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Fermentation
Processes
Fermentation
• The most common meaning of fermentation is the conversion of a sugar
into a organic acid or an alcohol.
• Fermentation occurs naturally in many foods and humans have
intentionally used it since ancient times to improve both the
preservation and organoleptic properties of food.
• However, the term “fermentation” is also used in a broader sense for
the intentional use of microorganisms such as bacteria, yeast, and fungi
to make products useful to humans (biomass, enzymes, primary and
secondary metabolites, recombinant products, and products of
biotransformation) on an industrial scale.
Types of Fermentation Processes

There are basic types of fermentation processes

1. Submerged Cultivation
2. Solid Substrate Fermentation (SSF)
1. Submerged Cultivation
• Submerged cultivation of microbial cells in bioreactors guarantees a
controlled environment for the efficient production of high-quality
end products and to achieve optimum productivity and yield.
• Industrial bioreactors operated in i) batch, ii) fed-batch, or iii)
continuous mode are utilized to culture different types of
microorganisms producing a wide range of products.
i) Batch Cultivation

• Batch culture represents a closed system in which the medium,

nutrients, and inoculum are added to the bioreactor, mostly under
aseptic conditions, at the beginning of cultivation.
• The volume of the culture broth in the bioreactor is theoretically
constant during cultivation (practically, small deviations in culture
volume are caused by a low feed rate of acid/base solutions to keep
the pH at a desired level and by sampling or introducing air/gas into
the culture; on balance, such changes are usually ignored due to their
small value relative to the total working volume of the bioreactor).
Cont’d…
• Typically, at the beginning of batch cultivation, a known number of viable cells are
inoculated into the bioreactor that is already filled with sterilized medium
containing all nutrients.
• After inoculation, the cell culture follows the classical growth curve which is divided
into four main phases.
1. As the lag phase is an “inefficient” stage of culture (even though the cells are
metabolically active—they are adapting their enzymatic apparatus to a new
environment; no significant increases in biomass concentration, substrate
consumption, or product synthesis are observed), it is desirable to shorten it as
much as possible. The length of the lag phase is influenced mainly by the
concentration of cells in the inoculum and their physiological state, the
composition of the inoculation and cultivation medium (mainly the source of
carbon and energy, pH, and temperature), and the size of the inoculum.
Cont’d…
2. The exponential (logarithmic growth) phase is characterized by
rapid cell proliferation (biomass concentration is an exponential
function of time), constant specific growth rate, which is equal to
the maximum specific growth rate of the culture under conditions
of absence of growth limitation (growth rate is not limited because
all nutrients are present in excess, while also not attaining growth-
inhibiting concentrations), fast consumption of the source of carbon
and energy, and a high rate of primary metabolite production.
Cont’d…
3. The depletion of nutrients by the end of the exponential phase (in
the case of aerobically grown cultures, these are signaled by a rapid
increase in dissolved oxygen concentration) causes a progressive
reduction in the specific growth rate and a transition to the
stationary phase, characterized by the stagnation of growth and
utilization of endogenous reserves of carbon and energy; this phase
is important for the synthesis of secondary metabolites.
Cont’d…
4. Death Phase: As nutrients become less available and waste
products increase, the number of dying cells continues to rise. In
the death phase, the number of living cells decreases exponentially
and population growth experiences a sharp decline. As dying cells
lyse or break open, they spill their contents into the environment
making these nutrients available to other bacteria. This helps spore
producing bacteria to survive long enough for spore production.
Spores are able to survive the harsh conditions of the death phase
and become growing bacteria when placed in an environment that
supports life.
The bacterial growth curve represents the number
of living cells in a population over time.
Cont’d…
• Most industrial bioreactors are operated in batch mode due to the
relative simplicity of this process. The whole batch operation consists
of several steps, including medium formulation, filling the bioreactor,
sterilization in place (SIP systems), inoculation, cultivation, product
harvesting, and bioreactor cleaning in place (CIP systems).
• For efficient performance of batch operation, it is important to
minimize all nonproductive steps (all steps listed above except
cultivation), achieve a high rate of product synthesis, optimize
productivity, and maximize the yield of the end product.
Cont’d…
• Therefore performance of any particular batch operation is thus
influenced by the type of end product—an extension of exponential
growth is advantageous for the efficient production of biomass
(baker’s yeasts, feed biomass) or primary metabolites (ethanol, acetic,
citric, or lactic acids), whereas in the case of secondary metabolite
production, the exponential phase is shortened (by the limitation of
one nutrient, usually the source of nitrogen) and the stationary phase
is prolonged to achieve the maximum yield of the product.
Applications of Submerged Batch
Cultivation
• Submerged batch cultivation can be used for the production of
alcoholic beverages (beer, wine, and distilled spirits such as whisky,
brandy, rum, and others), organic acids used in the food industry
either as acidifiers or as preservatives (citric, acetic (vinegar), and
lactic acids), and amino acids used as flavor enhancers (e.g.,
monosodium glutamate) or sweeteners (e.g., aspartate).
ii) Fed-Batch Cultivation
• Fed-batch culture represents a semi-open system in which one or more nutrients
are aseptically and gradually added to the bioreactor while the product is retained
inside, that is, the volume of the culture broth in the bioreactor increases within
this time.
• The main advantages of fed-batch over batch cultures are:
a) the possibility to prolong product synthesis,
b) the ability to achieve higher cell densities and thus increase the amount of the
product, which is usually proportional to the concentration of the biomass,
c) the capacity to enhance yield or productivity by controlled sequential addition
of nutrients, and
d) the feature of prolonged productive cultivation over the “unprofitable periods”
when the bioreactor would normally be prepared for a new batch.
Cont’d…
• There are many methods of adding a substrate to the bioreactor (either as a
concentrated solution of a sole carbon and energy source or as a medium
containing carbon plus other nutrients); the proper choice of the nutrient feeding
rate can enhance the culture performance considerably since it influences cellular
growth rate, cell physiology, and the rate of product formation.
• The common feeding strategies are:
• (a) discontinuous feeding, achieved by regular or irregular pulses of substrates and
• (b) regular continuous feeding of nutrients designed according to a pre-calculated
profile or based on the feedback control of online measured variables associated
with cell growth and metabolism, for example, dissolved oxygen concentration, pH,
CO2 , evolution rate, and biomass concentration.
Applications of Fed-batch
Fermentation
• The typical food fed-batch fermentations are large-scale production of
baker’s yeast, pure ethanol, which is further utilized for alcoholic
beverages produced by mixing ingredients such as liquors or cordials,
and submerged acetification for vinegar production.
iii) Continuous Cultivation
• Continuous culture represents an open system in which nutrients are
aseptically and continuously added to the bioreactor, and the culture broth
(containing cells and metabolites) is removed at the same time, that is, the
volume of the culture broth is constant due to a constant feed-in and feed-
out rate.
• Frequently, continuous culture is used as a synonym for a chemostat,
represented by a constant specific growth rate of cells, which is equal to the
dilution rate and is controlled by the availability of the limiting nutrient,
although other types of continuous operation such as turbidistat (a
constant concentration of biomass controlled by the dilution rate) or
nutristat (a constant parameter related to cell growth controlled by the
dilution rate) can be employed.
Cont’d…
• The main advantages of continuous culture (chemostat) over the
batch mode are
a) the possibility to set up optimum conditions for maximum and long-
term product synthesis,
b) the ability to achieve stable product quality (the steady state is
characterized by a homogeneous cell culture represented by a
constant concentration of biomass and metabolites),
c) a distinct reduction in “unprofitable” periods of the bioreactor
operation.
Cont’d…
• There are also several problems that hamper the extensive utilization
of continuous operation on a large scale.
• These include
a) increased risk of contamination due to the pumping of the medium
in and out of the bioreactor,
b) the danger of genetic mutations in the production strain in a long-
term operation,
c) additional investments may be required for technical facilities.
Applications of Continuous
Cultivation
• In food applications such as in modern distilleries, a semi-continuous
fermentation operated in a series of fermenters is usually used
instead of genuine continuous fermentation.
• The current estimates are that only about 16% of ethanol in North
America is produced in a continuous mode due to problems with
contamination. Nevertheless, both batch and semi-continuous mode
fermentations permit continuous distillation, which is the reason why
the whole distillery production is often considered as continuous.
2. Solid Substrate Fermentation
(SSF)
• The term “solid substrate fermentation” (SSF) or “solid substrate
cultivation” (SSC) is used for systems where microorganisms are
cultured on the surface of a concentrated water-insoluble substrate
(usually containing polysaccharides as a carbon and energy source)
with a low level of free water.
• This technique was developed in the Eastern countries, where it has
been used for centuries for the production of traditional foods such as
soy sauce, koji, miso, or sake, using different substrates and
microorganisms. In the Western countries, it has not been widely
exploited and its application is limited mainly to the production of
industrial enzymes, certain food products, or feed supplements.
Typical Features of Solid Substrate
Fermentation
• Solid substrate fermentation is characterized by very low water activity thus, the main features of
this system are substantially different compared with classical submerged cultivation.
• There are several advantages of SSF over the conventional submerged technology such as
a) the use of a concentrated medium, resulting in a smaller reactor volume and lower capital
investment costs,
b) the lower risk of contamination with yeasts and bacteria due to low moisture levels and
substrate complexity,
c) the simplicity of the technology and low production of effluent water from the process,
d) the higher product yield and easier product recovery,
e) the use of agricultural wastes as substrates for certain applications (e.g., feed supplements and
cellulolytic enzymes).
Main Differences between Solid Substrate Fermentation and
Submerged Cultivation
Microorganisms and Substrates
Used in SSF Processes
• Filamentous fungi are preferable for SSF processes, mainly due to their abilities to
a) grow on substrates with reduced water activity,
b) penetrate their hyphae into the solid substrate, and
c) produce exoenzymes (e.g., amylolytic and cellulolytic enzymes), which decompose
the polysaccharides (the main carbon source often present in solid substrates).
• When using yeast, it is necessary to integrate a material pretreatment step (such as
steam explosion, acid or alkali treatment, followed by enzymatic digestion or a
combination of these) into the process or to use a mixed culture, wherein the
complex cellulosic or starchy material is first degraded by other organisms (usually
molds) that are able to produce extracellular enzymes and the released glucose is
then consumed by yeasts (or less frequently by bacteria) yielding the desired
product.
Efficiency of SSF
The efficiency of SSF is highly influenced by the selection of the solid substrate. The substrates
suitable for SSF should ideally meet the following requirements:
a) have a porous solid matrix with a large surface area per unit volume (103–106 m2/cm3),
b) should sustain gentle compression and mixing,
c) should contain biodegradable carbohydrates,
d) its matrix should absorb water in the proportion of 1 to several times its dry weight,
e) should have relatively high water activity on the solid/gas interface to support microbial
growth,
f) should absorb the additionally added nutrients such as nitrogen sources (ammonia, urea,
and peptides) and mineral salts.
On the basis of these characteristics, the ideal material consists of small granular or fibrous
particles that do not break or stick together. The commonly used solid substrates include wheat,
wheat bran, soybean, rice barley, oats, and other cereals.
Limitations and Challenges of Solid
Substrate Fermentation
• Although SSF has many advantages over liquid cultivation, the main challenges
are poor heat and mass transfers within the substrate, and limited potential to
monitor, online, key cultivation parameters (temperature, pH, dissolved oxygen,
nutrient concentrations, or water content), and thus an inability to precisely
control the microbial environment.
• The heat generated by microbial metabolism is a major bottleneck for process
scale-up; the released heat can reach up to 3000 kcal from 1 kg of assimilated
substrate, causing a radial gradient of 5°C/cm at the center of the reactor.
• Low water content in the system and poor heat conductivity of SSF substrates
promote the heat gradient, which is accentuated by limited agitation (the molds
are very sensitive to shear stress) and is reduced mostly by evaporative cooling,
which can conversely exacerbate further water loss.
Cont’d…
• The pH gradient caused mainly by the production of organic acids and
the utilization of proteins poses another challenge; pH monitoring and
control is difficult because no existing pH electrodes can operate in
the absence of free water.
• Therefore, the variations in pH should be prevented by increasing
substrate buffering capacity (e.g., by the addition of CaCO3 ) or by the
addition of urea, which can counteract acidification. Oxygen gradients
can be reduced by aeration with moist air, which also plays a role in
the desorption of carbon dioxide and the regulation of temperature
and moisture levels.
Industrial Applications of SSF

• In Europe, SSF in the food and feed contexts is usually used for the
production of mold and ripening cheeses, production of fermented
vegetables (e.g., pickled cucumbers) or silage (preserved cattle feed), and in
Asia for the production of fermented products from soya, rice, or corn.
• An example of Oriental SSF is red yeast rice (red rice, angkak, anka, ankak,
benikoji, and other names), which is a product obtained after SSF of rice
with different Monascus species, in the most usual case with Monascus
purpureus. This product has been known in various Asian countries (China,
Japan, Thailand, Indonesia, Taiwan, and Philippines) for centuries and can
be used for food coloring, such as red koji (for the production of Kaoliang
liqueur) or as a food supplement with an anticholesterolemic effect.
Cont’d…
• SSF has also been described as a protein enhancement factor for
cereal grain and potato residues. The protein content of wheat bran
was increased fourfold by SSF of Aspergillus terreus.
• Among a wide range of SSF applications, the processes yielding
protein-enriched agro-industrial materials that can be used as animal
feed play an important role. there are reports on the production of
protein enriched animal feeds by SSF using starchy materials as
substrates.