) and capillary electrophoresis (CE) are the most common separating techniques
used in combination with MS, which allow maximum separation of metabolites in a
complex biological sample (Villas-Bôas et al. 2003; Ramautar et al. 2011;
Theodoridis et al. 2012). However, direct infusion (DI) is also widely used for
metabolite profiling, which is usually referred to as metabolic footprinting or fin-
gerprinting, depending on whether the analysis is of extra- or intracellular
metabolites (Villas-Bôas et al. 2005; Han et al. 2009). Because of the development
of interfacing systems such as atmospheric pressure ionisation (API), DI-MS can be
used to analyse a sample to obtain mass spectra of the metabolites within a few
seconds (Villas-Bôas et al. 2005). The requirement for a small amount of sample is
the major advantage of using DI-MS. Moreover, no derivatisation is required for
this analysis, and more metabolites are detected by DI-MS than by GC-MS, making
this technique best suited for high throughput non-targeted metabolite profiling
(Mas et al. 2007). However, DI-MS shows poor reproducibility when analysing
complex mixtures because of the matrix effect. The identification of metabolites by
DI-MS is also very troublesome, and stereoisomers cannot be resolved using this
technique (Villas-Bôas et al. 2005; Glinski and Weckwerth 2006; Pope et al. 2007).
GC is one of most efficient separation techniques in metabolomics, allowing the
separation of hundreds of metabolites within a single analysis and requiring a very
small sample volume (1–2 lL). The coupling of GC with MS is a perfect match
because metabolites in an inert gas phase can be ionised much more easily at the
8 Metabolomics: Applications to Food Safety and Quality Research 237

MS ion source, making GC-MS the best combination of separation technique with
mass spectrometry detection. The instrumentation has been developed considerably
during the last 50 years. GC-MS is a highly sensitive analytical platform, which
also provides excellent instrument repeatability, around 5 % or below (Villas-Bôas
et al. 2005). However, an extra step of sample derivatisation is required for the
analysis of semi- and non-volatile metabolites, and GC-MS has been widely used
for the last 50 years for the analysis of a wide range of metabolites present in foods
(Table 1). LC is another powerful separation technique that allows rapid analysis of
small amounts of sample. The main advantage of this separation technique over GC
is that no previous derivatisation of the sample is required to analyse the
non-volatile compounds (Scalbert et al. 2009). This technique is often coupled with
MS and sometimes also with NMR (Shulaev 2006; Zhou et al. 2012). For LC-MS, a
wide range of different detectors is used, ranging from ultra-high resolution MS
such as FTICR or orbitrap to low-resolution MS such as ion traps and triple quads
and hybrid systems. The most recent addition is the ion-mobility TOF-MS system
(Lanucara et al. 2014). The development of methods depends on the nature of the
metabolites to be analysed. LC-MS has been applied widely in metabolite profiling
(both targeted and non-targeted) of complex biological samples (Berg et al. 2012;
Theodoridis et al. 2012). CE is an efficient and rapid technique that can separate a
wide range of charged metabolites within a single analytical run (Villas-Bôas et al.
2005). CE is often coupled to MS and it is considered a promising analytical
instrument in metabolomics (Shulaev 2006). CE-MS has high resolving power and
requires very small volumes of samples (1–20 nL). Thus, it has been used for both
targeted and non-targeted high throughput analysis of metabolites (Nevedomskaya
et al. 2010; Sato and Yanagisawa 2010; Ramautar et al. 2011). The major drawback
for CE-MS is its poor sensitivity; thus the detection limit is several magnitudes
higher than those of chromatographic methods (Table 1). The introduction of a
small volume of sample is inadequate for the detection of many metabolites (Cai
and Henion 1995). Moreover, low recovery and irreversible adsorption of analytes
onto the capillary wall also can occur in CE. For these reasons, CE-MS is mostly
used in metabolomics as a combination of different protocols targeting different
groups of metabolites combined with sample preparation steps to concentrate the
metabolites in the samples, making it a lower throughput technology in metabo-
lomics and certainly more suitable for targeted analysis.
Fourier transform infrared spectroscopy (FTIR) is another analytical technique
that is extensively applied in the food industry because it is rapid, highly automated,
reproducible, non-destructive and cost-effective (Bauer et al. 2008; Versari et al.
2010). Nevertheless, FTIR is insensitive for complex liquid samples and water in
the samples often increases the background noise, increasing the limit of detection
and reducing linearity (Achá et al. 1998). The data obtained from FTIR are also
very complex and there few databases available for assisting with identification of
metabolites (Berthomieu and Hienerwadel 2009).
Table 1 Comparisons of different analytical instruments used in food safety and quality research
238

Analytical Advantages Disadvantages Application in food safety and quality research

technique
GC-MS • High chromatographic • Derivatisation is Gilbert-López et al. (2010), Anizan et al. (2012), Canellas et al. (2012),
resolution required for Banerjee et al. (2013), Caligiani et al. (2013), Cao et al. (2014), Guo
• Sensitive and robust non-volatile et al. (2014), Duedahl-Olesen et al. (2015a, b), Giri et al. (2015),
• Simultaneous analysis of metabolites Grimalt et al. (2015), Janssens et al. (2015), Walorczyk et al. (2015)
different groups of metabolites • Unable to analyse
• Large linear range, availability thermo-labile
of commercial and in-house MS compounds
libraries
LC-MS • High sensitivity • Average to poor Da Rosa et al. (2012), García-Gómez et al. (2012), Russo et al. (2012),
• Derivatisation not usually chromatographic Gottfried and Herebian (2013), Boutsiadou-Theurillat et al. (2014), Fan
required resolution et al. (2014), Zeng et al. (2014), Arroyo-Manzanares et al. (2015),
• Large sample capacity • De-salting may be Juan-Borrás et al. (2015), Khan (2015), Squadrone et al. (2015), Zhang
• Thermo-stable compounds can required et al. (2015), Han et al. (2016)
be analysed • Limited commercial
libraries
• Tough restrictions on
LC eluents
• Matrix effects
CE-MS • High resolution, small volume • Poor reproducibility Font et al. (2008), Lara et al. (2008), Nevedomskaya et al. (2010)
of sample required • Poor sensitivity
• Rapid analysis • Buffer incompatibility
• Usually no derivatisation with MS
required • Difficulty in interfacing
with MS
• Limited commercial
libraries
(continued)
F.R. Pinu
Table 1 (continued)
Analytical Advantages Disadvantages Application in food safety and quality research
technique
Direct MS • High resolution depending on • Interference of salts and Ackerman et al. (2009), Self and Wu (2012), Shen et al. (2012), Farré
the mass analyser strong ions et al. (2013), Self (2013), Doué et al. (2015)
• Sensitive • Limited identification
• Rapid analysis power
• Poor reproducibility
NMR • Rapid analysis • Low sensitivity Spraul et al. (2009a, b), Balan et al. (2013), Osselaere et al. (2013),
• Non-destructive • More than one peak per Badilita et al. (2014), Lima et al. (2014), Qi et al. (2015), Ragone et al.
• Minimal sample preparation component (2015), Tomita et al. (2015), Wan et al. (2015)
• Quantitative • Identification is
laborious because of
complex matrix
GC-MS Gas Chromatography and Mass Spectrometry; LC-MS Liquid Chromatography and Mass Spectrometry; CE-MS Capillary Electrophoresis and Mass
Spectrometry and NMR Nuclear Magnetic Resonance
8 Metabolomics: Applications to Food Safety and Quality Research
239
240 F.R. Pinu

6 Application of Metabolomics in Food Safety and Quality

Research

Although the main aim of metabolomics is to generate hypotheses based on data

using an unbiased and non-targeted approach, both targeted and non-targeted
methodologies are frequently applied in food safety and quality research (Table 1).
It is especially true when metabolomics has been used for the analysis of chemical
contaminants including pesticides in different types of food materials (Forsberg
et al. 2011; Arroyo-Manzanares et al. 2015; Chatterjee et al. 2015). However, when
the main purpose of a study is to determine biomarkers either for microbial spoilage
or other sort of contamination, it is better to choose a non-targeted approach that
might help in finding novel compounds or metabolic pathways relevant to a food
system. The two main areas in food safety where metabolomics has been widely
applied are in determining either chemical or microbiological hazards present in a
food or food processing system. However, consumers these days are also concerned
about food prepared using GM ingredients, as the long-term effects of consumption
of such foods are still not adequately known; therefore, a growing research trend is
observed in this area.

6.1 Chemical Food Safety

A large number of chemicals have entered our food chain as a result of the wide
application of different growth-promoting agents (e.g. clenbuterol in pigs, recom-
binant growth hormones in fish and steroids in bovine animals), antibiotics and
pesticides that help us to produce large amounts of agricultural products. Therefore,
both food ingredients and the environment are facing the burden of chemical
exposures, many of which are unwanted and pose a threat to human health.
Moreover, deliberate contamination of food ingredients with unwanted chemicals
(e.g. melamine) also poses a serious risk to consumers. Therefore, these chemicals
should be banned or their use should be limited as set by the regulatory authorities.
To achieve this, development of high throughput methods that would enable study
of these contaminants is urgently needed. Both targeted and non-targeted meta-
bolomics demonstrate enormous potential in detecting and identifying these
chemicals in our food. For instance, targeted analysis using a suitable analytical
platform (e.g. NMR and MS) is beneficial for the study of already-known chemical
contaminants in food. However, it is more problematic when the contaminants are
either not known or are unknown breakdown products of a familiar compound. An
untargeted metabolomics approach could be a better way to study those novel
compounds and also to determine and validate candidate biomarkers that could be
used for tackling illegal practices in food production (Dervilly-Pinel et al. 2012).
Although NMR was initially the method of choice for detecting and identifying
chemical contaminants in food because of its capacity to quantify and elucidate the
8 Metabolomics: Applications to Food Safety and Quality Research 241

structure of molecules (Dieterle et al. 2011), MS is currently more popular due to its
sensitivity, and robustness of coupling with various separation systems (e.g. GC,
LC and CE). Moreover, tandem MS/MS or MSn experiments can now elucidate the
structure of any unknown metabolites, and thus are useful for the development of
multi-residue methods to detect and identify chemical contaminants accurately.
Many LC-MS-based methods have been published recently that have been used to
study chemical contaminants (Table 1) in different crops, including herbal teas,
nutraceuticals, rice and maize, and also in animals (Castro-Puyana and Herrero
2013). For instance, 255 veterinary drug residues and other chemical contaminants
were determined within 10 min using a UHPLC-MS/MS method (Zhan et al.
2012). In addition, high resolution MS/MS has been widely applied for the analysis
of pesticides, toxins and antibiotics. More than 500 pesticides were screened in
fruits and vegetables using an orbitrap tandem MS/MS method (Alder et al. 2011).
De Dominicis et al. (2012) also reported an orbitrap-MS/MS-based protocol for
analysing pesticides and toxins simultaneously in bakery and other foods. In
addition to analysing pesticides and other xenobiotic molecules, attempts have also
been made to develop methods using LC-MS to identify other contaminants in
food, including melamine in infant formula (Inoue et al. 2015). Many other
LC-MS/MS protocols have already been published that analysed a wide range of
food and fermented products including fruits, vegetables, wine, baby foods and
cereals (Lacina et al. 2012; Pérez-Ortega et al. 2012; Fan et al. 2014).
While LC has been the choice of separation technique for the analysis of con-
taminants in food materials, GC still remains the mostly used system coupled with
high resolution MS for the determination of environmental pollutants in food such
as polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs) or
polybrominated diphenyl ethers (PBDEs) in various samples including fruits,
vegetables, cereals, teas, fish muscle, dietary supplements and sheep milk
(Mastovska and Wylie 2012; Storelli et al. 2012; Banerjee et al. 2013; Cao et al.
2014; Duedahl-Olesen et al. 2015a, b; Walorczyk et al. 2015). For example,
Sapozhnikova and Lehotay (2013) accurately quantified over 80 pollutants (PCBs,
PAHs and pesticides) in fish within 9 min using a low pressure GC-MS/MS
method. In addition to tandem MS used with a GC system, two-dimensional GC
(GCxGC) analysis is also gaining popularity for the analysis of pollutants, which
allows determination of these compounds in very small amounts (0.1 µg/kg)
(Kalachova et al. 2012, 2013; Giri et al. 2015).
Some applications of food safety in terms of chemical contaminant determina-
tion also make use of direct MS analysis either as direct analysis in real time
(DART) or as desorption electrospray analysis (DESI). Rapid and high throughput
analysis of food samples can be obtained using DART-MS, as it directly analyses
samples from the surface, thus decreasing sample preparation time significantly
compared with those for the other coupled MS systems (Castro-Puyana et al. 2013).
DART-MS combined with high resolution analysers has been applied to analyse
pesticides in fruits and grains (Schurek et al. 2008; Farré et al. 2013). Similarly,
DESI-MS has been used for the determination of pesticides in fruit peels and
vegetables (García-Reyes et al. 2009; Zhang et al. 2009).
242 F.R. Pinu

6.2 Early Detection of Food Pathogens and Food Spoilage

Microorganisms

Food pathogens are one of the major threats for food safety, and large numbers of
people around the world suffer from diseases including diarrhoea, dysentery and
other forms of food poisoning that are directly caused by various food pathogens,
such as Salmonella spp., Shigella spp., Listeria monocytogenes, Campylobacter
jejuni and E. coli. Moreover, food spoilage microorganisms (e.g. Pseudomonas
spp., Acinetobacter spp., Botrytis spp.) are not necessarily pathogenic for people,
but can cause severe economic damage because of the spoilage of a wide range of
food materials. If there was a way to detect these pathogens or spoilage microbes in
the early stages of their growth in food products, it would be possible to reduce
dramatically the numbers of food-borne outbreaks and subsequent significant losses
of food by undertaking appropriate measurements to control their further growth.
Traditional identification and cultural methods for pathogens or food spoilage
microbes are time consuming, and therefore it is necessary to develop techniques
that would enable the rapid detection of microbes soon after the contamination
occurred in a food system (Xu et al. 2010). The metabolomics approach has already
shown huge potential for developing analytical methods to detect food pathogens in
their early stages of growth in a food product (Li et al. 2011; Beale et al. 2014). In
this approach, an experiment is performed using both a contaminated and a
non-contaminated food, and the metabolites from both are analysed. Multivariate or
discriminant analyses are performed to determine a group of potential candidate
biomarkers that can distinguish between the two conditions. Once validated, these
biomarkers can be used for the determination of pathogens in a real food sample, or
a simpler screening technique (e.g. enzymatic or colorimetric) can be developed to
be used routinely by food auditors.
GC-MS has been the choice of analytical instrument for determining pathogenic
growth in a food, as this technique is very efficient in determining the volatile
organic compounds (VOC) produced by microorganisms. Food spoilage microbes
generally produce many VOCs as part of their metabolism, and this causes alter-
ation in the sensory properties of the food (Li et al. 2011). VOC analysis is
attractive for food sampling and is also advantageous because of rapid and
non-invasive sample preparation. Once collected in the headspace using an
appropriate SPME cartridge, VOCs produced by spoilage or pathogenic microor-
ganisms are ready to be analysed by GC-MS. Xu et al. (2010) published a
VOC-based GC-MS metabolite profiling approach to determine the potential
biomarkers of spoilage of pork by S. typhimurium, and identified 16 metabolites
that clearly distinguished the naturally spoiled from S typhimurium-contaminated
pork meats. Moreover, Li et al. (2011) demonstrated the capability of metabolomics
in assigning biomarkers to the spoilage microbes, Botrytis allii and Burkholderia
cepacia, and they identified 16 volatile metabolites related to post-harvest onion
spoilage. In addition to volatile metabolites, other primary metabolites including
sugars and amino acids (dextrose, glycine, tyrosine and histidine) were also
8 Metabolomics: Applications to Food Safety and Quality Research 243

characterised as potential biomarkers that could be used for the early detection of
E. coli O157:H7 and of different strains of Salmonella in ground beef and chicken
(Cevallos-Cevallos et al. 2011).
Among other MS-based techniques, Matrix-assisted laser desorption/ionisation
coupled to time of flight-MS (MALDI-TOF-MS) also has been used to identify
different strains of pathogenic microbes in biological samples, including food and
beverages (Böhme et al. 2010, 2012; Picariello et al. 2012; Ojima-Kato et al. 2014;
Beale et al. 2014; Jadhav et al. 2015). MALDI-TOF-MS is very useful for iden-
tifying microbial strains using either whole cells or cell extracts and has been
applied to epidemiological studies, biological warfare agents, detection of antibiotic
resistance pathogens, detection of water- and food-borne pathogens and entero-
toxins (Tsilia et al. 2012). This technique is rapid, sensitive and inexpensive, and
thus well received by microbiologists (Singhal et al. 2015). Recently, rapid
detection and source tracking of L. monocytogenes was carried out using
MALDI-TOF-MS in Australian dairy products (Jadhav et al. 2015). Staphylococcus
aureus strain characterisation was also performed in Italian dairy products using
MALDI-TOF-MS fingerprinting (Böhme et al. 2012). However, the main drawback
of using this technique is the inability to identify new species, as the identification
process depends on the existing spectral database that contains the peptide mass
fingerprints of the type strains (Singhal et al. 2015).

6.3 Microbial Food Toxins

In contrast to chemical contaminants, food toxins that pose serious health hazards
for consumers are mainly produced naturally by different microorganisms (fungi,
algae and others) growing on food substrates or during the food preparation and
storage. Many of these toxins are produced as by-products of fungal metabolism
(mainly Penicillium, Aspergillus and Fusarium) in contaminated foods including
beer, wine, bread, rice and maize, and are thus known as mycotoxins. The deter-
mination of mycotoxins is very similar to that of chemical contaminants and mainly
carried out by multi-residue analysis (Giacometti et al. 2013; Gottfried and
Herebian 2013; Aniołowska and Steininger 2014; Pizzutti et al. 2014;
Rodríguez-Carrasco et al. 2015). In addition to mycotoxins, toxins produced by
some algae are also of interest for food safety. This type of toxin is usually found in
contaminated seafood and fish and may affect people, such as fish and different
types of shellfish poisoning (e.g. neurotoxic, paralytic, diarrhoeic and amnesic). It is
important to study algal toxins to know about their modes of action and toxic doses
in detail, to help to take appropriate precautions to prevent an outbreak. MS-based
metabolomics approaches have been developed to detect and quantify several
groups of algal toxins, such as ciguatoxins and domoic acid (Yogi et al. 2011;
Beach et al. 2014; Stewart and McLeod 2014).
244 F.R. Pinu

6.4 Study of GM Crops and Food Materials

GM or transgenic crops have gained popularity since the first commercial plantings
in 1996. GM technology has allowed us to produce crops and food materials with
enhanced nutritional properties, increased yield; and moreover, these crops can be
resistant to various pests and diseases, and adverse environments, including drought
and salinity (Parrott et al. 2010). A wide range of GM crops are now commercially
cultivated around the world, including soybean, maize, cotton, canola, potatoes and
tomatoes. Although the new traits in GM crops are mainly from the introduction of
small RNAs or some regulatory proteins, it is still unknown if even that small
amount of change could cause an overall alteration in other metabolic pathways,
and thus adverse modification of cellular downstream products (Delaney 2015). The
effects of growing transgenic plants on the environment have also been well doc-
umented (Liu et al. 2012; Brookes and Barfoot 2013; Knight et al. 2013), which has
in turn raised concerns about the long-term consumption of GM food materials on
human and animal health.
As the long-term effects of consuming GM food ingredients are still under
question, it is time to do more research on toxicological aspects that could provide
assurance to consumers about their safety. A few research projects have already
been carried out that addressed the effects of consumption of GM feed ingredients
by animal models. For instance, Sheng et al. (2014) evaluated the toxicity and
allergenicity of GM rice as expressed in human serum albumin in rats, and Qi et al.
(2015) assessed the safety of consumption of similar GM rice by rats using their
urine metabolome. Although both sets of authors found significant differences in
profiles between GM and non-GM rice-consuming rats, they concluded that GM
rice could be considered safe. However, both these studies were performed for a
limited period of time (only 90 days), and therefore it is still too soon to conclude if
long-term consumption (e.g. 5–10 years) of such GM rice by the rats would have
any adverse effects on their health.
Much research has been undertaken in last 10 years to acquire more knowledge
regarding the compositional differences between GM and non-GM crops and food
materials (Barros et al. 2010; Asiago et al. 2012; Cao et al. 2012; Liu et al. 2012;
Clarke et al. 2013; Kusano et al. 2014). This provides valuable insight into the
nutritional properties compared with those of conventional crops, and if any
unintended chemicals or proteins are present in that given crop that might pose a
threat to consumers. Metabolomics (both targeted and non-targeted), especially
metabolite profiling, is very useful in this regard, as it allows the generation of data
on the comprehensive composition of any GM crop (Rischer and
Oksman-Caldentey 2006). For example, Kusano et al. (2014) recently published a
study using metabolomics and ionomics approaches to determine the chemical
diversity of a soybean lineage representing 35 years of breeding. The authors used
different analytical platforms, including CE-TOF-MS, GC-TOF-MS and
LC-qTOF-MS, to determine the global metabolite profiles of soybeans, and found
that newer varieties are completely different from older ones. However, they found
8 Metabolomics: Applications to Food Safety and Quality Research 245

no significant differences in metabolite composition between conventional and GM

soybeans, thus indicating that GM soybeans could be safe as food and feed; their
findings were in accordance those of with Clarke et al. (2013). Similar studies have
been performed for other GM crops (e.g. maize and potato). Using a hierarchical
metabolomics approach, it has been shown that metabolite composition is very
similar between conventional and GM potato crops (Catchpole et al. 2005).
However, it is noteworthy that compositional differences also exist among con-
ventional crops, depending on their origin, environmental conditions, and genetics
(Reynolds et al. 2005; Harrigan et al. 2007). The use of ‘omics’ approaches,
including metabolomics, in determining the safety aspect of GM food materials still
has scope for improvement, and more research should be undertaken to highlight
the toxicological aspects in addition to the compositional studies.

6.5 Food Quality and Traceability

Both targeted and non-targeted metabolomics have been widely applied to determine
food quality by analysing any adulteration during food processing, or to confirm
authenticity of a food or beverage. Adulteration is usually performed deliberately to
gain more profit, using unwanted substances, such as the addition of melamine to
milk powder, synthetic dyes in spices, and the use of horsemeat in other meat
products (Senyuva et al. 2015). Some of these adulterations might cause serious
health issues (Chu and Wang 2013) and some of them are related to economic
adulterations to consumers (Everstine et al. 2013). However, in all cases, it is
essential to know the overall quality of the food materials and also to determine if the
food product is adulterated or not before it can be sold in the market. Authentication
of food is a useful step, and attempts have been made to determine potential markers
in different food matrices that can differentiate between adulterated and normal food.
For example, both targeted and untargeted metabolomics approaches using
LC-MS/MS have been used to authenticate Indian citrus fruits, and several
metabolites (didymin, rhoifolin, isorhoifolin, neohesperidin, hesperidin, naringin,
narirutin, limonin glucoside, and vicenin) were characterised as potential markers
(Jandrić et al. 2015). Arias et al. (2016) recently published a metabolomics study
using UHPLC–QTof-MS (ESI + mode) to distinguish the control (non-medicated
pigs) and pigs treated with ronidazole, dimetridazole and metronidazole where they
have identified at least four ionic features that could be used as potential biomarkers
of illegal 5-nitroimidazole abuse. GC-MS is another popular analytical platform that
has been successfully implemented to determine the quality of various food products.
For instance, GC-MS has been used to monitor the kimchi fermentation process (Park
et al. 2016). Moreover, fatty acid profiling using GC-MS has also been found to be
useful in determining adulteration in flaxseed oil (Sun et al. 2015). Recently,
Isotope-ratio MS (IRMS) along with either GC or LC has become another technique
that can be used for authentication of foods including wines, essential oils in man-
darin, and flavoured strawberry foods (Schipilliti et al. 2010; Guyon et al. 2011;
246 F.R. Pinu

Schipilliti et al. 2011). In addition to MS-based techniques, other spectroscopic

instruments and imaging systems have also been used successfully to authenticate
different foods. NMR-based metabolomics was applied to determine adulteration in
foods and beverages, including saffron and fruit juice (Balan et al. 2013; Ordoudi
et al. 2015). NMR metabolomics approach has also been successfully used to
determine the freshness of shucked mussels and a significant increase of many pri-
mary metabolites including acetate, lactate, succinate, alanine, branched chain amino
acids and trimethylamine was observed during storage of mussels between 0 and 4 °
C (Aru et al. 2016). Yang et al. (2016) recently reported the use of both NMR and
LC-MS-based metabolomics study of milk adulteration and they identified some
metabolites (choline and succinic acid) that can distinguish Holstein milk from that of
other cows. Moreover, the use of Raman imaging could also identify the adulteration
agents (e.g. melamine and dihydrocyanide) in powdered food (Qin et al. 2014).
One major area where much attention has been paid and which helps to prevent
adulteration is the determination of geographic origins of foods and beverages. In
many countries, food and condiment preparation is considered an art (e.g. wine-
making) and strong regulation already exists to ensure the quality of the product
before it can be marketed under protected designation of origin (PDO) or protected
geographic indication (PGI). This is especially true for many European food
products (balsamic vinegars, olive oils and cheeses). Traditional balsamic vinegars
are important product from Italy, produced in many regions; however, Modena is
famous for production of balsamic vinegars with PGI certification. GC-MS analysis
of volatile compounds was found to be very useful for distinguishing balsamic
vinegar samples with PGI (Chinnici et al. 2009; Cirlini et al. 2011). Extra virgin
olive oil is another product for which PDO status is very important. Many pro-
ducers follow fraudulent practices to obtain PDO for low-quality olive oils for
which consumers will pay comparatively higher prices. Pizarro et al. (2011)
identified volatile markers related to the geographic origin of Spanish extra virgin
olive oils using headspace-SPME-GC-MS analysis. GC-MS along with multivariate
data analysis has also been used to study the origin and quality of Italian buffalo
mozzarella cheese (Pisano et al. 2016). Moreover, different types of NMR
experiments (1H, 13C, 31P) have also been successfully employed for quality
assessment and authentication of olive oils (Dais and Hatzakis 2013).

7 Conclusion

Metabolomics has already been employed widely in food science, especially to

address issues of food safety and quality. Although recent analytical developments
allow us to analyse over 1000 metabolites in a single analysis (or using multiple
analytical instruments), a big challenge still exists in the handling of large data sets
(Skov et al. 2014). This is where all the ‘omics’ approaches currently have major
difficulties and, frequently, the huge amount of data generated cannot be
8 Metabolomics: Applications to Food Safety and Quality Research 247

appropriately interpreted biologically because of the inability to analyse the data.

Therefore, genuine efforts should be undertaken to improve the data analysis
platforms, to improve the overall quality of research in the metabolomics area. In
addition, ongoing technical improvement should lead to more cost-effective,
user-friendly and high throughput methods that could be used for the analysis of
various food matrices. Other aspects that need attention include the creation of more
food-based databases to make identification and discrimination of foods much
easier. It would be also easier to formulate regulatory actions to maintain the quality
and safety of foods and food products. We have already seen a tremendous
improvement in food safety and quality research in the last few years, and the
application of metabolomics will allow us to look at a holistic overview of the food
system, thus improving the overall objectives of ensuring food safety and quality.

Acknowledgements The author would like to thank Dr Damian Martin, Megan Jones and
Science and Publishing Office of Plant and Food Research Ltd for their support during the writing
of this manuscript.

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481
Chapter 9
Microbial Metabolomics in Biomass Waste
Management

Avinash V. Karpe, David J. Beale, Ian H. Harding

and Enzo A. Palombo

1 Introduction

Plant biomass has a complex structure that consists of cellulose and hemicellulose
surrounded by lignin (Fig. 9.1a) together with trace amounts of protein and fatty
acids (Eriksson et al. 1990; Hori et al. 1985; Sánchez 2009). Cellulose is the most
abundant and most studied polysaccharide and it forms the basic structure of cell
walls of all plants and algae. Cellulose is predominantly found within the primary
layer of the plant cell wall and its presence decreases in the secondary and tertiary
cell wall layers. It occurs as an aggregated, lateral, microfibril network that forms
highly complex mesh-like structures. To date, cellulose has only been partially
understood using advanced technologies such as nuclear magnetic resonance
(NMR) spectroscopy, X-ray crystallography and electron microscopy (O’Sullivan
1997; Fernandes et al. 2011; Harris et al. 2012).
Cellulose is an almost linear molecule composed of β-D-glucopyranose, linked
by β-1, 4-polyanhydroglucose, with cellobiose as the smallest repetitive unit
(Fig. 9.1b), and is thus a β-glucan (Kumar et al. 2008; O’Neill et al. 2004; Stone
1958). Hemicelluloses form the secondary structural component of the lignocel-
lulose complex (Fig. 9.1c). They are composed of hetero-structures of acelyted and
other derived monosaccharides, mostly pentoses. Various hemicelluloses have been
reported to be directly or indirectly associated with cellulose. They include sugars
such as xylans, xyloglucans, mannans, pectins, homogalacturonans, rhamnogalac-
turonans, arabinans and galactans.

A.V. Karpe (&)
I.H. Harding
E.A. Palombo

Department of Chemistry and Biotechnology, Swinburne University of Technology,
PO Box 218, Hawthorn, Melbourne, VIC 3122, Australia
e-mail: [email protected]
A.V. Karpe
D.J. Beale
Land and Water, Commonwealth Scientific and Industrial Research Organization
(CSIRO), PO Box 2583, Brisbane, QLD 4002, Australia

© Springer International Publishing Switzerland 2016 261

D.J. Beale et al. (eds.), Microbial Metabolomics,
DOI 10.1007/978-3-319-46326-1_9
262 A.V. Karpe et al.

Fig. 9.1 a–d Complex structure of Plant biomass. a Schematic characterization of plant biomass
structure comprising lignocellulose complex. b Fragment of cellulose with the reducing,
non-reducing and cellobiose component of cellulose. c Representative example of hemicellulose
(β-glucans). d Structures of (i) Hydroxycinnamyl alcohols-p-coumaryl alcohol, coniferyl alcohol
and sinapyl alcohol and (ii) derived structural units H, G and S
9 Microbial Metabolomics in Biomass Waste Management 263

Lignin is the second most abundant biopolymer. In most plants, especially in

higher plants, it functions as the prominent component of xylem. Due to its
hydrophobic nature, lignin aids in water transport throughout the plant system. This
property also makes lignin tolerant towards biodegradation (Boerjan et al. 2003).
Lignin biosynthesis and deposition occurs in the secondary cell wall after the cell
has completed its growth. During primary growth, cellulose and hemicellulose are
deposited on the cell wall, followed by sequential deposition of lignin in the sec-
ondary walls. However, most lignin deposition takes place in the next stage, which
is followed by cell death (Baucher et al. 1998). Lignins are made up of hydrox-
ycinnamyl alcohols, i.e, p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol
(Fig. 9.1d) and their methoxy derivatives (Boerjan et al. 2003; Higuchi 2006;
Vanholme et al. 2010). The main methoxy products of these three hydroxy cin-
namyl alcohols, which form the structural units of lignin polymers, are known as
general p-hydroxyphenyl units (H), general guaiacyl units (G) and general syringyl
unit (S) (Baucher et al. 1998; Boerjan et al. 2003; Higuchi 2006; Ralph et al. 2004;
Vanholme et al. 2010). However, due to the very complex nature of the lignin
polymer, they have not been successfully isolated and characterised from natural
sources (Vanholme et al. 2010). Vascular plants have greater amounts of G and S
units, while grasses and other monocots are rich in H units (Baucher et al. 1998;
Boerjan et al. 2003; Yang et al. 2010a).
The complex structure and sheer volume of biomass makes it a major waste
management challenge. According to recent estimates from the “Food and
Agriculture Organisation of the United Nations”, the global agricultural output
during the year 2011 was 5.6 × 109 metric tonnes (FAO 2015). A majority of this
agricultural production was from cereals such as wheat, rice, barley, sorghum and
maize among others, comprising up to about 4.3 × 109 metric tonnes. During the
same year, biomass waste generation from all agricultural activities was estimated
at about 1.06 × 109 metric tonnes. It has long been observed that vast quantities of
biomass generated from agricultural activities is burned (ca. 7.43 × 108 metric
tonnes in 2012) (FAO 2015).

1.1 Biomass Waste Management

Biomass waste management comprises various approaches including the conver-

sion of biomass to commercially/industrially useful products. The process involves
pre-treatment designed to break several linkages and bonds between cellulose,
hemicellulose and lignin; thus exposing considerable amounts of hemicellulose and
cellulose to subsequent enzyme/microbial based degradation (Sarkar et al. 2012).
There are several physical, chemical and biological methods of biomass
pre-treatment, which are used according to the biomass type, source and type of
264 A.V. Karpe et al.

product recovery required. No single pre-treatment by itself is adequate for com-

plete biomass conversion. However, a mixture of two or more pre-treatments (e.g.,
physical–chemical, physical–biological and chemical–biological) has been
observed to generate higher biodegradation conversion in a range of biomass
matrices (Khuong et al. 2014; Kovacs et al. 2009; Lu et al. 2013; Maeda et al. 2011;
Pribowo et al. 2012).
Physical treatment methods are generally applied to increase the overall surface
area of biomass, thus exposing higher amounts of cellulose and hemicellulose
residues. This leads to a decrease in recalcitrance, enabling subsequent chemical,
enzymatic or microbial treatment to be more effective due to the availability of a
higher proportion of the substrate. There are various types of physical
pre-treatments reported and they can be broadly categorised as milling (Karimi et al.
2013), irradiation (Budarin et al. 2010; Ma et al. 2014) sonication (Karimi et al.
2013; Rehman et al. 2013), extrusion (de Melo et al. 2014) and hydrothermal
treatment (Karpe et al. 2014).
Physicochemical methods use a combination of physical disruption and chem-
ical treatment techniques to decompose/degrade the lignocellulose complex.
Standalone physical methods such as milling and pyrolysis require high amounts of
input energy to break down the lignocellulose complex sufficiently for degradation
to occur. To achieve enhanced degradation, physicochemical techniques utilise
physical disruption attributes (temperature and pressure) along with the chemical
(aqueous, acids and alkaline) treatments to either separate biomass components or
alter the recalcitrant structure to make it more degradable. Hydrothermal treatment
(HT) (Ando et al. 2000; Goto et al. 2004; Karpe et al. 2014), steam explosion
(SE) (Duarte et al. 2012; Shevchenko et al. 2000) and ammonia fibre explosion
(AFEX) (Sarkar et al. 2012) are some of the most common physicochemical
methods used at laboratory and industrial scales.
In a steam explosion (SE) experiment where 3 % H2SO4 was applied at 120 °C,
the release of about 14–36 % pentoses and 18–27 % hexoses (Shevchenko et al.
2000) and up to 90 % hemicellulose removal was reported (Karimi et al. 2013).
Similarly, an application of 2 % NaOH in combination with ultrasonic treatment for
20 min at 50 °C has been reported to remove about 81 % hemicellulose and 91 %
lignin from sugarcane bagasse. Follow-up bacterial treatment with Cellulomonas
flavigena (MTCC 7450) resulted in about 91 % glucose yield (Velmurugan and
Muthukumar 2012).
Organosolvent treatments use organic solvents in either standalone (e.g. 100 %
methanol) mode or in combination (e.g. methanol: acetonitrile: acetone in a v/v ratio
of 40:20:40) to degrade biomass. A Pinus radiata substrate treated with an
acetone-water mixture at 195 °C for 5 min resulted in an ethanol yield of
approximately 99.5 %. The disadvantages of this treatment method are its high
costs and volatility of non-polar molecules which are flammable and explosive at
elevated treatment temperatures (Haghighi et al. 2013).
9 Microbial Metabolomics in Biomass Waste Management 265

Biological pre-treatments rely on microbial cells such as bacteria and fungi to

degrade biomass. Fungi such as Trichoderma spp., Aspergillus spp., Phanerochaete
spp., Trametes spp., Phlebia spp., Postia spp. and Laetiporus spp. are widely used
in biomass treatment (Alvira et al. 2010; Karpe et al. 2015a; Liu et al. 2012; Zuroff
et al. 2012). The process is cost-effective compared to chemical and physical
pre-treatments, however the overall effectiveness of standalone biological
pre-treatment is still low without physical or chemical pre-treatments. In combi-
nation, the pre-treatment process of milling and enzyme (extracted from
Acremonium spp.) has been found to be highly effective and has reported to yield as
much as 90 % hexose and 77 % pentose degradation (Sarkar et al. 2012). Khuong
et al. (2014) reported that the subsequent applications of 5 % NaOH and the fungus
Phlebia sp. MG-60 resulted in an ethanol yield of approximately 66 %.
Thermophilic bacteria such as Clostridium thermocellum, C. thermohydrosulfu-
ricum and Thermoanaerobacter ethanolicus have high rates of bioethanol produc-
tion with an increased tolerance towards ethanol of up to 5 % (Georgieva et al. 2007)
and 8 %, respectively (Rani and Seenayya 1999). Additionally, in an independent
experiment, it was observed that a combination of T. brockii β-glucosidase and C.
thermocellum cellulosome in ammonia soaked rice straw for 7 days at 60 °C
resulted in the conversion of about 91 % glucan to glucose (Waeonukul et al. 2012).

1.2 Biomass Degradation/Conversion Methods

Submerged fermentation/shake flask fermentation (SmF) is an aqueous phase fer-

mentation process where the medium-to-substrate ratio is generally high (in excess
of 10:1). The technique is well-established for wine and related alcohol production,
and has been industrially applied in Western countries since the late 1940s for
enhanced penicillin production from Penicillium chrysogenum. The basic methods
have now been greatly optimised due to the requirement for scaling up (>3000–
5000 litres) for agricultural use. Such optimization includes supplementation of
oxygen, heat exchange, agitation and foaming and culture load prevention
(Humphrey 1998). However, even with such a long history, and with this more
recent optimization, SmF methods are not fully adequate for either enhanced pro-
duction of lignocellulolytic enzyme or effective degradation of the lignocellulose
complex—as evidenced in several experimental reports (Hideno et al. 2011; Juhász
et al. 2005; Merino et al. 2007).
Solid State Fermentation (SSF) is a microbial bioprocessing method conducted
at very low levels of free water (Hölker and Lenz, 2005a). The technique has been
traditionally used for a long time in bread making, and for at least 3000 years for
food processing in Asian countries. For example, the Koji process for food fer-
mentation utilises Aspergillus oryzae. Production of sake is another example of SSF
and utilises Trichoderma spp. Both of these applications are well-known in
Japanese and Chinese culture. In Europe, SSF is also used to make traditional
French blue cheese (Couto and Sanromán 2006; Hölker and Lenz 2005b).
266 A.V. Karpe et al.

More recently, SSF has been developed for a number of industrial bioprocesses
such as biomass conversion (Bak et al. 2009; Brethauer and Studer 2014; Cheng
and Liu 2012). Degradation and bioconversion of the lignocellulose complex from
various biomass sources has increased the production of industrial metabolites,
biofuels and secondary metabolites which can be used, for example, as medicinal
compounds. The use of biomass addresses issues related to managing the consid-
erable amounts of waste material generated during numerous agricultural and allied
processes. SSF employs a mixture of different organisms and/or enzymes in a single
step to generate considerably higher bioconversion of biomass as compared to SmF
methodologies (Brijwani et al. 2010; Kausar et al. 2010; Lee 1997; Sarkar et al.
2012).
Recently, Cheng and Liu (2012) have shown the effects of pre-treatment in
hydrogen production from milled cornstalk. SSF meditated by T. reesei Rut-30 was
applied to this substrate, followed by sludge seeding at 35 and 55 °C. It was
observed that within 4 days of seeding, the 6-day pre-treated substrate generated
about 200 mL H2 when incubated at 55 °C. Additionally, considerable amounts of
low molecular weight fatty acids and ethanol were produced. In a similar fashion,
rice straw pre-treated with Phanerochaete chrysosporium displayed considerable
lignin peroxidase (LiP) and manganese peroxidase (MnP) activities. It was
observed that after 30 days of pre-treatment, these enzymes had degraded about
33 % of the lignin. Follow-up enzymatic degradation resulted in the degradation of
about 17 % of the glucan and 3 % of the xylan (Bak et al. 2009).
Consolidated bioprocessing (CBP) (Fig. 9.2) is a relatively new methodology
for improving biomass conversion to products of commercial interest (Brethauer

Fig. 9.2 A conceptual design of a multi-species biofilm membrane (MBM) reactor for CBP to
generate ethanol from pre-treated wheat straw. Diagram taken from Brethauer and Studer 2014;
published by The Royal Society of Chemistry (Wiethölter et al. 2003) on behalf of the Centre
National de la Recherche Scientifique (CNRS) and the RSC
9 Microbial Metabolomics in Biomass Waste Management 267

and Studer 2014; Lynd et al. 2005). CBP combines the individual processes of
biomass hydrolysis and subsequent fermentation to generate products such as
ethanol in a single step. It eliminates the time-consuming separate biological
pre-treatment process. It also eliminates the need for separate fermentations of
different sugars, such as tetroses or pentoses, which cannot be fermented by general
industrial fermenters such as Saccharomyces cerevisiae (Lynd et al. 2005).
CBP relies on the development of cellulolytic organisms via strategies such as
metabolic engineering or genetic engineering of native species. The first strategy
has been reported to generate about 0.47 g ethanol/g hexose using the thermophilic
bacterium, Geobacillus thermoglucosidasius. Clostridium thermocellum and C.
cellulolyticum, two thermophilic biomass degrading bacteria, have been reported to
generate about 50 g/L ethanol on pre-treated cellulose. The second strategy
involves recombinant DNA technology to mediate heterologous expression of
cellulolytic enzymes in naturally occurring biomass degrading microorganisms.
These strategies have been successful at the laboratory scale; however, success
under industrial conditions has not yet been reported (Olson et al. 2012). In a fungal
(mushroom)-based CBP experiment, Kamei et al. (2014) described the use of spent
sawdust waste from Lentinula edodes cultivation as a substrate for white-rot fungus,
Phlebia spp. MG-60. They observed about 45 % ethanol production from the
mushroom sawdust waste in about 400 h due to increased saccharification.
Symbiotic fermentation may be carried out as part of CBP. The strategy is to
develop a symbiotic consortium of different microorganisms, or their lignocellu-
lolytic enzymes, in either a single batch or continuous batch fermentation. It has
been shown previously that fungi such as Trichoderma spp. and Aspergillus
spp. have the ability to co-culture during cellulose degradation (Brijwani et al.
2010; Gupte and Madamwar 1997). A similar approach has been investigated in
lignocellulose degradation by termites (Brune 2014) and lignin degradation by
co-culture of wood-rot fungi (Qi-he et al. 2011). Mixed microbiota taken from
biogas digested sludge inoculated onto wheat stalk was able to produce 37 mL/g of
H2 after 200 h of incubation. In addition, the concentration of volatile fatty acids,
such as acetate, propionate and butyrate, also increased considerably. Furthermore,
about 75 % of the cellulose was solubilized during this process (Chu et al. 2011).
Additionally, Cheng and Liu (2012) reported that corn stalk substrate pre-treated
with T. reesei RUT C30 by SSF and then seeded with winery sludge was able to
generate H2 gas up to 200 mL within 6 days.
An experiment displaying the synergistic properties of cellulolytic enzymes was
recently reported. Crude, filtered and dialysed fungal enzyme samples obtained
from SSF were mixed to an already operating SmF biodegradation sample. The
resulting symbiosis of Trichoderma and Aspergillus enzymes caused degradation of
cellulose and hemicellulose, as evidenced from a conversion of up to 64 % biomass
to reducing sugars. Under anaerobic conditions, this enzyme mix in combination
with S. cerevisiae was able to generate up to 43 g/L ethanol (about 84 % of the
theoretical maximum) within 7 h (Pirota et al. 2014).
268 A.V. Karpe et al.

2 Microbial Process Metabolomics and Its Application

to Biomass

The study of metabolomics has the potential to provide biochemical information

related to fungal biomass degradation that increases our understanding of the
microbial processes and characterises the various mechanisms utilised. Previously,
metabolomic techniques have been applied to investigate bacterial processes related
to preventative health (Bi et al. 2013; Marcinowska et al. 2011), environmental
pollution (Beale et al. 2013), food (Beale et al. 2014) and fungal metabolism on
various substrates including benzoic acid and Chardonnay grape berries (Hong
et al. 2012; Matsuzaki et al. 2008). However, within the context of fungal-mediated
biomass degradation, its application has been limited compared to other sectors.
Analysis of the metabolic flux, however, has been shown to have the capability to
enable a good understanding of the nature, time-dependence and substrate-based
limitations in regard to the metabolism of fungal cells. Metabolomics also has the
potential to assist in extricating the correlation between cell phenotypes and their
metabolic patterns and stoichiometry (Meijer et al. 2009). Metabolic flux studies
have previously been applied in toxicology and medicine (Maier et al. 2009; Niklas
et al. 2010; Saunders et al. 2015), plant proteomes (Nelson et al. 2014) and
microbial respiratory systems (Driouch et al. 2012; Pedersen et al. 1999). Numerous
methods are available that monitor the flux (change) of metabolite
production/consumption over time (i.e. metabolic flux analysis). The majority of
these methods involve the use of heavy isotopes due to their superior tracking
abilities. In commonly utilised analyses, such as nuclear NMR and gas
chromatography-mass spectrometry (GC-MS), metabolite tracking is performed
using isotopes such as 1H, 2H, 13C, 14N, 15N and 18O. While each isotope has its
advantages and shortcomings, 13C has been used by most researchers (Nelson et al.
2014), probably because of its wide-spread use in general chemistry. 2H is a much
more abundant element in nature, comprising about half of the peptide atomic
population (Yang et al. 2010b). It is much more economical compared to other
isotopes, has a comparatively higher invasive rate and a slower decay rate (Kim
et al. 2012). Complete 2H labelling is not feasible, however, due to the limited
tolerance of multicellular organisms, thereby resulting in partial labelling, ranging
from 8 to 30 % (Yang et al. 2010b; Kim et al. 2012).

2.1 Classification of Metabolomic Approaches

Various fungi such as those from the divisions of Ascomycota and Basidiomycota
have been reported as effective biomass degraders. They generate an array of
enzymes including endo- and exo-glucanases, β-glucosidase, xylanases, arabino-
furanosidases and pectinases (Brink and Vries 2011; González-Centeno et al.
2010). The resultant degradation caused by these enzymes generates useful
9 Microbial Metabolomics in Biomass Waste Management 269

industrial and medicinal biomolecules such as ethanol, flavonoids, phenolic com-

pounds, anthocyanins and hydroxybenzoic acid (Arvanitoyannis et al. 2006;
Sánchez 2009; Strong and Burgess 2008). Additionally, fungi such as Penicillium
spp. and Phanerochaete spp. can be used for lignin mineralization during the
degradation process (Arora and Sharma 2010; Rodríguez et al. 1994).
The application of metabolomics in environmental and non-clinical areas is still
in a nascent stage as compared to other ‘omic’ based approaches such as genomics,
transcriptomics and proteomics. Unlike the other approaches, metabolomics deals
with the complete metabolic behaviour of an organism by profiling the substrates
and their metabolic products (e.g. saccharides, lipids, amino acids, organic acids,
vitamins and phenolics) thereby providing a direct functional output of the bio-
chemical behavioural pattern of the living system under the study (Fiehn 2002).
Largely, due to this, in recent years, metabolomics has arguably been the most
rapidly emerging field in biosciences (Roessner and Bowne 2009). However, due to
the highly complex physiology, biochemistry and behaviour of fungal (or eukary-
otic) cells, metabolomic studies of these organisms has presented considerable
difficulties as compared to prokaryotic systems (Niklas et al. 2010). The metabolic
behaviour of the fungus, therefore, needs to be elucidated by multiple approaches in
either an individual or combined fashion. These approaches include untargeted and
targeted metabolic profiling, metabolic fingerprinting and metabolic flux analysis.

2.2 Metabolic Profiling: Targeted

and Untargeted Approaches

Metabolic profiling involves qualitative and quantitative analysis of the complete

range of metabolites involved in an organism’s metabolism (Roessner and Bowne
2009). This approach, therefore, may be considered as a precursor to either meta-
bolic fingerprinting or metabolic flux analysis (Karpe et al. 2015b; Roessner and
Bowne 2009). Because of its wide scope of application, metabolic profiling has
been widely used in the study of environmental (Beale et al. 2013; Jones et al. 2014,
2015), bioprocess (Zha et al. 2014; Karpe et al. 2015c) and other industrial
microbiology and treatment sectors (Ding et al. 2012; Matsuzaki et al. 2008; Beale
et al. 2016). Besides these areas, it has also been used in clinical research (Ng et al.
2012; Kim et al. 2012; Kouremenos et al. 2010), food processing (Beale et al. 2014)
and plant–microbial interactions (both parasitic and commensal) (Draper et al.
2011; Hong et al. 2012). Overall, the field of metabolic profiling is vast; however,
this chapter will only be considering the metabolic profile of bioprocessing,
specifically related to fungal/bacterial biomass conversions.
Microbial biomass processing is an emerging domain application with respect to
most other fields in which metabolic profiling approaches have been used. One of
the earlier reports that considered the potential of metabolomics was the perspective
article by Dixon et al. (2006) detailing the application of metabolomics approaches
270 A.V. Karpe et al.

in agriculture. While the Dixon et al. report considered issues such as data acqui-
sition, data accuracy and a number of gaps in metabolic pathway information, it is
apparent that the same issues have relevance in biomass related metabolomics. For
example, in biomass metabolomics, the analyst has to deal with differential con-
centrations of larger substrates such as oligo- and polysaccharides, peptones and
smaller, but varied, metabolites such as sugars, amino acids, fatty acids and aro-
matics. One of the reported studies considering an untargeted metabolomic
approach in biomass degradation was the rumen bacterial mediated degradation of
birch wood xylan and apple pectin (Villas-Bôas et al. 2006). The bacterial species
Clostridium proteoclasticum, Butyrivibrio fibrisolvens and Streptococcus bovis
were used. The biomass degradation quantified by GC-MS indicated the presence
of about 40 different sugars, sugar acids, fatty acids and acids of lignin and tannin
degradation including gallic acid, coumaric acid and quinic acid (among others). In
our research, winery biomass was degraded using ascomycete fungi such as T.
harzianum, A. niger, P. chrysogenum, P. citrinum and S. cerevisiae (as a negative
control). An untargeted approach using GC-MS based metabolomic analysis was
used which yielded 233 metabolite peaks. These consisted of biomass degraded
mono- and oligosaccharides, sugar alcohols, sugar acids, fatty acids, amino acids,
organic acids and aromatic acids, although the major metabolites present in the
biomass degrading fungal profile were pentoses, hexoses and to a lesser degree fatty
acids and amino acids. The results were in agreement with considerable enzyme
activities of cellulases, xylanases and β-glucosidase in the biomass degrading fungi
(Karpe et al. 2015c). In further research, the winery biomass was degraded by a
symbiotic mixture of all the above mentioned fungi, except S. cerevisiae; and the
degradation level increased considerably, indicated primarily by the higher enzyme
activities, especially xylanase. The enhanced enzyme activity affected overall
metabolic output where the higher metabolite concentration consisted of fatty acids
such as oleanolic acid, glyceric acid, heptadecanoic acid and tetradecanoic acid.
Additionally, the mixed fungal degradation was also able to yield some industrially
useful secondary metabolites such as gallic acid, inositol, β-sitosterol and mannitol
in higher concentrations (Karpe et al. 2015a).
By comparison to the above untargeted approach, targeted metabolomics deals
with quantitative analysis of a limited number of the metabolites which represent
the complete metabolome output of an organism. On a superficial level, targeted
metabolomics resembles metabolic fingerprinting, however they are significantly
different. While, the fingerprinting approach classifies different metabolic (func-
tional) groups and identifies signature metabolic patterns to each organism (quali-
tatively or quantitatively), the targeted approach selectively screens any
non-targeted metabolites and quantifies only targeted metabolites. The presence
of targeted metabolites or metabolic groups in an organism’s metabolome is
therefore a prerequisite for targeted metabolomics (Shulaev 2006). The quantitative
approach combined with selectivity increases accuracy and sensitivity of the
analysis (for those specific metabolites). The main issues associated with wider use
of a targeted approach are the current incomplete knowledge of organism specific
metabolic pathways, the identification of intermediate metabolites, and their
9 Microbial Metabolomics in Biomass Waste Management 271

structural elucidation and availability of metabolites in pure form that can be used
as a reference standard and, in general, an increased level of complexity. A targeted
approach, therefore, has been used less compared to both non-targeted and fin-
gerprinting metabolomic approaches. It has been argued that targeted approaches
are largely limited to either genetic behavioural studies or clinical applications, as
indicated in a recent review by Putri et al. (2013).
In spite of these limitations, targeted metabolomics methods are becoming more
prominent in studies that encompass multiple ‘omics’ approaches. One recent study
on targeted metabolomics involved the quantification of sugars, sugar phosphates,
organic and amino acids within the S. cerevisiae metabolome (Kato et al. 2012). On
a GC-MS system, around 99 metabolites were targeted using mass-to-charge (m/z)
channel based screening of a mass spectrum range of 89–299 m/z units. Based on
the data collected, the samples were then quantitatively re-analysed on an
LC-QQQ-MS. The combined approach was able to detect metabolites concentra-
tions of as low as to 1.4 femtomole in the yeast metabolome to a very high accuracy
(R2 = 99.94 %) (Kato et al. 2012). Another reported work involved the quantifi-
cation of the Clostridium thermocellum metabolic profile. The thermophilic bac-
terium was grown on cellobiose and its metabolic output was analysed by
LC-QQQ-MS/MS (using 13C-isotopes). About 45 metabolites including amino
acids and organic acids were quantified, some present in concentrations as low as
0.8 ng/mL of detection limit and 2.7 ng/mL of quantification level (Cui et al. 2013).
The targeted approach has also been applied in the assessment of metabolites
that may be responsible for development of fungal tolerance in horticulture crops.
A good example is the polyphenol assessment of wine-grape varieties displaying
tolerance towards powdery and downy mildew; the fungal infections are caused by
Uncinula necator and Plasmopara viticola, respectively. The analysis of secondary
metabolites belonging to phenolic acids, dihydrochalcones, stilbenes, flavanols and
flavanols was performed by the combination of two different UPLC-QQQ-MS
systems. About 55 metabolites including gallic and coumaric acids, resveratrol,
procyanidine, cathechins and naringenin were quantified with results ranging from
30 ng/mL to 203 μg/mL (Ehrhardt et al. 2014).
An interesting application of combined (targeted and non-targeted) metabolomic
approaches was recently demonstrated by Godelmann et al. (2013). This group
performed untargeted and targeted metabolic profiling using GC-TOF-MS and
1
H-NMR, respectively, to differentiate the grape variety, its vintage and geo-
graphical distribution. Specifically, 16 metabolites were quantified which included
organic acids such as lactate, citrate, malate, succinate, acetate, fumarate and tar-
trate; alcohols such as methanol, ethanol, 3-methylbutanedioland glycerol; and
secondary metabolites such as shikimate, caftarate and acetone. The concentrations
ranged from 79.3 mg/L for shikimate to 120.2 g/L for ethanol. Chemometric sta-
tistical analysis was able to classify the geographic regions and vintage year to
about 90 and 97 % accuracy, respectively. The experiment provides not only
quality parameters for wine grapes, but also indicates the potential of applying
specific fungal/bacterial species to extract targeted metabolites on a commercial
scale from those substrates.
272 A.V. Karpe et al.

Fig. 9.3 The general structure of chief antibacterial metabolites secreted by P. communes fungus.
The structures represent a Lovastatin and b 3-isobutyl hexahydropyrrolo[1, 2-a]pyrazine-1,
4-dione (α-Ketoisovaleryl-Leu-Pro lactam)

A metabolomics approach can also be applied to prevent or minimise process

inhibition, either competitive or product, as shown in recent research using
GC-MS-based analysis of yeast (Zha et al. 2014). It was observed that biomass
pre-treatment products such as hydroxymethyl furfural (HMF) and furfural pro-
longed the log-phase of yeast growth, thereby causing considerable growth inhi-
bition. Similarly, other metabolites such as aldehydes, phenolics, vanillin and sorbic
acid negatively affected the growth rate. S. cerevisiae was found to counter the
effects of HMF and furfural by converting these molecules to their respective acids.
It was also noted that amino acids, when initially present in considerable amounts,
compensated for the inhibitory and toxic effects of various molecules during fer-
mentation (Zha et al. 2014).
In related research, the satins and anti-pathogenic metabolites from environ-
mental Penicillium commune were analysed by a combination of analytical tech-
niques such as thin layer chromatography (TLC), Fourier transformed infrared
spectroscopy (FTIR), direct insertion probe/electron ionisation/ion trap detection
(DIP/EI/ITD) mass spectrometry and GC-MS. The multiple techniques identified
lovastatin (Fig. 9.3a) and 3-isobutyl hexahydropyrrolo[1, 2-a]pyrazine-1, 4-dione
(or α-Ketoisovaleryl-Leu-Pro lactam) (Fig. 9.3b) (Diblasi et al. 2015). These
metabolites or the fractions containing these metabolites were shown to be effective
antibacterial agents; the lovastatin containing fraction was shown to be effective
against Pseudomonas aeruginosa and the fraction containing 3-isobutyl hexahy-
dropyrrolo[1, 2-a]pyrazine-1, 4-dione was observed to be inhibitory towards
Staphylococcus aureus.

2.3 Metabolic Fingerprinting

Metabolic fingerprinting can be defined as the quantification of selective metabo-

lites from a larger metabolic output of an organism at a particular given period and
grouping them together for potential further assessment. As this approach considers
9 Microbial Metabolomics in Biomass Waste Management 273

the measurement of a subset of metabolites and is focused on profiling metabolites

associated with the organism(s) under experimental conditions, it provides more
accurate tracking of metabolic behaviour of the organism relative to its surround-
ings (Kosmides et al. 2013). Currently, most of the methods used for metabolic
fingerprinting are NMR based, with some studies using GC-MS and LC-MS
(Roessner and Bowne 2009).
As previously stated, there has been limited metabolomics-based research related
to biomass conversion compared to clinical applications. Nevertheless, a number of
useful studies, especially in the area of metabolic fingerprinting, have been recently
described. For example, the supramolecular effects of pre-treatment and successive
microbial degradation of rice straw using NMR were described by Ogura et al.
(2013). This biomass was pre-treated by ZrO2 ball milling for 6 h followed by
metabolic analysis using 2D 13C-1H heteronuclear correlation spectra (HETCOR) in
solid-state NMR and thermogravimetric analyses. The pre-treated biomass was
supplemented with paddy soil and the products were analysed by 1H-NMR spec-
troscopy. The presence of syringyl, guaiacyl, hydroxyphenyl and coumarate
derivatives suggest considerable lignin linkage breakdown. Similarly, sugars and
sugar acids resulting from degradation of cellulose and hemicellulose were detec-
ted. Deformation of cellulose was noticed from the shift in position of the C4 peak
(NMR spectroscopy) of crystalline cellulose and the increasing intensity of amor-
phous cellulose. With respect to other pre-treatments, the activation energy of ball
milling reduced considerably from 166 to 96 kJ/mol. This suggests an increase in
amorphous cellulose. Successive microbial degradation of this biomass resulted in
further generation of pentoses, hexoses and disaccharides (from 3 to 4 ppm) and
short chain fatty acids (SCFAs), especially butyrate (up to 2.2 ppm). Principal
component analysis (PCA) of the data suggested the termination of degradation at
21 days (Ogura et al. 2013). In an extension to this experiment, bacterial cellulose
generated by Gluconacetobacter xylinus was degraded by anaerobic sludge and the
products were analysed by solid state-, solution state- and gas state- NMR. While
solid-state NMR showed decreasing crystallanity of cellulose over 84 h,
solution-state NMR indicated that the major metabolite output consisted of SCFAs
and ethanol. These were generated during the early phase of fermentation and most
were re-utilised after 60 h of fermentation. Similarly, CO2 and CH4 were generated
by oxidation and reduction of acetate, as detected by gas-state NMR (Yamazawa
et al. 2013).
Another method recently used for metabolic fingerprinting was FTIR. The
experiment applying this method of metabolome discrimination used various rye
grass varieties of Lolium perenne as animal feed. Metabolites generated in rumen
over 24 h were periodically analysed by FTIR to build up a preliminary metabolite
fingerprint for the fermentation process of each grass variety. The major metabolic
groups discriminated by FTIR based multivariate analyses indicated the presence of
numerous sugars, amides, fatty acids and alkanes in cow rumen. Production of
ammonia and volatile fatty acids (VFAs) was the primary indicator of biomass
fermentation. VFAs such as acetate, propionate, butyrate, pentanoate, hexanoate
and heptanoate were observed at different concentrations at various sampling times.
274 A.V. Karpe et al.

Although the FTIR approach was unable to evaluate individual metabolites, it could
separate the overall metabolite spectra into several groups providing a defined
analysis pathway. The authors suggested using a more specific metabolic flux
analysis to obtain a specific fingerprint related to fermentation of rye grass biomass
by rumen microbiota. The study also indicated the likelihood of increasing protein
utilisation by increasing the availability of soluble carbohydrates to rumen micro-
biota. The authors also indicated the use of FTIR as a preliminary
non-discriminatory technique to establish a rapid understanding of metabolite
interactions, which can then be studied in detail by a secondary flux analysis
(Kingston-Smith et al. 2013). The study, albeit a preliminary investigation, indi-
cated that the metabolic fingerprint and flux pattern in the fermentation of plant
biomass by rumen microbiota were specific to the type of plant source.
Another widely used method for metabolite fingerprinting is liquid chro-
matography with electrospray ionisation-mass spectrometry (LC-MS). Due to
protonation/de-protonation during ionic adduct formation, the analyte is observed
as a molecular ion. The mass-to-charge ratio (m/z) calculation by ESI is then used
to predict the putative metabolite in numerous types of samples (Draper et al. 2013).
Secretome studies of Trichoderma hamatum GD12 in relation with Arabidopsis in
the presence of other fungi showed the presence of cellulose binding-motif (CBM).
Further analysis by combined 1H-NMR and direct infusion-ESI-MS (DI-ESI-MS)
showed the presence of 30 metabolites of at least fivefold change, based on
unbiased positive and negative ionisation mode discrimination (Studholme et al.
2013). Similarly, in a recent study of Aspergillus nidulans mediated degradation of
Quercus suber cork, the secretome analysis showed ca. 28 active protein species, of
which ca. 12 % comprised plant cell wall degradation proteins. These were further
classified as β-glucosidases, arabinosidase, alcohol oxidase and oxidoreductases.
The metabolic output of this secretome was analysed by ultra-high-performance
liquid chromatography coupled to electrospray ionisation-high resolution mass
spectrometry (UHPLC-ESI-HRMS). The organic extracts that resulted from A.
nidulans mediated degradation displayed the presence of signature metabolites such
as low molecular weight aromatics (e.g. ferrulates, veratraldehyde and cinnamate),
about five β-aryl ether linked metabolites and a pinoresinol group compound
(Martins et al. 2014). Secretome analysis of Fusarium spp. Q7-31T by ESI coupled
with tandem mass spectrometry (LC-MS/MS) indicated the presence of ca.
10 glycoside hydrolase enzymes out of a total of 28, most of them responsible for
producing smaller glucan metabolites by hydrolysing biomass substrates (Tian et al.
2015). A further downstream metabolic effect on the sugar release due to Fusarium
spp. based biomass degradation was analysed by FTIR metabolite fingerprinting.
The results showed 13 % decrease in glucose and about 11 % increase in uronic
acids for Fusarium circinatuum mediated rotting of Pinus pinaster biomass.
Additionally, the content of methyl esterified polysaccharide in pectins also
increased following post-Fusarium culturing (Vivas et al. 2014). It has been
observed that fingerprinting of biomass degradation has been mostly focused
towards proteome/secretome analysis, as indicated from some recent research
(Kosmides et al. 2013; Martins et al. 2014; Studholme et al. 2013; Tian et al. 2015;
9 Microbial Metabolomics in Biomass Waste Management 275

Vivas et al. 2014; Cattaneo et al. 2014). Additional elucidation of the metabolic
output of these secretomes still needs to be applied in the context of biomass
conversion, as currently is applied in plant–fungal parasitic or symbiotic and
microbiome relations (Draper et al. 2011; Kingston-Smith et al. 2013).

2.4 Metabolic Flux (Fluxomics)

Metabolic profiling can be applied to obtain metabolic flux information on

metabolite production/consumption rates (fluxomics), in which metabolic engi-
neering tools such as “induction of expression” can be utilised. These techniques
not only decrease the overall inhibition, but also aid in developing systems which
generate desired products. This has been reported recently where a Zymomonas
mobilis ethanolic pathway system was expressed in Sphingomonas spp. A1 to
convert alginate to ethanol (Takeda et al. 2011).
Due to the complex nature of biomass composition and the allosteric nature of
most lignocellulolytic enzymes, standard biochemical tests have proven less
effective compared to metabolomic approaches for deciphering biomass conversion.
Metabolic profiling indicates signature metabolites and critical pathway points
which can be exploited to not only improve the biomass degradation process, but
also to convert this biomass to the products of industrial and medicinal interests.
In recent developments, flux studies of A. niger and P. chrysogenum-mediated
SSF of grape biomass waste during an 8 day period were performed. The untar-
geted metabolic flux analysis of A. niger was studied with the aid of 2H2O. The
process not only helped to identify the key points in the A. niger metabolism of
lignocellulose, but was also able identify the onset of inhibition caused by specific
by-products and competitive inhibitors. Overall, 37 unique metabolites were
characterised during this flux study, spread across 17 pathways including fatty acid,
amino acid and chitin synthesis. It was observed that although the fungal growth
rate was constantly increasing over this period, enzyme activities were inhibited
from day 5 onwards. Metabolic pathway analysis also showed the potential of
applying metabolic engineering in order generate biofuels such as 2-butanol. This
could be achieved by termination of the amino acid pathway using enzymes such as
2-keto-acid decarboxylases followed by alcohol dehydrogenases (Karpe et al.
2015b). Similarly, an untargeted metabolic flux analysis of P. chrysogenum indi-
cated the presence of 94 signature metabolites spread over 28 different pathways,
including xylitol synthesis, lignin and tannin degradation and pentose degradation.
The analysis indicated an onset of autolysis from day 6. Interestingly, the pathway
analysis also displayed pentose metabolism, possibly leading to ethanol generation
via xylose biosynthesis pathway, a property absent in other biomass degrading
ascomycetes investigated under similar conditions. Additionally, the fungus also
displayed tannin and lignin mineralization, leading to the generation of syringate
over 3–5 day SSF. The antioxidant metabolites syringate or gallate (amongst oth-
ers) have been identified to possess medicinal properties (Alonso et al. 2002, 2004).
276 A.V. Karpe et al.

Some of the earlier metabolism flux related research was performed on the
galactoglycome of T. reesei during its growth on lactose. The work showed that
lactose, when used as a carbon source, acted an initial inducer for galactokinase and
aldose reductase XYL1. These enzymes further form the oligosaccharides which
serve as inducers for cellulase formation (Hartl et al. 2007; Seiboth et al. 2007).
A more downstream quantitative metabolic flux analysis of this fungus used
sophorose as an initial inducer for cellulase formation and activity. The induction
did not affect any balance between intracellular and extracellular proteins, the latter
of which consist of cellulases and hemicellulases, a critical factor for industrial
application of this fungus (Rautio et al. 2006). A similar, but a more amino acid
focussed experiment, was conducted to optimize the growth medium composition
for the basidiomycete Pleurotus sapidus by using 13C-glucose based metabolic flux.
It was observed that the isotopic glucose contributed to fungal amino acid synthesis,
ranging from 22 (asparagine/aspartate) to 92 % (alanine). In addition, the threonine
aldolase enzyme based mechanism to generate glycine from threonine was
observed to be inactive or absent in this fungus. Additionally, glucose was utilised
at a rate of 0.24 mmol g/hour, with about 35 % of its output going to the pentose
phosphate pathway (Fraatz et al. 2014).
Similar approaches have been reviewed by Kubicek (2013) and asserts the
necessity of multi ‘Omics’ approach for understanding cellulolytic properties of T.
reesei for industrial applications. It also indicated a limited number of approaches
used in metabolome studies of T. reesei and other fungal enzyme outputs during
biomass degradation as compared to genomics, transcriptomics and proteomics.

3 Metabolic Engineering Approaches

As traditional manufacturing sectors transition (e.g. automotive manufacturing), we

find ourselves on the verge of the fourth industrial revolution catalysed by a wave
of disruptive technological advances and the creation of lean start-ups. The fourth
industrial revolution comprises technological and service based advancements such
as the ‘internet of things’, to 3-D manufacturing and synthetic biology/biological
engineering (Maynard, 2015). Biological engineering in particular is relevant to
metabolomics and omics-based platforms. It is an emerging interdisciplinary field
representing the convergence of diverse domains, such as biotechnology, evolu-
tionary biology, molecular biology, systems biology, physics, chemistry, genetic
engineering and informatics (Wagner and Alper 2016). It is widely acknowledged
that there is already a number of established metabolic engineering and synthetic
biology tools available that develop high-performance cell factories (Carbonell
et al. 2014). However, relative to the potential opportunities, the number of suc-
cessful applications has been limited due to the complexity of exploring the
metabolic space efficiently. As an outcome of such work, biological engineering has
the potential to generate value from traditional wastes and natural systems biology
processes that can benefit society within a range of sectors. For example, biological
9 Microbial Metabolomics in Biomass Waste Management 277

engineering can provide novel in silico design and fabrication materials, the con-
struction of new biological parts, devices, systems and machines, as well as the
re-design of existing, natural biological systems for useful purposes (Wagner and
Alper 2016; Maynard 2015).
The application of metabolomics to biological engineering will become
increasingly important for the production of chemicals of value from agricultural
wastes, which are either high-value fine chemicals (e.g. medicinal products) or bulk
low-value commodities (e.g. fuels) (Ellis and Goodacre 2012). The integration of
metabolomics and fluxomics at each stage of biological engineering projects will
enable metabolic pathways with a desired outcome to become the focus of bio-
engineering design, where the genetic/metabolic optimisation of those pathways
will result in more efficient conversion of low-cost starting materials into highly
desirable products (Ellis and Goodacre 2012; Chen 2015). For example, this
emerging field that has already been identified as a sustainable solution for pro-
ducing an alternative source of Omega-3 fatty acids that is not reliant on wild fish
harvests or aquaculture; work has already been performed to create recombinant
sources of Omega-3 fatty acids in a canola and yeast (Adarme-Vega et al. 2012).
Bioprocessing of polyols using lactic acid bacteria has been investigated as an
alternative to current industrial food production approaches (Ortiz et al. 2013). The
major challenge for all bioengineering studies is to balance the flux in the pathway
of interest in order to obtain high yields and maintain productivity of the target
microorganism (Yuan et al. 2013).

3.1 Metabolic Engineering to Develop Fungal

Bioprocessing Abilities

Although lignocellulosic biomass is found in abundance, it is relatively inexpensive

and has been used in the past as feedstock. It is also considered a renewable source
for bioethanol production. As previously stated, lignocellulosic biomasses are
complex mixtures of different fermentable sugars, as well as inhibitors that impact
the performance of the target microorganism (Rumbold et al. 2009). However, it
should be noted that for all its potential benefits, producing bioethanol from biomass
is still not economically viable when compared to other substrates (such as corn
starch and cane juice) (Hasunuma and Kondo, 2012). This challenge may have been
overcome by Hasunuma and Kondo (2012) through consolidated bioprocessing
(CBP). CBP combines enzyme production of engineered microorganisms, saccha-
rification and fermentation in a single step (Hasunuma and Kondo 2012). However,
the overproduction and misfolding of heterologous and endogenous proteins can
trigger cellular stress, increasing the metabolic burden and retarding growth
(Hasunuma et al. 2015). Furthermore, exposure of various lignocellulose-derived
inhibitors disrupts the efficiency of hexose and pentose co-fermentation (Hasunuma
et al. 2015; Wang et al. 2014). To investigate this further, Wang et al. (2014)
278 A.V. Karpe et al.

measured the metabolic response of a xylose-fermenting S. cerevisiae 424A

(LNH-ST) and its parental strain 4124 with and without three typical inhibitors
(furfural, acetic acid and phenol). It was concluded through metabolomics that the
424A (LNH-ST) strain had a lower capacity to buffer redox changes caused by
inhibitors compared to strain 4124. Furthermore, a lower ethanol yield in glucose
and xylose co-fermentation was observed in strain 424A when compared to glucose
fermentation in strain 424A (LNH-ST). Lastly, xylose utilisation of strain 424A
(LNH-ST) was more significantly reduced by inhibitors than glucose utilisation.
This suggests that xylose catabolism and energy supply, rather than xylose uptake,
were the limiting steps in xylose utilisation in the presence of inhibitors (Wang et al.
2014).
A systems biology approach including disruptome screening, transcriptomics
and metabolomics has been recently exploited to gain insight into the molecular and
genetic traits involved in tolerance and adaptation to fermentation inhibitors using
direct and inverse metabolic engineering (Fig. 9.4) (Hasunuma and Kondo 2012). It
is noteworthy to mention that both direct and inverse metabolic engineering
approaches tend to reach the same outcome, principally because they use both a
rational design and evolutionary design strategy applied to native strains, albeit in
different combinations. A rational design strategy involves selecting and modifying
preselected pathways for specific outcomes. Conversely, an evolutionary design
strategy involves harnessing the natural evolutionary processes that resulted in the
expression of the desired pathway (i.e. tolerance, production, enhanced growth).
Irrespective of the approach, metabolomics (and the other omics based platforms)
play a prominent role in achieving the desired outcomes.

Fig. 9.4 Design strategy of rational and evolutionary direct and inverse metabolic engineering
experiments. Adapted from Hasunuma and Kondo (2012)
9 Microbial Metabolomics in Biomass Waste Management 279

Papini et al. (2012) described a rational metabolic engineering strategy that was
used to express the fungal genes of the phosphoketolase pathway in S. cerevisiae
and the effects of the expression of this recombinant route in yeast using intra-
cellular flux analysis based on 13C labelling. Several bacterial species and fila-
mentous fungi utilise the phosphoketolase pathway for glucose dissimilation as an
alternative to the Embden-Meyerhof-Parnas pathway. However, through the use of
fluxomics, Papini et al. (2012) observed yeast using the recombinant pathway and it
was observed that the utilisation of the phosphoketolase pathway did not interfere
with glucose assimilation through the Embden-Meyerhof-Parnas pathway.
Furthermore, the expression of the recombinant pathway contributed to an increase
in the acetyl coenzyme supply, therefore enabling future potential metabolic
engineering strategies to be considered that utilise acetyl CoA as precursor for the
biosynthesis of industrially relevant and value-added compounds (Papini et al.
2012). Huang et al. (2015) applied biological evolutionary engineering to activate
the glycerol utilisation pathway for fumaric acid production. In this study, an
evolved strain G80 was selected that was tolerant to high concentrations of crude
glycerol to produce fumaric acid.
While A. niger is an effective organism in the biodegradation of biomass and has
been widely applied for the production of high value-added products (Karpe et al.
2015b; Krull et al. 2010; Meijer et al. 2009), the morphology of this fungus, the
transport phenomena and the related metabolic activity result in unreliable yield
within industrial bioprocessing facilities (Krull et al. 2010). As such, further
characterization of the molecular and cell biology of A. niger, in particular char-
acterising interactions between the growth conditions, cell morphology,
spore-hyphae-interactions and value-added product formation, is of great impor-
tance for ensuring a sustainable bioprocessing industry (Krull et al. 2010).
Conversely, Rumbold et al. (2009) used a substrate-oriented approach as opposed
to a product-oriented approach towards the selection of a microbial production host
that avoids the need for extensive metabolic engineering. In this work, six
microorganisms were evaluated and the performance of two bacteria (Escherichia
coli and Corynebacterium glutamicum), two yeasts (S. cerevisiae and Pichia
stipitis) and two fungi (A. niger and T. reesei) were assessed for their ability to
degrade lignocellulosic hydrolysates. The study was particularly focused on their
ability to utilise monosaccharides, characterising the resistance against lignocellu-
losic hydrolysates inhibitors and assess growth and utilisation rates on different
feedstock (with feedstocks thermally pre-treated under mild acidic conditions fol-
lowed by enzymatic hydrolysis and non-enzymatic treatments). Rumbold et al.
(2009) concluded that carbon source versatility and inhibitor resistance were the
major discriminators of the microorganisms tested, with P. stipitis and A. niger
found to the moist effective. C. glutamicum and S. cerevisiae were shown to be the
least adapted to renewable feedstock. These results highlight the complexity of
substrate-orientated approaches that remove the over engineering of biological
systems and utilising natural metabolic pathways present in substrate matched
microorganisms.
280 A.V. Karpe et al.

As stated previously, P. chrysogenum has been applied to the industrial-scale

process of penicillin production (Ding et al. 2012). Metabolic engineering
methodologies have stimulated new efforts toward optimising fungal production
strains of P. chrysogenum. Meinert et al. (2013) devised a protocol for fungal
metabolomics, intended to inform metabolic engineering of industrial processes
using P. chrysogenum. In this work, metabolites were quantified directly from
biomass extracts using automated sampling and fast filtration methodologies and
the use of a 13C tracer (Meinert et al. 2013). The use of 13C tracers is advantageous
when investigating complex media containing multiple substrates (Antoniewicz
2013).
Some of the most productive metabolic engineering strategies involve genetic
modifications that cause severe metabolic burden to the host cell (Williams et al.
2015). Growth-limiting genetic modifications can be more effective if they are
‘switched on’ after a population growth phase has been completed. Williams et al.
(2015) demonstrated this concept through the engineered dynamic regulation of a
synthetic quorum sensing circuit in S. cerevisiae, whereby the circuit autonomously
triggers gene expression at a high population density through the shikimate path-
way for the production of p-hydroxybenzoic acid (PHBA) (Williams et al. 2015).
Lastly, while experimental work is needed to confirm desired or undesired meta-
bolic expression of studied microorganisms, computational methods are increas-
ingly being used to extract direct genotype to phenotype information that can be
used to predict metabolic function (Udatha et al. 2013).

4 Future Research and Conclusions

Considerable advances have been made in biomass treatment processes in recent

years. However, metabolic studies regarding fungal-mediated processes still remain
in a nascent phase. Unlike bacteria, fungal cellular systems (including yeasts) are
highly complex and are not yet fully understood. This complexity is further
increased by various interactions within the fungi (commensal, symbiotic or par-
asitic) and their substrates (timber, general agriculture and food processing wastes).
In spite of the nascent nature of fungal metabolomics, especially with respect to
bioprocessing, rapid advances have been recently observed. These developments
have improved not only our basic understanding of biomass degrading fungi, but
have also provided the key points to improve their metabolism to generate
metabolites of commercial interest. Additionally, some of the very recent works
have demonstrated the application of these improvements in yeasts to enhance their
efficiency in production of various metabolites including food grade alcohols,
enzymes and pharmaceutical metabolites.
With continuous developments of different databases such as the Kyoto
Encyclopaedia of Genes and Genomes (KEGG), the Joint Genome Initiative (JGI),
the Chemical Entities of Biological Interest (ChEBI) and the Human Metabolome
Database (HMDB) (amongst others), metabolome studies in the near future are
9 Microbial Metabolomics in Biomass Waste Management 281

expected to show an exponential growth, as observed with genomic research in the

previous decade. A more complete development in metabolomics is also evident
from the emergence of a ‘multi-omics’ approach (a combination of genomics,
transcriptomics, proteomics and metabolomics). Another recent approach is the
construction of entire novel fungi specifically designed for targeted metabolite
production. Although this method is still in its infancy, even from a bacterial system
development perspective, a complete ‘multi-omics’ approach will aid researchers to
achieve this development in fungal systems. These approaches are expected to not
only close a number of gaps in our current understanding of fungal processes, but
will provide an elaborate view towards improving biomass conversion to obtain
greater efficiency in metabolite yield.

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Chapter 10
Beyond Metabolomics: A Review
of Multi-Omics-Based Approaches

David J. Beale, Avinash V. Karpe and Warish Ahmed

1 Introduction

‘Omics’-based techniques comprise a suite of tools and approaches, each with their
own specific protocols and frameworks for setting minimum data and reporting
standards (Field et al. 2008; Morrison et al. 2006; Sansone et al. 2007; Sumner et al.
2007; Orchard and Kerrien 2010). This suite of techniques primarily comprises
metagenomics, transcriptomics, proteomics and metabolomics. There are also a
number of other specialized ‘omic’-based approaches that are included under the
broader ‘omics’ banner, such as lipidomics, fluxomics (metabolic flux analysis),
toxicogenomics, nutrigenomics and foodomics. However, these additional ‘omics’
approaches are not considered within the context of this chapter, as they are subcat-
egories of the aforementioned suite of techniques. In addition, for completeness, it
should be noted that within the context of this chapter, (meta)genomics has been
defined as the analysis of genetic material recovered from an organism or environ-
mental samples (Handelsman 2004); (meta)transcriptomics is the analysis of RNA
molecules, including messenger RNA (mRNA), Ribosomal RNA (rRNA), transfer
RNA (tRNA) and other non-coding RNA produced by an organism or a population of
organisms (Pascault et al. 2015); (meta)proteomics is the analysis of proteins produced
by an organism or population of organisms, and their function (Douterelo et al. 2014);
and lastly, metabolomics is the analysis of the small chemical compounds produced
and consumed by an organism or a population of organisms (Beale et al. 2016a).

D.J. Beale (&)
A.V. Karpe
W. Ahmed

Land and Water, Commonwealth Scientific and Industrial Research Organisation
(CSIRO), Ecosciences Precinct, 41 Boggo Road, Dutton Park, QLD 4102, Australia
e-mail: [email protected]
A.V. Karpe
Department of Chemistry and Biotechnology, Faculty of Science, Engineering
and Technology, Swinburne University of Technology, P.O. Box 218
Hawthorn, VIC 3122, Australia

© Springer International Publishing Switzerland 2016 289

D.J. Beale et al. (eds.), Microbial Metabolomics,
DOI 10.1007/978-3-319-46326-1_10
290 D.J. Beale et al.

The application of these ‘omics’ techniques in isolation has been demonstrated

to be beneficial in the understanding and characterization of numerous biological
systems within the environment, engineered and industrial systems and treatment
processes, in addition to providing insight into human health and clinical investi-
gations (Cohen et al. 2015). It is when these ‘omics’-based techniques are applied in
combination, however, that their real power can be utilized. These techniques
enable researchers to identify and characterize the entire microbial community
present at greater depth (i.e. metagenomics) and, in combination with the other
techniques studied in parallel, further enable researchers to identify what that
community is doing, in terms of gene expression (i.e. transcriptomics), protein
production (i.e. proteomics) and the community metabolism (i.e. metabolomics)
(Turnbaugh and Gordon 2008). Figure 1 illustrates the chronological order (i.e. the
magnitude of information that can be obtained) of these principal ‘omics’ based
techniques, which inform researchers on the microbial potential, functionality, and
activity starting at the metagenome through to the metabolome. This multiple
‘omics’-based approach has been coined “multi-omics”.
The objective of this chapter is to highlight examples in the literature that go
beyond metabolomics only studies, giving rise to a greater depth of systems biology
research that is multi-omics. While multi-omics research is not new, it is a growing
area of research that has increased in popularity amongst the scientific community
in recent years. Furthermore, the underlying aim of this review is not to provide a
detailed chronology of multi-omics or a detailed guide on how to integrate
multi-omics datasets; this has been the focus of recent reviews published

Microbial Potential

Microbial Function Microbial Activity

1 2 3 4

(meta)Genome (meta)Transcriptome (meta)Proteome Metabolome

Overview of multi-omics approaches

Fig. 1 Chronical order of ‘omics’ based techniques commonly used in multi-omics studies.
Adapted from Abram (2015)
10 Beyond Metabolomics: A Review of Multi-Omics-Based Approaches 291

(Barh et al. 2013; Blanchet and Smolinska 2016; Fondi and Liò 2015; Kohl et al.
2014; Schneider and Orchard 2011; Zhang et al. 2010). Instead, the aim of this
chapter is to provide an overview of the different approaches used in multi-omics-
based research and the tools used to integrate multi-omics data, using examples from
the literature from a range of environmental, industrial and biomedical applications
to highlight the value of extending beyond metabolomics only research.

2 The Multi-Omic Data Analysis Challenge

Multi-omics based techniques are inherently data-rich studies. For example, the
human genome comprises ca. 20,000–25,000 protein coding genes (Pertea and
Salzberg 2010) and the human metabolome is estimated to comprise over 40,000
metabolites (Forsythe and Wishart 2009). As such, the challenge in all ‘omic-based
investigations’, which is only compounded further when applying a multi-omics
approach, is the handling of these large and complex datasets (Kohl et al. 2014;
Röling et al. 2010). Röling et al. (2010) characterized the data processing and
quantitative comprehension of multi-omic information as a bottleneck in the overall
workflow, requiring input and interpretation of ‘systems biologists’ and microbi-
ologists. In addition, the authors of this review propose that it needs further input
from the bioanalytical chemist/biochemist/biostatistician to first evaluate the quality
and validity of the study (experimental) design, as well as the quality of the data
acquired from the instrument, before even attempting to integrate and synthesize
findings (indeed the old adage of poor data in equals poor data out applies). In any
case, assuming the data obtained are of high quality and are valid [following each
‘omics’ specific protocol and frameworks for minimum data and reporting stan-
dards as highlighted by the various societies (Field et al. 2008; Sansone et al. 2007;
Sumner et al. 2007)], there are a number of approaches to analyzing and inter-
preting multi-omics data, namely: post-data analysis integration and integrated data
analysis techniques.
In a post-data analysis approach, datasets are analyzed in isolation of each other
and key features are networked in a post analysis exercise through the synthesis of
significant features at joint nodes in the overall model metabolic pathway. This
approach has been used in previous studies that focused on characterizing and
assessing biological wastewater treatment systems (Beale et al. 2016b), the
microbial resistance of marine sediments after an oil spill (Kimes et al. 2013) and
characterizing permafrost (Hultman et al. 2015). In contrast, an integrated
multi-omics approach employs specialized tools to merge datasets prior to under-
taking any data analysis and interpretation (Kuo et al. 2013), thus enabling simi-
larities of each omic approach to be statistically derived, as opposed to relying on
human interpretation. The principal differences between the post-data analysis and
integrated analysis approaches are graphically presented in Fig. 2.
In addition to post-data analysis integration and integrated data analysis tech-
niques, a third model-based integration approach has been identified. However, a
292 D.J. Beale et al.

Fig. 2 Principal differences between the post-data analysis and integrated analysis approaches

model-based approach is considered unobtainable, in a practical sense, as stated by

Kamburov et al. (2011). Model-based integration methods rely on a well-defined
understanding of the system being investigated in order to compare new experi-
mental findings against modelled predictions. That’s not to say that a complete
multi-omics model-based integration approach is not obtainable as suggested. There
are already examples in the literature of its use. For example, Noecker et al. (2016)
used a model-based approach to integrate taxonomic and metabolomics data to
predict the effects of community ecology on metabolite concentrations and evalu-
ated these predictions with measured metabolomic profiles from the vaginal
microbiome. It was concluded that predicted species composition correlated with
identified putative metabolic mechanisms underlying these predictions (Noecker
et al. 2016). However, it is noteworthy to mention that a model-based integration
approach was achieved primarily because of the vaginal microbiome investigated
had already been well defined and studied beforehand, utilizing previously pub-
lished data and publically available datasets (Erickson et al. 2012; Srinivasan et al.
2015; Theriot et al. 2014; Jansson et al. 2009; Jozefczuk et al. 2010). As such, the
real challenge for model-based integration approaches is not that they are princi-
pally unobtainable, but that not all the systems studied are yet fully characterized as
per the example presented by Noecker et al. (2016). Therefore, until a systems
biology approach is taken for all studied systems (i.e. a multi-omics approach in
10 Beyond Metabolomics: A Review of Multi-Omics-Based Approaches 293

order to obtain baseline data), a model-based integration approach is limited to only

those systems that are already well defined.
Regardless of the multi-omics approach being applied, there are numerous tools
and approaches that assist researchers to integrate omics-based datasets. As illus-
trated in Table 1, there are tools that have been developed that are context specific
(i.e. targeted towards the integration of omics data from specific animal models,
medical and clinical studies and selected plant species). There are also tools that are
unspecified in terms of the studied system but provide users a set of statistical tools
for data normalization and transformation, and a range of chemometric models for
interpreting data once integrated.

3 Application of Multi-Omics

There are many examples in the literature of multi-omic studies, with various levels
of integration. Some studies comprise simple levels of integrations (i.e. combing
two different -omics datasets) through to more comprehensive and computationally
demanding studies (i.e. integration of multiple omics datasets). Typically, a
two-omics integration study combines either metagenomics or transcriptomics data
with proteomics (Sunagar et al. 2016; Wildburger et al. 2015) or metabolomics
datasets (Tokimatsu et al. 2005b; Garcia-Alcalde et al. 2011; Kamburov et al. 2011)
or combines proteomics and metabolomics datasets (Xu et al. 2015). These studies
demonstrate how predicted functional metabolism (metagenomics) or gene
expression (transcriptomics) relate to actual protein and metabolite expression and,
by extension, provide a means to self-validate findings through cross-referencing
experimental findings (Fondi and Liò 2015). The following section provides some
examples of multi-omics studies applied to various fields of research, all of which
provided an extension beyond a metabolomics only study (or other singular
omics-based approaches) and enabled further depth of analysis that would other-
wise not be achieved.

3.1 Environmental Contaminants

Marine subsurface environments comprise an abundant and diverse microbial

community (El-Serehy et al. 2016; Yanagawa et al. 2014). These communities have
the ability to bio-transform and mineralize numerous contaminants (Kimes et al.
2013). The application of omics-based research has been used in the past to
characterize and understand sediment and marine environment dynamics. Chariton
et al. (2014) used metagenomics to investigate benthic invertebrate diversity in
exposed sediments with elevated concentrations of triclosan (antibacterial and
antifungal agent). However, a single omics-based approach only provides limited
information. As is this case, a metagenomics approach will only provide
Table 1 Summary of multi-omic tools
294

Software tool Omics integrated Domain Functionality License References

3Omics – Transcriptomics Medical (human) – Correlation network analysis Open Kuo et al. (2013)
– Proteomics – Co-expression analysis
– Metabolomics – Phenotype generation
– KEGG/HumanCyc pathway enrichment
– GO enrichment
Biofomics – Transcriptomics Biofilms – Experiment library Open Lourenço et al. (2012)
– Proteomics – Data depository
– Metabolomics
Escher – Genomics Unspecified – Web application for visualizing data on Open King et al. (2015)
– Proteomics biological pathways
– Metabolomics – Rapidly design new pathway maps based on
user data and genome-scale models
– Visualize data related to genes or proteins on
the associated reactions and pathways
– Identify trends in common genomic data
types
GIM3E (gene inactivation moderated – Transcriptomics Unspecified – Establishes metabolite use requirements with Open (Python and Machado and
by metabolism, metabolomics and – Metabolomics metabolomics data requires a COBRApy Herrgård (2014)
expression) – Model-paired transcriptomics data to find 0.2.x.)
experimentally supported solutions
– Calculates the turnover
(production/consumption) flux of metabolites
IMPaLA (integrated molecular – Transcriptomics or proteomics Medical and clinical – Enrichment analysis Academic only Kamburov et al.
pathway level analysis) – Metabolomics – Pathway analysis (2011)
Ingenuity pathway analysis – Metagenomics Medical (human) and – Metabolic pathway analysis Commercial Krämer et al. (2013)
– Transcriptomics clinical – Network visualization
– Proteomics – Data integration
– Metabolomics – Upstream regulator analysis
– Mechanistic networks
– Causal network analysis
– Downstream effects analysis
(continued)
D.J. Beale et al.
Table 1 (continued)
10

Software tool Omics integrated Domain Functionality License References

INMEX (integrative meta-analysis of – Transcriptomics Medical and clinical – Meta and integrative analysis of data Open Xia et al. (2013)
expression data) – Metabolomics – Pathway analysis
IOMA (integrative omics-metabolic – Proteomics Unspecified – Integrates proteomic and metabolomic data to Open Yizhak et al. (2010)
analysis) – Metabolomics predict flux distributions
KaPPA-view – Transcriptomics Plants – Integrates transcriptomics and metabolomic Open Tokimatsu et al.
– Metabolomics data to map pathways (2005a)
MADMAX (management and – Metagenomics Plants, medical and – Integrates omics data Open Lin et al. (2011)
analysis database for – Transcriptomics clinical – Statistical analysis and pathway mapping
multiple * omics experiments) – Metabolomics
MapMan – Metagenomics Plants (developed for – Compare data across these two species Open Thimm et al. (2004),
– Transcriptomics use with Arabidopsis. – KEGG classification Usadel et al. (2005)
– Metabolomics Includes more – Classification into KOG clusters
species) – Mapping expression responses
MarVis-Pathway (marker – Metagenomics Unspecified – Toolbox for interactive ranking, filtering, Academic only Kaever et al. (2015)
visualization pathway) – Transcriptomics combination, clustering, visualization and
– Metabolomics functional analysis of data sets containing
intensity-based profile vectors
MassTrix – Transcriptomics Unspecified – Integration of data Open Wagele et al. (2012)
– Metabolomics – Generation of colored pathway maps KEGG
data analysis
MetScape 2 – Transcriptomics Medical and clinical – Integrates data from KEGG and EHMN Open Karnovsky et al.
– Metabolomics databases (2012)
mixOmics (R package) – Metagenomics Unspecified – Integration of data Open Günther et al. (2014)
– Transcriptomics – Chemometric analysis (similarity/difference)
– Proteomics
– Metabolomics
Beyond Metabolomics: A Review of Multi-Omics-Based Approaches

Cytoscape with MODAM and – Transcriptomics Unspecified – Multi-Omic Data Miner and OmicsAnalyzer Open Enjalbert et al. (2011),
Cytoscape with OmicsAnalyzer – Proteomics were designed as an accessible and handy Xia et al. (2010)
– Metabolomics Cytoscape plugin that facilitates omics
– Fluxomics analysis
– Compile all biologically-relevant information
regarding the model system through web link
association
– Map the network components with
multi-omics data
– Model omics data
295

(continued)
Table 1 (continued)
296

Software tool Omics integrated Domain Functionality License References

PaintOmics – Transcriptomics 100 top species of – Integration and visualization of Open Garcia-Alcalde et al.
– Metabolomics different biological transcriptomics and metabolomics data (2011)
kingdoms
PathVisio 3 – Transcriptomics Unspecified – Integration of omics data Open (Apache) Kutmon et al. (2015)
– Proteomics – Visualize omics data based on common data
– Metabolomics nodes and interactions in the pathway
ProMeTra – Transcriptomics Medical and Clinical – Interactive visualizations of metabolite Open Neuweger et al.
– Metabolomics concentrations together with transcript (2009)
measurements mapped on the pathways and
GenomeMaps
SIMCA – Metagenomics Unspecified – Integration of data Commercial Wheelock and
– Transcriptomics – Chemometric analysis (similarity/difference) Wheelock (2013)
– Proteomics
– Metabolomics
VANTED (visualization and analysis – Metagenomics Unspecified – Comparison of multiple omics data sets Open Junker et al. (2006)
of networks with related – Transcriptomics – Visualization of metabolic maps
experimental data) – Proteomics – Correlation networks analysis
– Metabolomics
VitisNet – Metagenomics Grapes – Integration of data Open Grimplet et al. (2009)
– Transcriptomics – Visualization of connectivity
– Proteomics
– Metabolomics
D.J. Beale et al.
10 Beyond Metabolomics: A Review of Multi-Omics-Based Approaches 297

information of the microbial diversity present (and by extension, a theoretical

analysis of their functionality). Investigating beyond metagenomics would provide
a greater depth to the analysis by measuring the actual function of the studied
population using proteomics or metabolomics. Hook et al. (2014) investigated
contaminated sediments using a similar approach, however, they used transcrip-
tomics and metabolomics in order to better understand the modes of toxic action
within contaminated ecosystems. The inclusion of metabolomics in such a study
enabled the assessment of changes in the biochemical profiles of microbial com-
munities living in contaminated sites (Jones et al. 2014; Llewellyn et al. 2015). In
the study by Hook et al. (2014), the function of transcripts with altered abundance
in Melita plumulosa (an epibenthic amphipod) following whole-sediment exposure
to a series of common environmental contaminants was investigated. Contaminants
included in the study comprised porewater ammonia, bifenthrin and fipronil (pes-
ticides), diesel and crude oil (petroleum products) and metals (Cu, Ni and Zn).
Subsequent data integration and hierarchical cluster analysis demonstrated grouped
transcriptome and metabolome expression profiles by contaminant class. Many of
the transcriptional changes observed were consistent with patterns previously
described in other crustaceans (Osborn and Hook 2013).
Furthermore, following the Deepwater Horizon (DWH) oil spill in the Gulf of
Mexico, researchers used metagenomic analysis and metabolomic profiling of
deep-sea sediment samples (Kimes et al. 2013). Post-data analysis integration of the
two datasets identified the presence of aerobic microbial communities and their
associated functional genes among all the samples collected, whereas, a greater
number of Deltaproteobacteria and anaerobic functional genes were found in the
sediments closest to the point of oil contamination. Metabolic profiling revealed a
greater number of putative metabolites in sediments surrounding the contamination
site relative to background sites. These putative metabolites were identified as a
series of benzylsuccinates (with carbon chain lengths from 5 to 10), suggesting that
increased exposure to hydrocarbons enriched the Deltaproteobacteria, which are
known to be capable of anaerobic hydrocarbon metabolism. Lastly, through a
combined multi-omics approach, it was surmised that the sediment samples col-
lected at the site of contamination comprised an active indigenous microbial
community capable of metabolising aromatic hydrocarbons in deep-sea sediments
of the Gulf of Mexico (Kimes et al. 2013).
Hultman et al. (2015) undertook a similar study investigating the microbial
metabolism of permafrost. They used several ‘omics approaches, combined with
post-data analysis in order to determine the phylogenetic composition of microbial
communities of intact permafrost, the seasonally thawed active layer and thermo-
karst bog (surfaces of marshy hollows). The multi-omics strategy revealed good
correlation of process rates for methanogenesis (the dominant process), in addition
to providing insights into novel survival strategies for potentially active microbes in
permafrost (Hultman et al. 2015).
298 D.J. Beale et al.

The advancement of omics-based techniques and their integration have con-

tributed towards marine biologists and molecular biologists pushing the boundaries
of our understanding of marine molecular biology (Thakur et al. 2008). This is best
evident in a global holistic approach to the study of marine ecosystems presented by
Karsenti et al. (2011). In this study, the application of multi-omics was extended by
including additional meta-data, linking biogeography with ecology, genetics and
morphology datasets to provide a global perspective to marine systems.

3.2 Food and Nutrition

Metagenomics-based characterization of microbial communities provides a very

promising and powerful approach to food safety testing, with research to date
undertaken focusing on foodborne pathogen biology (Stasiewicz et al. 2015).
However, transcriptomics, proteomics and metabolomics approaches have also
been demonstrated to have the potential for food safety applications (Valdés et al.
2013; Jadhav et al. 2014, 2015; Beale et al. 2014b). To date, transcriptomics,
proteomics and metabolomics have become the three most commonly used tech-
niques in food and nutrition-based ‘omics’ research (Kato et al. 2011).
Takahashi et al. (2014) combined gene expression profiles using DNA
microarrays with proteomic and metabolomics data in order to assess the
anti-obesity effects of coffee in mice. The underlying premise was that coffee
consumption may reduce the risks of developing obesity and diabetes. As such,
gene expression, protein and metabolite profiles in the livers of C57BL/6J mice fed
a high-fat diet containing three types of coffee (caffeinated, decaffeinated and green
unroasted coffee) were investigated. The data were integrated using an in-house
software tool and visualized in KEGG pathways (summarized in Fig. 3). Takahashi
et al. (2014) concluded that the alterations within and around the urea cycle were
found to be highly consistent between transcripts and metabolites, suggesting that
expression of the genes related to the urea cycle were downregulated by a high-fat
diet and up-regulated by coffee consumption. These findings were also consistent
when integrated post-data analysis of each omics dataset.
For nutrition-based research, to further advance the application of multi-omics
research and disseminate current findings more broadly for other researchers to use,
a database of nutrition-focused omics (genomics and DNA microarray data) has
been created (Nutrigenomics Database) (Saito et al. 2005). Similar approaches have
been applied to other areas of research. For example, VitisNet was created in order
analyze omics-based data relating to common wine grapes enabling the integration
of large datasets, streamlining biological functional processing and improving the
understanding of dynamic processes in systems biology experiments (Grimplet
et al. 2009). PaintOmics has similar functionality but is targeted towards a defined
list of plants (Garcia-Alcalde et al. 2011).
10 Beyond Metabolomics: A Review of Multi-Omics-Based Approaches 299

Fig. 3 Integrated analysis of transcriptomics, metabolomics and proteomics of liver tissue

samples collected from mice fed a high-fat diet containing high levels of coffee. Adapted from
Takahashi et al. (2014)

3.3 Water and Wastewater Systems

The application of metagenomics to characterize microbial populations in

wastewater treatments systems (Talbot et al. 2008; Vanwonterghem et al. 2014; Pap
et al. 2015; Kovács et al. 2015; Ma et al. 2014; Albertsen et al. 2012) and pipes
(Vincke et al. 2001; Santo Domingo et al. 2011; Neria-González et al. 2006) is not
new. However, to the authors’ knowledge, the application of multi-omics approa-
ches is limited. Gomez-Alvarez et al. (2012) analyzed the whole-metagenome to
determine the microbial composition and functional genes associated with biomass
harvested from sections of a corroded wastewater pipe. Taxonomic and functional
analysis demonstrated that approximately 90 % of the total diversity was associated
with the phyla Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria.
Furthermore, the biofilm was found to have an abundance of sulphur-oxidizing
bacteria and sulphate-reducing bacteria. Combined with transcriptomics,
Gomez-Alvarez et al. (2012) also demonstrated an enrichment of genes associated
with heavy metal resistance, virulence (protein secretion systems) and stress
response in the biofilm analyzed.
Mohan et al. (2014) analyzed hydraulic fracturing source water and wastewater
produced during fracking using metagenomic and metabolomic techniques.
300 D.J. Beale et al.

Fig. 4 Post-data analysis approach applied to a multi-omics study investigating operational

shocks within laboratory scale digesters treating municipal waste. Adapted from Beale et al.
(2016b)

The post-data analysis of the multi-omic datasets revealed a relative increase in

genes responsible for carbohydrate metabolism, respiration, sporulation and dor-
mancy, iron acquisition and metabolism, stress response and sulphur metabolism in
the produced water samples from a diverse microbial community (Mohan et al.
2014). These results suggest that microbial communities in potable water have an
increased genetic ability to handle stress, which has significant implications for
infrastructure management, such as biofilm control and combating microbial
influenced corrosion.
Another multi-omics study conducted by Beale et al. (2016b) investigated
operational shocks in laboratory scale Anaerobic Digesters (AD) treating municipal
wastewater (Fig. 4). The laboratory scale digesters were operated to simulate
potential shocks to the AD process experienced at a 350 ML/day wastewater
treatment plant. The shocks included high (42 °C) and low (32 °C) temperature
(either side of the optimum temperature of 37 °C) and a 20 % loading of fats, oil
and grease (20 % w:v). These variables were explored at two sludge retention times
(12 and 20 days) and two organic loading rates (2.0 and 2.5 kg TS/m3 day).
Metagenomics and metabolomics approaches were then used to characterize the
impact of operational shocks in regard to temperature and fats, oil and grease
addition, as determined through monitoring of biogas production, the microbial
profile and their metabolism. Results showed that AD performance was not greatly
affected by temperature shocks. Post-data analysis of the metagenomics and
metabolomics data indicated that methanogens and methane oxidizing bacteria were
low in relative abundance, and that the ratio of oxidizing bacteria (methane, sul-
phide and sulphate), with respect to sulphite reducing bacteria had a noticeable
influence on biogas production. Furthermore, increased biogas production corre-
lated with an increase in short chain fatty acids, a product of the addition of 20 %
fat, oil and grease.
As demonstrated by the works of Gomez-Alvarez et al. (2012), Mohan et al.
(2014), Beale et al. (2016b), the application of omics-based techniques to charac-
terize the microbial community and their function provides insight to the resilience
10 Beyond Metabolomics: A Review of Multi-Omics-Based Approaches 301

of crucial microbial populations within water sources, pipes and treatment process.
In addition, it is anticipated that through multi-omics approaches, new insights into
microbial populations in terms of diversity, resilience and activity when exposed to
shocks and stresses (such as illegal discharges within sewer networks and opera-
tional shock during treatment) will be obtained. Furthermore, multi-omics may
provide insight into driving higher biogas and methane production during treatment
and collection systems for reuse.

3.4 Biofilms and Biofilms Known to Influence Corrosion

The safety and quality of potable water is often assumed and taken for granted by
consumers in most developed countries. However, our understanding of potable
water biofilms is limited, partly as they are difficult to access and traditional
microbiology approaches fail to provide sufficient information on their composition
and activity (Douterelo et al. 2014). To date, numerous researchers have applied
omics-based techniques to characterize and assess aquatic biofilms, in a range of
environments. For example, Shaw et al. (2014), Chao et al. (2013) assessed the
community structure of potable water biofilms using metagenomics when exposed
to different water treatment strategies. Metagenomics was also used to investigate
intertidal marine biofilm communities (Zhang et al. 2013) and black fungal biofilms
growth in domestic water fixtures (Heinrichs et al. 2013). Metabolomics has also
been used to investigate biofilms found on the surface of copper pipes (Beale et al.
2010; Beale et al. 2012) and within potable water distribution networks (Beale et al.
2013). However, the application of multi-omics approaches has been limited. Leary
et al. (2014) used a metagenomic and metaproteomic approach to analyze the
composition and function of marine biofilms; others have investigated microbial
communities in extreme environments using metagenomics and proteomics (Singer
2012; Schneider and Riedel 2010). Nevertheless, it has been identified that there is a
need for a multi-omics approaches to assess biofilms. This has resulted in the
recently proposed term “Biofomics” and web-based interface for biofilm data
(Lourenço et al. 2012). Biofomics provides a framework, database depository and
selection of statistical tools for researchers to follow, merge and examine data from
different approaches such as metagenomics, transcriptomics, proteomics and key
metabolites (metabolomics) (Nozhevnikova et al. 2015). The biofilm data results
from experiments involving several types of bacterial genera such as Salmonella
spp., Escherichia coli and Candida spp., and is supported by the minimum infor-
mation about a biofilm experiment (MIABiE) initiative (Lourenço et al. 2012). To
date, the majority of biofomics work has been related to the biomedical and clinical
fields.
An issue of particular concern to infrastructure asset managers is biofilms that
cause biocorrosion/biodeterioration (also known as MIC). Due to their potential
financial and economic impact to infrastructure, there has been a considerable
amount of research published on the role of microorganisms in promoting
302 D.J. Beale et al.

corrosion. The majority of this work has been undertaken to address the problem of
MIC in offshore oil and gas pipelines, and concrete structures with some prelimi-
nary research on microbial/metal surface interactions in water pipes. As such, a
number of extensive reviews have been compiled on MIC mechanisms over the
past 20 years (Videla and Herrera 2009; Edyvean and Videla 1991; Videla 2003;
Beech and Gaylarde 1999; Flemming 1994; Beech et al. 2014).
Corrosion is the result of a series of chemical, physical and (micro) biological
processes leading to the deterioration of materials. The mechanisms of MIC and
MIC inhibition are not completely understood, because they cannot be linked to a
single biochemical reaction or specific microbial species or group (Kip and van
Veen 2015). As such, MIC biofilm communities can be studied at both their
compositional and functional levels through the use of multi-omics. A number of
traditional techniques, such as clone libraries and genetic fingerprinting, along with
more recent metagenomics and transcriptomics, are being used to characterize and
understand MIC biofilms (Chakraborty et al. 2014).
Relatively few metabolomic-MIC studies have been reported. The role of cor-
rosion products on MIC of carbon steel has been investigated by gas
chromatography-mass spectrometry (GC-MS) (Liu et al. 2000), where S2− and
organic acids were found to destroy the protective layer and promote hydrogen
permeation. Furthermore, GC-MS-based metabolomics has also been explored as a
potential tool in monitoring MIC in copper pipes in water distribution systems
(Beale et al. 2010, 2012). It was found that the biofilm metabolites of bacteria
causing copper pipe MIC comprised a combination of organic acids, amino acids
and lipids. These are common in biological metabolic processes, specifically those
relating to soluble monomers and sulphite reducing substrates. In addition, the
range of carboxylic acids released from the isolates (Bosea, Methylobacterium,
Paenibacillus, Sphingomonas and Variovorax) suggests that the corrosion potential
of these biofilms varies, which would account for localized pitting corrosion
commonly observed in metallic pipes (Beale et al. 2014a). Kouremenos et al.
(2014) investigated the metabolic profile of Pseudomonas putida in potable water
exposed to high and low doses of soluble and insoluble iron using LC-TOFMS and
identified metabolites that included nucleobases, nucleosides, dipeptides, amino
acids, fatty acids, sugars and phospholipids as a response to exposure. While not
directly related to MIC, the approach by Kouremenos et al. (2014) and the pre-
liminary work of Beale et al. (2010, 2012) demonstrate the feasibility of GC and
LC-based metabolomics techniques to assess microbial populations exposed to
metals or isolated from biofilms known to cause MIC in potable water networks. In
a study by Booth et al. (2011), the difference in response to metal stress between
sessile and planktonic bacterial populations was characterized. The planktonic
population had an oxidative stress response to copper ion exposure, whereas the
same species within a biofilm environment responded by shifts in
exo-polysaccharide-related metabolism. This finding suggests that microbial
responses pertinent to corrosion are different between sessile and planktonic pop-
ulations, and through more research using metabolomic-based techniques, linkages
between the metabolite activity of both sessile and planktonic populations and their
10 Beyond Metabolomics: A Review of Multi-Omics-Based Approaches 303

release of extracellular metabolites from a biofilm can be achieved. While biofilms,

in general, and their influence on corrosion, in particular, have been subject to
extensive research, there has been limited experimental work performed on the
in situ characterization of the organic compounds within biofilms (Beech et al.
2014; Graeber et al. 2014).
With DNA sequencing costs decreasing, omics-based techniques are enabling an
increasing number of laboratories to taxonomically and functionally classify a wide
range of the organisms that are present in biofilms, and the extension of proteomics
and metabolomics techniques enables the assessment of biofilm microbiological
communities more broadly (Douterelo et al. 2014). The study of functional genes
involved in metabolic pathways is essential when attempting to link microbial
diversity with specific ecological functions. In the context of biofilms, better
knowledge of the role microorganisms play in MIC and MIC processes such as
biofilm formation and corrosion is required and through the application of
multi-omics research, such knowledge will be realized.

3.5 Medical and Clinical

Metabolomics studies have been used to investigate the gut microbial population
structure associated with a wide range of human diseases (Alam et al. 2014; Garrett
2015; Gevers et al. 2014; Goldman et al. 2014; Ley et al. 2006; Smith et al. 2013).
Expression profiling studies have contributed significantly to the understanding of
underlying molecular mechanisms of several diseases. In the context of cancer
research, this has resulted in the improved classification of tumour subtypes
(Karnovsky et al. 2012). However, the lack of early diagnostic markers still remains
a problem (Diamandis 2010). Proteomics and metabolomics have the potential to
provide additional biological insight for solving this problem (Enjalbert et al. 2011).
The multi-omics approach has been applied to identify markers related to
retinoblastoma which is caused by the RB1 gene inactivation. The miRNA pathway
analysis, coupled with miRNA microarray indicated a presence of 18 novel
miRNAs responsible for the onset of this type of retinal cancer (Guha et al. 2015).
In addition to cancer studies, multi-omics approach has been applied to other
studies such as asthma-COPD overlap syndrome (Trentacoste et al.), autism dis-
orders, sickle cell anemia and kidney disorders among many (Megan et al. 2016;
Zeidan-Chulia et al. 2014; Goodman et al. 2016; Cisek et al. 2015). A very recent
research conducting ACOS pattern among asthma patients recorded a trend of
increased Immunoglobulin E (IgE) antibody. A differential gene expression of
Toll-like receptor 10 (TLR10), an asthma related gene was observed. Also, a further
study of single nucleotide polymorphisms (SNPs) indicated a role of another gene
IL21R in ACOS. Based on pathway analysis, the pattern was also observed to affect
the activities related to cytochrome P450 system (Megan et al. 2016).
A recent review by Higdon et al. (2015) indicated numerous domains (genes,
RNA and proteins) related to autism spectrum disorders (ASD) such as fragile-X
304 D.J. Beale et al.

mental retardation protein (FMRP) and chromatin modifying genes and postsy-
naptic and embryonic development proteins. Expression analysis models such as
linear models for microarray analysis (LIMMA) and significance analysis of
microarrays (SAM) have been used to identify differential expressions based on
several databases and networks such as KEGG, HMDB, BioCyc, Panther among
many others. The former modelling approach (LIMMA) was used to identify
Alzheimer’s related genes and signal transduction pathways such as NOTCH and
Wnt in mitochondrial expressional system of ASD patients (Zeidan-Chulia et al.
2014).
Overall, it has been observed that multi-omics research has been at fairly
advanced levels in cancer research, and has made inroads into other clinical
researches. It is expected that a rapid growth in those studies will be observed in
near future, with a further expansion in other related fields, thereby leading to
‘personalized medicine’ development.

3.6 Biofuels

In recent years, one of the prominent fields of multi-omics applications is algal

origin biofuels. Although, the studies involving these applications have been
generally referred to as ‘metabolic engineering’, they involve more than two
approaches (mostly, genomics or transcriptomics and metabolomics). A recent
study reported by Trentacoste et al. (2013) was conducted to improve the lipid
accumulation in microalga Thalassiosira pseudonana. Transcriptomic analysis
indicated the role of hydrolase Thaps3_264297. A knocking out of this gene by
lipase enzyme overexpression by RNAi and antisense approach resulted in a 3-5
fold increased output of lipids.
Another approach of multi-omics in improving lipid accumulation is by nitrogen
starving of algal cells. The responses based on triacylglycerol (TAG) synthesis by
Chlamydomonas reinhardtii showed a switching on of gluconeogenic phase
(  30 min), followed by a transition to glycolytic phase (  4 h). Additionally, a
transduction to Carbon–Nitrogen responsive pathways occurred (due to alterations
in cw15 proteome), causing increased carbon assimilation and nitrogen metabolism.
This led to an increased TAG synthesis in two stages of ‘before TAG synthesis
(BTS)’ and ‘after TAG synthesis (ATS)’ (Park et al. 2015). Similar approaches
have been previously reported for alga mediated high density biofuels (biodiesel
fuel) (Beer et al. 2009; Hossain et al. 2008), indicating an estimated 13 % in energy
surplus (Dassey et al. 2014).
Similar results to that of algal research mentioned above have also been observed
in other organisms regarding biofuel production capabilities of microbial cells.
A recent example is that of metabolome and proteome analysis of the yeast
Yarrowia lipolytica. Nitrogen depletion in this yeast induced an alteration in 133
phosphorylation sites, thereby enriching phosphorylating proteins, causing
up-regulation of lipid synthesis (Pomraning et al. 2016).
10 Beyond Metabolomics: A Review of Multi-Omics-Based Approaches 305

4 Conclusion

Enhanced knowledge through the application of ‘omics’-based techniques provides

a holistic opportunity to measure biological systems and the impact of these sys-
tems under stress. Furthermore, through the application of multi-omics, researchers
are able to obtain a greater depth of knowledge that otherwise would not be
achieved through a single ‘omics’-based study. Combining metagenomic and
transcriptomic data with proteomics and metabolomic datasets will provide
researchers and clinicians details on the microbial population present and their
metabolic functions. In addition, multi-omics research provides a path for
self-validating findings through a combined parallel ‘omics’ approach, and will
ultimately result in the accelerated understanding of these complex populations and
processes, driving better diagnostic techniques and drug therapies, environmental
remediation strategies and the development of innovative synthetic/engineered
biological products.
It is anticipated that the rise of multi-omics research will enable biological
systems to be well characterized, enabling model-based assessments to be generated
and future research undertaken with only a subset of the data needed for clinical and
experimental research, resulting in cost-effective more affordable research (through
data mining of available literature and datasets and reducing the amount of
experimental data needed). Although it is currently considered to be a nascent field,
various potential applications and data integration tools are either currently
underway or are expected to appear on the scientific horizon in the near future. It is
expected that the exponential development in overall ‘omics’ knowledge coupled
with rapidly developing technology in this field, will assist in the exploration of
various multi-omics applications and related scientific discoveries in the fields of
environmental, industrial and clinical biology.

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Index

Note: Page numbers followed by f and t indicate figures and tables respectively

A Bacteria, 149–150
ABC transmembrane proteins (ATP-binding bacterial soil inoculants, 166–167
cassette), 6 and metal toxicity, 204
Abiotic stress adaptation, 167 biofilm versus planktonic response to
Adipose tissue form (ATF), 92 copper, 208
Adulteration of food material, 231, 245–246 investigations of biofilms and
After TAG synthesis (ATS), 304 morphological variants, 207–208
Akkermansia muciniphila, 24, 28, 58 methodological considerations and
Alkylphosphocoline, 95 conclusions, 209
Aminosugars, 93 tellurite resistance, 205–206, 205f
Ammonia fibre explosion (AFEX), 264 Bacteroidetes, 14
Anaerobic digesters, 300 Bayesian probabilistic approach, 214
Anaerobic digestion, 209, 210 Before TAG synthesis (BTS), 304
Anaerobic reactors, 210 β-oxidation inhibitors, 97
Analytical instruments in metabolomics, Bifidobacteria, 16
advancements of, 234–239 Bifidobacterium, 24, 28
Anthropogenic effects on soil microbes, Biodefense and Emerging Infections Research
174–175 Resources Repository (BEI Resources), 61
climatic change effects, 181–183 Biofertilizers, 166
engineered nanomaterials (ENM), 175–177 Biofilms, 162, 178, 301–303
heavy metal contamination, 177–179 and morphological variants, 207–28
organic contaminants, 179–181 versus planktonic response to copper, 208
Antibiotics, 27, 67, 162, 167, 173, 180–181, Biofomics, 294t, 301
241, 243 Biofuels, 304
Antimonials, pentavalent and trivalent, 94 Biomarkers, 233, 240, 242–243, 245
Arabidopsis–Pseudomonas rhizosphere model, Biomass waste management, microbial
164 metabolomics in, 261, 263–265
Arabidopsis thaliana, 93, 164–165, 167, 169 biomass degradation/conversion methods,
Arbuscular mycorrhizal fungi (AMF), 161, 265–267
163, 170–171, 178, 180, 181–182 future research, 280–281
Arginine deprivation response (ADR), 91 metabolic engineering approaches, 276
Aspergillus nidulans, 274 to develop fungal bioprocessing
Aspergillus oryzae, 265 abilities, 277–280
Aspergillus spp., 265, 267 microbial process metabolomics and its
Atmospheric pressure ionisation (API), 236 application to biomass, 268
Autism spectrum disorders (ASD), 14, 28, 303 classification of metabolomic
approaches, 268–269
B metabolic fingerprinting, 272–275
Bacillus subtilis, 134, 174, 181 metabolic flux (fluxomics), 275–276

© Springer International Publishing Switzerland 2016 313

D.J. Beale et al. (eds.), Microbial Metabolomics,
DOI 10.1007/978-3-319-46326-1
314 Index

metabolic profiling: targeted and potential industrial uses of, 209

untargeted approaches, 269–272 anaerobic waste conversion
Biopesticides, 180–181 applications, 209–211
Bodonidae, 83 ecosystems biology, integrated
Botrytis allii, 242 approach for, 213–214
Burkholderia cepacia, 242 state of the science, 211–213
Butyrivibrio fibrisolvens, 270 soil science, 201–204
upcoming challenges, 215–216
C data storage and sharing, 217–218
13
C-acetate, 131, 159 knowledge transfer to applied research
Cadmium contamination, 178–179 outcomes, 218–219
13
C-amino acids, 99, 101 meta-data, importance of, 217
cAMP, 33 need for better reference samples, 216
Capillary electrophoresis and mass Consolidated bioprocessing (CBP), 266, 267,
spectrometry (CE-MS), 125, 236, 237 277
Carbohydrates, dietary, 21–22 Corrosion, 301–303
Carbon fullerenes and nanotubes, 176 Corynebacterium glutamicum, 279
13
C-cellulose, 131, 132 Coxiella brunetti, 87
Cellulomonas flavigena, 264 Crenarchaeota, 212
13 13
C-fatty acids, 99, 101 C-ribose, 131
13
C-glucose, 101, 131, 276 CRISPR (clustered regularly interspaced short
13
CH4, 131 palindromic repeat)-Cas9 system, 93
Challenges, for microbial metabolomics, 1–2, Critical control points (CCPs), 227
5–6 Crohn’s disease (CD), 31
Chemical contaminants and xenobiotic Cryptococcus neoformans, 87
molecules, 229 Cultivation-based multiplex phenotyping, 59
Chemical food safety, 240–241 Culture-dependent analysis of gut microbiota,
Chicory, 20 57–61
13
CH3OH, 132 Culturomics, 59–60
13
C-labeling approaches, 103–104 Cutaneous infections (CL), 83–84
13
C-labelled carbon sources, 203 Cutting-edge applications, 7
13
C-labelled glucose, 183 assessing metabolic state of microbial
Claudin-1, 31 community, 7
13
C-lignin, 131 future perspective, 8
Climatic change effects, on soil microbes, host–microbe interactions, 7
181–183 new diagnosis of microbial infection
Clostridium cellulolyticum, 267 through metabolomics, 7–8
Clostridium difficile VPI 10463, 59 Cutting-edge technologies, 1
Clostridium proteoclasticum, 270 Cytoscape with MODAM and Cytoscape with
Clostridium thermocellum, 265, 267, 271 OmicsAnalyzer, 295t
Clostridium thermohydrosulfuricum, 265
13
C-naphthalene, 132 D
Codes of good practices (CGP), 227 Data integration, 138
Codex Alimentarius Commission (CAC), 226 Data storage and sharing, 217–218
Colorectal cancer (CRC), 16–17, 29, 49, 50 Defensins, 15
Community metabolomics, 199, 201, 204 Desorption electrospray analysis (DESI), 241
bacteria and metal toxicity, 204 Deuterium oxide, 103
biofilm versus planktonic response to Deutsche Sammlung von Mikroorganismen
copper, 208 und Zellkulturen (DSMZ) GmbH, 61
investigations of biofilms and Dietary fibre, 19–23, 34
morphological variants, 207–208 and gut health, 16–19
methodological considerations and and short chain fatty acids, 26–29
conclusions, 209 interaction with gut microbiota, 23–26
tellurite resistance, 205–206, 205f Dietary fibre hypothesis, 17
Index 315

Diffuse cutaneous infections (DCL), 83, 84 F

Direct analysis in real time (DART), 241 Faecalibacterium prausnitzii, 66, 69–71
Direct infusion (DI), 236 Faecal microbial transplantation (FMT), 28
Direct infusion-ESI-MS (DI-ESI-MS), 280 Fast food, 225
Direct insertion probe/electron ionisation/ion Fatty acid methyl esters (FAMEs), 177
trap detection (DIP/EI/ITD) mass Fatty acid β-oxidation, 89
spectrometry, 274 Fermentable Oligo- Di- and Monosaccharides
Dissolved organic matter (DOM), 122, 125 And Polyols (FODMAP), 24
DNA sequencing, 56 Firmicutes phylum, 14
D2O, 103 FISH-microautoradiography, 134
Drug resistance, 88, 94 Flavonoids, 161
Fluorescence in situ hybridisation (FISH), 212
E Fluxomics, 276–277
EcoGenomics, 215 Food adulteration, 231, 245–246
Ecological intensification, 147 Food and nutrition, 298
Eco-metabolomics, 157 Food-borne illness, 230
Ecosystems biology, 213–214 Food-borne pathogens, 231
integrated approach for, 213–214 Food metabolome, workflow for analysis of,
Electrospray ionization (ESI), 125 235
Embden-Meyerhof-Parnas pathway, 279 Food metabolomics, 233, 233–234
Engineered nanomaterials (ENM), 175–177 Foodomics, 233
Enteroids/colonoids, 63 Food safety and current practices, 226–229
Enteropathogenic microbes, 28 Food safety and quality research, 225, 229–230
Environmental contaminants, 293–298 application of metabolomics in, 240
Environmental metabolomics, 216 chemical food safety, 240–241
Era pre-metabolome, 3t food pathogens and food spoilage
Escher, 294t microorganisms, 242–243
Escherichia coli, 2, 28, 230 food quality and traceability, 246–247
Metabolome Database (ECMDB), 4 microbial food toxins, 243
O157:H7, 15 study of GM crops and food materials,
transconjugants, 68 244–245
Essential oil (EO), 166 current state, 230
Euglena mutabilis, 128–129 pathogenic microbes and their stress
European Prospective Investigation into Cancer responses, 231–232
and Nutrition (EPIC), 16 study of food composition and its
Exometabolomics, 119 importance, 231
for analysis of metabolomics and, 233
soil organic matter, 120–126 food metabolomics, 233–234
whole microbial communities, 126–128 recent advancements of analytical
characterizing metabolism of individual instruments in metabolomics,
members, 128–131 234–235
future outlook, 136–138 Food safety regulatory model, 227f
metabolite imaging, 133–136 Fourier transform infrared spectroscopy
stable isotope probing (SIP) techniques, (FTIR), 235, 237, 272
131–133 Fourier transform ion cyclotron resonance
Exopolysaccharides, 162 (FTICR), 236
Expression analysis models, 304 Fourier transform-ion cyclotron
Extracellular enzymes (EEs), 173–174 resonance-mass spectrometry
Extracellular polymeric substance (EPS), 210 (FT-ICR-MS), 125
Exudates, 162 Fragile-X mental retardation protein (FMRP),
rhizoengineering, 163–164 304
316 Index

Fructooligosaccharides (FOS), 20, 23–24 structure-function capacity of human gut

Fructose 1,6-bisphosphatase (FBPase), 92 microbiota, 53–57
Fructose 6-phosphate, 93
Fungal bioprocessing abilities, metabolic H
1
engineering to develop, 277–280 H-nuclear magnetic resonance spectroscopy
Fungal soil inoculants, 167 (1H-NMR), 150t, 173, 174–175, 178, 274
Fungi, 265, 267, 268–269, 274, 279, 280–281 -based community metabolic profiling, 202
Fusarium circinatuum , 274 metabolites found in soil-related samples
Future perspective, 8 using, 150t–156t
2
H2O, 103–106, 275
G “Habitat simulating” media, use of, 58
Galactooligosaccharides (GOS), 23–24 Hazard Analysis Critical Control Point
Gamma amino butyric acid (GABA), 15 (HACCP), 227, 228f
Gas chromatography and mass spectrometry Heavy metal contamination, 177–179
(GC-MS), 4, 123, 126, 169, 208, 231, Heavy water, 103, 104
236–237, 242, 245, 272 Hemicelluloses, 19, 20
Genetically modified (GM) food products, 225, High-throughput screening approaches, 63
229 Histone deacetylases (HDACs), 30
Genetic knockouts, using metabolite profiling, Holobiont concept, 51–53
92–93 Host cells, 85–86, 86, 96
Geobacillus thermoglucosidasius, 267 Host–microbe interactions, 7
Geobacter sulfurreducens, 212 Human colonic microbiota, 13
GIM3E, 294t Human holobiont, 51–53
Glucagon-like peptide-2 (GLP-2), 32 Human Microbiome Project (HMP), 51
Gluconacetobacter xylinus, 273 Humic substances, 120
Gluconeogenesis, 89 Humus, 168–169
Glucosamine (GlcN), 93 Hydrothermal treatment (HT), 264
Glucosamine 6-phosphate deaminase (GND), Hydroxymethyl furfural (HMF), 272
93
Glutamic acid, 172 I
Glycolysis, 89 IMPaLA (integrated molecular pathway level
Glycosomal succinate fermentation analysis), 294t
(GSF) pathway, 97 Inflammatory bowel disease (IBD), 27, 28, 31,
GM crops and food materials, study of, 49, 50
244–245 Ingenuity pathway analysis, 294t
G-protein-coupled receptors (GPCRs), 30 INMEX, 295t
Green revolution, 229 Intestinal barrier, 31
Growth rate, 96, 104 Inulin, 20–21, 23
Gut-associated disorders, 14 IOMA, 295t
Gut intestinal barrier, 31 Ion-mobility spectrometry (IMS), 236
Gut microbiota, 14 Isobaric tags for relative and absolute
bioactive landscape of (see Gut microbiota, quantification/liquid chromatography-mass
bioactive landscape of) spectrometry/mass spectrometry
dietary fibres interaction with, 23–26 (iTRAQ/LC-MS/MS), 91
Gut microbiota, bioactive landscape of, 49 Isotope labelling, 183
culture-dependent analysis, 57–61 Isotope-ratio MS (IRMS), 245
human holobiont, 51–53
NF-κB suppressive gut microbes J
bioprospecting for, 69–71 Juvenile spondyloarthritis, 70
genetic-based strategies to identify,
66–69 K
in vitro approaches to identify, 62–64 KaPPA-view, 295t
metabolomic-based strategies to Kinetoplastida, 83
identify, 64–66 Klebsiella mobilis CIAM 880, 177
Index 317

Knowledge transfer to applied research Liquid chromatography and mass spectrometry

outcomes, 218–219 (LC-MS), 94, 125, 126, 166, 209, 236–237
Koji process, 265 Liquid chromatography with electrospray
Kyoto Encyclopedia of Genes and Genomes ionisation-mass spectrometry, 274
(KEGG), 88 Listeria monocytenes, 230
Listeria monocytogenes, 87, 232, 243
L Lolium perenne, 273
Lactones, 163, 173 Lubricus rubellus, 203–204
Labelling techniques, 174–175 Lubricus terrestris, 203–204
Laetiporus spp., 265 Lutzomyia, 85
Laser ablation electrospray ionization (LAESI),
8 M
LdAAP3, 91 Macroinvertebrate communities, 218
LeishCyc, 88 Macrophages, 32, 87, 93
Leishmania, 83 MADMAX (management and analysis
central carbon metabolism in, 98f database for multiple * omics
characterization of genetic knockouts using experiments), 295t
metabolite profiling, 92–93 MAM (microbial anti-inflammatory molecule)
characterizing metabolism of, 88 protein, 70
genome studies and metabolism mapping, Manganese peroxidase, 266
88–89 MapMan (software tool), 295t
insights into metabolism using Marine subsurface environments, 293
metabolomics, 91–92 MarVis-Pathway (marker visualization
key differences between developmental pathway), 295t
stages, 97 Mass Spectrometry (MS), 2, 8, 123, 134, 136,
flux analyses, 99–102 235, 236
footprinting approaches, 97–98 MassTrix, 295t
intracellular metabolite levels, 98–99 Matrix-assisted laser desorption/ionisation-time
and leishmaniasis, 83–84 of flight mass spectrometry
lifecycle, 85–87 (MALDI-TOF-MS), 60, 61, 243
stages, 105f Matrix-assisted laser desorption/ionization
studying a drug’s mode of action and the (MALDI), 8
development of resistance in, 94–96 Matrix effect (ME), 233
studying metabolism in vivo, 102–106 Metabolic engineering approaches, 276, 278
transcriptomic and proteomic studies, to develop fungal bioprocessing abilities,
89–91 277–280
treatment of leishmaniasis, 84–85 Metabolic fingerprinting, 272–275
Leishmania aethiopica, 83 Metabolic flux (fluxomics), 275–276
Leishmania amastigotes, 87, 104 Metabolic footprinting, 98, 122
Leishmania amazonensis, 83, 93 Metabolic profiling, targeted and untargeted
Leishmania braziliensis, 83 approaches, 269–272
Leishmania chagasi, 83 Metabolic state of microbial community,
Leishmania donovani, 83 assessing, 7
Leishmania guyanensis, 84 Metabolite imaging, 133–136
Leishmania infantum, 83, 89, 94 Metabolite profiling, characterization of genetic
Leishmania major, 83, 106 knockouts using, 92–93
Leishmania mexicana, 83, 87, 91, 99, 101, 104 Metabolites found in soil-related samples using
Leishmania peruviana, 83 1H-NMR, 150t–156t
Leishmania spp., 87, 92 Metabolome, 2–4, 3t
Lentinula edodes, 267 Metabolomics, 1, 7–8, 297, 300, 303
Lignin, 21, 261, 263 classification of, 268–269
Lignin peroxidase (LiP), 266 experimental setup, 200f
Linear models for microarray analysis insights into Leishmania metabolism using,
(LIMMA), 304 91–92
318 Index

Metabolomics (cont.) Monocarboxylate transporter-1 (MCT-1), 30

revealing key differences between Most probable number (MPN) approach, 59
Leishmania developmental stages, 97 Mucocutaneous leishmaniasis (MCL), 83
flux analyses, 99–102 Multi-omic data analysis challenge, 291–293
footprinting approaches, 97–98 Multi-omics-based approaches, 289
intracellular metabolites levels, 98–99 applications, 293
to identify NF-κB suppressive gut biofilms and corrosion, 301–303
microbes, 62–64 biofuels, 304
untargeted, 93 environmental contaminants, 293–298
Meta-data, importance of, 217 food and nutrition, 298
Metagenomes, 125–126, 300, 301 medical and clinical, 303–304
Metagenomic DNA, 215 water and wastewater systems, 299–301
Meta-metabolomics, 201 challenges for, 291–293
Metaparental mating, 66–68 tools, 294t–296t
Methionine sulfoxime (MSO), 101 Mycobacterium tuberculosis, 93
Methyl chloroformate (MCF) derivatization, 4
MetScape 2 (software tool), 295t N
MFS (Major Facilitator Superfamily), 6 N-acetylglucosamine acetyltransferase
MIAME (Minimal Information about a (GNAT), 93
Microarray Experiment) standard, 217 Nanostructure-initiator mass spectrometry
MIC biofilms, 302, 303 (NIMS), 134
Microbes, metabolites and health, 13 Nascent field, 157–159
dietary fibre, 19–23 Nitrogen, in rhizosphere, 170–171
and gut health, 16–19 Nitrogen starving of algal cells, 304
and short chain fatty acids, 26–29 NMR metabolomics, 174
interaction with gut microbiota, 23–26 N-nitroso compounds (NOC), 29
future prospects, 34–35 Non-digestible oligosaccharides, 20
short chain fatty acids, inflammation and Non-starch polysaccharides (NSP), 16, 19, 20,
allergy, 29–33 22, 26
Microbial biomass processing, 269 “Non-targeted” community metabolomics, 204
Microbial food toxins, 243 Nuclear factor-kappa B (NF-κB) suppressive
Microbial fuel cells (MFCs), 209, 210, 211 gut microbes, 50, 53
and anaerobic waste conversion bioprospecting for, 69–71
applications, 209–211 genetic-based strategies to identify, 66–69
Microbial interactions through space, 133–136 in vitro approaches to identify, 62–64
Microbial systems, 1 metabolomic-based strategies to identify,
Microcoleus vaginatus, 129 64–66
Mid-infrared (MIR) spectroscopy, 127 Nuclear magnetic resonance (NMR), 123, 126,
Mid-infrared spectroscopy (MIR), 174, 203 127, 231, 235–236
MIENS (Minimal Information about an Nutrients in rhizosphere, 170–172
Environmental Sequence), 217
MIGS (Minimal Information about a Genome O
Sequence), 217 Obesity, 49, 50
Miltefosine, 95 role of the microbiota in, 14
MIMS (Minimal Information about a Oil organic matter (SOM) metabolites, 148
Metagenome Sequence), 217 Oligofructose, 21
Mini-guts, 63 Omics-based techniques, 289
Minimum information about a Biofilm Omics information, 213
experiment (MIABiE) initiative, 301 Orbitrap, 236
MiRNA pathway analysis, 303 Organic contaminants, 179–181
Mitogen activated protein kinase 2 (MAPK2)- Organosolvent treatments, 264
dependent signaling pathway, 91 Orthogonal partial least square-discriminant
MixOmics (R package), 295t analysis (OPLS-DA), 206
Model-based integration methods, 291–293
Index 319

P Pseudomonas pseudoalcaligenes, 157

PaintOmics (software tool), 296t, 298 Pseudomonas pseudoalcaligenes KF707, 205
Parasitic disease, 84 Purine salvage, 95
Partial least squares discriminant analysis Pyrimidine synthesis, 89
(PLS-DA), 207 Pyruvate, 97
Particulate organic matter (POM), 204
Pathogenic microbes and their stress responses, Q
231–232 Quadrupole (Q), 236
PathVisio 3 (software tool), 296t Quadrupole ion-trap (QIT), 236
pEHR5 vector system, 67 Quality control, 226. See also Food safety and
Penicillium chrysogenum, 265, 280 quality research
Penicillium commune, 272 Quantitative microbial metabolomics, 4
Pentavalent and trivalent antimonials, 94 Quercus suber, 274
Pentose phosphate pathway (PPP), 89 Quorum sensing (QS), 162, 163
Persistent organic pollutants (POP), 180
Pesticide reduction, 180 R
Pesticides, 229, 240, 241 Rare biosphere, 213
Phanerochaete chrysosporium, 266 Ready-to-eat food, 225
Phanerochaete spp., 265 Replica extraction transfer, 134
Phenolic compounds, 161 Resistant starch (RS), 17–18, 20, 23–24
Phlebia spp., 265 REX-NIMS approach, 136
Phlebotomus , 85 Rhizoengineering, 163–164
Phosphoenolpyruvate (PEP), 97 Rhizosphere, 149, 159
Phospholipid fatty acids (PLFAs), 131, 176 map of, 159–161
Phosphorus, in rhizosphere, 170 metabolomics, 161–162
p-hydroxybenzoic acid (PHBA), 280 mRNA pyrosequencing, 165
Phytotoxin, 163 nutrients in, 170–172
Plant biomass, 261 Ribosomal Database Project, 214
Plant growth-promoting rhizobacteria (PGPR), Rugose small-colony variants (RSCVs), 207
166, 171 Ruminococcus bromii, 25, 27
Plant metabolomics, 2
Plasmopara viticola, 271 S
Plate wash PCR, 59 Saccharomyces cerevisiae, 2, 4, 93, 267, 271,
Pleurotus sapidus, 276 272, 279
Polybrominated diphenyl ethers (PBDEs), 241 Salinity, 182
Polychlorinated biphenyls (PCBs), 164, 180, Salmonella enterica, 232
241 Salmonella spp., 230
Polycyclic aromatic hydrocarbons (PAHs), Salmonella typhimurium, 242
180, 241 Sampling (quenching) methods, 5
Polymerase chain reaction (PCR)-amplifying Saprotrophic microbes, 161
16S rRNA genes, 211 Schewanella oneidensis MR-1, 134
Polymorphonuclear leucocytes (PMNs), 85, 97 Secondary ion mass spectrometry (SIMS), 8,
Post-data analysis versus integrated analysis 134
approaches, 292f Separation techniques, 234, 237–238, 241
Postia spp., 265 Shigella spp., 87
Principal component analysis (PCA), 127, 207, Short chain fatty acid (SCFA), 17, 18, 24, 27,
273 29–33, 273
Promastigote, motile, 85 and NF-κB activation, 64–65
Promastigote to amastigote differentiation, 91 Short chain fatty acids, inflammation and
ProMeTra, 296t allergy, 29–33
Protected designation of origin (PDO), 246 Significance analysis of microarrays (SAM),
Protected geographic indication (PGI), 246 304
Proteomics, 89–91, 303 SIMCA (software tool), 296t
Pseudomonas fluorescens, 207, 208 Single-cell metabolomics, 8
320 Index

Single nucleotide polymorphisms (SNPs), 303 Synechococcus sp. PCC 7002, 128
16S rRNA gene, 53–54, 59–61, 211–212 Systems biology, 214, 278
Small-colony variants (SCVs), 207
Sodium fluoroacetate (NaFAc), 101 T
Soil, complexity of, 147–157 T5 cells, 206
Soil community, 128–131 Targeted metabolomics methods, 271
Soil exometabolomics studies, 122, 127 Tellurite resistance, 205–206, 205f
Soil inoculants, 165–168 Tellurium oxide, 205
Soil matrix, 148 Terroir, 169
Soil microbial metabolomics, 147 Thermoanaerobacter ethanolicus, 265
anthropogenic effects on soil microbes, Thermophilic bacteria, 265, 267
174–175 Thin layer chromatography (TLC), 272
climatic change effects, 181–182 3Omics, 294t
engineered nanomaterials (ENM), Time of flight (ToF), 236
175–177 Time-of-flight secondary ion mass
heavy metal contamination, 171–173 spectrometry (TOF-SIMS), 136
organic contaminants, 179–181 Toll-like receptor 10 (TLR10), 303
complexity of soil, 147–157 Toll-like receptors (TLRs), 62, 63
extracellular enzymes, 173–174 Total dietary fibre (TDF), 17
exudates, 162 Toxoplasma gondii, 87
rhizoengineering, 163–164 Trametes spp., 265
humus, 168–169 Transmembrane protein family PulD, 6
LC–MS analyses, 158t Tree litter, 172
metabolite coverage over the lifespan of Triacylglycerol (TAG) synthesis, 304
plants, 165 Trichoderma, 167, 168
nascent field, 157–159 Trichoderma hamatum, 274
rhizosphere, 159 Trichoderma reesei, 276
map of, 159–161 Trichoderma spp., 265, 267
metabolomics, 161–162 “Trojan Horse” strategy, 86
nutrients in, 170–172 “Trojan rabbit” model, 86
soil inoculants, 165–168 Trypanosoma, 83
terroir, 169 Trypanosoma brucei, 83, 92
tools for, 183–184 Trypanosoma cruzi, 83, 87
Soil microorganisms, 119, 133 Trypanosomatidae, 83
metabolomics (see Soil microbial Type IV secretion systems (T4SS), 6
metabolomics)
Soil organic matter (SOM), 120, 122 U
exometabolomics for analysis of, 120–126 Ulcerative colitis (UC), 27
Soil science, 201–204 Ultra-high-performance liquid chromatography
Solid phase extraction (SPE), 234 coupled to electrospray ionisation-high
Solid phase microextraction (SPME), 234 resolution mass spectrometry
Solid state fermentation (SSF), 265–266 (UHPLC-ESI-HRMS), 274
Stable isotope labeling approaches, 99, 103 Ultrasensitive analytical methods, 8
Stable isotope probing (SIP) techniques, Uncinula necator, 271
131–133, 134 Upflow anaerobic sludge blanket (UASB)
Steam explosion (SE) experiment, 264 reactor, 210
Streptococcus bovis, 270
Streptomyces coelicolor, 134 V
Submerged fermentation/shake flask VANTED (visualization and analysis of
fermentation (SmF), 265 networks with related experimental data),
SWCNT (single-walled carbon nanotubes), 176 296t
Symbiotic fermentation, 267 Visceral leishmaniasis (VL), 83
Index 321

VitisNet (software tool), 296t, 298 X

Volatile fatty acids (VFAs), 273 xcp secretion pathway, 6
Volatile organic compounds (VOC), 242
Y
W Yarrowia lipolytica, 304
Water and wastewater systems, 299–301 Yeast Metabolome Database (YMDB), 4
Water extractable organic matter (WEOM),
122 Z
Water soluble fibres, 21 Zwitterionic HILIC chromatography, 125
Web-based tools, 4 Zymomonas mobilis, 275
Whole microbial communities,
exometabolomics for analysis of, 126–128
Wrinkly spreaders (WS), 207