BIOCHEMICAL ENGINEERING

BIOCHEMICAL ENGINEERING CHP583E
Course outline and objectives
References
1. Biochemical Engineering, 2nd edition (1973) by Shuichi Aiba, Arthur

Humhrey and Nancy Mills, Academic press
2. Principles of Fermentation Technology (1984) by Stranbury P.F. and

Whitaker A.,
Pergamon Press
3. Biochemical engineering, unit processes in Fermentation (1958) by

Steel R. (editor) Heywood and Co. Ltd
4. Biochemical Engineering (1964) by Webb F.C., Van Nostrand

Company
1.0 Introduction
1.1 Historical Background
About 150 years ago, Louis Pasteur pointed out the important role of
living micro organisms in biochemical processes. In 1928, a major
breakthrough was achieved by Alexander Fleming in discovering
penicillin. The urgent need of penicillin throughout the 2nd world war led
to microbiologists, biochemists and chemical engineers in a “crash”
programme of developing and designing processes in areas which
were hitherto unfamiliar to them. Three American companies led the
way-Merck, Pfizer, and Squibb. Since then micro organisms have been
known to be used in the manufacture of a host of complex chemicals,
antibiotics, enzymes and vitamins.
In the presidential address to the institutions of Chemical Engineers in

1952, Sir Harold Hartley made a first public reference to the term
Biochemical Engineering. He recognized Biochemical Engineering as
an emerging branch of chemical engineering. He further underscored
the imminent contribution of biological substances and processes in
industrialisation. This inevitably led to among the first series of
Biochemical lectures being offered at the University of Manchester,
where George Davis gave his first lectures in Chemical engineering.
However, most of the Biochemical fundamental principles had been in
application since antiquity.
And so, what is Biochemical Engineering? Biochemical engineering is

the interaction of two disciplines-Biological sciences and chemical
engineering. Biochemical engineering is concerned with conducting
biological processes on an industrial scale, providing the link between
Biological and Chemical engineering. Biological sciences will include
microbiology, biochemistry and genetics. The heart of Biochemical
engineering lies in the scale up and management of the cellular
processes. Thus, therefore there is a precise need for the chemical
engineer to understand the relative dynamic nature of the biological
catalysts.
In order to develop biological units on an industrial scale, three distinct

disciplines of chemical engineering, biochemistry and microbiology are
required combined with specific trade technology. The experiences
gained from chemical engineering are handy at the development of
this rather fast growing branch of engineering. Some interesting
comparisons can be drawn between chemical engineering and
Biochemical engineering:
Basic concepts of material and energy transfer and fluid flow are
common in both but biological processes are notoriously
associated with a narrow range of temperature and non-
Newtonian materials
Many unit of operation are common only for the details to differ
The scope of biochemical engineering

1. Industrial fields in fermentation
2. Operations of food processing; pasteurization, sterilization
and preservation
3. Industrial solvents, organic acids
4. Manufacture of sera and vaccines
5. Commercial enzymes
6. Extraction processes for insulin and other hormones
7. Processing of forests and crop products
8. Effluent disposal
Among the above activities are some that may attract the prophet
and visionary.
However, it is important to note that a broad base of technologies

must be included in Biochemical engineering, which might require
additional information and knowledge. For example, vaccine
manufacture requires in depth medical information while aspects of
hygiene and sterilization are rather unfamiliar to a traditional chemical
engineer. The awareness of Biological background is essential in
Biochemical engineering. This course assumes that the students have
basic knowledge in biological sciences.
The organization of this course is in such a way that it prepares students

to work in the existing industries as well as applying their basic
knowledge to the unborn industries of the future.
Role of a biochemical engineer
Design and operation of absolutely pure and mixed cultures

Prevention of contamination – providing sterile conditions as well
as “contaminant proof” environment
Design of auxiliary systems of air compression, delivery systems,
instrumentation and control
Demonstration of pilot plant equipment and its subsequent
scale-up to the production stage
Separation and isolation of the product using well known
techniques such as filtration, extraction, adsorption and
concentrations
Primarily, the laboratory scientists, microbiologists, biochemists,

genecists must continue in the discovery and advancement of
desirable interactions between micro organisms and their environment.
The biochemical engineer must control and translate the laboratory
results to the production scale operation in an economic manner.
1.2 Exploitation of Biological processes
Potential source of proteins e.g. the use of bacteria to

breakdown n-alkane
Co-oxidation of substrates that do not support their growth
resulting into more useful ones e.g. p-xylene can be oxidized to
dimethyl-cis and cis-muconic acid
Production of enzymes-some of the enzymes are used in
detergent manufacture, hydrolytic reactions and as analytic
tools
Production of polymers- Microbial cells excrete little slime outside
the cell wall. For example glucan which is used as plasma
substitute
Pollution control-treatment of wastes with high BOD(Biological
oxygen demand)
Types of Biological materials
It is not easy to define a living process and it may become

increasing difficult as further biochemical advances are made. This
course is designed primarily for the micro-organisms and enzymes
but it is important to gain a broader sense of other forms of life-the
similarities and differences between the unicellular organisms and
larger animals and plants.
Industrial applications of micro-organisms can be broadly classified

into:
Those in which great care is taken to use only one selected strain of
a particular organism, typified by manufacture of antibiotics and
some vaccines
Those that effort is made to maintain a fairly constant mixture of
strains of the same species e.g. in brewing and wine making
Processes which are chiefly depend on adventitious flora of mixed
species e.g. curing bacon in the manufacture of margarine, tea,
coffee etc. these activities are by large dependent on tradition and
experience.
1.3 MICRO-ORGANISMS
Types of micro-organisms
Micro organisms exist as single cells or at most in relatively

unspecialized multi-cellular colonies, with no capacity to control
cellular temperatures.
Micro organisms may be classified into four main groups

Bacteria
Viruses
Fungi including yeast and actinomycetes
Protozoa including algae
Bacteria
These are single cells, in the form of cocci, rods and spirals capable
of independent growth.
Bacteria are ubiquitous in nature, in aerobic and anaerobic

environments containing water. In the genera, synthetic abilities
range from those of autotrophic species, which require only
inorganic compounds for growth, to those of heterotrophic species,
which may have synthetic ability and must be grown in tissue
culture.
Due to the diverse abilities, bacteria may be exploited industrially to

accumulate both intermediate and end products of metabolism.
Viruses
Viruses are the smallest microbes, obligate intracellular parasites of

animals, plants, insects, fungi, algae or bacteria. They contain no
water and have little or no synthetic or metabolic activity
themselves. Growth and multiplications take place intracellular. This
frequently results in the damage and subsequent death of the host
cells.
The genetic material of viruses may either be ribonucleic (RNA) acid

or the deoxyribonucleic acid (DNA).
Myxoviruses show helical symmetry, with nucleic protein helix

enclosed by a lipoprotein sheath. Bacteriophages are viruses that
are parasitic on bacteria. These phages are possible contaminants
of biological systems.
Fungi
Fungi are widely spread in environments of lower relative humidity

than which favour bacteria. The metabolism of fungi is essentially
anearobic, they form long filamentous, nucleated cells(Hyphae)
between 4 u to 20µ wide.
Generally fungi are free living saprophytes but a few are parasitic
on animals and many are serious pathogens of plants.
Actinomycetes are intermediate between fungi and bacteria.
Industrially, this group is extremely important as a source of powerful

antibiotics.
Protozoa
These are widely distributed in fresh and salty water, in soil and in
animals. They may be unicellular or multi cellular and exhibit a wide
range of morphological forms. Protozoa are either photosynthetic or
non-photosythetic while algae are capable of photosynthesis.
Protozoa are handy in removing bacteria from waste water in

trickling filters and activated sludge plants.
1.4 Requirements for Growth and Formulation of Media
Requirements for growth

Detailed investigation is prerequisite to establish the most suitable
medium for individual biological processes, but certain requirements
must be met by any such medium. All micro organisms require
water, sources of energy, carbon, nitrogen, mineral elements and
possibly vitamins plus oxygen if aerobic. It is important to appreciate
that the cultural conditions that achieve maximum cell mass may
not be necessarily those that give maximum yield of some products
of metabolism. On a small scale, it is relatively simple to devise
medium, although supporting satisfactory growth may not be
suitable for use in large scale.
On large scale, one must normally use resources of cheap nutrients

to create medium which will meet as many as possible of the
following criteria:
It will produce maximum yield of products and biomass per
gram of substrate used
It will produce maximum concentration of the products
It will permit maximum rate of product formation
There will be minimum yield of undesirable products
It will be cheap and of consistent quality and readily
available throughout the year
It will cause minimum problems in other aspects of production
process particularly aeration and agitation, extraction,
purification and waste management
Some of the cheap sources of nutrients include cane molasses, beet

molasses, cereal grains, glucose, sucrose and lactose(Carbon sources)
while ammonium salts, urea, nitrates, corn steep liquor, Soya bean
meal, slaughter house waste and fermentation residues(nitrogen
sources).The medium selected will affect the design of equipment and
processes.
The best temperature for cultivation varies with species but organisms
occurring in the soil naturally grow best at temperatures between 25°c
and 30°c while those isolated from animals grow best at 37°c. Some
organisms are actually thermophilic e.g. those used in bio-digesters
(Lactobacillus), which grow best at 40-45°c.
The products of microbial metabolism often cause major shifts in PH. It is

important to maintain desirable PH level. Sometimes the optimum PH
for product formation may not be the optimum PH for growth. When
acid by-products accumulate in the medium, causing unwanted fall in
the PH, ammonia is slowly fed to the culture, so supplying nitrogen for
growth while maintaining PH. Calcium carbonate may also be used for
the PH control in conditions where the required product is water
soluble. When cell mass or some insoluble metabolite is required, acidic
products are conveniently neutralised by adding sodium hydroxide.
Micro organisms vary in their need for oxygen. On one hand fungi,
algae and a few bacteria are obligate anaerobes. On the other hand,
a few bacteria are strict anaerobes and many bacteria and many
bacteria can grow in both situations (facultative anaerobes).
The physical conditions of temperature and PH will have a profound

effect on microbial growth. The useful range of temperature over
which the metabolic processes of a micro-organism proceed at
significant rate is quite narrow, usually not more than -20°c.
temperature is important in controlling the flavour balance of many
fermented beverages and food. There is usually an optimum PH range,
which is normally limited with complete inactivation or death on either
extreme.
1.5 FORMULATION OF MEDIA
This is an essential part in the design of successful laboratory

experiments, pilot-scale development and the manufacturing
processes. The constituent of media must satisfy the elemental
requirements for the biomass and metabolite production and there
must be adequate supply of energy for the biosynthesis and cell
maintenance. The first step to consider is an equation based on the
stoichiometry for growth and product formation.
Thus,
Carbon + energy + nitrogen + other requirements ⎯environmen

⎯⎯⎯ ⎯tal
→ cell biomass + products
+ CO2 + H 2 O + Heat
A quantitative treatment of the above equation is desirable in the

economical design of the media if compound wastage is to be
minimized. This calls for the elemental composition (K, N, O, Mg etc),
whose data is not readily available. Sometimes, it is important to have
excess quantity of some elements. For example, the concentration of
phosphorous is deliberately raised in some culture to offer a buffering
effect in addition.
The carbon substrate has dual role in the biosynthesis and energy
generation. The carbon requirement under aerobic conditions may be
estimated from the cellular yield coefficient(Y) which is defined as
quantity of cell dry matter produced

γ =
quantity of carbon substrates utilised
Analyses made to determine how the observed conversion of the
carbon sources to the product compares with the theoretical
maximum yield.
1.6 CONSTITUENT OF CULTURE MEDIA
WATER
Water is a major component. Clean water of consistent composition is

therefore required in large quantities from a reliable source. Some of
the factors needed to consider include PH, dissolved solids and effluent
contamination. For example, the mineral content of water is important
in the brewing industry; with hard waters containing CaSO4 more
suitable for pilsner type lagers. It is advisable to deionise the water to
make it more adaptable for the biological processes.
Energy sources
Energy for growth comes from either the oxidation of medium

components or from light. Most industrial micro-organisms are chemo-
organotrophs, therefore the commonest sources of energy will be
carbon sources such as carbohydrates, lipids and proteins.
Carbon sources
The following are some of the examples of carbon sources:
Starch from maize grains, potatoes, cassava etc

Sucrose from sugar cane or sugar beet
Lactose from milk whey powder
Commercial vegetable oils-both as a carbon source and anti
foaming agent
Corn steep liquor- a by-product of starch extraction from maize
Simple organic acids and alkanes
Factors influencing the choice of carbon sources
The main products of the fermentation- determine the cost of the

processes
Impurities of the carbohydrate sources
Government legislation e.g. in the EU, the beet sugar and
molasses are encouraged compared to the cane sugar and
molasses
Local laws especially in some countries where specific acts forbid
the use of some ingredients. For instance, in France, many wines
may only be called by a certain name if producing vineyard is
within a limited geographical area or locality. These names
include vin de bordeaux, which is cultivated around the south
East of France
Method of media preparation e.g. it is often best to sterilise
sugars separately because they may react with ammonium ions
and amino acids to form black nitrogen containing compounds
The influence of carbon sources on product formation
Of great significance is the rate at which the carbon source is

metabolized. This will influence the formation of biomass or production
of primary metabolites.
Nitrogen sources
Most industrially used micro organisms can utilise inorganic or organic

sources of nitrogen. In organic sources may include ammonia gas,
ammonium salts and nitrates. Most organic sources of nitrogen are
mainly supplied as amino acids, protein or urea. Organic sources are
relatively expensive.
Factors influencing the sources of nitrogen sources:
The nitrogen sources have been shown to influence the fermentation

pattern. Antibiotic production may be inhibited by a rapidly utilized
nitrogen source. For example, in the production polygene antibiotics,
soybean meal is considered a good nitrogen source because of
balance of nutrients.
Minerals
In many media magnesium, phosphorous, sulphur, calcium and
chlorine are considered as essential. These are added as a distinct
component. Others such as cobalt, copper, iron, manganese,
molybdenum and zinc are also essential but usually present as
impurities in major ingredients.
Vitamins sources
While many of the natural carbon and nitrogen sources contain all or
some of the required vitamin, any vitamin deficiency may be
eliminated by a careful blend of materials.
Nutrient recycle
In cases of large scale continuous culture fermenters, there is always
need for appropriate adjustment of nutrient levels. Phosphoric acid is
used as a reagent for flocculating bacteria.
Buffers
These are added to control the PH. The buffers include calcium
carbonate, phosphates, sodium hydroxide or sulphuric acid.
Precursors/inhibitors/inducers
Precursors help in the regulation of the product rather than support the
growth of micro organisms. Examples of precursors include corn steep
liquor which increases the yield of penicillin since it contains phenyl
ethylamine.
Inhibitors-sodium bisulphite in the production of glycerol

Inducers-use of yeast in streptomycin production
Oxygen
This is not added in the initial media, but nevertheless an important

parameter in controlling the growth of micro organisms. The medium
may influence the oxygen availability in a number of ways including:
1. Fast metabolism- oxygen limited
2. Rheology-affects aeration and agitation
3. Antifoam- reduces the oxygen transfer rates
ANTI FOAMS
Foaming is a common problem in most Biological systems. This is

caused by the proteins in the media, which denature at the air-broth
interface and form a skin which does not rupture readily. Foaming may
cause the removal of cells from the medium which may lead in the
autolysis and further release of microbial cell protein.
There are two approaches in combating foaming in the fermenters:
Partial purification of some complex nutrients and modification

of some physical parameters
Use of antifoam
An ideal antifoam need to have the following properties

Disperse easily
Active in low concentrations
Long acting to prevent new foams from foaming
Non-toxic to the micro-organisms
Non toxic to animals and humans
Should not cause problems in the extraction of the products
Should not cause handling hazards
Should be cheap
Should not affect oxygen transfer
Should be heat sterilizable
Examples of antifoaming agents:

Alcohols, stearyl and octyl decanol
Esters
Fatty acids and derivatives particularly glyerides
Silicones
Sulphonates
Propylene glycerol
1.7 Changes in the composition of cells with age and with

growth rate
In a batch culture, cells multiply in a closed system until some nutrient is

exhausted or some product accumulate to toxic levels. The developing
population passes through a number of phases, namely:
1. Lag phase, in which cell mass increase but no division occurs

2. Logarithmic phase-cell numbers increase at a constant rte
3. Stationary phase-rate of death and multiplication are equal
4. Decline phase-rate of death is faster than the rate of
multiplication
It is important to appreciate that in a batch system, the environmental

conditions are not constant, even during the phase of constant
growth.
Development of inocula for industrial Fermentation
It is essential that a culture used to inoculate fermentation processes

satisfies the following main criteria:
It must be healthy- in an active state to minimize the length of

the lag phase
It must be available in sufficiently large volumes to provide an
inoculum of optimum size
It must be in a suitable morphological form
It must be free of contamination
It must retain its product forming capabilities
The quantity of the inoculum normally used is between 3 and 10% of

the medium volume. This implies that starting from a stock-culture; the
inoculum must be built up in a number of stages to produce sufficient
biomass to inoculate the production stage fermenter. The master
culture is reconstituted and plated on a solid medium; to form colonies
of sub master culture.
The Isolation, preservation and improvement of the industrial micro

organisms
The first stage is screening for micro organisms of potential industrial

application in their isolation.
Isolation involves obtaining of either pure or mixed cultures followed by
their assessment to determine which can carry out the desired
reactions or produce the desired products.
The criterion for the choice of organisms is a follows:
1. The nutritional characteristics of the organisms-so that the

process may be carried out using very cheap medium
2. The optimum temperature of the organism- the choice of an
organism having an optimum temperature of above 40°c
reduces on the cooling costs of a large scale fermentation
3. The reaction of the organism with the equipment to be
employed and the suitability of the organism to the type of
process to be used
4. The productivity of the organism, in terms of ability to convert
substrate into the product and to give high yield of product per
a unit time
5. The stability of the organism and its sensibility to genetic
manipulation
6. The ease of product recovery from the culture
Before the process may be put into commercial operations, the toxicity
of the product and the organism must be checked and assessed. The
above account implies that cultures must be isolated from the natural
environments. However, the industrial microbiologists may also isolate
micro organisms from culture collections. It is probably cheaper to buy
a culture than isolate from nature.
The ideal isolation procedure starts with an environmental source

(frequently soil) and incorporates a simple test to distinguish the most
desirable types. Selective pressure may be used in the isolation of the
micro organisms which grow on particular substrates, in the presence
of certain compounds or under cultural conditions adverse for these
types. In other cases, it may be possible to design a procedure to
select microbial strain which is known to show certain characteristics at
a relatively high frequency e.g. the production of antibiotics by the
Streptomycetes. Another method may apply random isolation or
isolation by taxa.
Isolation methods utilizing selection of the desired characteristics
Enrichment of liquid culture
The process involves taking a mixed population and providing

conditions either suitable for the growth of the desired type or
unsuitable for the growth of the other types e.g. providing a particular
substrates and inclusion of inhibitors. The enriched culture is inoculated
in a fresh medium and subsequently the procedure is repeated several
times before the dominant organism is isolated by spreading a small
inoculum of the enriched culture in a solid medium. This is normally
hampered by the time of transfer and selection on the basis of specific
growth rate. This may be overcome by the use of continuous process.
Preservation of industrially important organisms
The isolation of a suitable organism for commercial process may be

long and very expensive procedure and it is therefore essential that the
isolated species retains the desired characteristics that led to its
selection. Also the culture must be viable and free from contamination.
Industrial cultures must be stored in a such away as to

1. Eliminate genetic change
2. Protect against contamination
3. Retain viability
Methods of Micro organism preservation
1. Storage at reduced temperature
Some of the common mean include
Storage on agar slopes, which are refrigerated at 5°c or
frozen at -20°c and sub cultured at intervals of about 6
months
Storages of spores in water in which it’s suspended in a
sterile distilled water and stored at 5°c. this technique is
limited in application
Storage under liquid nitrogen- the metabolic activities of
the micro organisms may be reduced considerably by
storage at very low temperatures(-150°c to -196°c), which
is achieved by liquid nitrogen refrigeration
2. Storage at dehydrated form
Soil culture-inoculated before being allowed to dry for a
period of about 2 weeks, thereafter refrigerated
Lyophilization- the culture is frozen followed by its drying under
vacuum which results in the sublimation of the cell water. This
culture may remain stable for upto 10 years.
Quality control of preserved stock cultures

Each batch of newly preserved stock culture should be routinely
checked to ensure their quality in terms of viability, purity and
productivity.
The viability of a micro organism is the ability to reproduce.
Reasons for controlling Micro organisms

It is interesting to consider a few reasons for wishing to reduce
adventitious contamination:
1. Avoid spoilage of a product after a period of storage
2. Avoid pathogenic organisms
3. Avoid unwanted end products
4. Maintain the yield of the desired products
Sources of contaminants
Present in raw materials
Airborne contamination
packing supplies
personnel
The multiplication of the contaminants is prevented by:
design of buildings to prevent cross-infection and avoid crevices

and ledges
design of equipment to maintain hygienic standards
control of storage and holding conditions at all stages from raw
materials to the ultimate consumer
choice of operating procedures and conditions
The use of advance methods for prevention and control of

contaminants will be discussed under sterilisation.
Variations of micro-organisms
When a micro-organism is sub cultured so as to give a number of

generations of progeny, the final culture obtained contains many
individuals differing from the first parents. This is a result of mutations or
the relatively permanent change in the genetic apparatus of the cell.
This is mainly associated with genetic changes. Though most changes
in genes are disadvantageous or even lethal to the organisms, enough
survive with higher incidences of variants after a few generations. Thus,
every culture of micro-organisms loses its original character by sub-
cultivation. To avoid this, a few generations should be allowed to
intervene between the tested master- culture (obtained from single
colony isolation) and the production stage.
In addition, a number of arbitrary treatments such as heat- and cold-

shock, pasteurisation and sub-cultivation on special selective media
may be employed. Besides its deleterious effect on the yield of
product, mutation makes itself manifest in a number of ways:-
Alter the degree of pigmentation e.g. the pale-green normal
penicillin Chrysogenum throws white, dark green, yellow orange
and other coloured mutants
The power to sporulate may disappear
The new organism may lose the power to synthesis essential
amino acids or vitamins
The colony form may change as well as growth rate
However, mutations have a beneficial side also, and to this end may
be produced artificially. A suspension of organisms is subjected to
treatment and desirable properties may be sought by examination of
individuals in the resultant population. The principal methods of
artificially induced mutations include:-
1. Exposure to ultra violet light
Ultra violet light is absorbed by the nucleoprotein material, which forms
the genetic material, resulting in the resonance and leads to
destructive changes. This method is not highly selective and is
accompanied by a good deal of damage to the rest of the cell.
2. Mustard gas
Mustard gas is a more drastic agent reacting with generic centres and
disrupts chromosomes. Once the generic centres are changes,
information on heredity is interrupted.
3. X-rays and Gamma rays
X-rays act almost the same way as the mustard gas, giving rise to free
radicals in the ambient medium. The free radicals are responsible for
further changes in genetic information. Gamma rays are the more
recent.
4. Use of radioactive substances
The use of radioactive materials is known to give higher mutants rates
with a lower killer rate. The classical isotope used is the sulphur-35 which
decays in 87 days to give chlorine-35. The major advantage of this
method is that mutations may occur without appreciable cell trauma.
It can also be simple to control. It also enjoys higher rates of survival,
almost about 100%. From the mutated cultures, conventional testing
techniques are used to obtain a higher yield of antibiotic or other
desired quality. For example, the yield of penicillin has been raised from
10units/ml in 1942 to over 2000units/ml in 1960. As an example of the
commercial use of mutant strain, Escherichia Coli has been mutated in
lysine fermentation.
Hybridization
The selective sexual breeding is finding limited application in micro-

organisms.
STERILIZATION OF AIR, MEDIA AND EQUIPMENT
Definition: Sterilization is the destruction of all forms of life in a medium

and environment. In the laboratory, this is achieved by the use of heat;
the bacteriological medium is held at temperatures of about 120°c for
20 minutes. Dry heating in an oven is yet another method commonly
used to sterilize equipment in the laboratory. Besides, the use of heat,
there are other methods which can be used to sterilize the equipment,
air and media. These methods include:-
a) Use of ultra violet light
b) Irradiation by chemical regents or high frequency
c) Filtration especially for the sterile air
STERILIZATION OF EQUIPMENT AND MEDIA
The sterilization of equipment and media together is achieved by the

use of steam. This is a simple operation in which a jacket or a coil is
fitted to the bioreactor is supplied with pressure steam at a
temperature and duration suitable for the destruction of life. This is
followed by subsequent cooling to the fermentation temperature. A
vent is provided through which air is expelled from the bioreactor and
the space above the medium is filled with steam. A supply of sterile air
must be connected to the fermentor to prevent formation of a
vacuum when the system is cooled.
STERILIZATION OF THE BIOREACTOR AND MEDIUM SEPARATELY
The procedure of sterilizing an empty bioreactor is exactly as described

in the last section. There are however, several methods of sterilizing the
medium separately:-
1) Batch cooker
A vessel fitted with coils or a jacket for heating and cooling. An agitator
may be fitted to aid heat exchange. The interconnecting piping
between the cooker and the main bioreactor must be sterilized at the
same time so that sterile medium may be transferred. This method
reduces the time in which the main reactor is unoccupied between
fermentations; against this is the higher cost of the extra equipment
involved, and the increased steam usage.
2) Continuous sterilization
The medium is sterilized as it is pumped from the medium make-up

vessel to a fermentor which has been sterilized empty. This method
offers flexibility in choice of time temperature conditions in which the
medium is exposed and advantage taken of lower sterilizing
temperatures or shorter holding periods if the medium has low PH.
Increased yield have been reported in cases where continuous
sterilization is employed.
There are two principle that may be applied in continuous sterilization
a) Heat Exchange Principle
This is a three section plate type heat exchange. This methods realizes
some important steam saving economy. It is necessary to avoid any
leakage or short-circuiting between the non-sterile medium entering
and the sterile medium leaving the system.
b) Continuous Retention Tube
In this system, medium is made up in a mixing vessel and is pumped

from this into one end of a long retention tub. At this end, steam is
injected to heat the medium to the desired sterilizing temperatures for
the requisite time, before being passed to a cooling section and
thereafter a bioreactor; in the design of the retention tube, it is
important to ensure that there is no end-to-end mixing. This would result
in the non-sterile medium by-passing sterilizing temperatures zone and
infecting the rest of the sterile system.
In sterilization, there are principles of securing and maintaining sterile

and pure culture conditions. These include:-
1. No direct connection should be made permanently between

non-sterile and sterile parts of the system
2. Welded construction should be used where possible and
convenient
3. Where joints have to be used they should be of high quality finish,
employing rubber or any other impervious material as the seal
4. The type of valve used should be easily sterilizable and serviced
when necessary
5. After sterilization, all parts of the system which are to be kept
sterile should be kept under a positive pressure, either with sterile
air or sterile liquid
6. Each part of the system should be capable of independent
sterilisation, without interfering with operation of the rest of the
plant
7. Isolated danger spots, which are difficult to sterilize at the star of
a batch, should be provided with steam connections for
continuous or intermittent use.
STERILIZING OF AIR
Since the time of Pasteur, air has been recognized as a source of
microbial contamination. At this time, it was discovered that a plug of
cotton wool would allow air to reach the culture but would prevent
other micro-organisms from contaminating it. Over the years, the
demand for pure air for industrial operation has been on the increase.
This calls for the sterilizing of air in terms of thousands of cubic feet of air
per hour. The most surely effective method is heating the air, but the
cost is prohibitive when large volumes of air have to be sterilized.
Therefore, other methods of sterilizing the air have been applied:-
1. Electrostatic precipitation
2. Exposure to UV light
3. Filtration through columns packed with glass wool, slug wool,
cotton wool, activated carbon or other filtering media coupled
or without counter-current scrubbing through phenols, caustic
soda, acids or other germicidal agents.
Direct chemical treatment is not possible because of the danger of

carry-over into the culture. Most antibiotic plants use filters packed with
fibrous media such as glass or slug wool or granular material such as
activated carbon.
The efficiency of filtration is calculated from
100( N 1 − N 2 )
effeciency % =
N2
Where N1 is the number of micro-organisms per unit volume of air
before filtration and N2 is the number of micro-organism per unit
volume of air after the filtration.
The mechanism of filtration is not a simple sieving action but is due to

retention of airborne particles on the fibres or granules of the filter
media. Therefore, the efficiency of the filter media, and the efficiency
of filtration would therefore depend on a number of factors like
Diffusion
Inertial impingement
Electrostatic attraction
A mathematical evaluation on the dimensioning of filters for sterilisation

of air a addressed as assignment and will be dealt during tutorial
session.
CHEMICAL STERILIZATION
Sterilization can sometimes be achieved by the use of chemicals. There

are numerous terms used in connection with the control of
contaminants. These terms include antiseptic, disinfectant,
prophylactic, sanitizer, germicide, bactericide, fungicide.
Due to the large spectrum of sterilizing agents, different methods of
testing sterilising agnts have been developed.
1. Rideal_walker test
This depends on a comparison between the inhibitory effect of phenol

and the other agent on the growth of selected micro-organism, under
closely monitored condition
2. Chick-Martin test
Due to the inadequacy of the comparison test (Rideal-Walker) in terms
of variations dependent on the organic nutrient present, dried yeast is
used in a more standardises procedure.
3. Reddish and other official tests
A number of detail modifications have been introduced into the chick-
Martin test to make results of wider applicability.
In spite of these modifications, no method has been found which

consistently reflects the behaviour of sterilising agent under actual
working conditions. There is a growing tendency for each user to devise
tests to suit their own requirement.
Factors affecting chemical sterilisation

I. Presence of other matter- the presence of some other matter
may interfere with the sterilising action e.g. the presence of
grease can offer a large measure of physical protection to the
organisms unless the agent can penetrate it. In addition, grease
can offer nutrients for the organisms. This has dual effect; allows
undesirable faster multiplication and bringing organisms into an
actively mitosing condition in which there are vulnerable to
attack.
II. Rate of destruction of micro-organisms: the use of a single
sterilising chemical agent on a single strain follows approximately
logarithmic course in respect with time
2.303 N
N t = N o e −κt or κ = log o
t Nt
where No, Nt are the concentration of organisms initially and at a
time t respectively, k is reaction velocity constant and t is time
usually in minutes
the hourly basis for air change has been calculated by
138 N
KA = log o
t Nt
III. the level of initial contamination- assuming the logarithmic
relationship, then it follows without doubt that absolute sterility
can only be approached asymptotically and arbitrary final level
must be quoted. The higher the initial contamination, the higher
the probability of including pathogenic or resistant micro-
organisms in th sterile environment.
IV. Concentration of the sterilizing agent- a general relationship
between the concentration of the sterilizing agent and the time
to reach a low count can be expressed
C1n t1 = C 2n t 2
Where n is considered as the concentration coefficient
V. Decay rates of the disinfectants-sterilising agent can
progressively loose their effectiveness as a result of chemical
change, adsorption and various other factors. For instance
hypochlorite (jik) is quite unstable or highly reactive compound
whose KA value approaches 20.
VI. Physiological effect of sterilising agents-unless there is clear
evidence to the contrary, it should be assumed that any
compound capable of disorganising a unicellular organism as to
cause its death, will also have a significant physiological effect
on larger creatures including man. These effects include allergy,
cumulative effect of ingestion in small amounts over a long
period of period. Caution needs to be exercised at all times!
Detergents
Detergents in a broad sense are the foundation of industrial cleaning

procedures and may be included in the final products either
deliberately or fortuitously as residues. The chosen detergent may
occasionally be relatively pure compound, but is usually a mixture,
either blended on site or purchased from a specialized company
There are important aspects of detergency, which must be considered

in the choice of the most efficacy detergent:-
a. Surface and interfacial tensions are reduced, allowing
emulsification of grease. But this poses of a negative side effect
of increasing wettability of the surface
b. Dirt and grease should be held in suspension
c. Calcium and magnesium are held in control
Form soluble Ca and Mg salts
Form true soluble complexes with Ca++ and Mg++
Phosphates and other builders may combine with these
ions to form insoluble but finely divided precipitates
d. A high PH level, increasing detergency
Uses of sterilising agents

The use can be considered under the following headings
Sterilising water
Cleaning equipment
Preservation of food and cosmetics
Pharmaceutical preparations
FERMENTATION PROCESSES
ANAEROBIC FERMENTATION PROCESSES: ACETONE-BUTANOL
Historically, this type of fermentation was developed as a source of n-

butanol which could be used in the preparation of butadiene, a major
ingredient in the manufacture of synthetic rubber. After the world I, this
process was used as a source of acetone. The acetone was used in the
manufacture of cordite. After some years, the demand for acetone
reduced and the pressure reverted to be a source of butanol. This time,
the butanol was the main raw material for the production of butyl
esters, particular solvents of nitro-cellulose lacquers.
The organism used for this fermentation is the Clostridium

Acetobutylicum, which by definition is an anaerobic spore forming
bacterium. The name acetobutylicum implies that it produces
acetone and butanol. It does this in a medium of molasses, made up of
about 6% sugars (calculated as sucrose). From a good fermentation it
produces a solution mixed solvents composed of about 65% butanol,
30% acetone and 5% ethanol. This solution is distilled and fractionated
to obtain particular products. There is also considerable gas produced,
a mixture of CO2 and hydrogen gas. Animal feed may be prepared
from the residue, largely composed of the bacteria.
AEROBIC FERMENTATION PROCESSES: PENECILIN
The principal advances in the technology of aerobic fermentation

were made in the course of development of penicillin fermentation.
Technically, penicillin is a generic name applied to a group of
compounds. Therefore, a large number of penicillin occur naturally or
could be artificially prepared. For example there exist penicillin G and
Penecillin V, which are distinguished by the side chain-benzyl and
phenoxymethyl group respectively. However, most penicillin are labile
in the acid form and they are normally prepared as much stable salts
or esters e.g. sodium benzyl penicillin.
Penicillin are formed by several different organisms; chiefly aspergi and
penicillia. Penicillium Chrysogenum group is known to give the highest
yields. At the industrial scale, the penicillium spores are developed in a
flask of sterile corn in sufficient quantity to provide inoculum for the
seed mash.
After inoculation the seed mash is maintained at temperatures optimal

for the germination of the spores and the growth of the organisms. It is
necessary to aerate and agitate vigorously to promote metabolism
and growth. This may take between 24 and 48 hours depending on the
size of the vessel and the inoculum. The termination is usually based on
some empirical criterion such as
Age of the culture
The exhaustion of the sugars
Tendency of the PH to rise
The seed is then transferred along the sterile connection into the
fermentor charged with mash. Aeration, agitation and temperature
control of the mash commences at least as soon as the seed is
received into a fermentor and continuous until the mash is harvested
between 3 and 5 days later.
The first step in harvesting consists of separating the mycelium from the
medium by the use of rotary vacuum filter in which the mycelium is
continuously stripped as felt.
YEASTS
Yeasts have uniquitous distribution throughout the plant and the animal
kingdom and also in the soils. From the industrial viewpoint, there re two
main genera of interest, the broad range of saccharomycetes and the
more limited candida or torulopsis yeasts. Industrial yeasts can be
classified broadly into six groups
a. Beer or ale yeasts-these are referred as top fermentation. They
rise to the surface of the wort as the head of foam, which can be
skimmed off for re-use
b. Lager yeasts- brewing lager is carried out at a much lower
temperature than beer and for longer periods. This yeast must
withstand the conditions and also settle out to the bottom of the
vessel towards the end of the fermentation
c. Distiller’s yeasts- they are selected strains of the beer/ale yeast
adapted for high sugar and alcohol tolerance and capable of
giving high conversion ratio.
d. Baker’s yeast-while its selected from beer/ale yeast, the baker’s
yeast is bland in flavour, lacking hop bitterness from beer, is more
rapid in action and has extended shelf life
e. Wine’s yeast
f. Food and fodder yeast
BREWING
Large scale brewing has been developed empirically in various parts of

the world. There are differences also in raw materials and the methods
evolved to deal with them.
Barley malting-clean and uniform is selected and stored for a resting

period before it can be germinated. For malting, it is steeped in several
changes of water for about 2days at 50°f, taking care to avoid
germination. The final water is then drained off and the soaked barley
is spread on the malting floor for about a week to sprout. During this
period, various enzymes are activated and potentially able to convert
up to 75% of the solid contents into soluble sugars, mainly maltose,
dextrins and some polypeptides. The next stage is kilning, which stops
further activity by reducing water availability. This is then milled.
Preparation of wort- All the ingredients are broken down to an

intermediate size by a roller mill. The mixture is fed with hot water
through a mashing machine. Mashing carries out several functions
namely:-
The soluble fractions of malted grains is extracted
The amylotic enzymes present have an opportunity to hdyrolyse
unattacked strch still in malt
The smaller proportion of proteolytic enzymes must be allowed to
break down proteins into polypeptides
The balance of ingredients and technique are arranged so that
the final wort has the correct specific gravity
Fermentation- The temperature of fermentation depends on the type
of beer required and the yeast strains in use. The cooled wort is pitched
in a cream made from yeast taken from previous brews and the
fermentation allowed to proceed for about 3 days. During this time, the
temperature is maintained by passing cold or hot water through
immersed pipes, known as attemperators.
Industrial Alcohol
Although a large proportion of industrial alcohol in the world is made
from petrochemicals, considerable amounts of fermentation spirits are
still made through the world the raw materials include grain, beet/cane
molasses etc.
After mashing the final wort is adjusted to conditions to suit the chosen
yeast. Temperatures are in the range of 23°c to 30°c and the initial PH is
adjusted to 4.5 to 6.0. The balance of nutrients and total concentration
are adjusted t provide only a limited growth but maximum conversion
of carbohydrates to ethanol.
Utilisation of by-products
Carbon dioxide-about ¾ is recovered for sale as compressed liquid or
dry ice. Since pure grade is required, the CO2 is scrubbed through
water, from which ethanol is recovered, then sulphuric acid and finally
deoderized an activated charcoal.
Fermentation residues- these are insoluble material filtered off and
dried to be used as animal feed.
Alcohol distillation: The filtered liquor after fermentation contains not

only ethanol and water but also small amounts of volatile components
(acetahyde, esters, fusel oil). After distillation, the ethanol obtained is
good enough for certain sales outlets. However, some dehydrated
form of ethanol may be required. The methods used including 1)
absorb it as water of crystallisation on a mixture of sodium and
potassium acetates 2) azeotropic distillation – use of a solvent
(benzene) to form a ternary azeotrope with the water and a small part
of the ethanol.
In the production of industrial alcohol, the alcohol is denatured for
purposes of excise duty control; to avoid reaching the public in
potable forms. Depending on the intended final use, it is normally
denatured by methanol. Methanol is highly neurotic.
CONTINUOUS FERMENTATION
Applied relatively fast chemical reactions or physical processes,

continuous fermentation methods offer very great advantages
compared to batch methods, provided the demand of the products is
high enough. These advantages are:-
Non-productive time i.e. time spent cleaning, filling, heating,
cooling and emptying are drastically reduced
This affects the useful loading of the plant, so that smaller
equipment and buildings can be used
Labour can significantly reduced, partly because it is easier to
mechanize some operations
Automatic controls and warning systems are easier to install and
they in turn can reduce the requirements of skilled supervision
The product can be held at optimum reaction conditions,
unaffected by heating and cooling, producing better yields
Product uniformity should be improved
However against these advantages may be listed certain possible

disadvantages:-
Although the plant may be smaller, instrumentation and
mechanical handling devices may make it equally expensive
The design and operation of such equipment requires the
services of highly educated staff
The shift work necessary may increase some costs, highly hourly
rates, provision of steam, laboratory services etc
It is rare for manual efficiency of the night workers to be as high
as that of day workers
In practice, a careful balance is struck between these factors, often a

compromise is decided on e.g. large scale operations are carried out
continuously while minor operations e.g. cleaning of the equipment is
carried out during the day shift. Experience with continuous
fermentation indicates that there are quite severe limitations to the
time an operation may be run. These factors that limit include:-
Adventitious contamination can become serious after even a
few days
There is a tendency for fast growing strains of organisms to
become dominant
Some organisms develop the morphological and biochemical
mutation under continuous culture
Physical problems such as removal of fungal mycelium limit to
the operation
The yield of the product and/or the concentration significantly
reduces
THEORY OF CONTINUOUS FERMENTATION
Interest has been focussed on the continuous stirred-tank reactors

(CSTR), used singly or a few in sequence. The essential feature of this
form of reactor is that stirring is sufficiently vigorous so that the
composition of the efficient is identical with the bulk of the tank
contents; the danger of short-circuiting of vital nutrients from feed to
the effluent must be taken into account and conditions have to be
selected to keep this minimum.
Mathematical analysis of such continuous fermentor is much easier if

the following assumptions are made:-
The growth rate of the organism is held constant
Only a single strain of organism is present which maintains its growth
characteristics
Therefore, the density of the organism within a single vessel can be

described by the following equation
dN F F
= Nf + κN − N .
dt V V
Rate of change of the fermentor = Increase due to organism in feed +

increase by growth- loss of effluent
N= Concentration of organisms, Nf= organisms of feed, F= flow into and
out of the fermentor, V= volume of fermentor, k= specific growth
The dimensions of the equipment may be eliminated by referring the

dilution rate, D= F/V
SINGLE VESSEL STERILE FEED

Under steady-state conditions
dN
= 0 and the first term, NfD disappears
dt
F
therefore, κN = N = ND OR κ = D
V
for the population to remain steady, the holding time 1/D, must be
equal to the generation time or reciprocal of growth rate. The specific
growth factor, k may be identical to the value found on the logarithms
part of the batch growth curve. In practice it may be well below this
value because.
The feed rate of limiting nutrients is insufficient to maintain maximum
growth
The organism may be subject to environmental factors more nearly
corresponding to early stationary phase
If avalue of kn is taken as logarithmic value, then by definition the
maximum achievable, then D=km, thus the process may be treated as
batch.
Thus, F = Vκ m
If this flow rate is exceeded i.e. F is greater than km, then clearly the
organisms are carried out in the efficient than they can grow in the
fermentor.
The classical work of Monod, showed that the growth rate of many
organisms can be related to the concentration of the limiting
component by formula similar to Michaeli’s enzyme equation.
S
κ = km ( )
K+S
Where k= saturation constant, numerically equal to Km/2
The dilution rate
⎛ S ⎞ ⎛ D ⎞
D = κm⎜ ⎟ where S = κ ⎜⎜ ⎟⎟
⎝K+S⎠ ⎝κm − D ⎠
MICROBIAL GROWTH KINETICS
Microbial growth is defined as the orderly increase of all chemical

constituents of an organism. It may result from the synthesis and
accumulation of a cellular material.
As dictated by the type of the product being produced, fermentation

may be carried out as batch, continuous and fed-batch processes.
Batch Culture- This is a cloud culture which contains an initial, limited

amount of nutrients. In this process, the lag phase must be limited as
much as possible, while the exponential phase is encouraged. The
exponential phase is described by the equation
dX
= µX ………………………………………………………………1
dt
X=concentration of microbial biomass, t= time(hrs) and µ is the specific
growth rate, hrs-1
Equation 1 is intergrated as follows
X t =Xoeµt
Xo is the original biomass concentration
On taking the natural logarithms,
ln X t = ln X o + µt
During exponential growth, the micro-organism is growing at its

maximum growth, µmax for the prevailing conditions.
However, the nature of limitation of the growth ay be exposed by

growing the micro-organisms in the presence of a range of substrates
concentrations. The situation may be described as
X = Y (S R − S )
Where X IS THE BIOMASS produced, Y is the yield factor, SR is the original
substrate concentration and S is the residual substrate concentration.
CONTINUOUS CULTURE
A media displaces an equal volume of culture from the vessel to
achieve continuous production. The flow of medium into the vessel is
related by the term dilution rate, D, defined as
F
D=
V
The net change in cell concentration over a time period may be
expressed as
dX
= growth − output
dt
dX
= µX − DX
dt
Under steady state conditions, the cell concentration remains
constant, thus dX/dt =0,
Then
µX + DX
and
µ=D
Thus under steady state condition, the specific growth is controlled by
the dilution rate.
For the substrate

dS X⎛ S ⎞
= DS R − DS − µmax ⎜⎜ ⎟
dt Y ⎝ K S + S ⎟⎠
Fed-batch culture- there are batch system which are fed continuously.
THE RECOVERY AND PURIFICATION OF FERMENTATION PRODUCTS
The recovery and fermentation products may be difficult and costly to

ensure good recovery or purification, speed of operation may be the
overriding factor because of the labile nature of the product. The
choice of the recovery process is based on the following criteria:-
a. The intracellular or extra-cellular location of the products
b. The concentration of the product in the broth
c. The physical and chemical properties of the desired products
d. The intended use of the product
e. The minimum acceptable standard of purity
f. The impurities of the fermentor broth
g. The marketable price of the product
The main objective of the first stage for the recovery of an extra-cellular
product is the removal of large solid particles and the microbial cells b
centrifugation or filtration. In the next stage the broth is fractionated or
extracted into major fractions using adsorptions or ion-exchange
chromatography liquid-liquid solvent extraction or precipitation, further
more precise chromatographic and crystallisation.
It may be possible to modify the handling characteristic of the broth so

that it can be handled much faster with simpler equipment making use
o a number of techniques
Selection of the micro-organisms which does not produce
pigments or undesirable metabolites
Modification of the fermentation conditions to reduce the
production of undesirable metabolites
Precise timing of harvesting
PH control
Temperature treatment after harvesting
Addition of flocculating agents
Use of enzymes to attack cell walls
Removal of microbial cells and other solid matter.
Due to the small size of microbial cells, filter aids are applied to improve
filtration rates while heat and flocculation are employed as techniques
for increasing sedimentation rates in centrifugation.
a. Foam separation- this is done by exploiting the differences in
surface activity of materials. The material is selectively adsorbed
or attached to the surface of gas bubbles, concentrated and
finally removed by skimming
b. Precipitation- It is possible to obtain some products from the
broth, either by adding a compound which leads to the
formation of insoluble complexes or salts or by adding a suitable
organic solvent.
c. Filtration- this is the commonest method of removing suspended
particles from a liquid or gas, using a porous medium which
retains the particles but allows the liquid or gas to pass. The
following factors influence the choice of the most suitable
filtration equipment to be used:-
Properties of the filtrate, viscosity and density
Nature of solid particles especially size, shape and size
distribution
Solids:liquid ratio
The need for recovery of the solid or liquid fraction or
both
The scale of operation
The need of batch or continuous operations
The need for aseptic conditions
The need for pressure/vacuum suction to ensure an
adequate flow rate of the liquid
d. Centrifugation-micro-organisms and other similar sized prticles
can be removed from broth by using centrifuge when filtration is
not satisfactory separation method. Although a centrifuge may
be expensive when compared with a filter it may be essential
when:-
a. Filtration is slow and difficult
b. The cells or other suspended matter must be obtained free
of filter aids
c. Continuous separation to a high standard of hygiene is
required
e. Liquid-liquid extraction- this is the separation of a component
from liquid mixture by treatment with a solvent in which the
desired component is preferentially soluble. The solvent is then
recovered by distillation.
f. Chromatography- this is used to isolate and purify relatively low
concentrations of metabolic products. This will involve the
passage and separation of different solutes as the liquid is
passed through a column. Depending on the mechanism by
which solutes may be differentially held in a column, the
technique can be grouped as
Adsorption chromatography-binding of the solute to
the solid phase primarily by weak van der waal forces
Ion-exchange-reversible exchange of ions between
liquid phase and solid phase which is not
accompanied by radical change in the solid structure
Gel-filtration-separates on the basis of size in which the
small particles diffuse through the gel rapidly
Affinity chromatography-depends on the interactions
between pairs of biological materials such as enzymes-
substrate
g. Ultra-filtration- the process in which solutes of high molecular
weights are retained when the solvent and the low molecular
weight solutes are forced under hydraulic pressure through a
membrane of very fine pore size.
h. Drying- this is often the last stage, which involves the removal of
water from the heat sensitive material ensuring that there is
minimal losses of viability, activity or nutritional value. Drying is
undertaken because:-
The cost of transport can be reduced
The material is easier to handle
The material can be more conveniently stored in dry
state
A spray drier is the most widely used for drying biological materials
when starting materials is in the form of a liquid or paste which can
be initially atomized into small droplets through a nozzle or by
contact with a rotating disc.
i. Crystallisation- This is best applied in the initial recovery of
organic acids and amino acids
FERMENTATION ECONOMICS
If a fermentation process is to yield a product at a competitive price,

the chosen micro-organism should give the desired end products is
predictable, and economically commonly used in developing a
successful process:-
a) The capital investment should be confined to minimum
b) Raw materials should be a cheap as possible and utilized
efficiently
c) The highest yielding strain of micro-organism should be used
d) There should be saving in labour whenever possible
e) Growth cycle must be short as possible for batch fermentors
f) Recovery and purification procedures should be simple and
rapid as possible
g) The effluent discharge should be efficiently
h) Space requirement should be kept minimum
There a number of steps in a biological process, which need to be

carefully analysed in terms of cost. The major steps are as follows:-.
1. Isolation of micro-organisms-Isolation programmes are

unfortunately time consuming, expensive and may be
something of a gamble e.g. Pfizer spent about £1430000
on screening programme for a broad spectrum antibiotic
producer during which terramycin was detected in 1952.
In search for suitable micro-organisms for use for single-cell
protein production, many objectives will have an
economic basis.
2. Strain improvement- The strain improvement by a mutation

selection for improving an established process can be very
cost effective. Obviously time and money worth spending
on mutation/selection programme will depend on the size
of the manufacturing process.
3. Media-The cost of the various components of a production

medium can have a profound effect on the overall cost of
fermentation. The carbon source is usually the most
expensive contributing to the cost of the process. In
addition, the price of natural material may fluctuate due
to other competing demands annual variations in
quantities harvested. Problems concerned with storage,
handling and mixing of media should not be neglected.
4. Air Sterilisation- although air sterilization by heating is

technically possible, it has generally been regarded as too
costly for full-scale operation of a plant. Filtration of air
through deep bed of fibrous or granular material is
preferred. While the capital costs are dependent on the
size of the plant, the operating costs will be estimated on
the life of the filters.
5. Heating and cooling-A biochemical process may include

heating and cooling in the following major areas:-
Sterilization or boiling of medium to 120°c followed
by cooling to 35°c
Heating of fermenter and other ancillary
equipment
Removal of water from products
The cooling costs of micro-organisms can be minimized with the use of
micro-organisms with a higher optimal growth temperature.
6. Aeration and agitation-Fermentation having high oxygen

demand must be agitated with sufficient power to
maintain uniform environment and to disperse the stream
of air introduced by aeration. Some companies have
introduced air-lift principle, with benefits of simple design
and reduced energy costs.
7. Batch-process cycle times- For shorter growth cycles such

as baker’s yeast (14 to24 hrs), the turn around time will be
as important as the time between inoculation and
harvesting while when production cycle is long e.g.
penicillin(6 to 7 days),a few extra hours of turn around will
be insignificant on the total cost.
8. Recovery cost-A number of factors contribute to the

recovery costs:-
Yield losses
High energy and maintenance associated with the
recovery and purification e.g. depreciation accounts for
80% of the overall cost of a large-scale rotary filtration
High costs of solvents and other raw materials used in the
recovery and refining of the products
9. Water usage and recycling- As the charges for water

increase, many of the biological process will become
vulnerable to cost escalation because of relatively large
volumes of water required per unit volume of product.
There is a wide spread interest in reducing the overall
consumption.
10. Effluent treatment-In the majority of fermentation

processes it is impossible to dispose of effluents at zero
cost. The various alternative disposal procedures may be
compared using economic considerations. Before
deciding on the most economic form of treatment, the
water column, the organic and solids loading, range of PH
variation, nutrient level, temperature fluctuaction,
company finance policy, the site location and
government legislation for waste disposal must be known.
11. Plant and equipment- It is most logical to build equipment

as large as possible because of the economy of scale.
However, there are a number of restraints which have to
be considered before deciding on scale of operation such
as restriction include:-
Cooling and aeration requirements
Methods of fermentation vessel construction
Due to the capital investment and operational costs there is
now a trend of fermentor design to consider unconventional
designs of simple construction with every efficiently oxygen
transfer to be used for specific purposes e.g. single protein
cell. A more detailed guide is prepared by the Institution of
Chemical Engineers.
MARKET POTENTIAL
It is necessary to estimate the size of the present and potential market

and increase in the demand for a compound. The life expectancy of
the compound will have to be predicted when covered by a patent.

BIOCHEMICAL ENGINEERING

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BIOCHEMICAL ENGINEERING CHP583E

Course outline and objectives

1. Biochemical Engineering, 2nd edition (1973) by Shuichi Aiba, Arthur

2. Principles of Fermentation Technology (1984) by Stranbury P.F. and

3. Biochemical engineering, unit processes in Fermentation (1958) by

4. Biochemical Engineering (1964) by Webb F.C., Van Nostrand

1.1 Historical Background

In the presidential address to the institutions of Chemical Engineers in

And so, what is Biochemical Engineering? Biochemical engineering is

In order to develop biological units on an industrial scale, three distinct

The scope of biochemical engineering

However, it is important to note that a broad base of technologies

The organization of this course is in such a way that it prepares students

Role of a biochemical engineer

 Design and operation of absolutely pure and mixed cultures

Primarily, the laboratory scientists, microbiologists, biochemists,

1.2 Exploitation of Biological processes

 Potential source of proteins e.g. the use of bacteria to

It is not easy to define a living process and it may become

Industrial applications of micro-organisms can be broadly classified

Micro organisms exist as single cells or at most in relatively

Micro organisms may be classified into four main groups

Bacteria are ubiquitous in nature, in aerobic and anaerobic

Due to the diverse abilities, bacteria may be exploited industrially to

Viruses are the smallest microbes, obligate intracellular parasites of

The genetic material of viruses may either be ribonucleic (RNA) acid

Myxoviruses show helical symmetry, with nucleic protein helix

Fungi are widely spread in environments of lower relative humidity

Industrially, this group is extremely important as a source of powerful

Protozoa are handy in removing bacteria from waste water in

1.4 Requirements for Growth and Formulation of Media

Requirements for growth

On large scale, one must normally use resources of cheap nutrients

Some of the cheap sources of nutrients include cane molasses, beet

The products of microbial metabolism often cause major shifts in PH. It is

The physical conditions of temperature and PH will have a profound

1.5 FORMULATION OF MEDIA

This is an essential part in the design of successful laboratory

Carbon + energy + nitrogen + other requirements ⎯environmen

A quantitative treatment of the above equation is desirable in the

quantity of cell dry matter produced

1.6 CONSTITUENT OF CULTURE MEDIA

Water is a major component. Clean water of consistent composition is

Energy for growth comes from either the oxidation of medium

The following are some of the examples of carbon sources:

 Starch from maize grains, potatoes, cassava etc

Factors influencing the choice of carbon sources

 The main products of the fermentation- determine the cost of the

The influence of carbon sources on product formation

Of great significance is the rate at which the carbon source is

Most industrially used micro organisms can utilise inorganic or organic

Factors influencing the sources of nitrogen sources:

The nitrogen sources have been shown to influence the fermentation

Inhibitors-sodium bisulphite in the production of glycerol

This is not added in the initial media, but nevertheless an important

Foaming is a common problem in most Biological systems. This is

There are two approaches in combating foaming in the fermenters:

 Partial purification of some complex nutrients and modification

An ideal antifoam need to have the following properties

Examples of antifoaming agents:

1.7 Changes in the composition of cells with age and with

In a batch culture, cells multiply in a closed system until some nutrient is

Design and operation of absolutely pure and mixed cultures

Potential source of proteins e.g. the use of bacteria to

Starch from maize grains, potatoes, cassava etc

The main products of the fermentation- determine the cost of the

Partial purification of some complex nutrients and modification

It must be healthy- in an active state to minimize the length of

design of buildings to prevent cross-infection and avoid crevices