High Performance Biorefinery Processing Of Lignocellulosic Agricultural Residues For Ethanol And L-lactic Acid Production

Today, world is facing long-term energy shortage due to ever increasing energy demands and dwindling conventional fuel resources, plus environmental and global warming concerns. Lignocellulosic biorefinery is an important and sustainable alternative for renewable energy development to lessen the petroleum dependence and food crop usage for production of fuels and other value added commodities. Currently, high cost in lignocellulosic processing and operation is the major technical barrier in its commercialization. In this thesis, several practical solutions were developed to overcome the difficulties in lignocellulosic biorefinery processes for ethanol and lactic acid production, including the replacement of pure sugars by corn stover for yeast cell culture, the dried distillers’grains and solubles (DDGS) usage to replace expensive fermentation nutrients, the improved ethanol yield by evolutionary adaptation of yeast fermenting strains, open lactic acid fermentation under unsterilized conditions, and cofermentation of glucose and xylose to ethanol.Dried distillers’grains and solubles (DDGS) is a major by-product of corn based ethanol production and generally used as animal feed due to its rich nutrient contents, including protein, fibers, oil, yeast cells, glucan, starch, and amino acids. This could be excellent cost effective nutrient for ethanol production. In this thesis, (Chapter 3 and 5) evaluated the utilization of DDGS as cost effective nutrient for ethanol production from adapted yeasts. In future, DDGS will be used in all SSF studies as inexpensive nitrogen source.Solid seeds culture by using freshly pretreated corn stover as a carbon source instead of pure glucose reduced a 22% of yeast seeds culture preparation cost. In addition, yeast cell growth and ethanol fermentation performance did not show any difference when the yeast seeds were cultured by using glucose, the corn stover hydrolysate liquid, and the pretreated corn stover solids as carbon sources, respectively. The solid-liquid separation step, which is required in preparation of hydrolysate was eliminated. Moreover, additional bioreactor required for enzymatic hydrolysis was directly cut. During solid seeds culture, unused cellulase bound to solid materials could be recycled during ethanol fermentation step.Pretreatment generates a wide range of toxic compounds, those reduce the cellulase activity, cell growth, and ethanol productivity. Therefore, evolutionary adaptation of fermenting strain could improve the fermentability and inhibitors tolerance. Evolutionary adaptation of a routine yeast strain S. cerevisiae DQ1 in corn stover hydrolysate was performed for consecutive 65 days that significantly improved ethanol fermentation performance in both corn stover hydrolysate and simultaneous saccharification and fermentation (SSF) at high solids content. About 50% more ethanol titer and yield were obtained from adapted strain over parental strain. The ethanol titer and yield of 71.40 g/L and 80.34% were obtained, respectively, at the optimum SSF conditions without any wastewater generation from pretreatment to fermentation. In addition, yeast extract replacement with cost effective DDGS also reduced the cellulosic ethanol production cost to large extent. The cost of DDGS was only 0.5% comparing to that of yeast extract.A pediocin producing strain Pediococcus acidilactici was employed for open L-lactic fermentation under completely unsterilized condition in synthetic medium, corn stover hydrolysate, and in high solids content SSF. The added contaminant bacteria were also inoculated in the fermentation system to confirm the antibacterial function, and contaminant strain did not grow due to the existence of antibacterial pediocin produced by Pediococcus acidilactici. High lactic acid titer, productivity, and yield of 97.30 g/L,1.47 g/L/h, and 69.34% were obtained, in high solids content SSF, respectively. The results provided a simple, robust and cost effective fermentation process for L-lactic acid using lignocellulose feedstock. Furthermore, labor consuming and energy intensive step was directly reduced.A xylose fermenting strain Saccharomyces cerevisiae NAN-127 experienced evolutionary adaptation. Adapted strain produced 41.82 g/L ethanol corresponding to 91.01% yield by cofermentation of glucose and xylose in the fresh corn stover hydrolysate. More than 90% xylose was converted into the ethanol. Ethanol concentration and yield were reached to 74.14 g/L and 74.01% by adapted strain under industrial relevant conditions. Ethanol titer is very close to corn based ethanol 10% v/v (78.9 g/L). Further optimization of the strain is in process, we are pretty sure to achieve the target 10% v/v ethanol concentration from lignocellulosic biomass.The research works presented in this thesis show considerable contributions towards cost reduction and technical advantages of lignocellulose biorefinery. Each approach proposal in this thesis reduced processing cost for ethanol and or L-lactic acid production at certain range. These advancements certainly provide helps on the commercialization of lignocellulosic biorefinery. Further efforts are required for development of fast, efficient, and cost effective biorefinery process.