Optimization of seed production for a simultaneous saccharification cofermentation biomass-to-ethanol process using recombinantZymomonas

The five-carbon sugard-xylose is a major component of hemicellulose and accounts for roughly one-third of the carbohydrate content of many lignocellulosic materials. The efficient fermentation of xylose-rich hemicellulose hydrolyzates (prehydrolyzates) represents an opportunity to improve significantly the economics of large-scale fuel ethanol production from lignocellulosic feedstocks. The National Renewable Energy Laboratory (NREL) is currently investigating a simultaneous saccharification and cofermentation (SSCF) process for ethanol production from biomass that uses a dilute-acid pretreatment and a metabolically engineered strain ofZymomonas mobilis that can coferment glucose and xylose. The objective of this study was to establish optimal conditions for cost-effective seed production that are compatible with the SSCF process design. Two-level and three-level full factorial experimental designs were employed to characterize efficiently the growth performance of recombinantZ. mobilis CP4:pZB5 as a function of nutrient level, pH, and acetic acid concentration using a synthetic hardwood hemicellulose hydrolyzate containing 4% (w/v) xylose and 0.8% (w/v) glucose. Fermentations were run batchwise and were pH-controlled at low levels of clarified corn steep liquor (cCSL, 1-2% v/v), which were used as the sole source of nutrients. For the purpose of assessing comparative fermentation performance, seed production was also carried out using a “benchmark” yeast extract-based laboratory medium. Analysis of variance (ANOVA) of experimental results was performed to determine the main effects and possible interactive effects of nutrient (cCSL) level, pH, and acetic acid concentration on the rate of xylose utilization and the extent of cell mass production. Results indicate that the concentration of acetic acid is the most significant limiting factor for the xylose utilization rate and the extent of cell mass production; nutrient level and pH exerted weaker, but statistically significant effects. At pH 6.0, in the absence of acetic acid, the final cell mass concentration was 1.4 g dry cell mass/L (g DCM/L), but decreased to 0.92 and 0.64 g DCM/L in the presence of 0.5 and 1.0% (w/v) acetic acid, respectively. At concentrations of acetic acid of 0.75 (w/v) or lower, fermentation was complete within 1.5 d. In contrast, in the presence of 1.0% (w/v) acetic acid, 25% of the xylose remained after 2 d. At a volumetric supplementation level of 1.5–2.0% (v/v), cCSL proved to be a cost-effective single-source nutritional adjunct that can support growth and fermentation performance at levels comparable to those achieved using the expensive yeast extract-based laboratory reference medium.     

PHOTOREACTIVATION IN THE EXTREME HALOPHILIC ARCHAEBACTERIUM Halobacterium cutirubrum

Abstract— Photoreactivation in the extreme halophilic archaebacterium Halobacterium cutirubrum was studied both in vivo and in vitro. Cells irradiated with ultraviolet (UV)-fluences up to 350 J/m² could be completely photoreactivated, indicating very efficient repair of pyrimidine dimers in UV-irradiated DNA. Dark repair is apparently absent in Halobacterium since liquid holding under non-growth conditions did not influence the survival of UV-irradiated cells, while cells remained completely photoreactivable with no change in the kinetics of photoreactivation. Experiments with Halobacterium isolates of different carotenoid content indicated that carotenoids do not influence either UV-inactivation or photoreactivation. Small differences in the rates of UV-inactivation and photoreactivation could be assigned to the occurrence of gas vesicles. Flash experiments and the temperature dependence of photoreactivation indicated an enzymatical reaction. This was confirmed by in vitro experiments with partially purified photoreactivating enzyme. The in vivo action spectrum of photoreactivation showed a main band in the 400-470 nm region with a maximum at 440 nm. Comparison with action spectra of other microorganisms classified the Halobacterium enzyme as a 8-hydroxy-5-deazaflavin type photoreactivating enzyme.     

The structures and vibrational spectra of crystals and glasses in the silica-alumina system

Solar furnace melting and fast-quench techniques have been used to prepare SiO₂Al₂O₃ glasses to high alumina content (near 60 mol% Al₂O₃), which have been studied by Raman spectroscopy. These spectra may not be simply interpreted. The structures of crystalline compounds in the SiO₂Al₂O₃ system are discussed in relation to their vibrational spectra. On the basis of this discussion and other considerations, a structural model for the silica-alumina glass system is proposed, which is consistent with the stable or metastable immiscibility suggested along this join. The essential features of this model include a modified silica structure at low alumina content, and “structure-broken” regions at high alumina compositions, with silicon in tetrahedral coordination, but aluminium assuming a variety of bonding geometries. These are proposed to include aluminate tetrahedra with higher polymerization than simple corner-sharing, and less well-defined polyhedra of higher average coordination number.     

AN APPROXIMATE PROJECTION SCHEME FOR INCOMPRESSIBLE FLOW USING SPECTRAL ELEMENTS

An approximate projection scheme based on the pressure correction method is proposed to solve the Navier–Stokes equations for incompressible flow. The algorithm is applied to the continuous equations; however, there are no problems concerning the choice of boundary conditions of the pressure step. The resulting velocity and pressure are consistent with the original system. For the spatial discretization a high-order spectral element method is chosen. The high-order accuracy allows the use of a diagonal mass matrix, resulting in a very efficient algorithm. The properties of the scheme are extensively tested by means of an analytical test example. The scheme is further validated by simulating the laminar flow over a backward-facing step.     

金属卟啉催化条件下有机化合物与二氧化碳的电羧化反应Ⅰ. 金属卟啉对苄基氯电羧化催化活性的研究

本文研究了系列金属卟啉对苯基氯(phCH~2Cl)和CO~2电羧化反应的催化活性.用熔点,MS,IR,UV鉴别产物为苯乙酸卟脂,利用高效液相色谱(HPLC)定量分析了羧化产物,选出最佳电羧化电解电位为-1.6v(相对于饱和甘汞电极,vs.SCE,下同). 并对催化活性较高的钴卟啉系列配合物进行了深入研究. 探讨了金属卟啉具有催化活性的原因,认为能形成M(Ⅰ)中间体的金属卟啉配合物.具有较高的催化活性.     

Enzyme production, growth, and adaptation of T. reesei strains QM9414, L-27, RL-P37, and rut C-30 to conditioned yellow poplar sawdust hydrolysate

National Renewable Energy Laboratory (NREL) has developed a conditioning process that decreases acetic acid levels in pretreated yellow poplar hydrolysate. Trichoderma reesei is sensitive to acetic acid and this conditioning method has enabled applied cellulase production with hardwoods. T. reesei strains QM9414, L-27, RL-P37, and Rut C-30 were screened for growth on conditioned hydrolysate liquor. Tolerance to hydrolysate was found to be strain-dependent. Strain QM9414 was adapted to grow in 80% (v/v) conditioned hydrolysate (40 g/L of soluble sugars and 1.6 g/L acetic acid from pretreated poplar). However, enzyme production was highest at 20% (v/v) hydrolysateusing strain L-27. Cellulasetiters of 2–3 International Filter Paper Units (IFPU)/mL were achieved using pretreated yellow poplar liquors and solids as the sole carbon sources.     

Pressure-induced structural transformations of the Zintl phase sodium silicide

The high-pressure behaviour of NaSi has been studied using Raman spectroscopy and angle-dispersive synchrotron X-ray diffraction to observe the onset of structural phase transformations and potential oligomerisation into anionic Si nanoclusters with extended dimensionality. Our studies reveal a first structural transformation occurring at 8–10 GPa, followed by irreversible amorphisation above 15 GPa, suggesting the formation of Si–Si bonds with oxidation of the Si⁻ species and reduction of Na⁺ to metallic sodium. We have combined our experimental studies with DFT calculations to assist in the analysis of the structural behaviour of NaSi at high pressure.     

Crystal-liquid phase relations in silicon at negative pressure

The stable and metastable melting relations for silicon in the diamond and Si136 clathrate-II structures at positive and negative pressures are calculated by molecular dynamics computer simulation. The simulated liquid and crystalline clathrates undergo cavitation at approximately -3 and -12 GPa. Between these limits a stretched crystal would transform directly to gas in response to a mechanical instability. Most importantly, the clathrate-II crystal becomes thermodynamically stable over the diamond at negative pressure below -1 GPa at the melting point. Si136 should then crystallize from a slightly stretched liquid, which would have the same volume as a diamond-structure crystal.     

The coherent scattering function of the reptation model: Simulations compared to theory

We present results of Monte Carlo simulations measuring the coherent structure function of a chain moving through an ordered lattice of fixed topological obstacles. Our computer experiments use chains up to 320 beads and cover a large range of wave vectors and a time range exceeding the reptation time. For additional information we also measured the coherent structure function of internal pieces of the chain. We compare our results i) to the predictions of the primitive chain model, ii) to an approximate form resulting from Rouse motion in a coiled tube, and iii) to our recent evaluation of the full reptation model. i) The primitive chain model can fit the data for times , where T ₂ is the Rouse time of the chain. Besides some phenomenological amplitude factor this fit involves the reptation time T ₃ as a second fit parameter. For the chain lengths measured, the asymptotic behavior is not attained. ii) The model of Rouse motion in a tube, which we have criticized before on theoretical grounds, is shown to fail also on the purely phenomenological level. iii) Our evaluation of the full reptation model yields an excellent fit to the data for both total chains and internal pieces and for all wave vectors and all times, provided specific micro-structure effects of the MC dynamics are negligible. Such micro-structure effects show up for wave vectors of the order of the inverse segment size and enforce the introduction of some phenomenological, wave-vector-dependent prefactor. For the dynamics of the total chain our data analysis based on the full reptation model shows the importance of tube length fluctuations. Universal (Rouse-type) internal relaxation, however, is unimportant. It can be observed only in the form of the diffusive motion of a short central subchain in the tube. Finally, we present a fit formula which in a large range of wave vectors and chain lengths reproduces the numerical results of our theory for the scattering from the total chain.Received: 9 July 2003, Published online: 11 November 2003PACS: 83.10.Kn Reptation and tube theories - 82.35.Lr Physical properties of polymers - 83.10.Rs Computer simulation of molecular and particle dynamics