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Biodiesel production from cotton-seed cake (CSC) and the pretreatment of the remaining biomass for dark fermentative hydrogen production was investigated. The direct conversion to biodiesel with alkali free fatty acids neutralization pretreatment and alkali transesterification resulted in a biodiesel with high esters content and physicochemical properties fulfilling the EN-standards. Blends of cotton-seed oil methyl esters (CME) and diesel showed an improvement in lubricity and cetane number. Moreover, CME showed good compatibility with commercial biodiesel additives. On the basis of conversion of the remaining CSC to sugars fermentable towards hydrogen, the optimal conditions included removal of the oil of CSC and pretreatment at 10% NaOH (w/w dry matter). The extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed good hydrogen production, 84–112% of the control, from NaOH-pretreated CSC and low hydrogen production, 15–20% of the control, from the oil-rich and not chemically pretreated CSC, and from Ca(OH)2-pretreated CSC.

Increased hydrogen (H2) levels are known to inhibit H2 formation in Caldicellulosiruptor saccharolyticus. To investigate this organism's strategy for dealing with elevated H2 levels the effect of the hydrogen partial pressure ( P H 2 ) on fermentation performance was studied by growing cultures under high and low P H 2 in a glucose limited chemostat setup. Transcriptome analysis revealed the upregulation of genes involved in the disposal of reducing equivalents under high P H 2 , like lactate dehydrogenase and alcohol dehydrogenase as well as the NADH-dependent and ferredoxin-dependent hydrogenases. These findings are in line with the observed shift in fermentation profiles from acetate production to the production of acetate, lactate and ethanol under high P H 2 . Moreover, differential transcription was observed for genes involved in carbon metabolism, fatty acid biosynthesis and several transport systems. In addition, presented transcription data provide evidence for the involvement of the redox sensing Rex protein in gene regulation under high P H 2 cultivation conditions.

Integrating of lignocellulose-based and starch-rich biomass-based hydrogen production was investigated by mixing wheat straw hydrolysate with a wheat grain hydrolysate for improved fermentation. Enzymatic pretreatment and hydrolysis of wheat grains led to a hydrolysate with a sugar concentration of 93.4 g/L, while dilute-acid pretreatment and enzymatic hydrolysis of wheat straw led to a hydrolysate with sugar concentration 23.0 g/L. Wheat grain hydrolysate was not suitable for hydrogen production by the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus at glucose concentrations of 10 g/L or higher, and wheat straw hydrolysate showed good fermentability at total sugar concentrations of up to 10 g/L. The mixed hydrolysates showed good fermentability at the highest tested sugar concentration of 20 g/L, with a hydrogen production of 82–97% of that of the control with pure sugars. Mixing wheat grain hydrolysate with wheat straw hydrolysate would be beneficial for fermentative hydrogen production in a biorefinery.

The main objective of this study was to use the fermentability test to investigate the feasibility of applying various dilute acids in the pretreatment of barley straw for biological hydrogen production. At a fixed acid loading of 1% (w/w dry matter) 28–32% of barley straw was converted to soluble monomeric sugars, while at a fixed combined severity of −0.8 30–32% of the straw was converted to soluble monomeric sugars. With fermentability tests at sugar concentrations 10 and 20 g/L the extreme thermophilic bacterium Caldicellulosiruptor saccharolyticus showed good hydrogen production on hydrolysates of straw pretreated with H3PO4 and H2SO4, and to a lesser extent, HNO3. The fermentability of the hydrolysate of straw pretreated with HCl was lower compared to the other acids but equally high as that of pure sugars. At sugar concentration 30 g/L the fermentability of all hydrolysates was low.

The objective of this work was to investigate the feasibility of combining low severity dilute-acid pretreatment of barley straw and decreased enzyme loading hydrolysis for the high production of fermentable substrates and the low release of inhibitory compounds. For most of the pretreatments at 160 and 180 degrees C, the sugar production with 15 FPU (filter paper unit)/g straw was equally high compared to higher enzyme loadings. For the pretreatments at 170 degrees C an enzyme loading higher than 15 FPU/g straw was necessary to achieve a carbohydrate conversion of 50% or higher. The effect of acid loading on sugar production was discernible only in the experiments with 15 FPU/g straw or higher. The concentration of 5-hydroxymethylfurfural (HMF), levulinic acid and formic acid was kept below 0.7, 0.6 and 0.8 g L-1, respectively, with all experiments. The release of acetic acid and furfural reached toxic levels with experiments at 170 degrees C and experiments at 180 degrees C, respectively. Decreasing the enzyme loading did not have a major effect on the release of HMF, furfural and formic acid but resulted in decreased release of acetic acid and levulinic acid at 170 and 180 degrees

The gene coding for xylose isomerase from the thermophilic bacterium Fervidobacterium gondwanense was cloned and overexpressed in Escherichia coli. The produced xylose isomerase (XylA), which closely resembles counterparts from Thermotoga maritima and T. neapolitana, was purified and characterized. It is optimally active at 70 degrees C, pH 7.3, with a specific activity of 15.0 U/mg for the interconversion of glucose to fructose. When compared with T. maritima XylA at 85 degrees C, a higher catalytic efficiency was observed. Divalent metal ions Co2+ and Mg2+ were found to enhance the thermostability.

The diazotroph Azotobacter vinelandii possesses three distinct nitrogenase isoenzymes, all of which produce molecular hydrogen as a by-product. In batch cultures, A. vinelandii strain CA6, a mutant of strain CA, displays multiple phenotypes distinct from its parent: tolerance to tungstate, impaired growth and molybdate transport, and increased hydrogen evolution. Determining and comparing the genomic sequences of strains CA and CA6 revealed a large deletion in CA6's genome, encompassing genes related to molybdate and iron transport and hydrogen reoxidation. A series of iron uptake analyses and chemostat culture experiments confirmed iron transport impairment and showed that the addition of fixed nitrogen (ammonia) resulted in cessation of hydrogen production. Additional chemostat experiments compared the hydrogen-producing parameters of different strains: in iron-sufficient, tungstate-free conditions, strain CA6's yields were identical to those of a strain lacking only a single hydrogenase gene. However, in the presence of tungstate, CA6 produced several times more hydrogen. A. vinelandii may hold promise for developing a novel strategy for production of hydrogen as an energy compound., J.N. was the recipient of a 3-year National Science Foundation Graduate Research Fellowship. This project was supported by the North Carolina State University Department of Microbiology (now Plant and Microbial Biology). The University of North Carolina-Chapel Hill Core facility is supported by grant number NIH P30 DK34987.

Hydrogen produced from biomass by bacteria and archaea is an attractive renewable energy source. However, to make its application more feasible, microorganisms are needed with high hydrogen productivities. For several reasons, hyperthermophilic and extremely thermophilic bacteria and archaea are promising is this respect. In addition to the high polysaccharide-hydrolysing capacities of many of these organisms, an important advantage is their ability to use most of the reducing equivalents (e.g. NADH, reduced ferredoxin) formed during glycolysis for the production of hydrogen, enabling H2/hexose ratios of between 3.0 and 4.0. So, despite the fact that the hydrogen-yielding reactions, especially the one from NADH, are thermodynamically unfavourable, high hydrogen yields are obtained. In this review we focus on three different mechanisms that are employed by a few model organisms, viz. Caldicellulosiruptor saccharolyticus and Thermoanaerobacter tengcongensis, Thermotoga maritima, and Pyrococcus furiosus, to efficiently produce hydrogen. In addition, recent developments to improve hydrogen production by hyperthermophilic and extremely thermophilic bacteria and archaea are discussed.

The araA gene encoding an L-arabinose isomerase (L-AI) from the psychrotrophic and food grade Lactobacillus sakei 23K was cloned, sequenced and over-expressed in Escherichia coli. The recombinant enzyme has an apparent molecular weight of nearly 220 kDa, suggesting it is a tetramer of four 54 kDa monomers. The enzyme is distinguishable from previously reported L-AIs by its high activity and stability at temperatures from 4 to 40 degrees C, and pH from 3 to 8, and by its low metal requirement of only 0.8 mM Mn(2+) and 0.8 mM Mg(2+) for its maximal activity and thermostability. Enzyme kinetic studies showed that this enzyme displays a high catalytic efficiency allowing D-galactose bioconversion rates of 20% and 36% at 10 and 45 degrees C, respectively, which are useful for commercial production of D-tagatose. (C) 2010 Elsevier Ltd. All rights reserved.

The expression of arginine metabolism in Lactococcus lactis is controlled by the two homologous transcriptional regulators ArgR and AhrC. Genome sequence analyses have shown that the occurrence of multiple homologues of the ArgR family of transcriptional regulators is a common feature of many low-G + C Gram-positive bacteria. Detailed studies of ArgR type regulators have previously only been carried out in bacteria containing single regulators. Here, we present a first characterization of the two L. lactis arginine regulators by means of gel retardation and DNase I footprinting. ArgR of L. lactis was shown to bind to the promoter regions of both the arginine biosynthetic argCJDBF operon and the arginine catabolic arcABD1C1C2TD2yvaD operon, but in an arginine-independent manner. Surprisingly, AhrC alone was unable to bind to DNA. Arginine-dependent DNA binding was obtained by mixing the two regulators in gel retardation assays. With both regulators present, the addition of arginine led to increased binding of ArgR-AhrC to the biosynthetic argC promoter but also to diminished binding to the catabolic arcA promoter. Footprinting showed ArgR-AhrC protection of regions containing ARG box operator sequences preceding argC. In the absence of AhrC, ArgR protected sites in the arcA promoter region with similarity to ARG box half-sites, here called ARC boxes. We propose a model for repression of arginine biosynthesis and activation of catabolism by anti-repression, involving arginine-dependent interaction between the two L. lactis regulator proteins, ArgR and AhrC.