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4.1 Product Harvesting

Harvesting microorganisms from submerged fermentation is often difficult due to the low concentration of the products, their thermolabile nature and in some cases their poor stability. Stabilizing adjuvants may have to be incorporated immediately post-harvest to prevent spore death and/or germination. Rapid drying or the addition of specific biocidal chemicals may be required to prevent growth of microbial contamination in the broth or centrifuge slurry (Soper and Ward, 1981).

Spore-forming Bacillus thuringiensis are usually concentrated prior to drying by centrifugation or filtration. Centrifugation using a continuous centrifuge concentrates the product from 2-3 % suspended solids to 15-20 %. Centrifugation may result in some loss of suspended solid as well as loss of dissolved materials. Such losses may not be acceptable and concentration using this technique can often be omitted. Following concentration, one of the technique mixes the crystal/spores slurry with lactose, adjuvants such as wetting agents, spreader-stickers or dispersing agents, and the whole product is spray-dried at 175^oC (Dulmage, 1981). The dry product is blended and/or mixed with additional formulation adjuvants before packaging and/or use. The lactose added may act as a cryoprotectant or it may help to prevent clumping Dulmage and Rhodes, (1971). Dulmage at al. (1970) (see chapter 2) developed an alternative drying technique for laboratory preparations where spray drying facilities are not available; this technique of recovery of B. thuringiensis is based on the lactose-acetone processing (see 2.1.1). Many patents exist such as a foam flotation process for separating B.t. sporulation products.

Fungal blastospores obtained in submerged culture are much less stable than conidia and are consequently difficult to process after harvesting. Laboratory cultures are frequently freeze-dried with or without protectants, but even dried product may have short viability. Verticillium lecanii freeze-dried blastospores, for example, have a half-life at 5, 20 and 30^oC of 11, 4 and 2 days, respectively. Blachere et al.(1973) harvested Beauveria brongniartii by centrifugation before mixing with silica powder, osmotically active materials (such as sucrose and sodium glutamate), anti-oxidizing agents (sodium ascorbate) and a mixture of liquid paraffin-polyoxyethylene glycerin oleate. The resultant paste was then dried at 4^oC in ventilated drying closet. Blastospores dried in this fashion were viable for 8 months at 4^oC (Blachere et al., 1973).

Belova (1978) dried Beauveria bassiana product in five different ways: vacuum, freeze, spray-drying, drying by mixing with an inert filler, and in a fluidized bed with an inlet temperature of 40^oC and an outlet of 30^oC. The virulence of the fluidized bed-dried material is enhanced and the process accelerated by precipitation using a calcium carbonate, surfactant, silica gel mixture. In addition, a sulphite liquor is mixed in. Drying in a vacuum desiccator produced samples of high viability and virulence, some remaining active for 1 year at 4^oC storage. Spray-dried B. bassiana spores together with the culture media led to a complete loss of viability. However, using a 2 % molasses mixture as a protective medium permitted retention of viability and efficacy, although to a lesser extent than for the freeze-dried product.

Globa (1980) recovered Beauveria bassiana from fermenter broth by precipitation with calcium carbonate. Fargues et al. (1979) spray-dried B. brongniartii conidiospores coated with bentonite clay, yielding 50-70 % viable spores. However, blastospores were too sensitive for this technique and these were lyophilized with powdered milk and glycerin. The spray-dried conidia showed no loss of viability after 18 months storage at 5^oC: lyophilized blastospores were still viable after 8 months.

4.2 Formulation

Angus and Luthy (1971), Couch and Ignoffo (1981) mentioned that the development of microbial insecticide formulation closely paralleled that of chemical insecticides. Pesticide formulation is the process of transforming a pesticide chemical into a product which can be applied by practical methods to permit its effective, safe and economic use. Important specific differences, of course, do exist because microbial insecticides do not directly depend on the effect of a poisonous chemical but exploit the activity of living (or self-replicating) entities. An exception is the enterotoxinosis caused by Bacillus thuringiensis where a pre-formed toxic glycoprotein is essential for infection to occur.

The aim of the formulator then is to avoid practices that might inhibit or harm the pathogen and wherever possible to enhance the possibility of infection. Thus, not only must one avoid agents in any way antimicrobial but also, with B. thuringiensis, any compounds capable of denaturing the glycoprotein comprising the toxic crystal. Any attempts to utilize a particular species of micro-organism in an insecticide formulation should be based on an intimate knowledge of the host-parasite relationships. Generally, however it is the multi-plication of a microorganism in the host tissues that leads to disease and death.

A microbial pesticide formulation is a physical mixture of living entities with inert ingredients which provides effective and economic control of pests.

Formulation of a pathogen product with an extensive shelf-life (>18 month) is critical to industrialization.

In commercial development of a basic formulation of an entomopathogen, technology concerns maintaining pathogen viability and virulence during the production process and developing a product form which preserves or enhances these properties. To do this, knowledge of the biology of pathogen and target insect is essential. Effect of temperature, humidity and media (inert carrier) on the entomopathogen can turn out to be the most important.

Spreaders or wetting agents are added to the water diluent to ensure "wetting" of the surface to be sprayed. Many materials have been used including dried milk, powdered casein, gelatin, saponins, soaps etc. In so far as microbial insecticides are concerned, it is essential that the compound used should encourage premature growth or germination and that it should not inhibit successful establishment of the pathogen. Some factors are likely to be rather subtle, e.g. although detergents such as sodium dodecyl sulphate do not inactive the crystal toxin of B.t, they open up the structure of the crystal and make it more sensitive to destruction by other means.

Table Additives that have been added to preparations of microbial insecticides.

Talc B.t., Tung oil B.t.
Celite B.t., Molasses B.t.
Starch B.t., Powdered skin milk B.t., V
Synthetic silicates B.t., F Methocel B.t., V
Bentonite B.t., Corn syrup B.t., V
Lactose B.t., V Latex D B.t.
Microcell B.t., Casein B.t.
Attagel B.t., Neosil A B.t., F
Pyrax B.t., Folicote B.t.

Alkyl fenols B.t., Tween 80 B.t., V, F
Sandovit B.t., 9 D 207 B.t., V
Novémol B.t., Pinolene 1882 B.t.
Petro AG B.t., Span 80 B.t., V
Colloidal X77 B.t., V Triton N60 B.t., F
Triton X100 V Triton GR7M B.t., F
Triton 155 B.t., Atlox 848 B.t.
Tween 20 F Atlox 849 B.t.
Tween 80 F Atlox 3404/849 F
Triton X45 V Atplus 448 F
Triton X114 B.t., Atplus 300 F, V

Water Citrus pulp
Preformed oil in water emulsion Corn cob
Preformed water in oil emulsion Corn meal
Edible oil, Wheat bran
Corn oil, Grape pomace
Crude sorbitol Apple pomace
Aromatic spray oils, Rice hulls
Emulsified cottonseed oil, Cracked corn

Bentone 38
CAB-O-SIL
SOLOID

4.3 Oil Suspension Formulation

Uniform suspension is prepared by preliminary wetting of the dry microbial insecticide with an emulsifer water mixture before final dispersion in oil carrier. Edible oil is suitable for microbial pesticide formulation. The storage of fungal spores under edible oil preserve its viability for sufficient time. Advantage of edible oil is its nonphytotoxicity and its residua might be acceptable.

4.4 Dusts or Wettable Powder

In the case of one B.t., Formulation, the pathogen is cultured on a semi-solid medium so that it is preferable to process it as a dust or wettable powder rather than attempt to separate the spores and crystals from the medium solids. When grinding and mixing material containing the pathogens to obtain a sufficiently fine powder, care should be taken to avoid increase in temperature or physical damage that would harm the pathogen.

The choice of the filler for use in a dust formulation is subject to the general provision that it is nontoxic to the pathogen being used.

The decision as to the most desirable form (spray or dust) varies with the crop being protected, the target insect, climatic conditions and, of course, the particular pathogen being used.

4.5 Suspension Concentrates (sc)

Are also known as flowable emulsions, colloidal suspensions or dispersions. Biological agents are not soluble and dispersing medium is edible oil, which is not aggressive to biological agents.

A flowable must have a satisfactory viscosity for handling purposes; it should disperse spontaneously or with slight stirring when poured into water before application.

Simplest method to produce flowables is by adding thickeners and thixotropic agents to biological agent in powder form. The use of thixotropic agents enables the production of flowables which at the application point do not exceed 2,000 MPa`s. Commercially available flowables have viscosities ranging from 200 to 2,000 MPa`s. This results then in an excellent self dispersibility. Flowables should disperse spontaneously when poured into water, but roping my be tolerated if the flowable disperses rapidly upon agitation.

The coarse and wide range of particle sizes produced by this technique may cause sedimentation and ultimately claying or cauling, due to the absence of interfacial forces preventing close packing.

In commercial flowable production, "wet milling" techniques are preferred as they provide the most economical and direct means of producing the desired average particle size of 1-5 microns in diameter. Possible mills include attritors, sand mills, and ball (roller) mills for batch or continuous process. The use of an attritor or sand-mill requires the preparation of a premixture, which usually consists of all the ingredients (such as siloxyl) at their required concentrations.

Surfactants play an important role as basic component in flowable formulations. They are responsible for wetting of the pesticide particles before and during the milling process. They help to liquefy the unmilled premixture in the milling chamber. They help to stabilize the micronised particles in the dispersing medium. The role of the surfactant in every step of the production process can be described as follows:

When a dry pesticide is mixed with water during flowable production process, the surfaces of individual particles must be wet and the air between the particles must be displaced to make efficient wet milling possible. As little air entrainment as possible is desired by the formulator, because the air remaining on the surfaces after milling, causes flocculating when bubbles of the neighbouring particles coalesce. This coalescence reduces the dispersion stability.

The most widely used wetting agents are ethoxylated alcohols and ethoxylated nonylphenols, which should have very low foaming properties. Foam formulation should always be avoided during the production process and also during spraying on the field. Foaming can reduce the uniformity and the effectiveness of the pesticide in the field.

The concentration on wetting agent varies usually from 0.5 % to 3 % depending on the concentration, the morphology and the surface properties of the active ingredient.

During the milling process, surfactants help to rewet and disperse the newly formed particles. Bad rewetting will result in paste formulation and the whole milling chamber will be blocked. During the milling process, the temperature can easily rise up to 60^oC. As known, desorption of the surfactant from the particles take place at the cloud point. Therefore it is necessary to use wetting and dispersing agents with a sufficiently high cloud point. Usually, nonionic surfactants are selected with a cloud point at least 10^oC above the milling temperature and the maximum required storage temperature.

Because of the high loading and the small particle size of the active ingredient, the dispersed particles have an inherent tendency to irreversibility flocculate due to the London-Van der Waals forces of attraction. Adsorption of a dispersing agent on the particles will generate repulsive forces between the particles or eliminate the attractive forces between the particles.

Electrostatic repulsion occurs when ionic surfactants are adsorbed onto particles. The charge imparted by the surfactant causes particles to repel each other. Electrostatic repulsion is most important in aqueous flowables.

Steric hindrance results from the adsorption on nonionic surfuctants having long chains which are soluble in the dispersing medium. When two particles approach, the solvated chains interact to prevent irreversible agglomeration. This type of repulsion is important in both aqueous and oil-based flowables.

PREMIXTURE (wet grinding mills) Ideally, the premixture should consist of all the ingredients at the required concentration and yet be pumpable. The ingredients are:

1) dispersing medium (water, edible oil)
2) active biological agent (conidia, blastospores, spores and crystals)
3) wetting agent (Atlox 4862)
4) thickening or viscosity modifying agents (Atlox 1086 or Bentone 38 [0.1-1.0 % w/v] or Rhodopol 23 [0.05-0.3 w/v] or other type of Atlox (1096, 4868 B)
5) stabilizer (if necessary)

The active biological agents

B.t. - spores and crystals
Fungi - Metarhizium anisopliae, conidia strong hydrophobic
         - Beauveria bassiana, conidia medium hydrophobic
         - Verticillium lecanii, conidia or blastospores hydrofility

These particles are under 20 microns but all of them aggregate and must be milled. Polyethylene glycol preserves aggregation and can be used during wet milling.

Renex 702 / Atlox 1045 A Atlox 4884 / Atlox 1045 A
Atlox 4856 B / Atlox 4885 Atlox 4856 B / Atlox 3386 B
Atplus 300 F

The good emulsification of the oil when added to the water.

The required stability of the emulsion in the spray tank.

Furthermore the combination ATPLUS 300F/oil improves the efficiency and the selectivity of the applied pesticide. As a thickener "BENTONE 38" (NL Industries USA) can be used. This thickener is activated by a polar solvent to ensure a good thickening effect. The polar solvent is generally added whilst preparing the master solution.

250 - 100 g powder mix of spores and crystal B.t. (or conidia)
30 g Atplus 300F
68 g Bentone 38
to 1 litre of water or oil

4.6 Suggested Evaluation Techique of Flowables

The mechanical stability is tested by shaking the flowable on shaker for half an hour. A good flowable will not thicken or gel under the influence of shaking.

Suspensibility, or dilution stability is the degree to which a flowable stays suspended when diluted in water. It is expressed as the volume percent of setting in a dilution mixture at various time intervals. The degree of flocculating is noted by the number of inversions of the test cylinder required to redisperse the sediment.

Samples are stored in sealed bottles at -10^oC, room temperature, 40^oC for several months. Afterwards, they are periodically inspected for:

a) bleeding; the amount of liquid separation on top of the flowable is expressed as a percent of the total sample depth. The bleeding should be minimal.
b) thickening; the absence of thickening is checked by probing with a glass rod or by measuring the viscosity
c) sedimentation; the absence of packing is checked by inserting into the flowable a glass rod to check the bottom of the container for any sedimentation. Packing should be absent and any non-uniformity of the suspension should be easily correctable with mild agitation.

The viscosity is measured at one day, one week and one month intervals. A minimum variation in viscosity is tolerated. The main criterion is to maintain pourability.

While conducting the suspensibility test, the bloom is observed and rated qualitatively on a three point scale: good, fair and poor I.O.U.. The scale is ranging from total spontaneity to no spontaneous dispersion.

The biological activity is measured at one day, one week and every month intervals during storage under evaluated temperature to way: Test of germination of spores, LD50 on target pest in lab.

4.7 Evaluation of Separation Process "Recovery"

Each process for the isolation and purification of bioproduct from microorganisms consists of a sequence of individual process steps. The choice of the individual process steps is governed by the properties of the by-product to be isolated.

The yield of the step: The yield of the step, given in amounts (e.g., g or kg), or in units (or mega units = millions of units, documents the scale of the process step and the batch size and permits conclusions concerning the equipment used.

               units (or amount) after purification
100 . --------------------------------------------- =% of efficiency
               units (or amount) before purification

The common evaluation of enrichment factor and percentage yield of the step permits a statement concerning the efficiency of the technology used in the process step. The product of purification factor times percentage yield of the step is termed the efficiency.

Economic aspects must also be included, especially the manufacturing costs of the bioproduct. For calculating these, all types of costs, such as:

- material costs - costs for waste treatment,
- personnel costs - overheads
- energy costs - costs of repair
- depreciations must be added and the result divided by the yield of the step.

      total costs                           total costs
.------------------  or  -------------------------------
    kg of product             mega units of bioproduct

and they integrate the economic and technical parameters of the process step.

The evaluation of the total process results from the evaluation of the individual process steps. Thus, the percentage total yield may be obtained by multiplying all the percentage step yield, and the manufacturing costs of the end product by adding all the types of costs of all process steps and dividing by the overall yield.

The technical and economic classification of a process into process steps does not only permit an optimization of the process but also an immediate adaptation to necessary changes in the process even when they are independent of the process itself.

ANGUS, T.A., and LUTHY, P. 1973: Formulation of microbial insecticides. In: Burges H.D. and N.W. Hussey: Microbial control of insects and mites. Acad. Press, London and New York., pp.623-636.

BELOVA, R.N., 1978: Proc. 1st Joint US/USSR Conf. Prod. Selec Stand. Entomopath. Fungi, Jurmula (Riga) Latvia SSR, 20-21 May, pp. 102-119.

BLACHERE, H., CLAVEZ, J., FERRON, P., CORRIE, G. and PERINGER, P. 1973: Ann. Zool. Ecol. Anim. 5, 69-79.

DULMAGE, H.T. and Cooperators, 1981: In: Microbial control of pests and plant diseases. Ed. H.D. Burges,pp. 191-220. Acad. Press, London and New York.

DULMAGE, H.T. and RHODES, R.A. 1971: In:Microbial control of insects and mites. Ed. H.D. Burges and N.W. Hussey, pp. 507-540. Acad. Press, London and New York.

FARQUES J., ROBERT, P.H., and REISINGER, O. 1979: Ann. Zool.

Ecol. Anim. 11 (2) 247-257.Ferron P.,1978: Annu. Rev. Entomol. 23: 409-442.

GLOBA, L., 1980: U.S.S.R. Patent SU 73 85 71.

SOPER, R.S. and WARD, M.G. 1981: Beltsville Symposia in Agricultural Research. Vol.5: Biological Control in Crop Production, pp. 161-180.

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