Douglas S. Clark

Metagenomic discovery of biomass-degrading genes and genomes from cow rumen.

Metagenomic sequencing of biomass-degrading microbes from cow rumen reveals new carbohydrate-active enzymes. The paucity of enzymes that efficiently deconstruct plant polysaccharides represents a major bottleneck for industrial-scale conversion of cellulosic biomass into biofuels. Cow rumen microbes specialize in degradation of cellulosic plant material, but most members of this complex community resist cultivation. To characterize biomass-degrading genes and genomes, we sequenced and analyzed 268 gigabases of metagenomic DNA from microbes adherent to plant fiber incubated in cow rumen. From these data, we identified 27,755 putative carbohydrate-active genes and expressed 90 candidate proteins, of which 57% were enzymatically active against cellulosic substrates. We also assembled 15 uncultured microbial genomes, which were validated by complementary methods including single-cell genome sequencing. These data sets provide a substantially expanded catalog of genes and genomes participating in the deconstruction of cellulosic biomass.

Ionic liquid pretreatment of cellulosic biomass: Enzymatic hydrolysis and ionic liquid recycle

Ionic liquids (ILs) are promising solvents for the pretreatment of biomass as certain ILs are able to completely solubilize lignocellulose. The cellulose can readily be precipitated with an anti‐solvent for further hydrolysis to glucose, but the anti‐solvent must be removed for the IL to be recovered and recycled. We describe the use of aqueous kosmotropic salt solutions to form a three‐phase system that precipitates the biomass, forming IL‐rich and salt‐rich phases. The phase behavior of [Emim][Ac] and aqueous phosphate salt systems is presented, together with a process for recycling the [Emim][Ac] and enzymatically hydrolyzing the cellulose. This process reduces the amount of water to be evaporated from recycled IL, permitting efficient recycle of the IL. Material balances on the process, with multiple recycles of the [Emim][Ac], quantify the major components from a Miscanthus feedstock through the pretreatment, separation, and enzymatic hydrolysis steps. A more rapid and higher yielding conversion of cellulose to glucose is obtained by use of the three‐phase system as compared to the cellulose obtained from biomass pretreated with IL and precipitated with water. The addition of a kosmotropic salt during the precipitation results in partial delignification of the biomass, which makes the substrate more accessible, enhancing the enzymatic hydrolysis. Biotechnol. Bioeng. 2011; 108:511–520.

Pressure effects on intra- and intermolecular interactions within proteins

The effects of pressure on protein structure and function can vary dramatically depending on the magnitude of the pressure, the reaction mechanism (in the case of enzymes), and the overall balance of forces responsible for maintaining the proteins structure. Interactions between the protein and solvent are also critical in determining the response of a protein to pressure. Pressure has long been recognized as a potential denaturant of proteins, often promoting the disruption of multimeric proteins, but recently examples of pressure-induced stabilization have also been reported. These global effects can be explained in terms of pressure effects on individual molecular interactions within proteins, including hydrophobic, electrostatic, and van der Waals interactions, which can now be studied in greater detail than ever before. However, many uncertainties remain, and thorough descriptions of how proteins respond to pressure remain elusive. This review summarizes basic concepts and new findings related to pressure effects on intra- and intermolecular interactions within proteins and protein complexes, and discusses their implications for protein structure-function relationships under pressure.

Integration of chemical catalysis with extractive fermentation to produce fuels

Nearly one hundred years ago, the fermentative production of acetone by Clostridium acetobutylicum provided a crucial alternative source of this solvent for manufacture of the explosive cordite. Today there is a resurgence of interest in solventogenic Clostridium species to produce n-butanol and ethanol for use as renewable alternative transportation fuels. Acetone, a product of acetone–n-butanol–ethanol (ABE) fermentation, harbours a nucleophilic α-carbon, which is amenable to C–C bond formation with the electrophilic alcohols produced in ABE fermentation. This functionality can be used to form higher-molecular-mass hydrocarbons similar to those found in current jet and diesel fuels. Here we describe the integration of biological and chemocatalytic routes to convert ABE fermentation products efficiently into ketones by a palladium-catalysed alkylation. Tuning of the reaction conditions permits the production of either petrol or jet and diesel precursors. Glyceryl tributyrate was used for the in situ selective extraction of both acetone and alcohols to enable the simple integration of ABE fermentation and chemical catalysis, while reducing the energy demand of the overall process. This process provides a means to selectively produce petrol, jet and diesel blend stocks from lignocellulosic and cane sugars at yields near their theoretical maxima.

High-throughput cellular microarray platforms: applications in drug discovery, toxicology and stem cell research

Cellular microarrays are powerful experimental tools for high-throughput screening of large numbers of test samples. Miniaturization increases assay throughput while reducing reagent consumption and the number of cells required, making these systems attractive for a wide range of assays in drug discovery, toxicology, stem cell research and potentially therapy. Here, we provide an overview of the emerging technologies that can be used to generate cellular microarrays, and we highlight recent significant advances in the field. This emerging and multidisciplinary approach offers new opportunities for the design and control of stem cells in tissue engineering and cellular therapies and promises to expedite drug discovery in the biotechnology and pharmaceutical industries.

Long-term antibacterial efficacy of air plasma-activated water

Indirect air dielectric barrier discharge in close proximity to water creates an acidified, nitrogen-oxide containing solution known as plasma-activated water (PAW), which remains antibacterial for several days. Suspensions of E. coli were exposed to PAW for either 15 min or 3 h over a 7-day period after PAW generation. Both exposure times yielded initial antibacterial activity corresponding to a ~5-log reduction in cell viability, which decreased at differing rates over 7 days to negligible activity and a 2.4-log reduction for 15 min and 3 h exposures, respectively. The solution remained at pH ~2.7 for this period and initially included hydrogen peroxide, nitrate and nitrite anions. The solution composition varied significantly over this time, with hydrogen peroxide and nitrite diminishing within a few days, during which the antibacterial efficacy of 15 min exposures decreased significantly, while that of 3 h exposures produced a 5-log reduction or more. These results highlight the complexity of PAW solutions where multiple chemical components exert varying biological effects on differing time scales.

Enzymatic Oxidation of Cholesterol Aggregates in Supercritical Carbon Dioxide

Fundamental studies of enzyme-solvent interactions can be conducted with supercritical fluids because small changes in pressure or temperature may bring about great changes in the properties of a single solvent near its critical point. Cholesterol oxidase is active in supercritical carbon dioxide and supercritical carbon dioxide-cosolvent mixtures. Variations in solvent power caused by pressure changes or by the addition of dopants affected the rate of enzymatic oxidation of cholesterol by altering the structure of cholesterol aggregates.

Can immobilization be exploited to modify enzyme activity

Immobilization has long been recognized as a useful tool for retaining enzymes in bioreactors and enabling the continuous operation of enzymic processes. However, the potential of immobilization for modifying enzyme activity in an advantageous, if not predictable, manner is less-well appreciated. This review summarizes selected studies that have used immobilization to tailor the catalytic properties of enzymes, and highlights the application of immobilization to the rational design of biocatalysts.

A Mechanistic Model of the Enzymatic Hydrolysis of Cellulose

A detailed mechanistic model of enzymatic cellulose hydrolysis has been developed. The behavior of individual cellulase enzymes and parameters describing the cellulose surface properties are included. Results obtained for individual enzymes (T. reesei endoglucanase 2 and cellobiohydrolase I) and systems with both enzymes present are compared with experimental literature data. The model was sensitive to cellulase‐accessible surface area; the EG2–CBHI synergy observed experimentally was only predicted at a sufficiently high cellulose surface area. Enzyme crowding, which is more apparent at low surface areas, resulted in differences between predicted and experimental rates of hydrolysis. Model predictions also indicated that the observed decrease in hydrolysis rates following the initial rate of rapid hydrolysis is not solely caused by product inhibition and/or thermal deactivation. Surface heterogeneities, which are not accounted for in this work, may play a role in decreasing the hydrolysis rate. The importance of separating the enzyme adsorption and complexation steps is illustrated by the models sensitivity to the rate of formation of enzyme–substrate complexes on the cellulose surface. Biotechnol. Bioeng. 2010;107: 37–51.

Ozone correlates with antibacterial effects from indirect air dielectric barrier discharge treatment of water

Ambient-condition air plasma produced by indirect dielectric barrier discharges can rapidly disinfect aqueous solutions contaminated with bacteria and other microorganisms. In this study, we measured key chemical species in plasma-treated aqueous solutions and the associated antimicrobial effect for varying discharge power densities, exposure times, and buffer components in the aqueous medium. The aqueous chemistry corresponded to air plasma chemistry, and we observed a transition in composition from ozone mode to nitrogen oxides mode as the discharge power density increased. The inactivation of E. coli correlates well with the aqueous-phase ozone concentration, suggesting that ozone is the dominant species for bacterial inactivation under these conditions. Published values of ozone-water antibacterial inactivation kinetics as a function of the product of ozone concentration and contact time are consistent with our results. In contrast to earlier studies of plasma-treated water disinfection, ozone-dependent bacterial inactivation does not require acidification of the aqueous medium and the bacterial inactivation rates are far higher. Furthermore, we show that the antimicrobial effect depends strongly on gas-liquid mixing following plasma treatment, apparently because of the low solubility of ozone and the slow rate of mass transfer from the gas phase to the liquid. Without thorough mixing of the ozone-containing gas and bacteria-laden water, the antimicrobial effect will not be observed. However, it should be recognized that the complexity of atmospheric pressure plasma devices, and their sensitivity to subtle differences in design and operation, can lead to different results with different mechanisms.