Comparative Efficiency and Driving Range of Light- and Heavy-Duty Vehicles Powered with Biomass Energy Stored in Liquid Fuels or Batteries

This study addresses the question, When using cellulosic biomass for vehicular transportation, which field-to-wheels pathway is more efficient: that using biofuels or that using bioelectricity? In considering the question, the level of assumed technological maturity significantly affects the comparison, as does the intended transportation application. Results from the analysis indicate that for light-duty vehicles, over ranges typical in the United States today (e.g., 560-820 miles), field-to-wheels performance is similar, with some scenarios showing biofuel to be more efficient, and others indicating the two pathways to be essentially the same. Over the current range of heavy-duty vehicles, the field-to-wheels efficiency is higher for biofuels than for electrically powered vehicles. Accounting for technological advances and range, there is little basis to expect mature bioelectricity-powered vehicles to have greater field-to-wheels efficiency (e.g., kilometers per gigajoule biomass or per hectare) compared with mature biofuel-powered vehicles

Computational Design and Characterization of a Temperature-Sensitive Plasmid Replicon for Gram Positive Thermophiles

Temperature-sensitive (Ts) plasmids are useful tools for genetic engineering, but there are currently none compatible with the gram positive, thermophilic, obligate anaerobe, Clostridium thermocellum. Traditional mutagenesis techniques yield Ts mutants at a low frequency, and therefore requires the development of high-throughput screening protocols, which are also not available for this organism. Recently there has been progress in the development of computer algorithms which can predict Ts mutations. Most plasmids currently used for genetic modification of C. thermocellum are based on the replicon of plasmid pNW33N, which replicates using the RepB replication protein. To address this problem, we set out to create a Ts plasmid by mutating the gene coding for the RepB replication protein using an algorithm designed by Varadarajan et al. (1996) for predicting Ts mutants based on the amino-acid sequence of the protein

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Rates of phosphorolytic cleavage of -glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temper- atures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by \u3e20-fold for both cellobiose and cellopentaose over a 10-fold range of -glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of -glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of Vmax values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent Km for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM)

Acceptable Risk

Perhaps the topic of acceptable risk never had a sexier and more succinct introduction than the one Edward Norton, playing an automobile company executive, gave it in Fight Club: “Take the number of vehicles in the field (A), multiply it by the probable rate of failure (B), and multiply the result by the average out of court settlement (C). A*B*C=X. If X is less than the cost of the recall, we don’t do one.” Of course, this dystopic scene also gets to the heart of the issue in another way: acceptable risk deals with mathematical calculations about the value of life, injury, and emotional wreckage, making calculation a difficult matter ethically, politically, and economically. This entry will explore the history of this idea, focusing on its development alongside statistics into its wide importance today

Identification of the [FeFe]-Hydrogenase Responsible for Hydrogen Generation in Thermoanaerobacterium saccharolyticum and Demonstration of Increased Ethanol Yield via Hydrogenase Knockout

Three putative hydrogenase enzyme systems in Thermoanaerobacterium saccharolyticum were investigated at the genetic, mRNA, enzymatic, and phenotypic levels. A four-gene operon containing two [FeFe]-hydrogenase genes, provisionally termed hfs (hydrogenase-Fe-S), was found to be the main enzymatic catalyst of hydrogen production. hfsB, perhaps the most interesting gene of the operon, contains an [FeFe]-hydrogenase and a PAS sensory domain and has several conserved homologues among clostridial saccharolytic, cellulolytic, and pathogenic bacteria. A second hydrogenase gene cluster, hyd, exhibited methyl viologen-linked hydrogenase enzymatic activity, but hyd gene knockouts did not influence the hydrogen yield of cultures grown in closed-system batch fermentations. This result, combined with the observation that hydB contains NAD(P)+ and FMN binding sites, suggests that the hyd genes are specific to the transfer of electrons from NAD(P)H to hydrogen ions. A third gene cluster, a putative [NiFe]-hydrogenase with homology to the ech genes, did not exhibit hydrogenase activity under any of the conditions tested. Deletion of the hfs and hydA genes resulted in a loss of detectable methyl viologen-linked hydrogenase activity. Strains with a deletion of the hfs genes exhibited a 95% reduction in hydrogen and acetic acid production. A strain with hfs and ldh deletions exhibited an increased ethanol yield from consumed carbohydrates and represents a new strategy for engineering increased ethanol yields in T. saccharolyticum

Natural Competence in Thermoanaerobacter and Thermoanaerobacterium Species

Low-G+C thermophilic obligate anaerobes in the class Clostridia are considered among the bacteria most resistant to genetic engineering due to the difficulty of introducing foreign DNA, thus limiting the ability to study and exploit their native hydrolytic and fermentative capabilities. Here, we report evidence of natural genetic competence in 13 Thermoanaerobacter and Thermoanaerobacterium strains previously believed to be difficult to transform or genetically recalcitrant. In Thermoanaerobacterium saccharolyticum JW/SL-YS485, natural competence- mediated DNA incorporation occurs during the exponential growth phase with both replicating plasmid and homologous recombination-based integration, and circular or linear DNA. In T. saccharolyticum, disruptions of genes similar to comEA, comEC, and a type IV pilus (T4P) gene operon result in strains unable to incorporate further DNA, suggesting that natural competence occurs via a conserved Gram-positive mechanism. The relative ease of employing natural competence for gene transfer should foster genetic engineering in these industrially relevant organisms, and understanding the mechanisms underlying natural competence may be useful in increasing the applicability of genetic tools to difficult-to-transform organisms

Formation and Characterization of Non-Growth States in Clostridium Thermocellum: Spores and L-Forms

Clostridium thermocellum is an anaerobic thermophilic bacterium that exhibits high levels of cellulose solublization and produces ethanol as an end product of its metabolism. Using cellulosic biomass as a feedstock for fuel production is an attractive prospect, however, growth arrest can negatively impact ethanol production by fermentative microorganisms such as C. thermocellum. Understanding conditions that lead to non-growth states in C. thermocellum can positively influence process design and culturing conditions in order to optimize ethanol production in an industrial setting

Increase in Ethanol Yield via Elimination of Lactate Production in an Ethanol-Tolerant Mutant of Clostridium Thermocellum

Large-scale production of lignocellulosic biofuel is a potential solution to sustainably meet global energy needs. One-step consolidated bioprocessing (CBP) is a potentially advantageous approach for the production of biofuels, but requires an organism capable of hydrolyzing biomass to sugars and fermenting the sugars to ethanol at commercially viable titers and yields. Clostridium thermocellum, a thermophilic anaerobe, can ferment cellulosic biomass to ethanol and organic acids, but low yield, low titer, and ethanol sensitivity remain barriers to industrial production. Here, we deleted the hypoxanthine phosphoribosyltransferase gene in ethanol tolerant strain of C. thermocellum adhE*(EA) in order to allow use of previously developed gene deletion tools, then deleted lactate dehydrogenase (ldh) to redirect carbon flux towards ethanol. Upon deletion of ldh, the adhE*(EA) Δldh strain produced 30% more ethanol than wild type on minimal medium. The adhE*(EA) Δldh strain retained tolerance to 5% v/v ethanol, resulting in an ethanol tolerant platform strain of C. thermocellum for future metabolic engineering efforts

Enhanced Microbial Utilization of Recalcitrant Cellulose by an Ex Vivo Cellulosome-Microbe Complex

A cellulosome-microbe complex was assembled ex vivo on the surface of Bacillus subtilis displaying a miniscaffoldin that can bind with three dockerin-containing cellulase components: the endoglucanase Cel5, the processive endoglucanase Cel9, and the cellobiohydrolase Cel48. The hydrolysis performances of the synthetic cellulosome bound to living cells, the synthetic cellulosome, a noncomplexed cellulase mixture with the same catalytic components, and a commercial fungal enzyme mixture were investigated on low-accessibility recalcitrant Avicel and high accessibility regenerated amorphous cellulose (RAC). The cellbound cellulosome exhibited 4.5- and 2.3-fold-higher hydrolysis ability than cell-free cellulosome on Avicel and RAC, respectively. The cellulosome-microbe synergy was not completely explained by the removal of hydrolysis products from the bulk fermentation broth by free-living cells and appeared to be due to substrate channeling of long-chain hydrolysis products assimilated by the adjacent cells located in the boundary layer. Our results implied that long-chain hydrolysis products in the boundary layer may inhibit cellulosome activity to a greater extent than the short-chain products in bulk phase. The findings that cell-bound cellulosome expedited the microbial cellulose utilization rate by 2.3- to 4.5-fold would help in the development of better consolidated bioprocessing microorganisms (e.g., B. subtilis) that can hydrolyze recalcitrant cellulose rapidly at low secretory cellulase levels

Winter Rye as a Bioenergy Feedstock: Impact of Crop Maturity on Composition, Biological Solubilization and Potential Revenue

Background: Winter annual crops such as winter rye (Secale cereale L) can produce biomass feedstock on seasonally fallow land that continues to provide high-value food and feed from summer annuals such as corn and soybeans. As energy double crops, winter grasses are likely to be harvested while still immature and thus structurally different from the fully senesced plant material typically used for biofuels. This study investigates the dynamic trends in biomass yield, composition, and biological solubilization over the course of a spring harvest season. Results: The water soluble fraction decreased with increasing maturity while total carbohydrate content stayed roughly constant at about 65%. The protein mass fraction decreased with increasing maturity, but was counterbalanced by increasing harvest yield resulting in similar total protein across harvest dates. Winter rye was ground and autoclaved then fermented at 15 g/L total solids by either (1) Clostridium thermocellum or (2) simultaneous saccharification and cofermentation (SSCF) using commercial cellulases (CTec2 and HTec2) and a xylose-fermenting Saccharomyces cerevisiae strain. Solubilization of total carbohydrate dropped significantly as winter rye matured for both C. thermocellum (from approximately 80% to approximately 50%) and SSCF (from approximately 60% to approximately 30%). C. thermocellum achieved total solubilization 33% higher than that of SSCF for the earliest harvest date and 50% higher for the latest harvest date. Potential revenue from protein and bioethanol was stable over a range of different harvest dates, with most of the revenue due to ethanol. In a crop rotation with soybean, recovery of the soluble protein from winter rye could increase per hectare protein production by 20 to 35%. Conclusions: Double-cropping winter rye can produce significant biomass for biofuel production and feed protein as coproduct without competing with the main summer crop. During a 24-day harvest window, the total carbohydrate content remained relatively constant while the early-harvest yielded much higher carbohydrate solubilization for both C. thermocellum fermentation and SSCF. C. thermocellum fermentation achieved higher carbohydrate solubilization than SSCF across all growth stages tested. Although winter rye’s yield, composition, and biological reactivity change rapidly in the spring, it offers a substantial and stable income across the harvest season and thus flexibility for the farmer