PS803: PHARMACEUTICAL BIOTECHNOLOGY

B. Pharmacy IV Year II semester

UNIT - I
a. Fermentation Technology: Isolation, Selection and Screening of Industrially important microbes,
Strain improvement. Types of fermentations, optimization of fermentation process.
Types, design & operation of Bioreactor.
b. Specific Fermentations: Selection of organism, fermentation & purification of various
antibiotics - penicillin, streptomycin, tertacycline, erythromycin
vitamins - cyanocobalamin
aminoacids - glutamic acid,
organic acids - citric acid
solvents - alcohol
biomass - Lactobacillus sporogenes

a. Fermentation Technology

Fermentation, chemical process by which molecules such as glucose are broken down anaerobically.
More broadly, fermentation is the foaming that occurs during the manufacture of wine and beer, a
process at least 10,000 years old. The frothing results from the evolution of carbon dioxide gas, though
this was not recognized until the 17th century. French chemist and microbiologist Louis Pasteur in the
19th century used the term fermentation in a narrow sense to describe the changes brought about
by yeasts and other microorganisms growing in the absence of air (anaerobically); he also recognized
that ethyl alcohol and carbon dioxide are not the only products of fermentation.

Anaerobic Breakdown of Molecules

In the 1920s it was discovered that, in the absence of air, extracts of muscle catalyze the formation of
lactate from glucose and that the same intermediate compounds formed in the fermentation of grain are
produced by muscle. An important generalization thus emerged: that fermentation reactions are not peculiar to
the action of yeast but also occur in many other instances of glucose utilization.
Glycolysis, the breakdown of sugar, was originally defined about 1930 as the metabolism of sugar into lactate.
It can be further defined as that form of fermentation, characteristic of cells in general, in which the six-carbon
sugar glucose is broken down into two molecules of the three-carbon organic acid, pyruvic acid (the nonionized
form of pyruvate), coupled with the transfer of chemical energy to the synthesis of adenosine
triphosphate (ATP). The pyruvate may then be oxidized, in the presence of oxygen, through the tricarboxylic
acid cycle, or in the absence of oxygen, be reduced to lactic acid, alcohol, or other products. The sequence from
glucose to pyruvate is often called the Embden–Meyerhof pathway, named after two German biochemists who
in the late 1920s and ’30s postulated and analyzed experimentally the critical steps in that series of reactions.

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 Isolation, Selection and Screening of Industrially important microbes
Microorganisms or microbes are microscopic organisms that exist as unicellular, multicellular, or cell clusters.
Microorganisms are wide spread in nature and are beneficial to life, but some can cause serious harm. They can be divided into
six major types: bacteria, archaea, fungi, protozoa, algae, and viruses

PRODUCT ISOLATION
It 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, ultra-filtration, and precipitation are some of the unit operations
involved. (go through in detail)
SELECTION & STRAIN IMPROVEMENT:
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
phenol type 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.

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 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) Auxano-graphy:
This technique is largely employed to for detecting microbes able to produce growth factors
extracellular. 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 heterogeneous
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, bromo-thymol 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 organism 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.

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 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) Checking 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.
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:
b)Turbidity Analysis and cell yield determination:
c) Spectrophotometric Assay:
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:
b)Turbidimetric and growth assays:
c) Endpoint determination assays:
d)Metabolic response assays:
e) Enzymatic assay:
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.

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 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) Auxo-trophic 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 who 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 homo-serine. On the other hand wild strain does not
need homo-serine & fails to excrete lysine.
2) There are some mutant strains with enzymes that offer resistance to feedback 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 feedback 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 anti-metabolite 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 increases in the production of
chlortetracycline over the original strain.

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 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. 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.
2) 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.
3) 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%.
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.

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 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.

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
F is the flow rate in dm3/hr
V is the volume in dm3
There are 2 types of continuous culture:
a) 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)
b) 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.

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 3. Fed-Batch Culture:
Yoshida (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: Variable volume fed batch culture and Fixed volume fed batch culture
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)
xt =biomass concentration after time, t in hours x0 = inoculum concentration
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

Types of Fermentations
The following points highlight the five main types of fermentation. The types are:
1. Alcoholic Fermentation 2. Lactic Acid Fermentation
3. Propionic Acid Fermentation 4. Butyric Acid — Butanol Fermentation
5. Mixed Acid Fermentation.
Type # 1. Alcoholic Fermentation:
Alcoholic fermentation generally means production of ethanol (CH3CH2OH). Commonly yeasts, particularly
Saccharomyces cerevisiae, are used for production of various alcoholic beverages, as well as industrial alcohol.
Yeasts are essentially aerobic organisms, but they can also grow as facultative anaerobes. The energy-yield
under anaerobic conditions is much lower and hence the growth is slower with much lower cell-yield. When
grown with aeration, the cell-yield increases dramatically, but alcohol production falls. Thus, oxygen inhibits
fermentation.
This is known as Pasteur-effect.Conversion of pyruvic acid to ethanol proceeds in two steps: pyruvic
acid to acetaldehyde and acetaldehyde to ethanol.
The first step is catalysed by pyruvic acid decarboxylase which requires TPP as coenzyme, and the second step
by alcohol dehydrogenase which requires NADH2 as coenzyme.
NADH2 is thereby oxidized to NAD which can be reused for reduction of GAP to DPGA in the EMP:

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Type # 2. Lactic Acid Fermentation:
Lactic acid fermentations are of two types: Homo-fermentative & Hetero-fermentative.
First type, Homo-fermentative, lactic acid is produced as the sole product by reduction of pyruvic acid
with the help of the enzyme lactic acid dehydrogenase. The reaction regenerates NAD from NADH2 which is
reused for oxidation of GAP to DPGA in the glycolytic pathway.
As one molecule of lactic acid is formed from one molecule of pyruvic acid, two molecules of lactic acid are
produced from each molecule of glucose, when it is dissimilated through EMP. Some representative species of
homo-fermentative lactic acid bacteria are Lactococcus lactis, L. cremoris, L. diacetilactis, L. thermophilus,
Lactobacillus lactis, L. bulgaricus, L. acidophilus etc. Homo-lactic fermentation is the simplest of all
fermentations, involving only a single step in which pyruvic acid is reduced to lactic acid. Lactic acid is formed
also in muscles by a similar reaction.

Second type, hetero-fermentative type, the products are lactic acid and ethanol or acetic acid and
CO2. The hetero-fermentative lactic acid bacteria dissimilate glucose via PPC. They produce lactic acid from
one-half of the glucose molecule, and ethanol or acetic acid and CO2 from the other half.
Representatives of hetero-fermentative type include Lenconostoc mesenteroides, Lactobacillus brevis,
Bifidobacterium bifidum etc. There is also a spore-forming lactic bacterium, Sporolactobacillus.
Lactic acid bacteria are both morphologically and physiologically diverse. The lactic acid bacteria prefer
anaerobic conditions for optimal growth as they do not have cytochromes or catalase, though they can also grow
in micro-aerophilic environment.
The hetero-fermentative lactic acid bacteria lack two vital enzymes of the glycolytic pathway — aldolase and
triose phosphate isomerase. Hence, they are unable to use EMP. As an alternative, they employ the pentose
phosphate pathway. An intermediate of this pathway is xylulose 5-phosphate.
The hetero-fermentative bacteria cleave xylulose 5-phosphate by a TPP-linked pentose phosphate ketolase into
glycerin aldehyde phosphate (GAP) and acetyl phosphate. GAP is then converted to pyruvic acid by the usual
EMP enzymes, while acetyl phosphate is reduced either to acetic acid or to ethanol. From pyruvic acid, lactic
acid is formed by the lactate dehydrogenase activity. Leuconostoc mesenteroides produces from one molecule
of glucose, one molecule of lactic acid, one molecule of ethanol and one molecule of CO2. On the other hand,
Lactobacillus brevis produces acetic acid in place of ethanol.
Both heterofermentative and homo-fermentative lactic acid bacteria are used as ‘starter’ for production
of fermented food. Some of these bacteria are Lactococcus cremoris, L. lactis, L. thermophilus, Lactobacillus
bulgaricus, L. plantarum, L. brevis, Leuconostoc mesenteroides, Pediococcus cerevisiae etc.
Lactic acid bacteria are widely used for production of various fermented food throughout the world. The
bacteria ferment the milk sugar (lactose) to produce lactic acid which curdles milk protein. Various species are
used to yield products of variable consistency, taste and aroma. In different countries the products are variously

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known as yogurt in Europe and America, dadhi or dahi in India, Kefir in Russia, Kumiss, butter milk,
acidophilus milk etc.
Lactic acid bacteria are also employed in producing fermented vegetable products, like sauerkraut
(fermented cabbage), cucumber pickles and fermented olive. These bacteria are also used for production of
sausages from beef and pork.
The hetero-fermentative pathway is shown in Figure:

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Type # 3. Propionic Acid Fermentation:

Propionic acid (CH3-CH2-COOH) is produced by several anaerobic bacteria among which are the
coryneform Propionibacterium, and Veillonella, Clostridium, Selenomonas etc. Propionibacterium
acidipropionici and P. freudenreichii are the main propionic acid fermenters. Propionibacteria possess
cytochromes and catalase and can tolerate some amount of oxygen. They are natural inhabitants of rumen of
herbivorous cattle.
The propionic acid bacteria dissimilate glucose via EMP and produce pyruvic acid. By a biotin- linked
carboxylation reaction pyruvic acid is converted to oxalacetic acid which is then reduced in two steps to
succinic acid through reversal of TCA cycle reactions.
Succinic acid is then converted to succinyl-CoA, also by a reverse step of the TCA cycle. Next,
succinyl-CoA produces methyl malonyl- CoA by the action of a vitamin B12-linked enzyme methyl malonyl
mutase which catalyses an intra-molecular rearrangement. Methyl malonyl-CoA is then decarboxylated to
propionyl-CoA.
In the final step, propionyl-CoA yields propionic acid, and CoA is transferred to succinic acid by an
enzyme, CoA-transferase.

Together with lactic acid bacteria, the propionic acid bacteria are used for commercial production of Swiss
cheese. Propionic acid contributes to the special flavour of this cheese.

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Type # 4. Butyric Acid — Butanol Fermentation:
The bacteria carrying out butyric acid-butanol fermentation are all obligately anaerobic spore- forming bacteria
belonging to the genus Clostridium. Besides butyric acid and n-butanol, several other products of this
fermentation are acetic acid, ethanol, isopropanol and acetone depending on species.

For example, C. butyricum, C. lactoacetophilum, C. pasteurianum etc. produce butyric acid together with acetic
acid, while C. butylicum and C. acetobutylicum produce butyric acid, acetic acid and isopropanol or acetone.
Also, as a fermentation product, CO2 is always present.
Clostridia dissimilate glucose by the EMP to form pyruvic acid which by decarboxylation produces acetyl-CoA.
The latter acts as the key intermediate in the butyric-butylic fermentation and gives rise to all the products by
different pathways as shown in Figure:

In the pathway leading to butyric acid in C. butyricum,

two molecules of acetyl-CoA are condensed by the action of the enzyme thiolase to produce acetoacetyl
CoA with liberation of one CoA.
Acetoacetyl CoA is then dehydrogenated by β-hydroxybutyryl-CoA dehydrogenase to form P-
hydroxbutyryl CoA with NADH2 acting as H-donor.
The dehydrogenated product next gives rise to crotonyl-CoA through the action of the enzyme
crotonase.
Crotonyl-CoA undergoes another step of reduction catalysed by butyryl-CoA dehydrogenase which is
FADH2-linked producing butyryl-CoA.
The latter is finally converted to butyric acid by removal of CoA and addition of water.
Under alkaline conditions, butyryl CoA is converted by C. acetobutylicum to n-butanol through two steps
catalysed by butyryl-CoA dehydrogenase and butyryl aldehyde dehydrogenase.

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C. acetobutylicum also produces isopropanol by reduction of acetone under alkaline conditions. Acetone is
produced by decarboxylation of aceto acetic acid.
Clostridia always produce molecular hydrogen as one of the fermentation products. Hydrogen originates from
phosphoroclastic cleavage of pyruvate.
This cleavage produces acetyl phosphate, molecular hydrogen and CO2 as shown:

During such cleavage, hydrogen is at first transferred to an iron-containing protein called ferredoxine which is
thereby reduced. Molecular hydrogen is liberated from the reduced compound through the action of
hydrogenase, and ferredoxine is oxidized.

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Type # 5. Mixed Acid Fermentation:
This type of fermentation occurs characteristically in bacteria belonging to the family
Enterobacteriaceae. These bacteria can grow either aerobically carrying out oxygen respiration or anaerobically
carrying out fermentation.
The type of fermentation is called mixed-acid, because, as products, several different organic acids and
neutral compounds are produced. A characteristic acid of mixed fermentation is formic acid, though it is by no
means the major product.
Depending on species, a number of-different substance is formed, like acetic acid, succinic acid, lactic
acid, ethanol, acetoin, butanediol, CO2 and molecular hydrogen.
On the basis of fermentation products, the enterobacteria can be divided into two groups: one group
having Escherichia coli-type fermentation, and the other having an Enterobacter aerogenes type. One very
significant difference in these two types is the formation of acetoin and butanediol (2, 3-butylene glycol) from
pyruvic acid by Enterobacter aerogenes. In E. coli type of fermentation these are absent. Both types dissimilate
glucose to pyruvic acid.
Mixed acid fermentation is sometimes called formic acid fermentation. Under anaerobic condition, E. coli
cleaves pyruvic acid to acetyl-CoA and formic acid.

The reaction is catalysed by the enzyme, pyruvate-formic acid lyase as shown:

Formic acid so formed is then cleaved by another lyase, formic acid-hydrogen lyase to molecular hydrogen and
CO2 which are liberated in gaseous form.

Formic acid is also produced in Enterobacter-type of fermentation, but in a different way. The reaction is
catalyzed by a TPP-linked enzyme. In this type, pyruvic acid is cleaved into TPP-linked “active” acetaldehyde
(hydroxyethyI-Tpp.Enz.) and formic acid.

Formic acid then breaks into CO2 and H2:

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Enterobacter-type of fermentation produces acetoin (acetylmethyl carbinol) and butanediol which are not
formed by E. coli-type of fermentation. The detection of acetoin and butanediol forms the basis of Voges-
Proskauer reaction. Hence, E. coli is Voges-Proskauer negative.

Formation of acetoin and butanediol in Enterobacter proceeds via acetolactate pathway. The TPP- linked active
acetaldehyde produced from pyruvic acid, described above, reacts with another molecule of pyruvic acid to
form acetolactate.

The reaction is catalysed by acetohydroxyl acid synthase. Acetolactate so formed, is then decarboxylated by the
enzyme acetolactate decarboxylase to produce acetoin. The latter is reduced by butanediol dehydrogenase to
2,3-butylene glycol (butanediol), NADH2 acting as H-donor.
The reactions are:

Other products of enteric bacteria fermentations include acetic acid, ethanol, lactic acid and succinic acid.
AcetyI-CoA produced in pyruvic acid-formic acid lyase reaction in E.coli can be used in several ways.

It can be converted acetyI phosphate and from it either ethanol may be produced via acetaldehyde or it
may form acetic acid as shown:

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Lactic acid is formed directly from pyruvic acid through the action of lactate dehydrogenase. Succinic acid is
produced also from pyruvic acid by carboxylation with the help of a biotin enzyme to oxalacetic acid. The latter
leads to formation of succinic acid by reversal of steps of the TCA cycle.

Formation of different products of mixed-acid fermentations is summarized:

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 FERMENTATION PROCESS OPTIMIZATION
Ref: Bibhu Prasad Panda , Mohd. Ali and Saleem Javed , 2007. Fermentation Process
Optimization. Research Journal of Microbiology, 2: 201-208
URL: https://scialert.net/abstract/?doi=jm.2007.201.208
INTRODUCTION
For an industrial fermentation process fermentation medium and fermentation process condition
plays an critical role because they effect the formation, concentration and yield of a particular
fermentation end product thus effecting the overall process economics therefore it is important to
consider the optimization of fermentation medium and process conditions in order to maximize the
profits from fermentation process.
There are many challenges associated with optimization of fermentation process, it is laborio us,
expensive, open ended and time consuming process involving many experiments. In bioprocess industry
it is often needs to conduct optimization experiments because new mutants and strains are continuously
being introduced.
In fermentation process optimization different combinations and sequence of process conditions and
medium components are needs to be investigated to determine the growth condition that produces the
biomass with the physiological state best constituted for product formation. The present review explores
the different well-known and newer optimization method applied in fermentation process.

OPEN AND CLOSE ENDED SYSTEMS FOR PROCESS OPTIMIZATION

In close-ended system, a fixed number and type of component parameters are analyzed for
optimization, this is the simplest strategy but many different possible components/parameters which are
not considered, could be beneficial in the medium.
In open-ended system any number and type of components/parameters are analyzed for
optimization of fermentation process. The advantage of this system is that it makes no assumption of
which components/parameters are best for fermentation process.
The ideal method would be to start with an open-ended system, select the best
components/parameters for optimization of fermentation process then move to the close -ended system.

DIFFERENT METHODS FOR FERMENTATION PROCESS OPTIMIZATION

1) Borrowing
this is an open-ended system for process optimization. The medium components and process
conditions are obtained from the literatures and what other workers were used to grow the same
genus, species or strains are analyzed. The problem with this method is that there are too many
options for a given fermentation process. Therefore short listing is necessary and advantage of this
method is that it is simple, easy and requires no mathematical skill.
2) Component-Replacing
This is an open-ended system for process optimization and only used to compare the component
of one type in a fermentation medium. In this method, one of component of the medium was
replaced by a new one at same incorporation level. However this method does not consider the
components interactions. But this method can useful for screening differe nt carbon, nitrogen and
other source for improving the medium utilization. Screening of suitable carbon source for

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 mevastatin and citric acid production by solid-state fermentation was carried out by component
replacing techniques.
3) Biological-Mimicry
Biological mimicry is a close-ended system for fermentation process optimization. This method is
useful for optimization of various components of fermentation media and based on concept that
cell grow well in a medium that contains every things it needs in right proportion (mass balance
strategy). The medium is optimized based on elemental composition of microorganisms and
growth yield. The limitation of this method is measuring elemental composition of
microorganisms is expensive, laborious and time consuming moreover it does not consider the
component interaction however this method gives an idea about different micro and macro
elements level require in the media for optimal growth of microorganisms.
4) One-factor-at-a-time
One-factor-at-a-time is a close-ended system for fermentation process optimization. This method
can be applied for optimization of medium components as well as for process condition and it is
based on the classical method of changing one independent variable while fixing all other at a
certain level. This strategy has the advantage that it is simple, easy and the individual effects of
medium components and process condition can be seen on graphs but the limitations of this
method are interaction between the components are ignored, extremely time consuming, expensive
for large number of variable as it involves a relatively large number of experiments. Because of its
easy and convenience one-factor-at-a-time method has been the most popular method for
improving the fermentation medium and process condition.
5) Factorial-Design
Factorial design is a close-ended system for process optimization. In this method, level of
factors/parameters are independently varied, each factor at two or more levels. This effects that
can be attributed to the factors and their interactions are assessed with max imum efficiency in
factorial design more over it allow for the estimation of the effects of each factor and interaction.

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 BIOREACTORS
The six types of bioreactors used in bioprocess technology. The six types are:
(1) Continuous Stirred Tank Bioreactors (2) Bubble Column Bioreactors
(3) Airlift Bioreactors (4) Fluidized Bed Bioreactors
(5) Packed Bed Bioreactors and (6) Photo-Bioreactors.
Type # 1. Continuous Stirred Tank Bioreactors:
A continuous stirred tank bioreactor consists of a cylindrical vessel with motor driven central shaft that supports
one or more agitators (impellers). The shaft is fitted at the bottom of the bioreactor (Fig. 19.1 A). The number of
impellers is variable and depends on the size of the bioreactor i.e., height to diameter ratio, referred to as aspect
ratio.

The aspect ratio of a stirred tank bioreactor is usually between 3-5. However, for animal cell culture
applications, the aspect ratio is less than 2. The diameter of the impeller is usually 1/3 rd of the vessel diameter.
The distance between two impellers is approximately 1.2 impeller diameter. Different types of impellers
(Rustom disc, concave bladed, marine propeller etc.) are in use.
In stirred tank bioreactors or in short stirred tank reactors (STRs), the air is added to the culture medium under
pressure through a device called sparger. The sparger may be a ring with many holes or a tube with a single
orifice. The sparger along with impellers (agitators) enables better gas distribution system throughout the vessel.
The bubbles generated by sparger are broken down to smaller ones by impellers and dispersed throughout the
medium. This enables the creation of a uniform and homogeneous environment throughout the bioreactor.
Advantages of STRs:
There are many advantages of STRs over other types. These include the efficient gas transfer to growing
cells, good mixing of the contents and flexible operating conditions, besides the commercial availability of
the bioreactors.

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Type # 2. Bubble Column Bioreactors:
In the bubble column bioreactor, the air or gas is introduced at the base of the column through perforated pipes
or plates, or metal micro porous spargers (Fig. 19.1B). The flow rate of the air/gas influences the performance
factors —O2 transfer, mixing. The bubble column bioreactors may be fitted with perforated plates to improve
performance. The vessel used for bubble column bioreactors is usually cylindrical with an aspect ratio of 4-6
(i.e., height to diameter ratio).

Type # 3. Airlift Bioreactors:

In the airlift bioreactors, the medium of the vessel is divided into two interconnected zones by means of
a baffle or draft tube. In one of the two zones referred to a riser, the air/gas is pumped. The other zone that
receives no gas is the down comer. The dispersion flows up the riser zone while the down flow occurs in the
down comer. There are two types of airlift bioreactors.
Internal-loop airlift bioreactor (Fig. 11.1C) has a single container with a central draft tube that creates
interior liquid circulation channels. These bioreactors are simple in design, with volume and circulation at a
fixed rate for fermentation.
External loop airlift bioreactor (Fig. 19.1D) possesses an external loop so that the liquid circulates
through separate independent channels. These reactors can be suitably modified to suit the requirements of
different fermentations. In general, the airlift bioreactors are more efficient than bubble columns, particularly
for more denser suspensions of microorganisms. This is mainly because in these bioreactors, the mixing of the
contents is better compared to bubble columns.
Airlift bioreactors are commonly employed for aerobic bioprocessing technology. They ensure a
controlled liquid flow in a recycle system by pumping. Due to high efficiency, airlift bioreactors are sometimes
preferred e.g., methanol production, waste water treatment, single-cell protein production. In general, the
performance of the airlift bioreactors is dependent on the pumping (injection) of air and the liquid circulation.
Two-stage airlift bioreactors:
Two-stage airlift bioreactors are used for the temperature dependent formation of products. Growing cells from
one bioreactor (maintained at temperature 30°C) are pumped into another bioreactor (at temperature 42°C).
There is a necessity for the two-stage airlift bioreactor, since it is very difficult to raise the temperature quickly
from 30°C to 42°C in the same vessel. Each one of the bioreactors is fitted with valves and they are connected
by a transfer tube and pump (Fig. 19.2A). The cells are grown in the first bioreactor and the bioprocess proper
takes place in the second reactor.

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Tower bioreactors:
A pressure-cycle fermenter with large dimensions constitutes a tower bioreactor (Fig. 19.2B). A high
hydrostatic pressure generated at the bottom of the reactor increases the solubility of O2 in the medium. At the
top of the riser, (with expanded top) reduces pressure and facilitates expulsion of CO2. The medium flows back
in the down comer and completes the cycle. The advantage with tower bioreactor is that it has high aeration
capacities without having moving parts.
Type # 4. Fluidized Bed Bioreactors:
Fluidized bed bioreactor is comparable to bubble column bioreactor except the top position is expanded to
reduce the velocity of the fluid. The design of the fluidized bioreactors (expanded top and narrow reaction
column) is such that the solids are retained in the reactor while the liquid flows out (Fig. 19.3A). These
bioreactors are suitable for use to carry out reactions involving fluid suspended biocatalysts such as
immobilized enzymes, immobilized cells, and microbial flocs.

For an efficient operation of fluidized beds, gas is spared to create a suitable gas-liquid-solid fluid bed. It is also
necessary to ensure that the suspended solid particles are not too light or too dense (too light ones may float
whereas to dense ones may settle at the bottom), and they are in a good suspended state. Recycling of the liquid
is important to maintain continuous contact between the reaction contents and biocatalysts. This enable good
efficiency of bioprocessing.

Type # 5. Packed Bed Bioreactors:

A bed of solid particles, with biocatalysts on or within the matrix of solids, packed in a column constitutes a
packed bed bioreactor (Fig. 19.3B). The solids used may be porous or non-porous gels, and they may be
compressible or rigid in nature. A nutrient broth flows continuously over the immobilised biocatalyst. The
products obtained in the packed bed bioreactor are released into the fluid and removed. While the flow of the
fluid can be upward or downward, down flow under gravity is preferred. The concentration of the nutrients (and
therefore the products formed) can be increased by increasing the flow rate of the nutrient broth. Because of
poor mixing, it is rather difficult to control the pH of packed bed bioreactors by the addition of acid or alkali.

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However, these bioreactors are preferred for bioprocessing technology involving product-inhibited reactions.
The packed bed bioreactors do not allow accumulation of the products to any significant extent.

Type # 6. Photo-Bioreactors:
These are the bioreactors specialised for fermentation that can be carried out either by exposing to sunlight or
artificial illumination. Since artificial illumination is expensive, only the outdoor photo-bioreactors are
preferred. Certain important compounds are produced by employing photo-bioreactors e.g., p-carotene,
asthaxanthin.
The different types of photo-bioreactors are depicted in Fig. 19.4. They are made up of glass or more commonly
transparent plastic. The array of tubes or flat panels constitute light receiving systems (solar receivers). The
culture can be circulated through the solar receivers by methods such as using centrifugal pumps or airlift
pumps. It is essential that the cells are in continuous circulation without forming sediments. Further adequate
penetration of sunlight should be maintained. The tubes should also be cooled to prevent rise in temperature.

Photo-bioreactors are usually operated in a continuous mode at a temperature in the range of 25-40°C.
Microalgae and cyanobacteria are normally used. The organisms grow during day light while the products are
produced during night.

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 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).

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

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 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.

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