Biphasic Fermentation Is an Efficient Strategy for the Overproduction of δ-Endotoxin from Bacillus thuringiensis

Abstract

This study illustrates a biphasic solid-state fermentation (SSF) strategy for the overproduction of δ-endotoxin from Bacillus thuringiensis subsp. kurstaki (Btk) and also purification of δ-endotoxin from the solid-fermented medium. The fermentation strategy had two phases (biphasic); i.e., the first short phase was semisolid state (12 h), and the remaining long phase was strict SSF. To achieve the biphasic SSF, after 12 h (150 rpm, 37 °C) fermentation of the medium [Luria-Bertani (LB) supplemented with 30 % (w/v) raw soybean flour (phase I)], the supernatant in it was completely centrifuged out (1,000 × g, 10 min) aseptically for harvesting the extracellular enzymes as by-product. The resultant wet solid matter without free-flowing liquid but with embedded Btk was incubated 60 h more (phase II) for enhancing δ-endotoxin production at static condition (37 °C). Coupled with this, δ-endotoxin was purified by the modified phase separation method, and its purity was physically confirmed by both staining and microscopic techniques. The maximum δ-endotoxin yield from solid medium (48 h) was 15.8 mg/mL (recovery was 55-59 %) LB-equivalent, while that of LB control (recovery was 95 %) was only 0.43 mg/mL (72 h), i.e., thus, in comparison, 36.74-fold more yield in solid medium obtained by 24 h less gestation period. The purified crystal proteins showed apparent molecular weights (MWs) of 45, 35, and 6 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Briefly, this unique study physically demonstrates how Btk δ-endotoxin is purified (95-99 % purity) from solid-fermented matter for the first time, coupled with its overproduction at the expense of only 21.5 % higher production cost.

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“…) and 30% soya bean flour (Jisha et al. ) supplemented LB medium (solid‐state fermentation), respectively, against unsupplemented LB liquid medium. Polymorphic δ ‐endotoxin crystals were produced by the Bt strain reported in this study; similarly bipyramidal, cuboidal and spherical crystals or the combinations of these structures were produced by various strains of Bt (Attathom et al.…”

“…Upon ingestion of δ ‐endotoxin, the alkaline condition in the insect gut facilitates the solubilization of the crystal and subsequent cleavage; thus, the inactive δ ‐endotoxin crystal form (protoxin) is converted into an active and toxic form (Jisha et al. ).…”

“…However, higher quantities of d-endotoxin yield were reported from solid media, for instance, our group showed sixfold and 37-folds enhancement in the yield of d-endotoxin with Bt subsp. kurstaki on 10% potato flour ) and 30% soya bean flour (Jisha et al 2014) supplemented LB medium (solid-state fermentation), respectively, against unsupplemented LB liquid medium. Polymorphic d-endotoxin crystals were produced by the Bt strain reported in this study; similarly bipyramidal, cuboidal and spherical crystals or the combinations of these structures were produced by various strains of Bt (Attathom et al 1995).…”

“…Similarly, the use of purified and activated crystals entails a greater risk of lesser activity and its decline during storage, than the use of a crude spore and d-endotoxin mixture. Upon ingestion of d-endotoxin, the alkaline condition in the insect gut facilitates the solubilization of the crystal and subsequent cleavage; thus, the inactive d-endotoxin crystal form (protoxin) is converted into an active and toxic form (Jisha et al 2014). We adopted the leaf disc method for rearing the mites, wherein artificial diet feeding assay system was followed (Payne et al 1993).…”

“…Endospore production pattern of the isolate in LB was monitored at an interval of 12 h. Endospores were visualized by coomassie brilliant blue staining (Jisha et al. ) and observed with the aid of phase contrast microscope (Zeiss, China).…”