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.
