Solid State Fermentation

Advances in Biochemical Engineering/Biotechnology 169
Series Editor: T. Scheper
Susanne Steudler
Anett Werner
Jay J. Cheng Editors
Solid State
Fermentation
Research and Industrial Applications
169
Advances in Biochemical
Engineering/Biotechnology
Series Editor
T. Scheper, Hannover, Germany
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W. Zhou, Shanghai, China
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Susanne Steudler • Anett Werner • Jay J. Cheng
Editors
Solid State Fermentation

Research and Industrial Applications
With contributions by
R. C. Alnoch M. Berovic A. Biz L. F. de Lima Luz Jr.
G. S. Dias A. T. J. Finkler M. A. Fraatz N. Krieger
D. A. Mitchell A. Orban L. O. Pitol S. C. Robinson
M. Rühl S. Steudler R. C. Van Court T. Walther
Z. Wen A. Werner H. Zhou
Editors
Susanne Steudler Anett Werner
Institut für Naturstofftechnik Institut für Naturstofftechnik
Technische Universität Dresden Technische Universität Dresden
Dresden, Sachsen, Germany Dresden, Sachsen, Germany
Jay J. Cheng
Department of Biological and Agricultural
Engineering
North Carolina State University
Raleigh, NC, USA
ISSN 0724-6145 ISSN 1616-8542 (electronic)

Advances in Biochemical Engineering/Biotechnology
ISBN 978-3-030-23674-8 ISBN 978-3-030-23675-5 (eBook)
https://doi.org/10.1007/978-3-030-23675-5
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Preface
Solid-state fermentation (SSF) is a well-established technology for cultivation of

different microbes on solid substrates without free-flowing aqueous phase. Filamen-
tous fungi, such as Ascomycota and Basidiomycota species, are the most commonly
used microorganisms for this type of cultivation. The fungi grow in nature on pieces
of wood, leaves, roots and soil, mostly solid substrates with various moisture
contents but no free water. The SSF is an important alternative to suspended
fermentation for the production of value-added products such as pharmaceutical
drugs, enzymes, organic acids, pigments, biopesticides and flavour supplements.
SSF is also applied in bioleaching, biodigestion, bioremediation, biopulping and
mushroom cultivation in the last decades.
SSF technology has obvious advantages over suspended fermentation. First
of all, SSF processes can utilize solid waste materials such as agricultural
residues and wood chips as the primary substrates for the microorganisms.
This provides a mechanism to recover resources from the waste materials for
sustainable development. In addition, SSF technology combined with tray
reactors and/or moving-bed reactors (such as drum reactors) can substantially
improve heat transfer of the materials and prevent blocking during the fermen-
tation processes. SSF also has some disadvantages, especially in modelling and
scaling up. Determination of microbial cell mass and growth kinetics on solid
substrates during SSF processes is particularly difficult. Another important
challenge for the SSF is characterization of the substrate consumption during
the fermentation processes.
This book presents recent development in SSF technology. Innovative SSF
processes have been described in the book for their applications in food processing,
enzyme production and biofuel generation. The book includes cultivation of special
microbial culture and cells through SSF processes. Technical potential, limitations
v
vi Preface
and research needs of the SSF technology are also discussed in the book. The book is
designed as a reference book for scientists and engineers who are interested in solid-
state fermentation.
Dresden, Germany Anett Werner

Dresden, Germany Susanne Steudler
Raleigh, NC, USA Jay J. Cheng
Contents
Part I Cultivation of Microbes Under Solid-State Fermentation

Cultivation of Medicinal Mushroom Biomass by Solid-State
Bioprocessing in Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Marin Berovic
Design and Operation of a Pilot-Scale Packed-Bed Bioreactor
for the Production of Enzymes by Solid-State Fermentation . . . . . . . . . . 27
David Alexander Mitchell, Luana Oliveira Pitol, Alessandra Biz,
Anelize Terezinha Jung Finkler, Luiz Fernando de Lima Luz Jr.,
and Nadia Krieger
It Is the Mix that Matters: Substrate-Specific Enzyme Production
from Filamentous Fungi and Bacteria Through
Solid-State Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
Susanne Steudler, Anett Werner, and Thomas Walther
Part II Application of Solid-State Fermentation for Product Generation

Aroma Profile Analyses of Filamentous Fungi Cultivated
on Solid Substrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 85
Axel Orban, Marco A. Fraatz, and Martin Rühl
Stimulating Production of Pigment-Type Secondary Metabolites
from Soft Rotting Wood Decay Fungi (“Spalting” Fungi) . . . . . . . . . . . . 109
R. C. Van Court and Seri C. Robinson
Fermented Solids and Their Application in the Production of Organic
Compounds of Biotechnological Interest . . . . . . . . . . . . . . . . . . . . . . . . . 125
Nadia Krieger, Glauco Silva Dias, Robson Carlos Alnoch,
and David Alexander Mitchell
vii
viii Contents
Solid-State Anaerobic Digestion for Waste Management

and Biogas Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 147
Haoqin Zhou and Zhiyou Wen
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169
Part I
Cultivation of Microbes Under Solid-State
Fermentation
Adv Biochem Eng Biotechnol (2019) 169: 3–26
DOI: 10.1007/10_2019_89
Published online: 4 March 2019
Cultivation of Medicinal Mushroom

Biomass by Solid-State Bioprocessing
in Bioreactors
Marin Berovic
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
2 Solid-State Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
3 Differences Between Submerged and Solid-State Bioprocessing . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4 Bioreactor Types Used in SSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.1 Tray Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.2 Packed-Bed Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11
4.3 Rotating Drum Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
4.4 Stirred Aerated Beds Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5 Substrates for Medicinal Mushrooms Cultivation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14
6 Cultivation of Medicinal Mushroom in Solid-State Bioreactors . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
6.1 Cultivation of Ganoderma lucidum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
6.2 Cultivation of Grifola frondosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
6.3 Cultivation of Trametes versicolor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
6.4 Cultivation of Hericium erinaceus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20
6.5 Cultivation of Cordyceps militaris . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
7 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Abstract Basidiomycetes of various species and their wide range of pharma-

ceutically interesting products in the last decades represent one of the most attractive
groups of natural products in Asia and North America. Production of fungal fruit
bodies using farming technology is hardly covering the market. Comprehensive
solid-state technologies and bioreactors are the most promising part for fast and large
amount of cultivation of medicinal fungi biomass and its pharmaceutically active
products. Wood, agriculture, and food industry wastes represent the main substrates
that are in this process delignified and enriched in proteins and highly valuable
M. Berovic (*)
Faculty of Chemistry and Chemical Technology, University of Ljubljana, Ljubljana, Slovenia
e-mail: [email protected]
4 M. Berovic
pharmaceutically active compounds. Research in physiology, basic and applied

studies in fungal metabolism, process engineering aspects, and clinical studies in
the last two decades represent large contribution to the development of these
potentials that initiate the development of new drugs and some of the most attractive
over-the-counter human and veterinary remedies. Present article is an overview of
the achievements in solid-state technology of the most relevant medicinal mushroom
species production in bioreactors.
Graphical Abstract
Keywords Fungal biomass, Medicinal mushrooms, Solid-state bioreactors, Solid-

state cultivation, Substrates
List of Symbols and Abbreviations
ψ Water potential
aw Water activity
EPS Exopolysaccharide
M Water molecular mass
NGF Nerve growth factor
Cultivation of Medicinal Mushroom Biomass by Solid-State Bioprocessing. . . 5
P Water vapor pressure

P0 Water vapor pressure of pure water under the same condition
PSK Polysaccharide krestin from Trametes versicolor
PSP Polysaccharopeptide from Trametes versicolor
R Gas constant (8.31 J/mol K)
SmB Submerged bioprocessing
SSC Solid-state cultivation
T Absolute temperature
1 Introduction
According to Wasser [1], the number of mushrooms on Earth is estimated at

140,000, yet maybe only 10% of species are known and named. Basidiomycetes
mushrooms comprise a vast but largely untapped source of new pharmaceutical
products in fruit bodies, cultured mycelium, and cultured broth. The practice of using
medicinal mushrooms in Chinese traditional medicine dates back into antiquity and
has been recorded in ancient Chinese manuscripts [2]. Increased scientific and
medical research in recent years published in peer-reviewed journals, especially in
Japan, Korea, and China and more recently in the USA, is increasingly confirming
the medicinal efficacy and identifying the bioactive molecules [3–5]. Recent
advances in chemical technology have allowed the isolation and purification of
some of the relevant compounds especially polysaccharides which possess strong
immunomodulation and anticancer activities. The bioactive polysaccharides isolated
from mushroom fruit bodies, submerged cultured mycelial biomass, or liquid culture
broths are either water-soluble β-D-glucans; β-D-glucans with heterosaccharide
chains of xylose, mannose, galactose, or uronic acid; or β-D-glucan-protein com-
plexes, i.e., proteoglycans [6]. While many are orally bioavailable, others are mainly
effective only by intraperitoneal injection. All of these compounds are currently
produced by Asian pharmaceutical companies. These medicinal polysaccharides are
primarily modifiers of biological response where these polymers interact with the
immune system to upregulate or downregulate specific aspects of the response of the
host, and this may result in various therapeutic effects [7]. Their ability to enhance or
suppress immune responses can depend on a number of factors including dosage,
route of administration, timing and frequency of administration, mechanism of
action, or the site of activity. Several of these compounds have been shown to
potentiate the host’s innate (non-specific) and acquired (specific) immune responses
and to activate many kinds of immune cells that are important for the maintenance of
homeostasis, e.g., host cells (such as cytotoxic macrophages, monocytes, neutro-
phils, natural killer cells, dendritic cells) and chemical messengers (cytokines such as
interleukins, interferon, colony-stimulating factors) that trigger complement and
acute-phase responses [4, 8]. They can also be considered as multi-cytokine inducers
capable of modulating gene expression of various immunomodulatory cytokines via
specific cell membrane receptors [9, 10]. Lymphocytes governing antibody produc-
tion (β cells) and cell-mediated cytotoxicity (T cells) are also stimulated.
6 M. Berovic
Mushroom polysaccharides do not attack cancer cells directly but produce their
antitumor effects by activating different immune responses in the host. Their mecha-
nisms of action involve them being recognized by several immune cell receptors as
nonself molecules, so the immune system is stimulated by their presence. Structurally
different β-glucans have different affinities toward receptors and thus generate differ-
ent host responses [11]. Immunomodulating and antitumor activities of these metab-
olites are related to immune cells such as hematopoietic stem cells, lymphocytes,
macrophages, T cells, dendritic cells, and natural killer cells, involved in the innate and
adaptive immunity, resulting in the production of biologic response modifiers [12].
Clinical evidence for antitumor and other medicinal activities come primarily
from some commercialized purified polysaccharides that have undergone extensive
anticancer clinical trials, such as lentinan from shiitake – Lentinula edodes, krestin
(PSK polysaccharide-K) and PSP (polysaccharopeptide) from Trametes versicolor,
grifolan and grifron-D from Grifola frondosa, and schizophyllan from
Schizophyllum commune [1, 11], but polysaccharide preparations of some other
medicinal mushrooms also show promising results [13].
2 Solid-State Cultivation
Bioprocessing of various natural sources can be divided generally into processing of

liquid – submerged and solid media sources – solid-state cultivation. SSC involves
the growth and metabolism of microorganisms in beds of moist solid materials in
which the interparticle spaces contain a continuous gas phase and little or no free
water. The main sources of water, carbohydrates, phosphorous, nitrogen, and sulfur
are intrapartically bounded; therefore the microbial culture applied has to possess the
abilities to access the water and essential element sources out the solid matrix [14].
Solid-state cultivation is a three-phase heterogeneous process, comprising solid,
liquid, and gaseous phases, which offers potential benefits for the microbial culti-
vation for bioprocesses and product development. Over the last two decades, SSC
has gained significant attention for the development of industrial bioprocesses,
particularly due to lower energy requirement associated with higher product yields
and less wastewater production with lesser risk of bacterial contamination [15]. Its
historical importance for human kind dates from thousands of years ago, mainly for
food processing, both in western (bread and cheese) and eastern (koji) countries.
Considering the last century and the recent decades, it is used for production of
important biomolecules and products for many industries, including food, pharma-
ceutical, textile, biochemical, and bioenergy, among others [15, 16].
An important advantage of solid-state cultivation over other techniques is that a
concentrated product can be obtained from a cheap substrate, such as an agricultural
residue with little pretreatment or enrichment. On the other hand, the use of an
undefined medium, such as sawdust, might make the product purification more
difficult. For this reason, solid-state cultivation seems to be most appropriate for
the production of pharmaceutically active animal feed supplements, for which the
whole fermented substrate can be used as the product.
In recent years, substantial credibility in employing solid-state cultivation (SSC)

technique has been witnessed owing to its numerous advantages over submerged
bioprocessing (SmB). In spite of enormous advantages, true potential of SSC
technology has not been fully realized at industrial scale [17].
Solid-state cultivation in principal involves the growth and metabolism of fungi
in beds of moist solid materials in which the interparticle spaces contain a continuous
gas phase and little or no free water. The water activity (aw) of substrates is an
important aspect of fungal cultivation, because the growth of fungi is controlled by
the level of aw. The water activity of a substrate is not the same parameter as its water
content but refers to free, unbound, or active water that can support the growth of
fungi. The water activity simply represents the ratio of the water vapor pressure (P)
in any substrate system to the water vapor pressure of pure water (P0) under the same
condition [18].
aw ¼ P=P0 ð1Þ
Water activity is therefore defined as the equilibrium relative humidity divided by

100, and the scale of aw extends from 0 (completely dry) to 1.0 (pure water). The
water potential ψ can be calculated from the water activity as follows [18]:
ψ ¼ RT=M ln P=P0 ð2Þ
where R is the gas constant (8.31 J/mol K), T is the absolute temperature, M is the
molecular mass of water, and P/P0 is the water activity.
Water activity has its most useful application in predicting the growth of fungi. It
has been established that most fungi cease to grow at water activities below 0.60.
This means that the water activity of the substrate determines the lower limit of
available water for fungal growth [18].
A major difference between submerged cultivation and solid-state cultivations is
therefore the amount of free liquid in the substrate. In the case of submerged
cultivation, the amount of dissolved solids rarely exceeds 5–10% by weight (approx.
50–100 g/l), whereas in solid-state cultivation, the solids typically represent between
60 and 80% of the total substrate mass.
The upper limit of moisture content for solid-state cultivations is determined by
the absorbency of the solid, which varies between substrates, although for most
substrates, a free water becomes apparent before 80% moisture level is reached.
The main mechanism of hyphal growth on surface of solid matrix joins the
following steps (Fig. 1):
• Production of fungal polysaccharides on the apical tip of hyphae
• Gluing the apical tip of hyphae on the surface of the lignocellulosic matrix with
polysaccharide gel material
• Transferring lignocellulosic enzymes through gel anchorage to the lignocellulosic
surface
8 M. Berovic
Fig. 1 The main mechanism of hyphal growth on surface of solid matrix. (a) Fungal polysaccha-
rides on the apical tip of hyphae, (b) cross section of glued apical tip of hyphae on the surface of the
lignocellulosic matrix, (c, d) cross section of fungal growth, (e) the void space in overgrown solid
matrix, (f) fungal mycelia on solid surface [14]
• Depolymerization of lignocellulose and mechanical penetration through the

solid matrix
Penetration into substrates is an important phenomenon which has been observed
experimentally but has not yet received modeling attention.
3 Differences Between Submerged and Solid-State

Bioprocessing
Benefits and the main differences between solid-state and submerged cultivations are
summarized in Table 1.
Table 1 Comparison of solid-state and submerged cultivation [14]

Solid-state cultivation Submerged cultivation
Some products can only be produced well under A wide range of products can be produced,
low moisture conditions. For other products, if from a wide range of microorganisms and
the producing organisms require free water, fungi. Many products are produced best under
solid-state cultivation cannot be used submerged cultivation
The medium is relatively simple (e.g., grain) The medium often contains more highly
and unrefined. It may contain all nutrients nec- processed ingredients and is therefore more
essary for growth or only require wetting with a expensive. Unprocessed ingredients may need
mineral solution. Pretreatment can be as simple processing to extract and solubilize the nutri-
as cooking or grinding. However, the substrate ents. With defined media good reproducibility
composition and characteristics can be variable is possible
The low water availability helps to select and The water activity is usually very high, and
protect against growth of contaminants many contaminants can grow well
Media are concentrated and smaller bioreactors Media are dilute and therefore occupy larger
can be used, leading to higher volumetric pro- volumes, leading to lower volumetric
ductivities, even when growth rates and yields productivities
are lower
High substrate concentrations can enable high High substrate concentrations can cause rheo-
product concentrations logical problems. Substrate feeding systems
may be required
Aeration requires less power since pressures are High air pressures can be required. Gas transfer
lower. Gas transfer is easier since the particles from the gas to liquid phase is slow and can be
have a large surface area limiting
Mixing within particles is not possible, and Vigorous mixing can be used, and diffusion of
growth can be limited by the diffusion of nutrients is usually not limiting
nutrients
Ability to remove metabolic heat is restricted, High water content and more dilute nature
leading to overheating problems make temperature control easier
Process control can be difficult due to difficul- Many online sensors are available and more
ties in making online measurements and in are being developed. Additions of substances
measuring biomass. The addition of substances can be made to control the process
during the process is difficult
Downstream processing may be simpler since Downstream processing requires removal of
products are more concentrated. However, large volumes of water and is more expensive.
extracts can be contaminated with substrate However, with defined media, product purifi-
components cation may be easier
Liquid waste is not produced Usually large volumes of liquid wastes are
produced
Growth kinetics and transport phenomena have Much kinetic and transport information is
received little attention and are poorly available in literature, which can guide reactor
characterized design and operation
Research results and information from the In scaling up fungal submerged cultivation
solid-state cultivation can be scaled up or even processes, various technical problems need to
transferred and applied in liquid-state be solved, such as an increased broth viscosity
cultivation and oxygen supply
Solid-state cultivation of fungal mycelia is less Submerged cultivation is more demanding and
labor intensive labor intensive
10 M. Berovic
4 Bioreactor Types Used in SSC
SSC bioreactors could be categorized on the basis of the aeration and agitation
strategies used. They can be classified as bioreactors without mixing and bioreactors
with periodically or continuous mixing. These groups may appear quite different
physically but that operate in similar manners. The most applied bioreactors in solid-
state cultivation are presented in Fig. 2.
4.1 Tray Bioreactors
Tray bioreactors consist of rooms or cabinets containing a number of individual

trays. The cabinet of the bioreactor can be made similar to that of the forced air
circulation dryers or it can be in the form of a room built with tiled walls [19]. The
characteristics of tray bioreactors are a relatively thin layer of substrate is contained
within a tray; the bottom and sides of the tray may be perforated to promote oxygen
transfer, carbon dioxide transfer, and released water transfer; at larger scales, a large
number of trays are usually stacked in a room; the temperature and humidity of
which may be controlled; there is no forced aeration, although air may be circulated
around the tray; and the substrate is usually not mixed, although in some cases, the
substrate is turned intermittently. The trays holding a 2–5 cm thick layer of the
substrate are kept in suitable racks in a cabinet or chamber wherein optimum growth
Mixing
No mixing (or very infrequent) Continuous mixing, or frequent intermittent mixing
Aeration
No forced
aeration
(air passes
around bed)
Tray chamber Rotating drum Stirred drum
Forced
aeration
(air blown
forcefully
through
the bed)
Packed bed Gas-solid Stirred bed Rocking drum

fluidized bed
Fig. 2 Classification of SSC bioreactors according to the agitation and used aeration strategies [14]
Air out
Gaps between trays
Trays with perforated base
Air in
Substrate-
support
Fig. 3 Tray bioreactor and schematic diagram of inside tray layer and indidual tray [17]
parameters are maintained for obtaining higher yield. The tray chamber is humidified
by humidifiers or by forcing in humid air, and provisions are made for the ventilation
and control of humidity as well as temperature (Fig. 3).
Heat removal in tray bioreactors is by conduction through the substrate mass to
the substrate surface or to the tray itself and then convection from these surfaces to
the circulating air. Due to the fact that the air is only circulated around the tray
surfaces and not forcefully blown through the bed, mass transfer within the tray is
limited to diffusion and heat transfer to conduction. Heat and mass transfer consid-
erations can limit the depth of substrate to as little as 5 cm. With larger depths the
inner regions of the substrate may overheat or be deprived of oxygen.
Pleurotus djamor was grown on sunflower seed shell, grape wastes, or potato
peels as renewable cheap substrates producing lipases in solid-state cultivation
carried out in a tray bioreactor [20, 21].
4.2 Packed-Bed Bioreactors
The characteristics of packed-bed bioreactors are a static substrate mass packed into
a column resting on a perforated plate and a forced aeration with controlled temper-
ature and humidity air through the substrate bed, usually coming from below the bed
but sometimes from above. Many variations of this basic design are possible,
including perforated trays [22, 23]. Very often the design of a packed-bed bioreactor
consists of a glass or plastic column with entries at both top and bottom for aeration,
while the temperature is controlled either by putting the column in a temperature-
controlled room or by passing water through double jackets, fitted around the
column. The dominant heat removal mechanism in packed-bed bioreactors depends
12 M. Berovic
on the design and operation of the reactor. Tall thin beds increase the contribution of
radial heat conduction. However, for most packed-bed bioreactors, convection is the
dominant heat transfer mechanism. During active growth, the air temperature can be
lowered to below the optimum temperature for growth, to increase the rate of
convective heat transfer, although this only effectively cools the bottom of the
bed. If the air has low humidity, then the evaporative cooling becomes significant,
although the bottom of the bed tends to dry out. Humidity in the bioreactor can be
controlled by using moist air, which can be additionally manipulated to aid in the
regulation of the incubation temperature. Several researchers have been successfully
using packed-bed bioreactors for a variety of applications [23, 24].
4.3 Rotating Drum Bioreactors
Rotating drum bioreactors are characterized as cylindrical reactors lying horizontally

or slightly inclined, with low-pressure air fed into the reactor headspace. They are
partially filled with substrate and rotate around the central axis to promote mixing of
the substrate, which in some cases might be promoted by baffles. The rotation of the
drum is similar to a rotating cylinder in a washing machine – it moves the cultivated
solids along the drum. Distinct compartments within the drum are also possible. The
main feature of rotating drums is that the air is not forcefully blown through the bed
itself. Rather, it is blown through the headspace above the bed, and the gas exchange
between the headspace and bed is promoted by mixing of the bed, caused by
rotation.
Mixing provided in a rotating drum bioreactor is gentle and among all methods of
automated mixing causes the least damage to fungal hyphae and to the agglomera-
tion of substance particles. However, fungi which are highly sensitive to abrasive
damage may still be affected. In addition, temperature control is quite difficult, even
at small scale, since it is difficult to water-jacket the moving body of the drum,
although it is not impossible. These problems increase significantly with scale.
Heat removal from the substrate bed occurs by two main routes: transfer directly
to the headspace air by convection and evaporation and transfer through the biore-
actor wall by conduction followed by convective cooling of the bioreactor wall.
Mixing in rotating drum bioreactors allows the use of dry air to promote evaporation,
because water can be replenished by spraying a fine mist of water onto the bed, as it
is being mixed. If low humidity air is used for aeration, evaporative cooling
contributes to heat removal [25, 26].
4.4 Stirred Aerated Beds Bioreactors
Stirred aerated beds bioreactors fall into the categories of horizontal or vertical
stirred bioreactors, depending on the orientation (Fig. 4). Horizontal stirred tank
AIR OUT AIR IN
INOCULUM
AIR OUT
WATER
INOCULUM IN
WATER OUT
AIR IN
WATER IN WATER
OUT
Fig. 4 Horizontal stirred tank bioreactor [27]
CROSS SECTION
AIR
FILTER HUMIDITY HEATED
CONTROLLER FILTER
HEATER INOCULATION
AIR AIR
MOTOR
PUMP
STEAM GENERATOR
T = 35°
Fig. 5 Horizontal stirred tank reactor and equipment [29]
bioreactors are similar to rotating drum bioreactors, except that mixing is provided
by the internal scalpers or paddles attached to a central shaft, rather than by the
rotation of the whole body of the bioreactor. Agitation may be continuous or
intermittent. Low-pressure air is introduced into the bioreactor headspace [27, 28].
The horizontal stirred tank reactor, presented in Fig. 5, is filled to two-thirds
volume with a substrate. Air is introduced through a perforated central shaft embed-
ded in the substrate bed, upon which mixer blades are mounted. Principles of process
optimization, such as inoculation, sterilization, mixing, aeration, temperature, and
humidity, were studied and optimized in the production of pectinolytic enzymes by
Aspergillus niger [27]. This type of bioreactor has been later used for solid-state
14 M. Berovic
cultivations of medicinal mushrooms Ganoderma sp. [28, 39, 48] and Grifola
frondosa [29, 42], Trametes versicolor [46], and Hericium erinaceus [49].
Vertical stirred bioreactors often have similarities with packed beds, since they
are usually forcefully aerated. They differ from packed-bed bioreactors by the fact
that they are agitated, which may be continuous or intermittent. The advantage of
being well mixed helps in avoiding the temperature and moisture gradients, which
often occur in packed beds. In the design described by Mitchell et al. [24], three
screw agitators are mounted across the breadth of the reactor on a trolley above the
substrate bed, with the screws extending down into the substrate bed. The trolley
moves backwards and forwards along the length of the bioreactor, such that each
location in the bed is intermittently mixed.
5 Substrates for Medicinal Mushrooms Cultivation
In the wild, wood-degrading mushrooms grow primarily on hardwood of trees.

However, under artificial cultivation conditions, they thrive on various other sub-
strates containing lignin and cellulose and therefore have a high potential for
recycling organic waste materials of different types. As lignocellulose-containing
wastes are produced worldwide in large quantities and in many instances they pose a
threat to the environment, cultivation of edible and medicinal wood-degrading fungi
on lignocellulosic substrates offers almost unlimited possibilities and economically
viable potentials for large-scale commercial cultivation on a world scale [30]. Fol-
lowing groups of lignocellulosic substrates have been identified for artificial culti-
vation (Fig. 6).
Traditional cultivation of fruit bodies on wood logs has been known for centuries.
With time, cultivation methods have diversified, modified, and developed (Fig. 7).
Besides on wood logs, fruit bodies are being produced on sawdust substrates in trays
or beds and in sterilized plastic bags or in bottles. In addition, production of fungal
mycelia has been developed in bioreactors, utilizing submerged cultivation technol-
ogies in liquid media or solid-state substrates. In order to decrease the cultivation
time and to improve the quality, great efforts have been invested into the controlled
cultivation in bioreactors. Cultivation of pharmaceutically active biomass of higher
fungi in bioreactors with optimized substrate compositions and process parameters
enables a shorter cultivation time and a large-scale production under a full process
control.
Fig. 6 Lignocellulosic
substrates for artificial
cultivation of edible and
medicinal mushrooms
6 Cultivation of Medicinal Mushroom in Solid-State

Bioreactors
An important advantage of solid-state cultivation over other techniques is that a

concentrated product can be obtained from a cheap substrate, such as an agricultural
residue with little pretreatment or enrichment. On the other hand, the use of an
16 M. Berovic
Fig. 7 Technological possibilities of cultivating medicinal fungi fruit bodies or fungal biomass on
a commercial scale
undefined medium, such as sawdust, might make the product purification more
difficult. For this reason, solid-state cultivation seems to be most appropriate for
the production of medicinal mushroom, for which the whole bioprocessed substrate
can be used as the product.
Most of medicinal mushroom biomass is still produced using long-term farming
technology (e.g., 4 months for Ganoderma lucidum fruit bodies and 6 months for
G. frondosa), compared to 14 days of solid-state cultivation in the first and 18 days in
the second case for fungal mycelium type of fungal biomass production that is
sufficient for further product isolation. The most important and investigated medic-
inal mushrooms used in comprehensive cultivation in solid-state bioreactors are
Ganoderma lucidum, Grifola frondosa, Trametes versicolor, Hericium erinaceus,
and Cordyceps militaris. SSC of some other species in bioreactors is more in
development [1–3].
6.1 Cultivation of Ganoderma lucidum
Inoculation density is an important factor for the solid-state cultivation processes.

Control of inoculation density was significant for cell growth, morphology, and
production of polysaccharides and ganoderic acids [31–33].
An example of a solid substrate composition, used in the production of
G. lucidum mycelia in a bioreactor, was given by Habijanic et al. [33, 34], consisting
of beech sawdust, olive oil, (NH4)2SO4, KH2PO4, CaCl22H2O, MgSO47H2O,
FeSO47H2O, and distilled water. During cultivation, the content of extracellular
polysaccharides in the solids increased rapidly during the first 7 days, remained
relatively constant until 21 days, and then decreased, suggesting that the polysac-
charide was actually degraded in the latter stages of the process. The period during
which the polysaccharide content decreased corresponded with the time in which the
water mass fraction was falling rapidly, from the values of 70–80% that were
maintained during the first 21 days to 20% at 35 days [34]. However, it was not
clear whether there was a direct cause-and-effect relationship between these two
observations.
The feasibility of reusing soy residue for the production of G. lucidum was
investigated by Hsieh and Yang [35]. The study concluded that soy residue could
be successfully reused as a substrate for the solid-state cultivation. However, proper
mixing with other solid medium was necessary for adjusting the C/N ratio. Soy
residue was found successfully reused as a substrate. However, proper mixing with
other solid medium may be necessary for adjusting the C/N ratio. In the solid-state
cultivation of G. lucidum, the C/N ratio was found to be a crucial factor for the
growth rate of mycelium. C/N ratio has to be adjusted in the range of 70–80.
Song et al. [36] used a whey permeate as an alternative additive to a growth
medium for the cultivation of G. lucidum mycelia in a solid-state cultivation. The
optimum condition was found at pH 4.4 and at 29 C. The results showed that the
cultivation of G. lucidum mycelia could be a potential cost-effective solution for
treatment of cheese whey permeates.
Cultivation on a substrate consisting of oak sawdust and corn bran was studies by
Montoya et al. [36, 37]. Glucose, soy oil, and yeast extract were found as the best in
exopolysaccharide (EPS) production with G. lucidum (0.79 g/l EPS). Agitation was
found that strongly improved EPS production. The IPS content of the carpophores
varied from 1.4% up to 5.5% and 6% in G. lucidum and G. frondosa, respectively.
For G. lucidum solid-state cultivation in pilot-scale bioreactor, Postemsky et al.
[38] used a substrate consisting of 26.2% rice straw, 9.4% rice husk, and 1.9% rice
bran as well as solid substrate based on 32.5% sunflower seed hulls and 5.0% barley.
Substrates were imbibed in 0.5% CaCO3, 2.0% CaSO4 and Cu+2, and enough water
to achieve a final moisture of solid matrix of 60%. As an addition of growth
stimulants, 1% olive oil was applied. After 2 h pasteurization at 85 C, the substrates
were inoculated with grain spawn (8% rate).
18 M. Berovic
In a rare report available by Habijanic et al. [28], the cultivation was carried out in
a horizontal stirred tank reactor with a total working volume of 30 l. The conditions
were controlled as follows: temperature of 30 C, an airflow of 2 l/min, agitation rate
of 80 rpm for 2 min every second day during the first 7 days, and every day during
the latter stages of the cultivation. The effect of initial moisture content was
evaluated. At least aw 0.85 was necessary to give satisfactory rates of cell growth
and exopolysaccharide production. The control mechanisms of the fungal
exopolysaccharide production and consumption were not known; however, the
authors suggested that exopolysaccharides served to fasten the hyphae to the surface
of the solid particles and to protect the hyphae both from mechanical damage during
the agitation and from desiccation at low moisture contents.
Habjanic and Berovic [39] patented a process of growing G. lucidum on a
solid cultivation substrate using the solid-state cultivation in a horizontal stirred
bioreactor. The process enabled a precise leading and monitoring of the fungal
growth at sterile conditions. Periodically mixing of 80 rpm for 2 min/day was
applied. Production of fungal polysaccharides and fungal biomass on solid substrate
based on beech sawdust, olive oil, and mineral salts was studied. Optimal moisture
of the solid matrix in the range of 80 to 74% was found. When the moisture content
dropped below 57%, the growth of the mycelium and polysaccharide production
stopped, but it revived when wet air was applied in further process. Final concen-
tration of the intracellular was 4.5 mg/g and extracellular polysaccharides 1.05 mg/g
at biomass concentration of 0.68 mg/g solid substrate. Produced biomass could also
be used as a solid inoculum for further cultivation of G. lucidum [39].
6.2 Cultivation of Grifola frondosa
As the great demand for G. frondosa fruit bodies and/or mycelium biomass is in
constant increase, cultivation of fruit bodies in bags and biotechnological production
of biomass in bioreactors, especially for the production of polysaccharides with
medicinal properties, have become the fastest and most efficient ways to respond to
the increasing demands. However, several authors report that the quality and quan-
tity of physiologically active substances vary from strain to strain and also depend on
location, culture conditions, extraction, and processing procedures [37, 40].
Cheap substrates composed of secondary raw materials, such as agricultural,
food, and wood industry residues with little pretreatment or enrichment with mineral
salts (MgSO47H2O, K2HPO4, and MnSO45H2O), can be used for G. frondosa
cultivation. For example, Xing et al. [40] used the substrate consisting of (dry weight
basis) 75% of oak sawdust (25% humidity), 23% of corn bran (15% humidity), 1%
sucrose (2% humidity), and 1% of calcium carbonate. The moisture content was then
adjusted to 62%. Montoya-Barreto et al. [36, 37] assayed two different substrate
formulations. The first one consisted of (dry weight basis) 75% oak sawdust (25%
humidity), 23% corn bran (15% humidity), 1% sucrose (2% humidity), and 1%
calcium carbonate. In the second formulation, 75% oak sawdust was replaced by
50% oak sawdust and 25% coffee spent ground (70% humidity). The humidity was
calculated in relation to the components dry weight. It was found that the use of corn
Table 2 Cultivation of G. frondosa mycelium by solid-state cultivation in bioreactors

Cultivation
Cultivation Substrate/inoculation parameters Yield Reference
Solid-state Wheat bran mixed 6–12 days of culti- 5–25% (dry weight of [40]
cultivation with tap water 6:4 vation, 25 C, rela- mycelium per dry
(w/w), sterilized at tive humidity >97% weight of substrate)
121 C for 40 min,
inoculation by myce-
lium from the agar
slants
Horizontal 2,000 g of milled 27–40 days, 30 C, Biomass 0.20 mg [29]
stirred tank whole corn plant (Zea airflow 2 l/min. glucosamine/g solid
bioreactor mays) supplemented Periodic mixing. substrate. Polysac-
with 100 mg Moisture content charides (per g of
(NH4)2SO4, 400 mg 80 5%, lowest solid substrate)
KH2PO4, 100 mg 50% 30 mg/g extracellular
CaCl22H2O, 100 mg and 14.0 mg/g
MgSO47H2O, intracellular
300 mg
FeSO47H2O, 4 g
CaSO4, 40 ml olive
oil, 1,000 ml distilled
water, and 500 g of
olive press cake
Horizontal Food and wood 38 days, 28 C, aera- Dry biomass 35 g/l; [41]
stirred tank industry waste tion 5 l/min, periodic 3.8 mg/g extracellu-
reactor, materials mixing, 5 min/day lar, 0.70 mg/g intra-
30 l cellular
polysaccharides
grains as substrate for spawn production results an important factor for reducing crop
cycle time. A cold shock to 10 C was requisite for basidiome formation. Coffee
spent ground was a good substrate for mycelial growth, but not for mushroom
production. When using oak sawdust plus corn bran as substrate, consistent yields
with combined high biological efficiency (BE) (35.3%) were obtained in crop cycle
of 12–14 weeks.
Cultivation of G. frondosa mycelium by solid-state cultivation in a bioreactor is
still rare. Examples of cultivation parameters are given in Table 2.
6.3 Cultivation of Trametes versicolor
To cultivate Trametes versicolor, Knežević et al. [42] were using wheat straw and
oak sawdust as carbon sources and 10 ml of modified synthetic medium without
glucose, with NH4NO3 and pH 6.5, while Stoilova et al. [43] were using wheat bran
and oat straw carbon sources. The SSC was carried out using a medium consisted of
4.0 g wheat bran, 2.5 g oat straw, and 2.5 g beetroot press in 300 ml flasks. The moist
20 M. Berovic
of the substrate was adjusted to 60% by mineral salt solution containing 0.14%
(NH4)2SO4; 0.2% KH2PO4; 0.03% MgSO47H2O, 0.03% CaCl2, FeSO47H2O,
ZnSO47H2O, and MnSO47H2O; and 0.002% CoCl2 at pH 4.5.
In T. versicolor solid-state cultivation, the modified wheat straw was used by
Dinis et al. [44], while de Souza et al. [45] demonstrated that it was possible to
produce high laccase levels using the wheat bran as a substrate without any supple-
mentation in the fluidized bed reactor [44, 45].
Rakuš et al. [46] cultivated T. versicolor mycelia in solid-state cultivation on a
corn straw in 15 l horizontal stirred tank bioreactor. The moisture content of the
substrate before inoculation was 2.33 (w/w dry biomass). The inoculum consisted of
twenty-five 1 cm2 cuts mixed in a sterile grinder together with 700 ml of sterilized
distilled water. Both inoculation and cultivation were carried out at 24 C and the
airflow of 5 l/min. Periodical mixing of 80 rpm, per 2 min (in the first 7 days every
second day, and 2 min every day in the last part of cultivation) was used. 5.95 g/l
intracellular polysaccharides were isolated.
6.4 Cultivation of Hericium erinaceus
The potential of using several agricultural by-products as supplements of sawdust

substrate for the production of edible mushroom Hericium sp. was evaluated using
seven Hericium species. All the tested supplements (rice bran, wheat bran, barley
bran, Chinese cabbage, egg shell, and soybean powder) were found to be suitable for
the mycelial growth of all the tested species. In mycelial growth, soybean powder
was the best supplement for Hericium americanum, Hericium coralloides, and
Hericium erinaceus, while barley bran was the best for Hericium alpestre, Hericium
laciniatum, and Hericium erinaceus. For Hericium abietis, rice bran and Chinese
cabbage were the best. The possibility of mushroom production on oak sawdust
substrate with 20% rice bran supplement was demonstrated with H. coralloides,
H. americanum, and H. erinaceus, which showed 26–70% biological efficiency. Our
results also showed that strain selection is important to improve biological efficiency
and mushroom yield in Hericium cultivation [47].
Gerbec et al. [48, 49] cultivated Hericium erinaceus in a 15 l horizontal stirred
tank reactor (HSTR) of our own construction and design. The cultivation was carried
out at 24 C and 5 l/min airflow. Periodical mixing of 80 rpm for 2 min (in the first
7 days every second day, and every day in the last part of cultivation) was used.
Although solid substrate was intensively covered by fungal biomass, no erinacine A
production was detected. The main reason for this could be the lack of precursors
needed for erinacine A biosynthesis. Substrate used did not include enough starch to
support the fungal metabolism in sense of erinacine A synthesis. During cultivation
carbon dioxide was ventilated out of solid matrix, therefore eliminating its stimula-
tory effect, possibly affecting erinacine A biosynthesis [49].
Han [50] studied Hericium erinaceus solid-state cultivation on cornmeal
degrading starch and upgrading nutritional value. On the basal medium which
consisted of cornmeal and salt solution, H. erinaceus produced a strong α-amylase on

the 15th day after inoculation, which resulted in a 52% degradation of the starch. By
supplementation with 5–15 g soybean meal per 100 g cornmeal, the α-amylase
activity and degradation rate of starch were raised significantly. Prolongation of
fermentation time from 15 to 30 days did not increase significantly the degradation
rate of starch, though the α-amylase activity reached its maximum value of 179 U/g
on the 20th day after inoculation.
6.5 Cultivation of Cordyceps militaris
For the cultivation of Cordyceps militaris, substrates from rye seeds and spent
brewery grains were mixed in different proportions (9:1, 8:2, 7:3, 6:4, 5:5, 4:6).
Water was added to the mixture to achieve 65% moisture content and 100 g of
substrate weight. Spent brewery grains represent a readily available, low-price
substrate for cordycepin solid-state production. So far the highest reported concen-
trations (10.42 mg/g) obtained on solid-state substrates were reported [51].
7 Conclusions
Reports on pharmacological activity of solid-state bioreactors produced G. lucidum

biomass and extracts, partly purified and isolated compounds from fungal mycelia
biomass are convincing. There is abundant scientific evidence that triterpenoids,
polysaccharides, and proteoglycans produced in solid-state bioreactors have been
effective also in in vitro and in vivo testing [10, 11]. Synergistic effects of mixtures
of active components have been known, however, but their biological activities need
further assessment before they can be fully accepted not only by the traditional Asian
medicine but also by the Western science and medicine. In this respect, modern
biotechnological cultivation methods in solid-state bioreactors enable fast, efficient,
and economical production of G. lucidum biomass in sufficient quantities for
potential future pharmaceutical large-scale industrial production [39, 48].
G. frondosa can be regarded as a natural bioreactor for the production of healthy
nutritious food and pharmaceutical compounds with beneficial effects. The beauty of
its nature is that it is a gourmet and medicinal mushroom, willing to grow on simple
cellulose and lignin containing substrates, including waste materials from agricul-
tural production, as well as on liquid substrates in bioreactors, where its hyphae
excrete medicinal polysaccharides into the medium. Isolation is rather simple and is
based on precipitation with ethanol. Crude extracts show equal or stronger pharma-
cological activity as purified compounds, which suggests potential synergistic
effects of several naturally occurring compounds. Expensive high-tech purification
procedures for isolation of pure pharmaceutical substances may therefore not be
needed [29, 42]. However, from the viewpoint of Western science and medicine and
22 M. Berovic
pharmaceutical legislation and regulations, this might be one of the main obstacles
hindering the introduction of G. frondosa products as registered pharmaceuticals. In
any case, further research is needed to fully understand all mechanisms of pharma-
ceutical effects and to identify potential side effects of G. frondosa medicinal
preparations [52].
Convincing reports on in vitro and in vivo pharmacological activity of extracts,
partly purified preparations and isolated compounds from SSC medicinal mushroom
biomass as terpenoids and polysaccharide krestin (PSK) and polysaccharide-peptide
(PSP) of Trametes versicolor [43, 46] also with immunomodulation, antibody
production activities. In vitro and in vivo studies PSK and PSP shown as pure
substances and also as PS mixture shown of both highly affecting immune cell
proliferation and highly express antitumor activities [46, 53–58].
Among the compounds isolated from H. erinaceus fruit bodies and cultured
mycelia are polysaccharides and diterpenoids where the most interesting are the
low-molecular-weight compounds belonging to a group of cyathin diterpenoids
(erinacines A–K, P, and Q) [59–61]. Several of them, i.e., erinacines A–H, are
known to have a potent stimulating effect on nerve growth factor (NGF) synthesis
in vitro [59–65].
Great potential of pharmaceutical active compounds and Cordyceps militaris extract
contains many biological bioactive materials, such as the terpenoids cordycepin and
cordycepic acid, polysaccharides, sterols, and other compounds [66]. Cordyceps
militaris main active component is terpenoid cordycepin that inhibit the development
of cancer cells including antitumor, anti-metastatic, insecticidal, antiproliferative,
antibacterial, antileukemia, and antimalarial activities. The second main active com-
ponents are polysaccharides, which research have shown to be effective in regulating
blood sugar and also have anti-metastatic and antitumor properties [66–68].
Although solid-state cultivation of fungal biomass in bioreactors does not pro-
duce fungal fruit bodies, its products also represent great potential of delignified and
in proteins enriched biomass with various highly valuable pharmaceutically active
compounds that could be extracted and isolated or simply pulverized and applied in
veterinary use. In this respect, modern biotechnological cultivation methods in
bioreactors enable fast, efficient, and economical production of medicinal fungi
biomass in sufficient quantities that could be applied in pharmaceutical large-scale
industrial production.
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DOI: 10.1007/10_2019_90
Design and Operation of a Pilot-Scale

Packed-Bed Bioreactor for the Production
of Enzymes by Solid-State Fermentation
David Alexander Mitchell, Luana Oliveira Pitol, Alessandra Biz,

Anelize Terezinha Jung Finkler, Luiz Fernando de Lima Luz Jr.,
and Nadia Krieger
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2 Choice of Bioreactor Type and Design Features . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.1 Available Bioreactor Types . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
2.2 Choice of a Packed-Bed Bioreactor with Intermittent Agitation . . . . . . . . . . . . . . . . . . . . . . 29
2.3 Bioreactor Designs that Were Considered but Discarded . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.4 Aspects of the Final Design of the Bioreactor that Was Built . . . . . . . . . . . . . . . . . . . . . . . . 32
2.5 Design of the Air Preparation System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
2.6 Monitoring Equipment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
2.7 Sampling from the Bed . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
D. A. Mitchell (*)
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba,
Paraná, Brazil
L. O. Pitol and A. T. J. Finkler
Departamento de Engenharia Química, Universidade Estadual de Maringá, Maringá, Paraná,
Brazil
A. Biz
Department of Chemical Engineering and Applied Chemistry, Toronto, ON, Canada
L. F. de Lima Luz Jr.
Departamento de Engenharia Química, Universidade Federal do Paraná, Curitiba, Paraná, Brazil
N. Krieger
Departamento de Química, Universidade Federal do Paraná, Curitiba, Paraná, Brazil
28 D. A. Mitchell et al.
3 Preparation of Substrate, Bioreactor, and Inoculum for a Pilot-Scale Fermentation . . . . . . . . 39

3.1 Preparation of the Substrate and the Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39
3.2 Preparation of Sufficient Inoculum and Inoculation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
4 Production of Enzymes in the Pilot Bioreactor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
4.1 Production of Pectinases on Wheat Bran . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4.2 Production of Pectinases and Lipases in Beds with High Sugarcane Bagasse
Contents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
5 The Pilot Bioreactor and Mathematical Models in Scale-Up Studies . . . . . . . . . . . . . . . . . . . . . . . 46
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Abstract In this review, we describe our experience in building a pilot-scale

packed-bed solid-state fermentation (SSF) bioreactor, with provision for intermittent
mixing, and the use of this bioreactor to produce pectinases and lipases by filamen-
tous fungi. We show that, at pilot scale, special attention must be given to several
aspects that are not usually problematic when one works with laboratory-scale SSF
bioreactors. For example, it can be a challenge to produce large amounts of inoculum
if the fungus does not sporulate well. Likewise, at larger scales, the air preparation
system needs as much attention as the bioreactor itself. Sampling can also be
problematic if one wishes to avoid disrupting the bed structure. In the fermentations
carried out in the pilot bioreactor, when the substrate bed contained predominantly
wheat bran, the bed shrank away from the walls, providing preferential flow paths for
the air and necessitating agitation of the bed. These problems were avoided by using
beds with approximately 50% of sugarcane bagasse. We also show how a mathe-
matical model that describes heat and water transfer in the bed can be a useful tool in
developing appropriate control schemes.
Graphical Abstract
saturated air
Humidification
column
Filter
flowmeter
cool reservoir warm reservoir
Air preparation Bioreactor
Keywords Inoculum preparation, Intermittent mixing, Mathematical modeling,

Monitoring and sampling, Pectinases and lipases, Scale-up
Design and Operation of a Pilot-Scale Packed-Bed Bioreactor for SSF 29
1 Introduction
This review describes our experiences with building and operating a pilot-scale
packed-bed bioreactor for solid-state fermentation (SSF). It describes why we
chose this particular bioreactor design and our most recent results. Our aim is to
provide some insight into the thinking behind the design and operation of the
bioreactor and some of the key challenges that we faced; this type of information
is often not given in scientific papers. We hope that this description of our experi-
ences will be useful to other researchers intending to scale up their laboratory-scale
SSF bioreactors to pilot scale.
2 Choice of Bioreactor Type and Design Features

2.1 Available Bioreactor Types
When we decided to build a bioreactor with a capacity for several kilograms of solid
substrate, the first decision was which type of bioreactor to build. Bioreactor types
for SSF can be classified into four groups based on how the bed is agitated and how
the bed is aerated [1]:
1. Tray-type bioreactors – the bed remains static or is mixed infrequently (e.g., once
or twice per day) while air is circulated around the bed, not being forced to flow
through it.
2. Packed-bed bioreactors – the bed remains static or is mixed infrequently (e.g.,
once or twice per day), with air being blown forcefully into the bed, having to
flow through the bed in order to leave it.
3. Rotating- or stirred-drum bioreactors – the bed is agitated continuously or
frequently in a horizontal drum, with air being circulated through the headspace
above the bed, not being forced to flow through the bed itself.
4. Forcefully aerated agitated bioreactors – the bed is agitated continuously or
frequently, with air being blown forcefully into the bed, having to flow through
the bed in order to leave it.
2.2 Choice of a Packed-Bed Bioreactor with Intermittent

Agitation
We considered several factors in choosing a packed-bed bioreactor with provision

for intermittent agitation. The main consideration was that forced aeration would be
necessary in order to allow effective removal of waste metabolic heat from the bed.
This left the choice between a packed-bed bioreactor and a forcefully aerated
agitated bioreactor. The choice between these two bioreactor types was based on
considerations as to the frequency with which the bed should be mixed.
Two thoughts guided our decision to provide for intermittent agitation. Since
most of our work involved the cultivation of filamentous fungi, we wanted to leave
the bed static most of the time. Although some filamentous fungi can tolerate
continuous agitation, agitation significantly affects the growth morphology, with
aerial hyphae being squashed into a moist biofilm at the particle surface. A static
bed allows aerial hyphae to grow into the inter-particle spaces, where they are
surrounded by an air phase [2]. However, we were also aware that it might be
necessary to mix the bed several times during the fermentation. Mixing might be
necessary to enable the addition of water to the bed or to prevent the formation of
agglomerates and the related phenomenon of channeling.
Addition of water is often necessary because the forced aeration in a packed bed
tends to dry the bed out, even if saturated air is supplied at the air inlet. This occurs
because the air removes metabolic heat from the solid particles as it passes through
the bed: the resulting increase in the air temperature increases its water holding
capacity, creating a driving force for evaporation [3]. This evaporation can cause the
water activity of the solids in the bed to fall to values that cause a significant decrease
in the rate of growth and product formation, meaning that it becomes necessary to
replenish the water. It is not feasible to add water uniformly to a static bed; rather, the
water should be sprayed as a fine mist onto the surface of the bed as it is agitated.
Channeling occurs if cracks appear in the bed or the bed pulls away from the
walls, allowing the air to flow preferentially though the gaps, rather than through the
bed itself. The appearance of cracks or gaps is common in SSF processes with
filamentous fungi, resulting not only from a decrease in particle size as the bed dries
and as the solid particle shrinks as it is consumed by the fungus but also from the fact
that the fungus tends to bind the particles together into agglomerates, promoting
shrinking of the bed as a whole [3]. When channeling occurs, it is necessary to
agitate the bed, with the intention of breaking agglomerates and allowing the bed to
be “reseated,” with a layer of substrate particles of uniform height across the whole
bed. It should be noted that agitation is not always effective at breaking agglomer-
ates, such that it can be appropriate to undertake “preemptive” mixing events, with
the intention of preventing channeling from occurring in the first place [4].
2.3 Bioreactor Designs that Were Considered but Discarded
We considered several designs before settling on the packed-bed design. Two key
designs that were carefully considered, but later discarded, were a horizontal stirred
drum with a perforated bottom and a horizontal cylinder with air provided through
perforated walls and removed through a perforated pipe at the central axis (Fig. 1).
(a)
air in
air in
(b)
air
in
air
out
air
in
Fig. 1 Bioreactor designs that were considered but were discarded. (a) A horizontal stirred drum
with a perforated bottom. In the diagram on the right, the air chamber has been removed in order to
make the perforated bottom of the cylinder visible. (b) A horizontal cylinder with air provided
through a perforated outer wall and removed through a perforated pipe at the central axis. In both
cases, substrate bed is indicated in gray. Part (a) is reprinted from Mitchell et al. [3] with the kind
permission of Springer
The horizontal stirred drum with a perforated bottom was discarded because the
bed is not of a uniform height, due to the circular cross section of the drum (Fig. 1a).
During static periods of operation, the air would tend to flow preferentially through
the shallower parts of the bed; there would be little or no flow through the deeper
parts of the bed. In such a bioreactor, the whole bed would only be aerated
effectively if the bed were mixed continuously.
The horizontal cylinder with perforated walls and a perforated pipe at the central
axis had the potential to ensure uniform airflow through the bed (Fig. 1b) but was
discarded due to potential complications in design and operation. The first compli-
cation is that, in order to avoid preferential flow through some parts of the bed, such a
bioreactor would need to be always full, to ensure that the flow paths through the
bed, from the wall to the center, were of equal length. We considered that it might be
possible to divide the “air jacket” between the outer wall and the substrate bed into
compartments. It would then be possible to close off some compartments if the
bioreactor were used when only partially full, ensuring that air were only introduced
into the bed itself. However, it soon became clear that the engineering would be
unnecessarily complex. The second complication is that, with the cylinder being
operated completely full of substrate, it would be difficult to mix the bed if it were
necessary to add water or to reseat the bed after the appearance of cracks or gaps.
Having decided on a traditional packed bed, with the solid bed resting on a
perforated base plate, the next decision was whether or not to have internal heat
transfer plates, such as those used in the Zymotis bioreactor [5]. Simulations with a
mathematical model of this type of bioreactor had shown that, for the heat transfer
plates to be effective in removing the metabolic heat generated by a fast-growing
fungus, it would be necessary for the plates to be about 6 cm apart [6, 7]. Such a close
spacing would make it difficult to mix the bed if it were necessary to add water or to
reseat the bed after the appearance of cracks or gaps.
2.4 Aspects of the Final Design of the Bioreactor that Was

Built
The final design (Fig. 2) was a traditional packed bed, constructed in stainless steel
(AISI 306). The bed height can be varied as desired, up to a maximum of 50 cm, by
loading more or less substrate into the bed chamber. Since this bed chamber is
rectangular, the bed height is uniform, even if the bioreactor is not used at its full
capacity of 210 L (corresponding to a base of 70 cm by 60 cm and a bed height of
50 cm, Fig. 2b).
The question then arose as to how the intermittent agitation of the bed should be
done. Two potential designs were considered but discarded. First, a rotating agitator
could be inserted into the bed. Such a design has been used in the bioreactors of
INRA Dijon, with the use of traveling screw augers (which mix by lifting the
substrate and allowing it to fall) (Fig. 3a) or a non-traveling planetary-type mixer
(Fig. 3b) [8]. Second, either rotating agitators or static blades could remain in the
same position, with the whole bed being rotated, such as done by the group of PUC
in Chile (Fig. 3c) [9]. However, for these designs involving mechanical agitators in
the bed, it would be desirable to remove the agitator from the bed after the agitation
events, in order to avoid preferential air paths next to the stopped agitator during
static operation. This would have required careful mechanical design and signifi-
cantly increased the cost of our bioreactor. In our final design, the bed was mixed by
rotating the whole bioreactor around its central axis, enabling mixing without the
insertion of moving mechanical parts within the bioreactor itself. The bioreactor
rotation was achieved with an electric motor placed outside of the bioreactor. The
V-shaped wedge protruding into the headspace (Fig. 2a) was intended to help break
up any agglomerates of substrate particles during the rotation. It should be noted that
the bed height is not necessarily uniform after an agitation event, but it is an easy
matter to open the lid at the top of the bioreactor and then flatten the top of the bed
manually.
(a) (b)
60 cm
intermittently rotated
lid
air
closed
out rectangular
chamber 50 cm
for the bed
70 cm
wire mesh
air perforated
in base plate
(holes are 1 cm in diameter)
(c) (d)
lid open
h= 45
h= 33
height (cm)
h= 18
h=5
0 5 35 43 56 70
position (cm)
Fig. 2 Details of the pilot bioreactor. (a) Perspective of the bioreactor, with the lid closed. The
circular front face has been removed so that the bed chamber is visible (in darker gray). The bed is
agitated by rotating the whole bioreactor around its central axis. (b) Exploded view of the substrate
bed chamber. The wire mesh prevents small particles from falling through the 1 cm diameter holes
in the base plate. (c) Perspective of the bioreactor, as in (a) but with the lid open, allowing not only
loading and unloading of the bioreactor but also the removal of samples from the top of the bed
during the fermentation. (d) Positions of the thermocouple sleeves in the bed chamber. In this
diagram, the front face of the bed chamber is the same face that is visible at the left in (c) (i.e., the
bed chamber has been rotated slightly counterclockwise). Each sleeve contains four thermocouples
at different horizontal positions. Part (a) is adapted from Mitchell et al. [3] with the kind permission
of Springer
(a) motors mounted on a carriage (b)

that moves back and forth
motor
flexible water air
hoses and power out
cables
spray
nozzle headspace
intermittently
rotated
planetary
screw mixer
auger bed
wire mesh support air in

air box
air in
(c)
motor
nozzle
system
for water
addition
stationary blades
bed
basket with
seal perforated bottom
air in
air box
Fig. 3 Potential ways of providing intermittent mixing in a packed-bed bioreactor that were
considered but discarded. (a) A system of traveling screw augers [8]. (b) A planetary mixer
[8]. (c) A bed that moves past mixers that remain in place [9]. Parts (a), (b), and (c) are adapted
from Mitchell et al. [3] with the kind permission of Springer
Cost considerations also affected the design of the aeration system. We consid-
ered passing the inlet air pipe through the central axis of the bioreactor. This would
have allowed for the possibility of aeration during mixing, such that the bioreactor
could operate somewhere between a rotating drum (the third bioreactor group listed
in Sect. 2.1) and a forcefully aerated agitated bioreactor (the fourth bioreactor group
listed in Sect. 2.1). However, introducing air through the central axis would have
significantly increased costs.
2.5 Design of the Air Preparation System
Given the importance of the forced aeration in removing the metabolic heat from the
bed in a packed-bed bioreactor, the temperature, humidity, and flow rate at which the
air will be supplied are key considerations [3, 10]. Our air preparation system is
shown in Fig. 4.
The airflow rate is not difficult to control: if the compressor or blower is designed
to supply a high flow rate, then a butterfly valve can be used to regulate the flow.
Although such a valve could be controlled automatically, for example, in response to
temperatures measured in the bed, we manipulated our butterfly valve manually.
We used a blower that was already available to us. It was capable of giving
volumetric airflows of the order of 150 m3 h1. Based on our bioreactor design, this
saturated air
to bioreactor
Humidification
column
Butterfly Electronic
containing a bed
valve flowmeter
of Raschig rings
Filter
blower
cool reservoir warm reservoir
Fig. 4 Air preparation system for the pilot bioreactor. The humidification column can be fed from
either the cool reservoir or the warm reservoir. When one reservoir is being used, the pump of the
other reservoir is turned off, and the return valve of the other reservoir is closed. The temperatures of
the water in the reservoirs are maintained using heating coils controlled by thermostats
would give a nominal superficial velocity (volumetric flow rate divided by the total
cross-sectional area of the bed) of:

m3 106 cm3 1h
150 =ð60 cm 70 cmÞ 10 cm s1
h 1 m3 3;600 s
The air was passed through a HEPA (high-efficiency particulate air) filter;
however, no attempt was made to maintain the air line after the filter sterile, such
that the main role of the HEPA filter was to remove dust from the inlet air.
Theoretically, it would be possible to supply air of any desired combination of
temperature and humidity. This could be done by using electrical resistances to heat
the air and injecting steam into it or, alternatively, by mixing dry and saturated air at
the desired temperature [3]. However, these strategies are not simple to implement,
with both requiring equipment for measurement and control.
In the end, we decided to use a much simpler design for the air preparation
system, namely, a system with a humidification column, with this column being
supplied from one of two water reservoirs (Fig. 4), each of which has a volume of
1,200 L. One reservoir, the “warm reservoir,” was maintained at the optimal
temperature for growth, and the other reservoir, the “cool reservoir,” was maintained
at a temperature several degrees lower. The humidification column contained a
dispersion nozzle that showered water over a 70-cm-high bed of Raschig rings.
The water was then returned to the reservoir from which it came. The bed of Raschig
rings was oversized: it was designed by using a heat and mass balance model that
suggested that saturation of the air would be achieved with a 35-cm-high bed.
The idea was that, at the beginning of the fermentation, it would be necessary to
warm the bed to the optimal temperature for growth but that, once growth had
started, it would be necessary to pass cooler air through the bed, approximately 5 C
below the optimal temperature for growth, following the strategy suggested by
Mitchell et al. [3]. Since the optimal temperatures for growth of the fungi that we
were using (strains of Aspergillus and Rhizopus) were over 30 C and the room
temperature typically did not exceed 25 C, it was not necessary to refrigerate the
cool reservoir. In fact, during winter, it was typically necessary to heat the cool
reservoir to around 25 C.
The disadvantage of our air preparation system is that we are limited to using
saturated air at one of two temperatures and, therefore, we cannot promote evapo-
ration by using partially saturated air. However, we considered that it is not partic-
ularly desirable to use unsaturated air in a packed-bed bioreactor, especially if the
aim is to minimize the number of agitation events. Supplying unsaturated air to a
packed-bed reactor can lead to the bed drying out significantly.
2.6 Monitoring Equipment
Thermocouples are placed at the air inlet and air outlet. Originally, combined
temperature and relative humidity sensors were placed at these positions; however,
they were not able to cope with the saturated air and lost their function. Four
thermocouple sleeves cross the bed horizontally, at different vertical and horizontal
positions (Fig. 2d). Each sleeve contains four thermocouples. A piezoelectric dif-
ferential pressure sensor is connected at the air inlet and air outlet. The readings of
these sensors are logged every 18 s by LabVIEW. In some fermentations, we
measured the O2 and CO2 concentrations in the outlet air, using an equipment that
combines an electrochemical O2 sensor with an infrared CO2 sensor, with these
readings also being logged by LabVIEW.
There is also an electronic flowmeter in the inlet air line. It measures the pressure
drop across an orifice plate, but the reading is not logged by LabVIEW.
Although data is logged on the computer, due to cost considerations, the biore-
actor does not have any computer-controlled systems. The temperatures in the water
reservoirs for the humidification column are controlled automatically by thermostats
that control electrical resistances. Each reservoir works at a fixed setpoint; the
setpoint temperatures of the reservoirs are not manipulated during the process.
The control of other aspects of bioreactor operation is operator-dependent. Although
it would be possible to use an automated system to measure bed temperatures and
control the pumps feeding the humidification column and the valves in the pipes
returning the water from the humidification column to the reservoirs, in our case, this
switching is done manually by the operator (Fig. 4). Likewise, if it is decided that it is
necessary to rotate the bioreactor to mix the bed, the operator must disconnect the
inlet and outlet air hoses and the cables attached to the thermocouples and then turn
on the electric motor manually.
A perforated tube was placed in the headspace of the bioreactor, with the intention
of allowing water to be sprayed onto the bioreactor surface when the bioreactor was
in the upright position during a mixing event. The idea was that the water would be
added in several aliquots, with rotation of the bioreactor between additions. How-
ever, this tube did not give a sufficiently uniform water distribution. Water addition
was therefore done by opening the lid of the bioreactor and using a spray bottle to
mist water onto the top of the bed. Once again, the water was added in several
aliquots, with rotation of the bioreactor between additions.
2.7 Sampling from the Bed
Removing samples from packed-bed SSF bioreactors is problematic. The removal of

samples from within the bed can lead to the formation of preferential flow paths. We
were aware of this but were also interested in monitoring the development of axial
gradients of moisture content and product formation in the bed. We therefore
developed a sampler consisting of a screw auger inside a sleeve (Fig. 5a). During
a completely static fermentation, we used this sampler to remove a “core” from the
bed. We then took fresh substrate that had been prepared as for a fermentation, but
which had not been inoculated, and pressed it into the vertical hole that had been left
by the removal of the sample. However, when we opened the bioreactor later during
the fermentation to take another sample, the fresh substrate that we had pressed into
(a)
i ii
iii iv
(b)
top
middle
bottom
substrate bed
exploded view
Fig. 5 Sampling from the bed of the pilot bioreactor. (a) Attempts to remove a core from the bed
during the fermentation. Key: (i) Use of a screw auger to remove a core from the bed; (ii) vertical
hole in the bed after removal of the core; (iii) vertical hole packed with uninoculated substrate;
(iv) by the next sampling time, the uninoculated substrate had been blown out of the hole by the
forced aeration. (b) Mapping of the bed at the end of the fermentation. Three horizontal planes were
sampled, at the top, middle, and bottom of the bed. Five samples were removed from each plane, at
the locations marked
the hole was on top of the bed, and the hole was fully open, with the air flowing
preferentially through this hole, rather than through the bed. After this, we decided
not to try to remove samples from within the bed during unmixed fermentations. In
unmixed fermentations, we limited ourselves to removing samples from the top of
the bed. Of course, in a fermentation with intermittent agitation, it is possible to
remove samples from within the bed without disturbing the bed, if this is done
immediately prior to the mixing event.
At the end of the fermentation, it is possible to sample the bed destructively in
order to “map” gradients of moisture content and product levels (e.g., enzyme
activities) in three dimensions. Layers of substrate can be carefully removed, with
samples being removed from as many horizontal and vertical positions as is desired
(Fig. 5b).
In some fermentations, we had an interest in following the evolution of gradients

in a static bed during the fermentation. For this, it was necessary to undertake
replicate fermentations and sample each one destructively at a different fermentation
time. In other words, in order to characterize the spatial moisture content and enzyme
activity gradients within the bed, we undertook three different but identical fermen-
tations, stopping each one at a different time and sampling it destructively [11]. Two
such experiments were done. For a completely static bed, the three fermentations
were stopped at 12, 20, and 26 h. For a bed mixed three times (at 8, 10, and 12 h), the
three fermentations were stopped at 15, 20, and 26 h.
The effect of mixing on spatial homogeneity within the bed is discussed in Sect.
4.1. However, these experiments also gave an insight into the reproducibility
between identical fermentations carried out at different times. We plotted the
temporal profiles for the enzyme activity of samples removed from the top of the
bed. For the bed mixed three times, the temporal profiles for the fermentations
stopped at 15, 20, and 26 h were quite similar to one another. For the completely
static bed, there was significantly more variation among the profiles for the fermen-
tations stopped at 12, 20, and 26 h [11].
3 Preparation of Substrate, Bioreactor, and Inoculum

for a Pilot-Scale Fermentation
In order to undertake a fermentation, it is necessary to prepare both the substrate and

an inoculum in sufficient quantities and to prepare the bioreactor.
3.1 Preparation of the Substrate and the Bioreactor
The rectangular substrate chamber of the bioreactor has a base of 60 cm by 70 cm

and a height of 50 cm (Fig. 2b). When this chamber is full, the bed occupies 210 L.
Of course, the mass of fresh substrate that can be held within this volume varies,
depending on several factors: (1) the density of the substrate particles themselves;
(2) the size and shape of the substrate particles, which affect how they pack; (3) the
loading procedure, especially whether or not any pressure is applied to the substrate
bed during packing; (4) whether or not the substrate bed is mixed, as this affects the
packing of the particles; and (5) the water content used [12].
We used masses of up to 100 kg of dry substrate (50 kg soybeans plus 50 kg
wheat bran) plus 40 kg water in fermentations undertaken with Aspergillus oryzae
[13]. In fermentations undertaken to produce pectinases, we used smaller masses.
For example, for the production of pectinases by Aspergillus niger, we used 27 kg of
wheat bran and 3 kg of sugarcane bagasse plus around 50 kg of water, in a 40-cm-
high bed [14]. For the production of pectinases by Aspergillus oryzae, we used
7.3 kg of sugarcane bagasse, 7.7 kg of citrus pulp, and about 50 kg of water, again in
a 40-cm-high bed [15]. For the production of lipases by Rhizopus microsporus, we
used 7.5 kg of wheat bran and 7.5 kg of sugarcane bagasse (dry mass) and 25 kg of
water, which, once again, gave an initial bed height of 40 cm [16]. Obtaining and
processing such masses of substrate raise challenges that are usually not faced when
performing laboratory-scale SSF processes, which typically require the preparation
less than 2 kg of substrate.
Often, one would like to run several fermentations with the same lot of substrate,
in an attempt to avoid variation in bioreactor performance due to lot-to-lot variations
of substrate properties. In order to do this for fermentations in a pilot bioreactor, it
might be necessary to obtain a hundred or even several hundred kilograms of
substrate. Even if the substrate is a residue and available free of charge, this is still
problematic. A company will typically supply the residue free of charge at the
location where it is generated and in the particular physicochemical state in which
it is generated. It is then necessary to transport the substrate to the laboratory,
incurring transportation costs. The substrate may be moist or wet when generated
and, therefore, prone to natural fermentations that consume nutrients and generate
inhibitory metabolites. It is, therefore, essential to obtain the residue as soon as
possible after it has been generated, transport it immediately, and dry it without
delay. For many research laboratories, suitable equipment for drying hundreds of
kilograms of moist solids may not be easily available.
It may be necessary to process the substrate, for example, by chopping or
grinding. For laboratory-scale fermentations, it is common to sieve the substrate
by hand to ensure a uniform particle size (or range of particle sizes). To sieve many
kilograms of substrate for a pilot fermentation, a vibratory sieve with sufficient
capacity would be necessary. However, even if such equipment is available, one
needs to consider carefully whether sieving is appropriate; if the substrate is sieved, a
significant part of it may be wasted. The necessity of any other operations should
also be carefully considered. For example, when we use sugarcane bagasse as a bed
porosity modifier at laboratory scale, we normally wash the bagasse to remove
residual sugars, with the intention of preventing these sugars from causing catabolic
repression of enzyme production. However, this generates a large amount of residual
wash water. The necessity of washing must be evaluated. In other words, the gain in
fermentation performance due to removal of the residual sugars needs to be balanced
against the costs of treating the wash water.
Also, at laboratory scale, it is common to add nutrients to the solid substrate. For
some substrates, it is necessary to add a nitrogen source. Various inorganic salts may
also be added because they lead to better growth and product formation. However, the
real necessity for such additives needs to be evaluated. Pitol et al. [16] did this for
lipase production by R. microsporus in SSF. At laboratory scale, we had been using
washed sugarcane bagasse, impregnated with a medium containing soybean oil, urea,
lactose, K2HPO4, MgSO47H2O, and oligoelements (ethylenediaminetetraacetic
acid, MnCl24H2O, CoSO47H2O, CaCl22H2O, CuCl22H2O, and ZnSO47H2O)
[17]. Before undertaking pilot-scale studies, we investigated the possibility of using
a simpler medium. A medium composed of sugarcane bagasse, wheat bran, and urea
gave a price, per kg, that was a third of that of the medium that we had been using.
Lipase production was not adversely affected. Pitol et al. [16] also reduced the costs
of preparing the sugarcane bagasse, by eliminating the steps of washing, drying, and
sieving of the sugarcane bagasse that we had been using previously [17].
Of course, the substrate needs to be autoclaved before the process, and the
bioreactor also needs to be sterilized. Since our bioreactor was not designed as a
pressure vessel, the substrate is autoclaved in a 300 L autoclave. Between fermen-
tations, the bioreactor is cleaned by washing with neutral detergent. This is rinsed
off, and then the bioreactor surfaces are sprayed with a food-grade sanitizing product
containing peracetic acid and hydrogen peroxide. This is left for 30 min and then
rinsed off.
3.2 Preparation of Sufficient Inoculum and Inoculation
The production of inoculum for laboratory-scale SSF processes is typically easy.

However, when one needs to inoculate many kilograms of substrate, it can be a
challenge.
We inoculated our pilot fermentations with spore suspensions. This is the most
convenient form of inoculum for most filamentous fungi, although spore inocula
result in long lag times at the beginning of the fermentation, since the spores take
several hours to germinate.
The strains of Aspergillus that we used, A. niger and A. oryzae, sporulate easily.
We prepared the inoculum by SSF in Erlenmeyer flasks. In order to inoculate 30 kg
(dry mass) of substrate, Pitol et al. [14] and Finkler et al. [11] prepared a spore
inoculum of A. niger using 40 250-mL Erlenmeyer flasks, each containing 10 g (dry
mass) of a mixture of 30% sugarcane bagasse and 70% wheat bran (30:70 by mass).
This mixture was autoclaved and then wetted with an ammonium sulfate solution to
obtain a moisture content of 50% (m/m, wet basis). After inoculation, at 1 107
spores per gram of dry solid substrate, the flasks were incubated for 7 days, and then
the spores were suspended in sterile distilled water. This process resulted in a spore
concentration of 2 107 spores mL1. Likewise, in order to inoculate 15 kg (dry
mass) of substrate, Biz et al. [15] prepared a spore inoculum of A. oryzae using
20 500-mL Erlenmeyer flasks, each of which contained 20 g of a mixture of citrus
pulp and sugarcane bagasse (52:48 by mass). This mixture was autoclaved and then
wetted with an ammonium sulfate solution to obtain a moisture content of 60%
(w/w). After inoculation, at 4 107 spores per gram of dry solid substrate, the flasks
were incubated for 6 days, and then the spores were suspended in sterile distilled
water. The suspension was filtered through sterile gauze to remove residual sub-
strate. This process resulted in 6 L of spore suspension, with 1 108 spores per mL.
When we tried a similar strategy with R. microsporus, producing spores in
Erlenmeyer flasks with the fermentation substrate (a 50:50 mixture of wheat bran
and sugarcane bagasse), the fungus did not sporulate well. We therefore had to
develop a process to induce better sporulation [16]. The final process involved six
stainless-steel trays (each with a base of 19 cm 26 cm and a height of 3.5 cm), each
containing 183 g (dry mass) of a mixture of 86% parboiled rice and 14% rice husk.
The wetting solution, prepared by boiling diced potatoes (300 g per liter of water),
was added to give a low initial water content of 37.5% (w/w, wet basis), which
favors sporulation of this fungus. After autoclaving, a spore suspension was added
(1.7 107 spores per gram of dry solid substrate), and the inoculated substrate was
placed in the trays, with a 30 mm bed depth. After 7 days of incubation at 30 C, a
spore suspension was obtained by adding 5 mL of sterile Tween 80 solution (0.01%
w/v) per gram of dry substrate. The spore suspension was filtered through sterile
gauze to remove residual substrate.
Spreading the inoculum uniformly is essential to ensure that all particles are
inoculated, avoiding extra lag time due to the need for the fungus to colonize
uninoculated particles. However, adding the inoculum uniformly to a large mass
of solids can be challenging. One needs to be careful with the addition of wet
inoculum. If the liquid is added to the top of a mass of substrate and is given time
to absorb into the substrate before the bed is mixed, then the transfer of spores from
these top particles to the other particles during mixing may not be particularly
effective. Ideally, the inoculum should be applied as a fine spray onto the substrate
as it is being mixed. Potentially, this could be done within the bioreactor itself. In our
pilot bioreactor, one possibility would be to add the inoculum in various aliquots.
There would be repeated cycles in which a small volume would be sprayed onto the
top of the bed (either through a well-designed nozzle or manually with a spray bottle)
and then the bed would be mixed thoroughly. The addition of the inoculum could
also be done in a dedicated mixing vessel. In this case, the mixer would turn the
substrate bed continuously, with the addition of the inoculum as a continuous spray.
In our case, we added the inoculum manually. The substrate was autoclaved in
several bags, each containing a few kilograms of substrate. Appropriate aliquots of
the spore suspension were added directly to each bag, and then the contents were
immediately mixed, thoroughly, within the bag. The contents were then added to the
bioreactor. Once the bioreactor was fully loaded, it was rotated for several minutes to
mix the contents thoroughly. A manual procedure like this is feasible at pilot scale
but would soon become impractical as the scale is increased further, making it
essential to develop an automated system.
4 Production of Enzymes in the Pilot Bioreactor
This section describes our studies of the production of pectinases and lipases in the
pilot bioreactor. The focus is not on comparing our results with other reports of the
production of pectinases and lipases in SSF (for this, the reader should refer to
the original articles). Rather, we focus on operational issues, such as the formation of
gradients of temperature and moisture content within the bed, and how this is
affected by agitating the bed and by using an inert material, such as sugarcane
bagasse, to ensure a high porosity.
In this section, various different pectinase activities and lipase activities are given.
The definition of the units (U) of activity can be found in the original papers. These
definitions are not of key importance in the current context, but it is important to note
that the activity units can only be used to compare the different fermentations
undertaken by our group. Since other authors have used many different conditions
for their activity assays, especially in the area of pectinases, it is not possible to
compare our activity units with activity units given in the literature [18].
4.1 Production of Pectinases on Wheat Bran
Our first studies involved the production of pectinases by A. niger cultivated on

wheat bran, in fermentations lasting up to 26 h [11, 14]. In these fermentations, the
air entered the bioreactor at 150 m3 h1 (nominal superficial velocity of 0.1 m s1)
and at a temperature around 30 C.
We initially undertook fermentations without any attempt at control: we did not
agitate the bed, and we did not change the temperature of the water being fed to the
humidification column [14]. In the first fermentation, the substrate bed consisted
entirely of wheat bran (20 kg dry mass). Around 17 h of fermentation, the bed shrank
away from the bioreactor walls. With the preferential flow of air through the gaps
next to the wall, the bed was no longer properly aerated, and temperatures as high as
37 C were recorded in the bed [14]. At this time, since the air was passing around the
bed, not through it, the air temperature in the headspace above the bed was lower,
ranging between 30 and 32 C. This “inversion of temperatures,” namely, the bed
temperature being higher than the air temperature in the headspace, is a clear sign of
preferential flow and could be used in an automated system to trigger an alert,
informing the operator of the need to take corrective action. When the fermentation
was ended at 24 h, large compact agglomerates were encountered within the bed
during the unloading of the bioreactor.
In the second fermentation, we attempted to avoid the problem of bed shrinkage
by replacing 10% of the wheat bran with sugarcane bagasse (i.e., dry masses of 18 kg
wheat bran and 2 kg sugarcane bagasse). The addition of sugarcane bagasse
increased the porosity of the bed: whereas the bed height at the start of the first
fermentation was 23 cm, it was 27 cm at the start of the second fermentation. The
fermentation was successful, in that the bed did not shrink away from the wall and
the bed temperature was well controlled, not exceeding 31 C. Based on the poor
performance of the first fermentation, we decided to “map” the pectinase activities in
the bed at the end of this second fermentation, at 26 h (see Fig. 5b). Since the bed
conditions were well controlled, the pectinase activity did not vary significantly: the
values ranged from 17 to 20 U g1 [14].
Encouraged by the success of the addition of sugarcane bagasse, we decided to
use the same substrate mixture, but to increase the overall mass of the bed by 50%:
27 kg of wheat bran and 3 kg of sugarcane bagasse (dry masses), giving an overall
bed height of 40 cm. Our thinking was that a packed-bed bioreactor should have as
high a bed height as possible: for a given mass of substrate, a higher bed height
would give a smaller bioreactor footprint. However, with the increased bed height,
compaction problems returned. At 16 h, the bed shrank away from the bioreactor
wall, and temperatures as high as 43 C were measured in the upper regions of the
bed. With this high temperature, the pectinase activity of samples removed from the
top of the bed decreased, from a value of 23 U g1 at 14 h to 13 U g1 at 20 h. When
the bed was mapped at 26 h, the pectinase activities varied significantly: from 16 to
28 U g1 at the bottom of the bed, from 11 to 19 U g1 in the middle of the bed, and
from 13 to 20 U g1 at the top of the bed [14].
In a following study, we maintained the same substrate, microorganism, and
fermentation conditions but investigated whether agitation of the bed could prevent
the bed from pulling away from walls, thereby promoting uniformity of pectinase
levels [11].
Four mixing regimes were compared: (1) a completely static bed, (2) one mixing
event (at 10 h), (3) three mixing events (at 8, 10, and 12 h), and (4) five mixing
events (at 8, 10, 12, 14, and 16 h). Agitation of the bed enabled the bed temperature
to be controlled well: over a period of at least 3 h after a mixing event, the bed
temperature did not exceed 35 C [11], whereas in the absence of mixing, bed
temperatures over 40 C had been measured [14]. However, if the bed was left static
for longer periods (about 8 h) after an agitation event, evidence of the formation of
preferential flow paths appeared (i.e., bed temperatures that were higher than the
headspace temperature). Mixing promoted uniformity of temperatures across the
bed: in the mixed fermentations, the thermocouples within the same horizontal
sleeves gave readings that typically varied less than 2 or 3 C from one another.
On the other hand, after 15 h of fermentation in a static bed, thermocouples within
the same horizontal sleeve sometimes gave readings that were 5–17 C different [11].
As a result of the poor temperature control in the static fermentations, the
pectinase activities of samples removed from the top of the bed varied by up to
10 U g1 toward the end of the fermentation (from 13 to 23 U g1 after 14 h). On the
contrary, the pectinase activities of samples removed from the top of the bed for
fermentations with three or more mixing events were very close to each other up to
16 h. After 16 h, they varied more but still by less than 5 U g1 for samples removed
at the same fermentation time [11].
When the bed was mapped at the end of the 26-h fermentation, the fermentation
with five mixing events had the most uniform distribution of pectinase activities,
with the average pectinase activities at the bottom, middle, and top of the bed all
falling in the range of 22–23 U g1. This fermentation also gave the best uniformity
within horizontal planes, with sample standard errors (calculated for the five samples
collected at the same bed height) being below 2 U g1. However, the use of five
mixing events was slightly deleterious. The highest overall pectinase production at
26 h was obtained in the fermentation with three mixing events, although the
activities were less uniform within the bed. In this case, the average pectinase
activities ranged from 29 U g1 at the bottom of the bed to 22 U g1 at the top of
the bed, and the sample standard errors calculated for five samples removed from the
same horizontal plane ranged from 2 to 4 U g1 [11].
An important conclusion from the study of the effect of mixing is that, if the bed is
heterogenous, then removing samples only from the top of the bed (in order not to
disturb the bed structure) can lead to erroneous decisions about the best agitation
strategy and the best harvesting time. Based on samples removed from the top of the
bed, it would appear that the static fermentation might be best, since an activity of
23 U g1 was obtained at 14 h. However, due to the relatively poor uniformity within
the static bed, there is no guarantee that this value applies to the whole bed. In fact,
based on mapping of the whole bed at 20 h, the fermentation with three mixing
events had an average pectinase activity of 22 U g1, which was significantly higher
than the average pectinase activity of 18 U g1 for the static fermentation [11].
4.2 Production of Pectinases and Lipases in Beds with High

Sugarcane Bagasse Contents
We also did fermentations with high contents of sugarcane bagasse: the production
of pectinases by A. oryzae [15] and the production of lipases by R. microsporus
[16]. In both cases, the initial bed height was 40 cm. In the case of the production of
pectinases by A. oryzae, we used 7.3 kg of sugarcane bagasse and 7.7 kg of citrus
pulp (dry masses), with about 50 kg of water [15]. For the production of lipases by
R. microsporus, we used 7.5 kg of wheat bran and 7.5 kg of sugarcane bagasse (dry
masses), with about 28 kg of water [16].
With the high content of sugarcane bagasse in these two systems, we managed to
avoid the problems that Pitol et al. [14] encountered, namely, the formation of
compact agglomerates of substrate and bed shrinkage. As a consequence, we
avoided the formation of preferential flow paths and overheating of the bed. In
both cases, the temperatures throughout the substrate bed remained very close to the
inlet air temperature [15, 16]. It was, therefore, not necessary to use the cooler
reservoir to provide cool air to the bed, a strategy that Pitol et al. [14] had tried during
a static fermentation, unsuccessfully. The good temperature control of the bed had an
added benefit: saturated air was supplied to the bed and, in the absence of axial
temperature gradients, the water carrying capacity of the air did not increase as it
flowed through the bed. Consequently, the moisture content of the bed remained
close to the initial water content throughout the fermentation. In various fermenta-
tions reported by Pitol et al. [14] and Finkler et al. [11], the moisture content
decreased by over 10 percentage points during the fermentation. Of course, the
advantage of using inert materials that do not shrink during the fermentation in
packed-bed bioreactors has been known for many years [19].
In the production of pectinases by A. oryzae, the sugarcane bagasse was added
simply as a bed porosity modifier; it was not impregnated with nutrients. This inert
material represented half of the mass of the substrate bed, and the bulk density of the
bed was only about 90 kg of dry substrate per m3 [15], a half of the value of 180 kg of
dry substrate per m3 for the wheat bran medium used by Pitol et al. [14]. Potentially,
the use of a medium with a much lower bulk density would lead to a much lower
overall production of pectinases in the bioreactor. However, the pectinase activity
per gram of dry substrate obtained by Biz et al. [15], 37 U g1, was much higher than
the best value of 20 U g1 obtained by Pitol et al. [14]. As a result, Biz et al. [15]
obtained a total of 555 103 U in the bioreactor, which is more than 90% of the
value of 600 103 U obtained by Pitol et al. [14].
5 The Pilot Bioreactor and Mathematical Models in Scale-

Up Studies
Studies within a pilot bioreactor are not an end in themselves. Rather, these studies
should provide a basis that guides the design and operation of a full-scale, commer-
cial bioreactor that contains hundreds of kilograms, or even several tonnes, of
substrate.
Our pilot bioreactor allows for the use of substrate beds of heights up to 50 cm, a
bed height that has been used in some large-scale packed beds [20]. If the fermen-
tation in the pilot bioreactor is successful, then one possible scale-up strategy is to
maintain the same bed height and simply increase the width of the bioreactor. In this
case, phenomena that depend on the bed height, such as axial temperature gradients
and pressure drops across the bed, can be studied at pilot scale using the same bed
height that will be used at commercial scale.
If the same bed height is maintained, one possibility would be to use the same
design as we used and simply increase the length of the central axis of the cylinder.
In this case, a larger-scale bioreactor would be intermittently mixed using the same
tumbling action that we used for mixing in our pilot bioreactor. Of course, other
designs could be used, such as the circular beds used in the production of soy sauce
koji [20]. Such designs use mechanical agitators to mix the bed (Fig. 3).
However, it is also useful to consider using bed heights higher than 50 cm at
commercial scale, since for the same amount of substrate, a higher bed height will
give a smaller bioreactor footprint. The possibility of using higher bed heights than
those studied at pilot scale can be investigated using mathematical models of heat
and mass transfer. These models predict axial temperature and moisture gradients,
identifying the potential of high bed temperatures or low water activities occurring in
particular regions of the bed.
Various models have been proposed that can be used as tools for guiding the
scale-up of packed-bed bioreactors. The first models assumed that, as the air passes
through the bed, it remains in thermal and moisture equilibrium with the solids.
Effectively, this is equivalent to the assumption that, as the air heats up due to the
removal of metabolic heat from the solids, water evaporates from the solids to the air
to maintain the air saturated. When our research group first proposed scale-up
strategies for packed-bed SSF bioreactors, for both the traditional design and the
Zymotis design, we used models based on this assumption [6, 10]. However, Weber
et al. [21] showed that the air and solids are not necessarily in equilibrium in a
packed-bed bioreactor, especially during the periods of most rapid microbial growth:
in their studies, the relative humidity of the outlet air fell to values below 90%. When
we developed a model to be used as a tool in guiding the design and operation of our
forcefully aerated, intermittently agitated, pilot bioreactor, we recognized that the
solid phase and gas phase are not necessarily in equilibrium [22]. As such, we called
it a “two-phase model.”
Our “two-phase model” describes heat transfer only along the direction of the
airflow [22]. Casciatori et al. [23] extended the two-phase modeling approach by
including the radial coordinate. In other words, in addition to describing heat and
mass transfer by conduction, convection, and evaporation along the central axis of
the bioreactor (i.e., in the axial direction, parallel to the airflow), they also described
heat transfer by conduction from the central axis to the bioreactor wall (i.e., in the
radial direction, normal to the airflow). However, although it may be appropriate to
describe radial heat transfer in order to model the performance of a thin, water-
jacketed, laboratory-scale packed bed, when the aim is to use the model to guide the
scale-up of a traditional packed-bed bioreactor, there is no advantage in describing
heat transfer in the radial direction. At large-scale, traditional packed beds are
typically several meters wide, and conductive heat transfer radially to the bioreactor
wall influences only a few centimeters near the bioreactor wall, making a negligible
contribution to bed cooling. In fact, this consideration means that when one is using
a thin packed bed in studies aimed at guiding the design and operation of a traditional
large-scale packed-bed bioreactor, one should insulate the sides of the thin packed
bed rather than using a water jacket. On the other hand, describing heat transfer
normal to the airflow is useful in guiding the scale-up of Zymotis-type packed-bed
SSF bioreactors. A model of this type of bioreactor was developed by Mitchell and
von Meien [6] and used to show that the internal heat transfer plates need to be less
than 10 cm apart in order for the bed temperature to be well controlled [7]. This
model assumed thermal and moisture equilibrium between the air and solid phases.
Such a model could benefit from incorporating the two-phase approach.
The two-phase model of von Meien and Mitchell [22] was a useful tool for
investigating control strategies [24]. Two control strategies were investigated, PID
(proportional-integral-derivative) and DMC (dynamic matrix control). The aim was to
maintain the average bed temperature as close as possible to the optimal temperature for
growth. Two aeration strategies were compared: (1) maintaining the air essentially
saturated (relative humidity of 99%) while varying its temperature and (2) maintaining
the air at the optimal temperature for growth while varying its humidity. The former
strategy was predicted to give better control of the average bed temperature and,
therefore, a better predicted growth profile (representing the average biomass content
of the bed). DMC was predicted to give better growth than PID. A key conclusion of the
work was that the availability of the two-phase model allowed a relatively inexpensive
investigation of different control strategies for the pilot bioreactor. The costs in terms of
both material and time would be much greater if it were necessary to undertake this
work solely experimentally. Of course, it is essential to confirm that the most promising
control strategy identified in the simulation work does indeed lead to a high level of
performance of the bioreactor, but this confirmation can be done with a relatively small
number of experiments.
Computational fluid dynamics (CFD) can be used to model SSF processes in
bioreactors. It has been applied to describe the initial phase of heating of the
substrate bed during the lag phase in our pilot bioreactor [25] and the initial stages
of a fermentation itself [26]. Given the long simulation times, which can be of the
order of several weeks, CFD has most to offer in situations that cannot easily be
modeled with classical continuous differential equations. Continuous differential
equations (either ordinary or partial) can be used to model simple geometries, such as
cylinders, and smooth gradients in space (in axial or radial directions). CFD is useful
for complicated geometries, of the bioreactor itself, or of the air supply. It is also
useful when the bed contains obstacles and when the bed has irregular properties,
which might occur through nonuniform packing or the opening up of preferential
flow paths (in the form of cracks or gaps). Since CFD programs were not originally
designed for describing fermentations, it is necessary to use user-defined functions to
simulate many of the phenomena that occur during SSF, such as microbial growth
and particle shrinkage.
Mathematical models will only represent fermentations well if the correlations
and values used in them are correct, so it is important to characterize the properties of
the beds used in packed-bed bioreactors. The two-phase model of von Meien and
Mitchell [22] relies heavily on data obtained in studies of the drying of grains that are
different from the substrates that we have used for the production of pectinases and
lipases. Recently, hygroscopic properties have been published for substrates that we
used in our fermentations, including properties of orange pulp [27], sugarcane
bagasse, and wheat bran [28]. It is important to note that studies of the hygroscopic
properties of substrates used in SSF processes usually focus on characterizing the
original substrate, as prepared for a fermentation. However, the isotherms obtained
for the original substrate might not apply during the fermentation, since the biomass
can have hygroscopic properties that are significantly different from those of the
substrate [29]. Data have also been published for key structural properties of beds
packed with sugarcane bagasse, wheat bran, and orange pulp and peel and with
mixtures of these substrates [12]. These properties include the bulk packing density
and the bed porosity; they were characterized as functions of the bed moisture
content and the packing technique [12].
Acknowledgments The construction of the pilot solid-state fermentation bioreactor described in

this review was originally funded by the Brazilian-Argentinean Biotechnology Committee (CBAB,
Comitê Brasileiro-Argentino de Biotecnologia), using funds provided by the Brazilian Ministry of
Science and Technology (MCT, Ministério de Ciência e Tecnologia) and administered by the
Brazilian National Council for Scientific and Technological Development (CNPq, Conselho
Nacional de Desenvolvimento Científico e Tecnológico). Since then, the continued studies with
the bioreactor have been funded by several projects funded by MCT/CNPq. David Mitchell and
Nadia Krieger also thank CNPq for research scholarships.
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DOI: 10.1007/10_2019_85
Published online: 23 February 2019
It Is the Mix that Matters:

Substrate-Specific Enzyme Production
from Filamentous Fungi and Bacteria
Through Solid-State Fermentation
Susanne Steudler, Anett Werner, and Thomas Walther
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2 Enzyme Production Through Solid-State Fermentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
3 Important Extracellular Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.1 Selected Industrially Relevant Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55
3.2 Lignocellulolytic Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
4 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
Abstract Fungi have a diverse spectrum of extracellular enzymes. In nature, extra-

cellular enzymes primarily serve to procure nutrients for the survival and growth of
the fungi. Complex polymers such as lignocellulose and starch as well as proteins
and fats are broken down into their basic building blocks by extracellular enzymes
such as amylases, proteases, lipases, xylanases, laccases, and many more.
The abilities of these enzymes are made use of in diverse areas of industry,
including food technology, textiles, and pharmaceuticals, and they have become
indispensable for today’s technology. Enzyme production is usually carried out
using submerged fermentation (SmF). However, as part of the search for more
sustainable uses of raw materials, solid-state fermentation (SSF) has become the
focus of research.
The rate of enzyme formation depends on different factors, for example, micro-
organism, temperature, or oxygen supply. However, one of the most important
factors in enzyme production is the choice of substrate, which varies depending on
S. Steudler (*), A. Werner, and T. Walther

Institut für Naturstofftechnik, Professur für Bioverfahrenstechnik, Technische Universität
Dresden, Dresden, Germany
52 S. Steudler et al.
the desired target enzyme. Substrates with proven effectiveness include wheat bran
and straw, but unusual agricultural residues such as forage cactus pears and orange
peels have surprisingly positive effects on enzyme formation as well.
This review gives an overview of various technically relevant enzymes produced
by filamentous fungi and suitable substrates for the production of the enzymes
by SSF.
Graphical Abstract
Keywords Basidiomycetes, Extracellular enzymes, Filamentous fungi, Residues,

Solid-state fermentation, Substrate-specific enzyme formation, Sustainability
It Is the Mix that Matters: Substrate-Specific Enzyme Production from. . . 53
1 Introduction
Nature fascinates us with its incredible diversity and wide array of possibilities.
A variety of organisms has arisen through millions of years of evolution, generating
metabolic pathways and resulting products which are highly interesting for industry.
This review focuses on extracellular enzymes, predominantly those formed by
bacteria and fungi and used in different industries where the breakdown of complex
macromolecules or the improvement of digestibility, solubility, or viscosity is
desired. In nature, these enzymes are mainly used for substrate degradation and
nutrient extraction. Organisms are able to form special enzyme cocktails for each
specific substrate.
In order to obtain these specialized enzymes, it is sometimes necessary to
simulate the natural habitat of the organisms. One possibility for the implementation
of preferred terrestrial systems in industry is solid-state fermentation (SSF).
The term solid-state fermentation applies to all forms of fermentation involving
solid particles in the absence of a free liquid phase. This means the organism is
grown on a bed of solid particles, with the space between the particles consisting of a
continuous gas phase and liquid being retained by the solid particles, which fulfil the
following three important functions: carrier material, nutrient source, and moisture
reservoir. In addition to classical solid-state fermentation, various mixed forms have
also become established, such as solid-substrate fermentation. Solid-substrate fer-
mentation is a form of liquid cultivation (submerged fermentation, SmF) used
predominantly for industrial purposes, with the addition of solid particles as complex
substrates.
In addition to the cultivation method, various parameters have a significant
influence on enzyme formation. These include, among others, culture time, culture
temperature, inoculation level, humidity, initial pH, aeration, agitation (mixing
speed, mixing duration, and mixing rate), and particle size. However, the choice of
substrate remains an important factor, as different enzymes are specifically induced
by various complex solid substrates.
To provide an overview of the main enzymes produced by filamentous fungi,
along with their relevant substrates, this review evaluates the substrate-specific
formation properties of most studied extracellular enzymes. Various enzymes and
enzyme groups relevant for industry are compared and analyzed along with their
preferred substrate for fermentation, and results from our own work are presented.
2 Enzyme Production Through Solid-State Fermentation
Solid-state fermentation is ideal for cost-effective and substrate-optimized production

of extracellular enzymes by filamentous fungi, whose implementation usually
follows the same principle that has hardly changed in recent years.
First, the substrates were sterilized and inoculated. The incubation took place
over several days to weeks depending on the organism and the target enzyme. Most
of the studies were still carried out in static shake flasks (usually 250 mL) with
5–10 g of substrate filling [1–10], but investigations on a larger scale were also
conducted. In this case, the substrate bed either remained static (e.g., tray reactors)
[11–17] or was occasionally circulated (e.g., drum reactors) [18]. Also, oxygen
supply to the organisms used and heat dissipation were supported by partially
enhanced ventilation [19, 20].
The extracellularly formed enzymes were finally obtained as a crude solution
through enzyme extraction. To achieve this, the overgrown substrate was mixed with
water or buffer (e.g., phosphate buffer or acetate buffer) [2, 3, 6, 9, 21–24]. The
mixing was carried out on a laboratory scale by shaking or stirring, usually for
30 min to 2 h (time periods of 5 min to 4 h were also used), at 100–250 rpm [2, 9, 14,
15, 25–30]. Subsequently, the solid particles were separated by filtration or centri-
fugation. In some cases, additional centrifugation of the retentate was carried out
[5, 10, 31–33]. A crude enzyme solution was recovered which could be used for
further investigation or purification or used directly in the application. Additional
purification steps included precipitation with ammonium sulfate and chromato-
graphic purification steps [34, 35].
In recent years, a variety of different enzymes and their environmentally friendly
production by solid-state fermentation have been investigated. The relevant enzymes
can be roughly categorized into two major groups: (1) hydrolytic enzymes, such as
amylases, pectinases, and proteases, and (2) lignocellulolytic enzymes, such
as cellulases, xylanases, laccases, and various peroxidases. Other enzymes, such as
phytases or tannases, have also been investigated by some research groups [36–39].
The formation rate and amount varied depending on the substrate and organism
used. Preferred organisms were representatives of ascomycetes (e.g., Aspergillus sp.,
Trichoderma sp., Penicillium sp.) and bacteria (e.g., Bacillus sp.), as well as
basidiomycetes (e.g., Pleurotus sp., Trametes sp., Phanerochaete sp.) [1, 17, 21,
34, 40–46].
The substrates consisted mostly of residues from the agriculture, food, and
forestry industries, such as crop residues, wood chips, fruit and vegetable peels,
and other renewable raw materials. These substrates have high availability and are
inexpensive, meaning production costs and negative environmental effects (due to,
e.g., disposal) are reduced. In general, they can be divided into six categories:
(1) sugary, such as sorghum and sugarcane bagasse, for the production of cellulases
and xylanases [3, 4, 43]; (2) starchy, such as wheat bran, suitable for amylases
[2, 47]; (3) lignocellulosic, such as fruit peels, straw, and sawdust, suitable for
cellulases, xylanases, laccases, and peroxidases [5, 8, 46, 48]; (4) protein rich,
such as soya residues, suitable for proteases [29, 49, 50]; (5) oily, such as olive
pomace, for the production of lipases [19]; and (6) inert carriers, such as polyure-
thane foam [51, 52], used rarely or in combination with liquid fermentation.
In the following, some selected enzymes and their substrate-specific production
are discussed in more detail.
3 Important Extracellular Enzymes
3.1 Selected Industrially Relevant Enzymes
In the following, various industrially relevant enzymes which can be produced by

solid-state fermentation are presented. The main focus is on the selection of sub-
strates used. It turned out that, in addition to the production strain used, the substrates
have a considerable influence on the enzyme activity achieved. Most of all, the
composition of substrates plays an important role. Thus, the individual components,
as expected, have an inducing effect on the desired target enzymes.
3.1.1 Amylases
Amylases belong to the group of hydrolases and cleave the glycoside bonds of
polysaccharides (primarily starch), which is why they are also classified as glycosi-
dases. Amylases are subdivided into α-, β-, γ-, and iso-amylases and are produced by
various organisms such as bacteria, fungi, plants, and animals. In particular, mainly
the amylases of bacteria and fungi, especially α-amylase (EC 3.2.1.1), are used in
industry and technology [53, 54].
Amylase makes up approximately 25% of the world enzyme market [55]. The
main area of application is in the food industry. Here, amylases are used in the
mashing process in breweries and for the pretreatment and modification of flour (for
airier dough or a greater degree of browning) and starch, as well as in the production
of glucose/maltose syrup and maltodextrin. They are also used in pharmacy or in
dishwashing and laundry detergents for the removal of starchy stains and dirt, as well
as in the production of animal feed and biofuels [53–55].
Amylases are produced both by submerged fermentation and by solid-state
fermentation. Here, bacteria such as Bacillus sp. or fungi, especially ascomycetes,
are preferably used [54]. However, alongside the introduction of new production
strains, production has also been improved by the use of genetically modified or
engineered strains [54]. Basidiomycetes were not in focus for the production of
amylases.
Submerged fermentation usually uses expensive media. Costs can be reduced by
the use of residues in solid-state fermentation. Wheat bran has been a popular and
well-suited substrate for amylase production since the 1980s [47]. Recent studies
have shown that other substrates are also suitable for the production of amylases (see
Table 1). Substrates on which high enzyme yields were obtained were mostly those
which still contained starch residues. For example, different types of bran (residues
after the sieving of flour) have between 8% and 45% of starch (wheat 13–18%, oats
18–45%, rice 18–30%) [56]. However, in comparison to studies on wheat bran
(63.25 U/g [57] and 14.5 U/min/mL [47]), investigations on laboratory scale (sub-
strate use 5 g to 1.2 kg) showed that even substrates with small amounts of starch,
such as wheat straw (6,900 U/gds (gram dry substrate)) [58], banana peels (42 U/mg)
[59], mixtures of orange peels and cheese whey (220 U/mL) [1], and a mixture of soy
Table 1 Review of recent studies on amylase production on solid substrates (bold: best substrate)
including achieved amylase activity, used production strain, and cultivation size [1, 2, 18, 25, 47,
50, 57–59, 61]
Substrate Activity Microorganism Reactor Ref.
Orange peels and cheese 220 U/mL Bacillus Erlenmeyer [1]
whey amyloliquefaciens flasks
(250 mL)
Wheat straw, sugarcane 6,900 U/gds Bacillus [58]
bagasse, rice straw, and sp. BBXS-2
rice husk
Corn bran, barley bran, 2.8 U/mg protein Rhizoctonia Erlenmeyer 5g [2]
wheat bran solani AG-4 flasks
strain ZB-34 (250 mL)
Sago hampas 1.055 0.03 U/mL Aspergillus flavus Flasks 5g [25]
NSH9
Raw banana peel, sugar- 42 U/mg Microbacterium Flasks 5g [59]
cane bagasse, sawdust, foliorum GA2
potato peel, wheat bran,
rice bran, cassava peel,
orange peel
Wheat bran, corn straw, 63.25 U/g (6.32 U/mL) Gongronella Erlenmeyer 5g [57]
corn cob, rice peel, soy butleri flasks
bran (250 mL)
Maize pericarp, soybean 50.75 IU/g Bacillus sp. Erlenmeyer 5g [50]
meal, sunflower meal, TMF-1 flasks
maize bran, olive oil cake, (150 mL)
and wheat bran
Wheat bran, banana 14.5 0.1 U/mL/min Bacterial Erlenmeyer 10 g [47]
peels, rice bran and husk, co-culture: flasks
gram husk, mineral salt Bacillus cereus (250 mL)
solution and Bacillus
thuringiensis
Soy and bread waste 39.9103 U/gds Thermomyces Cylindrical 1.2 kg [61]
mixture (90:10) sp. ATCC- reactors
200065 and (4.5 L)
Geobacillus
sp. ATCC-31198
Corn bran 20.68 1.03 U/mL Bacillus Rotating 2.8– [18]
amyloliquefaciens drum biore- 5.6 L
NRRL B-645 actor (7 L)
and bread wastes (90:10) (39,900 U/gds) [14] are suitable for amylase production.
The substrates used have a low starch content (less than 3%) and a high non-starch
carbohydrate content (e.g., hemicellulose, cellulose, pectin, etc.) of up to 60%
(in banana peels) [60], which could also promote enzyme formation.
3.1.2 Proteases
Proteases cleave proteins through hydrolysis of peptide bonds. According to their

catalytic mechanisms, they are subdivided into exoproteases (cleavage of fewer
amino acids at the C- or N-terminus) and endoproteases (cleavage in the middle of
the polypeptide chain). In addition, a differentiation is made of exoproteases into di-
and tri-peptidyl proteases and endoproteases into serine- (subtilisin), cysteine-

(papain), aspartate- (pepsin), and metallo-proteases (thermolysin) [53].
The most important protease for industry is subtilisin, which is used in detergents
and cleaning agents (global (2010), 2000 t/a [54]; Europe (2002), 900 t/a [53]). This
enzyme is mainly produced in industry by bacteria of the species Bacillus subtilis,
B. amyloliquefaciens, and B. licheniformis [53, 54]. Further uses of proteases include
the production of protein hydrolysates and baby food, and they also offer an
environmentally friendly alternative in leather processing and the treatment of
industrial and domestic wastewater [53].
Just as for amylases, industrial production of proteases usually involves sub-
merged fermentation. However, the use of solid substrates is also possible. For this
purpose, Bacillus species are still cultivated as the main producer in research [29, 49,
50, 58, 62].
Above all, protein-rich substrates of soy residue (protein content up to 50% [63]),
wheat and rice bran (protein content 15–18% [56, 64]), or residues of animal origin
such as cuttlefish (protein content 16–18% [65]) or cow hairs were shown to be
suitable for the production of extracellular proteases (see Table 2). Thus, through
fermentation in a 50 L adiabatic packet bed reactor with a loading of 20 kg soy fiber
and 17.5 kg dehydrated sludge and cow hair, up to 33,374 U/gds of protease activity
were achieved [66]. In addition to the use of Bacillus species, proteases with an
activity of 463.5 U/mL could also be produced on a mix (3 kg) of cupuaçu exocarp
and rice bran by the basidiomycete Lentinus citrinus (analysis of fruiting bodies)
[24]. However, the yields are much lower than that achieved by the cultivation of
Bacillus sp.
3.1.3 Lipases
Lipases, also referred to as triacylglycerol hydrolases, degrade fats to glycerol and

free fatty acids or to mono- and diglycerides. They occur in fungi, bacteria, animals,
and plants. Their most important properties include their broad substrate specificity,
their high stability (including in organic solvents), as well as the conversion of many
substrates with high stereo- and enantioselectivity [53, 54].
This means there are many applications for lipases, ranging from pharmaceuticals
(e.g., ibuprofen production) to the production of enantiomerically pure fine
chemicals and fragrances for use in the food industry (e.g., transesterification of
wheat flour lipids or for the production of breast milk fat substitutes and cocoa butter
substitutes). In addition, lipases are used as a detergent additive, in leather
production (e.g., degreasing) and in the paper industry [53, 54].
Industrial production is carried out by fungi, preferably ascomycetes, such as
Aspergillus niger, Candida antarctica, Rhizomucor miehei, and Pichia sp. [54]. The
main issue in their production is usually the use of water-insoluble substrates, which
can cause enzymes to accumulate in the interfaces and reduce yields [54].
In recent years there have been efforts in research to produce lipases using solid-
state fermentation. Representatives of the ascomycetes continue to be preferred for
Table 2 Review of recent studies on protease production on solid substrates (bold: best substrate)
including achieved protease activity, used production strain, and cultivation size [7, 24, 29, 49, 50,
58, 62, 66–68]
Wheat straw, sugarcane 12,200 U/gds Bacillus [58]
bagasse, rice straw, and sp. BBXS-2
rice husk
Corn pericarp, soybean 50.5 IU/g Bacillus Erlenmeyer 5g [50]
meal, sunflower meal, sp. TMF-1 flasks
maize bran, maize peri- (150 mL)
carp, olive oil cake,
wheat bran
Wheat bran and rice 4,143 + 12.31 U/g Bacillus Erlenmeyer 5g [62]
bran, green gram husk, sp. IND12 flasks
tapioca peel, banana peel
Cuttlefish waste and 1,218 27 U/g Bacillus cereus Erlenmeyer 5g [29]
cow dung + C-source IND5 flasks
(100 mL)
Wheat bran (WB) and 1193.77 U/g Brevibacterium Erlenmeyer 10 g WB [67]
rice flour (RF) luteolum flasks 0.5 g RF
MTCC 5982 (250 mL)
Cotton cake 728 U/mL Bacillus subtilis Erlenmeyer 10 g [49]
Gram husk 714 U/mL K-1 flasks
Mustard cake 680 U/mL (500 mL)
Soybean meal 653 U/mL
Wheat bran, chicken
feather maize bran, rice
husk, cane bagasse, corn
cob
Soybean bran, wheat 115.6 U/mL Aspergillus Erlenmeyer 10 g [7]
bran oryzae INCQS flasks
40068 and (250 mL)
other
ascomycetes
Soy fiber mixed with 800 U/gds Thermus Bioreactor 115 g [68]
wood chopsticks and sp. ATCC® (500 mL)
wood chips 670 U/gds 3,174 Bioreactor 2.3 kg
(10 L)
Cupuaçu exocarp + rice 463.55 U/mL Lentinus Polythene 3 kg [24]
bran, cupuaçu citrinus bags
exocarp + litter (fruiting bodies)
Agro-industrial wastes: 33,374 U/gds (SF) n.n. Adiabatic 3.8 kg [66]
Soy fiber, cow hair 23,541 U/gds (HS) packet bed SF + 2.8 kg
mixed with dehydrated reactor HS
sludge from wastewater (10 L)
treatment plant Adiabatic 20 kg
packet bed SF + 17.5 kg
reactor HS
(50 L)
lipase production (see Table 3). Basidiomycetes play only a minor role as lipase
producers.
As expected, lipid-rich substrates were used as preferred substrates, such as
coconut meal, olive pomace, and palm kernel cake. These were used separately or
in combination with nutrient-rich substrates, such as wheat bran. Wheat bran has a
fat content of only 4–5%, but it contains a lot of carbohydrates (13–18% starch,
Table 3 Review of recent studies on lipase production on solid substrates (bold: best substrate)
including achieved lipase activity, used production strain, and cultivation size [16, 19, 27, 33, 40,
69–74]
Lipid-rich agro- 41.1 0.4 U/mL/min Aspergillus niger, [40]
wastes: 169.5 U/mg mesophilic fungi
Coconut meal with
supplements, almond
meal, brassica meal,
sesame meal, rice,
wheat bran
Oil palm empty fruit 0.195 U/g Trichoderma sp.1 Erlenmeyer 3g [33]
bunch 0.211 U/g Hypocrea flasks
neorufa.1 (250 mL)
Soybean meal 72.6 2.4 U/g Yarrowia lipolytica Cylindrical 10 g [69]
Canola cake 93.9 2.9 U/g IMUFRJ50682 polypropylene
bioreactors
Wheat bran and 265 U/g Rhizopus Laboratory 10 g [73]
sugarcane bagasse microsporus column
CPQBA 312–07 bioreactor
113 U/g DRM Pilot packed- 15 kg
bed bioreactor
Rice bran, rice husk 38.67 U/g Aspergillus niger Erlenmeyer 20 g [27]
with olive oil flasks
(250 mL)
Olive pomace and 90.5 1.5 U/g Aspergillus ibericus Erlenmeyer 30 g [70]
wheat bran MUM 03.49, flasks
A. niger MUM (500 mL)
03.58,
A. tubingensis
MUM 06.152
Olive pomace and 223 5 U/gds Aspergillus ibericus Erlenmeyer 30 g [19]
wheat bran MUM 03.49 flasks
(500 mL)
Packed-bed 25 g
bioreactor
Palm kernel cake – Trichoderma viride Erlenmeyer 30 g [71]
SDTC EDF 002 flasks
44.43 U/g Aspergillus niger (500 mL)
SDTC SRW-4
42.05 U/g Aspergillus niger
DSMZ 2466
Rice bran with 19.844 U/g Aspergillus niger Cylindrical 40 g [72]
glycerol flasks
Rice bran 13.267 U/g (500 mL)
Rice husk, soybean
meal, wheat bran
Palm kernel cake 34.20 U/gds Rhizopus sp. Erlenmeyer 50 g [74]
flasks
(250 mL)
Olive pomace with 120 U/g Aspergillus ibericus Tray-type 300– [16]
wheat bran MUM 03.49 bioreactors 500 g
45–50% non-starch carbohydrates) for rapid growth [56, 64], so that high lipase
activities are possible (e.g., 223 U/gds by Aspergillus ibericus on olive pomace and
wheat bran [19]).
3.1.4 Pectinase
Pectinases are used to break down pectin. Pectin is a common heterogeneous

polysaccharide of galacturonic acids with varying proportions of D-galactosyl,
L-arabinosyl, or L-rhamnosyl residues in the plant kingdom [53]. Pectinases are
subdivided into polygalacturonases, galacturonases, and pectin methylesterases as
well as pectin and polygalacturonate lyases.
Just as in the production of amylases, proteases, and lipases, basidiomycetes play
only a minor role in the production of pectinases. Pectinases are mainly produced by
various plant pathogenic fungi or bacteria [75]. The main application field is the
clarification of fruit juices, which has been carried out since about 1930 [53]. Here,
further side effects are viscosity reduction and increased fruit juice yields. However,
pectinases are also involved in olive oil production, in the production of fruit and
vegetable purées, in the treatment of sugar beets, and in the fermentation of apple
cider and coffee and cocoa beans. In addition to their use in the food industry,
pectinases are mainly used in the textile industry for washing textiles as well as in
detergents for dishwashers, in the production of vegetable fibers, and for the
treatment of wastewater in the paper and pulp industry [53, 54].
For the production of pectinases on solid substrates, various ascomycetes, such as
Aspergillus sp. or Fusarium sp., have been the focus of recent research (see Table 4).
A broad spectrum of usable substrates was found to exist. The highest enzyme
activities have been achieved, inter alia, on fruit or vegetable peels. However, studies
by Kaur et al. [10] showed that not all fruit and vegetable residues are equally
suitable. By comparing different studies, it was found that orange peels in particular
appear to be suitable for the production of pectinases (e.g., 3,315 U/gds produced
by Bacillus subtilis [10]). Wheat bran was also successfully used in pectinase
production (e.g., 1828 U/gds produced by Bacillus tequilensis [76]). With a mix of
different citrus peels and wheat bran, up to 73,000 U/gds of endopectinase activity
was achieved [17]. The good suitability of citrus peels can be attributed to their
high pectin content of up to 30% [77, 78], which may induce enzyme formation.
Wheat bran also has a high fiber content (hemicellulose, cellulose, and pectin) of
about 45% [56, 64]. In comparison, apples (whole fruit), for example, contain only
0.5–1.5% pectin [78, 79].
3.2 Lignocellulolytic Enzymes
Lignocellulolytic enzymes include cellulolytic enzymes such as cellulases and

xylanases as well as lignolytic enzymes such as laccases and various peroxidases.
In nature, these enzymes are formed for nutrient supply through the degradation of
lignocellulose, the scaffold of lignified plants, by various microorganisms. Among
the best-known representatives are the so-called white rot and brown rot producers,
which a large portion of basidiomycetes belong to [75].
Table 4 Review of recent studies on pectinase production on solid substrates (bold: best substrate)
including achieved pectinase activity, used production strain, and cultivation size [10, 15, 17, 20,
50, 76, 80–85]
Jojoba mill solid waste 656.6 U/gds Aspergillus [80]
oryzae FK-923
Tomato processing 53.57 U/mL Fusarium Erlenmeyer 2g [81]
by-products solani pisi flasks
(50 mL)
Soybean meal, sunflower 64.90 IU/g Bacillus Erlenmeyer 5g [50]
meal, maize bran, maize sp. TMF-1 flasks
pericarp, olive oil cake, (150 mL)
wheat bran
Orange peel and coconut 3,315 U/gds Bacillus Erlenmeyer 5g [10]
fiber agro-residues: wheat (pectinase) subtilis flasks
bran, rice bran, paddy 10.5 U/gds (pectin SAV-21 (250 mL)
straw, corn cob, sugarcane lyase)
bagasse, mustard oil cake,
sawdust, mustard straw,
cotton straw, groundnut
peel, wheat straw, cotton-
seed oil cake Fruit peel
wastes: lemon peel,
mosambi peel, pineapple
peel, papaya peel, banana
peel, mango peel, coconut
fiber, pomegranate peel,
orange peel, cheeku peel,
kinnow peel
Wheat bran, orange and 73 kU/gds (endo-PG) A. giganteus Erlenmeyer 5g [17]
lemon peel mix, citrus 197 U/gds (PG) NRRL 10, and flasks
peel, lemon peel, apple 101 U/gds (PGM) 11 others (100 mL)
pomace Aspergillus sp. Static tray- 100 g
type
bioreactor
Green algae (Ulva lactuca 1,432 1.46 U/mg Bacillus Flask 10 g [82]
and Codium tomentosum), licheniformis
brown algae (Dictyopteris KIBGE-IB2
polypodioides, Sargassum bis B4
wightii, and Dictyopteris Aspergillus
divaricata) flavus KIBGE-
IB34
Aspergillus
terreus
KIBGE-IB35
Wheat bran, rice bran, 1828.13 U/gds (pectin Bacillus Erlenmeyer 10 g [76]
cotton seed cake, corn cob, lyase) tequilensis flasks
coconut cake, ground nut 105.55 U/gds (PG) SV11-UV37 (250 mL)
cake
Sugarcane bagasse and 33–41 U/g A. oryzae Glass col- 8g [83]
citrus pulp CPQBA 394– umns
12 DRM 01 (4 21 cm)
Pilot-scale 15 kg
bioreactor
Orange peels 8.0 U/g Penicillium Columns 20 g (+ 7 g [84]
minioluteum (25 4 cm) plastic
N3C2, pieces)
and other
ascomycetes
Wheat bran 298 U/gds Aspergillus Tray-type 50–100 g [85]
sojae ATCC solid-state
2023 reactor
(continued)
Table 4 (continued)
Wheat bran and sugar- 1,840 U/kgds/h Aspergillus Pilot-scale 12–30 kg [15]
cane bagasse niger CH4 packed-bed
bioreactor
Wheat bran and sugar- 22 U/g Aspergillus Pilot-scale 27 kg [20]
cane bagasse niger CH4 packed-bed WB + 3 kg
bioreactor SCB
(200 L)
The use of these enzymes in industry is diverse and is set to continue expanding.
To ensure cost-effective enzyme production, continuous development is necessary.
This includes research into new enzyme producers as well as suitable substrates.
In own studies, 41 basidiomycetes were investigated for their suitability as
enzyme producers (see Fig. 1). The focus was on their ability to produce laccases,
peroxidases, cellulases, and xylanases. The screening was carried out in triplicate on
malt agar plates (malt extract agar, Roth, Karlsruhe, Germany) with various addi-
tives at room temperature (24–26 C). In order to detect the formation of desired
enzymes, for lignolytic enzymes Lev-Blue dye (1 g/L), ABTS (2,20 -azino-bis
(3-ethylbenzothiazoline-6-sulphonic acid; 0.1 g/L) and guaiacol (100 μL/L) and
for cellulolytic enzymes CMC (carboxymethyl cellulose; 5 g/L) and beechwood
xylan (5 g/L), as well as pine sawdust and beech sawdust (10 g/L), were added to the
agar. Lignolytic activity could be observed directly via the development of a colored
halo (ABTS, guaiacol) or by decolorization (Lev-Blue). Cellulolytic activity was
determined indirectly by staining with a 1% Congo red solution and the formation of
a halo. Thus, it was possible to characterize the different basidiomycetes with regard
to their enzymatic performances and their growth rate and to use them for further
investigations.
3.2.1 Cellulases
Cellulases degrade cellulose, the main component of the plant cell wall. The term
cellulases encompasses several different enzymes, including the three enzymes
endoglucanase (endo), exoglucanase (exo), and ß-glucosidase (ß-glu). The different
enzymes act synergistically and can be secreted into the system as free enzymes or
bound as a multienzyme complex (cellulosome). Endoglucanases preferentially
hydrolyze amorphous regions of the cellulose, thereby resulting in oligosaccharides,
cellobiose, and glucose. Exoglucanases cleave cellobiose from the nonreducing end,
while β-glucosidase predominantly hydrolyzes cellobiose [53, 54].
Cellulases are produced by bacteria, fungi, and plants, and the most important
producers belong to the species of Trichoderma. The most well-known field of
application for cellulases is the fermentation of vegetable waste or other renewable
raw materials for the production of bioethanol. They are also used in the production
of silage. A further application is their use in detergents, where they degrade
superficial lint and thus improve the color intensity and softness of laundry. These
Fig. 1 Depiction of screening and categorization of basidiomycetes with regard to their ability to
produce lignocellulolytic enzymes as basis for further investigations
effects are also used in the textile industry, where cellulases are used as softeners, for
desizing processes and to produce a stone-wash effect. They are also used in the
paper industry for removal of colors from waste paper [53, 54].
In recent studies on the production of cellulases, ascomycetes, such as Aspergillus
sp. and Trichoderma sp., were used [3, 4, 11, 12, 21, 26, 28, 31, 32, 41–43, 48, 84,
86–91]. However, several basidiomycetes, such as Pleurotus ostreatus and Trametes
versicolor, also achieved good results on solid substrates [41, 86, 92]. Residues from
the agricultural industry have also been shown to be suitable, such as wheat bran,
sugarcane bagasse, or corn straw, which have high levels of cellulose (wheat bran,
15%; sugarcane bagasse, 50%; corn straw, 41%) [93–95] (see Table 5). For example,
Table 5 Review of recent studies on cellulase production on solid substrates (bold: best substrate)
including achieved cellulase activity, used production strain, and cultivation size [3–5, 11–13, 21,
26, 28, 31, 32, 41–43, 48, 50, 81, 84, 86–89, 91, 92, 100–103]
Citrus sinensis 264 U/gds Bacillus subtilis Flask [100]
bagasse, 12 different
agro-industrial wastes
Wheat bran 0.40 IU/g (FPase) Aspergillus oryzae Lab-scale bio- [26]
123.64 IU/g reactor, 16
(Endo) columns
18.32 IU/g (20 2.5 cm)
(ß-Glu)
Carnauba straw, sug- 0.9 U/g (FPase) Trichoderma reesei Flasks [91]
arcane, green coconut, 13 U/g (CMCase) CCT2768
cashew
Sugarcane bagasse 11 IU/g (FPase) Aspergillus flavus, Erlenmeyer 2g [41]
Trichoderma viride, flasks (250 mL)
and Pleurotus
ostreatus
Tomato processing 39.50 U/mL Fusarium solani pisi Erlenmeyer 2g [81]
by-products flasks (50 mL)
Rice by-products: 1.31 U/gds Trichoderma reesei Erlenmeyer 5g [21]
Rice bran, rice husk, CECT 2414 flasks (150 mL)
and rice straw
Wheat bran, wheat 26.3 IU/gds Aspergillus niger Conical flasks 5g [3]
straw, rice straw, rice (Endo) NFCCI (250 mL)
husk, sugarcane tops, 2.11 IU/gds
sugarcane bagasse, (FPase) 34.3 IU/
corn cob, carrot grass gds
(ß-Glu)
Wheat bran 8 0.2 IU/g Aspergillus niger Erlenmeyer 5g [32]
(FPase) RCKH-3 flasks (250 mL)
17 0.1 IU/g
(CMCase)
90 4.0 IU/g
(ß-Glu)
Biomass sorghum 30.64 U/g (Exo) Aspergillus niger Erlenmeyer 5g [43]
and wheat bran 41.47 U/g (Endo) SCBM1 and Aspergil- flasks (250 mL)
54.90 U/g (ß-Glu) lus fumigatus SCBM6
Maize bran, almond 14.23 IU/gds Bacillus subtilis MS Flasks 5g [101]
shells (5.93 IU/mL) 54
Maize bran, maize 1.19 IU/g Bacillus sp. Erlenmeyer 5g [50]
pericarp, soybean TMF-1 flasks (150 mL)
meal, sunflower meal,
olive oil cake, wheat
bran
Wheat straw and cot- 298.12 U/gds Sporotrichum thermo- Erlenmeyer 5g [5]
ton oil cake (CMCase) phile BJAMDU5 flasks (250 mL)
Wheat bran, rice bran, 280.13 U/gds
mustard oil cake, sug- (FPase)
arcane bagasse, sesame 6.67 U/gds
oil cake, rice straw, (ß-Glu)
kitchen waste, peanut
peel, sawdust
Oil palm frond 2.57 U/g (FPase) Aspergillus niger Erlenmeyer 5g [28]
flasks (250 mL)
Corn cob 33.0 U/mL P. pulmonarius Erlenmeyer 5g [92]
(ß-Glu) DBUI002 flasks (250 mL)
Sawdust 15.0 U/mL Pleurotus ostreatus
(Endo) DBUI 14
Rice bran 13.0 U/mL (Exo)
(continued)
Table 5 (continued)
Sweet sorghum 30.32 0.05 U/g Trichoderma Erlenmeyer 10 g [4]
bagasse, sweet sor- harzianum HZN11 flasks (250 mL)
ghum stalks, wheat
bran, sugarcane
bagasse, groundnut
shells, corn cobs, rice
bran, rice husk, saw-
dust, groundnut oil
cakes, coconut oil
cakes, leaf litter
Forage cactus pear 3456.91 U/g Aspergillus niger Erlenmeyer 10 g [89]
1630.07 U/g Rhizopus sp. flasks (250 mL)
Rice husks 17.2 0.7 IU/g Aspergillus oryzae Erlenmeyer 10 g [87]
Peanut shells 42.9 0.7 IU/g ATCC 10124 flasks (125 mL)
Olive pomace 9.99 1.39 U/g Aspergillus Erlenmeyer 10 g [86]
sp. (10 different flasks (500 mL)
species)
46 3 U/g Trametes versicolor
Cassava residue, 34.0 2.8 Penicillium oxalicum Erlenmeyer 10 g [102]
corncob, rice husk, rice FPU/gds EU2106 flasks (500 mL)
straw, sugarcane
bagasse, wheat bran
Prickly palm cactus 4.165 U/mL Aspergillus niger Erlenmeyer 10 g [88]
husk (CMCase) flasks
30.923 U/mL
(FPase)
7.859 U/mL Rhizopus sp.
(CMCase)
13.571 U/mL
(FPase)
Sawdust 22.97 U/g (ß-Glu) Aspergillus sp., Erlenmeyer 15 g [48]
138.77 U/g Bacillus sp. flasks (250 mL)
(Endo)
32.16 U/g (Exo)
Brevibacillus sp.
Oil palm fronds 12 IU/g Aspergillus sp. and Steriplan™ 15 g [42]
Trichoderma sp. petri dish
(100 15 mm)
Orange peels 6.5 U/g Penicillium Columns 20 g (+ [84]
minioluteum N3C2, (25 4 cm) 7g
Trichoderma reesei plastic
ATCC 26921, Fusar- pieces)
ium sp. N6C6,
Cladosporium
oxysporum N1C1,
Mucor racemosus
N9C1
Wheat bran 959.53 IU/gds Trichoderma reesei Erlenmeyer 5g [11]
RUT C-30 flasks (250 mL)
457 IU/gds Tray reactor 50 g
Wheat straw 8.583 0.025 IU Trichoderma viride Conical flasks 100 g [31]
(1,000 mL)
Agave atrovirens 12860.8 U/g Trichoderma spp. Tray reactor 200 g [12]
fibers (Endo)
3144.4 U/g (Exo)
384.4 U/g (ß-Glu)
(continued)
Table 5 (continued)
Wheat bran 375 IU/gds Trichoderma Shallow 500 g [13]
(CMCase) citrinoviride aluminum tray
695 IU/gds AUKAR04
(ß-Glu)
Coffee husk Specialized Erlenmeyer 90 g [14]
Wood chips were consortium (compost) flasks (500 mL)
added as bulking agent 10 FPU/gds Packed-bed 1,2 kg
reactor (4,5 L)
48 4 FPU/gds Packed-bed 15,2 kg
reactors (50 L)
959.5 IU/gds was obtained by Trichoderma reesei RUT C-30 on wheat bran
[11]. However, enzyme activities were usually found to be far lower.
Interestingly, very high levels of enzyme activity were found to occur on special
substrates such as forage cactus pears and agave atrovirens fibers, with 3,457 U/g
(Aspergillus niger on forage cactus pears) [89] and 12,861 U/g (endo) and 3,144 U/g
(exo) (Trichoderma sp. on agave fibers) [12]. Both substrates are succulents and rich
in trace elements, vitamins, and dietary fibers. Agave leaf fibers contain 70–80%
cellulose and have a low hemicellulose content of 3–30% and low lignification
[96]. In comparison, wheat bran has a cellulose content of only 15% and a hemicel-
lulose content of 38% [93]. Competition between the formation of cellulases and
hemicellulases is thus probably shifted in favor of cellulases when using succulent
fibers.
In our investigations, 14 basidiomycetes were tested on different substrate com-
binations. Round cups (diameter 4 cm, height 11.5 cm, membrane cap) were filled
with 35 g of substrate, made up of one part beech wood chips to one part either hemp
litter, corn cob bedding, cotton seeds, or wheat straw. The highest cellulase activity
of 627 U/L was achieved by Flammulina velutipes, followed by Lentinus tigrinus
with 603 U/L and Gloeophyllum trabeum with almost 500 U/L. The preferred
substrate in this case was the mixture of beech wood chips and corn cob bedding
when cultivating the two brown rot fungi F. velutipes and G. trabeum, while the
white rot fungus L. tigrinus preferred a mixture of beech wood chips and hemp litter
for the production of cellulases. Corn cob bedding, with a cellulose content of
around 35% [97], and beech wood chips with around 47% [98], have a similar
composition to usual suitable substrates, such as wheat bran. In a further experiment
with pure corn cob bedding (25 g in 500 mL Erlenmeyer flask) with F. velutipes and
Trametes hirsuta, cellulase activity was even increased to 1,642 U/L for F. velutipes.
The cellulase activity of T. hirsuta was only 382 U/L.
Unlike corn cob bedding, hemp litter has a high cellulose content of up to 70%
[99], but the use of hemp litter as an additive only produced good cellulase activities
for L. tigrinus. This shows how important it is to not only find a suitable producer but
also its preferred substrate. For example, T. hirsuta was cultivated on 3.5 kg pine
wood chips or corn silage in an SSF reactor (rotating drum, working volume 10 L,
developed by the Research Center for Medical Technology and Biotechnology,
fzmb GmbH, Bad Langensalza, Germany). Despite the theoretical suitability of corn
silage for the production of cellulases, hardly any cellulase activity (0.33 U/L on pine
wood chips, 1.2 U/L on corn silage) was observed.
3.2.2 Xylanase
Xylanases, like cellulases, encompass several distinct enzymes. They hydrolyze

xylan, the most abundant hemicellulose. Xylanases include endoxylanases (hydro-
lysis of glycosidic linkages in xylan backbone), β-xylosidases (cleavage of
xylose at nonreducing end), α-L-arabinofuranosidases, α-glucuronidases, and acetyl
xylan esterase (cleavage of side chains). The main xylanase producers are filamen-
tous fungi, such as Aspergillus sp., Penicillium sp., Humicola sp., and Trichoderma
sp. In industry, xylanases are produced by submerged fermentation. The induction
takes place using insoluble carbon sources, such as xylan, xylobiose, or
sophorose [54].
The main application of xylanases is in the animal feed industry. The enzymes
ensure a better digestibility of animal feed, especially for pig and poultry farming.
They are also used in the food industry to degrade arabinoxylan, thereby increasing
yields in grain flour production, improving processability of doughs, and reducing
water requirements. In addition, xylanases are used in the extraction of coffee, oils,
and starches, as well as in combination with pectinases for the clarification of fruit
juices. Other fields of use are the production of fibers from flax, jute, and hemp, as
well as the production of cellulose fibers in the paper and pulp industry and the
treatment of the resulting wastewater [53, 54].
In recent research, ascomycetes such as Aspergillus sp. and Trichoderma
sp. remain the preferred producers of xylanases. Cultivation is carried out with
residues from the agricultural industry, preferably bran or straw (see Table 6).
Wheat bran, wheat straw, and corn cobs seem to stimulate xylanase production the
most. Xylanase activities of 55,000 IU/gds on wheat bran by Trichoderma
citrinoviride AUKAR04 [13] or 2,919 U/g by Aspergillus niger CCUG33991
[104], as well as 1,189 U/gds on wheat straw by Botryotinia fuckeliana CECT
20518 [46] and 795 U/g on corn cobs by Sporotrichum thermophile BJAMDU5
[5] and 587 U/gds by Rhizomucor pusillus [105], were achieved. Another interesting
result was achieved with the use of empty oil palm bunches, on which Aspergillus
niger USM SD2 yielded xylanase activities of 3,246 IU/gds [106]. No direct
conclusions can be drawn regarding substrate composition. The hemicellulose
contents vary between 20% (wheat straw) and 43% (corn cob), while the cellulose
content is between 24% (wheat bran) and 65% (oil palm empty bunches) with a
lignin content (with the exception of corn cobs 5%) between 13% and 27% [64, 94,
97, 107].
As already described for the investigations on cellulase formation, 14 basidiomy-
cetes were tested on different substrate combinations in round cups (diameter 4 cm,
height 11.5 cm, membrane cap) filled with 35 g of substrate, consisting of one part
beech wood chips to one part either hemp litter, corn cob bedding, cotton seeds, or
Table 6 Review of recent studies on xylanase production on solid substrates (bold: best substrate)
including achieved xylanase activity, used production strain, and cultivation size [5, 6, 13, 23, 28,
41–43, 46, 48, 84, 86, 89, 91, 104, 105, 108–110]
Corn bran mixed 33.4 U/gds (acetyl xylan Aspergillus niger Glass columns [108]
with polyurethane esterase) PCS6
as support (75:25),
wheat bran, coffee
pulp
Carnauba straw, 99.5 U/g Trichoderma reesei Flasks [91]
sugarcane, green CCT2768
coconut, cashew
Cocoa meal 72 U/g Aspergillus [109]
awamori IOC-3914
Oil palm empty 3,246 IU/gds Aspergillus niger [106]
bunches USM SD2
Sugarcane bagasse 180 IU/g Aspergillus flavus, Erlenmeyer 2g [41]
Trichoderma viride, flasks (250 mL)
and Pleurotus
ostreatus
Corn cob, agro- 587.6 U/gds Rhizomucor Tray bioreactor 3g [105]
industrial wastes (packed-bed) pusillus and packed-bed
bioreactor
Biomass sorghum 300.07 U/g (Xyl) Aspergillus niger Erlenmeyer 5g [43]
and wheat bran 41.47 U/g (ß-Xyl) SCBM1 and Asper- flasks (250 mL)
gillus fumigatus
SCBM6
Wheat straw and 795.12 U/g Sporotrichum ther- Erlenmeyer 5g [5]
cotton oil cake mophile BJAMDU5 flasks (250 mL)
Wheat bran, rice
bran, mustard oil
cake, sugarcane
bagasse, sesame oil
cake, rice straw,
kitchen waste, pea-
nut peel, sawdust
Wheat straw, grape 1,189 U/gds Botryotinia Petri dishes 5g [46]
pomace, orange fuckeliana CECT
peels, rice husk 20518, Aspergillus
awamori CECT
2907, Trichoderma
reesei CECT 2414,
Phanerochaete
chrysosporium
CECT 2798
Oil palm frond 4.12 U/g Aspergillus niger Erlenmeyer 5g [28]
flasks (250 mL)
Olive pomace 19.44 3.85 U/g Aspergillus sp. (ten Erlenmeyer 10 g [86]
different species) flasks (500 mL)
100 15 U/g Trametes
versicolor
Wheat bran, rice 24.66 U/mL Streptomyces Erlenmeyer 10 g [110]
bran, sugarcane hygroscopicus flasks (250 mL)
molasses, cellulose
paper powder
Wheat bran, sug- 139 U/mL Aspergillus spp. Erlenmeyer 10 g [6]
arcane bagasse, rice flasks (250 mL)
bran, corn cob,
wheat straw,
sawdust
Forage cactus pear 355.56 U/g Aspergillus niger Erlenmeyer 10 g [89]
204.57 U/g Rhizopus sp. flasks (250 mL)
(continued)
Table 6 (continued)
Oil palm fronds 109 IU/g Aspergillus sp. and Steriplan™ 15 g [42]
Trichoderma sp. petri dish
(100 15 mm)
Sawdust 104.96 U/g Bacillus sp., Asper- Erlenmeyer 15 g [48]
gillus sp., flasks (250 mL)
Brevibacillus sp.
Corn cob, agro- 12.30–48.63 U/g Aspergillus niger, Erlenmeyer 25 g [23]
wastes Aspergillus flasks (250 mL)
fumigatus, Asper-
gillus flavus,
Trichoderma
longibrachiatum,
Botryodiplodia sp.
Orange peels 16.5 U/g Penicillium Columns 20 g [84]
minioluteum N3C2, (25 4 cm) (+ 7 g
Trichoderma reesei plastic
ATCC 26921, pieces)
Fusarium sp. N6C6,
Cladosporium
oxysporum N1C1,
Mucor racemosus
N9C1
Wheat bran, sor- 2,919 174 U/g Aspergillus niger Erlenmeyer 5g [104]
ghum stover, corn CCUG33991 flasks (250 mL)
cob, and soybean Tray bioreactor 100 g
meal
Wheat bran 55,000 IU/gds Trichoderma Shallow 500 g [13]
citrinoviride aluminum tray
AUKAR04
wheat straw. By far the highest xylanase activities, 23,063 U/L and 7,344 U/L, were
achieved by F. velutipes and by Schizophyllum commune, both brown rot fungi.
Here, the preferred substrate was also a mixture of beech wood chips and corn cob
bedding, a substrate already classified as suitable in the literature [23, 104, 105]. The
good suitability of F. velutipes for the production of xylanases, which was at the
same level of known producer strains [13, 104, 106], was also confirmed in
subsequent experiments with pure corn cob bedding (25 g in 500 mL Erlenmeyer
flask). In this case, xylanase activity of 16,739 U/L was achieved.
3.2.3 Laccases and Peroxidases
Lignin is a heterogeneous biopolymer consisting of aromatic phenolic building

blocks in the plant cell wall. It causes lignification and thus an increase in the
stability of plant material. Lignin degradation is very slow due to its high complex-
ity, and it is preferably carried out by representatives of white rot fungi, especially
basidiomycetes. It is an oxidative process and cannot be carried out by a single
enzyme, so a consortium of different enzymes that interact with each other is needed.
These include oxidative enzymes such as laccases, peroxidases, lignin peroxidases,
and manganese peroxidases [75].
Laccases belong to the multicopper oxidases and are mainly formed by filamentous
fungi. These include, in particular, various basidiomycetes, such as Agaricus bisporus,
Cerrena unicolor, and Trametes versicolor. They are used in the pulp and paper
industry (e.g., paper bleaching), the textile industry (e.g., denim bleaching), cosmetics
(e.g., hair bleaching), the wood industry, the food industry, the production of biofuels,
and the pharmaceutical industry (e.g., transformation of antibiotics) [111].
Peroxidases are a diverse group of enzymes that reduce peroxide, usually hydro-
gen peroxide. Manganese and lignin peroxidases as well as non-specific peroxidases,
mainly produced by fungi, are used especially for lignin degradation [75].
As already mentioned above, the main producers of lignolytic enzymes are
filamentous fungi, especially the basidiomycetes. This is also reflected in the organ-
isms studied for the production of these enzymes on various solid substrates in the
literature. Again, various residues from the agricultural industry were used (see
Table 7).
Substrates with a high lignin content were popular, in other words lignified
materials, such as sawdust or vineyard trimmings. However, only moderate enzyme
activities were achieved, for example, 305 U/gds on sawdust by Fusarium equiseti
VKF-2 [35]. The use of fruit peels or leaves proved much more successful. For
example, 1,200 U/g was observed for laccase activity of Phanerochaete
chrysosporium CECT 2798 on orange peel [46] and 1,633 U/ml of Pleurotus
ostreatus strain NCIM 1200 on pineapple leaves [112], as well as 10,800 U/L for
peroxidase activity of Pleurotus eryngii IJFM 169 on banana peels [9]. Also, the use
of pretreated substrates, such as steam-exploded corn stalks (2,600 U/g laccase
activity of T. versicolor [45]), or Parthenium sp., a belligerent noxious herbaceous
wasteland weed (34,444 U/gds laccase activity of Pseudolagarobasidium acaciicola
[30]), showed a considerable increase in laccase activity. This effect may be attrib-
uted to the availability of vegetable ingredients, such as essential oils in orange
peels or parthenolide in the wasteland weed, whose aromatic structures similar to
the degradation products of lignin may promote the formation of laccases and
peroxidases.
In addition to known basidiomycetes, such as Pleurotus eryngii [9] and
Marasmius sp. [114], two fungi species, Lentinus tigrinus and Trametes hirsuta,
were also investigated in our studies due to the good suitability of basidiomycetes,
especially white rot fungi, for the production of lignolytic enzymes.
First investigations in round cups (diameter 4 cm, height 11.5 cm, membrane cap)
filled with 35 g of substrate, consisting of cotton seeds, pine wood chips or wheat
straw, showed the investigated fungi (P. eryngii, Marasmius sp., L. tigrinus) to be
suitable for laccase formation in principle. As in the studies on cellulase and
xylanase activity, the activities achieved differed depending on the producer and
substrate used. However, it was found that by far the highest activity of laccases for
all three fungi tested was achieved on wheat straw (P. eryngii, 131 U/L; Marasmius
sp., 117 U/L; L. tigrinus, 70 U/L). The lignin content of wheat straw with max. 25%
Table 7 Review of recent studies on production of lignolytic enzymes on solid substrates (bold:
best substrate) including achieved activity, used production strain, and cultivation size [8, 9, 30, 35,
41, 42, 44–46, 48, 112–114]
Laccase
Sawdust, ground- 305 U/gds Fusarium equiseti Flask [35]
nut oil cake, neem VKF-2
oil cake, rice bran
Peanut shell 5 U/g Pycnoporus Flasks (250 mL) [113]
sp. SYBC-L3
Vineyard 0.34 mU/mg Lentinus edodes [44]
trimmings protein
Wheat straw 0.08 mU/mg
protein
Sugarcane 10 IU/g Aspergillus flavus, Erlenmeyer flasks 2g [41]
bagasse Trichoderma (250 mL)
viride, and
Pleurotus
ostreatus
Peels of citrus Lysinibacillus sp. Flasks (250 mL) 5g [8]
fruits, soybean
meal, tofu dreg,
lignin monomers,
tea leaves, peels of
onion and kiwi,
paper, dying
industry effluents
Steam-exploded 2600.33 81.89 Trametes Erlenmeyer flasks 5g [45]
corn stalk U/g versicolor (250 mL)
Rice bran, rice 1.4 U/mL Marasmius Conical flasks 5g [114]
straw, sugarcane sp. BBKAV79 (250 mL)
bagasse, sawdust,
pigeon pea waste
Parthenium sp. 34,444 U/gds Pseudolagarobas- Erlenmeyer flasks 5g [30]
(belligerent nox- idium acaciicola (250 mL)
ious herbaceous LA 1
wasteland weed),
rice straw, wheat
straw, sugarcane
bagasse
Orange peels, 1,200 U/g Phanerochaete Petri dishes 5g [46]
wheat straw, grape chrysosporium
pomace, rice husk CECT 2798,
Botryotinia
fuckeliana CECT
20518,
Aspergillus
awamori CECT
2907,
Trichoderma
reesei CECT 2414
(continued)
Table 7 (continued)
Pineapple leaves 1632.63 IU/mL Pleurotus Erlenmeyer flasks 5– [112]
ostreatus strain (250 mL) 25 g
NCIM 1200
Sawdust 71.18 U/g Bacillus sp., Erlenmeyer flasks 15 g [48]
Aspergillus sp., (250 mL)
Brevibacillus sp.
Peroxidase
Banana peel 10,800 U/L Pleurotus eryngii Erlenmeyer flasks 6g [9]
36 U/gds IJFM 169 (250 mL)
Sawdust 729.12 U/g Aspergillus sp., Erlenmeyer flasks 15 g [48]
Bacillus sp., (250 mL)
Brevibacillus sp.
Manganese peroxidase
Vineyard 2.21 mU/mg Lentinus edodes [44]
trimmings protein
Wheat straw 0.60 mU/mg
protein
Sawdust 47.73 U/g Bacillus sp., Erlenmeyer flasks 15 g [48]
Aspergillus sp., (250 mL)
Brevibacillus sp.
Lignin peroxidase
Oil palm fronds 222 IU/G Aspergillus Steriplan™ petri 15 g [42]
sp. and dish
Trichoderma sp. (100 15 mm)
and 9% of cotton seeds is lower than that of pinewood chips, which is around 27%
[94, 115, 116]. The lower lignin content and the simplified accessibility might favor
laccase production on wheat straw or cotton seeds, as opposed to the use of untreated
pinewood chips.
In a further study, the versatile white rot fungi T. hirsuta was cultivated on
various substrates in an SSF reactor (rotating drum, working volume 10 L, devel-
oped by the Research Center for Medical Technology and Biotechnology, fzmb
GmbH, Bad Langensalza, Germany) (see Table 8).
Once again, enzyme activity was confirmed to be strongly dependent on the
substrate used. It was shown again that substrates with high lignin content, such as
pinewood chips, do not necessarily result in better enzyme production. In contrast,
substrate mixtures can significantly improve enzyme production. Thus, the positive
effect on enzyme formation when using orange peels, as also described in the
literature [46], was confirmed and amplified by the mixture with pinewood chips,
leading to greater structural integrity and improved air exchange in the substrate bed
(compare laccase activity in Table 8).
Table 8 Comparison of lignolytic enzyme activities achieved by T. hirsuta cultivated on different

substrates in an SSF rotating drum reactor
Pinewood Orange Pinewood chips + orange peel Corn
Substrate chips peels + xylidine silage
Enzyme
Laccase U/L 106 742.5 1,881 346
Unsp. U/L 11.4 99.8 433 51
peroxidase
Manganese U/L 5.6 – 24.37 473
peroxidase
Lignin mU/L 4 – 6.5 153
peroxidase
4 Conclusions
Solid-state fermentation is a cost-effective and sustainable alternative for the pro-

duction of technically relevant extracellular enzymes by selected filamentous fungi
and bacteria on industrial residues. This review has shown that, in addition to the
engineering aspects, the combination of producer strain and substrate is crucial for
effective enzyme production.
The substrates used were mainly residues from the agricultural industry, the food
industry, and the forestry. In most cases, the choice of substrate was made based on
its composition and the natural function of the target enzyme. For example, starch-
containing substrates were preferably used for the production of amylases. However,
the example of lignolytic enzymes showed that it is not always sufficient to make the
component that is to be degraded available in large quantities. It was found that ease
of accessibility is also a significant factor in ensuring good induction of enzyme
formation.
Wheat bran, either alone (e.g., for the production of amylases) or in combination
with other substrates (e.g., for the production of lipases), is a very versatile and
popular substrate for enzyme production. Various types of bran, as well as straw and
fruit peel, were found to be suitable for the production of amylases despite their low
starch content. As expected, proteases were preferably produced on protein-rich
substrates, such as soy residues, wheat and rice bran or animal-origin residues such
as cuttlefish. Lipid-rich substrates, such as coconut meal, olive pomace, and palm
kernel cake, partly in combination with wheat bran, were successfully used for lipase
production. For the formation of pectinase, citrus fruit peels were especially suitable.
Wheat bran, sugarcane bagasse, and corn straw are often used for cellulase produc-
tion. However, it turned out that especially the use of succulent fibers, such as forage
cactus pears and agave atrovirens fibers, had a very positive effect on the formation
of cellulases. For the formation of xylanases, a very diverse spectrum of substrates
could be used, such as wheat bran, wheat straw and corn cobs, empty oil palm
bunches, and corn cob bedding. Lignolytic enzymes, such as laccases and various
peroxidases, were formed primarily on substrates with relatively readily available
aromatic compounds, such as fruit peels (e.g., orange peels) or leaves, and not, as
expected, on substrates with very high lignin contents.
Finally, it was shown that there is not just one ideal substrate for each enzyme but
that the choice of substrate needs to take into account the preferred substrate of the
producer chosen for the enzyme.
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Part II
Application of Solid-State Fermentation
for Product Generation
DOI: 10.1007/10_2019_87
Aroma Profile Analyses of Filamentous

Fungi Cultivated on Solid Substrates
Axel Orban, Marco A. Fraatz, and Martin Rühl
Contents
1 Aroma Profile Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87
1.1 Methods for Volatile Organic Compound Extraction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88
1.2 Detection of Volatile Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
2 Fungal Volatile Organic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
2.1 Fungal VOC in Food Manufacturing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
2.2 VOC Produced During Non-food SSF of Ascomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
2.3 VOC Produced During SSF of Basidiomycetes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97
3 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 102
Abstract Filamentous fungi have been used since centuries in the production of
food by means of solid substrate fermentation (SSF). The most applied SSF
involving fungi is the cultivation of mushrooms, e.g., on tree stumps or sawdust,
for human consumption. However, filamentous fungi are also key players during
manufacturing of several processed foods, like mold cheese, tempeh, soy sauce, and
sake. In addition to their nutritive values, these foods are widely consumed due to
their pleasant flavors. Based on the potentials of filamentous fungi to grow on solid
substrates and to produce valuable aroma compounds, in recent decades, several
studies concentrated on the production of aroma compounds with SSF, turning
A. Orban and M. A. Fraatz

Justus Liebig University Giessen, Institute of Food Chemistry and Food Biotechnology,
Giessen, Germany
M. Rühl (*)
Justus Liebig University Giessen, Institute of Food Chemistry and Food Biotechnology,
Giessen, Germany
Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Project Group
“Bioresources”, Giessen, Germany
86 A. Orban et al.
cheap agricultural wastes into valuable flavors. In this review, we focus on the
presentation of common analytical methods for volatile substances and highlight
various applications of SSF of filamentous fungi dealing with the production of
aroma compounds.
Graphical Abstract
Keywords Ascomycetes, Basidiomycetes, Fermented food, SSF, Volatile organic

compounds
Aroma Profile Analyses of Filamentous Fungi Cultivated on Solid Substrates 87
Abbreviations
6-PP 6-Pentyl-α-pyrone
AAO Aryl alcohol oxidase(s)
ADA Aroma dilution analysis
AEDA Aroma extract dilution analysis
CAR Carboxen
DHS Dynamic headspace
DM Dry matter
DVB Divinylbenzene
FD Flavor dilution factor
FID Flame ionization detector
GC Gas chromatography
HS Headspace
LLE Liquid-liquid extraction
MS Mass spectrometer
O Olfactometry
OAV Odor activity value
ODP Olfactory detection port
PA Polyacrylate
PDMS Polydimethylsiloxane
PEG Polyethylene glycol
SAFE Solvent-assisted flavor evaporation
SBSE Stir bar sorptive extraction
SmF Submerged fermentation
SPME Solid-phase microextraction
SSF Solid substrate fermentation
TD Thermal desorption
VOC Volatile organic compound(s)
1 Aroma Profile Analysis
The analysis of aroma active compounds in food started in the 1960s with
the possibility to separate complex aroma mixtures by means of capillary gas
chromatography (GC) (reviewed in [1]). With this analytical method, new
possibilities arose to determine the volatile composition of a sample. Nevertheless,
the impact of each volatile organic compound (VOC) on the human nose still
remained unknown until GC-olfactometry (GC-O) entered the aroma research.
GC-O enables the correlation between a VOC and its perception using the human
nose as a detector [1–3]. In most cases, the outlet of the GC capillary column is
installed into a column flow splitter. The column flow splitter possesses two outlets
directing the gas flow into a destructive detector (e.g., flame ionization detector
88 A. Orban et al.
Fig. 1 GC-MS-O equipped with an autosampler and an ODP (highlighted)
(FID) or mass spectrometer (MS)) and into a nondestructive olfactory detection port
(ODP) (Fig. 1). With this equipment, it is possible to directly assign an odor
impression to an MS spectrum or FID peak, respectively [3, 4].
1.1 Methods for Volatile Organic Compound Extraction
Prior to the GC analysis, VOC have to be extracted from the analyte’s matrix.
Among others, this can be food, a plant, or a microbial culture, and the method of
extraction depends on the matrix as well as on the VOC to be analyzed.
1.1.1 Solvent Extraction
Several extraction methods for aroma analysis exist, each having different pros
and cons. This makes the decision for an optimal analytical method difficult.
Continuous extraction of volatile organic compounds from a solid matrix using a
Soxhlet extractor followed by a concentration of the extract is a basic method for
gaining an aroma extract. In 1964, Likens and Nickerson improved this method by
inventing an apparatus for simultaneous solvent extraction and distillation, reducing
the thermal load on the sample [5]. Nevertheless, elevated temperatures during
distillation and extraction may lead to artefact formation. Thermal stress during
extraction can be overcome by using a liquid-liquid extraction (LLE). Generally, the
solid matrix is dispersed in an aqueous phase and extracted exhaustively using
an organic solvent. For an efficient extraction and the subsequent analysis, different
properties of the solvent including polarity, density, solubility, and potential
reactivity regarding the analytes have to be taken into account [5]. The disadvantage
of LLE is the often occurring concurrent extraction of nonvolatile compounds
resulting in difficulties during GC analysis. This disadvantage as well as thermal
stress can be reduced to a minimum by applying the solvent-assisted flavor
evaporation (SAFE) method. The core part of this procedure is the SAFE distillation
unit [6], which is evacuated by a high vacuum pump. The distillation starts by
dropping the sample extract into the evaporation flask. The immediately formed
vapor is transported into the distillation head, where nonvolatile compounds are
trapped. Volatile compounds condense in a sample collection flask, which is cooled
in a Dewar using liquid nitrogen [6]. Nonetheless, the demand for environment-
friendly as well as more easy and fast extraction methods led to the development of
solvent-free procedures.
1.1.2 Dynamic Headspace
One of the first dynamic headspace (DHS) systems was developed in the 1970s to
establish a routine procedure for volatile extraction and analysis by means of
GC [7]. DHS involves passing a defined flow of (inert) gas through a container
holding the sample. The VOC inside the container are carried by the steady gas flow
into a sorbent, a cryogenic container, or a solvent, where they are trapped
[8]. When the gas flow is led through a liquid sample, the method is also referred
to as “purge and trap” [9]. DHS can be conducted in a circular system by using a
closed-looped stripping apparatus, where the extraction gas flows in a closed
circuit [10, 11]. The sample might be heated, stirred, or supplemented with salts to
increase the volatility of the analytes [9, 12]. With DHS, exhaustive extraction of the
sample is possible, since the analytes are permanently removed from the headspace
(HS), and therefore no equilibrium between the matrix and the gas phase is
established [13]. In addition to the necessity to elicit the right gas flowrate, the
extraction time and temperature as well as the type of sorbent in the trap are crucial
parameters. Various trapping materials are commercially available, including acti-
vated charcoal, poly(2,6-diphenyl-p-phenylene oxide) (Tenax®), silica-based mate-
rials (Chromosorb®), carbon molecular sieves (Carboxen®), and graphitized carbon
(Carbotrap®). Traps containing different types of sorbents are used frequently in
order to achieve the extraction of a wider range of substances [14]. Depending on the
sorbent applied, desorption of the VOC can be performed with a solvent or thermally
in the GC [15]. For thermal desorption (TD) applications, Tenax® is often used due
90 A. Orban et al.
Fig. 2 Scheme of a laboratory installation for DHS during SSF of a fungus. MFC mass flow
controller
to its high thermostability, its low water adsorption capacity, and its low bleed
characteristic [8, 15, 16]. Nonetheless, Tenax® has some disadvantages: e.g., it has
only a small surface area, resulting in a low adsorption capacity, and it is limited to
the extraction of more nonpolar analytes because of its low affinity for polar
compounds [17].
Generally, DHS is an important extraction method for volatile compounds since it
reduces or even eliminates the need of solvents. It offers the possibility of multiple
trapping approaches as well as the usage of various sorbent materials [18]. During
solid substrate fermentation (SSF), DHS is applied in our laboratory to extract VOC
produced during the cultivation of filamentous fungi (Fig. 2).
1.1.3 Microextraction Methods SPME and SBSE
Further options for the extraction of volatile compounds comprise solid-phase

microextraction (SPME) and stir bar sorptive extraction (SBSE) [19]. SPME was
presented to the scientific world in the early 1990s as a new possibility to perform
sample extractions in a more environment-friendly way [20]. SPME is a
non-exhaustive extraction technique in which a fiber coated with sorbent materials
is exposed to the sample [20, 21]. Commercial SPME fibers often consist of fused-
silica as a carrier modified with different absorbent or adsorbent materials, including
polydimethylsiloxane (PDMS), polyethylene glycol (PEG), polyacrylate
(PA), divinylbenzene (DVB), carboxen (CAR), as well as combinations thereof
[22–24]. This diversity of available coatings makes SPME the method of choice in
a broad range of applications, such as in the food [25], aroma [26], medical [27],
environmental [28], or bioanalytical [29] sector. Extraction can be performed in the
HS by incubating the fiber in the vapor phase above the sample (Fig. 3) or by direct
immersion of the fiber into the matrix [30].
The benefits of SPME compared to LLE are the absence of solvents, the higher
sensitivity, the demand for less sample material, and a faster and convenient
handling [19, 20]. Furthermore, the entire extraction process at a specific
Fig. 3 SPME applied

during SSF of a fungus
temperature for a given period can be conducted automatically by using GC systems

equipped with multipurpose autosamplers (Fig. 4). Nonetheless, SPME suffers from
drawbacks regarding the stability of the fiber and coating, the capacity for analytes,
and the durability in organic solvents [23, 31]. It is worth mentioning that new
materials for SPME fibers and coatings are on the rise, reducing the aforesaid
problems [22, 32].
SBSE was developed around 20 years ago as a new microextraction method
[33]. For SBSE, a magnetic stir bar, covered with a layer of sorbent material,
usually PDMS [34], is used. In contrast to SPME, the coating is 50–250 times
thicker, resulting in a higher capacity and sensitivity [35]. Extraction can be
performed in the HS above a sample or, more common, in an aqueous sample by
stirring the sample under controlled conditions [36]. The extraction process is
followed by the desorption of the analytes which can be accomplished thermally
or by rinsing the stir bar with a solvent [37]. In most SBSE GC applications, the TD
is preferred due to easier handling. TD is usually conducted at a temperature range
of 150–300 C. Compared to SPME, desorption time can be quite long, up to
15 min, due to the thicker coating of the stir bar. In this case, compounds
might desorb too slowly, leading to insufficient chromatographic separation of
the analytes. To ensure an adequate analysis, cryofocusing of the substances in
the GC inlet is necessary [37]. Cryotrapping enables quantitative transfer of the
analytes (with considerable increase in sensitivity) and minimizes chromatographic
peak width [38]. Besides the need for special equipment, during the early days of
SBSE, one drawback was that only PDMS as a coating material was commercially
available. This made the analysis less suitable for more polar compounds. In recent
years, new PEG- and PA-containing sorbents for SBSE were developed, thus
making this method attractive for a broader range of applications [39]. Besides
SPME and SBSE, further microextraction techniques like single-drop
microextraction and microextraction by packed sorbent offer solutions for a wide-
spread field of analytical problems [28, 40]. It is important to keep in mind that
every method leads to a unique aroma profile and that there is no universal
extraction technique available [41].
92 A. Orban et al.
Fig. 4 Automatically
performed SPME GC-MS
analysis of SSF with
filamentous fungi in 20 mL
vials (height 7.5 cm; width
2 cm)
1.2 Detection of Volatile Compounds
Generally, the identification of volatile compounds is based on retention indices

(RIs) and mass spectral analysis. RIs of the analytes obtained on two columns of
different polarity have to be compared with those of the corresponding authentic
reference compounds. In addition, the mass spectra of the analytes have to
correspond to the mass spectra of the standard substances [42, 43]. Linear RIs
modified after Kováts [44] should be calculated according to the retention times of
homologous n-alkanes by linear interpolation. Additionally, it is common to com-
pare the odor quality of the analyte with that of the authentic standard by olfactory
detection at comparable concentration levels [43]. Although single compounds can
be identified with this approach, the impact of each VOC on the overall aroma
profile is still unknown. Especially in fermented products, several hundreds of VOC
are present, i.e., more than 800 in coffee [1, 45]. To determine the compounds
representing the sample, the so-called character impact compounds, an aroma extract
dilution analysis (AEDA) using GC-O is carried out [45]. A stepwise dilution
(generally 2n) of an extract is performed, and each dilution is analyzed by means
of GC-O until no compound is perceived at the ODP [45]. The highest dilution at
which a flavor compound can still be perceived is defined as the so-called flavor
dilution factor (FD) [45]. Thus, it is assumed that aroma compounds with the highest
FD factors mainly contribute to the overall aroma. By focusing on these substances,
the key odor compounds can be identified [4, 46]. In recent studies as an alternative
to AEDA, an aroma dilution analysis (ADA) is performed without the need of
preparing an extract due to the usage of SPME or SBSE. The stepwise dilution is
achieved by adjusting the split ratio of the carrier gas flow [47, 48].
Generally, the FD factor of a VOC is meaningless concerning the impact of this
compound on the overall olfaction. For this purpose, the odor activity value (OAV)
can be determined [43]. Besides the concentration of a specific compound, the odor
threshold of a standard substance of this volatile compound has to be determined by
several panelists. The OAV is then calculated by dividing the concentration of the
volatile compound in the sample with the determined threshold of the specific
substance. Only VOC with an OAV >1 are taken into account.
2 Fungal Volatile Organic Compounds
Higher fungi are a remarkable source of aroma compounds highly valued

by humans since ancient times: (1) mushrooms are an esteemed repository for
nutrition, appreciated for their medical benefits and their unique as well as delicious
flavors, and (2) ascomycetes, like Saccharomyces cerevisiae, are essential in food
processing, ensuring the quality and taste, e.g., in bread or beer. This review focuses
on the aroma compounds produced by filamentous fungi during SSF within various
applications. Besides the discussion on different VOC produced, the various
extraction methods used within the works listed below should depict the possibilities
of aroma analysis during SSF.
2.1 Fungal VOC in Food Manufacturing
Ascomycetes such as Aspergillus oryzae and Aspergillus sojae have been applied to
ferment soy bean, rice, and wheat to hydrolyze starch and proteins. The product of
this SSF is called koji and is used as a starter culture for subsequent fermentation of
the material into soy sauce, miso, or sake [49]. Ito et al. investigated the volatile
compounds during the production of koji for the sake manufacturing process, which
is rice fermented with A. oryzae [50]. Husked rice grains were soaked in water for
2 h, steamed for 30 min, cooled, and drained. The rice was inoculated with conidia
(asexual spores), and incubation was performed at 36 C with a relative humidity of
95%. Volatiles were extracted using a DHS system equipped with a Tenax® trap,
desorbed via TD, and analyzed by means of GC-FID and GC-MS. In total,
17 compounds were identified, including alcohols (e.g., ethanol, 3-methyl-1-buta-
nol, butanol, oct-1-en-3-ol, and octan-1-ol), aldehydes (e.g., acetaldehyde), ketones
(e.g., acetone, butanone, and octan-3-one), and the ester ethyl acetate. Their presence
within the headspace of the active koji culture varied during cultivation. After 22 h in
the mid-log phase of the SSF, a grassy impression was dominant, whereas after 38 h,
the grassy fragrances decreased, and oct-1-en-3-ol increased resulting in the
mushroom-like odor in the stationary phase of koji making. The dependence of
94 A. Orban et al.
Fig. 5 Surface of a 3-day-old Rhizopus oligosporus culture on soy beans (left) and cross section of
the tempeh (right); photos kindly provided by Andrea Sabbatini
VOC on the stage of koji production enables process control of SSF on the basis of
VOC analysis. This is depicted in the work of Kum et al. [51], where a soybean-
based koji paste named doenjang is produced by means of SSF over a period of
8 weeks. The authors conducted a principal component analysis of the analyzed
VOC, which revealed a distinct clustering of the calculated VOC components in
relation to the cultivation period. Another soybean-based food is the traditional East
Asian tempeh. Generally, for tempeh production, soybeans are soaked in water,
cooked, and used as solid substrate for fungal colonization by the filamentous
zygomycete Rhizopus oligosporus. After fermentation a structured matrix is formed,
known as tempeh (Fig. 5). The aroma of fresh tempeh is specified by mainly
moldy, mushroom-like, earthy, and boiled potato-like aroma compounds, such as
2-methylpropanal, oct-1-en-3-one, and 3-methylsulfanylpropanal (methional)
[52, 53]. LLE of VOC from tempeh with subsequent SAFE and GC-MS
analysis, followed by the determination of the corresponding OAV, revealed
2-methylpropanal and oct-1-en-3-one as main aroma compounds after 1 day of
fermentation. After 5 days of fermentation, the boiled potato odor methional ensued
[53]. In a HS analysis of tempeh with Tenax® as adsorbent [54], it was not possible
to detect methional nor the typical mushroom odor oct-1-en-3-one found in liquid
extracts, but other C8 volatiles were present. Interestingly, the key aroma compound
2-methlypropanal was detected in the fermented soybeans as well as in the
non-fermented control, although in the latter only in small concentrations [54].
An SSF for food manufacturing with filamentous fungi adapted in Europe is the
production of soft cheese including camembert and blue cheese, e.g., Roquefort and
Gorgonzola. Filamentous fungi play an important role in the maturation of these
cheeses, contributing to the unique flavor, texture, and product composition
[55]. Camembert, a mold-ripened cheese originating from France, is traditionally
manufactured from raw cow milk [56]. During production, the curd is molded with
Penicillium camemberti on the surface, imparting the cheese its pronounced flavor.
Analysis of the camembert aroma was performed using solvent and static HS
extraction by means of GC-MS followed by AEDA and calculation of FD values
[57] or OAV [58]. In both works, 42 VOC were identified of which dimethyl sulfide,
methional, and especially methanethiol were key odorants of the sulfurous, garlic
note. The C8 volatiles oct-1-en-3-one and oct-1-en-3-ol contributed to the
mushroom-like odor, whereas 3-methylbutanal could be linked to the malty scent of

the camembert, and 2-phenylethyl acetate was related to a floral bouquet of the
cheese. In contrast to the cultivation on curd, no sulfur-containing volatiles were
detected when P. camemberti was cultivated on potato dextrose agar or Czapek’s
agar [59]. Similar to the curd fermentation, C8 compounds, including oct-1-en-3-ol,
octan-3-ol, and octan-3-one, have been extracted with a DHS device equipped with
Tenax® traps and identified in agar plate cultures. Another Penicillium species used
for blue cheese manufacturing is P. roqueforti. Similar to Camembert fabrication,
fungal conidia are either directly added to the milk or sprayed on the curd during
blue cheese production. Usually, ripening is performed for about 90 days at 10 C
and a relative humidity of at least 90% [60, 61]. P. roqueforti has a high tolerance
regarding physical parameters. It can even grow in an oxygen-poor environment
and in a broad pH range of 3–10. This enables, by adjusting suitable conditions, a
selective growth of the fungus inside the cheese [62]. During maturation, several
peptidases and lipases are expressed by P. roqueforti, leading to extensive
degradation of proteins (up to 35%) and fats [60, 63, 64]. Portions of the released
fatty acids are converted to 2-methyl ketones via oxidation to β-keto acids and a
decarboxylation step [65]. 2-Methyl ketones have a strong impact on the character-
istic aroma of blue cheese, contributing with fruity, floral, and musty notes
[66, 67]. A comparative study of 55 P. roqueforti strains, using a DHS system
with a Tenax® trap and analysis via GS-MS, resulted in the identification of 52 VOC.
Several substances known from Camembert, like sulfur compounds (e.g.,
methanethiol) and alcohols (e.g., oct-1-en-3-ol), were detected. With 50–75% of
the total volatiles, 2-methyl ketones were the most abundant analytes [61]. It is
notable that the composition of the 2-methyl ketones in the cheese depends strongly
on the stage of ripening [68], which again illustrates the growth phase-dependent
VOC production.
2.2 VOC Produced During Non-food SSF of Ascomycetes
Biotransformation of industrial side streams into valuable products, such as volatile

organic compounds, by means of fungal solid-state, respectively, solid-substrate
fermentation, is done since ages. In most cases, filamentous fungi are used for
SSF, but even yeasts have been applied on solid substrates. Rossi et al. [69] analyzed
the aroma profile of the plant pathogen Ceratocystis fimbriata cultured in SSF on
citric pulp, a residue of the citrus juice-producing industry. Citric pulp as substrate
was tested solely or supplemented with soya molasses, sugarcane molasses, soya
bran, or urea and different concentrations of saline solution (KH2PO4, CaCl2, and
MgSO4). HS analysis was performed by means of GC-FID to identify VOC
produced by the fungus. Acetaldehyde, ethanol, ethyl acetate, propyl acetate, ethyl
isobutyrate, hexan-2-one, hexan-2-ol, and 3-methylbutyl acetate were identified. The
addition of soya bran as N-source, sugarcane molasses as C-source, as well as saline
solution to the citric pulp resulted in the highest production of total volatiles.
96 A. Orban et al.
The amount of 3-methylbutyl acetate, a valuable banana aroma, increased 5.5-fold

compared to the citrus culture without supplementation. SSF of C. fimbriata on
cassava bagasse supplemented with either leucine or valine resulted in strong banana
aroma, which was probably due to the presence of 3-methylbutyl acetate [70]. In
contrast, SSF of cassava bagasse supplemented with urea showed a similar growth
rate, but only a slight production of VOC. The high 3-methylbutyl acetate concen-
tration in cultures supplemented with leucine might derive from an active
Ehrlich pathway, yet not known for C. fimbriata but described for the ascomycete
S. cerevisiae [71]. Here, leucine would be transformed into 3-methylbutanol,
which can be converted into 3-methylbutyl acetate by an alcohol acyltransferase
[72]. Contradictory to this assumption, valine would react via 2-methylpropanol into
2-methylpropyl ethanoate (isobutyl acetate) not detected by the authors [70]. On
coffee residues, C. fimbriata showed a different VOC pattern during SSF [73]. Cof-
fee pulp and coffee husk supplemented with glucose were used as substrates. After
inoculation with spores, the volatiles in the HS were analyzed by means of GC-FID
over a time period of 10 days. After 48 h, the highest amounts of volatile compounds
were detected. The dominant volatiles in the HS of the coffee husk and coffee
pulp samples were ethyl acetate (84.7 and 69.6%), ethanol (7.6 and 20.0%),
and acetaldehyde (2.0 and 2.1%). Esters including ethyl propionate, propyl acetate,
isobutyl acetate, ethyl isobutyrate, and ethyl butyrate contributed to the fruity odor of
the culture, but no 3-methylbutyl acetate was detected.
In addition to the impact of substrates on the composition of VOC, the cultivation
type, SSF or submerged fermentation (SmF), respectively, shaken or static cultures,
has an effect on the overall productivity of VOC, e.g., for the coconut-like aroma
compound 6-pentyl-α-pyrone (6-PP) in cultures of Trichoderma species. Kalyani
et al. compared the production of 6-PP in potato extract medium supplemented with
glucose in shaken and static cultures [74]. Over a period of 5 days, samples were
harvested every day. Aliquots of the culture supernatant were extracted with
dichloromethane, dried over sodium sulfate, concentrated, and 6-PP was
quantified with GC-FID. After 96 h, the highest 6-PP concentration of
455 mg L 1 was obtained in the static cultures, whereas in the shaken cultures, the
maximum amount of 187 mg L 1 was already reached after 48 h. In SSF cultures of
T. harzianum on sugarcane bagasse, the 6-PP concentration was even higher with
933 mg L 1 at the end of cultivation after 10 days [75]. The productivity of 6-PP by
T. harzianum was almost 14 times higher in SSF (171 mg L 1 day 1) than in SmF
cultures (12.5 mg L 1 day 1) [75]. Sugarcane bagasse was also used in several
other studies for 6-PP production. Araujo et al. screened 95 fungal isolates for
the occurrence of 6-PP [76]. One Trichoderma species showed the highest product
concentration with 3 mg 6-PP g 1 dry matter (DM) after 5 days of cultivation,
accounting for approximately 940 mg 6-PP L 1 of supernatant [76]. Another
Trichoderma species, T. viride, was cultivated on sugarcane bagasse for a period
of 12 days. The highest 6-PP concentration (3.6 mg g 1 DM) was also achieved at
day 5 [77]. It is worth mentioning that the usage of 6 g sugarcane bagasse instead of
4.5 g resulted in a significant decrease in 6-PP production (1.7 mg g 1 DM).
Improvement of the cultivation conditions for 6-PP production by SSF of
T. harzianum on green coir powder was performed by Souza Ramos et al. [78] by
altering the culture conditions regarding the composition of the supplements, the
moisture content, the amount of spores for inoculation, and the temperature. After
7 days of fermentation, the HS was analyzed with SPME (PDMS) and GC-FID. The
highest amount of 6-PP was achieved at 28 C by using per 100 g coir: 3 g sucrose,
0.24 g NaNO3, 0.18 g (NH4)2SO4, 0.1 g KH2PO4, an inoculum of 2.2 106 spores,
and water to reach a final moisture level of 55%. The fermentation under the selected
conditions led to a six times higher 6-PP production (5.0 mg g 1 DM) than the initial
one (0.8 mg g 1 DM).
Another interesting fruity flavor is the pineapple odor ethyl hexanoate. Yamauchi
et al. compared the production of ethyl hexanoate in SSF of Neurospora sp. on
pregelatinized rice, wheat bran ( fusuma), corn grits, and spent grain, supplemented
with different additives [79]. Cultivation on pregelatinized rice with 5% malt broth
resulted after 2 weeks in 180 mg kg 1 ethyl hexanoate, whereas cultures with
fusuma, corn grits, and spent grain only reached 10 mg kg 1 or less. This SSF of
pregelatinized rice with Neurospora sp. led to a koji used for the sake production and
resulted in a fruity perception of the sake. Ethyl hexanoate is also a substantial flavor
compound in traditional Chinese liquor, obtained by distilling SSF of grains
fermented with daqu, which is a starter culture consisting of a diverse microbiome,
including inter alia Lactobacillus, Bacillus, Aspergillus, and Saccharomycopsis.
Zhang et al. [80] analyzed volatiles appearing during the fermentation process of
daqu on a mixture of sorghum, corn, wheat, and rice. Cultivation was conducted at
30 C, and samples were harvested on days 1, 10, 23, 34, 48, 59, and 70. Aromatic
esters were extracted with ethanol and quantified by GC-FID. Ethyl hexanoate firstly
appeared on day 59 (93.3 μg g 1) and only slightly increased until day
70 (96.1 μg g 1). Such combinations of microorganisms can result in a diverse
pattern of VOC. A mixture of orange pulp molasses, potato pulp, whey, brewer’s
spent grain, and malt spent rootlets was fermented by a kefir community consisting
of symbiotic consortia of fungi and bacteria [81]. Volatiles in the HS of these
cultures were extracted with SPME and identified using GC-MS. In the HS of
kefir cultures, the highest total VOC concentrations have been determined with
ε-pinene being the most abundant aroma compound (4,208 mg kg 1).
2.3 VOC Produced During SSF of Basidiomycetes
Members of the phylum Basidiomycota, which most mushrooms belong to and

referred to as basidiomycetes, have been cultivated since centuries by humans for
the purpose of food production. The origin of mushroom production is in China,
with the first handwritten instruction for mushroom cultivation dating back to the
seventh century [82]. Generally, mushroom cultivation, which is probably the largest
industrial SSF sector, is performed on agroforestry wastes, like straw, sawdust, reed
grass, banana and bamboo leaves, tree bark and stems, husks, and scrubs [82–84].
98 A. Orban et al.
In addition, side streams of the food-producing industry, like wheat straw, citrus
peels, and cocoa shells, can be a valuable substrate source [85]. Worldwide, 950 dif-
ferent mushrooms are consumed and 50 different species are cultivated. China is the
biggest commercial producer with an annual production of 7 million tons in 2013,
which accounts for more than 70% of the worldwide production [86]. Several
articles already reviewed the variety of aroma compounds present in fruiting bodies
of fungi of the phylum Basidiomycota [87–89]. In the following, the focus is on the
aroma compounds produced by the vegetative mycelium. The influence of different
cultivation parameters, including substrate preparation, inoculation, and incubation
time on the production of different aroma compounds, is highlighted. Moreover, also
the genotype of the cultivated mushroom can have an impact on the production
process and yield [90]. In addition, recent results on VOC production during the
process of fructification are presented.
2.3.1 Volatiles Produced During Vegetative Growth
The fruiting bodies of the oyster mushroom Pleurotus ostreatus are known as a
delicate food with a pleasant aroma and derive from SSF on lignocellulosic residues.
When cultivated in liquid media or on an artificial solid substrate, the aroma
composition of P. ostreatus cultures alters [91]. Kabbaj et al. compared VOC derived
from P. ostreatus liquid cultures, agar plate cultures, and SSF on sugarcane
bagasse with its fruiting bodies produced on wheat straw [91]. VOC were extracted
with a DHS system equipped with a Tenax® trap. Afterward, volatiles were
thermally desorbed, cryofocused, and analyzed via GC-MS. The volatile profile of
the mycelium varied significantly between the different cultivation conditions. In
fruiting bodies, octan-3-one contributed to 80% of the integrated peak areas, followed
by octan-3-ol with about 14%. During SSF with sugarcane bagasse or on agar surface
cultures, mainly octan-3-one (72.5% and 67.4%, respectively) was detected, with
similar concentrations as in the fruiting bodies, whereas in liquid culture, it only
contributed with 36.2% to the detected volatiles. In liquid cultures, oct-1-en-3-ol
(38.5%) was the dominant compound but only found in small amounts in the SSF
with sugarcane bagasse (0.3%) and agar medium (1%). It is worth mentioning that the
approach with liquid medium contained relatively high quantities (16.2%) of
2-methylbutanol, resulting in a spicy and repellent note of the culture. This illustrates
the impact of the culture conditions on the volatile composition. In addition to the
culture conditions, the substrate itself has an effect on the VOC produced. When
P. ostreatus was cultivated on spent leaves of Eucalyptus cinerea derived from the
production of essential oils, the spectrum of VOC was influenced by the substrates
[92]. Cultivation of P. ostreatus on spent E. cinerea leaves was carried out in
propylene bags in darkness at 25 C for 30 days to completely colonize the substrate.
Afterward, the bags were removed, and the substrate block was further cultivated for
approximately 1 month at 20 2 C and 86–90% relative humidity as well as day and
night shifts to induce fructification. The volatiles of the substrate blocks after
colonization and after fructification were extracted by hydrodistillation in a
Clevenger-type apparatus, a distillation method used for the extraction of essential

oils. Analysis was performed with GC-FID and GC-MS. The amounts of various
volatiles including eugenol, globulol, α-cadinol, longifolene, and p-cymene
decreased during the fermentation of P. ostreatus on the spent leaves, whereas the
quantities of 1,8-cineole and β-caryophyllene increased significantly, and sabinene
hydrate appeared. The increase of the monoterpenes and β-caryophyllene might be
due to an active release of these compounds by P. ostreatus. 1,8-Cineole was
hydroxylated by the fungus to 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octan-6-ol and
further oxidized to 1,3,3-trimethyl-2-oxabicyclo[2.2.2]octan-6-one.
Wu et al. [93] analyzed the volatile biotransformation products of vine tea, a
product derived from the vine species Ampelopsis grossedentata, during SSF with
the medical mushroom Wolfiporia cocos (synonym: Poria cocos). The fungus was
cultivated on moistened tea leaves at 28 C for 15 days. Unfermented vine tea inhered
a strange taste, whereas fermented tea exhibited a pleasant aroma. Every third day,
cultures were harvested for LLE with water and dichloromethane. The extracts were
distilled for 5 h, subsequently concentrated, and analyzed by means of GC-MS.
During the fermentation remarkable amounts of methyl 2-methylpentanoate were
produced, resulting in the fruity note of the processed vine tea. Aroma compound
production of the medically valuable mushroom Antrodia camphorata was observed
during cultivation on millet for 25 days at 28 C [94]. On days 10, 15, 20, and
25 volatiles in the HS were analyzed by means of SPME (CAR/PDMS) and GC-MS.
In total, 124 compounds were detected not present in the unfermented millet. The
VOC profile varied remarkably during fermentation. In early stages, C8 compounds,
such as oct-1-en-3-ol, octan-3-one, and octan-1-ol, accounted for approximately
50% of all detected VOC. In later stages, octan-3-one (32–38%) was the most
abundant substance, followed by methyl 2-phenylacetate (13.1–16.6%). The relative
amount of oct-1-en-3-ol decreased rapidly over the time (down to 0.9%), whereas
the quantity of sesquiterpenes and lactones, including 5-butyloxolan-2-one,
5-heptyloxolan-2-one, and 6-heptyloxan-2-on, increasingly contributed to a fruit-
like flavor with herbal fresh notes.
As already shown, basidiomycetes are mainly cultivated on residues from
industrial or agricultural processes for human consumption. In general, the substrate
used consists of lignocellulose, which can be metabolized by all commercially
cultivated mushrooms using their diverse enzymatic system to a certain extent.
This so-called lignocellulolytic system can also generate some volatile side products
and thus might be an unexploited source for aroma compounds. The major class of
lignin degrading enzymes are class II fungal peroxidases, which require H2O2 as a
cofactor. The H2O2 is provided by different alcohol and glucose oxidases, such as
the aryl alcohol oxidases (AAO) [95]. As a substrate, AAO demand aryl alcohols,
such as benzyl alcohol, which are converted by the AAO into the almond flavor
benzaldehyde [96]. Bonnarme and coworkers had a closer look into this system
comparing cultivation conditions and their impact on VOC production and
enzymatic activity [97, 98]. They used the white rot fungus Bjerkandera adusta
for SSF and SmF for the production of benzaldehyde, benzyl alcohol, and benzoic
100 A. Orban et al.
acid [97, 98]. When SmF cultures of B. adusta were supplemented with
polyurethane foam cubes, the benzaldehyde concentration increased significantly
(8.3-fold) as did the AAO activity (4.3-fold). On the other hand, the maximum
benzyl alcohol concentration was higher in non-immobilized SmF cultures (1.5-
fold). This emphasizes the possible usage of lignocellulolytic enzymes for the aroma
biosynthesis. In a similar study, the effect of different solid supports was investigated
concerning the production of aryl metabolites. In comparison to the inert carrier
perlite, the utilization of lignocellulosic wheat bran for SSF of B. adusta resulted in a
remarkable production increase of benzyl alcohol and benzaldehyde (up to tenfold).
Similar to the SmF, the AAO activity was only detected in SSF on wheat bran and
not in cultures where perlite was used [97].
Although the substrate can have an influence on the aroma compounds produced
during SSF, the aroma profile of fruiting bodies derived from different substrates
remained unchanged for the delicious black poplar mushroom Agrocybe aegerita
[99]. A. aegerita was cultivated on 100% wheat straw, 100% cocoa shells, as well as
wheat straw supplemented with either cocoa shells (17%), citrus pellets (17%),
carrot mesh (17%), or black tea pomace (17 and 45%). Straw-based substrates
showed comparable growth of A. aegerita, whereas on 100% cocoa shells, only
marginal mycelium growth and no fruiting bodies were observed. Fruiting bodies
grown on 100% wheat straw and on wheat straw supplemented with black tea
pomace (45%) were compared in regard to their aroma profile. After addition of
methanol and water, homogenized fruiting bodies were extracted by means of LLE.
The concentrated extracts were analyzed via GC-MS/MS-O. Eleven VOC were
identified in the fruiting bodies, and the volatile composition of the two different
SSF cultivations did not exhibit considerable differences. C8 compounds, including
oct-1-en-3-ol, oct-1-en-3-one, and octan-3-one, contributed to the typical mushroom
odor, whereas 2-phenylethanol added a rose-like note to the aroma profile.
2.3.2 Volatiles Produced During Fructification
In basidiomycetes, sexual reproduction consists of a complex, multipolar mating-

type system, involving a haploid, monokaryotic stage [100]. Monokaryotic
mycelium of matching mating types can fuse, resulting in a dikaryotic stage. Out
of this stage, fruiting bodies derive to start a new reproduction cycle. Freihorst et al.
[101] compared the volatile profile during SSF of two sexual compatible
monokaryotic strains of Schizophyllum commune with the volatile composition of
a dikaryon obtained from mating both analyzed monokaryons. All three strains
were cultivated on solid complex medium for 7 days at 28 C. At the end of
cultivation, volatiles were extracted using HS-SPME (DVB/CAR/PDMS) for 1 h
and analyzed with a GC-MS system. Methyl 2-methylbutanoate was the dominant
VOC in both monokaryotic cultures (80%) and in the dikaryotic samples (31%).
Apart from that, the VOC profile of monokaryons and dikaryons differed tremen-
dously. The volatilome, the aggregate of all VOC, of the monokaryons contained
Fig. 6 Extraction of VOC with SPME during fructification of A. aegerita in SSF on malt extract
agar using modified crystallizing dishes; photos kindly provided by Sabrina Herold
methyl 2-methylpropanoate, not found among the volatiles of the dikaryon.

Vice versa, oct-1-en-3-ol, octan-3-one, S-methyl thioacetate, 3-methylbutan-1-ol,
isobutyl acetate, and β-bisabolol were only detected in the HS of the dikaryon.
Ethyl 2-methylbutanoate had a comparable ratio in the volatiles of all samples
(monokaryons 1–4%, dikaryon 4%).
All these results demonstrate that cultivation phases as well as the genetic
background of the analyzed specimens have a remarkable influence on the volatile
composition of basidiomycetes during SSF. In a recent work of our research
group (unpublished), we investigated the role of both factors on the volatilome of
A. aegerita in SSF. Therefore, strains were cultivated on 1.5% MEA in modified
crystallizing dishes (Fig. 6), allowing an efficient extraction of VOC by means of
SPME. This system had the advantage to ensure aeration of the culture, necessary for
the development of fruiting bodies. Furthermore, without harvesting the mushrooms,
it was possible to analyze the changes of volatile composition in the HS of the same
sample over the time, avoiding disturbance of the system and thus the formation of
VOC artefacts due to disruption of cell compartments. Four monokaryotic strains,
representing members of the “mycelium,” “initials,” “elongated,” and “fruiter”-type,
classified by Herzog et al. [102], and one dikaryotic strain were grown for 28 days.
Every second day, beginning with day 10 (agar plates were fully overgrown),
volatiles were extracted in the HS using SPME (DVB/CAR/PDMS) and analyzed
with GC-MS. The volatile profiles of all strains varied distinctively over the time. For
the dikaryon, raised VOC concentrations were detected. In the HS of the dikaryotic
102 A. Orban et al.
mycelium, mainly alcohols and ketones, like oct-1-en-3-ol, 2-methylbutan-1-ol,

acetone, and cyclopentanone, were identified. The composition of the VOC changed
significantly with the occurrence of fruiting bodies and during the sporulation phase.
Here, sesquiterpenes, especially Δ6-protoilludene, α-cubebene, and δ-cadinene, were
the dominant substances. After sporulation, the amount of sesquiterpenes decreased,
while additional VOC, mainly octan-3-one, appeared.
3 Conclusion
In addition to cultivation of mushrooms for food production, filamentous fungi are

promising candidates for SSF applications in the aroma sector. They are able to grow
on different substrates, which can result in a variable VOC profile, offering the still
not fully exploited potential of gaining a whole range of valuable flavors.
The diversity in these volatile profiles also depends on the developmental and
reproductive stage of filamentous fungi, which facilitates the process control of
SSF by observing the VOC production.
Acknowledgments We gratefully acknowledge the support by the Deutsche Forschungsgemeinschaft

RU 2137/1 and by the excellence initiative LOEWE within the project “AROMAplus” financed by the
Hessian Ministry of Science and Art.
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DOI: 10.1007/10_2019_93
Stimulating Production of Pigment-Type

Secondary Metabolites from Soft Rotting
Wood Decay Fungi (“Spalting” Fungi)
R. C. Van Court and Seri C. Robinson
Contents
1 Part I. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
1.1 Fungal Pigments: Useful Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110
1.2 Spalting Fungi . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
1.3 Soft Rotting Fungi and Their Pigments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
1.4 Inducing Pigment Production: Wood Species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 115
1.5 Inducing Pigment Production: Incubation Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
1.6 The Challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
2 Part II. Batch Culture: Amended Malt Agar Plates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 116
3 Part III. Batch Culture: Liquid Stationary Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 117
4 Part IV. Batch Culture: Liquid Shake Culture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
5 Part V. Applications and Future Developments of Spalting Technology . . . . . . . . . . . . . . . . . . 119
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 121
Abstract A small group of soft rotting wood decay fungi produce extracellular
pigments as secondary metabolites in response to stress and as a means of resource
capture. These fungi are collectively known as “spalting fungi” and have been used
in wood art for centuries. The pigments produced by these fungi are finding
increasing usage in industrial dye applications and green energy but remain prob-
lematic to grow in batch culture. Additionally problematic is that the pigments,
especially the blue-green pigment known as xylindein, produced by Chlorociboria
species, have yet to be fully synthesized. In order to further research development of
these pigments and find success in areas such as textile and paint dyeing, wood UV
R. C. Van Court and S. C. Robinson (*)

Wood Science & Engineering, Oregon State University, Corvallis, OR, USA
e-mail: [email protected]; [email protected]
110 R. C. Van Court and S. C. Robinson
protection, and organic photovoltaic cells, methods must be developed to mass

produce the pigments. To date, three distinct methods have been developed, with
varying degrees of success depending upon the fungal species (amended malt agar
plates, shake liquid culture, and stationary liquid culture). This chapter details these
three methods, their history, advantages and disadvantages, as well as their potential
for industrial scale-up in the future.
Graphical Abstract
Keywords Chlorociboria spp., Fungal dyes, Fungal pigments, Scytalidium

cuboideum, Scytalidium ganodermophthorum, Secondary metabolites, Spalting,
Zone lines
1 Part I. Background
1.1 Fungal Pigments: Useful Secondary Metabolites
Fungal species produce a range of pigments, with colors spanning the full spectrum
of the rainbow. These organic compounds include carotenoids, melanins, flavins,
phenazines, quinones, monascins, violacein, and indigo [1]. Production of pigments
is widespread across fungal species and lifestyles, ranging from a red produced by
insect-pathogenic Cordyceps variety [2] to blues from terrestrial Crocinoboletus spp.
[3, 4].
Fungal pigments (always referred to as “pigments,” even if they act more like
dyes) are considered secondary metabolites – low molecular weight compounds not
directly necessary for growth and development. Secondary metabolites are generally
Stimulating Production of Pigment-Type Secondary Metabolites from Soft. . . 111
understood to confer on the organism a competitive advantage, increasing their

adaption to ecological niches [5]. This has been demonstrated by the work of Rohlfs
et al. [6], who showed that loss of secondary metabolites by mutation leads to
decreased resistance to fungivory of Aspergillus nidulans (Eidam) G. Winter com-
pared to wild type. The activity of many of these fungal compounds has led to their
utility in industry, such as production of antibiotics, immunosuppressants, and
statins [7, 8].
While the specific roles performed by many pigments are still unknown, those
roles that have been identified are exceptionally diverse. Carotenoids, which are a
range of colors from yellow to red, have a protective role against oxidative stress and
UV and visible light, in addition to being used as intermediaries in compound
synthesis [9]. Other pigments are antagonistic. The red-colored cercosporin pro-
duced by Cercospora spp. is a particularly interesting example, playing a role in the
pathogenicity of the organism. A photosensitizer, when exposed to bright light,
generates active oxygen species leading to rapid damage of the membranes of plant
cells it infects, and its broad-spectrum toxicity has also been demonstrated against
fungi, mice, and bacteria [10]. Fungal pigments produced by Monascus ruber have
also been shown to have antimicrobial activity against bacteria, with different
pigments providing varying inhibitory effects [11].
The activity of fungal pigments has led to their use in a variety of industrial
applications, most prominently in the food industry and medical fields. In the food
industry, pigments have been used as food colorants. Monascus species in particular
have been traditionally used in Asia to produce red colors and are being further
refined for use as industrial food colorants [12–16]. In the medical field, fungal
pigments are under investigation for use as anticancer and antimicrobial agents,
antioxidants, and bioindicators [17].
These species used for pigment production on industrial scale are generally
saprobic fungi, deriving nutrients from dead organic matter. This has allowed for
straightforward pigment production using liquid cultures, which is industry standard.
However, there is a small group of wood decay fungi, known as soft rots, some of
which release very specific types of pigments into wood and are not easily grown in
liquid batch cultures. These naphthoquinone pigments have unusual UV stability,
hydrophobicity, and color stability, making them ideal not just for historic wood
coloration but also for textile dyes, paint colorants, decking protection, and organic
photovoltaics. The remaining hurdle for industrial use of these pigments is produc-
tion scale-up, as most have very specific growth requirements that must be met in
order to produce significant quantities of their pigments.
1.2 Spalting Fungi
Spalting is the colorization of wood by fungi, produced as a part of the normal life
cycle of certain species of decay fungi [18]. This can be due to either the breakdown
of colored compounds in the wood or through their production. There are three main
kinds of spalting: bleaching, zone line formation, and pigmentation.
Bleaching is caused by the breakdown of colored lignin from the wood cell wall,
generally by fungi classified as white-rotting, which results in a lightening of the
natural wood color [19, 20]. Heavy colonization by these fungi has a profound effect
on the structural integrity of the wood, making it softer and weaker. A lightening in
color can also be due to a buildup of white mycelium [21].
Zone line formation is characterized by production of darkly pigmented winding
lines in wood that are made up of melanized hyphae [22]. Zone lines are formed in
response to changes of moisture content in wood [23] and atmospheric conditions
such as presence of increased CO2 [24] and as a barrage reaction caused by
antagonism between dikaryons [25, 26]. Most fungi that produce zone lines are
basidiomycetes, though ascomycetes such as Xylaria polymorpha (Pers.) Grev. are
also known to do so [27]. Microscopic investigation of zone line formation across
selected species and wood types has shown two main forms of pigment deposition: a
melanin granule dense layer formed on the lumen of wood cell walls and a dense
packing of sclerotial hyphae fully obstructing the lumina [28, 29]. The sealing off of
lumina by fungi is considered a protective response to stress, be it from competition
or environmental change.
Pigmenting fungi are the final form of spalting fungi and produce internal
coloration of wood. Most pigmenting fungi are ascomycetes, from which basidio-
mycetes are considered to have evolved. The most common wood pigmentation is
“blue stain,” caused by exposure to airborne, soilborne, or beetle-borne fungi, and is
considered a defect in the lumber industry [30]. However, blue stain is not the only
form of pigmentation of wood; other vibrant colors such as red produced by
Scytalidium cuboideum (Sacc. & Ellis) Sigler & Kang, yellow produced by
Scytalidium ganodermophthorum Kang, Sigler, Lee & Yun, and blue-green pro-
duced by Chlorociboria spp. can also be seen.
Spalted wood has a history of use in fine wood artwork [18]. Chlorociboria spp.
blue-green stained wood has been used since the fifteenth century in intarsia, a form
of inlay, where the blue-green color was used for natural scenery to replicate the
color of leaves, for simulation of fabric, and to represent stone [31]. More recently,
zone line spalted wood was introduced to the woodturning community in the 1970s
by the Lindquist [32] and has become an exceptionally popular material to work
with. An example of a contemporary piece of wood turning showing both zone lines
and pigmentation can be seen in Fig. 1. The popularity and the rarity of pieces on the
market drive the high economic value placed upon spalted wood, making its
production not only an exercise in engendering natural beauty but also increasing
financial profit.
Fig. 1 Contemporary spalted wood art. Turned bowl with induced spalting by Dr. Seri Robinson,
with forms of spalting including blue, orange, pink, purple, and yellow pigmentation in addition to
white rot and zone lines. Image copyright northernspalting.com
1.3 Soft Rotting Fungi and Their Pigments
Pigmenting spalting fungal species are considered soft rotting ascomycetes,

producing a distinctive form of decay in wood. The name “soft rot” was applied
by Savory [33] to characterize a generally superficial form of decay associated with
wood discoloration and softening, in which cellulosic breakdown gives the wood a
cracked appearance when dried. Soft rot fungi are commonly found in conditions not
effectively colonized by more aggressive wood decay fungi, like basidiomycetes,
such as in exceptionally high moisture environments like water towers [34]. They
also show varying attack patterns, with carbohydrates degraded at different rates
depending on wood species [35].
Current research focuses on four species of pigmenting spalting fungi in the order
Helotiales for industrial applications. Two of these, Chlorociboria aeruginosa
(Oeder) Seaver ex C.S. Ramamurthi, Korf & L.R. Batra and Chlorociboria
aeruginascens (Nyl.) Kanouse ex Ram., Korf & Bat., are North American species
that both produce the blue-green pigment xylindein distinctive of the genus
[36, 37]. Scytalidium cuboideum (Sacc and Ellis) Singler and Kang produces the
red/pink pigment dramada in wood, which is a naphthoquinonic pigment (colloqui-
ally known as draconian red) and also a naturally occurring crystal [38]. Finally,
Scytalidium ganodermophthorum Kang, Sigler, Lee & Yun produces an as yet

undescribed yellow pigment. The extracted pigments have been physiologically
characterized by Vega Gutierrez and Robinson [39]. Scanning electron microscopy
(SEM) showed that the pigment from S. cuboideum created filament-like structures
that wrapped around substrates such as fibers to which it was applied, forming
flowerlike amalgamations. Pigment from C. aeruginosa was found to form a rela-
tively smooth amorphous film and S. ganodermophthorum a bumpy and uneven
surface.
Despite the structure of the pigment of S. ganodermophthorum being unknown,
its role in the ecology of the fungus is the best understood of all studied pigmenting
spalting fungi, indicated as playing a role in the pathogenicity of the fungus. This
species was identified as the causal agent behind yellow rot of reishi (Ganoderma
lucidum (Curtis) P. Karst), a popular cultivated medicinal, by Kang et al. [40] in
Korea. The authors theorized that the yellow pigment aided in pathogenicity,
commenting that culture isolates triggered lysis of G. lucidum cell membranes and
referencing a previous study by Oh et al. [41] where the pigment inhibited
G. lucidum growth. However, the fungus is also associated with wood decay and
produces pigment in wood like other species in the genus, fully completing its life
cycle without infection [40]. This suggests that while the pigment has utility as a
pathogenic agent, this likely developed from pigment already in production that may
confer an additional adaptive advantage.
The role of the pigment of S. cuboideum, while in the same genus, is less
understood. Scytalidium cuboideum has been isolated from a variety of pink stained
lumber and has even been found to grow on red oak (Quercus rubra L.) treated with
sodium pentachlorophenol [42]. When grown with other fungi, S. cuboideum
is often less competitive despite being a fast grower alone, occasionally producing
pink zone lines that are likely a competitive response analogous to melanized zone
lines. The pigment may also be UV and light protective [43–46]. Interestingly,
S. cuboideum has been isolated from clinical samples submitted to the University
of Texas Health Science Center, though did not produce pink pigmentation on PDA
as would be expected of the fungi [47]. This may suggest that the pigment confers a
particular competitive advantage in plant-based substrate.
The blue-green pigment xylindein produced by Chlorociboria sp. is conserved
throughout the genus, suggesting it confers a powerful ecological advantage for the
fungi though there has been no conclusive evidence as to what it may be. One line of
thought is that the pigment may have a powerfully protective effect against the
environment, due to its incredible stability. Blue-green pigmented wood from the
fifteenth century retains coloration today [31], and recent experimentation has found
xylindein to be exceptionally photostable and heat stable, with film properties
unaffected up to 180 C and degradation occurring at temperatures as high as
210 C [48]. As a coating and exposed to a weathering chamber, xylindein showed
potential to protect wood from degradation [49].
Xylindein may also protect the fungi from other competitors. A 1913 patent
reported in Robinson et al. [18] was filed for use of the pigment as an antimicrobial
agent, and a more recent 2007 patent was filed for use as plant germination inhibitor
[50]. These suggest bioactivity. Interestingly, this use was described as more effec-
tive after exposure to strong UV light, which turned the pigment purple.
It is hypothesized that the production of these pigments is a form of resource
capture by the fungi based on variations in fungal growth as exemplified by
Chlorociboria spp. Chlorociboria species are found on heavily decayed wood
though do not cause significant decay suggesting they are late-stage colonizers.
With other fungi already in the wood, use of pigment to demarcate territory, similar
to the more focused deposition of melanin in zone lines, may be an effective
competitive strategy. Production of xylindein as a competitive response is suggested
by the loss of pigment production in nutrient-rich media over time that has been seen
in multiple publications [51, 52].
Pigment production as a response to competition is also hinted at by variable
responses to growth on white-rotted and sound wood. Chlorociboria sp. growth on
wood pre-treated with a white-rotting fungus showed a possible, but nonsignifi-
cant, stimulatory growth effect for some species when compared to untreated wood
[53–55]. Spalted aspen also showed somewhat faster Chlorociboria growth com-
pared to unspalted aspen when added to malt agar plates, though with time this
effect was no longer seen [56–58]. The increased sugars available in decayed
wood likely influence the increase in growth rate; however it is also possible that
the evidence of other fungi in the wood may stimulate xylindein production.
If the pigment is produced as a competitive or stress response, it is speculated that
antagonizing the fungi results in more pigment production, as the stressed fungi
attempt to “control” their environment. Working with this theory, addition of spalted
wood chips to solid media, variations in moisture and growth substrate, and agitation
of liquid media have been explored as methods to increase pigment yield. Increase in
yield is critical because of the relative rarity and slow growth of these fungi,
especially Chlorociboria spp., which makes garnering enough pigment to study a
time-consuming and laborious process. In order for there to be a chance for industrial
adoption, identification of a growth method that stimulates industrial-scale pigment
production is necessary.
1.4 Inducing Pigment Production: Wood Species
Initial investigation of spalting conditions focused on identification of fungal inter-

actions and wood species that could produce superior pigmentation. Numerous
studies have found sugar maple (Acer saccharum Marsh.) to be an ideal substrate
for the production of zone lines [43–46, 53–60]. For pigmenting fungi, research by
Robinson and Laks (2010) found that a Chlorociboria sp. showed increased pig-
mentation and growth on trembling aspen (Populus tremuloides Michx.) compared
to other tested species. Scytalidium cuboideum has been found to effectively colo-
nize a range of species, and though a strong competitor against other spalting fungi,
in wood block cultures, inoculation onto sterile media was required [61–63].
1.5 Inducing Pigment Production: Incubation Conditions
Ideal conditions for stimulating pigment production from spalting fungi differ from
those required to stimulate fungal growth. Replacing vermiculate for soil in standard
growth chambers was shown to improve pigmentation for tested fungal species on
sugar maple [64], and that placement of the wood block above rather than below the
level of vermiculite increased pigmentation for some fungi [61–63]. Further research
has also shown that copper compounds such as copper sulfate can be used to produce
exclusion areas [53–55] and stimulate spalting [61–63].
Ideal moisture content has also been investigated, with results variable by species.
Zone line-forming species such as Trametes versicolor and Xylaria polymorpha
were found to have increasing pigmentation at low initial moisture contents
(29–33% in sugar maple and 29–32% in beech (Fagus grandifolia L.), though
other zone line species such as Polyporus brumalis (Pers.) Fr. showed increased
pigmentation at high moisture contents (59–96% in sugar maple and 26–41% in
beech) [65].
1.6 The Challenges
While spalting fungi can be reliably grown on wood for decorative purposes, it is
much more difficult to grow them in culture specifically to collect and extract their
pigment. Chlorociboria spp. have been especially challenging. In addition to being
extremely slow growers, this genus often ceases production of xylindein when in
laboratory storage [51, 52], and its complexity has made it difficult to synthesize.
Work on xylindein synthesis through dimerization of lactone produced a
7,9-dideoxy analogue, though did not progress further [66, 67]. Later work by
Donner et al. [68] took a different approach and described a methodology to produce
a pyranonaphthoquinone corresponding to half of the xylindein framework. The
difficulty of synthesis has led to research specifically focused on generating pigment
production (not fungal growth) in batch culture specifically for spalting fungi
and investigations into the unique environments required to stimulate pigment
production.
2 Part II. Batch Culture: Amended Malt Agar Plates
Malt agar plates amended with wood chips have been found to be the superior
growing media for pigmenting spalting fungi. Chlorociboria spp. have historically
been particularly difficult to grow in culture, with 2 percent malt agar plate colonies
failing to fully pigment or colonize plates even after 8 months of growth [53–55]. In
order to improve growth in plate cultures, wood chips of different species were
added to malt media. The addition of Acer saccharum yielded increased growth and
pigmentation across multiple Chlorociboria aeruginascens isolates [56–58]. This
result was supported by Tudor et al. [28, 29], who additionally found that cultures
from apothecia and stroma resulted in similar growth responses across a variety of
solid media.
Wood chip-amended plates have also been found to be effective growing media
for S. cuboideum and S. ganodermophthorum. These fungi are much faster growing
than the Chlorociboria cultures and can fairly quickly produce a large amount of
pigment. However, due to the unique color changes of their pigments, cultures must
be closely monitored and harvested when at the desired color. It has been seen in
multiple publications that the red pigment produced by Scytalidium cuboideum
becomes blue under certain conditions. This was noted when first isolated by
Chidester [69] and was in wood block cultures older than 8 weeks [61–63]. This
was partially explained by Golinski et al. [70], who showed that when the pigment
was dissolved in basic or high polarity solvents, it turned blue. Research into the
effects of the pH of media found that the highest intensity blue coloration was seen on
wood samples with a pH of 8 and a superior red color at a pH of 6 [71]. Scytalidium
ganodermophthorum also demonstrates changes in pigmentation, with plates starting
a bright yellow color after inoculation and then becoming a deep red-brown color
with age. When extracted, the color of the pigment in solution can range from yellow
to olive green to deep red to purple. Investigations into exactly why this occurs are
ongoing, though the effect is likely polarity or pH driven as for S. cuboideum and may
also be related to concentration.
The use of wood chip-amended plates has also allowed for a new method of
extraction. After complete pigmentation of plates by fungal growth, the media can be
dried and blended to a powder from which pigments can be extracted using a
selected solvent [43–46]. The blending followed by extraction method cannot be
used on traditional malt agar plates, as the media will partially dissolve along with
the pigment. DCM has been identified as the most effective solvent fungal pigments,
giving a fast extraction from growth media and good solubilization of dried extracted
pigment [43–46].
3 Part III. Batch Culture: Liquid Stationary Culture
Spalting pigmenting fungi under investigation have been found to grow effectively
in liquid 2 percent malt media, although exhibit varying growth characteristics as
described by [72]. Chlorociboria spp. were found to be slow growing, with myce-
lium forming discreet fluffy clumps and the minimal xylindein production aggre-
gating and adhering to the glass of culture flasks. Recent investigations have since
not seen any evidence of extracellular pigment production by Chlorociboria sp. in
liquid cultures. However, it was noted other strains of Chlorociboria have been
observed to grow as mycelial mats near the top of liquid culture media. Both
S. cuboideum and S. ganodermophthorum form mycelial mats when stationary, with

pigments not adhering to the glass but staying in solution.
Extraction of pigment from liquid cultures consists of blending hyphae and media
with a small electric blender, combining this with DCM, shaking, and using a
separatory funnel to collect the pigmented DCM layer. Blending of solution before
extraction yielded visibly more pigment compared to just extracting the water-
solubilized pigment [72]. Blending is particularly important to extract pigment
from Chlorociboria spp. and to some extent for S. ganodermophthorum which had
increased hyphal pigmentation when compared to S. cuboideum.
Static cultures seem to allow for increased growth for Chlorociboria spp. over
shake cultures, making them preferred for xylindein production. Blending of mature
Chlorociboria cultures produces such concentration of pigment that the dark green
color appears almost black. Further investigations into variations of liquid medias
used for Chlorociboria growth are ongoing.
4 Part IV. Batch Culture: Liquid Shake Culture
Shaking has been used as a method to stimulate pigment development, working under
the assumption that this stresses the fungi and invokes pigment production. Shake
culture was identified as the most effective form of liquid media growth by [72], citing
faster media pigmentation when compared to static cultures. Chlorociboria spp. in
shake culture at 110 rpm were shown to grow in clumps similar to static cultures,
though there was less adherence of xylindein to the culture glassware and hyphal mats
took longer to form. Scytalidium cuboideum and S. ganodermophthorum in the same
conditions grew dispersed throughout the volume of the flasks and showed good
production of pigment. Scytalidium cuboideum showed fast pigmentation of media in
2 to 3 days, with an initial pink-red coloration that became deep red with time.
Interestingly, pigmentation declined after day 3, not increasing again until between
16 and 48 days. Scytalidium ganodermophthorum cultures showed pigmentation of
media in 2 to 3 days like S. cuboideum, with coloration quickly increasing until day
8 and then slowing. The color of the culture changed with time, starting at light yellow
and moving to a yellow/red/brown coloration with increased pigment concentration
similar to that seen in solid media.
These results suggest that shake culture is the most effective liquid media
technique for the tested Scytalidiums, especially S. cuboideum, as extracellular
pigment is produced and can be harvested directly. This allows for relative ease of
maintaining culture and the possibility of continuous fermentation, though shake
cultures do seem more prone to contamination and sterility of glassware is critical.
Chlorociboria spp. in liquid culture do pigment the media with time; however this
seems to be a result of hyphae breaking causing pigment release instead of pigment
secretion. When liquid cultures are filtered, collected hyphae are highly pigmented
and liquid only faintly so.
5 Part V. Applications and Future Developments

of Spalting Technology
Spalted wood, especially zone line spalted wood, is a highly priced wood product.
Spalting underutilized or low-value woods therefore increases their market value and
generates profit for the producer. Application of fungal pigments has been found to
mitigate the visual effects of blue stain in pine, normally a product associated with
economic loss [73], and effective coloration of bamboo has been demonstrated
[74]. On a commercial scale, large non-sterile logs have also been found to be an
effective spalting substrate, producing good zone lines and stain [75].
Direct use of pigment carried in DCM as a colorant has been compared to fungal-
induced spalting, and for most of the 16 species of wood tested, the use of pigments
from C. aeruginosa, S. cuboideum, and S. ganodermophthorum carried in DCM
yielded higher pigmentation than direct inoculation [43–46]. These extracted pigments
in DCM and liquid media have also been favorably compared to aniline dyes as wood
colorants [43–46].
DCM is the most effective solvent for solubilization of the pigments, due to their
limited solubility, but is a harsh chemical which makes applications more difficult.
This limited solubility complicates methods of application, in addition to making
characterization of the pigments and investigations into their purity more difficult.
Because of this solvents and carriers beyond DCM have also been investigated.
Acetonitrile worked moderately well at solubilizing pigments from C. aeruginosa
and S. cuboideum [76], and natural oils, like linseed oil, have also proven effective
[77]. Investigations into making artist paints using linseed oil are also ongoing, but
results indicate that the pigments react to the oil over time and decolor [78]. The
extracted red pigment when used as a stain on wood has also been found to be stable
in response to natural and artificial UV lights [56–58] and is under investigation as a
coating for decking.
The use of extracted fungal pigment has opened up a variety of applications
beyond colorizing wood, and one of the most promising is the dying of textiles. The
current textile dying industry is associated with significant environmental impacts,
and interest is growing in more sustainable and less toxic natural pigments. Extracted
pigments from Scytalidium cuboideum, Scytalidium ganodermophthorum, and
Chlorociboria aeruginosa have been found to have strong potential as fabric dyes
[79], with xylindein and dramada showing good colorfastness without mordants
[80, 81] and in some cases outperforming commercial dyes [82].
In addition to use as coloring agents, one of the most exciting potential applica-
tions of spalting fungal pigments is their use in organic photovoltaic cells and other
(opto)electronics. Photovoltaic technology based on organic semiconductors has
great potential as a low-cost, highly flexible, and renewable technology, though at
present has low efficiency [83] making development of new materials important.
While pigments from Scytalidium cuboideum and Scytalidium ganodermophthorum
were found to have poor semiconductive capability, the blue-green pigment from
Chlorociboria has been found to have promising optoelectronic properties and
Fig. 2 Variety of colors produced by increasing concentrations of red pigment from S. cuboideum
in acetone, ranging from 0.024 mM (left) to 19 mM (right). Image copyright R. C. Van Court
impressive stability [48, 84] and is under development as a component in organic

photovoltaics.
In addition to these applications, recent development of a methodology to produce
a solid form of the pigments should allow for new developments and further refine-
ment of existing technologies. When the DCM of pigmented solutions is evaporated
off, the pigments bind to the glass of their containers instead of forming a collectable
particulate. This has prevented further commercialization, as the amount of pigment
required to produce color change could not be measured. However, recently a method
to crystalize the red pigment from S. cuboideum has been developed [38] which has
allowed for an association to be developed between the molarity of the solvent and its
color [85]. Very small amounts of pigment produced a variety of colors ranging from
deep red to bright orange to light pink, suggesting final coloration of products can be
fine-tuned and economically favorable (Fig. 2). The production of a solid, pure form
of one spalting pigment suggests that this will also be possible for the other pigments,
allowing for easier product development and study.
The ability to relate concentration to produced color will also allow for more
specific studies on the toxicity of the pigments. As none of the pigments are water-
soluble (with very slight solubility by S. cuboideum being the exception), when
applied to wood or other substrates, they are not likely to break down and cause
environmental concerns even if somewhat toxic. Toxicology studies may also
further inform about the activity of the pigments and their ecological roles.
The final and most pressing challenge facing spalting fungi is overcoming their
limited speed of fungal growth. Despite research into liquid fermentation, thus far
solid-state fermentation methods of production are preferred for ease and efficiency,
mirroring other studies that have found pigment production to be the most efficient
in solid state [86, 87]. Future research into media variations and diving into the world
of genetic analysis and manipulation will increase production of these pigments and
drive future adoption of new technologies.
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DOI: 10.1007/10_2019_88
Fermented Solids and Their Application

in the Production of Organic Compounds
of Biotechnological Interest
Nadia Krieger, Glauco Silva Dias, Robson Carlos Alnoch,

and David Alexander Mitchell
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
2 Use of Dry Fermented Solids in Biodiesel Synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
2.1 Fermented Solids as a Cheaper Source of Lipases for Enzymatic Biodiesel . . . . . . . . 127
2.2 Proof of Concept: Production of Biodiesel Esters Using Dry Fermented Solids . . . . 131
2.3 Production of Biodiesel by Transesterification Using Bacterial Fermented Solids . . 131
2.4 Production of Biodiesel Esters by Esterification Using Bacterial Fermented Solids 133
2.5 Production of Biodiesel Using Rhizopus microsporus Fermented Solids . . . . . . . . . . . 137
2.6 Production of Biodiesel Using Rhizomucor miehei Fermented Solids . . . . . . . . . . . . . . . 138
3 Miscellaneous Applications of Dry Fermented Solids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 139
3.1 Use of Fermented Solids in the Resolution of Racemic Mixtures . . . . . . . . . . . . . . . . . . . 139
3.2 Use of Fermented Solids in Interesterification Reaction to Produce Modified Fats . 141
3.3 Use of Fermented Solids for the Production of High-Value Sugar Esters . . . . . . . . . . . 141
3.4 Use of Fermented Solids in the Production of D-Galacturonic Acid . . . . . . . . . . . . . . . . . 142
4 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 142
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
Abstract We review the application of dry fermented solids (DFS) containing

naturally immobilized enzymes as catalysts in synthesis and in hydrolysis
reactions. The most studied application is the use of DFS containing lipases in
the synthesis of biodiesel esters, by transesterification of oils or by esterification
of fatty acids with short-chain alcohols in solvent-free reaction media. Other
applications of DFS that have been studied include the use of DFS containing
N. Krieger (*)
Departamento de Química, Universidade Federal do Paraná, Curitiba, Paraná, Brazil
G. S. Dias, R. C. Alnoch, and D. A. Mitchell
Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Paraná, Curitiba,
Paraná, Brazil
126 N. Krieger et al.
pectinases to liberate D-galacturonic acid from pectin and the production of high-
value compounds by DFS containing lipases, such as the synthesis of sugar esters
and the production of pure enantiomers by resolution of racemic mixtures. To date,
studies are limited to proof of concept, and there are still many challenges to be
faced in the development of industrial-scale processes using DFS as catalysts.
A key challenge is the relatively low activity of DFS compared to commercial
enzyme preparations. Attention needs to be given to scale up, not only of the
bioreactor for the application of the DFS but also for the production of the
fermented solids. There is also a need for economic feasibility studies to determine
whether the production of DFS and their use as catalysts can be competitive at
industrial scale.
Graphical Abstract
Products
Dried fermented
solids
Substrates
Solid-state Biocatalytic
fermentation application
Keywords Biodiesel, Dry fermented solid, Esterification, Solid-state fermentation,

Transesterification
1 Introduction
At the time of harvesting of a solid-state fermentation (SSF) process, the solid

medium contains residual substrate, microbial biomass, and microbial metabolites.
This medium has been referred to as “fermented solids.” The chemical composition
and the physical characteristics of these fermented solids can be quite different from
those of the original substrate. This is not only due to the presence of the microbial
biomass but also due to the degradation of substrate components during the growth
of the microorganism.
Among the metabolites produced in SSF, microbial enzymes are of key interest.
Many processes for the production of enzyme preparations using SSF involve
several steps, including the fermentation process in which the enzymes are produced
by the microorganism and subsequent processing steps in which the enzymes are
Fermented Solids and Their Application in the Production of Organic. . . 127
extracted and then purified, if a purified enzyme is necessary. In the final application
of the enzymes, they are often immobilized. The advantage of using enzymes in
immobilized form is that, in batch reactors, they can easily be removed from the
reaction medium and reused in a subsequent batch; in continuous bioreactors, they
can be confined within the bioreactor, allowing catalysis of the reaction over long
periods. Since an immobilized enzyme can catalyze the conversion of a large amount
of substrate, the contribution of the enzyme to the overall process costs is potentially
lower than if a free enzyme were used: a free enzyme would be limited to catalyzing
the reaction in a single batch, since it is not feasible to recover it after the reaction.
However, the potential cost savings gained by using immobilized enzymes must be
balanced against the costs of the immobilization procedure.
Faced with the costs of the classical route for producing immobilized enzymes for
biocatalytic processes, we had the idea of producing the enzyme by solid-state
fermentation, but not extracting it. Rather, the fermented solids would be dried
and would act as a natural immobilization support for the enzyme. It should be
noted that this is only feasible in those applications in which it is possible to use
crude enzyme preparations, as all enzymes produced by the microorganism are
present. Also, for this idea to be practical, the enzymatic activity contained within
the DFS must be sufficiently stable over repeated batches and typically should not
leach out into the reaction medium.
In our initial studies, we used lipases produced on corn bran to catalyze esterifi-
cation and transesterification reactions in organic media [1]. While these studies
were in progress, the paper of Nagy et al. [2] appeared; these authors used fermented
solids containing lipases to catalyze the resolution of racemic mixtures of secondary
alcohols. In this review, we describe the applications that have been developed since
then, by our group and by other groups, involving the use of dry fermented solids as
biocatalysts in processes for the production of organic compounds. We will start
with the application of dry fermented solids in biodiesel synthesis, given that this is
the most studied application.
In this review, we will refer to “dry fermented solids containing enzymes” simply
as “fermented solids” and use the acronym DFS. Also, enzyme activities of the DFS
are presented in U g1 (units per gram of DFS).
2 Use of Dry Fermented Solids in Biodiesel Synthesis

2.1 Fermented Solids as a Cheaper Source of Lipases
for Enzymatic Biodiesel
Biodiesel is a mixture of esters that is normally produced by the transesterification of

vegetable oils with short-chain alcohols. The most commonly used alcohol is
methanol, due to its high reactivity, although ethanol is less toxic and can be
produced from renewable resources by fermentation (Scheme 1). The chemical
Scheme 1 Transesterification of a triacylglycerol with an alcohol, producing glycerol and

biodiesel esters
route for biodiesel synthesis uses a strong base as catalyst, such as KOH or NaOH. It
gives a high productivity, with the conversion being complete in less than 1 h.
However, the chemical route has several drawbacks. First, it is necessary to use
relatively high-quality vegetable oils, with free fatty acid contents below 3%;
otherwise the free fatty acids form soaps with the catalyst, with these soaps making
purification of the biodiesel more difficult and leading to a more impure glycerol
by-product, decreasing its value. Second, even with high-quality feedstocks, it is
necessary to remove the catalyst after the process, and this requires relatively large
volumes of wash water [3, 4].
The enzymatic route using lipases (EC 3.1.1.3) is a more environmentally
friendly alternative for biodiesel synthesis. The biodiesel and the glycerol are
produced in purer forms, and the generation of waste wash water is avoided
[5]. However, the enzymatic process also has significant disadvantages in relation
to the chemical process: it has a lower productivity, and the costs of the enzyme are
quite high. It is, therefore, essential to decrease the costs of the enzyme. Enzyme
costs can be decreased by finding new enzymes that have high activity and stability
in organic media, allowing their reuse over repeated cycles. In order to be reused, the
enzymes must be immobilized. As mentioned above, traditional processes for
immobilizing enzymes involve the steps of extraction, concentration, and immobi-
lization. The use of fermented solids potentially allows a cheaper way of producing
lipases for use as biocatalysts for biodiesel synthesis.
Motivated by the potential to reduce costs of biodiesel production, our group and
other groups have studied the use of DFS containing lipases for the production of
biodiesel in transesterification and esterification reactions (Table 1) [1, 6–20].
Although studies were initially undertaken in the presence of cosolvents, such as
n-hexane or n-heptane, more recent efforts have focused on the use of solvent-free
systems, in which the initial reaction medium contains only the substrates (short-
chain alcohols and either oils or fatty acid mixtures). Solvent-free systems have the
potential to reduce costs further by allowing higher volumetric productivities to be
obtained and by eliminating the need to separate solvents from the final reaction
mixture and recycle them. The major challenge with solvent-free systems is to
overcome the deleterious effect of short-chain alcohols on the enzyme. In solvent-
free media, these alcohols can strip the water molecules from the aqueous shell
around the lipase, leading to denaturation. Additionally, the high alcohol concentra-
tions can lead to a significant degree of competitive inhibition of the lipase. Further
details are discussed in the following subsections.
Table 1 Studies of the direct application of dry fermented solids for biodiesel production
Substrate used to produce Alcohol and acid/oil Conversion
Type of reaction the dry fermented solid Microorganism (molar ratio) Solvent Bioreactor in time taken Reference
Esterification Corn bran and corn oil Burkholderia Ethanol and oleic acid (5:1) n-heptane Shake 94% in 18 h [1]
contaminans flasks
Perlite and nutrient Rhizopus sp. Ethanol and oleic acid (5:1) n-hexane 10-mL 98% in 1 h [6]
solution stirred
reactor
Sugarcane bagasse and Burkholderia Ethanol and fatty acids from Solvent- Packed 92% in 31 h [7]
sunflower seed meal contaminans soybean soapstock acid oil (3:1) free bed
Babassu cake Rhizomucor Ethanol and Acrocomia Solvent- Shake 91% in 8 h [8]
miehei aculeata acid oil (1:1) free flasks
Wheat bran, sugarcane Rhizopus Ethanol and oleic acid (1.5:1) Solvent- Shake 69% in 48 h [9]
bagasse, and urea microsporus free flasks
Sugarcane bagasse and Burkholderia Ethanol and olein (1.5:1) Solvent- Packed 88% in 24 h [10]
sunflower seed meal contaminans free bed
Babassu cake Rhizomucor Ethanol and palm and soybean Solvent- Shake 67–74% in [11]
miehei fatty acid distillates (1:1) free flasks 6h
Babassu cake Rhizomucor Ethanol and Acrocomia Solvent- Shake 85% in 96 h [12]
miehei aculeata acid oil (6:1) free flasks
Sugarcane bagasse and Rhizopus Ethanol and oleic acid (10:1) Solvent- Shake 98% in 48 h [13]
nutrient solution microsporus free flasks
Fermented Solids and Their Application in the Production of Organic. . .
Cottonseed meal Rhizomucor Ethanol or methanol and oleic Solvent- Shake 85% in 4 h [14]
miehei acid (2:1) free flasks
Transesterification Corn bran and corn oil Burkholderia Ethanol and corn oil (5:1) n-heptane Shake 95% in [1]
contaminans flasks 120 h
Sugarcane bagasse and Burkholderia Ethanol and soybean oil (3:1) Solvent- Packed 95% in 46 h [15]
sunflower seed meal contaminans free bed
Sugarcane bagasse and Burkholderia Ethanol and soybean oil (4:1) tert- Shake 86% in 96 h [16]
sunflower seed meal cenocepacia butanol flasks
129
(continued)
Table 1 (continued)
130
Substrate used to produce Alcohol and acid/oil Conversion

Type of reaction the dry fermented solid Microorganism (molar ratio) Solvent Bioreactor in time taken Reference
Sugarcane bagasse and Burkholderia Ethanol and soybean oil (4.3:1) tert- Shake 91% in 96 h [17]
sunflower seed meal cenocepacia butanol flasks
Sugarcane bagasse and Rhizopus Ethanol and corn oil (3:1) Solvent- Shake 68% in 72 h [18]
nutrient solution microsporus free flasks
Babassu cake Rhizomucor Butanol and Acrocomia Solvent- Shake 80% in 70 h [19]
miehei aculeata oil (5.47:1) free flasks
Sugarcane bagasse and Burkholderia Ethanol and palm oil (5:1) Solvent- Packed 89% in 30 h [20]
soybean oil contaminans free bed
Cottonseed meal Rhizomucor Ethanol or methanol and Solvent- Shake 76% in 24 h [14]
miehei Acrocomia aculeata oil (2:1) free flasks
N. Krieger et al.
2.2 Proof of Concept: Production of Biodiesel Esters Using

Dry Fermented Solids
In the early studies of our group about the use of fermented solids, we used a
bacterial strain (LTEB11) that was originally identified as Burkholderia cepacia
but which was recently reclassified as B. contaminans, based on genomic analysis
(unpublished data). Hereafter, it will be referred to as B. contaminans LTEB11, even
in those cases in which the original work referred to it as B. cepacia LTEB11. This
strain produces a lipase when cultivated in submerged fermentation. After immobi-
lization on Accurel (a commercial preparation of microscopic polypropylene beads
which are hydrophobic and have a high specific surface area), this lipase is quite
active and stable in organic media, catalyzing ester synthesis with yields comparable
to those that have been reported using commercial enzymes, such as the lipase from
Pseudomonas cepacia from Amano [21, 22].
In 2007, we published a proof of concept, showing that the DFS produced by
cultivating B. contaminans LTEB11 on a mixture of corn bran and corn oil (5% v/m)
could be used to catalyze the synthesis of biodiesel esters by esterification or
transesterification [1]. The reactions were done in a reaction medium containing n-
heptane as a cosolvent, with the intention of minimizing denaturation of the lipase.
This work made two key contributions. First, we described the use of a bacterium
that produces relatively high amounts of lipase in SSF (108 U of pNPP-hydrolyzing
activity per gram of dry solids after 72 h). At that time, most papers dealing with the
production of lipases by SSF had involved filamentous fungi. Second, we demon-
strated that it was possible to use lyophilized fermented solids containing lipases to
catalyze ester synthesis reactions.
Soon afterward, the paper of Martínez-Ruiz et al. [6] appeared. These authors
cultivated Rhizopus sp. on perlite impregnated with a nutrient solution containing
olive oil, lactose, and urea, as major components, and polyvinyl alcohol and various
mineral salts, as minor components. After the fermentation, the solids were either
lyophilized or air dried, with both methods giving DFS with similar olive-oil-
hydrolyzing activities. These DFS were used to catalyze the esterification of oleic
acid with ethanol using n-hexane as a solvent. With an ethanol to oleic acid molar
ratio of 5:1 (250 mmol L1 ethanol and 50 mmol L1 oleic acid), a conversion of
98% was obtained after 60 min at 45 C.
2.3 Production of Biodiesel by Transesterification Using

Bacterial Fermented Solids
Fernandes et al. [1] used the DFS produced by B. contaminans LTEB11 to catalyze
ester synthesis by transesterification of corn oil with ethanol, in a medium containing
n-heptane as a cosolvent. The reactions were done with 15 mL of reaction medium
initially containing 70 mmol L1 of corn oil. The best conversion after 120 h of
reaction at 37 C was 94.7%, obtained with an ethanol to oil molar ratio of 4.5:1 and
the addition of DFS containing 43.9 U of pNPP-hydrolyzing activity. Since the corn
oil concentration in n-heptane was relatively low, it limited productivity of the
system. Salum et al. [15] therefore investigated the possibility of carrying out the
transesterification in a solvent-free medium.
The transesterification reactor of Salum et al. [15] was a column packed with 3 g
of DFS that was produced by cultivating B. contaminans LTEB11 on a mixture of
sugarcane bagasse and sunflower seed meal. This substrate mixture gave a pNPP-
hydrolyzing activity of 234 U g1 after 96 h of cultivation, this being more than
twice the activity of the DFS used by Fernandes et al. [1]. The column reactor was
operated in batch mode, with closed-loop recirculation of a solvent-free reaction
medium initially consisting of ethanol and soybean oil. A conversion of 95% after
46 h of reaction was obtained, at 50 C, using a molar ratio of ethanol to oil of 3:1 (the
ethanol was added in two steps, at 0 and 7 h), with the addition of 1% (m/m) of water
to the reaction medium. This stepwise addition of ethanol helps to avoid high
ethanol concentrations in solvent-free media, thereby minimizing inhibition and
denaturation [23, 24].
The conversion of 95% in 46 h obtained by Salum et al. [15] corresponds to an
ester productivity of 152 mg g1 h1 (mass of esters produced per mass of DFS used
per hour). This was a significant improvement over the process of Fernandes et al.
[1], with the same conversion being achieved in less than half the time and with a
more concentrated medium (i.e., a solvent-free medium). When Salum et al. [15]
reused the DFS in successive 47-h transesterification cycles, conversions of over
95% were maintained for three cycles, but in the seventh cycle, the conversion was
less than 60%.
Other groups have used DFS as biocatalysts to produce biodiesel by trans-
esterification, but they have not obtained results as good as those of Salum et al.
[15]. Liu et al. [16] produced DFS by cultivating Burkholderia cenocepacia on a
mixture of sugarcane bagasse and sunflower cake (5:3 m/m). Their DFS had an
olive-oil-hydrolyzing activity of 72 U g1. They used their DFS to catalyze the
transesterification of soybean oil with ethanol in a reaction medium containing tert-
butanol (at a level of 40% (v/v) in relation to the oil content) (Table 1). Their best
conversion was 86% after 96 h. They reused the DFS in three 96-h reaction cycles
(giving a total of 288 h), obtaining a conversion of 67% in the last reaction cycle.
They later optimized this reaction using a Box-Behnken factorial design. The best
result was a conversion of 91% after 96 h, achieved at 44 C using a molar ratio of
ethanol to oil of 3:1, a DFS concentration of 1.63 g mL1, with the addition of 4.6%
(m/m) water and 20% (v/v) tert-butanol [17]. This result corresponds to an ester
productivity of 87 mg g1 h1.
Recently, our group investigated the transesterification of palm oil with ethanol
in a solvent-free medium [20]. The production of the fermented solid by
B. contaminans LTEB11 was optimized, giving 160 U g1 of triolein-hydrolyzing
activity. In shake flasks, a conversion of 90% in 48 h was obtained, using a molar
ratio of ethanol to palm oil of 4.5:1 and 8% (m/m) of DFS in relation to the palm oil,
with the ethanol being added in three steps in equal aliquots. In a packed-bed
bioreactor containing 12 g of DFS (12%, m/m, in relation to the palm oil mass), with
recirculation of the reaction medium, we obtained a conversion of 89% in 30 h, this
being achieved using a molar ratio of ethanol to palm oil of 5.5:1, with the addition
of ethanol in four steps. The DFS was reused in successive 30-h cycles, with
washing of the DFS between the cycles with n-hexane and tert-butanol. A conver-
sion of 66% was obtained in the fifth cycle.
2.4 Production of Biodiesel Esters by Esterification Using

Bacterial Fermented Solids
Most studies of the production of biodiesel by the enzymatic route have involved
transesterification of oils. However, recently, there has been interest in lipase-
catalyzed esterification of fatty acids, within the context of the so-called
hydroesterification processes [7, 8, 25–28] (Scheme 2). The hydroesterification
route for biodiesel production includes two steps. In the first step, triacylglycerols
are hydrolyzed, either enzymatically or chemically, with subsequent recovery of
fatty acids by distillation. In the second step, the fatty acids are esterified with a
short-chain alcohol; likewise, this step can be catalyzed either chemically or enzy-
matically. Hydroesterification has several potential advantages over the traditional
transesterification route for biodiesel production. First, lower-quality raw materials
can be used, containing significant amounts of free fatty acids or water. These raw
materials are significantly cheaper than the high purity vegetable oils typically used
in transesterification processes [26, 27]. Second, glycerol is removed after the first
step of the hydroesterification process, thereby avoiding the sorption of glycerol onto
Scheme 2 Production of biodiesel esters in a hydroesterification process. In the first step, a

triacylglycerol is hydrolyzed to produce glycerol and fatty acids. In the second step, the fatty
acids are esterified with an alcohol
solid catalysts, which can lead to decreased reaction rates due to mass transfer
limitations [29, 30].
In our initial esterification studies with fermented solids, Fernandes et al. [1] used
the DFS produced by B. contaminans LTEB11 to catalyze the esterification of oleic
acid with ethanol in 5 mL of reaction medium containing 70 mmol L1 of oleic acid
dissolved in n-heptane as a cosolvent. After optimization, a conversion of 94% was
obtained in 18 h at 37 C, using a molar ratio of ethanol to acid of 5:1 and with the
addition of DFS containing 60 U of pNPP-hydrolyzing activity (corresponding to
0.55 g of DFS, which represented 2.8% m/m in relation to the oleic acid). Since the
oleic acid concentration was relatively low, although high conversions were obtained,
the productivity of this system was relatively low, only 22 mg g1 h1. Subsequent
esterification studies were done using solvent-free media.
Later, Soares et al. [7] used DFS of B. contaminans LTEB11, produced as
described by Salum et al. [15], to catalyze the esterification of fatty acids with
ethanol in a solvent-free medium. The fatty acids had been obtained through the
hydrolysis of soybean soapstock acid oil in subcritical water. The reaction medium
was circulated, in a closed-loop batch system, from a reservoir through a packed-bed
bioreactor containing 12 g of DFS. The best conversion was 92% in 31 h, obtained at
50 C.
The work of Soares et al. [7] brought several advantages over the results for
transesterification of soybean oil reported by Salum et al. [15]. First, a lower-grade
starting material was used, namely, soybean soapstock acid oil. Second, the esteri-
fication process was 15 h shorter than the 46 h required for the transesterification
process. Third, the DFS were obtained through a simple air drying, whereas Salum
et al. [15] had lyophilized the fermented solids.
Soares et al. [7] reused the DFS in 48-h esterification cycles, maintaining
conversions of over 80% during seven cycles. This performance is better than that
which Salum et al. [15] obtained for repeated 47-h transesterification cycles with the
same DFS, with less than 60% conversion in the seventh cycle.
Two important phenomena occurred in the studies of Soares et al. [7]: First,
although the reaction medium was originally monophasic, a second liquid phase
formed during the reaction, presumably due to the production of water by the
esterification reaction and, second, part of the reaction medium remained sorbed
by the fermented solid when the bioreactor was drained at the end of the experiment.
These phenomena were then studied in greater depth by Soares et al. [31], who
determined the compositions of the aqueous and organic phases of the bulk reaction
medium and also of the phase sorbed onto the DFS, this being done for three
experiments with different initial overall molar ratios of ethanol to fatty acid, 1:1,
1.5:1, and 3:1, designated as MR1:1, MR1.5:1, and MR3:1, respectively.
In the bulk reaction medium, the time required for the aqueous phase to appear
increased as the initial molar ratio of ethanol to fatty acid increased. This is not
surprising, as a higher concentration of ethanol in the initial organic reaction medium
increases the miscibility of water in this medium. Once it formed, this aqueous phase
contained almost no fatty acids (for which the mole fraction remained below
0.035%) and esters (for which the mole fraction remained below 1.2%). Although
this is the first time that the formation of an aqueous phase was observed for a system
involving DFS, this phenomenon has been previously observed in other solvent-free
systems in which immobilized lipases have been used to catalyze esterification of
fatty acids [32–35].
An even more interesting result was that the phase sorbed onto the DFS
represented up to 30% of the overall reaction medium and was predominantly
polar: the water contents of the sorbed phase for MR1:1 and MR1.5:1 remained
above 50%, while the ethanol content of the sorbed phase for MR3:1 remained
above 65% [31]. On the other hand, at all times in all three experiments, the sorbed
phase had a fatty acid content of less than 8 mol% of fatty acids. As a result of these
sorption phenomena, the sorbed phase had molar ratios of ethanol to fatty acid that
were significantly higher than those found in the bulk reaction medium: For MR1:1,
the molar ratio in the sorbed phase remained above 2:1; for MR1.5:1, the molar ratio
in the sorbed phase remained above 4:1; finally, for MR3:1, the molar ratio in the
sorbed phase remained above 27:1. The formation of a sorbed phase on immobili-
zation supports had been observed previously. For example, nonpolar organic
components sorb onto the poly-(methyl methacrylate) beads used in Novozym
435 [36], while water sorbs onto the macroporous anionic resin (Duolite A568)
used in Lipozyme [32]. However, the sorption of medium components on DFS had
not previously been studied.
The high ethanol contents found by Soares et al. [31] for the phase sorbed on the
DFS may explain the results obtained by Dias [37], who used a single-pass
continuous system, comprising three packed-bed bioreactors in series, each
containing 40 g of DFS, produced using B. contaminans LTEB11. The solvent-
free reaction medium fed to the system had a molar ratio of ethanol to fatty acid of
3:1, in an attempt to maximize conversion. However, although the first reaction
medium to pass completely through the system had a conversion of 46% (after a total
residence time of about 4 h), 14 h later, the conversion of the medium exiting the
system had fallen to around 10%. The most likely explanation is that, as the medium
passed through the columns, a significant amount of ethanol sorbed onto the DFS,
favoring denaturation of the lipase.
Given the poor performance of the single-pass continuous system, Dias et al. [10]
scaled up the process for the production of biodiesel by using a closed-loop batch
system with recirculation, as Soares et al. [7] had done. The packed-bed bioreactor
contained 120 g of DFS produced by B. contaminans LTEB11, while the initial
reaction medium contained 245 g of ethanol and 1,000 g of olein, which contains
60% oleic acid and 22% linoleic acid. This reaction medium had an initial molar
ratio of ethanol to fatty acid of 1.5:1. A conversion of 88% in 24 h was achieved,
with the use of a decanting reservoir with two compartments. In this reservoir, the
reaction medium returns to the first compartment and then overflows into a second
compartment from which the medium is recirculated back to the packed-bed biore-
actor. When the system becomes biphasic, the denser aqueous phase is retained at
the bottom of the first compartment, thereby decreasing the water content of the
medium fed back to the bioreactor. This system was used in six successive 48-h
batches with the same DFS, in which a total of 4.6 kg of ester was produced [10]. To
date, this is the largest scale at which biodiesel has been produced using DFS, via
either esterification or transesterification.
Serres et al. [38] developed a mathematical model to describe the reaction profiles
obtained by Soares et al. [31]. This model recognized that the reaction involves both
adsorption and reaction steps. Adsorption was described by Langmuir isotherms in
multicomponent systems. In this case, the number of moles of component i sorbed
onto the DFS (denoted Si) is given by
ai Bi
Si ¼ Pn
1þ j¼1 b jB j
where Bi is the number of moles of component i in the bulk phase, a and b are
sorption parameters, and n is the number of medium components, including both
substrates and products. A sensitivity analysis showed that the sorption of the
products was not important in the denominator, such that the equation describing
sorption simplified to:
ai Bi
Si ¼
1 þ bFA BFA þ bEt BEt
There were four such equations, one each for the fatty acid (FA), ethanol (Et),
water (W), and Ester (Es).
The rate of reaction (r) was expressed in terms of the concentrations of the
components in the sorbed phase. After the sensitivity analysis, the “Ping Pong bi
bi” equation was simplified to
ðkcat SFA SEt αSW SEs ÞE

r¼
D1 SEt þ SFA SEt þ D2 SEs
In this equation, kcat is the turnover number of the enzyme, and the constants α,
D1, and D2 are fitting parameters that represent combinations of fundamental kinetic
constants. E is the fraction of active enzyme (relative to the initial amount of active
enzyme). It denatures according to first-order decay kinetics:
dE
¼ k d E
dt
The first-order denaturation constant, kd, was assumed to depend on the sorbed
ethanol according to a sigmoidal relationship:
kdmax ðSEt Þm
kd ¼
K D þ ðSEt Þm
In this equation, kdmax, KD, and m are empirical fitting constants.

With a single set of parameters, this model was able to fit well to the experimental
data profiles of Soares et al. [31]. It not only described the profile for overall
percentage conversion, but it also described the profiles for the compositions of
the bulk phase and sorbed phases. This opens up the possibility of using the model as
a tool to guide the scale-up and optimization of operation of the closed-loop
bioreactor system. However, in order to describe the separation of phases in the
two-compartment decanting reservoir, it is necessary to characterize the phase
behavior of the system components. The necessary phase diagrams were obtained
for the system FA-SSAO (fatty acids from soybean soapstock acid oil), ethanol,
water, and esters by Serres et al. [39]. However a complete model of the system has
not yet been developed.
2.5 Production of Biodiesel Using Rhizopus microsporus

Fermented Solids
Dry fermented solids produced using fungal strains have also been used to produce
biodiesel. Our investigations into the use of fungal strains were motivated by the fact
that B. contaminans belongs to the Burkholderia cepacia complex, which is a group
of species of Burkholderia that are opportunistic pathogens of humans. Although the
fermented solids produced by B. contaminans showed good potential in biodiesel
synthesis, as described in the previous section, the production and application of
fermented solids containing B. contaminans would require special containment
procedures and equipment in order to protect the process workers. This would
significantly increase process costs. Our reasoning was that the need for special
containment could be avoided by using a nonpathogenic lipase-producing fungus,
thereby reducing costs.
In our initial studies, we grew a strain of Rhizopus microsporus (CPQBA 312-07
DRM) on the same 1:1 mixture (on a dry mass basis) of sugarcane bagasse and
sunflower seed meal that Salum et al. [15] had used to produce fermented solids with
B. contaminans [18]. The tricaprylin-hydrolyzing activity of these DFS was
91 U g1. However, a much higher tricaprylin-hydrolyzing activity of 183 U g1
was obtained with an improved medium comprised of sugarcane bagasse impreg-
nated with an emulsion. This emulsion was prepared using soybean oil and a nutrient
solution containing urea, lactose, and various mineral salts. The improved DFS gave
a conversion of 68% in 72 h for the transesterification of corn oil with ethanol in a
solvent-free medium, with stepwise addition of the ethanol (three equal aliquots at
0, 24, and 48 h). This performance is significantly lower than the 95% conversion
after 46 h that we obtained previously for the transesterification of soybean oil in
solvent-free medium using the DFS of B. contaminans [15]. The lipase activity in the
R. microsporus DFS was highly sensitive to denaturation by the ethanol in the
transesterification reaction medium, with final conversions below 10% being
obtained when the ethanol was added in a single step at the beginning of the reaction.
The same improved R. microsporus DFS gave better conversions for biodiesel
synthesis via esterification [13]. With an overall molar ratio of ethanol to fatty acid
of 10:1 (equal aliquots added at 0 and 24 h), the conversions at 48 h were 98% for
oleic acid and 86% for fatty acids obtained through the hydrolysis of soybean
soapstock acid oil in subcritical water.
The good results that we obtained for the esterification motivated us to scale up
the production of the DFS [9]. We used a pilot-scale SSF bioreactor [40] containing
15 kg of a 1:1 mixture (on a dry mass basis) of wheat bran and sugarcane bagasse,
with the addition of urea as an additional nitrogen source. The peak olive-oil-
hydrolyzing activity of the DFS produced in the pilot bioreactor was 113 U g1
(at 20 h), which is much lower than the value of 265 U g1 obtained (at 18 h) in a
laboratory-scale packed-bed bioreactor containing only 10 g (dry mass) of the same
substrate. Despite this difference in hydrolytic activities, both fermented solids gave
a conversion of 69% in 48 h for the esterification of oleic acid with ethanol in a
solvent-free system [9]. This conversion is lower than the value of 98% in 48 h
obtained by Botton et al. [13] for the improved DFS produced at laboratory-scale
(see the previous paragraph); however, we have not yet used the improved solid
medium in the pilot bioreactor.
2.6 Production of Biodiesel Using Rhizomucor miehei

Fermented Solids
A group at the Federal University of Rio de Janeiro has investigated the production
of biodiesel esters using DFS produced by cultivating the fungus Rhizomucor miehei
on babassu cake. These DFS will hereafter be referred to as DFSRM-BC. Initially, they
developed a hydroesterification process in which acid oil from macaúba (Acrocomia
aculeata) pulp was hydrolyzed using a lipase from dormant seeds of the castor bean
(Ricinus communis), with the liberated fatty acids being esterified with ethanol in a
solvent-free system using DFSRM-BC [8]. In the esterification step, a conversion of
91% was obtained in 8 h, using stepwise addition of ethanol (at 0, 1 and 2 h).
DFSRM-BC gave conversions of over 60% when it was reused in eight successive 6-h
esterification reactions.
DFSRM-BC was also used to catalyze the esterification of palm fatty acid distillate
and soybean fatty acid distillate, these raw materials being by-products of the
refining of palm oil and soybean oil, respectively. For both raw materials, conver-
sions around 70% were obtained in 6 h in solvent-free medium, with either ethanol
or methanol as the alcohol [11]. In the studies of the reutilization of DFSRM-BC in
successive 6-h cycles, Aguieiras et al. [11] washed the DFSRM-BC between cycles
with a solvent. The idea was to remove the residual reaction medium from the
DFSRM-BC, since sorbed free fatty acids might cause mass transfer limitations,
decreasing the performance of the biocatalyst. For both raw materials, when
DFSRM-BC was washed with n-hexane, conversions around 70% were maintained
over five cycles. On the other hand, when DFSRM-BC was washed with ethanol
between cycles, conversions around 70% were maintained over five cycles only for
the esterification of soybean fatty acid distillate; for palm fatty acid distillate, the
conversion in the fifth cycle was around 35%. Aguieiras et al. [11] explained these
results by pointing out that ethanol can remove unsaturated free fatty acids, which
represent 75% of the fatty acids in the soybean fatty acid distillate, but not palmitic
acid, which represents 47% of the fatty acids in the palm fatty acid distillate.
DFSRM-BC have also been used to catalyze the transesterification of fatty acid
esters from macaúba acid oil with several alcohols (methanol, ethanol, isopropanol,
and n-butanol) in a solvent-free system, with ultrasound irradiation [19]. The best
result was obtained for butyl ester, with 79% conversion after 72 h. The DFS kept
55% of their initial activity after four cycles of reuse.
Finally, DFSRM-BC have been used to catalyze the simultaneous esterification and
transesterification of macaúba acid oil with anhydrous ethanol in a solvent-free
system [12]. After 96 h, the conversion was 79%, with the free fatty acids having
decreased from an initial value of 29% to 3%. The used DFSRM-BC were removed,
and a new batch of DFSRM-BC was added to the reaction medium. After a further 96 h
reaction cycle, the conversion had increased to 91%. Better conversions might be
possible if this system were optimized, or the product could be purified to remove
residual mono- and diglycerides. Simultaneous transesterification and esterification
of macaúba acid oil in a solvent-free system was also achieved with a fermented
solid produced by cultivating R. miehei on cottonseed meal [14]. Conversions of
about 76% were achieved in 24 h with either methanol or ethanol as the alcohol.
3 Miscellaneous Applications of Dry Fermented Solids
Although most studies of the application of DFS have focused on the synthesis of
biodiesel, there have been some studies into other applications aimed at producing
organic compounds (Table 2) [2, 41–44]. These other applications include synthesis
reactions, such as the kinetic resolution of racemic alcohols [2, 42], the synthesis of
modified lipids [41], and the synthesis of sugar esters [44], and hydrolytic reactions,
such as the hydrolysis of pectin to produce D-galacturonic acid [43]. DFS have
also been used to hydrolyze lipids in high-fat wastewaters [45–50]; however, this
application is beyond the scope of this review.
3.1 Use of Fermented Solids in the Resolution of Racemic

Mixtures
Nagy et al. [2] screened DFS produced with 38 strains of filamentous fungi for lipase
activity and enantioselectivity against secondary racemic alcohols. The fermented
solids were produced using a mixture of wheat bran and olive oil (9:1 m/m), wetted
140
Table 2 Studies of the direct application of dry fermented solids to obtain different products
Substrate used to
produce the
fermented solid Microorganism Application Substrates of the reaction Solvent Bioreactor Conversion in time taken Reference
Wheat bran Various fila- Kinetic resolu- rac-enylethanol or rac-1- n-hexane Shake Conversions as high as 50% [2]
mentous fungi tions of racemic cyclohexylethanol or rac-1-(naphth-2- flasks obtained, with enantioselectivity
secondary yl)ethanol and vinyl aa for the R-isomer (ER) over 100
alcohols rac-1-phenylethanol or rac-1-
cyclohexylethanol or rac-1-(naphth-2-
yl)ethanol and vinyl acetate
Sugarcane Rhizopus Interesterification Palm stearin, palm kernel oil and a Solvent-free Shake Solid fat content (at 35 C) as [41]
bagasse and sun- oryzae concentrate of triacylglycerols flasks low as 2.3% obtained in 24 h
flower seed meal enriched with ω-3 polyunsaturated
fatty acids
Sugarcane Rhizopus Kinetic resolu- rac-1-phenylethanol and isopropenyl n-heptane Shake 23% in 96 h with [42]
bagasse and nutri- microsporus tion of rac-1- acetate flasks enantioselectivity for the
ent solution phenylethanol S-isomer (ES) of 26
Sugarcane Aspergillus Hydrolysis of Concentrated pectin solution Solvent-free Shake 60% of the D-galacturonic acid [43]
bagasse and oryzae pectin to liberate (10% m/v) flasks present in the pectin liberated in
orange peels D-galacturonic 48 h
acid
Sugarcane Burkholderia Regioselective Methyl-α-D-glucopyranoside and Solvent-free Shake 76% in 72 h [44]
bagasse, nutrient contaminans modification of vinyl acetate flasks
solution, and soy- carbohydrates
bean oil
N. Krieger et al.
with a salt solution to a moisture content of 60% or 70% (m/m). All strains produced
DFS that were enantioselective, most for the R isomer, following the Kazlauskas rule
for secondary alcohols [51]. For these strains, conversions of the secondary alcohol
(R,S)-1-phenyl-1-ethanol were near 50%, with enantiomeric ratios (ER ¼ (kcatR/
KMR)/(kcatS/KMS)) above 200. The exception was the DFS produced with Mucor
hiemalis, which was enantioselective for the S isomer. In this case, the conversion of
(R,S)-1-phenyl-1-ethanol was 14%, with an ES value ((kcatS/KMS)/(kcatR/KMR)) of 15.
Recently, our group also produced a DFS that has preference for the S isomer of
secondary alcohols [42]. This DFS was produced by cultivating R. microsporus on
sugarcane bagasse impregnated with olive oil and urea, with an initial moisture
content of 80% (wet basis). Through optimization of the resolution reaction, we
increased the conversion of (R,S)-1-phenyl-1-ethanol from 3% to 23%, achieving an
ES value of 26. This result is better that the results obtained with (R,S)-1-phenyl-1-
ethanol by Nagy et al. [2] using the DFS of M. hiemalis (conversion of 14%,
ES ¼ 15).
3.2 Use of Fermented Solids in Interesterification Reaction

to Produce Modified Fats
Taking advantage of the fact that the lipases produced by the genus Rhizopus are sn-
1,3 specific and that species of this genus are considered GRAS, Rasera et al. [41]
used DFS produced by R. oryzae to catalyze the interesterification of palm stearin,
palm kernel oil, and a concentrate of triacyclglycerols enriched with omega-3
polyunsaturated fatty acids (ω-3 PUFAs) (EPAX 4510TG), in a solvent-free
medium. The aim was to incorporate ω-3 PUFAs into the palm stearin and palm
kernel oil, thereby reducing the solid fat content at 35 C (SFC35 C) of the blend. For
table margarines, this value must be as low as possible, to ensure that the margarine
melts in the mouth, avoiding a coarse and sandy texture [52]. The initial blend had an
SFC35 C value of 12.3%. The best conditions for interesterification were 65 C, a
palm stearin content of 38% and an EPAX content of 15%. Under these conditions,
after 24 h, the product had an SFC35 C of 2.3%, making it suitable for the
production of margarines and shortenings [41]. Despite this success, a key challenge
is to reduce the reaction time, since similar reductions can be obtained in as little as
30 min using commercial lipases such as Lipozyme TL IM [53].
3.3 Use of Fermented Solids for the Production of High-

Value Sugar Esters
Recently, Villalobos et al. [44] showed that the DFS produced by B. contaminans
LTEB11 can be used to produce high-value sugar esters. The DFS were produced on
sugarcane bagasse impregnated with a solution containing urea, lactose, and a
micronutrient solution and 20% of soybean oil (w/w, on a dry basis). They had an
olive-oil-hydrolyzing activity of 160 U g1, which is 1.5-fold higher than the activity
of the DFS produced by Soares et al. [7] with the same bacterium cultivated on a
mixture of sugarcane bagasse and sunflower seed meal. The DFS were used to
catalyze the transesterification of methyl-α-D-glucopyranoside (α-MetGlc) with
vinyl acetate in a solvent-free medium. In initial studies done with 4 mL of a reaction
medium containing 200 mg of DFS, 4.5 mg of α-MetGlc, and vinyl acetate, the main
product obtained was methyl 6-O-acetyl-α-D-glucopyranoside (6-OAc-α-MetGlc),
with a conversion of α-MetGlc to 6-OAc-α-MetGlc of 65% at 72 h. This system was
scaled up to 1 L of reaction medium. A conversion of 76% was obtained at 72 h, with
a calculated yield of 1.0 g (4.43 mmol) of 6-OAc-α-MetGlc. This is the largest scale
reported to date for production of sugar esters by enzymatic catalysis, in terms of
moles of sugar ester formed, indicating that the use of DFS has good potential in this
system.
3.4 Use of Fermented Solids in the Production of D-

Galacturonic Acid
Leh et al. [43] produced DFS containing pectinase activity by cultivating Aspergillus
oryzae on a mixture of ground sugarcane bagasse and orange peels (30:70 m/m).
The DFS were added to a 10% m v1 pectin solution, giving a concentration of
247 mmol L1 of D-galacturonic acid at 35 h. This is the highest D-galacturonic acid
concentration reported for enzymatic pectin hydrolysis to date and represents a
conversion of 60%, based on the D-galacturonic acid content of the original pectin.
This opens the possibility of a cheap route for producing D-galacturonic acid from
pectin in a citrus waste biorefinery. The D-galacturonic acid can then serve as the
basis for the production of several useful organic chemicals [54].
4 General Considerations
As our review has shown, DFS containing naturally immobilized enzymes (lipases
and pectinases) have been produced using both bacteria and fungi and then used in
synthesis and hydrolysis reactions to produce organic compounds of biotechnolog-
ical interest. To date, the main application of DFS has been in the production of
biodiesel, by transesterification and esterification, in solvent-free media. Although
many of the results reported in this review are promising, there are several hurdles to
overcome before the processes can be scaled up successfully to industrial scale.
We discuss the main hurdles below.
In general, processes involving DFS require longer reaction times than do
processes that use commercial enzyme preparations. Although DFS are cheaper,
the resulting low volumetric productivities will diminish the competitivity of pro-
cesses using DFS as the biocatalyst. One key challenge is to decrease reaction times
by increasing the enzymatic activity produced in the solids. Another key challenge is
to improve the stability of the DFS so that they can be reutilized over many cycles.
To date, most processes have shown a significant drop-off in performance over ten or
fewer reaction cycles.
The drying of the fermented solids should be done by air-drying, as lyophilization
will increase the costs of producing DFS significantly. Air-drying protocols need to
be optimized to minimize activity losses during this step. To reduce process costs,
one possibility is to dry the solids directly in the bioreactor used for the synthesis or
hydrolysis process. The stability of the DFS during storage also needs to be studied.
In hydrolytic reactions performed in aqueous media, the enzymes can leach from
the DFS into the reaction medium. This is acceptable if simultaneous extraction and
hydrolysis is desired. However, if one desires to maintain the enzyme in the DFS, an
in situ immobilization procedure will be necessary. To date, there are no reports of
such a procedure having been used with DFS.
To date, processes involving DFS have been applied at relatively small scales,
involving, at most, little more than 100 g of DFS. Processes need to be developed not
only to produce several kilograms of DFS but also to apply kilogram quantities in the
biocatalytic process. However, one should carry out an economic feasibility study
before one decides to scale up a process based on DFS. Only with such studies will it
be possible to confirm whether the low costs of producing DFS offset their relatively
low activities when compared to commercial enzymes. Such studies should also take
into account the need to dispose of the DFS adequately after they have been used.
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DOI: 10.1007/10_2019_86
Solid-State Anaerobic Digestion for Waste

Management and Biogas Production
Haoqin Zhou and Zhiyou Wen
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148
2 Feedstocks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 149
3 Inoculum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4 Factors Affecting Solid-State AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4.1 Nutrients . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 151
4.2 Feedstock-to-Inoculum Ratio . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4.3 pH . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 152
4.4 Temperature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
4.5 Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 153
4.6 Mixing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 154
5 Process Operations of SS-AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
5.1 Batch vs. Continuous Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155
5.2 Single-Stage vs. Multistage Operations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 156
6 Enhancement of Digestion Performance in SS-AD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
6.1 Feedstock Pretreatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157
6.2 Co-digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161
6.3 Additives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 162
7 Conclusion and Perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 163
Abstract Solid-state anaerobic digestion (SS-AD) is commonly used to treat feed-

stocks with high solid content such as municipal solid waste and lignocellulosic
biomass. Compared to liquid state anaerobic digestion (LS-AD), SS-AD has
multiple advantages including high organic loading, minimal digestate generated,
and low energy requirement for heating. However, the main disadvantages limiting
the efficiency of SS-AD are long solid retention time, incomplete mixing, and an
H. Zhou and Z. Wen (*)

Department of Food Science and Human Nutrition, Iowa State University, Ames, IA, USA
148 H. Zhou and Z. Wen
accumulation of inhibitors. For a successful and efficient SS-AD, it is important

to control operation parameters such as nutrient levels, C/N ratio, feedstock-to-
inoculum ratio, pH, temperature, and mixing. Biogas production in SS-AD perfor-
mance can be enhanced by feedstock pretreatment, co-digestion, and supplement of
additives such as biochar. The aim of this chapter is to provide a comprehensive
summary of the current development in SS-AD as an effective way for treating solid
waste materials.
Graphical Abstract
Keywords Biogas, Co-digestion, Pretreatment, Solid-state anaerobic digestion,

Solid wastes
1 Introduction
With the growth of world population and economics, the production of solid wastes
is increasing tremendously. A large quantity of these waste materials is biodegrad-
able agricultural residues and municipal solid wastes (MSW). It is estimated that the
annual production of MSW will reach 2.2 billion tons by 2025 [1]. These abundant
Solid-State Anaerobic Digestion for Waste Management and Biogas Production 149
materials can be used as a feedstock for anaerobic digestion (AD) to produce energy
while solving waste disposal problems.
AD is a process in which microorganisms decompose organic matters to produce
biogas in the absence of oxygen. An AD process typically consists of four stages:
hydrolysis, acidogenesis, acetogenesis, and methanogenesis. In the hydrolysis stage,
macromolecules such as cellulose, starch, proteins, and lipids are decomposed into
monomers such as sugars, amino acids, and fatty acids. Those monomers are then
converted into C2–C5-based volatile fatty acids (VFAs) and alcohols, as well as H2
and CO2 in the acidogenesis stage. In the acetogenesis stage, VFAs and alcohols are
converted into acetate. In the methanogenesis stage, methane (CH4) is produced
through the conversion of acetate to CH4 and CO2 (acetoclastic methanogenesis) or
the reduction of formate or CO2 to CH4 (hydrogenotrophic methanogenesis). Among
these four steps, hydrolysis is commonly the rate-limiting step particularly when the
feedstock is the complex organic substrates. When easily digestible organic matters
are used as a feedstock, methanogenesis becomes the limiting step [2]. The biogas
produced from an AD process usually contains 60–70% CH4 and 20–30% CO2 with
trace amounts of ammonia, hydrogen sulfide, and hydrogen. The biogas can be
combusted to generate heat and/or electricity or upgraded and refined into
transportation fuels. Meanwhile, the digestate rich in nutrients, such as nitrogen
and phosphorus, can be recycled as fertilizers or processed into biochar that can be
used as soil amendment [3].
Based on the total solid (TS) content, AD can be defined as liquid state AD
(LS-AD) with TS less than 15% or solid-state AD (SS-AD) with TS greater than
15% [4]. LS-AD is used to treat high moisture substrates such as animal manures and
sewage. However, the large amount of water used in LS-AD process leads to a
decreased volumetric CH4 productivity and creates the problem of disposing large
amount of digestate [5]. On the contrary, SS-AD can handle feedstocks with high
organic loading with minimal water demand and results in a high volumetric CH4
productivity. The wastewater generated and heating energy required in SS-AD are
also reduced. However, due to inadequate mass transfer, SS-AD has disadvantages
such as longer retention time, high cost, and a tendency to accumulate inhibitors
[6]. In the past decade, a steady increase of publications in SS-AD indicates a great
interest in this area (Fig. 1). The aim of this chapter is to provide a comprehensive
review of the recent advances of SS-AD including feedstock, inoculum, factors
affecting SS-AD performance, operation mode, and digestion process enhancement.
2 Feedstocks
Feedstocks with high moisture content, such as animal manure or municipal sewage,
have been traditionally treated with LS-AD. Recent development of AD has
expanded to feedstocks with high solid content such as agricultural residues (e.g.,
corn stover, wheat straw, and rice straw), industrial wastes, and municipal solid
wastes; SS-AD has been increasingly used to treat these feedstocks. Corn stover,
450
400
Number of publications
350
300
250
200
150
100
50
0
2007 2008 2009 2010 2011 2012 2013 2014 2015 2016 2017 2018
Year
Fig. 1 Number of publications with keywords, “solid-state anaerobic digestion,” via Google
Scholar. The search included patents but did not include citations. Updated on September 16, 2018
with a 1:1 weight ratio of residue to grain [7], is the most abundant agricultural
residue in the United States, with approximately 80 million dry tons of corn stover
produced each year [8]. It has been reported that SS-AD (18% TS) treatment of corn
stover produced a higher CH4 yield than LS-AD (5% TS) [9, 10]. SS-AD of wheat
straw, another abundant agriculture residue, also resulted in a much higher CH4 yield
than LS-AD [8, 11].
The microstructure of the fibrous feedstock significantly affects SS-AD
performance. Cui et al. [10] examined the fiber structure in wheat straw by scanning
it with an electron microscope (SEM). Compared to the raw wheat straw with long
and smooth intact fibers, the spent wheat straw with rough fiber and serrations at the
edge was more digestible. Similarly, corn stover treated by sodium hydroxide was
more digestible than the raw corn stover [11].
The organic municipal solid wastes (OMSW), such as food and yard wastes, have
also been commonly used as feedstock for SS-AD. It is estimated that 1.3 billion tons
per year of food wastes are produced worldwide [12]. In the United States, food
waste accounts for 12% of total municipal solid waste [13]. Food waste composition
varies widely depending on geographical locations and the eating habits of local
populations. In general, food wastes containing soluble organic matters can be easily
converted into VFAs, which may inhibit the subsequent CH4 formation if VFAs are
overproduced. A two-phase SS-AD can successfully overcome this problem
[14]. Among the 31 million dry tons per year of yard wastes generated in the United
States, more than 60% were treated through composting, during which energy was
wasted as respiration heat [5]. SS-AD as an alternative to composting can recover
energy. However, the types of yard wastes affect the methane yield due to different
TS, VS, and C/N in those materials [15, 16].
3 Inoculum
Inoculum brings the microbes, nutrients, and water to SS-AD reactors. Typical
inoculum in SS-AD includes sewage sludge, ruminant cultures, and digested manure
[17]. Since most solid feedstock does not naturally contain methanogens,
methanogens-rich inoculum is essential to a SS-AD process [15]. Characterization
of the microbial community in inoculum is important for an insightful understand-
ing, particularly the functional partitioning of a SS-AD process. Shi et al. [16]
studied the microbial community in SS-AD of corn stover using denaturing gradient
gel electrophoresis and found enriched archaeal and bacterial communities in
the system. In SS-AD of rice straw, a high-throughput sequencing analysis
revealed that Methanobacteria, Bacteroidia, Clostridia, Betaproteobacteria,
and Gammaproteobacteria were the primary species [18]. The acetoclastic
Methanosarcina and hydrogenotrophic Methanoculleus coexisted in this system.
For example, in the first 20 days of AD, Methanosarcina accounted around 86.5% of
microbial population, while Methanoculleus accounted 32.1% of microbial popula-
tion from days 7 to 45 [18]. Bacteria producing low temperature-adaptive lipases,
Psychrobacter, was identified in SS-AD of a mixed kitchen waste, pig manure,
and the sludge [19]. In SS-AD of fruit waste, a three-stage system was developed
to accommodate the favorable conditions for hydrolysis, acidogenesis, and
methanogenesis [20]. Lactobacillaceae and Pseudomonadaceae were predominant
in the hydrolysis of carbohydrate into lactate and biomic acids, respectively.
In the acidogenesis stage, the most abundant bacteria were switched to
Porphyromonadaceae and Enterobacteriaceae, while methanogens were the dom-
inant species in the methanogenic stage [20].
4 Factors Affecting Solid-State AD
4.1 Nutrients
Anaerobic microbes need balanced nutrients such as carbon, nitrogen, phosphorous,

and minerals for their growth. Carbon, the primary energy source for cell growth, is
usually rich in organic materials. Nitrogen and phosphorous are also essential
for anaerobic microbes to synthesize proteins and nucleic acids, respectively.
Ammonium is the nitrogen form methanogens can utilize [21] but will inhibit the
microbial growth at high levels. The C/N ratio of the feedstock also plays an
important role in digestion process. C/N ratio ranging from 20:1 to 30:1 with an
optimal C/N ratio of 25:1 is recommended [22]. Too low C/N ratios increase the risk
of ammonia inhibition, resulting in insufficient utilization of carbon sources, while
an excessively high C/N ratio results in insufficient nitrogen to maintain microbial
growth and biogas production. The demand of phosphorus is usually 15% of that of
nitrogen [23].
Trace elements such as iron, cobalt, nickel, and sulfur are essential for methane
fermentation [24–27]. Iron is often supplemented in AD systems to activate enzymes
such as ATPase, PEP carboxylase, and serine transhydroxymethylase [28, 29]. Due
to its reduction capacity, iron often reacts with sulfur to form FeS precipitant,
reducing H2S generation and alleviating odor problem [30]. Nickel is an essential
element in coenzyme F430, hydrogenase, and CO dehydrogenase in methanogenic
microbes [31–33]. Cobalt is involved in the activity of methyl transferase and
CO dehydrogenase (CODH) in acidogenesis [31]. The addition of cobalt has
been reported to stimulate CH4 productivity in methanol LS-AD process
[25]. Molybdenum is present in CO2 reductase, a molybdoprotein that is responsible
for reducing CO2 to formate and subsequently reducing to CH4 [34].
4.2 Feedstock-to-Inoculum Ratio
Feedstock-to-inoculum ratio (F/I) is another important factor in SS-AD. Too high F/I
ratio could result in overproduction of VFAs due to excess organic loads, which
eventually leads to an acidic pH and inhibition on methanogens. Zhou et al. [35]
reported that the CH4 yield of rice straw SS-AD was inversely proportional to F/I due
to the VFAs accumulation and poor mass transfer. On the contrary, SS-AD of palm
oil mill residues achieved the highest CH4 production rates at the lowest F/I ratio
within the range of 2:1–5:1, while a rapid hydrolysis at F/I ratio of 4:1–5:1 resulted
in a VFAs accumulation and low CH4 yield [36].
4.3 pH
The pH of a SS-AD system also affects the digestion performance. The ideal pH for a
SS-AD process is within a narrow range of 6.8–7.2 [37]. However, different groups
of microbes in SS-AD have different optimal pH requirements. For example, the
optimal pH for acidogens is between 5.5 and 6.5, while methanogens are most active
at pH 6.5–8.2 with an optimum at pH 7.0 [38]. Due to this discrepancy of pH
requirement, two-stage SS-AD, i.e., separating acidogenesis and methanogenesis
into two reactors, is usually used [37].
During an AD process, pH is affected by many parameters. In a SS-AD of
OMSW with liquid digestate recirculation, the pH was low (<6.5) initially due to
high VFAs concentration and then gradually increased to 8 after VFAs decreased
from 12,000 to 1,000 mg/L within 1 week [39]. The buffer capacity of an AD system
to resist pH fluctuation is evaluated through alkalinity. For example, in a corn stover
SS-AD system with a less alkalinity (1,036 mg CaCO3/kg), pH dropped from nine to
below six rapidly with a decreased CH4 yield [16]. When the alkalinity of the system
was increased (>1,700 mg CaCO3/kg) through adjusting the F/I ratio, pH of the
same system was stabilized with only slight a decrease from 9 to 8.4 [16]. In order
to maintain a stable pH during SS-AD process, it is essential to balance VFAs

concentration and bicarbonate. In general, reducing organic loading, adding bases
or bicarbonates, and modifying F/I ratio are used to increase alkalinity in SS-AD
systems [37].
4.4 Temperature
SS-AD is commonly operated at mesophilic (~37 C) or thermophilic (~55 C)

conditions. Compared to the mesophilic AD, thermophilic AD has a shorter start-
up time and hydraulic retention time (HRT) due to accelerated feedstock hydrolysis.
The CH4 yield in thermophilic SS-AD is also higher as methanogenic microbes have
an optimal growth at 55 C [40]. Thermophilic AD can also produce pathogen-free
digestate. Pohl et al. [41] compared the performance of wheat straw SS-AD under
37 C and 55 C. The CH4 yield from the thermophilic AD was 36% higher than that
in mesophilic AD due to a faster disintegration and hydrolysis of the feedstock.
However, compared to mesophilic SS-AD, poor stability and reliability often
represent obstacles in thermophilic SS-AD. In general, microbes in thermophilic
conditions are more sensitive to environmental changes, exhibiting poor stability and
less diversity and richness in microbial community [38]. Also, the fast hydrolysis
of feedstock in thermophilic processes often results in a rapid VFAs production,
causing an imbalance between acidogenesis and methanogenesis. The higher
temperature also shifts NH3/NH4+ equilibrium toward the cytotoxic ammonia
[40]. Heating energy in theomorphic AD is also higher [42]. Due to those reasons,
theomorphic digesters are still not commonly used in commercial SS-AD.
4.5 Inhibitors
A variety of compounds have been reported inhibitory to SS-AD, causing an adverse

shift in microbial population, an instability of the process, and a decreased CH4 yield
[43]. In LS-AD, the inhibitor concentrations can be diluted, while inhibitory effects
in SS-AD cannot be alleviated and often cause severe inhibition to the system. The
easily digestible feedstock often leads to a rapid hydrolysis and acidification,
producing excessive VFAs which inhibit methanogens. For example, in SS-AD of
tomato residues, VFAs concertation (12.48 g/L) was much higher than the threshold
level (6 g/L) and caused CH4 production inhibition [44]. Compounds derived
from phenolic degradation, such as p-cresol, inhibit acetogenesis, resulting in
accumulation of VFAs [45].
The partial pressures of CO2 and H2 in SS-AD system also affect the CH4
production. Increasing CO2 partial pressure results in an increased dissolved CO2,
which causes acidification and inhibition of methanogenesis. An elevated H2 partial
pressure leads to an accumulation of dissolved H2, which inhibits the degradation of
VFAs [46]. In SS-AD of wheat straw, high H2 partial pressure also led to a strong
inhibition on the initial hydrolysis step [47]. Since both CO2 and H2 are needed to
produce acetate/CH4, a balanced CO2/H2 pressure in the headspace is essential to
prevent inhibition.
Ammonia is produced from the degradation of nitrogenous compounds (e.g.,
protein and urea) during AD process. A moderate amount of ammonia is essential for
bacterial growth and neutralizing VFAs to maintain a stable pH; however, excessive
ammonia can inhibit methanogenesis. Ammonia exists as an equilibrium between
ammonium ion (NH4+) and free ammonia (NH3) [43]. Free ammonia can permeate
cell membrane and cause proton imbalance and thus is inhibitory to microbial cells.
Animal manure usually contains excessive ammonia, resulting in process inhibition.
For example, in SS-AD of chicken manure, the digester was completely inhibited
when influent total Kjeldahl nitrogen (TKN) (mainly ammonia) was 8.2 g/L
[48]. After ammonia was removed from influent, the digester achieved a much
higher CH4 yield.
4.6 Mixing
A certain degree of mixing in SS-AD is necessary to enhance the transfer of organic

substrates to microbes, prevent the sedimentation of denser particles or floating
lighter materials, and facilitate the release of gas bubbles trapped in the solid
feedstock. In SS-AD of rice straw, intermittent mixing with a 5/25 min on/off
cycle at 160 rpm resulted in a good mass transfer while saving energy compared
to a continuous mixing [35]. Premixing of the feedstock with inoculum is also
needed before loading into SS-AD reactor [49, 50].
The methods of mixing in SS-AD can be liquid (leachate) recirculation, solid
mixing using augers, and biogas recirculation [4], among which the leachate
recirculation is commonly used. Leachate recirculation in SS-AD facilitates the
nutrient diffusion from substrates to microbial cells [51] and also reduces the amount
of inoculum as the microbe-containing leachate collected from the reactor can be
reapplied to the digestion systems [52].
In addition to mixing, leachate recirculation also provides other benefits to
SS-AD. For example, when leachate recirculation was used in the acidogenic reactor
of a two-stage hybrid solid-liquid AD system, the extraction of organic matters from
the feedstock was facilitated, and the pH was buffered [53]. In the SS-AD of hay and
soybean processing wastes, leachate recirculation accelerated the daily CH4
production to the peak value due to the enhancement of VFAs mass transfer from
acidogenic to methanogenic pockets [54]. However, leachate recirculation may also
lead to accumulation of VFAs and other inhibitors compounds; therefore, dilution of
leachate with fresh water may be needed [15]. A leachate recirculation rate also
needs to be carefully controlled to avoid irreversible acidification of the system [55].
5 Process Operations of SS-AD
5.1 Batch vs. Continuous Operations
Batch and continuous operations are two operation modes commonly used in
SS-AD. Table 1 compares the performance of batch and continuous operation of
SS-AD. Compared to continuous operation, batch operation is easier to maintain
because it needs less capital and operating costs with less process control
requirements. However, the biogas production in batch SS-AD is variable with
time, and the majority of biogas is produced only at peak production time. For
example, it was reported that in a 55-day batch SS-AD of corn stover, more than
80% of biogas was produced only at 36-day period of methanogenic phase
[22]. Another limitation in batch SS-AD is the requirement of a large amount of
inoculum (i.e., low F/I ratio). For example, Capson-Tojo et al. [56] reported that a
batch SS-AD of food waste and cardboard mixture can only produce biogas at a F/I
lower than 0.25; the biogas production had completely ceased when the F/I ratio was
above this ratio due to overproduction of VFAs. Similar results were obtained for a
batch operation of yard trimmings SS-AD process in which the highest CH4 yield
(244 L/kg VS) was obtained at the lowest level of the F/I ratio ranging from 0.2 to
2 [57]. The inoculum sources also significantly affected the batch SS-AD process.
Guendouz et al. [58] studied three successive batches of MSW SS-AD and found
that the second and third batches inoculated with the residue from the previous batch
shortened the lag phase and accelerated reaction, which was due to the adaptation of
the microbes to the digestion system.
Contrary to the batch operation, continuous SS-AD can consistently produce
CH4 at steady state. Organic loading rate (OLR), CH4 production, and solid
retention time (SRT) are the three main parameters in determining the interaction
between microorganisms and substrates and thus are used in designing and
evaluating a continuous SS-AD performance [51]. OLR represents the conversion
capacity of an AD system; a maximum OLR level in SS-AD depends on various
parameters such as reactor design, feedstock characteristics, microbial activity,
Table 1 Comparison of batch and continuous SS-AD systems

Parameters Batch systems Continuous systems
Investment Low High
Technical operation Simple Complex
Land acreage required Large Small
Organic loading rate (OLR) Low High
Inoculum High Low
Water consumption Low High
Biogas yield Uneven; low Even; high
temperature, pH, and toxicity level [59]. A high OLR is always preferred as it
means an improved utilization efficiency and reduced digester size. However, high
OLR can also lead to VFAs overproduction, causing an imbalance between
acidogens and methanogens. For example, in a batch SS-AD process of rice
straw, increasing TS loading from 20% to 24% prolonged the lag phase from
15 days to 20 days [35]. Similarly, increasing OLR from 2.3 to 9.2 kg VS/m3 day
in semicontinuous SS-AD of food waste slowed down bacteria acclimatization in
the new environment, resulting in a prolonged adaptation time from 2 days to
31 days [60]. In a co-digestion of chicken manure and poplar leaf, CH4 yield
decreased when OLR increased from 4.0 to 8.0 g VS/L day [61].
One important operational parameter in continuous SS-AD is solid retention
time (SRT); the time organic compounds stay in the digester. Due to slower mass
transfer, the SRT in SS-AD is usually longer than the HRT commonly used in
LS-AD [54]. The retention time needed for a complete degradation of solid
feedstock can be determined through biomethane potential (BMP) assay
[62]. An optimal SRT depends on many factors such as the feedstock, OLR, and
TS. Decreasing SRT leads to washing out of microorganisms and insufficient
substrate utilization. A longer SRT is usually not economical because it would
require larger reactor volumes and higher costs for maintenance. SRT has a
considerable impact on CH4 production. In SS-AD of organic waste containing
vegetable, fruit, and green waste, increasing SRT from 15 days to 35 days
increased methane yield from 360 to 454 mL/kg VS [63].
5.2 Single-Stage vs. Multistage Operations
SS-AD can be operated in a single stage or multiple stages. In a single-stage system,

the multiple steps of the conversion of organic substrates into biogas are
implemented in one reactor vessel. In a multiple-stage operation, different
conversion steps are implemented into different reactor vessels. A two-stage AD is
commonly used as a multiple-stage operation during which the hydrolysis/
acidogenesis is in the first reactor and the methanogenesis is in the second
reactor [64].
Compared to a two-stage operation, a single-stage reactor is easier to design and
build with less operating costs. However, the OLR in a single-stage digester is often
limited in order to avoid VFAs overproduction and rapid pH drop [4]. Unlike the
single-stage digester, two-stage systems can accommodate each conversion step,
such as acidogenesis and methanogenesis, at their own optimal conditions (pH,
temperature, OLR, and SRT). Two-stage systems generally perform better than a
single-stage system. For instance, SS-AD of brewery spent grain (BSG) in a single-
stage reactor was limited by the inhibitors, such as weak acids, furan derivatives, and
phenolic substances, generated in the degradation of lignocellulose in BSG
[65]. While in a two-stage SS-AD system, separating hydrolysis in one reactor and
Table 2 Biogas, methane production, and feedstock degradation of brewery spent grain SS-AD in
single-stage and two-stage processes with raw and acid pretreated feedstock [65]
Feedstock types Single stage Two stage
Biogas production (L/kg) Raw 87.4 89.1
Pretreated 89.1 103.2
Methane production (L/kg) Raw 51.9 58.7
Biodegradation % Raw 62.0 63.5
acidogenesis and methanogenesis in another granular-based reactor, both biogas

production and feedstock biodegradation were improved (Table 2).
In some occasions, AD systems with more than two stages, such as three stages,
are designed to create different favorable conditions for hydrolyzing bacteria,
acidogenic bacteria, and methanogens, with each group of microbes performing a
particular role. A three-stage system was used in the co-digestion of food waste and
horse manure in which the first-stage hydrolysis and second-stage acidogenesis were
operated as a solid state, while the methanogenesis was operated as a liquid state.
This hybrid system increased CH4 yield by 11.2–22.7% and the abundance of
methanogenic archaea by 0.8–1.28 times compared to the single-stage reactor.
It should be noted that despite the fact that multistage AD systems are
advantageous in improving AD performance, high capital and operating costs
are the main hurdles for implementing this type of systems at a commercial scale.
As a result, single-stage AD is still dominantly used. In Europe, for example, about
90% of the installed AD capacity is from single-stage systems, and only about 10%
is from multistage systems [4].
6 Enhancement of Digestion Performance in SS-AD
6.1 Feedstock Pretreatment
As hydrolysis is the rate-limiting step for SS-AD of most solid feedstocks, various
treatment technologies have been developed to accelerate the feedstock hydrolysis
so overall biogas yield can be enhanced. Those pretreatment methods are
summarized in Table 3.
6.1.1 Physical Treatment
Physical treatment such as milling and grinding reduces the particle size of the
feedstock and thus provides a greater surface area for microorganisms to access. Tian
et al. [66] reported a 29% increase in CH4 yield in SS-AD of rape straw when the
Table 3 Methods used for the feedstock pretreatment in SS-AD

Treatment CH4 yield Enhancement
method Processes Feedstock (L/kg VS) (%) References
Physical Milling/grinding Rice straw 188–243 29 [66]
Sonolysis OFMSW 186.4 16 [67]
Microwave Wheat straw 345 28 [68]
Steam MSW 248 N/A [69]
autoclaving
Low temperature High solid 99.3–116 11% [70]
sludge (biogas)
Chemical Alkaline Poplar leaf 156.7 N/A [61]
Corn stover 372.4 37 [71]
Peracetic acid Waste acti- 175 (biogas) 21 [72]
oxidation vated sludge
Ozonation OFMSW 227.9 37 [67]
Organosolv Pinewood 71.4 84 [73]
Biological Trichoderma Rice straw 214 78.3 [74]
reesei
Pleurotus Rice straw 263 120 [74]
ostreatus
Ceriporiopsis Yard 44.6 154 [75]
subvermispora trimmings
Albizia chips 123.9 270 [76]
Miscanthus 170–175 25 [77]
sinensis
Orange 275–330 N/A [78]
processing
waste
Stacking Corn stover/ 450 (biogas) 29.1 [79]
cow dung
Pre-aeration Rice straw 355.3 N/A [35]
Composting Rice straw 353 N/A [18]
Combined Acid-thermal Brewery spent 55.3 6.5 [65]
grain
Thermo-lime Spartina 218.1 N/A [80]
alterniflora
Milling-steam Birch wood 188.1 N/A [81]
explosion
feedstock size was reduced from 2–2.5 cm to 0.5 mm. However, a too fine particle
size may negatively affect the AD performance. For example, Motte et al. [82]
compared the SS-AD of straw at three particle sizes (0.25, 1, and 10 mm) and found
that the coarse particles (10 mm) resulted in the highest CH4 yield followed with the
medium size particles (1 mm) and the finest size (0.25 mm). The reason for this
phenomenon was due to rapid acidification of the substrate at smaller particle sizes,
which resulted in an overproduction of VFAs and rapid pH drop.
Thermal treatment is an effective treatment method for industrial SS-AD

[83]. In addition to enhancing the reaction rate, thermal treatment removes
pathogens, improves dewaterability, and decreases viscosity of the digestion system.
In the SS-AD of steam autoclaved MSW, the digestate passed all the criteria for
biosolids land application in the United States [69]. In the thermal treatment, an
appropriate combination of temperature and time is needed as the high energy
consumption often offsets the overall benefits. Liao et al. [70] studied the effect of
treatment temperature (60, 70, and 80 C) on the SS-AD of sewage sludge and found
that 70 C for 30 min was optimal for SS-AD. Under this condition, biogas yield
increased by 11% and SRT reduced from 22 to 15 days.
Other physically based treatments were also reported. For example, ultrasound
treatment generates both mechanical effects through cavitation and chemical effects
through formation of free radicals. OMSW treated with low-frequency ultrasound
released more soluble organic matters, resulting in a 16% increase in biogas
production in SS-AD [67]. Microwave treatment is related to structure modification
as well as thermal effects, contributing to increased sludge solubility [84], shortened
initial lag phase [85], and improved CH4 yield [68].
6.1.2 Chemical Treatment
Chemicals such as acids, alkaline, or oxidants can facilitate the breaking down of
recalcitrant structures of feedstock. The effectiveness of chemical treatment relies
on the feedstock characteristics and the reagents used. Feedstocks with easily
digestible carbohydrates such as starch are typically not suited for chemical
treatment because it accelerates starch degradation leading to VFAs overproduction
and accumulation [86].
Alkaline treatment is usually carried out at ambient temperature with lime,
sodium hydroxide, potassium hydroxide, and ammonium hydroxide as agents. The
mechanism of alkaline treatment is to remove lignin from lignocellulose, improving
the accessibility of hemicellulose and cellulose by the microbes and enzymes
[87]. Additionally, the presence of residue alkali neutralizes carboxylic acids
resulted from lignocellulose degradation in subsequent acidogenesis stage and pre-
vents pH drop [71]. Zhu et al. [71] reported a 37% increase in biogas yield in SS-AD
of corn stover treated with 5.0% NaOH compared to that of untreated corn stover.
Liew et al. [88] achieved a 24-fold higher CH4 yield in SS-AD of fallen leaves
treated with 3.5% NaOH. However, excessive alkali loading may inhibit AD due to
high pH or ion toxicity [89]. For instance, in SS-AD of corn stover, although the
lignin degradation of corn stover increased with NaOH loadings from 1.0% to 7.5%,
the biogas yield was not improved correspondingly; 7.5% NaOH loading actually
inhibited biogas production due to VFAs accumulation and acidification [71].
Compared to alkali treatment, acid treatment is more effective to break down the
recalcitrant lignocellulosic structure and produce reducing sugars [83]. However,
compounds such as furfural and hydroxymethylfurfural (HMF) can be produced
during acid treatment which inhibit the AD process [86]. Acid treatment also
requires additional bases to neutralize pH before starting SS-AD. Overall, acid

treatment is less preferable than alkaline treatment in SS-AD.
Ozonation is another chemically based treatment in SS-AD with no chemical
residues left in the system. As a strong oxidant, ozone decomposes into radicals and
reacts with the soluble and insoluble fractions of the substrates [90]. The optimal
ozone dosage is reported in the range of 0.05–0.5 g O3/g TS [86]. In SS-AD of
OMSW, a 37% increase in biogas yield was achieved with feedstock treated with
ozone at 0.16 g O3/g TS; however, higher ozone dosages (0.4 and 1.2 g O3/g TS) led
to a lower biogas yield, probably due to the formation of intermediate compounds
that are difficult to be digested [67].
Organic solvent is another chemical used in the treatment of lignocellulose-based
feedstock by removing lignin and thus improve degradability of lignocelluloses. For
example, in SS-AD of elm, pine, and rice straw, treating the feedstock with ethanol
prior to SS-AD enhanced CH4 production by 73%, 84%, and 32%, respectively [73].
6.1.3 Biological Treatment
Biological treatment relies on microorganisms and/or enzymes to break down the

recalcitrant structure of the feedstock. Enzymes such as peptidase, carbohydrase, and
lipase [86] are commonly added to the LS-AD system to speed up the digestion.
However, the practices of adding external enzymes to the SS-AD process have
not been widely reported. Microorganisms, such as white-rot fungi, capable of
decomposing lignin and altering the linkage between lignin and polysaccharides
are commonly used in SS-AD [91]. The fungi Pleurotus ostreatus and Trichoderma
reesei were used to decompose rice straw as an effective way to enhance CH4 yield
in SS-AD of this feedstock [74]. The white-rot fungus Ceriporiopsis subvermispora
is considered one of the most effective species to degrade lignin while preserving
cellulose [76]. Due to its selective degradation feature, C. subvermispora-treated
SS-AD led to a 20.9% lignin degradation of yard trimming and only 7.4% cellulose
degradation, achieving a 154% increase in CH4 yield in the subsequent SS-AD
[75]. When C. subvermispora was used to treat albizia chips, CH4 yield increased
3.7-fold compared to the untreated feedstock [76].
Composting, an aerobic process facilitated by bacteria and fungi, is another
biological treatment for SS-AD. Yan et al. [18] reported that composting rice
straw resulted in a decrease of 63.6% TS, while the total carbon did not reduce
significantly, proving that composting can effectively improve the biodegradability
of rice straw. In order to improve the composting efficiency, pre-aeration is often
used to generate enough self-heating to increase the temperature of OMSW for the
start-up of thermophilic AD without external heating [92]. Composting with
pre-aeration can also reduce the excessive organic compounds in feedstocks and
thus reduce the risk of VFA overproduction and acidification in the following
SS-AD [92]. However, excessive pre-aeration may cause toxic effect on
methanogens by introducing oxygen. For example, Zhou et al. [35] reported that
rice straw aerated for 2 days achieved the highest CH4 yield, while the CH4 yield
gradually decreased when the aeration times increased from 4 days to 8 days.
6.2 Co-digestion
Co-digestion of different feedstocks is commonly used to adjust carbon-to-nitrogen

(C/N) ratio of the substrates in SS-AD. Other advantages of co-digestion include
improved nutrient profiles, a more balanced microbial community, obtaining a
desired moisture content, and economic advantages by sharing equipment. However,
there are several drawbacks of co-digestion such as the extra logistical cost of the
different feedstock, premixing requirement, varied policy to control different waste
materials, and increased effluent COD [93].
The optimal C/N ratio for an AD process is in the range of 20:1–30:1. Most
lignocellulose has a higher C/N ratio than 30; therefore, blending lignocellulosic
feedstock with animal manures (with a C/N ratio less than 20) is a good approach to
balance C/N ratio of SS-AD system. Li et al. [61] reported co-digestion of poplar leaf
(C/N ¼ 35.4), and chicken manure (C/N ¼ 8.09) brought C/N ratio to the optimal
range (Table 4) and produced 15.28% more CH4 than digestion of poplar leaf only.
In addition to adjusting C/N ratio, co-digestion of different feedstocks also
provides other benefits such as better nutrients, diverse microorganism consortium,
Table 4 Co-digestion of different feedstock in SS-AD

Co- CH4
digestion C/N TS CH4 yield increment
Feedstock feedstock Mix ratio ratio (%) (L/kg VS) (%) Reference
Straw Pig slurry 1:3 41.3 20.7 240.8 N/A [94]
(weight)
Food waste Horse 1:1 20 20 370 N/A [20]
manure (weight)
Poplar leaf Chicken 2:1 (VS) 21.9 22 115.7 15.28 [61]
manure
Household Cow 1:1 11.1 15 247 10.7 [95]
organic waste manure (weight)
Tomato Dairy 13:33:54 25.1 20 415.4 50–1,020 [44]
residues/corn manure (weight)
stover
Straw Swine 1:0.23 20 27 300 N/A [96]
manure (weight)
Food waste Yard waste 1:9 (VS) 16.9 19.3 120 118 [97]
Food waste Distiller’s 1:8 (TS) 22.3 20 159.74 75.73 [98]
grains
Spent mush- Yard 1:1 (VS) 74.6 20 194 1,500 [99]
room trimmings
substrate Wheat straw 1:1 (VS) 71.9 20 269 2,200
Expired dog Corn stover 1:1 (VS) 32.3 22 304.4 229 [100]
food
Biological OFMSW 1:4 39.8 38.8 220.6 34 [101]
sludge (weight)
and stable pH and higher buffering capacity [61]. Khairuddin et al. [95] reported that
the co-digestion of household organic waste and cow manure, even with a low C/N
ratio of 11.1, still increased CH4 yield by 10.7% compared to digestion of household
organic waste only. Similarly, co-digestion of spent mushroom substrate and yard
trimmings (with a C/N ratio of 74.6) produced 16-fold higher CH4 yield than
digestion of spent mushroom only [99].
The ratio of the co-digested substrates is important for a successful SS-AD.
Li et al. [44] conducted a SS-AD of tomato residues, corn stover, and dairy manure
with eight mixing ratios. The authors reported that a mixing ratio of tomato residues,
corn stover, and dairy manure at 13:33:54 (TS based) achieved the highest CH4
yield, while digestion of tomato residues failed due to ammonia inhibition. Simi-
larly, co-digestion of food waste with distiller’s grains under four ratios (1:4, 1:6,
1:8, 1:10) showed that food waste vs. distiller’s grains ratio at 1:8 resulted in the
highest CH4 yield [98].
6.3 Additives
Various additives have been used to supplement AD systems to improve digestion

performance [102]. Biochar, a charcoal-like product of incomplete combustion
(pyrolysis) of organic materials, has been used as an additive in AD with multiple
functions. In a study of chicken manure AD, Liang et al. [103] found that adding
biochar reduced the lag phase by 41% and increased CH4 production rate by 18%
with reduced H2S. In another study of AD of sludge amended with biochar, average
CH4 content in biogas was up to 92.3%, corresponding to a CO2 sequestration by
66.2% [104]. A biochar addition also enhanced process stability through increasing
the alkalinity and alleviated free ammonia inhibition [104]. Qin et al. [105] used
magnetic biochar (a composite of biochar and magnetic medium) as an additive in
sludge AD and recorded 11.69% increase in CH4 production. The authors attributed
the enhancement to the selective enrichment of functional bacteria and methanogens
absorbed on magnetic biochar.
Materials promoting direct interspecies electron transfer (DIET) are also used as
additives to accelerate the conversion of organic substrate to CH4 [106]. For instance,
carbon cloth and granular activated carbon were used to stimulate CH4 production in
AD of dog food, tolerate high OLR, and recover from soured digester faster
[106]. Conductive materials were also effective in stimulating the syntrophic
conversion of ethanol to CH4 in upflow anaerobic sludge blanket reactor
[107]. The CH4 production rates increased by 30–45% with the addition of conduc-
tive materials at each OLR [107].
It should be noted that although various additives have been shown to be
beneficial to AD systems, few studies have been done to apply those additives to
SS-AD. Further studies are needed to evaluate the technical and economic feasibility
of using additive in SS-AD systems.
7 Conclusion and Perspectives
SS-AD has become a popular approach to digest organic wastes with high solid
content due to its inherent advantages such as high OLR, reduced reactor size, and
minimal amount of digestate generated. A variety of materials, from municipal solid
wastes to agricultural residues, can be used as feedstock for SS-AD. To ensure
a successful SS-AD, operation conditions such as nutrient levels, feedstock-to-
inoculum ratio, pH, temperature, and mixing need to be carefully controlled.
Moreover, reactor systems configured with different operation modes (batch vs.
continuous; one stage vs. multiple stage) have been applied based on diverse
characteristics of the feedstocks. To enhance SS-AD performance, pretreatment is
needed to make lignocellulosic feedstock more amenable for microorganism to
degrade. Co-digestion of different feedstocks and supplement external additives
such as biochar are also effective to improve biogas production.
Further studies on SS-AD should focus on several issues in order to develop an
effective commercial-scale process. First, feedstock pretreatment should be carefully
selected to address the operational costs, treatment effectiveness, and inhibitors.
Second, mass transfer limitation needs to be effectively overcome. Finally,
understanding the microbial consortium and metabolic pathways involved in
SS-AD processes is crucial to provide potential guidance to improve the digestion
performance. Solving these hurdles will facilitate the application of SS-AD as a
promising alternative to the traditional waste disposal process.
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Index
A enzymatic, 127–130
Amended malt agar plates, 116–117 esterification, 133–137
Amylases, 54–56, 60, 73 fermented solids
Aroma extract dilution analysis (AEDA), 92, 94 Rhizomucor miehei, 138–139
Aroma profile analysis Rhizopus microsporus, 137–138
Ceratocystis fimbriata, 95 synthesis, 127
fruiting bodies, 100 transesterification, 131–133
gas chromatography (GC) and volatile Biogas
organic compound (VOC), 87 CH4, 162
Ascomycetes, 54, 55, 57, 58, 60, 63, 67, 93, methanogenic phase, 155
95–97, 112, 113 microbial growth, 151
oxygen absence, 149
recirculation, 154
B solid-state anaerobic digestion (SS-AD),
Basidiomycetes 157, 159
amylases, 55 Bioreactors
and bacteria, 54 air preparation system, 35–36
enzymatic performances, 62 designs, 30–32
lignolytic enzymes, 70 final design, 32–35
mushrooms, 5 medicinal mushrooms (see Medicinal
oxidative process, 69 mushrooms)
pectinases, 60 monitoring equipment, 36–37
screening and categorization, 63 packed-bed bioreactor, 29–30
SSF (see Solid-state fermentation (SSF)) rotating drum, 12
VOC produced sampling, 37–39
fructification, 100–102 stirred aerated beds, 12–14
vegetative growth, 98–100 tray, 10–11
zone lines, 112 types, 29
Batch culture See also Solid-state bioreactors cultivation
liquid shake culture, 118
liquid stationary culture, 117–118
naphthoquinone pigments, 111 C
spalting fungi, 116 Chlorociboria spp., 112, 114–119
Biodiesel Co-digestion, 156, 157, 161–162
dry fermented solids (DFS), 131 Computational fluid dynamics (CFD), 48
169
170 Index
D challenges, 116
Dry fermented solid (DFS) DCM, 117
biodiesel production (see Biodiesel) incubation conditions, 116
cheaper source, 127–130 inducing production, 115
high-value sugar esters, 141–142 secondary metabolites, 110–111
modified fats, 141 soft rotting fungi, 113–115
proof of concept, 131 spalting, 111–113, 119
racemic mixtures, 139–141
Dynamic headspace (DHS), 89–90, 93, 95, 98
H
E Hydroesterification processes, 133, 138
Esterification, 128, 129, 131, 133–139, 142
Extracellular enzymes
bacteria and fungi, 53 I
industrially relevant Inoculum preparation
amylases, 55–56 Aspergillus, 41
lipases, 57–59 biomass, 18
pectinases, 60, 61 bioreactor (see Bioreactor)
proteases, 56–57 feedstock-to-inoculum ratio (F/I), 152
lignocellulolytic (see Lignocellulolytic filamentous fungi, 41
enzymes) microbe-containing leachate, 154
solid-state fermentation, 73 repeated cycles, 42
solid-state anaerobic digestion (SS-AD)
reactors, 151
F solid-state fermentation (SSF), 41
Feedstocks wetting solution, 42
co-digestion, 161 Intermittent mixing, 34, 154
high-quality, 128
liquid state anaerobic digestion
(LS-AD), 149
microstructure, 150 L
organic municipal solid wastes Lignocellulolytic enzymes
(OMSW), 150 basidiomycetes, 62
pretreated, 157 cellulases, 60, 62–67
pretreatment laccases, 69–73
biological, 160 peroxidases, 69–73
chemical, 159–160 xylanases, 62–69
physical, 157–159 Lipases, 11, 45–46, 57–59, 95, 127–130, 141,
solid-state anaerobic digestion 142, 151
(SS-AD), 149 Liquid state anaerobic digestion (LS-AD), 149,
Fermented food, 94, 97 150, 152, 153, 160
Filamentous fungi
cultivation, 30
inoculum, 41 M
soft cheese, 94 Mathematical modeling, 46–48, 136
SSF (see Solid-state fermentation (SSF)) Medicinal mushrooms
VOC (see Volatile organic compounds Asian pharmaceutical companies, 5
(VOC)) basidiomycetes, 5
Flavor dilution factor (FD), 92–94 packed-bed, 11–12
Fructification, 98, 100–102 polysaccharides, 6
Fungal biomass, 16, 18, 20, 22 submerged and solid-state bioprocessing,
Fungal dyes, 110, 111, 119 8, 9
Fungal pigments substrates, 14, 17
application, 119 Monitoring and sampling
Index 171
P microorganisms, 149
Packed-bed bioreactors mixing, 154
design and operation, 12 municipal solid wastes (MSW), 148
intermittent agitation, 29–30 nutrients, 151–152
pilot-scale (see Pilot-scale packed-bed pH, 152–153
bioreactor) single-stage vs. multistage operations,
substrate mass, 11 156–157
Pectinases, 39, 42–46, 54, 60, 142 temperature, 153
Pilot-scale packed-bed bioreactor Solid-state bioreactors cultivation
enzymes Cordyceps militaris, 21
pectinases, 43–45 Ganoderma lucidum, 17–18
sugarcane bagasse contents, 45–46 Grifola frondosa, 18–19
inoculation, 41–42 Hericium erinaceus, 20–21
inoculum, 41–42 medicinal mushroom biomass (see
and mathematical models, 46–48 Medicinal mushrooms)
substrate, 39–41 Trametes versicolor, 19–20
See also Bioreactors Solid-state cultivation (SSC)
Pretreatment, 9, 15, 18, 55, 157–160 agricultural residue, 6
Proteases, 54, 56–57, 60, 73 bioreactor types (see Bioreactors)
fungal biomass, 22
Ganoderma lucidum, 17
R Grifola frondosa, 19
Residues hyphal growth, 8
agricultural industry, 63, 67 inoculation density, 17
alkali neutralizes carboxylic acids, 159 medicinal mushroom, 16, 22
CH4 production, 152 SmB, 7
citrus juice-producing industry, 95 vs. submerged, 9
crop, 54 water activity, 7
G. lucidum, 17 Solid-state fermentation (SSF)
industrial/agricultural processes, 99 aroma analysis, 93
pectinases, 60 bioreactors, 46
pretreatment/enrichment, 6, 18 computational fluid dynamics (CFD), 48
protein-rich substrates, 57 DFS (see Dry fermented solids (DFS))
solid-state anaerobic digestion enzyme production, 53–54
(SS-AD), 162 fermented solids, 126
solid-state fermentation (SSF), 55 filamentous fungi, 30
Rotating drum bioreactors, 12, 13, 56 food manufacturing, 94
free liquid phase, 53
immobilized enzymes, 127
S laboratory-scale, 41
Scale-up, 46, 110, 111, 137 lipase, 131
Scytalidium cuboideum, 112–115, 117–120 microbial enzymes, 126
Scytalidium ganodermophthorum, 112, 114, nature fascinates, 53
117–119 packed-bed bioreactor, 29
Secondary metabolites, 110–111 pilot-scale bioreactor, 138
Solid-phase microextraction (SPME), 90–92, pine wood chips, 66
97, 99–101 R. microsporus, 40
Solid-state anaerobic digestion (SS-AD) solid-phase microextraction (SPME), 91
additives, 162 volatile organic compound (VOC)
batch vs. continuous operations, 155–156 produced, 95–102
co-digestion, 161–162 Solid wastes, 61, 148, 150
feedstocks (see Feedstocks) Spalting
feedstock-to-inoculum ratio (F/I), 152 applications, 119–120
inhibitors, 153–154 Chlorociboria spp., 116
inoculum, 151 fungi, 111–113
172 Index
Spalting (cont.) bacterial fermented solids, 131–133

interactions, 115 biodiesel esters, 131
pigments, 119 corn oil, 137
future developments, 119–120 esterification, 139, 142
ideal conditions, 116 fermented solids, 129–130
pigmenting, 113 in organic media, 127
zone lines and stain, 119 soybean oil, 134
Stir bar sorptive extraction (SBSE), 90–93 triacylglycerol, 128
Stirred aerated beds bioreactors, 12–14 vegetable oils, 127
Substrates wheat flour lipids, 57
agglomerates, 32 Tray bioreactors, 10–11, 68, 69
air jacket, 31 Two-phase model, 47, 48
composition and characteristics, 9
fungal biomass, 20
heat removal, 11 V
medicinal mushrooms cultivation, 14, 15 Volatile organic compounds (VOC)
pilot-scale fermentation (see Pilot-scale detection, 92–93
packed-bed bioreactor) dynamic headspace (DHS), 89–90
solid-state cultivation (SSC), 7, 18 fungal
temperature and humidity, 11 basidiomycetes (see Basidiomycetes)
water activity, 7 food manufacturing, 93–95
Substrate-specific enzyme formation non-food SSF, 95–97
extracellular enzymes (see Extracellular solvent extraction, 88–89
enzymes) solid-phase microextraction (SPME) and
production, 54 stir bar sorptive extraction (SBSE),
SSF (see Solid-state fermentation (SSF)) 90–92
T Z
Transesterification Zone lines, 112–116, 119

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Advances in Biochemical Engineering/Biotechnology 169

Series Editor: T. Scheper

Volumes are organized topically and provide a comprehensive discussion of

In references, Advances in Biochemical Engineering/Biotechnology is abbreviated

More information about this series at http://www.springer.com/series/10

Solid State Fermentation

ISSN 0724-6145 ISSN 1616-8542 (electronic)

© Springer Nature Switzerland AG 2019

Solid-state fermentation (SSF) is a well-established technology for cultivation of

Dresden, Germany Anett Werner

Part I Cultivation of Microbes Under Solid-State Fermentation

Part II Application of Solid-State Fermentation for Product Generation

Solid-State Anaerobic Digestion for Waste Management

Cultivation of Medicinal Mushroom

Abstract Basidiomycetes of various species and their wide range of pharma-

pharmaceutically active compounds. Research in physiology, basic and applied

Keywords Fungal biomass, Medicinal mushrooms, Solid-state bioreactors, Solid-

List of Symbols and Abbreviations

P Water vapor pressure

According to Wasser [1], the number of mushrooms on Earth is estimated at

Bioprocessing of various natural sources can be divided generally into processing of

In recent years, substantial credibility in employing solid-state cultivation (SSC)

Water activity is therefore deﬁned as the equilibrium relative humidity divided by

ψ ¼ RT=M ln P=P0 ð2Þ

• Depolymerization of lignocellulose and mechanical penetration through the

3 Differences Between Submerged and Solid-State

Table 1 Comparison of solid-state and submerged cultivation [14]

4 Bioreactor Types Used in SSC

4.1 Tray Bioreactors

Tray bioreactors consist of rooms or cabinets containing a number of individual

Tray chamber Rotating drum Stirred drum

Packed bed Gas-solid Stirred bed Rocking drum

Trays with perforated base

4.2 Packed-Bed Bioreactors

4.3 Rotating Drum Bioreactors

Rotating drum bioreactors are characterized as cylindrical reactors lying horizontally

4.4 Stirred Aerated Beds Bioreactors

AIR OUT AIR IN

Fig. 4 Horizontal stirred tank bioreactor [27]

Fig. 5 Horizontal stirred tank reactor and equipment [29]

5 Substrates for Medicinal Mushrooms Cultivation

In the wild, wood-degrading mushrooms grow primarily on hardwood of trees.

6 Cultivation of Medicinal Mushroom in Solid-State

An important advantage of solid-state cultivation over other techniques is that a

6.1 Cultivation of Ganoderma lucidum

Inoculation density is an important factor for the solid-state cultivation processes.

6.2 Cultivation of Grifola frondosa

Table 2 Cultivation of G. frondosa mycelium by solid-state cultivation in bioreactors

6.3 Cultivation of Trametes versicolor

6.4 Cultivation of Hericium erinaceus

The potential of using several agricultural by-products as supplements of sawdust

consisted of cornmeal and salt solution, H. erinaceus produced a strong α-amylase on

6.5 Cultivation of Cordyceps militaris

Reports on pharmacological activity of solid-state bioreactors produced G. lucidum

1. Wasser SP (2002) Medicinal mushrooms as a source of antitumor and immunomodulating

48. Berovic M, Habijanic J, Boh B, Wraber B, Petravic-Tominac V (2012) Production of Lingzhi or

Design and Operation of a Pilot-Scale

David Alexander Mitchell, Luana Oliveira Pitol, Alessandra Biz,