Growth kinetics of Bacillus thuringiensis batch, fed-batch and continuous bioreactor cultures

Cells of Bacillus thuringiensis during their late stationary growth phase produce both spores and the bioinsecticidal crystal protein, delta-endotoxin. B. thuringiensis cells also evolve depending on the environmental conditions. Thus, it is necessary to adopt a broad approach to study the growth kinetics of B. thuringiensis considering not only the typical cell growth but also the metabolic changes occurring while spores and crystal protein are being produced.; This study presents the results of cell growth kinetics study of Bacillus thuringiensis subspecies kurstaki, strain HD-1 (ATCC 33679), grown under batch and fed-batch growth conditions. In addition, B. thuringiensis growth was investigated under continuous steady-state growth conditions in a single stage 2-L bioreactor system and a two-stage continuous bioreactor system which consisted of a 2-L followed by a 15-L bioreactor system.; Optical microscopy and transmission electron microscopy (T.E.M.) were used extensively in this study to assess the morphology and metabolic state of B. thuringiensis cells at different growth conditions during batch, fed-batch and continuous steady-state experiments. Significant morphological differences in B. thuringiensis cells, as a function of growth time for the batch experiments and as a function of dilution rates in the steady-state continuous experiments were observed.; The separation of crystal protein from the fermentation broth for analytical purposes presents serious problems. In this study a new method to isolate the crystal protein was developed which produced crystal protein with a purity up to 95% ± 5%. Additionally, high performance liquid chromatography (HPLC) was employed to detect this protein.; Experimental results demonstrated the high dependence of the formation of spore and crystal protein with the type of culture. In batch cultures, the formation of spore and crystal protein caused a decrease in biomass growth rate, reducing the final biomass concentration. Whereas in the continuous culture, a reduction in biomass concentration was observed at low dilution rates. Finally, in fed-batch growth, no spores neither crystal protein were produced even though the limiting substrate was completely depleted.; An in depth analysis of the experimental data showed that the classical growth kinetic model cannot predict accurately the biomass concentration for the complete time span of a batch and at all the dilution rates of a continuous culture of B. thuringiensis. Thus, a new growth kinetic with sporulation model, which involved a term representing the specific spore formation rate, was developed and tested successfully with both batch and continuous experimental data.; Experimental results were also employed to determine new and reliable cell growth kinetic parameters (μ_max and K_S), as well as the growth yield and respiration rate coefficients for batch and continuous cultures.