10 mark questions:

1)Selection & strain improvement.

Some examples of characteristics which may be important in this context are:

i)Selection of stable strains:

The ability of the producing strain to maintain its high productivity during broth culture maintenance &

fermentation is very important quality.

The introduction of more than one mutation giving the same phenotype gives a more stable strain.

Evaluation of culture was made using the second slant of slant to slant culture transfer. If the culture

was unstable the yield would be poor and the strain is rejected.

This method is involved in selecting stable strain of Penicillium chrysogenum.

ii)Selection of strains resistant to infection:

Bacterial fermentations are affected by phage infections, resulting in lysis of bacteria.

Therefore bacterial strains must be resistant to phages which can be developed by recombinant

method.

Primrose suggested that inclusion of one or more “host-restriction & modification” (HRM) system may

achieve this objective.

iii)Selection of non-foaming strains:

Foaming during fermentation results in the loss of broth, cells & product via the air outlet, as well as

causes risk of contamination.

Thus foaming is normally controlled by either chemical/mechanical means, but this is made easier by

selecting non-foaming strains, obtained by mutant screening & recombination.

iv)Selection of strains resistant to components in the medium:

Some media components which are required for product formation may interfere with the growth of

the organism & therefore it is desirable to select strains which are resistant to medium components.

Analogues of repressing media components have been used to select resistant mutants.

Eg: Arsenate is used to isolate phosphate resistant mutants.

v)Selection of morphologically favorable strains:

Morphological form of a filamentous microbe will affect both aeration of the system and ease of

filtration of fermentation broth.

Genetically altered P.chrysogenum in a pelleted form is better rather than filamentous form which gives

rise to much lower broth viscosity, resulting in lower power consumption.

vi)Selection of strains which are tolerant to low oxygen tension:

Provision of oxygen is frequently the limiting factor. Therefore selection of an organism which are

capable of producing the product at lower oxygen tension.

vii)Elimination of undesirable product from a production strain:

Along with large quantities of desirable metabolite certain amount of unwanted metabolites are also

produced.

Therefore it is important to alter the strain such that undesirable product is no longer produced.

Eg: In penicillin producing strains, elimination of production of yellow pigment, chrysogenin is done.

Strain Improvement:

Use of high yielding strain is the most critical factor. Therefore strains require improvement and this is

accomplished by producing mutant fermentation strains with the help of physical/chemical methods

and by using recombinant DNA technology.

Mutation for strain improvement:

Mutants formed by mutation are grouped into 2 categories:

i) auxotrophic mutants &

ii)mutants resistant to analogues.

Microorganisms usually have regulatory mechanisms that control the amount of metabolites

synthesized, therefore suppression of regulatory mechanisms is necessary to develop the strains for

higher yields.

Microbial cultures which have multivalent mechanisms, concerted repression or feed-back

inhibition may be used for strain development

Mutants which have lost the ability to synthesize one of the end product capable of feed-back

inhibition or repression is selected.

Different types of industrially important mutants have been summarized:

1)A mutant strain of Corynebacterium glutamicum can excrete about 60g of lysine/l in a medium

based on glucose & minerals. This mutant strain needs homoserine. On the other hand wild strain does

not need homoserine & fails to excrete lysine.

2)There are some mutant strains with enzymes that offer resistance to feed back control. A

mutant strain produced, may have the enzyme with an altered regulatory site, such altered regulatory

site fails to interact with the inhibitor.

3)Use of an analogue in selection of industrially important strains:

An analogue can interact with the regulatory site associated with feed back inhibition. Such an analogue

exerts toxic effect. This toxicity eliminates all sensitive mutant cells in population. Eg: α-amino, β

hydroxyl valeric acid is analogue of threonine. Selection of a mutant strain using this antimetabolite is

done in 2 stages:

a)The analogue of an aminoacid, threonine is added during preparation of a nutrient agar & poured onto

a sterile petridish & allowed to solidify & a wedge has been set. When the wedge has set, a second layer

of the same medium, without analogue is poured onto it & allowed to set. After sometime, diffusion of

an analogue into upper layer of the medium takes place. As a result a concentration gradient is

developed at the surface. Now a culture previously treated with a mutagen is spread on the surface of

this medium and selection of any mutants offering resistance to high concentrations is done.

b)It is also important to find out resistant mutants capable of producing threonine. This is accomplished

by inoculating the mutants as point cultures, onto an agar medium seeded with a threonine dependent

culture. Growth of seeded culture (ie, threonine requiring culture) around each colony of threonine

excreting mutant strain may occur. Diameter of the zone of seeded culture growth depends on the

quantity of threonine produced by mutants. Thus analogue resistant mutants excreting higher yields of

threonine may be obtained. Using this technique, mutant strain of Brevibacterium flavum capable of

excreting threonine upto 12.6g/l is obtained.

4)Revertants from non-producing strains are high producers. Eg: a reversion mutant of

Streptomyces viridifaciens showed 6 fold increase in the production of chlortetracycline over the original

strain.

5)Reversion mutants of appropriate auxotrophs may be high producers. Eg: in case of

S.viridifaciens reversion mutants of an auxotrophic mutant requiring homocysteine showed 28% more

chlortetracycline.

6)Selection for resistance to the antibiotic produced by the organism itself may lead to

increased yields. Eg: Streptomyces aureofaciens mutants selected for resistance to 200-400 mg/l

chlortetracycline showed a 4 fold increase in production of this antibiotic.

7)Mutants with altered cell membrane permeability show high production of some metabolites.

A mutant E.coli strain has defective lysine transport; it actively excretes L-lysine into the medium to 5-

times high in concentration.

8)Mutants have been selected to produce altered metabolites, especially in case of

aminoglycoside antibiotics. For eg: Pseudomonas aureofaciens produces the antibiotic pyrrolnitrin; a

mutant of this organism yields 4’-fluoropyrrolnitrin.

Mutant selection has been the most successful approach for strain improvement, but major

advances are made in r-DNA technology.

Recombinant DNA technology for strain improvement:

This technique has been used to achieve the following 2 broad objectives:

(i)production of recombinant proteins, and

(ii)modification of the organism’s metabolic pattern for the production of new, modified or more

quantity of metabolites.

Recombinant Proteins: These are the proteins produced by the transferred gene or transgene;

they themselves are of commercial value. Eg: insulin, interferon, etc.,

Metabolic engineering: When metabolic activities of an organism are modified by introducing

transgene; it affects enzymatic, transport and/or regulatory function of its cells, it is known as

“metabolic engineering”. Various approaches are summarized below:

1)A transgene may be added, which encodes an enzyme to modify a metabolite produced by the

organism to yield a new product of interest. Eg: Acremonium chrysogenum produces cephalosporin C.

The gene encoding D-amino acid oxidase from Fusarium was introduced into the former. This enzyme

converts cephalosporin C into 7-amino cephalosporanic acid, a precursor of several semisynthetic

antibiotics.

2)The enzyme encoded by transgene may enable a better utilization of the substrate or even the

previously inaccessible components of the substrate. Eg: normal yeasts are unable to utilize

cyclodextrinspresent in malt; this increases the calorie content of the beer. Transgenic yeasts capable of

utilizing cyclodextrins are now commercially used to produce low calorie beer with 1% more alcohol

content.

3)All the genes of an entirely new biosynthetic pathway may be transferred to generate new

products. Eg: 2 genes are involved in conversion of acetyl-CoA to PHB, which is used to produce

biodegradable plastic. The 2 genes were transferred into E.coli from Alcaligenes eutrophus. Transgenic

E.coli, under appropriate conditions, accumulate PHB to upto 50% of their dry weight.

4)Several gene transfers have enhanced growth rates of the organisms, reduced their nutrient

requirements and enabled their growth to higher cell densities. Eg: transfer of gene encoding glutamate

dehydrogenase from E.coli to glutamate synthase deficient mutants of Methylophilus methylotrophus

increased the efficiency of carbon conversion from 4% - 7%.

5)In some cases conversion of an intermediate product to the end product is slow due to low

activity of the rate-limiting enzyme. In such cases the activity of rate limiting enzyme can be increased

by increasing its dosage. Eg: in case of C.acremonium the enzyme (encoded by the gene cefEF) that

converts penicillin N intermediate in the cephalosporin C biosynthesisis rate limiting. The dosage of

cefEF was increased leading to a 15% higher cephalosporin C yield.

2)Screening methods used to detect industrially important microbes.

1)Primary Screening:

This consists of some elementary tests required to detect and to isolate new microbial species exhibiting

the desired property. The techniques involved are:

i)The crowded plate technique:

Used to detect and isolate antibiotic producers.

Serial dilutions of soil sample are made and they are plated on nutrient agar.

After incubation the ability of the organism to exhibit antibiotic activity is indicated by the presence of a

zone of growth inhibition around the colony.

Such cultures are then selected, purified and maintained as pure culture.

ii)Auxanography:

This technique is largely employed to for detecting microbes able to produce growth factors

extracellularly. The two major steps are:

a)Preparation of first plate:

A filter paper strip is put across the bottom of petridish.

The nutrient agar is prepared and poured on the paper disc and allowed to solidify.

Soil sample is diluted and proper dilutions are inoculated and incubated.

b)Preparation of second plate:

A minimal medium lacking the growth factor is prepared and seeded with the test organism.

The seeded medium is poured onto a fresh petridish and the plate is allowed to set.

The agar in the first plate is then carefully lifted with the spatula and placed on the second plate

without inverting. The growth factors produced by the colonies present on the surface of the first layer

of agar can diffuse into the lower layer containing the test organism. The zones of stimulated growth of

the test organism around the colonies Is an indication that the organism produce growth factor

extracellularly.

iii)Enrichment culture technique:

This was first designed by Beijerinck to isolate the desired microorganism from a heterogenous

microbial population

Nutrient broth is inoculated with the microbial source material and incubated.

A small portion of the inoculums is plated on to the solid medium and well isolated colonies are

obtained

Suspected colonies from the plate are subcultured on fresh media and subjected for further testing.

iv)Use of an indicator dye:

This method is used to detect microbes capable of producing organic acids or amines which changes the

colour of the medium according to pH.

Examples of such dye are neutral red, bromothymol blue, etc.

2)Secondary screening:

This helps in detecting in really useful microorganisms in fermentation process.

This is accomplished by performing experiments in agar plates, in flasks or in bioreactors containing

liquid media.

Example: antibiotic producing Streptomyces sp is taken.

Streptomycal isolate is streaked as a narrow band on nutrient agar plates and plates are incubated.

Test organisms are then streaked from the edge of the plates without touching the streptomycal isolate

and the plates are then incubated.

At the end of incubation, growth inhibitory zones for each organisms are measured in millimeters.

Such organisms are again subjected to further testing by growing the culture in sterilized liquid media

and incubated at constant temperature in a mechanical shaker.

Samples are withdrawn at regular intervals under aseptic condition and are tested in quality control

laboratory.

The tests to be done include:

i)checking for contamination

ii)cheking for pH

iii)estimation of critical nutrients

iv)assaying of the antibiotic.

Some other determinations include:

i)screening of fermentation media in which high yield is obtained.

ii)determining whether the antibiotic is new

iii)determination of number of antibiotics accumulated in the broth.

iv)toxicity tests are to be done in mice.

v)the streptomycete is characterized and is classified into species.

3)Detection and assay of fermentation products:

Primary and secondary screening requires the use of good detection and assay procedures for

fermentation products. These procedures must be quick, simple, reliable and accurate. These assays

usually fall into 3 categories:

i)Physical-chemical assay:

Three methods are employed

a)Titration and gravimetric analysis:

Amount of organic acid such as lactic acid produced during fermentation is determined by adding a pH

indicating dye such as bromothymol blue,to sample of fermentation broth followed by titration with

alkali of known strength.

Electrometric titrations are also employed.

b)Turbidity Analysis and cell yield determination:

Turbidity analysis is used to measure the cell yield of a fermentation. Cells suspended in growth medium

are diluted to a turbidity range that can be measured as optical density with a calorimeter.

Portions of fermentation broth with their cells are centrifuged in graduated centrifuge tubes and the

volumes of sedimented cells are measured in cubic centimeter for cell yield determination.

If the medium contains sediment other than microbial cells , plate counting procedure is done.

c)Spectrophotometric Assay:

It is used to measure the amount of absorption of visible light by colored solutions at specific

wavelength, quantity of UV light absorbed by a compound or intensity of fluorescence emitted when a

compound is exposed to UV.

ii) Biological Assay:

These are more difficult to perform, provide greater error and less reproducible than chemical or

physical assays. This assay falls into 4 groups.

a)Diffusion assay:

Carried out on solid medium and the compound to be assayed is allowed to diffuse through medium in a

radial fashion.

The diameter of the area reflects the concentration of compound being assayed and compared with

standards.

b)Turbidimetric and growth assays:

These are the method in which the effect of compound under test in liquid culture is measured

associated with turbidity showing growth rate or total growth of microorganisms.

Turbidity can be determined using spectrophotometer.

Readings are made as OD/absorbance.

c)Endpoint determination assays:

A fermentation product that inhibits growth, such as antibiotic is inoculated with test organism.

Tubes are incubated and observed for growth.

Relative amount of fermentation product in original fermentation broth is determined by amount of

dilution which the fermentation product can withstand in the assay tubes and still able to inhibit growth

of test organism.

d)Metabolic response assays:

Used for assays of metabolic reactions such as acid production, Co2 evolution, oxygen absorption and

enzyme dehydrogenase activity.

e)Enzymatic assay:

Highly specific and quantitatively detect minute amounts of fermentation products.

Eg: L-glutamic acid can be assayed by adding washed cells of certain strains of E.coli which contains the

glutamic acid decarboxylase.

iii)Chromatographic Partition Assay:

This allows detection of compounds in either pure/impure states.

Paper and thin layer chromatography are examples of partition chromatography.

Solute/sample is partitioned continuously between a stationary phase, such as paper or silica gel of thin

layer plates and a mobile phase, consisting of mixture of solvents, as these solvents migrate across the

paper/silica gel layer.

Paper chromatography utilizes a good grade of chemically clean filter.

Thin layer chromatography requires thin layer of silica gel, aluminium oxide or others applied to the

surface of glass plate.

A binding compound is used such as calcium sulphate/starch to bind the layer to the glass.

Sample is placed as spot on the paper/glass and allowed to dry.

After drying, paper or glass containing the sample spot is called as chromatogram.

A mixture of solvents is allowed to migrate across the chromatogram, but not completely.

During solvent migration individual components of the spot also moves along the stationary phase.

The ratio of the distance from the origin that each compound has traveled to the total distance traveled

by the solvent provides ‘Rf value’ which is characteristic of each compound.

Solvent migration must be carried out in closed tanks

The compounds being separated will be observed when they are colored. Eg: aminoacids are detected

by spraying the chromatograms with ninhydrin to yield purple colored spots.

Antibiotics can be detected by placing the chromatogram for a short period on the surface of inoculated

agar. Antibiotic diffuses from the chromatogram to the agar and shows inhibition of growth of test

organism.

4)Mutation and r-DNA techniques used for strain improvement.

Use of high yielding strain is the most critical factor. Therefore strains require improvement and this is

accomplished by producing mutant fermentation strains with the help of physical/chemical methods

and by using recombinant DNA technology.

Mutation for strain improvement:

Mutants formed by mutation are grouped into 2 categories:

i) auxotrophic mutants &

ii)mutants resistant to analogues.

Microorganisms usually have regulatory mechanisms that control the amount of metabolites

synthesized, therefore suppression of regulatory mechanisms is necessary to develop the strains for

higher yields.

Microbial cultures which have multivalent mechanisms, concerted repression or feed-back

inhibition may be used for strain development

Mutants which have lost the ability to synthesize one of the end product capable of feed-back

inhibition or repression is selected.

Different types of industrially important mutants have been summarized:

1)A mutant strain of Corynebacterium glutamicum can excrete about 60g of lysine/l in a medium

based on glucose & minerals. This mutant strain needs homoserine. On the other hand wild strain does

not need homoserine & fails to excrete lysine.

2)There are some mutant strains with enzymes that offer resistance to feed back control. A

mutant strain produced, may have the enzyme with an altered regulatory site, such altered regulatory

site fails to interact with the inhibitor.

3)Use of an analogue in selection of industrially important strains:

An analogue can interact with the regulatory site associated with feed back inhibition. Such an analogue

exerts toxic effect. This toxicity eliminates all sensitive mutant cells in population. Eg: α-amino, β

hydroxyl valeric acid is analogue of threonine. Selection of a mutant strain using this antimetabolite is

done in 2 stages:

a)The analogue of an aminoacid, threonine is added during preparation of a nutrient agar & poured onto

a sterile petridish & allowed to solidify & a wedge has been set. When the wedge has set, a second layer

of the same medium, without analogue is poured onto it & allowed to set. After sometime, diffusion of

an analogue into upper layer of the medium takes place. As a result a concentration gradient is

developed at the surface. Now a culture previously treated with a mutagen is spread on the surface of

this medium and selection of any mutants offering resistance to high concentrations is done.

b)It is also important to find out resistant mutants capable of producing threonine. This is accomplished

by inoculating the mutants as point cultures, onto an agar medium seeded with a threonine dependent

culture. Growth of seeded culture (ie, threonine requiring culture) around each colony of threonine

excreting mutant strain may occur. Diameter of the zone of seeded culture growth depends on the

quantity of threonine produced by mutants. Thus analogue resistant mutants excreting higher yields of

threonine may be obtained. Using this technique, mutant strain of Brevibacterium flavum capable of

excreting threonine upto 12.6g/l is obtained.

4)Revertants from non-producing strains are high producers. Eg: a reversion mutant of

Streptomyces viridifaciens showed 6 fold increase in the production of chlortetracycline over the original

strain.

5)Reversion mutants of appropriate auxotrophs may be high producers. Eg: in case of

S.viridifaciens reversion mutants of an auxotrophic mutant requiring homocysteine showed 28% more

chlortetracycline.

6)Selection for resistance to the antibiotic produced by the organism itself may lead to

increased yields. Eg: Streptomyces aureofaciens mutants selected for resistance to 200-400 mg/l

chlortetracycline showed a 4 fold increase in production of this antibiotic.

7)Mutants with altered cell membrane permeability show high production of some metabolites.

A mutant E.coli strain has defective lysine transport; it actively excretes L-lysine into the medium to 5-

times high in concentration.

8)Mutants have been selected to produce altered metabolites, especially in case of

aminoglycoside antibiotics. For eg: Pseudomonas aureofaciens produces the antibiotic pyrrolnitrin; a

mutant of this organism yields 4’-fluoropyrrolnitrin.

Mutant selection has been the most successful approach for strain improvement, but major

advances are made in r-DNA technology.

Recombinant DNA technology for strain improvement:

This technique has been used to achieve the following 2 broad objectives:

(i)production of recombinant proteins, and

(ii)modification of the organism’s metabolic pattern for the production of new, modified or more

quantity of metabolites.

Recombinant Proteins: These are the proteins produced by the transferred gene or transgene;

they themselves are of commercial value. Eg: insulin, interferon, etc.,

Metabolic engineering: When metabolic activities of an organism are modified by introducing

transgene; it affects enzymatic, transport and/or regulatory function of its cells, it is known as

“metabolic engineering”. Various approaches are summarized below:

1)A transgene may be added, which encodes an enzyme to modify a metabolite produced by the

organism to yield a new product of interest. Eg: Acremonium chrysogenum produces cephalosporin C.

The gene encoding D-amino acid oxidase from Fusarium was introduced into the former. This enzyme

converts cephalosporin C into 7-amino cephalosporanic acid, a precursor of several semisynthetic

antibiotics.

2)The enzyme encoded by transgene may enable a better utilization of the substrate or even the

previously inaccessible components of the substrate. Eg: normal yeasts are unable to utilize

cyclodextrinspresent in malt; this increases the calorie content of the beer. Transgenic yeasts capable of

utilizing cyclodextrins are now commercially used to produce low calorie beer with 1% more alcohol

content.

3)All the genes of an entirely new biosynthetic pathway may be transferred to generate new

products. Eg: 2 genes are involved in conversion of acetyl-CoA to PHB, which is used to produce

biodegradable plastic. The 2 genes were transferred into E.coli from Alcaligenes eutrophus. Transgenic

E.coli, under appropriate conditions, accumulate PHB to upto 50% of their dry weight.

4)Several gene transfers have enhanced growth rates of the organisms, reduced their nutrient

requirements and enabled their growth to higher cell densities. Eg: transfer of gene encoding glutamate

dehydrogenase from E.coli to glutamate synthase deficient mutants of Methylophilus methylotrophus

increased the efficiency of carbon conversion from 4% - 7%.

5)In some cases conversion of an intermediate product to the end product is slow due to low

activity of the rate-limiting enzyme. In such cases the activity of rate limiting enzyme can be increased

by increasing its dosage. Eg: in case of C.acremonium the enzyme (encoded by the gene cefEF) that

converts penicillin N intermediate in the cephalosporin C biosynthesisis rate limiting. The dosage of

cefEF was increased leading to a 15% higher cephalosporin C yield.

5)Types of fermentation.

It is the process of energy production in the cell under anaerobic condition. It also relates to energy

generation by catabolism of organic compounds.

It includes 3 types: Batch culture ,Continuous culture & Fed- batch culture.

1)Batch culture:

This is a closed culture system which contains an initial, limited amount of nutrient. The inoculated

culture will pass through a number of phases.

After inoculation there is a period during which no growth takes place, referred as lag phase/time of

adaptation.

The growth rate of the cell gradually increases, cells grow at a constant, maximum rate & this period is

known as log/exponential phase.

Exponential phase is described by the equation:

dx/dt=µx -------- (1)

x is the concentration of microbial biomass

t is time in hours

µ is the specific growth rate per hour.

On integration it gives:

xt= x0eut ----------- (2)

x0 is original biomass concentration

xt is the biomass concentration after time interval t (hours)

e is the base of natural logarithm

On taking natural logarithm, equation becomes:

ln xt = ln x0 + µt --------- (3)

During exponential phase nutrients are in excess & the organism is growing at its maximum specific

growth rate ie., µmax for the prevailing condition. After consumption of nutrients and excretion of

microbial products the growth rate of culture decreases until growth ceases. Cessation of growth may

be due to the depletion of nutrients in the medium and accumulation of some autotoxic products in the

medium.

An increase in initial substrate concentration, which gives a proportional increase in biomass produced

at stationary phase can be given as:

x = Y(SR –s) ------------ (4)

x is the concentration of biomass produced

Y is the Yield factor

SR is the initial substrate concentration

s is the residual substrate concentration

Yield factor is a measure of the efficiency of conversion of any one substrate into biomass & it can be

used to predict the substrate concentration required to produce certain biomass concentration. Y is not

a constant & varies according to growth rate, pH, temperature, limiting substrate & concentration of

substrate in excess.

Decrease in growth rate, due to depletion of substrate is described by the relationship between µ &

residual growth limiting substrate & represented as:

µ = µmax s (Ks + s) ------------ (5)

s is the residual substrate concentration

Ks is the substrate utilization constant

The stationary phase in batch culture is the point where the growth rate has declined to zero. Bull

pointed out that during this phase organisms are still metabolically active & produce secondary

metabolites.

Pirt has discussed the kinetics of product formation by microbial cultures in terms of growth linked &

non-growth linked products. Growth linked products are considered as primary metabolites and non-

growth linked products are considered as secondary metabolites.

Formation of growth linked products is given as:

dp/dt = qpx -------------- (6)

p is the concentration of product

qp is the specific rate of product formation

The product formation is related to biomass production & is given by the equation:

dp/dx = Yp/x ------------ (7)

is the yield of product in terms of biomass. Multiply equation 7 by dx/dt, then

dx/dt.dp/dx = Yp/x.dx/dt

& dp/dt = Yp/x.dx/dt

But dx/dt = µx, therefore,

dp/dt = Yp/x.µx

& dp/dt = qpx as per the equation 6

Therefore qpx = Yp/x.µx

qp = Yp/x*µ ---------- (8)

From equation 8 it is seen that when product formation is growth associated, the specific rate of

product formation increases with specific growth rate. Thus, productivity in batch culture will be

greatest at µmax & improved product output will be achieved by increasing both µ & biomass

concentration.

Non-growth linked product formation ie, secondary metabolites are produced only under certain

conditions like limitation of a particular substrate. Thus batch fermentation may be used to produce

biomass, primary & secondary metabolites.

2)Continuous culture:

Exponential growth in batch culture may be prolonged by the addition of fresh medium to the vessel

which is made continuous culture. Medium has been designed such that growth is substrate limited and

exponential growth will proceed until additional substrate is exhausted.

The fermentor is fitted with an overflow device, such that the added medium displaces an equal volume

of culture from the vessel & continuous production of cells can be achieved.

If medium is fed continuously to such culture at a suitable rate, a steady state is achieved eventually by

formation of new biomass by the culture is balanced by the loss of cells from the vessel.

The flow of medium into the vessel is related to the volume of the vessel by the term “dilution rate”

defined as:

D = F/V ----------- (1)

F is the flow rate in dm3/hr

V is the volume in dm3

The net change in cell concentration over a time period may be expressed as:

dx/dt = growth-output or

dx/dt = µx-Dx ---------- (2)

Under steady state conditions the cell concentration remains constant thus, dx/dt = 0 and

µx = Dx --------------- (3)

& µ = D --------------- (4)

Thus, under steady state conditions the specific growth rate is controlled by the dilution rate, which is

an experimental variable.

There are 2 types of continuous culture:

1)Chemostat:

The growth of cells in continuous culture is controlled by the availability of growth limiting chemical

component of the medium and thus the system is described as “chemostat”. The mechanism underlying

the controlling effect of dilution rate is the relationship expressed as:

µ = µmaxs/(Ks+s) ------------ (5)

A t steady state, µ = D & therefore,

D = µmaxs/(Ks+s) ----------- (6)

Where s is the steady state concentration of substrate in the chemostat &

s = Ks D/( µmax-D) ------------ (7)

predicting that substrate concentration is determined by dilution rate

Thus, a chemostat is nutrient limited self balancing culture system which may be maintained in a steady

state over a wide range of sub-maximum specific growth rate.

The concentration of cells in the chemostat at steady state is described as:

x = Y(SR-s) ------------- (8)

Where x is the steady state cell concentration in chemostat.

2)Turbidostat:

This is a type of continuous culture, where the concentration of cells in the culture is kept constant by

controlling the flow of medium such that the turbidity of the culture is kept within certain, narrow limits.

This is achieved by monitoring the biomass with a photoelectric cell and feeding the signal to a pump

supplying medium to the culture such that the pump is switched on, if the biomass exceeds the set point

& is switched off if the biomass falls below the set point.

Systems other than turbidity may be used to monitor the biomass concentration, such as carbon dioxide

concentration, pH, etc.

3)Fed-Batch Culture:

Yoshida et al., (1973) introduced this term to describe batch cultures which are fed continuously or

sequentially with medium , without the removal of culture fluid. A fed batch culture is established

initially in batch mode and is then fed according to one of the following feed strategies:

i)the same medium is used to establish the batch culture is added, resulting in an increase in volume.

ii)a solution of limiting substrate at same concentration, as that in initial medium is added.

iii)a concentrated solution of limiting substrate is added at a rate less than (i) & (ii)

iv)a very concentrated solution of limiting substrate is added at a rate less than (i),(ii)&(iii).

There are 2 basic types:

1)Variable volume fed batch culture:

Developed by Dunn, Mor & Pirt.

Consider a batch culture in which is limited by the concentration of one substrate; the biomass at any

point in time will be described by the equation:

xt = x0+Y(SR-s) ------------ (1)

xt =biomass concentration after time, t in hours

x0 = inoculum concentration

The final biomass concentration produced when s=0, may be described as xmax & provided that x0 is

small compared with xmax.

xmax Y. SR ------------- (2)

If at the time when x= xmax, a medium feed is started such that the dilution rate is less than µmax & all the

substrates will be consumed as fast as it enters the culture, thus:

FSR µ(X/Y) -------------- (3)

F is the flow rate of medium feed

X is the total biomass in the culture, described by X=xV,

V is the volume of culture medium at time, t.

From equation 3, it may be concluded that input of substrate is equaled by consumption of substrate by

the cells. Thus ds/dt 0. The total biomass in the culture (X) increases with time, cell concentration (x)

remains constant ie., dx/dt 0 & therefore µ D. This situation is termed as quasi steady state. As time

progresses, the dilution rate will decreases as the volume increases & D is given as:

D = F/(V0+Ft) -------------- (4)

V0 is the original volume

Pirt has expressed the change in product concentration in variable volume fed batch culture as:

Dp/dt = qpx-Dp

Thus product concentration changes according to the balance between production rate and dilution of

the feed.

2)Fixed volume fed batch culture:

A batch culture is considered in which the growth of the process organism has depleted the

limiting substrate to a limiting level. If the limiting substrate is then added in a concentrated feed such

that the broth volume remains almost constant, then:

dx/dt = GY ------------ (1)

G is the substrate feed rate

Y the yield factor

But dx/dt = µx, thus substituting for dx/dt in equation (1)

µx = GY & thus

µ = GY/x --------- (2)

provided that GY/x does not exceed µmax then the limiting substrate will be consumed as soon as it

enters the fermenter & ds/dt 0. Biomass concentration is given by the equation:

xt = xa+GYt ----------- (3)

xt is the biomass after operating in fed batch for t hours

xa is the biomass concentration at the onset of fed batch

Pirt described the product balance in a fixed volume fed batch system as:

dp/dt = qpxt,

substituting for x from equation 3 gives

dp/dt =qp(xa+GYt)

6)Aeration & agitation system.

Aeration provides oxygen for microorganisms in submerged culture.

Agitation ensures uniform suspension of microbial cells in the medium.

Structural components in aeration and agitation are:

a)The agitator

b)Stirrer glands & bearings

c)Baffles

d)Aeration system (sparger)

The agitator: To achieve mixing of bulk fluid & gas-phase mixing, air dispersion, oxygen transfer, etc.

Types include:

• Disc turbines

• Vaned disc turbines

• Open turbines

• Propellers

b)Stirrer glands & bearings:

2 types: top & bottom entry. Seal assembly is the important factor which includes:

• Stuffing box

• Simple bush seal

• Mechanical seal

• Magnetic drive

c)Baffles: It is incorporated to prevent vortex and to improve aeration efficiency.

d)Aeration system(Sparger): This is a device for introducing air into the liquid in a fermenter.

Types of sparger:

• Porous sparger

• Orifice sparger

• Nozzle sparger

• A combined sparger-agitator (used in laboratory fermenters).

7)Monitoring & control systems used in a fermentation process.

Monitoring equipment produces information indicating fermentation progress as well as being linked to

a suitable control system

3 main types of sensors:

1. Sensors penetrating the interior of fermenter. Eg: pH electrodes

2. Sensors which operate on samples.Eg: exhaust-gas analysers

3. Sensors which do not come in contact with fermentation broth. Eg: tachometer.

Sensors are also classified as:

• In-line sensor

• On-line sensor

• Off-line sensor

Methods of measuring process variables:

A)Temperature:

1. Mercury in glass thermometer

2. Electrical resistance thermometers

3. Thermistors

B)Flow measurement & control:

1. Thermal mass flow meters

2. Rotameters

3. Magnetic flowmeter

C)Pressure measurement:

1. Bourdon tube pressure gauge

2. Nested diaphragm type

D)Measurement of dissolved oxygen:

1. Galvanic electrodes

2. Polarographic electrodes

E)Inlet and exit gas analysis:

1. Paramagnetic gas analyser

a)Deflection type

b)Thermal type

2. Infra red analyser for carbondioxide monitoring

3. Mass spectrometer

F)pH measurement & control:

1. Combined glass reference electrode

2. Calomel/mercury electrodes

3. Ingold electrodes

G)Redox:

Electrodes consist of gold, platinum or iridium welded to copper lead.

CONTROL SYSTEMS:

Control loop consists of 4 components:

1. A measuring element

2. A controller

3. A final control element

4. Process to be controlled.

Two types of control:

Manual & automatic control.

Automatic control system is classified into:

1. Two position control(ON/OFF)

2. Proportional controllers

3. Integral controllers

4. Derivative controllers

Computer applications:

1. Logging of process data

2. Data analysis

3. Process control.

8)Asepsis & containment requirements.

Aseptic operation involves protection against contamination.

Containment involves prevention of escape of viable cells from a fermenter or downstream

equipment.

Different assessment procedures are used depending upon whether or not the organism

contains foreign DNA. Once the hazards are assessed, an organism can be classified into a hazard group

for which there is an appropriate level of containment.

Non-genetically engineered organisms may be placed into hazard group using criteria to

assessrisk such as:

i)known pathogenecity of the organism

ii)disease caused by it

iii)number of organism required to initiate an infection

iv)routes of infection

v)known incidence of infection in the community

vi)amounts or volumes of organisms used in fermentation process

vii)techniques/process used

viii)ease of prophylaxis and treatment

Hazard group I organisms used on a large scale only require Good Industrial Large Scale

Practice(GILSP).

If the organism is placed in hazard group IV the stringent requirements of level 3 will have to be

met before the process can be operated.

Large scale processes fall into 2 categories, IB or IIB. IB processes require containment level B1

and are subjected to GILSP, whereas IIB processes are further assessed to determine the most

suitable containment level.

Most organisms used in industrial processes are in the lowest hazard group which only require

GILSP.

9)Types of fermenter.

Different types of fermenter include:

1)The Waldhof-type fermenter:

Made of carbon steel, clad in stainless steel, 7.9 in dm & 4.3 m high. operating volume is 100,000 dm3 of

broth without air.

2)Acetators & cavitators:

A self aspirating rotor sucks the air & broth & disperse the mixture through rotating stator.

3)Tower fermenter:

An elongated non mechanically stirred fermenter, through which there is a unidirectional flow of gases.

4)Cylindro-conical vessels:

It consists of a stainless steel vertical tube with a hemispherical top & a conical base.

5)Air-lift fermenters:

It is a gas-tight baffled riser tube connected to a downcomer tube. Air or gas mixture is introduced into

the base of riser by a sparger.

6)Deep-jet fermenter:

2 basic principles are used for gas entrainer nozzles. Injector: a jet of medium is surrounded by a jet of

compressed air. Ejector: liquid jet enters & entrains the gas around jet.

7)The cyclone column:

Developed for the growth of filamentous culture. The culture liquid was pumped from the bottom to the

top of the cyclone column through a closed loop.

8)The packed tower:

Appliction of immobilized cells. A vertical cylindrical column is packed with the pieces of inert materials

like twigs, coke polythene, etc.

9)Rotating disc fermenter:

Has been used in effluent treatment. This utilizes a growing microbial film on slow rotating discs to

oxidize the effluent.

10)Cheese making.

Curdling

The only strictly required step in making any sort of cheese is separating the milk into solid curds and

liquid whey. Usually this is done by acidifying (souring) the milk and adding rennet. The acidification can

be accomplished directly by the addition of an acid like vinegar in a few cases (paneer, queso fresco),

but usually starter bacteria are employed instead. These starter bacteria convert milk sugars into lactic

acid. The same bacteria (and the enzymes they produce) also play a large role in the eventual flavor of

aged cheeses. Most cheeses are made with starter bacteria from the Lactococci, Lactobacilli, or

Streptococci families. Swiss starter cultures also include Propionibacter shermani, which produces

carbon dioxide gas bubbles during aging, giving Swiss cheese or Emmental its holes.

Some fresh cheeses are curdled only by acidity, but most cheeses also use rennet. Rennet sets the

cheese into a strong and rubbery gel compared to the fragile curds produced by acidic coagulation

alone. It also allows curdling at a lower acidity—important because flavor-making bacteria are inhibited

in high-acidity environments. In general, softer, smaller, fresher cheeses are curdled with a greater

proportion of acid to rennet than harder, larger, longer-aged varieties.

Curd processing

At this point, the cheese has set into a very moist gel. Some soft cheeses are now essentially complete:

they are drained, salted, and packaged. For most of the rest, the curd is cut into small cubes. This allows

water to drain from the individual pieces of curd.

Some hard cheeses are then heated to temperatures in the range of 35 °C–55 °C (100 °F–130 °F). This

forces more whey from the cut curd. It also changes the taste of the finished cheese, affecting both the

bacterial culture and the milk chemistry. Cheeses that are heated to the higher temperatures are usually

made with thermophilic starter bacteria which survive this step—either lactobacilli or streptococci.

Salt has a number of roles in cheese besides adding a salty flavor. It preserves cheese from spoiling,

draws moisture from the curd, and firms up a cheese’s texture in an interaction with its proteins. Some

cheeses are salted from the outside with dry salt or brine washes. Most cheeses have the salt mixed

directly into the curds.

A number of other techniques can be employed to influence the cheese's final texture and flavor. Some

examples:

Stretching: The curd is stretched and kneaded in hot water, developing a stringy, fibrous body.

Cheddaring: (Cheddar, other English cheeses) The cut curd is repeatedly piled up, pushing more

moisture away. The curd is also mixed (or milled) for a long period of time, taking the sharp

edges off the cut curd pieces and influencing the final product's texture.

Washing: The curd is washed in warm water, lowering its acidity and making for a milder-tasting

cheese.

Most cheeses achieve their final shape when the curds are pressed into a mold or form. The harder the

cheese, the more pressure is applied. The pressure drives out moisture—the molds are designed to

allow water to escape—and unifies the curds into a single solid body.

Aging

A newborn cheese is usually salty yet bland in flavor and, for harder varieties, rubbery in texture. These

qualities are sometimes enjoyed—cheese curds are eaten on their own—but normally cheeses are left

to rest under carefully controlled conditions. This aging period (also called ripening, or, from the French,

affinage) can last from a few days to several years. As a cheese ages, microbes and enzymes transform

its texture and intensify its flavor. This transformation is largely a result of the breakdown of casein

proteins and milk fat into a complex mix of amino acids, amines, and fatty acids.

Some cheeses have additional bacteria or molds intentionally introduced to them before or during

aging. In traditional cheese making, these microbes might be already present in the air of the aging

room; they are simply allowed to settle and grow on the stored cheeses. More often today, prepared

cultures are used, giving more consistent results and putting fewer constraints on the environment

where the cheese ages. These cheeses include soft ripened cheeses such as Brie and Camembert, blue

cheeses such as Roquefort, Stilton, Gorgonzola, and rind-washed cheeses such as Limburger.

11)Microbes as source of food.

Single Cell Protein(SCP) are sources of microbes used as food. Can be prepared from cells of algae,

bacteria, yeasts & fungi.

It has high protein content of 43-85%

Algal SCP:

• Algae grows autotrophically & synthesize their food by utilizing energy from sunlight& carbon

from carbondioxide.

• Spirulina maxima & Spirulina platensis are used for SCP production.

Mass cultivation of Spirulina:

Can be done in2 systems:

1)Semi natural lake system

2)Artificially built cultivation system:

a)Clean water system

b)Waste water system

Requirements for growth of Spirulina:

• Substrates used: wheat straw, saw dust, milled corn cobs, ligno cellulosic wastes, etc.

• Algal tanks: circular/rectangular tanks

• Light: low light intensity

• Temperature: 35-40 degrees

• pH: 8.5-9.5

• Agitation: carried out using brush paddle power, wind power, rotators

• Harvesting: harvested using fine mesh, steel screens/nylon cloth

• Drying: Sun drying

• Yield: 8-12g/sq.m/day

Uses:

Protein Supplementary food containing proteins, vitamins, aminoacids, minerals & crude fibres.

As health food & baby food in the form of multi vitamin tablets and powder.

Bacterial SCP:

1. Bacteria used in SCP production include:

2. Hydrogen utilizing bacteria

3. Methane/methanol utilizing bacteria

4. N-paraffins utilizing bacteria

5. Photosynthetic bacteria

Yeast SCP:

Saccharomyces, Candida, Torulopsis.

Cultivated on solid organic wastes.

Fungal SCP:

Aspergillus oryzae, Rhizopus arrhizus are used.

12)Down stream processing:

Downstream processing refers to the recovery and purification of biosynthetic products, particularly

pharmaceuticals, from natural sources such as animal or plant tissue or fermentation broth, including

the recycling of salvageable components and the proper treatment and disposal of waste. It is an

essential step in the manufacture of pharmaceuticals such as antibiotics, hormones (e.g. insulin and

human growth hormone), antibodies (e.g. infliximab and abciximab) and vaccines; antibodies and

enzymes used in diagnostics; industrial enzymes; and natural fragrance and flavor compounds.

Downstream processing is usually considered a specialized field in biochemical engineering, itself a

specialization within chemical engineering, though many of the key technologies were developed by

chemists and biologists for laboratory-scale separation of biological products.

Downstream processing and analytical bioseparation both refer to the separation or purification of

biological products, but at different scales of operation and for different purposes. Downstream

processing implies manufacture of a purified product fit for a specific use, generally in marketable

quantities, while analytical bioseparation refers to purification for the sole purpose of measuring a

component or components of a mixture, and may deal with sample sizes as small as a single cell.

Stages in Downstream Processing

A widely recognized heuristic for categorizing downstream processing operations divides them into four

groups which are applied in order to bring a product from its natural state as a component of a tissue,

cell or fermentation broth through progressive improvements in purity and concentration.

Removal of insolubles is the first step and involves the capture of the product as a solute in a

particulate-free liquid, for example the separation of cells, cell debris or other particulate matter from

fermentation broth containing an antibiotic. Typical operations to achieve this are filtration,

centrifugation, sedimentation, flocculation, electro-precipitation, and gravity settling. Additional

operations such as grinding, homogenization, or leaching, required to recover products from solid

sources such as plant and animal tissues, are usually included in this group.

Product Isolation is the removal of those components whose properties vary markedly from that of the

desired product. For most products, water is the chief impurity and isolation steps are designed to

remove most of it, reducing the volume of material to be handled and concentrating the product.

Solvent extraction, adsorption, ultrafiltration, and precipitation are some of the unit operations

involved.

Product Purification is done to separate those contaminants that resemble the product very closely in

physical and chemical properties. Consequently steps in this stage are expensive to carry out and

require sensitive and sophisticated equipment. This stage contributes a significant fraction of the entire

downstream processing expenditure. Examples of operations include affinity, size exclusion, reversed

phase chromatography, crystallization and fractional precipitation.

Product Polishing describes the final processing steps which end with packaging of the product in a form

that is stable, easily transportable and convenient. Crystallization, desiccation, lyophilization and spray

drying are typical unit operations. Depending on the product and its intended use, polishing may also

include operations to sterilize the product and remove or deactivate trace contaminants which might

compromise product safety. Such operations might include the removal of viruses or depyrogenation.

A few product recovery methods may be considered to combine two or more stages. For example,

expanded bed adsorption accomplishes removal of insolubles and product isolation in a single step.

Affinity chromatography often isolates and purifies in a single step.