World Journal of Microbiology & Biotechnology 15: 747±749, 1999.
Ó 1999 Kluwer Academic Publishers. Printed in the Netherlands.
747
Short Communication
Enhanced growth, sporulation and toxin production by Bacillus thuringiensis subsp. kurstaki in oil seed meal extract media containing cystine Dipak Vora1,* and Y.I. Shethna2 1 Department of Microbiology, Ramnarain Ruia College, Matunga, Mumbai 400 019, India 2 Department of Life Sciences, University of Mumbai, Mumbai 400 098, India *Author for correspondence: E-mail:
[email protected] Received 19 April 1999; accepted 3 August 1999
Keywords: Bacillus thuringiensis, cystine, d-endotoxin production, growth media oil seed extracts, sporulation
Summary A mineral salts medium supplemented with peptone and 40 mg% cystine supported enhanced sporulation (1011 spores/ ml) and high yields of insecticidal crystal protein (17.2 mg/ml) in Bacillus thuringiensis subsp. kurstaki (3a3b) in 2.0 l baed aerated cylinders. These high yields could also be achieved with defatted soybean and groundnut seed meal extracts when supplemented with cystine. Introduction
Materials and methods
The nutritional aspects of Bacillus thuringiensis have been studied extensively by a number of workers (Nickerson & Bulla 1975; Rogo & Yousten 1969; Singer et al. 1966). It is well established that various strains of B. thuringiensis would not grow in so-called mineral salt medium unless certain growth factors such as glutamic acid and either aspartate or citrate are added to the medium. Further, the addition of a known mixture of amino acids or casein hydrolysate allowed rapid growth of B. thuringiensis but sporulation was poor unless glucose was added (Singer et al. 1966). Cystine or cysteine, when added to a mineral salt medium promoted vegetative growth of B. thuringiensis (Nickerson & Bulla 1975). They also reported the role of various amino acids on lipid metabolism of B. thuringiensis. Sporulation was inhibited as long as cystine was present in the medium (Rajalakshmi & Shethna 1977). Gangurde and Shethna (1995) have demonstrated the eect of defatted mustard meal on B. thuringiensis subsp. israelensis and B. sphaericus. The present study is concerned with the eect of cystine on the growth, sporulation and crystal formation of B. thuringiensis subsp. kurstaki. In addition the eect of alternate locally available defatted oil-seed-derived protein sources supplemented with cystine were studied with a view of enhancing the commercial potential of the organism. We also present comparative data on the use of defatted seed meal extracts in specially designed fermentation cylinders in the presence and absence of cystine.
Bacteria and growth B. thuringiensis subsp. kurstaki (3a3b) was obtained from Dr M. Lecadet, Institut Pasteur. The organism was maintained on nutrient agar slopes (HiMedia, Mumbai) throughout the study. The organism was grown in a basal medium consisting of the following at a ®nal concentration in g/l in distilled water: MgSO4 á 7H2O 0.5, MnSO4 á 7H2O 0.1, FeSO4 á 7H2O 0.001, CuSO4 á 5H2O 0.0005, ZnSO4 á 7H2O 0.0005, CaCl2 0.1, Glucose 10.0. The medium also contained potassium phosphate buer 50 mM, pH 7.2, supplemented with various defatted seedmeal extracts and cystine, where indicated. The mineral salts, glucose, buer and CaCl2 solutions were sterilized separately and mixed to give the complete medium (CBM). Defatted seed meal extracts when used, were prepared according to the method of Gangurde and Shethna (1995) and incorporated so as to correspond to 5.0 g protein/l in the mineral salt solution itself. The growth, sporulation and alkali soluble protein (dendotoxin) content in the fermentation medium were compared against media containing 5.0 g peptone/l as a laboratory benchmark because glucose and peptone together gave the best results under laboratory conditions. Cystine was suspended in 10.0 ml of phosphate buer and autoclaved. This suspension was vortexed and added to the other ingredients.
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D. Vora and Y.I. Shethna
Analytical methods
Scale-Up
Estimation of growth and sporulation The organism was grown on nutrient agar slopes for 24 h at 30 °C. The entire growth was washed o and suspended in 20.0 ml of saline and vortexed vigorously to obtain a homogenous suspension, which was added to 100 ml CBM at the rate of 1.0 ml per 100 ml CBM and corresponded to an inoculum of approx. 1 ´ 106 c.f.u/ml. The ¯asks, in duplicate, were incubated at 30 °C on a rotary orbital shaker (180 rev/min) having a stroke of 2.54 cm. The total viable cell counts were determined by the spread plate method using nutrient agar plates. Serial dilutions were prepared in sterile saline and aliquots of 0.1 ml of the appropriate dilution were spread on the plates in duplicate and each count was repeated. The total viable counts thus represent the sum of vegetative cells and spores present in the sample. Spore counts were determined by heating 1.0 ml of the freshly sampled homogenized aliquot of the fermented broth at 80 °C for 15 min (Dulmage 1970) in a water bath after which dilutions were prepared and plated as above. The plates were incubated at 30 °C for 24 h and only those plates containing 15 to 200 colonies were used for enumeration. The values of total cell count and the spore count reported are the mean of the two sets of readings performed in duplicate. The spore-crystal complex (SCC) was recovered by the acetone co-precipitation method (Dulmage et al. 1970). Routine estimation of the d-endotoxin was carried out by determining the net alkali soluble protein (ASP) content of the acetone dried powders (Liu et al. 1995). Protein content was determined by the Lowry method. All data are the results of duplicate determinations. The presence of d-endotoxin was con®rmed at various stages by phase contrast microscopy.
Data was obtained using stainless steel aerated baed fermentation cylinders designed at the University Department of Life Sciences (Vora 1998) of 2.0 l volume (height = 22.5 cm, diameter = 11.0 cm) and having three baes 120° apart, each 1.5 cm wide and running 9.0 cm upwards from the base. The cylinders had a 3.0 cm diameter opening on top which could be plugged with cotton, for introduction of media and withdrawal of samples. These aerated cylinders were ®lled with 1.5 l CBM containing various seed meal extracts supplemented with cystine, and placed on a orbital shaker (250 rev/min, stroke 2.54 cm) ®tted with special clamps and incubated at 30 °C for 36 h. The use of such aerated cylinders gave higher aeration, similar to that obtained in fermenters and thus resulted in enhanced growth, sporulation and ASP production. Results Amongst all the carbon sources tested (data not shown) glucose (10 g/l) was found to support the highest yields. When sucrose was used instead of glucose, sporulation occurred earlier, but ASP yields were lower. Table 1 indicates the results obtained in CBM containing various defatted seed meal extracts and peptone respectively corresponding to 5.0 g protein/l prepared as described earlier and containing various concentrations of cystine under shake ¯ask conditions. Use of casein or of casein hydrolysate as the nitrogen source (data not shown) resulted in good growth but sporulation was poor. Table 2 depicts growth in the baed aerated fermentation cylinders to study the eect during a single step scale up. It was observed that 99% sporulation and high d-endotoxin production was achieved using sucrose
Table 1. Eect of defatted seed meal extracts on the growth, sporulation and ASP content of Bacillus thuringiensis in the presence and absence of cystine in CBM in 500 ml Erlenmeyer ¯asks. Cy
0 100 200 300 400 500 600 700
Peptone
Cotton
Soybean
Linseed
Groundnut
TC
S%
ASP
TC
S%
ASP
TC
S%
ASP
TC
S%
ASP
TC
S%
ASP
8.3 8.4 8.6 8.9 9.2 8.6 8.5 8.5
6.8 6.8 6.9 7.0 8.3 7.0 7.0 6.9
516 521 921 1403 1526 1384 1225 1200
8.6 8.5 8.6 8.6 8.7 8.7 8.7 8.7
93 92 93 93 95 94 94 93
295 286 335 377 416 415 397 399
8.5 8.5 8.6 9.0 8.7 8.7 8.7 8.8
96 94 94 95 93 90 88 90
547 876 1285 1523 1234 811 656 665
8.5 8.5 8.5 8.5 8.7 8.6 8.6 8.6
80 86 88 88 91 90 90 89
289 293 316 334 353 330 328 329
8.7 8.7 8.7 8.9 8.9 8.8 8.7 8.6
91 92 92 93 96 94 93 90
598 622 936 1315 1489 903 879 877
The data with peptone represents the control for comparison. TC = Total cell count expressed as log c.f.u/ml of fermented broth. S% = Percent of TC present as spores. ASP = Net alkali soluble protein (lg/ml) of fermented broth. The sugar used was glucose. Cy = Cystine mg/l of CBM. The highest value in each column is printed in bold.
B. thuringiensis growth and toxin production
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Table 2. Eect of defatted seed meal extracts on the growth, sporulation and ASP content of Bacillus thuringiensis in the presence and absence of cystine in 2.0 l fermentation aerated cylinders. Nutrients added
Incubation period 24 h
Incubation period 36 h
Seed meal extract
Carbon Cy source
TC
S%
ASP
TC
Peptone Peptone Peptone Peptone Linseed Linseed Groundnut Groundnut Cotton Cotton Soybean Soybean
Glucose Glucose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose Sucrose
9.57 10.09 10.06 10.09 9.06 9.89 9.86 9.91 9.77 9.81 9.96 10.06
59.0 65 95 97.1 84.5 89 93 97 87 96 97 99
4100 9.54 96.0 6070 11.63 99.0 5890 9.58 97.5 9730 9.64 98.0 3890 9.00 86.0 8230 9.85 88.0 6190 9.88 95.0 10230 9.90 98.5 4850 9.76 90.0 7380 9.83 96.0 6590 9.98 99.0 10630 9.98 99.0
00 400 00 400 00 400 00 400 00 400 00 300
S%
the instant demands of the farmer or preserved and formulated for use later (Burges 1998). The reduced input and processing costs could help induce the farmer to use bioinsecticides instead of chemical insecticides.
ASP
Acknowledgements 6150 17240 832 10150 3700 7660 5680 7950 4690 7530 7230 9360
TC = Total cell count expressed as log c.f.u/ml of fermented broth. S% = Percent of TC present as spores. ASP = Net alkali soluble protein lg/ml of fermented broth. Cy = Cystine mg/l of CBM. Carbon sources at 1% concentration. All seed meal extracts incorporated at ®nal protein concentration of 5.0 g/l. Peptone at 5.0 g/l was used as a control for comparison. The highest value in each column is printed in bold.
as the carbon source in 24 h as compared to that obtained using glucose. Peptone combined with glucose gave high yields of ASP in 36 h. The SCC preparations were also demonstrated to possess insecticidal activity against Heliothis armigera and Earias vitella when tested by the protocol of Kulkarni and Amonkar (1988). Discussion The viable counts of 1010 to 1011, percentage sporulation of 99% and ASP content of 10±17 mg/ml observed in the fermentation cylinders compare well with those reported for large scale fermentations (Bernard & Utz 1993; Liu et al. 1995). These high yields could also be achieved with defatted soybean and groundnut seed meal extracts when supplemented with cystine. It is thus possible to produce the insecticide quite inexpensively using the technique described. This fermentation broth with high spore count and ASP content could be sprayed directly on crops. This is important in developing countries where the bioinsecticide could be produced in regional laboratories in sucient quantities to meet
We thank the University of Mumbai for research grant No. sch/2440 of 1996, Dr M. Lecadet for providing the culture B. thuringiensis subsp kurstaki (3a3b), Dr H.D. Rananaware, Scientist, Nuclear Agriculture Division, BARC, Mumbai for help in the bioassays.
References Bernard, K. & Utz, R. 1993 Production of Bacillus thuringiensis insecticides for experimental and commercial uses. In Bacillus thuringiensis, an environmental biopesticide: Theory and Practice, eds Entwistle, P.F., Cory, J.S., Bailey, M.J. & Higgs, S. pp. 255± 267. Chichester, UK: John Wiley and Sons. ISBN 0-471-93306-6. Burges, H.D. 1998 Formulation of microbial Biopesticides, Bene®cial Microorganisms, Nematodes and seed Treatments. Dordrecht, The Netherlands: Kluwer Academic Publishers. ISBN 0-41262520-2. Dulmage, H.T., Correa, J.A. & Martinez, A.J. 1970 Co-precipitation with lactose as a means of recovering the spore crystal complex of Bacillus thuringiensis. Journal of Invertebrate Pathology 15, 15±20. Gangurde, R.P. & Shethna, Y.I. 1995 Growth, sporulation and toxin production by Bacillus thuringiensis subsp. israelensis and B. sphaericus in media based on mustard-seed meal. World Journal of Microbiology and Biotechnology 11, 202±205. Kulkarni, U.V. & Amonkar, S.V. 1988 Microbial control of Heliothis armigera (Hb): Part I ± Isolation and characterization of a new strain of Bacillus thuringiensis and comparative pathogenicity of three isolates of B. thuringiensis against H. armigera. Indian Journal of Experimental Biology 26, 703±707. Liu, W.-M. & Bajpai, R.K. 1995 A modi®ed growth medium for Bacillus thuringiensis. Biotechnology Progress 11, 589±591. Nickerson, K.W. & Bulla, L.A. Jr. 1975 Lipid metabolism during bacterial growth, sporulation and germination: an obligate nutritional requirement in Bacillus thuringiensis for compounds that stimulate fatty acid synthesis. Journal of Bacteriology 123(2), 598± 603. Rajalakshmi, S. & Shethna, Y.I. 1977 The eect of amino acids on growth, sporulation and crystal formation in Bacillus thuringiensis var. thuringiensis. Journal of the Indian Institute of Science 59(12), 169±176. Rogo, M.H. & Youston, A.A. 1969 Bacillus thuringiensis: Microbiological considerations. Annual Review Microbiology 23, 357±386. Singer, S., Goodman, N.S. & Rogo, M.H. 1966 De®ned media for the study of Bacilli pathogenic to insects. Ann. NY. Acad. Sci. 139(16), 16±23. Vora, D. 1998 Biological control of Insect Larvae. Ph.D Thesis, University of Mumbai, Mumbai.
