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
 MODERN
White Biotechnology
• 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, P, Minerals), O2

requirement, temp, pH, salinity etc
• Synthetic media
• Semi synthetic media
• Natural media

• 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

Moist heat (121oC/15psi/20min),

Sterilization of equipment, radiation, ultrasonic treatment,
media and air chemicals, mechanical, gases (ozone),
filtration for sterilizing air

CULTURING

Avoidance of contamination can be achieved by

• 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 To be carefully monitored and maintained
O2 conc

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 micro-

organisms…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 Organism Use

Ethanol Saccharomyces Industrial solvents,

cerevisiae beverages
Glycerol Saccharomyces Production of
cerevisiae explosives
Lactic acid Lactobacillus Food and
bulgaricus pharmaceutical
Acetone and Clostridium Solvents
butanol acetobutylicum
-amylase Bacillus subtilis Starch hydrolysis
Some important fermentation products

16
Some important fermentation products
Some important fermentation products
Winemaking fermenter

19
 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 Biomass

Culture Cell
Stock Shake Seed fluid separation
culture flask fermenter

Cell free
supernatant
Medium STERILIZATION

Medium FORMULATION Product

extraction
Medium raw material
Product
purification
Effluent treatment

DOWNSTREAM
Product packaging 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: 25
http://web.ukonline.co.uk/webwise/spinneret/microbes/penici.htm)
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 time-

independent 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

• 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)