Unit 3: Formulations of biopesticides

I. Orientation: Microbial Biopesticide Formulation

A microbial biopesticide formulation is a stable preparation in which a living microorganism, its infective propagules, or biologically active products are combined with carriers and additives for storage, transport, and application. Common active agents include bacteria such as Bacillus thuringiensis, fungi such as Beauveria bassiana and Metarhizium anisopliae, baculoviruses such as nucleopolyhedroviruses, and entomopathogenic nematodes. Formulation converts laboratory-produced biomass into a practical product while preserving biological activity.

• Active ingredient: The biologically effective component may be bacterial cells or spores, fungal conidia, viral occlusion bodies, nematode infective juveniles, or microbial metabolites.
• Carrier: A solid or liquid material that distributes the active ingredient uniformly; examples include talc, kaolin, clay, starch, vegetable oil, and water.
• Adjuvant: An additive that improves spreading, adhesion, wetting, suspension, protection, or shelf life; examples include surfactants, stickers, humectants, and ultraviolet protectants.
• Viability: The proportion of microbial propagules capable of germination, multiplication, or infection must remain sufficiently high throughout storage.
• Potency: Biological activity is expressed through measures appropriate to the organism, such as colony-forming units per gram (CFU g⁻¹), viable spores per millilitre, occlusion bodies per millilitre, or infective juveniles per package.
• Stability: The formulation must resist moisture changes, temperature stress, oxidation, contamination, sedimentation, and premature germination.
• Compatibility: Ingredients must not inhibit the organism or interfere with application equipment, host infection, or the target pest’s exposure.
• Field performance: An effective formulation deposits an adequate dose on the target site and protects the agent from desiccation, rainfall, sunlight, and ultraviolet radiation.
• Quality control: Identity, purity, viable count, pathogenicity, moisture, pH, particle size, suspensibility, and storage stability are assessed before release.
• Biosafety: Production should use authenticated, non-pathogenic strains and prevent contamination by undesirable microorganisms or microbial toxins.

II. Formulation Process and Product Types

A. Preparation of different formulations of microbial biopesticides

Preparation involves producing the microbial active ingredient, separating or concentrating it when necessary, blending it with compatible formulation materials, and packaging the standardized product under conditions that preserve infectivity.

• Selection of microbial strain: A strain is chosen for virulence, host range, environmental tolerance, mass-production potential, and genetic stability.

  • B. thuringiensis strains are selected for insecticidal crystal proteins active against particular insect orders.
  • Beauveria and Metarhizium isolates are evaluated through conidial germination and insect bioassays.
  • Baculoviruses are selected for high infectivity against their specific host insects.

• Preparation of inoculum: A pure mother culture is multiplied through one or more seed stages to obtain enough viable inoculum for production.

  • Bacterial seed cultures are commonly developed in sterile nutrient broth.
  • Fungal inoculum may consist of conidia, blastospores, or actively growing mycelium.
  • Viral inoculum consists of purified occlusion bodies obtained from infected host larvae.
  • Aseptic transfer prevents competing bacteria, fungi, and bacteriophages from entering the production system.

• Mass production: The method depends on the reproductive biology of the active agent.

  1. In vitro production: Bacteria and many fungi are multiplied by submerged-liquid or solid-state fermentation.
  2. In vivo production: Baculoviruses are generally multiplied in susceptible insect larvae because replication requires living host cells.
  3. Nematode production: Entomopathogenic nematodes may be produced in insect hosts or in liquid culture with their symbiotic bacteria.

• Submerged fermentation: Microorganisms grow in an aerated liquid medium inside a fermenter, where temperature, pH, agitation, oxygen, and nutrient supply are controlled.

  • Bacterial fermentation produces vegetative cells, spores, and toxins.
  • Fungal fermentation commonly yields blastospores, submerged conidia, or mycelial fragments.
  • Biomass is recovered by centrifugation, filtration, or sedimentation and then concentrated.
  • Excessive shear, heat, or osmotic stress during recovery can reduce viability.

• Solid-state fermentation: Fungi grow on moist solid substrates such as rice, wheat bran, maize, or other sterilized grains.

  • The substrate supplies nutrients and a large surface for aerial conidium production.
  • After sporulation, the material is dried under controlled conditions, and conidia are separated by sieving.
  • Moisture must be reduced sufficiently to limit metabolism without causing lethal desiccation.

• Carrier-based powder formulation: Concentrated propagules are blended with a finely divided, inert carrier to form dusts or wettable powders.

  • Talc, kaolin, clay, diatomaceous earth, and starch are common carriers.
  • The carrier is dried, sieved, and sterilized or otherwise treated to reduce contamination.
  • A sticker or binder, such as a compatible cellulose derivative or gum, may improve adherence.
  • Wettable powders also contain wetting and dispersing agents so that the powder forms a uniform suspension in spray water.
  • Particle size must permit easy mixing while minimizing inhalable dust and nozzle blockage.

• Granular formulation: The active ingredient is applied to or incorporated into coarse particles such as clay, maize cob granules, bran, or starch-based granules.

  • Granules are useful for soil application because they carry the agent into pest habitats and reduce spray drift.
  • The microbial suspension may be sprayed onto preformed granules, followed by low-temperature drying.
  • Alternatively, propagules and carrier may be mixed into a moist mass, extruded, sized, and dried.
  • High pressure and temperature during granulation must be avoided because living agents are heat-sensitive.

• Water-based liquid formulation: Microbial cells or propagules are suspended in buffered water containing stabilizers and suspension aids.

  • Thickeners reduce rapid sedimentation, while surfactants improve wetting and dispersion.
  • Humectants such as glycerol can reduce desiccation, but their concentration must remain compatible with the organism.
  • Antifoaming agents may be required during manufacture and tank mixing.
  • Water-based products are easy to dilute but are comparatively susceptible to contamination and loss of viability during warm storage.

• Oil-based formulation: Dry conidia or concentrated biomass are dispersed in vegetable or mineral oils with compatible emulsifiers.

  • Oil protects fungal conidia against drying and may improve adhesion to the insect cuticle.
  • An emulsifiable suspension disperses as fine droplets when added to spray water.
  • The oil’s viscosity, oxidation stability, and toxicity to propagules must be evaluated.
  • Oil formulations are particularly valuable for entomopathogenic fungi used under low-humidity conditions.

• Suspension concentrate: Fine microbial particles are maintained at high concentration in a liquid phase using dispersants and rheology modifiers.

  • Milling or homogenization produces a uniform particle distribution, but mechanical treatment must not destroy spores or viral occlusion bodies.
  • The product should redisperse after storage with gentle shaking.
  • Sedimentation, hard caking, phase separation, and nozzle blockage are major formulation concerns.

• Emulsifiable and suspo-emulsion formulations: These products combine an oil phase with water-dispersible ingredients to improve handling and deposition.

  • An emulsifiable concentrate forms an oil-in-water emulsion after dilution.
  • A suspo-emulsion contains suspended solid microbial particles together with an emulsified liquid phase.
  • Emulsifier selection depends on microbial compatibility and the required emulsion stability.

• Encapsulated formulation: Microbial propagules or toxins are enclosed within protective matrices such as alginate, starch, gelatin, or other biodegradable polymers.

  • Encapsulation can protect the active ingredient from ultraviolet radiation, oxidation, moisture loss, and unfavorable pH.
  • In alginate encapsulation, microbial material is mixed with sodium alginate and dropped into a calcium salt solution, producing calcium-alginate beads.
  • Bead size controls handling and release; small capsules may be suspended for spraying, while larger beads suit soil placement.
  • Release occurs through matrix hydration, erosion, diffusion, or microbial degradation.

• Bacterial biopesticide preparation: Fermentation broth containing bacterial spores and insecticidal crystals is harvested and standardized before formulation.

  • In B. thuringiensis, sporulation and crystal formation are monitored because both spores and crystal proteins may contribute to activity.
  • The concentrate may be spray-dried or freeze-dried when the process does not cause unacceptable potency loss.
  • It is formulated as wettable powder, granules, suspension concentrate, or water-dispersible granules.
  • Potency is confirmed by bioassay against a susceptible reference insect rather than by viable count alone.

• Fungal biopesticide preparation: Conidia are harvested from solid substrates or fermentation liquids, cleaned, dried, and mixed with solid or oil carriers.

  • Conidial moisture and water activity are carefully controlled because high moisture accelerates deterioration.
  • Germination percentage is determined on a suitable medium after a specified incubation period.
  • Oil dispersions, wettable powders, and granules are widely used because they support contact between conidia and the insect cuticle.

• Viral biopesticide preparation: Susceptible larvae are infected with a standardized viral dose, collected after disease development, and processed to recover occlusion bodies.

  • Infected cadavers are homogenized in water and filtered to remove coarse insect material.
  • Occlusion bodies are concentrated by sedimentation or centrifugation and quantified microscopically.
  • Formulations may include feeding stimulants, stickers, and ultraviolet protectants such as lignin-based materials.
  • Because baculoviruses are host-specific, both production hosts and field targets must be correctly identified.

• Finishing and packaging: The standardized formulation is filled into moisture-resistant, light-resistant, and chemically compatible containers.

  • Labels specify the active organism, strain, potency, batch, storage conditions, expiry period, and application directions.
  • Foil laminates or opaque containers help protect dry products from moisture and light.
  • Liquid products require enough headspace for shaking and should not form irreversible sediment.

B. Applications and limitations

The value of each formulation depends on whether it delivers a viable and effective dose under the intended storage, application, and field conditions.

• Application matching: Wettable powders and suspension concentrates suit foliar spraying, granules suit soil pests, oil dispersions improve fungal performance in dry environments, and encapsulated products support protection or controlled release.
• Advantages: Formulation improves dosing accuracy, ease of transport, dispersal, adhesion, environmental persistence, and compatibility with conventional application equipment.
• Biological constraints: Living agents can lose viability through heat, ultraviolet exposure, desiccation, high moisture, unsuitable pH, oxidation, or toxic additives.
• Physical constraints: Settling, caking, poor wettability, phase separation, dust formation, and blocked spray nozzles can cause uneven field delivery.
• Ecological constraints: Rainfall may wash deposits away, dense foliage may prevent target contact, and low humidity may suppress fungal germination.
• Operational limitation: Microbial pesticides often act more slowly than conventional contact insecticides because infection, toxin ingestion, or pathogen replication requires time.
• Compatibility testing: Tank mixing with fungicides, disinfectants, highly alkaline materials, or incompatible surfactants may inactivate the microbial agent and therefore requires prior evaluation.
• Storage requirement: Cool, dry storage generally prolongs shelf life, but the exact temperature and humidity limits depend on the organism and formulation.
• Performance verification: A complete quality assessment combines physical tests, viable counts, purity checks, and target-insect bioassays because a high propagule count does not necessarily guarantee high virulence.