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Methods in
Molecular Biology 1859
Edward E.K. Baidoo

Editor
Microbial
Metabolomics
Methods and Protocols
METHODS IN MOLECULAR BIOLOGY
Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire AL10 9AB, UK
For further volumes:

http://www.springer.com/series/7651
Microbial Metabolomics
Methods and Protocols
Edited by
Edward E.K. Baidoo

Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Emeryville, CA, USA
Editor
Edward E.K. Baidoo
Joint BioEnergy Institute
Lawrence Berkeley National Laboratory
Emeryville, CA, USA
ISSN 1064-3745 ISSN 1940-6029 (electronic)

Methods in Molecular Biology
ISBN 978-1-4939-8756-6 ISBN 978-1-4939-8757-3 (eBook)
https://doi.org/10.1007/978-1-4939-8757-3
Library of Congress Control Number: 2018957074
© Springer Science+Business Media, LLC, part of Springer Nature 2019

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Preface
Since the term metabolomics was first coined, it was thought that comprehensive metabo-
lomic data sets would be the shape of things to come. However, the structural diversity that
exists within the metabolome would make it extremely difficult to achieve this with current
analytical technologies. This left scientists to ponder the questions, how much of the
metabolome was necessary to characterize metabolism and what aspects of metabolomics
data were truly meaningful to a research study? This book explores current approaches to
answering these two very fundamental questions in microbiological research.
The protocols in this volume represent the breadth of microbial metabolomics
approaches to both large-scale and small-scale experiments. The intention of this volume
is to cover protocols that can be used for applications ranging from environmental microbi-
ology to human disease. All protocols described in this volume utilize mass spectrometry as
their primary measurement tool, which is reflective of the popularity of this technique. This
volume covers four major areas: (1) microbial metabolomics, metabolism, and microbial
physiology, (2) metabolite sample preparation, (3) current analytical techniques used to
profile primary and secondary metabolites and lipids, and (4) establishing data analysis
workflows for targeted metabolomics, untargeted metabolomics, analysis of metabolic
fluxes, and genome-scale models.
Part II of this volume provides background information on metabolomics in the context
of microbial metabolism and physiology. The protocols described in this volume provide
guidance for both novice and advanced users. Extra information on the steps of each
protocol can be found in the notes section at the end of each chapter. The information
covered in these protocols can serve as reference material and can be adapted to similar
analytical platforms or customized to suit the needs of the researcher. I would like to thank
all the authors for their outstanding efforts and for kindly agreeing to contribute to this issue
on Microbial Metabolomics. I would also like to thank and acknowledge John M. Walker for
his invitation to edit this volume of Methods in Molecular Biology and for his invaluable advice
and encouragement during the preparation of the issue.
Emeryville, CA, USA Edward E.K. Baidoo
v
Contents
Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
1 Microbial Metabolomics: A General Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Edward E.K. Baidoo
PART I MICROBIAL METABOLOMICS, METABOLISM, AND PHYSIOLOGY
2 Mass Spectrometry-Based Microbial Metabolomics: Techniques,

Analysis, and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
Edward E.K. Baidoo and Veronica Teixeira Benites
3 Metabolomics: A Microbial Physiology and Metabolism Perspective. . . . . . . . . . . 71
Chijioke J. Joshua
PART II METABOLITE SAMPLE PREPARATION
4 Untargeted Soil Metabolomics Using Liquid Chromatography–Mass

Spectrometry and Gas Chromatography–Mass Spectrometry . . . . . . . . . . . . . . . . . 97
Tami L. Swenson and Trent R. Northen
5 Fatty Acid Metabolome Extraction from Mycobacterial Cells
for GC-MS Metabolomics Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
Ilse du Preez, Derylize Beukes, and Du Toit Loots
6 Total Metabolome Extraction from Mycobacterial Cells
for GC-MS Metabolomics Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Derylize Beukes, Ilse du Preez, and Du Toit Loots
7 High-Throughput Solid-Phase Microextraction–Liquid
Chromatography–Mass Spectrometry for Microbial Untargeted Metabolomics . 133
Fatemeh Mousavi, Barbara Bojko, and Janusz Pawliszyn
PART III CURRENT ANALYTICAL TECHNIQUES
8 Targeted Metabolomics of Xylose-Fermenting Yeasts Based

on Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
Christiane Gonçalves Campos, José Antônio de Aquino Ribeiro
João Ricardo Moreira de Almeida, Betania Ferraz Quirino,
and Patrı́cia Verardi Abdelnur
9 Exploiting High-Resolution Mass Spectrometry for Targeted
Metabolite Quantification and 13C-Labeling Metabolism Analysis . . . . . . . . . . . . 171
Zhucui Li, Yujing Li, Yinjie J. Tang, and Wenqing Shui
10 Quantitative Profiling of Endogenous Metabolites Using
Hydrophilic Interaction Liquid Chromatography–Tandem
Mass Spectrometry (HILIC-MS/MS) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
Attila Teleki and Ralf Takors
vii
viii Contents
11 Liquid Chromatography and Mass Spectrometry Analysis of Isoprenoid

Intermediates in Escherichia coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209
Edward E.K. Baidoo, George Wang, Chijioke J. Joshua
Veronica Teixeira Benites, and Jay D. Keasling
12 Determining the Mode of Action of Antimalarial Drugs Using
Time-Resolved LC-MS-Based Metabolite Profiling . . . . . . . . . . . . . . . . . . . . . . . . . 225
Simon A. Cobbold and Malcolm J. McConville
13 Use of Liquid Chromatography–Mass Spectrometry-Based
Metabolomics to Identify Biomarkers of Tuberculosis . . . . . . . . . . . . . . . . . . . . . . . 241
Juntuo Zhou and Yuxin Yin
14 Metabolomics Analysis of Leishmania by Capillary Electrophoresis
and Mass Spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253
David Rojo, Coral Barbas, and Ángeles Lopez-Gonzálvez
PART IV DATA ANALYSIS
15 A High-Throughput Targeted Metabolomics Workflow for the

Detection of 200 Polar Metabolites in Central Carbon Metabolism . . . . . . . . . . . 263
Yuping Cai and Zheng-Jiang Zhu
16 Cluster Analysis of Untargeted Metabolomic Experiments . . . . . . . . . . . . . . . . . . . 275
Joshua Heinemann
17 Machine Learning in Untargeted Metabolomics Experiments . . . . . . . . . . . . . . . . 287
Joshua Heinemann
18 Dynamic 13C Labeling of Fast Turnover Metabolites for Analysis
of Metabolic Fluxes and Metabolite Channeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . 301
Mary Abernathy, Ni Wan, Wenqing Shui, and Yinjie J. Tang
19 Genome-Scale 13C Fluxomics Modeling for Metabolic Engineering
of Saccharomyces cerevisiae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 317
David Ando and Héctor Garcı́a Martı́n
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347
Contributors
PATRÍCIA VERARDI ABDELNUR Brazilian Agricultural Research Corporation, Embrapa

Agroenergy, Brası́lia, DF, Brazil; Institute of Chemistry, Federal University of Goiás,
Goiânia, GO, Brazil
MARY ABERNATHY Department of Environmental, Energy and Chemical Engineering,
Washington University in St. Louis, St. Louis, MO, USA
JOÃO RICARDO MOREIRA DE ALMEIDA Brazilian Agricultural Research Corporation,
Embrapa Agroenergy, Brası́lia, DF, Brazil; Graduate Program on Chemical and
Biological Technologies, Institute of Chemistry, University of Brası́lia, Brası́lia, DF, Brazil
DAVID ANDO Biological Systems and Engineering Division, Lawrence Berkeley National
Laboratory, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA
JOSÉ ANTÔNIO DE AQUINO RIBEIRO Brazilian Agricultural Research Corporation,
Embrapa Agroenergy, Brası́lia, DF, Brazil
EDWARD E.K. BAIDOO Biological Systems and Engineering Division, Lawrence Berkeley
National Laboratory, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA
CORAL BARBAS Centro de Metabolomica y Bioanállisis (CEMBIO), Facultad de Farmacia,
Universidad CEU San Pablo, Campus Monteprı́ncipe, Madrid, Spain
DERYLIZE BEUKES Human Metabolomics, North-West University, Potchefstroom,
South Africa
BARBARA BOJKO Department of Chemistry, University of Waterloo, Waterloo, ON, Canada
YUPING CAI Interdisciplinary Research Center on Biology and Chemistry, Shanghai
Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, P. R. China;
University of Chinese Academy of Sciences, Shanghai, P. R. China
CHRISTIANE GONÇALVES CAMPOS Brazilian Agricultural Research Corporation, Embrapa
Agroenergy, Brası́lia, DF, Brazil; Institute of Chemistry, Federal University of Goiás,
Goiânia, GO, Brazil
SIMON A. COBBOLD Department of Biochemistry and Molecular Biology, Bio21 Institute of
Molecular Science and Biotechnology, The University of Melbourne, Parkville, Victoria,
Australia
HÉCTOR GARCÍA MARTÍN Biological Systems and Engineering Division, Lawrence Berkeley
JOSHUA HEINEMANN Environmental Genomics and Systems Biology, Lawrence Berkeley
CHIJIOKE J. JOSHUA Biological Systems and Engineering Division, Lawrence Berkeley
JAY D. KEASLING Biological Systems and Engineering Division, Lawrence Berkeley National
Laboratory, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA;
Department of Chemical Engineering, University of California, Berkeley, CA, USA;
Department of Bioengineering, University of California, Berkeley, CA, USA; Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kgs
Lyngby, Denmark; Center for Synthetic Biochemistry, Institute of Synthetic Biology
Research, Shenzhen Institutes of Advanced Technologies, Shenzhen, Guangdong, China
YUJING LI College of Life Sciences, Nankai University, Tianjin, China
ix
x Contributors
ZHUCUI LI iHuman Institute, ShanghaiTech University, Shanghai, China; Tianjin

Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, China;
University of Chinese Academy of Sciences, Beijing, China
DU TOIT LOOTS Human Metabolomics, North-West University, Potchefstroom,
South Africa
ÁNGELES LÓPEZ-GONZÁLVEZ Centro de Metabolomica y Bioanállisis (CEMBIO), Facultad
de Farmacia, Universidad CEU San Pablo, Campus Monteprı́ncipe, Madrid, Spain
MALCOLM J. MCCONVILLE Department of Biochemistry and Molecular Biology, Bio21
Institute of Molecular Science and Biotechnology, The University of Melbourne, Parkville,
Victoria, Australia
FATEMEH MOUSAVI Donnelly Centre for Cellular and Biomolecular Research, University of
Toronto, Toronto, ON, Canada; Department of Chemistry, University of Waterloo,
Waterloo, ON, Canada
TRENT R. NORTHEN DOE Joint Genome Institute, Walnut Creek, CA, USA;
Environmental Genomics and Systems Biology Division, Lawrence Berkeley National
Laboratory, Berkeley, CA, USA
JANUSZ PAWLISZYN Department of Chemistry, University of Waterloo, Waterloo, ON,
Canada
ILSE DU PREEZ Human Metabolomics, North-West University, Potchefstroom, South Africa
BETANIA FERRAZ QUIRINO Brazilian Agricultural Research Corporation, Embrapa
Agroenergy, Brası́lia, DF, Brazil; Graduate Program on Chemical and Biological
Technologies, Institute of Chemistry, University of Brası́lia, Brası́lia, DF, Brazil; Genomic
Sciences and Biotechnology Program, Universidade Catolica de Brasilia, Brası́lia, DF,
Brazil
DAVID ROJO Centro de Metabolomica y Bioanállisis (CEMBIO), Facultad de Farmacia,
Universidad CEU San Pablo, Campus Monteprı́ncipe, Madrid, Spain
WENQING SHUI iHuman Institute, ShanghaiTech University, Shanghai, China
TAMI L. SWENSON Environmental Genomics and Systems Biology Division, Lawrence
Berkeley National Laboratory, Berkeley, CA, USA
RALF TAKORS Institute of Biochemical Engineering, University of Stuttgart, Stuttgart,
Germany
YINJIE J. TANG Department of Environmental, Energy and Chemical Engineering,
Washington University in St. Louis, St. Louis, MO, USA
VERONICA TEIXEIRA BENITES Biological Systems and Engineering Division, Lawrence
Berkeley National Laboratory, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville,
CA, USA
ATTILA TELEKI Institute of Biochemical Engineering, University of Stuttgart, Stuttgart,
Germany
NI WAN Department of Environmental, Energy and Chemical Engineering, Washington
University in St. Louis, St. Louis, MO, USA
GEORGE WANG Biological Systems and Engineering Division, Lawrence Berkeley National
Laboratory, Berkeley, CA, USA; Joint BioEnergy Institute, Emeryville, CA, USA
YUXIN YIN Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking
University Health Science Center, Beijing, China
JUNTUO ZHOU Institute of Systems Biomedicine, School of Basic Medical Sciences, Peking
University Health Science Center, Beijing, China
ZHENG-JIANG ZHU Interdisciplinary Research Center on Biology and Chemistry, Shanghai
Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, P. R. China;
University of Chinese Academy of Sciences, Shanghai, P. R. China
Chapter 1
Microbial Metabolomics: A General Overview

Edward E.K. Baidoo
Abstract
In the biosciences, there has been growing interest in the elucidation of gene function. Consequently,
metabolomics has garnered a lot of attention of late due to its provision of metabolic information pertaining
to both function and phenotype. Furthermore, when metabolomics data is integrated with other “omics”
data, precise characterization of metabolic activity can be achieved. This chapter briefly introduces a few
important aspects of the metabolome, the challenges faced when acquiring metabolomic information and
the steps that are necessary to overcoming them. This chapter also briefly covers current analytical
technologies and some microbial metabolomic applications.
Key words Mass spectrometry, Metabolomics, Microbial, LC-MS, GC-MS, CE-MS, Metabolic
quenching, Metabolite extraction, Data analysis, Microbial communities, Human disease
Progression in biology related scientific research has always

depended on technological advances, which has enabled the study
of living organisms and the components the that are necessary for
life. Over past 70 years, concerted efforts have been made to
understand biological systems through the elucidation of chemical
structure, function, development, adaptation, and evolution. In the
early years, biochemical information was thought to flow direction-
ally from the transcription of genomic DNA to mRNA, the transla-
tion of the latter to proteins, which subsequently produce
metabolites [1]. From the 1980s onward, molecular biology
“omics” technologies explored the geneses, interactions, associa-
tions, and roles of various types of molecules that make up an
organism, to provide a deeper understanding of the principles of
life. This led to the discovery that the flow of biochemical informa-
tion is not only unidirectional but rather a complex system of
interactions between DNA, RNA, protein, and metabolite [1],
with the metabolome (the complete set of metabolites formed by
the cell in association with metabolism [2]) being able to influence
the preceding “omes” by many forms of regulatory control
Edward E.K. Baidoo (ed.), Microbial Metabolomics: Methods and Protocols, Methods in Molecular Biology, vol. 1859,
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Fig. 1 “Omics” overview. This is an illustration of the flow of molecular information from genes to metabolites
to function and phenotype, the interactions between the “omes” and the “omics” techniques used to measure
them. This diagram was adapted from Hollywood et al. [10]
mechanisms (feedback loops, chromatin regulation, global

biological regulation, etc.) (Fig. 1) [3–7].
Metabolites mediate regulation of microbial metabolism in
response to continual environmental changes [8]. These regulatory
mechanisms allow microorganisms to adapt to various ecological
situations (e.g., changes to nutrient supply, etc.) but must also be
reversible as the environment can revert to its original state. Since
metabolites show the fastest response times to changes in the
environment, it is clear to see why they are utilized in numerous
regulatory mechanisms. Aside from regulation, metabolites are
mainly known for participating in biochemical reactions (with envi-
ronmental nutrients) that are involved in maintaining the living
state of cells (metabolism) to generate energy (catabolism) for
Microbial Metabolomics: A General Overview 3
synthesis of the building blocks of cellular materials such as nucleic

acids, proteins, polysaccharides, and lipids (anabolism) [9]. These
biochemical reactions are also responsible for producing secondary
metabolites such as alkaloids and terpenoids, which are involved in
cell function and survival [9]. Thus, metabolites play a major role
in the response of a biological system to abiotic or biotic perturba-
tions, thereby linking genotype to phenotype (which is generated
by the flux of metabolites within a biological system) [8, 10]. It is
for these reasons that molecular information from the metabolome
is crucial to understanding metabolic networks and determining
genetic function.
Metabolomics, the newest member of the “omics” family of
techniques, seeks to measure the low molecular weight chemical
products of enzyme-catalyzed reactions (metabolites) in biological
systems (e.g., cells, compartments, tissues, organisms) at specific
points in time [2, 9]. The importance of metabolomics to
biological research is highlighted by the fact that biochemical infor-
mation from genes, transcripts, and proteins do not always corre-
late, due to not all genes being under transcriptional control and
the incomplete prediction of the proteome from the transcriptome
owing to post-translational modifications [9]. However, the bio-
polymeric nature of genes, transcripts, and proteins makes their
analyses easier than that of the metabolome because they encode
information based on sequences of well-defined monomeric
nucleotides and amino acids [1, 10]. By contrast, metabolites are
distinct chemical entities from biochemical transformations, which
complicates both the measurement and data analysis processes. At
present, there is no single analytical method that can measure the
entire metabolome of an organism and to achieve something close
to that, multiple methods would have to be used. Sample prepara-
tion, which is arguably the most critical step in metabolomics
experiments, is still challenging. Both the chemical diversity within
the metabolome and the complexity of cell wall structure across the
microbial diaspora complicate this process. Therefore, it is currently
not possible to extract all metabolites from a given biological
system with the same level of efficiency. This is further compounded
by the rapid change in metabolite levels due to their constant
formation and transformation in biochemical reactions within a
cell. Furthermore, some metabolites are required in small amounts
by their prospective reactions (e.g., signaling molecules such as
cyclic adenosine monophosphate, cAMP, which plays a role in
regulating gene expression in catabolite repression [11, 12]),
while others are needed in large amounts (e.g., adenosine triphos-
phate, ATP, which provides energy for physiological processes upon
hydrolysis) [2]. Thus, stopping (i.e., quenching) these biochemi-
cal reactions quickly is critical to capturing accurate, quantita-
tive metabolic information. Additionally, efficient extraction of
metabolites from biological sample matrices with appropriate
Fig. 2 Popularity of MS and NMR from 2000 to 2017. The popularity was based
on the number of publications obtained from Web of Science. The search criteria
are as follows: (1) metabolomics and mass spectrometry and years, and
(2) metabolomics and NMR and years. NMR was used instead of nuclear
magnetic resonance because it generated more publications
extraction agents (e.g., methanol, chloroform, water, etc.) is neces-

sary for obtaining quantitative metabolite measurements.
The two major measurement techniques used in microbial meta-
bolomic analyses are nuclear magnetic resonance (NMR) and mass
spectrometry (MS). Although the former can provide high-through-
put metabolic fingerprinting (i.e., analysis providing a fingerprint of
metabolites produced by a cell [2]), unambiguous identification of
metabolites through comprehensive structural elucidation, and non-
destructive metabolite measurements, it is rather costly, lacks sensi-
tivity, and is semiquantitative. On the other hand, MS-based
technologies can provide high-throughput accurate mass determina-
tion and structural elucidation, while operating at high sensitivities,
enabling the use of quantitative metabolite measurements. Further,
MS instrumentation can also be available at much lower costs,
depending on the level of analytical resolution required. It is for
these reasons that MS remains the most popular metabolomic mea-
surement technique to date (Fig. 2). MS systems are normally cou-
pled to separation devices via electron ionization (EI) or electrospray
ionization (ESI). Separation technologies such as liquid chromatog-
raphy (LC separates hydrophobic and hydrophilic compounds), gas
chromatography (GC separates volatile hydrophobic compounds),
and capillary electrophoresis (CE efficiently separates hydrophilic
compounds) improve metabolite identification and quantitation by
reducing the complexity of sample matrices. These separation tech-
nologies provide complementary (orthogonal) methods, and
because their devices are relatively inexpensive, are often purchased
together with MS instrumentation.
Over the past 14 years, there have been significant advances in
separation and MS system integration (i.e., better MS ion source
interfacing), chromatographic separation efficiency (e.g., UHPLC

and 2D-GC), MS resolution (e.g., time-of-flight and orbitrap),
detector sensitivity, data acquisition, system robustness, and
computational power. Furthermore, the multiplexing of LC tech-
niques has enabled scientists to achieve target-based metabolite
profiling across many different classes of organic compounds
[13]. These advancements have led to better metabolite identifica-
tion, quantitation, throughput, and reproducibility, and therefore
greater capture of metabolite information. But to achieve the
desired goal of global metabolite profiling, these boundaries need
to be pushed further in addition to developments in efficient auto-
mated microbial metabolite sample preparation (i.e., by improving
sampling and providing better, universally applied, metabolic
quenching and metabolite extraction methodologies).
Data analysis is still challenging though. While there are numer-
ous computational tools to automate the process, issues regarding
widely varying metabolite abundances, reliable detection, and mass
spectral identification [14], make it difficult to achieve precise
metabolic feature extraction and, therefore, rapid automated data
analysis. As more data is being generated, data analysis workstations
are constantly requiring upgrades in software, processing power,
and RAM management to meet these demands.
Untargeted and target metabolomics are the two major strate-
gies used to obtain metabolic information. In untargeted metabo-
lomics experiments, the available metabolite information
is obtained from a biological sample via comprehensive feature
extractions of measurable analytes [1] and treated with the relevant
statistical analyses to identify metabolic features that are unique
among sample types. This approach requires excellent chro-
matographic separation and high-resolution accurate mass spec-
trometry (i.e., HRAMS such as from the quadrupole time-of-
flight and orbitrap). The resulting accurate mass metabolic features
can then be identified by searching against known MS databases
and/or MS/MS spectral libraries. Even though untargeted meta-
bolomics can take a considerable amount of time and effort, it can
be used very effectively in discovery experiments and to identify
diagnostic biological markers. Targeted metabolomics, however,
measures specific groups of known metabolites (e.g., classes of
organic compounds, biosynthetic pathways, etc.), highlighting
only those portions of metabolomics data that are meaningful to a
research study, thus, providing more immediate answers to
biological questions. Other advantages of targeted metabolomics
experiments are that they can provide quantitative information (i.e.,
via external calibration curves and/or internal standardization), do
not require expensive HRAMS or elaborate chromatographic
separations, can be used with high-throughput separation-MS
methodologies, and achieve faster data analysis times. For these
reasons, targeted metabolomics has emerged as the favored
Fig. 3 Popularity of metabolomics in the fields of human biology, plant biology,

and microbiology from 2000 to 2017. The popularity was based on the number of
publications obtained from Web of Science. The search criteria are as follows:
(1) metabolomics and human and year, (2) metabolomics and plant and year,
and (3) metabolomics and microbial and year
approach of most laboratories. Algorithms are now able to overlay

metabolomics data on top of metabolic pathways. Furthermore,
the data from these approaches can be modeled to reveal fluxes in
metabolic pathways and to predict the outcomes of future
biological experiments.
At present, metabolomics data is more widely applied to human
related studies, followed by plant studies, and then microbiological
studies (Fig. 3). In systems microbiology research, multi-omics
technologies (including metabolomics) are being used to charac-
terize metabolic networks and microbial interactions. Metabolite
data from these experiments can also being leveraged to provide
signaling pathway information to strengthen predictive mathemat-
ical models. Advancements in systems biology “omics” technolo-
gies are leading to developments in synthetic biology research. In
synthetic biology studies, metabolomics is enabling researchers to
identify bottlenecks in engineered pathways (through pathway flux
assessments), identify components of cellular toxicity (e.g., accu-
mulating toxic intermediates or products), and assess strain varia-
tion (e.g., carbon utilization and energy production). In addition
to this, when metabolomics is utilized with the other “omics”
technologies and microbial physiology is incorporated in the design
of an experiment, the information gained can be used to identify
the microbial regulatory mechanisms that circumvent engineered
biosynthetic pathways [15]. This approach, together with machine
learning, can improve the rational design, development, and opti-
mization of engineered strains.
In the study of infectious human diseases, metabolomics is
helping researchers to identify biomarkers of infection and new
antimicrobial compounds. By understanding the mechanisms
involved in microbial infection, pathogenicity, and immunity,

researchers can develop more effective strategies to reduce the
occurrence of infectious disease. For the noninfectious category,
metabolomics is being used to find biomarkers of microbial
induced disease (diabetes, cancer, atherosclerosis, etc.) from micro-
organisms and human related tissues and bodily fluids. A deeper
learning of host–microbe interaction will provide a greater under-
standing of the factors influencing microbial growth and popula-
tion maintenance within host organisms. It will also allow us to
understand how the actions of these factors are countered by the
host immune system [16] and will be necessary to establish reliable
cultured isolates from patient specimens, which is required to
develop and hence implement effective treatments.
Metabolomics studies of the human gut microbiome are
enabling researchers to identify mechanisms that predispose indivi-
duals to disease and the roles the microbiome plays in drug metab-
olism, aging, and cancer. In the study of environmental microbial
communities, metabolomics is being used in combination with
other “omics” technologies to characterize community structure
and functional diversity. Knowledge from these studies can be used
to unravel microbial interactions within communities, the environ-
ment, and host organisms to address environmental challenges and
human disease.
Metabolomics has come a long way since its inception and is
now routinely applied to many biological experiments. As research-
ers continue to push the envelope in metabolomic technological
advancements, measurements are likely to become more reliable,
readily available, and comprehensive, and could be used to obtain
global metabolite information on a regular basis. Further, the
integration of multi-omic data (that includes high-quality metabo-
lomic measurements) can be used to generate accurate predictive
models, with the potential to revolutionize the biological sciences.
Acknowledgments
The authors would also like to acknowledge that this work was part
of the DOE Joint BioEnergy Institute (http://www.jbei.org) sup-
ported by the US Department of Energy, Office of Science, Office
of Biological and Environmental Research, through contract
DE-AC02-05CH11231 between Lawrence Berkeley National
Laboratory and the US Department of Energy.
References
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9. Baidoo EEK, Benke PI, Keasling JD (2012) 449:7164–7164
Mass spectrometry-based microbial
Part I
Microbial Metabolomics, Metabolism, and Physiology

Chapter 2
Mass Spectrometry-Based Microbial Metabolomics:

Techniques, Analysis, and Applications
Edward E.K. Baidoo and Veronica Teixeira Benites
Abstract
The demand for understanding the roles genes play in biological systems has steered the biosciences into
the direction the metabolome, as it closely reflects the metabolic activities within a cell. The importance of
the metabolome is further highlighted by its ability to influence the genome, transcriptome, and proteome.
Consequently, metabolomic information is being used to understand microbial metabolic networks. At the
forefront of this work is mass spectrometry, the most popular metabolomics measurement technique. Mass
spectrometry-based metabolomic analyses have made significant contributions to microbiological research
in the environment and human disease. In this chapter, we break down the technical aspects of mass
spectrometry-based metabolomics and discuss its application to microbiological research.
Key words Mass spectrometry, Metabolomics, Microbial, LC-MS, GC-MS, CE-MS, Metabolic
quenching, Metabolite extraction, Data analysis, Microbial communities, Human disease
1 Introduction
In recent years, there has been a push in the biosciences to charac-

terize the functions and phenotypes associated with genes. In this
regard, the metabolome (i.e., the complete set of metabolites from
a cell, compartment or tissue [1]) provides both functional and
phenotypic information since metabolites are the small molecule
products of enzyme-catalyzed metabolic reactions. The measure-
ment of metabolite profiles (metabolomics) can be achieved by
spectra obtained from mass spectrometric analyses. Mass spectro-
meters either induce or facilitate ionization of metabolites and
separate them based on the mass-to-charge ratios of their charged
forms. To date, mass spectrometry (MS) is the most popular mea-
surement technique for metabolomics studies due to its structural
elucidation capability, high sensitivity, and quantitative ability.
When MS is coupled to chromatographic or electrophoretic sepa-
ration, these features are further enhanced. However, since each
metabolite is structurally unique, at present, there is no single
Edward E.K. Baidoo (ed.), Microbial Metabolomics: Methods and Protocols, Methods in Molecular Biology, vol. 1859,
https://doi.org/10.1007/978-1-4939-8757-3_2, © Springer Science+Business Media, LLC, part of Springer Nature 2019
11
12 Edward E.K. Baidoo and Veronica Teixeira Benites
analytical method that can resolve all metabolites. Additionally,

metabolomics sample preparation is further complicated by the
varying rates at which metabolites are formed and transformed
and the chemical diversity within the metabolome. Consequently,
there are many metabolomics and sample preparation methods that
are designed to capture either a broad range of metabolites (e.g.,
for untargeted metabolomics experiments) or focus on a specific
subset (e.g., for targeted metabolomics experiments). Metabolo-
mic data mining and integration can also be challenging due to
varying metabolite abundances resulting from the data acquisition
process [2]; and while recent developments in data analysis tools are
designed to correct issues concerning variation and identification,
obtaining precise metabolic feature extraction can still be difficult.
Thus, the future of mass spectrometry-based metabolomics will
most likely center around resolving these issues and automating
much of the data acquisition and analysis processes.
2 Metabolic Quenching and Metabolite Extraction
Preparation of biological samples is of the upmost importance to

analytical measurements as it can preserve the metabolic integrity of
sampled biomass. Strategies involving efficient metabolic quench-
ing and metabolite extraction are necessary to capture accurate
metabolite information and should be determined during the
design of the experiment (simple approaches with the least number
of steps will often yield the best measurement precision). Metabolic
quenching can be described as the cessation of all cellular activity
that is governed by proteins and nucleic acids. It is achieved by
dramatically changing the cell environment through the process of
denaturation, in which proteins and nucleic acids lose their native
structure (i.e., quaternary, tertiary, and secondary structures)
[3, 4], by the application of a quenching agent (i.e., an external
stress such as temperature, pH, or a chemical such as an organic
solvent). To understand the immediate impact metabolic quench-
ing has on the metabolome, it is important to consider the roles
proteins play in the cell.
Proteins are required to maintain the structure, function, bio-
chemical interactions, regulation, and transport function of a cell
and can be generally categorized as structural proteins, regulatory
proteins, transport proteins, and enzymes. Structural proteins are
the largest class of proteins and, as suggested by their name, confer
rigidity to cell components and are frequently involved in
motility [3]. Transport proteins aid the passage of nutrients, meta-
bolites, and other proteins in and out of the cell via the cell mem-
brane. These proteins have a high degree of specificity for the
substance being transported [5]. They are often used to excrete
metabolites that are underutilized or toxic to the cell to prevent the
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Mass Spectrometry-Based Microbial Metabolomics 13
intracellular accumulation of these compounds. Regulatory pro-

teins can bind to determined sequences on a DNA strand (e.g.,
TATA binding protein) to turn genes on and off, thus, playing an
important role in gene expression [6]. Lastly, enzymes are directly
responsible for the biochemical interactions and molecular trans-
formations in a cell. Enzyme catalysis directly results in the genera-
tion of a new metabolite or an intermediate to a final compound
from a metabolic pathway in a cell. Enzymatic activity depends on
the availability of substrates and cofactors and can also be affected
by their own products and the products of other enzyme catalyzed
reactions via feedback and feedforward cellular control mechan-
isms. Enzymes can recompose themselves after catalyzing sub-
strates into products, which not only maintains their activity but
cell metabolism as well. While most researchers often think of
enzymes when designing sample preparation procedures, it is clear
to see the importance of all classes of proteins to maintaining
metabolism and hence the metabolome and why quenching their
activities negatively impacts the metabolic processes in a cell.
High metabolic quenching efficiency is crucial to obtaining
instantaneous metabolic information. However, this is often diffi-
cult to achieve due to the rapid turnover of certain metabolites,
some of which have half-lives of 1 s and others that are inter-
mediates of several metabolic reactions [1]. Furthermore, the
quenching agent needs to rapidly transit the cell membrane barrier
and affect the intracellular environment, which provides another
major obstacle. Since microorganisms have varying morphologies,
it also is difficult to devise a single effective quenching and metabo-
lite extraction strategy for them all. Some of the fastest quenching
methods directly transfer the microbial culture to a quenching
agent, followed by the separation of biomass from the culture
medium by either centrifugation or filtration. The disadvantage of
this is that intracellular metabolites from microorganisms with weak
cell walls, such as gram-negative bacteria, can be leaked from the
cell [1]. On the contrary, Koning and Dam found evidence to
suggest that S. cerevisiae cells do not leak metabolites when the
culture is quenched with 40 C methanol [7]. Methods that
separate biomass from the culture medium first, on average, do
not quench metabolism as efficiently but are not as prone to
metabolite leakage. In the past, this has been especially true of
centrifugation, whereas fast filtration can provide good quenching
efficiency [8, 9]. In any case, the goal of the research study (and,
therefore, the type of metabolites targeted) will dictate the type of
quenching strategy to use. Metabolic quenching efficiency is often
determined via the adenylate energy charge (i.e., [ATP] + 0.5
[ADP]/[ATP] + [ADP] + [AMP]) as ATP is rapidly utilized by
the cell as soon as it is formed [4]. Since the energy charge of most
cells falls within 0.8–0.95, obtaining values within this range nor-
mally indicates that the quenching process is efficient [4].
Fig. 1 Sample preparation workflow. Biomass can be separated from the culture medium before or after
quenching and extraction. Prepared samples are then run on the data acquisition system
Extracellular metabolites can simply be obtained by separating

the microbial biomass from the culture medium (by centrifugation
or filtration) and collecting the latter (followed by the addition of
quenching agents if extracellular enzymes are suspected) (Fig. 1).
Since most proteins are found intracellularly or bound to cell
membranes, metabolic quenching of the broth is not as significant,
but it is still recommended by many researchers. The exometabo-
lome can provide metabolic footprints of excreted metabolites
[10], whereas the endometabolome (intracellular metabolites)
yields metabolic fingerprints of metabolites produced by a
cell [1]. While these approaches are normally qualitative, high
quality extra- and intracellular extracts can be used for quantitative
experiments. Of the two, however, it is far more difficult to extract
intracellular metabolites.
To extract metabolites from a cell, factors influencing cell lysis
such as permeability, disruption, and degradation of the cell mem-
brane and wall should be considered. It is, therefore, important for
researchers to understand the physical characteristics and chemical
composition of the microbial cell membrane and wall under study,
as they will confer different barrier strengths to each microorgan-
ism. For example, gram-positive bacteria are more difficult to lyse
than gram-negative as they contain a thick peptidoglycan layer
outside of the cell membrane. Further, Yeast cells are also difficult
to lyse because they contain a thick cell wall comprised of manno-
proteins, glucans, and chitin. In these cases, harsher extraction
agents may be required. Organic solvents are the most widely
used extraction agents due to their affinity for most cell membrane
structures, as they enable cell permeabilization and the efficient
release of cell contents. Additionally, knowing the chemical and
physical properties of the analytes of interest will help to improve
extraction efficiency (i.e., the extent to which metabolites are recov-
ered from a cell) as methods can be tailored to those metabolites.
For example, in methanol–water–chloroform extraction of micro-
bial metabolites, methanol and water can interact with polar meta-
bolites while chloroform interacts with the nonpolar fraction
[11–15]. The multipurpose nature of methanol and chloroform
allows them to be used to disrupt cell membranes and quench
metabolism. As well as being used as quenching agents, most
organic solvents tend not to degrade metabolites because of their
pH neutrality and weak oxidizing capabilities. However, when they
are used in conjunction with extreme pH variation (e.g., via per-
chloric acid) and high temperatures (e.g., boiling ethanol), meta-
bolites could be degraded or deformed, leading to reduced yields.
Of the numerous extraction methods, methanol-based extraction
procedures appear to be the most popular for intracellular metabo-
lites. This is due to methanol’s quenching and extraction (causing
cell lysis and interacting with polar to mid-polar metabolites) cap-
abilities, and lack of artifact production [2]. Cell lysis can also be
brought about via enzymatic (e.g., lysozyme) and mechanical (e.g.,
sonication and bead-beating) cell wall/membrane disruption tech-
niques [1, 2]. Enzymatic techniques should not be used with
quenching agents as enzyme activities can be arrested. Care should
also be taken with mechanical disruption techniques, as they can
often raise the temperature of sample lysates, leading to the possible
degradation of thermally labile compounds (in such cases the sam-
ple disruption technique can be performed at low temperatures).
Thus, efficient metabolite extraction from microbial cells is
achieved by complete cell lysis, strong analyte-extracting agent
interaction, and minimal loss and degradation of analytes.
Clean-up strategies can be employed to enhance the overall
quality of the metabolite extract by reducing its complexity and
ensuring reproducibility by minimizing interferences from highly
abundant sample matrix ions [16]. There are numerous examples of
metabolite sample cleanup strategies. Molecular weight cut-off fil-
tration (MW/CO) is a separation process using a membrane filter
under centrifugation to remove macromolecules from a biological
extract. Liquid-liquid extraction uses solvent phase separation to
purify metabolites and remove interfering compounds. The best
example of this is the previously described methanol–water–chloro-
form extraction procedure [11–15]. Solid phase extraction (SPE)
allows the retention of metabolites on a solid stationary phase
support (via chromatography), while unretained substances are
washed off. Metabolites can then be eluted from the SPE column/
cartridge via a solvent or solution it has a strong interaction with
[16, 17]. Solid phase microextraction (SPME) purifies metabolites

by their adsorption on a fiber coated with an extraction phase.
Retained metabolites are transferred to the injection port of the
separating device, where they are subsequently desorbed and trans-
ferred to the mass spectrometer [18]. Cleanup strategies are becom-
ing more desirable to analytical laboratories as they become part of
automated sample preparation platforms that are helping to stream-
line metabolomic workflows and improve the consistency of analyt-
ical measurements [18].
3 Analytical Separation Technologies
3.1 Introduction It is without doubt that separation, whether chromatographic or

to Separation electrophoretic, is one of the most important components of meta-
bolomic measurements. By separating metabolites in in real-time,
they will elute from the separating device at different times, thereby
reducing the complexity of biological extracts before MS detection.
Without separation, all sampled metabolites would enter the MS
simultaneously, making it difficult to distinguish between those of
similar chemical structure (e.g., isomeric and isobaric compounds).
Further, when multiple unseparated compounds enter the MS ion
source, they compete for available charges [19]. Consequently, the
most ionizable volatile compounds yield the best detector responses
and dominate the acquired mass spectra. This is especially true of
sample matrix components involved in the now infamous ion sup-
pression phenomenon. Such components affect the detection capa-
bility, precision, and accuracy of analytical methods. Separation
reduces these effects and, therefore, improves upon quantitation.
Chromatography is the most popular separation technique in
metabolomic analyses and relies on the differential distribution and
equilibration of sample components (according to their distribution
coefficients) between two phases [20]. The phases employed are
typically a moving mobile (liquid or gas) phase and a stationary
phase adsorbent (i.e., microscopic particles on which analytes are
retained by physicochemical interactions). The mobile phase carries
components from the sample through the absorptive stationary
phase material. The extent of analyte partitioning between the
phases determines the length of time spent in the chromatography
column. Individual components are detected as they leave the col-
umn and the eluted compound is observed as a Gaussian distribu-
tion plot of the detector response as a function of elution time and
resides within a chromatogram (a graphical representation of detec-
tor response versus time for the duration of the analytical run). The
area under the Gaussian distribution plot is used to calculate the
peak area for quantitative purposes. The amount of time a com-
pound is retained in chromatography column is known as the reten-
tion time (RT). Acquisition and analysis software typically measure
the RT from sample injection to the elution of the compound from

the column and is normally measured at the apex of a chro-
matographic peak. The RT helps to differentiate between com-
pounds with similar structures (e.g., isomers) and, when coupled
to MS detection, the two can be used to reliably determine the
identity of a compound.
Liquid chromatography (LC) separation is dependent on inter-
actions between the analyte, stationary phase, and the liquid mobile
phase. A change in the composition of the liquid mobile phase can
alter analyte chemistry, interaction, and separation. Gas chroma-
tography (GC) separation is dependent on analyte volatility and
interaction with the stationary phase. An unfortunate consequence
of the GC temperature gradient (which is employed to encourage
volatilization), is its adverse effects (i.e., degradation and/or
decomposition) on thermally labile compounds. Low GC tempera-
tures are needed to analyze these compounds. Where LC excels is in
its versatility. LC is generally used to separate nonpolar, polar, and
charged analytes, which span the entire spectrum of functionalities
within the metabolome, whereas GC is ideal for nonpolar, volatile
analytes. This is, in part, due to the broad stationary phase portfolio
of LC, which spans numerous chromatography methods to suit any
metabolomic application. LC can cover a wider range of metabolite
classes without the need for the time-consuming analyte derivati-
zation steps (which are required to render nonvolatile compounds
volatile) found in GC methods. Conventional GC methods, how-
ever, provide better separation efficiency and reproducibility than
their LC counterparts. Further, the simple construction of a GC
system (i.e., a column housed in an oven) creates a very cost-
effective, robust, easy-to-use separation device. GC is also easier
to couple to MS, since it utilizes a temperature-controlled carrier
gas mobile phase as opposed to the liquid mobile phase of
LC. Hence, volatiles are transferred directly in the gas phase from
GC to MS, thereby simplifying the coupling of these two systems.
On the other hand, LC requires elaborate evaporation/desolvation
procedures to produce gaseous ions before they enter the MS.
Capillary electrophoresis (CE) utilizes an applied electric field
to separate charged analytes, by the process electrophoresis,
through a fused silica capillary (FSC) filled with a conductive liquid
medium (e.g., a buffer or electrolyte) [2]. Migration and elution
order largely depend on the charge-to-size ratios of analytes, which
is complementary to MS detection. Consequently, the electropho-
retic mobility of an analyte relies on its charge state (q), its radius
(r), and the viscosity of the liquid separation medium (η), which can
be described as follows: μEP ¼ q/6πηr [21]. Like other electropho-
retic techniques, size and charge play key roles in analyte elution
order, with smaller highly charged ions migrating faster than larger
ions with lower charge states [2]. The time it takes for the analyte to
migrate through the FSC and be detected is known as the
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into a relatively stable equilibrium, would arrest development.
Evidently organic evolution, whether individual or general, must
always and everywhere have been subordinate to these physical
necessities. Though natural selection, beginning with minute
portions of protoplasm, must all along have tended to establish a
molecular composition apt to undergo this differentiation of surface
from centre to the most favourable extent, yet it must all along have
done so while controlled by this process of direct equilibration.
Contrariwise, the many and great unlikenesses among the dermal
structures of creatures inhabiting the same element, cannot be
ascribed to any such cause. The contrasts between naked and
shelled Gastropods, between marine Worms and Crustaceans,
between soft-skinned Fishes and Fishes in armour like the
Pterichthys, must have been produced entirely by natural selection.
Environing forces are, as before, the ultimate causes; but the forces
are now not so much those exercised by the medium as those
exercised by the other inhabitants of the medium; and they do not
act by modifying the surface of the individual, but by killing off
individuals whose surfaces are least fitted to the requirements: thus
slowly affecting the species. Still the dermal skeleton bristling with
spines, which protects the Diodon or the Cyclichthys from enemies it
could not escape, comes within the general formula of an outer
tissue differentiated from inner tissues by the outer actions to which
the creature is exposed: the differentiation having gone on until
there is equilibrium between the destructive forces to be met and
the protective forces which meet them.
If we venture to apportion the respective shares which mediate
and immediate actions have had in differentiating outer from inner
tissues, we shall probably not be far wrong in ascribing that part of
the result which is alike in all animals, mainly to the direct actions of
their media, while we ascribe the multitudinous unlikenesses of the
results in various animals, partly to the indirect actions of the media,
and partly to the indirect actions of other animals by which the
media are inhabited. That is to say, while assigning the specialities of
the differentiations to the specialities of converse with the agencies
in the environment, most of them organic, we may assign to the
constant and universal converse with its inorganic agencies, the
universal characteristic of tegumentary structures—their growth
outwards from a layer lying below the surface which continually
produces new substance to replace the substance worn away or cast
off.
Here let me add a piece of evidence which strengthens the
general argument, at the same time that it justifies this
apportionment. When ulceration has gone deep enough to destroy
the tegumentary structures, these are never reproduced. The
puckered surface formed where an ulcer heals, or where a serious
burn has destroyed the skin, consists of modified connective tissue,
which, as the healing goes on, spreads inwards from the edges of
the ulcer: some of it, perhaps, growing from the portions of
connective tissue that dip down between the muscular bundles. This
connective tissue is normally covered by the epidermis and thus
sheltered from environing actions. What has happened to it? It has
now become the outermost layer. And how does it comport itself
under its new conditions? It produces a superficial substance which
plays the part of the epidermis and grows outwardly. For since the
surface, subject to friction and exfoliation, has to be continually
renewed, there must be a continual reproduction of an outermost
layer from a layer beneath. That is to say, the contact of this deep-
seated tissue with outer agencies, produces in it some approach
towards that character which we find universally characterizes outer
tissue. But while we see under this exposure to the conditions
common to all integument, a tendency to assume the structure
common to all integument, we see no tendency to assume any of
the specialities of tegumentary structure: no rudiments of glands or
hair sacs make their appearance.
Analogous conclusions may be drawn respecting the processes of
differentiation by which from the outer layer nervous tissue and
finally a nervous system are evolved. Here, also, both direct and
indirect equilibration appear to have operated. Two reasons may be
assigned for the belief that the transformation of certain superficial
cells into sensitive cells was initiated by exposure to external stimuli.
The first is that, extremely unstable as protoplasm is, disturbances
received by the outer side of a specially-exposed cell could scarcely
fail to cause changes passing through it towards the interior mass of
the body, and that perpetual repetition of such changes would tend
to generate channels of easy transmission through the protoplasm.
The second reason is that, if we do not assume this process of
initiation but assume that survival of the fittest was the sole agency,
then no reason can be assigned why the nervous system should not
have been at the outset formed internally instead of being initiated
externally and then transferred to the interior: the roundabout
process would be inexplicable. At the same time the production of a
central nervous system by introversion of superficial sensitive cells
cannot be ascribed to the differentiating effects of external stimuli,
but must be ascribed to natural selection. No perpetual repetition of
outer disturbances would cause the sinking inwards, and covering
up, of the specially-sensitive area and the plexus below it. But it is
manifest that since these nervous structures, at once all-important
and easily injured, would be safer if removed from the surface,
survival of the fittest, continually preserving those in which they
were more deeply seated, would tend to produce an arrangement in
which all parts but the actual receivers of external stimuli became
internal.
Hence, contemplating generally these two fundamental
differentiations of inner from outer tissues, we may conclude that
though their first stages resulted from direct equilibration, their
subsequent and higher stages resulted from indirect equilibration.
CHAPTER VII.
DIFFERENTIATIONS AMONG THE OUTER TISSUES OF ANIMALS.
§ 291. The outer tissues of animals, originally homogeneous over

their whole surfaces, pass into a heterogeneity which fits their
respective parts to their respective conditions. So numerous and
varied are the implied differentiations, that it is impracticable here to
deal with them all even in outline. To trace them up through classes
of animals of increasing degrees of aggregation, would carry us into
undue detail.
Did space permit, it would be possible to point out among the
Protozoa, various cases analogous to that of the Arcella; which may
be described as like a microscopic Limpet, having a sarcode body of
which the upper surface has become horny, while the lower surface
with its protruding pseudopodia, retains the primitive jelly-like
character. That differentiations of this kind have been gradually
established among these minute creatures through the unlike
relations of their parts to the environment, is an inference supported
by a form which, while the rest of the body has a scarcely
distinguishable coating, “agrees with Arcella and Difflugia in having
the pseudopodia protrusible from one extremity only of the body.”
Many parallel specializations of surface among aggregates of the
second order might be instanced from the Cœlenterata. In the
Hydra, the ectoderm presents over its whole area no conspicuous
unlikenesses; but there usually exist in the hydroid polypes of
superior types, decided contrasts between the higher and lower
parts. While the higher parts retain their original characters, the
lower parts excrete hard outer layers yielding support and
protection. Various stages of the differentiation might be followed.
“In Hydractinia,” says Prof. Green, this horny layer “becomes
elevated at intervals to form numerous rough processes or spines,
while over the general surface of the ectoderm its presence is almost
imperceptible.” In other types, as in Cordylophora, it spreads part
way up the animal’s sides, ending indefinitely. In Bimeria it “extends
itself so as to enclose the entire body of each polypite, leaving bare
only the mouth and tips of the tentacles.” While in Campanularia it
has become a partially-detached outer cell, into which the creature
can retract its exposed parts.
But it is as needless as it would be wearisome to trace through
the several sub-kingdoms the rise of these multiform contrasts, with
the view of seeking interpretations of them. It will suffice if we take
a few groups of the illustrations furnished by the higher animals.
§ 292. We may begin with those modifications of surface which

subserve respiration. Though we ordinarily think of respiration as the
quite special function of a quite special organ, yet originally it is not
so. Little-developed animals part with their carbonic acid and absorb
oxygen, through the general surface of the body. Even in the lower
types of the higher classes, the general surface of the body aids
largely in aërating the blood; and the parts which discharge the
greater part of this function are substantially nothing more than
slightly altered and extended portions of the skin.
Such differentiations, marked in various degrees, are to be seen
among Mollusca. In the Pteropoda the only modification which
appears to facilitate respiration, is the minute vascularity of one part
of the skin. Higher types possess special skin-developments. The
Doris has appendages developed into elaborately-branched forms—
small trees of blood-vessels covered by slightly-changed dermal
tissues. And these arborescent branchiæ are gathered together into
a single cluster. Thus there is evidence that large external respiratory
organs have arisen by degrees from simple skin: as, indeed, they do
arise during the development of each individual having them. Just as
gradually as in the embryo a simple bud on the integument, with its
contained vascular loop, passes by secondary buddings into a tree-
like growth penetrated everywhere by dividing and subdividing
blood-vessels; so gradually has there probably proceeded the
differentiation which has turned part of the outer surface into an
organ for excreting carbonic acid and absorbing oxygen.
Certain inferior vertebrate animals present us with a like
metamorphosis of tissues. These are the Amphibia. The branchiæ
here developed from the skin, are covered with cellular epidermis,
not much thinner than that covering the rest of the body. Like it they
have their surfaces speckled with pigment-cells; and are not even
conspicuous by their extra vascularity—where they are temporary at
least. They facilitate the exchange of gases in scarcely any other
way than by affording a larger area of contact with the water, and
interposing a rather thinner layer of tissue between the water and
the blood-vessels. Those very simple branchiæ of the larval
Amphibia that have them but for a short time, graduate into the
more complex ones of those that have them for a long time or
permanently; showing, as before, the small stages by which this
heterogeneity of surface accompanying heterogeneity of function
may arise.
In what way are such differentiations established? Mainly, no
doubt, by natural selection; but also to some degree, I think, by the
inheritance of direct adaptations. That a portion of the integument
at which aëration is favoured by local conditions, should thereby be
led to grow into a larger surface of aëration, appears improbable.
Survival of those individuals which happen to have this portion of the
integument somewhat more-developed, seems here the only likely
cause.
§ 293. Among the conspicuous modifications by which the

originally-uniform outer layer is rendered multiform, are the
protective structures. Let us look first at the few cases in which the
formation of these is ascribable mainly to direct equilibration.
Already reference has been more than once made to those
thickenings that occur where the skin is exposed to unusual pressure
and friction. Are these adaptations inheritable? and may they, by
accumulation through many generations, produce permanent dermal
structures fitted to permanent or frequently-recurring stress? Take,
for instance, the callosities on the knuckles of the Gorilla, which are
adapted to its habit of partially supporting itself on its closed hands
when moving along the ground. Shall we suppose that these
defensive thickenings are produced afresh in each individual by the
direct actions; or that they are inherited modifications caused by
such direct actions; or that they are wholly due to the natural
selection of spontaneous variations? The last supposition does not
seem a probable one. Such thickenings, if spontaneous, would be no
more likely to occur on the knuckles than on any other of the
hundred equal areas forming the skin-surface at large; and the
chances against their simultaneous occurrence on all eight knuckles
would be incalculable. Moreover, the implication would be that those
slight extra thicknesses of skin on the knuckles, with which we must
suppose the selection to have commenced, were so advantageous as
to cause survivals of the individuals having them, in presence of
other superiorities possessed by other individuals. Then that
survivals so caused, if they ever occurred at all, should have
occurred with the frequency requisite to establish and increase the
variation, is hardly supposable. And if we reject, as also unlikely, the
reproduction of these callosities de novo in each individual (for this
would imply that after a thousand generations each young gorilla
began with knuckles having skin no thicker than elsewhere), there
remains only the inference that they have arisen by the transmission
and accumulation of functional adaptations. Another case which
seems interpretable only in an analogous way, is that of the spurs
that are developed on the wings of certain birds—on those of the
Chaja screamer for example. These are weapons of offence and
defence. It is a familiar fact that some birds strike with their wings,
often giving severe blows; and in the birds named, the blows are
made more formidable by the horny, dagger-shaped growths
standing out from those points on the wings which deliver them. Are
these spurs directly or indirectly adaptive? To conclude that natural
selection of spontaneous variations has caused them, is to conclude
that, without any local stimulus, thickenings of the skin occurred
symmetrically on the two wings at the places required; that such
thickenings, so localized, happened to arise in birds given to using
their wings in fight; and that on their first appearance the
thickenings were decided enough to give appreciable advantages to
the individuals distinguished by them—advantages in bearing the
reactions of the blows if not in inflicting the blows. But to conclude
this is, I think, to conclude against probability. Contrariwise, if we
assume that the thickening of the epidermis produced by habitual
rough usage is inheritable, the development of these structures
presents no difficulty. The points of impact would become indurated
in wings used for striking with unusual frequency. The callosities of
surface thus generated, rendering the parts less sensitive, would
enable the bird in which they arose to give, without injury to itself,
more violent blows and a greater number of them: so, in some
cases, helping it to conquer and multiply. Among its descendants,
inheriting the modification and the accompanying habit, the
thickening would be further increased in the same way: survival of
the fittest tending ever to accelerate the process. Presently the
horny nodes so formed, hitherto defensive only in their effects,
would, by their prominence, become offensive—would make the
blows given more hurtful. And now natural selection, aiding more
actively, would mould the nodes into spurs: the individuals in which
the nodes were most pointed would be apt to survive and
propagate; and the pointedness generation after generation thus
increased, would end in the well-adapted shape we see.
But if in these cases the differentiations which fit particular parts
of the outer tissues to bear rough usage are caused mainly by the
direct balancing of external actions by internal reactions, then we
may suspect that the like is true of other modifications that occur
where special strains and abrasions have to be met. Possibly it is
true of sundry parts that are formed of hardened epidermis, such as
the nails, claws, hoofs, and hollow horns of Mammals; “all of which,”
says Prof. Huxley, “are constructed on essentially the same plan,
being diverticula of the whole integument, the outer layer of whose
ecderon has undergone horny metamorphosis.” Leaving open,
however, the question what tegumentary structures are due to direct
equilibration, furthered and controlled by indirect equilibration, it is
tolerably clear that direct equilibration has been one of the factors.
§ 294. Dermal structures of another class are developed mainly, if

not wholly, by the actions of external causes on species rather than
on individuals. These are the various kinds of clothing—hairs,
feathers, quills, scales, scutes. Though it is no longer thought as at
one time that all these various tegumentary structures are
homologous with one another, yet it is unquestionable that sundry of
the more conspicuous ones are. Those which are extremely unlike
may be seen linked together by a long series of graduated forms. A
retrograde metamorphosis from feathers to appendages that are
almost scale-like, is well seen in the coat of the Penguin. There is
manifest a transition from the bird-like covering to the fish-like
covering—a transition so gradual that no place can be found where
an appreciable break occurs; and if the scale-like appendages are
not truly scales yet they exemplify an extreme metamorphosis. Less
striking, perhaps, but scarcely less significant, are the modifications
through which we pass from feathers to hairs, on the surfaces of the
Ostrich and the Cassowary. The skin of the Porcupine shows us hairs
and quills united by a series of intermediate structures, differing
from one another inappreciably. Even more remarkable are certain
other alliances of dermal structures. “It may be taken as certain, I
think,” says Prof. Huxley, “that the scales, plates, and spines of all
fishes are homologous organs; nor as less so that the tegumentary
spines of the Plagiostomes are homologous with their teeth, and
thence with the teeth of all vertebrata.”
Further details concerning these tegumentary structures are not
needful for present purposes, and are indeed but indirectly relevant
to the subject of physiological development. Here they are of
interest to us only by involving the general question—What physical
influences have brought them into existence? Still with a view to
definite presentation of the problem, it will be well to contemplate
the mode of development common to the most familiar of them.
Suppose a small pit to be formed on the previously flat skin; and
suppose that the growth and casting off of horny cells which goes on
over the skin in general, continues to go on at the usual rate over
the depressed surface of this pit. Clearly the quantity of horny
matter produced within this hollow, will be greater than that
produced on a level portion of the skin subtending an equal area of
the animal’s outside. Suppose such a pit to be deepened until it
becomes a small sac. If the exfoliation goes on as before, the result
will be that the horny matter, expelled, as it must be, through the
mouth of the sac, which now bears a small proportion to the internal
surface of the sac, will be large in quantity compared with that
exfoliated from a portion of the skin equal in area to the mouth of
the sac: there will be a conspicuous thrusting forth of horny matter.
Suppose once more that the sac, instead of remaining simple, has its
bottom pushed up into its interior, like the bottom of a wine-bottle—
the introversion being carried so far that the introverted part reaches
nearly to the external opening, and leaves scarcely any space
between the introverted part and the walls of the sac. It is easy to
see that the exfoliation continuing from the surface of the
introverted part, as well as from the inside of the sac generally, the
horny matter cast off will form a double layer; and will come out of
the sac in the shape of a tube having within its lower end the
introverted part, as the core on which it is moulded, and from the
apex of which is cast off the substance filling, less densely, its
interior. The structure resulting will be what we know as a hair.
Manifestly by progressive enlargement of the sac, and further
complication of that introverted part on which the excreted
substance is moulded, the protruding growth may be rendered larger
and more involved, as we see it in quills and feathers. So that
insensible steps, thus indicated in principle, carry us from the
exfoliation of epidermis by a flat surface, to the exfoliation of it by a
hollow simple sac, an introverted sac, and a sac further complicated;
each of which produces its modified kind of tegumentary
appendage.
But now, after contemplating this typical illustration, we return to
the general question. What are the agencies which have been
operative in developing these skin-structures? Indirect equilibration
must have worked almost alone in producing them. No direct
incidence of forces can have developed the enamelled armour of the
Lepidosteus or the tesselated plates of the Glyptodon and its modern
allies. Survival of the fittest must here and in multitudinous other
cases be regarded as the sole cause.
§ 295. Among many other differentiations of the outer tissues, the

most worthy to be noticed in the space that remains, are those by
which organs of sense are formed. We will begin with the simplest
and most closely-allied to the foregoing.
Every hair that is not too long or flexible to convey to its rooted
end a strain put upon its free end, is a rudimentary tactual organ; as
may be readily proved by touching one of those growing on the back
of the hand. If, then, a creature has certain hairs so placed that they
are habitually touched by the objects with which it deals, or amid
which it moves, an advantage is likely to accrue if these hairs are
modified in a way that enables them the better to transmit the
impressions derived. Such modified hairs we have in the vibrissæ, or,
as they are commonly called, the “whiskers” possessed by Cats and
feline animals generally, as well as by Seals and many Rodents.
These hairs are long enough to reach objects at considerable
distances; they are so stiff that forces applied to their free ends,
cause movements of their imbedded ends; and the sacs containing
their imbedded ends being well covered with nerve-fibres, these
developed hairs serve as instruments of exploration. By constant use
of them the animal learns to judge of the relative positions of
objects past which, or towards which, it is moving. When stealthily
approaching prey or stealthily escaping enemies, such aids to
perception are obviously important: indeed their importance has
been proved by the diminished power of self-guidance in the dark,
that results from cutting them off. These, then, are dermal
appendages originally serving the purpose of clothing, but
afterwards differentiated into sense-organs.
That eyes are essentially dermal structures seems scarcely
conceivable. Yet an examination of their rudimentary types, and of
their genesis in creatures that have them well developed, shows us
that they really arise by successive modifications of the double layer
composing the integument. They make their first appearance among
the simpler animals as specks of pigment, covered by portions of
epidermis slightly convex and a little more transparent than that
around it. Here their fundamental community of structure with the
skin is easy to trace; and the formation of them by differentiation of
it presents no difficulty. Not so far in advance of these as much to
obscure the relationship, are the eyes which the Crustaceans
possess. In every fishmonger’s shop we may see that the eyes of a
Lobster are carried on pedicles; and when the Lobster casts its shell,
the outer coat of each eye, being continuous with the epidermis of
its pedicle, is thrown off along with the rest of the exo-skeleton.
Beneath the transparent epidermic layer, there exists a group of eyes
of the kind which we see in an insect; and these, according to a high
authority, are inclosed in the dermal system. Describing the
arrangement of the parts, M. Milne Edwards writes:—“But the most
remarkable circumstance is, that the large cavity within which the
whole of these parallel columns, every one of which is itself a perfect
eye, are contained, is closed posteriorly by a membrane, which
appears to be neither more nor less than the middle tegumentary
membrane, pierced for the passage of the optic nerve; so that the
ocular chamber at large results from the separation at a point of the
two external layers of the general envelope.” Thus too is it, in the
main, even with the highly developed eyes of the Vertebrata. “The
three pairs of sensory organs appertaining to the higher senses,”
says Prof. Huxley—“the nasal sacs, the eyes, and the ears—arise as
simple cœcal involutions of the external integument of the head of
the embryo. That such is the case, so far as the olfactory sacs are
concerned, is obvious, and it is not difficult to observe that the lens
and the anterior chamber of the eye are produced in a perfectly
similar manner. It is not so easy to see that the labyrinth of the ear
arises in this way, as the sac resulting from the involution of the
integument is small, and remains open but a very short time. But I
have so frequently verified Huschke’s and Remak’s statement that it
does so arise, that I entertain no doubt whatever of the fact. The
outer ends of the olfactory sacs remain open, but those of the ocular
and auditory sacs rapidly close up, and shut off their contents from
all direct communication with the exterior.” That is to say, the eye
considered as an optical apparatus is produced by metamorphoses
of the skin: the only parts of it not thus produced, being the
membranes lying between the sclerotic and the vitreous humour,
including those retinal structures formed in them. All is tegumentary
save that which has to appreciate the impressions which the
modified integument concentrates upon it.
Thus, as Prof. Huxley has somewhere pointed out, there is a
substantial parallelism between all the sensory organs in their modes
of development; as there is, too, between their modes of action. A
vibrissa may be taken as their common type. Increased impressibility
by an external stimulus, requires an increased peripheral expansion
of the nervous system on which the stimulus may fall; and this is
secured by an introversion of the integument, forming a sac on the
walls of which a nerve may ramify. That the more extended sensory
area thus constituted may be acted upon, there requires some
apparatus conveying to it from without the appropriate stimulus; and
in the case of the vibrissa, this apparatus is the epidermic growth
which, under the form of a hair, protrudes from the sac. And that the
greatest sensitiveness may be obtained, the external action must be
exaggerated or multiplied by the apparatus which conveys it to the
recipient nerve; as, in the case of the vibrissa, it is by the
development of a hair into an elastic lever, that transforms the slight
force acting through considerable space on its exposed end, into a
greater force acting through a smaller space at its rooted end.
Similarly with the organs of the higher senses. In a rudimentary eye,
the slightly modified sense cell has but a rudimentary nerve to take
cognizance of the impression; and to concentrate the impression
upon it, there is nothing beyond a thickening of the epidermis into a
lens-shape. But the developed eye shows us a termination of the
nerve greatly expanded and divided to receive the external stimulus.
It shows us an introverted portion of the integument containing the
apparatus by which the external stimulus is conveyed to the
recipient nerve. The structure developed in this sac not only conveys
the stimulus, but also, like its homologue, concentrates it; and in the
one case as in the other, the structure which does this is an
epidermic growth from the bottom of the sac. Even with the ear it is
the same. Again we have an introverted portion of the integument,
on the walls of which the nerve is distributed in the primitive ear.
The otolithes contained in the sac thus formed, are bodies which are
set in motion by the vibrations of the surrounding water, and convey
these vibrations in an exaggerated form to the nerves. And though it
is not alleged that these otolithes are developed from the epidermic
lining of the chamber, yet as, if not so developed, they are
concretions from the contents of an epidermic sac, they must still be
regarded as epidermic products.
Whether these differentiations are due wholly to indirect
equilibration, or whether direct equilibration has had a share in
working them, are questions that must be left open. Possibly a short
hair so placed on a mammal’s face as to be very often touched, may,
by conveying excitations to the nerves and vessels at its root, cause
extra growth of the bulb and its appendages, and so the
development of a vibrissa may be furthered. Possibly, too, the light
itself, to which the tissues of some inferior animals are everywhere
sensitive, may aid in setting up certain of the modifications by which
the nervous parts of visual organs are formed: producing, as it must,
the most powerful effects at those points on the surface which the
movements of the animal expose to the greatest and most frequent
contrasts of light and shade; and propagating from those points
currents of molecular change through the organism. But it seems
clear that the complexities of the sensory organs are not thus
explicable. They must have arisen by the natural selection of
favourable variations.
§ 296. A group of facts, serving to elucidate those put together in
the several foregoing sections, has to be added. I have reserved this
group to the last, partly because it is transitional—links the
differentiations of the literally outer tissues with those of the truly
inner tissues. Though physically internal, the mucous coat of the
alimentary canal has a quasi-externality from a physiological point of
view. As was pointed out in the last chapter, the skin and the
assimilating surface have this in common, that they come in direct
contact with matters not belonging to the organism; and we saw
that along with this community of relation to alien substances, there
is a certain community of structure and development. The like holds
with the linings of all internal cavities and canals that have external
openings.
The transition from the literally outer tissues to those tissues
which are intermediate between them and the truly inner tissues, is
visible at all the orifices of the body; where skin and mucous
membrane are continuous, and the one passes insensibly into the
other. This visible continuity is associated not simply with a great
degree of morphological continuity, but also with a great degree of
physiological continuity. That is to say, these literally outer and
quasi-outer layers are capable of rapidly assuming one another’s
structures and functions when subject to one another’s conditions.
Mucous surfaces, normally kept covered, become skin-like if exposed
to the air; but resume more or less fully their normal characters
when restored to their normal positions. These are truths familiar to
pathologists. They continually meet with proofs that permanent
eversion of the mucous membrane, even where it is by prolapse of a
part deeply seated within the body, is followed by an adaptation
eventually almost complete: originally moist, tender to the touch,
and irritated by the air, the surface gradually becomes covered with
a thick, dry cuticle; and is then scarcely more sensitive than ordinary
integument.
Whether this equilibration between new outer forces and reactive
inner forces, which is thus directly produced in individuals, is
similarly produced in races, must remain as a question not to be
answered in a positive way. On the one hand, we have the fact that
among the higher animals there are cases of quasi-outer tissues
which are in one species habitually ensheathed, while in another
species they are not ensheathed; and that these two tissues, though
unquestionably homologous, differ as much as skin and mucous
membrane differ. On the other hand, there are certain analogous
changes of surface, as on the abdomen of the Hermit-Crab, which
give warrant to the supposition that survival of the fittest is the chief
agent in establishing such differentiations; since the abdomen of a
Hermit-Crab, bathed by water within the shell it occupies, is not
exposed to physical conditions that directly tend to differentiate its
surface from the surface of the thorax. But though in cases like this
last, we must assign the result to the natural selection of variations
arising incidentally; we may, I think, legitimately assign the result to
the immediate action of changed conditions where, as in cases like
the first, we see these producing in the individual, effects of the
kinds observed in the race.
However this may be, the force of the general argument remains
the same. In these exchanges of structure and function between the
outer and quasi-outer tissues, we get undeniable proof that they are
easily differentiable. And seeing this, we are enabled the more
clearly to see how there have, in course of time, arisen those
extreme and multitudinous differentiations of the outer tissues which
have been glanced at.
CHAPTER VIII.
DIFFERENTIATIONS AMONG THE INNER TISSUES OF ANIMALS.
§ 297. The change from the outside of the lips to their inside,
introduces us to a new series of interesting and instructive facts,
joining on to those with which the last chapter closed. They concern
the differentiations of those coats of the alimentary canal which, as
we have seen, are physiologically outer, though physically inner.
These coats are greatly modified at different parts; and their
modifications vary greatly in different animals. In the lower types,
where they compose a simple tube running from end to end of the
body, they are almost uniform in their histological characters; but on
ascending from these types, we find them presenting an increasing
variety of minute structures between their two ends. The argument
will be adequately enforced if we limit ourselves to the leading
modifications they display in some of the higher animals.
The successive parts of the alimentary canal are so placed with
respect to its contents, that the physical and chemical changes
undergone by its contents while passing from one end to the other,
inevitably tend to transform its originally homogeneous surface into
a heterogeneous surface. Clearly, the effect produced on the food at
any part of the canal by trituration, by adding a secretion, or by
absorbing its nutritive matters, implies the delivery of the food into
the next part of the canal in a state more or less unlike its previous
states—implies that the surface with which it now comes in contact
is differently affected by it from the preceding surfaces—implies, that
is, a differentiating action. To use concrete language;—food that is
broken down in the mouth acts on the œsophagus and stomach in a
way unlike that which it would have done had it been swallowed
whole; the masticated food, to which certain solvents or ferments
are added, becomes to the intestine a different substance from that
which it must have otherwise been; and the altered food, resolved
by these additions into its proximate principles, cannot have those
proximate principles absorbed in the next part of the intestine,
without the remoter parts being affected as they would not have
been in the absence of absorption. It is true that in developed
alimentary canals, such as the reasoning here tacitly assumes, these
marked successive differentiations of the food are themselves the
results of pre-established differentiations in the successive parts of
the canal. But it is also true that actions and reactions like those
here so definitely marked, must go on indefinitely in an undeveloped
alimentary canal. If the food is changed at all in the course of its
transit, which it must be if the creature is to live by it, then it cannot
but act dissimilarly on the successive tracts of the alimentary canal,
and cannot but be dissimilarly reacted on by them. Inevitably,
therefore, the uniformity of the surface must lapse into greater or
less multiformity: the differentiation of each part tending ever to
initiate differentiations of other parts.
Not, indeed, that the implied process of direct equilibration can be
regarded as the sole process. Indirect equilibration aids; and,
doubtless, there are some of the modifications which only indirect
equilibration can accomplish. But we have here one unquestionable
cause—a cause that is known to work in individuals, changes of the
kind alleged. Where, for instance, cancerous disease of the
œsophagus so narrows the passage into the stomach as to prevent
easy descent of the food, the œsophagus above the obstruction
becomes enlarged into a kind of pouch; and the inner surface of this
pouch begins to secrete juices that produce in the food a kind of
rude digestion. Again, stricture of the intestine, when it arises
gradually, is followed by hypertrophy of the muscular coat of the
intestine above the constricted part: the ordinary peristaltic
movements being insufficient to force the food forwards, and the
lodged food serving as a constant stimulus to contraction, the
muscular fibres, habitually more exercised, become more bulky. The
deduction from general principles being thus inductively enforced,
we cannot, I think, resist the conclusion that the direct actions and
reactions between the food and the alimentary canal have been
largely instrumental in establishing the contrasts among its parts.
And we shall hold this view with the more confidence on observing
how satisfactorily, in pursuance of it, we are enabled to explain one
of the most striking of these differentiations, which we will take as a
type of the class.
The gizzard of a bird is an expanded portion of the alimentary
canal, specially fitted to give the food that trituration which the
toothless mouth of a bird cannot give. Besides having a greatly-
developed muscular coat, this grinding-chamber is lined with a thick,
hard cuticle, capable of bearing the friction of the pebbles swallowed
to serve as grindstones. This differentiation of the mucous coat into
a ridged and tubercled layer of horny matter—a differentiation
which, in the analogous organs of certain Mollusca, is carried to the
extent of producing from this membrane cartilaginous plates, and
even teeth—varies in birds of different kinds, according to their food.
It is moderate in birds that feed on flesh and fish, and extreme in
granivorous birds and others that live on hard substances. How does
this immense modification of the alimentary canal originate? In the
stomach of a mammal, the macerating and solvent actions are
united with that triturating action which finishes what the teeth have
mainly done; but in the bird, unable to masticate, these internal
functions are specialized, and while the crop is the macerating
chamber, the gizzard becomes a chamber adapted to triturate more
effectually. This adaptation requires simply an exaggeration of
certain structures and actions which characterize stomachs in
general, and, in a less degree, alimentary canals throughout their
whole lengths. The massive muscles of the gizzard are simply
extreme developments of the muscular tunic, which is already
considerably developed over the stomach, and incloses also the
œsophagus and the intestine. The indurated lining of the gizzard,
thickened into horny buttons at the places of severest pressure, is
nothing more than a greatly strengthened and modified epithelium.
And the grinding action of the gizzard is but a specialized form of
that rhythmical contraction by which an ordinary stomach kneads
the contained food, and which in the œsophagus effects the act of
swallowing, while in the intestine it becomes the peristaltic motion.
Allied as the gizzard thus clearly is in structure and action to the
stomach and alimentary canal in general; and capable of being
gradually differentiated from a stomach where a growing habit of
swallowing food unmasticated entails more trituration to be
performed before the food passes the pylorus; the question is—Does
this change of structure arise by direct adaptation? There is warrant
for the belief that it does. Besides such collateral evidence as that
mucous membrane becomes horny on the toothless gums of old
people, when subject to continual rough usage, and that the
muscular coat of the intestine thickens where unusual activity is
demanded of it, we have the direct evidence of experiment. Hunter
habituated a sea-gull to feed on grain, and found that the lining of
its gizzard became hardened, while the gizzard-muscles doubled in
thickness. A like change in the diet of a kite was followed by like
results. Clearly, if differentiations so produced in the individuals of a
race under changed habits, are in any degree inheritable, a structure
like a gizzard will originate through the direct actions and reactions
between the food and the alimentary canal.
Another case—a very interesting one, somewhat allied to this—is
presented by the ruminating animals. Here several dilatations of the
alimentary canal precede the true stomach; and in them large
quantities of unmasticated food are stored, to be afterwards
returned to the mouth and masticated at leisure. What conditions
have made this specialization advantageous? and by what process
has it been established? To both these questions the facts indicate
answers which are not unsatisfactory. [Creatures that obtain their
food very irregularly—now having more than they can consume, and
now being for long periods without any—must, in the first place, be
apt, when very hungry, to eat to the extreme limits of their
capacities; and must, in the second place, profit by peculiarities
which enable them to compensate themselves for long fasts, past
and future. A perch which, when its stomach is full of young frogs,
goes on filling its œsophagus also; or a trout which, rising to the
fisherman’s fly, proves when taken off the hook to be full of worms
and insect-larvæ up to the very mouth, gains by its ability to take in
such unusual supplies of food when it meets with them—obviously
thrives better than it would do could it never eat more than a
stomachful. That this ability to feed greatly in excess of immediate
requirement, is one that varies in individuals of the same race, we
see in the marked contrast between our own powers in this respect,
and the powers of uncivilized men; whose fasting and gorging are to
us so astonishing. Carrying with us these considerations, we shall
not be surprised at finding dilatations of the œsophagus in vultures
and eagles, which get their prey at long intervals in large masses;
and we may naturally look for them, too, in birds like pigeons,
which, coming in flocks upon occasional supplies of grain,
individually profit by devouring the greatest quantity in a given time.
Now where the trituration of the food is, as in these cases, carried
on in a lower part of the alimentary canal, nothing further is required
than the storing-chamber; but for a mammal, having its grinding
apparatus in its mouth, to gain by the habit of hurriedly swallowing
unmasticated food, it must also have the habit of regurgitating the
food for subsequent mastication. This correlation of habits with their
answering structures, may, as we shall see, arise in a very simple
way. The starting point of the explanation is a familiar fact—the fact
that indigestion, often resulting from excess of food, is apt to cause
that reversed peristaltic action known as vomiting. From this we
pass to the fact, also within the experience of most persons, that
during slight indigestion the stomach sometimes quietly regurgitates
a small part of its contents as far as the back of the mouth—giving
an unpleasant acquaintance with the taste of the gastric juices.
Exceptional facts of the same class help the argument a step further.
“There are certain individuals who are capable of returning, at will, a
greater or smaller portion of the contents of the digesting stomach
into the cavity of the mouth.... In some of these cases, the expulsion
of the food has required a violent effort. In the majority it has been
easily evoked or suppressed. While in others, it has been almost
uncontrollable; or its non-occurrence at the habitual time has been
followed by a painful feeling of fulness, or by the act of vomiting.”
Here we have a certain physiological action, occasionally happening
in most persons and in some developed into a habit more or less
pronounced: indigestion being the habitual antecedent. Suppose,
then, that gregarious animals, living on innutritive food such as
grass, are subject to a like physiological action, and are capable of
like variations in the degree of it. What will naturally happen? They
wander in herds, now over places where food is scarce and now
coming to places where it is abundant. Some masticate their food
completely before swallowing it, while some masticate it
incompletely. If an oasis, presently bared by their grazing, has not
supplied to the whole herd a full meal, then the individuals which
masticate completely will have had less than those which masticate
incompletely—will not have had enough. Those which masticate
incompletely and distend their stomachs with food difficult to digest,
will be liable to these regurgitations; but if they re-masticate what is
thus returned to the mouth (and we know that animals often eat
again what they have vomited), then the extra quantity of food
taken, eventually made digestible, will yield them more nourishment
than is obtained by those which masticate completely at first. The
habit initiated in this natural way, and aiding survival when food is
scarce, will be apt to cause modifications of the alimentary canal.
We know that dilatations of canals readily arise under habitual
distensions. We know that canals habitually distended become
gradually more tolerant of the contained masses that at first irritated
them. And we know that there commonly take place adaptive
modifications of their surfaces. Hence if a habit of this kind and the
structural changes resulting from it, are in any degree inheritable, it
is clear that, increasing in successive generations, both immediately
by the cumulative effect of repetitions and mediately by survival of
the individuals in which they are most decided, they may go on until
they end in the peculiarities which Ruminants display.
§ 298. There are structures belonging to the same group which

cannot, however, be accounted for in this way. They are the organs
that secrete special products facilitating digestion—the liver,
pancreas, and various smaller glands. All these appendages of the
alimentary canal, large and independent as some of them seem,
really arise by differentiations from its coats. The primordial liver
consists of nothing more than bile-cells scattered along a tract of the
intestinal surface. Accumulation of these bile-cells is accompanied by
increased growth of the surface which bears them—a growth which
at first takes the form of a cul-de-sac, having an outside that
projects from the intestine into the peri-visceral cavity. As the mass
of bile-cells becomes greater, there arise secondary lateral cavities
opening into the primary one, and through it into the intestine; until,
eventually, these cavities with their coatings of bile-cells, become
ramifying ducts distributed through the solid mass we know as a
liver. How is this differentiation caused?
Before attempting any answer to this question, it is requisite to
inquire the nature of bile. Is that which the liver throws into the
intestines a waste product of the organic actions? or is it a secretion
aiding digestion? or is it a mixture of these? Modern investigations
imply that it is most likely the last. The liver is found to have a
compound function. Bernard has proved to the satisfaction of
physiologists, that there goes on in it a formation of glycogen—a
substance which is transformed into sugar before it leaves the liver
and is afterwards carried away by the blood to eventually disappear
in the active organs, chiefly the muscles. It is also shown,
experimentally, that there are generated in the liver certain biliary
acids; and by the aid either of these or of some other compounds, it
is clear that bile renders certain materials more absorbable. Its effect
on fat is demonstrable out of the body; and the greatly diminished
absorption of fat from the food when the discharge of bile into the
intestine is prevented, is probably one of the causes of that pining
away which results. But while recognizing the fact that the bile
consists in part of a solvent, or solvents, aiding digestion, there is
abundant evidence that one element of it is an effete product; and
probably this is the primary element. The yellow-green substance
called biliverdine in herbivora and bilirubin in man and carnivora,
which gives its colour to bile, is a product the greater part of which
is normally cast out from the system continually, as is shown by the
contrast between the normal and abnormal colours of fæcal matters,
and as is still more strikingly shown by the effects on the system
when there is a stoppage of the excretion, and an attack of jaundice.
Hence we are warranted in classing biliverdine as a waste product,
and we may fairly infer that the excretion of it is the original function
of the liver.
One further preliminary is requisite. We must for a moment return
to those physico-chemical data set down in the first chapter of this
work (§§ 7–8). We there saw that the complex and large-atomed
colloids which mainly compose living organic matter, have extremely
little molecular mobility; and, consequently, extremely little power of
diffusing themselves. Whereas we saw not only that those absorbed
matters, gaseous and liquid, which further the decomposition of
living organic matter, have very high diffusibilities, but also that the
products of the decomposition are much more diffusible than the
components of living organic matter. And we saw that, as a
consequence of this, the tissues give ready entrance to the
substances which decompose them, and ready exit to the
substances into which they are decomposed. Hence it follows that,
under its initial form, uncomplicated by nervous and other agencies,
the escape of effete matters from the organism, is a physical action
parallel to that which goes on among mixed colloids and crystalloids
that are dead or even inorganic. Excretion is a specialized form of
this spontaneous action; and we have to inquire how the
specialization arises.
Two causes conspire to establish it. The first is that these
products of decomposition are diffusible in widely different degrees.
While the carbonic acid and water permeate the tissues with ease in
all directions, and escape more or less from the exposed surfaces,
urea, and other waste substances incapable of being vaporized,
cannot escape thus readily. The second is that the different parts of
the body, being subject to different physical conditions, are from the
outset sure severally to favour the exit of these various products of
decomposition in various degrees. How these causes must have co-
operated in localizing the excretions, we shall see on remembering
how they now co-operate in localizing the separation of morbid
materials. The characteristic substances of gout and rheumatism
have their habitual places of deposit. Tuberculous matter, though it
may be present in various organs, gravitates towards some much
more than towards others. Certain products of disease are habitually
got rid of by the skin, instead of collecting internally. Mostly, these
have special parts of the skin which they affect rather than the rest;
and there are those which, by breaking out symmetrically on the two
sides of the body, show how definitely the places of their excretion
are determined by certain favouring conditions, which corresponding
parts may be presumed to furnish in equal degrees. Further, it is to
be observed of these morbid substances circulating in the blood,
that having once commenced segregating at particular places, they
tend to continue segregating at those places. Assuming, then, as we
may fairly do, that this localization of excretion, which we see
continually commencing afresh with morbid matters, has always
gone on with the matters produced by the waste of the tissues, let
us take a further step, and ask how localizations become fixed.
Other things equal, that which from its physical conditions is a place
of least resistance to the exit of an effete product, will tend to
become established as the place of excretion; since the rapid exit of
an effete product will profit the organism. Other things equal, a
place at which the excreted matter produces least detrimental effect
will become the established place. If at any point the excreted
matter produces a beneficial effect, then, other things equal, survival
of the fittest will determine it to this point. And if facility of escape
anywhere goes along with utilization of the escaping substance,
then, other things equal, the excretion will be there localized still
more decisively by survival of the fittest.
Such being the conditions of the problem, let us ask what will
happen with the lining membrane of the alimentary canal. This,
physiologically considered, is an external surface; and matters
thrown off from it make their way out of the body. It is also a
surface along which is moving the food to be digested. Now, among
the various waste products continually escaping from the living
tissues, some of the more complex ones, not very stable in
composition, are likely, if added to the food, to set up changes in it.
Such changes may either aid or hinder the preparation of the food
for absorption. If an effete matter, making its exit through the wall
of the intestine, hinders the digestive process, the enfeeblement and
disappearance of individuals in which this happens, will prevent the
intestine from becoming the established place for its exit. While if it
aids the digestive process, the intestine will, for converse reasons,
become more and more the place to which its exit is limited. Equally
manifest is it that if there is one part of this alimentary canal at
which, more than at any other part, the favourable effect results,
this will become the place of excretion.
Thus, then, reverting to the case in question, we may understand
how a product to be cast out, such as biliverdine, if it either directly
or indirectly serves a useful purpose, when poured into a particular
part of the intestine, may lead to the formation of a patch of
excreting cells on its wall; and once this place of excretion having
been established, the development of a liver is simply a question of
time and natural selection.
§ 299. A differentiation of another order occurring in the

alimentary canal, is that by which a part of it is developed into a
lateral chamber or chambers, through which carbonic acid exhales
and oxygen is absorbed. Comparative anatomy and embryology
unite in showing that a lung is formed, just as a liver or other
appendage of the alimentary canal is formed, by the growth of a
hollow bud into the peri-visceral cavity, or space between the
alimentary canal and the wall of the body. The interior of this bud is
simply a cul-de-sac of the alimentary canal, with the mucous lining
of which its own mucous lining is continuous. And the development
of this cul-de-sac into an air-chamber, simple or compound, is merely
a great extension of area in the internal surface of the cul-de-sac,
along with that specialization which fits it for excreting and
absorbing substances different from those which other parts of the
mucous surface excrete and absorb. These lateral air-chambers,
universal among the higher Vertebrata and very general among the
lower, and everywhere attached to the alimentary canal between the
mouth and the stomach, have not in all cases the respiratory
function. In most fishes that have them they are what we know as
swim-bladders. In some fishes the cavities of these swim-bladders
are completely shut off from the alimentary canal: nevertheless
showing, by the communications which they have with it during the
embryonic stages, that they are originally diverticula from it. In other
fishes there is a permanent ductus pneumaticus, uniting the cavity
of the swim-bladder with that of the gullet: the function, however,
being still not respiratory in an appreciable degree, if at all. But in
certain still extant representatives of the sauroid fishes, as the
Lepidosteus, the air-bladder is “divided into two sacs that possess a
cellular structure,” and “the trachea which proceeds from it opens
high up in the throat, and is surrounded with a glottis.” In the
Amphibia the corresponding organs are chambers over the surfaces
of which there are saccular depressions, indicating a transition
towards the air-cells characterizing lungs; and accompanying this
advance we see, as in the common Triton, the habit of coming up to
the surface and taking down a fresh supply of air in place of that
discharged.
How are the internal air-chambers, respiratory or nonrespiratory,
developed? Upwards from the amphibian stage, in which they are
partially refilled at long intervals, there is no difficulty in
understanding how, by infinitesimal steps, they pass into complex
and ever-moving lungs. But how is the differentiation that produces
them initiated? How comes a portion of the internal surface to be
specialized for converse with a medium to which it is not naturally
exposed? The problem appears a difficult one; but there is a not
unsatisfactory solution of it.
When many gold-fish are kept in a small aquarium, as with
thoughtless cruelty they frequently are, they swim close to the
surface, so as to breathe that water which is from instant to instant
absorbing fresh oxygen. In doing this they often put their mouths
partly above the surface, so that in closing them they take in
bubbles of air; and sometimes they may be seen to continue doing
this—the relief due to the slight extra aëration of blood so secured,
being the stimulus to continue. Air thus taken in may be detained. If
a fish that has taken in a bubble turns its head downwards, the
bubble will ascend to the back of its mouth, and there lodge; and
coming within reach of the contractions of the œsophagus, it may be
swallowed. If, then, among fish thus naturally led upon occasion to
take in air-bubbles, there are any having slight differences in the
alimentary canal that facilitate lodgment of the air, or slight nervous
differences such as in human beings cause an accidental action to
become “a trick,” it must happen that if an advantage accrues from
the habitual detention of air-bubbles, those individuals most apt to
detain them will, other things equal, be more likely than the rest to
survive; and by the survival of descendants inheriting their
peculiarities in the greatest degrees, and increasing them, an
established structure and an established habit may arise. And that
they do in some way arise we have proof. The common Loach
swallows air, which it afterwards discharges loaded with carbonic
acid.
From air thus swallowed the advantages that may be derived are
of two kinds. In the first place, the fish is made specifically lighter,
and the muscular effort needed to keep it from sinking is diminished
—or, indeed, if the bubble is of the right size, is altogether saved.
The contrast between the movements of a Goby, which, after
swimming up towards the surface, falls rapidly to the bottom on
ceasing its exertions, and the movements of a Trout, which remains
suspended just balancing itself by slight undulations of its fins,
shows how great an economy results from an internal float, to fishes
which seek their food in mid-water or at the surface. Hence the habit
of swallowing air having been initiated in the way described, we see
why natural selection will, in certain fishes, aid modifications of the
alimentary canal favouring its lodgment—modifications constituting
air-sacs. In the second place, while from air thus lodged in air-sacs
thus developed, the advantage will be that of flotation only if the air
is infrequently changed or never changed, the advantage will be that
of supplementary respiration if the air-sacs are from time to time
partially emptied and refilled. The requirements of the animal will
determine which of the two functions predominates. Let us glance at
the different sets of conditions under which these divergent
modifications may be expected to arise.
The respiratory development is not likely to take place in fishes
that inhabit seas or rivers in which the supply of aërated water never
fails: there is no obvious reason why the established branchial
respiration should be replaced by a pulmonic respiration. Indeed, if a
fish’s branchial respiration is adequate to its needs, a loss would
result from the effort of coming to the surface for air; especially
during those first stages of pulmonic development when the extra
aëration achieved was but small. Hence in fishes so circumstanced,
the air-chambers arising in the way described would naturally
become specialized mainly or wholly into floats. Their contained air
being infrequently changed, no advantage would arise from the
development of vascular plexuses over their surfaces; nothing would
be gained by keeping open the communication between them and
the alimentary canal; and there might thus eventually result closed
chambers the gaseous contents of which, instead of being obtained
from without, were secreted from their walls, as gases often are
from mucous membranes. Contrariwise, aquatic vertebrates in which
the swallowing of air-bubbles, becoming habitual, had led to the
formation of sacs that lodged the bubbles; and which continued to
inhabit waters not always supplying them with sufficient oxygen,
might be expected to have the sacs further developed, and the
practice of changing the contained air made regular, if either of two
advantages resulted—either the advantage of being able to live in
old habitats that had become untenable without this modification, or
the advantage of being able to occupy new habitats. Now it is just
where these advantages are gained that we see the pulmonic
respiration coming in aid of the branchial respiration, and in various
degrees replacing it. Shallow waters are liable to three changes
which conspire to make this supplementary respiration beneficial.
The summer’s sun heats them, and raising the temperatures of the
animals they contain, accelerates the circulation in these animals,
exalts their functional activities, increases the production of carbonic
acid, and thus makes aëration of the blood more needful than usual.
Meanwhile the heated water, instead of yielding to the highly
carbonized blood brought to the branchiæ the usual quantity of
oxygen, yields less than usual; for as the heat of the water
increases, the quantity of air it contains diminishes. And this greater
demand for oxygen joined with smaller supply, pushed to an
extreme where the water is nearly all evaporated, is at last still more
intensely felt in consequence of the excess of carbonic acid
discharged by the numerous creatures congregated in the muddy
puddles that remain. Here, then, it is, that the habit of taking in air-
bubbles is likely to become established, and the organs for utilizing
them developed; and here it is, accordingly, that we find all stages of
the transition to aërial respiration. The Loach before-mentioned,
which swallows air, frequents small waters liable to be considerably
warmed. The Amphipnous Cuchia, an anomalous eel-shaped fish,
which has vascular air-sacs opening out at the back of the mouth, “is
generally found lurking in holes and crevices, on the muddy banks of
marshes or slow-moving rivers”; and though its air-sacs are not
morphological equivalents of those above described, yet they equally
well illustrate the relation between such organs and the environing
condition. Still more significant is the fact that the Lepidosiren, or
“mudfish” as it is called from its habits, though it is a true fish
nevertheless has lungs. But it is among the Amphibia that we see
most conspicuously this relation between the development of air-
breathing organs, and the peculiarities of the habitats. Pools, more
or less dissipated annually, and so rendered uninhabitable by most
fishes, are very generally peopled by these transitional types. Just as
we see, too, that in various climates and in various kinds of shallow
waters, the supplementary aërial respiration is needful in different
degrees; so do we find among the Amphibia many stages in the
substitution of the one respiration for the other. The facts, then, are
such as give to the hypothesis a vraisemblance greater than could
have been expected.

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Methods and Protocols

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ISSN 1064-3745 ISSN 1940-6029 (electronic)

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Emeryville, CA, USA Edward E.K. Baidoo

PART I MICROBIAL METABOLOMICS, METABOLISM, AND PHYSIOLOGY

2 Mass Spectrometry-Based Microbial Metabolomics: Techniques,

PART II METABOLITE SAMPLE PREPARATION

4 Untargeted Soil Metabolomics Using Liquid Chromatography–Mass

PART III CURRENT ANALYTICAL TECHNIQUES

8 Targeted Metabolomics of Xylose-Fermenting Yeasts Based

11 Liquid Chromatography and Mass Spectrometry Analysis of Isoprenoid

PART IV DATA ANALYSIS

15 A High-Throughput Targeted Metabolomics Workflow for the

PATRÍCIA VERARDI ABDELNUR  Brazilian Agricultural Research Corporation, Embrapa

ZHUCUI LI  iHuman Institute, ShanghaiTech University, Shanghai, China; Tianjin

Microbial Metabolomics: A General Overview

Progression in biology related scientific research has always

mechanisms (feedback loops, chromatin regulation, global

synthesis of the building blocks of cellular materials such as nucleic

extraction agents (e.g., methanol, chloroform, water, etc.) is neces-

interfacing), chromatographic separation efficiency (e.g., UHPLC

Fig. 3 Popularity of metabolomics in the fields of human biology, plant biology,

approach of most laboratories. Algorithms are now able to overlay

involved in microbial infection, pathogenicity, and immunity,

Microbial Metabolomics, Metabolism, and Physiology

Mass Spectrometry-Based Microbial Metabolomics:

In recent years, there has been a push in the biosciences to charac-

analytical method that can resolve all metabolites. Additionally,

2 Metabolic Quenching and Metabolite Extraction

Preparation of biological samples is of the upmost importance to

intracellular accumulation of these compounds. Regulatory pro-

Extracellular metabolites can simply be obtained by separating

[16, 17]. Solid phase microextraction (SPME) purifies metabolites

3 Analytical Separation Technologies

3.1 Introduction It is without doubt that separation, whether chromatographic or

the RT from sample injection to the elution of the compound from

§ 291. The outer tissues of animals, originally homogeneous over

§ 292. We may begin with those modifications of surface which

§ 293. Among the conspicuous modifications by which the

§ 294. Dermal structures of another class are developed mainly, if

§ 295. Among many other differentiations of the outer tissues, the

§ 298. There are structures belonging to the same group which

§ 299. A differentiation of another order occurring in the

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PATRÍCIA VERARDI ABDELNUR Brazilian Agricultural Research Corporation, Embrapa

ZHUCUI LI iHuman Institute, ShanghaiTech University, Shanghai, China; Tianjin