BE UNIT-4

Fermentation
Modes of bioreactor operation;
batch, continuous and fed batch,
Mixing and aeration, operation,
measurement of parameters and
control of bioreactors; Preparation
and sterilization of medium for
fermentation; study of product
formation kinetics in a fermentation
process
A suitable microorganism : critical requisite for any fermentation
process.
A wide variety of microorganisms : property of producing some specific
compounds - isolated from the pool of microbes found in a culture.
The most suitable organism : screening (identifying) from a population
or creating specific strains of microorganisms : yield high quantities of
the desired product by genetic manipulations.
Bacteria,actinomycetes, fungi and algae.
Found in air, water ,soil, on the surfaces of the plants and animals, and
in plant and animal tissues.
Good sources for isolation :soils, lakes and river muds.
Isolation by : enrichment techniques like
soil treatment (UV irradiation, air drying or heating at 70-1200C,
filteration or continuous percolation, washings from root system,
treatment with detergents (or) alcohols, pre incubation with toxic
agents), selective inhibitors (antimetabolites, antibiotics, etc.,),
nutritional variations (specific C and N sources), variations in pH,
temperature, aeration etc.
• The right microorganism : produces a large amount
of novel product : selected and screened.
• The selected strain : relatively stable characterstics
and the ability to grow rapidly and vigourously
• economically important strain : genetically stable,
but amenable to genetic modification.
• The strain : be non-pathogenic,non-producer of any
unwanted by-products or toxins.
• An ideal producer (strain) : be amenable to long-
term conservation and the risk of contamination
should be minimal under the optimum conditions.
• Screening of Industrially important Microbes :
• Screening : the use of highly selective procedures to allow the
detection and isolation of microorganisms producing the
desired metabolite from among a large microbial population.
• Primary Screening :
• Primary screening : the detection and isolation of
microorganisms that posses potentionally interesting
industrial applications.
• Primary screening : followed by secondary screening
• Secondary screening : qualitative or quantitative in its
approach.
• Secondary screening : conducted on agar plates, in flasks or
small fermenters containing liquid media, or as a combination
of these approaches.
• Qualitative approach : spectrum or range of microorganisms
which are sensitive to a newly discovered antibiotic.
• Quantitative approach : the yeild of antibiotic which can be
expected when the microorganism is grown in various media.
• Secondary screening
• Secondary screening : types of information which are
needed in order to evaluate the true potential of a
microorganism for industrial usage.
• The microorganisms are actually producing new
chemical compounds not previously described.
• reveal whether there are pH, aeration, or other critical
requirements associated with particular
microorganisms, both for the growth of microorganism
and for the formation of chemical products.
• Whether a product resulting from the microbial
fermentation occurs in the cultured broth in more than
one chemical form, and whether it is an optically or
biologically active material.
• Whether microorganisms are able to chemically alter or
even destroy their own fermentation products
Fermentation based commercial products
Fermentation based commercial products
Fermentation based commercial products
• Strain Improvement :
• Strain improvement or strain development (or) strain evaluation :
techniques and approaches used to genetically modify strains of
industrial importance to increase the production of desired
product.
• The utility of strain improvement : because of the existence of
rate-limiting steps within all metabolic pathways. (Drawback)
• The conventional method of strain improvement : to induce
mutations by using mutagens (chemical mutagens or UV light) and
screening the mutants for products.
• method 2 : protoplast fusion in which two strains with two
independent desirable characteristics could be fused to get a
hybrid with both the characteristics, : better growth from one and
better product yield from another strain.
• The modern approach to strain improvement : largely based on
molecular techniques and recombinant DNA technology.
• In this approach two things can be achieved, (i) Overproduction of
a gene product by molecular techniques,
• (ii) insertion of new genes into a known good strain to further
improve it.
• Mutation :
• Mutants : generated by using either chemical or
physical treatments in order to modify the genome of
the target organism.
• The agent responsible for mutation : known as
mutagen.
• Different mutagens : different mechanism of action,
such as genetic attraction by base transitions or by
frame shifts.
• During a long-term strain improvement program :
advisable to change mutagens periodically to take
advantage of these different mechanism of action.
• A small population of these cells : one which produces
large amount of bioactive components of interest.
• Major mutations : marked changes in the biochemical
characters and are useful in strain improvement.
• Example- the original strain of Streptomyces
griseus produced small amounts of streptomycin
and large amounts of mannosido streptomycin
which has low antibiotic activity.
• A major mutant isolated from this strain produced
less amount of manosido streptomycin and much
larger quantities of streptomycin.
• Similarly, a mutant strain (S-604) of Streptomyces
aureofaciens produces 6-demethyl tetracycline in
place of tetracycline; this demethylated form of
tetracycline is the major commercial form of
tetracycline.
• Recombination :
• Recombination :for the production of vaious industrial
strains.
• Recombination : when ever new gene arrangements
are formed through exchange, elimination or insertion
of DNA.
• Sexual reproduction by means of conjugation in some
bacteria and actinomycetes leads to the formation of,
usually,partial diploids in which crossing over produces
recombinant genotypes.
• In parasexual cycle, the nuclear fusion and gene
segregation could take place outside the sexual organs
of fungus, Aspergillus nidulans, Pencillium
chrysogenum, etc.
• heterokaryon formation takes place by nuclear fusion
of genetically indifferent nuclei.
• Protoplast fusion :
• A protoplast : defined as the cell which is devoid of
the cell wall.
• Protoplasts of bacteria, actinomycetes and fungi :
isolated by subjecting the cells to the action of
wall-degrading enzymes in isotonic solutions.
• The protoplasts require an osmoticum for stability
and fusion is induced by PEG (polyethlene glycol)
treatment.
• Example : Protoplast fusion between non-
producing strains of Streptomyces griseus and
Streptomyces tenjimariensis, has yielded a strain
that produces indolizomycin, a new indolizine
antibiotic.
• Recombinant DNA Technology :
• Recombinant DNA technology has been : achieve
the objectives like production of recombinant
proteins by transfering the genes of commercial
values into bacteria and modification of the
organism's metabolic pattern for obtaining new,
modified or more quantity of metabolites.
• This process of modifying the metabolic pattern :
known as metabolic engineering.
Recombinat DNA technology mediated changes in the
production of various metabolites.
• A fermentor : to the containment system for the
cultivation of prokaryotic cells (bacteria), while in a
bioreactor grows the eukaryotic cells (Mammalian,
insect, plant).
• Industrial fermentors : designed to provide the best
possible growth and biosynthesis conditions for
industrially important microbial cultures, and to
allow ease of manipulation for all operations
associated with the use of the fermentors
• Batch Bioreactor
lower fixed cost, flexibility, operational simplic-
ity. Operational costs , smaller operations, specialty
products, long growth periods, operations in which
flexibility is vital, unsteady-state processes, and
experimental development.
• Continuous bioreactor: more produc-
tion optimization, automation, and capital utilization.
• the larger initial investment can be easily offset by
lower variable costs and faster production times :
preferred for high volume production, processes using
mutation-resistant bacteria, and processes that do
not require a sterile media.
• The production of alcohols, solvents, vinegar, or baker’s
yeast, and wastewater treatment
challenging problem :the mode of
operation of a bioreactor which is optimal
for achieving the desired objective of
either
•maximizing the metabolite yield
• the bioreactor productivity.
•cell mass production with either
constant (or variable ,
•cellular yield and metabolite production
with kinetics
• A good fermentor :
• Heat and oxygen transfer settings
• Sterilization processes and foam control,
• a fast and thorough cleaning system
• A proper monitoring and control system
• The majority of fermentations : close systems to
prevent contamination.
• constructed of non-toxic and corrosion-resistant
materials.
• Small fermentor with a capacity of just a few liters :
glass or stainless steel.
• Large fermentor : mild steel, and then lined with
plastic or glass to cut down on costs.
• If an aseptic process : required the pipelines that
transport inoculum, air and ingredients for
fermentation have to be sterilized, normally with
steam.
• automated with spray jets and are referred to as
Cleaning in Place (CIP) located inside the vessel.
• Strong enough to hold large volumes of culture broth and
pressure that could be generated at times due to gas
production.
• Completely free from leakages, otherwise long-term
operations will be difficult and contaminations will occur.
• Adequate aeration and agitation of the fermenting broth :
microbial metabolism to proceed at the optimum level.
• System for monitoring and regulating temperature, pH.
• Allow monitoring and / or control of dissolved oxygen.
• Internal vertical plates called baffles to prevent vortexing
• Connected to inlet pipes to receive culture medium and
inoculum of the microorganism.
• Facility for sampling and should require a minimum of labour
in maintenance, cleaning, operating and harvesting
operations
• Constructed using the cheapest materials and
should be adequate service provisions for
individual plants.
• The size and the internal volume of the bioreactor
: designed by bio-engineers with proper
knowledge of fluid dynamics, and the
microbiologists should maintain the sterility of the
entire system.
Major components of fermentor
• Types of Fermentors (Bioreactors) :
• The most widely used bioreactors in industries are
• Stirred tank bioreactor
• Tower bioreactors
• Air lift bioreactors
• Packed - bed bioreactors
• Fluidized bed bioreactors
• Photo bioreactors
At the lateral side of the fermentor, :an opening through which culture
medium is being pumped into the fermentor.
Another opening : as the outlet for harvesting the products at the base
of the fermentor.
Volume of the vessel is about 30 to 50% larger than the required culture
volume leaving a head space-allowing disengagement of the liquid
droplets from the exhaust gas and room for foaming.
A heating coil :to raise the temperature inside the vessel.
Sterile air : pumped into the fermenter through a pipeline found just
• Tower bioreactors :
• Huge bioreactors : designed for the brewing
industry.
• The cells tend to aggregate and settle at the bottom
of the vessel inspite of the upflow of the fluid.
• A high hydrostatic pressure generated at the
bottom of the reactor increases the solubility of
oxygen in the medium.
• Tower fermentors are of different types based on
their design.
• Bubble-up fermentors, Bubble column tower
fermentors, Multistage tower fermenters, Vertical-
tower Beer fermentors
• The high density of cells may create anaerobic
condition which is advantageous to alcohol
fermentation.
• Tower bioreactor has high aeration capacities
without having moving parts.
• Bubble column tower fermentors : used for citric
acid and tetracycline production and for a range of
other fermentations based on mycelial fungi.
• Vertical-tower beer fermentors : designed to
maximise yeast biomass yields and for beer
production.
• Multistage tower fermenters : used for continuous
culture of E. coli, S. cerevisiae and activated sludge.
Tower reactor
• Air lift bioreactors :
• In airlift bioreactors, the medium is circulated
around two nested columns within a long tubular
tower.
• The rate of airflow of the reactor : depends on the
volumetric mass transfer co-efficient in the reactor
system.
• The incoming air forces the medium up the inner
column, or riser, and it then descends down the
outer column, or down comer tube.
• The riser tube may be placed within the down
comer tube or it may be externally located and
connected to the latter.
• Air lift bioreactors are of two types.
• (i) Internal-loop airlift bioreactor,
• (ii)External-loop airlift bioreactor.
• Internal-loop airlift bioreactor : a single container
with a central draft tube that creates interior liquid
circulation channels.
• External-loop airlift bioreactor : an external loop
so that the liquid circulates through separate
independent channels.
• An internal heat exchanger coil : located at the
bottom loop connecting the riser and downflow
tubes; it maintains the temperature at 300 degree
C.
• Airlift bioreactors : used for aerobic bioprocessing
technology.
• These bioreactors : mainly employed in the production
of single cell proteins, methanol production and waste
water treatment
• A Typical air lift bioreactor
Airlift bioreactors with internal loop
• Packed - bed bioreactors :
• A packed-bed bioreactor : filled about 2/3rd with solid particles.
• usually installed with a forced aeration device.
• A column which is filled with a solid matrix, traps microorganisms
within it.
• The solid matrix may be gels absorbed with the biocatalyst
(enzyme/cells), or more rigid particles, e.g. compressible
polymeric particles or particles of silica.
• The temperature adjustment between the top and bottom of the
substrate depends on the thickness of the bed and the aeration
rate.
• A nutrient broth is continuously poured over the immobilized
biocatalyst.
• The products obtained in the bioreactor - released into the fluid
and removed.
• The concentration of the nutrients - increased by increasing the
flowrate of the nutrient broth.
• The depth of the packed bed : limited by factors like pH, gradient
formation, nutrient concentration and oxygen requièrent.
A packed-bed fermentor with forced aeration
• Fluidized bed bioreactors :
• In the fluidized bed bioreactor, the solid substrate :
fluidized by upward flow , the solids are retained in
the rector while the liquid flows out
• A biological film developed on particles which are
suspended in an upward flow of liquid in which they
are then free to circulate.
• Fluidized beds are designed using a biocatalyst such
as immobilized enzyme or cells adsorbed to
particles.
• The support particles can be solids such as sand or
glass beads or porous such as plastic or stainless
steel mesh.
• The solid particles carrying the biocatalyst are
suspended in the liquid substrate. For an efficient
operation of fluidized beds, gas is sparged to create
a suitable gas-liquid-solid fluid bed.
• The upflowing stream of nutrient (substrate) is
used to fluidize the solid particles which get
dispersed in the liquid.
• The top of the reactor is kept broad and the
bottom narrow so that the particles concentrate
more on the lower narrow region.
• These bioreactors : used in conjuction with
immobilized cells or enzyme system and are
operated continuously
Fluidized bed bioreactor Fluidized bed recycle reactor
• Photo bioreactors :
• The photosynthetic organisms (Cyanobacteria, Microalgae) :
light for the production of important products (e.g :
Carotene, single cell protein and astaxanthin)
• Bioreactors designed for photosynthetic organisms :
photobioreactors.
• In addition to light, the cells require CO2 which can be
provided by dissolving bicarbonate.
• Bioreactors comprise an array of transparent tubes (glass or
clear plastic tubes), that are placed horizontally or vertically.
The array of tubes or flat pannels constitute light receiving
system.
• The culture is circulated through the light receiving system
solar receivers by centrifugal pumps or airlift pumps.
• The light penetration depends on the density of the biomass
and the temperature is maintained at 22-37 degree celcius .
Types of photobioreactors (A) Continuous run tubular loop (B) Multiple
parallel tube (C) Helical wounder tubular loop (D) Flat panel configuration.
• Design and operation of bioreactor :
• Industrial fermentors : the best possible growth and
biosynthesis for industrially important microbial
cultures, and to allow ease of manipulation for all
operations associated with the use of fermentors.
• fermentors : some provision for the control of or
prevention of the growth of contaminating
microorganisms.
• Conventional bioreactors : cylindrical vessels with
domed top and bottom.
• The reaction vessel, surrounded by a jacket, is
provided with a sparger.
• The agitator shaft is connected to a motor at the
bottom. The reaction vessel has side ports for pH,
temperature and dissolved oxygen sensors
• The bioreactor :designed to work at high temperature,
high pressure.
• provision for inoculation and sampling, as well as for
charging and discharging the vessel.
• In order to maintain aseptic operation the mechanical
integrity of the plant should be maintained.
• Entry points to the aseptic region of the plant such as
mechanical seals for agitators and pump shafts, valve
closers, probe insertions, sample codes and joints are
loci of contamination risks and should be as few in
number as possible.
• A good aseptic fermentor should not have direct
connections between sterile and non-sterile parts of a
system and minimise flange connections
• If the industrial fermentations are aerobic, the
fermentor : mechanical stirrers to mix the solution,
baffles to increase turbulence and ensure adequate
mixing, and forced aeration to provide needed
oxygen.
• The fermentor : provision for the intermittent
addition of antifoam agent.
• The fermentor : designed in such a way that it
should avoid stagnant regions, dead spaces,
pockets, pipe branches and crevices which cannot
only collect stagnant liquid and microorganisms but
also difficult to sterilize effectively.
• Diaphragm valves : needed to regulate fluid flow
in the fermentor or a piston valve for aseptic
conditions with a steam sealed gland is used.
• Porous spargers made up of sintered glass,
ceramics or a metal : used to introduce air into
the fermentor.
• Antifoaming agents like alcohols (sterile and octile
decanol); esters, fatty acids and their derivatives,
silicones, sulphonates and miscellaneons
compounds like oxaline and polypropylene glycol
are used to prevent or to decrease the foaming
during fermentation.
Different types of agitators. : A. Disc turbine; B. vaned disc; C.
open turbine, variable pitch; and D. marine propeller agitators.
Inoculation process of a fermentor vessel.
• For successful fermentation process: Sterilization can be in
situ sterilization and continuous heat sterlization.
• In in situ sterilization : the whole system is heated to about
1200 degree Celsius and held at this temperature for about
20 minutes.
• In continuous heat sterilization, the medium is rapidly heated
to 1400 degree Celsius for a short period, by injecting the
pressurised steam. Later, inoculation of the specific industrial
microorganism into the growth medium is done.
• The size of the incolum : genereally 1-10% of the total
volume of the medium.
• During the fermentation process, samples are regularly drawn
from the bioreactor and checked for contamination (if any).
• Adequate aeration and agitation of the fermenting broth
should be provided for the microbial metabolism to proceed
at the optimum level.
• Impellars are used for agitation, and they rotate
through a motor.
• Spargers are structures used for aeration. Aeration may
be air-lift system of aeration or stirred system of
aeration.
• Bioreactors meant for growing mycelial fungi may
require different kinds of spargers with holes of larger
diameter about 8 to 10 times the diameter specified for
the unicelled organism.
• Impellars (agitators) distribute the air bubbles released
from the spargers uniformly in the culture vessel
medium.
• The number of impellars in a vessel depends on the
vessel volume, usually 2 to 3 impellars may be
connected to a single shaft at a difference of about 1.2
times the impellar diameter.
• pH of the broth at the optimum level (pH control-can
be done by adding alkali or acid, after testing a
sample at intervals.
• Temperature control is absolutely essential for a good
fermentation process. Rapidly growing
microorganisms can generate a large amount of heat
that must be dissipated to prevent inactivation of
enzymes.
• Cooling coils are often employed in fermentors to
regulate temperature and to maximize the rate of
product accumlation.
• Some thermophilic organisms : require high
temperature, and in such cases, it may be necessary
to pass steam to raise the temperature using internal
coils.
• Oxygen is sparingly soluble in water and it should
be 0.0084 g/L at 250 degree Celsius (dissolved
oxygen).
• During the fermentation process, because of
aeration and agitation, foam is formed. The
antifoams are added in small quantities only, other
wise they will reduce oxygen transfer within the
broth and may affect product formation.
• After completion of the fermentation process, the
bioreactor is harvested and is prepared for the
next round of fermentation after cleaning
• Types of fermentations
• Solid substrate (solide state) fermentation (SSF) :
Solid-state fermentation (SSF) : a microbial process
in which a solid material is used as the substrate or
the inert support of microorganisms growing on it.
• SSF : developed for the manufacturing of
traditional foods and alcoholic beverages, its
application has been extended to the
pharmaceutical and biochemical industries.
• The most commonly used solid substrates for SSF :
cereal grains, wheat bran, saw dust, wood shavings
and several other plant and animal materials.
• Fungi and actinomycetes are the best suited for SSF,
because of their larger biomass and reach by means
of hyphae.
• SSF techniques are also being used for the
production of high-value products such as enzymes
and toxins.
• Most enzymes from fungi are now being cheaply
produced through SSF. More recently, this approach
has been used for the production of extracellular
enzymes, certain valuable chemicals, fungal toxins,
and fungal spores (used for biotransformation).
• In solid substrate fermentation, the microbial
distribution occurs on the solid surface, and microbial
growth and product formation also occur mainly on
the surface.
• The moisture content required for SSF : normally low,
depending on the physical or chemical characteristics
of the substrate.
• Heat derived from the metabolism and growth of the
microorganism raises the temperature of the solid
substrate bed and causes the loss of moisture.
• The air supply and temperature of the solid substrate
bed : controlled by forced aeration, in which the
large surface area of the solid substrate promotes
heat transfer and gas exchange of oxygen and
carbondioxide.
• The bioreactors used in SSF are tray fermentors
without any agitation, drum fermentors with
continuous or staggered slow agitation, and column
fermentors with forced aeration.
• The tray type fermentors consists of wooden, metallic
(aluminium, iron), or plastic trays with perforated
bottom. The trays are sterlized and filled with a layer
of substrate mixed with inoculum.
• The trays are stacked one above the other to a
convenient height. A humid atmosphere is created
inside the chamber, and the temperature is controlled
by cool or warm air. After fermentation process is
completed, the trays are removed and the fermented
mash is pooled for down stream processing for
product recovery.
Solid state fermentation : A set up for surface culture in trays. 1. Air
inlet, 2. air filter, 3. air moistener, 4. air heater, 5. fermentation
chamber with stacked trays, 6. culture collectror, 7. air outlet.
• Submerged fermentation
• (i) Batch culture (or) Batch fermentation :
• The growth of the fermentation organism : in a closed system in a
batch culture.
• A microbe grows in the medium until the nutrients are exhausted
or toxic metabolites secreted by it reach to inhibitory level.
• Important nutrients given to the microorganisms :utilized for the
reproduction and the accumulation of the metabolic products.
• This results in continually changing conditions in the culture in
which the composition of the cell wall and the concentrations of a
number of cytoplasmic components vary as growth of the
microorganisms proceed. The incubation is carried out under
optimal physiological conditions (pH, temperature O2 supply etc.).
• After inoculation, the culture enters lag phase, during which there
is increase in the size of the cells and not in their number.
• It is the period of adaptation to new conditions or prior to active
growth.
• The length of the lag phase is variable and is mostly determined by
the new set of physiological conditions, and the phase at which
the microorganisms were existing when inoculated
• The time : for the population to double in size is
referred as "doubling time", or mean generation time,
which is the average time taken by a single cell to
divide into two daughter cells.
• The two parameters : doubling time and mean
generation time are identical under favourable
conditions, but if some daughter cells are non-viable
the mean generation time is less than the observed
doubling time.
• The growth rate of the microbes also decreases due to
the nutrient limitation because of the depletion in the
substrate coupled with accumulation of toxic
metabolites. This phase is known as stationary phase.
The biomass remain almost constant during this
phase.
• [ii]fed-batch culture- the culture is subsequently fed with fresh
nutrient medium without removing the growing microbial
culture and allows one to supplement the medium with such
nutrients that are depleted or that may be needed for the
terminal stages of the culture.
• [iii] Continuous culture (or) Continuous fermentation :
Continuous culture : well defined cultivation conditions for
genetic, biochemical and physiological characterization of cells
and allows independent variation of growth parameters,
enabling reliable kinetic studies of cell growth and metabolism
for process optimization.
• Continuous cultures : used for production of some primary
metabolites (e.g, ethanol and organic acids), in waste water
treatment, and fermented foods and for some reactions
catalyzed by enzymes ; for the production of monoclonal
antibodies and recombinant proteins by animal cell cultures.
Schematic diagram of four types of continuous culture
A typical setup of a bench-scale continuous-culture system.
• Anaerobic fermentation
• One of the largest areas of industrial microbiology is anaerobic digestion of
wastes, and anaerobes that are having an increasing role in detoxicification
of hazardous wastes and pollutants.
• Anaerobic microorganisms do not utilise molecular oxygen in biosynthesis
and are not capable of using oxygen as a terminal electron acceptor.
• They use diverse array of organic and inorganic electron donors and
acceptors in their energy metabolism.
• In anaerobic fermentations, the production of biomass is less than aerobic
organisms. This is due to their characteristic energy metabolism and
consequent formation of small quantities of ATP. With less biomass
production, more carbon can be converted to end-products and the product
yeild is higher.
• In case of acetogens and other gas utilising bacteria, oxygen free sterile CO2
or other gases are bubbled through the medium.
• The culture vessel (fermentor) required for acetogens is 400 lts size and 3 kg
cells could be harvested in each run. Anaerobes can use a variety of
substrates including polysaccharides, molasses, sugars and other complex
substrates which may be obtained from agricultural waste streams and
reduce the overall cost of the fermentation process.
• The fermentation usually produces high yeilds of CH4 , organic acids or
other compounds by fermenting complex organic substrates such as
polysaccharides or proteins.
• Anaerobic organisms of industrial importance includes a wide
range of genera in the domains of bacteria and archaea.
• The genus Clostridium : important medically and for the
production of metabolites including enzymes and solvents.
• Toxigenic Clostridia : employed in production of human and
animal vaccines and for human theraupatics.
• Lactic acid bacteria (Lactococcus, Lactobacillus,
Streptococcus, Propionebacterium and Leuconostoc) :
important in food fermentations and also in the production
of organic acids.
• Sulphate reducing bacteria (Desulphovibrio, Desulphobacter
and Desulphotomaculum) : used in biotransformation of
organic substrates including hazardous compounds and toxic
wastes.
• These bacteria are industrially important in oil and gas
industry; in corrosion of iron, steel and concrete; and in
sewage treatment.
• Anoxygenic phototrophic bacteria are employed for
industrial production of vitamins, single cell
proteins, enzymes and in waste treatment.
• Aerobic Fermentation
• If the growth of the fermentation microorganism is
to occur aerobically, then provision must be made
for rapid incorporation of sterile air into the
medium in such a manner that the oxygen of this
air is dissolved in the medium and, therefore,
readily available to the microorganism, and the
carbondioxide resulting from microbial metabolism
is largely flushed from the medium.
• sterile air is supplied to the fermentor.
• The fermentors mostly employed in aerobic
fermentations include stirred tank type and air lift
type.
• The fermentor should provide aseptic means for
the withdrawal of culture samples during the
fermentation as well as for the introduction of
inoculum at the initiation of the fermentation.
• Unstructured kinetic models (UKMs) :From simple
experimental data, we can obtain information to represent
cellular growth with unstructured kinetic models
• The correlation among cell growth, substrate consumption
and inhibition or description of the substrate profiles within
the reactor during expression of extracellular proteins is the
central goal of the model process.
• The UKMs, which are unstructured, unsegregated, are based
on the monitoring of cell and nutrient concentration and
describe the fermentation process as an average of the
species under ideal conditions.
• UKMs consider the apparent rate obtained by metabolic
processes, which are carried out by microorganisms.
• These models are based on conservation equations for cell
mass, nutrients, metabolites, and species generation/
consumption rates. Most of the UKMs can be divided into
three terms: rate expressions for cell growth, rate
expressions for nutrient uptake, and rate expressions for
metabolite production.
• In the case of exponential growth phase,
which is the simplest representation of
microbial growth, nutrient concentration
profiles and decrease rate in several cases are
not almost considered.

where r is the reaction rate, X represents biomass,

μ is specific growth rate, kD is the death rate, α is
the stoichiometric factor, and Yi is the yield.
• UKMs is Monod’s model :This is one of the simplest
models to deal with microbial growth, physiology,
and biochemistry. The Monod equation describes
the proportional relationship between the specific
growth rate and low substrate concentrations

where μMAX is the maximum specific growth rate, [S]

is the substrate concentration, and KS is the
saturation constant.
 UNIT-5
Micobial
UNIIT-5
Reactors
Different types of microbial
reactors, Bioreactor operations
for industrial important
biological products, case studies
•Microbial cultures : highly developed specialized
capabilities such as increased demand for
aeration and agitation.
•The high growth rates : rapidly changing
conditions which require fast response times,
accurate sensors and reliable controls for pH,
DO, temperature in order to control the culture
conditions.
•A bioreactor is a vessel that allows controlled
growth of a microbial culture
• The major types are:
• (1) Continuous Stirred Tank Bioreactors
• (2) Bubble Column Bioreactors
• (3) Airlift Bioreactors
• (4) Fluidized Bed Bioreactors
• (5) Packed Bed Bioreactors
• bioreactors : advantages of providing a controlled environment where it is
possible to control critical process parameters to optimize the microbial
bioremediation process
• Another advantage : flexibility in design of the bioreactor (size and
configuration) to suit application or intended purpose of the
• bioremediation in bioreactors if operated ex situ, requires relocation of
pollutant, a process which can involve excavation for soils and sediments,
transportation and possible containment or controlled handling of the
contaminated media thus making the process expensive
• There is a potential for exposing other environments to the contamination.
• The advantages of microbial bioremediation : it has public
acceptance, as it is a natural process .
• It is a low cost technology in most cases when compared to other
clean-up methods for hazardous waste.
• It can be done in situ and ex situ, instead of contaminants being
transferred from one form to another or one medium to another,
complete destruction of target organic pollutants is possible.
• Disadvantages : bioremediation takes relatively long to achieve
treatment goals, may not be effective on all contaminants, some
products of biodegradation maybe more toxic or persistent than the
parent compound, specificity of the biological processes with
respect to microbial populations, pollutant and environmental
limitations
• specialized expertise are required in designing and implementing.
• Bioremediation using microbial bioreactors : application in soil,
air and water environments including:
• Waste water and industrial effluent treatment:
• Microorganisms : the primary agents of any biological
wastewater treatment.
• Microorganisms are already present in waste water systems
and feed on complex substances in the wastewater converting
them to simpler substances thus assisting in achieving the
treatment.
• Trickling filters, membrane bioreactors, slurry phase reactors
and upflow anaerobic sludge blanket bioreactors (UASB) :
some of the reactors that are used in waste water and
industrial effluent treatment.
• Oil and land treatment:
• Contaminants successfully treated include
• diesel fuel, fuel oils, oily sludge, wood-preserving
wastes (PCP, PAHs, and creosote), coke wastes, and
certain pesticides
• Soil bioremediation : proven most successful in treating
petroleum hydrocarbons and other less volatile,
biodegradable contaminants.
• Slurry phase, stirred tanks, biofilters, partitioning phase
and packed microbial reactors find application in
contaminated soil remediation.
• Control of air pollution
• Microorganisms : used in the bioremediation of organic and inorganic air
pollutants in spent gases before release or escape into the atmosphere.
• Microorganisms oxidize pollutants such as H2S, SO2, VOCs, and reduce
pollutants such as NOx to nitrate and this assist to prevent likely
environmental, health hazards and nuisances.
• Bioscrubbers and biofilters are some of the bioreactor : used in control of
air pollution.
• Solid waste management
• Microorganisms : chiefly responsible for the biodegradation of organic
wastes in nature and they drive the decomposition processes that occur
in landfills and composts.
• Anaerobic digesters : often applied mostly in the biotreatment solid
waste.
• Factors affecting microbial bioreactor performance
• A number of issues are at play in all bioremediation technologies
including when bioreactors are used.
• These are those that concern the contaminant, microbial community and
the design, optimization and monitoring of the process.
• In the design and operation of bioreactors in remediation, many of these
factors have to be optimized and controlled for best reactor performance.
• Variables that affect the operation and efficiency of a microbial bioreactor
relate to biotic and abiotic factors that affect microbial growth and those
factors that relate to the reactor design and configuration.
• Factors that affect microbial growth and activities in bioreactors include;
environmental factors (temperature, pH, moisture), pollutant mix,
pollutant concentration, macronutrient. Factors on reactor design include;
size, configuration and mode of operation.
• Environmental related factors
• Environmental conditions (temperature, pH, oxygen
availability/electron, and salinity) affect growth; the metabolic
activities of microorganisms and to some extent the behavior of the
pollutant such as solubility and volatility.
• In any process optimization for biodegradation, it is always
necessary to investigate the effects of the environmental conditions
and optimize the process in relationship to all the relevant
environmental conditions.
• Temperature
• always a temperature range at which microorganisms grow and
survive (i.e., minimum, optimum and maximum survival temperature).
• In addition, there is always a temperature optimum at which
biochemical processes take place to achieve required bio treatment
by each microorganism.
• Extremes of temperature (too low or too high) affect both
microbial growth and microbial enzyme catalyzed reactions.
• With an increase in temperature within appropriate range,
microbial metabolism increases and thus the rate of the
bioremediation processes.
• Increased temperatures lead to higher solubility of many
chemicals, and increased fluidity and diffusion rates.
• For example with pollutants, such as PAHs and heavy metals,
their solubility and in turn bioavailability increases with
temperature. Temperature is thus a critical factor in the optimum
operating efficiency of bioreactors to achieve best biotreatment
results. Often specialized bioreactors are designed with
provision for temperature control.
• pH
• Similar to temperature, pH affects microbial growth and metabolic
processes.
• pH influences microbial cell ionic properties thus microbial growth.
• Microorganisms have minimum, optimum and maximum pH of growth with
most bacteria for example growing optimally at pH 6–7.5, though there are
some which thrive best at acidic pHs (acidophiles) or at alkaline pH
(alkaliphiles).
• Fungi generally grow at pHs lower than that of bacteria.
• Reactor operating pH : set to provide the best pH conditions for growth
and enzyme activities.
• Behavior of pollutants is also influenced by pH thus affecting their
bioremediation.
• For example with metals,
• pH affects the redox and solubility of metals, different forms and
valence have different effects on microorganisms
• Metal solubility increases with a decrease in medium pH and
alkaline pH favor metal ion precipitation.
• Often lower pH values are required for metal attachment to the
microbial cell surface.
• Microorganisms that produce acids result in increased solubility
of the metal ions .
• To provide for best pH conditions, buffers are used in media
formulations, acids and bases can be added during the
bioreactor process .
• Nutrients
• Nutrients are required for growth and metabolism of the microorganisms.
Several elements are required in biosynthesis and energy production.
• Carbon is the most basic element of living forms and is needed in greater
quantities than other elements.
• Other elements : important in ensuring a balanced nutritional bioreactor
environment depending on the type of microorganism include hydrogen,
oxygen, nitrogen, sulfur, phosphorus, iron, calcium and magnesium.
• All necessary macro- and micro- nutrients requirements : provided in
reactor media.
• Microorganisms can use the pollutants they are degrading as primary
energy sources or a primary source of energy is provided to the
microorganism in the case of co metabolism of the pollutants.
• Moisture
• Water is required to support microbial growth and catalysis.
• Cellular chemical reactions occur in aqueous conditions and water is
required to ensure the correct osmotic pressure is maintained for
microbial growth.
• The amount of water available for microbial growth is called (aW).
• Most microorganisms grow at water activities of 0.98 or higher,
osmotolerant species can however grow at a range of low aw
• Electron acceptors
• The presence of electron acceptors, e.g., oxygen in aerobic microbes and
NO31−, SO42− and Fe (III) oxides in case of anaerobic microbes, also
affects the biodegradation processes.
• Reactor design related factors
• Bioreactors : provide for the best conditions for microbial growth and
biochemical process to occur.
• The reactor size, configuration and mode of operation : key factor for
design
• The reactor : provide favorable physical, biological and the combined
physical-chemical conditions for the best biological remediation processes
to be achieved.
• In designing the bioreactor, favorable physical conditions for transport of
gases and liquids and solids over time that ensure that the physical entity
of the bioreactor is favorably adapted to the biological system that
performs the bioreactions are required.
• Need to ensure that the biophysical and biochemical events taking operate
at optimum levels under real situation application.
• Polluted samples for remediation can be fed into the reactor either
as dry or slurry matter.
• Pollutants with hydrophobic properties are often unavailable for
microbial degradation, particularly if they are bound to soil matrix.
• Their degradation : limited by their transfer to liquid.
• Minimizing mass transfer resistance was found to be a key factor
in the degradation of hexachlorocyclohexane (HCH) in slurry
batch bioreactors .
• Despite the rapid development of bioreactors due to their
widespread use in biotechnology, the aspects of maintaining
stability and rates of bioprocesses are still areas to be addressed.
• Poor bioreactor construction and design, leading to inadequate
mixing, may expose the stability and performance of the process
• Mixing prevents thermal stratification,
• help maintain uniform conditions in the reactor,
• ensure good contact between microbial culture and media
reactants.
• The importance of mixing in bioreactor cannot be over
emphasized, poor mixing affect microbial process efficiency.
• Hydraulic retention times (HRT) required to achieve the
necessary remediation goals in the bioreactor have to be
determined and optimized.
• Longer HRTs result in poor substrate loading which diminishes
the microbial population, whereas shorter ones do not allow
microorganisms to effectively degrade the pollutant and can
result in microbial wash out from the system.
• Organism related factors
• population density, composition, inter and intraspecific interaction.
• A whole range of environments ranging from aerobic, anaerobic, acidic,
alkaline, and low to high temperature : utilized as sources of
microorganisms for bioremediation.
• Only certain species of bacteria and fungi have proven their ability as
potent pollutant degraders.
• In the natural environment degradation of pollutants is often achieved
through complex microbial population interactions.
• Single or mixed microbial cultures are used for pollutant remediation in
bioreactors.
• In the event where bioagumentation is applied the introduced organisms
need to be able to co-exist with indigenous residents.
• Different microorganisms : different metabolic capabilities, to this
extend the evaluation of several strains of different microbial
players have to be investigated in order to come up with the best
degraders.
• In screening and comparison of the bio-degradation of PAHs by
white rot fungi , found out that newly screened white rot fungi
strains had higher or comparable degradation capacity to the
model well applauded P. chrysosporium, and these strains did not
accumulate the metabolite quinone which accumulates as a dead
end metabolite in P. chrysosporium.
• microorganisms : certain inherent physiological characteristic, e.g.,
metabolism of known substrate with structural similarity to
xenobiotics of interest and/or adaptation to certain environmental
conditions can be selected.
• Pollutant related factors
• Factors that affect bioremediation in bioreactors that are related
to the pollutant include: nature of pollutant, i.e., the physical and
chemical properties including solubility, volatility, molecular
complexity, concentration and toxicity.
• Investigations for most pollutant biodegradation have centered
on how different concentrations, mixed pollutants, solubility and
molecular structure can affect microbial bioremediation.
• In the case of PAHs, degradation decreases in the order alkane>
branched chain alkanes>low molecular weight aromatics>
cycloalkanes.
• Some pollutants are resistant to biodegradation (recalcitrant, i.e.,
resistant to degradation) they are degraded at very low pace
even if the right microbial population and conditions are present.
• Microbial bioreactors in bioremediation
• Bioreactor technologies may offer effective means for treatment
of many contaminants in groundwater, soil and air .
• The bioreactor type of choice : easy to operate and maintain for
the selected purpose and application.
• Flexibility to design bioreactor tailor made for different processes
and remediation applications makes the use of bioreactors in
bioremediation attractive.
• The design should accommodate high biomass from cell growth,
supply of necessary nutrients and also removal of waste
components from the system
• Slurry phase bioreactors
• Slurry phase bioreactors : treats polluted media that is within a slurry
phase. Slurry bioreactors offer an ex situ environmentally friendly way
for remediating mostly soils and sediments from petrochemical
hydrocarbons, tars, creosotes, chlorinated solvents, herbicides,
pesticides and explosives or when a solid substrate that is formulated
into a slurry is used.
• Hydrophobic nature of most persistent chemicals makes them sorb to
soil or sediments and not easily accessible for biodegradation.
• Operation of the slurry reactor can be in batch, semi-continuous and
continuous mode, with the batch process being the most common one
• Water is mixed with the contaminated solid matrix in suitable ratios
and this enhances contact between microorganisms, pollutant, media
and oxygen.
• Pollutants that are solubilized become more bioavailable.
Simplified slurry reactor
• The ability of specific microorganisms to produce specialized enzymes and
proteins has been exploited for many purposes in industry.
• Industrial microorganisms : used to produce many things, including food,
cosmetics, pharmaceuticals and construction materials.
• Industrial microbiology includes the use of microorganisms to manufacture food
or industrial products in large quantities.
• Numerous microorganisms are used within industrial microbiology; : naturally
occurring organisms, laboratory selected mutants, or even genetically modified
organisms (GMOs).
• Example : Archaea : specific types of prokaryotic microbes that exhibit the
ability to sustain populations in unusual and typically harsh environments.
Those suriving in the most hostile and extreme settings are known
as extremophile archaea. The isolation and identification of various types
of Archaea, particularly the extremophile archaea, have allowed for analysis of
their metabolic processes, which have then been manipulated and utilized for
industrial purposes.
• Extremophile archaea species : of particular interest due to the
enzymes and molecules they produce that allow them to sustain life
in extreme climates, including very high or low temperatures,
extremely acid or base solutions, or when exposed to other harmful
factors, including radiation.
• Specific enzymes which have been isolated and used for industrial
purposes include thermostable DNA polymerases from the Pyrococcus
furiosus.
• This type of polymerase is a common tool in molecular biology; it is
capable of withstanding the high temperatures that are necessary to
complete polymerase chain reactions.
• Additional enzymes isolated from Pyrococcus species include specific
types of amylases and galactosidases which allow food processing to
occur at high temperatures as well.
• orynebacteria are characterized by their diverse origins.
• found in numerous ecological niches and are most often used in industry for
the mass production of amino acids and nutritional factors.
• The amino acids produced by Corynebacterium glutamicum include the amino
acid glutamic acid.
• Glutamic acid is used as a common additive in food production, where it is
known as monosodium glutamate (MSG).
• Corynebacterium can also be used in steroid conversion and in the
degradation of hydrocarbons.
• Steroid conversion is an important process in the development of
pharmaceuticals.
• Degradation of hydrocarbons is key in the breakdown and elimination of
environmental toxins. Items such as plastics and oils are hydrocarbons; the
use of microorganisms which exhibit the ability to breakdown these
compounds is critical for environmental protection
• Xanthomonas, a type of Proteobacteria, : known for its ability to cause disease in
plants.
• The bacterial species which : classified under Xanthomonasexhibit the ability to
produce the acidic exopolysaccharide commonly marketed as xanthan gum, used
as a thickening and stabilizing agent in foods and in cosmetic ingredients to
prevent separation.
• various species of Aspergillus. This genus includes several hundred types of
mold.
• Aspergillus : a key component in industrial microbiology, where it is used in the
production of alcoholic beverages and pharmaceutical development.
• Aspergillus niger : commonly used to produce citric acid, which is used in
numerous products ranging from household cleaners, pharmaceuticals, foods,
cosmetics, photography and construction.
• Aspergillus is also commonly used in large-scale fermentation in the production
of alcoholic beverages such as Japanese sake.
1.Beverages
2. Antibiotics
3. Amino Acids
4. Organic acids
5. Vitamins
6. Enzymes
7. Organic solvants
8. Single Cell Protein (SCP)
9. Steroids
10. Vaccines
11. Pharmaceutical Drugs
12.Dairy products
•Properties of useful industrial microorganism:
• Produces spores or can be easily inoculated
• Grows rapidly on a large scale in inexpensive
medium
• Produces desired product quickly
• Should not be pathogenic
• Amenable to genetic manipulation
• Food additives
• Ex- Guar Gum, Artificial Sweeteners, Sodium Benzoate
• Metabolites
• Ex- ethanol, lactic acid, resins
• Alcoholic and non-alcoholic beverages
• Ex- whiskey, fruit juices
• Biofuels
• Ex – butanol, ethanol
• Biofertilizers
• Ex- Azolla,
• Several chemicals
• Enzymes, catalysts, and other bioactive molecules
• Vaccines and antibiotics to kill pathogenic microorganisms or slow their growth
• Beverages –
• These microorganisms play an essential role in the fermentation process of many foods.
• fermentation in industrial processes : fermented beverages, malt grains, broths, fruit juices, and
antibiotics.
• Yeasts : used microorganisms to manufacture liquids like rum, wine, beer, whiskey, brandy, etc. Yeasts
are eukaryotic, single-celled microorganisms of the Kingdom Fungi. Saccharomyces cerevisiae, species
of yeasts, typically referred to as Brewer’s Yeasts, ferment fruit juices and malted cereals to supply
ethanol.
• after the fermentation, those liquids are distilled to supply alcoholic and non-alcoholic liquids
consisting of whiskey, bourbon, brandy, gin, rum, etc.
• Organic Acids –
• Microorganisms :used in the industrial production of some organic acids. Citric acid :first organic acid
discovered through microbial fermentation of citrus fruit- lemon. Organic acids are also made directly
from glucose.
• Acetobacter sharp, Aspergillus Niger, and Lactobacillus are some microorganisms : industrial
production of organic acids.
• These compounds can be produced directly from glucose (e.g. gluconic acid) or formed as end
products from pyruvate or ethanol.
• Examples of acids producing microorganisms are Aspergillus Niger (a fungus) of Citric acid,
Acetobacter acute (a bacterium) of Acetic Acid, Lactobacillus (a bacterium) of lactic acid and many
others.
• Amino acids such as Lysine and Glutamic acid are used in the food industry as
nutritional supplements in bread products and as flavor enhancing compounds
such as Monosodium Glutamate (MSG).
• Amino acids are generally synthesized as primary metabolites by microbes.
However, when the rate and amount of synthesis of some amino acids exceed
the cell’s need for protein synthesis, then cell excrete them into the
surrounding medium.
• Enzymes
• natural biological catalysts : used for controlling the biochemical
reactions in living systems.
• Enzymes : used extensively in both medical as well as non-medical
fields.
• enzymes can also be obtained from some microorganisms called
microbial enzymes. Micro-organisms are primarily used to produce
industrial enzymes through safe gene transfer methods.
• Many microbes synthesize and excrete large quantities of enzymes
into the surrounding medium. These include Amylase, Cellulase,
Protease, Lipase, Pectinase, Streptokinase, and many others.
• Enzymes are extensively used in food processing and preservation,
washing powders, leather industry, paper industry and in scientific
research.
• Vitamins and Antibiotics –
• Vitamins : organic compounds that can perform many essential functions in
our bodies.
• These are essential micronutrients that the body needs in small amounts for
its metabolism like thiamine, folic acid, vitamin b12, etc.
• Many antibiotics : produced by microorganisms, predominantly by
Actinomycetes in the genus Streptomycin (e.g. Tetracycline, Streptomycin,
Actinomycin D) and by filamentous fungi (e.g. Penicillin, Cephalosporin)
Vitamins
• Vitamins are some organic compounds which are capable of performing many
life-sustaining functions inside our body. These compounds cannot be
synthesized by humans, and therefore they have to be supplied in small
amounts in the diet.
• Microbes are capable of synthesizing the vitamins and hence they can be
successfully used for the commercial production of many of the vitamins e.g.
thiamine, riboflavin, pyridoxine, folic acid, pantothenic acid, biotin, vitamin
b12, ascorbic acid, beta-carotene (pro-vitamin A), ergosterol (provitamin D).
• INDUSTRIAL USES OF MICROBES –
• Saccharomyces cerevisiae (brewing and baking bread )
• E. coli (bacteria such as recombinant protein)
• Aspergillus niger (a fungus for the production of citric acid and enzymes)
• Clostridium butyclicum (bacteria used in yogurt and cheese)
• Xanthomonas campestri (xanthan gum-producing bacteria)
• Deinococcus radiorans (bacteria for soil and water restoration)
• MICROORGANISMS IN FOOD PRODUCTION
• Microorganisms are used in all sorts of food production : Dairy production,
meat production, alcohol production, health food production.
INDUSTRIALLY IMPORTANT MICROORGANISMS –
• They are important for converting chemicals that help reduce pollution and
the production of various metabolites such as ethanol, butanol, lactic acid,
and riboflavin. For example, microbes can be used to make biofertilizers or
to reduce metal contaminants. Microbes can also be used to make some
non-microbial foods, such as insulin for diabetes
• Making cheese is not possible without starter culture. As the
culture grows in milk, it converts sugar-lactose to lactic acid.
This provides the right level of acidity and moisturizes the
cheese. As the cheese ripens, culture gives it a balanced
aroma, flavor, and texture as the cheese ripens. It is also the
cause of “holes” in cheese such as Emmental.
• Meat Production –
• Starter cultures are used to make dry fermented foods such as
salami, pepperoni, chorizo ​, and dried ham. Lactobacilli develop
the taste and color of food. In addition, various shapes are used
to ripen the surface of the sausage, preserving the natural
properties of the product and regulating the development of
flavor.
• Alcohol Production –
• Yeast is responsible for the fermentation process that produces
alcohol in wine. Lactic acid bacteria also play an important role in
converting the unstable malic acid naturally present in wine
into stable lactic acid. These strains give the stability properties of
high-quality wines, which are enhanced during storage.
• Health Food Production –
• Lactobacilli are used in the health food industry in a variety of
tablets and sold as supplements. The busy lifestyle of modern
people often leads to an imbalance in the intestinal flora. Travel and
medical treatment are the two main causes. This balance can be
restored by taking supplements containing probiotics, which can
improve your quality of life.
• Biofuels
• Organic solvents such as ethanol, acetone, butanol, and glycerol are some
very important chemicals that are widely used in petrochemical industries.
These chemicals can be commercially produced by using microbes and low-
cost raw materials (e.g. wood, cellulose, starch).
• Yeast (Saccharomyces cerevisiae) is used for commercial production of
ethanol. This alcohol is used as motor fuel and is often referred to as green
petrol.
• Single Cell Protein (SCP)
• Single Cell Protein (SCP) : serve as an alternate source of energy when a larger
portion of the world is suffering from hunger and malnutrition.
• Single cell proteins : microbial cells that are rich in protein content and can be
used as protein supplements for humans and animals.
• Microbes like Spirulina can be grown easily on materials like waste water from
potato processing plants (containing starch), straw, molasses, animal manure,
and even sewage, to produce large quantities and can serve as food rich in
protein, minerals, fats, carbohydrate, and vitamins
• Steroids
• These are a very important group of chemicals, which are used as anti-
inflammatory drugs, and as hormones such as estrogens and progesterone,
which are used in oral contraceptives.
• Steroids are widely distributed in animals, plants, and fungi like yeasts. But,
producing steroids from animal sources or chemically synthesizing them is
difficult, but microorganisms can synthesize steroids from sterols or from
related, easily obtained compounds.
• Vaccines
• Vaccines are also a product of industrial microbiology. Many antiviral vaccines
are mass-produced in chicken eggs or cell cultures.
• The production of vaccines against bacterial diseases usually requires the
growth of large amounts of the bacteria. Recombinant DNA technology is
increasingly important in the development and production of subunit vaccines.
• Pharmaceutical Drugs
• Many pharmaceutical drugs are also produced by microbes e.g. Cyclosporin A,
that is used as an immunosuppressive agent in organ-transplant patients, is
produced by the fungus Trichoderma polysporum.
• Neutron structure of the immunosuppressant cyclosporin A. Statins produced
by the yeast Monascus purpureus have been commercialized as blood-
cholesterol lowering agents. It acts by competitively inhibiting the enzyme
responsible for the synthesis of cholesterol.
• Gluconic acid, : valuable organic acid, useful in medicine as a carrier for
calcium, because gluconic acid is easily metabolized in the body leaving a store
of calcium for distribution.
• produced from carbohydrates by A. niger and species of the bacterium
Gluconobacter cultivated in fermentation tanks.
• Calcium gluconate is also added to the feed of laying hens to provide calcium
that strengthens the eggshells.
• Certain properties make microorganisms well suited for industrial processes.
Microorganisms : broad variety of enzymes to make an array of chemical
conversions possible
• have a relatively high metabolic activity that permits conversions to take place
rapidly.
• a large surface area for quick absorption of nutrients, and release of end-
products.
• usually multiply at a high rate, as evidenced by the 20-minute generation time
for Escherichia coli under ideal conditions.
• In the industrial process, microorganisms act like chemical factories.
• To be effective, they should liberate a large amount of a single product that can
be efficiently isolated and purified.
• The microorganisms should be easy to maintain and cultivate, and should have
genetic stability with infrequent mutations.
• Their value : enhanced if they can grow on an inexpensive, readily available
medium that is a by-product of other industrial processes
• Microbiologists inoculate the mold to a medium of corn meal, molasses, salts,
and inorganic nitrogen in huge shallow pans or fermentation tanks.
• The absence of a Krebs cycle enzyme in the mold prevents the metabolism of
citric acid into the next component of the cycle, and the citric acid accumulates
in the medium.
• Another important microbial product is lactic acid, a compound employed to
preserve foods, finish fabrics, prepare hides for leather, and dissolve lacquers.
Lactic acid is commonly produced by bacterial activity on the whey portion of
milk. Lactobacillus bulgaricus is widely used in the fermentation because it
produces only lactic acid from lactose.
 UNIT-6
Introduction to downstream
processing
Recovery and the purification of
biosynthetic products
• The efficiency and successful working of a
bioreactor : on adequate upstream operations,
and the recovery of the product requires a series
of careful and meticulous steps collectively
referred to as downstream processing.
• These include purification, economic analysis,
legal and regulatory affairs, and production on
larger scales.
• The products of fermentation may be whole cells
(biomass), enzymes, amino acids, organic acids,
solvents,antibiotics, therapeutic proteins,
vaccines, gums etc., depending on the end
product, different separation principles are
required.
• filtration, centrifugation, flotation, disruption
(separation operation); solubilization, extraction,
thermal processing, membrane filtration,
precipitation (concentration operation);
crystallization, chromatography (purification
operations); and drying, packing and storage
(marketing operations)
• Downstream processes : knowledge in chemistry,
biology, chemical engineering molecular
engineering : the extraction and purification of the
product from the spent medium will be possible.
• The cost of downstream processing : more than
50% of the manufacturing cost, and there is product
loss at each step of DSP
• The various steps involved in down-stream
processing are as follows :
• 1. Solid-liquid separation.
• 2. Disintegration of cells (release of intracellular
products)
• 3. Extraction
• 4. Concentration
• 5. Purification
• 6. Formulation
• Solid-Liquid Separation
• The first step in DSP is the separation of solid, usually cells,
from the liquid medium.
• Centrifugation :
• used to separate bacteria :
• Based on the principle of density differences between the
particles to be separated and the medium.
• In continuous flow industrial centrifuges, there is a
continuous feeding of the slurry and collection of clarified
fluid, while the solids deposited can be removed
intermittently.
• disc centrifuge, scroll centrifuge, tabular bowl centrifuge and
multichamber centrifuge.
• Centrifugation difficulties arise due to small differences in the
densities of the particles and the medium.
• The disadvantages of the centrifugation are equipment cost,
power consumption, temperature etc.
• Flocculation and Flotation :
• The formation of large aggregates (floccules) of cells
to settle down for easy removal : as flocculation.
• sedimentation rate of a particle increases with size,
flocculated cells can be recovered by centrifugation.
• Flotation : used in alcoholic beverages industry.
• Gas bubbles can be created by sparging,release of
overpressure or electrolysis; and adhere to cells or
flocs, making them float at the surface of the liquid
from where they are skimmed off.
• Collector substances like long chain fatty acid
amines facilitates stable foam formation during
flotation process
• Filtration :
• Separation of biomass and culture filtrate is usually
carried out by filtration.
• Filtration uses pressure created by over pressure or
vacuum, and : rate depends on filter area, fluid
viscosity and the resistance generated by filter-
cake, which increases with time.
• (a) Cake filtration :
• The solid particles are retained as a cake in the
filter medium.
• The filters : made up of sintered metal, glass wool,
cloth, fibers, ceramics, cellulose etc.
• (b) Rotary vacuum drum filters :
• Useful in separating mycelia of filamentous fungi.
Rotary vacuum drum filters : proved to be quite
satisfactory for continuous or semi-continuous
separation of cells from whole broth.
• The filter is in the form of drum and a portion of
the drum rotates through the medium which is
partially immersed in a tank of broth
• (c) Ultrafiltation :
• It is a pressure driven membrane separation process for dissolved
and suspended materials.
• Two types of products can be processed by ultrafiltation,
• namely,
• (a) dissolved macromolecules
• (b) cellular products.
• Substances with molecular weights ranging from 500-1,000,000 :
separated by ultrafiltation,
• eg: substances like peptides, proteins, vaccines, viruses etc.
Ultrafiltration : used for
• (a) purification of culture medium prior to bioconversion (or)
fermentation,
• (b) removal of cell debris after lysis,
• (c) harvesting of cells after fermentation and
• (d) concentration of product.
• Ultrafiltation is mainly employed in pharmaceutical, chemical and
food processing industries.
Biological tissues
Microorganisms
Microorganisms
Animal cell supernatants
Biological liquids

Homogenization disruption
Extraction
Clarification
Precipitation
Ultrafiltration
Separation
Chromatography
Electrophoresis
Purification

Pure
protein
Principal steps in protein DSP
• (d) Membrane filtration :
• Millipore membrane filters : available in different pore sizes.
• Membranes are prone to choking and blocking.
• Membranes with asymmetric pores (asypore filters) : more
efficient.
• Membranes and membrane processes : effective tool in
modern biotechnology for down stream processing of
bioreactor constituents, sterilization of feed streams, or
immobilization of enzymes.
• Mass-scale separation : achieved physically at ambient
temperature without any chemical change is one of the
advantages of membranes.
• In cross flow membrane filtration, the broth is pumped in a
crosswise fashion across the membrane.
• Factors influencing the efficiency of the cross flow technique
include permeate flux, voltage and degree of turbulence.
• 2. Disintegration of cells (release of intracelluar
products) :
• If the desired product is intracellular, the cell
biomass can be disrupted so that the product
should be released.
• Disruption of microbial cells : usually difficult due to
their small size, strong cell wall and high osmotic
pressure inside the cells.
• There are three methods of cell disruption.
• They are enzymatic, chemical and physical methods
Methods of cell disruption to release intracellular products
• (i) Enzymatic Methods :
• Cell disruption by enzymatic methods : advantages
ie., lysis of cells occurs under mild conditions in a
selective manner.
• Enzymatic methods : use of egg white lysozyme to lyse
gram negative bacteria.
• Lysozyme : commercially available and it hydrolyses β-
1,4-glycosidic bonds of the mucopeptide in bacterial
cell walls.
• Lysozyme plus EDTA is used for the disruption of
Pseudomonas fluorescens cells to release acylamidase.
As the cell wall gets digested by the lysozyme, the
osmotic effects break the periplasmic membrane to
release the intracellular contents.
• (ii) Chemical Methods :
• Alkali treatment : used for the extraction of some bacterial
proteins.
• Alkali lysis : used at pH-11 for 20 minutes, using sodium or
potassium hydroxide for the release of L-asparagine from
Erwinia chrysanthemi. Organic solvents like methanol,
ethanol, isopropanol, butanol can be used in cell disruption.
• The use of detergent : method of cell lysis.
• The detergents may be ionic (sodium dodecyl sulphate),
anionic (sodium cholate), cationic (acetyl trimethyl
ammonium bromide) or non-ionic (Triton-X-100, Tween 80).
• The ionic detergents are more reactive than non-ionic
detergents, and can cause denaturation of many proteins, Ex
: triton-X- 100 is used to release cholesterol oxidase from
Nocardia species, sodium cholate is used for the release of
pullulanase (pullulan 6-glucon hydrogenase), a membrane
bound enzyme from Klebsiella pneumoniae.
Recovery process for intracellular product of fermentation
• (iii) Physical Methods :
• Mechanical methods : sonication, grinding, glass bead mills, the french
press, and the Manton-Gaulin homogeniser.
• These methods : destroy proteins as well as cells and requires optimization
of the release of active protein.
• Periplasmic proteins can be selectively liberated by osmotic shock, which
ruptures only the outer membrane by sudden transfer from hyper to
hypertonic medium.
• Gram negative bacteria such as Salmonella typhimurium and E.coli : easily
lysed by osmotic shock.
• Osmotic shock involves washing the cells in a buffer, suspending them in
20% sucrose and resuspending them in distilled water at 40 degree C.
Grinding the cells by mixing with glass beads are subjected to a very high
speed in a reaction vessel.
• Dynomill is an apparatus used for the release of proteins from cells by
abrasion.
• It is a chamber containing glass beads and a number of fixed and rotating
impeller discs. A dynomill can process 5 kg of bacteria per hour.
• The french press is a standard tool for lysis of some tonnes of grams of wet
cell paste by forcing cells through a small orifies at high pressure. It is
necessary to precool the cell suspension before homogenization.
• 3. Extraction :
• Compound recovery process from a mixture.
• The purification of microbial metabolite : wide range of
techniques since these compounds have very diverse
chemical structures. range : from extremely water-
soluble compounds to very solvent extractable
substances that have all types of ionic character and a
wide range of molecular weights.
• The most difficult compounds to purify : the very
water-soluble, neutral, or amphoteric substances. T
• To recover solvent extractable metabolites, a typical
extraction can be accomplished by mixing two volumes
of the fermentation filtrates with one volume of ethyl
acetate, separating the organic layer and repeating the
process.
• If the metabolite is an acid, the extraction : more
efficient if carried out under acidic conditions, and
the opposite effect is observed for basic
metabolites.
• Solvent extractable and some near-solvent-
extractable metabolites : to adsorb the metabolite
from the fermentation filtrate with a reverse-phase
resin such as Amberlite XAD-2, Diaion HP- 20, or
Diaion HP-21.
• The aqueous two phase separation method :
precipitation with polyethylene glycol (PEG) and
dextran, PEG and ammonium sulphate, followed by
centrifugation. : mainly employed for extraction of
compounds with high molecular weight.
• 4. Concentration :
• After extraction, both intracellular and
extracellular proteins are usually concentrated
from the cell-free broth.
• The commonly used techniques for concentrating
biological products
• are ultrafilteration or adsorption, evaporation and
membrane filteration, using ion-exchange
• resins. Ultrafilteration can be carried out at small
scale in centrifugally driven cells, or at
• larger scale in stirred cells or tangential flow units.
Evaporation is generally used in cases of
• solvent extraction. The evaporators have a heating
device for supply of steam, and unit for
• 5. Purification :
• Final step in the recovery of the products.
• Chromatography : used in the purification of proteins.
• In Industrial downstream processing, selection of the matrix for
chromatography : important as the process involves large scale
purification.
• The matrix chosen should be porous, rigid, stable, inert and
reusable.
• The commonly used matrices and their trade names are :
• (i) Cross linked dextran (Sephadex), (ii) Cross linked polyacrylamide
(biogen-P), (iii) Agarose (Sepharose, ultragel-A), (iv) Cross linked
agarose (Sepharose-CL, Superose), (v) Porous silica-silicaceous
particles coated with agarose (Spherosil), (vi) Kieselguhr
(Macrosorb), (vii) rigid organic polymers (Monobeads), (viii)
Polysternedivinyl benzene (Poros), and (ix) cellulose (Ultragel-A,
Whatman TM, Cellufine, cellex).
• Chromatography : selective adsorption of proteins on
the surface of porous particles, through interactions
that can be broadly classified as
• ion exchange, hydrophobic and affinity.
• Chromatography operations : classified as low-
pressure, high-pressure (HPLC), medium-pressure
(including Pharamacia's popular FPLC), depending on
the pressure used to force liquid through the packed
bed of adsorbent particle
• Apparatus used for chromatography : a column into
which the particles are packed (glass, plastic or steel),
• a pump to drive liquid flow,
• some method for introducing the sample into the flow
before the column (a switch wall or manual pipetting),
• a fraction collector that deposits the emerging,
separated proteins into different vessels.
Types of Chromatography
Chromatography techniques and principles
• 6. Formulation :
• Formulation : For antibiotics and acids like citric acid, by
crystallization by adding salts.
• Crystallization may be initiated by changing pH or temperature at
constant salt concentration.
• The formulation of low molecular weight products (solvents,
organic acids) : achieved by concentrating them with removal of
most of the water.
• Stabilizers like sugars, salts, polymers addition : Increase the shelf-
life of proteins
• Proteins may be formulated in the form of solutions, suspension or
dry powder.
• Drying is an essential component of product formulation.
• Drying : product suitable for handling and storage.
• In freeze drying, very low pressure is maintained to promote
sublimation and the energy needed for sublimation is provided by
heated plates and radiation onto the surface.
• The wide range of products such as diagnostic food stuffs,
pharmaceuticals are freeze dried.
• Effluent treatment (Biological waste treatment)
• The type of microorganisms and their quantity : vary
depending on the nature of the waste water.
• Most industrial processes waste waters : varying
amounts of salts and organic matter.
• waste waters associated with the industrial
fermentation industries : spent media, waste waters
and waters accumulating in various steps of product
recovery.
• The manufacturing industries : releases substances
which are biodegradable. These wastes can be utilized
and recycled by microorganisms and plants.
• Agriculture and Dairy industries : crop residues and
manures, which are biodegradable.
• The waste waters resulting from industrial
fermentation processes : water-soluble, colloidal, and
suspended wastes.
• If a plant or animal pathogen has been employed in the
fermentation, the fermentation wastes : require-sterilization
before undergoing waste treatment.
• The spent media or media residues also : preliminary
filtration before further treatment to remove the larger solids
and masses of microbial cells.
• Bacteria present in the sewage : coliforms, faecal
streptococci, anaerobic spore forming bacilli, proteus group
etc.
• A fermentation company, depending on its size and on the
type of waste waters that it produces, either may process its
own waste waters or arrange with the local muncipal
sewerage for treatment at the local sewage treatment plant.
• The natural microbial flora of the waste waters include
heterotrophic bacteria, to certain extent,actinomycetes and
fungi. Protozoa also are very active in organic matter
decomposition.
• Anaerobic bacteria : helpful in the anaerobic-digestion of
organic matters.