Biochemical Engineering Journal 13 (2003) 127135

Some engineering aspects of solid-state fermentation

K.S.M.S. Raghavarao a , T.V. Ranganathan a , N.G. Karanth b,
b

a Department of Food Engineering, Central Food Technological Research Institute, Mysore 570 013, India
Department of Fermentation Technology and Bioengineering, Central Food Technological Research Institute,
Mysore 570 013, India

Received 18 March 2002; accepted after revision 24 July 2002

Abstract
Solid state fermentation which involves growth of microorganisms on moist solid substrates in the absence of free flowing water, has
gained considerable attention of late due its several advantages over submerged fermentation. Solid-state fermentation is also finding
increased application in the production of enzymes, antibiotics, surfactants, biocides etc. as also for the production of value-added products
from wastes. There have been significant additions to the science and engineering knowledge of solid-state fermentations in recent years.
This paper aims to present an overview of these developments emphasizing important aspects such as mass and heat transfer, design,
scale-up, monitoring and control.
2002 Elsevier Science B.V. All rights reserved.
Keywords: Solid-state fermentation; Microorganisms; Mass transfer; Heat transfer; Bioreactor design; Monitoring and control

1. Introduction
Solid-state fermentation (SSF) involves the growth of microorganisms on moist solid substrates in the absence of free
flowing water. The necessary moisture in SSF exists in absorbed or complex form within the solid matrix, which is
likely to be more advantageous for growth because of the
possible efficient oxygen transfer process. In SSF, the water content is quite low and the microorganism is almost in
contact with gaseous oxygen in the air, unlike in the case
of submerged fermentation (SmF). The water activity in the
substrate is also important. The SSF process in the context
of this article mainly refers to one that is conducted under
controlled conditions and is useful in producing valuable
products like enzymes or secondary metabolites [1,2]. The
principles and engineering aspects of SSF have been earlier
reviewed by Moo-Young et al. [3] and Ramanamurthy et al.
[4].
In recent years, research interest in solid substrate fermentation has addressed several problems on the production of protein-enriched feed from starchy materials, single
cell protein (SCP) production from a variety of wastes,
ethanol from cassava roots and sugar beets, enzymes, organic acids, biogas, antibiotics, surfactants, bioremediation
agents, mushrooms, microbial polysaccharides, biocides,
Corresponding author. Tel.: +91-821-513-658; fax: +91-821-517-233.
E-mail address: ferm@cscftri.ren.nic.in (N.G. Karanth).

and mycotoxins from corn, etc. The use of mixed culture on

cellulosic wastes to improve the flavor and protein content
of such food products is a new interesting approach. SSF is
often simpler and requires less processing energy than SmF.
The low volume of water present in the media per unit mass
of substrate can substantially reduce the space occupied by
the fermentor without severely sacrificing the yield of the
product. Aeration and mixing requirements may also be
easily met. On the negative side, SSF processes are slower
than the liquid fermentations due to the additional barrier
from the bulk solid. They also present heat dissipation problems, which can be limited by inter as well as intraparticle
resistances and are more difficult to control due to the lack
of adequate sensors and efficient solid handling techniques
especially for continuous operations.
Many microorganisms are capable of growing on solid
substrates, but only filamentous fungi can grow to a significant extent in the absence of free water. Bacteria and
yeasts grow on solid substrates at 4070% moisture level,
such as in composting, anaerobic and aerobic ensiling, but
the growth and propagation of single cell organisms always
require free water.
While there have been reviews on various aspects of SSF
by Ramanamurthy et al. [4], Mitchell et al. [5] and Pandey
et al. [6], this paper presents an overview of the recent developments with special emphasis on bioreactor design and
control. An attempt has also been made to enumerate the
applications of SSF in various phases of development.

1369-703X/02/$ see front matter 2002 Elsevier Science B.V. All rights reserved.
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K.S.M.S. Raghavarao et al. / Biochemical Engineering Journal 13 (2003) 127135

2. Mass transfer aspects

The mass transfer processes involved in SSF can be divided into micro-scale phenomena and macro-scale phenomena. At the micro-scale, the mass transfer processes depend
on the nature of the micro-organisms, i.e. whether growth
occurs as mycelium or a biofilm of unicellular organisms as
also on their response to changes in the environmental conditions. Growth of the microorganisms is dependent on the
inter- and intraparticle diffusion of gases like O2 and CO2
as also enzymes, nutrients and products of metabolism.
At the macro-scale, mass transfer processes occurring
include:
(a) The bulk flow of air into and out of the bioreactor and
as a consequence changes in the sensible energy and
concentrations of O2 , CO2 and water.
(b) Natural convection, diffusion and conduction taking
place in a direction normal to the flow of air during
unforced aeration.
(c) Conduction across the bioreactor wall and convective
cooling to the surroundings.
(d) Shear effects caused by mixing within the bioreactor,
including damage to either the microorganism or the
integrity of the substrate particles.
2.1. Diffusion of oxygen and nutrients
The transfer of oxygen from the void fraction within the
solid phase to the growing microorganism is the interparticle
mass transfer [3]. The volume occupied by the air within the
substrate gives the void fraction, which itself is dependent on
the substrate characteristics and the moisture content. The
moisture content should be optimal. If it is too high, the void
space is filled with water and the air driven out, which creates
anaerobiosis. At the other extreme, if the moisture content
is too low, the growth of microorganism will be hindered.
Mixing and aeration provide means of achieving interparticle
oxygen transfer, the efficiency of which is influenced by the
void fraction and moisture content of the solid substrate.
However, at high values of the void fraction, mixing and
aeration may not be critical as the voids contain enough
oxygen to sustain the growth of cells.
Intraparticle mass transfer refers to the transfer of nutrients and enzymes within the substrate solid mass [7]. The
main aspects that need to be considered here are the diffusion of oxygen into the substrate containing the biomass and
the degradation of solid substrate by enzymes secreted by
the growing microorganisms. In dealing with intraparticle
mass transfer, the effectiveness factor (Ef ) is a very useful
concept. It is defined as the ratio of the observed reaction
rate (robs ) to the rate in the absence of any substrate concentration gradients. This concept, which helps in quantifying
the diffusional limitations in heterogeneous catalysis, is also
applicable to SSF systems. An important parameter required
for the evaluation of the effectiveness factor is the Theile

modulus () which is a measure of the ratio of biochemical

reaction rate to the rate of differential mass transfer within
the solid. By making use of this concept, a criterion was developed to evaluate intraparticle mass transfer limitation for
the case of first-order reaction rate kinetics [8].
Mitchell et al. [9,10], studied the diffusional limitation of
glucoamylase in a gel substrate in which starch was embedded. They showed that due to the diffusional limitation, the
enzyme concentration tended to be more at the surface, resulting in a rapid utilization of starch at the surface. This resulted in a drastic reduction in the rate of glucose production,
reaching a value as low as 20% of the activity that would be
expected if all the enzymes were in contact with the starch.
In their work, the biomass had no structure and diffusion of
glucose within the biomass layer was not considered. Furthermore, the role of oxygen diffusion and consumption at
the intraparticle level was not considered. Rajagopalan and
Modak [11] developed a model for the growth of a unicellular organism in a biofilm of constant density. They concluded that oxygen is more likely to limit the growth within
the film than lack of glucose even under conditions where
the outer edge of biofilm is high in oxygen concentration
[11,12].
Bischoff [13] developed an extended hypothesis to assess
the mass transfer limitations by defining a generalized form
of the Theile modulus valid for any reaction order and particle shape. This could be very useful since in most of the
cases the rates of the biochemical reactions are highly nonlinear. Experimentation to check the probable application of
these criteria to SSF as well as the development of more appropriate criteria suitable to SSF would be of immense use.
Oxygen diffusion into mold pellets has been extensively
studied in SmF. While the mass transfer phenomenon in SSF
is different, involving growth of microorganisms on and into
the solid substrate particles, the analysis of oxygen diffusion in mold pellets is useful in understanding the situation
in SSF. This is because of the fact that intraparticle concentration gradients, coupled with mass transfer limitations
affect the performance of a process. Oxygen is an important
substrate for fungal growth. In SSF, as the fungal mycelium
develops on a solid surface, the void spaces between the
hyphae can either be fully or partially filled with water. In
the former case, the situation is similar to SmF, resulting
in severe oxygen limitation leading to anaerobic conditions
affecting the performance of SSF [14]. Moo-Young et al.
[3] and Mitchell et al. [10] studied the growth of Rhizopus
oligosporus on model substrates in SSF and showed that
diffusive process limits the rate of growth, especially within
the substrate. This is because, to reach the interior, oxygen
must pass through the actively respiring biomass at the substrate particle surface and then diffuse through the aqueous
phase within the substratum. Literature information on oxygen transfer capabilities of fermentors involving complex
heterogeneous three-phase systems such as SSF is sparse
[15,16]. This is partly due to the inadequacy of the existing techniques for measuring the oxygen transfer coefficient

K.S.M.S. Raghavarao et al. / Biochemical Engineering Journal 13 (2003) 127135

(kL a) in these complex situations. Andre et al. [17] have suggested an improved method for the dynamic measurement
of mass transfer coefficients for SSF systems. Durand et al.
[18], adapting a method used in liquid culture, used sulfite
oxidation rates to estimate the mass transfer coefficient in
a packed bed bioreactor. Gowthaman et al. [19] reported a
method for the estimation of overall mass transfer coefficient
in SSF on the basis of the inlet and outlet oxygen concentrations, assuming that the decrease in oxygen concentration
within the liquid film was always 10% of the saturation concentration. For mycelial growth in SSF, the fungal hyphae
exposed directly to the air can probably take up oxygen directly from the air. It is well reported that the productivity in
SSF is much higher than in SmF. One of the main reasons is
the high interfacial area per unit volume. But this alone cannot explain completely the observed effect. There is no free
flowing water in SSF and the water content is just sufficient
to moisten the substrate. It is practically impossible to have
water film uniformly around all the substrate particles or the
substrate clusters or lumps. It was hypothesized that the microorganism takes oxygen directly in the places of substrate
where the water film is not present and hence much less
mass transfer resistance and in turn higher productivity.
In systems with forced aeration, oxygen transfer to these
aerial hyphae is less likely to be rate-limiting [20]. In this
case, nutrient movement within the aerial hyphae layer might
be the factor controlling growth. Diffusion is the most probable mechanism for translocation of at least some nutrients,
such as glucose and orthophosphate, within the hyphae of
some fungi, including Rhizopus nigricans [21]. Oostra et al.
[14] studied the effect of intraparticle oxygen limitation on
the growth of R. oligosporus on different media. Results
indicated that optimal oxygen transfer and hence aerobic
growth in SSF is dependent on increasing the gasliquid interfacial area as also the thickness of the wet fungal layer.
The interfacial area could be increased by either reducing the
particle size of the substrate or by some pretreatment techniques such as steaming, puffing, extrusion, etc. that help to
increase the pore size of the particles. This not only aids easy
oxygen transfer but also helps in making the substrate more
accessible for the action of enzymes of the fungi. However,
the optimum particle size needed for such an effect needs to
be experimentally found. The thickness of the fungal layer
depends on the moisture content of the substrate. Hence,
optimum intraparticle oxygen transfer could be achieved by
controlling the moisture content of the particle, which in
turn is dependent on the flow rate and relative humidity of
the incoming air.
2.2. Diffusion of enzymes
Diffusion of enzymes and substrate fragments is another
important aspect of intraparticle mass transfer in SSF. For
the most part, the substrate is water insoluble, whereas the
organism can utilize only water-soluble substrate for growth
[2224]. For this reason, the action of extracellular enzymes

129

in degrading the solid substrate into soluble fragments is a

very important step in SSF. If the mass transfer resistance
is very high, this could even be the rate-controlling step.
The diffusion of enzymes is facilitated by the open pore
structure of the substrate, and the degradation can happen
inside the substrate. In this case, the water-soluble fragments
of the substrate will have to diffuse out of the solid matrix
into the bulk region, where further enzymatic action will take
place and metabolizable compounds are formed. However,
when the porosity of the substrate is low, a major portion of
the degradation will occur at the outer surface of the substrate [25,26]. In either mode of enzymatic action, the solid
and polymeric materials are modified so that they enter the
cell and serve as carbon or energy sources. Thus, utilization of solid substrates by the microorganisms is affected by
factors that are relatively unimportant for the growth of microorganisms in SmF where the substrate is soluble and can
penetrate the cell membrane. These studies indicate that as
the substrate cannot reach the microorganism, by circulation
currents caused by mixing, as in the case of SmF, the substrate bed needs to be mixed intermittently, but very gently
so that the hyphae is not broken to a very great extent.
These theoretical studies on mass transfer indicate the
following directions for the design and operation of the
bioreactor:
(1) Since the O2 transfer is mainly by diffusion (which is
a slow process), superimposition of convective flow enhances the mass transfer (by forced convection of air
through the SSF bed). This enhances the O2 gradients
very effectively.
(2) Similarly CO2 dissipation could be easily achieved by
forced circulation.
(3) The SSF bed needs to be of a smaller thickness, instead
of one very thick bed, in order to overcome the problems
of reduction in porosity with progress of fermentation.

3. Heat transfer aspects

In general, during SSF, considerable amount of heat is
evolved, which is a function of the metabolic activities of
the microorganisms [27]. In the initial stages of fermentation, the temperature and oxygen concentrations are the
same at all the locations of the SSF bed. As the fermentation
progresses, oxygen diffuses and undergoes bioreactions, liberating heat, which is not easily dissipated due to the poor
thermal conductivity of the substrate. With the progress of
the fermentation, shrinkage of the substrate bed occurs and
the porosity also decreases, further hampering the heat transfer. Under these circumstances, temperature gradients develop in the SSF bed, which can sometimes be steep, giving
rise to high temperatures. For example, in the case of composting in heaps, temperature can rise to as high as 70 C.
The transfer of heat into or out of the SSF system is closely
associated with the metabolic activity of the microorganism,

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as well as the aeration of the fermenting system. The temperature of the substrate is very critical in SSF. High temperatures affect spore germination, growth, product formation
and sporulation [28], whereas low temperatures are not favorable for growth of the microorganisms and for the other
biochemical reactions. The low moisture content and poor
conductivity of the substrate make it difficult to achieve good
heat transfer in SSF. Significant temperature gradients are
reported to exist even when small depths of the substrates
are employed [29]; hence it is very difficult to control the
temperature of the fermentors on a large scale. In fact, limiting the heat dissipation is one of the major drawbacks of
SSF in comparison with conventional SmF, where good mixing provides for efficient dispersal of sparged oxygen also
serves to give better temperature control. Mixing not only
aids homogeneity of the bed but also ensures an effective
heat and mass transfer. Thus water addition coupled with
continuous mixing is advantageous for simultaneous control
of temperature and moisture control in large-scale SSF.
Unfortunately, few attempts have been made to develop
special equipment in order to achieve good heat transfer in
SSF.
The conventional techniques and concepts used for temperature control in SmF are not easily adaptable to SSF.
Conventionally, temperature control in SSF is primarily
accomplished by adjusting the aeration rate. If the temperature is too low, then decreasing the aeration rate enables
the temperature to rise due to the respiration of microorganisms. However, enough care has to be taken in order
to prevent the oxygen concentration from falling below the
critical level that would adversely affect metabolic activity
of the cells. On the other hand, if the temperature of the
substrate is high, increasing the aeration rate promotes cooling of the substrate, which is not favorable for the growth
of the organism. To compensate for this, air that is partially
saturated is used for aeration. This method, called evaporative cooling, of the substrate or the biomass, is effective
if uniform aeration exists. Airflow causes variation in the
water content profile and hence affects the biomass production and substrate consumption. Studies carried out by
Sargantanis et al. [30], on the growth of R. oligosporus on
corn grits in a rocking drum bioreactor, show that temperature of the substrate bed could be effectively controlled by
evaporative cooling, for an improved biomass production.
The importance of evaporative cooling and moisture content of the substrate on the performance of SSF bioreactor
has also been highlighted by Nagel et al. [31,32]. Experiments were conducted on Aspergillus oryzae grown on wheat
grains as also an experimental membrane-based model system. During scale-up, removal of the metabolic heat of the
actively growing microorganisms by conduction in static
beds is hampered due to the poor thermal conductivity of the
substrate as also due to the lack of heat exchange surfaces.
Evaporative cooling has not only been found to effect the removal of heat but has also been found to cause drying of the
substrate/biomass leading to a fall in the bioreactor perfor-

mance. Water activity could also drop due to accumulation

of solutes such as glucose, amino acids, etc. This could be
prevented by spraying of water on to the solid substrate coupled with the mixing. Water addition also aids the growth
of new fungal cells. Studies show that the growth of fungi is
hampered due to limited water availability even if the total
water content of the fermenting mass increases with time.
This is because water is rapidly taken by the growing spores
and as a result the residual water outside the cells, or in other
words the water activity of the substrate, becomes limiting.

4. Bioreactor design
The bioreactor is the heart of a fermentation process,
wherein the raw material, under suitable conditions is converted to the desired product. Maximization of the rate of
formation and yield of product within the bioreactor is a key
part of optimizing the production process. In contrast to SmF
systems, SSF bioreactor systems are yet to reach a high degree of development, mainly due to the problems associated
with solid beds like poor mixing and heat transfer characteristics and material handling. Some of the desired features
of a solid-state bioreactor system may be given as follows:
(a) Containment of the substrate bedthe material of construction of SSF bioreactors must be strong, resistant to
corrosion and must be nontoxic to the process organism.
It should also have an affordable cost.
(b) Prevention of the entry of contaminants into the process
as well as the uncontrolled release of the process organism into the environment. The former is practically difficult due to the need for solids handling which cannot
be pumped as in case of liquids in SmF and consequent
problems in creating contamination-free closed systems.
The latter is an equally important prerequisite considering that most of the SSF processes involve fungal spores,
which might be pathogenic and cause health hazards in
the surrounding environment. This could be achieved by
incorporation of filters on outlet air stream and by a careful design of seals and filtration of the inlet air stream,
which, however, adds to the cost of the equipment.
(c) Effective regulation of aeration, mixing and heat removal to control the operational parameters of temperature, water activity and gaseous oxygen concentration.
Often, solid substrate fermentations suffer from problems of an ineffective heat removal or evaporative loss
of water from the substrate bed affecting the yield and
quality of the desired product.
(d) Maintenance of uniformity within the substrate bed
this is achieved by an effective mixing which is also
crucial to minimize thermal gradients, a factor that is
particularly important in SSF.
(e) The overall SSF process involves substrate preparation,
sterilization of the substrate initially and of the biomass
after product recovery, inoculum preparation, loading

K.S.M.S. Raghavarao et al. / Biochemical Engineering Journal 13 (2003) 127135

and unloading of the bioreactor as also product recovery. A bioreactor system designed to facilitate all the
above operations is highly desirable.
4.1. Classification of SSF bioreactors
SSF processes could be operated in batch, fed-batch or
continuous modes, although batch processes are the most
common. An important aspect to be considered during the
construction of a bioreactor is the sensitivity of the substrate
and/or the microorganism to the shear forces generated by
mixing. In a study conducted on the growth of A. oryzae
ACM 4996 on an artificial gel-based substrate, Stuart et al.
[33] noted a steady decrease in the final protein content
of the fermented substrate in a rotating drum bioreactor.
This was observed when the speed of rotation was increased
from 10 to 50 rpm. This might be attributed to the shear
sensitivity of the gel substrate as also to the damage caused
to the penetrative hyphae of the fungus due to the shear.
Intermittent mixing with long intervening periods of static
operation might also cause disruption of the hyphae that
extend between particles during the static periods.
Another phenomenon that is likely to occur in the case
of microbially degradable substrates, is the shrinking of the
substrate bed pulling it away from the bioreactor walls [34].
This might result in an undesirable channeling of the air
through the gaps between the bed and walls. This could
be avoided by using an inert support or by using a natural
substrate in which the solid structure is not attacked by the
microorganism [35].
The substrate bed of SSF bioreactor can: (a) either be left
static or subjected to mixing and (b) either be aerated over or
through the bed. Accordingly, the bioreactors used for SSF
can be broadly classified as: (1) tray bioreactors, (2) packed
bed bioreactors, (3) rotating drum bioreactors, (4) gassolid
fluidized bed bioreactors, (5) stirred aerated bed bioreactors
and (6) rocking drum bioreactors. As regards the design of
bioreactors, some of the recent developments include:
(1) Recently, a novel and efficient design of integrated solid
matrix bioreactor called the PLAFRACTORTM has been
reported [36]. This bioreactor is a computer-controlled
compact device wherein all the operations including
sterilization of the substrate, inoculation, control of fermentation conditions as also extraction of the product
from the substrate and post-sterilization of the substrate
could be possible. This makes it uniquely useful for the
production of cytotoxic pharmaceuticals like mycophenolic acid and lovastatin [36].
(2) Another modern SSF bioreactor consisting of a rotating
bed in the form of a bucket with provision of mixing
of the substrate by ribbon-shaped baffles is being manufactured and marketed by M/s Fujiwara, Japan. In this
bioreactor, operations like substrate sterilization and inoculation are automated. Slow rotation of the helical
screws ensures minimal damage to growing hyphae and

131

also provides an effective mixing of the substrate bed.

This coupled with a slow rotation of the basket helps in
eliminating temperature gradients within the substrate
bed. The forced aeration of humid air from the bottom
helps in alleviating oxygen gradients without lowering
the moisture content of the bed [37].

5. Monitoring and control of bioreactors

The variables associated with any type of bioreactor operation can be either state variables or operating variables.
The basic goal of an on-line control system is to take the
values of some of the state variables measured on-line, and
to use these values to manipulate the values of the operating variables in order to influence the values of various state
variables (which may or may not be directly measurable) in
such a way as to optimize the growth and product formation.
The key objective of a control system for an SSF bioreactor is typically to control the temperature and water content
of the substrate bed at values that lead to optimal growth
and product formation [38]. The operating variables, which
can be manipulated to achieve this depend on the design of
the bioreactor and they include the temperature, flow rate
and humidity of the inlet air, and variables associated with
the agitation system such as the frequency and intensity of
agitation. Manipulation of the temperature and flow rate of
the circulating air or water passing through jackets or heat
exchangers is also possible. If water or aqueous nutrient or
pH-correcting solutions need to be added to the bioreactor,
the timing and quantity of these additions can be controlled.
The state variables that can be measured on-line and
can therefore readily be used in such control systems are
temperatures, and O2 and CO2 concentrations in the outlet gas stream. On-line temperature measurements can be
made with thermocouples, and it is normal practice to
have a number of them located at different positions within
the bioreactor. Outlet gas concentrations can be measured
on-line most conveniently with paramagnetic oxygen analyzers and infrared carbon dioxide analyzers [39,40]. Other
on-line measurements that might be possibly used include
a gas chromatograph with an automatic sampler to measure
the volatile end product concentrations in the head-space
gases [41], on-line sensors to measure relative humidities
in the outlet stream [42], pH electrodes for measuring the
pH depending on the substrate properties [43] and pressure
drop measurements (especially for packed bed bioreactors)
for indirectly measuring the amount of growth [44].
5.1. Advanced control techniques
The degree of control achievable by the aforementioned
simple control schemes is limited due to the complexity of
the processes occurring within the SSF bioreactors. In the
case of a rocking drum bioreactor, where maintenance of
the substrate temperature and the water content are crucial,

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it has been reported that water levels could be best controlled by using a multiple-input multiple-output scheme
in which both the total weight of the bioreactor and the
carbon dioxide evolution rate are used to control the dry
air flow rate and the water replenishment rate [45]. On-line
measurements invariably contain noise, which may come
from either variations in the process itself or from the measuring equipment [40]. In general, it is necessary to process
data before they can be used in control algorithms, using
mathematical filtering procedures like Kalman filtering or
Butterworth filtering to eliminate noise [43,46,47]. The
control schemes, which control the operating variables such
as inlet temperature, flow rate and humidity usually cannot
prevent significant variations from occurring [38]. Further
smoothing algorithms may also be required to account for
such variations in the values of these operating variables
while processing data from on-line measurements [46].
5.1.1. Application of off-line measurement techniques
Off-line measurements of state variables are more difficult to integrate into control schemes due to the typically
long delays in sample analysis. However, they are crucial
in post-fermentation analysis of the performance of the system and can be used in the validation of both predictive and
interpretive models. They may also be important to provide
a check on on-line measurements as they suffer from less
noise than on-line methods. Some of the most important
off-line measurements are as follows.
5.1.1.1. Measuring water content and water activity. Water content measurements, which are easier than those for
water activity, include vacuumoven drying, moisture evolution analysis, Karl Fischer titration, gas chromatography,
infra red analysis and nuclear magnetic resonance. Of all
the above methods, oven drying is the simplest and least expensive method. Water activity of the samples of solid bed
is generally measured using hygrometers, which are usually
accurate to within 2%.
5.1.1.2. Measuring pH. Although flat-ended electrodes
have been used to make off-line measurements directly
on the surface of solid substrates [48,49], measurements
of pH are usually made by measuring the pH of aqueous
suspensions or extracts of the solid sample.
5.1.1.3. Estimation of biomass. In spite of its importance
in SSF processes, a satisfactory on-line method for biomass
estimation is still unavailable although efforts have been
made to develop on-line sensors based on Fourier transform
infrared (FTIR) spectroscopy [50]. Off-line measurements
of biomass are normally carried out by a direct separation of
the biomass from the solid matrix or indirectly by measuring
the biomass components without separation or by measuring
the metabolic activities. With a few substrates like starch, an
enzymatic digestion could be used for filtration and recovery
of the biomass. Alternatively for unicellular organisms, the

organisms can be washed off the substrate and estimated by

viable count.
The most useful metabolic activities that can be used to
follow growth indirectly are O2 consumption and CO2 evolution, which result from microbial respiration. Pena y Lillo
et al. [51] have developed a model to predict the average bed
temperature and water content using the rate of CO2 production and inlet air conditions. In certain cases the production
of extracellular enzymes could be used as an index of the
amount of growth [52]. Protein measurements are simple if a
predominantly starchy substrate is used, but cannot be used
if the substrate contains significant amounts of protein.
In the case of fungi, several compounds specific to fungi
or subgroups of fungi can be used for monitoring biomass
like N-glucosamine [53], ergosterol [54], etc. However, it
has been found that the proportions of these components as
a part of the biomass used for estimation vary with the age
of the biomass [5557]. Membrane culture methods where
the fungus grows on a membrane overlaid on an artificial
medium and in which the biomass can be readily removed,
mimics SSF more closely and can be used for calibration
[56]. Other methods that find mention in the literature include measurement of the differential light scattering due
to change in color of the substrate [58] and direct measurement of biological activity in terms of ATP [59]. Dubey
et al. [60] have developed an enzyme-linked immunosorbent assay, which quantifies the reactivity of an antibody
raised against the mycelia of Aspergillus niger towards the
cell wall of a growing mycelium. This method is reported
to be rapid, genus-specific, and sensitive, quantitatively determining even small changes in the substrate colonization.
This method could be applied for an effective monitoring of
Koji fermentations.

6. Scale-up aspects
There has been a significant advance in the understanding of scale-up of SSF bioreactors since the review by
Lonsane et al. [61]. These advances have been achieved by
applying mass and energy balances to describe the operation of SSF bioreactors. The basic approach to using mass
and energy balances as a scale-up tool was pointed out by
Saucedo-Castenada et al. [62]. According to this approach,
the most important parameters that need due consideration
during scale-up of an SSF bioreactor include: (a) agitation,
(b) aeration and oxygen transfer, (c) temperature of the
substrate bed, (d) moisture content of the bed and (e) humidity of the bioreactor. The most practical solution which
is beneficial, efficient, and reliable is to combine the rate
of aeration and the moisture content of the substrate bed
by passing humidified air. This strategy, apart from controlling the temperature and moisture content of the substrate
bed also helps in meeting the oxygen demand of the microorganisms. According to this if the dynamic balance
equations for water and energy can be equated to zero, then

K.S.M.S. Raghavarao et al. / Biochemical Engineering Journal 13 (2003) 127135

the temperature and moisture content of the substrate bed

will be constant at pre-determined set values, at any given
time during the course of fermentation. Furthermore, if this
equality with zero can be maintained as the scale increases,
then the large-scale bioreactor should operate equally as
well as the small-scale bioreactor. The key is therefore to
find operating conditions for the bioreactor that will allow
the water and the energy balances to remain at a constant
value as scale increases.
As regards the prevention of undesirably high temperatures within the bioreactor, a relatively simple approach is
to concentrate on the time of peak heat generation. This
approach was studied by Mitchell et al. [63] for packed
bed bioreactors without internal heat transfer plates and by
Hardin et al. [64] for rotating drum reactors.
For packed beds without internal heat transfer, Mitchell
et al. [63] proposed a modified Damkoehler number (DaM )
assuming logistic growth kinetics without maintenance
metabolism. The first step in using the DaM number is to
identify a temperature, which must not be exceeded within
the bioreactor during the fermentation. Allowable bioreactor height can then be calculated by assuming that the ratio
of superficial velocity of the flowing air to that of the bioreactor height is a constant. However, this is likely to lead to
unacceptable pressure drops as the scale increases. More
knowledge is therefore required on the effects of pressure
drop on packed bed operation.
A similar approach applied to rotating drum bioreactor
yields a dimensionless design factor (DDF) [38]. According
to this model, maintaining the DDF constant would maintain the maximum temperature within the bed constant with
scale, although this might lead to unreasonably high airflow
rates.
Regarding water-balance, Weber et al. [35] applied a similar simplified approach in a packed bed. They concluded
that the rate of evaporation would be relatively constant
along the bioreactor axis, provided that the substrate bed is
at sufficiently high initial water content. This ensures that
the substrate water activity does not fall to inhibitory levels
during the fermentation.
The utility of mathematical models to guide the design
and operation of large-scale bioreactors have been theoretically demonstrated for different bioreactors. For packed
beds bioreactors without internal heat transfer plates, the
model predictions are similar to those given by the DaM
number [63]. In Zymotis type packed beds, to achieve
higher productivities, the best strategy suggested is to minimize the distance between any two heat exchanger plates,
increase the superficial air velocity to the maximum and
vary the temperature of cooling water, during fermentation
in response to the bed temperature [65]. The importance
of pressure drop considerations in influencing the scale-up
process was not investigated. In rotating drum bioreactors,
the importance of evaporative heat removal is predicted to
increase with scale [5]. However, it is desirable that models
be developed also for the maintenance of water-balance in

133

substrate beds, as under extreme conditions, evaporative

loss of water from the substrate bed might lead to a decrease in the performance of a bioreactor. These quantitative
methods are potentially powerful tools in guiding the design and operation of large-scale bioreactors. Use of these
methods is also cost-effective. Unfortunately, many of the
mass and heat transfer coefficients are poorly characterized
for SSF bioreactors. There is a need for experimental work
to determine these parameters for SSF bioreactors as also
to test these theoretical approaches by applying them in the
development of real large-scale SSF bioreactors.

7. Applications of SSF
SSF has been used in two areas of applications. These
include:
(a) applications for environmental control, including the
production of compost and animal feed from solid
wastes, bioremediation and biodegradation of hazardous
compounds, biological detoxification of industrial
wastes; and
(b) applications for value-addition, such as the nutritional
enrichment of crops or crop-residues by biotransformation, biopulping, production of fermented foods,
enzymes, pigments, antibiotics, biopesticides, organic
acids and flavor compounds.
Pandey et al. [66] have reviewed some developments in
the application of solid-state fermentation. The recent and
promising developments in this area include the following.
7.1. Enzyme inhibitors and other high value biomolecules
Work on SSF for production of enzyme inhibitors is
a recent development. Sekhar Rao et al. [67] mention
the production of an acetylcholine esterase inhibitor by
Chrysosporium sp. grown on wheat bran using SSF. Guo
et al. [68] have isolated cytonic acids A and B, which are
novel tridepside inhibitors of human cytomegalovirus protease (hCMV protease) by SSF of the endophytic fungi
Cytonaema sp. Immuno-suppressants like lovastatin are
also produced by SSF.
7.2. Biopulping
Chen et al. [69] have studied the degradation of steamexploded wheat straw, a new source for biopulp making
using Phanerochaete chrysosporium.
7.3. Enzymes
Development of a lab-scale reactor for the production
of tannase from coffee industrial waste is reported by Van
de Lagemaat and Pyle [70]. Hatvani and Mecs [71] report

134

K.S.M.S. Raghavarao et al. / Biochemical Engineering Journal 13 (2003) 127135

the production of laccase and manganese-peroxidase from

malted barley waste using Lentinus edodes. Fujian et al. [72]
produced lignin peroxidase and manganese-peroxidase from
steam-exploded straw using P. chrysosporium. Designing of
a rotating drum bioreactor for the production of the same by
Phanerochaete chrusosporium on an inert support of nylon
sponge has been reported by Dominiguez et al. [73]. Singh
and Soni [74] have attempted the production of starch-gel
digesting amyloglucosidase by A. oryzae HS-3 using SSF.

8. Conclusions and suggestions for future work

Many aspects pertaining to the bioreactor design are yet
to be studied in detail. To date, there has been little experimental effort to measure nutrient concentration gradients
within particles. An effort in this direction will aid in a better understanding of the micro-scale phenomenon of intraparticle diffusion. Further, in the case of tray bioreactor, it
is quite probable that the natural convection process might
occur within the bed in response to an increase in temperature, simultaneously aiding transfer of heat and CO2 , oxygen and water vapor transfer [75]. This phenomenon has,
however, attracted little attention. Similarly, in the case of
the Zymotis design, attention is required to analyze the pressure drops, change in bed structure during the fermentation
and the flow patterns of air through the bed. In the case of
the gassolid fluidized bed bioreactors, little information is
available about design and operation, which needs detailed
studies. Greater automation of the SSF process is needed for
its increased industrial exploitation.
SSF offers a viable alternative for many operations including waste disposal and value-added products from these
wastes. In the case of enzymes, thanks to the very low water
content of the product, the downstream processing of the enzyme produced by SSF is more efficient as compared to its
SmF counterpart. Some of the applications which appear to
have a promising future include the production of enzymes,
colors, biocides and flavors.

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