Abstract
Water stress and limited aeration imparted by solid-state fermentation (SSF) were reported as crucial factors for the enhancement of endospore production by Bacillus thuringiensis (Bt); and thus, more δ-endotoxin could be produced concomitantly with reduced time. Therefore, Bt subsp. kurstaki (Btk) was employed in the present study to evaluate its efficiency for the concomitant production of endospores and δ-endotoxin in LB medium supplemented with various naturally available agricultural products, i.e., flours of soybean, Bengal gram or jack seed at various concentrations (10%, 20%, 30%, 40%, 50%, 60%, 80% or 100%, all w/v). After 12 h fermentation, the supernatant in it was centrifuged off aseptically to obtain solid substrate for subsequent SSF. Of them, soybean (30%) supplemented medium was the best for the enhanced production of endospore and δ-crystals. The maximum yield of endospores during solid-state fermentation was observed 48 h, i.e., compared to submerged fermentation in LB, it was 24 h less gestation period. In control sample, the endospores achieved the maximum length (1.10 ± 0.13 μm) and diameter (0.63 ± 0.07 μm) at 72 h; while in soybean supplemented medium, the maximum length (2.10 ± 0.16 μm) and diameter (1.63 ± 0.16 μm) were at 48 h and 72 h, respectively. Upon staining, acridine orange specifically stained the endospores; malachite green-saffranin stained both δ-crystals and endospores; and coomassie brilliant blue specifically stained δ-endotoxin. Briefly, normal gestation period or harvest time for Btk is 72 h, which could be reduced to 48 h, if SSF is employed as demonstrated in this study.
Highlights
Bacteria of the gram positive genus Bacilli include a variety of commercially important species responsible for the production of a range of products including enzymes, antibodies and insecticides
This study makes a comparison of the concomitant production of endospore and δ-endotoxin by Bt subsp. kurstaki (Btk) in LB against 30% (w/v) soybean flour supplemented LB, 30% (w/v) Bengal gram flour supplemented LB and 10% (w/v) jack seed flour supplemented LB
Three staining techniques were used, viz., malachite green-safranin, acridine orange and coomassie brilliant blue; the former two were specific for visualizing endospores and the latter was for δ-endotoxin
Summary
Bacteria of the gram positive genus Bacilli include a variety of commercially important species responsible for the production of a range of products including enzymes, antibodies and insecticides. Bacillus thuringiensis Berliner (Bt) is a ubiquitous Gram-positive and sporulating bacterium producing insecticidal crystal proteins (the δ-endotoxin) juxtaposed to the endospores in the bacterium (sporangium) during the stationary phase of its growth cycle [1]. SSF has been developed in eastern countries over many centuries for the large scale production of microbial products including primary and secondary metabolites [4]. Commercial production of Bt toxin has been achieved by submerged or liquid fermentation (SmF), or by batch or fed-batch process [5], but advantages of SSF for the production of both primary and secondary metabolites of microbial origin have well been appreciated by many investigators [6] [7]. Compared to SmF, SSF received more attention recently, as it uses simpler fermentation medium and requires a smaller space, easier to aerate, lower waste water output, lower energy requirement and contamination, higher productivity, extended stability of the products and low production costs [4]
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