Unit 3: Formulations of biopesticides - Subjective Questions

Definition: A microbial biopesticide formulation is a stable preparation containing a viable microorganism or its biologically active products, together with carriers, adjuvants, protectants, and other additives, in a form suitable for storage, handling, and field application.

Major objectives of formulation:

• Maintain viability and virulence: Protects the microorganism from loss of infectivity during production and storage.
• Improve shelf life: Reduces damage caused by moisture, temperature, oxidation, and contamination.
• Facilitate application: Converts microbial biomass into forms such as wettable powders, granules, suspensions, or oil dispersions.
• Improve field persistence: Protectants reduce inactivation by ultraviolet radiation, desiccation, and rainfall.
• Enhance adhesion and coverage: Stickers and spreaders help the microbial agent remain on insect or plant surfaces.
• Ensure uniform dosage: A standardized formulation delivers a known number of viable cells, spores, conidia, or occlusion bodies.
• Increase safety and convenience: It minimizes dustiness, contamination, and operator exposure while simplifying transport and use.

The general preparation procedure includes the following steps:

1. Selection of strain: A virulent, host-specific, genetically stable, and environmentally adapted microbial strain is selected.
2. Preparation of pure culture: The strain is purified and maintained as a mother or master culture.
3. Inoculum development: The microorganism is multiplied through one or more seed-culture stages under aseptic conditions.
4. Mass production: Production is carried out by submerged fermentation, solid-state fermentation, or in vivo multiplication, depending on the organism.
5. Harvesting: Cells, spores, conidia, crystals, or occlusion bodies are separated by filtration, centrifugation, sieving, or sedimentation.
6. Concentration and stabilization: Excess moisture is removed, and stabilizers or protectants are added.
7. Formulation: The active material is blended with suitable carriers, surfactants, stickers, UV protectants, and preservatives.
8. Drying or homogenization: Solid formulations are dried carefully, whereas liquid formulations are mixed to obtain a uniform suspension.
9. Quality control: Viability, virulence, microbial count, purity, moisture, pH, and physical properties are tested.
10. Packaging and storage: The final product is packed in moisture- and light-resistant containers and stored under recommended conditions.

Carriers:

• Provide bulk and permit uniform distribution of the active microorganism.
• Absorb microbial biomass and improve handling.
• Common examples include talc, kaolin, bentonite, peat, starch, bran, and vegetable oils.

Surfactants:

• Reduce surface tension and improve wetting and spreading.
• Help hydrophobic spores disperse uniformly in water.
• They include wetting agents, dispersants, and emulsifiers.

Stickers:

• Improve adhesion of the microbial agent to foliage or insect cuticle.
• Reduce loss due to wind and rainfall.
• Examples include gums, starch derivatives, and certain polymers.

Protectants:

• Protect microbes from ultraviolet radiation, heat, oxidation, and desiccation.
• Examples include lignin, optical brighteners, carbon-based pigments, and selected oils.

Preservatives:

• Suppress unwanted contaminants in liquid formulations.
• They must be compatible with the active microorganism and used at non-toxic concentrations.

A successful additive should improve product performance without reducing microbial viability or pathogenicity.

A talc-based wettable powder can be prepared by the following procedure:

1. Produce the microorganism: Grow the selected bacterium in liquid medium or the fungus by liquid or solid-state fermentation.
2. Harvest the propagules: Collect bacterial cells and spores or fungal conidia after maximum viable yield is obtained.
3. Prepare the carrier: Use finely powdered, sterilized talc with suitable particle size and low moisture content. The pH may be adjusted to a range compatible with the microorganism.
4. Add formulation aids: Mix the carrier with a wetting agent, dispersant, sticker, and protectant as required.
5. Blend the active material: Mix the concentrated microbial biomass uniformly with the carrier under hygienic conditions.
6. Dry carefully: Reduce moisture at a temperature that does not damage the microorganism. Excessive heating must be avoided.
7. Pulverize and sieve: Break lumps and obtain a free-flowing powder of uniform particle size.
8. Standardize: Adjust the product to the required viable count, such as a specified number of colony-forming units or conidia per gram.
9. Pack and label: Use moisture-proof, light-resistant packages with directions for storage and application.

The finished powder should wet readily, remain suspended during spraying, and retain adequate viability throughout its shelf life.

The most suitable formulation depends on the microorganism, intended application method, storage environment, and target pest.

A suspension concentrate contains a high concentration of microbial cells, spores, conidia, crystals, or viral occlusion bodies dispersed in a liquid phase.

Preparation:

1. Produce and harvest the microbial active ingredient.
2. Remove coarse residues and concentrate the biomass by filtration, centrifugation, or sedimentation.
3. Add the concentrated biomass to sterile or suitably treated water.
4. Incorporate wetting agents, dispersants, suspending agents, antifoaming agents, and compatible preservatives.
5. Adjust pH and viscosity to values that maintain microbial viability and physical stability.
6. Homogenize gently to obtain uniform particle distribution without damaging propagules.
7. Fill the concentrate into clean, airtight, light-resistant containers.

Stabilization measures:

• Use thickeners or suspending agents to slow sedimentation.
• Maintain a suitable particle-size distribution.
• Include humectants to prevent harmful drying at the container surface.
• Use antioxidants and UV-protective packaging where required.
• Ensure that any preservative does not inhibit the active organism.
• Design the formulation so that settled material can be readily redispersed by shaking.

Quality tests include viscosity, pH, sedimentation, redispersibility, viable count, contamination, and bioefficacy.

Preparation:

• Produce abundant, infective conidia of fungi such as Beauveria bassiana or Metarhizium anisopliae.
• Harvest dry conidia and remove substrate particles by sieving or air separation.
• Determine conidial purity, viability, and moisture content.
• Disperse the conidia in a compatible vegetable or mineral oil.
• Add suitable emulsifiers and dispersants if the product is intended to form an emulsion after dilution with water.
• Homogenize the mixture to prevent clumping and obtain a uniform conidial concentration.
• Pack in airtight, light-resistant containers and store at a recommended temperature.

Advantages:

• Oil improves adhesion of conidia to the hydrophobic insect cuticle.
• It can improve spreading over the insect body.
• Oil formulations may reduce rapid loss of conidial moisture.
• They can provide better performance under relatively dry environmental conditions than simple aqueous suspensions.
• Oil may offer partial protection from ultraviolet radiation.
• Some oil formulations are suitable for ultra-low-volume application.

The selected oil and emulsifier must not reduce conidial germination or infectivity.

Preparation of granules:

1. Select a granular carrier such as clay, corncob, bran, starch, alginate, or another biodegradable material.
2. Prepare a concentrated suspension of cells, spores, conidia, toxins, or viral occlusion bodies.
3. Apply the microbial suspension to the carrier by spraying, adsorption, coating, extrusion, or encapsulation.
4. Add binders to maintain granule integrity and protectants to improve stability.
5. Dry the granules at a mild temperature until a safe moisture level is reached.
6. Sieve to obtain a uniform granule size and remove dust or oversized material.
7. Standardize the microbial content and package in moisture-resistant containers.

Uses and advantages:

• Suitable for soil application against soil-dwelling pests.
• Can be placed in plant whorls, insect breeding sites, or localized feeding zones.
• Reduces drift compared with fine powders or sprays.
• Provides gradual release and can protect microbes from immediate environmental stress.
• Allows easy mechanical application and accurate placement.

Granules must have good flowability, mechanical strength, microbial viability, and predictable disintegration or release after application.

Submerged fermentation is commonly preferred for bacteria such as Bacillus thuringiensis, whereas solid-state fermentation is widely used to produce aerial conidia of entomopathogenic fungi.

The preparation of a Bacillus thuringiensis formulation involves the following stages:

1. Strain selection: Select a strain producing insecticidal crystal proteins effective against the target pest.
2. Seed culture preparation: Develop a pure and active inoculum from the master culture.
3. Submerged fermentation: Inoculate a sterilized nutrient medium and maintain suitable aeration, agitation, pH, and temperature.
4. Sporulation and crystal formation: Continue fermentation until a high proportion of cells have sporulated and released spores and parasporal crystals.
5. Harvesting: Concentrate the spore-crystal complex by centrifugation, filtration, or other separation methods.
6. Formulation: Mix the concentrate with carriers and additives. It may be prepared as a wettable powder, suspension concentrate, granule, or water-dispersible granule.
7. Protection and stabilization: Add wetting agents, dispersants, stickers, UV protectants, and stabilizers compatible with the bacterium and its toxins.
8. Standardization: Determine viable spore count and insecticidal potency through laboratory bioassays. Potency is more meaningful than cell count alone.
9. Quality testing: Examine contamination, moisture, pH, suspensibility, particle size, and storage stability.
10. Packaging: Pack in moisture- and light-resistant containers carrying information on target pests, dose, storage, and expiry.

The formulation must preserve both the viable spores and the biological activity of the insecticidal crystal proteins.

The major steps are:

1. Selection and purification: Select a virulent fungal isolate and confirm its purity and identity.
2. Starter culture: Grow the isolate on a suitable agar medium and prepare a uniform inoculum.
3. Mass production: Produce conidia by solid-state fermentation on sterilized grains or other substrates, or use a suitable liquid fermentation system.
4. Incubation: Maintain proper temperature, aeration, and moisture until abundant sporulation occurs.
5. Drying: Dry the colonized substrate under controlled conditions. High temperature and excessively rapid drying can reduce conidial viability.
6. Harvesting: Separate conidia from the substrate by sieving, brushing, or air classification.
7. Standardization: Measure conidial concentration, purity, moisture content, and germination percentage.
8. Formulation: Blend conidia with talc or another solid carrier for a wettable powder, or disperse them in a compatible oil.
9. Addition of adjuvants: Add wetting agents, dispersants, stickers, anti-caking agents, and UV protectants where needed.
10. Packaging and storage: Use sealed, moisture-proof, light-resistant containers and store under cool conditions.

A high-quality product should contain infective conidia that germinate rapidly after reaching the insect cuticle.

Baculoviruses such as nucleopolyhedroviruses are generally multiplied in vivo in susceptible insect larvae, although cell-culture production is possible for some viruses.

Preparation procedure:

1. Rear healthy host insects under hygienic conditions.
2. Prepare a purified inoculum containing infective viral occlusion bodies.
3. Infect larvae by feeding them contaminated diet or treated plant material.
4. Maintain infected larvae until characteristic viral disease develops.
5. Collect dead or moribund larvae before decomposition or contamination becomes excessive.
6. Homogenize the larvae in clean water or buffer.
7. Filter the homogenate to remove large insect tissues.
8. Purify and concentrate occlusion bodies through sedimentation, centrifugation, or differential centrifugation.
9. Determine occlusion-body concentration and confirm pathogenicity by bioassay.
10. Add stickers, feeding stimulants, UV protectants, and stabilizers to prepare an aqueous suspension, wettable powder, or encapsulated formulation.
11. Package in opaque, well-sealed containers and store at a cool temperature.

Strict quality control is essential to prevent contamination and to ensure that the occlusion bodies remain infective.

Ultraviolet protectants:

• Baculovirus occlusion bodies can be rapidly inactivated by sunlight, particularly ultraviolet radiation.
• UV protectants absorb, scatter, or block harmful radiation and thereby increase field persistence.
• Materials investigated for this purpose include lignin-based substances, optical brighteners, carbon-based pigments, and selected plant-derived protectants.
• The protectant must not interfere with larval feeding or viral infectivity.

Feeding stimulants:

• Baculoviruses usually infect insects after being ingested.
• Feeding stimulants encourage larvae to consume treated foliage and therefore increase the dose of occlusion bodies received.
• Sugars, molasses, plant extracts, and phagostimulant materials may be used, depending on the target insect.

Additional formulation support:

• Stickers retain occlusion bodies on leaf surfaces.
• Spreaders improve coverage.
• Rainfastness agents reduce wash-off.

Together, these additives increase the probability that a susceptible larva consumes an infective dose before the virus is degraded or removed from the crop surface.

Microencapsulation is the enclosure of microbial propagules, toxins, or viral occlusion bodies within a thin protective matrix or coating.

General preparation:

1. Prepare a concentrated suspension of the active microbial ingredient.
2. Mix it with a compatible wall material or polymer such as alginate, starch, gelatin, gum, or another biodegradable material.
3. Produce capsules by methods such as extrusion, spray drying, coacervation, or emulsion-based gel formation.
4. Harden or stabilize the capsule wall under mild conditions.
5. Separate, dry, and grade the capsules if a solid product is required.
6. Test release behavior, viability, and bioefficacy.

Advantages:

• Protects the active agent from UV radiation, oxidation, moisture fluctuations, and heat.
• Reduces direct contact with incompatible formulation ingredients.
• Allows controlled or delayed release at the target site.
• Can reduce dust and improve handling.
• May increase persistence on foliage or in soil.

Limitations:

• Production can be expensive and technically demanding.
• Harsh drying or chemical cross-linking may reduce viability.
• Capsule size and release rate must match the intended application method.

A microbial bait formulation combines a microbial active ingredient with an attractive food material that is readily consumed by the target insect.

Preparation:

• Select a microbial agent that is effective through ingestion, such as Bacillus thuringiensis or an appropriate insect virus.
• Select a bait base preferred by the target pest, such as bran, cereal material, sugar, molasses, or another feeding attractant.
• Mix the microbial concentrate uniformly with the bait material.
• Add binders, humectants, preservatives, and protectants that are compatible with the microorganism.
• Prepare the product as a moist bait, dry bait, pellet, or granule.
• Standardize the amount of active ingredient per unit weight and test feeding acceptance.

Uses and benefits:

• Delivers the pathogen directly through feeding.
• Reduces the quantity of active ingredient required for broadcast treatment.
• Allows localized application in pest feeding or sheltering sites.
• Can protect the microorganism from immediate sunlight exposure.

Limitations: Bait performance may be reduced by drying, rainfall, competition from natural food, poor attractiveness, or contamination. The formulation must remain palatable while preserving microbial activity.

Moisture content strongly influences both microbial survival and the physical stability of a solid formulation.

• Excess moisture can promote unwanted microbial growth, chemical degradation, caking, and premature germination.
• Very severe drying may damage cell membranes, spores, conidia, or viral components.
• The optimum moisture level varies with the organism and carrier.

Drying methods:

• Air drying, vacuum drying, fluidized-bed drying, freeze drying, and spray drying may be used.
• Freeze drying is relatively protective but expensive.
• Spray drying is rapid and scalable, but inlet and outlet temperatures must be carefully controlled.
• Mild air or vacuum drying is often used for fungal conidia and carrier-based products.

Water activity:

• Water activity represents the amount of water available for biological and chemical reactions rather than total water alone.
• Lower water activity generally reduces contaminant growth and metabolic deterioration.
• However, an excessively low value or rapid dehydration can reduce viability in sensitive propagules.

Therefore, drying conditions must be optimized to achieve storage stability without sacrificing germination, infectivity, or potency.

Quality control includes biological, microbiological, physical, chemical, and safety-related tests.

Biological tests:

• Viable cell, spore, or conidial count.
• Germination percentage of fungal conidia.
• Concentration of viral occlusion bodies.
• Pathogenicity or potency through bioassay against a susceptible target insect.

Microbiological tests:

• Purity of the production strain.
• Absence or acceptable limits of contaminating microorganisms.
• Confirmation of strain identity where required.

Physical tests:

• Moisture content and particle size.
• Wettability, suspensibility, dispersibility, and flowability.
• Sedimentation and redispersibility of liquid formulations.
• Granule strength, dustiness, viscosity, and emulsion stability, as applicable.

Chemical tests:

• pH, compatibility of additives, and stability of active metabolites or toxins.

Storage and packaging tests:

• Shelf-life assessment under recommended and accelerated conditions.
• Container integrity and resistance to light and moisture.

A formulation should not be approved merely on microbial count; it must also demonstrate acceptable virulence, physical performance, purity, and stability.

Shelf life is determined by interactions among the microorganism, formulation ingredients, packaging, and storage environment.

Important factors include:

• Type of propagule: Endospores and dry conidia are generally more stable than fragile vegetative cells or blastospores.
• Temperature: High temperatures accelerate loss of viability and degradation of toxins.
• Moisture and water activity: Excess moisture encourages contamination and metabolic deterioration.
• Oxygen: Oxidation may damage microbial membranes and active compounds.
• Light: UV radiation can inactivate fungal conidia, bacteria, and viruses.
• pH: Extreme pH may reduce viability or destabilize the active ingredient.
• Carrier compatibility: Toxic impurities or unsuitable oils and surfactants can reduce survival.
• Contamination: Unwanted organisms may compete with or degrade the active agent.
• Packaging: Moisture-proof, airtight, and opaque containers often improve stability.
• Initial quality: Products with high purity, adequate viable count, and low physiological stress generally store better.

Shelf life is established by periodically measuring viability, potency, contamination, and physical properties under specified storage conditions.

Although all three require standardized potency and careful storage, their formulation strategies differ because their active biological units respond differently to drying, water, heat, light, and application conditions.

Formulation connects laboratory pathogenicity with practical field performance.

Influence on efficacy:

• Dose delivery: A uniform formulation ensures that the target pest receives an effective concentration of propagules or toxins.
• Coverage and spreading: Surfactants improve distribution over foliage and insect surfaces.
• Adhesion: Stickers prevent rapid loss due to wind, irrigation, or rainfall.
• Persistence: UV protectants, oils, and encapsulating materials reduce environmental inactivation.
• Infection efficiency: Oil formulations may improve contact between fungal conidia and the insect cuticle.
• Ingestion: Feeding stimulants and baits increase consumption of bacterial toxins or viral occlusion bodies.
• Release pattern: Granules and capsules can release the agent gradually or at a specific target site.
• Application compatibility: Appropriate viscosity, particle size, and suspensibility prevent nozzle blockage and uneven spraying.
• Storage stability: A stable product retains adequate potency until field use.

Thus, even a highly virulent microbial strain may perform poorly if its formulation does not protect it, deliver it uniformly, or place it where the target insect will contact or ingest it.