UC Riverside

ABSTRACT OF THE THESIS

Consolidated Bio-Processing of Cellulosic Biomass for Efficient Biofuel Production Using Yeast Consortium

by

Garima Goyal

Master of Science

Chemical and Environmental Engineering Graduate Program

University of California, Riverside, March 2011

Dr. Wilfred Chen, Chairperson

Fossil fuels have been the major source for liquid transportation fuels for ages. However, decline in oil reserves and environmental concerns have raised a lot of interest in alternative and renewable energy sources. One promising alternative is the conversion of plant biomass into ethanol. The primary biomass feed stocks currently being used for the ethanol industry have been food based biomass(corn and sugar cane). However, interest has recently shifted to replace these traditional feed-stocks with more abundant, non-food based cellulosic biomass such as agriculture wastes (corn stover) or crops (switch grass). The use of cellulosic biomass as feed stock for the production of ethanol via bio-chemical routes presents many technical challenges not faced with the use of corn or sugar-cane as feed-stock. Recently, a new process called consolidated Bio-processing (CBP)has been proposed.This processcombines simultaneous saccharification of lignocellulose with fermentation of the resulting sugars into a single process step mediated by a single microorganism or microbial consortium. Although there is no natural microorganism that possesses all properties of lignocellulose utilization and ethanol production desired for CBP, some bacteria and fungi exhibit some of the essential traits.

The yeast Saccharomyces cerevisiae is the most attractive host organism for the usage of this strategy due to its high ethanol productivity at close to theoretical yields (0.51g ethanol/g glucose consumed), high osmo- and ethanol- tolerance, natural robustness in industrial processes, and ease of genetic manipulation.Introductionof the cellulosome, found naturally in microorganisms, has shown new directions to deal with recalcitrant biomass. In this case enzymes work in synergy in order to hydrolyze biomass more effectively than in case of free enzymes. A microbial consortium has been successfully developed, which ensures the functional assembly of minicellulosome on the yeast surface composed of four yeast populations. These yeast populations include: one displaying scaffoldin on its surface and three populations secreting three different cellulases in the medium to hydrolyze the cellulose. The modular nature of the consortium system allows for the fine-tuning of each population by changing their initial inoculum ratio, thereby optimizing the cellulose hydrolysis and hence ethanol production. When comparing the optimized consortium with equal ratio consortium, the optimized one produced almost double the amount of ethanol (1.87 g/l) with a yield of 0.475 g ethanol/g cellulose. To further evaluate the feasibility of using consortium for CBP, it was grown at very low optical density (OD) under anaerobic conditions. Under stressful conditions like low OD and no oxygen, the consortium system was proficient in assembling the cellulosome on its surface and growing on the PAS-avicel as sole carbon source and concomitantly producing ethanol with a yield of 87% of the theoretical value. For the dynamic study of yeast consortium system, quantitative real time PCR was used to enumerate the individual yeast population in the mixed culture. At the end of the cultivation, ratios of each population in this consortium maintained similar number as the initial inoculums ratios, which further confirms the consortium system is suitable for the application of CBP.