## 13 英文概要

Project Code : 3K123007

Research Title : Biofuel Production from Glycerol Discharged after Bio-Diesel Manufacturing Process

Research Institution : University of Tsukuba

Principal Investigator : Toshiaki Nakajima-Kambe

Biodiesel fuel (BDF) is a biomass fuel that is used as an alternative fuel of diesel oil. BDF, a carbon neutral fuel, is also made from vegetable oils and animal fats. Almost all BDF production is carried out by transesterification with methanol in the presence of an alkaline catalyst. However, BDF waste has a high alkaline pH, and contains a large amount of crude glycerol as a by-product. Since almost all BDF-derived glycerol is waste oil-derived, it has no applied use, and is almost always incinerated. Therefore, the treatment of BDF-derived crude glycerol is an environmental problem. Bacteria that can utilize glycerol as a sole carbon source are known to produce some valuable metabolites. Among them, ethanol is particularly valuable as its biomass energy is similar to that of BDF. The production of bio-ethanol is an efficient use of waste and decreases burdens on the environment.

We isolated a glycerol-assimilating bacterium, Klebsiella variicola strain TB-83D. Strain TB-83D was able to grow in the presence of 7% (v/v) ethanol and it showed higher ethanol production. Examination of various culture conditions revealed that yeast extract was essential for ethanol production and bacterial growth. In addition, ethanol production was elevated by the addition of yeast extract and corn steep liquor (CSL), with a production of 34 g/L.

We also tried to acquire some formic acid tolerant mutants because it was proven that the ethanol production of strain TB-83D was strongly inhibited by formic acid which is a major by-product. UV and proton suicide methods were used to get mutants that are resistant to formic acid. Some of the mutants showed higher ethanol productivity than their wild type (TB-83D). Then, a metabolic flux analysis was performed and its metabolic flow from glycerol to ethanol was clarified. As a result, it was revealed that the suppression of the lactic acid production pathway is most effective to maximize ethanol production.

Based on the results of the metabolic flux analysis and the culture condition experiments, it was shown that suppression of formic acid and lactic acid are necessary to increase the ethanol production by strain TB-83D. At first, a formic acid degrading enzyme (formate dehydrogenase: FDH) and its activator gene were reinforced by gene manipulation, but, in both cases, the mutants were not able to get enough expression. Then, the FDH gene derived from S. cerevisiae was introduced into strain TB-83D. As a result, the ethanol production of the mutant was increased.

Furthermore, a feasibility study of this project was performed. Comparison of the initial parameter settings of the feasibility study with current achievement of our study revealed that it was able to clear a break-even point at least on the laboratory scale.

Finally a pilot-scale fermentation was performed. A 300-L class anaerobic fermentor was built and ethanol production by strain TB-83D was performed using batch culture method. As a result, approximately similar productivity (glycerol consumption and ethanol production) with the laboratory scale experiment was shown in the pilot-scale fermentation.