The cell growth and sporulation of Bacillus thuringiensis var. kurstaki were investigated under various culture modes: batch culture, fed-batch culture, total cell retention culture (TCRC) and two-stage continuous culture. In latter two culture modes, TCRC and two-stage continuous culture, an internal ceramic filter was used to improve spore concentration and volumetric productivities.
In batch culture, culture media based on semidefined GYS medium were optimized to increase cell mass and spore concentration. Acetic acid produced from glucose during exponential growth was further catabolized in the early stationary phase of growth. As concentration of glucose was increased from 1 to 16 g/L, pH was sharply decreased and poor sporulation occurred. When 8 g/L of yeast extract was added, $6.6＼times10^8$ of spore was obtained and pH drop was prevented. Spore concentration measured at the time when complete sporulation had occurred linearly increased from $1.1＼times10^9$ to $7.6＼times10^9$ spores/mL as the concentration of glucose was increased form 10 to 50 g/L. However, further increase of concentration of glucose strongly inhibited cell growth and sporulation.
Fed-batch culture was carried out for increasing the production of spore of B. thuringiensis. Constant fed-batch culture (CFBC) and intermittent fed-batch culture (IFBC) were employed in this study. In CFBC, the production of spore decreased with an increase of the feeding glucose concentration. In IFBC, during which fast growth rate was maintained, spore concentration of $1.25＼times10^{10}$ spores/mL was obtained with maximum cell mass of 72.6 g/L, and was about three times higher than that of constant fed-batch culture when total glucose concentration of 100 g/L was consumed. It was found that the maintenance of fast growth rate was important in order to obtain the high concentration and volumetric productivity of spore and IFBC was more suitable than CFBC from the viewpoint of the higher spore concentration.
The production of spore of B. thuringiensis was investigated in a bioreactor incorporating a ceramic membrane filter to improve spore concentration and volumetric productivity. Two cultivation methods were used in this study: a total cell retention culture (TCRC) and a two-stage continuous culture with partial cell bleeding. In the TCRC fed by 50 g/L of glucose a spore concentration of $1.6＼times10^{10}$ CFU/mL was obtained with a spore percentage greater than 95% and a maximum cell mass of 82.2 g/L. The volumetric productivity was four times higher than for batch cultivation. In the two-stage continuous culture with partial cell bleeding spore concentration was strongly dependent on the bleed ratio. The spore concentration of $1.8＼times10^9$ CFU/mL was obtained at the second stage with a bleed ratio of 0.33, a dilution rate of 0.23 $h^{-1}$ and a spore percentage of 70%.
Growth and sporulation kinetics of B. thuringiensis in batch and fed-batch culture are mathematically modelled and the model was compared with experiments. The processes of growth and sporulation have been assumed to compete with each other in the vegetative organism. In batch culture, the conventional unstructured model based on Monod kinetics was in good agreement with the experiment because the formation of sporulated cells did not appear during exponential growth. In glucose-limited fed batch culture the modified unstructured model presented can be applicable to the case of the feeding of low glucose concentration, while the conventional unstructured model describes the behavior of the system fairly well in the case of the feeding of high glucose concentration.
