| Dilute acid pretreatment followed by enzymatic hydrolysis of cellulose and sugar fermentation is a promising technology for converting lignocellulosic biomass to fuel ethanol. Two of these steps, enzymatic hydrolysis and fermentation, are ultimately catalyzed by protein. The broad goal of this study is to develop a more definite understanding of the physical barriers to performing these two process steps economically. The major foci of this work are high-solids enzymatic saccharification of pretreated lignocellulosic biomass (corn stover) and protein expression profiling of a metabolically engineered bacterium fermenting glucose and xylose, the major biomass sugars.; While high-solids enzymatic hydrolysis of cellulose is advantageous for reducing capital and operating costs, operating an enzymatic saccharification reactor at high insoluble solids levels presents a unique set of physical and reactor-dependent challenges such as mass transfer, temperature control, mixing, pH control, and sugar inhibition. This work partially focused on characterizing the effects of these problems. It was determined in saccharification characterization studies that slurries of pretreated corn stover (PCS) containing between 10% and 15% insoluble solids by weight represent the approximate upper limit for enzymatically hydrolyzing cellulose in a stirred tank reactor (2--10 L scale fermenter). As this work demonstrates, this maximum limit is ultimately derived from mixing limitations that cause difficulties with temperature control, as well with uniformly distributing enzymes. Using an offline optimal control algorithm in conjunction with a process model, a bench-scale stirred tank reactor was operated at 15% insoluble solids in fed-batch mode, while demonstrating sugar concentrations and yields equivalent to what would be found if operating at 25% initial insoluble solids. Further work applied 2-D gel electrophoresis and hierarchical cluster analysis to track transient protein abundance levels in during the course of mixed substrate fermentation of the two principle sugars contained in lignocellulosic biomass. Of particular significance the saccharification work was able to demonstrate for the first time that high glucose concentrations (>140 g/L or 80% cellulose conversion) are achievable in high-solids enzymatic saccharification reaction systems, and that high-solids enzymatic saccharification presents a viable reaction step option in lignocellulosic biomass conversion technologies. |
