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.

India’s federal system is distinguished by tax and expenditure assignments that result in large vertical fiscal imbalances, and consequent transfers from the central government to the state governments. Several channels are used for these transfers: the Finance Commission, the Planning Commission, and central government ministries. We use panel data on center-state transfers to examine how the economic and political importance of the states influences the level and the composition of per capita transfers to the states, as well as differences in temporal patterns of Planning Commission and Finance Commission transfers. We find evidence that states with indications of greater bargaining power seem to receive larger per capita transfers, and that there is greater temporal variation in Planning Commission transfers.

India’s federal system is distinguished by tax and expenditure assignments that result in large vertical fiscal imbalances, and consequent transfers from the central government to the state governments. Several channels are used for these transfers: the Finance Commission, the Planning Commission, and central government ministries. We use panel data on center-state transfers to examine how the economic and political importance of the states influences the level and the composition of per capita transfers to the states, as well as differences in temporal patterns of Planning Commission and Finance Commission transfers. We find evidence that states with indications of greater bargaining power seem to receive larger per capita transfers, and that there is greater temporal variation in Planning Commission transfers.

This thesis endeavors to showcase this fragmented cosmopolitanism in Global South through a comparative analysis of national identity and urban cosmopolitan space in the novels The Ministry of Utmost Happiness (2017) by the Indian author Arundhati Roy, How to get filthy rich in Rising Asia (2013) by Pakistani Mohsin Hamid, Fábula Asiática (2017) by Guatemalan Rodrigo Rey Rosa and El sueño del retorno (2013) by the Salvadoran Horacio Castellanos Moya’s. It explores the questions such as rooted and unrooted cosmopolitanism, and homelessness, and how these dynamics are dependent on social, political and geographical boundaries and how does it, in turn, shapes national identities? If there is a trend that can be traced across boundaries and if neoliberal politics and political unrest are part of a larger design? And lastly, how the artists and literary figures in the global south have been expressing their dissent against the nation?

Antibodies are immune system&ndashrelated proteins called immunoglobulins (IgGs) which have applications for medical diagnostics and research. However, their purification from different sources has always been a challenge because of low antibody concentration and higher purity requirements for usage. The unique capability of Elastin like Proteins (ELPs) to reversibly precipitate at a relatively modest temperature has been utilized for purification of antibodies. This feature of ELPs to purify antibodies has already been explored using larger fusion domains such as Protein L and Protein G. However the usage of larger Protein G/L fusions with ELP resulted in 10&ndashfold lower expression when compare to ELP or ELP fusions with shorter peptides.

In the current work, ELP fusions with a small IgG&ndashbinding peptide i.e. zz domain (a synthetic IgG binding domain derived from the Staphylococcus aureus protein A) were generated. The production of ELP[VPGVG]&ndash78&ndashzz fusion in E. coli ( &sim 500 mg/L) was found to be five-fold higher than ELP[VPGVG]&ndash78&ndashProL (&sim100 mg/L). In addition, the ELP[VPGVG]&ndash78&ndashzz fusion showed excellent binding affinity toward human, mouse, and rabbit IgGs, enabling simple purification of the different antibodies by reversible thermal precipitation. In order to recover antibody from the ELP&ndashzz&ndashIgG complex, different elution conditions were investigated. Close to 90% recovery was achieved using 0.5 M arginine pH 3.8 buffer.

To further increase the production of ELP-zz fusion protein, three different ELP domains (VPGXG)&ndash40 (where X= K:V:F=1:8:1); (VPGXG)&ndash60 (where X= K:V:F=1:8:1), (VPGXG)&ndash80 (where X= K:V:F=1:8:1), were generated. Production of ELP(KV8F)&ndashzz fusions were increased by two fold, while maintaining similar binding affinity for IgGs. Due to its high level production and affinity for different IgGs, we believe that these ELP-zz fusions will be useful as an economical, highly efficient, and universal platform for the purification of antibodies.