Biofertilizers: Mass Multiplication and Field Application

Pseudomonas striata: Mass Multiplication and Field Application

Pseudomonas striata is an important phosphate-solubilizing microorganism widely used as a biofertilizer in sustainable agriculture. Phosphorus is a major essential nutrient required for plant growth, playing a vital role in root development, flowering, seed formation, and energy transfer through ATP synthesis. Although soils contain large amounts of phosphorus, most remains in insoluble forms like tricalcium phosphate and rock phosphate. Pseudomonas striata converts these into soluble forms (H₂PO₄⁻ and HPO₄²⁻) that plants can readily absorb, thereby enhancing soil fertility and crop productivity.

Taxonomy and General Characteristics

Pseudomonas striata belongs to the kingdom Bacteria and the genus Pseudomonas. It is a Gram-negative, rod-shaped, motile, and non-spore-forming aerobic bacterium. It inhabits the rhizosphere, where it produces organic acids—such as gluconic, citric, and oxalic acid—to dissolve insoluble soil phosphates.

Mass Multiplication Process

The goal is to produce a high population of viable cells (10⁸–10⁹ cells/g). Optimal conditions include a temperature of 28–30°C, pH 6.5–7.0, proper aeration, and moisture.

• Mother Culture: Prepared on nutrient agar slants and incubated for 24–48 hours.
• Growth Medium: Nutrient broth or Pikovskaya’s broth (containing glucose, ammonium sulfate, and minerals) is sterilized at 121°C.
• Inoculation and Incubation: Inoculum is transferred to sterile broth and incubated in a shaker at 100–150 rpm for 3–5 days.
• Large-Scale Production: Broth is transferred to fermenters for controlled growth.
• Carrier Mixing: The culture is mixed with sterilized carriers like peat, lignite, or vermiculite (35–40% moisture).
• Curing and Packing: The mixture is cured for 24–48 hours, packed in polyethylene bags, and stored at 4–15°C.

Field Application

• Seed Treatment: Biofertilizer is mixed with a jaggery solution to create a slurry, coated on seeds, and shade-dried.
• Seedling Root Dip: Seedlings are dipped in a biofertilizer suspension for 20–30 minutes before transplanting.
• Soil Application: Mixed with farmyard manure (FYM) or compost and broadcasted (2–5 kg/acre).

Benefits and Limitations

While it improves phosphorus availability and crop yield, its efficiency is limited by temperature fluctuations, UV radiation, and a shelf life of 4–6 months.

Cyanobacterial Inoculum: Isolation and Mass Multiplication

Isolation and Purification

Cyanobacteria are isolated from freshwater, rice fields, or wet rocks. The process involves:

1. Sample Collection: Sterile collection of soil or water.
2. Enrichment: Using BG-11 or Chu’s medium under light (12–16 hrs/day) at 25–30°C.
3. Purification: Achieved through repeated subculturing, antibiotic treatment, or filtration to ensure axenic cultures.

Mass Multiplication Methods

• Tank Method: Controlled production in cemented tanks (2m x 1m x 0.3m). Involves adding soil, superphosphate, and starter culture.
• Pit Method: A low-cost, traditional method using shallow soil pits, ideal for rural farmers.

Rhizobium: Symbiotic Nitrogen Fixation

Rhizobium is a symbiotic bacterium forming root nodules in legumes. It converts atmospheric N₂ into ammonia. Isolation involves surface-sterilizing pink, healthy nodules and streaking on Yeast Extract Mannitol Agar (YEMA). Mass multiplication follows similar steps to Pseudomonas, using YEMA broth and carrier materials.

Azolla: Mass Cultivation

Azolla is a free-floating aquatic fern that hosts Anabaena azollae. It is cultivated in pits (2m x 1m x 20cm) with cow dung slurry and superphosphate. It is applied to rice fields via dual cropping or as green manure.

Vermicomposting

Vermicomposting is a bio-oxidative process using earthworms (e.g., Eisenia fetida) to convert organic waste into nutrient-rich humus. It involves pre-composting, bed preparation, inoculation, and harvesting. It improves soil structure, aeration, and microbial diversity.

Azospirillum and Azotobacter

These are free-living or associative nitrogen-fixing bacteria. Azospirillum is isolated using nitrogen-free semi-solid malate medium, while Azotobacter is isolated on Ashby’s Mannitol Agar. Both are mass-multiplied in nitrogen-free media and applied via seed treatment or soil application.

Mycorrhizae

Mycorrhizae are symbiotic associations between fungi and plant roots. Ectomycorrhizae form a mantle around roots (common in forest trees), while Endomycorrhizae (AM) penetrate root cells (common in crops). They significantly enhance phosphorus uptake and stress tolerance.

Biofertilizer Storage and Shelf Life

To maintain viability, biofertilizers should be stored at 4–15°C in the dark. Shelf life is enhanced by using high-quality carriers, protective substances like glycerol, and proper packaging that allows gas exchange.