Hydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes. Two of the biggest challenges for its commercialization are: effective release of fermentable sugars from pretreated biomass, and preparations of thermostable enzymes with low-cost. The hydrolysis performance of 21 reconstituted bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase, family 9 Clostridium phytofermentans processive endoglucanase, and family 48 Clostridium phytofermentans cellobiohydrolase was investigated on microcrystalline cellulose (Avicel) and regenerated amorphous cellulose (RAC). The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was most important for high cellulose digestibility regardless of substrate type. These results suggested that the hydrolysis performance of reconstituted cellulase cocktail strongly depended on experimental conditions. Thermobifida fusca YX was hypothesized to have a thermophilic polyphosphate glucokinase. T. fusca YX ORF Tfu_1811 encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was over expressed in Escherichia coli. By a simple one-step immobilization, the half-life time increased to 2 h, at 50 °C. These results suggest that this enzyme was the most thermostable PPGK reported. My studies would provide important information for the on-going project: high-yield hydrogen production from cellulose by cell-free synthetic enzymatic pathway. / Master of Science

Enzyme-catalyzed biofuel cells would be a great alternative to current battery technology, as they are clean, safe, and capable of using diverse and abundant renewable biomass with high energy densities, at mild reaction conditions. However, currently, three largest technical challenges for emerging enzymatic fuel cell technologies are incomplete oxidation of most fuels, limited power output, and short lifetime of the cell. Synthetic pathway biotransformation is a technology of assembling a number of enzymes coenzymes for producing low-value biocommodities. In this work, it was applied to generate bioelectricity for the first time. Non-natural enzymatic pathways were developed to utilize maltodextrin and glucose in enzymatic fuel cells. Three immobilization approaches were compared for preparing enzyme electrodes. Thermostable enzymes from thermophiles were cloned and expressed for improving the lifetime and stability of the cell. To further increase the power output, non-immobilized enzyme system was demonstrated to have higher power densities compared to those using immobilized enzyme system, due to better mass transfer and retained native enzyme activities. With the progress on pathway development and power density/stability improvement in enzymatic fuel cells, a high energy density sugar-powered enzymatic fuel cell was demonstrated. The enzymatic pathway consisting of 13 thermostable enzymes enabled the complete oxidation of glucose units in maltodextrin to generate 24 electrons, suggesting a high energy density of such enzymatic fuel cell (300 Wh/kg), which was several folds higher than that of a lithium-ion battery. Maximum power density was 0.74 mW/cm2 at 50 deg C and 20 mM fuel concentration, which was sufficient to power a digital clock or a LED light. These results suggest that enzymatic fuel cells via synthetic pathway biotransformation could achieve high energy density, high power density and increased lifetime. Future efforts should be focused on further increasing power density and enzyme stability in order to make enzymatic fuel cells commercially applicable. / Ph. D.