SOLID STATE

FERMENTATION
WITH BIOREACTOR DESIGNS

SAKETH BODDU
2012A1PS422P
What is fermentation?
 Technique for the biological conversion of complex substrates into
simple compounds by various microorganisms such as bacteria and
fungi.
 Release of additional compounds known as secondary metabolites,
or bioactive compounds since they possess biological activity.
Include peptides, enzymes, antibiotics, and growth factors.
 For our study, fermentation refers to the bulk growth of organisms on
a growth medium, with the goal of producing a specific chemical
product.
 Growth medium or culture medium is the liquid or gel designed to
support the growth of microorganisms, cells, or small plants.
How does it happen?
 Fermentation takes place in the lack of oxygen (when the electron transport
chain is unusable) and becomes the cell’s primary means of ATP (energy)
production.
 It turns NADH and pyruvate produced in the glycolysis step into NAD+ and
various small molecules depending on the type of fermentation.
 In the presence of O2, NADH and pyruvate are used to generate ATP
in respiration. This is called oxidative phosphorylation, and it generates much
more ATP than glycolysis alone.
 The first step, glycolysis, is common to all fermentation pathways:
 C6H12O6 + 2NAD+ + 2ADP + 2Pi → 2CH3COCOO− + 2NADH + 2ATP + 2
H2O + 2H+
 TYPES
(based on the substrate used)
 Submerged Fermentation (SmF):–
 Uses free flowing liquid substrates, such as molasses and broths. The
substrates are used up very rapidly and need to be replenished constantly.
 They are best suited for microorganisms that require high moisture content.
 The bioactive compounds are secreted into the fermentation broth.
 Primarily used in the extraction of secondary metabolites that are required in
the liquid form.
 Has the advantage of easier purification of products.
 TYPES
(based on the substrate used)
 Solid-State Fermentation (SSF) (Koji Process):-
 Defined as the growth of microbes on solid materials without the presence of any
liquid. It utilizes solid substrates such as bran, bagasse, paper pulp.
 Substrates are utilized very slowly, and so the same substrate can be used for long
periods of fermentation.
 This method has the advantage of easy recycling of nutrient-rich waste materials.
 Best suited for microorganisms and fungi requiring small amount of moisture.
 However, it cannot be used for organisms having a high water activity (ratio of
vapor pressure of an aqueous solution to that of pure water at the same
temperature)
Advantages
 Higher volumetric productivity.
 Simpler with lower energy requirements (reduces production cost)
 Easier to meet aeration requirements.
 Resembles natural habitat of some fungi and bacteria
 Easier downstream processing.
 Substrate usually provides all nutrients required for growth.
 Bioreactors with simple design and few spacial requirements can be used due
to concentrated nature of substrates.
 Low levels of polluting effluents.
 Facilitates production of specific compounds due to low moisture availability.
Drawbacks
 Substrate requires pretreatment by grinding, or enzymatic hydrolysis.
 Microorganisms requiring high moisture levels cannot sustain easily.
 Monitoring parameters such as pH, moisture content, substrate oxygen
difficult due to solid nature leading to build-up.
 Agitation to be caused is not easily done.
 Possibility of contamination with unwanted fungal species.
 Temperature control not easy, because of the heat generated by the
metabolic activities of the micro-organisms, and the low thermal
conductivity of the substrate. Heat generation beyond a certain level leads
to product denaturation.
Mechanism
 Inoculating a solid substrate with microorganisms, which is then left inside
a temperature controlled room for a few days.
 Initially, the substrate and the solid culture compounds are non-soluble
compounds composed of complex molecules broken down by the fungi to get
C and N nutrients.
 Fungal organism produces metabolites required for it’s growth.
 Composition of the growth medium decides the metabolism of the
microorganism towards the enzyme production.
 So some amount of selectivity is possible in the substrate selection.
 Air flow monitoring is done as it impacts temperature, oxygen supply and
moisture.
Production of Citric Acid
 Cavassa bagasse was used as the substrate. This was thermally treated to
gelatinize it by adding 110mL of distilled water to 100 gm of bagasse.
 Bagasse was supplemented with a nutritive solution containing (g/L) urea,
KH2PO4 and FeSO4.7H2O, which was sterilized at 121ºC for 15 minutes. After that
cooling, methanol was added under sterile conditions.
 SSF on trays was carried out with the initial moisture adjusted to 70% with the
nutritive solution. Substrate was then inoculated with the spore suspension.
 A strain of Aspergillus Niger was used to produce spores of the same using potato
dextrose agar solution. The suspension was stored at 4 ºC for 7 days.
 The pre-inoculated substrate was distributed on trays to obtain bed thicknesses
of 2, 4 and 6 cm of dry substrate.
 The trays were placed in a room with controlled temperature and a humidity. The
fermentation was carried out for 120 h.
Leudeking – Piret Equations
 Relates the cell growth to the product formation, that is heavily used in solid
state fermentation.
 The product formation rate allows this correlation between cell mass and
product concentration.

 P = Product concentration
X = Concentration of cells
α and β represent coefficients depending on the fermentation process. For
growth associated product formation: α = 0; β ≠ 0
Bioreactors
 A bioreactor is a vessel in which a chemical process is carried out
which involves organisms or biochemically active substances derived
from such organisms.
 For SSF, the bioreactor has two main functions:
 To hold the substrate bed and provide a barrier against both the
release of the organism to the surroundings and the contamination of
the substrate bed by organisms in the surroundings.
 To control, to the degree that is possible, the key environmental
conditions, such as the bed temperature and water activity, at values
which are optimal for growth and product formation by the
microorganisms.
Macroscale view
 An SSF bioreactor has 3 phases:
 The bioreactor wall that prevents mass transfer. Matter enters
only through pores present in the wall.
 A headspace full of gas, the extent of which depends on the
bioreactor type. It allows space for bed expansion and
disengagement of particles and air.
 A substrate bed, composed of particles and air within the inter-
particle spaces.
Classification
 On the basis of manner of operation, bioreactors for SSF can be classified largely into
four groups:
 Group I: Bioreactors in which the bed is static, or mixed only very infrequently (i.e.,
once or twice per day) and air is circulated around the bed, but not blown forcefully
through it. These are often referred to as “tray bioreactors”
 Group II: Bioreactors in which the bed is static or mixed only very infrequently (i.e., once
per day) and air is blown forcefully though the bed. These are typically referred to as
“packed-bed bioreactors”.
 Group III: Bioreactors in which the bed is continuously mixed or mixed intermittently
with a frequency of minutes to hours, and air is circulated around the bed, but not blown
forcefully through it. Two bioreactors that have this mode of operation, using different
mechanisms to achieve the agitation, are “stirred drum bioreactors” and “rotating drum
bioreactors”.
 Group IV: Bioreactors in which the bed is agitated and air is blown forcefully through the
bed.
What happens in the bioreactor?
 Within the substrate bed:-
 Metabolic heat production, conduction, diffusion, convection, evaporation.
 Within the headspace:-
 Typically the headspace gases are flowing, since, even in those bioreactors
in which the bed itself is not forcefully aerated, air is typically circulated
through the bioreactor such that it moves transversely across the bed
surface.
 Within the bioreactor wall:-
 Heat will be transferred across the bioreactor wall by conduction if there
is a temperature gradient across it.
How should it be operated?
 Continuous mode of operation is more preferred as it leads to giving us a more
uniform product.
 Batch processes suffer a lag time that causes the biological activity of the
microorganisms to deplete.
 Parts of the bed located to the left of the mixer are mixed soon after the start of
mixing while parts located to the right of the mixer are mixed only after a lag time
that depends on the bed length and the speed with which the mixer travels back and
forth along the bed.
 However in continuous mode, the mixer stays in place as the substrate is moved past
it and all of the fermenting solids are mixed or wetted at the same time interval after
their entrance into the bioreactor, leading to more uniform product.
 But the continuous mode is more susceptible to contamination as the operations
cannot necessarily be done aseptically. But time saved and volume productivity is
much higher in continuous mode.
When to use solid–state fermentation?
 When the product needs to be in a solid form (e.g., fermented foods)
 When a particular product is only produced under the conditions of SSF or, if
produced in both SmF and SSF, is produced in much higher levels in SSF.
 When the product is produced in both SmF and SSF, but the yield is much
higher in SSF. For example, Monascus pigment and many fungal spores are
produced in much higher yields in SSF.
 When it is imperative to use a solid waste in order to avoid the
environmental impacts that would be caused by its direct disposal.
 When the product is produced in both SSF and SmF, but the product
produced in SSF has desirable properties which the product produced in
SmF lacks.
Applications of SSF
 Agro-industrial residues conversion into value added and protein enriched end
products for poultry and cattle feed. Residues like coffee pulp, husk, soybeans, and
bagasse, are bio-converted into ethanol, single cell protein, organic acids like citric and
lactic acids, amino acids, pigments, antibiotics etc.
 SSF is increasingly applied in environmental control and monitoring. Bioremediation
and biodegradation of hazardous compounds. Biological detoxification of industrial
wastes are the latest.
 Enzyme production. Enzymes like amylase, protease, xylaxase, lipase etc were
produced by SSF using cheap carbon sources like cassava bagasse and soybean defatted
cake etc.
 Bio insecticides for pest control in crops.
 Work is also being carried out-on enzyme inhibitors and bio molecules production
through SSF.
References
 Paper :- Solid state fermentation for the production of industrial enzymes.
Authored by: Ashok Pandey, P. Selvakumar. School of Biological and Chemical
Sciences, University of Ulster, Coleraine, Ireland
 Paper:- An overview of Engineering aspects of solid state fermentation.
Authored by: Prabhakar A, Krishnaiah K, Janaun J. University of Malaysia, Sabah
 Paper:- Solid state Fermentation Bioreactors – Fundamentals of design and operation
David A. Mitchell, Nadia Krieger, Springer Publications
 Paper:- Design aspects of Bioreactors for solid state fermentation:- H. Kh. Q. Ali, M.M.D.
Zulkali,
School of Bioprocess Engineering, University of Malaysia, Perlis
 Paper:- An overview of Solid state fermentation:- S. Bhargav B.P. Panda, M. Ali, S. Javed
Pharmaceutical Biotechnology Laboratory, New Delhi
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