FORMULATION OF BIOINOCULANTS

 FORMULATION OF BIOINOCULANTS

Biofertilizers can be applied to the crops by different formulations like (1) Carrier-based inoculant, (2) Liquid-based inoculant and (3) Alginate bead based inoculant.

1) CARRIER BASED INOCULANT

·         Carrier is defined as the medium in which microorganisms allowed to multiply.

·         Various types of material are used as Carrier for Seed or Soil inoculation.

·         For preparation of Seed inoculant, the Carrier material is milled to fine powder with particle size of 10 - 40 μm.

·         Peat is the most frequently used Carrier material for Seed inoculation.

·         For soil inoculation, Carrier material with granular form (0.5 – 1.5 mm) is generally used. Granular forms of Peat, Perlite, Charcoal, Talcum powder or Soil aggregates are suitable for soil inoculation.

Carrier materials used for Biofertilizers

a)     Celite

b)     Cellulose powder

c)      Charcoal

d)     Cheese whey

e)     Coal

f)       Coconut shell

g)     Compost/Vermicompost material

h)     Diatom

i)       Kaolin

j)       Leaf manure

k)     Lignite

l)       Mineral soils/ Soil aggregates

m)   Oxalic acid

n)     Peat

o)     Perlite

p)     Porosil

q)     Pressmud

r)      Rice husk

s)      Sugarcane bagasse

t)      Talcum powder

u)     Vermiculite

v)     Wastewater sludge

w)   Wheat bran

Characteristics of good Carrier material

a)     Non – toxic to Inoculant microbial stain and Plants.

b)     Good moisture adsorption capacity.

c)      Easy to sterilize by Autoclaving or Gamma – irradiation.

d)     Easy access for mixing with Bioinoculants.

e)     Available in adequate amounts.

f)       Low cost.

g)     Good adhesion to seeds.

h)     Good pH buffering capacity.

i)       Organic matter content should be around 40 %.

j)       Water holding capacity should be more than 50 %.

Preparation of Carrier materials for Seed or Soil inoculation

a) Drying and Grinding of the Carriers

·         Sundry upto 5 %.

·         Grind and pass through 100 – 200 µ Sieve.

·         Survival of microorganisms is poor in Coarse material.

b) Pre-treatment of the Carriers

·         Mix with Calcium carbonate (CaCO₃) powder, neutralize and pH is adjusted to 6.5 to 7.0.

·         The amount of CaCO₃ can be added according to the Acidity of the Carrier material.

 c) Sterilization of the Carrier materials

·         Sterilization of Carrier material is essential to keep high number of inoculant bacteria on carrier for long storage period.

·         Gamma-irradiation is the most suitable way of Carrier sterilization, because the sterilization process makes almost no change in physical and chemical properties of the material. Carrier material is packed in thin-walled polyethylene bag, and then gamma-irradiated at 50 kGy (Kilogray).

·         Another way of carrier sterilization is Autoclaving. Carrier material is packed in partially opened, thin-walled polypropylene bags and autoclaved for 60 min at 121 °C. It should be noted that during autoclaving, some materials changes their properties and produce toxic substance to some bacterial strains.

d) Inoculation of microorganisms to the Carrier materials

·         Prepare starter culture for inoculation. Optionally, appropriately dilute with sterile water for moisture and cell number adjustment.

·         Inject the culture to the carrier package using a sterile disposable plastic syringe with a needle.

·         Keep the package at appropriate temperatures for maturation and storage. Although the temperatures suitable for maturation and storage are dependent on the inoculant microorganisms, 30 °C for maturation and 20 °C - 30 °C for storage will be suited for inoculants in most cases.

Advantages of Carrier based Inoculants

a)     Low cost

b)     Easy to produce

c)      Less investment

Disadvantages of Carrier based Inoculants

a)     Low Shelf – life

b)     Temperature sensitive

c)      Contamination prone

d)     Low cell counts

e)     Less effective than Liquid based inoculants

2) LIQUID BASED BIOINOCULANT

• Respective Culture broth was prepared and mixed in combination with different additives to increase the survival of Bioinoculants in a Liquid formulation.
• Wetter like Triton & Tween with 0.5, 1.0 and 1.5 % concentration; Stickers like Carboxy methyl cellulose (CMC) & Gum Arabic with 0.5, 1.0 and 1.5 %; Humectants – Glycerol, Trehalose & Polyvinyl pyrollidone (PVP) with 0.5, 1.0 and 1.5 % were used to increase the survival of microbial inoculants.
• One ml of log phase culture of Bioinoculant was inoculated as single inoculant in respective broth and the flasks were incubated at room temperature.
• The formulation was analyzed for viable cell population at 1 month interval upto 12 months.

Advantages of Liquid based Inoculants

a)     Longer Shelf – life (12 to 24 months)

b)     No contamination

c)      No loss of properties due to storage upto 45 °C

d)     Product can be 100 % sterile

e)     Better survival population on Seed and Soil

f)       More effective than Carrier based inoculants

g)     Very easy to produce

h)     Very easy to use by farmers

i)       Temperature tolerant

j)       High export potential

k)     High commercial revenues

Disadvantages of Liquid based Inoculants

a)     High cost

b)     High investment for production unit

3) ALGINATE BEAD BASED BIOINOCULANT

• The microbial inoculants were grown in respective Culture broth.  
• Two grams of Sodium alginate was added to 100 ml of Culture broth of Microbial inoculants and it was mixed for 30 mins in a Magnetic stirrer.
• The mixture was added drop wise through a 10 ml syringe into 100 ml sterile 0.1N CaCl₂ to obtain uniform Alginate beads.
• One gram of material contained 16 to 17 beads, each bead approximately weighing 60 mg.
• The beads were washed twice in sterile distilled water and incubated in respective broth containing microbial inoculants for seven days in an incubator at room temperature to allow microbial inoculants to multiply inside the beads.
• The beads were again washed in sterile distilled water and air dried in Laminar air flow chamber under aseptic condition. The alginate beads were then stored in Polythene bags at room temperature upto 6 months.

 

BIOFERTILIZERS

·         Generally, the term "Fertilizer" is used for "Fertilizing material or Carrier", meaning any substance which contains one or more of the essential elements (nitrogen, phosphorus, potassium, sulphur, calcium, magnesium, iron, manganese, molybdenum, copper, boron, zinc, chlorine, sodium, cobalt, vanadium and silicon). Thus, fertilizers are used to improve the fertility of the land.

·         Biofertilizers may be defined as “Substances which contains living strains of microorganisms (Bacteria, Fungi and Algae) that colonize the Rhizosphere or the interior of the plants and promote growth by increasing the supply or availability of primary nutrients to the target crops, when applied to soils, seeds or plant surfaces”.

·         Biofertilizers infuse nutrients by natural processes such as synthesis of Growth promoting substances, Fixing nitrogen and Solubilizing phosphorous in the Rhizosphere.

NEED OF BIOFERTILIZERS

·         The heavy use of synthetic fertilizers for past many decades has led to depletion of essential nutrients from soil, contamination of the soil with harmful and non-degradable substances, pollution of water resources and destruction of friendly insects and essential microorganisms from the soil.

·         The global demand for fertilizers is much higher than the availability. The costs of chemical fertilizers are also increasing every other day, making them unaffordable by marginal and small farmers.

TYPES OF BIOFERTILIZERS

1)     Nitrogen fixers

a)     Free living or Asymbiotic or Non – symbiotic Nitrogen fixers

·         Aerobic Heterotrophs– Azotobacter sp., Achromobacter sp.  and Beijerinckia sp.

·         Aerobic Autotrophs– Nostoc sp., Anabaena sp., Colothrix sp. and Blue Green Algae (BGA)

·         Anaerobic Heterotrophs – Clostridium sp., Klebsiella sp. and Desulfovibrio sp.

·         Anaerobic Autotrophs – Chlorobium sp., Chromnaticum sp., Rhodospirillum sp. and Methanobacterium sp.

b)     Symbiotic Nitrogen fixers – Rhizobium sp., Bradyrhizobium sp., Azhorhizobium sp., Frankia sp., Blue Green Algae and Anabaena azollae

c)      Associative symbiotic Nitrogen fixers – Azospirillum sp. and Herbaspirillum sp.

d)     Endophytic Nitrogen fixers – Gluconacetobacter sp. and Burkholderia sp.

 

2)     Phosphorous solubilizers

a)     Bacteria – Bacillus megaterium var phosphaticum, Bacillus subtilis, Bacillus circulans, Bacillus polymyxa and Pseudomonas striata.

b)     Fungi – Aspergillus awamori and Penicillium sp.

3)     Phosphate mobilizers

a)     Arbuscular mycorrhiza (AM) – Glomus sp., Gigaspora sp., Acaulospora sp., Scutellospora sp. and Sclerocystis sp.

b)     Ectomycorrhiza – Laccaria sp., Pisolithus sp., Boletus sp. and Amanita sp.

c)      Ericoid mycorrhiza – Pezizella ericae

d)     Orchid mycorrhiza – Rhizoctonia solani

4)     Potassium mobilizers – Frateuria aurentia

5)     Silicate and Zinc solubilizers – Bacillus sp.

6)     Manganese solubilizers – Penicillium citrinum

7)     Silicate solubilizers – Bacillus mucilaginous

8)     Plant Growth Promoting Rhizobacteria (PGPR) – Pseudomonas fluorescence

ADVANTAGES OF BIOFERTILIZERS OVER CHEMICAL FERTILIZERS

1)     Biofertilizers have replaced the chemical fertilizers as chemical fertilizers are not beneficial for the plants. Chemical fertilizers decrease the growth of the plants and make the environment polluted by releasing harmful chemicals.

2)     Plant growth can be increased if biofertilizers are used, because they contain natural components which do not harm the plants.

3)     If the soil will be free of chemicals, it will retain its fertility which will be beneficial for the plants as well as the environment, because plants will be protected from getting any diseases and environment will be free of pollutants.

4)     Biofertilizers destroys harmful components from the soil which cause diseases in the plants.

5)      Biofertilizers are not costly and even poor farmers can make use of them.

6)      Biofertilizers are environment friendly and protect the environment against pollutants.

7)     Biofertilizers provides plant nutrients at very low cost.

8)     Biofertilizers helps for the survival of beneficial microorganisms in soil.

9)     Biofertilizers helps to get high yield of crops by making the soil rich with nutrients and useful microorganisms necessary for the growth of the plants.

10)Biofertilizers are also known to provide better nourishment to plants than chemical fertilizers.