The role of manganese peroxidase in biomass conversion technologies

Graduation date: 2011Currently, the technologies used to separate lignocellulosic biomass into its\ud component parts (cellulose, hemicellulose, and lignin) and enzymatically hydrolyze\ud the cellulose to glucose for conversion to ethanol could be improved economically and\ud in terms of efficiency. A major impediment to utilizing the biomass is the presence of\ud residual lignin. The residual lignin "poisons" the cellulase enzymes and as a result, a\ud lower glucose yield is obtained. A higher titer of cellulases could be used to increase\ud the yield of glucose, but cellulases are expensive. A possibility to reduce the cost of\ud cellulase enzymes and improve the efficiency, economics, extent and/or rate of\ud hydrolysis is to use lignin-degrading fungal heme peroxidase such as manganese\ud peroxidases.\ud An efficient and economical glucose assay was developed to monitor the rate\ud and extent of enzymatic hydrolysis of cellulose. The glucose assay is based on the\ud glucose oxidase horseradish peroxidase enzymatic method, but uses bulk enzymes\ud making it more economical than commercial kits. The glucose assay also has a higher\ud throughput rate and is more economical than HPLC.\ud In order to evaluate possible synergistic effects between cellulases and\ud manganese peroxidase (MnP), rMnP was produced in high cell density fed-batch\ud cultivations by the genetically engineered yeast, P. pastoris. In addition, a mathematic\ud model was developed to describe the temperature dependant growth of P. pastoris and\ud consumption of glucose and the production and temperature dependent degradation of\ud rMnP in the bioreactor broth. The model successfully predicted the cell growth,\ud substrate consumption, and rMnP production for the base case and also for cultivations\ud with varying fed-batch air flow rates (k[subscript L]a) and temperatures.\ud The production of rMnP requires cultures amended with exogenous heme and\ud there are several sources of heme. Through shake flask experiments and bioreactor\ud cultivations it was determined that 0.1 g/L of heme was necessary for producing high\ud titers of rMnP (4,500 U/L). It was also determined that not all types of heme will yield\ud the same rMnP titer.\ud The water soluble fraction post pretreatment of lignocellulosic biomass\ud contains inhibitors to fermentation such as 5-hydroxymethyl furfural (HMF) and\ud furfural. rMnP was shown to degrade HMF (1 g/L) and furfural (1 g/L) and detoxify\ud medium containing these inhibitors. The rMnP reduced furfural and HMF, measured\ud by absorbance at 276 and 286 nm respectively and the degree of absorbance decrease\ud was dependent on rMnP concentration. Furfural was more readily degraded by rMnP\ud than HMF. Growth assays using S. cerevisiae indicated rMnP treatment detoxified\ud medium containing furfural and HMF.\ud The optimal conditions (temperature, pH, and buffer) for enzyme activity were\ud determined for both AccelleraseTM 1000 (commercially available combination of\ud cellulases) and rMnP using filter paper as a substrate. Woody biomass and corn stover\ud were pretreated and then exposed to simultaneous or sequential treatment with rMnP\ud and AccelleraseTM 1000. The results for the sequential treatment of Ponderosa pine\ud and corn stover with rMnP and AccelleraseTM 1000 was inconclusive as to whether or\ud not rMnP effected the production of glucose due to the high variability between\ud replicates.\ud Finally, part of my doctoral program was significant mentoring and facilitation\ud of undergraduate research. A significant portion of my time was dedicated toward\ud senior laboratory teaching assistance, serving as a mentor for high school students, and\ud participating in research mentoring with 24 undergraduate students over five years, 19\ud of whom were women