Development of a cost effective medium for enhanced production of Bacillus thuringiensis δ-endotoxin

Abstract

The control of pest populations by using bacteria as insect pathogens has been an attractive alternative to the application of chemical pesticides. As chemicals cause damage to the environment, biological control is preferable and Bacillus thuringiensis (Bt) which produces insecticidal δ-endotoxin (crystal protein) have been most widely used as biopesticide in agriculture. But to date no broad based target oriented work on Bt mass production has been initiated in Bangladesh. The present study was aimed at enhancing the δ-endotoxin synthesis in a suitable medium by Bacillus thuringiensis subsp. kurstaki (Btk) HD-73 harboring potential cry genes active against lepidoptera insects by regulating some key components of medium such as carbon and nitrogen sources, amino acid (cystine) and basal salts affecting fermentation. In this regard, the growth, sporulation and δ-endotoxin synthesis by Btk HD-73 were examined in the culture of different media at 30ºC in shake and bioreactor culture conditions. The experiment was carried out under both monophasic submerged fermentation (SmF) and then biphasic solid state fermentation (SSF) so as to which facilitates sporulation under its stressed conditions. In SmF condition, the conventional Luria-Bertani (LB) medium which was enriched with nitrogen source (10% defatted soybean meal) supported 28.57% sporulation and 125% endotoxin increase over LB (alone). In biphasic SSF condition although the sporulation increased but the endotoxin yield was decreased when compared with monophasic SmF condition. The effect of cystine on sporulation and endotoxin synthesis was highly pronounced in LB-soybean medium (LBS) with a range of 19.54% and 131.35% higher endotoxin yield respectively in SmF condition. The basal salts-soybean-cystine (SMc) medium resulted in 7.65% decrease in endotoxin production than in LB-soybean-cystine (LBSc) medium, but it is comparable. Addition of molasses balanced the C:N ratio in the SMc medium thus helping 84.85% higher endotoxin synthesis after 24 hours fermentation. Substitution of basal salts with cost effective sea water yielded about 21% less endotoxin. For large scale production, use of soybean extract than that of soybean mass in the culture medium supported better performance. The optimum medium thus obtained consisting of soybean extract-molasses-cystine with sea water was used in 3L bioreactor cultivation for endotoxin synthesis by Btk HD-73 under 30% saturation of dO2 through cascade of agitation and aeration. The production rate obtained was 1.67 fold higher in bioreactor than in shake culture. The present results may successfully be used for large scale production of biopesticide in Bangladesh.