Effect of Cry+ Bacillus thuringiensis cells and fermentation condition on consistent production of δ-endotoxin

Abstract

The method for preparation of the competent Cry+ Bacillus thuringiensis strain stock culture to overcome inconsistent production of δ-endotoxin in stirred tank bioreactor was investigated. The selection of Cry+, which has stable production of δ-endotoxin was achieved by repeated isolation of colonies until stable types of B. thuringiensis were obtained. The polymerase chain reaction (PCR) approach was used to analyse the presence of cry genes in the isolates. Cry+ cells were freeze-dried to prepare competent stock culture in a powderized form. Results indicate that inoculum prepared using the powderized form managed to achieve high maximum cell concentration (˃1.0 x 10¹² cfu/ml) and spore count (˃3 x 10¹¹ spore/ml), but a longer cultivation time (16 h) was required. The Cry+ strain was able to form larger aggregates during the growth phase contrary to the Cry- strain. Glucose concentration and dissolved oxygen tension (DOT) level in the culture significantly affected spore and  δ-endotoxin formation during the cultivation of Cry+ B. thuringiensis in a 5 L stirred tank bioreactor. In comparison to cultivation without glucose supply and a high dissolved oxygen tension (DOT) level, cultivation with 8 g/L glucose and controlled DOT at 80% saturation produced the highest viable cells (1.3 x 10¹² cfu/ml), with a high sporulation rate and the existence of δ-endotoxin.