GOOD LABORATORY PRACTICES

Good laboratory practice (GLP) within which laboratory studies are planned, performed, monitored, recorded, reported and achieved. These studies were undertaken to generate data by which the hazards and embodies a set of principle that provides a framework risks to users, consumers and third parties, including the environment, can be assessed for pharmaceuticals (only preclinical studies) ,agrochemicals, cosmetics, feed additives and contaminants, novel foods, biocides, detergents etc.

Definition:“ GLP is a quality system concerned with the organizational process and conditions under which non-clinical health and environmental safety studies are planned, performed, monitored, recorded, achieved and reported “.

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Standard operating procedures (SOP’S) Statistical procedures for data evaluation. Instrumentation validation. Reagent /materials certification Analyst certification Laboratory facilities certification specimen/simple tracking Laboratory door and window should be kept closed when work is in progress . Protective laboratories clothing must be worn for work in the lab All technical procedure should to be performed in a way that minimizes the formation of aerosols and droplets. After finishing work and before living the laboratory hand must be washed if infectious material have been handled should be disinfected. Working space should kept clean and cleared up. 1

2. BIOFERTILIZERS
 Biofertilizer is a Natural organic fertilizer known that helps to provide all the nutrients required by the plants and helps to increase the quality of the soil moss.Biofertilizer Contains a wide range of naturally chelated plant nutrients and trace elements, carbohydrates, amino acids and other growth promoting substances.  Kelp acts as a soil conditioner by stimulating microbial activity in the soil which results in improved air water relationships in soil, improved fertility and makes soil less prone to compaction and erosion. Organic Growers who use kelp in their regular fertility program report increases in yield, quality, shelf-life and resistance to environmental stresses such as drought, extreme heat, early frost, pest and disease problem. This blend makes an excellent foliar fertilizer. Besides being a nutritionally complete fertilizer (containing even calcium), the nutrients are readily absorbed by the leaf. This is because the nitrogen in fish is in the form of amino acids which plants take in and use directly– unlike inorganic fertilizers in which the nitrogen needs to be converted into a usable form first. Additionally, because the micronutrients in the fish and in the kelp are in a naturally chelated form they are quickly and readily absorbed into the leaf surface. Foliar applications on a regular basis can increase the health, vigor and yield of plants due to this easily absorbed additional nutrients

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Organic fertilizers differ from chemicals fertilizers in that they feed your plants while adding organic material to the soil. Soils with lots of organic matter remain loose and airy, hold more moisture and nutrients, foster growth of soil organisms, and promote healthier plant root development. If only chemicals are added the soil gradually loses its organic matter and microbiotic activity. As organic matter is used up, the soil structure deteriorates, becoming compact, lifeless and less able to hold water and nutrients. This results in increased amounts of chemical fertilizers needed to feed plants. We also like organic fertilizers because they're made from renewable resources; chemicals are not. The Biofertilizer, is a premium natural fertilizer composed just with certified organic ingredients special of nutrient-poor Western soils. This organic fertilizers is unequaled in its ability to nourish the beneficial micro-organisms in the soil greatly increasing the soil’s humus content and improving its ability to sustain and nurture healthy, more colorful plants. Use by the handful when planting individual plants, broadcast and mix it deeply into the soil when planting flower beds or spread it around established plants and scratch it into the soil. It is also excellent for use in vegetable gardens, container plantings and as a compost-pile activator Bio Fertilizer: The Best Economic Value: Proven, top-quality product. Stick with biofertilizer the one that always has and always will give you top quality and the best value immediately for your investment and much more profits at long term. Research winner. Growing trials conducted by an independent research center at a professional greenhouse compared various soils amended with peat, coir,compost and blends. The conclusion:-"Sphagnum peat can be considered the best overall performer as a soil amendment and substrate: it is homogenous, easy to handle and has shown the best growth results; all of this at a highly competitive price." 3

At present in India there is a gap of about 10 million tones of plant nutrients between removal bicrops and replenishment through fertilizer. Supply of nutrient from organic manure has not so far been able to fill up this gap and also in years to come nutrient supply from the source is unlikely to improve due to competitive demand for alternate uses like fuel and fodder. Of late, biofertilizers are being promoted as an important component in supplementing plant nutrient need of the country.

2.1 SYMBIOTIC RELATIONSHIP A symbiotic relationship is a relationship between two entities which is mutually beneficial for the participants of the relationship. Thus there is a positive-sum gain from cooperation. This is a term commonly used in biology to explain the relationship between two entities that need each other to survive and prosper. The bumblebee and the flower would be an example. The bumble bee extracts the flower's pollen for protein and its nectar for energy. The bumblebee, while collecting these sources, inadvertently brushes pollen from one flower to another to ensure the flower's reproduction process begins. The bumblebee needs the flower to survive, the flower needs the bumblebee to survive. These are positive sum relationships. Other relationships in biology, especially with respect to the food chain, are not so forgiving, being a zero sum relationship, where one player clearly benefits from the other (typically consumes the other). In this type of relationship, it is still essential that both players (as species not individuals) survive, or the dominant player will lose its food supply, and therefore die. If the foxes kill all the chickens, the foxes die as they lose their source of survival. If the predator kills all its prey, the predator will die.

2.2 NON-SYMBIOTIC RELATIONSHIP
In non-symbiotic relationship the species do live together nor are dependent on each-other, the relationship is facultative or opportunistic but does not profit the organisms when together. 4

Azotobacter, Azomonas, Azotococcus, Mycobacterium spp., Methylosinum trichosporium, Thiobacillus ferooxidans, Chlorobium thiosulfatophilum, Chromatium vinosum. C. minutissinllm. Bacillus polymixa, B. macerans, Enterobacter aerogenes (Aerobacter aerogenes), Escherichia intermedia, E. coli, Klebsiella spp., Rhodospirillum rubrum, Rhodomicrobium, Rhodopseudomonas, Clostridium spp., Desulfovibrio spp. exemplify the bacteria (free living) actively participating in non-symbiotic biological nitrogen fixation. Azotobacter spp. (aerobic) are the main nitrogen fixing free living bacteria. Clostridium spp., (anaerobic) stand next. The free living cyanobacteria are considered to be fairly important nitrogen-fixers. They may fix ten times as much nitrogen as the other free living bacteria fix under suitable conditions. There are claims that the cyanobacteria are mainly responsible for maintaining the fertility and productivity of rice fields, Anabaena, Nostoc are the good examples.

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Similarly blue green algae are needed to grow rice while Acetobacter is used to grow sugarcane. Nitrogen is a necessary component which is used for the growth of the plant. The type of the crops also determines the level f nitrogen. Azotobacteria is used for the non legume crops. These biofertilizer have an important role play in rain fed agriculture primarily because they are low in cost input. Phosphate solubilizing and mobilizing microorganism and compost etc. Rhizobium is needed for the legume crops. Fr example. The nitrogen-fixing organisms All the nitrogen-fixing organisms are prokaryotes (bacteria). Plants need a limited amount of nitrogen for their growth. Some crops need more nitrogen for their growth while some crops need fewer amounts. Some of them live independently of other organisms .g. Of this nearly 50% is under unsure rainfall condition. protozoa).the so-called free-living nitrogen-fixing bacteria. i. Others live in intimate symbiotic associations with plants or with other organisms (e. Examples are shown in the table below Examples of nitrogen-fixing bacteria (* denotes a photosynthetic bacterium) Free living Aerobic Azotobacter Beijerinckia Klebsiella Cyanobacteria (some)* (some) Anaerobic Clostridium Desulfovibrio Purple Purple sulphur non-sulphur bacteria* Rhizobium bacteria* (some) Frankia Azospirillum Symbiotic with plants Legumes Other plants Green sulphur bacteria* 6 . The type of the soil also determines that which type of biofertilizers is needed for this crop.3 TYPES OF BIOFERTILIZERS Biofertilizer can be divided into Nitrogen fixing bacteria. Nitrogen Biofertilizers :This type of biofertilizers helps the agriculturists to determine the nitrogen level in the soil. some of these micro-organism are also being reported to produce plant growth promoting substance. It means almost all the crops need different types of biofertilizers depending on their needs.2. In India about 70% of the cultivated area is under rain fed condition.

and Acetobacter can be used for all crops. maturation of fruits. The microorganisms in phosphorus biofertilizer solubilize fixed forms of phosphorus already present in the soil. application can be used in any location. iii. therefore. This product is not crop specific. Low levels of soil phosphorus make it hard for plants to absorb the vital nutrients from biofertilizers.ii. organic and inorganic. According to the Regional Biofertilizer Development Centre website. If the soil is phosphorus deficient. Phosphorus biofertilizer is not crop specific. and for reproduction. 50- Types of Bacteria yield Fodders give better results. Phosphorus is a naturally occurring element that helps promote healthy plant growth. Benefits Usually Remarks Seen like 10-35% increase. thus making it available for plants. enriched compost biofertilizer comes in two varieties. it still requires the use of soil phosphorus for effectiveness. Compost Biofertilizers :Organic and synthetic fertilizers contain potassium. Enriched compost biofertilizer acts similar to phosphorus by speeding up the composting process and enriching the nutrient values of the other two elemental fertilizers. thus limiting the positive effects of the solution. Azotobacter. states the Regional Biofertilizer Development Centre website. therefore Rhizobium. thus making them ineffective. Cellulolytic fungal culture and Phosphotika and Azotobacter culture. and is found in all fertilizers. Azospirillum. which helps the absorption of nitrogen and phosphorus in plants. Although enriched compost helps maximize the nutrients available in the soil. Name Rhizobium strains Crops Suited Legumes pulses. the organisms will have a hard time extracting the trace amounts in the soil. Symbiotic bacteria Leaves residual N 7 . Phosphorus biofertilizers :Phosphorus is needed for plant photosynthesis.

Azotobacter 200 kg N/ha. inoculated to Fungi Bacteria give biomass up as feed. barley. some 30-50% crops. It can be applied to legumes as co-inoculants Phosphate Solubilizers Blue-green algae and Azolla application 5-30% increase yield Can be mixed with rock Phosphate phosphate. sorghum. Free Bacteria living soybean Soil treatment for 10-15% nonlegume crops including increaseadds 20-25 kg N/ha yield A fodder gives Free Bacteria living Azospirillum dry land crops Non-legumes like 10-20% maize. They have growth Tonnes and fix hormonal effects. yield Also controls certain diseases. rice etc. can be Symbiotic Azolla can to 40-50 30-100 kg N/ha used for fishes promoting TNAU has developed high yielding Azolla Hybrids.groundnut. Produces growthpromoting substances. Soil for all crops Rice/wet lands oats. millet. in the soil. yield Usually seedlings. Mycorrhizae (VAM) Many trees. and ornamental plants increase some enhances uptake of P. S and Water COMPARATION BETWEEN CHEMICAL AND BIOFERTILIZER Factors Chemical Fertilizer Biofertilizers 8 . solubilizing Bacteria 20 -30 kg N/ha. increase higher/enriches fodder response. Zn. Reduces soil alkalinity. Sugarcane.

Centralized Biological. 9 . small scale or Decentralized Process Chemical Biological Effect on subsequent Crop For nitrogen nil or low Residual effect for nitrogen Shelf life Pollution effect Long exists due to indiscriminate use short for bacteria. and growth. long for BGA pollution free Irrigation more useful to irrigated field useful for both irrigated and dry farming Cost high cost input low cost input Soil health indiscriminate use deteriorates the soil health improves soil health ADVANTAGES   Germination increase up to 20 percent. Improved seedling emergence Increase yield from 10 to 40 percent.Production Industrial.

They are compatible with organic manures. 10 . Biofertilizers are also produce growth promoting substances. They are non. Leaves no harmful residues in plants or soil. fertilizers and agro-chemicals. Increase the availability and up take of N and P in plants. It is safe to handle and easy to apply. Saving of 25 to 35 percent inorganic fertilizers. Suppress harmful and pathogenic soil micro-organism.         Improve nitrogen and phosphorus fertilizer efficiency.polluting and eco-friendly. Higher population of beneficial micro-organism in soil increase nutrient retention and availability leading to improve yields. Composting waste matter and produce organic manure. Improve the status of soil fertility maintain good soil health and crop productivity.     Improve the quality of fruit and keeping quality.

Biofertilizer packets need to be stored in cool and dry place away from direct sunlight and heat. Should use for the specified crop only. Damage the environment example about 10% of the ground water sample in Punjab contained more nitrite than the maximum permissible limit prescribed by World Health Organization. Are costly. (Rhizobium) APPLICATION 11 .   Right combinations of biofertilizers have to be used. Other chemicals should not be mixed with the biofertilizers Biofertilizers are live product and require care in the storage.     Slow action. Utilize petroleum (nitrogenous fertilizer).DISADVANTAGES     Are short in supply.

A BRIEF STUDY OF BACTERIAL SPECIES i. Obviously most interest among biofertilizer surround Rhizobium inoculants because of their ability to fix nitrogen in association with leguminous plants which result not only in meeting the nitrogen required of the plant but also the symbiotic system leaves behind sizeable amount of air over a hectare of land contain approximately 80000 tones of inert nitrogen which is not available to plants and animals as 12 . RHIZOBIUM  It belongs to Rhizobiaceae family and fixed 50-100 kg atmospheric nitrogen per hectare.

The soyabean Rhizobium will only infectand form nodules on soyabean plantand not on groundnut plant. the fixation of N2 must occur under conditions which are anaerobic at least locally. .It means that Rhizobium of pea group will only pea plant and fix nitrogen and not with soyabean plant. This wastes carbohydrate. Oil seeds: soybean. In Azotobacter. blackgram. Therefore. lucern. Facultative organisms such as purple photosynthetic bacteria or Klesbsiella fix N2 only when anaerobic.such but can be utilized by plants in association with bacteria having capability to fix atmospheric nitrogen. This shows that Rhizobium is specific for each leguminous plant. For anaerobes there is no problem. cowpea. Increase in yield: 10-35% ii. It produces 13 . screened. In cyanobacteria O2 is actually generated by photosynthesis. greengram. It lives in soil and enter into symbiosis only with leguminous plants by infecting their roots and forming nodules on them.  Since nitrogenase is inactivated by O2. the O2 concentration inside the cell is held down by partial uncoupling of a highly active respiratory chain. Other organisms have protective mechanisms. pigeonpea. Rhizobium is very specific in choosing its symbiotic partners and each Rhizobium species infect limited no of legumes. the competitive. Recommended for Pulses: Chickpea. Can you guess whether the same Rhizobium can form nodules and fix nitrogen with all the leguminous plant. the rhizobium bacteria present in the nodules of these crops are not always efficient. soybean and groundnut occupy nearly 25 million hectare in India and the bacteria present in nodule of this plants fix the atmospheric nitrogen to meet their needs. but if growth is limited by absence of nitrogen compounds then this is justifiable. It produces growth promoting substances which improve seed germination and growth of extended root system. AZOTOBACTER It belongs to azotobacteriaceae. However. There are man species of Rhizobium present in the soil. efficient bacteria are isolated. selected and produced as carrier based inoculants  This bacterium was isolated for the first time in 1838. It lives inside the nodules and fixed atmospheric nitrogen for plants. The answer is no. Fixation of N2 occurs in special cells known as heterocysts which do NOT photosynthesize but are devoted solely to N2 fixation. lentil. groundnut. The use of azotobacter help in saving 10 to 20 kg N/ha. Pulses. Fodders: berseem. an obligate aerobe. pea.

PHOSPHATE SOLUBILIZING BACTERIA Phosphorus is one of the most important plant nutrients and may be critical nutrient the optimum growth of plants. other cereals. Recommended for Rice. The proliferation of efficient strain of phosphate solubilization micro-organism. cotton. The use of this biofertilizer will also increase the availability of phosphate from rock phosphate applied directly even to neutral to alkaline soil or when used for preparation of phosphor-compost. sunflower. flowers. millets. vegetable. Increase in yield: 20 to 30% iii. In the Rhizosphere of crops will render insoluble soil phosphate available to plants due to production and secretion of organic acid by them. mustard. Azotobacter suppresses the growth of saprophytic and pathogenic micro-organism near the root system of crop plants. Phosphate solubilizing micro-organism include efficient strain of bacteria. wheat. Most of our soils are in available forms of phosphorus required phosphate application.polysaccharides which improve soil aggregation. fungi. In a country like India where the application of chemical fertilizer in rain fed cultivated area is low and in irrigated area its application is much less as compared to many developed country its benefit have been reported. yeast and actinomycetes in that order Increase in yield: 10 to 20%  14 .

+ O2 -> 2 NO3- 15 . Nitrosomonas bacteria first convert nitrogen gas to nitrite (NO2-) and subsequently Nitrobacter convert nitrite to nitrate (NO3-). during the nitrification process. Nitrogen in the gaseous form cannot be absorbed and used as a nutrient by plants and animals. Plants can use ammonia as a nitrogen source. during the nitrogen fixation process. so that it can enter food chains as a part of the nitrogen cycle. The gaseous form of nitrogen (N2) makes up 78% of the troposphere.2. it must first be converted by nitrifying bacteria. but unfortunately it does not work that way. a plant nutrient. proteins and DNA. During the conversion of nitrogen cyanobacteria will first convert nitrogen into ammonia and ammonium. One might think this means we always have plenty of nitrogen available. such as amino acids.4 MECHANISM OF NITROGEN FIXATION  Nitrogen is a part of vital organic compounds in microrganisms. the ammonia and ammonium that is formed will be transferred further.  Nitrification 2 NH3 is + carried 3O2 out > according 2 NO2 to + the 2 following H+ + 2 reactions: H2O 2 NO2. Nitrogen fixation is carried out according to the following reaction:   N2 + 3 H2 -> 2 NH3  After ammonium fixation. Aerobic bacteria use oxygen to convert these compounds.

+ H2O (nitrate ion) (nitrite ion) 16 . nitrogen is often the limiting factor for growth and biomass production in all environments where there is suitable climate and availability of water to support life. making the molecule almost inert. which is required in large amounts as an essential component of proteins. and none is more important than nitrogen. Animals cannot absorb nitrates directly. In order for nitrogen to be used for growth it must be "fixed" (combined) in the form of ammonium (NH 4) or nitrate (NO3) ions. during a process called denitrification.+ 2e. However. specialized decomposing bacteria will start a process called ammonification. N2 is unavailable for use by most organisms because there is a triple bond between the two nitrogen atoms.nearly 79% in the form of N2 gas. anaerobic bacteria will convert them back into nitrogen gas. such as amino acids and DNA. Role of nitrogen in the biosphere The growth of all organisms depends on the availability of mineral nutrients. Nitrogen fixation reactions  N2 + 6 e. nucleic acids and other cellular constituents. After the nutrients are converted back into ammonia.+ CH2O + H+ -> ½ N2O + CO2 + 1½ H2O  Finally. There is an abundant supply of nitrogen in the earth's atmosphere . They receive their nutrient supplies by consuming plants or plant-consuming animals.+ 2H+ -----------> NO2. after which they are converted into nitrogen-containing organic molecules. The weathering of rocks releases these ions so slowly that it has a neglible effect on the availability of fixed nitrogen. The whole process starts over after release. So. Plants absorb ammonium and nitrate during the assimilation process. nitrogen is released into the atmosphere again.+ 8H+ ---> 2 NH4+ (ammonium ion)  NO3. to convert them back into ammonia and watersoluble ammonium salts. Denitrification is carried out according to the following reaction:   NO3. When nitrogen nutrients have served their purpose in plants and animals.

(nitrate ions)  HNO2 --------> H+ + NO2. Three processes are responsible for most of the nitrogen fixation in the biosphere: • Atmospheric fixation by lightning 17 .+ 2H+ ----------> NH4+ + 2 H20  NO2.(nitrite ions) Types of Nitrogen fixation processes The nitrogen molecule (N2) is quite inert. NO2. To break it apart so that its atoms can combine with other atoms requires the input of substantial amounts of energy.+ 2H+ ----------> NH4+ + 2 H2O  2 NO + O2 ---------------> 2NO2  2 NO2 + H2O -------> HNO3 + HNO2  HNO3 --------> H+ + NO3.+ 6e.+ 6e.

or 106 metric tons per year) about 50 about 20 about 10 about 80 about 90 about 50 about 35 about 175 18 .• • Industrial fixation Biological fixation by certain microbes — alone or in a symbiotic relationship with some plants and animals Type of fixation Non-biological Industrial Combustion Lightning Total Biological Agricultural land Forest and non-agricultural land Sea Total N2 fixed (1012 g per year.

Azoferredoxin is modified by the NifM protein. or "nitrogenase" Azoferredoxin = component II. This MoFe cofactor is unique to nitrogen fixation and distinct from the Mo-pterin cofactor of other Mo proteins (e. a flavoprotein.g. Here we consider nitrogenase from Klebsiella. These must be supplied with reducing equivalents by other proteins that vary. Fe protein. In Klebsiella there is no ferredoxin and flavodoxin (NifF protein) is used all the time. The reaction N2 + 3H2 Æ 2NH3 actually releases energy. as ATP. Azoferredoxin transfers electrons from reduced flavodoxin (or ferredoxin) to molybdoferredoxin. If there is an excess of azoferredoxin then ATP tends to be 19 . Molybdoferredoxin from one genus can often interact with azoferredoxin from another genus to give active enzyme. the activation energy needed to break the NºN triple bond is very high and in practice energy. or nitrogenase reductase Nitrogenase is not very fast (the turnover number is around 50 moles/min per mole of Mo) and so about 2-5% of the total cell protein is nitrogenase. These two proteins have several alternative names: Molybdoferredoxin = component I. In most bacteria electrons are passed from NAD(P)H or pyruvate to ferredoxin. nitrate reductase. a close relative of E.Structure and Operation of Nitrogenase Nitrogenase contains the two proteins molybdoferredoxin and azoferredoxin. Azoferredoxin is a dimer of identical subunits encoded by nifH and contains a single Fe4S4 group per dimer. Molybdoferredoxin is an alpha2/beta2 tetramer. The alpha and beta subunits are similar but distinct and are encoded by genes nifK and nifD. an FeS protein. coli where the accessory proteins are flavodoxin and pyruvate flavodoxin reductase. If iron is in short supply ferredoxin is replaced by flavodoxin. MoFe protein. Each tetramer contains 2 Mo and several FeS groups. The molybdenum is part of a low molecular weight cofactor containing Mo bound to an Fe7S8 cluster and to homocitrate. is consumed by NifH protein (azoferredoxin). xanthine oxidase). However.

wasted. In Klebsiella nifHDK form an operon that keeps the ratio of components constant. Oxygen.D = Molybdoferredoxin nifB.E. Carbon monoxide.R = regulation nifU.N. at least under normal conditions) Mechanism of Nitrogenase Nitrogenase will reduce many small molecules with triple bonds in addition to nitrogen. Biological nitrogen fixation (BNF) occurs when atmospheric nitrogen is converted to ammonia by an enzyme 20 .W. which is triple-bonded inactivates nitrogenase. another triply bonded molecule is a competitive inhibitor.S = metal center biosynthesis nifX.Z = MoFe cofactor synthesis nifY = MoFe cofactor insertion nifQ = Molybdenum uptake nifA.L. The Nif (nitrogen fixation) proteins are often referred to by their gene names: NifJ = Pyruvate Flavodoxin Reductase nifF = Flavodoxin nifH = Azoferredoxin nifM = processing of NifH protein nifK.V.T = function unknown (not necessary.

This mechanism also explains why acetylene. most N2 fixing bacteria contain hydrogenase which uses gaseous H2 to reduce NAD(P). Further transfer of two electrons activates the Fe of the MoFe cofactor in the active site.e. is a non-competitive inhibitor of N2 fixation. some of which will fix N2 chemically (but very inefficiently). Although N2 fixation wastes reducing power when H2 is evolved. Mo in active site is reduced from Mo6+ to Mo5+ to Mo4+ by sequential electron transfer from Azoferredoxin.(plus 2H+) from the active site Mo4+. ATP is no longer needed to hype up the redox potential) since only step requires extreme reducing power. Acetylene reduction discharges nitrogenase before it ever reaches full activation. N2 binds end on to the Fe[H]2 complex and releases H2. The bound N2 is reduced to HN=NH by sideways transfer of 2e.called nitrogenase. Conversion of N2H2 to 2NH3 requires two further 2e.       21 . Semi-activated Nitrogenase can reduce easy substrates such as Acetylene.[1] The formula for BNF is: N2 + 8 H+ + 6 e− → 2 NH3 + H2 Proposed Steps In Nitrogenase Mechanism :The mechanism is largely based on work with non-protein MoFe complexes. Hence they recycle the hydrogen at least partly.steps. which carries 2[H]. C2H2. but partial activation of the enzyme is sufficient (i.

NtrA is an alternative sigma factor used by RNA polymerase to recognize many genes involved in nitrogen metabolism which are not recognized by the standard sigma factor. Other organic N-sources will also repress nitrogenase. such as Azotobacter. nitrogenase is made in the presence of O2 (but is inactivated by O2). 22 . vnf and anf genes are very similar. the NifL protein binds to NifA and prevents it from activating the other nif genes. The nifL gene is required for O2 repression. The nifA gene encodes a protein required for switching on all of the nif genes except the regulatory genes nifLA themselves. Alternative Nitrogen Fixation Systems When Mo is absent some N-fixing bacteria.the even less efficient anf system. In the absence of NifL protein. The nitrogen regulators NtrC (= GlnG). its function is to activate the other nif genes. NtrC-P then binds to the upstream region of the nifLA operon and activates transcription. make an alternative nitrogenase in which vanadium is used instead of Mo. and NtrB determine whether or not the nifLA operon is expressed (depending on the presence of ammonia or organic nitrogen). When oxygen is present. coli to which the nif genes of Klebsiella have been transferred can fix N2. The nif genes are regulated by the nifLA operon. If vanadium is also absent Azotobacter can make a third nitrogenase which uses only iron . E. In the absence of ammonia or organic nitrogen the NtrC protein is phosphorylated by the NtrB protein.Regulation of Nitrogen Fixation All the nif genes in Klebsiella are clustered and coordinately regulated. The sequences of the nif. coli nitrogenase is expressed only in the absence of both O2 and NH3 in the growth medium. This is encoded by a duplicate set of vnf genes which make VFe cofactor as well as the corresponding nitrogenase proteins. The better the N-source the greater the repression. In both the original Klebsiella and the E. if NifA protein is made. which is needed for expression of the nifLA operon and the nif structural genes. Mo. if available represses the vnf system which is less efficient. NtrA (= GlnF = RpoN = s54) is the nitrogen sigma factor.

8% share in domestic production of Urea achieved in the country during 2009-10. Urea. IFFCO :    Established in 1967 Backed by five mega plants More than 39824 members societies More than 70 lakh metric tonnes of Fertilizer Production per annum.NPK. a Schedule A and Mini Ratna Company. Gujarat and Maharashtra. is the second largest producer of Nitrogenous Fertilizers in the Country with 15. The present production capacity of different biofertilizers production units in the country is about 4500 tones per annum . The actual production of biofertilizers during 1994-1995 was about 2000-2500 tones.NP and Biofertilizer Products  NFL : NFL.The maximum production capacity is in agro industries corporation followed by state agriculture universities and private sector.  They produce Nitrogen Biofertilizers and Phosphorous Biofertilizers. 23 . RCF : RCF is one of the leading producers of Fertilizers in India.2.DAP.5 INDUSTRIAL ANALYSIS OF BIOFERTILIZER India is one of the important countries in biofertilizer production and consumption in the world. UP. Among the different states the maximum production capacity is in Tamil Nadu followed by MP.

 Urea. Cyclohexanone. Methyl Ethyl Ketoxime. Dimethyl Formamide. Bio-Fuels. BVFCL : The Brahmaputra Valley Fertilizer Corporation Limited located on the bank of the river Dilli in the south-western border of Dibrugarh District in Assam is the first factory of its kind in India to use associated natural gas as basic raw material for producing nitrogenous fertilizer.C. Ammonium Sulphate. Sulphuric Acid. Gypsum. Anhydrous Ammonia. Microla and Biola are its major fertilizers. Dimethylacetamide in India.  Ammonia is used as a intermediate product which is used to make Urea. Ammonium bicarbonate.F is the only manufacture of DMF in India Main biofertilizer producing companies are :GSFC : GSFC today stands for superior quality with many of its products being ISO 9001 certified. Suphala 15:15:15. Methylamines. Oleum.   Today R. Argon Gas. Water Soluble Fertilizers. Sodium Nitrate. Sodium Nitrite. Nylon-6. Caprolactam. 24 . APS. RCF pioneered the manufacture of basic chemicals such as Methanol.  BVFCL has only one finished product ie Prilled Urea (Brand name : Mukta Urea). Melamine. Plant Tissue Culture seeds . NPK. Ujjwala. Bio-fertilizers. Sujala. Suphala 20:20:0. DAP.

Ministry of Chemicals and Fertilizers. Madras Fertilizers Limited has been serving the Nation for the past 40 years since plant commissioning in 1971 and is proud to be part of Green Revolution. Madras Fertilizers Limited is a Public Sector Undertaking under administrative control of the Department of Fertilizers.MFL : Established in 1966.Complex (17:17:17) (14:28:14) (19:19:19) (20:20:0:13) NK Mixture MOP (Imported) DAP (Imported) (20:0:10) Biofertilizers Azospirillum (Paddy) Azospirillum (Other crops) Azospirillum (Plantation Crops) Rhizobium Rhizobium (Groundnut) (Pulses) (All Crops) Phospho Bacteria NP Bio (All Crops) 25 . • Products :- Chemical fertilizers Urea NPK .

72 thousand tones.00 560.44 lakh tones. These are well manage by experienced agricultural graduates to provide the technical know-how.71 450. Organized promotional and marketing strategy for successful biofertilizers business is very important.43 450. most of the biofertilizer units lack in this respect.No the .00 737. The recent estimated potential demand of different kind of biofertilizers by government of Tamil Nadu is Rhizobium 35000 tones.38 thousand tones and phosphate solubilizer 275.8 26 .17 550.00 296. mainly because they did not indicate phosphate solubilizer and there estimates for Azospirillum were also low. Azolla 20. PSU/Coo p. blue green algae 267.03 450.the national biofertilizers development centre (NBDC) Ghaziabad and biotech consortium India ltd (BCIL) have estimated the total requirement of biofertilizers to be about 5. GSFC has established its own distributor network. Production technology of biofertilizers is relatively simple and its installation cost is very low compare to chemical fertilizer plants. MLF and SPIC have also well organized themselves in this respect.07 and 3.51 thousand tones. The total of all these amounts come out to be 12. Azospirillum 482 thousand tones. conduct the demonstration and give before and after sale service to the farmers. Azotobacter 162.  PRODUCTION STATISTICS OF BIOFERTILIZER Name of S.61 thousand tones.44 lakh tones which is significantly higher than the estimates of NBDC and BCIL. In order to provide biofertilizers upto village level.00 775. Fertilizer company like GSFC has more than 200 farm information centres-cum-depots suitated in remote areas. Based on the area under different crops and dose of biofertilizers to be applied .00 602. KRIBHC O n y n y n y n y n Capacity/Production (in MT) 250. 2002-03 2003-05 2005-06 2007-08 2009-10 Capacit Productio Capacit Productio Capacit Productio Capacit Productio Capacit Productio y 1.

95 0.10 195.00 150.00 240.34 84.00 240.91 105.00 400.21 3.00 394. 5.54 6. 4.00 339.00 100. 7.92 4.80 58.00 100.00 133.00 0. 6.27 165.00 0.00 431.84 8. REGI South North East West North- 27 .00 150.00 400.00 240.00 100.00 150.00 150. IFFCO NFL MFL FACT RCF BVFCL 240.25 125.15 0.39 213.00 240.00 100.00 30.06 .43 0. 3.00 0.00 3.12 143.14 130.2.00 7.00 100.00 100.00 400.23 186.00 150.39 228.00 150.68 0.00 150.00 400.50 115.00 0.00 54.75 39.00 150.00 136.95 234.00 150.00 150.

3 MATERIALS AND METHODS REQUIREMENTS :28 .

RHIZOBIUM Rhizobium is a genus of Gram-negative soil bacteria that fix nitrogen. The solid inoculants carry more number of bacterial cells and support the survival of cells for longer periods of time. The bacteria colonize plant cells within root nodules. Rhizobium forms an endosymbiotic nitrogen fixing association with roots of legumes and Parasponia. here the bacteria convert atmospheric nitrogen to ammonia and then provide organic nitrogenous compounds such as glutamine or ureides to the plant. The organic carrier materials are more effective for the preparation of bacterial inoculants. Biofertilizers are formulated usually as carrier based inoculants. The plant provides the bacteria with organic compounds made by photosynthesis 29 .1 STRAIN SELECTION Biofertilizers are carrier based preparations containing efficient strain of nitrogen fixing or phosphate solubilizing microorganisms.GLASSWARES Petri Plates Test Tubes Conical Flasks Beakers Spreader Glass Rod Measuring Cylinder EQUIPMENTS Autoclave Laminar Air Flow Incubator Hot Air Oven 3.

2-3. convex. Fastgrowing. slow (Bradyrhizobium) Doubling time : fast grower 2-4 hours slow grower 6-12 hours. mono and disaccharide. The percent G+C ranges from 59-64%. C source : supplied by legume through photosynthesis. Cells stain Gram negative. Symbiotic with legume. Granules of polyhydroxybutyrate common.Identifying characterstics                Strictly aerobic. Contains the enzyme nitrogenase. semi-translucent. N source : fixed from atmosphere. Physiological properties        Nature : Chemoheterotrophic. Respiration : aerobic. Chemoorganotrophic. Non-spore forming. Growth : fast ( Rhizobium).0 um in size. 0. Growth media : YEMA 30 . Rod shaped cells. Forms root nodules with legumes and undergoes nitrogen fixation. Host specific. Mobile by a single polar flagellum or two to six peritrichous flagella.5-0. raised and mucilaginous. Colonies are white pigmented.9 um x 1. Colonies are circular.

1 atm. Rhizobium is surrounded by a slimy capsule made of exopolysaccharide. Rhizobia are predominantly aerobic chemoorganotrophs and grow well in the presence of oxygen and utilize a wide range of relatively simple carbohydrates and amino compounds.leguminosarum R. Their cellular morphology and biochemical characteristics are very similar to those of nonsymbiotic nitrogen-fixing bacteria called Azotobacter.phaseoli R.0.0-7. Table of important Species :S.Taxonomy of Rhizobia All rhizobium are root nodulating bacteria and are gram negative. Vicia.No Rhizobium sps. many strains are able to grow well under microaerophillic conditions at oxygen tensions of less than 0. non spore forming medium sized rod shaped cells that contain the enzyme complex called nitrogenase and are typically motile. The primary distinguishing characteristic of Rhizobium species is their ability to nodulate leguminous plants. And also helps the bacterium stick to root hairs during various stages of its life cycle. Trigonella 5 6 7 R.japonicum R. Lens Phaseolus Trifolium Melilotus.meliloti Cross inoculation group Pea group Bean group Clover group Alfalfa group Legume types Pisum.sps Lupini group Soybean group Cowpea group Lupinus Glycine Vigna.) Optimal growth of most strains occurs at a temperature range of 25-30 C and at a pH of 6. 31 . Arachis Medicago.trifolii R.lupini R. Despite their usual aerobic metabolism. 1 2 3 4 R. (With the exception of a few strains. which protects it from drying out. they have not been found to fix N in the free-living form except under special conditions.

plant proteins similar to human Hemoglobins help to provide oxygen for respiration while keeping the free oxygen concentration low enough not to inhibit nitrogenase activity. decreased carbon supply to nodules. ammonium (NH4+).Nodule formation and functioning Infection threads grow to the nodule. or reduced oxygen supply to nodules that fix less nitrogen. infect its central tissue and release the rhizobia in these cells where they differentiate morphologically into bacteroids and fix nitrogen from the atmosphere into a plant usable form. Recently. although called "partner choice" by the authors. Because several unrelated strains infect each individual plant. wild lupine plants allocate less resources to nodules containing less-beneficial rhizobia. The partner choice hypothesis proposes that the plant uses prenodulation signals from the rhizobia to decide whether to allow nodulation and chooses only non-cheating rhizobia. Similarly. suggesting the existence of alternative communication signals other than Nod factors. Leghaemoglobins. This is consistent with the definition of sanctions just given. a classic tragedy of the commons. other studies have found no evidence of plant sanctions and instead support the partner choice hypothesis. There are two competing hypotheses for the mechanism that maintains legume-rhizobium symbiosis (though both may occur in nature). But this form of cheating should be equally tempting for all strains. it was discovered that a Bradyrhizobium strain forms nodules in Aeschynomene without producing Nod factors. early nodule death. and sufficient oxygen so as not to interfere with the fixation process. The sanctions hypothesis suggests that plants police cheating rhizobia. any one strain could redirect resources from nitrogen fixation to its own reproduction without killing the host plant upon which they all depend. limiting rhizobial reproduction inside. which reduce rhizobium reproduction (perhaps by limiting oxygen supply) in nodules that fix less nitrogen. Sanctions could take the form of reduced nodule growth.[ The legume–rhizobium symbiosis is a classic example of mutualism—rhizobia supply ammonia or amino acids to the plant and in return receive organic acids (principally as the dicarboxylic acids malate and succinate) as a carbon and energy source—but its evolutionary persistence is actually somewhat surprising. There is evidence for sanctions in soybean plants. AZOTOBACTER 32 . utilizing the enzyme nitrogenase. In return the plant supplies the bacteria with carbohydrates. However. proteins.

The first representative of the genus. Lipman described Azotobacter vinelandii. binding atmospheric nitrogen. In 1903. nigricans and Azotobacter nigricans subsp. Russian microbiologist Nikolai Krasilnikov identified the species of Azotobacter nigricans KRASIL'NIKOV. They are found in neutral and alkaline soils. Azotobacter chroococcum. and releasing it in the form of ammonium ions into the soil. Taxonomy Scientific classification Domain: Bacteria Kingdom: Bacteria Phylum: Proteobacteria Class: Gammaproteobacteria Order: Pseudomonadales Family: Pseudomonadaceae/Azotobacteraceae Genus: Azotobacter The Azotobacter genus was discovered in 1901 by the Dutch microbiologist and botanist Martinus Beijerinck. it is used by humans for the production of biofertilizers. which he named in honor of Beijerinck. 1949 which was divided in 1981 by Thompson Skerman into two subspecies of Azotobacter nigricans subsp. 1904. Apart from being a model organism. was discovered and described in 1901 by the Dutch microbiologist and botanist Martinus Beijerinck. and a year later Azotobacter beijerinckii LIPMAN. Azotobacter are Gram-negative bacteria.Azotobacter is a genus of usually motile. who was one of the founders of environmental microbiology. achromogenes. in water and in association with some plants. They are aerobic. food additives and some biopolymers. In 1949. free-living nitrogen fixer. which is inaccessible to plants. in 33 . He selected and described the species Azotobacter chroococcum – the first aerobic. free-living soil microbes which play an important role in the nitrogen cycle in nature. oval or spherical bacteria that form thick-walled cysts and may produce large quantities of capsular slime.

FA8 Azotobacter nigricans Azotobacter paspali Azotobacter salinestris Azotobacter tropicalis Azotobacter vinelandii Identifying characterstics       Aerobic but can grow under reduced oxygen conditions. Large rod of varying length down to coccoid morphology. but then were transferred to the family Pseudomonadaceae based on the studies of nucleotide sequences 16S rRNA. Thompson and Skerman described the Azotobacter armeniacus THOMPSON SHIVPRASAD 1991 which was dependent on sodium ions. but do form thick-walled cysts. or in clumps. Azomonas and Pseudomonas are related and might be synonyms. a phylogenetic study revealed that Azotobacter vinelandii belongs to the same clade as the bacterium Pseudomonas aeruginosa. AR Azotobacter beijerinckii Azotobacter chroococcum Azotobacter sp. In 1991. in chains. 34 . representatives of the genus were assigned to the family Azotobacteraceae PRIBRAM. Can occur singly. Page and Shivprasad reported a microaerophilic and air-tolerant type Azotobacter salinestris PAGE AND Earlier.[64] AND SKERMAN.[65] and in 2007 it was suggested that the genera Azotobacter. 1981. No endospores are formed. In 2004. DCU26 Azotobacter sp. may be almost the size of yeasts. Azotobacter agilis Azotobacter armeniacus Azotobacter sp.the same year. 1933. Gram negative. 2 um or more in diameter.

but they can be partially replaced by vanadium ions. thus this growth mode may occur in nature.0–7. alcohols and salts of organic acids as sources of carbon and can fix at least 10 micrograms of nitrogen per gram of glucose consumed. Optimum temperature range between 20 and 30°C. It can benefit crops by Nitrogen fixation. but growth is sustained in the pH range from 4.5.0 – 7.8 to 8. which may form films in liquid nutrient media. 35 substances. ammonium ions or amino acids. Azotobacter can use a variety of carbohydrates. green and other colors. Nitrogen fixation requires molybdenum ions. Azotobacter produce flat. The source of nitrogen can be nitrates. using organic compounds as electron donors. Copious amounts of capsular slime may be formed.66 %. The growth is favored at a temperature of 20–30 °C Benefits :    It improves seed germination and plant growth Azotobacter are tolerant to high salts. receiving energy from redox reactions.8. this growth mode is hydrogen dependent. fungi static growth.[24] While growing.5.     May be mobile by peritrichous flagella or non-mobile. fluorescent or red-violet/brownishblack. slimy. growth promoting substances.[23] Azotobacter can also grow mixotrophically.5.   Growth range is between pH 5. but optimum pH is 7. The optimal pH for the growth and nitrogen fixation is 7.5. Physiological properties Azotobacter respire aerobically. either yellow-green. Non-symbiotic nitrogen fixers. Catalyse positive.5 . in a nitrogen-free medium containing mannose. The colonies can have dark brown. or may be colorless. Hydrogen is available in the soil. paste-like colonies with a diameter of 5–10 mm. The percent G+C = 63 . Azotobacter is heaviest breathing organism and requires a large amount of organic carbon for its . depending on the species. Can produce a water soluble pigment.

However. It is poor competitor for nutrients in soil and hence its growth promoting substances. There are some micro-organism which stimulate the Azotobacter population in soil thereby increasing the nitrogen fixation by Azotobacter. Similarly.. Azotobacter is less effective in soils with poor organic matter content. There are different strains of Azotobacter each has varied chemical. so also it controls plant diseases due to above substances produced by Azotobacter. Azotobacter also requires calcium for nitrogen fixation. Besides carbon. biological and other characters. For example cephallosporium is most commonly found organisms in soil which restricts the growth of Azotobacter. a medium used for growth of Azotobacter is required to have presence of organic nitrogen. indol acitic acid and giberalin. Riboflavin. On the other hand there are some micro-organisms which adversely affect the Azotobacter population and hence nitrogen fixation process is hampered. Azotobacter also produces fixation of Thiomin. These various species survive in soil while maintaining a balance of population is between various microbial species within certain limits. A normal population of Azotobacter could be about than 10 thousand to 1 lakh/g of soil and it is mostly influenced by other micro-organisms present in soil. It thrives even in alkaline soils. Azotobacter uses carbon for its metabolism from simple or compound substances of carbonaceous in nature. When Azotobacter is applied to seeds. some strains have higher nitrogen fixing ability than others. Nicotin. Azotobacter in Soil Soils are habited by a very large number of microbial species. Importance 36 . micronutrients and salt in order to enhance the nitrogen fixing ability of Azotobacter. seed germination is improved to a considerable extent.   Functions of Azotobacter :Azotobacter naturally fixes atmospheric nitrogen in the rhizosphere. The co-existence of the relative populations of each one of the species is determined by ecological factors prevailing in the soil. fungistatic substance.

mash bean. such as 2. Applications Owing to its ability to fix molecular nitrogen and therefore increase the soil fertility and stimulate plant growth. They also facilitate the mobility of heavy metals in the soil and thus enhance bioremediation of soil from heavy metals. In fact. is native to Bangladesh. They comprise of one of the several species to have been moved from the genus Phaseolus to Vigna. in the food industry as an additive to ice cream. these beans are eaten either whole or as bean sprouts. India. Taiwan. and green soy. Pakistan. Mung beans are commonly used in the preparation of Chinese dishes. They are also used to make soups and desserts. mongo. and Southeast Asia.Nitrogen fixation plays an important role in the nitrogen cycle.6-trichlorophenol.4. thereby stimulating plant growth. mung beans are called đậu xanh. also known as green bean. moog dal. The dal is green in color along with the husk and yellow when the husk is removed. Azotobacter also synthesize some biologically active substances. Mung beans are small and ovoid in shape. Azotobacter are widely used in agriculture. mercury and lead. moong. Particularly in nitrogen biofertilizers such as azotobacterin.[61] and in the biosorption of metals. Some kinds of Azotobacter can also biodegrade chlorine-containing aromatic compounds. the English word ‘mung’ has been derived from the Hindi word ‘moong’. mung. Korea. India and Pakistan. the bean is known as moong dal in India. They are also used in production of alginic acid (E400). The starch extracted from 37 . The latter was previously used as an insecticide. golden gram. green gram. They are also used extensively in Thailand. It is the seed of Vigna Radiata and when split. such as cadmium. In Vietnam. where they are called lǜ dòu. MOONG BEAN Mung bean. Japan. munggo or monggo. which is applied in medicine as an antacid. fungicide and herbicide but later found to have mutagenic and carcinogenic effects. puddings and creams. including some phytohormones such as auxins. Generally.

38 .them is used to make jellies and noodles. along with their nutritional value. Mung beans are known to be very healthy and packed with a variety of nutrients. Read on to know the health benefits of eating mung beans.

Kingdom: (unranked): (unranked): (unranked): Order: Family: Genus: Species: Plantae Angiosperms Eudicots Rosids Fabales Fabaceae Vigna V. radiata Binomial name Vigna radiata Health and Nutrition Benefits Of Eating Moong bean 39 .

studies show that it may have negative effects on health. which is essential in a metabolic process known as methylation cycle. or hypoglycemia. fiber and protein. high fiber in green beans keeps blood sugar from rising after mealtime. green beans are popularly used for hot. which helps one to lower the high cholesterol level in the blood system. magnesium and vitamin B6 which help promote heart health. green beans contain thiamin (vitamin B-1). A high level of homocysteine in the blood is attributed to heart attack. Herbalists use them for all hot. which aid digestion. It is a rich source of protein and fiber. peripheral vascular disease and stroke. Since moong dal or green bean is a low carb diet. Aids in weight loss ie Moong dal or green beans provide great source of complex carbohydrates. they provide an excellent source of folates. inflammatory conditions. This makes green beans a great choice for people with insulin resistance. Lowers cholesterol levels. they are an excellent source of molybdenum and folic acid. In Chinese medicine. fiber. vitamin-C and vitamin-B6 (pyridoxine). in Chinese medicine. iron and potassium The bean is popular as the perfect food for reducing weight. Promote heart health. folic acid. as it has a very low fat content. Complex carbs are also effective in stabilizing blood sugar and prevent its rapid rise after meal consumption. Mung bean sprouts contain rich quantities of Vitamin A. mung bean sprouts are considered as a cooling food. Provides resistance against infectious diseasesand also. apart from keeping body’s energy at a balanced level. The high fiber content of mung beans yields complex carbohydrates. It prevents neural-tube defects during pregnancy. C and E. such as calcium. inflammatory conditions. Vitamin B6 and folic acid lower homocysteine levels in the body. 40           . It is recommended as a food replacement in many slimming programs. They are also known to be an excellent source of many minerals. Contain anti-cancer properties. Green beans have antioxidants properties. regular consumption of green beans helps to reduce cholesterol since they are rich in fiber. They were considered to have anti-cancer properties and were also used as a cooling diet. such as hypertension. Prevent age related muscular diseases. Those who suffer from diabetes or high cholesterol are recommended frequent consumption of mung bean. They provide a good nutrition for diet ers since they are low in fat. B. containing anti-cancer properties. According to recent studies. a great nutrition during preconception. Regular consumption of diets rich in Vitamin-B6 help you develop resistance against contagious agents that cause diseases. Also. ranging from systematic infections to heat stroke and even hypertension. especially with regards to weight loss.

The growth mediums used for mass culturing of different bacterial biofertilizers used by us are Yema Medium and Jensen Medium. CULTURING OF MICROORGANISMS Although many bacteria can be used beneficially as a biofertilizer the technique for production of Rhizobium and Azotobacter are discussed here.  The media in 50 mL flask is inoculated with efficient bacterial strain of Rhizobium and Azotobacter under aseptic conditions. 200 mL and 250 mL) containing the media.i. 41 .  The broth is checked for the population of inoculated organism and contamination if any at the growth period.  Observe flasks for growth in culture and estimate the population. 100 mL and 250 mL conical flasks and sterilize. which serves as the starter culture. INOCULUM PREPARATION  Prepare appropriate medias for specific to the bacterial inoculant ( Rhizobium and Azotobacter) in 25 mL.the above media for Rhizobium and Azotobacter is prepared in large quantities and kept ready for use. 50 mL.  After obtaining growth in each flask.  Keep flasks under room temperature in Rotary Shaker (200 rpm) for 2.  Using the starter culture (at log phase) inoculate the larger flasks (100 mL. ii.4 days.

iii. one of the colony is picked up and put in the bottles containing media in order to scale up the culture. STREAKING ON PLATES For Rhizobium In this step streaking of Rhizobium is done using an inoculation loop on its respective media (Yema media). 42 . the plates are kept in the Incubator for 24-48 hrs at 37 degree centigrade.Bacterial colonies are picked up and and scaling up of culture is done. Now when after 48 hrs bacterial colonies become visible.After the completion of streaking. For Azotobacter Streaking of Azotobacter is also done using inoculation loop on the media (Jensen media). After streaking is done the petriplates are kept in the Incubator for about 24-48 hrs at 37 degree centigrade.

Vermicompost is a preferred nutrient source for organic farming. lignite. organic material containing adequate quantities of N. friability and vulnerability. The neutralized peat soil/lignite are found to be better carrier materials for biofertilizer production The following points are to be considered in the selection of ideal carrier material. • Cheaper in cost • Should be locally available • High organic matter content • No toxic chemicals • Water holding capacity of more than 50% • Easy to process. PROCESSING OF CARRIER MATERIAL The use of ideal carrier material is necessary for the production of good quality biofertilizer. PREPARATION OF CARRIER MATERIAL VERMICOMPOST Vermicompost is an organic manure (bio-fertilizer) produced as the vermicast by earth worm feeding on biological waste material. It is eco-friendly. farmyard manure and soil mixture can be used as carrier materials. v. vermiculite.iv. charcoal. Peat soil. consumes low energy input for composting and is a recycled biological product. This compost is an odorless. 43 . P. non-toxic. plant residues. K and several micronutrients essential for plant growth. press mud. clean.

vi. This thick slurry is now placed evenly in trays which are kept for drying. resulting in a geometric progression of the concentration in a logarithmic fashion. It should be noted that during autoclaving. Gamma-irradiation is the most suitable way of carrier sterilization. carrier material is packed in thin-walled polyethylene bag. because the sterilization process makes almost no change in physical and chemical properties of the material. vii. some materials changes their properties and produce toxic substance to some bacterial strains. In brief. 44 . Usually the dilution factor at each step is constant.The process of drying may take 2-3 days as these trays are kept in sunlight. Carrier material is packed in partially opened. BINDING OF CULTURE WITH CARRIER In this step the Carrier (vermicompost) in mixed with respective bacterial cultures (Rhizobium and Azotobacter) with the help of a Glass Rod until a thick slurry is formed. STERILIZATION OF CARRIER Sterilization of carrier material is essential to keep high number of inoculant bacteria on carrier for long storage period. Another way of carrier sterilization is autoclaving.Complete drying is necessary as this slurry has to be beaten to convert into powdered form. viii. thin-walled polypropylene bags and autoclaved for 60 min at 121 ºC. and then gamma-irradiated at 50 kGy (5 Mrads). SERIAL DILUTION METHOD FOR COUNTING OF BACTERIAL COLONIES Serial dilution is the stepwise dilution of a substance in solution.

Serial dilutions are used to accurately create highly diluted solutions as well as solutions for experiments resulting in concentration curves with a logarithmic scale.16-fold (100. A tenfold dilution for each step is called a logarithmic dilution or log-dilution and a 3. Make sure the solution is uniformly mixed. Materials  Buffer used to dissolve the sample  The sample  Multiple tubes  Pipette or graduated cylinder  Stirring rod Procedure  Shake the solution by hand or use the stirring rod to swirl the solution. Besides the more conventional uses described above. serial dilution may also be used to reduce the concentration of microscopic organisms or cells in a sample. 45 .5-fold) dilution is called a half-logarithmic dilution or half-log dilution.

-6. Take half of the solution out to a new tube and add equal amount of buffer into it to dilute the solution concentration to half of the original solution.  Keep plate in incubator for 1 to 2 days for result and then count the colonies.-8 -10) and take the tube for measuring are 10 (-4 and -6) and spread on media plates.  In this procedure we take 10 (-2. analysis is not necessary. Result On YEMA media for Rhizobium Measuring plate 10(-4) = 10 colonies Plate 10(-6) = 7colonies On Jensen media for Azotobacter Measuring plate 10(-4) = 17 colonies Plate 10(-6) = 11 colonies 46 .  Continue the process until reaching the desired concentration of the solution. Therefore.  Take half of the newly made solution to another new tube and add equal amount of buffer into it to dilute the solution concentration to one quarter of the original solution.-4. Analysis Serial dilution is a process of solution preparation.

Now after 24 hrs sprouts are seen. FIELD TREATMENT Moong Dal seeds are taken and grown for two days until sprouts come out of them.These pots need to be watered everyday. vermicompost containing Rhizobium and Azotobacter cultures is mixed and Moong Dal seeds are sown into these pots.ix. 47 . Now pots are taken . SEED PREPRATION FOR INOCULATION Moong dal seeds are taken and dipped in water for about two days.After two days all the seeds are taken out of water and tied in piece of cloth for a 12-24 hrs.The cloth is watered 3-4 times to keep it wet. are filled by soil. x.

There should be minimum yield of an undesired product. but the resulting medium although supporting satisfactory growth may be un suitable for use in large scale process. It should permit the maximum rate of product formation.2 MEDIA USED FOR BIOFERTILIZER PRODUCTION All micro organisms require water. 4. • The population of inoculant in the carrier inoculant packet may be determined at 15 days interval. It should produce the maximum yield of product or biomass per gram of substrate used. There should be more than 109 cells per gram of inoculant at the time of preparation and107 cells per gram on dry weight basis before expiry date. 48 . On a large scale one must normally use sources of nutrients to create a medium that will meet all of the following criteria:1. 2. 3. STORAGE OF BIOFERTILIZER PACKET • The packet should be stored in a cool place away from the heat or direct sunlight. • The packets may be stored at room temperature or in cold storage conditions in lots in plastic crates or polythene/gunny bags. source of energy carbon. nitrogen. It should be of a consistent quality and be available throughout the year.XI. 3. minerals element and possibly vitamins plus oxygen if aerobic. On a small scale it is relatively simple to devise a medium containing pure compound.

phosphorous. growth hormones and ions.The constituents of a medium must satisfy the element requirement for cell biomass and metabolite production. lipids and proteins.Vegetable oils (olive. linseed. Micronutrients and it includes trace elements. Macronutrients and it includes carbon. hydrogen. cleaning and rinsing. maize. ii. Some microorganisms can also use hydrocarbon or methanol as carbon sources. When assessing the suitability of water supply it is important to consider pH. soyabean) 49 . magnesium and potassium. vitamins.energy from growth comes from either the oxidation of medium components or from light.it is common practice use carbohydrates as the carbon source in microbial fermentation processes. The qualitative and quantitative nutritional requirements of cell are always determine to optimize growth and product formation. Glucose Molasses Corn stew liquor Lactose b) Oils and fats :. growth hormones and certain metabolic precursors. oxygen. dissolved salts and effluent contamination. Microorganisms need following components for their growth :WATER: . Following carbohydrates are used as carbon sources :• • • • • • Starch – Starch is obtained from maize grain and other cereals. Most industrial microorganisms are chemorganotrophs. CARBON SOURCES: a) Carbohydrates: . therefore the commonest source of energy will be the carbon source such as carbohydrates. cooling. Clean water of consistent composition is therefore required in large quantities from reliable permanent sources. Sucrose – Sucrose is obtained from sugarcane and sugar beet.water is the major component of all fermentation media and is needed in many of the ancillary services such as heating. ENERGY SOURCES: . Nutrients required by microbial cells are classified as:i. nitrogen. there must be an adequate supply of energy for biosynthesis and cell maintenance. potatoes and cassava. cottonseed. Most of the fermented products formed by organism are produce as a result of their response to the environmental condition such as nutrients. sulfur.

ammonium salt or nitrate. P. fine chemicals and agricultural chemicals. BUFFERS: . Others such as Co. GROWTH FACTORS: . Fe. In many media. they must be added as distinct component. K.5 0.all microorganisms require certain mineral elements for growth and metabolism. pharmaceuticals. Mn. fatty acids or sterols.K2PO4 etc are also included in the media as raw materials. Inoganic nitrogen maybe supplied as ammonia gas.Hydrocarbons and their derivatives might have potential role in higher value products such as intermediates.the buffers are used to control the pH of media. Mo and Zn are essential but are usually present as impurities in other major ingredients. The growth factors most commonly require vitamins but there maybe also be need for specific amino acids. Ca and Cl are essential components.most industrially used microorganisms can utilize inorganic or organic sources of nitrogen. MINERALS: . Buffers like sodium acetate.0 20. Mg. Ammonia has been used for pH control and as the.1 7. Many of natural carbon and nitrogen sources used in media formulations contain all or some of the required growth factors.2 0.c) Hydrocarbons and their derivatives: .0 1000ml Jensen’s Media for Azotobacter:Composition Ingredients gms / litre 50 . of and because of the concentration required.0 1. YEMA Media (Yeast Extract Mannitol Agar Media) for Rhizobium :Composition Ingredients Magnesium sulphate Sodium chloride Mannitol Yeast Extract Agar Distilled Water gms / litre 0. Cu. S. NITROGEN SOURCES :.some microorganisms cannot synthesize a full component of a cell component and therefore require performed compounds called growth factors.

000 0. At present the use of chemical fertilizers is far below the recommended level. amino-acids etc. On the other hand biofertilizers supply the nitrogen continuously throughout the entire period of crop growth in the field under favourable conditions. RESULT AND CONCLUSION Biofertilizers improve soil fertilitily and enhance nutrient uptake and water uptake in deficient soil thereby aiding in better establishment of plants. Biofertilizers also secrete growth substances and antifungal chemicals as well as improve seed germination and root growth.500 0. vitamins. which harm the environment and deplete non renewable energy sources.Sucrose Dipotassium phosphate Magnesium sulphate Sodium chloride Ferrous sulphate Sodium molybdate Calcium carbonate Agar 20.000 15. OBSERVATION 5. The deleterious effects of chemical fertilizers are that they are toxic at higher doses. Chemical fertilizers supply over nitrogen whereas Biofertilizers provide inaddition to nitrogen certain growth promoting substances like hormones. Thus the use of biofertilizers will effectively enrich the soil and will cost less then chemical fertilizers.500 0.100 0. crops have to be provided with chemical fertilizers repeatedly to replenish the loss of nitrogen utilized for crop growth. It may be borne in mind but biofertilizers are no substitute for chemical fertilizers. QUALITY CONTROL REQUIRED FOR BIOFERTILIZER 51 . Continuous use of chemical fertilizers adversely effect the soil structure whereas biofertilizers when applied to soil improve the soil structure.000 1. Therefore the aim and object of spread of biofertilizer technology as a industry has to build up efficiency in use of chemical fertilizers supplemented by low cost inoculants to the extent possible. Biofertilizers have definite advantage over chemical fertilizers.000 4. biofertilizers however have no toxic effects. The dual effects of phosphorus mobilizing fungi and specific nitrogen fixing bacteria can cater to the needs of current coffee plantation sector.005 2.

3. mask and apparen is necessary to avoid contamination. date of manufacture. The packet has to be used before its expiry. batch number and instructions for use. 52 . only for the specified crop and by the recommended method of application. the color depending upon the color of the carrier. Right combinations of biofertilizers have to be used. Other chemicals should not be mixed with the biofertilizers.5 and 7. As Rhizobium is crop specific.• • • • • • • Colors: Carrier based. Peat soil. Carrier and its particle size: Peat lignite. Contamination: The product should be free from all types of contamination upto 105 dilutions. date of expiry. pH: The pH should be between 6. name and address of the manufacturer.3 PRECAUTIONS             Surface Sterilization of LAF is very important. Moisture: Carrier should be in moist form with 30-40% moisture. It is important to use biofertilizers along with chemical fertilizers and organic manures. name of the crop for which intended. Biofertilizers are live product and require care in the storage Both nitrogenous and phosphatic biofertilizers are to be used to get the best results. Expiry period: Maximum of 6 months from the date of manufacture. Total viable count: Total number of viable cells during the entire period of shelf life should be at minimum of 1x10^8 g of carrier on dry mass basis.5. Biofertilizer packets need to be stored in cool and dry place away from direct sunlight and heat. Nails should be properly cut. Wearing gloves. one should use for the specified crop only. While purchasing one should ensure that each packet is provided with necessary information like name of the product. Humus are similar material in the form of powder capable of passing through 150-212 micron in sieve.

EQUIPMENTS In biofertilizers production industry equipment are the major infrastructure which involve 70% of capital investment.        Autoclave Laminar Air Flow Incubator pH Strips Inoculation Loop Spirit Lamp Hot air Oven Autoclave 53 .V. some of the instrument can be replaced with a culture room fitted with a U. The correct use of equipment will give uninterrupted introduction with quality inoculums. lamp Autoclave. Any compromise on the usage of the following mentioned equipments may finally decline in the quality of biofertilizers. Biofertilizers are not replacement of fertilizers but can supplement plant nutrient requirements. Hot air oven. After studying the principle behind the use of all instruments. Incubator and sealing machine are indigenously made with proper technical specification.

It is aapparatus in which materials are sterilized by air free saturated steam ( under pressure ) at a temp above 00 oC . Culture transfer and inoculation can be done here. This uniform flow of Air will prevent settling of particle in the work area. the temp will rise to 121 oC . Normally all growth medium sterilized under autoclave . Laminar Air flow LAF chamber provide a uniform flow of sterilized air. Air borne contamination is avoided in the chamber. This is sufficient to destroy all vegetative cell. If the steam pressure inside the autoclave is increased to 15 psi. Incubator 54 .

S. Multiplication of starter culture can be done in this instrument. Lee. B. and Wilson..60 (1942). Proc. and Wilson.. S. Sot. S. Dry heat is used in this apparatus to sterilised the materials.. and Wilson. 265 (1942). temp. Biol. H. W. W. P. J. Normally 180 oC is used for 2 hrs for sterilizing glass ware BIBLOGRAPHY • • • Phelps.. 47. Bad. 55 ... B. Lee. P. A. Chem. 473 (1941). and Med. humidity) required for the growth and development of microorganism. P. Exp.. Burris. R... Wilson.. Biol. 43.Incubator providing controlled condition (light. B. Hot Air Oven Hot air oven is meant for sterilizing all glass ware materials. 144. W. J.. J.

Ithaca. pp 275-287 (2003). A. and Jain. (2002) Vermiculture in India . S. Cytryn. 1996. ISBN.. 1981. Ministry of Environment and Forests.. Cambridge University Press. CABI Publishing. and Engineering Service (NRAES) (1992) On-farm composting. Kaushik. Meerut (1996). Vol. Sammerfield and A.. Avarre.An eco. Studies on Azotobacter species in soil.H. V. Kumar Vibhas. M. Rynk. N. USA.• Giraud. P. Pal A. R. crops and soil fertility: concepts and research methods. Prentice Hall. Barbe. R. 309–319. V. New Delhi. Kew England.K. Caister Academic Press The Nature and Properties of Soil" by Nyle C. R. and E. 11th Edition The Nitrogen Fate and Transformations Game". American Naturalist 156:567-576 Allen. Gibson.. Rastogi & Co. Govt of India. Environmental Pollution Control Journal. Vallenet. NRAES Cooperative Extension. NJ 07458.C. Jaubert. 1980. E. Das.K. 26 Alagwadi and Gaur (1988) Trees. M. Moir.1126/science. R. JWB (editor) (2011).. D et al.E. D. Sharma. Simon. F. "Legumes symbioses: absence of Nod genes in photosynthetic bradyrhizobia. Environmental Challenges of the 21st Century. Legume sanctions and the evolution of symbiotic cooperation by rhizobia. Gupta. Brown. Enviro News. Natural Resource. Allen. et al. April-June. doi:10.J.(1999).9. Biofertilizers: An eco-friendly alternative to chemical fertilizers. Journal of Natural Resources and LIfe Sciences Education. Nitrogen Fixation. Brady and Ray R. ^ a b c d e Postgate.online training material.K. Weil. Comparison of media and techniques for counting Azotobacter in soil.K. PMID 17540897.K. • • • • • • • • • • • • • • • • • • • Denison. Science 316 (5829): 1307–12. Host determinant in nodulation and nitrogen fixation. 2000. Eds..P. Pune. New Delhi (2005). Royal Botanic Gardens. University of Wisconsin Press. Upper Saddle River. Agriculture. I. Vol. Bunting p69. Biofertilizer. Elements of Biotechnology.1139548. Gupta. Inc. 1962. Jackson. 56 . Plant and Soil 17.M.0851995934. L. R. and Saxena. O. Nov-Dec. 3rd Edition. edited by R. Jambhhekar H. JC. S. (2007). ISBN: 0-02-313371-6. Maharashtra. J (1998). The Leguminosae. Cambridge UK.. India..K.K. Nitrogen Cycling in Bacteria: Molecular Analysis.friendly fertilizer. Agricultural Bioteks.H.. APH Publishing Corporation.". Burlingham. Eric. P. In: Advance in legume Science.

1997. WEBSITES 57 .• • Clementi. Critical Review in Biotechnology 4 Environmental Microbiology Vol.. Alginate production by Azotobacter vinelandii. F.70.

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