1

SECTION-1

1. Microbiology laboratory and its equipments and machineries2. Nutritional media and their preparations and methods of sterilization3. Isolation of beneficial microbes: From legume root nodules, rhizospheric soil, plant samples, etc.4. Metabolic characteristics and growth identification of different microbes

1. Laboratory Equipments, glassware, other necessitiesThe minimum equipments needed in a microbiological laboratory are as follows:

1.1. Equipments

1. Compound biological microscope with low, high and oil-immersion lenses, measuring micrometersand haemocytometers for microscopic examination, dimension and counting of vegetative andcyst/spore/buds etc.

2. Autoclave for sterilization of media3. Hot-air oven for sterilization of glassware4. Incubator (25 - 40°C range) for microbial growth5. Colony counter for cfu counts in agar plates6. Water bath controlled temperature operations like melting of solid agar media7. Refrigerator for storing strains etc8. pH meter for measuring acidity / alkalinity of media9. Weighing balance for weighing of ingredients of media10. Bunsen burner / sprit lamp

1.2. Glassware and other necessary materials

1. Petri dishes for holding molten media2. Conical flasks, 50 - 1000 ml range size3. Test tubes or culture tubes without rim or culture tubes with caps (Biju bottles)4. Funnels for pouring media5. Pipettes ranging from capillary size to all graduated size up to 10 ml6. Measuring cylinders7. Beakers, 50 - 1000 ml range8. Glass slides and cover slips9. Bent glass rods (spreaders)10. Inoculating loops and inoculating needles11. Dropping bottles for stains and reagents12. Saucepans for boiling media13. Wire baskets for holding tubes and Petri plates14. Filter papers15. Wax pencil / markers16. Metal container for sterilized pipettes and Petri dishes17. Cotton wool18. Cheese cloth19. Scalpel and forceps20. Bottles containing disinfectants21. Detergent powder22. Necessary chemicals

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1.3. CLEANING GLASS WARE

New Glassware

Use hot liquid detergent to clean glassware like test tubes, Petri dishes, pipettes and flasks; rinsethoroughly with tap water and finally with sterilized water; dry before use.

New Microscope Slides and Cover Slips

Use alcohol containing HCl (3%) for several hours; rinse thoroughly with tap water and finally withsterilized water; dry with sterilized soft cotton cloth. Hold the slides by their edges. Keep the slides in acovered jar till use.

2. Aseptic methods and SterilizationPhysical Chemical

(denature and coagulateproteins and generalprotoplasmic poisons)

Heat Radiation (dimerization-pyrimidines defusion)

Filtration

Dry heat Moist heat Ionizing Non-ionizingHot air(160°C for 1.5 to 2 hr;180oC for 0.5 hr)

Steam under pressure [15 lb psi(2 atm) or 121.6°C for 15-20min]

γ rays(60Co)

UV rays(240-280 nm)

Sievingmicro-filters(0.1-0.2 μdia)

• Acid• Alcohol• Phenol (carbolic acid)• Formaldehyde (60-80oC) for

2-3 hr• Ethylene oxide for plastic

ware (<50oC): 800-1000 mg/lat 55 - 60°C for 3-4 hr or0.5-1.0% at 4-10oC for 1 hr

• Sod. hypochlorite 1% (pH 6-8)• Merc. chloride 0.1% or H2O2

3-5% for 4-5 min for surfacesterilization of live samples

Infra red heating(180oC on movingconveyor belt)

Tyndallization / Fractionalsterilization (in cane): (100 oCfor 15-20 min for 3 days)- forheat resist. SporesPasteurization (LTLT/HTST)quick heat and cooling at 60 or72oC for 30 min/30 sec or at140oC for 2-3 sec- for liquidfoodsHot oil (silicone) bath at 160oC

3. Nutritional media and their preparation

Culture medium or growth medium is a liquid or gel designed support for the growth of microorganisms.Culture media gives artificial environment simulating natural conditions necessary for growth ofmicroorganisms. Microbial culture media can be of different types, depending on the nutritional growthrequirements of the microorganisms.Microorganisms require about 10 macro-elements namely (C, O, H, N, S, P, K, Ca, Mg and Fe). The first6 components are used in the synthesis of organic components like Carbohydrates, Lipids, Proteins andNucleic acids, and the remaining 4 exist in the cell as cations and play a variety of roles. In addition, allmicroorganisms require several micro-elements like (Mn, Zn, Cu, Co, Mo and Ni). These are generallypart of enzymes and cofactors. Microorganisms also require growth factors, which are organiccompounds.

3.1. Basic requirements for culture media are:

Energy source Optimum pHCarbon source Adequate oxidation-reduction potentialNitrogen source Growth factorsSalts

3.2. Application of ingredients

The ingredients are applied on the basis of idea of C:P:N ratio of exponentially growing cellsC limited cells: 32:6.4:1N limited cells: 34:9.2:1P limited cells: 172:16:1Average: 45:7.4:1

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3.3. Conditions

Each of the six conditions (FAT TOM) that foster the growth of microorganisms are

F Food: Sufficient nutrients to promote the growth of microorganisms. Protein-rich foods, such as meat,milk, eggs and fish are most common.

A Acidity (pH): For bacteria at or near neutral pH 6.0-8.0; for Fungi 5.5-6.5 or even neutrality; foractinomycetes pH 6.5-7.5

T Time: For Bacteria 3-4 days even a week; for Fungi and Actinomycetes 7-10 days even 3-4 weeks.T Temperature: Psychrophilic at 0-15 oC; Mesophilic at 20-40 oC (30±2 oC ) and Thermophilic 40-60 oC

or aboveO Oxygen: Aerobic at 10-12% O2; Anaerobic at no O2;M Moisture: Water in terms of water activity (aw = 0.95-1.0 or even <0.85) as a measure of the water

available for life support within the microbial cell

Additionally, environmental pressure such as osmotic pressure and atmospheric pressure are alsoimportant

3.4. Components of culture media:

• Water– Source of hydrogen and oxygen.• Electrolytes– NaCl and other electrolytes.• Peptone– Mixture of partially digested proteins (animal or vegetable). Commercial peptone.• Meat extract, yeast extract – Protein degradation products/carbohydrates/Inorganic salts/Growth

factors.• Blood– It enriches media. 5-10 % defibrinated horse or sheep blood (serum).• Agar–Sea-weed (Algae-geledium species) contains carbohydrates / protein-like material / fatty acids.

Applied @ 1.5-2.0% (w/v). It melts at 950C and solidifies at 420C, acts merely as solidifying agent ofculture medium and does not provide any nutrition to the bacteria.

3.5. Classification of media:

Media have been classified in many ways:1.Solid / Liquid / Semisolid2.Simple / Complex / Synthetic / Special3.Aerobic / Anaerobic

Simple media:It is also called basal media. It consists of meat extract, peptone, sodium chloride and water.

Complex media:There are ingredients for special purposes or for bringing out certain characteristics or providing specialnutrients required for the growth of certain organisms.

Synthetic / defined media:These media are prepared solely from pure chemical substances and the exact composition of medium isknown. They are used for research purposes.

Special media:Enriched media. growth enhancers like blood, serum or egg is added to basal medium to allow fastidiousorganisms to flourish.(Blood agar & chocolate agar)Enrichment broth. encourage the growth of small number of a particular organism while suppressing otherflora present.(Alkaline peptone water)Selective media. support the growth of one type of microbe over another. It may contain inhibitorysubstances (antibiotics or other inhibitory chemicals) to prevent the growth of microbes. MacConkey agarfor enteric Gram-negative bacilli.Differential media. allow grouping of microbes on the basis of different characters displayed on themedium. (Non-selective or selective)

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Indicator media. contain an indicator, which changes color when bacteria grow in them.(Sugar medium isan indicator medium)Transport media. special media required for transport of delicate microorganisms, like gonococci whichmay not survive the time taken for transportation or overgrown by non-pathogen. Stuart’s mediumAnaerobic media. Used for anaerobic organisms. (Robertson’s cooked meat medium)

Fig. Differential growth on selective media MacConkey agar plate: E. coli colonies lactose fermentor (pinkcolour), and Serratia marcescens lactose non-fermentor (cream colour) both are streaked on themedium

Selective media: The media that allow the growth of certain types of micro-organisms is termed asselective media. This is achieved by1. excluding a critical nutrient unfavourable for most other microorganisms. e.g. Jensen’s nitrogen free

agar medium of Azotobacter.2. addition of some inhibitory substance that prevent growth of certain type of microorganism. e.g.

Penicillin to prevent growth of Gram ‘+’ bacteria.

Differential media: Where contain certain substances (e.g. dyes) that differentiate one species fromanother. e.g. methylene blue agar which is used to differentiate Azospirillum from other Gram ‘-’ bacteria

3.6. Procedure for preparation and inoculation of media

1. Suspend all the ingredients of the medium in 1 litre of purified water in conical flask.2. Heat with frequent agitation and boil for 1 minute to completely dissolve the medium.3. Take the molten medium in test tube (1/3 of volume) for slant, if required.4. Autoclave at 121° C for 15 minutes.5. Cool to 45 to 50°C and pour into sterilized Petri dishes (12-15 ml).6. Streak the specimen onto the agar medium in Petri plates with a sterile inoculating loop / glass

spreader in order to obtain isolated colonies.7. Incubate the plates at 30±2°C in an inverted position (agar side up) with increased humidity.8. Cultures should be examined at least 3-4 days for bacteria, weekly or even 3-4 weeks for fungal and

actinomycetes growth before being reported as negative.

3.7. Growth media for bacteria, fungi and actinomycetes

A sufficient number of growth media with their composition are available in literature for getting thecharacteristic growth of common bacteria, fungi, actinomycetesBacteria: Sodium albuminate agar,Fungi: V-8 vegetable juice-dextrose-yeast extract agar (VDYA) and Rose Bengal-streptomycin agar(RBSA).Antibiotics like Chloramphenicol and/or tetracycline acts as broad spectrum antimicrobials to inhibit thegrowth of a wide range of gram-positive and gram-negative bacteria. Gentamicin is added to furtherinhibit the growth of gram-negative bacteria.Actinomycetes: Starch-caseinate agarAntibiotic like Nystatin (fungicidin) / Actidione (cycloheximide) @ 50 μg/lit are added to the cooled mediaafter autoclaving.

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Pretreatment: Prior to making suspension-dilution for isolation, the soil samples are heated at 121°C for1 hr (to enrich the isolates of actinomycetes). In serial dilution method, serial dilution upto 10-6 is carriedout.

Fig. Growth and morphology of (a) bacteria, (b) fungi and (c) actinomycetes

Characteristic growth parameters of microorganismsS.No. Microorganism and source

of isolate/strainGrowth medium pH Temp (oC) Incubation Growth characteristics

1 Rhizobium (slow grower)Strain: Local (root nodule)

YEM (yeast extractmannitol)

6.8-7.5 28±2 5-7 days Whitish-light yellow heavysurface growth; light yellowmedium with turbidity (gummy)

Smell: Light sour milk (matha) No foam formation

2 AzospirillumStrain: Local (soil)

Spirillum N free / N-free semisolid malicacid (Dobereiner'smalic acid)

7.2-7.4 30±2 3-4 days White thin surface growth;light blue colour medium withturbidity

Smell: Light decomposed Acidic pH confirms no growth

of Azospirillum3 Azotobacter

Strain: Local (soil)Ashby’s mennitol /Waksman mediumNo.77 (N-freemannitol)

7.0-7.2 30±2 2-3 days Off white-ash colour, thinsurface growth; light whitecolour medium with turbidity

Smell: Light sour milk (matha) The pigmentation of aged

culture of Azotobacter ismelanin from oxidation oftyrosine by tyrosinasecontaining copper.

The pigment characteristicsmay be different by differentspecies of Azotobacter

4 BGA (hetero sp.)Strain: Local(Narmada river bed)

Tank culture /Fogg’s medium /BG 11(for strainsmaintenance)

7.0-7.2 30-35oC(Hot-humid)

1.0-1.5months

Surface thick scum growth onaquatic phase

Soil based: Soil colour (BGAflakes ground : Soil 50:50) orbrownish-black when mixedwith compost / lignite 25-50%

Smell: No5 Trichoderma

Strain: LocalPotato dextrose /Malt extract

7.0-7.2 25±2 7 days(12L:12D)

Thick cottony white surface

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growth, turns greenish anddevelopment of black sporeswith subsequent growths;coppery brown colour mediumwith no turbidity

Smell: No6 PSB (Bacillus sp.)

Strain: Local (soil)Pikovskaya’s 7.0-7.2 28±2 3-4 days Light orange surface growth;

medium dark orange colourwith turbidity

Smell: Light sour milk7 PGPR (Pseudomonas

fluorescens)Strain: Local (PlantPathology)

King’s B 7.0-7.2 26±2 1-2 days Whitish-yellow thick surfacegrowth; orange colour heavyturbid medium

Smell: Very light decomposedsmell

8 Common Bacteria (soil) Nutrient medium /Tryptic Soy Broth

6.0-8.0 28±2 oC 3-4 days Miscellaneous, glistening,convex on solid agar medium

Turbid growth in liquid medium9 Common Fungi (soil) Sabouraud dextrose /

Rose bengal5.5-6.5 37±2 oC 7-10 days Wooly colouful growth on solid

agar medium Surface scum growth in liquid

medium10 Common Actinomycetes

(soil)Starch casein / LuriaBertani

6.5-7.5 37±2 oC 7-10 days Chalky white deep seatedgrowth on solid agar medium

Surface isolated colony growthin liquid medium

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SECTION- 2

Mass production technologies of biofertilizers

For successful and effective biofertilizer preparation, authentic strains of desired beneficialmicroorganism is must to isolate from various most probable habitats viz., soil, compost, water, even plantsurface, etc. for free living microorganisms, and plant root nodule, root itself, leaf, flower, fruit, etc. forendophytic microorganisms.

Fig. Isolation of microbial strainNote: For maintaining purity, sub-culturing of the microorganism at every 15-20 days intervals is

essential; also take necessary steps to check vertical mutation.Once the effective strain of a beneficial microorganism is isolated in pure form, this can be

multiplied and mass produced in shape of biofertilizer either in powder carrier base or liquid formulation(Fig. ).

Fig. Production techniques for carrier based and liquid biofertilizer production

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SECTION-3

Preparation of media for high demand biofertilizers (PSB, PGPR and Trichoderma), their respectiveinoculations and incubations

3.1. Biofertilizers

Definition: Biofertilizer can be defined as a substance which contains living microorganisms and/or itsdormant cysts/spores which colonizes the rhizosphere or the interior of the plant and promotes growth byincreasing the supply or availability of nutrient (N2 fixers and nutrient soulbilizers and/or mobilizers) and/orgrowth stimulus (PGPRs and phytostimulators) to the target crop, when applied to seed, plant surfaces,or soil.Since 2015, the Microbes Research & Production Centre (MRPC) is producing either 2-6 tonnes of carrierbased or 400-800 litre of liquid based of various biofertilizers of the following 15 types with theirimportance in crops.

S.No.

Biofertilizer Recommended crop Nutrient saving(Kg ha-1)

Increase inproduction (%)

1 Jawahar Rhizobium Legumes (crop specific) 40-90 N 15-302 Jawahar Azotobacter Non-legumes (vegetables) 20-25 N 07-123 Jawahar Azospirillum Non-legumes (cereals) 20-30 N 10-254 Jawahar Acetobacter Sugarcane and other crops 10-12 N 10-155 Jawahar BGA Paddy 40-60 N 15-206 Jawahar PSB All crops 15-30 P2O5 10-177 Jawahar Mycorrhiza All crops, vegetables, and trees 15-25 P2O5 05-158 Jawahar KSB All crops 10-25 K2O 10-259 Jawahar ZSB All crops 1.5-2.5 Zn 15-2510 Jawahar Pseudomonas All crops Vegetative growth,

crop quality-

11 Jawahar Biofertisol Fruit bearing all plants (fruits,vegetables, etc.)

Plants’ fertility, fruityield

-

12 Jawahar Trichoderma All crops Plants’ resistance,waste decomposer

-

13 Jawahar Biodigester-I(Fungal)

Waste decomposer Waste decomposer -

14 Jawahar Biodigester-II(Bacterial)

Waste decomposer Waste decomposer -

15 Jawahar EM Culture All crops phyto-probiotics,waste decomposer

Nutrient availability,waste decomposer

-

Reputations of phytostimulators and biofertilizers has been frequently hampered due to thevariability and inconsistency of results between laboratory, greenhouse, and field studies. The responsesof biofertilizers application usually depend on several environmental factors.

1. The type of soil as measured by its water holding capacity, its levels of other nitrates, phosphate andeven calcium and molybdenum that help in protein synthesis in Rhyzobia and the alkalinity, salinityand acidity of soil, all affect the response. Higher dose of mineral N as starter suppresses nodulation,reducing response of Rhyzobium but phosphate deficiency can be an inhibitor also.

2. The inadequacy of organic matter especially common in dry-land and agriculture is a deterrent more forthe non-symbiotic strains, which essentially depend on soil organic matter for energy. Phosphobactrinresponse was found to be positive only in soils with high organic content and low availablephosphorous.

3. Soil water deficit and high temperature (hyper-thermia) are prominent abiotic factors that affect N-fixation in dry land agriculture.

4. Native microbial population opposes the inoculants. In general, predatory organisms, often alreadypresent in the soil are more adapted to the environment and out compete the inoculated population.

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3.2. Preparation of media for high demand biofertilizer (PSB), its respective inoculations andincubations

Phosphorus (P), the second important plant growth-limiting nutrient after nitrogen, is abundantly availablein soils in both organic and inorganic forms (Fig. 3) (Khan et al., 2009). Despite of large reservoir of P, theamount of available forms to plants is generally low. This low availability of phosphorous to plants isbecause the majority of soil P is found in insoluble forms, while the plants absorb it only in two solubleforms, the monobasic (H2PO4

-) and the diabasic (HPO4-2 ) ions (Bhattacharyya and Jha, 2012). The

insoluble P is present as an inorganic mineral such as apatite or as one of several organic forms includinginositol phosphate (soil phytate), phospho-monesters, and phosphotriesters (Glick, 2012). To overcomethe P deficiency in soils, there are frequent applications of phosphatic fertilizers in agricultural fields.Plants absorb fewer amounts of applied phosphatic fertilizers and the rest is rapidly converted intoinsoluble complexes in the soil (Mckenzie and Roberts, 1990). Therefore, P deficiency results in stuntedgrowth, dark leaves, and inhibition of flowering and root system development. In most plants, thesesymptoms will appear when P concentration in the leaves goes below 0.2%.Certain soil microorganisms have inherent capacity to dissolve part of the fixed phosphorus (P) and makeit available to the crop by secreting certain organic acids. Bacillus megaterium, B. circulans, B. subtilis, B.polymyxa, B. sircalmous, Pseudomonas striata, and Enterobacter could be referred as the mostimportant strains.

Typically, the solubilization of inorganic phosphorus occurs as a consequence of the action of lowmolecular weight organic acids which are synthesized by various soil bacteria (Zaidi et al., 2009). Theseorganic acids (dominantly gluconic acid) including siderophores dissolve phosphatic minerals and / orchelate cationic partners of the P ions directly, releasing P into solution.

Conversely, the mineralization of organic phosphorus occurs through the synthesis of a variety ofdifferent phosphatases, catalyzing the hydrolysis of phosphoric esters (Glick, 2012). Importantly,phosphate solubilization and mineralization can coexist in the same bacterial strain (Tao et al., 2008).Though a number of theories have been proposed to explain the mechanism of phosphate solubilizationbut two are more important: Acid production theory Proton and enzyme theory (release of protons accompanying respiration or assimilation of ammonium /

nitrate)

Jawahar PSB culture are useful for all the crops i.e. cereals, cash crops, leguminous crops, horticulturalcrops, vegetables, etc.

Advantages

The culture of PSB increases the level of available P in soil through its solubilzation 30 to 40% ofphosphate deposit.

With the increase in available P level, overall plant growth can be increased. In certain condition they also exhibit anti-fungal activities and thereby fungal diseases may be controlled

indirectly. About 10 to 15% increase of crop yield can be achieved with the use of this culture.

Doses and packing

A. Carrier based Jawahar PSB cultureDoses (seed treatment): 250 g /10-12 kg seeds withlight sprinkling of waterSoil application: 3-4 kg culture mixed with 50 kg FYMfor one acre and broadcast

B. Liquid Jawahar PSB cultureDose (seed treatment): 100 ml 10 kg-1 seeds with light sprinkling of water

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3.3. Preparation of media for high demand biofertilizer (PGPR), its respective inoculations andincubations

PGPR (plant growth promoting rhizobacteria) is a mutifinctional agent, effects on plant physiology andgrowth, induced systemic resistance, biocontrol of plant pathogens, biofertilization, and potential greenalternative for plant productivity, viability of coinoculating, plant microorganism interactions, andmechanisms of root colonization. By virtue of their rapid rhizosphere colonization and stimulation of plantgrowth, there is currently considerable interest in exploiting these rhizosphere bacteria to improve cropproduction. The main groups of PGPR can be found along with the phyla Cyanobacteria, Actinobacteria,Bacteroidetes, Firmicutes, and Proteobacteria. Various species of bacteria like Pseudomonas,Azospirillum, Azotobacter, Klebsiella, Enterobacter, Alcaligenes, Arthrobacter, Burkholderia, Bacillus andSerratia have been reported to enhance the plant growth.

PGPR can affect plant growth by different direct and indirect mechanisms. Some examples ofthese mechanisms, which can probably be active simultaneously or sequentially at different stages ofplant growth, are (1) increased mineral nutrient solubilization and nitrogen fixation, making nutrientsavailable for the plant; (2) repression of soil borne pathogens (by the production of antibiotics, hydrogencyanide, siderophores, and/or competition for nutrients i.e, competitive exclusion); (3) improving plantstress tolerance to drought, salinity, and metal toxicity/bioremediation [against abiotic stress: triggeringInduced Systemic Resistance (ISR); and against biotic stress: competitive exclusion for substrate,producing allelo-chemicals and triggering ISR; bioremediation: bacterial siderophore binds iron withextremely high affinity and plants are able to take up and utilize the iron from these complexes, and alsocadmium in some cases]; and (4) production of phytohormones such as indole-3-acetic acid (IAA) in rootsdevelopment and ACCD enzyme. The enzyme 1-aminocyclopropane-1-carboxylate (ACC) deaminasehydrolyses ACC, the immediate precursor of ethylene in plants. By lowering ethylene concentration inseedlings and thus its inhibitory effect, these PGPR stimulate seedlings root length.

Induced systemic resistance (ISR) and Systemic acquired resistance (SAR) are two differentphenomena but both represent active plant defence responses against stresses (abiotic / biotc) includingphytopathogen attack. ISR activated by non-pathogenic microbes is similar to hypersensitive responseinclude rapid senescence, ripening and abscission of infected tissue like sudden fall of leaves or fruits.While as SAR against pathogen is inherent immunity of plant system developing primarily from tissuenecrosis (infection site) caused by pathogen. SAR is signaling activates of biochemical-genetics nature tothe distal ends of leaves/branches/stems/roots for acquiring immunity through lignin formation anddeposition creating efficient barrier to infecting agents.

Fig. Direct and indirect mechanisms mediated by plant growth-promoting rhizobacteria (PGPR) with beneficial effectson host plants

The microbes in Jawahar PGPR can grow on plant leaves and roots where they can contribute to plantgrowth. It also degrade pollutants. Some species present biocontrol properties by producing secondarymetabolites including antibiotics (like mupirocin), protecting the seeds and roots from fungal infectionagainst parasitic microbes such as Fusarium, Pythium, Alternaria, Sclerotium, Rhizoctonia,Macrophomina, Curvularia as well as some phytophagous nematodes.

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Advantages

The bacteria might induce systemic resistance in the host plant, so it can better resist attack by a truepathogen.

The bacteria might outcompete pathogenic soil microbes, e.g., by siderophores (pyoverdin), giving acompetitive advantage at scavenging for iron.

Certain isolates of P. fluorescens produce the anti-fungal secondary metabolite 2,4-diacetylphloroglucinol and its derivatives phloroglucinol, phloroglucinolacaboxylic acid, responsible for anti-phytopathogenic and biocontrol properties in these strains.

Doses and packing

A. Carrier based Jawahar Pseudomonas cultureDoses (seed treatment): 250 g /10-12 kg seeds with light sprinkling of waterSoil application: 3-4 kg culture mixed with 50 kg FYM for one acre and broadcast

B. Liquid Jawahar Pseudomonas cultureDose (Seed treatment): 100 ml 10 kg-1 seeds with light sprinkling of water.

3.4. Preparation of media for high demand biofertilizer (Trichoderma), its respective inoculationsand incubations

Trichoderma grows rapidly in rhizosphere preferably under sufficient aerobic conditions andenhance the root growth of plants using competition exclusion over other non-beneficial microorganisms.Increased activity of Trichoderma may also be resulted from solubilization of nutrients like phosphates,micronutrients and mineral cations as envisaged that the fungus thrives better under acidic conditions.

The control response of the fungal isolate against disease caused by soil bornephytopathogenic fungus might be due to their cell wall degradable by the hydrolytic enzymes andcompetition for space and nutrients. The biocontrol agent generally grows in its natural habitat on seedand root surfaces, and so affects root disease in particular.

Advantages

Disease control: Trichoderma is a potent biocontrol agent. It has been used successfully againstvarious pathogenic fungi belonging to various genera, viz. Fusarium, Phytopthara, Scelerotia.

Plant growth promoter: Trichoderma strains solubilize phosphates and micronutrients. Its applicationincreases the number of deep roots, thereby increasing the plant's ability to resist drought.

Biochemical elicitors of disease resistance: Trichoderma strains are known to induce resistance inplants. These compounds also induce ethylene production, hypersensitive responses and otherdefence related reactions in plant cultivates.

Bioremediation: Trichoderma strains play an important role in the bioremediation of soil that arecontaminated with pesticides and herbicides.

Dose and packingCarrier based Jawahar Trichoderma culture

Doses (seed treatment): 250 g /10-12 kg seeds with waterSoil application: 2-3 kg culture mixed with 50 kg FYM/ acre

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SECTION- 4

Fermentation techniques in production of biofertilizers

4.1. Fermentation and fermentation process

Fermentation

In applied microbiological sense, fermentation is a process in which microorganisms are involved evenif the process involves strong aeration and is respiratory in nature as in the antibiotic fermentations.Energetically, fermentations are poor energy yielding processes and the products have lower energycontent than the substrates. In terms of bond energy, fermentation may be viewed as a process in whichthe H and O atoms in an organic molecule are regrouped to yield a carboxyl group or CO2. In a broadsense, fermentation is a reaction in which organic compounds serve both as electron donors andelectrons acceptors.Since there is no net oxidation in a fermentation, the number of moles of C, H and O remain the same inthe products as in the substrate, as it occurs in the conversion of glucose to lactic acid or ethanol.

C6H12O6 2C3H6O3Glucose Lactic acid

orC6H12O6 2CH3CH2OH + 2CO2Glucose Ethanol Carbon dioxide

Fermentation differs from anaerobic respiration, in the latter oxygen is replaced by an inorganic electronacceptor such as NO3

−, or SO4=. Two typical fermentations that have been studied in details are (i) the

ethanol fermentation with yeast, and (ii) the lactic acid fermentation with lactic bacteria.

Fermentation process

Depending on the circumstance, the term fermentation can have three meanings. In a physiologicalsense, fermentation refers to a process that produces energy by breaking down of energy-richcompounds under anaerobic conditions. This is the basis of food fermentation, which is commonly usedin the food industry in order to produce diverse food products and also as a food preservation method. Ina biotechnological context though, this term is used in a much broader sense. There, fermentationmeans a process in which microorganisms that are cultured on a large-scale under aerobic oranaerobic conditions, convert a substrate into a product which is useful to man.

This article attempts to provide an overview of the industrial fermentation process, mainly focusing onvarious types of fermentations, the basic steps in a typical fermentation process and the major industrialapplications of fermentation.

4.2. Basic Steps of Industrial Fermentation

Any industrial fermentation operation can be broken down into three main stages, viz, upstreamprocessing, the fermentation process and downstream processing.

4.2.1. Upstream processing includes formulation of the fermentation medium, sterilisation of air,fermentation medium and the fermenter, inoculum preparation and inoculation of the medium.

The fermentation medium should contain an energy source, a carbon source, a nitrogensource and micronutrients required for the growth of the microorganism along with waterand oxygen, ifnecessary.

A medium which is used for a large scale fermentation, in order to ensure the sustainability of theoperation, should have the following characteristics;

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1. It should be cheap and easily available2. It should maximise the growth of the microorganism, productivity and the rate of formation of the

desired product3. It should minimise the formation of undesired products

Usually, waste products from other industrial processes, such as molasses, lignocellulosic wastes,cheese whey and corn steep liquor, after modifying with the incorporation of additional nutrients, are usedas the substrate for many industrial fermentations.Sterilisation is essential for preventing the contamination with any undesired microorganisms. Air issterilised by membrane filtration (0.2-2 μ) while the medium is usually wet heat sterilized (121oC for15-20 min). Any nutrient component which is heat labile is filter-sterilised and later added to thesterilised medium. The fermenter may be sterilised together with the medium or separately.Inoculum build up is the preparation of the seed culture in amounts (5% v/v) sufficient to be used in thelarge fermenter vessel. This involves growing the microorganisms obtained from the pure stockculture in several consecutive fermenters. This process cuts down the time required for the growth ofmicroorganisms in the fermenter, thereby increasing the rate of productivity. Then the seed cultureobtained through this process is used to inoculate the fermentation medium.

4.2.2. The fermentation process involves the propagation of the microorganism and production ofthe desired product. The fermentation process can be categorised depending on various parameters.

It can be either aerobic fermentation, carried out in the presence of oxygen or anaerobic fermentation,carried out in the absence of oxygen. Many industrial fermentation are carried out under aerobicconditions where a few processes such as ethanol production by yeast require strictly anaerobicenvironments.

The fermentation process can also be divided into three basic systems, namely batch,continuous or fed-batch, depending on the feeding strategy of the culture and the medium into thefermenter. Each of these processes has their own advantages and disadvantages. In a batch operation,the medium and the culture are initially fed into the vessel and it is then closed. After that, no componentsare added apart from oxygen (in an aerobic process) and acid or alkali for the pH adjustment. Thefermentation is allowed to run for a predetermined period of time and the product is harvested at the end.In a continuous process, fresh medium is continuously added and the products, along with the culture isremoved at the same rate, thus maintaining constant concentrations of nutrients and cells are maintainedthroughout the process. A fed-batch system is a combination of these two systems where additionalnutrients are added to the fermenter as the fermentation is in progress. This extends the time of operationbut the products are harvested at the end of the production cycle as in a batch fermenter.

The process can also be categorised as solid state fermentation (SSF) or submergedfermentation (SmF), depending on the amount of free water in the medium. In a solid statefermentation, the medium contains no free flowing water. The organisms are grown in a solid substratewhich is moistened. This is used in certain industrial process such as ‘koji’ fermentation from soybeans,production of amylase and protease by Aspergillusoryzae on roasted soybeans and wheat,bioremediation, detoxification of agro-industrial wastes, etc. Submerged fermentation is in whichmicroorganisms grow submerged in a liquid medium where free water is abundant. This is the method ofchoice for many industrial operations over SSF although SSF is also rapidly gaining interest in thepresent.

4.2.3. Downstream Processing includes the recovery of the products in a pure state and the effluenttreatment. The entire downstream process is accomplished in five stages are: (1) Solid-LiquidSeparation (2) Release of Intracellular Products (3) Concentration (4) Purification byChromatography and (5) Formulation.

Stage # 1. Solid-Liquid Separation:

The first step in product recovery is the separation of whole cells (cell biomass) and other insolubleingredients from the culture broth (Note: If the desired product is an intracellular metabolite, it must bereleased from the cells before subjecting to solid-liquid separation). Several methods for solid-liquid

14

separation are flotation (using collector substances facilitating stable foam formation e.g., long chain fattyacids, amines), flocculation (flocculating agents: inorganic salt, organic polyelectrolyte, mineralhydrocolloid), filtration (micro-, ultra- and hyper-filtration/reverse osmosis depending on size cells/virusesand MW molecules) and centrifugation (laboratory or industrial continuous flow filtration).

Stage # 2. Release of Intracellular Products:

As already stated, there are several biotechnological products (vitamins, enzymes) which are locatedwithin the cells. Such compounds have to be first released (maximally and in an active form) for theirfurther processing and final isolation. The microorganisms or other cells can be disintegrated or disruptedby physical (ultra-sonication, high pressure homogenization, grinding with glass beads or heatshock/thermolysis), chemical (alkaii, organic solvelnts or detergents) or enzymatic (lysozyme, glucanase,mannanase or protease) methods. The selection of a particular method depends on the nature of thecells, since there is a wide variation in the property of cell disruption or breakage. For instance, Gram-negative bacteria and filamentous fungi can be more easily broken compared to Gram-positive bacteriaarid yeasts.

Stage # 3. Concentration:

The commonly used techniques for concentrating biological products to be free from water (80-98%) areevaporation, liquid-liquid extraction (physical, dissociative, reactive or supercritical fluid extraction),membrane filtration, precipitation and adsorption. The actual procedure adopted depends on the nature ofthe desired product (quality and quantity to be retained as far as possible) and the cost factor.

Stage # 4. Purification by Chromatography:

The biological products of fermentation (proteins, pharmaceuticals, diagnostic compounds and researchmaterials) are very effectively purified by chromatography like gel-filtration, ion-exchange, affinity, orhydrophobic interaction chromatography.

Stage # 5. Formulation (product polishing):

Formulation broadly refers to the maintenance of activity and stability of a biotechnological productsduring storage and distribution. The formulation of low molecular weight products (solvents, organicacids) can be achieved by concentrating them with removal of most of the water by drying, spary-dryingor freeze drying. For certain small molecules, (antibiotics, citric acid), formulation can be done bycrystallization by adding salts. Proteins are highly susceptible for loss of biological activity; hence certainstabilizing additives are added to prolong the shelf life of protein. The stabilizers are sugars (sucrose,lactose), salts (sodium chloride, ammonium sulfate), polymers (polyethylene glycol) and polyhydricalcohols (glycerol). Proteins may be formulated in the form of solutions, suspensions or dry powders.Finally, the effluents are treated by chemical, physical or biological methods.

Fermentation Products

Commercially important products of fermentation can be described in five major groups as follows.

1. Biomass (Baker’s yeast, SCP, Starter cultures, animal feed, etc.)2. Primary metabolites (amino acids, organic acids, vitamins, polysaccharides, ethanol, etc.)

and secondary metabolites (antibiotics, etc.)3. Bioconvertion or biotransformation products (steroid biotransformation, L-sorbitol etc.)4. Enzymes (amylase, lipase, cellulase, etc.)5. Recombinant products (some vaccines, hormones such as isulin and growth hormones etc.)

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4.3. Fermentor and its control systems

4.3.1. Basic components

These versatile systems are finding increased application as available capital, time and space becomemore limited. For these reasons, fermentation systems for multipurpose operation should be consideredwhen planning and scaling up bioprocesses. Critical consideration should be given to:

• Vessel design• Process piping• Jacket service piping• Agitation and mixing• Instrumentation and controls

It is also worthwhile to evaluate various design options, to ensure that added features are both beneficialand cost effective, with respect to long-term operations and maintenance (Figure 1).

Fig. Diagrammatic presentation of inner and outer part of a fermentor

Basic components of a fermentor

Part PurposeTop plate cover (made of steel)Seal mechanical seals (lower- carbon, upper- tungsten) separates top plate

from vessel to prevent air leakageClamp top plate compressed onto vessel using clampVessel steel (SS 316L), jacketed (SS 304) with ports for various outputs, inputs,

probes, etc.Drive motor used to drive mixing shaft

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Drive shaft mixes the medium evenly with its impellerMarine impeller on shaft for giving movement of mediumBaffles on inner wall ofvessel

for circulation of air throughout the vessel prevent sedimentation on sidesand proper mixing

Sparger with diaphragmfilter, rotameter, valves(BV, NRV, PRV)

air supplier / after filtration via membranes – ensures efficient dispersal –by attached to impeller

Exit gas cooler like condenser remove as much moisture as possible from exhaust (usedin pharmaciteucals)

Inoculation port port to add inoculumsPt100 temperature sensor (platinum resistance electrode)Foam breaker to break the excess foam formation from the toppH electrode gel filled pH probe, senses pHO2 sensor polarographic high temperature SS probe with transmitter, monitors

dissolved oxygen levelHeater pad directly heats the mediumCooling system after direct heating – used to cool the vessel contents with the help of

chillerRotameter variable air flow meter – indicates rate of air flow into vessel – attached to

air spargerPneumatic pressure valve(5) and manual valve withdiaphragm (3)

attached to rotameter for safer operation automatically through PLC andsafe individual vessel operation, respectively

Bottom valves withdiaphragms (3)

outlet the sample after sterilization, drainage of water during washing

Compressor pump withmicron filters (2 & 5 μ)

supply of air

Peristaltic pump to pump in medium, acids, bases, antifoamFilter housing with valves air outletMotor and gear box withdigital indicator for speedin rpm, set point throughPLC

stirrer system

4.3.2. Control systems

1. PLC and SCADA system

PLC (programmable logic control) is an industrial computer that monitors inputs, makes decisionsbased on its programme and controls outputs to automate the process of fermentor system. It has a built-in operating system (OS). This OS is highly specialized to handle incoming events in real time, or at thetime of their occurrence. It is a sort of digital computer designed for multiple inputs and outputarrangements/ parameters/ events like temperature, pressure, OD, pH, etc. The PLC has input lineswhere sensors with actuators are connected to notify upon events and output lines to signal any reactionto the incoming events. It uses a programmable memory to store the instructions and specific functions(for set values) that include on/off control, timing counting, sequencing, arithmetic and data handling.

SCADA is “supervisory control and data acquisition” - a real time industrial process controlsystem used to centrally monitor and control remote or local equipments like different components of afermentor such as motors, valves, pumps, relays, etc. It is a system that gathers information on events(such as a leakage on a pipe line, a change in air/steam pressure), transfers the information back to acentral site, then alerts the home station that an event has occurred, carrying out necessary analysis andcontrol, such as determining if the event is critical and displaying the information in a logical andorganized fashion. It locates the faulty site of the system using a graphical processor.

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2. Sensors and actuators

A sensor is transducer that converts a physical stimulus from one form into a more useful form tomeasure the stimulus. In the existing system both analog and discrete (binary and digital) sensors areused.

Actuator is a hardware device that converts a controller command signal into a change in aphysical parameter. The change is usually mechanical (example position or velocity of steam/air/water).An actuator is also a transducer because it changes one type of physical quantity into some alternativeform. It is usually activated by a low level command signal, so an amplifier is required to provide sufficientpower to drive the actuator.

In the existing fermentors models Biosac 200 and Biosac 700 along with utility items (steamboiler, chiller and compressor) manufactured by M/s Andel equipments, Chandigarh have been used.Temperature is an important parameter of fermentation, since during cultivation of microorganisms inbroth, the temperature deviation by a couple of degrees can diminish dramatically the growth andbiosynthesis productivity. For temperature measurements, stainless steel Pt100 sensors are normallyused. A heater is located inside the bioreactor vessel, and cooling is ensured by thin-wall pipes located inthe upper cover, which are connected with an electromagnetic / pneumatic valve with the cooling water.Heating and cooling proceed through a thermostat, and this thermostatted water with the help of a pumpcirculates through the bioreactor jacket. pH is one of the most important chemical environmentalmeasurements used to indicate the course of the fermentation process. It indicates the presence of somespecific chemical factors that influence growth, metabolism, and final product.

5. Processing of carrier based biofertilizers

Response from application of biofertilizers and phytostimulators may be variabile and inconsist observedunder laboratory, greenhouse, and field studies. The reason for these discrepancies lies in the incompleteunderstanding of the complex relationships established between the components of the system: the plant,the microorganisms, and the environmental conditions, particularly that of soil.

In addition, the lack of correct formulations and the expensive and time-consuming proceduresare also among the factors holding back the use of PGPM on a wider scale. The development oftechniques for the production of large quantities of pure inocula, with high infectivity potential, is the mainissue to be tackled in order to allow a wide use of biofertilizers.

The key aspects in the inoculation technology are the use of a proper formulation of inoculapreparations, the selection of an adequate carrier, and the design of correct delivery methods.

5.1. Inocula Formulation

The kinds of carrier utilized defines the physical form of the biofertilizer.

A. Carrier based inocula

The production of selected bacteria and yeasts in pure cultures is a quite common practice making use offermentors. Therefore, once the particular strain(s) for the inoculum have been selected, an industrialstandardized process of production can be defined. However, in case of biofertilizers, unlike that ofbiopesticides, the cost of production is an important constraint considering that the price of the fertilizershall not exceed that of conventional ones to assure a market sustainability.The carrier is the major portion (by volume or weight) of the inoculant that helps to deliver a suitableamount of PGPM in good physiological condition. The materials constituting the carrier can be of variousorigins: organic, inorganic, or synthesized from specific molecules. Availability and cost are the mainfactors affecting the choice of a carrier. A good carrier should therefore posses as much as the followingproperties:

• should provide a suitable microenvironment for the microbes and should assure a sufficient shelf life ofthe product (at least 2-3 months for commercial purposes, possibly at room temperature).

• should allow an easy dispersion or dissolution in the volume of soil near the root system.• good moisture absorption capacity, easy to process and non-lump forming materials, easy to sterilize

by autoclaving or by other methods (e.g., gamma-irradiation), and good pH buffering capacity• low cost and availability in adequate amounts for carriers that shall be used for seed coating, a good

adhesion to seeds is also important.

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Other characteristics that are affecting the carrier appropriateness:

• standardized composition ensuring chemical and physical stability, suitability for desired microbialspecies and strains, the possibility of mixing with other compounds (i.e., nutrients or adjuvants), andbeing composed of biodegradable and nonpolluting compounds

• for seed coating, the carrier shall assure the survival of the microbes on the seed since normally seedsare not immediately sown after seed coating. Survival of the microbes is important both during thestorage period of the bioproduct and after being introduced into the soil. The latter is fundamental fordefining the application technology and dosing the product.

• the inoculant has to compete with native soil microorganisms for the nutrients and habitable niches, andhas to survive against grazing protozoa.

• carrier materials that make available nutrients and/or habitable micropore to the microbes, particularlyin case of bacteria, would then be more suitable.

Dry inoculants

• It can be produced using different kinds of soil materials (peat, coal, lignite, clays, inorganic soil),organic materials (composts, soybean meal, wheat bran, sawdust, etc.), or inert materials (e.g.,vermiculite, perlite, kaolin, bentonite, silicates).

• Natural Carriers like peat, plant waste materials, composts, sawdust, sludge; Lignite, coal, clays, andinorganic soils (i.e., volcanic pumice or diatomite earths), vermiculite, perlite, and bentonite and mixtureof organic and inorganic materials may also be used.

• Standard sizes of the powder material may vary from 75 μm to 0.25 mm. Powder-type inoculants canbe used to coat seeds or suspended in a liquid to form a slurry that is directly applied to the furrow or,alternatively, the seeds/plants are dipped in it just prior to sowing/planting.

• The size of granules and beads ranges from 100–200 μm to 3-4 mm indiameter.• However, the method of inclusion has some drawbacks, particularly in relation to the survival of the

microorganisms and their protection during transport, storage, and handling.

Reasons for failure of carrier based biofertilizers

• Short Shelf life• Sensitivity to temperature• Dosage contradictory to farmers• Lack of identifiable character• Lack of instant visual effects on application• Unavailability of good carrier in local area• Quality in consistency of carriers• Poor cell protection• Poor moisture retention capacity• Bulk sterilization problem in terms of economy and facilities• Problem of proper packing• High transport cost• Labour intensive• Restriction on use carrier (wood charcoal) as a measure of conservation• For quality control takes more time• Very slow adoption by farmers

B. Liquid Biofertilizers (Formulations)

Liquid formulation comprises aids for preserving organisms, delivering them to their targets and oncethere, for improving their activities. It contains not only the desired microorganisms and their nutrients,but also special cell protectants or substances that encourage formation of resting spores or cysts forlonger shelf life and tolerance to adverse conditions.

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Advantages of Liquid Biofertilizers

• Contain special cell protectants or substances that encourage formation of resting spores or cysts,thereby better shelf life.

• Contain special nutrients for longer shelf life and better survival on seeds and soil and tolerance toadverse conditions.

• Liquid formulations are easy to handle and apply.• No loss of properties due to storage up to 45oC.• Greater potentials to fight with native population.• Dosages are 10 times lesser than the carrier-based biofertilizers.• Very high enzymatic activity since contamination is nil.• Identified by typical fermented smell• Cost saving on carrier material and its pulverization, neutralization, sterilization, packing and transport• Easy to quality control

Liquid inoculants

This can be based on broth cultures, mineral or organic oils, or on oil-in-water suspensions. Liquidinoculants may of two types

Dormant Aqueous SuspensionGenerally, growth suppressants and contaminant suppressant are used: like sodium azide, sodiumbenzoate, butanol, acetone, etc. for the long term viability. These formulations are not suitable for shortduration crops, where actions are required immediately as the organisms take prolonged reactivation time(around 7 days).

Dormant Oil Suspension

Microorganisms can be suspended in oil at high concentration with various degrees of dehydration forlonger viability. Similar to the case of dormant aqueous suspension, the oil suspension (mainly in coconutoil) are not so promising as claimed and also has the limitation in reactivating the bacteria. It generallytakes 10-20 days to regenerate and the initial requirement of the plant is not fulfilled by them. Again itinvolves additional cost of oil used.

Advantages of liquid biofertilizers are

• Better shelf life• No effect of high temperature• Identified by typical fermented smell and/or colour• No loss of properties due to sub-culturing• Better survival on seeds and in soil• Cost saving on carrier material, pulverization, neutralization, sterilization, packing and transport• No contamination etc.• Easy to quality control• Dosages is less than carrier biofertilizers• Greater potentials to fight with native population

Composition (by wt) of Liquid Formulations

Liquid formulations contains (by wt): Desired microorganisms: 10-40%; Suspended ingredient: 1-3%;Dispersant: 1-5%; Surfactant: 3-8%; Liquid carrier (or water): 35-65%

Technique of Liquid Formulations

To achieve a sterile product free from contaminants, fermentation is carried out in more sophisticatedfermentors. This is necessary for a high shelf life, since contaminants can spoil the product. Propercontrol over environmental conditions in the fermentor is very important. Mixing (agitation), aeration,temperature, pH and dissolved oxygen are crucial parameters. When all these conditions are metproperly, a cell count of as high as 1013 cfu is achievable.

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Heatless concentration techniques as cross-flow microfiltration are ideal for concentratinginoculants, since they ensure that a majority of the cells remain viable. This process also partiallyremoves the residual nutrients in the medium and makes them unavailable to the cells, thereby fosteringthe transformation of the cells into the dormant phase.

Suitable preservatives are added to the cell concentrate. The preservatives are safe for theproduction cultures, but preventive for spoilage cultures. Packing is carried out under aseptic conditions,either manually using laminar flow workbench or using an automatic filling machine. Pre-sterilized bottlesare used for filling. Preferred method of bottle sterilization is by radiation.

Facilities required for production of Liquid Biofertilizers / Bio-control Agents (1 lakh litre perannum)

Production Plant consists of four Sections

A. Manufacturing section1. Fermentors with pH and temperature sensors2. Storage tanks3. Blenders4. Conveyors, piping and instrumentation - as required

B. Utilities equipments for operating fermentors1. Air compressor2. Boiler and cooling tower for temp. control water system3. Water softener and purifier for boilers and fermentors4. Pumps and vacuum pumps

C. Filling room1. Cross-flow microfilter / Reverse osmosis system2. Bottle/pouch filling machine (automatic) with sterilized pipelines3. Air filter system (0.2 micron) to supply pressurized air to filling room

D. Culture laboratory with standard testing and culture propagation equipments1. Laminar air flow cabinets (horizontal type with 0.2 micron German made heppa filter) - 2 nos.2. Phase contrast Microscope3. BOD Incubators5. Horizontal autoclaves6. Orbital Shakers7. Refrigerators8. Demineralised water plant

Deterioration of Liquid Formulation

The principal causes of deterioration are high temperature, presence of free water (as opposed tomolecularly bound water), adverse pH, enzymes (particularly proteases), and combinations of thesefactors. Some substances, e.g., certain sugars and amino acids, stimulate bacterial spores to germinateand lose their innate resistance to adverse factors.

Due to water evaporation once applied and effects both cell viability and release kinetics, use ofwater-in-oil emulsion is better. But the drawback is the problem of cell sedimentation and thickening theoil phase during storage. Use of hydrophobic silica nanoparticles significantly reduced cell sedimentationand improved cell viability during storage. Other common problems with the overall liquid formulation are

• High temperature: deterioration in fermentor can be minimized by cooling• Presence of free water (not molecularly bound water): by using high molecular osmotic molecules as

adjuvant.• Adverse pH: save by lowering pH to a minimum of 6.0-6.9 with food grade stabilizers, e.g., ascorbic

acid, it simultaneously prevents growth of contaminant microorganisms.• Enzymes (particularly proteases), surfactants and combinations of these factors.• Some substances e.g., certain sugars and amino acids, stimulate bacterial spores to germinate and

lose their innate resistance to adverse factors.

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• Time elapses between stopping the production process and the preservation by drying.• The above conditions are ensured positively during stabilization of broth material with base material

and left for curing up to 72 hours.

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SECTION- 5

6. Entrepreneurships in BFs production: Production of BFs in different scales (small, medium andlarge) with techno-economic viabilities, constraints, marketing of BFs

6.1. Financial outlay and return

Facilities / infrastructure required1.Fixed Assets

a. Building: Approx. 768 sq m @ Rs. 8000.00/sq m, estimate Rs. 61.44 lakhs with other charges

S.No.

Components Dimensions (ft) Area (sq ft.)

1 Media preparation room 20 x 18 3602 Sterilization room 20 x 18 3603 Fermentor hall 20 x 18 3604 Mixing Room 20 x 18 3605 Packing Room 20 x 18 3606 Store 20 x 18 3607 Sale room and Office 20 x 18 3608 Tin shade* (for storing & pulverization of carrier material) 20 x 100 200

Total 2,520 sq ft(768 sq m)

* Tin shade for lignite storage cost extraCost of construction (Rs. in lakh) = 61.44

b. List of Equipments

S.No

Equipments Quantity(No)

Total cost(Rs. in lakh)

Justification for

1 Balance 01 0.10 Weighing2 pH meter 01 0.15 Measuring pH of liquid media3 Inoculation chamber 02 0.20 Inoculation microorganisms4 Autoclave (Vertical) 02 1.00 Sterilization of liquid media5 Incubator (BOD) 02 1.20 Incubation of inoculated samples6 Refrigerator 02 0.25 Storage of microbial samples7 Rotary Shaker 02 1.50 Fermentation of cultures

Total 4.40

2. Operational Cost (per year)

S.No.

Particular Amount(Rs. in lakh)

1 Glassware 2.002 Chemicals and carrier materials 20.003 Salaries (2 Research Fellows with payment @ Rs. 15,000/- PM) 1.204 Daily wage workers (10, @ Rs. 300/- per person per day) 5.405 Miscellaneous (electricity, packing material, etc) 2.00

Total (Rs. in lakh) 20.60

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Total Cost (for 3 years)

S.No.

Particular I year(Rs. in akh)

II year(Rs. In lakh)

III year(Rs. in lakh)

Total(Rs. in lakh)

1 Building and Equipments 65.84 Nil Nil 65.842 Operational cost for 3 years (working capital) 20.60 20.60 20.60 61.80

Total (Rs. in lakh) 86.44 20.60 20.60 127.64

TECHNO-ECONOMIC VIABILITY / COST BENEFIT ANALYSIS

(to include cash flow, working capital management, pay back period, etc.)Working out the average sale price of 90 tonnes of biofertilizers per annum (5.00 lakh packets / annumeach containing 200 g biofertilizer) subjected to full production, supply and realization of money out ofsold packets and lifting of the packets from the production unit:

S.No.

Particular Acctt.(Rs. in lakh)

I. Input costs on NRC+RC items1. Fixed cost (building / equipments): 65.842. Variable cost: 20.60II. Production Cost (for 5 lakh packets / annum as target)1. Depreciation in building cost (Rs. 61.44 lakh)

@ 5% on total cost of building3.07

2. Depreciation in equipments cost (Rs. 4.40 lakh)@ 10% on total cost of equipments

0.44

3. Operational cost 20.60Total (Rs. in lakh)(of Production Cost of 5.00 lakh pkts)

24.11

Production Cost of 100 pkts of Biofertilizer (average) Rs. 482(~Rs. 500)

III. Selling Price1. Production Cost / 100 pkts 5002. Risk coverage for unsold pkts @ 25% 1253. Charges for Transport and Publicity 2004. Profit margin 100

Total Selling Price per 100 pkts 825(~Rs. 800)

Tentative Bio-preparation production target

(subjected to full supply, realization of money out of sold packets and lifting of the packets from theproduction unit)

S.No.

Yr Tentative target Production Supply Production Cost Anticipated Receipt(Rs. in lakhs)

Net Profit(Rs. in lakhs)

1. 1st R & D and estt. of theproduction unit

Nil Nil Nil Nil Nil

2. 2nd R & D and Production 90 tonne 89 tonne(pkts 4.88 lakh

each 200 g)

Rs. 24.40 lakh@ Rs. 5.00 / pkt

Rs. 39.04 lakh@ Rs. 8.00 / pkt

Rs. 14.64 lakh

3. 3rd R & D and Production 90 tonne 89 tonne(pkts 4.88 lakh

each 200 g)

Rs. 24.40 lakh@ Rs. 5.00 / pkt

Rs. 39.04 lakh@ Rs. 8.00 / pkt

Rs. 14.64 lakh

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6.2. MARKET OPPORTUNITY OF BIOFERTILIZERS

Madhya Pradesh is the second largest producer and consumer of bio-fertilizers in India, but with regard toantagonists, bio-control agents and disease management there is no such documented units in MadhyaPradesh. The use of biofertilizers is confined to elite farmers in certain pockets only.

Biofertilizer requirement of M.P. (million tonne)

Crop / BFs Rhizobium Azotobacter /Azospirillum

PSB Total

1. Cereals - 24,609 24,609 49,2182. Pulses 4,593 - 13,779 18,3723. Oil Seeds 4,430 - 13,290 17,7204. Fruits and Vegetables - 632 632 1,264

Total 9,023(45.11)

25,241(126.2)

52,310(209.2)

86,574(380.51)

Requirement is based on area under cultivationRhizobium, Azotobacter, and Azospirillum 200 g, and PSB 250 g per packetFigure in parenthesis is in million packets

Government policy

The total production of various biofertilizer in M.P. is 1,756 metric tone which is limited to <2% of theactual requirement of 86,574 metric tones in the state. Further, by the application of quality biofertilizers,the crop yield could be increased by 15 to 20% with an additional increase of the protein and/or oilcontent by 5 to 10%. Deptt. of Agriculture and Cooperation (GOI) has launched a Central SectorSchemes and National Project on Organic Farming (NPOF) with an outlay of crores of rupees since 10thPlan period for promotion, production, certification and market development of organic farming in thecountry.

Main components of the scheme are

1. Certification of organic produce with prescribed national standards (BIS) for organic farming2. Capacity building of organic farming through service providers3. Support with financial assistance to the commercial production units of organic inputs like –

i. biofertilizers production unitsii. hatcheries for vermin-cultureiii. fruits and vegetables waste compost units

4. Providing training to the personnel engaged in production, promotion, certification, etc.5. Field demonstrations on organic inputs and technology for organic farming.6. Market development and promotion of organic farming

• A Service Provider serves farmers in cluster of villages for capacity building.• The service providers could be State Govt. agencies/lCAR/SAUs/KVKs/NGOs/Pvt. Agencies / Agri-

Clinics, who could also act as retail input suppliers to farmers.• Every year financial feedback support to each Service Provider is provided by the Government for

serving the farmers in clusters.

Field demonstrations (organized under NPOF)

• 1. Field demonstrations on organic inputs and farmers’ fair• 2. Field demonstrations on utilization of enriched biogas slurry• 3. Setting up of model organic farms

Market Development and Promotion

In order to encourage organic farming, it is required to carry out both the development of technologypackage through research institutions, as well as market assessment and consumer behaviour throughmarket intelligence and outsourcing on the following items:

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1. Development of Technology Packages2. Market Development3. Development of new initiatives4. Dissemination of proven technology5. National/Regional Conferences and Publicity

6.3. Business and Commercial Potential

In India the formulations of inoculants for nitrogen management available in the market are ineffective forits assured quality and response to crops. The strains of Jawahar cultures are developed looking to thecauses for failure in the eco-system and better competence against the native strains.

ScalabilityFormulation has been successfully made on small scale to large scale using different capacityfermentors. Refinement of the technology would be possible for high scale commercialization.

Limiting factors for large scale commercialization

• Batch wise slight variation in qualitative and quantitative parameters of the fermented cultures.• Low shelf life.• Low level of awareness among farmers.• Limited market.

Business Potential: The technology will have wide acceptance as it can be used to manage nutritionand plant protection of various legumes and other crops. Farmers can reduce the use of chemicalfertilizers with application of the low cost input technology.

Market Potential: (for all biofertilizers /biopesticides together)

Sell of culture (pkts/yr) Revenue realizeda

(Rs. in lakh)Cost and Profit statement

Total project costb(Rs. in lakh)

Profit(Rs. in lakh)

C:B ratio

For Small Scale Production3,60,000 72.00 33.12 38.88 1:1.17For Medium Scale Production6,30,000 126.11 59.00 67.11 1:1.29For Large Scale Production12,02,850 240.57 99.00 141.57 1:1.43a Revenue realized = Sell of culture / Rate of culture sold (Rs. 20)b Total project cost (only for Ist yr) = NRC+RC+Tech. Comm. Fee

Target Market / Customer

MP State Agril. Deptt, JNKVV: Research Farms/Stations, KVKs, Biofertilizer Companies andCooperatives, Agri-input traders, Farmer Cooperatives, NGOs, Pvt Companies, Organic farming inputsuppliers, Farmers

Social impact of the technology

Substituting/decreasing use of inorganic fertilizers. Promotion of organic farming. Increase in productivityof crops. Increase in income of farmers

Any other relevant information

It is a green technology. Unemployed youth can start small production unit through BPD support and helpfarming system and themselves.

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FinancialsPlant parameters Cost Small scale

preparationMedium scalepreparation

Large scalepreparation

Land & site - Facilities Rs. in lakh Rs. in lakh Rs. in lakhNo. of shifts 1 Non-recurring:Working days/yr 180 Basic infrastructure & facilities - 10.00 20.00Land required(sq.ft)

3000-5000

Equipments/machineries

20.00 30.00 50.00

Manpower Total 20.00 40.00 70.00Managerial 1 Recurring:Skilled 1-2 Electrical power 3.00 5.00 10.00Unskilled 3-10 Manpower 3.12 (1+1+3) 5.00 (1+2+6) 7.00 1+2+10)Utilities Consumable, transport, trade

margin, etc.5.00 7.00 10.00

Electrical power(KVA)

10-50 Total 11.12 17.00 27.00

Tech. commercialization fee perproduct

2.00 2.00 2.00

Total (value of technology) 33.12 59.00 99.00Economic analysis:Profit (net) 38.88 76.11 141.57Cost:Benefit ratioafter 1 yr onward

1:1.17 1:1.29 1:1.43