Production of delta endotoxins by Bacillus thuringiensis using complex substrates

Abstract

The Bacillus thuringiensis delta endotoxins are widely used as insecticidal proteins. These crystalline inclusions show a wide range of specificity for different insect orders such as Lepidoptera, Coleoptera, Diptera and also to nematodes. Moreover, productivity of crystal proteins can be regulated by optimizing the concentration of complex substrates yielding an economic medium. On the other hand,agro-industrial raw materials and waste products are constantly produced being their final disposal sometimes associated with several environmental problems. Thus, bioconversion of these cost-effectivesubstrates that are also locally available is turning in an interesting and a useful approach. In this connection, we previously applied a sequential optimization strategy involving a Plackett-Burman and a full factorial experimental design to maximize the production of crystal proteins; as a result the followingculture medium formulation was obtained (in g/l): milk serum 7.5, starch 3.0 and soybean meal 10.0.In this work, both the supplementation of 10.0 g/l of vinasse (X1) and the ratio reactor volume/workingvolume (X2) were evaluated using a full factorial design. The native Bacillus thuringiensis RT from our own culture collection was used throughout this study. The crystal protein (Cry) concentration was determined by the method of dye elution in SDS-isopropanol using bovine serum albumin as a standard.Fermentations were carried out during 72 h at 30 °C and 200 rpm. Our results indicated that X1 has a significant and a positive effect on the production of both Cry 1 Ac (p=0.008) and Cry 2 Ab (p<0.001).While, X2 (p<0.001) and the interaction X1-X2 (p<0.001) only have a significant impact on the Cry 2 Ab production. In addition, the adequacy of each model was verified by the R2 (> 88.67) and the Adj R2 (> 80.17) coefficients indicating the percentage of variability that is explained by the model. Thus, our finding revealed that a careful balance of culture conditions should be established to increase delta endotoxins production. In addition, the biological activity of the improved product against Spodoptera frugiperda was also discussed.