Improvement of bioinsecticides production through adaptation of Bacillus thuringiensis cells to heat treatment and NaCl addition.

AIMS: The present work aimed to increase yields of delta-endotoxin production through adaptation of Bacillus thuringiensis cells to heat shock and sodium chloride and to investigate their involvements in bioinsecticides production improvement. METHODS AND RESULTS: Growing B. thuringiensis cells were heat treated after different incubation times to study the response of the adaptative surviving cells in terms of delta-endotoxin synthesis. Similarly, adaptation of B. thuringiensis cells to sodium chloride was investigated. Adaptation to combined stressors was also evaluated. When applied separately in the glucose-based medium, 20-min heat treatment of 6-h-old cultures and addition of 7 g l(-1) NaCl at the beginning of the incubation gave respectively 38 and 27% delta-endotoxin production improvements. Heat shock improved toxin synthesis yields, while NaCl addition improved delta-endotoxin production by increasing the spore titres without significant effect on toxin synthesis yields. Cumulative improvements (66%) were obtained by combination of the two stressors at the conditions previously established for each one. Interestingly, when the similar approach was conducted by using the large scale production medium based on gruel and fish meal, 17, 8 and 29% delta-endotoxin production improvements were respectively, obtained with heat shock, NaCl and combined stressors. CONCLUSIONS: Heat treatment of vegetative B. thuringiensis cells and NaCl addition to the culture media improved bioinsecticides production. Heat treatment increased toxin synthesis yields, while addition of NaCl increased biomass production yields. Cumulative improvements of 66 and 29% were obtained in glucose and economic production media, respectively. SIGNIFICANCE AND IMPACT OF THE STUDY: Overproduction of bioinsecticides by B. thuringiensis could be obtained by the combination of heat treatment of vegetative cells and addition of NaCl to the culture medium. This should contribute to a significant reduction of the cost of B. thuringiensis bioinsecticides production and utilization, and also manage for higher toxin content in the bioinsecticides, which is very interesting from a practical point of view because fewer spores would be disseminated into the ecosystem.

Improvement of bioinsecticides production through mutagenesis of Bacillus thuringiensis by u.v. and nitrous acid affecting metabolic pathways and/or delta-endotoxin synthesis.

AIMS: The present work aimed to obtain bioinsecticide over-producing mutants through classical mutagenesis of vegetative cells of Bacillus thuringiensis (Bt) by using u.v. and nitrous acid, and to evidence the involvement of cell metabolism in delta-endotoxin synthesis. METHODS AND RESULTS: Vegetative cells of Bt were treated by nitrous acid (0.17 mg ml(-1)) or exposed to u.v. rays (emitted at a wave length of 240 nm). The isolated survivors were screened on the basis of the production of delta-endotoxins and biomass in glucose and/or in gruel-based media at two aeration conditions. Bioinsecticide over-producing mutants were obtained with high frequencies because random mutations were shown to affect cell metabolism at different pathways related to the regulation of delta-endotoxin synthesis. CONCLUSIONS: Classical mutagenesis of Bt cells lead to the isolation of a large variety of delta-endotoxin over-producing mutants that could be classified into six groups based on the location of the mutations, particularly in metabolism pathways and delta-endotoxin synthesis. SIGNIFICANCE AND IMPACT OF THE STUDY: High frequencies of delta-endotoxin over-producing mutants of Bt could be obtained through classical mutagenesis of vegetative cells. This should contribute to a significant reduction of production and utilization costs of Bt bioinsecticides.