1

BIOPROCESS TECHNOLOGY
 Bioprocess Technology
2

A bioprocess is any process that uses

complete living cells or their components (e.g.,
bacteria, enzymes, chloroplast) to obtain
desired products. This process is commonly
referred to as Fermentation.
 Bioprocessing
3

The entire process can be divided in three stages.

Stage I :
Upstream processing which involves preparation of liquid medium, separation of particulate and inhibitory
chemicals from the medium, sterilization, air purification etc.

Upstream processes include selection of a microbial strain characterized by the ability to synthesize a specific product having
the desired commercial value.

This strain then is subjected to improvement protocols to maximize the ability of the strain to synthesize economical amounts
of the product.

Included in the upstream phase is the fermentation process itself which usually is carried out in large tanks known as
fermenters or bioreactors.

In addition to mechanical parts which provide proper conditions inside the tank such as aeration, cooling, agitation, etc., the
tank is usually also equipped with complex sets of monitors and control devices in order to run the microbial growth and
product synthesis under optimized conditions.

The processing of the fermentation reactions inside the fermenter can be done using many modifications of engineering
technologies. One of the most commonly used fermenter types is the stirred-tank fermenter which utilizes mechanical
agitation principles, mainly using radial-flow impellers, during the fermentation process.
 Bioprocessing
4

Stage II:
Fermentation which involves the conversion of substrates to desired product with the help of biological agents
such as microorganisms.

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.

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.
 Bioprocessing
5

Stage III:

Downstream processing which involves separation of cells from the fermentation broth, purification and
concentration of desired product and waste disposal or recycle.

Downstream processing, the various stages that follow the fermentation process, involves suitable techniques and methods
for recovery, purification, and characterization of the desired fermentation product. A vast array of methods for downstream
processing, such as centrifugation, filtration, and chromatography, may be applied. These methods vary according to the
chemical and physical nature, as well as the desired grade, of the final product.
 6

Culture fluid
biomass
Cell separation

Stock Shake Cell free supernatant

Seed fermenter
culture flask
Product extraction
Effluent treatment
Medium sterilization
Product purification

Medium raw material Product packaging

 FERMENTATION
7

 The term fermentation is derived from the latin verb

fervere, to boil, describing the appearance of the
action of yeast on extracts of fruits or malted grain.
 Biochemical meaning relates to the generation of
energy by the catabolism of organic compounds
 Meaning in industrial microbiology: much broader
 The range of fermentation processes
8

There are five major groups of commercially

important fermentations:
i) Those that produce microbial cells (or biomass) as the
product
ii) Those that produce microbial enzymes
iii) Those that produce microbial metabolites
iv) Those that produce recombinant products
v) Those that modify a compound which is added to the
fermentation-the transformation process
Parts of a fermentation process
9

 Medium formulation

 Sterilization

 Inoculum preparation

 Growth of the organism under optimum condition

 Extraction of the product and it’s purification

 Disposal of effluent produced by the process

 Microbial biomass
10

The commercial production of microbial biomass may

be divided into two major processes:
 The production of yeast to be used in the baking

industry
 The production of microbial cells to be used as
human or animal food
 Bioreactor design
11

 The design and mode of operation of a bioreactor are based on the production of an organism, optimum conditions required
for desired product formation, product value, and its scale of production.

 A good bioreactor design will help to improve productivity and provide higher quality products at lower prices.

 A bioreactor is a device that consists of various features such as an agitator system, an oxygen delivery system, a foam control
system, and a variety of other systems such as temperature & pH control system, sampling ports, cleaning, and sterilization
system, and lines for charging & emptying the reactor.

 The material used for the construction of a bioreactor must have the following important properties:
 It should not be corrosive.
 It should not add any toxic substances to the fermentation media.
 It should tolerate the steam sterilization process.
 It should be able to tolerate high pressure and resist pH changes.

 The sizes of the bioreactor vary widely depending on the application.

 Some bioreactors are designed for small scale fermenters and some for large scale industrial applications from the microbial
cell (few mm3) to shake flask (100-1000 ml) to the laboratory-scale fermenter (1 – 50 L) to pilot level (0.3 – 10 m3) to plant
scale (2 – 500 m3) for large volume).
 12

BIOREACTORS
Bioreactor is any device or vessel that is used to carry out
one or more biochemical reactions to convert any starting
material

The sizes of a bioreactor can vary over several orders of

magnitudes.
Microbial cell (1-10 µm)
Shake flask ( 100-1000 ml)
Laboratory fermenter ( 1 – 50 L)
Pilot scale (0.3 – 10 m3)
Plant scale ( 2 – 500 m3)
The choice of bioreactor depends on a number of
factors

Reactor and processing Culture and culture medium

• Area or application (research, • Metabolic state (aerobic, micro aerobic,
production) anaerobic)
• Value of desired product • Physiological state (viable, dead, growing,
• Processing (batch, fed- batch, resting
continuous) • Morphology (spherical, filamentous, flocs,
• Subsequent downstream processing films)
• Product formation kinetics • Sterility (sterile, non-sterile)

Properties of fermentation medium / broth

• Physicochemical (gaseous, liquid, solid, multiphase)

• Biological (inhibition, mechanical stress)
• Rheological (high or low viscosity)
• Bubbles (promoting or inhibiting coalescence)
13
• Foam formation
 Basic functions of fermenter
14

 Aseptic operation of the vessel

 Adequate aeration and agitation should be provided

 Mixing should not cause damage to the organism

 Power consumption should be as low as possible

 A system of temperature control should be provided

 A system of pH control should be provided

 Sampling facilities should be provided

  Evaporation losses from the fermenter should not be excessive

 The vessel should be suitable for a range of process

 The vessel should be constructed to ensure smooth internal

surface
 The vessel should be of similar geometry to both smaller and
larger vessels in the pilot plant or plant to facilitate to scale- up
 The cheapest materials which enable satisfactory results should be
used
 There should be adequate service provisions for individual plants

15
 Bioreactor

16
17
 Fermentation Equation
Ethanol Fermentation
glucose → 2 ethanol + 2 carbon dioxide
C6H12O6 → 2 C2H5OH + 2 CO2

Lactic Acid Fermentation

glucose → 2 lactic acid
C6H12O6 → 2 C3H6O3

Products of Fermentation
While there are a number of products from fermentation, the most common are ethanol, lactic acid, carbon dioxide, and
hydrogen gas (H2).
These products are used commercially in foods, vitamins, pharmaceuticals, or as industrial chemicals.
In addition, many less common products still offer commercial value.
For example, the production of acetone via the acetone – butanol – ethanol fermentation was first developed by the Jewish
chemist Chaim Weizmann and was important to the British war industry during Word War I.

18
 1. Fermenter Vessel
A fermenter is a large cylinder closed at the top and bottom connected with various pipes and valves.

The vessel is designed in such a way that it allows to work under controlled conditions.

Glass and stainless steels are two types of fermenter vessels used.

The glass vessel is usually used in small-scale industries. It is non-toxic and corrosion-proof.
Stainless steel vessel is used in large scale industries. It can resist pressure and corrosion.

2. Heating and Cooling Apparatus

The fermentor vessel’s exterior is fitted with a cooling jacket that seals the vessel and provides cooling water.

Thermostatically controlled baths or internal coils are generally used to provide heat while silicone jackets are used to remove
excess heat.

A cooling jacket is necessary for sterilization of the nutrient medium and removal of the heat generated during fermentation in the
fermentor.

3. Aeration System
An aeration system is one of the very important parts of a fermentor.
It is important to choose a good aeration system to ensure proper aeration and oxygen availability throughout the culture.
It contains two separate aeration devices (sparger and impeller) to ensure proper aeration in a fermentor.
The stirring accomplishes two things:
It helps to mix the gas bubbles through the liquid culture medium and
It helps to mix the microbial cells through the liquid culture medium which ensures the uniform access of microbial cells to
the nutrients.

19
 4. Sealing Assembly
•The sealing assembly is used for the sealing of the stirrer shaft to offer proper agitation.
•There are three types of sealing assembly in the fermenter:
Packed gland seal
Mechanical seal
Magnetic drives

5. Baffles
The baffles are incorporated into fermenters to prevent a vortex improve aeration in the fermenters.
It consists of metal strips attached radially to the wall.

6. Impeller
Impellers are used to provide uniform suspension of microbial cells in different nutrient mediums.
They are made up of impeller blades attached to a motor on the lid.
Impeller blades play an important role in reducing the size of air bubbles and distribute them uniformly into the fermentation media.
Variable impellers are used in the fermenters and are classified as follows.
Disc turbines
Variable pitch open turbine
7. Sparger
A sparger is a system used for introducing sterile air to a fermentation vessel. It helps in providing proper aeration to the vessel.
The sparger pipes contain small holes of about 5-10 mm, through which pressurized air is released.
Three types of sparger are used
Porous sparger
Nozzle sparger
Combined sparger–agitator
8. Feed Ports
They are used to add nutrients and acid/alkali to the fermentor.
Feed ports are tubes made up of silicone.
In-situ sterilization is performed before the removal or addition of the products.

20
 9. Foam-Control
•The level of foam in the vessel must be minimized to avoid contamination, this is an important aspect of the fermentor.
•Foam is controlled by two units, foam sensing, and a control unit.
•A foam-controlling device is mounted on top of the fermentor, with an inlet into the fermentor.

10. Valves
•Valves are used in the fermentor to control the movement of liquid in the vessel.
•There are around five types of valves are used, that is,
 globe valve,
butterfly valve,
a ball valve, and
diaphragm valve.
A safety valve is built-in in the air and pipe layout to operate under pressure

11. Controlling Devices for Environmental Factors

A variety of devices are utilized to control environmental elements like temperature, oxygen concentration, pH, cell mass,
essential nutrient levels, and product concentration.

12. Use of Computer in Fermenter

For an efficient process, monitoring, and data collecting, fermentors are generally coupled with modern automated and semi-
automated computers and databases.

21
GENERALIZED VIEW OF BIOPROCESS
RAW MATERIALS

UPSTREAM PROCESSES

Media Formulation
Inoculum Equipment
and
Preparation Sterilization
Sterilization

BIOREACTOR - FERMENTER

Reaction Kinetics Transport Phenomena Instrumentation

and Bioactivity and Fluid Properties and Control

DOWNSTREAM PROCESSES

Recovery and Waste Recovery,

Separation
Purification Reuse and Treatment

THE BOTTOM LINE

REGULATION ECONOMICS HEALTH AND SAFETY 22

TYPICAL BIOPROCESS FLOW SHEET
PR E PA R A T IO N
O F B IO M A S S FOAM CONTROL pH CONTROL
In n o c u lu m S ta g e s A n t if o a m A d d it i o n A c i d -A l k a l i A d d it io n

PRO DUCT RECO VERY

C E L L S E P A R A T IO N
B IO R E A C T O R In t r a c e l l u la r
product
E x t r a c e ll u l a r
1 ). C E L L D IS T R U P T IO N p ro d u c t
2 ). P R O D U C T E X T R A C T IO N

F re e C e lls ,
Im m o b liz e d C e lls PRODUCT
or C O N C E N T R A T IO N
PROCESS
E n z y m e B io re a c to r

PRODUCT
S E P A R A T IO N

P U R IF IC A T IO N

S T E R IL IZ A T IO N
D R Y IN G

R A W M A T E R IA S A ir F IN A L P R O D U C T
N u trie n ts a n d R e a c ta n ts
in A q u e o u s S o lu tio n 23
( m a y c o n t a i n in s o l u b l e
o r g a n i c a n d / o r in o r g a n i c
m a t e r ia l s )
 BASIC BIOREACTOR DESIGN CRITERIA

• Microbiological and Biochemical Characteristics of the Cell System (Microbial, Mammalian,

Plant)

• Hydrodynamic Characteristics of the bioreactor

• Mass and Heat Transfer Characteristics of the Bioreactor

• Kinetics of the Cell Growth and Product Formation

• Genetic Stability Characteristics of the Cell System

• Aseptic Equipment Design

• Control of Bioreactor Environment (both macro- and micro-environment)

• Implications of Bioreactor Design on Downstream Products Separation

• Capital and Operating Costs of the Bioreactor

• Potential for Bioreactor Scale-up

24
SUMMARY OF BIOREACTOR SYSTEMS

Bioreactor Design Cell Systems used Products

Air-Lift Bioreactor Bacteria, Yeast and other SCP, Enzymes, Secondary

fungi metabolites, Surfactants

Fluidized-Bed Bioreactor Immobilized bacteria, yeast Ethanol, secondary

and other fungi, activated metabolites, wastewater
sludge treatment

Microcarrier Bioreactor Immobilized (anchored) Interferons, Growth factors,

mammalian cells on solid Blood factors, Monoclonal
particles antibodies, Vaccines,
Proteases, Hormones
Surface Tissue Mammalian, tissue growth Interferons, Growth factors,
Propagator on solid surface, tissue Blood factors, Monoclonal
engineering antibodies, Vaccines,
Proteases, Hormones

25
 Bioreactor design Cell systems used Products

Membrane Bioreactors, Bacteria, Yeasts, Mammalian Ethanol, Monoclonal

Hollow fibers and cells, Plant cells antibodies, Interferons,
membranes used, Rotor- Growth factors, Medicinal
fermentor products

Modified Stirred Tank Immobilized Bacteria, Yeast, Ethanol, Monoclonal

Bioreactor Plant cells antibodies, Interferons,
Growth factors

Modified Packed Bed Immobilized Bacteria, Yeasts Ethanol, Enzymes, Medicinal

Bioreactor and other fungi products

Tower and Loop Bioreactors Bacteria, Yeasts Single Cell Protein (SCP)

26
 Various types of bioreactors

27

1. Stirred Tank Bioreactor

Stirred tank bioreactor uses baffles and

agitator for optimal mixing

They offer excellent mixing and reasonably

good mass transfer rates

The cost of operation is lower and the

reactors can be used with a variety of
microbial species
 2. Fluidized bed reactor

28

Most of the fluidized bed reactors developed for biological system involving
cells as biocatalysts are of three phase system (gas, liquid and solids )

The volumetric productivity attained in these reactors is usually higher than

in stirred tank and packed bed bioreactors
 3. Bubble column bioreactor

Sterile air or pure gases is used for

agitation and oxygenation
Low operating cost (no mechanical
agitation)

Mostly used for large scale production

Low viscosity fermentation where oxygen

demand is low (40-60 mM/h) KLa < 600 /h

29
 4. Hollow fiber bioreactors

30

Specially designed for immobilized enzymes

and cells

Enzymes are immobilized with polymeric

membrane and used in hollow fiber
bioreactors. Sometimes enzymes are just
adsorbed and this is called soluble
immobilized enzymes.

Various monoclonal and polyclonal

antibodies are produced in hollow fiber
reactor on large scale.

Continuous mode of production takes place.

Production of the desired products with

simultaneous purification is possible.
 Principle configuration of a bioprocess
upstream fermentation primary recovery

Substrate tank Fermenter Storage tank Centrifuge Cooling

downstream processing

homogeniser centrifuge filtration dilution IEX chromato- Gel filtra-

graphy tion
product
Ultra/diafiltration

31
 Types of fermenters
32

 Batch fermenter (BF)

 Continuous stirred tank fermenter (CSTR)
 Tubular fermenter (TF)
 Fluidized bed fermenter (FBF)
 Batch fermenter (BF)
33
Substrate

Initial concentration

Concentration
substrate
8

Time
Batch fermenter (BF)
 Continuous stirred tank reactor (CSTR)
34

Substrate Substrate Substrate

FEED
FEED

Concentration
Substrate FEED
Substrate
Substrate
8
8

(1) (2) 8(3)

(1) (2) (3)

Continuous stirred-
stirred-tank reactor train Product vessel
(CSTR)
 Tubular fermenter
35

Product

Inlet concentration

Concentration

Axial distance

Substrate
 Fluidized bed fermenter (FBF)
36
Substrate concentration and time profile for FBF
Product

Inlet concentration

Concentration

Axial distance

Substrate
 Fermentation vessels for microbes
37
 The packed tower
 Tower fermenter
 The Waldhof-type fermenter
 Acetators and Cavitators
 The cyclone column
 Cylindro-conical vessels
 Air lift fermenter
 Rotating-disc fermenter
 Deep-jet fermenter
 Packed tower
38

 A vertical cylindrical column was packed with pieces of some relatively inert material
eg wood shavings, twigs, coke or polythene.

 Initially both medium and cells are fed into the top of the reactor

 Once the cells have adhered to the support and are growing well as a thin film, fresh
medium is added at the top of the column and fermented medium is removed from the
bottom.

 The reactor is having high surface area

 Eg. 1. Vinegar generation in which ethanol was oxidized to acetic acid by strains of
Acetobacter

2. Sewage and effluent treatment

 Tower fermenter
39

 Tower fermenter is an elongated non mechanically stirred fermenter.

 Simple type which consists of a tube into which the substrate is pumped and
air is sparged . Eg. For citric acid production on a lab scale

 Water jacketed tubes of various dimensions inclined at an angle of 9 to 900 to

the horizontal eg Used in brewing industry

 Tower fermenter with perforated plates at intervals in the tower to maintain

maximum biomass production. Eg used in yeast production

 Multistage tower fermenter is also there. Eg used for brewing beer,

continuous cultures of E. coli and baker’s yeast.
 Cyclone column
40

 Dawson (1974) developed the cyclone column.

 The culture liquid was pumped from the bottom to the top of the
cyclone column through a closed loop. Nutrients and air were fed in
near the base of the column while the exhaust gases left at the top of
the column.

 Good gas exchange, lack of foaming and limited wall growth.

 Good for filamentous culture.

 Has a number of potential bacterial, fungal and yeast applications

including batch production of a vaccine for scours in calves.
 Cyclone column fermenter
41

Recirculating
limb

Cyclone column

Circulating
pump
 Cylindro-conical vessel
42

 Reduced process time.

 Primary fermentation and conditioning may be

carried out in the same vessel.

 The sedimented yeast may be easily removed

since its separation is good.

 The maturing time may be reduced by gas

washing with CO2.
 Air-lift fermenter
43

There are two types of air lift fermenters

1. Air lift fermenter with outer loop

2. Air lift fermenter with inner loop

 Effluent gas exit

44
Liquid level Culture exit
Air sparger Bubble breakup device
Down comer Riser

Direction of flow
Heat exchanger
Air/ Ammonia
sparge pipes

Sterile medium inlet

Air-lift fermenter outer loop

 Exhaust gas

45 Gas disengagement space

Up flow tube with baffles

Down flow tube

Cooling water

Compressed air

Air lift fermenter with inner loop

 Rotating-disk fermenter
46

 It is used for effluent treatment

 Utilizes a growing microbial film on slow rotating
disc
 A range of filamentous fungi, species of Aspergillus,
Rhizopus, Mucor and Penicillium could be grown on
polypropylene discs
 Citric acid has been produced by Aspergillus niger
using this fermenter
47

REFERENCE: Principles of Fermentation Technology by

Stanbury, Whitaker and Hall