As the price of oil continues to increase and the concern over global warming heightens, finding alternative renewable sources of energy becomes more and more imperative. Considering the abundance of lignocellulosic biomass, the potential significance of its conversion to fuel such as ethanol has long been recognized. Ethanol production from lignocellulosic biomass requires the breakdown of the solid material to simple sugars that can be consumed by microorganisms. The breakdown of lignocelluloses includes the important step of hydrolyzing cellulose, which are β-1, 4 linked polymers of glucose, by a group of enzymes collectively termed cellulase. The cost of cellulase production profoundly influences the economics of the entire ethanol production process. Trichoderma reesei Rut C-30 is the most commonly used fungal strain for industrial cellulase production. With an overall goal of decreasing the cellulase production cost, this thesis work was focused on two topics: (1) investigation of the performance of different cellulase-inducing substrates in T. reesei Rut C-30 fermentation and (2) development of a more advanced model to describe the culture behaviors of T. reesei Rut C-30 grown on lactose-based media. The cellulase-inducing substrates investigated in this work included the acid-treated hydrolysate, lactose, and the sophorolipids produced by the yeast Candida bombicola. In addition to the above focused topics, the effects of culture conditions on cellulase production were also investigated.
The acid-treated hardwood hydrolysates used in the study were prepared by a two-stage process, with different durations of boiling and acid concentrations. The results indicated that the inducing ability of the hydrolysates decreased with increasing boiling time (less oligomers). The observation was attributed to the lower amounts of inducing oligomers remaining after the longer boiling in acid. When compared with cultures growing on mixed carbon substrates of cellulose and glucose, the culture growing on hydrolysates showed a longer lag phase of about 24 hours (before the active cell growth began), but produced adequately comparable final cellulase activity.
The study with lactose as an inducing substrate was conducted in both batch and continuous culture systems with lactose and other relevant substrates (glycerol, glucose and galactose) as the carbon source. Instead of direct ingestion, lactose is believed to be hydrolyzed by extracellular enzymes to glucose and galactose, which are then taken up by the cells. The study results indicated that glucose strongly represses the galactose metabolism: Cells started to consume galactose only after the glucose had been depleted. A mathematical model incorporating all important metabolic activities was developed to describe the culture behaviors. All of the experimental results obtained were used in model fitting to generate a set of best-fit model parameters. The study provided significant conceptual and quantitative insights to the lactose metabolism and cellulase production by T. reesei Rut C-30.
This study was also the first to hypothesize and demonstrate the use of sophorolipids as the inducing substrate for cellulase production. A unique process for cellulase production using a mixed culture of T. reesei Rut C30 and Candida Bombicola growing on glycerol-based media was investigated. Hypothetically, the sophorolipids produced by C. bombicola were hydrolyzed to form sophorose, which then served as the inducer for cellulase production by T. reesei. Further study to optimize the sophorolipid-induced cellulase production process is recommended.