Strategies for enhancement of isobutanol production by saccharomyces cerevisiae wild type

Abstract

Isobutanol is one of the effective biofuels that can be produced from renewable biomass through microbial fermentation. This alcohol is a better alternative compared to bioethanol in order to replace gasoline as transportation fuel. Yeast naturally produces isobutanol as an end product of valine catabolism via the Ehrlich pathway. However, production of isobutanol using yeast as production host produces low alcohol titers. Therefore, this study was conducted to increase the isobutanol production using several strategies on selected yeast based on the preliminary studies. Yeasts including Saccharomyces cerevisiae, Kluyveromyces lactis GG799, Pichia pastoris KM71H, Pichia pastoris GS115 and Pichia pastoris X33 were investigated in respect of production yield and alcohol tolerance. The result of preliminary study showed that Saccharomyces cerevisiae produced isobutanol titers continuously during 96 hours of fermentation and has high alcohol resistance compared to other strains thus it was selected as the best producer. In order to improve the isobutanol production, optimization of medium compositions and operating conditions for fermentation was carried out using central composite design. Medium components affecting the isobutanol production were screened using fractional factorial design and the optimized medium compositions obtained are glucose of 140 g/l, yeast extract of 8 g/l and peptone of 8 g/l with the yield of isobutanol of 172 mg/l. The fermentation was further optimized in order to evaluate the effects of operating conditions. The optimum isobutanol of200 mg/l was obtained with temperature of 28 oC, pH of 7, agitation of 179 rpm and inoculum size of 10%. Amino acids, keto acid and vitamins were added into the fermentation medium in order to enhance the alcohol production yield as isobutanol is the by-product of amino acid catabolism. Vitamins were required in the first step of alcoholic fermentation, thus by adding these materials into the fermentation medium, the alcohol yield could be increased. Valine, leucine and 2-ketoisovalerate with concentration of 0.05 to 1.5 g/l were added into the fermentation medium and the concentration of 1.5 g/l contributes a significant effect towards isobutanol production. The highest isobutanol concentration (856 mg/l) was obtained with the addition of 1.5 g/l 2-ketoisovalerate. Further investigation on the interaction effect of amino and keto acids was carried out and the highest production of isobutanol was 1058 mg/l which is 10-fold higher compared to the control, obtained with supplementation of valine and 2-ketoisovalerate combination. An amount of 0.05 to 0.5 g/l of biotin, niacin, thiamine pyrophosphate and paraaminobenzoic acid (PABA) was supplemented into the fermentation medium. Isobutanol concentration decreased with the addition of these vitamins individually. Supplementation of combination of vitamins mixture showed that the combination of niacin-PABA and biotinniacin- PABA contributed to highest isobutanol production of 402 mg/l. Supplementation of amino acids and vitamins simultaneously into the medium broth resulted in the highest isobutanol production yield of 1027 mg/l with the addition of valine-2-KIV-niacin-PABA. The kinetics on microbial growth and product formation were studied using Monod and Leudeking-Piret equations. The values of ^max and ks obtained were 0.75 h-1 and 58 g/L, respectively. The isobutanol yield (Y p /s ), biomass yield (Y x / s ) and the correlation between isobutanol production and yeast growth (Yp /x ) were found to be 5.19 mg/g, 0.1 g/g and 50.0 mg/g, respectively. Throughout the study, it can be concluded that Saccharomyces cerevisiae is the best isobutanol production host and the alcohol titers can be increased via optimization and supplementation of amino acids and vitamins. With the improvement of technologies nowadays, the isobutanol production is expected to be increased in the future, thus encouraging the usage of this fuel in the transportation industry worldwide.