Dr Diptendu Sarkar

diptendu81@gmail.com

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

2

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.

3

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

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

© 2011 Pearson Education, Inc.12/10/2019 4DS/ABBS/BTP401

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

© 2011 Pearson Education, Inc.12/10/2019 5DS/ABBS/BTP401

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© 2011 Pearson Education, Inc.

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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

© 2011 Pearson Education, Inc.12/10/2019 8DS/ABBS/BTP401

2 ADP 2 P i 2 ATP

Glucose Glycolysis

2 Pyruvate

2 CO22 NAD

2 NADH

2 Ethanol 2 Acetaldehyde

(a) Alcohol fermentation

2 H

Figure 9.17a

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(b) Lactic acid fermentation

2 Lactate

2 Pyruvate

2 NADH

Glucose Glycolysis

2 ADP 2 P i 2 ATP

2 NAD

2 H

Figure 9.17b

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11

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.

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.12/10/2019 13DS/ABBS/BTP401

Fermenter

14

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

15

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

cerevisiae

Industrial solvents,

beverages

Glycerol Saccharomyces

cerevisiae

Production of

explosives

Lactic acid Lactobacillus

bulgaricus

Food and

pharmaceutical

Acetone and

butanol

Clostridium

acetobutylicum

Solvents

-amylase Bacillus subtilis Starch hydrolysis

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Fermentation Process

Fermentation Raw Materials Production Microorganism

Fermentation

Product Purification

ProductEffluent Wastes

Downstream

Processing

Upstream Processing

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Upstream Processing

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

Fermenter Continuous flow

Fermenter12/10/2019 24DS/ABBS/BTP401

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.12/10/2019 27DS/ABBS/BTP401

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

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•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.

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 reaction12/10/2019 DS/ABBS/BTP401 33

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.12/10/2019 36DS/ABBS/BTP401

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 systemsA 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 controlSteam valve to regulate the temperature of water flowing through

a pipe

Human operator instructed to control

temperature within set limits

Manual adjustment

of valve

Visual awareness

Steam

Valve

(Final control

element)

WaterPipe

Thermometer

EXPENSIVE

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Automatic controlSimple automatic control loop for temperature control

Controller

Signal to operate valve

Measured valve

Steam

Control

Valve

WaterPipe

Thermocouple

Set-point

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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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Temperature

controller

Pressure line

to valve

Hot

waterPressure

regulated

valve

Heating

Jacket

Water

outletThermocouple

A fermenter with a temperature-controlled

heating jacket

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Automatic controlProportional 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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Printout

VDU

Data store

Graphic unit

Alarms

Clock

Dedicated

mini-computer

Mainframe

computer

Interface

Analogue to

digital converter

Meter

Reservoir

Analogue to

digital converter

Pump

Sensor

Computer-controlled fermenter with control loop

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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, S12/10/2019 55DS/ABBS/BTP401

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.12/10/2019 60DS/ABBS/BTP401

Transfer of Heat in Bioreactors

To maintain a constant temperature in the

fermenter, heat is either supplied or

removed from the fermentation broth

during the course of fermentation.

In fixed 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

69

Molecules

Protein 30-60 %

Carbohydrate 5-30 %

Lipid 5-10 %

DNA 1 %

RNA 5-15 %

Ash (P, K+, Mg2+, etc)

Elements

C 40-50 %

H 7-10 %

O 20-30 %

N 5-10 %

P 1-3 %

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 controlling12/10/2019 70DS/ABBS/BTP401

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

Selection and

Improvement

Media

P

R

O

C

E

S

S

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What’s it all about?

Substrate

Organism

Process Product

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)

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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 shown12/10/2019 80DS/ABBS/BTP401

Crude and Defined Media

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

Media can be loosely assigned two types

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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.)

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Defined Media – Bad Properties

Expensive

Need to define and supply all growth factors. Only mineral salts present

Yields and volumetric productivity can be poor:

Cells have to “work harder”…proteins etc. are not present

Missing growth factors…amino acids etc.

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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.

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Crude Media – Good Properties

Cheap

Provide growth factors (even “unknown”

ones)

Good yields and volumetric productivity

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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.

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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)

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Crude Media - Status

In spite 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.

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Crude Media - Accessibility Problems

Plant cellular structure “wraps up” nutrients.

Alignment of macromolecules (e.g. cellulose, starch).

Solutions (pre-treatments):

Grinding.

Heat treatment (cooking, heat sterilization).

Chemical treatments.

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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.

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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.

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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 92DS/ABBS/BTP401

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

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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 94DS/ABBS/BTP401

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 95DS/ABBS/BTP401

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

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Carbon Sources - Glucose

Solid or syrup (starch derived)

Readily used by almost all organisms

Catabolite repression can cause

problems

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Carbon Sources –Vegetable Oils

Olive, cotton seed, linseed, soya bean etc.

High energy sources

Increased oxygen requirement.

Increased heat generation.

Antifoam properties (see later).

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Nitrogen Sources - Inorganic

Ammonium salts

Ammonia

Nitrates

Yeasts cannot assimilate nitrates

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Nitrogen Sources - Organic

Proteins – completely or partially

hydrolysed.

Some organisms prefer peptides to amino

acids.

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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 101DS/ABBS/BTP401

Vitamins and Growth factors

Pure sources expensive

Often supplied by crude ingredients:

Pharmamedia

Cornsteep powder

Distillers solubles

Malt sprouts

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Minerals and Trace Elements

Found in crude ingredients.

Use inorganic sources if necessary.

Inorganic phosphates.

Also act as buffering agents.

Excessive levels depress secondary

metabolite formation.

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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

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Precursors

Help direct metabolism and improve yields

Examples:

Precursor Organism Product

Glycine Corynebacterium

glycinophilum

L-Serine

Chloride Penicillium

griseofulvin

Griseofulvin

Phenylacetic

acid

Penicillium

chrysogenum

Penicillin-G

12/10/2019 105DS/ABBS/BTP401

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 106DS/ABBS/BTP401

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

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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 108DS/ABBS/BTP401

What Happens...

Acetaldehyde + NADH2 → Alcohol

Sodium bisulphite complexes and

removes acetaldehyde

12/10/2019 109DS/ABBS/BTP401

What Happens....

This leaves the cell with an excess of

NADH2

Dihydroxyacetone phosphate is used as

an alternative hydrogen acceptor:

NADH2

NAD

Dihydroxyacetone phosphate

Glycerol 3 Phosphate Glycerol

12/10/2019 111DS/ABBS/BTP401

Foaming problems and Antifoams

What Causes foam to form?

Aeration

Certain surface active compounds

(proteins):

In the medium

Product

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Problems caused by foam

Sub-optimal fermentation

Poor mixing

Cells separated from medium

Product denatured

Contamination

Loss of bioprocessor contents

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Dealing with foaming problems

Avoid foam formation

Choice of medium

Modify process

Use a chemical antifoam

Use a mechanical foam breaker

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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

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Desirable Antifoam Properties

Effective

Sterilisable

Non toxic

No interference with downstram

processing

Economical

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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 117DS/ABBS/BTP401

Antifoams - Examples

Silicones

Non metabolisable

More expensive

More persistant

Less needed.

Could interfere with downstream processing

Often formulated with a metabolisable oil

“carrier”

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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

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Ultrasonic Whistles

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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 122DS/ABBS/BTP401

Stages in Downstream Processing

1) Removal of insoluble's

2) Product Isolation

3) Product Purification

4) Product Polishing

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Operational diagram of large-scale batch

fermentation system

Preculture Preparation of Fermentation Recovery of enzyme-

inoculum containing medium

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Downstream Processing

Primary ‘unit operations’ of Downstream

Processing

Cell recovery/removal

Centrifugation

Dewatering

Ultrafiltration

Precipitation

Spray drying12/10/2019 125DS/ABBS/BTP401

Downstream ProcessingSecondary ‘unit operations’

Protein purification

Adsorption chromatography

Gel permeation chromatography

Protein processing

Immobilisation

Beading/Prilling

Protein packaging

Sterilisation

Bottling etc12/10/2019 126DS/ABBS/BTP401

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

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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 128DS/ABBS/BTP401

Industrial centrifuges

Tubular bowl Chamber Disc

12/10/2019 129DS/ABBS/BTP401

Properties of industrial centrifuges

Tube

High centrifugal force

Good dewatering

Easy to clean

Chamber

Large solids capacity

Good dewatering

Bowl cooling possible

Disc type

Solids discharge

No foaming

Bowl cooling possible

Limited solids capacity

Foams

Difficult to recover protein

No solids discharge

Cleaning difficult

Solids recovery difficult

Poor dewatering

Difficult to clean

12/10/2019 130DS/ABBS/BTP401

Centrifugation properties of different cell types

Bacteria

Small cell size

Resilient

Yeast cells

Large cells

Resilient

Filamentous fungi

Mycelial

Resilient

Cultured animal cells

Large cells

Very fragile

High speed required

Low cell damage

Lower speed required

Low cell damage

Lower speed required

High water retention in

pellet

Very susceptible to

damage12/10/2019 131DS/ABBS/BTP401

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 132DS/ABBS/BTP401

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 133DS/ABBS/BTP401

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 134DS/ABBS/BTP401

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

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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 136DS/ABBS/BTP401

Product Isolation

• reducing the volume of material to be

handled and concentrating the

product.

• the unit operations involved

-Solvent extraction

-ultra filtration

-precipitation

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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 138DS/ABBS/BTP401

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 139DS/ABBS/BTP401

Acetone Precipitation Protocol 1. Cool the required volume of

acetone to -20°C.

2. Place protein sample in acetone-

compatible tube.

3. Add four times the sample volume

of cold (-20°C) acetone to the

tube.

4. Vortex tube and incubate for 60

minutes at -20°C.

5. Centrifuge 10 minutes at 13,000-

15,000 x g

6. Decant and properly dispose of the

supernatant, being careful to not

dislodge the protein pellet.

.

Optional: If additional cycles of

precipitation are necessary to

completely remove the

interfering substance, then

repeat steps 2-5 before

proceeding to step 7.

7. Allow the acetone to evaporate

from the uncapped tube at room

temperature for 30 minutes. Do

not over-dry pellet, or it may not

dissolve properly.

8. Resuspend in appropriate

buffer.

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

volume 100 ul

2. Add 400 ul methanol

3. Vortex well

4. Add 100 ul chloroform

5. Vortex

6. Add 300 ul H2O

7. Vortex

8. Spin 1 minute @ 4,0000 g

9. Remove top aqueous

layer (protein is between

layers)

10. Add 400 ul methanol

11. Vortex

12. Spin 2 minutes @

14,000g

13. Remove as much

Methanol as possible

without disturbing pellet

14. Speed-Vac to dryness

15. Bring up in 2X sample

buffer for PAGE

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 143DS/ABBS/BTP401

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

(anionic) ion

exchange matrix

Net negatively

charged (cationic)

protein at selected pHProtein binds to matrix

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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 145DS/ABBS/BTP401

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 146DS/ABBS/BTP401

Affinity chromatography

Matrix Spacer arm

Affinity

ligand

+

Active-site-bound enzyme

1. Substrate analogue affinity chromatography

Matrix Spacer arm

Antibody

ligand

+

Antibody-bound enzyme

2. Immunoaffinity chromatography

Protein epitope

Enzyme

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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 148DS/ABBS/BTP401

Gel Filtration Chromatography

12/10/2019 149DS/ABBS/BTP401

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 150DS/ABBS/BTP401

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

12/10/2019 153DS/ABBS/BTP401

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 154DS/ABBS/BTP401

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.

12/10/2019 DS/ABBS/BTP401 155

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 156DS/ABBS/BTP401

Fermentation

Culture supernatant

Centrifugation

to remove cells

Liquid preparation

to animal feed

market

Fermentation

Culture supernatant

Fermentation

Cell pellet

Intracellular fraction

Animal feed enzyme Analytical enzyme Therapeutic protein

Centrifugation

to remove cells

Centrifugation

to remove

medium

Protein

precipitation

Cell

lysis Centrifugation

Protein fraction

Protein

precipitation

Protein fraction

1 or 2 purification

steps

Semi-purified

protein 3-4 purification

steps

Homogeneous

protein

Sterile

bottling

To pharmaceuticals market

Lyophilisation

Bottling

To chemicals market

12/10/2019 157DS/ABBS/BTP401

References

12/10/2019 DS/ABBS/BTP401 158

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.

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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)

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