| The present research aims to employ and enhance biological routes of corn stover conversion to ethanol. An attempt has been made to combine three upstream unit operations: storage, pretreatment, and hydrolysis, in a simple and robust solid substrate fermentation. Ensilage was chosen to be the preferred storage method due to low dry matter losses. White rot fungi which preferentially degrade lignin over cellulose were grown on stover to pretreat the biomass. Cellulase and xylanase producing Trichoderma species were employed in an attempt to convert crystalline cellulose and hemicellulose to glucose and xylose molecules, respectively.;Initially, the effect of drying, freezing and refrigeration of corn stover prior to ensilage was studied. This first experiment was essential to see if lab studies being conducted on frozen stover can be extrapolated to real world scenarios where stover is ensiled fresh or dry. Dry and frozen corn stover produced comparable results to the control after ensilage; however refrigeration changed biomass properties considerably. Based on these results, we selected freezing as our method of storing substrate between harvest and experimentation, with the expectation that real world results would be as good as or better than our experimental trials.;As demonstrated in this initial sample pre-storage study, several parameters are useful to understand the effects of treatments and ensilage on the biomass. These parameters include pH, dry matter loss, water soluble carbohydrates, fiber content (cellulose, hemicellulose, and lignin), monosaccharides (mainly glucose, xylose, mannose, and arabinose), and organic acids (lactic, acetic, propionic, isobutyric, and butyric acids). The methods developed to study these variables are highly dependent on sample handling techniques and extraction procedures. In a second methods-development investigation, the effects of particle size, post ensilage biomass drying temperatures, and modified phenol-sulfuric acid methods were evaluated to identify the best approach for post ensilage parameter measurement methods.;After developing pre- and post-ensilage methods, an experiment was designed to combine anaerobic ensilage treatment with an aerobic lignin degradation process in a two step procedure. The following four factors were considered as issues to be addressed in developing an efficient pretreatment-storage process. (a) Sterilization was evaluated as an experimental treatment. (b) Two time periods, 7 and 14 days, were tested to study lignin degradation levels along with dry matter losses. (c) Two storage phases, aerobic and anaerobic, were tested in both possible orders. (d) Three white rot fungi: Phanerochaete chrysosporium, Ceriporiopsis subvermispora, and Pluerotus ostreatus were tested.;Sterilization through autoclaving decreased initial pH values by 1 pH unit, suggesting a mild pretreatment effect. However, the final pH values were significantly higher, presumably due to elimination of indigenous lactic acid producing bacteria during sterilization (p < 0.01). Surprisingly, sterilized biomass showed significantly higher dry matter loss (p < 0.0001). This could be due to freely available sugars and lack of competition in the biomass encouraging high metabolic rates for the surviving and inoculated organisms. Contrary to expectations, introducing an aerobic phase after ensilage did not increase pH values (p > 0.5) indicating no loss of organic acids due to oxidation. As expected, 14 days of an aerobic phase caused higher dry matter loss of biomass compared to 7 days, but also produced higher water soluble carbohydrates (p < 0.0001).;In addition to biological pretreatment, hydrolysis can also be considered during storage. To explore this option, Trichoderma reesei Rut C30 was tested in a sequenced aerobic and anaerobic system. The optimal hydrolysis temperature is 50°C, while maximum enzyme production for T. reesei RutC30 occurs at 30°C. Accordingly, a higher temperature, 50°C, was tested for 7 days after 7 days of incubation at 30°C. As expected, higher temperatures almost doubled the dry matter loss, but did not produce significantly higher water soluble carbohydrates (p > 0.1).;The final experiment attempted to combine the three unit operations, storage, pretreatment and hydrolysis into a single stage, two phase process. For this study, two co-cultures: co-culture A (P. ostreatus with T. reesei Rut C30) and co-culture B (P. ostreatus with T. reesei wild type organism) were tested to identify the more robust hydrolytic fungi combinations. The ideal results, i.e., lowest pH values, lowest dry matter loss and highest sugar production occurred at 30°C, 40% moisture, and 7 days of aerobic phase. Extending the aerobic phase to 14 days produced higher sugar concentration, but by 21 days, the sugar levels started to fall. A statistical model was developed to describe dry matter loss along with lignin degradation (ADL) based on the results from the factorial experiment. (Abstract shortened by UMI.)... |
