| Summary: | 碩士 === 國立東華大學 === 生物技術研究所 === 87 === Bacillus thuringiensis is a gram-positive flagellated rod shaped microorganism. It is a spore-forming bacillus characterized by the formation of para-sporal protein crystals in the course of sporulation. The crystals which exhibit a high specific insecticidal activity can be produced by fermentation process.
In this study, we used the B. thuringiensis A3-4, a local strain and design a series of media formulation to propagate B. thuringiensis in shaker flask culture. We adjusted the carbon and nitrogen ratio by changing the concentration of glucose and yeast extract to determine the optimum medium. When the carbon and nitrogen ratio was 8, we got the highest spore yield and the crude crystal protein concentration. The spore concentration obtained was 2 e9 spore/mL of broth.
A modified airlift reactor with a net draft tube, which had a well flow direction, better gas and liquid dispersion, was used to investigate the production of the d-endotoxin in this studty. We changed the concentration of optimum medium (B-1, B-2, B-3) and conducted batch fermentation. The results showed that in the B-3 medium, spore concentration obtained was the highest (4.5 e9 spore/mL) and the crude crystal protein yield was 65.3 mg protein/g-carbon. The yield value was lower than that of B-1(86.4 mg protein/g-carbon) and B-2(86 mg protein/g-carbon) media, the reason could be the high glucose concentration of B-3 culture medium, it resulted in the inhibition of synthesis function.
Beside the batch fermentation, in this study we used the fed-batch fermentation to produce B. thuringiensis d-endotoxin. In the beging of the process, we used the lower glucose concentation mediun. Glucose was fed by different feeding rate during the fermentation. The results showed that the crude crystal protein yield was increased depending on the feeding rate. When the feeding rate was 0.667 g/min, the highest yield of the crude crystal protein, 105.5 mg protein/g-carbon was achieved.
The morphology of electron microscopy study indicated that batch and fed-batch fermentation both had well-grown endospores and parasporal crystalline endotoxin; however, the batch process had an earlier releasing of spore and crystal. It corresponded well with the results of cell, spore, and glucose studies in the fermentor.
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