UPSTEAM PROCESSING

INTRODUCTION
 Biochemical engineering is concerned with conducting biological processes on an industrial
scale.

 This area links biological sciences with chemical engineering.

 The role of biochemical engineers has become more important in recent years due to the
dramatic developments of biotechnology

 The laboratory technology for the genetic manipulation within living cells is known as genetic
engineering.

 A major objective of this technique is to splice a foreign gene

 for a desired product into circular forms of DNA (plasmids), and then to insert them into an
organism, so that the foreign gene can be expressed to produce the product from the organism.

IMPORTANCE
 For example, in a laboratory scale of 100 mL, a small Erlenmeyer flask on a shaker can
be an excellent way to cultivate cells, but for a large-scale operation of 2,000 L, we
cannot make the vessel bigger and shake it.

 We need to design an effective bioreactor to cultivate the cells in the most optimum
conditions.

 Therefore, biochemical engineering is one of the major areas in biotechnology

important to its commercialization

To carry out a bioprocess on a large scale, biochemical engineers need to work together
with biological scientists:

1. to obtain the best biological catalyst (microorganism, animal cell, plant cell, or enzyme)
for a desired process

2. to create the best possible environment for the catalyst to perform by designing the
bioreactor and operating it in the most efficient way

3. to separate the desired products from the reaction mixture in the most economical
TYPICAL BIOPROCESS
Biological processes have advantages and disadvantages over traditional chemical processes.

The major advantages are as follows:

1. Mild reaction condition

2. Specificity

3. Effectiveness

4. Renewable resources

5. Recombinant DNA technology

However, biological processes have the following disadvantages:

1. Complex product mixtures

2. Dilute aqueous environments

3. Contamination

4. Variability

THE RANGE OF FERMENTATION PROCESSES

There are five major groups of commercially important fermentations:

(i) Those that produce microbial cells (or biomass) as the product.

(ii) Those that produce microbial enzymes.

(iii) Those that produce microbial metabolites.

(iv) Those that produce recombinant products.

(v) Those that modify a compound which is added to the fermentation the transformation process

THE COMPONENTS PARTS OF THE FERMENTATION PROCESS

(i) The formulation of media to be used in culturing the process organism during the development
of the inoculum and in the production fermenter.

(ii) The sterilization of the medium, fermenters and ancillary equipment.

(iii) The production of an active, pure culture in sufficient quantity to inoculate the production vessel.

(iv) The growth of the organism in the production fermenter under optimum conditions for product
formation.
(v) The extraction of the product and its purification.

(vi) The disposal of effluents produced by the process.

ISOLATION

The first stage in the screening for microorganisms of potential industrial application is their isolation.

Isolation involves obtaining either pure or mixed cultures followed by their assessment to determine
which carry out the desired reaction or produce the desired product.

1. The nutritional characteristics of the organism. It is frequently required that a process be carried out
using a very cheap medium or a pre-determined one, e.g. the use of methanol as an energy source.
These requirements may be met by the suitable design of the isolation medium.

2. The optimum temperature of the organism. The use of an organism having an optimum temperature
above 40° considerably reduces the cooling costs of a large-scale fermentation and, therefore, the use of
such a temperature in the isolation procedure may be beneficial.

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 stability of the organism and its amenability to genetic manipulation.

5. The productivity of the organism, measured in its ability to convert substrate into product and to give
a high yield of product per unit time.

6. The ease of product recovery from the culture

Enrichment culture is a technique resulting in an increase in the number of organism relative to the
numbers of other types in the original inoculum.

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 organisms

Eg., by the provision of particular substrates or the inclusion of certain inhibitors

Enrichment liquid culture

 Modification of the medium

 Sub-culturing several times

 The time of sub-culture is critical

 Corresponds to the point at which the desired organism is dominant

 the dominant organisms will be the fastest growing

Continuous enrichment techniques are especially valuable in isolating organisms to be used in a

continuous flow commercial process.

Organisms isolated by batch enrichment and purification on solid media frequently perform poorly in
continuous culture ,whereas continuous enrichment provides an organism, or mixture of organisms,
adapted to continuous culture.

NOT UTILIZING SELECTION OF DESIRED CHARACTER

(i) Developing procedures to favour the isolation of unusual taxa which are less likely to have been
screened previously

(ij) Identifying selectable features correlated with the unselectable industrial trait thus enabling an
enrichment process to be developed

Huck et al. (1991) used the statistical stepwise discrimination analysis (SDA) technique to design media
for the positive selection of antibiotic producing soil isolates.

This was achieved by characterizing a collection of eubacterial and actinomycete soil isolates according
to 43 physiological and nutritional tests.

Certain features were identified which, when used as selective factors enhanced the probability of
either isolating actinomycetes or antibiotic producing actinomycetes.

SCREENING METHODS
 Early screening strategies tended to be empirical, labour intensive and had relatively low
success rates.
  As the number of commercially important compounds isolated increased, the success rates of
such screens decreased further

 The evolution of antibiotic screens serves as an excellent illustration of the development of

more precise, targeted systems.

 The progress in molecular biology, genetics and immunology has also contributed extensively
to the development of innovative screens, by enabling the construction of specific detector
strains, increasing the availability of enzymes and receptors and constructing extremely
sensitive assays.

 Bull (1992) summarized the major contributions as follows:

 The provision of test organisms that have increased sensitivities, or resistances, to known
agents. For example, the use of super-sensitive strains for the detection of β-lactam antibiotics.

 The cloning of genes coding for enzymes or receptors that may be used in inhibitor or binding
screens makes such materials more accessible and available in much larger amounts.

 The development of reporter gene assays. A reporter gene is one which codes for an easily
assayable product so that it can be used to detect the activation of a control sequence to which
it is fused. Such systems have been used in the search for metabolites that disrupt viral
replication.

 Molecular probes for particular gene sequences may enable the detection of organisms capable
of producing certain product groups.

 This information may be used to focus the search on these organisms in an attempt to find novel
representatives of an already known commercially attractive chemical family.

 The development of immunologically based assays such as ELISA

PRESERVATION
 The culture used to initiate an industrial fermentation must be viable and free from
contamination. Thus, industrial cultures must be stored in such way as to eliminate genetic
change, protect against contamination and retain viability.

 An organism may be kept viable by repeated sub-culture into fresh medium, but, at each cell
division, there is a small probability of mutations occurring and because repeated sub-culture
involves very many such divisions, there is a high probability that strain degeneration would
occur
LOW TEMPERATURE

Storage on agar slopes

Cultures grown on agar slopes may be stored in a refrigerator (5°) or a freezer (-20°) and sub-cultured at
approximately 6-monthly intervals. The time of sub-culture may be extended to I year if the slopes are
covered with sterile medicinal grade mineral oil

2. Storage under liquid nitrogen

The metabolic activities of micro-organisms may be reduced considerably by storage at the very low
temperatures (-150° to -196°) which may be achieved using a liquid nitrogen refrigerator.

 Snell (1991) claimed that this approach is the most universally applicable of all preservation
methods.

 Fungi, bacteriophage, viruses, algae, yeasts, animal and plant cells and tissue cultures have all
been successfully preserved.

 The technique involves growing a culture to the maximum stationary phase, resuspending the
cells in a cryoprotective agent (such as 10% glycerol) and freezing the suspension in sealed
ampoules before storage under liquid nitrogen.

 Some loss of viability is suffered during the freezing and thawing stages but there is virtually no
loss during the storage period.

 Although the equipment is expensive the process is economical on labour. However, the
method has the major disadvantage that liquid nitrogen evaporates and must be replenished
regularly.

DEHYDRATED FORM

1. Dried cultures

 Dried soil cultures have been used widely for culture preservation, particularly for sporulating
mycelial organisms.

 Moist, sterile soil may be inoculated with a culture and incubated for several days for some
growth to occur and then allowed to dry at room temperature for approximately 2 weeks.

 The dry soil may be stored in a dry atmosphere or, preferably, in a refrigerator.

 The technique has been used extensively for the storage of fungi and actinomycetes and
Pridham et al. (1973) observed that of 1800 actinomycetes dried on soil about 50% were viable
after 20-years storage

2. Lyophilization
  Lyophilization, or freeze-drying, involves the freezing of a culture followed by its drying under
vacuum results in the sublimation of the cell water.

 The technique involves growing the culture to the maximum stationary phase and resuspending
the cells in a protective medium such as milk, serum or sodium glutamate.

 A few drops of the suspension are transferred to an ampoule, which is then frozen and
subjected to a high vacuum until sublimation is complete, after which the ampoule is sealed.

 The ampoules may be stored in a refrigerator and the cells may remain viable for 10 years or
more (Perlman and Kikuchi, 1977).

STRAIN IMPROVEMENT
MEDIA FOR INDUSTRIAL
FERMENTATION
• All micro-organisms require water, sources of energy, carbon, nitrogen, mineral element and
vitamin plus oxygen in their growth medium.
 • On a small scale, it is simple to device a medium containing pure compounds, but the resulting
medium although satisfy the growth, may be unsuitable for use in a large scale process.

• On a large scale one must use sources of cheap nutrient to create a medium which will meet as
many as possible of the following criteria:

• It will produce a maximum yield of product at biomass per gram of substrate used.

• It will produce a maximum concentration of product or biomass.

• It will permit the maximum rate of product formation.

• It will be the minimum yield of undesired product.

• It will be cheap and of a consistent quality and is readily available throughout the year.

• It will cause minimal problem in other aspects of production and agitation, extraction,
purification and waste treatment.

• The medium selected will affect the design of the fermenter to be used.

• A laboratory medium may not ideal in a large fermenter with a low gas-transfer pattern.

• Media with a high viscosity will also need a higher power input for effective stirring.

• Besides the requirement for growth and product formation, medium may also influence pH
variation, foam formation, oxidation-reduction potential and the morphological form of the
organisms.

• It may also be necessary to provide precursors or metabolic inhibitors.

MEDIA FORMULATION
 Medium formulation is an essential stage in the

• design of

• ~successful laboratory experiments

• ~pilot-scale development

• ~manufacturing processes

 The constituents of a medium must satisfy

• ~the elemental requirements for cell biomass and metabolite production

• ~must be an adequate supply of energy for biosynthesis and cell maintenance

  Equation based on the stoichiometry for growth and product formation.

 Carbon and energy source + nitrogen source + other requirements cell biomass +
products + CO2

+ H2O + heat

 This equation should be expressed in quantitative terms, which is economical design of media if
component wastage is to be minimal.

 It should be possible to calculate

~the minimal quantities of nutrients which will be needed to produce a specific amount of biomass

~the substrate concentration in order to produce required

product yield

 A knowledge of the elemental composition of a named micro-organisms, which should include

the content of C, H, O, N, S, P, Mg and K, is required in the solution of the elemental balance
equation.

 Some micro-organisms cannot synthesize specific nutrients such as amino acid, vitamins or
nucleotides.

 Once a specific growth factor has been identified it can be incorporated into a medium in
adequate amounts as a pure compound or as a component of a complex mixture.

 The role of carbon substrate involved in

~biosynthesis

~energy generation

 The carbon requirement under aerobic condition may be estimated from the cellular yield
coefficient (Y) which is defined as:

= Quantity of cell dry matter produced

Quantity of carbon substrate utilized

 Thus, for bacteria

Y of glucose = 0.5 = 0.5 g cells g-1 glucose

= the quantity of glucose needed to obtain 30 g dm-3

= 30/0.5
 = 60 g dm-3 glucose

thus, medium would also need to contain approximately

3.0 g dm-3N, 1.0 g dm-3P and 0.3 g dm-3S.

 An adequate supply of the carbon source is also essential for a product forming fermentation
process.

 In a critical study, analyses are made to determine how the observed conversion of the carbon
source to the product compares with the theoretical maximum yield.

 Cooney (1979) has calculated theoretical yield for penicillin G biosynthesis on the basis of
material and energy balances using a biosynthetic pathway based on reaction stoichiometry.

 The stoichiometry for the overall synthesis is:

 Solution of this equation yields:

 In this instance it was calculated that the theoretical yield was 1.1 g penicillin G g-1 glucose.

 The other major nutrient which will be required is oxygen which will be provided by aerating the
culture.

 The design of medium will influence the oxygen demand of a culture in that the more reduced
carbon sources will result in higher oxygen demand.

Water

 Major component of all fermentation media

  Factors need to be considered

 pH

 Dissolve salt

 Effluent contamination

 Mineral water content of water is very important in brewing, and most critical in the mashing
process, and historically influenced the siting of breweries and the type of beer produced.

 Nowadays, the water may be treated by deionization or other techniques and salts added, or pH
adjusted, to favour different beers so that breweries are not so depend on the local water
sources.

 In large continuous-culture SCP plants it is common practice to recycle the water streams.

 It may even be possible to reuse all the water in the fermenter fed with appropriate adjustment
of nutrient levels.

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 the carbon source such as carbohydrates, lipids and proteins.

 Some micro-organisms can use methane or methanol as carbon and energy sources.

CARBON SOURCES
Carbon requirement for the medium is normally provided by:

Barley grains

 It may be partially germinated and heat treated to give material known as malt.

 Malt:

i. Contains a variety of sugar besides starch

ii. Is the main substrate for brewing in many countries.

iii. Malt extract may also be prepared from malted grain.

Sucrose

• Obtained from sugar cane and sugar beet

 • Commonly used in fermentation media in very impure form as beet or cane molasses

Lactose and crude lactose(milk whey)

• Now is extremely limited in media formulation since the introduction of continuous-feeding

process.

• Whey is used as a substrate for biomass production

Vegetable oil

• Example: olive, maize, cotton seed, linseed, soya bean, etc.

• Source of carbon : oleic, linoleic and linolenic acid.

• As an antifoam in association with a surface active agent.

NITROGEN SOURCES
 Inorganic nitrogen may be supplied as ammonia gas, ammonium salts or nitrates.

 Ammonia has been used for pH control and as the major nitrogen source in a defined medium
for the commercial production of human serum albumin by Saccharomyces ceriuisiae

 Organic nitrogen may be supplied as amino acid, protein or urea. In many instances growth will
be faster with a supply of organic nitrogen, and a few microorganisms have an absolute
requirement for amino acids.

 In lysine production, methionine and threonine are obtained from soybean hydrolysate since it
would be too expensive to use the pure amino acids

 Other proteinaceous nitrogen compounds serving as sources of amino acids include corn-steep
liquor, soya meal, peanut meal, cotton-seed meal, Distillers' solubles, meal and yeast extract.

 The antibiotic production only begins to increase in the culture broth after most of the nitrogen
source has been consumed.

 In gibberellin production the nitrogen source has been shown to have an influence on directing
the production of different gibberellins and the relative proportions of each type

 Some of the complex nitrogenous material may not be utilized by a micro-organism and create
problems in downstream processing and effluent treatment.

CHELATORS
 The problem of insoluble metal phosphate(s) may eliminated by incorporating low
concentrations chelating agents such as ethylene diamine tetraa,ceticacid (EDTA), citric acid,
polyphosphates, etc., into medium.

 These chelating agents preferentially complexes with the metal ions in a medium. The ions then
may be gradually utilized by the organism.

 It is important to check that a chelating agent does not cause inhibition of growth the micro-
organism which is being cultured.

GROWTH FACTORS
Some micro-organisms cannot synthesize a full complement of cell components and therefore
require preformed compounds called growth factors. The growth factors most commonly required
are vitamins, but there may also be a need for specific amino acids, fatty acids.

Calcium pantothenate has been used in one medium formulation for vinegar production

In processes used for the production of glutamic acid, limited concentrations of biotin must be
present in the medium

Some components of a fermentation medium help to regulate the production of the product rather
than support the growth of the micro-organism. Such additives include precursors, inhibitors and
inducers, all of which may be used to manipulate the progress of the fermentation.

ANTIFOAMS
The most common cause of foaming is due to proteins in the medium, such as corn-steep liquor,
Pharmamedia, meal, soybean meal, yeast extract or meat extract

The foaming can cause removal of cells from the medium which will lead to autolysis and the
further release of microbial cell proteins will probably increase the stability of the foam.

These include reduction in the working volume of the fermenter due to oxygen exhausted gas
bubbles circulating in the system , changes in bubble size, lower mass and heat transfer rates, invalid
process data due to interference at sensing electrodes and incorrect monitoring and control

The biological problems include deposition of cells in upper parts of fermenter, problems of sterile
operation with the air filter exits of the fermenter becoming wet, and there is danger of microbial
infection

If excessive foaming is encountered there are three ways of approaching the problem:

1. To try and avoid foam formation by using a defined medium and a modification of some of the
physical parameters (pH, temperature, aeration and agitation). This assumes that the foam is
due to a component in the medium and not a metabolite.
 2. The foam is unavoidable and antifoam should be used. This is the more standard approach.

3. To use a mechanical foam breaker.

An ideal antifoam should have the following properties:

1. Should disperse readily and have fast action on an existing foam.

2. Should be active at low concentrations.

3. Should be long acting in preventing new foam formation.

4. Should not be metabolized by the microorganism.

5. Should be non-toxic to the micro-organism.

6. Should be non-toxic to humans and animals.

7. Should not cause any problems in the extraction and purification of the product.

8. Should not cause any handling hazards.

9. Should be cheap.

10. Should have no effect on oxygen transfer.

11. Should be heat sterilizable.

MEDIA OPTIMIZATION
 Medium optimization by the classical method changing one independent variable (nutrient,
antifoam, pH, temperature, etc.) while fixing all the others at certain level can be extremely time
consuming expensive for a large number of variables.

 Industrially the aim is to perform the minimum number of experiments to determine optimal
conditions.

 large number of experiments, xn where x is the number of levels and n is the number variables.

 This may be quite appropriate for three nutrients at two concentrations (23 trials) but not for six
nutrients at three concentrations. In this instance 36 (729) trials would be needed.

 When more than five independent variables are to be investigated, the Plackett-Burman design
may be used to find the most important variables in a system.

 This technique allows for the evaluation of X-I variables by X experiments. X must be a multiple
of 4, e.g. 8, 12, 16, 20, 24, etc.

 Any factors not assigned to a variable can be designated as a dummy variable.

  Alternatively, factors known to not have any effect may be included and designated as dummy
variables

 These can then be used in the design to obtain an estimate of error. Normally three dummy
variables will provide an adequate estimate of the error.

The effects of the dummy variables are calculated in the same way as the effects of the experimental
variables. If there are no interactions and no errors in measuring the response, the effect shown by a
dummy variable should be 0.

This procedure will identify the important variables and allow them to be ranked in order of importance
to decide which to investigate in a more detailed study to determine the optimum values to use.

1. Determine the difference between the average of the H (high) and L (low) responses for each
independent and dummy variable.

Therefore the difference = ∑A (H) - ∑A (L).

Thus the effect of

This value should be near zero for the dummy variables.

2. Estimate the mean square of each variable (the variance of

effect).
 For A the mean square will be =

. The experimental error can be calculated by averaging the mean squares of the dummy effects of E
and G.

Thus, the mean square for error =

The final stage is to identify the factors which are showing large effects.

In the example this was done using an F-test for

RESPONSE SURFACE METHODOLOGY

 To determine the optimum level of each key independent variable

 This may be done using response optimization techniques which were introduced by Box and
Wilson

 Response surfaces are similar to contour plots or topographical maps.

 The axes of the contour plot are the experimental variables and the area within the axes is
termed the response surface.

 Points giving the same results (equal responses) are then joined together to make a contour
line.

 It is important to appreciate that both variables are changed in the experimental series, rather
than one being maintained constant, to ensure that the data are distributed over the response
surface.

Hendrix proposed the following strategy to arrive at the optimum in an incremental fashion:

1. Define the space on the plot to be explored.

2. Run five random experiments in this space.

3. Define a new space centred upon the best of the five experiments and make the new space smaller
than the previous one, perhaps by cutting each dimension by one half.

4. Run five more random experiments in this new space.

5. Continue doing this until no further improvement is observed, or until you cannot afford any more
experiments!

The more sophisticated applications of the response surface technique use mathematical models to
analyse the first round of experimental data and to predict the relationship between the response and
the variables.

Several computer software packages are now available which allow the operator to determine the
equations underlying the responses and, thus, to determine the likely area on the surface in which the
optimum resides.

McDaniel et al. (1976), Fig. 4.3. The variables under investigation were cerelose and soybean level, with
the analysis indicating the optimum to be 6.2% cerelose and 3.2% soybean.

Saval et al. (1993). The medium for streptomycin production was optimized for four components
resulting in a 52% increase in streptomycin yield, a 10% increase in mycelial dry weight and a 48%
increase in specific growth rate

STERILIZATION
BATCH STERILIZATION
Liquid medium is most commonly sterilised in batch in the vessel where it will be used. The liquid is
heated to sterilisation temperature by introducing steam into the coils or jacket of the vessel;
alternatively, steam is bubbled directly into the medium, or the vessel is heated electrically.
Depending on the rate of heat transfer from the steam or electrical element, raising the
temperature of the medium in large fermenters can take a significant period of time.

Once the holding or sterilisation temperature is reached, the temperature is held constant for a
period of time thd.

Cooling water in the coils or jacket of the fermenter is then used to reduce the medium
temperature to the required value.

Cell death occurs at all times during batch sterilisation, including the heating-up and cooling-down
periods.

The holding time thd can be minimised by taking into account cell destruction during these periods.