Industrial Biotechnology
OLD
• Use of micro-organisms for large scale industrial
processes
• Oldest form of microbiology and biotechnology which
was used to make wine, beer, sake, bread with use of
bacteria and yeasts without knowing scientific basis
• Production of ethanol, lactic acid, butanol using
microbes and Enzymes like amylase, protease, invertase
were used during early 20th century
• Pencillin was produced during WWII and other amino
acids, nucleotides, enzymes were also produced later
White Biotechnology
MODERN
• Now use in biotransformations of chemicals, genetic
engineering of MO for non microbial products (insulin,
interferons, HGH, vaccines)
• Microbial Fermentations (organic acids, amino acids,
vitamins, antibiotics, enzymes) and fermented foods
(dairy, meat, plant, breads, alcoholic beverages)
• Use in increase of crop productivity
• Biofertilizers and Biopesticides
• Use Microbes as food: single cell protein
• Bioenergy and Bioremediation
• Mining and metallurgy
Isolation and culturing Micro-organisms
Sources
• Soils, lakes, oceans, river, plant, animal, air, non living
objects
Isolation methods
• Serial dilution, spread plate, gradient pour plate, streak
plate
• Filtration, centrifugation
• Importance of eliminating other organisms (antibiotics,
heating)
Isolation and culturing Micro-organisms
Growth Media
• MO require nutrients (C, N,
requirement, temp, pH, salinity etc
• Synthetic media
• Semi synthetic media
• Natural media
P,
Minerals),
O2
• Media needs to be economical for large scale
productions, consistent quality and available throughout.
Raw material can be pre-treated if required
• Cheap C and N2 sources can be used
Isolation and culturing Micro-organisms
Sources of nutrition
Carbon: sugarcane molasses, beet molasses, vegetable
oil, starch, cereal grains, whey, glucose, sucrose, lactose,
malt, hydrocarbons
Nitrogen: corn steep liquor, slaughter house waste,
urea, ammonium salts, nitrate, peanut granules, soyabean
meal, yeast extract etc
Growth factors: vitamins and amino acids are added
when MO cannot synthesize them
Isolation and culturing Micro-organisms
Sources of nutrition
• Trace elements: Zn, Mo, Mn, Cu, Co required for metabolism or in
metallo-enzymes or in proteins (Hb)
• Inducers, precursors, repressors: for enzymes to function in
metabolic processes inducers are required. Sometimes presence of
presursors enhances production of a secondary metabolite or production
an enzyme can be repressed due to repressors. Eg streptomycin is nduced
by yeast extract, Sec metabolites can be repressed due to some cpds.
• Antifoams: sunflower oil, olive oil to prevent foaming
• Water: clean water of consistent composition, dissolved
chemicals, pH is measured. Also required for cleaning, washing,
rinsing, cooling, heating etc.
Culturing methods for Micro-organisms
Sterilization: devoid of MO (aseptic conditions)
Contamination free seed
culture
Sterilization of equipment,
media and air
Moist heat (121oC/15psi/20min),
radiation, ultrasonic treatment,
chemicals, mechanical, gases (ozone),
filtration for sterilizing air
CULTURING
Avoidance of contamination can be achieved by
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Use pure inoculum to start fermentation
Sterilize the media
Sterilize fermenter vessel
Sterilize all materials to be added to the fermentation during the process
Maintaining aseptic conditions during the fermentation
Control of environmental conditions for Microbial growth
Temperature
pH
Agitation
O2 conc
To be carefully monitored and maintained
Acidic pH: fungi and yeast
Psychrophiles, acidophiles etc
Sterilization:
Elimination of threads and welding of pipes and tubes to reduce
contamination
Fermenters have pipes which flush steam into the system
Media along with fermenter is sterilized
Among the several factors that influence killing are temperature, pH,
osmotic pressure, shear, mass transport, and concentrations of extraneous
substances that also react with the killing agent. These factors operate
synergistically, and temperature plays roles other than simply affecting
the kinetics of a reaction
Aeration and Mixing
Shake culture: flasks are kept on a shaker for required rotations
Fermenters: Stirrers for O2 mixing and baffles for increasing turbulence
V shaped notch
• Incrs turbulence
• Incr eff of O2 transfer
• Improves growth of MO
Fermentation
Fervere: to boil
In Microbiology
Any process for the production of useful products through mass culture of MO
In Biochemistry
The numerous O-R reactions in which organic compounds used as carbon and
energy act as acceptors and donors of H2 ion. The organic cpd gives rise to
various products of fermentation which accumulate in the growth medium
Takes place in absence of O2
Now term industrial fermentation for large scale cultivation of microorganisms…most of them is aerobic
Bioprocess technology (plants and animal cells) replaces fermentation
technology (microbial use)………..not rigid
Fermenter or bioreactor
• A biorector is a device in which the organisms are cultivated and
motivated to form a desired product
• Closed vessel or containment designed to give a right environment for
optimal growth and metabolic activity of the organism
• Fermenter: for microbes/ Bioreactor : for eukaryotic cells
• Size variable ranging from 20-250 million litres or more.
• Large scale production (10-100L to1000-million L capacity)
• Helps to meet requirements of:
pH
temp
aeration
agitation
drain or overflow
control systems
sensors
cooling to achieve maximum microbial yield
What is fermentation technique (1)?
Techniques for large-scale production of microbial
products. It must both provide an optimum
environment for the microbial synthesis of the
desired product and be economically feasible on a
large scale.
They can be divided into surface (emersion) and
submersion techniques. The latter may be run in
batch, fed batch, continuous reactors
In the surface techniques, the microorganisms are
cultivated on the surface of a liquid or solid
substrate. These techniques are very complicated
and rarely used in industry
What is fermentation technique (2)?
In the submersion processes, the microorganisms
grow in a liquid medium.
Except in traditional beer and wine fermentation,
the medium is held in fermenters and stirred to
obtain a homogeneous distribution of cells and
medium.
Most processes are aerobic, and for these the
medium must be vigorously aerated.
All important industrial processes (production of
biomass and protein, antibiotics, enzymes and sewage
treatment) are carried out by submersion processes.
Some important fermentation products
Product
Ethanol
Glycerol
Lactic acid
Acetone and
butanol
-amylase
Organism
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Lactobacillus
bulgaricus
Clostridium
acetobutylicum
Bacillus subtilis
Use
Industrial solvents,
beverages
Production of
explosives
Food and
pharmaceutical
Solvents
Starch hydrolysis
Some important fermentation products
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Some important fermentation products
Some important fermentation products
Winemaking fermenter
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General Aspects of
Fermentation Processes
Fermenter
The heart of the fermentation process is the
fermenter.
In general:
• Stirred vessel, H/D  3
• Volume 1-1000 m3 (80 % filled)
• Biomass up to 100 kg dry weight/m3
• Product 10 mg/l –200 g/l
Component parts of a fermenter
1. Formulation of media to be used in culturing the organism during
development of inoculum and in the production fermenter
2. Sterilization of the medium, fermenter and ancillary equipment
3. Production of an active, pure culture in sufficient quantity to inoculate
the production vessel
4. The growth of the organism in the production fermenter under
optimum conditions for product formation
5. The extraction of the product and its purification
6. Disposal of effluents produced by the process
Production
fermenter
Stock
culture
Shake
flask
Biomass
Culture
fluid
Seed
fermenter
Cell
separation
Cell free
supernatant
Medium STERILIZATION
Medium FORMULATION
Medium raw material
Product
purification
Product packaging
Product
extraction
Effluent treatment
DOWNSTREAM
PROCESSING
Cross section of a fermenter for Penicillin production
( Copyright: http://web.ukonline.co.uk/webwise/spinneret/microbes/penici.htm)
Cross section of a fermenter for Penicillin production
( Copyright:
http://web.ukonline.co.uk/webwise/spinneret/microbes/penici.htm)
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Flow sheet of a multipurpose fermenter and its
auxiliary equipment
Basic modes of operations of a fermenter
1. Batch culture
Batch fermentation refers to
• a partially closed system in which most of the materials required
are loaded onto the fermentor, decontaminated before the
process starts and then, removed at the end.
• The only material added and removed during the course of a
batch fermentation is the gas exchange and pH control solutions.
• In this mode of operation, conditions are continuously changing
with time, and the fermentor is an unsteady-state system,
although in a well-mixed reactor, conditions are supposed to be
uniform throughout the reactor at any instant time.
The principal disadvantage of batch processing is the high
proportion of unproductive time (down-time) between batches,
comprising the charge and discharge of the fermenter vessel, the
cleaning, sterilization and re-start process
Basic modes of operations of a fermenter
2. Continuous culture
Continuous culture is a technique involving feeding the microorganism
used for the fermentation with fresh nutrients and, at the same
time, removing spent medium plus cells from the system
A unique feature of the continuous culture is that a timeindependent steady-state can be attained which enables one to
determine the relations between microbial behavior (genetic and
phenotypic expression) and the environmental conditions.
Basic modes of operations of a fermenter
3. Fed-batch processes
The fed-batch technique was originally devised by yeast producers in the
early 1900s to regulate the growth in batch culture of Saccharomyces
Yeast producers observed that in the presence of high concentrations of
malt, a by-product - ethanol - was produced, while in low concentrations of
malt, the yeast growth was restricted. The problem was then solved by a
controlled feeding regime, so that yeast growth remained substrate
limited.
The concept was then extended to the production of other products,
such as some enzymes, antibiotics, growth hormones, microbial cells,
vitamins, amino acids and other organic acids.
3. Fed-batch processes
Basically, cells are grown under a batch regime for some time, usually until
close to the end of the exponential growth phase.
At this point, the reactor is fed with a solution of substrates, without the
removal of culture fluid.
This feed should be balanced enough to keep the growth of the
microorganisms at a desired specific growth rate and reducing simultaneously
the production of by-products (that can be growth or product production
inhibitory and make the system not as effective).
By products may lead to cell death
A fed-batch is useful in achieving high concentration of products as a result of
high concentration of cells for a relative large span of time.
Two cases can be considered: the production of a growth associated product and
the production of a non-growth associated product. In the first case, it is
desirable to extend the growth phase as much as possible, minimizing the
changes in the fermenter as far as specific growth rate, production of the
product of interest and avoiding the production of by-products.
For non-growth associated products, the fed-batch would be having two phases:
a growth phase in which the cells are grown to the required concentration and
then a production phase in which carbon source and other requirements for
production are fed to the fermenter.
This case is also of particular interest for recombinant inducible systems: the
cells are grown to high concentrations and then induced to express the
recombinant product
Types of Bioreactors
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Simple fermenters (batch and continuous)
Fed batch fermenter
Air-lift or bubble fermenter
Cyclone column fermenter
Tower fermenter
Fluidized bed bioreactors
Packed bed bioreactor
photobioreactor
• Other more advanced systems, etc
The size is few liters (laboratory use) - >500 m3 (industrial
applications)