Browsing by Author "Liao, Hehuan"

Now showing 1 - 2 of 2
  • Thumbnail Image
    High-Yield Cellulosic Hydrogen Production by Cell-Free Synthetic Cascade Enzymes: Minimal Bacterial Cellulase Cocktail and Thermostable Polyphosphate Glucokinase
    Liao, Hehuan (Virginia Tech, 2011-04-27)
    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.
  • Thumbnail Image
    Improving microbial fate and transport modeling to support TMDL development in an urban watershed
    Liao, Hehuan (Virginia Tech, 2015-04-30)
    Pathogen contamination, typically quantified by elevated levels of fecal indicator bacteria (FIB), remains the leading cause of surface water-quality impairments in the United States. Continuous watershed-scale models are typically employed to facilitate Total Maximum Daily Load (TMDL) restoration efforts. Due to limited understanding of microbial fate and transport, predictions of FIB concentrations are associated with considerable uncertainty relative to other water-quality contaminants. By focusing on a data-rich instrumented urban watershed, this study aims to improve understanding of microbial fate and transport processes. Weekly FIB concentrations in both the water column and streambed sediments were monitored for one year, and statistical correlations with hydrometeorological and physicochemical variables were identified. An intensive six storm intra-sampling campaign quantified and contrasted loading trends of both traditional regulatory FIB and emerging Microbial Source Tracking (MST) markers. Together, these intensive monitoring efforts facilitated evaluation of the impacts of bacteria-sediment interactions on the predictions of daily FIB concentrations in Hydrological Simulation Program-Fortran (HSPF) over multiple years. While superior overall model performance was demonstrated as compared to earlier efforts, the inclusion of bacteria-sediment interactions did not improve performance. Large wet-weather microbial loading appears to have dwarfed the effects of FIB release and resuspension from sediment. Although wet-weather loading is generally considered as a primary source of waterbody microbial loads, dry-weather periods are more directly associated with public health concern, which may be a more suitable area for future model-refinement efforts. Site evaluation is critical to determine whether the added model complexity and effort associated with partitioning phases of FIB can be sufficiently offset by gains in predictive capacity. Finally, a stochastic framework to translate simulated daily FIB concentrations into estimates of human illness risks is presented that can be can be readily integrated into existing TMDLs. As even small concentrations of FIB from human sources are associated with great risk, and monitoring efforts indicated moderate/high levels of human-associated MST marker in this watershed, remediation efforts to protect public health would be best directed toward infrastructure improvements. Uncertainty analysis indicates more site-specific knowledge of pathogen presence and densities would best improve the estimation of illness risks.