FORMULATION OF BIOINOCULANTS

FORMULATION OF BIOINOCULANTS OR MICROBIAL INOCULANTS

  

• Microbial Inoculants can be applied to the crops by different formulations like

                          I.          Carrier based inoculant

                        II.          Liquid based inoculant

                     III.          Alginate bead based inoculant.

I) 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 Microbial Inoculants

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

Step 1: 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.

Step 2: 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.

Step 3: 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.

Step 4: Inoculation of microorganisms to the Carrier materials

• Prepare microbial 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

ü  Low cost

ü  Easy to produce

ü  Less investment

Disadvantages of Carrier based Inoculants

ü  Low Shelf – life

ü  Temperature sensitive

ü  Contamination prone

ü  Low cell counts

ü  Less effective than Liquid based inoculants

II) LIQUID BASED MICROBIAL INOCULANTS

·    Respective Culture broth was prepared and mixed in combination with different additives to increase the survival of Microbial Inoculants 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 Microbial Inoculants 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 Microbial Inoculants

ü  Longer Shelf – life (12 to 24 months)

ü  No contamination

ü  No loss of properties due to storage upto 45 °C

ü  Product can be 100 % sterile

ü  Better survival population on Seed and Soil

ü  More effective than Carrier based inoculants

ü  Very easy to produce

ü  Very easy to use by farmers

ü  Temperature tolerant

ü  High export potential

ü  High commercial revenues

Disadvantages of Liquid based Inoculants

ü  High cost

ü  High investment for production unit

III) ALGINATE BEAD BASED MICROBIAL INOCULANTS

·       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.