HU201657B - Process for producing insecticide compound with cultivating bacillus thuringiensis var. kurstaki - Google Patents

Abstract

A selectively lethal eutomo-pathogenic insecticide is prepd. from a toxic metabolites contg. fermentation brew resulting from culturing suitably selected Bacillus thuringiensis var. kurstaki.

Description

FIELD OF THE INVENTION The present invention relates to the production of a bacterial endotoxin-containing bacterial agent selectively acting on plant pests, in particular dwarf worms. by fermentation of kurstaki.

The active ingredient was selected from Bacillus thuringiensis var. kurstaki strain by fermentation. The product of this process is an active ingredient of a biological pesticide which is suitable for the control of dwarf pests in various plant cultures. The product obtained can be used directly in the form of juice, concentrate or powdered preparation, or it can be mixed with the usual excipients according to the needs of crop protection and application technology.

Efforts are being made to develop and implement economical, complex plant protection technologies that protect beneficial members of agrobiocenosis. These are processes characterized by the following criteria (B. Darvas, I. Seprős, J. Szántó: Biological control. MÉM NAK., Bp. 1979, 53. PP ·):

- use combined defense operations (agro-technical, physical, chemical, biological),

- reducing the quantity and quality of pesticide loads (pest management system),

- natural elements of flora and fauna are also used to control pests (integrated pest management technologies).

There are two main objections to commonly used chemicals:

- non-selective, killing beneficial members of biocenosis,

- over time, resistant types are selected.

The insecticidal activity of Bacillus thuringiensis was discovered by Ishiwata (1901). [Ishiwata, S.: 1901. On kind of severe flacherie (sotto disease), (I.) Dainikon Sanshi Keiho, 9: 1-5.]. The first detailed description of Berliner E. (1911) on a culture of B. thuringiensis by Ephestia kühniella Zcll. isolated from patient larvae. (Berliner E .: 1911. Über die Schlaffsucht der Mehlmattenrauge. Z. Gesante Getreidewes. 252: 3160-62.).

The first entomotoxic laboratory preparation was first practiced by Mctalnyikov (1930) (cit. Misusztinin E. N, "Emcev V. T .: 1978. Microbiology. Colossus. Moscow. 313-314).

No initial success in biological control was predicted by meager results (Paillot 1933, cit. Yamvrias C .: 1962. The effect of Bacillus thuringiensis Berliner on mealworm caterpillars. Entomophaga, Vol. VII, No. 2, n. 2). 101-159.). In the United States, Dulry (1940) attempted to use entomopathogens with moderate success against plant pests (Cit. Yamvrias 1962). Significant development work has been carried out in this field at the Pasteur Institute in Europe (Lemoigne and Bonnefoi, 1957: Yamvrias 1962).

By 1961, preparations of B. thuringiensis were marketed in many countries. The initial application was accompanied by many failures. For example, the "Sporein" formulation was not effective against Anagasta larvae at a concentration of 1.7 wt% (Kricg 1961, cit. Yamvrias,

1962).

The pathogen activity spectrum of B. thuringiensis was examined by HempclAngus (1956, 1959, cit. Yamvrias, 1962).

The QV of Yamvrias, quoted in the Biological Values, is discussed in detail. Burges et al. (1981) summarized the results of some 10 years of basic research and development in a very detailed study. [Burges, H. D. (ed.), Microbiol Control of Pests and Plant Diseases 1970-1980. Acad. Press. Inc. (London), Ltd. London, 1981.]. The technical knowledge regarding the production and application of entomocidal metabolites can be summarized as follows: Darvas et al., 1979, Burges 1981, Misusztin-Emcev 1986, Whiteley-Schnapf: 1986. Ann. Port. Microbiol. 1986. 40: 459-75., V. V. Smimov et al., 1986. Aerobic spore-forming bacteria. Medicine. Pp. 205 p. based on his work.

Bacilli that produce crystalline entomocidal metabolites (toxin crystals) are quite common in nature. The toxin-producing strains can be isolated from dead forms of insects of very different taxonomic groups, but can also be found in soil (Russel, S. et al., 1987. Sclectivcproccs for Efficiency of Bacillus sp. Appl. And Envir. Microbiol. Jan 1263-1266).

B. thuringiensis has 17 subspccicsc variances. Most of these correspond to the serotype defined by the H antigen. The most important are:

HD Serotype (Howard-Dulmage Collection)

B.thuringiensis var. thuringiensis HD-120,290

B.thuringiensis var. finitimus a B.thuringiensis var. alesti a, b B.thuringiensis var. kurstaki (Kurstak, 1962) a, b B.thuringiensis var. kurstaki HD-1 a, b B.thuringiensis var. sotto a, b B.thuringiensis var. dendrolimus a, c B.thuringiensis var. kenyae a, b B.thuringiensis var. gallery HD-8 a, c B.thuringiensis var. canadicnsis a B.thuringiensis var. subtoxieus a, b B.thuringiensis var. entomocidus HD-9

B.thuringiensis var. aizawai

B.thuringiensis var. morrisoni

B.thuringiensis var. tolworth HD-13

B.thuringiensis var. darmstadienis HD-498 a, b B.thuringiensis var. toumanoffi

B.thuringiensis var. tompsoni HD-542 14 B.thuringiensis var. israclcnsis HD-567 21 B.thuringiensis var. colmeri S

B.thuringiensis var. wuhancnsis

Certain serotypes may also differ in their enzymatic activity spectrum, which may have industrial significance in fermentation (eg starch degradation, sucrose fermentation).

Crystalline inclusions are proteinaceous. The crystals of B. thuringiensis protein have 18 amino acids, mainly glutamic and aspartic, irrespective of the type. Because of the latter, the crystalline proteins have a low isocytric point. Based on the cystine content, disulfide bonds are likely to be present. The protein crystals are soluble in 0.1 N NaOH, 8 M guanidine chloride, 8 M urea, alkaline thioglycol.

Upon crystallization of protein crystals, fragments of high molecular weight (> 800,000 Daltons) and low molecular weight (> 10,000 Daltons) are formed, and high molecular weight fragments are toxic. The so-called dclta-endotoxin is heat-sensitive, harmless to the higher ones. B. thuringiensis var. kurstaki crystals are only toxic to scaly swans.

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AB. thuringiensis var. israelelsis toxic parasporal inclusions in turn at 10 ^'4 gg / mL of the larvae of Aedes aegypti (yellow fever), Culex pipiens subsp. quinaquefasiatus (encephalitis) as well as larvae of AnophcIes albimanus (malaria). It was surprisingly effective against resistant strains of Simulium damnosum chlorphoxime ("blackfly"), which are vectors of onchoccrciasis (, / iver blindness). (Lüthy P., Studer, D .: 1986. Control of Simulid Blackflies and Mosquitoes with B. thuringiensis subsp. Israelensis. MIRCEN Journal, 2, 91-99.).

A broader overview of the impact spectrum is as follows:

I. Insects Sensitivity

1. Cockroaches

2. They are rectangular

3. Bladders

4. Chewing Teeth

5. Bugs

6. Equinoxes

7. Beetles

8. Butterflies

Cobwebs ++++

Plum moth ++++

Cabbage moth ++++

Peach Mole +

Apple Moth +++

Almailoncra ++++

Oak bud twig ++++

Corn moth ++++

Great winter mower ++++

Cabbage butterfly +

Woolly butterfly ++++

American white mcdvelcpke +++

Large cabbagecpkc ++++

9. Two-winged +

10. Mucous membranes

Lcvlaraz ++ +++

Saddle-saws Metal-saws

Π, they are spider-like

The composition clearly demonstrates the biocenosis-sparing effect of the drug product. Other entomocidal metabolites may be formed in cultures of B. thuringiensis; alpha-exotoxin, phospholipase, thermostable, soluble beta-exotoxin. The latter is an AMP-type compound, which inhibits RNA polymerase, and is mainly produced by serotype 1 (berliner) strains, which are effective against the house fly. Broader spectrum of action than endotoxin. LD ₅₀ (cger) can be harmful intraperiloneically at 18 gg / kg body weight, subcutaneously at 16.6 gg / kg body weight, and warmth blood. The larvicidal effect:

The alkaloids of the lymph nodes dissolve the absorbed toxin crystal and the spore germinates. The toxin paralyzes peristalsis and the larvae stop feeding food. The bclbolyhs degenerate, decay. Phospholipase dissolves the chitin membrane, bacterial cells invade the body fluid, causing sepsis. Moral development takes 2-8 days. In the meantime, there will be no significant damage. B. thuringiensis formulations are effective against intensively feeding forms (larvae). Viable spores in the formulation support the larvicidal effect due to the above

Particularly advantageous properties;

- does not cause acute toxicity in mammals,

- no cumulative effect is observed,

- no mutagenic or teratogenic effects,

- harmless to birds and fish at concentrations in insects,

- sparing pollinating insects,

- preserves beneficial ecto- and endoparasites.

It is known that B. thuringiensis strains show significant variability in laboratory cultivation: S-R dissociation, changes in carbon utilization ability, appearance of pigment formation, appearance of asporogenic variants, change in proliferation dynamics, cell wall composition.

From the industrial point of view, the fact that subcultures may lose their endotoxin crystal and spore-forming ability during laboratory cultivation is very significant (Smimov, 1986). Loss of virulence and pathogenicity (Müllcr-Kogler E .: 1965. Pilzkranheiten bei Inscktcn. Paul Parey, Berlin) can thwart the practical introduction of the defense agent into industrial production. In many cases, they do not experience a complete loss of pathogenicity with laboratory maintenance, but a very significant reduction. However, this can also frustrate economical industrial production (Müller-Koglcr, 1965).

Various methods for biomass production with B. thuringiensis cultures are known. U.S. Patent 3,076,922 (Mcchals, 1963) discloses SSF (Semis Solid Fermentation). In the process, the carrier is a mixture of wheat bran pcrlit, flowing with steam for 60 minutes before stcrileznck inoculation. The salt base contains NaCl and CaCl ₂ . Soybean meal is used as a source of nitrogen and glucose is used as a source of carbon. A particular disadvantage of the process is the cumbersome and costly process. Humidified wheat bran tends to converge even with perlite, drastically reducing the aeration efficiency. The flow of air dries the SSF bed, while the water content control in combination with the humidified and dry air is complicated, costly and inefficient. Dulmage and Rhodes (1971) attempted to make the above procedure more economical by introducing "clean" but not sterile air into the SSF bed. However, the air in the clumps of wheat bran formed channels, the efficiency of aeration was reduced, and the culture was often infected. (Dulmage, Η. T. and Rhodes, RA: 1971. In: Microbial Control of Insects and Mitcs, cd. Burges and Hussey. Acad. Press London, 507-540). Bulla and Youstcn (1979) report data on an entomocidal product ("Biotrol XK") allegedly produced by SSF. The producing strain is B. thuringiensis var. kurstaki HD-1. Activity: 7500 IU / mg.

however, it is not apparent from the publication that the activity relates to the fermented material or to a diluted preparation (Bulla, L.A. and Yousten, A.A., 1979). In: "Microbial Biomass" (A.A. Rose, ed.) Acad. Press New

York).

U.S. Patent 3,073,749 to Megna J.C., 1963 discloses the production of B. thuringiensis biomass by submerged fermentation. It uses beet molasses as a source of carbon in inoculum media as well as in the production medium, with dried corn jam and cotton seed flour as a nitrogen source.

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Although batch submerge fermentation is significantly more advantageous than the SSF process, the usefulness of a single charge of about 50 g / l of nutrient is questionable. The simultaneous presence of sucrose starch carbon sources can lead to quite a significant amount of diauxia. Culture synchronization is hardly possible.

U.S. Patent 3,087,865 to Drake and Smythe,

1963) describes a submerged culture method for B. thuringiensis which uses 6-12% by weight of the total nutrient input. Given the growth and enzymatic activity of B. thuringiensis, it is difficult to imagine the expedient use of such carbon and nitrogen sources in a batch system.

In the USSR, VASHNIL has developed methods for the preparation of several entomocidal products with different products and slightly different media (Quinlan R.J., "Lisansky S.G., 1983, Microbial Insecticides. Biotechnology, Vol. 3, Verlag Chemie, Basel. 233-254).

The aforesaid processes are not the object of the present invention.

It is an object of the present invention to provide a stably entomotoxic bacillus metabolite by fermentation suitable for killing dwarf pests in various plant cultures.

The present invention is based on the discovery that the entomotoxic metabolite (crystal delta-endotoxin) can be quantitated by selective staining under a light microscope and in known volumes.

The present invention is further based on the discovery that morphometric analysis can be used to directly select for dclta-endotoxin-producing strains of B. thuringiensis, to monitor the usefulness of the fermentation precursor and the main inoculants, and the main fermentation toxin and spore production.

The object and method of the present invention are as set forth in Table I. Μ. N.

G., NCAIM B 001022, deposited on November 11, 1987, with B. thuringiensis var. kurstaki K-CS 0019 ft. s. strain by submerged fermentation.

Strain Description: Identification is provided by The Prokaryotes, ed. Μ. P. Starr et al., Springer Verlag, Berlin, Heidelbcrg, New York, 1981, and Bergey's Manual of Derminological Bacteriology, The Williams and Wilcins Co., Baltimore. 1974. VIII. Ed.

Colony morphology: 5-7 mm diameter, barely prominent, white, smooth colonies on TGE agar in 72-hr pctric culture. HP soup is white, cloudy, fluffy. Wrinkled pcllicula on surface. Does not form pigment. Gram: +

Microscopic morphology: rounded rods, 1.0-1.5 x 2-8 µm. Spore: central, ellipsoidal. Parasporal body: +. Flagella: +. Capsula: Biological properties:

acid Training

D-glucose: +

L-arabinose: D-mannitol: Gas formation

From D-glucose: Indole: Acetoin: Catalase: +

Growth pH: 5.7 + pH: 6.8 +

Growth

NaCl 2% w / w: +

NaCl 5% by weight: +

NaCl 7% by weight: Growth

5'C: 10 'C: +' C: + 'C: +' C: Na acetate breakdown: +

Na-formate breakdown: +

Na-oxalate breakdown: Citrate breakdown: +

decarboxylase

Omitin: Lysine: Arginine: Esculin Breakdown: Nitrate Reduction: +

V. P. trial: +

Glucose breakdown: +

Starch breakdown: +

Casein resolution: +

Zsclatin resolution: +

Sucrose resolution: +

Salicin breakdown: +

Mannose resolution: +

Hemolysis: alpha, greening

Arginine dihydrolase: +

Urea resolution: +

It produces an entomotoxic dipyramidal toxin crystal.

Based on the above, the strain B. thuringiensis var. kurstaki.

B. thuringiensis var. kurstaki K-Cs 0019 The strain can be maintained by any of the methods known in the art, for example, by cryophilization, cultured on solid medium and inoculated every 30 days, sporulated on sterile soil, or other suitable means. Lyophilization was found to maintain the most preferred strains.

According to the present invention, the selection of strains producing an entomotoxic dipyramidal toxin crystal is as follows. B. thuringiensis var. kurstaki K-Cs 0019 shear-submerged cultures of sporulated inocula produce 0.1-6.0% w / v carbohydrate, preferably 1.5% glucose, as the sole carbon source, organic biofuel source, preferably yeast extract, organic nitrogen spun, preferably casein hydrolyzate, medium containing salts. pH 5.0-8.0, preferably 7.0 Culture temperature: 20-35 'C, preferably 30' C, incubation time: 8-72 hours, preferably 30 hours. Well-separated colonies from the sporulated inoculum thus obtained are prepared in a manner known per se, e.g. This medium may be the one described above, or alternatively, to test the effect of various media components and parameters on the entomotoxic components. The solid media cultures are incubated at 20-30 C, preferably 30 C, for 16-72 hours, preferably 48 hours. Alternatively, the sporulated inoculum may be treated with a mutagen in a known manner. The developed colonies are described in the above-4Ί

HU 201 657 Β is maintained in subcultures by inoculation on solidified slanted sterile medium. The assay for entomotoxic components (delta-endotoxin crystal, spore) is as follows. Heat-fixed slide preparations are prepared from colonies or subcultures developed from plate casting or solid or submerged cultures. Stains used for microscopic preparations, preferably carbol fuxin, are stained for 2 to 6 minutes, preferably 4 minutes.

After washing with water, drying is performed in a homogeneous immersion system under a microscope with Köhler illumination, Hl 100 / 1.3 x MFK 1: 5 to 1: 10, preferably MFK 1: 8. Using a green light filter (lambda = 0.5 gm), the above optical system has a resolution of A = 0.5 / 1.3 to 0.38 gm, suitable for identifying entomotoxic components (dipyramide crystal, spores). According to the method, the dipyramide crystal is intensely stained, the cell is well stained, and the spore hardly stained. Alternatively, using an ocular grid calibrated in the optical system, the number of morphological elements per unit volume can be determined with a selected sample thickness.

Proceeding as described in the first step of selection (colonies developed from die casting), variants that do not produce delta-endotoxin and / or spore are thus eliminated and have no entomotoxic effect. In production subcultures, particularly in submersible cultures, delta-endotoxin and / or spore formation can be continuously monitored.

The strains thus selected are used to produce entomotoxic biomass by fermentation.

B. thuringiensis var. kurstaki strain K-Cs 0019 is prepared by submerged culture containing an inoculum containing from 0.5 to 2.5% by weight of carbohydrate, preferably 1.5% by weight of glucose, an organic nitrogen source, preferably a protein hydrolyzate, a natural source of bios, preferably yeast extract and inorganic salts. medium, pH 5.0-8.0, preferably 7.0. Breeding temperature: 20-35 'C, preferably 30' C. Incubation time:

8 to 72 hours, preferably 12 hours. carbohydrate, preferably 3.0% w / v wheat flour, 0.5-5.0% w / v organic nitrogen source, preferably 2.0% w / v enzyme hydrolyzed sunflower meal, natural bicycle source. preferably hydrolyzed feed yeast and medium containing inorganic salts. pH 5.0-8.0, preferably 7.0. The culture temperature is 20-35 'C, preferably 30'C. Aeration: 0.5-2.0 v / v / min, preferably 1.0 v / v / min. Stirring: 200 n / min-600 n / min, preferably 400 n / min. Fermentation time: 8-96 hours, preferably 32 hours. Entomotoxic drug production is evaluated by the morphometric and / or biological assay described (Pieris brassicae, Anagasta kuchniclla). Activity: ALCjq (Pieris) 0.5-2.0 vol% (24 h exp.) In the culture medium. [Entomophaga, Tom. VII. No. 2.101-159 (1962)]. The resulting fermentation broth is concentrated by filtration and / or centrifugation, preserved, formulated with humectants, cmulsifiers, glues, heat protective agents in a known manner, sprayed and / or dusting grains, optionally powdered. The compositions obtained in this way are primarily active against Lepidoptera. Active ingredient: 25-30 trillion spores and crystals / g. Catable dose: 0.5-2.0 kg / ha.

Alternatively, the concentrated and doped fermentation broth may be used without entrapment in powder form.

In order to carry out the process of the present invention, the following embodiments are provided:

Example 1

B. thuringiensis var. kurstaki K-Cs 0019 (National Collection of Agricultural and Industrial Microorganisms, NCAIM B 001023) from a 5-day slant of tryptone glucose yeast extract (TGE) with 0.02% Tween-80 (oxyethylated anhydrosorbite monodeate) in sterile solution preparing a spore suspension. Prepare a shake culture with 2 to 2 ml of the spore suspension in the following medium.

MgSO ₄ .7H ₂ O 0.3 g FCSO4.7H2O 0.02 g ZnSO ₄ . zH ₂ O 0.02 g CaCO ₃ 1.0g Tripton (DIFCO) 5.0 g Pepton (WITTE) 2.0 g Yeast Extract (DIFCO) 2.0 g glucose 15.0 g Distilled water 1000 ml

pH: 7.0

Dosage: 50 ml / 300 ml E. flask

Sterilization: 30 minutes 0,981.10 ⁵ Pa

Incubation temperature: 30 'C. Speed: 300 rpm. Incubation time: 30 hours. Virtually spores are found in the culture in the order of 10'-10 ⁹ db / ml. A dilution series of 0.021% Tween 80 was prepared from the spore culture at 10 'increments. From 10 to ⁶ dilutions, 5 to 5 replicate plates are prepared on a 2.0 wt% agar agar solidified version of the above medium. The plates were incubated at 30 ° C for 48 hours. The colonies developed are inoculated on tryptone-glucose yeast extract slant, incubated at 30 ° C for 48 hours, and the cultures are stored at + 4 ° C. At the time of quenching, a heat-fixed slide preparation is prepared from the colonies. Carbolfuxin is aggressively stained for 4 minutes. After washing with water, homogenous immersion systems were examined with Hl 100 x MFK 1: 8 optics and Köhler illumination. Dipyramide delta-endotoxin crystals are intensely stained in the formulations, vegetative cells are well stained, and released spores are hardly stained. Microscopic selection is made, subcultures not producing the asporogenic toxin crystal are not maintained. Well-sporulated delta-endotoxin crystal production lines were checked for entomotoxic activity in shaken submerge culture.

Example 2

By shaking submerge culture according to Example 1, a sporulated inoculum of B. thuringiensis var. well-sporulated, selected toxin crystal subculture of K-Cs 0019 strain kurstaki. 2 to 2 ml of the sporulated inoculum are inoculated with the following shake culture:

MgSO ₄ .7H ₂ O 0.3 g

FcSO ₄ .7H ₂ O 0.02 g

ZnSO ₄ .7H ₂ O 0.02 g

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MnSO ₄ 0.02 g

CaCOs 3.0 g

Soya flour (extracted, roasted) 30.0 g

Corn flour 25.0 g

Tap water 1000 ml

The polyhydroxy compounds of the plant components were fluidized with heat-tolerant alpha-amylase (0.1 ml / 1000 ml medium from 120 KNU / g activity) for 95 minutes for 30 minutes. pH: 7.0

Dosage: 50 ml / 300 ml Erlenmayer flask Strain: 1 at 30 min Incubation temperature: 30 ° C Rotation: After 300 n / pcrc hour fermentation, the activity measured in a 5.5 wt% dry matter suboptimal aeration shake culture is also: Pieris) = 1.0 vol% / 24 h. exp.

Example 3

Submerged culture by shaking as in Example 1 produces a sporulated inoculum of B. thuringiensis var. well-sporulated toxin crystal producing K-CS 0019 strain of kurstaki.

80 ml of sterile pooled inoculum is inoculated with BIOTEC-FL110 type. Sterilized medium prepared in a 10 liter laboratory fermenter, containing: Inorganic salt base: as in Example 1

Sunflower meal, extracted 20.0 g

Wheat meal 30.0 g

Yeast hydrolyzate 5.0 g

Tap water 1000 ml

Enzyme treatment: as in Example 2. Fill: 8000 mL pH 7.0-7.2

Stcrilczés: 30 minutes, ⁴ 0,981.10 ⁵ Pa

For the antifoaming, ethyl alcohol was used in sterile form as needed.

Aeration: 8 liters / minute Mixing: paddle mixer / 400n / pcrc Temperature: 30'C, ± 1'C pH control: 10% w / w by addition of HCl or NaOH (sterile) Biological test for 96 hours fermentation: ALC ₉₀ (Pieris) = 1.0% v / v (24h exp.) Of the native medium. Lyophilized: standard fermentation broth: ALC _J0 (Pieris) = 1.5 ppm / 24 h. exp.)

Example 4

B. thuringiensis var. kurstaki strain K-CS 0019 was shaken by submerged culture as in Example 1 to produce a sporulated inoculum. 80 ml of sterile pooled inoculum is inoculated with BIOTECH-FL110 type. Sterile medium prepared in a 10-liter laboratory fermenter containing:

Inorganic salt base: as in Example 1

Wheat germ (GTHSZ 18-8) 5.0 g

Yeast extract (DIFCO) 2.0 g

Pepton (Witte) 2.0 g

Glucose 15.0 g

Tap water 1000 ml pH 7.2

Fill: 8000 ml

Sterilization: As described in Example 3, except that the glucose is separately sterilized in a non-hot autoclave, dissolved in 500 ml of distilled water, and after sterilization is added to the filling. The other parameters of fermentation are the same as those described in Example 3.

The result of the biological assay of the 96-hour culture was: ALC0O (Pieris) = 4.31% v / v (24 h exp.) Of native culture medium. Lyophilized Standard: ALC _M (Pieris) = 1.596 ppm (24h exp.)

Claims (4)

CLAIMS 1. A process for the preparation of a fermentation product containing a bacterial endoloxin which selectively fatales insects, in particular dwarf caterpillars, characterized in that Bacillus thuringiensis var. a selective staining and morphometric analysis of the K-CS 0019 strain of kurstaki to select a highly sporulating and highly delta-endotoxin producing line, preferably a carbon source of 0.1-6.0 wt% water soluble carbohydrate, organic nitrogen source, organic bios and inoculating the medium containing inorganic salts with aerobic, submerged culture, the resulting inoculum being suitably inoculated with 0.5-8.0% water / soluble carbohydrate and organic nitrogen spinning, organic biofuel spinning, and inorganic salt medium. The resulting fermentation broth is cultured in a known manner. 2. The process of claim 1 wherein the water-soluble carbon source is a carbohydrate or a carbohydrate by-product. 3. The process according to claim 1, wherein the organic nitrogen spinning is a protein of plant and / or animal origin or by-products containing them. The process according to claim 1, wherein the organic biofuel spinning is an edge extract and / or a biomass extract.