SEMESTER IV INDUSTRIAL BIOPROCESSING

MODULE 1

1. A REVIEW OF INDUSTRIAL FERMENTATION AND ENZYMATIC PROCESSES

AND PRODUCTS

Industrial fermentation is the intentional use of fermentation by microorganisms such

as bacteria and fungi to make products useful to humans. Fermented products have applications
as food as well as in general industry. Some commodity chemicals, such as acetic acid, citric
acid, and ethanol are made by fermentation. The rate of fermentation depends on the
concentration of microorganisms, cells, cellular components, and enzymes as well as
temperature, pH and for aerobic fermentation oxygen. Product recovery frequently involves the
concentration of the dilute solution. Nearly all commercially produced enzymes, such as lipase,
invertase and rennet, are made by fermentation with genetically modified microbes. In some
cases, production of biomass itself is the objective, as in the case of baker's yeast and lactic acid
bacteria starter cultures for cheesemaking. In general, fermentations can be divided into four
types:

 Production of biomass (viable cellular material)

 Production of extracellular metabolites (chemical compounds)
 Production of intracellular components (enzymes and other proteins)
 Transformation of substrate (in which the transformed substrate is itself the product)

The organisms used may be bacteria, yeasts, molds, algae, animal cells, or plant cells.
Special considerations are required for the specific organisms used in the fermentation, such as
the dissolved oxygen level, nutrient levels, and temperature.

In most industrial fermentations, the organisms are submerged in a liquid medium and in
others fermentation takes place on the moist surface of the medium. To avoid biological process
contamination, the fermentation medium, air, and equipment are sterilized. Foam control can be
achieved by either mechanical foam destruction or chemical anti-foaming agents. Several other
factors must be measured and controlled such as pressure, temperature, agitator shaft power, and
viscosity. An important element for industrial fermentations is scale up. This is the conversion of
a laboratory procedure to an industrial process.

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In general, what works well at the laboratory scale may work poorly or not at all when
first attempted at large scale. It is generally not possible to take fermentation conditions that have
worked in the laboratory and blindly apply them to industrial-scale equipment. Similarly many
parameters have been tested for use as scale up criteria, there is no general formula because of
the variation in fermentation processes. The most important methods are the maintenance of
constant power consumption per unit of broth and the maintenance of constant volumetric
transfer rate.

Phases of microbial growth

When a particular organism is introduced into a selected growth medium, the medium is
inoculated with the actual organism. Growth of the inoculum does not occur immediately, but
takes a little while. This is the period of adaptation, called the lag phase. Following the lag phase,
the rate of growth of the organism steadily increases, for
a certain period—this period is the log or exponential
phase. After a certain time of exponential phase, the rate
of growth slows down, due to the continuously falling
concentrations of nutrients and/or continuously
increasing (accumulating) concentrations of toxic
substances. This phase, where the increase of the rate of
growth is checked, is the deceleration phase. After the deceleration phase, growth ceases and the
culture enters a stationary phase or a steady state. The biomass remains constant, except when
certain accumulated chemicals in the culture lyse the cells (chemolysis). Unless other micro-
organisms contaminate the culture, the chemical constitution remains unchanged. If all of the
nutrients in the medium are consumed, or if the concentration of toxins is too great, the cells may
become scenescent and begin to die off. The total amount of biomass may not decrease, but the
number of viable organisms will decrease.

Fermentation medium

The microbes used for fermentation grow in (or on) specially designed growth medium
which supplies the nutrients required by the organisms. Varieties of media exist, but invariably
contain a carbon source, a nitrogen source, water, salts, and micronutrients.
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Carbon sources are typically sugars or other carbohydrates, although in the case of
substrate transformations (such as the production of vinegar) the carbon source may be an
alcohol or something else altogether. For large scale fermentations inexpensive sources of
carbohydrates, such as molasses, corn steep liquor, sugar cane juice, or sugar beet juice are used
to minimize costs.

Fixed nitrogen sources are required for most organisms to synthesize proteins, nucleic
acids and other cellular components. Depending on the enzyme capabilities of the organism,
nitrogen may be provided as bulk protein, such as soy meal; as pre-digested polypeptides, such
as peptone or tryptone; or as ammonia or nitrate salts. Cost is also an important factor in the
choice of a nitrogen source.

Phosphorus is needed for production of phospholipids in cellular membranes and for the
production of nucleic acids. The amount of phosphate which must be added depends upon the
composition of the broth and the needs of the organism, as well as the objective of the
fermentation.

Growth factors and trace nutrients are included in the fermentation broth for organisms
incapable of producing all of the vitamins they require. Yeast extract is a common source of
micronutrients and vitamins for fermentation media. Inorganic nutrients, including trace
elements such as iron, zinc, copper, manganese, molybdenum and cobalt are typically present in
unrefined carbon and nitrogen sources. To prevent foam from occurring or accumulating,
antifoaming agents may be added. Mineral buffering salts, such as carbonates and phosphates,
may be used to stabilize pH near optimum. When metal ions are present in high concentrations,
use of a chelating agent may be necessary.

Production of biomass
Microbial cells or biomass is sometimes the intended product of fermentation. Examples
include single cell protein, bakers yeast, lactobacillus, E. coli, and others. In the case of single-
cell protein, an alga is grown in large open ponds which allow photosynthesis to occur.

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Production of extracellular metabolites

Microbial metabolites can be divided into two groups: those produced during the growth
phase of the organism, called primary metabolites and those produced during the stationary
phase, called secondary metabolites. Some examples of primary metabolites are ethanol, citric
acid, glutamic acid, lysine, vitamins and polysaccharides. Some examples of secondary
metabolites are penicillin, cyclosporin A, gibberellin, and lovastatin.[8]

Primary metabolites

Primary metabolites are compounds made during the ordinary metabolism of the
organism during the growth phase. A common example is ethanol or lactic acid, produced during
glycolysis. These compounds are produced during the normal function of the cell and released
into the environment. There is therefore no need to rupture the cells for product recovery.

Secondary metabolites

Secondary metabolites are compounds made in the stationary phase; penicillin prevents
the growth of bacteria which could compete with Penicillium molds for resources. Some
bacteria, such as Lactobacillus species, are able to produce bacteriocins which prevent the
growth of bacterial competitors as well. These compounds are used to prevent the growth of
bacteria, either as antibiotics or as antiseptics. Typically secondary metabolites are not produced
in the presence of glucose or other carbon sources which would encourage growth, and released
into the surrounding medium without rupture of the cell membrane.

Production of intracellular components

Of primary interest among the intracellular components are microbial enzymes: catalase,
amylase, protease, pectinase, glucose isomerase, cellulase, hemicellulase, lipase, lactase,
streptokinase and many others. Recombinant proteins, such as insulin, hepatitis B vaccine,
interferon, granulocyte colony-stimulating factor, streptokinase and others are also made this
way. The largest difference between this process and the others is that the cells must be ruptured
(lysed) at the end of fermentation, and the environment must be manipulated to maximize the
amount of the product.

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Transformation of substrate
Substrate transformation involves the transformation of a specific compound into another
(such as in the case of phenylacetylcarbinol, and steroid biotransformation, or the transformation
of a raw material into a finished product) in the case of food fermentations and sewage treatment.

Food fermentation

Ancient fermented food processes, such as making bread, wine, cheese, curds, idli, dosa,
etc., can be dated to more than seven thousand years ago. They were developed long before man
had any knowledge of the existence of the microorganisms involved. Some foods such as
Marmite are the byproduct of the fermentation process, in this case in the production of beer.

Ethanol fuel

Fermentation is the main source of ethanol in the production of ethanol fuel. Common
crops such as sugar cane, potato, cassava and corn are fermented by yeast to produce ethanol
which is further processed to become fuel.

Sewage treatment

In the process of sewage treatment, sewage is digested by enzymes secreted by bacteria.

Solid organic matters are broken down into harmless, soluble substances and carbon dioxide.
Liquids that result are disinfected to remove pathogens before being discharged into rivers or the
sea or can be used as liquid fertilizers. Digested solids, known also as sludge, is dried and used
as fertilizer. Gaseous byproducts such as methane can be utilized as biogas to fuel electrical
generators. One advantage of bacterial digestion is that it reduces the bulk and odor of sewage,
thus reducing space needed for dumping. The main disadvantage of bacterial digestion in sewage
disposal is that it is a very slow process.

Agricultural feed

A wide variety of agroindustrial waste products can be fermented to use as food for
animals, especially ruminants. Fungi have been employed to break down cellulosic wastes to
increase protein content and improve in vitro digestibility.

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2. ROLE OF A BIOPROCESS ENGINEER IN BIOPROCESS INDUSTRY

Biotechnology-derived products already affect human health, nutrition, and

environmental improvement and will grow to provide new products and employment in new
industries.

Bioprocess Technology is the sub-discipline within Biotechnology that combines living

matter, in the form of organisms or enzymes, with nutrients under specific optimal conditions to
make a desired product. It is responsible for translating discoveries of life sciences into practical
and industrial products, processes and techniques that can serve the needs of society. Bioprocess
Technology is thus the backbone of the biotechnology industry that translates the research
and development to the industries. The stages involved in Bioprocess includes the preparation
stage of raw materials, substrates and media, the conversion state, biocatalysts, downstream
processing, volume production, purification and final product processing.

Most of the applications and potential applications of bioprocessing related to renewable

and nonrenewable resources involve large-scale operations and products of relatively low value.

 Bioprocess engineers will be employed in applying the new biology to producing smaller
molecules and specialty bioproducts. These include the formation of value-added products
and to engineer large-scale, integrated processes that use agricultural and forestry-based
materials and other renewable resources.

 To develop a suitable, efficient and economical manufacturing facility can be designed and
built.

 To develop a method for the processing of renewable resources (biomass) and used for the
production of industrial chemicals and of liquid and gaseous fuels.

 The processing of renewable resources must have high national priority in the coming
decade, so that the necessary know-how and production infrastructure for its practical
implementation can be developed.

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 Bioprocessing in space presents unique opportunities, particularly in bioregenerative life

support and as a research platform for the study of new types of manufacturing processes.

 The costs of processing have to be low and the decision to use bioprocessing for raw
materials must be made with care.

 In petroleum industry bioprocess engineer also can take a role, especially on oil recovery
process. By using bioprocessing the oil production is enhanced and recovered large amount
of oil than by conventional technology.
 Most of the major oil companies have in-house staff investigating and perfecting microbial-
enhanced oil recovery (MEOR), the method is low cost and appeal more to small-field
operators, who have already pumped and sold the easy-to-get component of their fields. But
this method is not predictable; like the use of microorganisms for hazardous-waste
remediation, the use of microorganisms for oil recovery is site-specific. Individual oil
deposits have unique characteristics that affect the ability of microorganisms to mobilize and
displace oil. An understanding of the microbial ecology of petroleum reservoirs is a
prerequisite to the development of any MEOR process, whether microbial or not, because an
inappropriate design might accelerate the detrimental activities of microorganisms (e.g.,
corrosion, reservoir souring, and microbial degradation of crude oil).
 The impact of bioprocessing on environmental remediation and industrial waste control
could be tremendous over the longer term.

3. OUTLINE OF THE VARIOUS UNIT OPERATIONS INVOLVED IN THE

UPSTREAM AND DOWNSTREAM OPERATIONS OF A BIOPROCESS PLANT

Industrial fermentations comprise both upstream (USP) and downstream processing

(DSP) stages (Fig. 1). USP involves all factors and processes leading to and including the
fermentation and consists of three main areas: the producer organism, the medium and the
fermentation process. DSP encompasses all processes following the fermentation. The
complexity of DSP is determined by the required purity of the product which is in turn
determined by its application. The products of biotechnology include whole cells, organic acids,
amino acids, solvents, antibiotics, industrial enzymes, therapeutic proteins, vaccines, gums etc.
The primary objective in industrial fermentation processes is to recover the product efficiently,
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reproducibly and safely to its required specification, while achieving maximum product yield at
minimum recovery costs.
Fermentation factors affecting DSP include the properties of the microorganisms
(particularly morphology, flocculation characteristics, size & cell wall rigidity). These factors
impact filterability, sedimentation and homogenization efficiency. The presence of fermentation
by-products, media impurities & additives like antifoam may interfere with DSP steps.
As the products vary greatly in size and nature, different separation techniques are
required for their recovery & purification. The sensitivity of many of the bioproducts,
particularly proteins, to the environmental conditions, places further demands on the
characteristics of the separation processes used for their production. The concentration of the
product in the starting material is a major factor in the overall cost of production. For optimum
economic viability, the number of steps required to attain the desired product specifications
should be minimized. In addition, some biochemical purification processes require substantial
analytical back-up support and some biological materials require complicated and time
consuming assay procedures. Such analytically intensive steps should be avoided if possible as
the analytical cost component may result in the purification step being cost-ineffective.

Upstream process, include selection of a microbial strain characterized by the ability to

synthesize a specific product having the desired commercial value. This strain then is subjected
to improvement protocols to maximize the ability of the strain to synthesize economical amounts
of the product. Included in the upstream phase is the fermentation process itself which usually is
carried out in large tanks known as fermenters or bioreactors. In addition to mechanical parts
which provide proper conditions inside the tank such as aeration, cooling, agitation, etc., the tank
is usually also equipped with complex sets of monitors and control devices in order to run the
microbial growth and product synthesis under optimized conditions. The processing of the
fermentation reactions inside the fermenter can be done using many modifications of engineering
technologies. One of the most commonly used fermenter types is the stirred-tank fermenter
which utilizes mechanical agitation principles, mainly using radial-flow impellers, during the
fermentation process.

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Fig 1. An outline of upstream and downstream processing application

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Downstream processing, the various stages that follow the fermentation process, involves
suitable techniques and methods for recovery, purification, and characterization of the desired
fermentation product. A vast array of methods for downstream processing, such as
centrifugation, filtration, and chromatography, may be applied. These methods vary according to
the chemical and physical nature, as well as the desired grade, of the final product.

4. PROCESS FLOW SHEETING

Process flowsheeting is the use of computer aids to perform steady-state heat and mass
balancing, sizing and costing calculations for a chemical process. It is an essential and core
component of process design. A process flow diagram (PFD) is a diagram commonly used
in chemical and process engineering to indicate the general flow of plant processes and
equipment. The PFD displays the relationship between major equipment of a plant facility and
does not show minor details such as piping details and designations. Another commonly used
term for a PFD is a flowsheet.

The block flow diagram (BFD) is a very simple diagram that can condense an entire
process onto as little as a single sheet. More detailed information can be found in the process
flow diagram (PFD). The PFD is used by plant designers to conduct initial layout studies of a
plant’s process systems and major pipe work. P&IDs (Piping and instrumentation diagrams)
provide the most detail of the three types of diagrams, using standard nomenclature and symbols
to fully describe the process. The main purpose of each document is to convey the right amount
of process information, as needed, during the various stages of the bidding, engineering, design,
procurement, construction, operation, and decommissioning phases of a facility’s lifecycle.

Block flow diagrams (BFDs)

The beauty (and advantage) of a BFD (figure 1) is its ability to outline the complete process on
little more than a single sheet. These diagrams usually resemble an organizational chart,
containing mainly text enclosed by boxes, inter connecting lines and the process commodities
they transport, and flow arrows to indicate process flow directions.

A good BFD typically contains the following:

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 large individual pieces of equipment, or equipment as part of a combined process, that are
denoted by a single symbol, typically a rectangle clear labels illustrating function (since
no equipment or package numbers appear on this document)
 The order of process flow arranged from left to right and, if possible, with a gravity bias,
i.e., if hydrocarbons are shown entering a separation process, then gas leaving the process
should be shown exiting from the top of the block and condensate from the bottom
 Lines linking equipment or processes to show flow direction
 Wherever more than one line leaves a process, then the processed commodity in each line
should be clearly marked.

Fig 2. A block flow diagram can illustrate an entire process on one sheet.

Process flow diagrams (PFD)

Process flow diagrams (Figure 3) carry more information than the block flow diagrams
from which they are derived. They show more detail about major equipment and subsystems and
the flow of product between them.

PFDs include information on the pressures and temperatures of feed and product lines to
and from all major pieces of equipment, such as vessels, tanks, heat exchangers, pumps, etc.
Also indicated are main headers and points of pressure, temperature and flow control, plus the
main shutdown points in the system.

For rotating equipment, PFDs carry important information, such as pump capacities and
pressure heads, and pump and compressor horsepower. For tanks, vessels, columns, exchangers,
etc., design pressures and temperatures are often shown for clarity.

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Figure 3. Process flow diagrams illustrate major equipment and subsystems and the product flow
between them.
A typical PFD shows the following items:

• process piping
• process flow direction
• major equipment represented by simplified symbols
• major bypass and recirculation lines
• control and process-critical valves
• processes identified by system name
• system ratings and operational values
• compositions of fluids
• Connections between systems.

Piping and instrumentation diagrams (P &IDs)

A P&ID (Figure 4) carries a wealth of information that spans engineering disciplines to

defi ne a process. It is the best way of accurately documenting the operation of a process, and it
is truly a coordinating document. P&IDs are also required health and safety documents. The U.S.
Dept. of Labor’s Occupational Safety and Health Administration (OSHA) mandates that P&IDs
be accurate and kept up-to-date throughout the life of the plant. As a result, P&IDs take center
stage in discussions of plant and process operations and product lifecycle management. P&IDs
take the conceptual aspects of the PFD and expand them by adding:

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•detailed symbols • relief and safety valves

•detailed equipment information • instrumentation
•equipment order and process sequence • controls
•process and utility (non-process) piping • types of process component connections
•process flow direction • vendor and contractor interfaces
•major and minor bypass lines • skid and package interfaces
•design requirements for hazardous operations. • hydrostatic vents and drains
•isolation and shutoff valves • maintenance vents and drains
 line numbers, pipe specifications, and pipe sizes

Figure 4. A piping and instrumentation diagram serves as an important reference that is useful at
any stage of the process lifecycle.

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5. ISOLATION, PRESERVATION AND IMPROVEMENT OF INDUSTRIAL STRAINS

1. ISOLATION OF INDUSTRIAL STRAINS

Strategies that are adopted for the isolation of a suitable industrial microorganism from
the environment can be divided into two types, “shotgun” and objective approaches. In the
shotgun approach, samples of free living microorganisms, biofilms or other microbial
communities are collected from animal and plant material, soil, sewage, water and waste streams
and particularly from unusual man-made and natural habitats. These isolates are then screened
for desirable traits. The alternative is to take a more objective approach by sampling from
specific sites where organisms with the desired characteristics are considered to be likely
components of the natural microflora. For example, when attempting to isolate an organism that
can degrade or detoxify a specific target compound, sites may be sampled that are known to be
contaminated by this material. These environmental conditions may select for microorganisms
able to metabolize this compound.

Once the samples have been collected, a major problem is deciding on the growth media
and cultivation conditions that should be used to isolate the target microorganism(s). An initial
step is often to kill or repress the proliferation of common organisms and encourage the growth
of rare ones. Enrichment cultures may then be performed in batch culture, or often more
suitably in continuous systems. This encourages the growth of those organisms with the desired
traits and increases the quantity of these target organisms, prior to isolation and screening.
However, this mode of selection is suitable only for cases where the desired trait provides a
competitive advantage for the organisms.

Subsequent isolation as pure cultures on solid growth media involves choosing or

developing the appropriate selective media and growth conditions. Once isolated as pure
cultures, each must be screened for the desired property, production of a specific enzyme,
inhibitory compound, etc. However, at this stage the level of activity or concentration of the
target product per se is not of major concern, as strain development can normally be employed to
vastly improve performance. Selected isolates must also be screened for other important features,
such as stability and where necessary, non-toxicity.

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These isolation and screening procedures are more easily applied to the search for a
single microorganism. However, it is much more difficult to isolate consortia which together
have the ability/characteristic that is sought and whose composition may vary with time. Such
groups can be more efficient, particularly where the ability to degrade a complex recalcitrant
compound is involved.

2. PRESERVATION OF INDUSTRIAL STRAINS

The isolation of a suitable organism for a commercial process may be a long and very
expensive procedure and it is therefore essential that it retains the desirable characteristics that
led to its selection. Also, the culture used in the industrial fermentation must be viable and free
from contamination. Thus, industrial cultures must be stored in such a way as to eliminate
genetic change, protect against contamination and retain viability. An organism may be kept
viable by repeated subculture into fresh medium, but, at each cell division, there is a small
probability of mutations occurring and because repeated subculture involves very many such
divisions, there is a high probability that strain degeneration would occur. Also, repeated
subculture carries with it the risk of contamination. Thus, preservation techniques have been
developed to maintain cultures in a state of ‘suspended animation’ by storing either at reduced
temperature or in a dehydrated form.

2.1. Storage at reduced temperature

Storage on agar slopes

Cultures grown on agar slopes may be stored ina refrigerator (5 oC) or a freezer (-20oC) and
subcultured at approximately 6 months intervals. The time of subculture may be extended to
1year if thye slopes are covered with sterile medicinal grade mineral oil.

Storage under liquid nitrogen

The metabolic activities of microorganisms may be reduced considerably by storage at very low
temperature (-150o to -196o) which may be achieved by using a liquid nitrogen refrigerator.
Fungi, bacteriophages, viruses, algae, yeast, animals and plant cells and tissue cultures have been
successfully preserved. The techniques involves growing a culture to the maximum stationary
phase, resuspending the cells in a cryoprotective agent (10% glycerol) and freezing the

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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. Thus, viability may be predicted after a period of many years. The equipment is
expensive but the process is economical on labour. The major disadvantage of this method is that
liquid nitrogen evaporates and must be replenished regularly. If it is not done, the consequences
are the loss of the collection.

2.2. Storage in a dehydrated form

Dried cultures

Dried soil cultures have been used widely for the preservation of 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
store in adry atmosphere or in a refrigerator. Silica gel and porcelain beads are used as
alternatives for soil.

Lyophilization

Lyophilization or freeze drying involves the freezing of a culture followed by its drying
under vacuum, which result in sublimation of cell water. The techniques involves the 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 suspension are transferred to an
ampoule, which is then frozen and subjected to high vacuum until sublimation is complete, after
which the ampoule is sealed. The ampoules are stored in the refrigerator and the cells are viable
for 10 more years. It is very convenient for service culture collections because once dried the
culture need no further attention and the storage equipment (a refrigerator) is very cheap and
reliable. The freeze dried cultures are tedious to open and revitalize and several subcultures may
be needed before the cells regain their typical characteristics.

Quality control of preserved stock cultures

Whichever technique is used for the preservation of an industrial culture it is essential to be

certain of the quality of the stocks. Each batch of newly preserved cultures should be routinely
checked to ensure their quality. A single colony of the culture to be preserved is inoculated into a

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shake flask and the growth of the culture observed to ensure a typical growth pattern. After a
further shake flask subculture the broth is used to prepare a large number of storage ampoules.
At least 3% of the ampoules are reconstituted and the cultures assessed for purity, viability and
productivity. If the samples fail any of these tests the entire batch should be destroyed. Thus, by
the use of such quality control system stock cultures may be retained and used with confidence.

3. IMPROVEMENT OF INDUSTRIAL STRAINS

Irrespective of the origins of an individual microorganism, it should ideally exhibit:

1. Genetic stability.
2. Efficient production of the target product, whose route of biosynthesis should preferably
be well characterized.
3. Limited or no need for vitamins and additional growth factors.
4. Utilization of a wide range of low-cost and readily available carbon sources.
5. Amenability to genetic manipulation.
6. Safety, non-pathogenicity and should not produce toxic agents, unless this is the target
product.
7. Ready harvesting from the fermentation.
8. Ready breakage, if the target product is intracellular.

In biotechnological processes the aim is primarily to improve/optimize the particular

characteristics sought in an organism. Advances have been achieved in this area by using
screening and selection techniques to obtain better organisms. In a selection system, all rare or
novel strains grow while the rest do not. In a screening system, all strains grow but certain strains
or cultures are chosen because they show the desired qualities required by the industry in
question.

3.1 GENETIC MANIPULATION OF MICROORGANISMS

Genetic manipulations are used to produce microorganisms with new and desirable
characteristics. The classical methods of microbial genetics play a vital role in the development
of cultures for industrial microbiology.

3.1.1 Mutation

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Once a promising culture is found, a variety of techniques can be used for culture
improvement, including chemical mutagenesis and ultraviolet light. However, such methods
normally lead only to the loss of undesirable traits or increased production due to loss of control
functions. It has rarely led to the appearance of a new function or property. Thus, an organism
with a desired feature will be selected from the natural environment, propagated and subjected to
a mutational programme, then screened to select the best progeny. As an example, the first
cultures of Penicillium notatum, which could be grown only under static conditions, yielded low
concentrations of penicillin. In 1943 a strain of P. chrysogenum was isolated – strain NRRL
1951 – which was further improved through mutation (using X-ray treatment, UV and mustard
gas the yield was increased from 120 IU to 2 580 IU). Today most penicillin is produced with P.
chrysogenum grown in aerobic stirred fermenters, which gives 55-fold higher penicillin yields
than the original static cultures.

In recent years industrial genetics has come to depend increasingly on two new ways of
manipulating DNA – protoplast and cell fusion and recombinant DNA technology (genetic
engineering).

3.1.2 Protoplast Fusion

Protoplast fusion is now widely used with yeasts and moulds. Most of these
microorganisms are asexual or of a single mating type, which decreases the chance of random
mutations that could lead to strain degeneration. To carry out genetic studies with these
microorganisms, protoplasts are prepared by growing the cells in an isotonic solution while
treating them with enzymes, including cellulose and ß-galacturonidase. The protoplasts are then
regenerated using osmotic stabilizers such as sucrose. If fusion occurs to form hybrids, desired
recombinants are identified by means of selective plating techniques. After regeneration of the
cell wall, the new protoplasm fusion product can be used in further studies.

A major advantage of the protoplast fusion technique is that protoplasts of different

microbial species can be fused, even if they are not closely linked taxonomically. For example,
protoplasts of Penicillium roquefortii have been fused with P. chrysogenum. Even yeast
protoplasts and erythrocytes can be fused.

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One of the most exciting and commercially rewarding areas of biotechnology involves a form of
mammalian cell fusion to the formation of monoclonal antibodies. In 1975, pure monoclonal
antibodies were produced from the fusion product of (hybridoma) of ß-lymphocytes and
myeloma tumour cells. The monoclonal antibody technique changes antibody secreting cells
(with a limited life span) to cells that are capable of continuous growth (immortalisation) while
maintaining their specific antibody secreting potential (Fig. 5). Monoclonal antibodies are now
widely applied in many diagnostic techniques which require a high degree of specificity. Specific
monoclonal antibodies have been combined into diagnostic kits in health care, in plant and
animal agriculture and food manufacture.

Stage 1: myeloma cells and antibody-producing cells (derived from immunized animal or man)
are incubated in a special medium containing polyethylylene glycol, which enhances fusion.

Stage 2: the myeloma spleen hybridoma cells are selected out and cultured in closed agar dishes.

Stage 3: the specific antibody-producing hybridoma is selected and propagated in culture vessels
(in vitro) or in an animal (in vivo) and monoclonal antibodies are harvested.

Fig. 5: The formation of antibody-producing hybridomas by fusion techniques.

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3.1.3 Genetic Engineering

Genetic recombination occurs during normal sexual reproduction and as a result of the
breakage and rejoining of DNA molecules of the chromosomes, there is reassortment of genetic
material – but this is restricted to close taxonomic relatives. Recombinant DNA technology or
genetic engineering offers unlimited opportunities for creating new combinations of genes. These
techniques allow the splicing of DNA molecules of quite diverse origin and when combined with
the techniques of genetic transformation, facilitate the introduction of foreign DNA into other
organisms (Fig. 6). DNA can be isolated from plants, animals or microorganisms (the donors),
and fragmented into groups of one or more genes. Such fragments can then be couples to another
piece of DNA (the vector) and then passed into the host or recipient cell, becoming part of the
genetic complement of the new host. The host cell can then be propagated in mass to form novel
genetic properties and chemical abilities that were unattainable by conventional ways of breeding
or mutation.

Short lengths of chemically synthesized DNA sequences can be inserted into recipient
microorganisms by the process of site-directed mutagenesis. This can create small genetic
alterations leading to a change of one or several amino acids in a target protein. Such minor
amino acid changes have been found to lead, in many cases, to unexpected changes in protein
characteristics and have resulted in new products such as more environmentally resistant
enzymes and enzymes that can catalyze desired reactions. These approaches are part of the field
of protein engineering. Enzymes and bioactive peptides with markedly different characteristics
(stability, kinetics, and activities) can be created. The molecular basis for the functioning of these
modified products also can be better understood. One of the most interesting areas is the design
of enzyme-active sites to promote the modification of “unnatural substrates”. This approach
may lead to improved transformation of recalcitrant materials, or even the degradation of
materials that have previously not been amenable to biological processing.

Strategies involved in genetic engineering

STRATEGY METHOD
Formation of DNA fragment Extracted DNA can be cut into smaller fragments
by specific enzymes -restriction endonucleases
found in many species of bacteria.
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Splicing of DNA into vectors
Introduction of vectors into host cells
Selection of newly acquired DNA
The small sequences of DNA can be joined or
spliced into the vector DNA molecules by an
enzyme DNA ligase, creating an artificial DNA
molecule.
The vectors are either viruses or plasmids and are
replicons and can exist in an extra-chromosomal
state; they can be transferred normal transduction or
transformation.
Selection and ultimate characterisation of the
recombinant clone.

Fig : 6. Recombinant DNA: the technique of recombining genes from one species with those of
another.

6. RECOMBINANT DNA TECHNOLOGY FOR OVER PRODUCTION OF PRIMARY

AND SECONDARY METABOLITES

The microbial metabolites are produced through fermentation and their overproduction is
of prime importance to shorten the production time and space and at the same time reducing the
product cost. Overproduction of metabolites depends on the genetic makeup of microbial strain
and the environmental conditions under which the fermentation is carried out. To increase the

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production of fermentation product, different approaches applied and among that genetic
improvement of strain becomes essential.

Recombinant DNA technology is used for modification of biochemical pathways which

ultimately overproduce a particular metabolite and better utilization of media components. The
bioinformatics tools now being used in metabolic engineering for closer understanding of the
gene networks in biochemical pathways to determine the hotspots that could be modified to
increase the metabolite yield. Metabolic engineering involves directed improvement of cellular
properties through the modification of specific biochemical reactions or the introduction of new
genes using recombinant DNA technology with the goal to increase process productivity, such as
the production of antibiotics, biosynthetic precursors or polymers.

Overproduction of Primary Metabolites

Primary metabolites are microbial products made during the exponential phase of growth
whose synthesis is an integral part of the normal growth process. They include intermediates and
end products of anabolic metabolism, which are used by the cell as building blocks for essential
macromolecules (e.g., amino acids, nucleotides) or are converted to coenzymes (e.g., vitamins).
Other primary metabolites (e.g., citric acid, acetic acid and ethanol) result from catabolic
metabolism; they are not used for building cellular constituents but their production, which is
related to energy production and substrate utilization, is essential for growth. Overproduction of
Primary Metabolites based on genetic engineering is regulated by feedback inhibition by the end
product of a particular pathway is suppressed by generation of auxotrophs (i.e. mutation to cause
accumulation of metabolite of interest), mutants resistant to antimetabolites through modification
of enzyme structure at allosteric site, modification of operator or regulator gene to express the
enzyme constitutively.

Overproduction of Secondary Metabolites

Overproduction of Secondary Metabolites based on genetic engineering is regulated by

the structural genes (directly participating in their biosynthesis), regulatory genes, antibiotic
resistance gene (immunizing responsible for their own metabolites) and genes involved in
primary metabolism (affecting the biosynthesis of secondary metabolites). Improvement strain

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advantages include increasing yields of the desired metabolite, removal of unwanted co-
metabolites, improving utilization of inexpensive carbon and nitrogen sources, alteration of
cellular morphology to a form better suited for separation of the mycelium from the product
and/or for improved oxygen transfer in the fermenter. Genetic engineering methods are divided
into two groups namely: Classical genetic methods and Molecular genetic improvement
methods.

7. REGULATION OF ENZYME ACTIVITY AND SYNTHESIS

REGULATION

Both catabolic and anabolic processes are regulated and metabolism is generally so
efficient that excess products are not formed. Strains with less efficient regulation can be
selected in a screening process. The strain development and the optimization of fermentation
conditions lead to a relaxation of regulation in the producing strains. Microbial metabolism is
controlled by the regulation of both enzyme activity and enzyme synthesis.

Regulation of enzyme activity

The activities of enzymes are controlled by different mechanisms. The mechanisms

which are significant for the process development are described below.

1. Feedback inhibition

In an unbranched biosynthetic pathway, the end products inhibit the activity of the first
enzyme of the pathway, a process called feedback inhibition. A confirmation change and hence
inactivation (allosteric effect) occurs when an end product is attached to a specific site of enzyme
(allosteric site). The end product thus inhibits the activity of the enzyme noncompetitively.

In a branched biosynthetic pathway, feedback inhibition of first common enzyme by

means of one of the end product would cause more than one end product to be affected. In
branched biosynthetic pathways, different kinds of feedback inhibition are found:

 The end products thus inhibit the first enzyme in each case after the branch point.
 The first step in the common synthesis path is catalyzed by several isoenzymes, each of
which can be regulated simultaneously.
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 The first common enzyme in branched biosynthetic pathway is influenced by each end
product only slightly or not at all; there must be an excess of all end products for
inhibition to occur.
 Each end product of a branched pathway act as an inhibitor; cumulative inhibition is the
effect of all the inhibitors.
2. Energy change

Feedback inhibition also regulates catabolite pathways in which ATP is the main
products. The relative concentrations of AMP, ADP and ATP in the cell are used in the
following formula to calculate the energy change (EC):

(ATP) + 0.5 (ADP)

EC =
(AMP) + (ADP) + (ATP)

The values of the EC calculated in this way lies between 0 and 1.0.

If the EC is high, the activities of enzymes involved in ATP synthesis are inhibited.
Conversely, the activities of anabolic enzymes which consume ATP, are stimulated by a high
energy charge. Thus, an alteration in the rate of catabolism; since ATP level and hence energy
charge, may cause an increase or decrease in the activity of a variety of enzymes.

3. Breakdown of enzymes
Enzymes which are no longer needed in metabolism may be broken down through the
action of highly specific proteases. One of the best known examples is the enzyme tryptophan
synthetase in Saccharomyces cerevisiae, which is broken down specifically when cells go into
stationary phase.
4. Modification of enzymes

The activity of some enzymes is controlled by conformational changes, such as

phosphorylation or adenylylation.

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Regulation of Enzyme Synthesis

Three mechanisms have been detected to regulate the synthesis of enzymes. Feedback
inhibition affects enzyme molecules that have been already made, the following mechanism
control the actual synthesis of enzyme production.

1. Induction

Some enzymes are formed irrespective of the culture medium; such enzymes are called
constitutive. Many catabolic enzymes are induced: they are not formed until the substrate to be
metabolized is present in the medium. The product of one enzyme can in turn induce the
synthesis of another enzyme.

2. Repression

Anabolic enzymes are generally present only when the end product is absent. The excess
end product suppresses enzyme synthesis, acting as a co-repressor.

3. Attenuation

This is a further mechanism for the control of gene expression which is involved in the
biosynthesis of aminoacids in bacteria.

According to attenuator model, the transcription rate of an operon is regulated by

secondary structure of leader sequence of the newly transcribed mRNA. The structure of this
leader sequence determines whether the transcription of the operon is continued by the RNA
polymerase or a termination occurs. If termination occurs, the mRNA transcription cease and the
enzyme or enzymes coded for that mRNA are not made.

8. CATABOLITE AND PHOSPHATE REGULATION

Catabolite regulation

Catabolite regulation is a general regulatory mechanism in which a key enzyme involved

in a catabolic pathway is repressed, inhibited, or inactivated when a commonly used substrate is
added. Substrates which have been found to bring about catabolite repression include both
carbon and nitrogen sources.

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Carbon sources: Biosynthesis of different secondary metabolites (antibiotics, gibberellins, ergot

alkaloids) is inhibited by rapidly fermentable carbon sources, particularly glucose. Depending on
the organism and metabolite, the basic mechanism of this carbon catabolite regulation is
different. One is the well-known carbon catabolite repression found in many bacteria, yeasts and
molds which involves a catabolite activator protein (CAP) that must combine at the promoter site
before RNA polymerase can attach. The CAP will only bind if it is first complexed with cyclic
adenosine monophosphate, cyclic AMP. Readily utilizable carbon sources such as glucose
stimulate an enzyme which causes the breakdown of cyclic AMP, thus rendering CAP inactive.
Thus, glucose inhibits the synthesis of the mRNA for any enzyme requiring CAP for its
biosynthesis.

In the manufacture of antibiotics, carbon sources other than glucose may be used or
glucose can be fed at low rates, to minimize catabolite repression.

Nitrogen sources: In several antibiotic fermentations it has been observed that ammonia or other
rapidly utilizable nitrogen sources act as inhibitors. The fundamentals of this regulation have not
yet been completely understood, although glutamine synthetase and glutamic dehydrogenase are
considered key enzymes. In enteric bacteria it has been established that glutamine synthetase has
a regulatory function in the synthesis of additional enzymes which are involved in nitrogen
assimilation.

Phosphate regulation:

In a culture medium inorganic phosphate (Pi) is required within a range of 0.3-300 mM

for the growth of prokaryotes and eukaryotes. However, a much lower phosphate concentration
inhibits the production of many secondary metabolites. The highest P. concentration which
allows unimpeded production of secondary metabolites is about 1 mM; complete inhibition of
production occurs at about 10 mM Pi. Phosphate regulation has been observed in the production
of alkaloids, gibberellins and particularly in several antibiotics. The phosphate regulation
mechanism is not yet fully understood. Pi controls the metabolic pathways which precede the first
stage of secondary metabolite formation, but also affects the biosynthesis of secondary
metabolites themselves.

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In one type of mechanism, phosphate stimulates primary metabolism. The consumption

of Pi in the culture medium seems to be one of the main causes for the metabolic change from the
trophophase to the idiophase. By adding Pi at the end of the trophophase, the idiophase can be
delayed; after addition of Pi in the idiophase, growth resumes and antibiotic synthesis steps. In a
second mechanism, phosphate inhibits or represses phosphatases, enzymes which are involved in
the biosynthesis of secondary metabolites. This mechanism has been demonstrated for the
biosynthesis of the aminoglycoside antibiotics. A third mechanism leads to a shift in the
carbohydrate metabolism from the pentose phosphate cycle or hexose monophosphate pathway
to glycolysis when excess phosphate is present. NADPH2 thus becomes a limiting factor in the
synthesis of antibiotics.

Finally, it has been shown that phosphate restricts the induction of secondary metabolite
production.

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