Elimination of Hydrogenase Active Site Assembly Blocks H2 Production and Increases Ethanol Yield in Clostridium Thermocellum

Background: The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2 , and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol. Results: H2 production in C. thermocellum is encoded by four hydrogenases. Rather than delete each individually, we targeted hydrogenase maturase gene hydG, involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe] hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in ΔhydGΔech was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of ΔhydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation was found in ethanol-tolerant C. thermocellum strain E50C, Δ hydG and ΔhydGΔech are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum. Conclusions: The dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes

The effect of lattice temperature on surface damage in fused silica optics

We examine the effect of lattice temperature on the probability of surface damage initiation for 355nm, 7ns laser pulses for surface temperatures below the melting point to temperatures well above the melting point of fused silica. At sufficiently high surface temperatures, damage thresholds are dramatically reduced. Our results indicate a temperature activated absorption and support the idea of a lattice temperature threshold of surface damage. From these measurements, we estimate the temperature dependent absorption coefficient for intrinsic silica

Structure of the sporulation histidine kinase inhibitor Sda from Bacillus subtilis and insights into its solution state

The crystal structure of the DNA-damage checkpoint inhibitor of sporulation, Sda, from Bacillus subtilis, has been solved by the MAD technique using selenomethionine-substituted protein. The structure closely resembles that previously solved by NMR, as well as the structure of a homologue from Geobacillus stearothermophilus solved in complex with the histidine kinase KinB. The structure contains three molecules in the asymmetric unit. The unusual trimeric arrangement, which lacks simple internal symmetry, appears to be preserved in solution based on an essentially ideal fit to previously acquired scattering data for Sda in solution. This interpretation contradicts previous findings that Sda was monomeric or dimeric in solution. This study demonstrates the difficulties that can be associated with the characterization of small proteins and the value of combining multiple biophysical techniques. It also emphasizes the importance of understanding the physical principles behind these techniques and therefore their limitations

Mitigation of Laser Damage Growth in Fused Silica with a Galvanometer Scanned CO2 Laser

At the National Ignition Facility (NIF) at the Lawrence Livermore National Laboratory (LLNL), mitigation of laser surface damage growth on fused silica using single and multiple CO{sub 2} laser pulses has been consistently successful for damage sites whose lateral dimensions are less than 100 {micro}m, but has not been for larger sites. Cracks would often radiate outward from the damage when a CO{sub 2} pulse was applied to the larger sites. An investigation was conducted to mitigate large surface damage sites using galvanometer scanning of a tightly focused CO{sub 2} laser spot over an area encompassing the laser damage. It was thought that by initially scanning the CO{sub 2} spot outside the damage site, radiating crack propagation would be inhibited. Scan patterns were typically inward moving spirals starting at radii somewhat larger than that of the damage site. The duration of the mitigation spiral pattern was {approx}110 ms during which a total of {approx}1.3 J of energy was delivered to the sample. The CO{sub 2} laser spot had a 1/e{sup 2}-diameter of {approx}200 {micro}m. Thus, there was general heating of a large area around the damage site while rapid evaporation occurred locally at the laser spot position in the spiral. A 30 to 40 {micro}m deep crater was typically generated by this spiral with a diameter of {approx}600 {micro}m. The spiral would be repeated until there was no evidence of the original damage in microscope images. Using this technique, damage sites as large as 300 mm in size did not display new damage after mitigation when exposed to fluences exceeding 22 J/cm{sup 2} at 355 nm, 7.5 ns. It was found necessary to use a vacuum nozzle during the mitigation process to reduce the amount of re-deposited fused silica. In addition, curing spiral patterns at lower laser powers were used to presumably ''re-melt'' any re-deposited fused silica. A compact, shearing interferometer microscope was developed to permit in situ measurement of the depth of mitigation sites

Functional Heterologous Expression of an Engineered Full Length Cipa from Clostridium Thermocellum in Thermoanaerobacterium Saccharolyticum

Background: Cellulose is highly recalcitrant and thus requires a specialized suite of enzymes to solubilize it into fermentable sugars. In C. thermocellum, these extracellular enzymes are present as a highly active multi-component system known as the cellulosome. This study explores the expression of a critical C. thermocellum cellulosomal component in T. saccharolyticum as a step toward creating a thermophilic bacterium capable of consolidated bioprocessing by employing heterologously expressed cellulosomes. Results:We developed an inducible promoter system based on the native T. saccharolyticum xynA promoter, which was shown to be induced by xylan and xylose. The promoter was used to express the cellulosomal component cipA*, an engineered form of the wild-type cipAfrom C. thermocellum. Expression and localization to the supernatant were both verified for CipA*. When a ΔcipA mutant C. thermocellum strain was cultured with a CipA*-expressing T. saccharolyticum strain, hydrolysis and fermentation of 10 grams per liter SigmaCell 101, a highly crystalline cellulose, were observed. This trans-species complementation of a cipA deletion demonstrated the ability for CipA* to assemble a functional cellulosome. Conclusion: This study is the first example of an engineered thermophile heterologously expressing a structural component of a cellulosome. To achieve this goal we developed and tested an inducible promoter for controlled expression in T. saccharolyticum as well as a synthetic cipA . In addition, we demonstrate a high degree of hydrolysis (up to 93%) on microcrystalline cellulose

In-situ monitoring of surface post-processing in large aperture fused silica optics with Optical Coherence Tomography

Optical Coherence Tomography is explored as a method to image laser-damage sites located on the surface of large aperture fused silica optics during post-processing via CO{sub 2} laser ablation. The signal analysis for image acquisition was adapted to meet the sensitivity requirements for this application. A long-working distance geometry was employed to allow imaging through the opposite surface of the 5-cm thick optic. The experimental results demonstrate the potential of OCT for remote monitoring of transparent material processing applications

Downstream Intensification Effects Associated with CO2 Laser Mitigation of Fused Silica

Mitigation of 351nm laser-induced damage sites on fused silica exit surfaces by selective CO{sub 2} treatment has been shown to effectively arrest the exponential growth responsible for limiting the lifetime of optics in high-fluence laser systems. However, the perturbation to the optical surface profile following the mitigation process introduces phase contrast to the beam, causing some amount of downstream intensification with the potential to damage downstream optics. Control of the laser treatment process and measurement of the associated phase modulation is essential to preventing downstream 'fratricide' in damage-mitigated optical systems. In this work we present measurements of the surface morphology, intensification patterns and damage associated with various CO{sub 2} mitigation treatments on fused silica surfaces. Specifically, two components of intensification pattern, one on-axis and another off-axis can lead to damage of downstream optics and are related to rims around the ablation pit left from the mitigation process. It is shown that control of the rim structure around the edge of typical mitigation sites is crucial in preventing damage to downstream optics

Results of applying a non-evaporative mitigation technique to laser-initiated surface damage on fused-silica

We present results from a study to determine an acceptable CO{sub 2} laser-based non-evaporative mitigation protocol for use on surface damage sites in fused-silica optics. A promising protocol is identified and evaluated on a set of surface damage sites created under ICF-type laser conditions. Mitigation protocol acceptability criteria for damage re-initiation and growth, downstream intensification, and residual stress are discussed. In previous work, we found that a power ramp at the end of the protocol effectively minimizes the residual stress (<25 MPa) left in the substrate. However, the biggest difficulty in determining an acceptable protocol was balancing between low re-initiation and problematic downstream intensification. Typical growing surface damage sites mitigated with a candidate CO{sub 2} laser-based mitigation protocol all survived 351 nm, 5 ns damage testing to fluences >12.5 J/cm{sup 2}. The downstream intensification arising from the mitigated sites is evaluated, and all but one of the sites has 100% passing downstream damage expectation values. We demonstrate, for the first time, a successful non-evaporative 10.6 {micro}m CO{sub 2} laser mitigation protocol applicable to fused-silica optics used on fusion-class lasers like the National Ignition Facility (NIF)