Unit – IV

PRODUCTION OF ENZYMES & OTHER BIOPRODUCTS

4.1.0 PRODUCTION OF ENZYMES

Microbial cells contain or produce a variety of enzymes and as we have
seen, these enzymes are the biological catalysts for the biochemical reactions leading to
microbial growth and respiration as well as to the formation of fermentation products. In
certain instances, however, some of these enzymes may in themselves become
fermentation products so that one then is specifically interested in obtaining high yields
of particular enzymes instead of high level activity of the enzymes as they mediate
fermentation process of the cells.
Enzymes are either adaptive or constitutive in nature. A constitutive
enzyme is always produced by the cell even in the absence of substrate but adaptive
enzymes are produced if required by the cell for degradation or utilization of the suitable
substrate.
Enzymes also may be either exocellular or endocellular. An endocellular
enzyme is produced within the cell or at the cytoplasmic membrane and normally does
not find its way into the fermentation medium surrounding the cell. Exocellular enzymes
are produced by microbial cell are liberated to the fermentation medium so that the
enzyme can hydrolytically attack and degrade substances which are too large or insoluble
or in some manner inaccessible to the cell. Examples for this exocellular enzymes
includes proteases, amylases, cellulases, lipases, etc.

4.1.1 PRODUCTION OF PROTEASES

Protease (also termed peptidase or proteinase) breaks down proteins. A protease is
any enzyme that conducts proteolysis, that is, begins protein catabolism by hydrolysis of
the peptide bonds that link amino acids together in the polypeptide chain forming the
protein. Proteases work best in acidic conditions.
Proteases occur naturally in all organisms. These enzymes are involved in a
multitude of physiological reactions from simple digestion of food proteins to highly-
regulated cascades (e.g., the blood-clotting cascade, the complement system, apoptosis
pathways, and the invertebrate prophenoloxidase-activating cascade). Peptidases can
either break specific peptide bonds (limited proteolysis), depending on the amino acid
sequence of a protein, or break down a complete peptide to amino acids (unlimited
proteolysis). The activity can be a destructive change, abolishing a protein's function or
digesting it to its principal components; it can be an activation of a function, or it can be a
signal in a signaling pathway.
Bacteria also secrete proteases to hydrolyse (digest) the peptide bonds in proteins
and therefore break the proteins down into their constituent monomers.
A secreted bacterial protease may also act as an exotoxin, and be an example of a
virulence factor in bacterial pathogenesis. Bacterial exotoxic proteases destroy
extracellular structures. Protease enzymes are also used extensively in the bread industry
in bread improver.
Proteases, also known as proteinases or proteolytic enzymes, are a large group of
enzymes. Proteases belong to the class of enzymes known as hydrolases, which catalyse
the reaction of hydrolysis of various bonds with the participation of a water molecule.
Proteases are involved in digesting long protein chains into short fragments,
splitting the peptide bonds that link amino acid residues. Some of them can detach the
terminal amino acids from the protein chain (exopeptidases, such as aminopeptidases,
carboxypeptidase www A); the others attack internal peptide bonds of a protein
(endopeptidases, such as trypsin, chymotrypsin, pepsin, papain, elastase).
Proteases are divided into four major groups according to the character of their
catalytic active site and conditions of action: serine proteinases, cysteine (thiol)
proteinases, aspartic proteinases, and metalloproteinases. Attachment of a protease to a
certain group depends on the structure of catalytic site and the amino acid (as one of the
constituents) essential for its activity.
Proteases are used throughout an organism for various metabolic processes. Acid
proteases secreted into the stomach (such as pepsin) and serine proteases present in
duodenum (trypsin and chymotrypsin) enable us to digest the protein in food; proteases
present in blood serum (thrombin, plasmin, Hageman factor, etc.) play important role in
blood-clotting, as well as lysis of the clots, and the correct action of the immune system.
Other proteases are present in leukocytes (elastase, cathepsin G) and play several
different roles in metabolic control. Proteases determine the lifetime of other proteins
playing important physiological role like hormones, antibodies, or other enzymes. This is
one of the fastest "switching on" and "switching off" regulatory mechanisms in the
physiology of an organism. By complex cooperative action the proteases may proceed as
cascade reactions, which result in rapid and efficient amplification of an organism's
response to a physiological signal. Proteases are also a part of many laundry detergents.
PRODUCTION
Proteases are produced by bacteria as well as by fungai. It is essential to
take care during their production, because they are relatively unstable and tend to lose
their activity during dehydration.
Bacteria like Bacillus subtilis, Bacillus licheniformis and fungai like Aspergillus
niger and Aspergillus oryzae are the organisms producing proteases in significant level.
There are two types of proteases. They are,
(a) Alkaline serine proteases
(b) Acid proteases
ALKALINE SERINE PROTEASE
This can be produced from the organism Bacillus licheniformis by sub-
merged fermentation process. The media used for the production contains the following
constituents.
Starch hydrolyzate 50 gm/ltr
Soya bean meal 20 gm/ltr
Casein 20 gm/ltr
Na2HPO4 3.3 gm/ltr
The temperature of fermentation in the range of 30-40 0C has been found to
be satisfactory. The pH of the production medium is kept 7.0 for satisfactory results. The
production of the enzyme begins when maximum cell growth is reached after 10-20
hours and this continues at an almost constant rate till the completion of fermentation. At
the end of the productive fermentation, protease is the only protein present in the
medium. The reason for this is the occurrence of hydrolysis of all the protein present in
the production medium by protease. The yield may be 10% of the initial protein content
of the medium. The enzyme is marketed primarily in the form of dust-free granules.
These granules contain 1-5% enzyme protein. The enzyme remains stable in liquid
preparations. Liquid preparation contain about 2% of the enzyme.
ACID PROTEASE PRODUCTION
These enzymes are mostly produced by fungai. The fungai employed for
producing these enzymes are Mucor pusillus, Mucer miehei, Aspergillus oryzae,
Aspergillus phoenicis, Aspergillus niger.
Acid protease may be produced by either semisolid-culture or submerged
culture depending upon the fungal species employed. For example, Mucor pusillus is
cultivated on a semisolid medium. The medium consists of 60% wheat bran with water.
The optimum temperature of the fermentation is 30 0C. The fermentation requires 3 days
for completion. The yield is 3,200 Soxhlet units per gramme of wheat bran. The yield of
the enzyme can be increased by the addition of ammonium salts. Finally, the enzyme is
extracted with water. On the other hand, Mucor miehei is grown by submerged culture
method. The production medium used for this purpose has the following composition,
Starch 4.0%
Soya bean meal 3.0%
Ground barley 10%
Calcium carbonat 0.5%
The optimum temperature of the fermentation is 300C. The fermentation is
completed in 7 days. The yield is about 3,500 Soxhlet units per milliliter of the broth.
The enzyme preparation are marked at concentrations of 10,000 - 1,50,000
Soxhlet units per milliliter. These preparations contain 0.2-3.0% of active enzyme.

4.1.2 PRODUCTION OF AMYLASES

Amylase is an enzyme that breaks starch down into sugar. Amylase is present in
human saliva, where it begins the chemical process of digestion. Foods that contain much
starch but little sugar, such as rice and potato, taste slightly sweet as they are chewed
because amylase turns some of their starch into sugar in the mouth. The pancreas also
makes amylase (alpha amylase) to hydrolyse dietary starch into disaccharides and
trisaccharides which are converted by other enzymes to glucose to supply the body with
energy. Plants and some bacteria also produce amylase. As diastase, amylase was the first
enzyme to be discovered and isolated. All amylases are glycoside hydrolases and act on
α(1-4) glycosidic bonds. It will start to denature at around 600C.
CLASSIFICATION OF AMYLASES
α-Amylase
The α-amylases are calcium metalloenzymes, completely unable to function
in the absence of calcium. By acting at random locations along the starch chain, α-
amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and
maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin.
Because it can act anywhere on the substrate, α-amylase tends to be faster-acting than β-
amylase. In animals, it is a major digestive enzyme and its optimum pH is 6.7-7.0.
In human physiology, both the salivary and pancreatic amylases are α-Amylases.
They are also found in plants (adequately), fungi (ascomycetes and basidiomycetes) and
bacteria (Bacillus).
β-Amylase
Another form of amylase, β-amylase is also synthesized by bacteria, fungi,
and plants. Working from the non-reducing end, β-amylase catalyzes the hydrolysis of
the second α-1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time.
During the ripening of fruit, β-amylase breaks starch into maltose, resulting in the sweet
flavor of ripe fruit.
Both α-amylase and β-amylase are present in seeds; β-amylase is present prior to
germination, whereas α-amylase and proteases appear once germination has begun.
Cereal grain amylase is key to the production of malt. Many microbes also produce
amylase to degrade extracellular starches. Animal tissues do not contain β-amylase,
although it may be present in microrganisms contained within the digestive tract.
γ-Amylase
In addition to cleaving the last α(1-4)glycosidic linkages at the non reducing
end of amylose and amylopectin, yielding glucose, γ-amylase will cleave α(1-6)
glycosidic linkages. Unlike the other forms of amylase, γ-amylase is most efficient in
acidic environments and has an optimum pH of 3.
Uses
Amylase enzymes finds use in bread making and to break down complex sugars
such as starch (found in flour) into simple sugars. Yeast then feeds on these simple sugars
and converts it into the waste products of alcohol and CO 2. This imparts flavour and
causes the bread to rise. While Amylase enzymes are found naturally in yeast cells, it
takes time for the yeast to produce enough of these enzymes to break down significant
quantities of starch in the bread. This is the reason for long fermented doughs such as
sour dough. Modern bread making techniques have included amylase enzymes (often in
the form of malted barley) into bread improver thereby making the bread making process
faster and more practical for commercial use.
When used as a food additive Amylase may be derived from swine pancreas or
mould mushroom.
Bacilliary amylase is also used in clothing and dishwasher detergents to dissolve
starches from fabrics and dishes.
Workers in factories that work with amylase for any of the above uses are at
increased risk of occupational asthma. 5-9% of bakers have a positive skin test, and a
fourth to a third of bakers with breathing problems are hypersensitive to amylase.
An inhibitor of alpha-amylase called phaseolamin has been tested as a potential
diet aid.
Blood serum amylase may be measured for purposes of medical diagnosis. A
normal concentration is in the range 21-101 U/L. A higher than normal concentration
may reflect one of several medical conditions, including acute inflammation of the
pancreas, macroamylasemia, perforated peptic ulcer, and mumps. Amylase may be
measured in other body fluids, including urine and peritoneal fluid.
PRODUCTION
Both concentrates of α and β amylases are prepared and used in a variety of
ways. These enzyme preparations must be carefully standardized for activity, according
to the purpose for which they are to be used.
α-Amylases are produced by the use of fungai (i.e. Aspergillus niger and
Aspergillus oryzae) as well as bacteria (i.e. Bacillus amyloliquefaciens and Bacillus
lichenformis). Therefore, α-Amylases are called either fungal α-amylases or bacterial
α-amylases according to the nature of the microorganism used for their production.
FUNGAL α-AMYLASE PRODUCTION
The α-Amylases producing fungai are grown on wheat bran (semisolid
culture). It is also possible to produce fungal α-Amylases by submerged culture,
employing the following medium,
Cornstarch 24 gm/ltr
 Corn-steep liquor 36 gm/ltr
KCl 0.2 gm/ltr
Na2HPO4 47 gm/ltr
CaCl2 1 gm/ltr
MgCl2.6H2O 0.2 gm/ltr
There is a problem for aeration and agitation because of a very high
viscosity of the medium due to the presence of mycelial body. Amylases biosynthesis is
inhibited when the medium contains glucose.

BACTERIAL α-AMYLASE PRODUCTION

Bacterial α-amylases are produced by the above mentioned two bacterial
species. Bacterial amylase is produced only by submerged-culture using the following
medium,
Soya bean meal 1.85%
Amber BYF 1.50%
Distillers dried solubles 0.76%
N-Z amine 0.65%
Lactose 4.75%
MgSO4.7H2O 0.04%
Antifoam agent 0.05%
Water 90.40%

A temperature in the range of 30-400C is satisfactory. The optimum pH for

the fermentation medium is 7.0. It is necessary to maintain the pH near neutrality, since
the amylase is denatured below 6.0. calcium carbonate is used as the buffer to maintain
neutral pH. The production of α-amylase begins when the bacterial count reaches 10 9-1010
cells per milliliter after about 10-20 hours and continues for another 100-150 hours.
Preservation of liquid preparations of bacterial α-amylase is done by 20% sodium
chloride. The most active preparations contain 2% active amylase protein. On the other
hand, the most active solid preparations contain 5% active amylase protein.
4.1.3 PRODUCTION OF LIPASE
A lipase is a water-soluble enzyme that catalyzes the hydrolysis of ester
bonds in water–insoluble, lipid substrates. Lipases thus comprise a subclass of the
esterases.
Lipases perform essential roles in the digestion, transport and processing of
dietary lipids (e.g. triglycerides, fats, oils) in most, if not all, living organisms. Genes
encoding lipases are even present in certain viruses.
Lipases are produced by plants, animals, bacteria and moulds. Plant
enzymes are not used commercially, while animal, bacterial and mould enzymes are used
extensively. Most important animal sources are cattle, sheep, pigs and dog pancreatic
lipases are most widely used.
Most lipases have an alkaline pH between 8-9. A few microbial lipases
prefer more hydrogen ion having a pH of 5-6. The optimum temperature is 30-40 0C;
however, it should be recognized that most enzymes stop catalysis at -10 0C because
liquid substrates become solid ice. Lipases act on solid emulsion; so the fats degraded
become rancid in the freezer. As indicated, lipases act on insoluble substrates and they
have next to no activity against soluble substrates; so the insoluble substrate must activate
the enzyme.

THE MICROORGANISM
Among bacteria Achromobacter sp., Acaligenes sp., Pseudomonas sp.,
Staphylococcus sp., and Chromobacterium sp. Have been exploited for the production of
lipases.
The chief fungal producers of commercial lipases are Aspergillus niger,
candida cylindracea, Humicola lanuginose, Mucor miehei, Rhisopus arrhizus, R.delemer,
R.japonicus, R.niveus and R.oryzae.

Structure and Catalytic Mechanism

While a diverse array of genetically distinct lipase enzymes are found in nature,
and represent several types of protein folds and catalytic mechanisms, most are built on
an alpha/beta hydrolase fold and employ a chymotrypsin-like hydrolysis mechanism
involving a serine nucleophile, an acid residue (usually aspartic acid), and a histidine.
Industrial uses
Lipases from fungi and bacteria serve important roles in human practices as
ancient as yogurt and cheese fermentation. However, lipases are also being exploited as
cheap and versatile catalysts to degrade lipids in more modern applications. For instance,
a biotechnology company has brought recombinant lipase enzymes to market for use in
applications such as baking, laundry detergents and even as biocatalysts in alternative
energy strategies to convert vegetable oil into fuel.
Medical applications
The inhibition of the human enzyme as a method of reducing fatty acid absorption
is being investigated for treatment for obesity. Lipases are used to acylate and deacylate
the hydroxyl group of castanospermine, a promising drug for the treatment of AIDS.
PRODUCTION
Microbial lipases are produced mostly by submerged culture, but solid state
fermentation methods can also be used. Immobilized cell culture has been used in few
cases. Many studies have been undertaken to define the optimal culture and nutritional
requirements for lipase production by submerged culture. Lipase production is influenced
by the type and concentration of carbon and nitrogen sources, the culture pH, the growth
temperature and the dissolved oxygen concentration. Lipid carbon sources seem to be
generally essential for obtaining a high lipase yield; however, a few authors have
produced good yields in the absence of fats and oils. For example, it has been observed
that the production of lipase enzyme from bacillus species in 1% olive oil in the culture
medium was normal. But the enzyme activity is observed in the absence of olive oil even
after prolonged cultivation. Fructose and palm oil were reported to be the best
carbohydrate and lipid sources, respectively for the production of an extracellular lipase
by Rhodotorula glutinis.
For nitrogen source, the peptone that is present in the culture mediumgives
considerable results in the extracellular lipase production. Nitrogen sources such as corn
steep liquor and soya bean meal stimulated lipase production but to a lesser extent than
peptone. Urea and ammonium sulphate inhibited lipase synthesis.
Lipase production was higher when metal ions such as magnesium, iron and
calcium were added to the production medium. Besides, the production of an
extracellular lipase is also enhanced when the medium was supplemented with mg 2+,
Ca2+, Cu2+ and Co2+.
COMPOSITION OF THE PRODUCTION MEDIA
Ammonium chloride 35 gm/ltr
Glycerol 10ml/ltr
K2HPO4 3 gm/ltr
KH2PO4 1 gm/ltr
MgSO4 0.1 gm/ltr
Glucose 2 gm/ltr
MgCl2 0.6 mM gm/ltr
Bacterial inoculums are prepared by growing the cells at 37 0C for 10 hours in
500ml shake flasks containing 100ml of LB medium. Lipase production is performed in a
tank fermenter. The fermentation condition is a three step procedure: batch, fed batch and
repeated fed batch. The conditions used in the batch phase are agitator speed 400rpm,
aeration rate 1vvm, temperature 37 0C and optimum pH control. Cell concentration is
measured by turbidimetry(600nm) and correlated with dry cell weight.
In solid state fermentation of fungai, the cell suspension is prepared as follows.
The organism is subcultured on potato dextrose agar medium and incubated for 72 hours
at 280C. The spore crop of each slant is scrapped into 5ml sterile water and shaken well
with sterile glass beads on a rotatory shaker for 30mins to break the spore chains and to
make uniform suspension. This suspension is filtered through sterile cotton to remove the
hyphal filaments. This spore suspension (5X106 spores per ml) is used as inoculum.
For the production of SSF, wheat bran moistened with mineral salt medium and oil
is sterilized at 1210C for 30 mins. After cooling, the substrates are inoculated with spore
suspension containing 106 spores from 7 day old culture. The medium is incubated at
300C for appropriate time.
4.1.4PRODUCTION OF CELLULASE
Cellulase refers to a class of enzymes produced chiefly by fungi, bacteria, and
protozoans that catalyze the cellulolysis (or hydrolysis) of cellulose. However, there are
also cellulases produced by other types of organisms such as plants and animals. Several
different kinds of cellulases are known, which differ structurally and mechanistically.
The EC number for this group of enzymes is EC 3.2.1.4.
Types and action
Five general types of cellulases based on the type of reaction catalyzed:
Endo-cellulase breaks internal bonds to disrupt the crystalline structure of
cellulose and expose individual cellulose polysaccharide chains
Exo-cellulase cleaves 2-4 units from the ends of the exposed chains produced by
endocellulase, resulting in the tetrasaccharides or disaccharide such as cellobiose. There
are two main types of exo-cellulases (or cellobiohydrolases, abbreviate CBH) - one type
working processively from the reducing end, and one type working processively from the
non-reducing end of cellulose.
Cellobiase or beta-glucosidase hydrolyses the exo-cellulase product into individual
monosaccharides.
Oxidative cellulases that depolymerize cellulose by radical reactions, as for
instance cellobiose dehydrogenase (acceptor).
Cellulose phosphorylases that depolymerize cellulose using phosphates instead of
water.
In the most familiar case of cellulase activity, the enzyme complex breaks down
cellulose to beta-glucose. This type of cellulase is produced mainly by symbiotic bacteria
in the ruminating chambers of herbivores. Aside from ruminants, most animals (including
humans) do not produce cellulase in their bodies, and are therefore unable to use most of
the energy contained in plant material. Enzymes which hydrolyze Hemicellulose are
usually referred to as hemicellulase and are usually classified under cellulase in general.
Enzymes that cleave lignin are occasionally classified as cellulase, but this is usually
considered erroneous.
Within the above types there are also progressive (also known as processive) and
non-progressive types. Progressive cellulase will continue to interact with a single
polysaccharide strand, non-progressive cellulase will interact once then disengage and
engage another polysaccharide strand.
Most fungal cellulases have a two-domain structure with one catalytic domain, and
one cellulose binding domain, that are connected by a flexible linker. This structure is
adaption for working on an insoluble substrate and it allows the enzyme to diffuse two-
dimensionally on a surface in a caterpillar way. However, there are also cellulases
(mostly endoglucanases) that lacks cellulose binding domain. These enzymes might have
a swelling function.
Mechanism of cellulolysis
The three types of reaction catalyzed by cellulases:
1. Breakage of the non-covalent interactions present in the crystalline structure of
cellulose (endo-cellulase)
2. Hydrolysis of the individual cellulose fibers to break it into smaller sugars (exo-
cellulase)
3. Hydrolysis of disaccharides and tetrasaccharides into glucose (beta-
glucosidase).
Uses
Cellulase is used for commercial food processing in coffee. It performs hydrolysis
of cellulose during drying of beans. Furthermore, cellulases are widely used in textile
industry and in laundry detergents. They have also been used in the pulp and paper
industry for various purposes, and they are even used for pharmaceutical applications.
Cellulase is used in the fermentation of biomass into biofuels, although this process is
relatively experimental at present. Cellulase is used as a treatment for Phytobezoars, a
form of cellulose bezoar found in the human stomach.
PRODUCTION
Many bacteria and fungai are cellulolytic, but preparations marketed for industrial
applications are derived only from Apergillus niger, Trichoderma viride, Neurospora and
some other organism. The Aspergillus enzyme exerts good activity on
carboxymethylcellulose (CMC), but fails to attack solid cellulose because it lacks C1-
cellulase. In contrast, Trichoderma viride produces an enzyme complex with high levels
of C1-cellulase, which extensively degrades insoluble cellulose.
Cellulase in fungai is an inducible enzyme. It is only produced when the cells are
grown on cellulose, on glucans of mixed linkages including the beta 1-4 bond and on a
few oligosaccharides. The inducing effect of cellulose is due to soluble hydrolysis
products of the beta 1-4 galactoside and sophorose, a beta 1-2 glucoside are the only
known cellulase inducers that do not have a beta 1-4 glucoside bond. The inductive
action of sophorose is limited to trichoderma viride. However, inspite of the impressive
inductive power of this rare sugar,the levels of enzyme produced are not equal to those on
cellulose.
In lower concentration (0.1%), cellobiose serves as an inducer of cellulase but, in
higher concentrations (0.5-1%) it represses cellulase formation and in addition it can also
act as an inhibitor of cellulase action.
Cellulase yields can be increased by various additives to the medium. Some
experiments have explained that, some surfactants like Tween80 and Tween40 doubled
the yield in Trichoderma cultures. The mechanism of action of these surfactants is not
understood clearly but may be related to increased permeability of the cell membrane.
In actual large scale fermentation Aspergillus niger ismostly cultured by the wheat
bran-tray method. This process has no problems and leads to high yields of cellulase. The
extraction of cellulase from solid substrate cultures is performed by percolation of the
dried mold bran with 0.02-0.1M lactic acid.
Neurospora and Trichoderma are grown by submerged culture. For continuous
culture it is advantageous that Trichoderma viride produces a suspension of short
mycelial threads, rarely forming pellets. Some scientists proposed a 2 stage operation in
continuous stirred tank reactors in which first stage utilizes glucose for biomass
production and the second stage utilizes pure spruce wood cellulose for enzyme
formation. A significant increase in enzyme productivity was obtained.
Bran, straw and other plant materials pretreated with alkali serve as cellulose
containing raw materials for submerged cultivations. Ammonium ions can be used as
suitable N source. A correct pH profile (3.0-3.5 for Trichoderma viride) is necessary to
give optimal enzyme yield in batch culture.
Recovery
Concentration and purification of the enzyme is carried out by precipitation,
adsorption or gel filtration techniques. Granulated preparations can be obtained by
mixing with salt hydrates and subsequent vacuum drying.

4.2.0 PRODUCTIN OF OTHER BIOPRODUCTS

4.2.1 Biological pesticide
The term biopesticide is used for microbial biological pest control agents that are
applied in a similar manner to chemical pesticides. Commonly these are bacterial, but
there are also examples of fungal control agents, including Trichoderma spp. and
Ampelomyces quisqualis (a control agent for grape powdery mildew). Bacillus subtilis
are used to control plant pathogens.
Biological insecticides include products based on:
Entomopathogenic fungi (e.g. Metarhizium anisopliae),
Entomopathogenic nematodes (e.g. Steinernema feltiae) and
Entomopathogenic viruses (e.g. Cydia pomonella granulovirus).
Biopesticides, key components of integrated pest management (IPM) programs,
are receiving much practical attention as a means to reduce the load of synthetic chemical
products being used to control plant diseases. In most cropping systems, biopesticides
should not necessarily be viewed as wholesale replacements for chemical control of plant
diseases, but rather as a growing category of efficacious supplements that can be used as
rotation agents to retard the onset of resistance to chemical pesticides and improve
sustainability. In organic cropping systems, biopesticides can represent valuable tools that
further supplement the rich collection of cultural practices that ensure against crop loss to
diseases.
Biopesticides for use against crop diseases have already established themselves on
a variety of crops. For example, biopesticides already play an important role in
controlling downy mildew diseases. Their benefits includes the ability to use under
moderate to severe disease pressure, and the ability to use as a tank mix or in a rotational
program with other registered fungicides.
A major growth area for biopesticides is in the area of seed treatments and soil
amendments. Fungicidal and biofungicidal seed treatments are used to control soil borne
fungal pathogens that cause seed rots, damping-off, root rot and seedling blights. They
can also be used to control internal seed–borne fungal pathogens as well as fungal
pathogens that are on the surface of the seed. Many biofungicidal products also show
capacities to stimulate plant host defenses and other physiological processes that can
make treated crops more resistant to a variety of biotic and abiotic stresses.
Advantages of biopesticides
1. Permenancy
2. Economical feasible
3. Environment safety
4. Absebce of development of resistance
Perceived disadvantages
High specificity, which will require an exact identification of the pest/pathogen
and may require multiple pesticides to be used often slow speed of action (thus making
them unsuitable if a pest outbreak is an immediate threat to a crop) often variable efficacy
due to the influences of various biotic and abiotic factors (since biopesticides are usually
living organisms, which bring about pest/pathogen control by multiplying within the
target insect pest/pathogen)
Biopesticide Production
Two processes are used commercially in the mass production of Bacillus
Thuringiensis:
(a) Submerged fermentation
(b) Semisolid fermentation
Submerged fermentation
Inoculum is prepared by passing B.thuringiensis cells through two media in
shake flasks and then to a seed fermentor. Nutrient broth can be used for the first flask.
The following medium is advisable for use in the second flask as well as seed fermentor,
- Beet molasses 1%
- Cornsteep solids 0.85%
- Calcium carbonate 1%
Each flask is to be incubated for 24hours. The seed tank is incubated until
vigorous vegetative growth is obtained. A typical production contains,
- Beet molasses 1.86%
- Cotton seed flour 1.40%
- Corn steep liquor 1.70%
- Calcium carbonate 0.10%
The production medium is designed so that the fermentable carbohydrate
and nitrogen available for growth present are exhausted at approximately the same time
after the commencement of sporulation. Apart from these constituents, K 2HPO4, KH2PO4,
Magnesium sulphate, ferrous sulphate, zinc sulphate and calcium carbonate wherever
required. The proflo and Soyabean meal media would be economical for mass production
of Bacillus thuringiensis.
 Submerged fermentation have been mainly used to produce flowable formulations
of the spore-crystal complex. These can be used as sprays, but, of course, are not
available as dusts. In the past, liquid formulations of the complex tended to deteriorate
and required refrigerated storage. At present, these preparations have been stabilized and
can be stored at room temperature.
Recovery:
Whole beer (pH 8.4-8.7)

Adjust to pH 7.0 with HCl

Discard supernatant Centrifuge

Suspend in 0.1-0.5 vol. 4-6% lactose

Stir 30 mins

Add slowly while stirring 4 vol. of acetone

Stirr 30 mins

Allow to stand for 10 mins

Discard filtrate Filter with suction

Residue

Stirr with small volume of acetone

Discard filtrate Filter with suction

Residue
Cont…..
Residue

Stirr with small volume of acetone

Discard filtrate Filter with suction

Residue

Allow to dry overnight at room temperature

BACILLUS THURINGIENSIS (Bacterial Insecticide)

The bacteria Bacillus thuringiensis (Bt) represents a major class of microbes used
for insect biocontrol. This Bt is used to kill wide range of insects like, moths, beetle,
mosquetoes, flies, insects, ants, butterflies and even some pathogenic fungai.

MECHANISM OF ACTION
Bt produce both exo and endotoxin. Most Bt strain endotoxin is a protein and is
called parasporal crystal. They are specific in their lethal activity against different insect
pests and not harmful to mammals, birds or beneficial insects. For this reason, Bt is being
exploited as an alternative to chemical pesticides. Usually these crystals will always be in
the inactive form. These crystalline proteins upon ingestion by the insect larvae are
solubilised under high alkaline conditions in midgut. The toxins are digested by the
enzyme called protease and converted into active fragments. These active fragments bind
to receptor proteins present in the gut epithelial membrane and those forms pores in the
gut membrane. As a result, osmotic equilibrium of the cell is disturbed and the cells
swells and burst. This result in the death of insect larvae. Initial results in laboratory have
shown that municipal wastewater sludge can support the growth of Bt and produce spore
and endotoxins. The toxicity of the spores and endotoxins were evaluated on spruce
budworm 3rd instar larvae. The entomotoxicity produced by different stains of Bt using
wastewater sludge as growth media was higher as compared to the entomotoxicity
produced using soy based commercial growth media.
VIRUS INSECTICIDES
Viruses of the family Baculoviridae are pathogenic to arthoropos viruses
contain lipid envelopes with circular double stranded DNA genome. Naked viral DNA is
infectious. Baculo virus are restricted on their host ranges. They do not infect vertebrates
non artho pod. They infect only a few arthropod species usually saw flies and lepi
elopterasps.

4.2.2 PRODUCTION OF BIOFERTILIZERS

'Biofertilizer' is a substance which contains living microorganisms which,
when applied to seed, plant surfaces, or soil, colonizes the rhizosphere or the interior of
the plant and promotes growth by increasing the supply or availability of primary
nutrients to the host plant.
Biofertilizers are not fertilizers. Fertilizers directly increase soil fertility by adding
nutrients. Biofertilizers add nutrients through the natural processes of fixing atmospheric
nitrogen, solubilizing phosphorus, and stimulating plant growth through the synthesis of
growth promoting substances. Biofertilizers can be expected to reduce the use of
chemical fertilizers and pesticides. The microorganisms in biofertilizers restore the soil's
natural nutrient cycle and build soil organic matter. Through the use of biofertilizers,
healthy plants can be grown while enhancing the sustainability and the health of soil.
These living organisms are free living bacteria that grow well on a nitrogen
free medium. These bacteria utilize atmospheric nitrogen for cell protein synthesis. As
soon as they die, this cell protein is mineralized in soil, thereby contributing towards the
nitrogen availability of crop plants. Azetobacter are known to fix on an average 10mg
N/gm of sugar in pure culture on a nitrogen free medium.
STEPS:
1. In a 1 ltr conical flask, 500ml broth of nitrogen free medium is added and
sterilized at 75lbs pressure. Then inoculated with mother culture aseptically and the flask
is transferred to shaker for 72-90 hours to get optimum growth. After 90 days, the
number per milliliter comes to 100 crores
2. Lignite is a substance that has high organic matter and water holding
capacity and supports the growth of organism. Lignite is ground into a fine powder with a
grinding machine. pH is adjusted with calcium carbonate and lignite is sterilized at 30lbs
for 30 mins. After this, the broth is mixed with lignite. After proper mixing using
galvanized trays, this mixture is filled in plastic bags of 250-500gm capacity.
3. As per the Bureau of Indian Standards, one gram biofertilizer should
have 10lakh bacteria. If it is stored at 15-200C, it will remain active for 6 months and for
2 years if it is stored at 0-40C

BENEFITS
1. Cost effective and increase crop yield upto 10-30%
2. As supplement, replaces chemical fertilizers upto 25%
3. Stimulate plant growth
4. Biologically activate the soil
5. Protects natural fertility of the soil
6. Provide protection against some soil borne diseases

TYPES

It is estimated that more than 100million tons of fixed nitrogen are needed for
global food production. The use of chemical/synthetic fertilizers is the common practice
to increase crop yields. Besides the cost factor, the use of fertilizers is associated with
environmental pollution.

This biofertilizers can be classified into,

1. Symbiotic nitrogen fixers
2. Asymbiotic nitrogen fixers
3. Phosphate solubilising bacteria
4. Organic fertilizers
SYMBIOTIC NITROGEN FIXERS
The Diazotrophic microorganisms are the symbiotic nitrogen fixers that
serve as biofertilizers.
Ex., Rhizobium sp and Bradyrhizobium sp.
The details on these bacteria with special reference to nitrogen fixation must be
referred now. Many attempts are being made to genetically modify the symbiotic bacteria
for improving nitrogen fixation.

Green Manuring: It is a farming practice where in the leguminous plants which

are benefited by the symbiotic nitrogen fixing bacteria are ploughed into the soil and a
non leguminous crop is grown to taken from the already fixed nitrogen. Green manuring
has been in practice in India for several centuries. It is a natural way of enriching the soil
with nitrogen and minimizing the use of chemical fertilizers. Rhizobium sp can fix about
50-100Kg nitrogen/hectare/annum.

ASYMBIOTIC NITROGEN FIXERS

The asymbiotic nitrogen fixing bacteria can directly convert the gaseous
nitrogen to nitrogen rich compounds. When these asymbiotic nitrogen fixers die, they
enrich the soil with nitrogenous compounds and thus serve biofertilizers.
Example: Azobacter sp and Azospirillum sp

Azolla: Azolla is an aquatic form which contains an endophytic cyanobacterium

Anabaena azollae in the leaf cavities providing a symbiotic relationship. Azolla with
Anaebaena is useful as biofertilizer. But, due to certain limitation, the use of Azolla has
not become popular.
- Azolla plant requires adequate supply of water
- It can be easily damaged by pest disease
- Azolla cultivation is labour intensive

PHOSPHATE SOLUBILISING BACTERIA

Certain bacteria are capable of converting non available inorganic
phosphorous present in the soil to utilizable (organic or inorganic) form of phosphate.
These bacteria can also produce sidorophores which chelates with bacteria. Thus, besides
making phosphate available, the plants are protected from disease causing
microorganisms.
Example: Thiobacillus sp and Bacillus sp.

Mycorrhiza: Mycorrhiza are the fungus roots with distinct morphological

structure. They are developed as a result of mutual symbiosis between certain root
inhabitating fungai and plant roots. Mycorrhizae are formed in plants which are limited
with nutrient supply. These plants may be herbs, shrubs and trees. For the development of
mycorrhizas, the fungus may be located on the root surface (ecto mycorrhiza) or inside
the root (endo mycorrhizas)
Example: Glomus sp.
LIMITATIONS:
1. Biofertilizers cannot meet total needs of the plants for nutritional supply.
2. They cannot produce produce spectacular results, as is the case with
synthetic fertilizers

4.2.3 PRODUCTION OF BIOPRESERVATIVE - NISIN

Nisin is a polycyclic peptide antibacterial with 34 amino acid residues used as a

food preservative. It contains the uncommon amino acids lanthionine (Lan),
methyllanthionine (MeLan), didehydroalanine (Dha) and didehydroaminobutyric acid
(Dhb). These unusual amino acids are introduced by post translational modification of the
precursor peptide. In these reactions a ribosomally synthesized 57-mer is converted to the
final peptide. The unsaturated amino acids originate from serine and threonine, and the
enzyme-catalysed addition of cysteine residues to the didehydro amino acids result in the
multiple thioether bridges.

PRODUCTION
Nisin is produced by fermentation using the bacterium Lactococcus lactis.
Commercially, it is obtained from the culturing of Lactoccus lactis on natural substrates,
such as milk or dextrose, and is not chemically synthesized.
It is used in processed cheese, meats, beverages, etc. during production to extend
shelf life by suppressing Gram-positive spoilage and pathogenic bacteria. While most
bacteriocins generally inhibit only closely related species, Nisin is a rare example of a
"broad-spectrum" bacteriocin effective against many Gram-positive organisms, including
lactic acid bacteria (commonly associated with spoilage), Listeria monocytogenes (a
known pathogen), etc.
However, when coupled with the chelator EDTA, Nisin has also been known to
inhibit Gram-negative bacteria, as well.
Nisin is soluble in water and can be effective at levels nearing the parts per billion
range. In foods, it is common to use Nisin at levels ranging from ~1-25ppm, depending
on the food type and regulatory approval.
Due to its naturally selective spectrum of activity, it is also employed as a selective
agent in microbiological media for the isolation of gram-negative bacteria, yeast, and
moulds. Subtilin and Epidermin are related to Nisin. All are members of a class of
molecules known as lantibiotics.

4.2.3 CHEESE MAKING

The word cheese ultimately comes from Latin caseus,[2] from which the
modern word casein is closely derived. The earliest source is from the proto-Indo-
European root *kwat-, which means "to ferment, become sour".

Cheese is a generic term for a diverse group of milk-based food products. Cheese is
produced throughout the world in wide-ranging flavors, textures, and forms.
Cheese consists of proteins and fat from milk, usually the milk of cows, buffalo, goats, or
sheep. It is produced by coagulation of the milk protein casein. Typically, the milk is
acidified and addition of the enzyme rennet causes coagulation. The solids are separated
and pressed into final form. Some cheeses have molds on the rind or throughout. Most
cheeses melt at cooking temperature.
Hundreds of types of cheese are produced. Their styles, textures and flavors
depend on the origin of the milk (including the animal's diet), whether they have been
pasteurized, the butterfat content, the bacteria and mold, the processing, and aging.
Herbs, spices, or wood smoke may be used as flavoring agents. The yellow to red color
of many cheeses is from adding annatto.
For a few cheeses, the milk is curdled by adding acids such as vinegar or lemon
juice. Most cheeses are acidified to a lesser degree by bacteria, which turn milk sugars
into lactic acid, and then the addition of rennet completes the curdling. Vegetarian
alternatives to rennet are available; most are produced by fermentation of the fungus
Mucor miehei, but others have been extracted from various species of the Cynara thistle
family.
Cheese is valued for its portability, long life, and high content of fat, protein,
calcium, and phosphorus. Cheese is more compact and has a longer shelf life than milk.
Cheesemakers near a dairy region may benefit from fresher, lower-priced milk, and lower
shipping costs. The long storage life of some cheese, especially if it is encased in a
protective rind, allows selling when markets are favorable.
Etymology
HISTORY
Cheese is an ancient food whose origins predate recorded history. There is no
conclusive evidence indicating where cheese making originated, either in Europe, Central
Asia or the Middle East, but the practice had spread within Europe prior to Roman times
and, according to Pliny the Elder, had become a sophisticated enterprise by the time the
Roman Empire came into being.
Proposed dates for the origin of cheese making range from around 8000 BC
(when sheep were first domesticated) to around 3000 BC. The first cheese may have been
made by people in the Middle East or by nomadic Turkic tribes in Central Asia. Since
animal skins and inflated internal organs have, since ancient times, provided storage
vessels for a range of foodstuffs, it is probable that the process of cheese making was
discovered accidentally by storing milk in a container made from the stomach of an
animal, resulting in the milk being turned to curd and whey by the rennet from the
stomach. There is a legend with variations about the discovery of cheese by an Arab
trader who used this method of storing milk.
Cheese making may have begun independently of this by the pressing and
salting of curdled milk in order to preserve it. Observation that the effect of making milk
in an animal stomach gave more solid and better-textured curds, may have led to the
deliberate addition of rennet.
The earliest archeological evidence of cheese making has been found in
Egyptian tomb murals, dating to about 2000 BCE. The earliest cheeses were likely to
have been quite sour and salty, similar in texture to rustic cottage cheese or feta, a
crumbly, flavorful Greek cheese.
Cheese produced in Europe, where climates are cooler than the Middle East,
required less salt for preservation. With less salt and acidity, the cheese became a suitable
environment for useful microbes and molds, giving aged cheeses their pronounced and
interesting flavors.
Modern era
Until its modern spread along with European culture, cheese was nearly
unheard of in oriental cultures, in the pre-Columbian Americas, and only had limited use
in sub-Mediterranean Africa, mainly being widespread and popular only in Europe and
areas influenced strongly by its cultures. But with the spread, first of European
imperialism, and later of Euro-American culture and food, cheese has gradually become
known and increasingly popular worldwide, though still rarely considered a part of local
ethnic cuisines outside Europe, the Middle East, and the Americas.
The first factory for the industrial production of cheese opened in Switzerland
in 1815, but it was in the United States where large-scale production first found real
success. Credit usually goes to Jesse Williams, a dairy farmer from Rome, New York,
who in 1851 started making cheese in an assembly-line fashion using the milk from
neighboring farms. Within decades hundreds of such dairy associations existed.
The 1860s saw the beginnings of mass-produced rennet, and by the turn of the century
scientists were producing pure microbial cultures. Before then, bacteria in cheesemaking
had come from the environment or from recycling an earlier batch's whey; the pure
cultures meant a more standardized cheese could be produced.
Factory-made cheese overtook traditional cheesemaking in the World War II era, and
factories have been the source of most cheese in America and Europe ever since. Today,
Americans buy more processed cheese than "real", factory-made or not.[12]
Making cheese
Milk
Acidification Lactic acid bacteria
Acidified Milk
Coagulum formation Rennet (chymosin)
Curd (coagulum)
Seperation Removal of whey
Curd subjected to salting
Ripening Protease, Lipases or microorganisms
Cheese
A required step in cheesemaking 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 35–55 °C
(95–131 °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 roles in cheese besides adding a salty flavor. It preserves cheese
from spoiling, draws moisture from the curd, and firms 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.
Other techniques influence a cheese's texture and flavor. Some examples:
 Stretching: (Mozzarella, Provolone) 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 time,
taking the sharp edges off the cut curd pieces and influencing the final product's
texture.
 Washing: (Edam, Gouda, and Colby) 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.
List of cheeses
Factors which are relevant to the categorization of cheeses include:
 Length of aging
 Texture
 Methods of making
 Fat content
 Kind of milk
 Country/Region of Origin
No one categorization scheme can capture all the diversity of the world's cheeses.
In practice, no single system is employed and different factors are emphasised in
describing different classes of cheeses.

NUTRITIONAL VALUE
In general, cheese supplies a great deal of calcium, protein, phosphorus and
fat. A 30-gram (1.1 oz) serving of Cheddar cheese contains about 7 grams (0.25 oz) of
protein and 200 milligrams of calcium. Nutritionally, cheese is essentially concentrated
milk: it takes about 200 grams (7.1 oz) of milk to provide that much protein, and
150 grams (5.3 oz) to equal the calcium.
 The calcium, protein, and phosphorus in cheese may act to protect
tooth enamel.
 Cheese increases saliva flow, washing away acids and sugars.
 Cheese may have an antibacterial effect in the mouth.
Compulsory pasteurization is controversial. Pasteurization does change the
flavor of cheeses, and unpasteurized cheeses are often considered to have better flavor, so
there are reasons not to pasteurize all cheeses. Some say that health concerns are
overstated, pointing out that milk pasteurization does not ensure cheese safety. This is
supported by statistics showing that in some European countries where young raw-milk
cheeses may legally be sold, most cheese-related food poisoning incidents were traced to
pasteurized cheeses.
4.3.0 PRODUCTION OF BIOPOLYMERS
Biopolymers are polymers produced by living organisms. Cellulose, starch
and chitin, proteins and peptides, and DNA and RNA are all examples of biopolymers, in
which the monomeric units, respectively, are sugars, amino acids, and nucleotides.
Cellulose is both the most common biopolymer and the most common organic compound
on Earth. About 33 percent of all plant matter is cellulose (the cellulose content of cotton
is 90 percent and that of wood is 50 percent)
Biopolymers versus polymers
A major but defining difference between polymers and biopolymers can be
found in their structures. Polymers, including biopolymers, are made of repetitive units
called monomers. Biopolymers often have a well defined structure, though this is not a
defining characteristic (example:ligno-cellulose): The exact chemical composition and
the sequence in which these units are arranged is called the primary structure, in the case
of proteins. Many biopolymers spontaneously fold into characteristic compact shapes
(see also "protein folding" as well as secondary structure and tertiary structure), which
determine their biological functions and depend in a complicated way on their primary
structures. Structural biology is the study of the structural properties of the biopolymers.
In contrast most synthetic polymers have much simpler and more random (or stochastic)
structures. This fact leads to a molecular mass distribution that is missing in biopolymers.
In fact, as their synthesis is controlled by a template directed process in most in vivo
systems all biopolymers of a type (say one specific protein) are all alike: they all contain
the similar sequences and numbers of monomers and thus all have the same mass. This
phenomenon is called monodispersity in contrast to the polydispersity encountered in
synthetic polymers. As a result biopolymers have a polydispersity index of 1.
Some biopolymers- such as polylactic acid (PLA), naturally occurring zein, and
poly-3-hydroxybutyrate can be used as plastics, replacing the need for polystyrene or
polyethylene based plastics.
Biopolymers (also called renewable polymers) are produced from biomass for use
in the packaging industry. Biomass comes from crops such as sugar beet, potatoes or
wheat: when used to produce biopolymers, these are classified as non food crops.
 Xanthan gum is a polysaccharide used as a food additive and rheology modifier. It
is produced by fermentation of glucose or sucrose by the Xanthomonas campestris
bacterium.
4.3.1 XANTHAN GUM
Xanthan are more frequently referred to as xanthan gum was the first
polysaccharide available commercially. It is well studied and most widely used
hexopolysaccharide.
APPLICATIONS
1. Xanthan gum is used as a food additive for the preparation of the food.
2. It is used in oil industry for enhancing oil recovery
3. Xanthan is useful for the preparation of tooth paste & water based paint.
BIOSYNTHESIS:
1. The monomer are bound to a carrier lipid molecule and then transferred
to a growing polymer chain.
2. The activated monosaccharide nucleotide supply energy for the
formation of glycosidic bonds between adjacent unit.
3. The biosynthesis of other exopolysaccharide is comparable with that of
xanthan.
PRODUCTION:
 Xanthan is commercially produced by the gram negative bacterium.
 Culture media usually consist of 4-5% of carbohydrate and salt.
 The pH is maintained around 7.0
 Fermentation is carried out by batch culture for 2-3 days
 Xanthan in the culture broth is precipitated by isopropanol or
methanol.
 These agent is killing the microorganism.
 The precipitated xanthan is dried and used for commercial purpose.

GENETIC ENGG’ OF XANTHOMONAS

 The wild type X.camperris can efficiently utilize glucose glucose, sucrose or
starch as a carbon source.
 They are unable to use lactose as a carbon substrate.
  Whey is a byproduct obtained in the manufacture of cheese.
 Disposal of large quantities of whey is a major problem in dairy industry.
 Whey is rich in lactose based containing small quantities of protein, vitamins and
minerals. It is used in fermentation industries.
 Genetically engineered X.comparris have been developed that can utilize lactose
production of xanthan.
 The E.coli were cloned under the transcriptional control of X.comparris
bacteriophage promoter.
 This construct was first introduced into E.coli and then transferred into
X.comparris.
 It is an example of successfully converting a waste product into commercially
important and valuable product (xantham gum).

4.3.2 POLY HYDROXY BUTYRATE

The pathway for the biosynthesis of Polyhydroxy butyrate has been
thoroughly investigated starting with acetyl CoA.
PHB is synthesized in 3 reaction step.
Acetyl CoA is converted into acetoacetyl CoA by the enzyme 3ketothiolase
This 3-ketothiolase reduced to 33-hydroxybutyryl CoA by acetoacetyl CoA
reductase.
The reducing equivalents are supplied by NADPH.
The enzyme PHA synthase is responsible for the addition of 3-hydroxy
butyrate residues to the growing PHB chain.
APPLICATION
 PHB can be implanted in the human body without rejection.
 PHB does not produce any immune response and it is biocompatible.
 PHB has several medical applications as curable bone implant
 PHB is used as degradable structures and other implant has not met
with success due to slow degradation of PHB.
PRODUCTION
1. PHB is mostly manufactured by batch culture.
2. PHB production occurs when there is an excess supply of carbon source.
 3. The production occurs by the limitation of some other essential nutrient
such as nitrogen, phosporous or sulfur source.
4. There are two distinct phase.
1. Growth phase
2. Polymer accumulation phase
5. The growth phase ceases due to nutritional exhaustion, synthesis of
polymer commences.
6. It is also possible to produce PHB by restricting the oxygen supply to
aerobic bacteria.
4.4.0 SINGLE CELL PROTEIN
Selection of an appropriate microorganism is essential to the success of any
single-cell protein production undertaking. The microorganism must be one that is edible
and can serve as a feedstock for humans and/or livestock. Of course, its culture must be
technologically and economically feasible. To satisfy the second condition: 1) the
organisms must grow rapidly and vigorously; 2) culture of the organism should involve
the use of relatively simple growth units and inexpensive nutrient sources (e.g.,
commercial crop fertilisers); 3) ideally, the organism could be grown in open culture, or
at least as an enrichment culture; and 4) because single-cell protein production is only
marginally economically feasible, the least “permissible” condition is the need to culture
the organism under sterile conditions, i.e., as a completely pure culture. However,
competition with other organisms is eliminated in sterile culture and rapidity of growth is
thereby increased. Moreover, contamination with possibly toxic organisms is avoided.
Most of the work on single-cell protein production has been focused on the
yeast, Candida utilis (Torula utilis). The yeast meets most of the requirements named in
the preceding paragraph. Not only is the yeast easily grown, it also is a good food and
fodder yeast. Although sterility is necessary, purity of culture is not essential.
The high nucleic acid content of bacterial proteins renders them less desirable
as feedstuff for man and animal. Additionally, some groups of bacteria are characterised
by the possession of endotoxins. The endotoxins could be incorporated in the feedstuff
product. There is also a possibility that certain bacterial feedstuffs can promote allergenic
reactions in humans who handle or ingest them. Finally, the much smaller size of bacteria
makes them more difficult to harvest than yeasts.
 The relation of single-cell protein production to the reclamation of useful nutrient
elements in waste is by way of the utilisation of sugars formed through hydrolization of
cellulosic substances in municipal waste. However, a separate hydrolysis step may be
bypassed by culturing the yeast directly on the cellulosic waste.
Indirect production
The production of C. utilis is an example of the indirect approach. With respect to
nutritional requirements, the sugars (glucose) satisfy the carbon needs. The other required
essential nutritional elements are nitrogen, phosphorus, and potassium, which must come
from an external source. Usually, nitrogen is added as an ammonium compound (e.g.,
ammonium sulphate); a phosphate is used for phosphorus; and a potassium sulphate or
hydroxide compound for potassium. Generally, it is not necessary to add the essential
trace elements.
Principal cultural conditions are a temperature at 20° to 35°C; and O 2, about 1.02
kg/kg cell mass-produced. The necessarily aerobic conditions are attained by
continuously agitating the culture. The volume of air applied to meet the oxygen demand
would be a rate of about 120 millimoles O2 absorbed per L-hr (3.84 g/L-hr). The yield to
be expected at such a rate is 3.66 g yeast per L-hr.
Under proper cultural conditions, the yield of the cell mass should be from 45% to
55% of the sugar consumed. The production rate under continuous conditions depends
upon a combination of cell mass and hydraulic detention time (culture volume/volume
feed medium/day). Maximum cell concentration is a function of the hydrolysate sugar
concentration multiplied by the sugar conversion efficiency of the yeast.
Direct production
Direct production differs from indirect production in that organisms are cultured
upon unhydrolyzed wastes. Indirect production involves two discrete steps (hydrolysis
and cell production); whereas in direct production, the two steps are neither spatially nor
always temporally discrete. Although of necessity, the steps are sequential (hydrolysis
must precede utilisation for cellular growth; both may involve the same microorganism).
In other words, an organism can degrade a cellulosic molecule and utilize the constituent
sugars to synthesise cellular mass. All sequences are not occurring simultaneously and,
collectively, they constitute a single unit process. Therefore, at least some of the
microorganisms must be cellulolytic, i.e., capable of breaking down cellulose molecules.
Preferably, most should be cellulolytic. A disadvantage is the inability to use submerged
culture in the absence of special adaptations.
Most of the experience with single-cell production from waste has been at the
laboratory- and pilot-scale levels and has been with paper and bagasse. Paper is from
40% to 80% cellulose, 20% to 30% lignin, and 10% to 30% hemicellulose and xylosans.
Bagasse is the residue remaining after the juice has been extracted from sugar cane by
milling. Inasmuch as the studies were limited to laboratory- and pilot-scale levels,
projections and estimates based on the studies must be considered in that light.
Among the cellulolytic microorganisms that have been studied are the yeasts, C.
utilis and Myrothecium verrucaria, and the bacteria, Cellulomonas flarigena.
The culture of M. verrucaria on a substrate composed of ball-milled newspaper, a
yield of crude protein amounting to 1.42 g/L was obtained. The organism used in the
investigation was C. utilis. The bagasse was pre-treated because without pre-treatment,
the soluble carbohydrate content of untreated bagasse is only about 2%; whereas after
treatment, it is almost 18%. Pre-treatment reduces the cellulose crystallinity of the
bagasse from almost 50% to only 10%. As stated earlier, pre-treatment generally takes
one or a combination of the following forms: fine milling and exposure to moderately
elevated temperature under either acid or alkaline conditions.
The bacteria C. flavigena and C. uda constituted the product in a pilot study in
which the feedstock was bagasse. The study confirmed the need to pre-treat bagasse
specifically, alkaline pre-treatment. Moreover, in the study, extent of conversion of
feedstock to cell mass was very modest despite a continuous fermenter efficiency of 75%
and an approximate 90% solubilization of bagasse. Supplementary nutritional needs
could be supplied by fertiliser and industrial chemicals. From 50% to 55% of the product
is crude protein that has a good amino acid balance.
Another pilot-scale study involved a mixed culture of Cellulomonas and
Alcaligenes faecalis. The cell density was 6.24 g/L. The crude protein composition was
as follows (in g/100 g protein): arginine, 9.21; histidine, 2.30; isoleucine, 4.74; leucine,
11.20; lysine, 6.84; methionine, 1.86; phenylalanine, 4.36; tyrosine, 2.67; threomine,
5.37; and valine, 10.71.
RECOVERY
Harvesting usually is done in two main stages: a concentration stage and a
concentrate processing stage.
First-stage concentration
This stage results in the formation of a concentrate that has a sludge-like
consistency and is in need of further processing. The need for the concentration step
arises from the relatively low concentration of cells and large volumes of material that
must be processed. The sludge (concentrate) is dewatered and dried. The concentration
step is beset with many and grave difficulties due to the microscopic size and the physical
characteristics of the cells, as well as their modest monetary value. The several
technologies available for accomplishing the concentration step can be grouped into the
categories of screening, filtration, settling (sedimentation), and centrifugation.
Screening and filtration
Screening and filtration are discussed under a single heading because they share a
common characteristic: separation of particles (cells) depends upon the difference
between the size of the particles and that of the openings (screen) or pores (filter
medium). The problem is that the screen or filter medium becomes clogged before a
workable “cake” can be accumulated.
Settling
Their small size, low specific gravity, density, and low settling velocity render
concentration by sedimentation impractical. The settling velocity of yeast cells is
approximately 1.1 x 10-5 cm/sec.
Significant advances in settling tank design and operation may enhance settling to
a point at which it becomes a feasible option. Another approach to settling or a
modification is to induce floc formation and thereby promote settling to a level at which
it might be practical. Floc formation can be induced by altering the surface charge of
yeast cells such that they agglomerate into floc particles. Surface charge can be altered by
introducing a polymer flocculant (either anionic or cationic) into the suspension.
Alteration can also be accomplished by passing the suspension through an ion exchange
column.
Centrifugation
Centrifugation is an effective concentration method. Unfortunately, it is expensive
in terms of equipment and power, and requires skilled personnel. A high-velocity rotor is
necessary because of the microscopic size and low specific gravity and density of the
cells and viscosity of the medium. A putative advantage is that the two separation stages
can be accomplished in a single operation.
Second stage - concentrate (sludge) processing
Treatment consists of dewatering and drying. Flash drying is a good approach. It is
rapid and is amenable to mass production and is successfully used in food and feedstuff
preparation. Moreover, it removes threats to human and animal health posed by chance
pathogens. Other options include pressure filtration and vacuum drying, such as is used in
sewage sludge conditioning.