Industrial Biotechnology- Definitions
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Industrial Biotechnology refers to the use of microorganisms or biological
substances such as enzymes to perform industrial or manufacturing
processes.
Industrial microbiology or Microbial biotechnology encompasses the
use of microorganisms in the manufacture of food or industrial products.
Fermentations and fermentation technology.
The term Fermentation is derived from the Latin verb ‘Fervere’, ‘to boil’,
describing the appearance of the action of Yeast on extracts of fruits or
malted grain, due to production of CO2 caused by anaerobic catabolism.
Biochemical definition
“ Generation of Energy by catabolism of organic compounds”
Industrial definition
Chemical changes or decompositions produced in organic substrates
through the activity of microorganisms.
Any process mediated by or involving microorganisms in which a product of
economic value is obtained is called fermentation (Casida, Jr., 1968).
Other definitions of fermentation
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A type of energy-converting metabolism in which the substrate is
metabolized without the involvement of an exogenous (external) oxidizing
agent. Typically, but not necessarily occurs anaerobically in the absence of
oxygen. Products are neither more nor less oxidized than the substrate.
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A process in which chemical changes are brought about in organic
substrates through the activity of microorganisms.
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Any chemical process mediated by microorganisms , which may be aerobic
or anaerobic.
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Carbohydrates are often essential materials for fermentation but organic
acids, amino acids, proteins, fats, sterols, alcohols, esters and organic
compounds are also fermentable.
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An anaerobic cellular process in which organic materials are converted into
simpler compounds, and chemical energy (ATP) is produced.
Fermentation products
• Fermentation products command large industrial
markets and are assured of market growth because
most can not be produced economically by other
chemical processes.
• Note: The development of modern industrial
fermentations is rooted in traditional fermentations,
with applications in the production of fermented foods
and beverages such as beers and wines, fermented
dairy products (yoghurt and cheese), fermented meats
and vegetables.
• More recently antibiotics, industrial ethanol, organic
acids, vitamins, and enzymes such as amylases,
proteases, cellulases and lipases have been
produced through fermentation processes.
Types of fermentation
Traditional
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Wine, beer, vinegar, bread, cheese, yoghurt.
Industrial (20th century).
There are 5 major groups of commercially important
fermentations:
• Microbial cells or biomass as the product, e.g. single cell
protein, baker’s yeast, lactobacillus sp. for starter cultures.
• Microbial enzymes; catalase, amylase, protease, pectinase,
glucose isomerase, cellulase, hemicellulase, lipase, lactase.
• Microbial metabolites:
- Primary metabolites; fuel/ industrial ethanol, citric acid, acetic
acid, glutamic acid, lysine, vitamins, polysaccharides.
- Secondary metabolites- antibiotics eg. penicillin
• Recombinant products (genetically engineered) - e.g insulin.
• Biotransformations e.g. steroid biotransformation.
Advantages of fermentations
• Complex molecules such as proteins can not be
produced by chemical means.
• Bioconversions give higher yields.
• Biological systems operate at lower temperatures and
near neutral pH.
• Can achieve exclusive production of an isomeric
compound.
Disadvantages of fermentations
• Can be easily contaminated with foreign unwanted
microorganisms (Minimize by aseptic operation of
fermenter).
• The desired product will usually be present in a complex
product mixture requiring separation.
• Slow when compared to chemical processes.
Essential features of a fermentation unit
• Fermenter.
• Culture collection laboratory- provides suitable
inocula for initiation of required microbiological
processes and makes routine sterility checks on
fermentation broths.
• Control lab - Monitors each fermentation process with
assays for reducing sugars, total hydrolysed sugars,
and available ammoniacal nitrogen. Also carries out
routine determinations of pH and mycelial weight to
provide a biochemical picture of the progress of each
batch. Fermentation and extraction yields are
determined by chemical and biological analyses of
samples taken at various points.
Fermentation unit (essential features cont’d)
Services
• Clean water for fermentation media.
• Cooling water for temperature control- treated to remove
hardness.
• Sterile compressed air for aeration.
• Boiler house to supply sufficient high pressure steam for
sterilization of ingredients.
• Adequate supply of electricity to run stirrer motors and
air compressors.
• Acceptable system of waste disposal.
Fermentation unit (essential features cont’d)
• Ingredients store: bulk storage for inflammable
solvents, sugars, cornsteep liqour, cornmeal and ionexchange resins.
• Investigation laboratory: conducts microbiological and
biochemical studies of the fermentation and extraction
process to increase the yield and efficiency of extraction.
• Extraction area.
Fermenter design
Fermenters for aerobic respiration
• Typically a closed vessel that can be sterilized, aerated, stirred,
and have the temperature of its contents regulated with a high
degree of accuracy.
• The shape is usually cylindrical, with a rounded base and
smooth interior to facilitate cleaning.
• A stirrer shaft runs through the center of the fermenter.
• Has impellers (protrusions from shaft) which are dependent on
fermenter height and intensity of agitation required.
• A cooling system required to remove heat generated by stirring
and metabolic heat generated by microbial cultures. The
optimum temperatures for penicillin and streptomycin
fermentations are 25oC and 28oC, respectively.
• Has a non-return air valve to the sparger at the bottom of the
vessel.
• A bacteriological filter in the exhaust pipe to the atmosphere
minimizes dissemination of microorganisms present in spent air.
• Addition of anti-foam prevents loss of broth as foam.
Fermenter design - Mixing of substrates
• Transfer of energy, substrates, and metabolites must be
brought about by a suitable mixing device. The efficiency
of the transport of any one substrate may be crucial to the
efficiency of fermentation.
• Microbial fermentation is a three phase system with liquidsolid, gas-solid, and gas-liquid interfaces.
• The liquid phase contains dissolved salts, substrates and
metabolites.
• The solid phase consists of individual cells, pellets,
insoluble substrates or precipitates of metabolic products.
• The gaseous phase provides a reservoir for oxygen
supply, CO2 removal, or for adjustment of pH with gaseous
ammonia.
Fermenter design- stirrers
• Stirring brings about dispersion of air in nutrient solution,
homogenization to equalize temperature and concentration of
nutrients throughout the fermenter, suspension of
microorganisms and solid nutrients, and dispersion of
immiscible liquids.
• Gas is distributed through pumps and by stirring. Air enters
fermentation liqour by an air-sparger at the bottom of the
fermenter, beneath the lowest impeller.
• The disc stirrer is the most widely used, where 4-8 radial
blades/ impellers project out from the edge of the disc.
Blades may be curved in turbine stirrers.
• Baffles transfer turbulence to fermenter walls, with four baffles
commonly installed in each fermenter.
• Foam separators- Foaming is frequently a problem in large
scale aerated systems. Anti-foam chemical agents cannot
always be added as they may have inhibitory effects on
fermentation. Foam can be broken down by mechanical
means e.g. rakes mounted on stirrers or by centrifugal force.
Construction materials/ anaerobic fermenters
Construction materials
• Select materials that can withstand repeated steam
sterilization.
• Stainless steel is mainly used for industrial and pilot
scale fermenters.
Anaerobic fermenters
Fermenters designed for anaerobic fermentations are
similar to aerobic fermenters but features for
aeration are unnecessary. The intensity of agitation
is sufficient only for mixing and maintenance of
temperature.
Process control
Defined environmental conditions for biomass and
product formation are critical for the success of
fermentations. Temperature, pH, oxygen concentration
etc. should be maintained by monitoring and correction
through control systems.
• Rapid changes in pH are minimized or controlled by
choice of carbon and nitrogen source, incorporation of
buffers, and addition of appropriate quantities of
ammonia, NaOH or acid.
• Foam levels are monitored with a stainless steel probe
inserted through the top plate of the fermenter. When
foam rises and reaches the level of the probe, a current
passes through the circuit with foam acting as an
electrolyte , and a signal is given off.
Temperature control
Temperature control
• Heat produced by microbial activity and mechanical
agitation may be removed from the system by chilled
water in external jackets.
• Extra heat may be provided by use of internal heating
coils or heating jackets. There may be overheating at
point source with the use of heating coils but efficiency
of heat transfer by use of heating jackets may be limited
in large vessels due to the surface area/ volume ratio.
Process parameters measured in fermentation
processes.
Physical
Chemical
Biological
Turbidity
Viscosity
Temperature
Weight of fermenter
Pressure
Agitator shaft power
Foam
Flow rate
Power consumption
pH
Redox potential
Exit gas analysis (O2, CO2)
Dissolved O2
Medium analysis
Substrate product concentration.
Active product
Enzyme activity
Protein content.
Scale-up
Scale-up is the operation of a fermentation process at
higher production levels. An efficient process at labscale may perform poorly when attempted on a large
scale as the fermentation conditions designed at labscale may not be applicable at industrial scale. It is
therefore important to evaluate scaled-up processes for
maximal yield, and minimal operating time and cost.
Aseptic operation
Aseptic operation is the protection of microbial media, cultures,
and equipment against contamination and subsequent
production of undesired metabolites . This may be achieved
through :
• Foam control
• Sterilization of fermenters, air supply, and nutrient media.
• Aseptic addition of inoculum, nutrients and supplements.
• Aseptic sampling- Sample tubes are closed by external valves
connected to steam lines for sterilization of the valve area
between samplings.
Sterile transfer between fermenters.
• Inoculum or substrate in a small fermenter may be transferred
to the main fermentation vessel by air pressure through
sterilized transfer lines. Transfer should be as rapid as
possible to prevent excessive aeration.
Maintenance of sterility in fermentations
• Minimum number of openings in fermenter.
• Small openings should be made leak- proof with O-rings
and larger openings with flat gaskets.
• Injection ports should be covered with steam –
sterilizable closures
• No direct connection between non-sterile and sterile
areas.
• Sterile pipes should be slanted to collect condensate and
to drain it.
Batch culture
• Batch culture is a closed system. Sterilized nutrient solution in the
fermenter is inoculated and fermentation is allowed to proceed
without the addition of new nutrient. The composition of the culture
medium, biomass concentration and metabolite concentration
change constantly as a result of cell metabolism.
• The four typical phases of growth in batch culture are lag, log,
stationary and the death phase.
• Biomass and primary metabolite production occur at the growth (log)
phase, and secondary metabolite production occurs during the
stationary phase i.e. conditions of substrate limitation.
• Batch culture may be used for biomass, primary and secondary
metabolite production.
• Examples of batch culture fermentations are- antibiotic production,
brewing, wine production, and dairy (lactic) fermentations.
Advantages of batch culture
Advantages of batch culture are:
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Production is intermittent and based on quantities
that are required.
Secondary metabolites are produced during the
stationary phase and therefore in batch processes
only.
Instability of strains requires regular renewal - in
batch culture fresh inoculum is used for each
production batch.
Continuous processes present technical difficulties ,
are labour intensive, and require constant attention.
Batch processes are less costly and are simple to
operate.
Growth phases in Batch culture
Lag phase
Phase of cell enlargement and adjustment to culture conditions. Duration varies
with culture conditions and with species, but inoculation with young actively
growing cells at optimal conditions minimises lag.
Exponential (log) phase.
• Cell division is most rapid at this phase.
• Examples of minimal generation times are 13 -17 minutes for E. coli and 6-18h
for M. tuberculosis.
Stationary phase
• There is a balance between cell division and cell death and the net population
is constant.
• Growth is limited by exhaustion of nutrient supply and the generation of toxic
wastes.
• For a species, a more or less uniform maximum population per unit volume is
usually attained e.g. max 1010/ml for E. coli.
Death phase
Decline in cell numbers as rate of cell death exceeds cell division due to
accumulation of waste and depletion of nutrients.
Fed-batch culture
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In a fed-batch culture, the initial culture is fed sequentially or continuously
with the same medium used to establish the culture, without removal of
culture fluid, resulting in an increase in volume.
A solution of the limiting substrate at the same concentration as that in the
initial medium may be added, resulting in a significant increase in volume.
A concentrated solution of the limiting substrate may be added, resulting in
a minimal increase in volume.
A very concentrated solution of the limiting substrate may be added
resulting in an insignificant increase in volume.
In fed-batch culture, the concentration of the limiting substrate can be
maintained at a low level, avoiding repressive effects of high substrate
concentration.
Fed batch culture is used in the production of bakers’ yeast as an excess of
malt leads to a high growth rate that results in oxygen depletion. Also used
in penicillin production in the slow growth phase (production phase) by a
controlled glucose feed
Continuous culture
• Microbial populations can be maintained in a state
of exponential growth by continuous culture, which
is an open system where fresh medium is
continuously supplied from a reservoir, and an
equal amount (excess medium) is removed
continuously from the system through a siphon.
• Ensure dilution rate is equal to the growth rate so
that the cell loss as a result of outflow is balanced
by growth so that there is no change in bacterial
mass (concentration) and the system is in a
steady state. If the dilution rate exceeds the
growth rate, there is progressive dilution of culture,
resulting in a washout or loss of culture.
Advantages/ disadvantages of
continuous culture
• Continuous fermentation processes have been
developed for the production of single cell protein,
starter cultures, organic solvents and ethanol.
• The advantages of continuous fermentation processes
are potential high yields of biomass and primary
metabolites as growth rates are maintained at optimal
levels in the log phase and production is continuous,
ensuring high production volumes.
• Disadvantages are the complexity of the system, high
cost, inflexible production schedule and the difficulty in
maintaining sterile conditions over a long period of
time. Mutant strains may also arise and overgrow
production strains.
Continuous culture in
chemostats/turbidostats
Chemostats
• The chemostat consists of a culture vessel and a reservoir
that supplies nutrient medium at a constant rate.
• Growth is controlled and maintained by monitoring the pH or
carbon dioxide concentration of the medium or by monitoring
the concentration of the limiting nutrient .
Turbidostats
• In turbidostats continuous culture is based on keeping the
turbidity and bacterial concentration constant.
• Nutrient flow is controlled by a turbidity probe switch
mechanism.
Calculation of media flow rate
The dilution rate (D) = F/ V
Where:
D is the volume change/ hour.
F = flow rate of medium into the fermenter, given as litres per hour.
V = volume of culture medium in litres
and specific growth rate is the number of generations per hour.
N.B. If the dilution rate exceeds growth rate, the culture becomes
progressively diluted to extinction and is said to have undergone a
washout.
Example
1. If the flow rate F is 30 ml/hr and V is 100 ml,
the dilution rate is (30/100)/h = 0.3/ h.
2. A vessel of 1000mL has a flow rate though it of 500ml/hr.
The dilution rate is 500/1000 = 0.5/h.
Problem 1
• If the specific growth rate of a culture in a
continuous fermentation is 0.8 generations
per hour and the vessel has a capacity of
500 L what is the maximum flow rate if
culture washout is to be avoided?
Industrial microrganisms : Selection
criteria
Selection of the culture to be used is a compromise between
productivity of the organism and the economic constraints of
the process. Criteria include
i.
Nutritional characteristics of the organism (The process
must be carried out using a low cost medium).
ii. The optimum temperature of the organism.
iii. Reaction of the organism with the equipment to be
employed.
iv. Stability of the organism and its amenability to genetic
manipulation.
v. Productivity and yield per unit time.
vi. Ease of product recovery from culture.
vii. Toxicity of the organism and product.
Isolation of Industrial
microorganisms
• The first stage in screening for microorganisms of potential industrial
application is their isolation, which involves obtaining either pure or
mixed cultures, followed by their assessment for ability to carry out
the desired reaction or produce the required product.
• Ideal isolation procedures start with an environmental source,
frequently soil, which is likely to be rich in the desired
microorganisms, and are designed to favour the growth of those
organisms possessing the industrially important characteristic.
• The desired characteristic is used as a selective factor and a simple
test is applied to distinguish the most desirable types.
Selective pressure may be used in the isolation of organisms which
will grow on particular substrates or under conditions that are
adverse to other types.
• Alternatively, select for a target species or taxon known to have
the desirable characteristic e.g. Antibiotic production by
Streptomycetes.
Preservation of industrial cultures
• As isolation is lengthy and expensive, it is essential that the
organism retains the desirable characteristics that led to its
selection, and should be free from contamination.
• Cultures should therefore be stored in a way which eliminates
genetic change, protects against contamination and retains
viability.
Storage on agar slopes
• May be stored refrigerated at 5oC or frozen at -20oC and subcultured at weekly or six-monthly intervals. Note: Subculturing may result in strain degeneration through mutation
and contamination.
Broth cultures
• May be stored as as glycerol/broth stocks at -30oC.
Storage in dehydrated form
Freeze-drying/ Lyophilisation
• Involves the freezing of a culture, followed by drying
under vacuum, which results in the sublimation of
water from cells.
• The culture is grown to the stationary phase and resuspended in a protective medium e.g. Serum , milk
or sodium glutamate.
• Cell suspensions are transferred to ampoules, frozen,
and subjected to a vacuum at slightly raised
temperatures until sublimation is complete.
• The ampoules are finally sealed, and the freeze-dried
culture stored at refrigeration temperatures with
minimal loss of viability for up to ten years.
• Improvement of industrial cultures
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Natural isolates usually produce commercially important products in low
concentrations, and as a mixture with closely related compounds.
Consequently, attempts are made to increase the productivity of the
selected organism through induced mutations and genetic recombination
(e.g. penicillin yield increased from 20 to 8000 units/ml between 1943 and
1955’ and is currently above 85 000 units/ml or 50g/L).
Recombinant DNA technology may result in organisms producing
compounds which they were not able to produce previously or improve
significantly the production of conventional fermentation products.
Directed mutation is relevant when genes to be modified are known and a
site targeted through in-vitro enzymatic cleavage and manipulation. A
knowledge of the biosynthetic route and control mechanism also enables
the prediction of a blue-print of the desirable mutant.
• Mutagenesis through radiation.
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Non-ionizing (Ultraviolet) radiation.
Short wavelength ultraviolet radiation is an effective mutagenic agent at
wavelengths between 200 – 300 nm, and an optimum of 254 nm, the
absorption maximum of DNA.
Long wavelength UV radiation at 300- 400 nm has less lethal and
mutagenic effects than short wavelength UV. However, LWUV may be
effective if carried out in the presence of DNA intercalating dyes e.g. 8 Methoxypsoralen.
Survivors of UV treatment may be twice as productive as parent strains.
In mutagenesis through UV irradiation, a strain is exposed to the mutagen,
and mutants with required characteristics are selected.
Important products of UV action are dimers, (T-T, T-C, C-C), formed
between adjacent pyrimidines or complementary strands, which results in
cross-links and induces transitions of GC-AT, transversions, frame-shift
mutations and deletions.
• Ionizing radiation
• Ionizing (high- energy) radiation includes X-rays, γ
and β rays, and is used when cell material is
impenetrable to UV rays.
• Single and double strand breaks occur with a
significantly higher probability than non-ionizing
radiation.
• Double-strand breaks result in major structural
changes e.g. translocation and inversion and
consequently the resultant mutants are often nonviable and do not survive the irradiation process.
• Mutagenesis with chemical agents
• Mutagenic chemical agents are generally classified into three
groups: mutagens which affect non-replicating DNA; base analogs;
and frameshift mutagens.
Mutagens affecting non-replicating DNA
A number of chemicals cause direct damage to non-replicating DNA
• Nitrous acid (HNO2) deaminates adenine to hypoxanthine and
cytosine to uracil, resulting in AT-GC transitions through the
changed pairing properties of the deamination products.
• Hydroxylamine (NH2OH) reacts with cytosine, and the derivative
from cytosine pairs with adenine, resulting in GC-AT transitions.
• Alkylating agents which include N-methyl-N-nitrosoguanidine (a
carcinogen), ethyl methanesulfonate, and mustard gas (Di-2clorethyl-sulfide) cause transitions, transversions, and deletions.
• Mutagenesis with chemical agents (cont’d)
Base analogs
Base analogs such as 5- bromouracil and 2- aminopurine are incorporated
into replicating DNA instead of the corresponding naturally occuring bases
thymine and adenine due to their structural similarity, resulting in AT-GC
transitions.
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Frameshift mutagens
Frameshift mutagens intercalate into the DNA molecule and cause errors
which result in the alterationof the reading frame, resulting in the formation
of faulty protein.
The most commonly used frameshift mutagens are the acridine dyes such
as acridine orange, proflavine and acriflavine. . which are inserted between
two neighbouring bases of DNA strands.
• Protoplast fusion
• Protoplasts (cells devoid of their cell walls) may be prepared by
subjecting cells to the action of wall degrading enzymes in isotonic
solutions.
• Cell fusion, followed by nuclear fusion, may occur between
protoplasts of strains that would otherwise not fuse, and the
resulting fused protoplast may regenerate a cell wall and grow as a
normal cell with characteristics of both parent cells.
• Protoplast fusion is used where a sexual reproductive phase is
absent in strains, and where conventional techniques have failed.
• Example – An asporulating, slow-growing , high antibiotic yield
Cephalosporium acremonium strain was crossed with a
sporulating fast- growing , low yield strain, resulting in good
sporulation, high growth rate and high antibiotic yields.
• Application of recombinant DNA techniques
• Targeted genetic material derived from one species may be
incorporated into another, where it may be expressed.
Requirements for transfer and expression of foreign DNA are:
i.
A vector DNA molecule (plasmid or phage), capable of entering
the host cell and replicating within it.
ii.
A method of splicing foreign genetic information into the vector.
iii. A method of introducing the vector and foreign DNA recombinants
into the host cell and selecting for their presence.
iv. A method of assaying for the required foreign gene product from
the population of created recombinants.
• Modification of strain properties other than yield
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Important characteristics which may be modified
include:
Strain stability i.e. avoidance of reverse mutants, and
maintenance of high yield.
Resistance to phage infection.
Tolerance to low oxygen tension.
Tolerance of high medium components such as high
phosphate levels.
Low foam production.
Favourable morphology for aeration and filtration.
Low production of undesirable product (e.g. elimination of
the yellow pigment chrysogenein in penicillin producing
strains).
• Media for industrial fermentations
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– Microbial cultures require growth factors e.g. vitamins,
specific amino acids and fatty acids which are provided by
fermentation media.
– Some media components or chemicals added as
supplements to media are directly incorporated into the
fermentation product and are known as precursors. Corn
steep liquor contains phenylethylamine which is
incorporated into the penicillin molecule to yield benzylpenicillin (Penicillin G)
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• Criteria to be met by media for industrial fermentations
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• Media should
– Produce the maximum concentration and yield of the product
or biomass.
– Allow the maximum rate of product formation.
– Produce a minimum yield of undesired product.
– Be of consistent quality and be readily available throughout the
year.
– Cause minimal problems during media making, sterilization,
aeration, agitation, extraction, purification and waste treatment.
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• Defined and undefined media
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• Defined media
– Pure defined chemicals of known chemical composition may be used for labscale fermentations and some industrial fermentations e.g. production of
vaccines for human use. Although composition is controlled, defined media
are expensive and not commonly used for industrial fermentations.
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Undefined media
– Low- cost complex undefined substrates and by-products of other industries
are mostly used for industrial scale fermentations.
– Cane and beet molasses, cereal grains, starch, glucose, sucrose, and lactose
are used as carbon sources.
– Urea, corn-steep liqour, soya-bean meal , nitrates and ammonium salts may be
used as nitrogen sources.
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• Advantages/ disadvantages of undefined media.
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• Advantages
– Low cost!
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• Disadvantages
– Undefined media have a variable concentration of components which may
vary with season and among different batches resulting in unpredictable
biomass and product yields.
– Impurities in natural materials may interfere with fermentations.
– Product recovery and effluent treatment may be problematic for undefined
media because not all components will be consumed by the organism. Some
residual components interfere with recovery and also contribute to the high
biological oxygen demand/ organic content of the effluent.
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• Medium formulation
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– Media should satisfy elemental requirements for cell biomass and
metabolic products, and there must be an adequate supply of energy
for biosynthesis and cell maintenance.
– Identified growth factors e.g. amino acids, vitamins, and nucleotides
which can not be synthesized by the culture microorganism should be
incorporated into media in adequate amounts.
• Role and choice of carbon substrates
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– Dual role of carbon substrate
– Biosynthesis
– Energy generation
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• Factors influencing choice of carbon source
– The rate at which the carbon source is
metabolised as this influences biomass formation
and production of primary or secondary metabolites.
– Price and availability.
– Purity of carbon source.
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• Carbon sources: Sucrose/ molasses/ glucose
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• Sucrose/ molasses
– Sucrose may be supplied in the pure form or as crude sugar molasses with
33.4% sucrose.
– Cane or beet molasses are residues left after crystallization of sugar solutions
in refining.
– Molasses are used in high-volume low-value products e.g. ethanol and singlecell protein and are also used in high-value products e.g. antibiotics.
– Use of crude molasses is more cost-competitive in comparison with pure
carbohydrates, but impurities will necessitate more expensive and
complicated extraction and purification.
• Grapes
• Grape juice/ must is used as a medium for wine production and contains
17% sugar (glucose, fructose) and 0.3% ash.
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• Carbon sources: Starch/ lactose
Starch based media
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Sources of starch include soya bean meal (35% carbohydrate), groundnut
meal, oat flour, rye flour, maize, wheat, sorghum , barley, potatoes and
cassava.
• Barley
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– Raw material for lager beer manufacture.
– Has a high carbon content but only 1.5% nitrogen.
– Barley is allowed to germinate under controlled conditions for partial
digestion of starch by the enzyme amylase in the malting process.
– The malted barley is mashed by warming with water and is sterilized by boiling
to give wort, the medium to be fermented by yeast to beer.
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• Sorghum
• Raw material for traditional beer in Southern
Africa.
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Lactose
• Whey powder is used as a lactose source
which is slowly metabolized for the
production of secondary metabolites.
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• Malt extract
• Sulfite waste liqours
• Carbon sources - Oils and Fats
• Vegetable oils e.g. Olive, maize, cotton seed, and soyabean
oil may be used as carbon sources, and also for their
content of the fatty acids oleic and linoleic acids which are
carriers for antifoams in antibiotic processes.
• Nitrogen sources
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– Most industrial microorganisms can utilize inorganic and organic
sources of nitrogen.
– Inorganic nitrogen is supplied as ammonia gas, ammonium salts
or nitrates.
– Ammonia is also used for pH control and is a major nitrogen
source in a defined medium for the production of human serum
albumin by Saccharomyces cerevisae.
– Organic nitrogen sources include urea.
– Complex undefined organic nitrogen sources include corn steep
liquor, soya meal, and cotton seed meal.
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• Factors influencing choice of nitrogen source
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– Ammonia or ammonium ion may be used for
biomass production.
– Antibiotic production is inhibited by a rapidly used
source e.g. ammonium, nitrate or amino acids.
Production of secondary metabolites begins after
depletion of the nitrogen source.
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• Best nitrogen sources for some secondary
metabolites
• Penicillin Corn steep liquor
• Bacitracin Peanut granules
• Riboflavin Pancreatic digest of gelatine
• Novobiocin Distillers’ solubles
• Rifomycin Pharmamedia
• Gibberelins Ammonium salt
• Polyene antibiotics Soybean meal
• Nitrogen sources: Corn Steep Liqour
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– CSL a by-product of starch and sugar production from maize.
– Sugars are extracted from maize by steeping in dilute aqueous
sulphur dioxide which establishes an acid pH and prevents
putrefaction by controlling the bacterial population.
– A well balanced source of carbon, nitrogen, sulphur and mineral
salts. Contains lactic acid ,some reducing sugars, and complex
polysaccharides.
– Phenyl ethylamine which is present in CSL is a precursor in
penicillin-G production.
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• Corn Steep Liquor cont’d
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– The corn sugar extraction process dissolves nitrogen rich substances and
minerals, and a natural fermentation by thermophilic Lactobacillus spp.
produces lactic acid.
– Extracted corn is drained and filtered before the filtrate is concentrated by
heat to 50% solids.
– CSL contains 4% w/v nitrogen. 25% of the nitrogen is found as alanine,
arginine and glutamic acid. Other amino acids include leucine, methionine and
cysteine.
– Riboflavin, niacin, biotin, and pyridoxine are also significant components, and
calcium, phosphorus and potassium are present at 1, 2.5 and 1.5%,
respectively.
– The acidic nature of corn steep liqour requires inclusion of calcium carbonate
(1% w/v) to provide a suitable pH for microbial growth.
•
•
• Nitrogen sources: Soya bean meal / Pharmamedia
•
• Soya bean meal
– Harvested soya bean seeds contain up to 40% protein,
18.5 - 22% oil, 35% carbohydrate, and 5% ash. Ash contains
potassium, phosphorus, sulphur, magnesium and iron.
– After heating ,flaking , and oil extraction, the residue soya
bean meal contains 8% nitrogen in complex form and is
used as a nitrogen source in industrial fermentations.
•
• Pharmamedia
– Pharmamedia is a finely ground powder made
from the embryo of cotton seed and contains 56%
protein, 24% carbohydrate, 5% oil and 5% ash.
– Used for the production of tetracycline.
•
•
• Nitrogen sources - Distillers’ solubles
•
• Distiller’s solubles
• The residue after distillation of alcohol from fermented
grain contains 6 – 8% solids, and is rich in protein and
the vitamin B complex. Suspended solids are removed
by screening and the effluent is concentrated to 35%
solids to give evaporator syrup and drum-dried to give
distillers solubles, which are a rich source of nitrogen
and accessory factors.
•
•
• Minerals
•
– Magnesium, phosphorus, potassium, sulphur, calcium and
chlorine are essential elements and may be added at
required concentrations as distinct components.
– Cobalt, copper, iron, manganese, molybdenum and zinc
essential but usually present as impurities in major
ingredients.
– Inorganic phosphate concentration influences production
of bacitracins, citric acid, and oxytetracycline. Monomycins
(antibiotics) are produced by Streptomyces at 0.1 mM
phosphate levels.
•
Penicillin G production process
•
• Penicillin G is produced using submerged processes
in 40 000 – 200 000 L fermenters. Larger tanks not
used due to resultant inadequate oxidation.
• Penicillin fermentation an aerobic process, with an
oxygen absorption rate of 0.4 – 0.8 mM/L per min,
requiring an aeration rate 0.5 – 1.0 vvm ( Air vol/ liquid
vol/ min)
• Optimal temperature is 25-27oC.
• Inoculum is propagated from lyophilized spores of
Penicillium chrysogenum (initially P. notatum) in seed
fermenters
• To begin the fermentation process, a number of spores
are introduced into a small (normally 250-500ml)
conical flask of corn steep liquor where it will be
incubated for several days.
• The culture is then transferred to a 1 or 2 litre benchtop reactor.
• Once this has been successful the process is scaledup again to a pilot-scale bioreactor. This reactor will be
similar in design to the bench-top reactor except it will
have a size of about 100-1000 litres. After about 24-28
hours, the material in the seed tanks is transferred to
the primary fermentation tanks.
Penicillin production cont’d
• A spore concentration of ~ 5x103/ml and pellet
formation is crucial for satisfactory subsequent
yield. The recommended inoculation rate is 10%
v/v.
• For optimal penicillin formation rates, pellets must
grow in a loose form and not as compact balls.
• Growth phase is typically 40- 60 hours in duration,
with a generation time of six hours. There is initial
rapid proliferation in the growth phase and
ammonia is released
• Oxygen supply is critical as increasing viscosity
hinders oxygen transfer.
Penicillin production cont’d
• After growth phase, penicillin production phase
commences. Growth rate reduced to 0.01 gen/hr.
• Production phase may be extended to 120 – 160
hours by feeding with glucose.
• Medium for penicillin fermentation by fed-batch
culture consists of corn steep liquor of 4-5% dry
weight, which may be replaced by Pharmamedia.
Additional nitrogen sources such as soy meal or
yeast extract, a carbon source (lactose) and
buffers are also added to supplement CSL. Corn
steep liquor contains phenylacetic acid, a
precursor of penicillin G.
Penicillin production cont’d
• Typical penicillin production medium composition is; lactose
3.5%, glucose 1%, cornsteep liquour solids 3.5%, calcium
carbonate buffer 1%, potassium dihydrogen phosphate buffer
0.4%, oils 0.25%,
pH 5.5 – 6.0.
• Slow glucose feeding (10%) of total volume in the production
phase increases yield by up to 25%. The pH is kept constant
at 6.5 and phenylacetic acid is fed continuously as a
precursor at 0.5 – 0.8% concentration.
• Penicillin is excreted into medium, with less than 1% being
mycelium bound.
• Current yields of ~50g/L are due to culture selection and
improvement.
Penicillin recovery
• Product recovery accomplished by two-stage
continuous counter-current extraction of fermenter
broth with amyl or butyl acetate.
• Broth filtrate mixed with butyl acetate and acidified
with phosphoric acid so that the non-ionised
penicillin concentrates in the solvent layer.
• The butyl acetate phase is then mixed with
phosphate buffer at pH 7.0.
• Ionized sodium salt of penicillin concentrated in
aqueous phase for final isolation.
Flow-chart : Penicillin recovery and
partial purification
1.
2.
3.
4.
5.
Harvest broth from fermenter
Chill to 5- 10oC
Filter off P. chrysogenum mycelium using rotary vacuum filter.
Acidify filtrate to pH 2.0 – 2.5 with sulphuric or phosphoric acid.
Extract penicillin from aqueous filtrate into butyl acetate in a centrifugal
counter-current extractor . Treat and dispose of aqueous phase.
6. Extract penicillin from butyl acetate into aqueous buffer (pH 7.0) in a
centrifugal counter-current extractor. Recover and recycle butyl acetate.
7. Acidify the aqueous fraction to pH 2.0 – 2.5 with sulphuric acid and reextract penicillin into butyl acetate as in 5.
8. Add potassium acetate to the organic extract in a crystallization tank to
crystallize the penicillin as the potassium salt.
9. Recover crystals in a filter centrifuge. Recover and re-use butyl acetate.
10. Further processing of penicillin salt.
Penicillin G biosynthesis
•
•
•
•
•
•
Over 100 biosynthetic penicillins have been produced by adding side-chain
precursors, but commercially, only penicillin G and V have been produced.
Biosynthesis is inhibited by high phosphate concentration and shows
catabolite repression by glucose.
In penicillin G biosynthesis the -lactam thiazolidine ring is constructed
from L- cysteine and L- valine.
Biosynthesis of the penicillin molecule occurs in a non-ribosomal process
through a tri-peptide composed of L- cysteine, L-valine and L-
aminoadipic acid (AAA).
The first product of cyclization is Isopenicillin from which benzylpenicillin is
produced by exchange of L --AAA with activated phenylacetic acid.
Overall, there is a total of three main and important steps to the
biosynthesis of penicillin-G (benzylpenicillin)
Penicillin-G biosynthesis cont’d
• The first step in the biosynthesis of penicillin G is the
condensation of three amino acids L-α-aminoadipic
acid, L-cysteine, L-valine into a tripeptide.
• Before condensing into a tripeptide, the amino acid Lvaline will undergo epimerization and become Dvaline. After the condensation, the tripeptide is named
δ-(L-α-aminoadipyl)-L-cysteine-D-valine, which is also
known as ACV.
• While this reaction occurs, we must add in a required
catalytic enzyme ACVS, which is also known as δ-(Lα-aminoadipyl)-L-cysteine-D-valine synthetase. ACVS
is required for the activation of the three amino acids
before condensation and the epimerization of L-valine
to D-valine.
• The second step in the biosynthesis of
penicillin G is to use an enzyme to change
ACV into isopenicillin N. The enzyme is
isopenicillin N synthase. The tripeptide on
the ACV will then undergo oxidation, which
then allows a ring closure so that a bicyclic ring is formed.
Penicillin-G biosynthesis cont’d
The last step in the biosynthesis of
penicillin G is the exchange of the sidechain group so that isopenicillin N will
become penicillin G. Through the catalytic
coenzyme isopenicillin – Nacyltransferase (IAT), the alphaaminoadipyl side-chain of isopenicillin N is
removed and exchanged for a
phenylacetyl side-chain.
Penicillin G biosynthesis
Citric acid production
• 99% of citric acid output is produced microbially
and 60% is used in the food and beverage
industry as an acidulant and for flavouring fruit
juices, ice-cream, candy and marmalade, and as
sodium citrate in processed cheese. Some is used
in the pharmaceutical industry for iron citrate
production and as a blood anti-coagulant.
• Mutants of Aspergillus niger are used for
commercial production. Strains are also selected
for suppression of side products e.g. oxalic acid,
isocitric acid and gluconic acid.
Production medium
• Nutrient medium consists of a 15-25% sugar solution
(sucrose or cane molasses purified by cation
exchangers or calcium hexacyanoferrate) or potato
starch. Each batch of molasses should be given a
preliminary fermentation test.
• Conversion of carbohydrate to citric acid is dependent
on intracellular enzymes. Sugar from nutrient fluid
should be able to enter cells of the mycelium, and
citric acid produced must be able to diffuse out into the
medium.
• Copper, manganese, magnesium, iron, zinc and
molybdenum are necessary in concentrations optimal
for yields.
Citric acid- production medium
cont’d
• pH generally at 5.0 at the beginning of the
growth phase, but falls to 3.0 at the
production phase due to metabolism of
ammonium ions. Low pH reduces
incidence of microbial contamination and
discourages oxalic acid formation.
• 80% of citric acid is produced by
submerged processes and 20% by
surface processes.
Production of inoculum
• Spore suspension is used as inoculum after growth for 10 –
14 days in glass bottles on solid substrates at 25oC.
• Both numbers and viability of the spore crop are critical.
• For submerged fermenters, spores are induced to germinate
in a preliminary fermentation.
• Nutrient solution containing 15% sugar from molasses is
used in the seed fermenter and cyanide ions added to induce
pellet formation from mycelial growth.
• Spores germinate and form pellets 0.2 – 0.5 mm in diameter
within 24 hours at 32oC.
• Pellets are then used as inoculum for production fermenters.
The efficiency in production fermenters is dependent on the
manner off spore and pellet production.
Submerged processes
• Submerged processes have the advantage of lower total
investment by 25%, savings in space requirements, and lower
labour costs.
• Disadvantages include greater energy costs, and the more
sophisticated control technology and highly trained personnel
required.
• Stainless steel acid resistant fermenters are required to
withstand low pH and liners are required for protection in
some small- scale fermenters of <1000L.
• If the mycelium is loose and filamentous, with limited
branches and no chlamydospores, little citric acid is produced
in the production phase. Mycelium for optimal production
rates consists of small solid pellets. The ratio of iron to copper
in a medium determines the mycelial structure.
Citric acid submerged processes
cont’d
• An oxygen concentration of 20 – 25% of the saturation
value is required throughout the fermentation.
• A foam chamber 1/3 the size of fermenter volume is
required. Anti-foam agents e.g. lard oil are added at
frequent intervals in the batch fermentation.
• The pH reaches 1.5 after 10 days. The maximum
titratable acidity attained is directly related to the
amount of sugar added to the fermentation medium.
• Growth maximum is reached 3-4 days before
harvesting.
Surface processes employing solid
substrates
Surface processes employing solid substrates
may use either wheat bran or pulp from sweet
potato starch production.
• pH is reduced to 4-5 before sterilization, after
which material is inoculated with spores, spread
on trays in layers 3-5 cm thick and incubated at
28oC for 5 days.
• Citric acid is extracted from the substrate with
hot water.
Surface properties using liquid media
• Account for up to 20% of the supply of citric acid.
• Sucrose is supplied as molasses and inoculation
is at 30-40oC by blowing dry spores on the
surface or by spraying to 5x 107 spores/m2.
• Temperature in the 8-10 cm deep trays is kept
constant at 30oC.
• Mycelium floats as a white layer on the nutrient
solution and yields 1.2-1.5 kg citric acid/m2 after a
14 day fermentation period.
• The presence of excessive concentrations of iron
results in oxalic acid production and a yellow
colouration.
Biosynthesis
• Citric acid is a primary metabolic product
formed in the tricarboxylic acid cycle.
• When pyruvate is decarboxylated with the
formation of acetyl coenzyme A, acetate
residue is channeled to the TCA cycle.
• Pyruvate carboxylase is the key enzyme
for citric acid production.
Citric acid recovery
• Citric acid is produced as the calcium salt is
precipitated from the fermentation liquor and
from washes of the mycelium by treatment
between 70 – 90oC at neutral pH for 3-4
hours.
• Oxalic acid is precipitated as calcium oxalate
at low pH (5.8) leaving citric acid in solution
as monocalcium citrate.
• Rotating filters or centrifuges are used to
separate the mycelium and precipitated
calcium oxalate.
• At pH 7.2 and 70-90oC, citric acid is precipitated
and separated by rotating filters and dried.
• Further purification is by adding sulphuric acid to
dissolve citric acid and re-precipitating as calcium
sulfate. Subsequent recovery steps include
treatment (decolourisation) of crude citric acid with
activated carbon, and removal of soluble iron with
anion exchangers or ferrocyanide and final
crystallization.
• Above 40oC, crystallization is in the anhydrous
form, and below 36oC, in the monohydrate form.
• Summary of citric acid recovery process.
1. Filter off A. niger mycelium from harvested broth using
a rotary vacuum filter.
2. Add Ca(OH)2 to filtrate until neutral and filter calcium citrate
precipitate.
3. Add sulphuric acid at 60oC to calcium citrate to give a
calcium sulphate precipitate and release free citric acid.
4. Filter on rotary vacuum filter to recover CaSO4.
5. Decolourise citric acid with activated charcoal.
6. Isolate on cation and anion exchange resins,
7. Evaporate to crystallization at 36oC.
8. Separate crystals of monohydrate citric acid in continuous
centrifuges and dry at 50 – 60oC.
• Production of streptomycin
• Most media for production of streptomycin by Streptomyces
griseus provide glucose as the principal source of
carbohydrate as the strains are unable to use sucrose. Typical
medium is glucose (2.5%), soybean meal (4%), distillers’
solubles (0.5%),sodium chloride (0.25%).
• Nitrogen is continuously but slowly available from complex
organic sources.
• Optimum conditions are:
i. Adequate concentration of glucose (~2.5%).
ii. Low concentration of inorganic phosphate (≤ 0.006%).
iii. Continuous but limited concentration of available nitrogen.
iv. Good aeration.
v. Temperature 27-29oC.
• Three phases in production are:
i. Release of ammonia, pH rises from 6.7
– 7.6 in the first 20 hrs.
ii. Productive phase – decrease in pH to
6.7.
iii. Mycelium disintegrates, pH rises to
8.5.
• Industrial ethanol
• May be produced by fermentation of any
carbohydrate material containing a fermentable
sugar (mainly cane or beet molasses).
• Yeasts used as fermentation cultures are
Saccharomyces cerevisae for hexoses , Candida
utilis for lactose and pentoses and
Saccharomyces kluyveromyces for lactose.
• Ethanol fermentations are also used to reduce
the biological/ biochemical oxygen demand (BOD)
of industrial effluent.