10.1007@978 3 319 25001 46

Solid-State Fermentation: Special
Physiology of Fungi 10
Javier Barrios-González and M. Rosario Tarragó-Castellanos
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 320
2 Solid-State Fermentation: Definition and Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 321
3 Modern SSF Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322
3.1 SSF Modern Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
3.2 Bioreactors for SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 323
3.3 Types of SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
3.4 Other Systems with Certain SSF-Type Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
4 Production of Primary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 325
5 Production of Enzymes and Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 326
6 Microbial Secondary Metabolites . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
6.1 General Aspects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
6.2 New Biological Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 327
7 Secondary Metabolites Production by SSF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
7.1 Secondary Metabolism in SSF: Physiological Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
8 Physiology of Solid Medium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
8.1 Growth Model . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
8.2 General Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331
8.3 Secondary Metabolism in SSF: Molecular Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 332
8.4 Enzyme and Protein Production in SSF: Molecular Studies . . . . . . . . . . . . . . . . . . . . . . . . . 335
8.5 Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 340
9 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 342
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 343
J. Barrios-González (*)
Departamento de Biotecnología, Universidad Autónoma Metropolitana–Iztapalapa, México DF,
Mexico
e-mail: [email protected]
M.R. Tarragó-Castellanos
Departamento de Biología de la Reproducción, Universidad Autónoma Metropolitana – Iztapalapa,
México DF, Mexico
e-mail: [email protected]
# Springer International Publishing Switzerland 2017 319

J.-M. Mérillon, K.G. Ramawat (eds.), Fungal Metabolites, Reference Series in
Phytochemistry, DOI 10.1007/978-3-319-25001-4_6
320 J. Barrios-González and M.R. Tarragó-Castellanos
Abstract
The evolution of higher fungi and actinomycetes took place on solid growth sub-
strates, so these microorganisms are perfectly adapted to grow in a solid environment.
This implies that their cultivation in liquid culture may impair their metabolic
efficiencies. However, conventional technology for the production of valuable fungal
products is liquid submerged fermentation. In recent times, solid-state fermentation
has become an alternative industrial production system to produce enzymes, primary
and secondary metabolites. There are several advantages in employing many solid-
state fermentation processes over the conventional submerged fermentation ones, like
higher yields of secondary metabolites or enzymes. Moreover, certain enzymes and
secondary metabolites can only be produced in solid-state fermentation. The main
advantages of this culture system are related to the special physiology displayed by
fungi when growing in solid culture. This chapter describes and analyzes recent
advances in our understanding of this special physiology (the physiology of solid
medium) and the underlying molecular mechanisms. It is also discussed how this
knowledge can be applied to create novel technological advances.
Keywords
Physiology of solid medium • Advantages • Gene expression differences •
Regulation • Applications
List of Abbreviations
Aw Water availability
cDNA Complementary DNA
CM Cellulase carboxymethyl cellulose
dsDNA Double-stranded DNA
GFP Green fluorescent protein
glaA Glucoamylase A
glaB Glucoamylase B
GSH/GSSG Redox balance
HSE Heat shock element
MSLC Membrane-surface liquid culture
PSM Physiology of solid medium
ROS Reactive oxygen species
SmF Submerged fermentation
SMs Secondary metabolites
SSF Solid-state fermentation
1 Introduction
Fungi are relatively simple eukaryotic organisms that perform biological processes
that are essentially based on the same principles as biological processes in higher
eukaryotes. These are interesting and useful microorganisms that show high levels of
protein secretion, metabolite production, and metabolic versatility.
10 Solid-State Fermentation: Special Physiology of Fungi 321
For these reasons, they are being increasingly used for the large-scale production
of enzymes and a wide range of (semi)bulk chemicals and pharmaceuticals, as well
as other products like high-value nutraceuticals (health and medical benefits).
Conventional technology for the production of these fungal products is liquid
submerged fermentation. However, the evolution of higher fungi and actinomycetes
took place on solid growth substrates, i.e., spent their evolutionary history as
terrestrial. In other words, they were designed by evolution to grow on moist solids,
so they are perfectly adapted to grow in a solid environment. This implies that their
cultivation in liquid culture may impair metabolic efficiencies and bring about
certain shortcomings.
Solid-state fermentation (SSF) is an ancient culture method that has been mod-
ernized to become an alternative industrial production system. SSF has been used to
produce enzymes, primary and secondary metabolites.
There are several advantages in employing many SSF processes over the con-
ventional submerged fermentation (SmF) ones, like higher yields of secondary
metabolites or enzymes. Moreover, certain enzymes and secondary metabolites
can only be produced in SSF. The main advantages of SSF are related to the special
or different physiology displayed by fungi and other microorganisms when growing
in solid culture. This physiology (i.e., a behavior that deviates from the one
displayed by the fungus in liquid medium) is sometimes referred to as “physiology
of solid medium.”
This physiology is just beginning to be understood, but recent advances show a
very wide and interesting scientific panorama with great technological potential.
In this chapter, general aspects of SSF are described, and how this different
physiology became apparent, by reviewing examples of proteins, enzymes, and
metabolites production. The last sections review recent advances in our understand-
ing of the molecular mechanisms underlying the physiology of solid medium, and
how this knowledge can be applied to create novel technological advances.
2 Solid-State Fermentation: Definition and Background
SSF is an alternative microbial culture system that has been used since antiquity, but
that has been modernized by research carried out in the last 25 years. By applying
modern concepts of microbiology, biochemistry, and biochemical engineering these
are more controlled processes that can be applied to more sophisticated ends.
Solid-state culture or solid-state fermentation (SSF) is defined as a microbial
culture that develops on the surface and at the interior of a solid matrix and in the
absence of free water [1].
At this point, it is important to clarify the use of the term fermentation, which in the
field of biochemistry is described as an anaerobic process: an energy production
process where the final acceptor of the electrons is not the oxygen but an organic
compound, pyruvate or a derivative. These compounds when reduced by the electrons
form useful products like ethanol or lactic acid. However, the term fermentation, in
industrial environments, is used as any type of microbial culture (aerobic or not).
The origins of SSF are related to the koji process in the Far East. The use of koji in
China was reported in the year 1000 BP but was probably used since 3000 BP [2],
and Buddhist priests took this tradition to Japan in the seventh century. Koji process
can be considered as a prototype of SSF. It consists of the cultivation of Aspergillus
oryzae on soybeans and other grains to produce proteases and amylases, which
degrade proteins and transform starch in sugars. In this way, the fermented material
is used for the production of soy sauce or, in a second stage, rice wine or saké.
In the West, the types of SSF had been used for the production of vinegar, gallic
acid for the tanning process. Even in the beginning of the twentieth century there was
production of primary metabolites such as enzymes and organic acids by microor-
ganisms in SSF.
Takamine [3] adapted the koji process, using wheat bran for the production of
taka-koji or “moyashi” obtained by drying the spent moldy bran. He later patented
the making of an alcoholic precipitate of a water extract of taka-koji [4] called
“distasic enzyme.” This precipitate was sold as a digestive aid called “taka-distase”
by Sakyo Co and was the pioneer of crude amylolytic extracts produced by SSF [5].
Underkofler [6] patented the use of rotating drums in order to produce amylase by
“moldy brans” or wheat bran fermented by A. oryzae. Underkofler et al. [7]
commented the advantage of koji moldy bran over malting or acid hydrolysis, to
improve the conversion of starch into ethanol. Later, Underkofler et al. [7] reported
the successful industrial scale-up of the moldy bran process, but using trays instead
of rotating drums because of the slow damage problems of fungal biomass by
mechanical stirring. The process was used to produce a crude amylolytic preparation
blended with cooked cereal grains that was the input for ethanol production. The
final ethanol yields were around 80 % of the theoretical yield. Hence, Takamine and
Underkofler planted the seeds of present bioethanol industries.
Although the earliest commercial production of enzymes depended on SSF
technology, the advent of sterile submerged culture techniques or liquid submerged
fermentation (SmF) in the 1940s displaced the solid-state methods in the western
countries. The discovery of penicillin in the 1930s, followed by streptomycin,
chloranphenicol, and tetracycline in the early 1950s, overshadowed the emerging
SSF process and emphasized on SmF.
In the 1970s, work performed by Hesseltine et al. [8–10] studied and described the
traditional SSF processes of the East, and informed the West about the great techno-
logical importance of these culture systems. These reports are probably responsible,
in great part, for the renewed interest in SSF observed during the last 30 years, with
important research woks that have transformed and modernized SSF.
3 Modern SSF Systems
As a consequence, solid-state fermentation (SSF) is being transformed for new

purposes, using new approaches. This has resulted in new SSF systems that often
present several advantages over submerged fermentation (SmF).
3.1 SSF Modern Applications
Recent years have witnessed a boom in the development of bioprocesses such as

production of feed (biotransformation of crops and crop residues for nutritional
enrichment), fuel, food, and industrial chemicals and biopesticides, as well as in
bioremediation, biobleaching, and biopulping [11].
Importantly, also the development of SSF production processes for high value,
low volume, high cost products like biopharmaceuticals or compounds of use in food
industry or agriculture, such as biologically active secondary metabolites including
antibiotics, cholesterol lowering drugs, alkaloids, plant growth factors, aroma com-
pounds, etc. Also, developments of applications for the production of proteins:
recombinant proteins as well as enzymes.
Lately, some problems of SmF, such as high-energy consumption and serious
pollution problems, are becoming increasingly notorious, significantly limiting the
sustainable development of fermentations.
Academic or industrial researchers are again taking notice of advantages of SSF
such as water saving, energy saving, and low costs. SSF has begun to play a role in the
chemical, pharmaceutical, and environmental fields, which points out a clear direction
for the sustainable development of the entire biological and chemistry industry [12].
As mentioned before, typical advantages of SSF process include lower capital
investment, low energy requirements, less water output, and very superior
productivity.
Nevertheless, large-scale applications are less abundant than expected due to
relative difficulty to control process parameters (pH, heat, nutrient conditions,
etc.), as well as less knowledge of the SSF process by western scientists.
Commercial operations using SSF processes have been developed in countries
such as Japan, India, U.S.A., and France. The successful operation of the companies
utilizing the SSF process ensured that the process gets its due attention.
Research on SSF is in great part devoted to search for substrates and optimization
of the production of different products, as well as potential new applications. The
deeper and more basic research is generally engineering oriented and studies fungal
physiology mainly from the standpoint of the effect of the main fermentation
parameters (T, moisture content, aeration, etc.) on growth. This has been the basis
for mathematical models that have been very useful for scale-up as well as for reactor
design and control.
However, much less attention has been paid to the special physiology shown by
fungi in SSF, i.e., a behavior that deviates from the one displayed by the fungus in
liquid medium and that includes the major advantages of this culture system, and
sometimes it is referred to as “physiology of solid medium.”
3.2 Bioreactors for SSF
The delay in SSF being the major mode of fermentation can be partially attributed to
the bioreactors initially available. During the initial phases mostly tray-type
fermenters were used with poor instrumentation support. Heat generated during the
culture was poorly dissipated.
However, research in this area has made important advances in fermenter design
and in developing sensors and measurements in SSF processes [13, 14].
Different bioreactor configurations include periodic pressure solid-state fermen-
ter, immersion, expanded bed and tray-type reactor, intermittent agitation rotating
drum type, and “Plafractor” [15], developed for production of enzymes, biocontrol
agents, and pharmaceuticals at industrial level.
3.3 Types of SSF
Besides the koji-type systems, SSF cultures are now performed on other amylasic
substrates like roots (cassava or potato flour), bananas, etc. Generally, agroresidues
including lignocellulosic materials, but also wastes of industries such as potato
chips, spent brewing grain, paper, and wood processing industries. This type of
SSF can be seen as a technique to utilize organic wastes as raw materials to produce
value-added products [16]. SSF on natural solid substrates has the advantage of
reducing process costs by using agricultural wastes, additionally contributing to
solve the environmental problems caused by their disposal.
Another type of SSF uses an inert support with absorbed liquid medium. The
support can be of natural origin like sugarcane bagasse, or artificial like polyure-
thane, amberlite or vermiculite, etc.
Hence, today, two types of SSF systems can be distinguished, depending on the
nature of the solid phase used: (a) SSF on natural solid substrates and (b) SSF on
impregnated inert supports [17].
This last SSF system was initially used for basic studies of SSF, since the
composition of the absorbed medium could be designed, and its constitution deter-
mined at any time of the culture. In addition, biomass concentration was more easily
quantified. Remarkably, production yields at least as high as the ones obtained in the
more common SSF on solid natural substrates are also reported in these systems
[17–20]. Moreover, its advantages for basic studies, as well as for industrial produc-
tion of high-value products such as metabolites, enzymes, and biocontrol agents,
have been assessed, as well as its economic feasibility [21].
In this way, in the SSF on inert support product recovery is less complicated
because the extracellular product can be easily extracted from the inert support and
products are obtained with fewer impurities [11], but medium costs are higher than in
the previous case.
3.4 Other Systems with Certain SSF-Type Behavior
Some culture systems have been developed as models to study basic aspects of SSF
or other biological phenomena, but also for applied purposes. These are systems
where the fungi display a physiology different from the one shown in SmF, although
not exactly the one in SSF; probably a midway physiology.
Interestingly, A. oryzae and other fungi, grown on a nylon membrane, placed over
an agar plate medium, show a SSF-type physiology. This has been very useful for
certain experiments and also because mycelia can be recovered and much more
easily prepared than from native SSF.
However, it has been shown that the presence of the membrane filter reduces
maximum respiration rate and biomass and α-amylase production [22]. This is
further supported by our observations that, although lovastatin specific production
increases in membrane cultures (in relation to normal petri dish cultures), this value
is still very far from the one displayed in real SSF [23] (see Sect. 8.4).
On the other hand, in what could be seen as applications of the physiology of
solid medium (PSM), some novel types of culture systems have been developed, for
applied purposes. Two interesting examples are the membrane-surface liquid culture,
and the so-called biofilm (liquid) reactors, both with potential industrial applications,
and are described in a later section.
4 Production of Primary Metabolites
Primary metabolites are those which have identifiable functions and play specific
roles in normal physiological processes, like amino acids and nucleic acids. These
include intermediates and end products of anabolic metabolism. Other primary
metabolites (e.g., citric acid, acetic acid, and ethanol) result from catabolic metab-
olism and their production is related to energy production [24].
Organic acids (like citric acid) are widely used in the food and beverage indus-
tries, but also find application as additive in detergents, pharmaceuticals, cosmetics,
and toiletries.
Recently, the importance of citric acid and fermentation (in the biochemical
sense) products, like ethanol, lactic acid, and others, is increasing due to the concepts
of white biotechnology and biorefineries. The idea of replacing oil and gas with
(renewable) biomass, mainly lignocellulosic agricultural residues, for biofuel pro-
duction and replacement of chemical synthesis by fermentation or biocatalysis
products, is very attractive. Functional groups that must be introduced by costly
oxidative process steps into naphtha are already present in organic acids, so these
compounds can be produced by fermentation and can then be used as building
blocks to obtain polymers, bioplastics, etc. Consequently, the field that investigates
microbial organic acid production is currently moving fast [25].
Examples are citric acid that has been on the market for some time, lactic acid,
which came to market in large scale only recently, and succinic acid, which (despite
the fact that a feasible industrial bioprocess has not yet been developed) has huge
potential.
The use of SSF is being reconsidered in this field. In fact, citric acid has been
produced under SSF conditions for many years, so this would facilitate the technol-
ogy transition needed to produce other organic acids by SSF. An important
advantage of SSF over SmF for citric acid production is that the presence of trace
elements does not affect the yield, as it does in SmF. Therefore, substrate
pretreatment is not required, saving time and energy [26].
Lactic acid and its derivatives are widely used in food, medicine, feed, chemicals,
and environmental protection. Recently, there has been increased interest because of
its applications in the production of polylactic acid that can be used to synthetize
biodegradable, biocompatible plastics and coatings. There have been reports of SSF
processes, using lactic acid bacteria or fungal strains (Rhizopus) by SSF, that claim
that the use of agroresidues in SSF could be significant in reducing production costs
[27, 28].
Itaconic acid is another promising building block for the polymer industries that
has also been produced by SSF on sugarcane bagasse with strains of Aspergillus
terreus [29].
5 Production of Enzymes and Proteins
Besides other applications, modern SSF systems have a record of successful appli-
cations for the production of microbial enzymes and secondary metabolites or
bioactive compounds [30].
There has been growing interest in SSF because the amounts of enzymes
(or heterologous proteins) secreted by filamentous fungi in this system are large
and very frequently exceed those secreted in submerged fermentation (SmF) [19, 20,
31, 32]. For example, it has been reported that in SSF on wheat bran Aspergillus
oryzae produced 500-fold higher yield of heterologous protein (chymosin) than in
SmF [33].
The enzymes produced by SSF include cellulases, hemicellulases, pectinases,
amylases, α- and β- galactosidases, caffeinase, tannase, proteases, etc. The major
support matrices used include brans (wheat, rice, barley), oil cakes (sesame, soy
olive, coconut, mustard), bagasse (sugarcane, cassava, orange) [34].
Regarding enzymes production, several evident differences between SSF and
SmF have been reported (Table 1). These include higher productivities and somehow
less regulated (probably higher regulation thresholds). In many cases, enzymes
produced in SSF have different characteristics in relation to the ones produced in
SmF: like higher optimal temperature or pH stability, different kinetic parameters, or
Table 1 Enzymes production. Special physiology shown by fungi in SSF that contrasts with the
one shown in SmF, in this table, characteristics related to enzymes production
1 Enzymes are generally produced in much higher concentrations in SSF
2 Some enzymes from SSF show different characteristics (molecular weight, kinetic
parameters, optimal conditions) in relation to the ones obtained in SmF
3 Some enzymes that are intracellular in SmF are extracellular in SSF
4 Strains that are good producers in SmF are not so good in SSF and vice versa
Modified from Ref. [23]
even enzymes that were intracellular in SmF that are secreted to the medium in SSF
[19, 35].
Moreover, in recent years SSF-specific enzymes like glucoamylase B or the
protease PepA have been identified (see Sect. 8.4).
6 Microbial Secondary Metabolites
6.1 General Aspects
Secondary metabolites are compounds with varied and sophisticated chemical

structures, produced mainly by actinomycetes and fungi, usually late in the growth
cycle. These compounds do not play a physiological role during exponential phase
of growth, so they have been described as SMs in opposition to primary metab-
olites that are essential for growth. Although antibiotics are the best-known
secondary metabolites (SMs), there are other such metabolites with an enormous
range of biological activities, hence acquiring actual or potential industrial
importance.
SMs are usually not produced during the phase of rapid growth (trophophase), but
are synthesized during a subsequent (production) stage: idiophase. From studies in
liquid medium, it is now known that production of SMs starts when growth is limited
by the exhaustion of one key nutrient: carbon, nitrogen, or phosphate source. For
example, penicillin biosynthesis by Penicillium chrysogenum starts when glucose is
exhausted from the culture medium and the fungus starts consuming lactose, a less
readily utilized sugar.
6.2 New Biological Activities
The last 25 years have been a phase of rapid discovery of new activities and
development of major compounds of use in different industrial fields. This new
stage has been driven by modern strategies to find microbial SM. Earlier whole cell
assay methods, like bioassays, are being replaced by new and sophisticated, target-
directed, mode-of-action screens. In this way, culture broths of new isolates are
tested in key enzymatic reactions or as antagonistic or agonistic of particular
receptors. This new approach relies on the knowledge of the biochemical and
molecular details of different diseases or physiological processes [36, 37].
This has allowed the development of major compounds of use in different
industrial fields, mainly pharmaceutical and cosmetics, food, agriculture, and farm-
ing. Some examples are anti-inflammatory, hypotensive, antitumor, anticholes-
trolemic, but also insecticides, plant growth regulators, and environmental-friendly
herbicides and pesticides. This growing wealth of bioactive compounds is usually
produced by SmF but many of these metabolites could be advantageously produced
by SSF.
7 Secondary Metabolites Production by SSF
In the production of SMs, SSF presents advantages like higher product yield, often in
shorter times and higher product stability, while some disadvantages are related to
more complicated scale-ups [38–40]. Perhaps an additional disadvantage or weak-
ness is the limited knowledge of PSM, so its full potential is still to be developed.
In any case, industrial SMs production is now a reality. Several years ago an
Indian company started industrial production of several SMs, and the Food and Drug
Administration approved this technology (SSF) for the production of metabolites for
human application [15].
In relation to production of SMs by SSF, the last years have witnessed not only an
increase in the number of publications but also by the increase in the proportion of
SMs with biological activities different from antibiotics. Another interesting feature
of this stage is the surprisingly high productivity of SMs obtained in the processes
designed in these studies, i.e., relatively high yields are quite common in recent
works. Interestingly, also the production of SMs from actinomycets, and even from
Bacillus species, have also been produced by SSF [17].
Other fungal SMs that have been produced in this culture system include the
cyclodepsipeptides dextruxins A and B, compounds that display insecticidal and
antiviral activity [41]. Also, the novel tetramic acid antibiotic conoicetin, which
shows a pronounced antibacterial and antifungal action, inhibiting even multidrug-
resistant strains of Staphylococcus aureus, has been produced by SSF. Interestingly,
the producing fungus, C. ellipsoida, synthesizes this antibiotic only in SSF, although
it grows well in SmF [42]. Another interesting case where new antibiotics were only
produced under SSF-type conditions was reported by Bigelis et al. [43].
Pyrrocidienes A, B and acremonidins A–E were only detected when the
corresponding fungal strains were grown on agar bearing moist polyester-cellulose
paper. Differences were also apparent in bioassays of the extracts against antibiotic-
resistant Gram-positive bacteria. These antibiotics that are produced only in SSF
remind the SSF-specific enzymes described in Sect. 8.4.
7.1 Secondary Metabolism in SSF: Physiological Studies
The physiological responses of fungi to growth in a solid environment could be

divided into responses that are similar to the ones displayed by the microbe in liquid
medium, and responses that are particular of growth in SSF. Although the use of the
concept PSM generally refers to the latter, the former are also integral part of this
physiology and have implications in the control of secondary metabolism in SSF.
7.1.1 Similarities with SmF

It has been observed that physiology of idiophase or secondary metabolism in solid
culture shares several basic characteristics with the well-known physiology studied
in liquid environment (SmF).
Using SSF on inert support, i.e., sugarcane bagasse impregnated with liquid
medium, our group performed basic studies on secondary metabolism in SSF. This
research showed that the same culture medium used for penicillin production by
Penicillium chrysogenum in SmF can be used to impregnate this kind of inert
support SSF, although using a higher medium concentration. The use of 2X concen-
trated medium caused a fivefold penicillin production increase in SSF, while it was
detrimental in SmF [44].
Moreover, respirometric studies together with glucose concentration kinetics in
this system showed that idiophase (in the case of penicillin and several other SMs) in
SSF also started exactly when growth was limited by exhaustion of one key nutrient,
as is the case in SmF. Results also suggested that the same mechanisms that regulate
penicillin biosynthesis in SmF (e.g., catabolite repression) regulated its production in
solid culture. In this way, C, N, or P regulation mechanisms are also operating
in SSF.
Moreover, experiments on cephalosporin C production have shown that pH
regulation of cephalosporin C is also active in SSF, and works in a similar way as
in SmF.
Studies in SmF have shown that cephalosporin C biosynthesis is regulated by pH,
and that it occurs in a relatively narrow pH range. Cuadra et al. [45] reported that in
SSF using sugarcane bagasse as inert support, biosynthesis of cephalosporin C only
took place in a defined range of pH. In a subsequent work, comparative experiments
SmF versus SSF showed that the antibiotic synthesis occurred at fairly the same pH
range (6.4–7.8) in both culture systems [46].
This basic knowledge has practical implications for SM production in SSF: the
same strategies of medium design, considering limiting nutrient, avoiding repressing
components, while including inducers and precursors, should be used in SSF [47, 48].
7.1.2 Particularities of Secondary Metabolism in SSF

Besides the nutritional and environmental factors that bring about metabolic reac-
tions similar to the ones observed in SmF, there are other factors in SSF conditions
that induce higher SMs production, or even biosynthesis of metabolites that are not
produced in SmF. These characteristics are part of the special physiology shown by
fungi in SSF, i.e., a behavior that deviates from the one displayed by the fungus in
liquid medium. Table 2 summarizes the main characteristics of this physiology in
relation to secondary metabolism. As can be seen, another apparent difference is the
effect of using concentrated media, which strongly stimulates the metabolite pro-
duction in SSF on impregnated support, while causing the opposite effect when used
in SmF.
A further confirmation that physiology required in solid medium is different from
the one in SmF came from the finding that enzymes or secondary metabolites
overproducing strains, generated for SmF, generally do not perform well in SSF
[49], and vice versa. In fact, this has dictated the need for methods to generate
overproducing strains particularly suited for SSF [17, 18, 39] particularly since
continuous improvement of the production strain(s) is a condition to make and
keep a fermentation industry competitive.
Table 2 Physiology of solid medium. Special physiology shown by fungi in SSF that contrasts
with the one shown in SmF, in this table, characteristics related to secondary metabolites production
1 Higher production, often in shorter times
2 Some secondary metabolites are only produced in SSF, like coniosetin, pyrrocidienes A, B
and acremonidins A-E
3 Concentrated medium increase production in SSF
4 Strains developed for SmF don’t perform well in SSF, indicating different functions are
needed to produce in SSF
5 Molecular differences in biosynthetic genes expression
8 Physiology of Solid Medium
The abovementioned cases, of different physiological responses to solid culture

conditions, raises questions the study of PSM strives to answer:
• Why are enzymes and secondary metabolites generally produced in much higher
concentrations in SSF?
• Why do some enzymes from SSF show different characteristics (molecular
weight, kinetic parameters, optimal conditions) in relation to the ones from
SmF; also, why many are enzymes that are intracellular in SmF extracellular in
SSF?
• Why are strains that are good producers of enzymes or metabolites in SmF not so
good in SSF and vice versa?
8.1 Growth Model
A current model of the microscopic events taking place during fungal growth in SSF
helps visualize some characteristics of growth in SSF. After germination, filamen-
tous fungi form hyphae that elongate at the tips and periodically form new branches.
Their morphology allows filamentous fungi to colonize the surface of the substrate
and penetrate into the substrate matrix in search of nutrients. The microbial biomass
inside and on the surface of the substrate secretes metabolites and enzymes and
consumes the liberated nutrients. Concentration gradients are needed to supply the
substrates and remove the products. Likewise, gradients in the concentration of
inducers and repressors may affect enzyme production [23, 50]. In this way, these
gradients are one of the noticeable differences between SSF and SmF and therefore
might be contributing to the observed differences in physiology [50].
It is considered that in SSF fungal hyphae form a layer of biomass on the substrate
particle, i.e., a three-dimensional net of hyphae with pores in between: (1) an upper
layer with aerial hyphae and air-filled pores and (2) a lower layer with densely
packed hyphae and liquid-filled pores; (3) underneath, hyphal tips penetrate the
substrate forming the third layer: penetrative mycelia [51] (Fig. 1). It has been
Aerial hyphae
Air liquid interface
Aerobic wet
hyphae layer
Anaerobic wet
Substrate hyphae layer
Penetrative
hyphae
Fig. 1 Model or schematic drawing of fungal growth in SSF, showing the different mycelial layers
(From Barrios-González [23])
proposed that A. oryzae might overcome oxygen limitation, in the deeper parts of the
wet mycelia layer, by forming abundant aerial mycelia, so rapid diffusion of oxygen
is expected there, but diffusion of nutrients and enzymes in the cytoplasm of the
hyphae is likely to be comparatively slow [50]. Recently, Viniegra-González
et al. [19] compared the productivity of three fungal enzymes using SSF and SmF
techniques. Thus, they proposed that the higher titers found in SSF than in SmF were
due to SSF cultivation works as a fed batch culture with fast oxygenation but slow
sugar supply.
8.2 General Physiology
A few works have studied deeper physiological differences that arise during the
growth of microbial cells in the two types of culture systems. In A. oryzae, low water
activity and osmotic stress are important environmental conditions particular of SSF
that bring about physiological differences. This is apparent from the accumulation of
polyols (glycerol, erythritol, and arabitol) in the cells under SSF conditions, while
only mannitol was accumulated under SmF conditions [52]. It is important to note
that glycerol and these other polyols are synthesized as a defense against osmotic
stress, suggesting that growth in SSF proceeds with this kind of stress. Moreover, we
have observed a strong expression of the osmotic stress defense gene gldB during
lovastatin SSF, indicating that Aspergillus terreus senses osmotic stress during the
course of SSF, but not in SmF [53].
Recently, we found that mycelium, growing in SSF, had a more reducing intra-
cellular environment than mycelium from SmF. Redox balance (GSH/GSSG) kinet-
ics were performed during lovastatin SmF and SSF. A high redox balance was
observed, in both culture systems, during the growth phase that dropped to a low
value during idiophase (around sixfold) [54]. It was later seen that this acute
reduction was related to regulation of lovastatin biosynthesis (see Sect. 7.1.2).
However, there was a difference between the culture systems, a fourfold higher
redox balance (in both phases) was observed in the mycelium cultured in SSF. The
meaning and consequences of this in the PSM remains to be unveiled.
8.3 Secondary Metabolism in SSF: Molecular Studies
Molecular studies related to secondary metabolism in SSF are very scarce. Our
group performed a series of studies at this level with the intention of obtaining a
deeper understanding of PSM, using lovastatin production as a model.
Lovastatin is a valuable SM produced industrially by Aspergillus terreus that
holds medical and industrial importance since it decreases cholesterol levels in
blood. In more recent research we developed a novel SSF production process on
polyurethane foam (artificial inert support). This system not only facilitates basic
studies but also induces very high lovastatin productivity. A 30-fold higher produc-
tion was obtained in SSF as compared with SmF conditions under analogous
conditions (Fig. 2). Specific production calculations revealed that each milligram
of (dry) biomass produced 815 μg of lovastatin in SSF vs 54.7 mg1 in SmF. In this
way, physiology of solid medium is clearly manifested in this system [55, 56].
Since higher SMs production is a clear characteristic of PSM, in these studies we
used this parameter to identify PSM, particularly specific production, to avoid
divergences only due to differences in growth between the culture systems.
20
0.7
A
Lovastatin mg/gdc
15 0.6
Lovastatin mg/ml
0.5
10 0.4 B
0.3
0.2
5
0.1
B 0
0 0 2 4 6 8 10
0 2 4 6 8 10
Time (days)
Time (days)
Fig. 2 Comparison of lovastatin production kinetics by Aspergillus terreus in SSF (a), and in SmF
(b), using the same culture medium. Lovastatin production in SmF (b) is shown again in the left,
using a much smaller scale of lovastatin concentration. Both cultures showed similar pH kinetics
(not show) (From Barrios-González [23])
8.3.1 Higher SMs Production and Higher Expression Levels

of the Biosynthetic Genes
A subsequent study was performed to establish the causes of the superior lovastatin
productivity in SSF. Results showed that the higher production in SSF was related to
a higher expression of the lovastatin biosynthetic genes and, most importantly, that
these higher transcription levels were the result of higher (4.6-fold) expression of the
regulatory gene lovE. This gene encodes the specific transcription factor for the
lovastatin biosynthetic genes cluster (Fig. 3). Moreover, it is very probable that this
higher expression of biosynthetic genes is an important underlying cause of the
higher production reported for other secondary metabolites [55].
These experiments showed that higher expression of the biosynthetic genes is an
important cause underlying higher SMs production.
In a later work we compared the cephalosporin C biosynthesis-related genes
expression in the SmF versus SSF. Results showed the existence of higher expres-
sion levels of the genes encoding for epimerase, expandase/hydroxylase, and metab-
olites exporting activities in SSF, probably explaining the increased antibiotic
production in this system [46].
a
SSF SmF
1 3 5 1 3 5 7 Time (days)
lovE
4.7 2.6 2.85 0.98 0.1 1 0.98 Densitometry
18S
b lovF
1.4 2.1 1.1 0 0 1 0.49 Densitometry
18S
Fig. 3 Northern blot analysis comparing the expression of genes lovE and lovF during the course
of lovastatin solid-state and submerged fermentations. Intensity of the bands was quantified by
densitometry and normalized values were expressed as a fraction of the highest expression value in
SmF, to which a value of one was assigned (From Barrios-Gonzalez [23])
These and other works indicate that there are certain environmental cues that
notify the fungus it is a solid medium. These stimuli must be sensed and then
transduced, triggering a number of events at a molecular level. These reactions
upregulate certain transcription factors, which in turn upregulate different groups
of genes. That is, groups of genes must be upregulated and others downregulated,
giving rise to physiology of solid medium [23].
8.3.2 Environmental Stimuli that Trigger PSM

In search for the solid culture environmental stimuli inducing PSM, Avila & Barrios-
González [57] showed that (1) direct contact with air, (2) the stimuli of the support,
and (3) water availability (Aw) are important environmental signals. This work
studied the effect of these stimuli on lovastatin specific production, which was
taken as the parameter indicative of either PSM or normal liquid environment
physiology. Results showed that direct contact with air is a major environmental
cue that induces the PSM, at least in relation to secondary metabolism.
It was also observed that the contact with a support (or with the “stimuli of the
support”) also induced a higher lovastatin specific production, although increases
were smaller than the ones observed with direct contact with air. It was concluded
that the added effects of stimuli of direct contact with air, the support, and in smaller
proportion low water availability, contribute to generate the PSM, i.e., the very high
lovastatin specific production obtained in SSF.
In relation to direct contact with air (identified as a major environmental cue), it
was considered that its effect on fungal physiology could be mediated by reactive
oxygen species (ROS).
This was an interesting idea since, although high ROS concentrations in the cell
are harmful, lower levels can function as signaling molecules in the cell. In recent
years, many authors have found evidence of a close association between ROS and
development and differentiation in fungi, for example, increased ROS levels have
also been detected during cell differentiation in A. nidulans [58, 59].
Regarding lovastatin biosynthesis, we recently showed a link between ROS and
lovastatin biosynthesis. It was shown that sod1 gene (oxidative stress-defense
enzyme) was intensely expressed during rapid growth phase (or trophophase), but
it was downregulated in production phase (idiophase). In that moment ROS levels
increased to high levels that remained during all production phase [54]. In a
subsequent work it was shown that ROS regulate lovastatin biosynthesis at a
transcriptional level, in both culture systems [60]. It is considered that transcription
factors, associated to signal transduction pathways related to oxidative stress, could
be the link between ROS and lovastatin biosynthetic genes.
Although ROS accumulation in idiophase happens in both culture systems, there
are differences that could explain higher lovastatin production in SSF: Surprisingly,
ROS concentration in idiophase was ten times higher in SmF in relation to SSF.
Probably equally important, ROS kept a much more steady level in SSF.
This agrees with the relatively lower sod1 expression level in SSF, as well as the
higher redox balance found in this culture system (see above).
Evidently, signal transduction pathways play a central role in sensing these

stimuli and triggering a chain of events resulting in the massive change in gene
expression observed in fungi growing in SSF. Our group has carried out studies in
this area (SAPK/MAPKinase and cAMP-PKA) and identified a different behavior of
some components of cAMP-PKA pathway (manuscript in preparation). LaeA is a
global regulator of secondary metabolism in fungi that is associated to this signaling
pathway. In a very recent work we overexpressed this gene in A. terreus and
increased lovastatin production was obtained. Lovastatin overproducing mutants
for SSF were much more abundant in the transformant population. In SSF, the
constitutive promoter-containing transformant T2laeAcons reached 30.6 mg of
lovastatin g dry culture1 [61].
We have performed research exploring the relation between ROS profiles and
lovastatin biosynthesis. Manipulation of ROS accumulation profile by genetic or
environmental means greatly impacted the metabolites production level. Silencing
genes encoding transcription factors involved in these signaling pathways generated
transformants displaying lovastatin production increases of 60 % in SmF and 70 %
in SSF (Pérez-Sanchez et al. Manuscript in preparation). The role of ROS in the
induction of the physiology of solid medium caused by direct contact with air is a
subject still under investigation.
8.4 Enzyme and Protein Production in SSF: Molecular Studies
Filamentous fungi are eukaryotic microorganisms commonly used for the produc-
tion of enzymes and metabolites. They are also considered as suitable hosts for
extracellular recombinant protein production, due to their high secretion potential
and their ability to perform posttranslational modifications [62].
As described in a previous section, Aspergillus oryzae is widely used in Japan and
other oriental countries to produce traditional fermented foods. Since it is known that
under SSF conditions it produces greater amounts of hydrolases (like amylases and
proteases) than in SmF, it is also used in commercial enzyme production in Koji-type
cultures.
Because of its industrial importance, it has been subjected to many molecular
biology studies that have significantly contributed to understand PSM. These studies
have contributed or helped to identify SSF-specific genes and given a deeper insight
in the genetic expression and regulation mechanisms involved, determining impor-
tant differences with SmF [23].
A series of studies on the glucoamylase glaB gene of A. oryzae became an
emblematic case, not only because it was the first SSF-specific enzyme identified
but because subsequent studies gave a deep and exciting view of its genetic
regulation, identifying environmental factors that induced its expression.
In SSF, this fungus produces great quantities of amylases and proteases [63], but
in SmF glucoamylase productivity is much lower. Although it was originally thought
that it was the same enzyme, Hata et al. [64] found that the enzyme produced in SSF
had a different amino acid sequence, and that this organism has two glucoamylase-
encoding genes: glaA and glaB. However, the expression of glaB is very high in SSF
but repressed in SmF, while glaA is mainly expressed in SmF. This meant that glaB is
a SSF-specific gene.
Ishida et al. [65] measured individual transcriptional efficiency of glaA and glaB
promoters under various culture conditions, by means of reporter genes (the glaB or
glaA promoter fused to the coding sequence of an easily detected gene product). As
expected both genes were induced by starch, but other physical factors were required
for maximal expression of glaB. They showed that this SSF-specific gene was also
induced by three environmental factors: (1) low water availability (Aw), (2) high
temperature, and (3) physical barriers to hyphal extension, that is, SSF-specific
conditions. In addition, it was shown that glucoamylase production in SSF was
regulated at a transcriptional level. It is important to note that during the koji
traditional process more amylase is generated at high temperature (40 C) than at
low temperature (30 C).
In relation to the third environmental factor, the authors observed that A. oryzae
grown on a nylon membrane, placed over an agar plate medium (physical barriers to
hyphal extension), showed significant induction of the SSF-specific glucoamylase
gene (glaB). Small pore size (0.2 mm) of the membrane (not letting the hyphaes pass
through) and high concentration of maltose (high Aw) in the medium were important
for strong induction.
This method has been useful because of the SSF-like conditions it simulates and
because mycelia can be recovered and more easily prepared than in a real SSF.
However, it is important to note that it has been shown that these model systems
present certain differences in metabolism and kinetics, in relation with SSF [23].
Interestingly, what these authors called “physical barriers to hyphal extension”
corresponds to the environmental cue that was called “support stimuli” in the work
on secondary metabolism (lovastatin production) described before. Moreover, in that
work, Aw was also identified as one of the environmental stimuli inducing PSM,
although its effect was smaller in the case of lovastatin.
It is worth remembering that a cis-acting factor is a short DNA sequence in the
promoter region of a gene, shown to be a binding site for a transcription factor
(regulatory protein), to control this gene’s expression.
In a later work of this group [66] cis-acting factors in the glaB promoter necessary
for high-level expression in SSF were identified. A deletion analysis indicated that
removal of a short region of the promoter (350 to 324) (Region A) produced fatal
loss of the promoter activity in SSF, but only loss of regulation in SmF. This region
contains two heat shock element motifs (HSE) (50 -AGAAN-30 ) and a GC box
(335 to 324). Substitution of the first HSE brought about lower promoter activity
although regulation was conserved, but substitution of the GC box caused lower
expression in SSF and induction by starch, Aw, and T was lost.
This region, together with the neighboring B Region, was used to construct
improved promoters for SSF. When eight copies of this 97-bp fragment (350 to
254) were tandemly fused to the glaB promoter, a 4.6-fold increase in promoter
activity was observed. This improved promoter showed a 4.1-fold increase in
recombinant glucoamylase production (glaA) in SSF, reaching 1,524 mg/kg-koji.
Table 3 Chronology of important events related to molecular basis of physiology of solid medium.
Year Secondary metabolites production

1990–2008 Classical regulatory mechanisms of secondary metabolites, like carbon or nitrogen
regulation, are also active in SSF. So, like in SmF, these mechanisms should be
by-passed (or taken advantage of) by manipulating the culture medium. However,
a higher medium concentration (at least 2X) is needed for high production of
secondary metabolites in SSF
2008 Lovastatin high production system: SSF on inert artificial support: 14-fold higher
specific production in SSF
2008 Higher lovastatin production in SSF is related to a fourfold higher expression of
pathway-specific transcription factor (lovE), and lovastatin biosynthetic genes
2011 Environmental stimuli for high lovastatin production in SSF include contact with
air, water activity (Aw) and physical barriers against hyphal extension
Enzymes and protein production
1997 Discovery of first SSF-specific enzyme: glucoamylase, and corresponding gene:
glaB
1998 Regulation of glaB gene: environmental stimuli that induce its production: low
water activity (Aw), physical barriers against hyphal extension and/or high
temperature (42 C)
2000 Identification of cis-acting factors (functional elements) in glaB promoter,
necessary for high-level expression in SSF: GC box and two heat shock elements
(f-HSE)
2002 Discovery of other SSF-specific enzymes like pepA gene. Its promoter does not
have a GC box, but it contains a HSE
2002–2010 Subtractive cloning, cDNA microarrays and proteomic analysis: This special
physiology is supported by a major change at a molecular level 4,628 genes are
differentially expressed between SSF and SmF. About half of the genes, expressed
only in SSF, are annotated as functionally unknown
2006 Protein production in SSF is regulated in a complex form, with different regulatory
circuits for different gene groups; one of which is similar to SmF. Other genes
groups respond exclusively to SSF- or SmF-environmental conditions, at
transcriptional and/or posttranscriptional levels
After that, other SSF-specific genes have been found. The pepA gene, which
encodes an acid protease, is also specifically expressed in SSF [67]; but unlike glaB
gene, pepA was not found to contain a GC box; but its promoter contains a HSE.
Hence, the molecular mechanisms for regulation of gene expression in SSF were not
as simple as originally thought (Table 3).
8.4.1 SSF-Specific Genes and Genes Expressed Differentially

Several years later (between 2002 and 2007), in the search for SSF-specific genes,
Japanese groups performed subtractive cloning on A. oryzae grown in SSF and SmF.
This method compares mRNAs from two conditions. cDNAs are generated and it
allows for PCR-based amplification of only cDNA fragments that differ between
both populations. The technique relies on the removal of dsDNA formed, by
hybridization between cDNAs common to both conditions, leaving only the cDNAs
that are differentially expressed.
Several SSF-specific genes were detected, but about half of them were annotated
as encoding functionally unknown proteins. This has been also found in other
studies and is indicative of the lack of knowledge in this field and also of the great
difference between these two physiologies.
On the other hand, about one half of genes expressed specifically in SSF encoded
for secreted or internal enzymes and for transport proteins. These transporters might
be necessary for the growth in vast amounts of the limited variety of raw materials,
with slower diffusion processes.
In 2005, a Dutch group [68] performed similar experiments with A. oryzae,
focusing on genes related to the different mycelial growth phenotype required in
SSF (polarized growth for colonization of the solid substrate). They identified
29 genes, strongly upregulated in SSF: Six encoded proteins were functionally
related to polarized growth, four encoded products involved in morphogenesis,
and three coded for cell wall composition proteins. The rest were unknown proteins.
The authors interpreted these findings as suggestive of important differences in the
organization of the cytoskeleton during growth in SSF, with potential impact on
intracellular distribution of several organelles and in polarized secretion of proteins.
Genes encoding proteins related to formation of aerial hyphae and attachment to
surfaces were also differentially expressed in solid culture.
These findings substantiate the idea that mycelium from solid culture is physio-
logically different from the one from SmF, and relate to the concept that support-
related stimuli are important environmental cues inducing PSM. In addition, these
advances have been the basis for genetic improvement methods described in
Sect. 8.5.
After this, and well into the omics era, many genes that are differentially
expressed in SSF have been identified by cDNA microarrays [69, 70] and proteomic
analysis [71]. More recently Wang et al. [72] applied a high-throughput
RNA-sequencing methodology (RNA-Seq) to a full-scale transcriptome analysis.
In this way, it was revealed that this special physiology is supported by a major
change at a molecular level: 4,628 genes are differentially expressed between SSF
and SmF.
These include 2,355 upregulated and 2,273 genes downregulated on SSF.
Upregulated genes were specifically located in the pathways of ribosome, DNA
replication, oxidative phosphorylation, and the TCA cycle. The authors interpreted
this as the capacities for protein translation/modification and energy production were
much more powerful in the fungus grown on SSF compared to SmF. They suggested
this could be due to hyphal differentiation and the faster growth observed in
SSF [23].
Again, about half of the genes that were expressed only in SSF are annotated as
functionally unknown.
Upregulated genes in SSF also suggested that protein folding is more efficient
under SSF conditions. In addition, results also indicated that the capacity for protein
glycosylation was greater under SSF conditions. These are all valuable
characteristics that could make SSF a competitive system for homologous and
heterologous protein production.
These findings can explain the results of Maruyama et al. [73] in an application of
SSF for the production of antigens for use in humans. They found that a better
glycosylation pattern in the pre-S2 antigen (hepatitis B virus) produced in SSF. In
SmF, A. oryzae secreted a heterogeneously glycosylated form of the fusion protein
that was partially degraded. Contrasting, wheat bran SSF resulted in the secretion of
a homogeneously glycosylated form of the whole fusion protein.
8.4.2 SSF: Proteomic Studies

Transcriptomic studies are of key importance to understand differences in gene
expression due to different cultural conditions. However, RNAs do not always
correlates with protein content. It is now known that mRNA is not always translated
into protein, and the amount of protein produced for a given amount of mRNA also
depends on other factors Proteomics confirms the presence of the protein and pro-
vides a direct measure of the quantity present. In addition, many proteins can
undergo a wide range of post-translational modifications. Many of these post-
translational modifications are critical to the protein's function. Hence, proteomics
provides a good complement to transcriptional analysis, since it reveals additional
information on posttranscriptional and secretion regulation.
Oda et al. [71] carried out a proteomic analysis comparing extracellular proteins
in SSF and in SmF. Although this work only focused on the secreted proteins
(secretome), it improved the global view of the complex way in which A. oryzae
controls protein production in response to solid-culture conditions. The authors were
able to identify 29 proteins. Taking into account the conditions under which they
were synthesized or produced, the authors divided these proteins into four groups:
Group 1 consisted of enzymes specifically produced in SSF, such as glucoamylase B
(glaB). Group 2 is formed by extracellular proteins specifically produced in SmF,
such as glucoamylase A (glaA). Group 3 consisted of proteins produced in both
culture conditions, such as xylanase G1. Group 4 consisted of proteins that were
secreted to the medium in the SSF, but trapped in the cell wall in the liquid culture,
such as amylase (TAA).
It was also observed that secretion of GlaB was regulated at transcriptional level,
while GlaA was regulated at the posttranscriptional level in SSF. This work revealed
that not only transcriptional regulation but also posttranscriptional regulation plays
important roles in protein production in SSF.
On the other hand, the regulation of Group 3 proteins substantiates the notion,
described before (Sect. 7.1.1), that one part of physiology of fungi in solid culture
shares a number of basic characteristics with the physiology displayed in liquid
environment (SmF).
Since then, different groups have reported several proteomic studies on this and
other fungal species. However, these studies have mainly been driven by the
“biorefinery” idea, and have focused on lignocellulosic degrading enzymes in the
secretome. Others have performed intra- and exaproteomics. Unfortunately, these
studies have been carried out either in SSF or in SmF, but very few have performed
comparative studies. In this way the panorama of this area has not changed substan-
tially. Comparisons of the whole proteome (intra- and exa-) in SSF vs SmF are badly
needed to complement the transcriptomic data available.
In a recent study Li et al. [74] compared cellulase activities and the secretomes of
Neurospora sitophila cultured in SSF and SmF using steam exploded wheat straw as
carbon source and enzyme inducer. The very high capacity of proteins secretion in
SSF was again seen. The total amounts of protein and biomass in SSF were
respectively 30 and 2.8 times of those in SmF. A great difference in these enzymes’
production was also confirmed. The CMCellulase, FPA, and β-glucoside activities in
SSF were 53–181 times of those in SmF. Many of the critical enzymes were
produced in both culture systems, although a β-xylosidase was exclusively identified
in SSF.
Interestingly, the nonenzyme proteins in SSF were involved in fungal mycelia
growth and conidiation while those in SmF were more related to glycosemetabolism
and stress tolerance. The authors discuss that SSF more likely serves as a natural
habitat for filamentous fungi to facilitate the enzyme secretion.
The fact that an important part of nonenzyme proteins, more actively produced in
SmF, were related to stress tolerance suggests that, against what has been thought in
the past, fungal life in SmF is more stressful that in SSF. This agrees with very recent
results of our group indicating that mycelium was subjected to cell wall stress in
SmF, but not in SSF (Bibián et al. manuscript in preparation).
8.5 Applications
Advancement in the understanding of PSM could be applied to better control

metabolism and direct product formation in SSF processes. In fact, some of these
basic findings are starting to be applied to the construction of strains, particularly
suited for SSF.
Based on the model of fungal growth in SSF, where there can be an oxygen
limitation, in the deeper parts of the wet mycelia layer, described in Sect. 8.1; Te
Biesebeke et al. [68] identified a hemoglobin domain of gene fhbA, encoding a
flavohemoglobin, i.e., a protein that can attract and bind O2. The authors cloned and
overexpressed this protein domain in A. oryzae. The transformants displayed slightly
higher growth on SSF, as well as higher amylase, protease, and particularly
glucoamylase activities. This indicated that this strategy could be used as a molec-
ular genetics strain improvement method for protein production under SSF.
Findings related to SSF-specific enzymes in A. oryzae have also been used in the
construction of improved promoters for SSF that can be used in the production of
enzymes or recombinant proteins.
Examples of applications, derived from the advances in the PSM in A. terreus,
include the overexpression of a component of a signal transduction pathway (cAMP-
PKA) apparently involved in transducing stimuli of solid medium. Transformants
overproducing lovastatin in SSF were very abundant and displayed important
lovastatin production increases (described in Sect. 7.1.2). In another work, the
silencing of a gene, encoding a transcription factor associated to the SAPK/

MAPKinase signaling pathway, changed the ROS accumulation profile during the
course of the culture, inducing 70 % higher lovastatin production in the transformant
(described in the same section).
A deeper understanding of the PSM can also be applied to the development of
new types of SSFs, hybrid SSF–SmF systems, or even novel SmF systems that will
include some solid culture stimuli. One example of the latter is the one developed by
Shoji et al. [75]. The authors found that higher enzyme productions were obtained
when whole barley grains were used in SmF, instead of milled whole barley
glucoamylase and α-amylase very probably involving support stimuli.
The group of Nakanishi has reported that fungi cultivated by membrane-surface
liquid culture (MSLC) show cultivation behaviors that are similar to those cultivated
on agar-plate culture [76–78]. They showed that neutral protease, α-glucosidase, and
kojic acid are produced at much higher levels by MSLC than by SmF, in a manner
similar to SSF. The authors claim that MSLC could be an efficient production
system [78].
Recently the term biofilm has been extended to the surface-associated growth of
filamentous fungi. In opposition to immobilized-cell reactors, in a fungal biofilm
reactor, biomass naturally adheres and colonizes the surface of an inert support in
contact with a liquid medium [79, 80]. In other words the cells will sense what was
called here the “support stimuli.”
In a very recent study, Zune et al. [81] tried to apply concepts of PSM to the
production of recombinant proteins by A. oryzae. Considering that BfR conditions
were at least partially similar to SSF the authors studied the production of a GlaA::
GFP (Green Fluorescent Protein) fusion protein, under the control of the glaB
promoter, that is, an SSF-specific promoter. In addition, they compared this system
with a typical SmF (stirred tank bioreactor). Although this work represents an
advance in the right direction, results were unpredicted. Unexpectedly, the fusion
protein was also produced in SmF, despite the use of the glaB promoter. Moreover,
the best yield was obtained in the classical stirred tank reactor, but involved
alteration of the recombinant product. On the other hand, production in the BfR
enhanced stability of the recombinant product. Expression of the glaB promoter in
SmF appears to be related to the shear rate, since when this reactor was operated at
200 rpm (instead of 800 rpm) no excretion of the fusion protein was observed.
In addition, it has become apparent that basic aspects of the PSM, found in studies
with A. oryzae, can be applied to other fungal species, even phylogenetically distant
fungi like Rhizopus. Xu et al. [82] found that R. chinensis produced a wide range of
lipases that were able to synthesize useful flavor esters from free fatty acids and
alcohols. Particularly, this fungus produced lipase Lip1 with high synthetic activity
and that turned out to be a solid-state specific enzyme, so it was produced in large
amounts in SSF [83]. As in the case of GlaB, low water activity played a significant
role in the induction of Lip1, and they were able to increase the expression level of
this lipase in SmF (20–46 mg g dm1) when decreasing water activity of the liquid
medium. Again, physical barriers against hyphal extension were found to be another
required factor. Expression of Lip1 was significantly enhanced (in agar plates) by
threefold, so enzyme production reached 388.4 mg g dm1. When this growth

barrier effect was combined with low water activity (in petri dish), specific produc-
tion increased to 921.5 mg g dm1 [84].
This study on enzymes production, together with others, and our own work on
SMs, indicate that the knowledge of physiology of solid medium, generated in
studies with A. oryzae or A. terreus, is of general nature and can be applied to
processes with other fungi.
9 Conclusions
Many primary metabolites show advantages when produced by SSF. However, the
most impressive results are seen in the area of SMs and enzymes production by SSF.
These compounds are often produced at much higher yields in SSF, and certain SMs
and certain enzymes are only produced under SSF conditions. This is considered to
be part of the special physiology displayed by fungi (and other microorganisms) in
SSF and that has been called physiology of solid medium [23].
These capacities have called the attention of researchers and made that recently
PSM has acquired importance in hot research topics like lignocellulosic residues
biotransformation (to biofuels or bulk chemicals), or the search for conditions to
awake SM biosynthetic gene clusters, which have been found by genomic mining.
Studies on the biosynthesis of secondary metabolites in solid culture, mainly
using the model of lovastatin production, have shown that the solid culture environ-
ment induces higher transcription of the specific transcription factor (LovE) as well
as the biosynthetic genes, and hence higher production. These studies showed that
important environmental factors, inducing this different physiology, are (1) direct
contact with air, (2) support stimuli (physical barriers to growth), and with a lower
impact, (3) low Aw.
The last two stimuli have also been identified in the field of enzymes (and
proteins) production. Research using SSF-specific enzyme glucoamylase GlaB as
a model identified physical barriers to growth and low Aw as inducing environmen-
tal cues. Moreover, the study of cis-regulatory elements, identified in the promoter of
this and other SSF-specific genes, together with research described below, have
given a deeper insight in the genetic expression and regulation in SSF.
Transcriptomic and proteomic studies have revealed that this special physiology
is supported by a major change in gene expression. An impressive figure of 4,628
genes was found to be expressed differentially between SSF and SmF. Also, that
protein production is controlled in response to solid-culture (or liquid) conditions.
Genetic expression is regulated in a complex form, with at least four different
regulatory circuits, one of which is similar to SmF, i.e., does not respond to culture
system, but only to medium components. Other gene groups respond exclusively to
SSF- or SmF-environmental conditions, at transcriptional and/or posttranscriptional
levels.
A great proportion of molecular studies were performed on A. oryzae, but this
knowledge has been found to explain the behavior of other fungi in SSF. These basic
findings have already found applications in technologies like genetic improvement

methods to generate overproducing strains for SSF. Moreover, it is also starting to be
applied to design novel culture systems, and other technological advances. Eventu-
ally, this understanding will surely start having an impact in other applications of
SSF like production of feed, fuel, food, and industrial chemicals.
Acknowledgment This work was financially supported by CONACYT, México. Project

CB-2013-01 222028.
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Solid-State Fermentation: Special

# Springer International Publishing Switzerland 2017 319

2 Solid-State Fermentation: Definition and Background

3 Modern SSF Systems

As a consequence, solid-state fermentation (SSF) is being transformed for new

3.1 SSF Modern Applications

Recent years have witnessed a boom in the development of bioprocesses such as

3.2 Bioreactors for SSF

3.3 Types of SSF

3.4 Other Systems with Certain SSF-Type Behavior

4 Production of Primary Metabolites

5 Production of Enzymes and Proteins

6 Microbial Secondary Metabolites

6.1 General Aspects

Secondary metabolites are compounds with varied and sophisticated chemical

6.2 New Biological Activities

7 Secondary Metabolites Production by SSF

7.1 Secondary Metabolism in SSF: Physiological Studies

The physiological responses of fungi to growth in a solid environment could be

7.1.1 Similarities with SmF

7.1.2 Particularities of Secondary Metabolism in SSF

8 Physiology of Solid Medium

The abovementioned cases, of different physiological responses to solid culture

8.1 Growth Model

8.2 General Physiology

8.3 Secondary Metabolism in SSF: Molecular Studies

8.3.1 Higher SMs Production and Higher Expression Levels

4.7 2.6 2.85 0.98 0.1 1 0.98 Densitometry

1.4 2.1 1.1 0 0 1 0.49 Densitometry

8.3.2 Environmental Stimuli that Trigger PSM

Evidently, signal transduction pathways play a central role in sensing these

8.4 Enzyme and Protein Production in SSF: Molecular Studies

Year Secondary metabolites production

8.4.1 SSF-Specific Genes and Genes Expressed Differentially

8.4.2 SSF: Proteomic Studies

Advancement in the understanding of PSM could be applied to better control

silencing of a gene, encoding a transcription factor associated to the SAPK/

threefold, so enzyme production reached 388.4 mg g dm1. When this growth

ﬁndings have already found applications in technologies like genetic improvement

Acknowledgment This work was ﬁnancially supported by CONACYT, México. Project

19. Acuña-Arguelles ME, Gutierrez-Rojas M, Viniegra-Gonzalez G, Favela-Torres E (1995) Pro-

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