Fed-batch fermentation studies with Bacillus thuringiensis H-14 synthesising endotoxin.

Cell yield and toxicity of B. thuringiensis H-14 was improved markedly by adopting a simple fed-batch fermentation technique based on controlling glucose concentration. Maintenance of steady glucose concentration (0.3-0.5%) in the culture medium was achieved by the continuous addition of concentrated glucose solution. Addition of glucose at 3 g/hr/l of culture starting from 3rd hr till 16th hr of fermentation was found to yield cell densities of 80 g/l (wet weight) which represented a nearly 3-fold increase over the batch mode. A fivefold increase in toxicity was obtained by fed-batch fermentation. Cultivation of B. thuringiensis H-14 to high cell densities had no negative effect on sporulation and toxin synthesis. The rate of pH drop and dissolved oxygen level were within manageable limits.

An enzyme linked immunosorbent assay for monitoring Bacillus sphaericus toxin.

Larvicidal proteins of B. sphaericus H5a5b (strain VCRC B42), purified by ion-exchange chromatography were used to raise antibodies in rabbits. The antibodies were specific in reacting to alkali-solubilized fractions from whole cells of toxic strains only. Ouchterlony immunodiffusion showed homology in toxin structure between strains of different serotype. A sandwich ELISA using avidin-biotin amplification was standardized. The lowest detectable limit was 6.25 ng/ml. Near linear quantitative binding of the antigen was found in the range 25-200 ng/ml. The growth, toxin level and LC50 values during various stages of fermentation of B. sphaericus strains 1593 and B42 were compared. There was significant correlation between LC50 values and toxin levels as measured by ELISA.

Evaluation of cyclosporine-A obtained from Tolypocladium sp. for immunosuppressive potential.

Cyclosporine-A obtained from an indigenous isolate of Tolypocladium sp. was evaluated for immunosuppressive potential by skin grafting in rats using the commercially available Sandimmune as reference at 15 and 30 mg/kg/day for a period of 14 days. The Sandimmune treated animal group showed 83 per cent acceptance of skin grafts while the test preparation treated group showed 67-83 per cent acceptance. The degree of nephro- and hepatotoxicity was comparable for both the preparations.

Evaluation of Bacillus sphaericus formulations against the vector of bancroftian filariasis.

Three different formulations of Bacillus sphaericus viz, Spherimos, Vectobac and Spherifix, were evaluated for their efficacy and residual activity against Culex quinquefasciatus breeding in polluted disused wells. Spherimos, a flowable concentrate formulation, exerted 96-100% control when treated at the dosage of 10 l/ha for 17 days, whereas the effective residual activity lasted up to 67 days at 15 l/ha. In the case of Vectolex, a granular formulation, the residual activity lasted up to 56 days with the dosage of 30 l/ha and up to 66-77 days with higher dosages of 45 and 60 l/ha. The residual activity of Spherifix, a floating controlled release formulation, lasted up to 67 days with a dosage of 10 kg/ha.

Effect of corn-steep liquor on growth and mosquito larvicidal activity of Bacillus thuringiensis var israelensis de Barjac 1978 and B. sphaericus Neide 1904.

Influence of corn steep liquor on the cell yield and toxicity of three strains of B. thuringiensis var israelensis and two strains of B. sphaericus was studied and compared with peptone-yeast extract using a laboratory fermentor. Large increase in the cell yield of all the three strains of B. thuringiensis var israelensis was observed when cornsteep liquor was used as the sole nitrogen source. Significant increase in toxicity was also observed in B. thuringiensis var israelensis strains B17 and B113. Among the two B. sphaericus strains tested, the strain 1593 showed no significant change in cell yield and toxicity, whereas the strain VCRC B42 showed increased cell yield and toxicity in this medium. The results indicate that cornsteep liquor can effectively replace both peptone and yeast extract in the media presently used for large scale multiplication of the two larvicidal bacilli.

Extra cellular hydrolytic enzyme secretion in Bacillus thuringiensis H14 & B. sphaericus & their significance in media design.

Level of extracellular proteolytic and amylolytic enzyme production was determined to assess the ability of these strains in utilizing complex carbon and nitrogen sources. Protease secretion reached maximum at around 12th h of growth in case of all the three B. thuringiensis strains and declined sharply thereafter. In B. sphaericus strains the protease level gradually increased and reached maximum at around 24th h of growth. Amylase activity was not detectable in the culture supernatants of B. sphaericus strains whereas all the three B. thuringiensis strains tested showed significant amount of amylase activity.