FERMENTATION

TECHNOLOGY

Source:
Fermentation Microbiology and Biotechnology By EMT Mansi et al
Industrial Microbiology : An Introduction By MJ Waites
Industrial Microbiology By HS Patel
Food and Industrial Microbiology By Raveendra Reddy

Dr Diptendu Sarkar
[email protected]
What is fermentation?
 Pasteur’s definition: “life without air”, anaerobe
red ox reactions in organisms
 New definition: a form of metabolism in which the
end products could be further oxidized
 For example: a yeast cell obtains 2 molecules of
ATP per molecule of glucose when it ferments it
to ethanol.
 Fermentation takes place in the absence of oxygen,
when the electron transport chain is unusable. It is
used by the cell not to generate energy directly,
but to recycle NADH into NAD+ so that glycolysis
can continue, as long as glucose is present.
2
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

3
 Fermentation and anaerobic
respiration enable cells to produce ATP
without the use of oxygen

• Most cellular respiration requires O2 to produce

ATP
• Without O2, the electron transport chain will
cease to operate
 In that case, glycolysis couples with
fermentation or anaerobic respiration to
produce ATP

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DS/ABBS/BTP401 4
  Anaerobic respiration uses an electron
transport chain with a final electron acceptor
other than O2, for example sulfate
 Fermentation uses substrate-level
phosphorylation instead of an electron
transport chain to generate ATP

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Fate of Pyruvate

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 Types of Fermentation
 Fermentation consists of glycolysis plus
reactions that regenerate NAD+, which can
be reused by glycolysis
 Two common types are
alcohol fermentation and
lactic acid fermentation
 In alcohol fermentation, pyruvate is
converted to ethanol in two steps, with the
first releasing CO2
 Alcohol fermentation by yeast is used in
brewing, winemaking, and baking
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  In lactic acid fermentation, pyruvate is
reduced to NADH, forming lactate as an end
product, with no release of CO2
 Lactic acid fermentation by some fungi and
bacteria is used to make cheese and yogurt
 Human muscle cells use lactic acid
fermentation to generate ATP when O2 is
scarce

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Figure 9.17a

2 ADP  2 P i 2 ATP

Glucose Glycolysis

2 Pyruvate

2 NAD  2 NADH 2 CO2

 2H

2 Ethanol 2 Acetaldehyde

(a) Alcohol fermentation

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Figure 9.17b

2 ADP  2 P i 2 ATP

Glucose Glycolysis

2 NAD  2 NADH
 2H
2 Pyruvate

2 Lactate

(b) Lactic acid fermentation

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What is fermentation techniques ?
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 etc) are carried out by
submersion processes.
11
 OPTIMIZATION OF
FERMENTATION PROCESS

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Introduction
 The function of the fermenter or bioreactor is to provide a
suitable environment in which an organism can efficiently
produce a target product—the target product might be
 · Cell biomass
 · Metabolite
 · Bioconversion Product

 The sizes of the bioreactor can vary over several

orders of magnitudes.
 The microbial cell culture (few mm3), shake flask
( 100 -1000 ml), laboratory fermenter
 ( 1 – 50 L), pilot scale (0.3 – 10 m3) to plant
scale ( 2 – 500 m3) are all examples of
bioreactors.
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 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

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Types of fermenter
 Simple fermenters (batch and continuous)
 Fed batch fermenter
 Air-lift or bubble fermenter
 Cyclone column fermenter
 Tower fermenter
 Other more advanced systems, etc
The size is few liters (laboratory use) - >500
m3 (industrial applications)

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Flow sheet of a multipurpose fermenter and
its auxiliary equipment

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 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

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DS/ABBS/BTP401
 Fermentation Process
Upstream Processing
Fermentation Raw Materials Production Microorganism

Upstream Processing
Fermentation

Downstream
Product Purification Processing

Effluent Wastes Product

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 Upstream Processing
• Three main areas:
A) Producer microorganism
• This include processes for
• obtaining a suitable microorganism
• strain improvement to increase the
productivity and yield
• maintenance of strain purity
• preparation of suitable inocullum
B ) Fermentation media
C) Fermentation Process
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Downstream Processing

The processes that follows fermentation:

A) Cell harvesting
B) Cell disruption
C) Product purification from cell extracts
or the growth medium

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 Types of Fermentation Process
1. Batch Fermentation
2. Continuous Fermentation
3. Fed batch
Batch reactors ,simplest type. Reactor is filled
with medium and the fermentation is allowed.
• Fermentation has finished, contents are
emptied for downstream processing.
• The reactor is then cleaned, re-filled, re-
inoculated and the fermentation process starts
again.

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 Batch
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Continuous flow 24
Batch Fermentation Process

 Dynamic processes that are never in a

steady state.
 Often , the critical parameter is gas
exchange or balance between
respiration rate and oxygen transfer.
 sterilized media components are
supplied at the beginning of the
fermentation with no additional feed
after inoculation.
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Batch Fermentation Process
 cells are grown in a batch reactor,
they go through a series of stages:
 Lag phase
 Exponential phase
 Stationary phase
 Death phase

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 Batch Fermentation Process
• Lag Phase
• microbial population remains constant as there is no
growth. However it is the period of intense metabolic
activity.
• Factors Influencing the Lag Phase
1. · Chemical composition of the fermentation media
influences the length of the lag phase.
2. Longer lag phase is observed if the inocullum is
transferred into a fresh medium of different carbon source.
3. · Age of the inocullum. If the inocullum is in exponential
growth phase, it will exhibit shorter lag in the fresh
medium.
4. · Concentration of the inocullum.
5. · Viability and morphology of the inocullum.
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Batch Fermentation Process

 Exponential Phase
 · Cell divides with increasing frequency
till it reaches the maximum growth rate
(μmax).
 · At this point logarithmic growth begins
and cell numbers or cell biomass
increase at a constant rate.

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 Stationary Phase
 · The specific growth rate of the microorganism
continues decelerating until the substrate is
completely depleted.
 · Overall growth rate has declined to zero and
there is no net change in cell numbers/ biomass ie.
rate of cell division equals rate of cell death.
 · Microorganisms are still metabolically active,
metabolizing intracellular storage compounds,
utilizing nutrients released from lysed cells and in
certain cases produce secondary metabolites.
 Death Phase
 · Cells die at constant rate and often undergo lysis.
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The generation time can be calculated from the
growth curve
•When growing exponentially by binary
fission, the increase in a bacterial
population is by geometric progression.
•If we start with one cell, when it
divides, there are 2 cells in the first
generation, 4 cells in the second
generation, 8 cells in the third
generation, and so on.
•The generation time is the time
interval required for the cells (or
population) to divide.

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 G (generation time) = (time, in minutes or hours)/n(number of
generations)
 G = t/n
 t = time interval in hours or minutes
 B = number of bacteria at the beginning of a time interval
 b = number of bacteria at the end of the time interval
 n = number of generations (number of times the cell population
doubles during the time interval)
 b = B x 2n (This equation is an expression of growth by binary fission
 Solve for m:
 logb = logB + nlog2
 n = logb - logB
log2
 n = logb - logB
0.301
 n = 3.3 logb / B , G = t/n
 Solve for G , G = t / 3.3 log b/B
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Continuous flow Fermenter.
 Here the raw materials are trickled in at the
top of a column in which there are
immobilised micro-organisms or enzymes
present.
 The product flows out the bottom in a pure
state.
 It does not need to be separated from the
catalyst.
 However this process can only be used for
reactions that are fast – possibly taking 10
minutes
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 Fed-batch culture or Fermentation
 Fed-batch culture is, in the broadest sense, defined
as an operational technique in biotechnological
processes ,where one or more nutrients (substrates)
are fed (supplied) to the bioreactor during cultivation
and in which the product(s) remain in the bioreactor
until the end of the run.
 It is also known as semi-batch culture.
 In some cases, all the nutrients are fed into the
bioreactor.
 The advantage of the fed-batch culture is that one can
control concentration of fed-substrate in the culture
liquid at arbitrarily desired levels ( in many cases, at
low levels).
 No control over rate of reaction
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 The types of bioprocesses for which fed-batch culture is
effective can be summarized as follows:

1) Substrate inhibition
2) High cell density (High cell concentration
3) Glucose effect (Crabtree effect)
4) Catabolite repression
5) Auxotrophic mutants
6) Expression control of a gene who has repressible
promoter in recombinant cell
7) Extension of operation time,
8) supplement of water lost by evaporation, and
decreasing viscosity of culture broth

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Key Factor of Fermenter design
 The performance of any fermenter depends on
the following key factors:
 · Agitation rate
 · Oxygen transfer
 · pH
 · Temperature
 · Foam production
 The design and mode of operation of a fermenter mainly
depends on the production organism, the optimal
operating condition required for target product formation,
product value and scale of production.
 The design also takes into consideration the capital
investment and running cost.

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Requirements of Bioreactors
 There is no universal bioreactor.
 The general requirements of the bioreactor are as follows:
A) The design and construction of bioreactors must keep
sterility from the start point to end of the process.
B) Optimal mixing with low, uniform shear.
C) Adequate mass transfer, oxygen.
D) Clearly defined flow conditions.
E) Feeding substrate with prevention of under or overdosing.
F) Suspension of solids.
G) Gentle heat transfer.
H) Compliance with design requirements such as: ability to
be sterilized; simple construction; simple measuring,
control, regulating techniques; scale-up; flexibility; long
term stability; compatibility with up- downstream
processes; antifoaming measures.
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 Why control fermentations?
 Success of a fermentation depends on the
maintenance of defined environmental conditions for
biomass and product formation

 Therefore many criteria or parameters need to be

kept in control

 Any deviations from optimum conditions need to be

controlled and corrected by a control system

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PROCESS OPTIMIZATION THROUGH MONITOR
AND CONTROL
KEY OBJECTIVE:

 Analyse process status

 Establish optimum conditions

MONITOR ; Sampling, on-, off-line, state and control variables, sensors, gate-way
sensors, biosensors

MEASURE; Factors significant in sensing, measurement and display, data capture and
storage

CONTROL; Key variables controlled, state and control / process variables, levels of
process control, automatic control

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 Control systems
A control system consists of three basic components

1. A measuring element (senses a process property and generates a

corresponding output signal)

2. A controller (compares the measurement signal with a pre-

determined desired value, the set point, and produces an output signal
to counteract any differences between the two

3. A final control element, which receives the control signal and

adjusts the process by changing a valve opening or pump speed
causing the controlled process to return to the set point

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CONTROL SYSTEMS - general

CONTROL BASED ON;

Event has occurred == FEED BACK CONTROL
Premise that an event will occur == FEED FORWARD

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Manual control EXPENSIVE
Steam valve to regulate the temperature of water flowing through
a pipe

Human operator instructed to control

temperature within set limits

Steam

Manual adjustment Visual awareness

Valve
(Final control of valve
element)

Thermometer

Water
Pipe
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 Automatic control
Simple automatic control loop for temperature control

Controller Set-point

Steam

Measured valve
Control
Valve
Signal to operate valve

Thermocouple

Water
Pipe
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Automatic control systems
Can be classified into 4 main types

1. Two-position controllers

2. Proportional controllers

3. Integral controllers

4. Derivative controllers

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 Automatic control
In complex control systems there are 3 different methods which
are commonly used in making error corrections
-proportional
-integral
-derivative
May be used singly or in combination

With electronic controllers the response to an error is represented

as a change in output current or voltage

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 A fermenter with a temperature-controlled
heating jacket
Temperature
controller Thermocouple Water
outlet

Pressure line
to valve

Hot Pressure Heating

water regulated Jacket
valve
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 Automatic control
Proportional control
the change in output of the controller is proportional to the input signal
produced by the environmental change

Integral control
output signal of an integral controller is determined by the integral of the
error input over the time of the operation

Derivative control
when derivative control is applied the controller senses the rate of
change of the error signal and contributes a component of the output
signal that is proportional to a derivative of the error signal

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 PROGRAMMABLE LOGIC
CONTROLLER / CHIP (PLC)
Each has an input section, output section and a central processing unit (CPU)
 Input- connect to sensors
 Output - connected to motors / valves etc.
 CPU - provides and executes instructions

May be linked to a Management Information System (MIS) resulting in a database of

production data.

A Laboratory Information Management System (LIMS) can also be interfaced giving all
test data (e.g. info on tests carried out on all samples)

ADVANTAGE;
 REPEATABILITY
 TRACEABILITY

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COMPUTERS IN
FERMENTATION
3 Main areas of computer control;

LOGGING OF PROCESS DATA

Amount of data generated very great - need electronic capture

DATA ANALYSIS [Reduction of logged data]

Data reduction very significant - generates trends (e.g. graphs)
Makes analysis, management of data easier
LIMS is a good example of the benefits from this area
Predictive Modelling and Expert systems would be other examples

PROCESS CONTROL

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Computer-controlled fermenter with control loop
Mainframe
computer
Analogue to
digital converter Printout

VDU

Dedicated
Analogue to Interface mini-computer
Meter Data store
digital converter

Graphic unit
Pump Reservoir

Clock

Alarms

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COMPUTERS IN FERMENTATION
PROCESS CONTROL

 Digital Set-point Control (DSC)

Computer scans set-points of individual controllers and takes corrective
action when deviations occur

 Direct Digital Control (DDC)

Sensors interfaced directly with the computer

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CONTROL / PROCESS
VARIABLES

1. Temperature
2. Pressure
3. Vessel contents
4. Foam
5. Impeller speed
6. Gas Flow rates
7. Liquid flow
8. pH
9. Dissolved and Gas phase Oxygen
10. Dissolved and Gas phase Carbon Dioxide
11. General gas analysis

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 TEMPERATURE CONTROL
HEAT BALANCE IN FERMENTATION

Q met = Heat ---> Microbial metabolism

Q ag = " ---> Mechanical agitation
Q aer = " ---> Aeration
Q evap = " ---> Water evaporation
Q sens = " ---> Feed streams
Q exch = " ---> Exchanger / surroundings

UNDER ISOTHERMAL CONDITIONS;

Q met + Q ag + Q aer = Q evap + Q sens + Q exch

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FERMENTATION
MEASUREMENT /monitoring;

PHYSICAL (e.g Temperature, Pressure etc.)

CHEMICAL ( e.g. pH, Redox, Ions etc.)

INTRACELLULAR ( Cell mass composition, enzyme levels etc.)

BIOLOGICAL ( e.g. Morphology, cell size, viable count etc.)

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TYPICAL PARAMETERS -
Penicillin fermentation

(1) Feeding rate of substrate / precursor

(2) Biomass conc. per litre and per fermenter (mass)
(3) Penicillin conc. and mass
(4) Growth rate
(5) Fraction of glucose --> Mass
Maintenance
Product
(6) Respiration rate
(7) Oxygen demand
(8) Total broth weight
(9) Cumulative efficiency
(10) Elemental balance of P, N, S
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 Models
• Series of equations used to correlate data and predict behavior.

• Based on known relationships

• Cyclical nature of models, involves formulation of a hypothesis, then

experimental design followed by experiments and analysis of results
which should further advance the original hypothesis

• Conceptual, Empirical, and Mechanistic models

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Control of Physicochemical Parameters
 A) Agitation:
 Agitation of suspended cell fermentations is performed in order to mix
the three phases within a fermenter
 liquid phase contains dissolved nutrients and metabolites
 gaseous phase is predominantly oxygen and carbon dioxide
 solid phase is made up of the cells and any solid substrates that may be
present.
 Mixing should produce homogeneous conditions and promote
 a) Nutrient transfer b) Gas transfer c) Heat transfer
 Heat transfer is necessary during both sterilization and for temperature
maintenance during operation.

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 Control of Physicochemical Parameters
 Automatic temperature control during the fermentation is
accomplished by injecting either cold or hot water into the outer
jacket and/or internal coils.
 In some circumstances alternative cooling media may be used,
e.g. glycol.
A. Mass transfer
 Transfer of nutrients from the aqueous phase into the microbial
cells during fermentation is relatively straightforward as the
nutrients are normally provided in excess.

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 Control of Physicochemical Parameters
 B. Transport of Oxygen
 To prevent the risk of contamination, gases
introduced into the fermenter should be passed
through a sterile filter.
 A similar filter on the air exhaust system avoids
environ-mental contamination.
 Sterile filtered air or oxygen normally enters the
fermenter through a sparger system,
 To promote aeration in stirred tanks, the
sparger is usually located directly below the
agitator.
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 Transfer of Heat in Bioreactors
 Tomaintain a constant temperature in the
fermenter, heat is either supplied or
removed from the fermentation broth
during the course of fermentation.

 Infixed bed microbial reactors heat

transfer takes place by natural convection
or phase change (evaporation-
condensation).
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 Heat Transfer Configurations:
 The primary heat transfer configurations in
fermentation vessels are:
 i. External jackets
 ii. Internal coils
 iii. External surface heat exchanger
 The internal coils though provide better heat transfer
capabilities, but they cause problems of microbial film
growth on coil surfaces, alteration of mixing patterns
and fluid velocities.

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 Stages of Downstream Processing

Stage Unit Operations

1. Separation of insolubles filtration, sedimentation,

extraction, adsorption

2. Isolation of Product extraction, adsorption,

ultrafiltration, precipitation

3. Purification chromatography,
crystallization, fractional
precipitation

4. Polishing drying, crystallization

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Excipients
 Substances added to final product to
stabilize it

 Serum albumin
 Withstands low pH or elevated temps
 Keeps final product from sticking to walls of
container
 Stabilize native conformation of protein

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 Excipients cont’d

 Amino acids

 Glycine – stabilizes interferon, factor VIII, stabilizes against heat

 Alcohols (and other polyols)

 Stabilize proteins in solution

 Surfactants
 Reduces surface tension; proteins don’t aggregate, so don’t denature

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Final product fill

 Bulk product gets QC testing

 Passage through 0.22 m filter for final
sterility
 Aceptically filled into final product
containers
 Uses automated liquid handling
systems
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Freeze drying cont’d
 Need to add cryoprotectors
 Glucose or sucrose
 Serum albumin

 Amino acids

 Polyols

 Freeze drying can be done in many

steps

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 Composition of biomass
Molecules  Elements
 Protein 30-60 %  C 40-50 %
 Carbohydrate 5-30 %  H 7-10 %
 Lipid 5-10 %  O 20-30 %
 DNA 1 %  N 5-10 %
 RNA 5-15 %  P 1-3 %
 Ash (P, K+, Mg2+, etc)  Ash 3-10%

Typical composition biomass formula: C1H1.8O0.5N0.2

Suppose 1 kg dry biomass contains 5 % ash, what is the amount
of organic matter in C-mol biomass?

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Summary
• Why fermentations need to controlled

• How to control fementations

• Use of computers in control of bioprocesses

• Difference between manual and automatic control

systems

• Process variables that need controlling

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MEDIA FOR INDUSTRIAL
BIOPROCESSES
 Introduction
 Properties of useful industrial microorganisms

 Finding and selecting microorganism

 Improving the microorganism’s properties

 Conquering the cell’s control systems…mutants,
feedback, induction etc.

 Storing industrial micro-organisms – the

culture collection

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 Overview

Organism  Media
Selection and
Improvement
P
R
O
C
E
S
S

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What’s it all about?

Substrate

Process Product

Organism
MONEY
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 What does the medium need to do?
 Grow the microorganism so it produces
biomass and product, and should not
interfere with down stream processing.

 Carbon and energy source +nitrogen source

+ oxygen + other requirements → biomass +
products + carbondioxide+ water + heat

 Yield coefficient, Y= (Quantity of cell dry

matter produced) / (Quantity of carbon
substrate utilized)
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Media for Industrial Bioprocesses -
 Crude and defined media:
 Crude media is made up of unrefined agricultural
products e.g. containing barley.
 Defined media are like those we use in the lab e.g.
minimal salts medium.
 Crude media is cheap but composition is variable.
 Defined media is expensive but composition is
known and should not vary.
 Crude media is used for large volume inexpensive
products e.g. biofuel from whey.
 Defined media is used for expensive low volume
products e.g. anticancer drugs.
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Media for Industrial Bioprocesses

 Typical medium ingredients:

 Carbon sources
 Nitrogen sources
 Vitamins and growth factors
 Minerals and trace elements
 Inducers
 Precursors
 Inhibitors e.g. KMS in beer medium
 Antifoams

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 Medium Need to Do....
 Supply the raw materials for growth and product
formation.
 Stoichiometry ( i.e. biochemical pathways) may
help us predict these requirements, but:
 Ingredients must be in the right form and
concentrations to direct the bioprocess to:
 Produce the right product.
 Give acceptable yields, titres, volumetric productivity etc.
 To achieve these aims the medium may contain
metabolic poisons, non-metabolisable inducers etc.

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Medium Need to Do....

 Cause no problems with:

 Preparation and sterilisation
 Agitation and aeration
 Downstream processing

 Ingredients must have an acceptable:

 Availability
 Reliability
 Cost (including transport costs)

12/10/2019 DS/ABBS/BTP401 79
Medium Can Be a Significant Proportion of
Total Product Cost
Elements of total product cost (%)

Raw materials costs range from 38-77% in the

examples shown
12/10/2019 DS/ABBS/BTP401 80
Crude and Defined Media
Media can be loosely assigned two types

 Defined media
 Made from pure compounds

 Crude media
 Made from complex mixtures (agricultural products)

 Individual ingredients may supply more than one

requirement
 May contain polymers or even solids

12/10/2019 DS/ABBS/BTP401 81
Defined Media – Good Properties

 Consistent
 Composition
 Quality
 Facilitate R and D
 Unlikely to cause foaming
 Easier upstream processing (formulation,
sterilisation etc.)
 Facilitate downstream processing (purification
etc.)

12/10/2019 DS/ABBS/BTP401 82
Defined Media – Bad Properties
 Expensive
 Need to define and supply all growth
factors. Only mineral salts present
 Yields and volumetric productivity can
be poor:
 Cellshave to “work harder”…proteins etc.
are not present
 Missing growth factors…amino acids etc.

12/10/2019 DS/ABBS/BTP401 83
Defined Media - Status
 Main use is for low volume/high value
added products, especially proteins
produced by recombinant organisms

NOTE: Some “defined” media may

contain small amounts of undefined
ingredients (e.g. yeast extract) to supply
growth factors.

12/10/2019 DS/ABBS/BTP401 84
Crude Media – Good Properties
 Cheap

 Provide growth factors (even “unknown”

ones)

 Good yields and volumetric productivity

12/10/2019 DS/ABBS/BTP401 85
Crude Media – Bad Properties
 Variability:
 Composition
 Quality
 Supply
 Cost (Agri-politics)
 Availability to organism

 Unwanted components….iron or copper

which can often be lethal to cell growth.

12/10/2019 DS/ABBS/BTP401 86
Crude Media – Bad Properties
 May cause bioprocess foaming

 Problems with upstream processing (medium

pre-treatment and sterilisation)

 Problems with downstream processing

(product recovery and purification)

12/10/2019 DS/ABBS/BTP401 87
Crude Media - Status
 Inspite of the problems to be overcome,
the cost and other good properties
make crude media the choice for high
volume/low value added products.
 More often used than defined media.

12/10/2019 DS/ABBS/BTP401 88
Crude Media - Accessibility Problems

 Plant cellular structure “wraps up”

nutrients.
 Alignment of macromolecules (e.g.
cellulose, starch).
 Solutions (pre-treatments):
 Grinding.
 Heattreatment (cooking, heat sterilization).
 Chemical treatments.

12/10/2019 DS/ABBS/BTP401 89
Crude Media - Accessibility Problems

 Polymers (eg starch, cellulose, protein).

 Solutions:
 Find or engineer organisms with
depolymerase enzyme.
 Pretreatments:
 Chemical depolymerisation (heat and acid
hydrolysis).
 Enzyme pretreatment.

12/10/2019 DS/ABBS/BTP401 90
Typical Ingredients

 NOTE: Crude ingredients often supply

more than one type of requirement, so,
for example the same ingredient may be
mentioned as a carbon source, nitrogen
source etc.

12/10/2019 DS/ABBS/BTP401 91
Carbon Sources
 Carbon sources are the major components of
media:
 “Building blocks” for growth and product formation
 Energy source
 Easily used carbon sources give fast growth
but can depress the formation of some
products
 Secondary metabolites - catabolite
repression…large amounts of glucose can repress
B galactosidase

12/10/2019 DS/ABBS/BTP401 92
Carbon Sources – Carbohydrates:
Starch :
 Cheap and widely available:
 Cereals
 Maize (commonest carbohydrate source)

 Wheat

 Barley (malted and unmalted)

 Potato
 Cassava
 Soy bean meal
 Peanut meal
 Sources may also supply nitrogen and growth factors

12/10/2019 DS/ABBS/BTP401 93
Carbon Sources –Starch

 Pre-treatments may be used to convert

starch to mono-and disaccharides:
 Acid or enzymes
 Malting and mashing

 Grain syrups are available (pre-

treatment already carried out)

12/10/2019 DS/ABBS/BTP401 94
Carbon Sources –Sucrose

 Derived from sugar cane and beet

 Variety of forms and purities
 Molasses can also supply
 Trace elements
 Heat stable vitamins
 Nitrogen

12/10/2019 DS/ABBS/BTP401 95
Carbon Sources – Lactose
 Pure or whey derived product
 Used as carbon source in production of
penicillin at STATIONARY PHASE
 Liquid whey
 Cheap
 Uneconomic to transport
 Used for biomass and alcohol production

12/10/2019 DS/ABBS/BTP401 96
Carbon Sources - Glucose
 Solid or syrup (starch derived)

 Readily used by almost all organisms

 Catabolite repression can cause

problems

12/10/2019 DS/ABBS/BTP401 97
 Carbon Sources –Vegetable Oils
 Olive, cotton seed, linseed, soya bean etc.

 High energy sources

 Increased oxygen requirement.

 Increased heat generation.

 Antifoam properties (see later).

12/10/2019 DS/ABBS/BTP401 98
Nitrogen Sources - Inorganic
 Ammonium salts

 Ammonia

 Nitrates
 Yeasts cannot assimilate nitrates

12/10/2019 DS/ABBS/BTP401 99
Nitrogen Sources - Organic

 Proteins – completely or partially

hydrolysed.

 Some organisms prefer peptides to amino

acids.

12/10/2019 DS/ABBS/BTP401 100

Nitrogen Sources - Organic
 8% nitrogen:
 Soybean meal.
 Groundnut (peanut) meal.
 Pharmamedia (cottonseed derived).
 4.5% nitrogen:
 Cornsteep powder (maize derived).
 Whey powder.
 1.5-2% nitrogen:
 Cereal flours.
 Molasses.

Highlight indicates sources of growth factors.

12/10/2019 DS/ABBS/BTP401 101
Vitamins and Growth factors
 Pure sources expensive
 Often supplied by crude ingredients:
 Pharmamedia
 Cornsteep powder
 Distillers solubles
 Malt sprouts

12/10/2019 DS/ABBS/BTP401 102

Minerals and Trace Elements
 Found in crude ingredients.
 Use inorganic sources if necessary.
 Inorganic phosphates.
 Alsoact as buffering agents.
 Excessive levels depress secondary
metabolite formation.

12/10/2019 DS/ABBS/BTP401 103

Inducers
 Enzyme substrates/inducers.
 Example: starch for amylase production.

 Non-metabolisable inducer analogues.

 Higher unit cost but only need small amount.
e.g. ITPG for B galactosidase

12/10/2019 DS/ABBS/BTP401 104

Precursors
 Help
direct metabolism and improve yields
Examples:
Precursor Organism Product
Glycine Corynebacterium L-Serine
glycinophilum
Chloride Penicillium Griseofulvin
griseofulvin
Phenylacetic Penicillium Penicillin-G
acid chrysogenum
12/10/2019 DS/ABBS/BTP401 105
 Phenylacetic acid is the precursor of the penicillin G
side chain. Feeding Phenylacetic acid increases the
yield of penicillin x3 and directs production toward
penicillin G.
12/10/2019 DS/ABBS/BTP401 106
Inhibitors
 Used to redirect the cells metabolism

 Example:Glycerol production by yeast.

 The method:
 Set up a normal alcohol-producing
fermentation
 When it is underway add a nearly lethal
dose of sodium sulphite

12/10/2019 DS/ABBS/BTP401 107

What Happens.....
 The sodium sulphite reacts with carbon
dioxide in the medium to form sodium
bisulphite

A key step in alcohol production is:

Acetaldehyde + NADH2 → Alcohol

12/10/2019 DS/ABBS/BTP401 108

What Happens...

Acetaldehyde + NADH2 → Alcohol

 Sodium bisulphite complexes and

removes acetaldehyde

12/10/2019 DS/ABBS/BTP401 109

What Happens....
 This leaves the cell with an excess of
NADH2
 Dihydroxyacetone phosphate is used as
an alternative hydrogen acceptor:

NADH2 Dihydroxyacetone phosphate

NAD Glycerol 3 Phosphate Glycerol

12/10/2019 DS/ABBS/BTP401 111

Foaming problems and Antifoams
 What Causes foam to form?
 Aeration
 Certain surface active compounds
(proteins):
 In the medium
 Product

12/10/2019 DS/ABBS/BTP401 112

Problems caused by foam
 Sub-optimal fermentation
 Poor mixing
 Cells separated from medium

 Product denatured

 Contamination
 Loss of bioprocessor contents

12/10/2019 DS/ABBS/BTP401 113

Dealing with foaming problems
 Avoid foam formation
 Choice of medium
 Modify process

 Use a chemical antifoam

 Use a mechanical foam breaker

12/10/2019 DS/ABBS/BTP401 114

Chemical Antifoams
 Surface active
compounds which
destabilise foam
structure at low
concentrations
 Part of the medium
and/or pumped in as
necessary
 Can decrease oxygen
transfer to the
medium
12/10/2019 DS/ABBS/BTP401 115
Desirable Antifoam Properties
 Effective
 Sterilisable
 Non toxic
 No interference with downstram
processing
 Economical

12/10/2019 DS/ABBS/BTP401 116

 Antifoams - Examples
 Fatty acids and derivatives (vegetable oils)
 Metabolisable

 Cheaper

 Less persistant
 Foam may reoccur : more has to be added.
 Used up before downstream processing

12/10/2019 DS/ABBS/BTP401 117

Antifoams - Examples
 Silicones
 Non metabolisable
 More expensive

 More persistant
 Less needed.
 Could interfere with downstream processing

 Often formulated with a metabolisable oil

“carrier”

12/10/2019 DS/ABBS/BTP401 118

Mechanical Foam Breakers
 Fast spinning discs or cones just above the
medium surface
 Fling foam against the side of the
bioprocessor and break the bubbles
 Can be used with or without antifoams

12/10/2019 DS/ABBS/BTP401 119

 Ultrasonic Whistles

12/10/2019 DS/ABBS/BTP401 120

DOWNSTREAM PROCESSING
What is Downstream Processing?
 Downstream – ‘after the fermentation
process’
 The various stages of processing that occur
after the completion of the fermentation or
bioconversion stage is called downstream
processing.
 This includes separation, purification, and
packaging of the product.

12/10/2019 DS/ABBS/BTP401 122

Stages in Downstream Processing

1) Removal of insoluble's
2) Product Isolation
3) Product Purification
4) Product Polishing

12/10/2019 DS/ABBS/BTP401 123

Operational diagram of large-scale batch
fermentation system

Preculture Preparation of Fermentation Recovery of enzyme-

inoculum containing medium

12/10/2019 DS/ABBS/BTP401 124

Downstream Processing
Primary ‘unit operations’ of Downstream
Processing
 Cell recovery/removal

Centrifugation

 Dewatering

Ultrafiltration

Precipitation

Spray drying
12/10/2019 DS/ABBS/BTP401 125
Downstream Processing
Secondary ‘unit operations’
 Protein purification

 Adsorption chromatography

 Gel permeation chromatography

 Protein processing

 Immobilisation

 Beading/Prilling

 Protein packaging

 Sterilisation

 Bottling etc DS/ABBS/BTP401

12/10/2019 126
Separation of cells and medium

Recovery of cells and/or medium (clarification)

 For intracellular enzyme, the cell fraction is
required
 For extracellular enzymes, the culture
medium is required
On an industrial scale, cell/medium separation
is almost always performed by centrifugation
 Industrial scale centrifuges may be batch,
continuous, or continuous with dislodging

12/10/2019 DS/ABBS/BTP401 127

Centrifugation

• use of the centrifugal force for the

separation of mixtures
• More-dense components migrate
away from the axis of the centrifuge
• less-dense components migrate
towards the axis.

12/10/2019 DS/ABBS/BTP401 128

Industrial centrifuges

Tubular bowl Chamber Disc

12/10/2019 DS/ABBS/BTP401 129

Properties of industrial centrifuges
 Tube
 High centrifugal force

 Good dewatering  Limited solids capacity

 Easy to clean
 Foams
 Difficult to recover protein
 Chamber
 Large solids capacity
 No solids discharge
 Good dewatering
 Cleaning difficult
 Bowl cooling possible
 Solids recovery difficult
 Disc type
 Solids discharge  Poor dewatering
 No foaming  Difficult to clean
 Bowl cooling possible

12/10/2019 DS/ABBS/BTP401 130

Centrifugation properties of different cell types
 Bacteria
 Small cell size  High speed required
 Resilient  Low cell damage
 Yeast cells
 Large cells  Lower speed required
 Resilient  Low cell damage
 Filamentous fungi
 Mycelial  Lower speed required
 Resilient  High water retention in
 Cultured animal cells
pellet
 Large cells

 Very fragile
 Very susceptible to
damage
12/10/2019 DS/ABBS/BTP401 131
Removal of insoluble's

• capture of the product as a solute in a

particulate-free liquid
• Example
separation of cells, cell debris or
other particulate matter from
fermentation broth containing an
antibiotic.

12/10/2019 DS/ABBS/BTP401 132

 Typical operations
Filtration
• A mechanical operation used for the
separation of solids from fluids (liquids
or gases) by interposing a medium to
fluid flow through which the fluid can
pass, but the solids in the fluid are
retained.

12/10/2019 DS/ABBS/BTP401 133

 Filter media

two main types of filter media are

• solid sieve which
-traps the solid particles
• bed of granular materials
-retains the solid particles

12/10/2019 DS/ABBS/BTP401 134

Points to be considered while
selecting the filter media:

• ability to build the solid.

• minimum resistance to flow the
filtrate.
• resistance to chemical attack.
• minimum cost.
• long life
12/10/2019 DS/ABBS/BTP401 135
Flocculation
• process where a solute comes out of solution
in the form of flocs or flakes.
• Particles finer than 0.1 µm in water remain
continuously in motion due to electrostatic
charge which causes them to repel each other.
• Once their electrostatic charge is neutralized
(use of coagulant) the finer particles start to
collide and combine together .
• These larger and heavier particles are called
flocs.

12/10/2019 DS/ABBS/BTP401 136

 Product Isolation
• reducing the volume of material to be
handled and concentrating the
product.
• the unit operations involved
-Solvent extraction
-ultra filtration
-precipitation

12/10/2019 DS/ABBS/BTP401 137

Cell disruption (for intracellular enzymes)
 Sonication
 Use of high frequency sound waves to disrupt cell walls and
membranes
 Can be used as continuous lysis method

 Better suited to small (lab-scale) operations

 Can damage sensitive proteins

 Pressure cells
 Apply apply high pressure to cells; cells fracture as pressure
is abruptly released
 Readily adapted to large-scale and continuous operations

 Industry standard (Manton-Gaulin cell disruptor)

 Enzymic lysis
 Certain enzymes lyse cell walls
 Lysozyme for bacteria; chitinase for fungi

 Only useful on small laboratory scale

12/10/2019 DS/ABBS/BTP401 138

Precipitation
• formation of a solid in a solution during a
chemical reaction.
• solid formed is called the precipitate
and the liquid remaining above the solid
is called the supernate.
• Use either liquid-liquid or solid-liquid
extraction process.

12/10/2019 DS/ABBS/BTP401 139

 Acetone Precipitation Protocol
1. Cool the required volume of Optional: If additional cycles of
acetone to -20°C. precipitation are necessary to
2. Place protein sample in acetone- completely remove the
compatible tube. interfering substance, then
3. Add four times the sample volume repeat steps 2-5 before
of cold (-20°C) acetone to the proceeding to step 7.
tube. 7. Allow the acetone to evaporate
4. Vortex tube and incubate for 60 from the uncapped tube at room
minutes at -20°C. temperature for 30 minutes. Do
not over-dry pellet, or it may not
5. Centrifuge 10 minutes at 13,000- dissolve properly.
15,000 x g
8. Resuspend in appropriate
6. Decant and properly dispose of the buffer.
supernatant, being careful to not
dislodge the protein pellet.

 . 12/10/2019 DS/ABBS/BTP401 140

TCA Precipitation Protocol
1.Add an equal volume of 20% TCA
(trichloroacetic acid) to protein sample.
2.Incubate 30 min on ice.
3.Spin in microfuge at 4 deg. For 15 min.
4.Carefully remove all supernatant.
5.Add ~300 ul cold acetone and spin 5
min at 4 degrees.
6.Remove supernatant and dry pellet.
7.Resuspend samples in desired buffer
12/10/2019 DS/ABBS/BTP401 141
 Chloroform/Methanol Precipitation
1. To sample of starting 10. Add 400 ul methanol
volume 100 ul 11. Vortex
2. Add 400 ul methanol 12. Spin 2 minutes @
3. Vortex well 14,000g
4. Add 100 ul chloroform 13. Remove as much
5. Vortex Methanol as possible
6. Add 300 ul H2O without disturbing pellet
7. Vortex 14. Speed-Vac to dryness
8. Spin 1 minute @ 4,0000 g 15. Bring up in 2X sample
9. Remove top aqueous buffer for PAGE
layer (protein is between
layers)
12/10/2019 DS/ABBS/BTP401 142
Product Purification

• To separate contaminants that

resemble the product very closely
in physical and chemical
properties.

• Expensive and require sensitive

and sophisticated equipment.

12/10/2019 DS/ABBS/BTP401 143

Protein purification
 Adsorption chromatography
 Ion exchange chromatography – binding
and separation of proteins based on
charge-charge interactions
 Proteins bind at low ionic strength, and are
eluted at high ionic strength
+ + - - + -
+
+ + + +
+ + +
+ +
+ + - -+ + -+ -+
+ +
Positively charged Net negatively
(anionic) ion charged (cationic) Protein binds to matrix
exchange matrix protein at selected pH
12/10/2019 DS/ABBS/BTP401 144
Liquid Column Chromatography Process
 Purge Air from System with Equilibration Buffer
 Pack Column with Beads (e.g. ion exchange,
HIC, affinity or gel filtration beads)
 Equilibrate Column with Equilibration Buffer
 Load Column with Filtrate containing Protein of
Interest in Equilibration Buffer
 Wash Column with Equilibration Buffer
 Elute Protein of Interest with Elution Buffer of
High or Low Salt or pH
 Regenerate Column or Clean and Store

12/10/2019 DS/ABBS/BTP401 145

Affinity chromatography
 Binding of a protein to a matrix via a protein-
specific ligand
 Substrate or product analogue

 Antibody

 Inhibitor analogue

 Cofactor/coenzyme

 Specific protein is eluted by adding reagent

which competes with binding

12/10/2019 DS/ABBS/BTP401 146

Affinity chromatography
1. Substrate analogue affinity chromatography
Affinity Enzyme
ligand

Matrix Spacer arm

Active-site-bound enzyme

2. Immunoaffinity chromatography
Antibody Protein epitope
ligand

Matrix Spacer arm Antibody-bound enzyme

12/10/2019 DS/ABBS/BTP401 147
 Gel permeation chromatography (GPC)
 Also known as ‘size exclusion
chromatography’ and ‘gel filtration
chromatography’
 Separates molecules on the basis of molecular
size
 Separation is based on the use of a porous
matrix.
 Small molecules penetrate into the matrix
more, and their path length of elution is longer.
 Large molecules appear first, smaller
molecules later.
12/10/2019 DS/ABBS/BTP401 148
 Gel Filtration Chromatography

12/10/2019 DS/ABBS/BTP401 149

Crystallization
• process of formation of solid
crystals,precipitating from a
solution, melt or more rarely
deposited directly from a gas.
• chemical solid-liquid separation
technique, in which mass transfer
of a solute from the liquid solution
to a pure solid crystalline phase
occurs.
12/10/2019 DS/ABBS/BTP401 150
 Process
 The crystallization process consists of two major
events, nucleation and crystal growth.
 Nucleation is the step where the solute molecules
dispersed in the solvent start to gather into clusters,
on the nanometer scale, that become stable under
the current operating conditions.
 These stable clusters constitute the nuclei.
 Such critical size is dictated by the operating
conditions (temperature, supersaturation, etc.).
 It is at the stage of nucleation that the atoms arrange
in a defined and periodic manner that defines the
crystal structure.

12/10/2019 DS/ABBS/BTP401 151

 The crystal growth is the subsequent growth of the
nuclei that succeed in achieving the critical cluster size.
 Nucleation and growth continue to occur
simultaneously while the supersaturating exists.
 Supersaturation is the driving force of the
crystallization.
 Once the supersaturation is exhausted, the solid–
liquid system reaches equilibrium and the
crystallization is complete.
 Many compounds have the ability to crystallize with
different crystal structures, a phenomenon called
polymorphism.
 polymorphism is of major importance in industrial
manufacture of crystalline products.
12/10/2019 DS/ABBS/BTP401 152
Product Polishing
• final processing steps which end
with packaging of the product in a
form ,that is stable, easily
transportable and convenient.
• Crystallization, desiccation,
lyophilization and spray drying are
typical unit operations

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lyophilization

• freezing the material

• reducing the surrounding pressure
and adding enough heat to allow
the frozen water in the material to
sublime directly from the solid
phase to gas.

12/10/2019 DS/ABBS/BTP401 154

Processing:
The fundamental process steps are:
 Freezing: The product is frozen. This provides a
necessary condition for low temperature drying.
 Vacuum: After freezing, the product is placed under
vacuum. This enables the frozen solvent in the
product to vaporize without passing through the liquid
phase, a process known as sublimation.
 Heat: Heat is applied to the frozen product to
accelerate sublimation.
 Condensation: Low-temperature condenser plates
remove the vaporized solvent from the vacuum
chamber by converting it back to a solid. This
completes the seperation process.
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 Downstream processing depends on
product use
1. Enzyme preparations for animal feed
supplementation (e.g., phytase) are not
purified
2. Enzymes for industrial use may be partially
purified (e.g., amylase for starch industry)
3. Enzymes for analytical use (e.g., glucose
oxidase) and pharmaceutical proteins (e.g.,
insulin) are very highly purified

12/10/2019 DS/ABBS/BTP401 156

 Animal feed enzyme Analytical enzyme Therapeutic protein
Fermentation Fermentation Fermentation

Centrifugation Centrifugation
Centrifugation to remove
to remove cells to remove cells
medium
Culture supernatant Culture supernatant Cell pellet
Protein Cell
precipitation lysis Centrifugation
Liquid preparation Protein fraction Intracellular fraction
to animal feed Protein
1 or 2 purification
market precipitation
steps
Semi-purified Protein fraction
protein 3-4 purification
steps
Lyophilisation
Bottling Homogeneous
protein
To chemicals market
Sterile
bottling
12/10/2019 DS/ABBS/BTP401 To pharmaceuticals market157
 References
1) Ladisch, Michael R. (2001). Bioseparations Engineering:
Principles, Practice, and Economics. Wiley. ISBN 0-471-24476-
7.
2) Harrison, Roger G.; Paul W. Todd, Scott R. Rudge and Demetri
Petrides (2003). Bioseparations science and engineering.
Oxford University Press. ISBN 0-19-512340-9.
3) Krishna Prasad, Nooralabettu (2010). Downstream Processing-
A New Horizone in Biotechnology. Prentice Hall of India Pvt.
Ltd, New Delhi. ISBN 978-81-203-4040-4.

12/10/2019 DS/ABBS/BTP401 158

12/10/2019 DS/ABBS/BTP401 159
Final product fill cont’d
 Freeze drying (lyophilization)
 Yields a powdered product
 Reduces chemical and biological
degradation of final product
 Longer shelf life than products in
solution
 Storage for parenteral products (those
administered intravenously or injected)
12/10/2019 DS/ABBS/BTP401 160