Modern pharmaceutical manufacturing techniques frequently rely upon biotechnology. The emergence of biotechnology over the past several decades has transformed the drug business and ushered in a host of new participants and several novel business models. Biotechnology (Biotech) is a field of applied biology that involves the use of living organisms and bioprocesses in engineering, technology, medicine and other fields requiring bio products. Biotechnology also utilizes these products for manufacturing purpose. Modern use of similar terms includes genetic engineering as well as cell and tissue culture technologies. The concept encompasses a wide range of procedures (and history) for modifying living organisms according to human purposes, going back to domestication of animals, cultivation of plants, and improvements to these through breeding programs that employ artificial selection and hybridization. As understanding of biological systems has forged ahead, pharmaceutical companies have made increasing use of biotechnology in discovering and manufacturing new medicines. Biotechnology helps the pharmaceutical industry to develop new products, new processes, methods and services and to improve existing ones.
This book explains about the Market survey, manufacturing processes and other details of various enzymes (e.g. pectolytic enzyme, lytic enzyme, etc.), antibiotics, biofertilizer, etc. It also provides applications of biotechnology in agriculture (bioinsecticides & biopesticides, hybrid seeds, tissue culture, etc.), animal husbandry, aquaculture, human health, population control, fuel & fodder, waste etc.
Comprehensive in scope, the book provides solutions that are directly applicable to the properties, manufacturing technology and other specific details of enzymes, antibiotics, biofertilizer, insulin and growth hormone, aqua culture, floriculture, tissue culture, bulk drug intermediate. Research scholars, professional students, scientists, new entrepreneurs, and present manufacturers will find valuable educational material and wider knowledge of the subject in this book.
The student of Biology or
Biochemistry will already have learned of the Universal association of enzymes
with living matter and the important part played by them in all types of vital
Enzymes are proteinous in nature.
It acts with different substrate to produce small molecules to smallest molecule
according to the reaction parameters like pH, temp, pressure and physical
condition of the substrate.
All the enzymes are not capable to
react alone with substrate to convert product. Sometimes there is requirement of
presence of heavy metals like iron as iron ion, cobalt as cobalt ion or organic
molecules, which are called co-factors.
There are two types of enzymes
according to their sphere of action:
Exo-enzymes (2) Endo-enzymes
Exo-enzymes are such enzymes,
which stimulate outside of the cells and react with substrate to convert
Endo-enzymes are such enzymes,
which stimulate inside the cells and convert substrate to product, inside the
Basically enzymes are like organic
catalyst produced by living cells. Live catalyst enzymes are capable of
accelerating chemical reaction by millions of times under normal room
temperature in â€˜softâ€™ solvents. They have another quite unique
ability-strict selectiveness in the presence of definite enzyme only one, the
only one, of all possible reactions will, as a rule proceed.
Basically major enzymes are
produced by microbial fermentation process. Microbes may be bacteria, yeast or
mold, it may be some times plant origin.
Production of a-Amylase
(By Bacterial use) :-
The above composition ingredients
dissolve in 25 times of water and sterilized at 115Â°C for 15 minutes. A
solution of 10% Sodium Carbonate in water was also sterilized at 115Â°C for 15
minutes. These two solutions were mixed to prepare a culture medium 250 ml in 1
litre shake flask. Use inocculum of Bacillus species NO-38-2 (A.T.C.C.21783).
Make medium pH-10 and shake culture at 37Â°C for 48 Hours. The cells were
removed from the culture by a centrifuge and at pH â€“ 9, 1 ml. of this culture
filtrate contained 2500 units of alkaline amylase.
This culture broth thoroughly
cooled and 3 volumes of acetone were added by which enzyme precipitated
quantitatively. This precipitate was thoroughly washed with acetone and dried in
air. About 11 grams brown powder were obtained from the 1 litre of the culture
broth. The sample obtained thereby has an optimum pH around 9.0 and it was found
to be an amylase.
Manufacturing Process of Fungal a-amylase:
The mold is cultivated on a
nutrient medium containing the following composition
20% extract of Malt sprouts 20% by volume
Preparation of Seed Culture:
Commercial viable strain of
Aspergillus oryzae is grown in a test tube containing nutrient media 7 ml
(previously sterile). Growth of strain in test tube is washed by sterile water
and transfer to a shake flask containing a litre media of composition.
Starch â€“ 60 gm, NaNO3
9 gm, MgSO4 1.0 gm, KH2PO4 1gm, KCI 0.5 gm,
FeSO4 0.03 gm, Mg (NO3) 0.2 gm, Mg (H2PO4)
gm, and 10% extract of malt sprouts 10% by volume.
The seeding culture is grown for 30 Hours on a shaker
reciprocating at a speed of 180 R.P.M. at a temperature of 30Â°C.
Now produce seed
culture (0.5%) is transferred in sterile condition into a fermentation tank
containing sterile media of composition.
And 20% extract of Malt sprouts 20% by volume.
9 C.F.M. First 24 Hours
180c fm-25-72 Hours
There will be foam in the
fermentor, which can be controlled by antifoam dip like sperm oil or any other
suitable antifoam oil.
After completion of fermentation
mycelium in the fermented broth be separated by filtration or centrifugal
The activity of in the culture
filtrate 20 units/ml. The filtrate is dialyzed with 0.03 M Phosphate bufter at
pH 7.05 for 12 Hours. The obtained dialyzed is divided into several equal
portions containing 1000 mg of protein each and each having the activity of a-amylase
of 10,000 units. Each portion of the dialyzed is passed through an individual
column packed with 10 gm of diethyl amine ethyl cellulose. The diameter of the
column is 35 M.M., the height 250 M. M. During this operation a-amylase
and other proteins are absorbed on the column packing.
Next the a-amylase
is eluted from the sorbert. The process is carried out in two steps. The first
elution is effect with 0.06 M phosphate buffer at pH â€“ 7.15. During this
operation only the accompanying proteins are eluted where as the a-amylase
remains on the column packing.
is eluted during the 2nd step when 0.11 M phosphate buffer at pH-7.15
containing 0.001 M solution of calcium chloride is passed through the column.
The buffer completely elutes the fraction containing a-amylase.
The field of a-amylase
is 300 mg. having total activity of 10,000 units that is the yield of a-amylase
after elution is 100%.
Dialysis, adsorption and elution
of protein is carried out at a temperature of +8Â°C. The specific activity of a-amylase
obtained according to this process was almost twice as great compared with the
activity of a-amylase
obtained by the known method.
The activity of the product is
expressed in international units (I.U.). This is expressed in I.U/CC when the
product is in solution and in I.U./gm when the product is solid.
Stabilization of a-amylase:
can be stabilize in presence of Sodium and Calcium salts in solution (especially
Amylase the saccharifying enzymes produces maltose from starch, glycogen
dextrins by selectivity removing maltose units at the non reducing ends of the
molecule chains, b-
Amylase activity also stops at the a
-D (1 6) branching linkages of amylopectins leaving
polysaccharides termed limited dextrin.
Process by Using Bacterial Culture
A medium containing 5% milk case
in, 2% corn steep liquor, 10% soluble starch and enough water to make 15 litres
was sterilized in jarfermentor, adjusted to pH 7.0-7.2 and inoculated with
commercial viable strain of B. Megaterium. The mixture was incubated at 30Â°C
for 2 days with stirring where upon the broth showed an enzyme activity of 25.5
The culture was centrifuged at
8000 R.P.M. for 20 minutes to remove the cells and the supernatant was mixed
with enough solid (NH4)2SO4 to precipitate a
crude enzyme product. It was dissolved in M/100 acetate buffer solution and the
solution was dialyzed against running tap water for 3 days. Impurities still
present were precipitated by adding 25% lead acetate solution drop by drop. The
precipitate was removed by centrifuging.
The enzyme was again slated out
with (NH4)2SO4, dissolved in M/100 acetate
buffer at pH-6 and the solution was heated 15 at 60Â°C. It was dialyzed and
enzyme from the further purified solution was absorbed on a molecular sieve
chromatographic medium (SE Sephadex G-25), eluted with M/2 sodium chloride
solution and get filtered to Sephadex G-100. The active fraction was freeze
dried and thus product dried b-
Amylase obtained activity of 100 units per mg and thus had 50% of the original
Pectolytic Enzyme :
This enzyme is
prepared by solid state fermentation using mold strain of commercial viable.
Preparation of culture :
viable strain of Aspergillus Carbarierius transfer to sterile test tube
containing Agar medium, then plate (Petridish) it to grow pure individual colony
and then pick up well defined colony and transfer it to a test tube containing
liquid nutrient media. Keep it for 24 hrs at 37Â°C and then transfer it to shake
flask containing 250ml solid nutrient media and keep it for growth of 24 hrs.
This growth is used as seed culture for the propogation.
Fertilizer a substance which helps
to grow plants rapidly and produce fruits, flower, and vegetable more quantity
in proper time, otherwise fertilizer be a compound, which fulfill the needed
minerals or elemerts require for the growth of plants and vegetables to grow and
fruits in proper time.
Fertilizer basically three types
(Inorganic fertilizer (2) Organic fertilizer (3) Bio fertilizer.
Bio fertilizer are natural fertilizers which are microbial
inoculants of bacteria, algae, fungi alone or in combination and they augment
the availability of nutrients to the plants. Rhizobium is the best known bio
fertilizer which fixes atmosphere nitrogen symbiotically with legumes.
Other bio-fertilizers are Azotobacter, Azospirillum, blue green algae and
Bio fertilizer actually counteract
the toxic effects of chemical fertilizers, offers economic and ecological
benefits by way of soil health and fertility to farmers. Bio fertilizers provide
a dependable continued source of supply of nutrients unlike chemical
fertilizers, which, besides cost, are depleting source and add upto the
The earliest production of
Rhizobium in oculant in India started in 1934, but commercial production was
undertaken in 1964 when soyabean was introduced in the country subsequently
there was increase in demand for bio fertilizers for other legumes also
production of other bio fertilizers such as Blue Green Algae, Azotobacter,
Azospirillum and Azolla is also undertaken in the country on large scale. There
is a good demand for Azospirillum from farmers of Tamil Nadu as they have
obtained significant response in rice and sugar cane.
Although the bio-fertilizers
cannot by themselves totally replace chemical fertilizers at least in the near
future, there is need for integrated nutrient supply to crops through a
judicious amalagam of biological, chemical and organic source of nutrient
Among the different types of
fertilizers available at present, Rhizobium is relatively more effective and
more orderly used particularly the small and marginal farmers who constitute
about 56% at the total farming community are reluctant to use chemical
fertilizers in legume cultivation resulting in very poor yields. Considering the
area cultivated under legume crop use of Rhizobium can be a promising input
which can substantially increase legume crops.
Agriculture to-day Consumes high
inputs of nitrogen. The present needs of nitrogen are largely met from synthetic
nitrogen fertilizers their consumption has been rising every year. These
fertilizers are quite expensive because of high cost of production consequently
their high inputs have considerably increased agricultural production in a
highly energy consuming process (13800 k cals per kg of nitrogen fixed).
Therefore in the present situation of energy constraint, attention has diverted
to tap alternatives to supplement nitrogen resources by directly utilizing
atmosheric nitrogen through biological nitrogen through biological fixation. In
the symbiotic nitrogen fixing system, photo synthetically stored energy is
utilized instead of fossil fules. In this process atmospheric nitrogen is
reduced to ammonia in presence of celetron donor ferredoxin and flavodoxin, ATP.
Generating system and biological catalytser (nitrogenase, hydrogenase and some
Biotechnology is an emerging
technology with great potential. The country has the necessary talent to harness
the potential of this technology. Technical innovation and resulting changes is
the key to the process of economic development. Determining the choice of future
technologies thus becomes important economic priority for the nation. Today we
are living in a society, which is witnessing results of technological
development at such unprecented rate, which could not have been imagined even 50
Biotechnology the technological
gap between advanced nations and to the development country. Biotechnology,
defined in a broad sense, is the application of biotechnological organism and
molecules to technical and industrial processes. The fermentation process which
were first established after the 2nd world war were used to produce
new products such as steroid, antibiotics, and fine chemicals. And the
biotechnology arises from a wave of innovation occurring within this
fermentation process and results from application of different techniques. It
generally implies the application of novel microbes and other living system,
altered or modified through various technologies like genetic engineering,
biotechnological tools enable us to manipulate the core of all living matters
i.e. the DNA is a manner resulting in enhanced or even totally new properties in
plants animals and micro organisms, thus providing vast scope for application in
many areas like accelerated food production disease control, environment
protection and improvement etc. In other words, its application is multi
Potential application area of biotechnology are:
Bioinsecticide and bio-pesticides
Stress resistant crops and plants
2. Animal husbandry
3. Aqua culture
4. The human health
5. Population control
6. Fuel and fodder
7. Biomass from various sources
9. Chemical feedstock.
With 7000 Kms of
coastline and 4.5 million hectares of water area as ponds, tanks and lakes,
aquaculture potential of India is yet to be harnessed fully. The potential areas
of application of biotechnology in Indian fresh water aquaculture are recycling
of organic matter, biological nitrogen fixation and genetic transfers. For
example, average yield per hectare per annum of prawns in countries like
Thailand, Philippines, Taiwan, Hong Kong etc. is 10-12 tonnes compared to
300-400 tonnes in India. Above levels of high yields is possible by systematic
use of marine biology and pisciculture knowledge.
biotechnological tools are being utilized for use in aquaculture for improving
productivity both in terms of quantity and quality or for providing value added
It is estimated that India has a brackish water area of 11.90 lakh
hectares. The major centers of brackish water prawn farming lying in the coastal
regions of maritime states of India. At present the land used for brackish water
aquaculture is limited to 82000 hectares from where only 62000 tonnes of prawn
are produced. The rest of the area is idle or unutilized.
Detailed engineering investigation
has to be carried out to collect sufficient field data comprising of soil
condition, water availability, topographical features, position of creeks
canals, availability of power and road links. Marigrove land should not be used
for culture and should be allowed for natural growth, which helps for soil
binding and nutrient cyclings. Similarly agricultural and also should not be
selected for brackish water shrimp culture. Only marginal land not used for
agriculture should be used for brackish water shrimp farming. After detailed
site survey topographic maps are prepared which are essential for planning lay
out of the farm. Information on total height of the region and leveling of
ground are very important. Subsoil investigation is necessary to obtain
engineering properties of soil, which are required for designing the foundation
of various farms structures. Testing of permeability of the soil is very
important to measure the rate at which water sweeps vertically. After completion
of engineering investigation the data used to prepare hydrographic and soil
profile plan. Shrimp farm layout when properly designed should strike a balance
considering economy, functionality and aesthetics. The basic principle is to
minimize the number of gates, the sizes and length of dikes and canals but it
should not sacrifice biological requirements of suitable environment of the
culture species. The shrimp farms are designed as farmed system, which suits for
semintensive culture practices.
is the start of the culture operation and involves planning and coordination of
schedules to maximize resources without compromising basic principals of shrimp
production. The pond should be prepared at least 30-45 days prior to water
culture. This is important to allow physical disintegration and chemical
decomposition of minerals and organic matters from soil and adjust pH to
slightly alkaline (7-8).
Non Acidic Sulphate Soil
The non acidic
sulphate soil has pH value of more than 6. The soil is recognized by a dark to
light brown colour and a clay to sandy clay texture.
Acidic Sulphate Soil
soil has very less pH (3-4). The soil is recognizable by the reddish colour (of
iron oxide) that may form on the pond bottom after flooding. In the process
concentration of iron and aluminium, which one harmful to the shrimp are
released from soil.
Pond preparation for reclaiming
acid sulphate soil to pH above 5 includes steps such as (a) drying, cultivating
and submerging and (b) refilling, flushing and liming. In case where a thin
layer of non acid top soil, overlay soil materials with high potential acidity,
it may be advisable to cut the pond bottom to a desired depth and fill back with
non acid top soil.
Components of Farm
The various farm components are
reservoir system of ponds comprising nursery rearing ponds, bio ponds, supply
canal dikes. Water control structures, pumps and supporting facilities like
buildings, pump house, storage sheds and roads.
layout is planned it is necessary to keep in mind the requirement of size and
shape of ponds such as pre growing pond, rearing pond and bio-pond. While
designing the farm, a minimum space of 500m (buffer zone) between adjacent farms
and drinking water source should be maintained.
are nursery pond used for rearing fry up PL 20 stage after which they are
transferred to rearing ponds. Pre growing pond will be in 10% of the total
production area, shape will be square or rectangular. Pre growing ponds can be
detected if PL 20 seeds are obtained from hatchey.
It is used for growing from PL 20
to marketable size at the harvest stage. Itâ€™s size will be 70% of the total
production area, itâ€™s shape will be rectangular of the length and breadth
ratio 1.5:1. Sometimes the shape of the ponds depends on the shape of site. The
size of the grow out pond range from 1 ha. But for management convenience ponds
beyond 5 ha is not preferred. Bottom should be free from projected rocks, tree
stumps etc. The pond bottom must have a gradual slope (3/1000) from the inlet
gate towards drainage gate.
It is for
satisfaction of pollution control authorities and to prevent environment
degradation, drainage water from the farm should be acceptable to flow to the
area or river system or sea. Bio ponds are necessary as part of the culture pond
system. Bio ponds are large setting where the rich nutritional affluent are
treated naturally. Bio ponds are also used for culture of fish, mussel etc. Bio
ponds constitute about 7-10% of the total farm area.
The entire farm
is provided with dikes or bands there are three types of dike :-
Is to protect the prawn farm from destruction due to
flood, storm and tide etc.
Smaller in size to the main peripheral dike, they are usually
provided on both sides of the main supply or drainage canal of and secondary
supply or drainage canal. The total height is obtained by providing suitable
free board of 0.3 m. The top secondary dike is usually 1.5 m and side slopes
Tertiary dikes are constructed
between ponds. They should be able to contain the desired depth of water in the
pond. The total slope of the top will be fixed in the same manner as the
Water Control Structure
The water flow in the pond system
are controlled by various control structures, which are (1). Main supply gate of
farm, (2). Secondary supply canal control gate (3). Supply canal drain gate (4).
Pond inlet gate (5). Pond outlet gate and (5). Farm main drainage gate.
Calculation of pump capacity
requirement will be based on parameters such as pond area, average water depth
in ponds, duration of pumping, water exchanges and efficiency of the pump.
Pumping requirement of farm is of high capacity and low head type.
Outlet Gate Screen
The most size of the screen is
related to average body weight (ABW) of the prawn. For prawn weight ranging from
0.5 gms to 15 gms, mesh size varies from 0.5 mm to 10 mm at the entrance gate.
Screen is necessary to prevent loss of culture prawn.
performed immediate after harvest of the pond. For preparing the pond, the
bottom soil of the pond has to be ploughed. It overturn the wet soil and oxidize
When the pH of the water is above
than 6.5, there is no requirement of liming. When the pH is below 6, CaCO3
or Ca (OH)2 may be used according to requirements.
Ca (OH)2 Kg/ha
6.5 and above