Abstract
The present study was aimed at designing a cost effective medium for increasing the δ-endotoxin (Cry protein) synthesis by Bacillus thuringiensis (Bt) kurstaki HD-73 and indigenous Bt JSc1 harboring potential cry genes active against Lepidoptera insect orders. In this regard, locally available cheap molasses as carbon source, soybean extract as nitrogen source, sea water as trace elements source, cystine as sporulation/growth factor were considered to design a cost effective medium. Molasses and soybean extract in place of glucose and peptone in glucose-peptone (GP) medium supported 78.85% increase in δ-endotoxin synthesis in shake flask culture. The effect of cystine on endotoxin synthesis was highly pronounced in two media with a range of 80.32 to 110% higher δ-endotoxin under comparable fermentation conditions. While, substituting basal salts with sea water, Btk HD-73 yielded satisfactory and comparable endotoxin (74.3% of yield with basal salts). It was detected that the rapid decrease of endotoxin synthesis in the culture after 24 h was due to the degradation by the endogenous protease, synthesized with the progress of fermentation. This degradation of the endotoxin was much better protected (1.23 mg/ml endotoxin versus 0.312 mg/ml) by adding 4% ammonium sulfate in the optimized medium. The medium thus formulated with molasses, soybean extract, ammonium sulfate, cystine and sea water was then used in 3.0 L bioreactor cultivation for endotoxin synthesis by both Btk HD-73 and Bt JSc1 under 30% saturation of dO 2 through cascade control of agitation and aeration producing a higher yield of δ-endotoxin (2.1 and 2.63 mg/ml, respectively). The present results may successfully be used for large scale production of Bt biopesticide in Bangladesh. Keywords: Bacillus thuringiensis biopesticide, cheap substrates, cystine, protease activity, ammonium sulfate.
Highlights
The control of pest populations by using biological pesticides has been an attractive alternative to the application of chemical pesticides (Glazer and Nikaido, 1995; Ciche and Ensign, 2003)
The present study reports the regulation of certain critical factors that affect the growth, sporulation and δ-endotoxin synthesis by reference B. thuringiensis kurstaki (Btk) HD-73 and indigenous Bacillus thuringiensis (Bt) strain JSc1
Industrial production of Bt biopesticide can facilitate its application in eco-friendly pest management for which higher yield in spore and δ- endotoxin production with low cost medium is one of the prerequisites to keep the product within farmers’ buying capacity
Summary
The control of pest populations by using biological pesticides has been an attractive alternative to the application of chemical pesticides (Glazer and Nikaido, 1995; Ciche and Ensign, 2003). Bt based microbial insecticides are available in world market, the high cost makes its application impracticable in large-scale in developing countries. The use of Bt as commercial insecticides would remain prohibitively and relatively expensive if it is not produced with high titer of insecticidal proteins by large-scale fermentation. More attention has been given to the regulation mechanisms that ensure the efficient production of the insecticidal proteins which could be achieved by application of an adequate fermentation technology (Zouari et al, 2002), essentially with use of appropriate media (Zouari and Jaoua, 1999), overcoming metabolic limitations (Zouari et al, 2002) etc. It was reported that the commercial application depends on the cost of raw materials, strain efficiency, fermentation cycle, maintenance of process parameters, bioprocessing of fermentation fluid, and formulation of the final product
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