School of Health Sciences & Technology

Department of Biotechnology
CUBTM 219: Fermentation Biotechnology

Chapter 2: UPSTREAM PROCESSING (Lecture 2 &3)

By

Dr. R. Kamusoko (DPhil, MSc, BSc)

INTENDED LEARNING OUTCOMES

 Demonstrate understanding on all the upstream

processes in fermentation

 Formulate and optimise media for fermentation

production

 Screen fermentation microbes from their natural

sources
CONTENT

 Overview of upstream processing

 Media formulation and optimisation

 Air, media and fermentation equipment sterilisation

 Seed culture and inoculum development

 Inoculation of the medium

 Introduction to process scale-up

OVERVIEW OF UPSTREAM PROCESSING

 Upstream processing involves identifying and extracting

the raw materials for fermentation

 This forms the initial process of fermentation

 Upstream process deals with:

 Inoculum preparation - screening of microbes, selection of

a suitable strain, genetic modification of the strain if
required and preparation of pure culture at lab-scale

 Preparation of media with suitable growth parameters at

lab-scale

 Sterilisation of medium, air and ancillary equipment

 Inoculation of the medium

 Scale-up of the entire process

FERMENTATION MEDIUM

 Microbes used for fermentation grow on/in a medium which

satisfies their nutritional needs for growth

 Complete analysis must be done to establish the most

favorable medium for growth of the fermentation microbes

 Formulating medium at lab-scale can be done by adding main

ingredients like water, C-source, N-source, minerals and other
supplements in pure form and in required quantities

 The same medium may not support satisfactory growth of the

same organism at industrial level
cont..

 At industrial scale, the following criteria need to be

satisfied for a material to be treated as a medium:

 It should give maximum yield of the product

 It should give minimum yield of undesired product(s)

 It should be consistently available throughout the year

 It should be cheap
cont..

 At industrial-scale, cane molasses, corn steep liquor

and sugar beet juice are utilised

 Some sensitive fermentation makes use of glucose,

sucrose and other carbohydrates in their pure form

 This ensures the purity and quality of the final product

 Sometimes starch will be added to the medium for

specific production of amylases
cont..

 Soy meal or ammonia or nitrate salts are added to

supplement a N-source

 Other elements including growth factors, vitamins, anti-

foaming agents, precursors, inducers, trace elements such
as Fe, Cu, Mn, Mo and Co are added

 Anti-foaming agents are also added

 In the presence of higher concentrations of metal ions,

chelating agents are added
cont..

 At lab-scale, peptone or tryptone or beef extract

which is a partially digested hydrolysate is utilised in
synthesis of proteins, components of nucleic acids and
other essential cellular components
Classification of fermentation medium

 Based on ingredients, the medium used for fermentation

can be classified as:

1. Defined/synthetic medium

2. Complex /undefined medium

3. Technical medium
Defined/synthetic medium:

 Consists of known quantities of all ingredients

 Provides trace elements and vitamins required by microbes, and

especially a defined C and N-source

 Glucose or glycerol are often used as C-sources, and ammonium

salts or nitrates as inorganic N-sources

 No yeast, animal, or plant tissue is present

 e.g. peptone water:

1% peptone + 0.5% NaCl in water

Complex/undefined medium:

 Composed of substrates with undefined composition, such as extracts

or hydrolysates of waste products

 Cheap substrates are mainly used in industrial production

 Relatively expensive substrates, such as yeast extract, brain heart

infusion, peptone, tryptic soy broth, tryptone, etc are often used as
complex medium

 A sugar, often glucose is added as the main C- and energy source

 A combination of extracts and sugar creates a medium which is rich in

minerals and organic nutrients
Technical medium:

 Used at industrial scale and it is cheaper

 Substrate sources can be derived from industrial wastes and

are often highly impure mixtures

 Pre-treatment before fermentation is necessary

 E.g. soy meal, whey, fishmeal, malt extract, and sulfite waste
liquor

 Wastewater from monosodium glutamate production, which

contains high levels of COD, sulphate, and ammonical N at a
low pH, has been used as a N and water source, with sugar
beet pulp as a C source for production of pectinases
Nutritional requirements of a fermentation medium

 Most fermentations require liquid media (broth) - SmFs

 SSFs are operated on solid medium

 Fermentation media must satisfy all the nutritional

requirements of a microbe and fulfill the technical objectives of
a process

 All microorganisms require water, sources of energy, C, N,

mineral elements and possibly vitamins and O2, if aerobic

 Nutrients should be formulated to promote the synthesis of a

target product
cont..

 In most industrial fermentations, several stages require

media

 The stages include inoculum (starter culture)

propagation steps, pilot-scale and the main bioprocess

 Technical objectives of inoculum propagation and the

main fermentation are often very different, which may be
reflected in differences in their media formulations
Medium formulation

 Medium formulation is an essential stage in a

manufacturing process

 The general equation for a manufacturing process is:

C, N & other energy sources + Biomass + products +

O2 + nutrients CO2 +H2O +heat

 Elemental composition of microorganism may be taken

as guide to formulate its medium
Elemental composition (%) of fermentation microbes:

Element (%) Bacteria Yeast Fungi

Carbon 50-53 45-50 40-63
Hydrogen 7 7 7
Nitrogen 12-15 7.5-11 7-10
Phosphorous 2-3 0.8-2.6 0.4-4.5
Sulphur 0.2-1.0 0.01-0.24 0.1-0.5
Potassium 1.0-4.5 1-4 0.2-2.5
Sodium 0.5-1.0 0.01-0.1 0.02-0.5
Calcium 0.01-1.1 0.1-0.3 0.1-1.4
Magnesium 0.1-0.5 0.1-0.5 0.1-0.5
Chloride 0.5 - -
Iron 0.02-0.2 0.01-0.5 0.1-0.2
Carbon source:
 A C-source is required for biosynthesis leading to reproduction,
product formation and cell maintenance

 In most fermentations, it serves as the energy source

 E.g. molasses, malted barley, starch and dextrins, sulphite waste

liquor, alkanes, alcohols, oils and fats

 Factors influencing a C-source

- cost of the product
- rate at which it is metabolised
- geographical location
- government regulations
- cellular yield
Nitrogen source:

 Most industrial microbes can utilize inorganic and organic N-

sources

 Inorganic N - ammonium salts, often ammonium sulphate and

di-ammonium hydrogen phosphate, or ammonia

 Ammonia can also be used to adjust pH of the fermentation

 Organic N sources include amino acids, proteins and urea in

corn steep liquor, yeast extracts, peptones, soya bean meal,
etc
Minerals:

 All microorganisms require certain

mineral elements for their growth and
metabolism

 In many media, Mg, P, K, S, Ca & Cl are

essential elements that must be added

 Trace elements - Co, Cu, Fe, Mn, Mb

and Zn are present in sufficient
quantities in water supplies and as
impurities in other media ingredients
Chelators:

 Many media cannot be prepared without precipitation

 Hence, some chelating agents are added to form complexes with

metal ions which are gradually utilised by microorganisms

 E.g. EDTA, citric acid, polyphosphates, etc

 Check the concentration of a chelator otherwise it may inhibit

the growth of the microorganism

 In many media, chelators are added separately after autoclaving

 Vitamins and growth factors:

 Many bacteria can synthesize all the necessary vitamins

from basic elements

 For other bacteria, fungi and yeasts, vitamins may be

supplemented to the fermentation medium

 Most natural C and N sources also contain at least

some of the required vitamins as minor contaminants
 Precursors:

 Substances added before or simultaneously

with the fermentation medium

 Incorporated without any major change into a

molecule of the fermentation product

 Serve to improve yield or quality of the

product

 They are required in certain industrial

fermentations

 Provided through crude nutritive constituents

e.g. corn steep liquor or by direct addition of

more pure compounds

Inducers and elicitors:

 An inducer or a structural analogue must be

added into the culture medium or added at a

specific point during fermentation

 Majority of enzymes of industrial interest are

inducable

 Inducers are often substrates e.g. starches or

dextrins for amylase

 In plant cell culture, production of secondary

metabolites, such as flavanoids and terpenoids

can be triggered by adding elicitors e.g. salicylic

acid, methyl salicylate, benzothiadiazole, benzoic

acid, chitosan, etc

Inhibitors:

 Used to re-direct metabolism

towards the target product and
reduce formation of other
metabolic intermediates

 e.g. sodium bisulphite

 Others halt a pathway at a

certain point to prevent further
metabolism of the target product
Water:

 All fermentations (except SSF), require vast amounts of water

 Water is also important for ancillary services like heating, cooling,
cleaning and rinsing
 A reliable source of large quantities of clean water with consistent
composition is essential
 Assessing suitability of water for pH, dissolved salts and effluent
contamination
 Re-use of water is important:
- It reduces water cost by 50%
- Effluent treatment cost by 10-fold
Oxygen:

 Required in aerobic fermentations - occurs normally at the

beginning of fermentation

 It may be supplied in the form of air containing about 21% (v/v)

oxygen or occasionally as pure oxygen when requirements are
particularly high

 The organism’s oxygen requirements may vary widely

depending on the C-source

 For most fermentations, the air or oxygen is filter-sterilised

before injection into the fermenter
Antifoam agents:

 Antifoam agents are also necessary

 Include insoluble oils, polydimethyl siloxanes and other

silicones, certain alcohols, stearates and glycols

 Foaming is largely due to medium proteins that become

attached to the air-broth interface where they denature to
form a stable foam “skin” that is not easily disrupted
 An ideal antifoam should have the following properties:
− Disperse readily and have fast action
− Active at low concentrations
− Long acting in preventing new foam
− Should not be metabolised
− Should not be toxic to microorganisms, humans, etc
− Should be cheap and not cause problems in fermentation
Optimisation of fermentation medium
 Medium optimisation is a process where components of medium or
different conditions are varied in concentration or changed so that
we can get better growth of the organisms for high productivity

 Different combinations and sequences of process conditions need

to be investigated to determine the growth conditions which
produce biomass with physiological state best constituted for
product formation

 There may be a sequence of phases, each with a specific set of

optimal conditions

 Increases the yield and activity of the desired product

Methods for medium optimisation
 Strategies to improve the efficiency of production medium include:
• Borrowing
• Component replacing
• Biological mimicry
• One factor at-a-time
• Factorial design
• Plackett-Burman design
• Response surface methodology
• Evolutionary operation
• Evolutionary operation factorial design
• Artificial neural network (ANN)
• Genetic algorithms
Borrowing:

 An open-ended system where all the medium components used for

analysis of product formation are taken from different authors

 It is very easy to see the ingredients of particular media from various

authors

 There are so many options and one has to select the appropriate
medium for particular fermentation process

Evolutionary operation:

 A method for obtaining high yield by using factorial design serially;

while changing variables of media used in factorial design until
improvement in results is greater than the estimated values
Component replacing:

 Also an open-ended system

 Ingredients of the different fermentation medium are

compared and others replace few of the components

 With this method, one cannot get idea about interaction

of different medium components, but different medium
component like C, N and other sources are screened for
medium optimisation
Biological mimicry:

 Different elemental compositions required by the microbes for best

growth are studied

 Medium formulation is based on composition and exact amount of

components required by microbes

 Such type of medium gives the best growth and high yield of product

 It is time consuming and expensive

 Not easy to analyse the elemental composition of microbes

 A close-ended system, where component interactions cannot studied

One factor at-a-time:

 A close-ended system for medium optimisation

 Optimisation of medium is carried out by changing any one of the

ingredients in the medium while keeping all other parameter constant

 It is time consuming and difficult to study the interactions among the

medium ingredients

 Useful method to study only few medium ingredients or parameters as

it requires large number of experimental sets which is time consuming
and expensive

 Very popular method as it is easy and suitable for a given medium at a

time
Factorial design:

 A close-ended system which involves variation in factors or

parameters at two or more levels

 Highly efficient in providing interaction among various factors and

allows to study the effect of each factor and its interactions, giving
maximum yield

 An equation is used to provide information regarding particular

factors like strain, medium components and other physical parameters
and their interactions that can change yield

 To make a full factorial search which would examine each possible

combination of independent variable at appropriate levels, a large
number of experiments, xn, is required, where x is the number of
levels and n is the number of variables
Plackett-Burman design:

 It is useful for more than five variables in screening the most important variable

 Here, the no. of experiments (n) will be conducted for n-1 variables

 n MUST be a multiple of 4 like 8,12,16,20…100

 Authors give a series of experimental designs known as balanced incomplete

blocks

 Variables not having influence in the process are dummy variables

 Dummy variables are required to estimate the error in experimentation

 Minimum of one or two dummy variables should be included in the

experimental set-up

 More can be included if the real variables are less

Response surface methodology:

 The quantity of different variables is also important in formulating

medium for optimal growth of organisms giving high yield

 Is a statistical experiment design, which gives information regarding

quantity of various variable used in Plackett-Burman method

 Response is the yield of product when a particular quantity of various

variables is added

 Mathematical calculations used for combinations of quantities of various

parameters and its effect which gives result is plotted

 A model is prepared by which one can predict the amount of variables

for medium optimisation
Evolutionary operation factorial design:

 The method is a hybrid of evolutionary operation and factorial

design technique

 It is a multi-variable sequential search technique where the

effects of n variable factors are studied and response is
statistically analysed

 This enables the selection of optimum conditions for individual

parameters for planning of the following experiments
Artificial neural network (ANN):
 Various experiments are performed regarding medium
optimisation

 Data generated due to such experiments are plotted in

mathematical equations and models are created

 ANN is a model in which sets of experimental data are

used to predict new data with the help of mathematical
equation
Genetic algorithms:

 A non-statistical method based on principle of genetic

manipulation which leads to desired organisms producing high
yield

 With the help of mutations, crossing over or recombination,

unique organisms are produced which can give better yield under
particular medium formulation conditions

 Replication of such strain produce high yield strains

 Main disadvantage - Not capable of storing the information

generated at each stage of the optimisation process
Problems in medium optimisation

 High labour cost as large numbers of experiments are involved

 Rarely, the data generated from the shake-flask media match

exactly with fermenter studies

 All shake-flask studies suffer from weaknesses such as:

uncontrolled pH, poor oxygen transfer, inadequate mixing and
considerable evaporation

 The industrial-scale medium suffer from problems such as batch-

batch variability, availability all around the year, fluctuations in the
price, stability during the transport time cost, problems
associated with bulk storage and time
cont..

 Microbes or cells are dynamic in nature with a lot of internal

control mechanisms, but most media optimisation studies treat
them as black-box or utilise solely for empirical data only

 Almost all the researchers encounter this problem, “when

should one stop applying further optimisation techniques or
which step is the end point of optimisation studies” at one stage
or other

 Medium optimisation is often tedious and never-ending

STERILISATION OF AIR, MEDIUM AND FERMENTER

 Sterilisation is needed to prevent contamination with undesired microbes

 Ensures that only the desired microbe is present to carry out fermentation,
products are made of predicted quality, the environment is protected from
undesirable contamination and microbial spoilage of product is prevented

 Effect of contamination on fermentation:

− Medium is consumed unnecessarily and affects the growth of desired

organism & outweigh the desired product

− Affects fermentation conditions and growth of the desired organism thus

outweighing the desired product

− Desired product is contaminated and interfere with downstream

processes
Sterilisation methods

Methods available for sterilisation include:

1. Chemical methods - preferred for heat-sensitive equipment
 ethylene oxide (gas)
 70% ethanol
 3% sodium hypochlorite
2. Exposure to radiation
 UV for surfaces
 X-ray for liquids
3. Filtration
4. Heating - involve the rupture of the cell membrane by increasing the
transmembrane electric field strength beyond a certain threshold
Air sterilisation:

 The number of microbial cells in air is ~ 103–104 per m3

 Methods of air sterilisation include:

− Heating (economically impractical)

− Radiation (UV rays)

− Use of germicidal sprays (e.g., phenol, ethylene oxide or

formalin)

− Filtration: membrane filtration commonly used

cont..

 Filter is used to sterilise off-gases leaving the fermenter -

pathogens are harmful to plant personnel or environment

 Two types of air filters:

1. Depth filter (fibrous filter) with pores that are bigger

than the size of the microorganism to be removed

2. Surface filter (absolute filter) with pores that are smaller

than the size of the microorganism to be removed
Medium sterilisation:

 Destruction or removal of all forms of microbial life from

the aqueous substrate

 For small-scale fermentations, both the medium and

fermenter are sterilised by steam under pressure in an
autoclave

 For large-scale, heating the medium is done using steam at

121°C for 15 min
cont..

 Filter method is used for medium containing heat-sensitive components

− Membrane made of cellulose esters or other polymers with pore

diameter of 0.2-0.45 μm is used

− Membrane itself must be sterilised by steam or radiation before use

− Bacteria and other particles with size greater than the pore size are
screened out and collected on the surface of the membrane

− Filtration is not as effective or reliable as heat sterilisation

− Any nutrient component which is heat-labile is filter-sterilised and later

added to the sterilised medium
Fermenter sterilisation:

 The fermenter may be sterilised together with the medium or

separately

 If the medium is sterilised in a separate batch cooker, or is

sterilised continuously, then the fermenter must be sterilised
separately before the sterile medium

 Sterilisation of vessel is done using steam at 121°C for 15 min

 Negative pressure should not develop inside the fermenter

since it may lead to contamination
cont..

 Achieved by heating the jacket or coils of the fermenter with

steam and passing steam into the vessel through all entries,
apart from the air outlet from which steam is allowed to exit
slowly

 Steam pressure is held at 15 psi in the vessel for 20 min

 Sterile air must be sparged into the fermenter after the cycle is
complete and a positive pressure is maintained to avoid
vacuum formation and drawing of unsterile air into the vessel
SCREENING OF FERMENTATION MICROBES

 The procedure for isolation, detection and separation of

microorganisms of interest from a mixed population in a
natural environment by using highly selective procedures is
called SCREENING

 Such an organism is called as a producer strain

 Purpose: To yield a product at a cheaper price and give

the desired product in a predictable and economically
adequate quantity
Characteristics of a producer strain

 A producer strain should possess the following characters:

− It should be able to grow on relatively cheap substrates

− It should grow well at an ambient temperature preferably

at 30-40°C. This reduces the cooling costs

− It should yield high quantity of the end product

− It should possess minimum reaction time with the

equipment used in a fermentation process
cont..

− It should possess stable biochemical characteristics

− It should yield only the desired product without

producing undesirable substances

− It should possess optimum growth rate so that it can

be easily cultivated on a large-scale

Generalised scheme for microbial screening:
Important things to consider when screening

1. CHOICE OF SOURCE:- Samples for screening are

taken from soil, water, air, milk, compost, etc

2. CHOICE OF SUBSTRATE:- Nutrients and growth

factors should be supplied for growth of a desired
microorganism

3. CHOICE OF DETECTION:- Proper isolation and

detection of desired microorganisms is important
Primary screening

 The detection and isolation of the desired microbe from a

natural environment containing mixed species

 Determines which microorganisms are able to produce

compounds

 Separates only a few microorganisms with commercial

value and discards valueless microorganisms

 Does not provide much idea on production or yield

potential of the microorganism
Primary screening techniques

 The techniques involved include:

a) The crowded plate technique

b) Auxanography

c) Enrichment culture technique

d) Use of an indicator dyes

e) Supplementing volatile and organic substrates

The crowded plate technique:

 Used to detect and isolate antibiotic-producers

 Serial dilutions of source sample are made and plated (pour and
spread) on nutrient agar (300-400 or more colonies/plate)

 The ability of the organism to show antibiotic activity is indicated

by the presence of a zone of growth inhibition around the colony

 Such cultures are then selected, purified and maintained as

pure/stock/master culture

 The purified culture is tested to find the Microbial Inhibition

Spectrum (MIS)
Auxanography technique:

 The technique is employed to detect microbes capable of producing

certain extracellular substances like growth stimulating factors (e.g.
vitamins, amino acids, etc)

 A test organism with a definite growth requirement for a particular

metabolite is used

 The two major steps are:

i) Preparation of first plate:

 A filter paper disc (strip) is put across the bottom of petridish

 Nutrient agar is poured on the paper disc and allowed to solidify

 Soil sample is serially diluted, inoculated and incubated

 ii) Preparation of second plate:

 A minimal medium lacking the growth factor is prepared and seeded

with the test organism

 The medium is poured onto a fresh petridish and allowed to set

 The agar in the first plate is then carefully lifted with a spatula and placed

on the second plate without inverting

 The growth factors produced by colonies present on surface of the first

layer of agar can diffuse into the lower layer containing the test organism

 Zones of stimulated growth of the test organism around the colonies is

an indication that the organism produced a growth factor

Enrichment culture technique:

 First designed by Beijerinck (1888) to isolate the desired microorganism

from a heterogeneous microbial population

 Generally employed to isolate microbes that are few in a sample

 Nutrient broth is inoculated with the microbial source material and

incubated

 A small portion of the inoculum is plated onto a solid medium and well
isolated colonies are obtained

 Suspected colonies from the plate are sub-cultured on fresh media and
subjected for further testing
Use of indicator dyes:

 pH indicator dyes are used to detect microbes capable of producing organic

acids or amines

 The dyes change the colour of medium according to pH – an indication of

organic acid or base production

 Examples of dyes are neutral red, bromothymol blue, etc

 Colonies are sub-cultured to make stock culture

 Further testing is needed since organic acids and bases are also metabolic
products of microbial growth

 Incorporation of CaCO3 in medium is also used to screen organic acid

producing microbes on basis of formation of clear zone of dissolved CaCO3
around the colony
Technique of supplementing volatile organic substances:

 Employed to screen microbes capable of using C-source from

volatile substrates like hydrocarbons, low molecular weight alcohols
and similar carbon sources

 Dilutions of a microbial source are spread onto surfaces of sterile

agar containing all nutrients except volatile substances

 The required volatile substrate is applied on to the lid of the petri

plates, which are incubated by placing them in an inverted position

 Vapours from volatile substrate spread to the surface of agar and

provide the required specific nutrient to the microbe, which grows
and form colonies by absorbing the supplemented nutrient

 These colonies are isolated, purified and stock cultures are made
Secondary screening
 A systematic screening intended to isolate industrially important
or useful microbes

 Useful to detect microbes that have real commercial value

 Accomplished by performing experiments in agar plates, flasks

or small bioreactors containing liquid media

 Microbes having poor applicability in fermentation are discarded

 Provides the information whether the product formed by

microorganisms is new or not. This may be accomplished by
paper or thin layer chromatography
cont..

 Shows whether the product possesses physical properties such

as UV light absorption or fluorescence or chemical properties
that can be employed to detect the compound during use of
paper chromatography

 Gives an idea about the economic position of the fermentation

 Helps in providing information regarding the product yield

potential of different isolates

 Determines the optimum conditions for growth or

accumulation of a product associated with a particular culture
cont..

 Chemical, physical and biological properties of a product

are also determined

 Reveals whether a product produced in the culture broth

occurs in more than one chemical form

 Detects gross genetic instability in microbial cultures

 Tells about the chemical stability of the fermentation

product
Example of secondary screening
Antibiotic-producing Streptomyces sp.:

1. Streptomyces isolates are streaked as a narrow band on nutrient agar

plates and incubated

2. Test organisms are then streaked from the edge of plates without
touching streptomyceal isolate and then the plates are then
incubated

3. At the end of incubation, growth inhibitory zones for each organism

are measured

4. Such organisms are subjected for further testing by growing the

culture in sterilised liquid media and incubating at constant
temperature in a mechanical shaker
INOCULUM DEVELOPMENT
 Preparation of microbes from a dormant stock culture to a final productive
stage is called “inoculum development”

 Inoculum production is a critical stage in industrial fermentations

 Involves screening of production strain, and genetic modification if required and

preparation of pure culture at lab-scale

 Inoculum is prepared as a stepwise sequence employing increasing volumes of

media

 Preparation may range in scale and purpose from a small inoculum for a
bioassay to 1 m3 for production of a vitamin or antibiotic in a 200 m3 fermenter

 Cuts down the time required for growth of microbes in a fermenter, thereby
increasing the rate of productivity
cont..
 Definition of inoculum :- A mixture of cultured microbes along with
media in which it is growing

 Inoculum preparation aims to:-

a) Minimise the loss of viable microbes during recovery from

dormancy

b) Obtain a genotypically identical copy of the population that was

stored

c) Increase biomass

d) Develop a culture to a physiological state suitable for performance

in the final production stage
cont..

 An inoculum should meet the following criteria:-

• It must be in an active and health status to minimise the

duration of lag phase in subsequent fermentation

• It must be available in sufficiently large volumes to provide an

inoculum of optimum size (0.1-10% to the medium volume)

• The inoculum must be in suitable morphological state

• It must be resistant to infection

• The inoculated biomass must retain its product forming

capabilities
Inoculum development steps:

 Involves both lab and plant-based steps

 Usually begins with the transfer of cells from an agar slant to a shaker
flask by means of an inoculating loop

 The “master culture” must be well conserved according to its

taxonomic and genetic conditions

 If a good inoculum process is developed at lab-scale, it could be scaled

up to higher production volumes

 Inoculum is transferred to the production plant, usually to a seed vessel

 Plant-based steps are more automated than lab-based

NB:

 Make sure “what is transferred is as consistent as possible

regarding size and quality so that the control of
fermentation is as automatic as possible”

 Samples are removed aseptically and subjected to quality

control where various parameters including optical density
(OD), live cell count, etc, are measured by technicians or
operators
Classical steps in inoculum development
Inoculation
 Transfer of seed material or inoculum into a fermenter

 Inoculation of a lab fermenter is done using pre-sterilised tubing and

a peristaltic pump

 On large-scale, inoculate by applying a positive pressure on seed

fermenter connected aseptically to the production fermenter

 Connecting lines are sterilised before use to transfer inoculum

 Heat susceptible substances such as amino acids and some vitamins

must be dissolved in small volumes of water, sterilised by filtration
and added separately to the final medium aseptically
PROCESS SCALE-UP

 The goal of scale-up is to identify and develop a process

that will successfully produce a desired product at
commercial scale

 To successfully move from the small-scale to the large-scale,

one must understand how size changes impact a number of
physical and chemical phenomena

 See next chapter for more details

Cont..
--THE END--