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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.

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.