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.

 

Industrial production of Acetic acid

Acetic acid is a crucial platform substance. Only 10% of the world's production, which is used to make vinegar, is made by bacterial fermentation. It is primarily created synthetically. Acetic acid can be produced by a variety of microorganisms, some of which also incorporate CO into the process. 

Definition:

   Although it may have been preceded by certain fermented foods derived from milk, acetic acid (also known as ethanoic acid or methyl carboxylic acid) has been generated since the invention of winemaking, which goes back to at least 10,000 BC. Given that the Latin term acetum denotes sour or sharp wine, it is generally accepted that the first vinegar, which is an aqueous solution of acetic acid, was produced as a byproduct of the damaged wine. It served as a drug at first and was perhaps the very first antibiotic ever discovered. For the majority of human history, all acetic acid was produced using the same traditional method, which involved fermenting sugar to ethyl alcohol and then oxidizing it by microbes to generate vinegar. 

Acetic acid, which has the molecular formula CH3COOH, is also referred to as ethanoic acid, ethylic acid, vinegar acid, and methane carboxylic acid. Vinegar gets its distinctive smell from acetic acid, a byproduct of fermentation. In water, vinegar contains 4-6 percent acetic acid.

Two stages of fermentation were used to create this product. The process begins with anaerobic fermentation, which is the alcoholic conversion of carbohydrates into ethanol by yeasts and continues with aerobic fermentation (oxidation of ethanol into acetic acid by AAB). Before alcoholic fermentation can begin, the basic materials - starch or complex carbohydrates - need to be saccharified in order to release fermentable sugars. Due to the growing demand for vinegar, technical advancements were required to produce the product. Currently, three main techniques are used to produce vinegar: the submerged process, the generator process, and the slow surface culture fermentation, which is known as the "Orleans" or "traditional process."

Acetic Acid Production:

i. Chemical-based method:

Acetic acid as an industrial chemical is produced from fossil fuels and chemicals by three processes: acetaldehyde oxidation, hydrocarbon oxidation, and methanol carbonylation.

ii. Biology-based method

    i. Aerobic process production:

Food-grade acetic acid is produced by the two-step vinegar process. The first step is the production of ethanol from a carbohydrate source such as glucose. This is carried out at 30°C using the anaerobic yeast Saccharomyces cerevisiae. The second step is the oxidation of ethanol to acetic acid. Although a variety of bacteria can produce acetic acid, only members of Acetobacter are used commercially, typically the aerobic bacterium Acetobacter aceti at 27-37°C. This fermentation is incomplete oxidation because the reducing equivalents generated are transferred to oxygen and not to carbon dioxide:

C6H12O6 + 2CO2 + 2CH3CH2OH.

The overall theoretical yield is 0.67 g acetic acid per gram of Glucose. At the more realistic yield of 76% (of 0.67, i.e., 0.51 g per gram glucose), this process requires 2.0 pounds of sugar or 0.9 pounds of ethyl alcohol per pound of acetic acid produced. Complete aeration and strict control of the oxygen concentration during fermentation are important to maximize yields and keep the bacteria viable. Submerged fermentation has almost completely replaced surface fermentation methods. The draw-and-fill mode of operation can produce acetic acid at concentrations up to 10% wt/wt in continuous culture at pH 4.5 in about 35 hours.

2CH3CH2OH + 02 -+ 2CH3COOH + 2H20.  

    ii. Anaerobic process production:

    In 1980 another process for the production of acetic acid emerged based on anaerobic fermentation using Clostridia. These organisms can convert glucose, xylose, and some other hexoses and pentoses almost quantitatively to acetate according to the following reactions:

C6H12O6 + 3CH3COOH, 2C5H10O5 + 5CH3COOH.

Typical acidogenic bacteria are Clostridium aceticum, Clostridium thermoaceticum, Clostridium formicoaceticum and Acetobacterium woodii. Many can also reduce carbon dioxide and other one-carbon compounds to acetate.

Parameter for AAB::

pH

pH The optimum pH for the growth of AAB is 5.5 to 6.3. However, these bacteria can survive at low pH values of between 3.0 and 4.0, and some strains have been isolated from an aerated media containing acetate that could grow at pH values as low as 2.0 to 2.2. It was postulated that there are three groups of strains that might exist in vinegar production, namely, acidophilic strains that grow only at a pH value of about 3.5, acetophobic strains that only grow at pH levels higher than 6.5, and acetotolerant strains that can grow at both these values. There may be a gradual development from acetophobic to acetotolerant strains and, with prolonged exposure to low pH and high acetic acid concentrations, to acetophilic strains. This suggests the development of a gradual acid resistance in these bacteria.

Temperature

The optimum growth temperature for most AAB is 25◦C to 30◦C. The maximum temperature for the growth of A. aceti wasfound to be about 35◦C. Thermotolerant AAB that are able to grow at 37◦C to 40◦C have also been isolated. These bacteria were able to oxidize ethanol at 38◦C to 40◦C at the same rate that mesophilic strains do at 30◦C, as well as being able to oxidize ethanol more rapidly than the mesophilic strains at the higher temperatures. AAB can also be active at lower temperatures, and weak growth was observed even at 10◦C.

Production of Acetic acid

Batch fermentations in 16-L vessels with automatic pH control were conducted as a basis for predicting continuous fermentor performance at different dilution rates. Continuous fermentations in 0.3-L chemostats were performed to determine for C. thermoaceticum. Batch fermentations were conducted in pH-, temperature-, and gas-controlled New Brunswick 16-L Microferm fermentors. These units had provisions for homogeneous mixing, constant headspace flushing with CO2, gas, sterile sampling, automatic pH control, and in-place sterilization. Chemostats with a working volume of 333 mL were set up for continuous work. The units contained provisions for flushing the headspace with gas and sparging with C02 or N,, plus pH control with 10% NaOH. The headspace of the feed medium reservoir was also flushed with N, to maintain its anaerobic state.

German Method 

In 1832, German chemist Schutzenbach established the German process which is one of the quick processes (trickling method). It is also one of the oldest methods of acetic acid production. Acetic acid bacteria are Gram-negative and aerobic bacteria. The acetic acid bacteria were grown and fermented thick slim coating beech wood shavings occurs in 5000-6000 liters of wood /steel tank while the alcoholic liquid distributes of spray mechanism covered with AAB. During, the pH was maintained in the range between 2.5-3.2 and they produced at 27-30◦C of 88-90% of acetic acid from ethanol and the remaining substrate is used in biomass production. Depending on the size of the acetifier, production capacity was 70000- 100000 at 10% acidity. The acetic acid yield is approximately 1-2 kg/m^3.h. A high yield of acetic acid was produced in surface culture of 57 611 mg/L with inoculum techniques. 

Parameters: 

    (i) mash circulation, 

    (ii) cooling water through heat exchanger, 

    (iii) amount of air passed through the system.

 The advantages is it requires low space requirements, Low costs, and high acetic acid concentration. Drawbacks, high risk of clogging because of cellulose and high loss of ethanol by evaporation, and difficulty to producing AA production.

Application:

1. Antibacterial property for injuries like burns. -  Therapeutic effects
2. Used to prevent growth, spoilage and preserve. - Beverages industry
3. Food condiment and preserving meats and vegetables. - Food industry
4. Defensive effect and decrease fatality - Cardiovascular diseases
5. Used for diabetic treatment and decrease blood sugar levels. - Antidiabetic effect
6. Used in paper coatings, emulsion, resins for paints. - Petrochemical industry
7. Laboratory washing. - Machineries
8. Used in laundry detergents - Homecare
9. Managing soft tissues injury - Orthopaedic surgery

Reference:

1. Cheryan, M., Parekh, S., Shah, M., & Witjitra, K. (1997). Production of Acetic Acid by Clostridium thermoaceticum. Advances in Applied Microbiology, 1–33. doi:10.1016/s0065-2164(08)70221-1 

2. Li, P., Li, S., Cheng, L., & Luo, L. (2014). Analyzing the relation between the microbial diversity of DaQu and the turbidity spoilage of traditional Chinese vinegar. Applied microbiology and biotechnology, 98(13), 6073-6084.

3. Bayan, L., Koulivand, P. H., & Gorji, A. (2014). Garlic: a review of potential therapeutic effects. Avicenna journal of phytomedicine, 4(1), 1.