Biological hydrogen formation by thermophilic bacteria

Hydrogen gas (H2) 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 H2 (bioH2) production process, known as dark-fermentation, fermentative microorganisms are able to generate H2 from renewable resources like carbohydrate-rich plant material or industrial waste streams. Because of their favourable biomass degrading capabilities and H2-forming features both Caldicellulosiruptor saccharolyticus and Thermotoga maritima have become model organisms in the study of thermophilic H2 production. Novel insights in substrate usability, associated fermentation pathways and the mechanism involved in H2 formation will provide steps forward in the application of these organisms for H2 production and sustainable biological H2 formation via dark fermentation in general. Elevated H2 levels are known to inhibit H2-formation during dark fermentations. The response of C. saccharolyticus to the exposure of elevated H2 levels is investigated in different chemostat cultivation setups. Analysis of the fermentation profiles and transcriptome data associated with low and high H2 levels provides insight into this organism’s strategy to deal with elevated H2 levels. In addition, several chemostat studies were performed to elucidate the effect of increased H2 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 H2 formation is discussed with respect to the dissolved H2 concentration. Overall the dissolved H2 concentration was shown to be a dominant process determinant in causing the fermentation profile to shift away from maximal H2 yields. To be able to uphold desirable features for a H2 production process under high sugar load conditions, in terms of high H2 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 bioH2 production process. The role of inorganic pyrophosphate (PPi) in the energy metabolism of C. saccharolyticus is investigated. In agreement with the annotated genome sequence PPi-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 PPi levels throughout batch growth is discussed. A genomic distribution profile of PPi-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 H2-formation by T. maritima is investigated. Growth on glycerol is demonstrated in both batch and chemostat cultivation setups.The observed H2 yields nearly reach the theoretical maximum of 3 H2 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 H­2 formation from glycerol in T. maritima are discussed. For the future application of bioH2 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 H2 concentrations or osmotic stress. Moreover, for the incorporation of these desirable traits the development of genetic systems is required.</p

Biohydrogen Production from Glycerol using Thermotoga spp

Given the highly reduced state of carbon in glycerol and its availability as a substantial byproduct of biodiesel production, glycerol is of special interest for sustainable biofuel production. Glycerol was used as a substrate for biohydrogen production using the hyperthermophilic bacterium, Thermotoga maritima and Thermotoga neapolitana. Both species metabolized glycerol to mainly acetate and hydrogen. At glycerol concentrations of 2.5 g/L, hydrogen was produced with a yield of 2.75 and 2.65 mol H2/mol glycerol consumed by T. maritima and T. neapolitana respectively. Additionally, the effect of initial pH (ranging between pH 5.0-8.5) and yeast extract concentrations (0.5, 1, 2, 4 g/L) on glycerol fermentation by T. neapolitana was investigated in batch systems. An initial pH value of around 7 was optimal for hydrogen production by T. neapolitana. Lower concentration of yeast extract resulted in a lower H2 production, however increasing the concentration from 2 to 4 g/L did not affect H2 productio

Glycerol fermentation to hydrogen by Thermotoga maritima: Proposed pathway and bioenergetic considerations

The production of biohydrogen from glycerol, by the hyperthermophilic bacterium Thermotoga maritima DSM 3109, was investigated in batch and chemostat systems. T. maritima converted glycerol to mainly acetate, CO2 and H2. Maximal hydrogen yields of 2.84 and 2.41 hydrogen per glycerol were observed for batch and chemostat cultivations, respectively. For batch cultivations: i) hydrogen production rates decreased with increasing initial glycerol concentration, ii) growth and hydrogen production was optimal in the pH range of 7–7.5, and iii) a yeast extract concentration of 2 g/l led to optimal hydrogen production. Stable growth could be maintained in a chemostat, however, when dilution rates exceeded 0.025 h-1 glycerol conversion was incomplete. A detailed overview of the catabolic pathway involved in glycerol fermentation to hydrogen by T. maritima is given. Based on comparative genomics the ability to grow on glycerol can be considered as a general trait of Thermotoga species. The exceptional bioenergetics of hydrogen formation from glycerol is discusse

Hydrogen production by hyperthermophilic and extremely thermophilic bacteria and archaea: mechanisms for reductant disposal

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 discusse

Hydrogen production by hyperthermophilic and extremely thermophilic bacteria and archaea: mechanisms for reductant disposal

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 discusse

Self-assembled monolayers of 1-alkenes on oxidized platinum surfaces as platforms for immobilized enzymes for biosensing

Alkene-based self-assembled monolayers grafted on oxidized Pt surfaces were used as a scaffold to covalently immobilize oxidase enzymes, with the aim to develop an amperometric biosensor platform. NH2-terminated organic layers were functionalized with either aldehyde (CHO) or N-hydroxysuccinimide (NHS) ester-derived groups, to provide anchoring points for enzyme immobilization. The functionalized Pt surfaces were characterized by X-ray photoelectron spectroscopy (XPS), static water contact angle (CA), infrared reflection absorption spectroscopy (IRRAS) and atomic force microscopy (AFM). Glucose oxidase (GOX) was covalently attached to the functionalized Pt electrodes, either with or without additional glutaraldehyde crosslinking. The responses of the acquired sensors to glucose concentrations ranging from 0.5 to 100 mM were monitored by chronoamperometry. Furthermore, lactate oxidase (LOX) and human hydroxyacid oxidase (HAOX) were successfully immobilized onto the PtOx surface platform. The performance of the resulting lactate sensors was investigated for lactate concentrations ranging from 0.05 to 20 mM. The successful attachment of active enzymes (GOX, LOX and HAOX) on Pt electrodes demonstrates that covalently functionalized PtOx surfaces provide a universal platform for the development of oxidase enzyme-based sensors. (C) 2016 Elsevier B.V. All rights reserved

Pyrophosphate as a central energy carrier in the hydrogen-producing extremely thermophilic Caldicellulosiruptor saccharolyticus

The role of inorganic pyrophosphate (PPi) as an energy carrier in the central metabolism of the extremely thermophilic bacterium Caldicellulosiruptor saccharolyticus was investigated. In agreement with its annotated genome sequence, cell extracts were shown to exhibit PPi-dependent phosphofructokinase and pyruvate phosphate dikinase activity. In addition, membrane-bound pyrophosphatase activity was demonstrated, while no significant cytosolic pyrophosphatase activity was detected. During the exponential growth phase, high PPi levels (approximately 4 ± 2 mM) and relatively low ATP levels (0.43 ± 0.07 mM) were found, and the PPi/ATP ratio decreased 13-fold when the cells entered the stationary phase. Pyruvate kinase activity appeared to be allosterically affected by PPi. Altogether, these findings suggest an important role for PPi in the central energy metabolism of C. saccharolyticu

A thermophile under pressure: Transcriptional analysis of the response of Caldicellulosiruptor saccharolyticus to different H2 partial pressures

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 (PH2) on fermentation performance was studied by growing cultures under high and low PH2 in a glucose limited chemostat setup. Transcriptome analysis revealed the upregulation of genes involved in the disposal of reducing equivalents under high PH2, 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 PH2. 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 PH2 cultivation condition

Biosensor comprising a modified metal surface and method for the modification of a metal surface

The present invention relates to a device for the detection of an analyte in a fluid, the device comprising: (a) a working electrode comprising a modified metal surface, wherein: (1) the metal is selected from the group consisting of Ru, Rh, Pd, Ag, Ir, Pt and Au; (2) an enzyme is covalently attached to the metal surface via an alkyloxy or an alkenyloxy moiety and, optionally, a linker moiety; (3)the alkyloxy or alkenyloxy moiety is covalently bonded to said metal surface via the alkyloxy or alkenyloxy O- atom; and (4) the linker moiety, if present, is covalently bonded to theenzyme and to the alkyloxy or alkenyloxy moiety; (b) a reference electrode; and (c) means for detecting an electricalsignal, the means being operationally coupled to at least working electrode (a) and reference electrode (b). The device according to the invention is also referred to as a biosensor. The invention also relates to a process for the modification of a metal surface and to a modified metal surface obtainable by the process. Furthermore, the invention relates to an electrode comprising said modified metal surface, and to a biosensor comprising said modified metal surface