Biological hydrogen formation by thermophilic bacteria

Hydrogen gas (H 2 ) is an important chemical commodity. It is used in many industrial processes and is applicable as a fuel. However, present production processes are predominantly based on non-renewable resources. In a biological H 2 (bioH 2 ) production process, known as dark-fermentation, fermentative microorganisms are able to generate H 2 from renewable resources like carbohydrate-rich plant material or industrial waste streams. Because of their favourable biomass degrading capabilities and H 2 -forming features both Caldicellulosiruptor saccharolyticus and Thermotoga maritima have become model organisms in the study of thermophilic H 2 production. Novel insights in substrate usability, associated fermentation pathways and the mechanism involved in H 2 formation will provide steps forward in the application of these organisms for H 2 production and sustainable biological H 2 formation via dark fermentation in general. Elevated H 2 levels are known to inhibit H 2 -formation during dark fermentations. The response of C. saccharolyticus to the exposure of elevated H 2 levels is investigated in different chemostat cultivation setups. Analysis of the fermentation profiles and transcriptome data associated with low and high H 2 levels provides insight into this organism’s strategy to deal with elevated H 2 levels. In addition, several chemostat studies were performed to elucidate the effect of increased H 2 levels on the fermentation profile of C. saccharolyticus with respect to i) growth on ammonium deficient media and ii) low/high substrate loads. Furthermore, the thermodynamics of H 2 formation is discussed with respect to the dissolved H 2 concentration. Overall the dissolved H 2 concentration was shown to be a dominant process determinant in causing the fermentation profile to shift away from maximal H 2 yields. To be able to uphold desirable features for a H 2 production process under high sugar load conditions, in terms of high H 2 yields and productivities, the dissolved hydrogen concentrations should be kept below the fermentation switch threshold. This is only achievable via proper reactor design and certainly required for the efficient up-scaling of this bioH 2 production process. The role of inorganic pyrophosphate (PP i ) in the energy metabolism of C. saccharolyticus is investigated. In agreement with the annotated genome sequence PP i -dependent phosphofructokinase, pyruvate phosphate dikinase and membrane bound pyrophosphatase activity can be detected in glucose-grown cultures. Pyrophosphate is demonstrated to inhibit pyruvate kinase activity. Furthermore, the dynamics in ATP and PP i levels throughout batch growth is discussed. A genomic distribution profile of PP i -dependent glycolytic enzymes and their genomic co-occurrence with soluble or membrane-bound pyrophosphatases in 495 fully sequenced genomes is given. An ab initio classification of enzyme-subtypes, which elaborates on known classifications systems and incorporates characterized protein features e.g. catalytic site residues and allosteric regulatory site residues, is presented. The potential functional role of the PPi-dependent enzymes and membrane-bound pyrophosphatases is discussed. Overall the presented data indicates that the involvement of pyrophosphate in glycolysis/gluconeogenesis is a widespread phenomenon throughout the three domains of life. Glycerol is formed as a by-product during biodiesel formation. Given the highly reduced state of carbon in glycerol this low cost substrate is of special interest for sustainable biofuel production. The use of glycerol for H 2 -formation by T. maritima is investigated. Growth on glycerol is demonstrated in both batch and chemostat cultivation setups.The observed H 2 yields nearly reach the theoretical maximum of 3 H 2 per glycerol, which is 3 times the yield generally observed for mesophilic conversions In addition, the route of glycerol fermentation and the exceptional bioenergetics associated with H­ 2 formation from glycerol in T. maritima are discussed. For the future application of bioH 2 production by C. saccharolyticusand T. maritima,research should focus on i) the factors limiting complex sugar degradation, ii) the specific nutritional requirements to sustain growth during high sugar loads, and iii) specific mechanisms to make the organism more robust against stresses like elevated dissolved H 2 concentrations or osmotic stress. Moreover, for the incorporation of these desirable traits the development of genetic systems is required.