1. Enzymes
2. Antibiotics
3. Biofertilizer
4. Insulin and Growth Hormone
5. Aqua Culture
6. Floriculture
7. Tissue Culture
8. Bulk Drug Intermediate

MANUFACTURING PROCESS

Basically major enzymes areproduced by micro bial fermentation process. Microbes may be bacteria, yeast ormold, it may be some times plant origin.

Production of a-Amylase(By Bacterial use) :-

Medium Composition:

The above composition ingredientsdissolve in 25 times of water and sterilized at 115°C for 15 minutes. Asolution of 10% Sodium Carbonate in water was also sterilized at 115°C for 15minutes. These two solutions were mixed to prepare a culture medium 250 ml in 1litre shake flask. Use inocculum of Bacillus species NO-38-2 (A.T.C.C.21783).Make medium pH-10 and shake culture at 37°C for 48 Hours. The cells wereremoved from the culture by a centrifuge and at pH – 9, 1 ml. of this culturefiltrate contained 2500 units of alkaline amylase.

This culture broth thoroughlycooled and 3 volumes of acetone were added by which enzyme precipitatedquantitatively. This precipitate was thoroughly washed with acetone and dried inair. About 11 grams brown powder were obtained from the 1 litre of the culturebroth. The sample obtained thereby has an optimum pH around 9.0 and it was foundto be an amylase.

Manufacturing Process of Fungal a-amylase:

The mold is cultivated on anutrient medium containing the following composition

Preparation of Seed Culture:

Commercial viable strain ofAspergillus oryzae is grown in a test tube containing nutrient media 7 ml(previously sterile). Growth of strain in test tube is washed by sterile waterand transfer to a shake flask containing a litre media of composition.

Starch – 60 gm, NaNO39 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 shakerreciprocating at a speed of 180 R.P.M. at a temperature of 30°C.

Commercial Process:

Now produce seedculture (0.5%) is transferred in sterile condition into a fermentation tankcontaining sterile media of composition.

And 20% extract of Malt sprouts 20% by volume.

There will be foam in thefermentor, which can be controlled by antifoam dip like sperm oil or any othersuitable antifoam oil.

After completion of fermentationmycelium in the fermented broth be separated by filtration or centrifugalseparation.

The activity of in the culturefiltrate 20 units/ml. The filtrate is dialyzed with 0.03 M Phosphate bufter atpH 7.05 for 12 Hours. The obtained dialyzed is divided into several equalportions containing 1000 mg of protein each and each having the activity of a-amylaseof 10,000 units. Each portion of the dialyzed is passed through an individualcolumn packed with 10 gm of diethyl amine ethyl cellulose. The diameter of thecolumn is 35 M.M., the height 250 M. M. During this operation a-amylaseand other proteins are absorbed on the column packing.

Next the a-amylaseis eluted from the sorbert. The process is carried out in two steps. The firstelution is effect with 0.06 M phosphate buffer at pH – 7.15. During thisoperation only the accompanying proteins are eluted where as the a-amylaseremains on the column packing.

a-Amylaseis eluted during the 2^nd step when 0.11 M phosphate buffer at pH-7.15containing 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-amylaseis 300 mg. having total activity of 10,000 units that is the yield of a-amylaseafter elution is 100%.

Dialysis, adsorption and elutionof protein is carried out at a temperature of +8°C. The specific activity of a-amylaseobtained according to this process was almost twice as great compared with theactivity of a-amylaseobtained by the known method.

The activity of the product isexpressed in international units (I.U.). This is expressed in I.U/CC when theproduct is in solution and in I.U./gm when the product is solid.

Stabilization of a-amylase:

Bacterial a-amylasecan be stabilize in presence of Sodium and Calcium salts in solution (especiallyCalcium Acetate).

b-Amylase:

b-Amylase the saccharifying enzymes produces maltose from starch, glycogendextrins by selectivity removing maltose units at the non reducing ends of themolecule chains, b-Amylase activity also stops at the a-D (1 6) branching linkages of amylopectins leavingpolysaccharides termed limited dextrin.

Manufacturing  Process by Using Bacterial Culture

A medium containing 5% milk casein, 2% corn steep liquor, 10% soluble starch and enough water to make 15 litreswas sterilized in jarfermentor, adjusted to pH 7.0-7.2 and inoculated withcommercial viable strain of B. Megaterium. The mixture was incubated at 30°Cfor 2 days with stirring where upon the broth showed an enzyme activity of 25.5unit/ml.

The culture was centrifuged at8000 R.P.M. for 20 minutes to remove the cells and the supernatant was mixedwith enough solid (NH4)2SO4 to precipitate acrude enzyme product. It was dissolved in M/100 acetate buffer solution and thesolution was dialyzed against running tap water for 3 days. Impurities stillpresent were precipitated by adding 25% lead acetate solution drop by drop. Theprecipitate was removed by centrifuging.

The enzyme was again slated outwith (NH4)2SO4, dissolved in M/100 acetatebuffer at pH-6 and the solution was heated 15 at 60°C. It was dialyzed andenzyme from the further purified solution was absorbed on a molecular sievechromatographic medium (SE Sephadex G-25), eluted with M/2 sodium chloridesolution and get filtered to Sephadex G-100. The active fraction was freezedried and thus product dried b-Amylase obtained activity of 100 units per mg and thus had 50% of the originalbroth.

Pectolytic Enzyme :

This enzyme isprepared by solid state fermentation using mold strain of commercial viable.

Preparation of culture :

Took commercialviable strain of Aspergillus Carbarierius transfer to sterile test tubecontaining Agar medium, then plate (Petridish) it to grow pure individual colonyand then pick up well defined colony and transfer it to a test tube containingliquid nutrient media. Keep it for 24 hrs at 37°C and then transfer it to shakeflask containing 250ml solid nutrient media and keep it for growth of 24 hrs.This growth is used as seed culture for the propogation.

BIOFERTILIZER

INTRODUCTION

Fertilizer a substance which helpsto grow plants rapidly and produce fruits, flower, and vegetable more quantityin proper time, otherwise fertilizer be a compound, which fulfill the neededminerals or elemerts require for the growth of plants and vegetables to grow andfruits in proper time.

Fertilizer basically three types(Inorganic fertilizer (2) Organic fertilizer (3) Bio fertilizer.

Bio fertilizer are natural fertilizers which are microbialinoculants of bacteria, algae, fungi alone or in combination and they augmentthe availability of nutrients to the plants. Rhizobium is the best known biofertilizer which fixes atmosphere nitrogen symbiotically with legumes. Other bio-fertilizers are Azotobacter, Azospirillum, blue green algae andAzolla.

Bio fertilizer actually counteractthe toxic effects of chemical fertilizers, offers economic and ecologicalbenefits by way of soil health and fertility to farmers. Bio fertilizers providea dependable continued source of supply of nutrients unlike chemicalfertilizers, which, besides cost, are depleting source and add upto thepollution problems.

The earliest production ofRhizobium in oculant in India started in 1934, but commercial production wasundertaken in 1964 when soyabean was introduced in the country subsequentlythere was increase in demand for bio fertilizers for other legumes alsoproduction of other bio fertilizers such as Blue Green Algae, Azotobacter,Azospirillum and Azolla is also undertaken in the country on large scale. Thereis a good demand for Azospirillum from farmers of Tamil Nadu as they haveobtained significant response in rice and sugar cane.

Although the bio-fertilizerscannot by themselves totally replace chemical fertilizers at least in the nearfuture, there is need for integrated nutrient supply to crops through ajudicious amalagam of biological, chemical and organic source of nutrientsupply.

Among the different types offertilizers available at present, Rhizobium is relatively more effective andmore orderly used particularly the small and marginal farmers who constituteabout 56% at the total farming community are reluctant to use chemicalfertilizers in legume cultivation resulting in very poor yields. Considering thearea cultivated under legume crop use of Rhizobium can be a promising inputwhich can substantially increase legume crops.

Agriculture to-day Consumes highinputs of nitrogen. The present needs of nitrogen are largely met from syntheticnitrogen fertilizers their consumption has been rising every year. Thesefertilizers are quite expensive because of high cost of production consequentlytheir high inputs have considerably increased agricultural production in ahighly energy consuming process (13800 k cals per kg of nitrogen fixed).Therefore in the present situation of energy constraint, attention has divertedto tap alternatives to supplement nitrogen resources by directly utilizingatmosheric nitrogen through biological nitrogen through biological fixation. Inthe symbiotic nitrogen fixing system, photo synthetically stored energy isutilized instead of fossil fules. In this process atmospheric nitrogen isreduced to ammonia in presence of celetron donor ferredoxin and flavodoxin, ATP.Generating system and biological catalytser (nitrogenase, hydrogenase and someminerals).

AQUA CULTURE

INTRODUCTION

Biotechnology is an emergingtechnology with great potential. The country has the necessary talent to harnessthe potential of this technology. Technical innovation and resulting changes isthe key to the process of economic development. Determining the choice of futuretechnologies thus becomes important economic priority for the nation. Today weare living in a society, which is witnessing results of technologicaldevelopment at such unprecented rate, which could not have been imagined even 50years back.

Biotechnology the technologicalgap between advanced nations and to the development country. Biotechnology,defined in a broad sense, is the application of biotechnological organism andmolecules to technical and industrial processes. The fermentation process whichwere first established after the 2^nd world war were used to producenew products such as steroid, antibiotics, and fine chemicals. And thebiotechnology arises from a wave of innovation occurring within thisfermentation process and results from application of different techniques. Itgenerally 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 mattersi.e. the DNA is a manner resulting in enhanced or even totally new properties inplants animals and micro organisms, thus providing vast scope for application inmany areas like accelerated food production disease control, environmentprotection and improvement etc. In other words, its application is multisectorol.

Potential application area of biotechnology are:

1. Agriculture

(a) Bio fertilizers

(b) Bioinsecticide and bio-pesticides

(c) Hybrid seeds

(d) Artificial seeds

(e) Photosynthesis improvers

(f) Stress resistant crops and plants

(g) Tissue culture

2. Animal husbandry

3. Aqua culture

4. The human health

(a) Vaccines

(b) Immunodiognistics

(c) Medicines

5. Population control

6. Fuel and fodder

7. Biomass from various sources

8. Waste

9. Chemical feedstock.

Aquaculture Techniques

With 7000 Kms ofcoastline 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 areasof application of biotechnology in Indian fresh water aquaculture are recyclingof organic matter, biological nitrogen fixation and genetic transfers. Forexample, average yield per hectare per annum of prawns in countries likeThailand, Philippines, Taiwan, Hong Kong etc. is 10-12 tonnes compared to300-400 tonnes in India. Above levels of high yields is possible by systematicuse of marine biology and pisciculture knowledge.

Variousbiotechnological tools are being utilized for use in aquaculture for improvingproductivity both in terms of quantity and quality or for providing value addedproducts.

Production Techniques

It is estimated that India has a brackish water area of 11.90 lakhhectares. The major centers of brackish water prawn farming lying in the coastalregions of maritime states of India. At present the land used for brackish wateraquaculture is limited to 82000 hectares from where only 62000 tonnes of prawnare produced. The rest of the area is idle or unutilized.

Site selection

Detailed engineering investigationhas to be carried out to collect sufficient field data comprising of soilcondition, water availability, topographical features, position of creekscanals, availability of power and road links. Marigrove land should not be usedfor culture and should be allowed for natural growth, which helps for soilbinding and nutrient cyclings. Similarly agricultural and also should not beselected for brackish water shrimp culture. Only marginal land not used foragriculture should be used for brackish water shrimp farming. After detailedsite survey topographic maps are prepared which are essential for planning layout of the farm. Information on total height of the region and leveling ofground are very important. Subsoil investigation is necessary to obtainengineering properties of soil, which are required for designing the foundationof various farms structures. Testing of permeability of the soil is veryimportant to measure the rate at which water sweeps vertically. After completionof engineering investigation the data used to prepare hydrographic and soilprofile plan. Shrimp farm layout when properly designed should strike a balanceconsidering economy, functionality and aesthetics. The basic principle is tominimize the number of gates, the sizes and length of dikes and canals but itshould not sacrifice biological requirements of suitable environment of theculture species. The shrimp farms are designed as farmed system, which suits forsemintensive culture practices.

Pond Preparation

Pond preparationis the start of the culture operation and involves planning and coordination ofschedules to maximize resources without compromising basic principals of shrimpproduction. The pond should be prepared at least 30-45 days prior to waterculture. This is important to allow physical disintegration and chemicaldecomposition of minerals and organic matters from soil and adjust pH toslightly alkaline (7-8).

Non Acidic Sulphate Soil

The non acidicsulphate soil has pH value of more than 6. The soil is recognized by a dark tolight brown colour and a clay to sandy clay texture.

Acidic Sulphate Soil

Acidic sulphatesoil has very less pH (3-4). The soil is recognizable by the reddish colour (ofiron oxide) that may form on the pond bottom after flooding. In the processconcentration of iron and aluminium, which one harmful to the shrimp arereleased from soil.

Pond preparation for reclaimingacid sulphate soil to pH above 5 includes steps such as (a) drying, cultivatingand submerging and (b) refilling, flushing and liming. In case where a thinlayer 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 withnon acid top soil.

Components of Farm

The various farm components arereservoir system of ponds comprising nursery rearing ponds, bio ponds, supplycanal dikes. Water control structures, pumps and supporting facilities likebuildings, pump house, storage sheds and roads.

Pond

Before pondlayout is planned it is necessary to keep in mind the requirement of size andshape of ponds such as pre growing pond, rearing pond and bio-pond. Whiledesigning the farm, a minimum space of 500m (buffer zone) between adjacent farmsand drinking water source should be maintained.

Pre-Growing Pond

Pre-growing pondare nursery pond used for rearing fry up PL 20 stage after which they aretransferred to rearing ponds. Pre growing pond will be in 10% of the totalproduction area, shape will be square or rectangular. Pre growing ponds can bedetected if PL 20 seeds are obtained from hatchey.

Rearing Ponds

It is used for growing from PL 20to marketable size at the harvest stage. It’s size will be 70% of the totalproduction area, it’s shape will be rectangular of the length and breadthratio 1.5:1. Sometimes the shape of the ponds depends on the shape of site. Thesize of the grow out pond range from 1 ha. But for management convenience pondsbeyond 5 ha is not preferred. Bottom should be free from projected rocks, treestumps etc. The pond bottom must have a gradual slope (3/1000) from the inletgate towards drainage gate.

Bio Ponds

It is forsatisfaction of pollution control authorities and to prevent environmentdegradation, drainage water from the farm should be acceptable to flow to thearea or river system or sea. Bio ponds are necessary as part of the culture pondsystem. Bio ponds are large setting where the rich nutritional affluent aretreated naturally. Bio ponds are also used for culture of fish, mussel etc. Bioponds constitute about 7-10% of the total farm area.

Dike

The entire farmis provided with dikes or bands there are three types of dike :-

1. Main dike

2. Secondary dike

3. Tertiary dikes

1. Main Dike

Is to protect the prawn farm from destruction due toflood, storm and tide etc.

2. Secondary Dike

Smaller in size to the main peripheral dike, they are usuallyprovided on both sides of the main supply or drainage canal of and secondarysupply or drainage canal. The total height is obtained by providing suitablefree board of 0.3 m. The top secondary dike is usually 1.5 m and side slopes1:1.5

3. Tertiary Dike

Tertiary dikes are constructedbetween ponds. They should be able to contain the desired depth of water in thepond. The total slope of the top will be fixed in the same manner as thesecondary dike.

Water Control Structure

The water flow in the pond systemare controlled by various control structures, which are (1). Main supply gate offarm, (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.

Pumping station

Calculation of pump capacityrequirement will be based on parameters such as pond area, average water depthin 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 isrelated to average body weight (ABW) of the prawn. For prawn weight ranging from0.5 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.

Flushing

Flush isperformed immediate after harvest of the pond. For preparing the pond, thebottom soil of the pond has to be ploughed. It overturn the wet soil and oxidizethoroughly.

Liming

When the pH of the water is abovethan 6.5, there is no requirement of liming. When the pH is below 6, CaCO3or Ca (OH)2 may be used according to requirements.