High-temperature fermentation (HTF) of ethanol can reduce costs of cooling, sterilization, and related equipment compared to the costs of general ethanol fermentation. To realize HTF, however, there are various issues to be considered, such as the fermentation temperature upper limit for ethanol-producing thermotolerant yeast, the size of a fermenter that does not require cooling, and the effective temperature for suppressing microbial contamination. This study focused on these issues and also on downstream processes that exploit the advantages of HTF at temperatures exceeding 40 °C. The permissible size of a fermenter without cooling was estimated by simulating heat generation and heat dissipation. Fermentation productivity at high temperatures when using the thermotolerant yeast Kluyveromyces marxianus and the inhibitory effect of high temperatures on the growth of contaminant microorganisms were examined. After fermentation, the recovery and concentration of ethanol were performed by reduced-pressure distillation (RPD) and membrane separation. These experiments demonstrate that efficient HTF can reduce the amount of saccharifying enzymes in simultaneous saccharification and fermentation and can shorten the transition time from the saccharification step to the fermentation step in separate saccharification and fermentation, that RPD at fermentation temperatures enables a smooth connection to the HTF step and can be performed with a relatively weak vacuum, and that membrane separation can reduce the running cost compared to the cost of general distillation on a compact scale.

Vip3A (vegetative insecticidal protein) is a representative member of the Vip3 family, which is widely used for lepidopteran pest control. This Vip3A protein, a non-growth-associated protein, is an effective bioinsecticide against insect pests, but there is relatively little information about its production processes at large scales. Hence, the effects of environmental factors on Vip3A production by Bacillus thuringiensis Bt294 (antifoam agents, shaking speeds, agitation and aeration rates), as well as controlling physical conditions such as the lowest point of dissolved oxygen and controlling of culture pH, were observed in shaking flasks and bioreactors. The results showed that antifoam agents, flask types and shaking speeds had significant effects on Vip3A and biomass production. Cultivation without pH control and DO control in 5 L bioreactors at lower agitation and aeration rates, which was not favorable for biomass production, resulted in a high Vip3A protein production of 5645.67 mg/L. The scale-up studies of the Vip3A protein production in a pilot-scale 750 L bioreactor gave 3750.0 mg/L. Therefore, this study demonstrated the significant effects of agitation, aeration rates and culture pH on Vip3A production by B. thuringiensis Bt294. Balancing of physical conditions was necessary for obtaining the highest yield of Vip3A by slowing down the production rate of biomass. Moreover, this Vip3A protein has high potential as a bioinsecticide for lepidopteran pest control in organic crops. This information will be important for significantly increasing the Vip3A protein concentration by the bacterium and will be useful for field application at a lower cost.