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B subtilis endospore stain1/15/2024 ![]() ![]() developed a quick and simple technique for the fluores- cent staining of bacterial endospores. Staining is the conventional method for distinguishing endospore from δ-crystals, and also for monitoring the time-dependent production pattern. Compared to SmF, SSF re- ceived 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. Convention- ally, commercial production of Bt toxin has been achieved by submerged or liquid fermentation (SmF), or by batch or fed-batch process, but advantages of SSF for the production of both primary and secondary meta- bolites of microbial origin have well been appreciated by many investigators. SSF has been developed in eastern countries over many centuries for the large scale production of microbial products including primary and secondary metabolites. The term solid-sate fermentation (SSF) denotes cultivation of microorganisms on solid, moist substrates in the absence of a free aqueous phase (water). Bt isolates are also found active against nematodes, mites and protozoa. The toxicity of Bt is due to its capacity for the production of a crystalline protein (the δ-endo- toxin) concomitantly with endospore and this observation led to the development of bioinsecticides, based on Bt for the control of certain insect species among the orders Lepidoptera, Diptera, and Coleoptera. 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. kurstaki, Spore, Solid-state fermentation, δ-endotoxinīacteria 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. 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.īacillus thuringiensis subsp. ![]() 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. 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. 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. Of them, soybean (30%) supplemented medium was the best for the enhanced production of endospore and δ-crystals. ![]() After 12 h fermentation, the supernatant in it was centrifuged off aseptically to obtain solid substrate for subsequent SSF. 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). 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. Received 17 July 2014 revised 19 August 2014 accepted 2 September 2014 This work is licensed under the Creative Commons Attribution International License (CC BY). Enzyme Technology Laboratory, Biotechnology Division, Department of Botany, University of Calicut, Kerala, IndiaĮmail: * © 2014 by authors and Scientific Research Publishing Inc. ![]()
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