CA1085326A - Method for the biosynthesis of a microbial insecticide - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE

The bacillus thuringiensis microorganism or a related bacillus is cultivated in a nutrient medium under formation of spores in such a way that the early lysis of the cells caused in the course of the submerged cultivation by the ventilation prior to the end of the formation of spores is prevented.

Description

10853Z6 ~

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_ __ __ _ _ __ _ _ __ _ _ _ __ __ METHOD ~OR ~HE ~IOSYNTH~SIS OF A
MICROBIA~ I~SE~TICIDE
______________________ ___________________________________________ . . : ' The present invention relates to a novel method for the bio-synthesis of a microbial insecticide containing spores and crystalline endotoxin, said method being characterized in that the bacillus thuringiensis microorganism or a related bacillus is cultivated in a nutrient medium under formation of spore~ in such I -a manner that the early ly~is of cells caused in the course of the submerged culti~ation by the ventilation prior to the termi-nation of the formation of spores is prevented.
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It is known that the early lysis of cells in the course of the cultivation leads to low yields and a reduced effectiveness of the bioinsecticide as well as to an increase of its toxicity.
,. ' The present i~vention pro~ides for an improved method of producing spores and crystalline endotoxin in the process in which the early lysis of bacteria cell~ normally occurs as a result o~
enzymatic actions which are induced by the ventilation.

~ ' : ' ~08S3Z6 The delay in the formation of spores during the vegetative growth of bacteria is attributed to the effect of one or several inhibitors of an albuminous nature. The proteolytic decomposition of suggested inhibitors initiates the formation of the spores. Furthermore, various theories relating to the -function of the protease have been submitted which is one of --the early manifestations connected with the begin of spore formation (J. MANDELSTAM and W.M. WAITES, Biochem. J., volume 109, pp. 793 to 801, 1968). According to SCHAEFFER, it has been suggested that in the course of growth of bacteria in a quickly metabolized nutrient substrate and in the presence of a usuable nitrogen source, metabolic products occur which would suppress the synthesis of extracellular proteases and an enzym that is specific to the formation of spores, possibly in a manner similar to what is happening in the cancer cycle (P. SCHAEFFER, Bacteriol. Rev., volume 33, pp. 48 to 71, 1969). The result of such a suppression is the accumulation of acetic acid and pyroracemic acid which causes a decline of the pH and the early lysis of bacteria cells in general.
Now, according to a preferred execution of the present invention it is possible in connection with the sporulation to prevent the early lysis of bacteria cells by carrying out the biosynthesis for 4 to 6 hours under anaerobic conditions, and maintaining the p~l in the course of the biosynthesis within the range of 6.3 to 7.
With the help of the present invention it was possible to recover, among other things, the parasporal protein crystal bodies (~-endotoxin), and to purify the same by removing therefrom the water-soluble exotoxin ( ~ -exotoxin) which must be removed because of its toxicity to mammals. ~he best method for removing the exotoxin, according to the present invention, com-prises the use of semi-permeable diaphragmq.

It is known that the bacillus thuringiensis-group of micro-organisms is characterized by the ~ormation of protein-containing parasporal endotoxin crystals. ~he insect-pathogenic nature of bacillus thuringiensis on a large row of lepidopterous larvae by ingestion is primarily to be attributed to the e~fect of endo-toxin. Certain strains o~ bacillus thuringiensis, in addition to intracellular endotoxin, form an extracellular, water-soluble exotoxin which is stable unaer heat. The term exotoxin relates in this case to an active substance of living cells which is dis-charged or secreted into the medium while the endotoxin contrary thereto is set free only after the cells have been completely dissolved.

So as to facilitate a better understanding of the present in-vention the characteristics of the above-mentioned toxins are deæcribed in greater detail as follows:
, ~he Cr~stalline Endotoxin A protein-containing crystalline inclusion has been isolated f~om the sporulated culture of bacillus thuringiensis by HANNAY
and ~I~Z-JAMES (C.~. EANNAY and P. ~ITZ-JAMES, Can J. Microbiol., volume 1, pp. 694 to 710, 1955). ~he electron-microscopic studies have sho~m that in the course of the formation of spores of bacillus thuringiensis bacteria, an asporal crystalline body was developed together with the spores~ It was concluded that a new cont~d.
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relation exists between the formation of the~ spores and the endotoxin crystals. Recent research indicated that the crystalline endotoxin is composed of a high molecular protein component and a Rilicon sl~eleton. ~he spores and the endotoxin crystals may be recovered from the culture medium in the sediment of a high-speed centrifuge. ~he separation of the crystalline endotoxin from the spores was achieved in different ways, e.g. by fractionated sedi-mentation, step-by-step sedimentation, and separation in a two-phase system.

The Exotoxin , . .

In addition to the crystalline endotoxin, certain bacillus thuringiensis strains secrete in the course of the vegetative gro~ing phase a toxic fraction into the medium which is chemically related to the adenine nucleotides. No relation exists between the generation of crystalline endotoxin and e~otoxin. According to tests, exotoxin can be formed only by defined generlc types of special bacillus thuringienæis varieties. ~he method of pro-ducing exotoxin has been developed by De ~ARJAC. According to said method, bacillus thuringiensis is cultivated in a medium containing mineral salts in addition to 0.75~ peptone and 1%
glucose, namely for 70 hours at 30~.

~ he preparation of microbial insecticides according to the present invention is preferably carried out in the following stages:

A) Cultivation ~ he cultures are obtained based on the spores of a strain of bacillus thuringiensis bacteria. (ATCC 10792 E.A. STEINHAUS-O. MATTES) (N.R. SMITH USDA 996~,FROM MEDITERRANEAN FLOUR MOTH~

EPHESTIA KUEHNIELA, USDA MISC., PUBL. 559:51(1946) USDA AGR.
MONOGR. 16:67 (1952).
cont~d.

-` -5-. The nutrient mediu~ for preparing the vegetative inoculum in shaking bottles contains the following components:

bacto-trypton (T7Difco") 0.8 % by weight glucose 2.5 % " ~' NgS04. 7H20 0.2 % " "
4 7 2 0.2 % ~
. ~e2(S04)3 0.2 ~ " "
~nS04. 5H20 0.1 ~0 " '~ . - . .

The medium for agar slant cultures was the same as above but including also an addition of 2% agar.
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Good results have been obtained with the cultivation in the following media:

. Medium I:
molasses (50~ solid material) 1~4 ~0 by weight yeast 0,3 ~O~t "
(~H4)2S04 0.1 ~0'' "
C.S.~. (maize-macerating liquid) 0.1 % " "
CaC03 0.1 ~o 11 n The vH after the sterilization was 6.8.

Or with the medium containing starch instead o~ molasses:

edium II:
starch 1.3 ~0 by weig~t yeast 1.0 ~o " "

2 4 0.4 % ll ~
C.S.~. (maize-maceratin~ liquid) 0.2 % " "
CaC03 0.8 ~0 "

The pH after sterilization was 6.8.

: cont'd.

The nutrient medium for the vegetative inoculum (300 ml) was contained in sterilizable bottles with flat bottoms and 1 liter filling capacity. The nutrient medium contained in said bottles was sterilized for 45 minutes at 121C. The glucose was sterilized separately according to the Tindali-zation method and aseptically added to the sterilized medium.
The nutrient medium for the cultivation in the fermentators was prepared and sterilized in portions in the fermentator for 45 minutes at 121C. Cultivation was carried out in fermen-tators made of stainless steel. The culture was rotated at150 r.p.m.; ventilated with 0.5 liter per liter of culture per minute; the incubation temperature was from 28 to 30C. The application of an "anaerobic shock" was carried out by dis-continuing the ventilation for 3 to 6 hours in the 12th hour of submerged cultivation. The timing for interrupting the ventilation was achieved with the help of an oxygen analyzer.
The cultivation may be carried out without any early lysis of cells and without applying the afore-stated "anaerobic shock"
if in the course of the cultivation the pH is regulated in such a way that it will not fall below 6.3. The formation of spores and the simultaneous setting free of endotoxin crystals ends after 35 to 40 hours of cultivation.
B. Separation and Purification After the bacteria have completely dissolved while spores and endotoxin crystals have been set free at the same time, the separation of the spores and the crystalline endotoxin from the exotoxin is carried out. Water-soluble exotoxin is preferably removed by means of dialysis. This method results in a mixture of spores and crystalline endotoxin which is not toxic to mammals. The presence or absence of water-soluble exotoxin was determined by the biological test described by BOND et al. A nutrient medium for the biological test was prepared by heating the mixture comprising 10 g pulverized agar, 500 ml fresh milk, and 7 g yeast. A 10 ml-sample was subsequently placed into a Petri dish having a diameter of 9 cm and filled already with 250 u liter of the test solution.
As soon as the gels had solidified, 20 eggs of musca domestica were added. The plates were kept for 48 hours in an incubator at 30C, and the development of larvae and the disturbance of the agar gel were observed (R.P.M. BOND, C.B.C. BOYCE and 10 S.J. FRENCH, Biochem. J., volume 114, pp. 477 to 488, 1969).
The resulting product, which is freed from water-soluble -exotoxin, may be processed and used in the customary manner for the preparation of insecticides.
C. Drying The drying step may be carried out according to any one of the following methods:
By admixing the resulting mixture comprising spores and endotoxin crystals with Bentonitic clays and placing the blend in the form of thin films in a ventilated dryer equipped with plates, at a temperature of about 40 to 45C;
by suspending the resulting mixture in water and drying in a rotating or pneumatic dryer;
by spraying the resulting mixture in a spray dryer.
D. Crushing and Screening If the mixture is dried in a spray dryer the resulting product requires no further crushing. When drying the mixture in ventilated dryers on plates, the product is crushed in a device of the Alpina type and screened using a sieve with a fine mesh conforming at least to mesh No. 180.

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E. Determination of the Effectiveness The effectiveness was determined according to the following methods: by counting the living spores present according to the plate method and by carrying out bio-tests on test insects.
The resulting products are normed according to the standards established by the French Laboratoires de Lutte Biologique et de Biocinetique de la Miniere (I.N~R.A.).
The present invention will be more clearly understood with the help of the description of the following examples which, however, do not in any way constitute any limitation of the present invention:

Based on the Berliner variety of the bacillus thuringiensis culture, the cultivation is carried out according to the method herein described using a liquid, ventilated medium, namely medium I (see above). The incubation temperature applied in the course of the submerged cultivation is about 28C. The culture is put into motion at 150 r.p.m.; the rate of venti-lation is 0.5 liter per liter per minute. The ventilation isinterrupted for 3 to 6 hours in the 12th to 14th hour of cultivation. The timing for interrupting the ventilation was determined with the help of an oxygen analyzer; ventilation was interrupted as soon as the percentage of solubility and the oxygen consumption decreased, and the pH fell to about 5.8. After from 3 to 6 hours, the ventilation was continued at the same rate of 0.5 liter per liter per minute. The culti-vation without early lysis may be carried out without anaero-biosis if the pH of the medium is maintained within the range of 6.3 to 6.5~ The submerged cultivation is continued until .

~0853269 -~ .,~

the spores and the crystalline endotoxin are yielded or trans-ferred into the medium (35 to 40 hours on the average). ~he culture medium is subsequently placed into the re^eptacle in whic~
the purification is carried out by removing the water-soluble exo toxin. Said purification takes place by means of dialysis. Eor this purpose it is possible to use a device as shown in the en-closed figure. ~he culture medium is transferred from the fermen-tator to receptacle A which is equipped ~lith an agitator e and a dialysis diaphragm a. This diaphra~m may be colloidal or of any type as customarily used in practical life. ~he dialysis ta~es place more rapidly if deionized water b is added to receptacle ~.
lhe presence of absence of water-soluble eYotoxin is determined by the afore-described bio-test. A powdery fillin~ material is added to the resulting mixture of spores and cr~stalline endo-toxins, and the mixture is then dried in a drying kiln on plates.
~he resulting product is gro~md in a crusher of the Alpina type, and then screened in order to obtain a powder.
. ' Example 2 ~ he method is carried out as defined in Example 1, however, the nutrient medium II (see above) is used for the submerged cul-tivation in the fermentator.
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` ~ Example 3 .~
~ he method is carried out as defined in Example 1, however~
after the submerged cultivation of the bacteria and execution of ¦ the dial~sis, i.e. after the exotoxin has been removed, the resul ting product in dried in a centrifugal dryer and subsequently ground in an Alpina mill.
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cont'd.
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The method is carried out as defined in Example 1, however, the product obtained after the submerged cultivation of the bacteria and execution of the dialysis is sus~ended again in a defined amount of water, and this suspension is spray-dried in a spray dryer. The temperature within the dryer was from 90 to 100C. The best suitable devices for this purpose are spray dryers equipped with disks which rotate at from 10,000 to 15,000 r.p.m. The product is a very fine powder which needs neither grinding nor screening. The exotoxin is not present in this end product.
The present invention may be applied or utilized also for other varieties of the same type of bacteria, for example the sotto variety of bacillus thuringiensis, the subtoxicus variety of bacillus thuringiensis. All these bacteria are described in detail in the relevant literature.
One advantage of the present invention lies in the higher effectiveness of the resulting product and the excellent purity of this product with respect to exotoxin combined with high yields of spores and crystalline endotoxins.

Ii 108S326 Insecticidal Activity of the Active Com~onent, BACTUCIDE
The insecticidal activity of the active component embracing the spores of the Bacillus thuringiensis bacteria bacteria and the crystal endotoxin can be regarded in two ways: ¦
- as the number of spores of Bacillus thuringiensis bacteria i a gram of the active component, and - as inte~national units (IU).
The active component as a part of our formulations, shows the following activity:
101 spores/g of active component;
6000 and 15,000 IU/mg of the active component.
1000 IU/mg is the value of the product showing the same insecticidal activity as a standard sample E-61 (a mixture of spores and crystals of endotoxin of Bacillus thuringiensis bacteria tested on the insect Ephestia Kuhniella). With the active component the following formulations are prepared:-Powdered formulation "BACTUCIDE-P"
4.0% of the active component 15,000 IU/mg 25.0% aerosil (vesalon) 5.0% jugopon 66.0% china clay Liquid formulati_n ''BACTUCIDE-S"
8.0% of the acti~e component 6000 IU/mg 1.0% carboxymethyl cellulose 0.3 emulgator

3.0~ petrol-hydrocarbon 87.7% water pH 6, 8-7, 2 cont'd.
': . ,,, .

~ , , ~085326 Formulation of BACTUCIDE-G granules 98.0% of granule (calcium carbonate) 2.0~ of the active component (2.4 109 spores/g) The biologic insecticide "~ACTUCIDE" is used especially against insects of the Lepidoptera group.
The quantity of liquid formùlation of BACTUCIDE is 0.8-1.5 litres per ha, in that the dilution of this has to occur ~before the use) in water quantity of 500-1000 litre. The dilution depends on the mode of application, the power of insects and the thickness of plants. The product can also be applied from air.
The powder formulation of BACTUCIDE-P has the same application as a liquid formulation in quantity of 0.6-l.O kg~ha.
The formulation in form of granule (BACTUCIDE-G) is exclusively applied against insects Pyrausta nubilalis (corn). The quantity for application is 30 kg/ha.
BACTUCIDE is easily miscible and is compatible with chemical insecticides.
The examples of mixing the chemical insecticides with biologic insecticide BACTUCIDE are as follows:
Malathio~ 0.2% + Bactucide 0.12%
Dimethoate O.1-0.2% + Bactucide 0.15%
Sevin O.1-0.2% + Bactucide 0.15%
The same effects can be obtained with lower concentrations of chemical insecticide in that the toxicity is lowered. Also other chemical insecticides are compatible with Bactucide, as for example methylparathion, diazinon, DDT, Pyrethrum, Phosdrin etc.
We tested approximately 50 insects. The following tables list the insects sensitive to BACTUCIDE and the results obtained on these insects.

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~0853Z6 . , i The activity of the liquid formulation of BACTUCIDE-S on some important insects.

Table_l.
The activity of BACTUCIDE on the on the insect Trycho~lusia ni (cabbage).

. _ Quantity% of killed larva Total of larva lit/haTESTING in days tested 0.2 20 27 90 180 0.5 63 80 112 220 1.0 75 92 200 222 Control -- -- --- 650 Table 2.
The activity of BACTUCIDE on the insect Hyponomeuta mallinellus (apple).
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Quantity Insectarium Field Tests - -- . - .
llt/ha ~ mortality corr. mort. ~ mort. corr. mort.

.. . ... ... _ , 0.4 15 13.8 12 11.05 0.6 32 31.2 31 30.2 1.0 85 84.2 86.0 85.2 1.5 100 100 100 100 ' ' ' ' " ' cont'd.

: . ., .: . .~ . , ~085326 Table 3.
The activity of BACTUCIDE on the insect Heliothis virescens and Heliothis zea (tobacco and cotton).

.. . . . _ _ _ , .. .. .... . _ Concentration Days Cotton Tobacco lit/ha _ __ %_mortal_ y _ % mortality 0.2 4 30 75 0.5 6 50 100 1.0 8 95 100 1.5 10 10~

The activity of formulation of granule BACTUCIDE-G
Table 4.
:
- The activity of BACTUCIDE-G on the insect Pyrausta nubilalis (corn).
Material Dose N %of plant Average Effective-with living N of ness of (formulation) kg/ha plant larva larva/ BACTUCIDE- .
plant G in %
.
Bactucide-G 30 80. 41.0 0.9 82 ~ ~

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Claims (3)

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:

1. A method for the biosynthesis of a microbial insecticide containing spores and crystalline endotoxin, which comprises cultivating, by submerged cultivation, a Bacillus thuringiensis microorganism in a nutrient medium under formation of spores and preventing, by anaerobic shock, early lysis of the cells resulting during the course of the submerged cultivation by ventilation prior to the completion of spore formation.

2. A method according to claim 1, wherein the Bacillus thuringiensis is cultivated without lysis prior to the end of the formation of spores by interrupting the ventilation for 3 to 6 hours in the twelfth hour of cultivation and carrying out the cultivation under anaerobic conditions during that time.

3. A method according to claim 1, wherein the cultivation without lysis of the cells of Bacillus thuringiensis prior to the end of the formation of spores is carried out in such a way that the pH is maintained in the course of the cultivation within the range of 6.3 to 7.