JOURNAL
OF INVERTEBRATE
Influence
PATHOLOGY
55,
147-151
of Media Composition on the Production of GEndotoxin by Bacillus thuringiensis var. thuringiensis AND A.N. RAGHUNATHAN
S.G.MUMMIGATTI Infestation
(1990)
Control
and Protectants
Discipline, Central Food Mysore 570 013, India
Technological
Research
Institute,
Received October 7, 1988; accepted May 26, 1989 Powders of edible leguminous seeds, greengram (Vigna radiata) or soybean (Glycine max), were used as the major protein source with different combinations of soluble starch and/or cane sugar molasses as the major carbohydrate source for the production of 8-endotoxin by Bacillus thuringiensis var. thuringiensis serotype 1 in submerged fermentation. The primary product (lyophilized with 6 g of lactose) yield was 8.7 to 9.1 g/liter from media with dehusked greengram powder and 9.7 to 10.3 g/Iiter from media with defatted soybean powder in basal medium. The toxicity of primary products was assayed against f&h-in&r Bombyx mori larvae by force-feeding. The primary product from the medium containing defatted soybean powder and soluble starch gave a maximum viable spore count of 91.3 x lO?mg, with a corresponding potency of 35,800 IU/mg, whereas the medium containing dehusked greengram powder and cane sugar molasses gave a spore count of 49.5 x lO%ng, with a highest potency of 38,300 IU/mg. Either legume protein in combination with cane sugar molasses yielded primary product 2.1 to 2.4 times more potent than the U.S. standard. The combined carbohydrate source consisting of soluble starch and cane sugar molasses, irrespective of the source of protein in the media, drastically reduced bendotoxin production, thereby reducing the potency of the primary products compared to the U.S. standard. o 1990academic Pmss,
Inc.
KEY WORDS: Bacillus
thuringiensis
var.
submerged fermentation: &endotoxin; Units; potency.
mori;
thuringiensis;
Vigna
radiata;
Glycine
max;
Bombyx
primary product; viable spore count; International
INTRODUCTION Bacillus thuringiensis is the only insect pathogen being commercially exploited extensively in different parts of the world. The commercial production of B. thuringiensis for use as a bioinsecticide necessitates the search for cheaper locally available media ingredients. At present, several by-products of agroindustry, such as cotton seed meal, soybean meal, corn-steep liquor, and cane sugar molasses, are being used in industrial fermentation. Salama et al. (1983) studied the use of leguminous seeds such as horsegram, kidney bean, lima bean, soybean, chickpea, lentil, and peanut as the protein source for the growth of B. thuringiensis var. kurstaki and var. entomocidus. Nagamma et al. (1972) reported the use of groundnut cake and tamarind kernel powder as media ingredients for sur-
face culture of B. thuringiensis. The use of groundnut cake aqueous extract, cowpeas (white and black varieties), soybean, and bambara bean along with cow blood powder for B. thuringiensis var. israelensis growth in submerged fermentation was reported by Obeta and Okafor (1984). The present study was undertaken to evaluate dehusked greengram powder and defatted soybean powder and either soluble starch or cane sugar molasses or both for Gendotoxin production by B. thuringiensis var. thuringiensis in a submerged fermentation medium. MATERIALS B. thuringiensis
AND METHODS
var. thuringiensis serotype 1, kindly supplied by Dr. H. de Barjac, Pasteur Institute, Paris, was grown on tryptose phosphate agar (Difco) slants at 30” & 2°C for 72 hr and stored at 4°C until use.
147 0022-201 l/90 $1.50 Copyright 63 1990 by Academic All lights of reproduction
Press, Inc. in any form lwawxi.
148
MUMMIGATTI
AND RAGHUNATHAN
Seed Culture
A loopful of the above B. thuringiensis culture was inoculated into 100 ml of tryptose phosphate broth in a 500-ml Erlenmeyer flask which was then covered with a cotton-mesh pad and incubated on a rotary shaker (142 rpm) at 30” -t 2°C for 24 hr. Three percent by volume of the cultured broth from the first-passage seed flask was used to inoculate the second-passage seed flask medium and was incubated as above for 24 hr to obtain vigorous vegetative growth of B. thuringiensis. Production
Flask
Six media, designated A to F, were used for 6-endotoxin production by B. thuringiensis. All media contained 0.2% yeast extract, 0.1% K,HPO,, and 0.1% KH*PO, as basal medium ingredients. To this, 1.0% of TABLE INFLUENCE
OF MEDIA
COMPOSITION
either dehusked greengram (Vigna radiata) powder or defatted soybean (Glycine man) powder and combinations of 0.5 or 1.0% soluble starch and cane sugar molasses were added (Table 1). The commercial samples of dehusked greengram and soybean, procured from the local market, were powdered using a table top dry grinder and then passed through 35mesh 1 inch screen (The Tyler Standard Screen Scale; U.S. series equivalent 40). Soybean powder was defatted before use by a Soxhlet extraction apparatus using petroleum ether (40”-6O”C) for 6 hr, followed by evaporation of the solvent overnight from the defatted powder. The final media were adjusted to pH 7.5 with 1 N NaOH and were distributed at 100 mYSOO-mlErlenmeyer flask. Flasks were covered with cotton-mesh pads and sterilized at 121°C for 20 min. The six media were inoculated with 2% by volume of the 1
ON THE GROWTH, TOXICITY, VAR. thuringiensis
A B C
D E F
U.S.
BMd + 1% greengram + 1% soluble starch BM + 1% greengram + 1% cane sugar molasses BM + 1% greengram + 0.5% soluble starch + 0.5% cane sugar molasses BM + 1% soybean + 1% soluble starch BM + 1% soybean + 1% cane sugar molasses BM + 1% soybean + 0.5% soluble starch + 0.5% cane sugar molasses standard HD-1-S-1980
thuringiensis
ratio
24 hr
48 hr
96 hr
4.8
6.5
7.0
7.5
39.7 (9.1)
133.9
14,900
3.7
7.0
7.5
7.5
49.5 (8.7)
52.1
38,300
4.2
6.5
7.0
7.5
38.5 (8.8)
195.7
10,200
2.4
6.5
8.0
8.5
91.3 (10.0)
55.7
35,800
1.8
7.5
8.5
8.5
38.3 (9.7)
57.1
34,900
2.1
7.0
8.0
8.5
88.6 (10.3)
168.1
11.800
1.24
124.9
16,000
C:N
Composition
OF Bacillus
VSC” (primary productb yield g/liter dry weight)
Broth pH after Media
AND POTENCY
a Viable spore count per milligram of product x 106. b Lyophilized with 100 ml of 6% lactose solution. c International Units. d Basal Medium containing yeast extract (0.2%), K,HPO, (O.l%), and KH$‘Q
LDso bdg body wt of Bombyx
mori)
Primary product potency (IU’/mg)
(0.1%) adjusted to pH 7.5
PRODUCTION
OF
S-ENDOTOXIN
B. thuringiensis broth from the secondpassage seed flask and incubated at 30” + 2°C on a rotary shaker (142 rpm). The pH of the broth was monitored at 24-hr intervals and adjusted with sterilized 1 N HCl to 7.5 whenever it increased. Broth samples taken periodically were subjected to microscopical examination for sporulation and crystal formation (Smimoff, 1962) until the end of the fermentation process. Recovery
When 90% of the sporangia had lysed, releasing the spores and crystals, the pH was adjusted to 7 with sterilized 1 N HCl, and the broth was centrifuged at 7500 r-pm at 10°C for 20 min. The spore-crystal complex was resuspended in 100 ml of 6% lactose (Dulmage et al., 1970)and lyophilized. The dry primary products were stored at 4°C for further studies. Estimation
of Viable Spore Count (WC)
One milligram of the primary products from each of the six media was placed in 10 ml of sterile, distilled water separately containing 0.02 ml of 1% Tween 80 solution and incubated in a water bath at 65°C for 15min to destroy the vegetative cells. The VSC was determined by counting serial dilutions plated on nutrient agar medium (Difco). Evaluation
of Toxicity
Bioassay of the primary products was made with fifth-instar larvae of the silk moth, Bombyx mod. Primary products (140 mg) from each of the six media and the U .S. standard HD-1-S-1980 of 16,000 IU/mg were suspended in 4 ml of sterile, distilled water separately containing 0.008 ml of 1% Tween 80 solution. Larvae were force-fed under a stereomicroscope with the help of a blunt, polished hypodermic 25-gauge needle on an Agla syringe (Ishiguro and Miyasono, 1979). Accurate administration of 1, 2, 4, 6, 8, 10, 12, 14, and 15 ~1 of sporecrystal suspensions was possible by this method. Twenty larvae were force-fed for each concentration of each sample. The
149
BY B. hringiensis
control larvae were force-fed with 15 ~1 of sterile, distilled water containing 0.03 ~1 of 1% Tween 80 solution. The VSC per 1 ul of all the suspensions was determined by plating on nutrient agar. After administration of the B. thuringiensis suspensions, the larvae were fed with fresh mulberry leaves ad libitum. Mortality was recorded after 24 hr. The LD,, values, and thereby the potencies, in terms of IU (Dulmage et al., 1981), of all the six primary products were calculated (Table 1). RESULTS
AND DISCUSSION
Cultures could be harvested after 96 hr of incubation from media D, E, and F, and after 120 hr from media A, B, and C. The product yield (dry weight basis, including 6 g of lactose) ranged from 8.7 to 9.1 g/liter for media A, B, and C for dehusked greengram powder and 9.7 to 10.3 g/liter for media D, E, and F for defatted soybean powder as the major protein source. This variation could be due to the cumulative effect of the quality and quantity of protein and carbohydrate and their ratio in the respective media. Table 1 shows that media A, B, and C had higher carbohydrate content, with C:N ratios ranging from 3.7 to 4.8, whereas the C:N ratio ranged from 1.8 to 2.4 in media D, E, and F. The C:N ratios of the media were calculated from data from different references of C and N content of the substrates (Anonymous, 1970; Gopalan et al., 1981; Hough et al., 1982; Norman, 1978; Paturau, 1969). The higher carbohydrate content of media A, B, and C might have prolonged the vegtative exponential phase of the organism, delaying sporulation and lysis compared to media D, E, and F cultures, which could be harvested earlier. Media D, E, and F turned more alkaline at the end of 48 and 96 hr of shake incubation, and hence required pH adjustment to 7.5. Because of the higher carbohydrate content in media A, B , and C , the pH remained near neutral throughout the fermentation process. The maximum VSC of 91.3 x 106/mgwas
150
MUMMIGATTI
AND
obtained from medium D, defatted soybean powder and soluble starch, and had a corresponding potency of 35,800 IU/mg (Table 1). However, the product from medium B, dehusked greengram powder and cane sugar molasses, having only 49.5 x IO6 VSC/mg, recorded the highest potency of 38,300 IU/mg, inferring that the quality of the &endotoxin produced differed with the nutritive value of the media. Both dehusked greengram and defatted soybean powders in combination with cane sugar molasses (media B and E, respectively) and defatted soybean powder with soluble starch (medium D) yielded products 2.1 to 2.4 times more potent than the U.S. standard. Media A, C, and F yielded less potent products than the U.S. standard. It is evident, that when both soluble starch and cane sugar molasses are combined as the carbohydrate source, as in media C and F, irrespective of the protein source, drastic reduction of toxicity of the primary products occurs. This could be due to the production and accumulation of B. thuringiensis 6-endotoxin biosynthesis inhibitors in the media. The primary product harvested from medium A, which contains the highest amount of carbohydrate (Table l), was also less toxic than the US standard. Salama et al. (1983) screened various legume seeds and reported that 2% soybean in the basal medium produced fewer spores from B. thuringiensis var. kurstaki and B. thuringiensis var. entomocidus than kidney bean and chick pea, both of which supported good spore production and endotoxin production. Obeta and Okafor (1984) reported that media containing bambara bean powder (0.75%) and cow blood powder (1.O%) in basal medium produced a highly toxic primary product of B. thuringiensis var. israelensis. In the present study, medium B containing dehusked greengram protein, which is cheaper than the defatted soybean protein and cane sugar molasses, gave the most toxic B. thuringiensis biocide. Potency of a B. thuringiensis product against a particular
RAGHUNATHAN
pest is known to vary with the media ingredients, the B. thuringiensis strain, and the growth conditions. The balance between the amino acids glutamic acid, aspartic acid, arginine, glycine, and proline, which are necessary for B. thuringiensis Gendotoxin biosynthesis (Mm-thy and Rana, 1980; Rajalakshmi and Sethna, 1977), and the amino acids lysine, threonine, isoleucine, leucine, valine, histidine, tyrosine, serine, and cystine, which inhibit 6-endotoxin biosynthesis (Conner and Hansen, 1967; Rajalakshmi and Sethna, 1980; Singer and Rogoff, 1968), in the fermentation medium determines the production of Gendotoxin and thus the toxicity and the potency of the B. thuringiensis primary product. The high potency of the primary product against a target pest determines its superiority over others. This study revealed the intricate role played by the individual components of the fermentation medium in supporting the growth of B. thuringiensis var. thuringiensis and the production of S-endotoxin. However, more detailed study is required for the possible utilization of legume seeds and their by-products as major protein sources in large-scale fermentation of B. thuringiensis varieties. ACKNOWLEDGMENTS We thank the Director, CPIRI, Mysore, and the Area Co-ordinator, Infestation Control and Protectants Discipline, CFIRI, Mysore, for providing the facility where this work was carried out. We are also grateful to Dr. S. C. Basappa, Scientist, Microbiology and Sanitation Discipline, CFI’RI, Mysore, for his critical reading of the manuscript.
REFERENCES 1970. “Pulse Crops of India.” Indian Council of Agricultural Research, New Delhi. CONNER, R. M., AND HANSEN, P. A. 1%7. Effects of valine, leucine and isoleucine on the growth of Bacillus thuringiensis and related bacteria. J. Invertebr. Pathol., 9, 12-18. DULMAGE, H. T., CORREA, J. A., AND MARTINEZ, A. J. 1970. Coprecipitation with lactose as a means of recovering the spore crystal complex of Bacillus rhuringiensis. J. Znvertebr. Pathol., 15, 15-20. ANONYMOUS
PRODUCTION
OF S-ENDOTOXIN
DULMAGE, H. T., ETAL. 1981. Insecticidal activity of
BY B. thuringiensis
151
NORMAN, A. G. (Ed.) 1978. “Soybean Physiology, isolates of Bacillus thuringiensis and their potential Agronomy and Utilization.” Academic Press, New for pest control. In “Microbial Control of Pests and York. Plant Diseases, 1970-1980” (H. D. Burges, Ed.), OBETA,J. A., ANDOKAFOR, N. 1984. Medium for the pp. 193-222. Academic Press, New York. production of primary powder of Bacillus thuringGOPALAN, C., RAMASASTRI, B. V., ANDBALASUBIU- iensis sub.sp. israelensis. Appl. Environ. MicroMANIAN,S. c. 1981. “Nutritive Value of Indian biol., 47, 863-867. PATURAU,J. M. 1969. “By-products of the Cane Foods.” Indian Council of Medical Research, New Delhi. Sugar Industry: An Introduction to Their Industrial HOUGH,J. S., BRIGGS,D. E., STEVENS, R., AND Utilization.” Elsevier, Amsterdam. YOUNG,T. W. 1982. Yeast growth. In “Malting and RAJALAKSHMI, S., ANDSETHNA,Y. I. 1977.The efBrewing Science,” Vol. 2, “Hopped Wort and fect of amino acid on the growth, sporulation and crystal formation in Bacillus thuringiensis var. thuBeer,” 2nd ed., pp. 615-643. Chapman & Hall, New York. ringiensis. J. Indian Inst. Sci., 59, 161176. ISHIGURO, T., ANDMIYASONO,M. 1979. A bioassay RAJALAKSHMI, S., AND SETHNA,Y. I. 1980. Spore for toxicity of Bacillus thuringiensis insecticide usand crystal formation in Bacillus thuringiensis var. thuringiensis during growth in cystine and cysteine. ing the force feeding method applied to the silkworm, Bombyx mori L. Japan. J. Appl. Entomol. J. Biosci., 2, 321-328. Zool., 23, 141-150. SALAMA,H. S., FODA,M. S., DULMAGE,H. T., AND MURTHY,K. G. K., ANDRANA,R. S. 1980. Role of EL-SHARABY, A. 1983. Novel fermentation media glutamic acid and glutamate dehydrogenase in for production of 8-endotoxins from Bacillus thuringiensis. J. Znvertebr. Pathol., 41, 8-19. sporulation of Bacillus thuringiensis var. thuringiensis in chemically defined media. Indian J. Biochem. SINGER, S., ANDROGOFF, M. H. 1968. Inhibition of Biophys., 17, 157-158. growth of Bacillus thuringiensis by amino acids in NAGAMMA,M. V., RAGHUNATHAN, A. N., ANDMAdefined media. J. Invertebr. Pathol., 12, 98-104. JUMDER, S. K. 1972. A new medium for Bacillus SMIRNOFF, W. A. 1962. A staining method for differthuringiensis Berliner. J. Appl. Bacterial., 35, 367entiating spores, crystals and cells of Bacillus thur370. ingiensis (Berliner). J. Insect Pathol., 4, 38k386.
OF INVERTEBRATE
Influence
PATHOLOGY
55,
147-151
of Media Composition on the Production of GEndotoxin by Bacillus thuringiensis var. thuringiensis AND A.N. RAGHUNATHAN
S.G.MUMMIGATTI Infestation
(1990)
Control
and Protectants
Discipline, Central Food Mysore 570 013, India
Technological
Research
Institute,
Received October 7, 1988; accepted May 26, 1989 Powders of edible leguminous seeds, greengram (Vigna radiata) or soybean (Glycine max), were used as the major protein source with different combinations of soluble starch and/or cane sugar molasses as the major carbohydrate source for the production of 8-endotoxin by Bacillus thuringiensis var. thuringiensis serotype 1 in submerged fermentation. The primary product (lyophilized with 6 g of lactose) yield was 8.7 to 9.1 g/liter from media with dehusked greengram powder and 9.7 to 10.3 g/Iiter from media with defatted soybean powder in basal medium. The toxicity of primary products was assayed against f&h-in&r Bombyx mori larvae by force-feeding. The primary product from the medium containing defatted soybean powder and soluble starch gave a maximum viable spore count of 91.3 x lO?mg, with a corresponding potency of 35,800 IU/mg, whereas the medium containing dehusked greengram powder and cane sugar molasses gave a spore count of 49.5 x lO%ng, with a highest potency of 38,300 IU/mg. Either legume protein in combination with cane sugar molasses yielded primary product 2.1 to 2.4 times more potent than the U.S. standard. The combined carbohydrate source consisting of soluble starch and cane sugar molasses, irrespective of the source of protein in the media, drastically reduced bendotoxin production, thereby reducing the potency of the primary products compared to the U.S. standard. o 1990academic Pmss,
Inc.
KEY WORDS: Bacillus
thuringiensis
var.
submerged fermentation: &endotoxin; Units; potency.
mori;
thuringiensis;
Vigna
radiata;
Glycine
max;
Bombyx
primary product; viable spore count; International
INTRODUCTION Bacillus thuringiensis is the only insect pathogen being commercially exploited extensively in different parts of the world. The commercial production of B. thuringiensis for use as a bioinsecticide necessitates the search for cheaper locally available media ingredients. At present, several by-products of agroindustry, such as cotton seed meal, soybean meal, corn-steep liquor, and cane sugar molasses, are being used in industrial fermentation. Salama et al. (1983) studied the use of leguminous seeds such as horsegram, kidney bean, lima bean, soybean, chickpea, lentil, and peanut as the protein source for the growth of B. thuringiensis var. kurstaki and var. entomocidus. Nagamma et al. (1972) reported the use of groundnut cake and tamarind kernel powder as media ingredients for sur-
face culture of B. thuringiensis. The use of groundnut cake aqueous extract, cowpeas (white and black varieties), soybean, and bambara bean along with cow blood powder for B. thuringiensis var. israelensis growth in submerged fermentation was reported by Obeta and Okafor (1984). The present study was undertaken to evaluate dehusked greengram powder and defatted soybean powder and either soluble starch or cane sugar molasses or both for Gendotoxin production by B. thuringiensis var. thuringiensis in a submerged fermentation medium. MATERIALS B. thuringiensis
AND METHODS
var. thuringiensis serotype 1, kindly supplied by Dr. H. de Barjac, Pasteur Institute, Paris, was grown on tryptose phosphate agar (Difco) slants at 30” & 2°C for 72 hr and stored at 4°C until use.
147 0022-201 l/90 $1.50 Copyright 63 1990 by Academic All lights of reproduction
Press, Inc. in any form lwawxi.
148
MUMMIGATTI
AND RAGHUNATHAN
Seed Culture
A loopful of the above B. thuringiensis culture was inoculated into 100 ml of tryptose phosphate broth in a 500-ml Erlenmeyer flask which was then covered with a cotton-mesh pad and incubated on a rotary shaker (142 rpm) at 30” -t 2°C for 24 hr. Three percent by volume of the cultured broth from the first-passage seed flask was used to inoculate the second-passage seed flask medium and was incubated as above for 24 hr to obtain vigorous vegetative growth of B. thuringiensis. Production
Flask
Six media, designated A to F, were used for 6-endotoxin production by B. thuringiensis. All media contained 0.2% yeast extract, 0.1% K,HPO,, and 0.1% KH*PO, as basal medium ingredients. To this, 1.0% of TABLE INFLUENCE
OF MEDIA
COMPOSITION
either dehusked greengram (Vigna radiata) powder or defatted soybean (Glycine man) powder and combinations of 0.5 or 1.0% soluble starch and cane sugar molasses were added (Table 1). The commercial samples of dehusked greengram and soybean, procured from the local market, were powdered using a table top dry grinder and then passed through 35mesh 1 inch screen (The Tyler Standard Screen Scale; U.S. series equivalent 40). Soybean powder was defatted before use by a Soxhlet extraction apparatus using petroleum ether (40”-6O”C) for 6 hr, followed by evaporation of the solvent overnight from the defatted powder. The final media were adjusted to pH 7.5 with 1 N NaOH and were distributed at 100 mYSOO-mlErlenmeyer flask. Flasks were covered with cotton-mesh pads and sterilized at 121°C for 20 min. The six media were inoculated with 2% by volume of the 1
ON THE GROWTH, TOXICITY, VAR. thuringiensis
A B C
D E F
U.S.
BMd + 1% greengram + 1% soluble starch BM + 1% greengram + 1% cane sugar molasses BM + 1% greengram + 0.5% soluble starch + 0.5% cane sugar molasses BM + 1% soybean + 1% soluble starch BM + 1% soybean + 1% cane sugar molasses BM + 1% soybean + 0.5% soluble starch + 0.5% cane sugar molasses standard HD-1-S-1980
thuringiensis
ratio
24 hr
48 hr
96 hr
4.8
6.5
7.0
7.5
39.7 (9.1)
133.9
14,900
3.7
7.0
7.5
7.5
49.5 (8.7)
52.1
38,300
4.2
6.5
7.0
7.5
38.5 (8.8)
195.7
10,200
2.4
6.5
8.0
8.5
91.3 (10.0)
55.7
35,800
1.8
7.5
8.5
8.5
38.3 (9.7)
57.1
34,900
2.1
7.0
8.0
8.5
88.6 (10.3)
168.1
11.800
1.24
124.9
16,000
C:N
Composition
OF Bacillus
VSC” (primary productb yield g/liter dry weight)
Broth pH after Media
AND POTENCY
a Viable spore count per milligram of product x 106. b Lyophilized with 100 ml of 6% lactose solution. c International Units. d Basal Medium containing yeast extract (0.2%), K,HPO, (O.l%), and KH$‘Q
LDso bdg body wt of Bombyx
mori)
Primary product potency (IU’/mg)
(0.1%) adjusted to pH 7.5
PRODUCTION
OF
S-ENDOTOXIN
B. thuringiensis broth from the secondpassage seed flask and incubated at 30” + 2°C on a rotary shaker (142 rpm). The pH of the broth was monitored at 24-hr intervals and adjusted with sterilized 1 N HCl to 7.5 whenever it increased. Broth samples taken periodically were subjected to microscopical examination for sporulation and crystal formation (Smimoff, 1962) until the end of the fermentation process. Recovery
When 90% of the sporangia had lysed, releasing the spores and crystals, the pH was adjusted to 7 with sterilized 1 N HCl, and the broth was centrifuged at 7500 r-pm at 10°C for 20 min. The spore-crystal complex was resuspended in 100 ml of 6% lactose (Dulmage et al., 1970)and lyophilized. The dry primary products were stored at 4°C for further studies. Estimation
of Viable Spore Count (WC)
One milligram of the primary products from each of the six media was placed in 10 ml of sterile, distilled water separately containing 0.02 ml of 1% Tween 80 solution and incubated in a water bath at 65°C for 15min to destroy the vegetative cells. The VSC was determined by counting serial dilutions plated on nutrient agar medium (Difco). Evaluation
of Toxicity
Bioassay of the primary products was made with fifth-instar larvae of the silk moth, Bombyx mod. Primary products (140 mg) from each of the six media and the U .S. standard HD-1-S-1980 of 16,000 IU/mg were suspended in 4 ml of sterile, distilled water separately containing 0.008 ml of 1% Tween 80 solution. Larvae were force-fed under a stereomicroscope with the help of a blunt, polished hypodermic 25-gauge needle on an Agla syringe (Ishiguro and Miyasono, 1979). Accurate administration of 1, 2, 4, 6, 8, 10, 12, 14, and 15 ~1 of sporecrystal suspensions was possible by this method. Twenty larvae were force-fed for each concentration of each sample. The
149
BY B. hringiensis
control larvae were force-fed with 15 ~1 of sterile, distilled water containing 0.03 ~1 of 1% Tween 80 solution. The VSC per 1 ul of all the suspensions was determined by plating on nutrient agar. After administration of the B. thuringiensis suspensions, the larvae were fed with fresh mulberry leaves ad libitum. Mortality was recorded after 24 hr. The LD,, values, and thereby the potencies, in terms of IU (Dulmage et al., 1981), of all the six primary products were calculated (Table 1). RESULTS
AND DISCUSSION
Cultures could be harvested after 96 hr of incubation from media D, E, and F, and after 120 hr from media A, B, and C. The product yield (dry weight basis, including 6 g of lactose) ranged from 8.7 to 9.1 g/liter for media A, B, and C for dehusked greengram powder and 9.7 to 10.3 g/liter for media D, E, and F for defatted soybean powder as the major protein source. This variation could be due to the cumulative effect of the quality and quantity of protein and carbohydrate and their ratio in the respective media. Table 1 shows that media A, B, and C had higher carbohydrate content, with C:N ratios ranging from 3.7 to 4.8, whereas the C:N ratio ranged from 1.8 to 2.4 in media D, E, and F. The C:N ratios of the media were calculated from data from different references of C and N content of the substrates (Anonymous, 1970; Gopalan et al., 1981; Hough et al., 1982; Norman, 1978; Paturau, 1969). The higher carbohydrate content of media A, B, and C might have prolonged the vegtative exponential phase of the organism, delaying sporulation and lysis compared to media D, E, and F cultures, which could be harvested earlier. Media D, E, and F turned more alkaline at the end of 48 and 96 hr of shake incubation, and hence required pH adjustment to 7.5. Because of the higher carbohydrate content in media A, B , and C , the pH remained near neutral throughout the fermentation process. The maximum VSC of 91.3 x 106/mgwas
150
MUMMIGATTI
AND
obtained from medium D, defatted soybean powder and soluble starch, and had a corresponding potency of 35,800 IU/mg (Table 1). However, the product from medium B, dehusked greengram powder and cane sugar molasses, having only 49.5 x IO6 VSC/mg, recorded the highest potency of 38,300 IU/mg, inferring that the quality of the &endotoxin produced differed with the nutritive value of the media. Both dehusked greengram and defatted soybean powders in combination with cane sugar molasses (media B and E, respectively) and defatted soybean powder with soluble starch (medium D) yielded products 2.1 to 2.4 times more potent than the U.S. standard. Media A, C, and F yielded less potent products than the U.S. standard. It is evident, that when both soluble starch and cane sugar molasses are combined as the carbohydrate source, as in media C and F, irrespective of the protein source, drastic reduction of toxicity of the primary products occurs. This could be due to the production and accumulation of B. thuringiensis 6-endotoxin biosynthesis inhibitors in the media. The primary product harvested from medium A, which contains the highest amount of carbohydrate (Table l), was also less toxic than the US standard. Salama et al. (1983) screened various legume seeds and reported that 2% soybean in the basal medium produced fewer spores from B. thuringiensis var. kurstaki and B. thuringiensis var. entomocidus than kidney bean and chick pea, both of which supported good spore production and endotoxin production. Obeta and Okafor (1984) reported that media containing bambara bean powder (0.75%) and cow blood powder (1.O%) in basal medium produced a highly toxic primary product of B. thuringiensis var. israelensis. In the present study, medium B containing dehusked greengram protein, which is cheaper than the defatted soybean protein and cane sugar molasses, gave the most toxic B. thuringiensis biocide. Potency of a B. thuringiensis product against a particular
RAGHUNATHAN
pest is known to vary with the media ingredients, the B. thuringiensis strain, and the growth conditions. The balance between the amino acids glutamic acid, aspartic acid, arginine, glycine, and proline, which are necessary for B. thuringiensis Gendotoxin biosynthesis (Mm-thy and Rana, 1980; Rajalakshmi and Sethna, 1977), and the amino acids lysine, threonine, isoleucine, leucine, valine, histidine, tyrosine, serine, and cystine, which inhibit 6-endotoxin biosynthesis (Conner and Hansen, 1967; Rajalakshmi and Sethna, 1980; Singer and Rogoff, 1968), in the fermentation medium determines the production of Gendotoxin and thus the toxicity and the potency of the B. thuringiensis primary product. The high potency of the primary product against a target pest determines its superiority over others. This study revealed the intricate role played by the individual components of the fermentation medium in supporting the growth of B. thuringiensis var. thuringiensis and the production of S-endotoxin. However, more detailed study is required for the possible utilization of legume seeds and their by-products as major protein sources in large-scale fermentation of B. thuringiensis varieties. ACKNOWLEDGMENTS We thank the Director, CPIRI, Mysore, and the Area Co-ordinator, Infestation Control and Protectants Discipline, CFIRI, Mysore, for providing the facility where this work was carried out. We are also grateful to Dr. S. C. Basappa, Scientist, Microbiology and Sanitation Discipline, CFI’RI, Mysore, for his critical reading of the manuscript.
REFERENCES 1970. “Pulse Crops of India.” Indian Council of Agricultural Research, New Delhi. CONNER, R. M., AND HANSEN, P. A. 1%7. Effects of valine, leucine and isoleucine on the growth of Bacillus thuringiensis and related bacteria. J. Invertebr. Pathol., 9, 12-18. DULMAGE, H. T., CORREA, J. A., AND MARTINEZ, A. J. 1970. Coprecipitation with lactose as a means of recovering the spore crystal complex of Bacillus rhuringiensis. J. Znvertebr. Pathol., 15, 15-20. ANONYMOUS
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NORMAN, A. G. (Ed.) 1978. “Soybean Physiology, isolates of Bacillus thuringiensis and their potential Agronomy and Utilization.” Academic Press, New for pest control. In “Microbial Control of Pests and York. Plant Diseases, 1970-1980” (H. D. Burges, Ed.), OBETA,J. A., ANDOKAFOR, N. 1984. Medium for the pp. 193-222. Academic Press, New York. production of primary powder of Bacillus thuringGOPALAN, C., RAMASASTRI, B. V., ANDBALASUBIU- iensis sub.sp. israelensis. Appl. Environ. MicroMANIAN,S. c. 1981. “Nutritive Value of Indian biol., 47, 863-867. PATURAU,J. M. 1969. “By-products of the Cane Foods.” Indian Council of Medical Research, New Delhi. Sugar Industry: An Introduction to Their Industrial HOUGH,J. S., BRIGGS,D. E., STEVENS, R., AND Utilization.” Elsevier, Amsterdam. YOUNG,T. W. 1982. Yeast growth. In “Malting and RAJALAKSHMI, S., ANDSETHNA,Y. I. 1977.The efBrewing Science,” Vol. 2, “Hopped Wort and fect of amino acid on the growth, sporulation and crystal formation in Bacillus thuringiensis var. thuBeer,” 2nd ed., pp. 615-643. Chapman & Hall, New York. ringiensis. J. Indian Inst. Sci., 59, 161176. ISHIGURO, T., ANDMIYASONO,M. 1979. A bioassay RAJALAKSHMI, S., AND SETHNA,Y. I. 1980. Spore for toxicity of Bacillus thuringiensis insecticide usand crystal formation in Bacillus thuringiensis var. thuringiensis during growth in cystine and cysteine. ing the force feeding method applied to the silkworm, Bombyx mori L. Japan. J. Appl. Entomol. J. Biosci., 2, 321-328. Zool., 23, 141-150. SALAMA,H. S., FODA,M. S., DULMAGE,H. T., AND MURTHY,K. G. K., ANDRANA,R. S. 1980. Role of EL-SHARABY, A. 1983. Novel fermentation media glutamic acid and glutamate dehydrogenase in for production of 8-endotoxins from Bacillus thuringiensis. J. Znvertebr. Pathol., 41, 8-19. sporulation of Bacillus thuringiensis var. thuringiensis in chemically defined media. Indian J. Biochem. SINGER, S., ANDROGOFF, M. H. 1968. Inhibition of Biophys., 17, 157-158. growth of Bacillus thuringiensis by amino acids in NAGAMMA,M. V., RAGHUNATHAN, A. N., ANDMAdefined media. J. Invertebr. Pathol., 12, 98-104. JUMDER, S. K. 1972. A new medium for Bacillus SMIRNOFF, W. A. 1962. A staining method for differthuringiensis Berliner. J. Appl. Bacterial., 35, 367entiating spores, crystals and cells of Bacillus thur370. ingiensis (Berliner). J. Insect Pathol., 4, 38k386.
