Hydrogen Turnover Research Articles

Tailor‐Made Modification of a Gold Surface for the Chemical Binding of a High‐Activity [FeFe] Hydrogenase

AbstractHydrogenases are iron–sulfur proteins that catalyze hydrogen turnover in a wide range of microorganisms. Three different classes have been described, and among these [FeFe] hydrogenases are the most active in H2 evolution. Hydrogenases are redox enzymes that have been shown to exchange electrons with graphite and modified noble metal electrodes. Making use of the latter, diffusible electron carriers are required to enable redox catalysis, as proteins do not specifically bind to the electrode surface. Diffusion‐limited electron transfer can be replaced by electron injection into immobilized hydrogenase by binding the redox mediator to the electrode surface. Here, we present the synthesis and spectroelectrochemical characterization of 1‐(10‐mercaptodecyl)‐1′‐benzyl‐4,4′‐bipyridinium dibromide (MBBP), which is used as redox‐active linker. CrHydA1, the high‐activity [FeFe] hydrogenase from Chlamydomonas reinhardtii, is immobilized on the linker‐modified gold electrode. Each surface modification step is controlled in situ by surface‐enhanced infrared absorption spectroscopy (SEIRAS). Functionality of the electrode–protein hybrid is demonstrated by recording the linker‐supported current. The specificcatalytic rate of hydrogen evolution by CrHydA1 (2.9 μmol H2 min–1 mg–1 hydrogenase) promises a valuable approach for further optimization of this novel bioelectrical interface.

Distribution Analysis of Hydrogenases in Surface Waters of Marine and Freshwater Environments

BackgroundSurface waters of aquatic environments have been shown to both evolve and consume hydrogen and the ocean is estimated to be the principal natural source. In some marine habitats, H2 evolution and uptake are clearly due to biological activity, while contributions of abiotic sources must be considered in others. Until now the only known biological process involved in H2 metabolism in marine environments is nitrogen fixation.Principal FindingsWe analyzed marine and freshwater environments for the presence and distribution of genes of all known hydrogenases, the enzymes involved in biological hydrogen turnover. The total genomes and the available marine metagenome datasets were searched for hydrogenase sequences. Furthermore, we isolated DNA from samples from the North Atlantic, Mediterranean Sea, North Sea, Baltic Sea, and two fresh water lakes and amplified and sequenced part of the gene encoding the bidirectional NAD(P)-linked hydrogenase. In 21% of all marine heterotrophic bacterial genomes from surface waters, one or several hydrogenase genes were found, with the membrane-bound H2 uptake hydrogenase being the most widespread. A clear bias of hydrogenases to environments with terrestrial influence was found. This is exemplified by the cyanobacterial bidirectional NAD(P)-linked hydrogenase that was found in freshwater and coastal areas but not in the open ocean.SignificanceThis study shows that hydrogenases are surprisingly abundant in marine environments. Due to its ecological distribution the primary function of the bidirectional NAD(P)-linked hydrogenase seems to be fermentative hydrogen evolution. Moreover, our data suggests that marine surface waters could be an interesting source of oxygen-resistant uptake hydrogenases. The respective genes occur in coastal as well as open ocean habitats and we presume that they are used as additional energy scavenging devices in otherwise nutrient limited environments. The membrane-bound H2-evolving hydrogenases might be useful as marker for bacteria living inside of marine snow particles.

Hydrogen is the central free intermediate during lignocellulose degradation by termite gut symbionts

The key role of free hydrogen in the digestion of lignocellulose by wood-feeding lower termites and their symbiotic gut microbiota has been conceptually outlined in the past decades but remains to be quantitatively analyzed in situ. Using Reticulitermes santonensis, Zootermopsis nevadensis and Cryptotermes secundus, we determined metabolite fluxes involved in hydrogen turnover and the resulting distribution of H(2) in the microliter-sized gut. High-resolution hydrogen microsensor profiles revealed pronounced differences in hydrogen accumulation among the species (from <1 kPa to the saturation level). However, flux measurements indicated that the hydrogen pool was rapidly turned over in all termites, irrespective of the degree of accumulation. Microinjection of radiotracers into intact guts confirmed that reductive acetogenesis from CO(2) dominated hydrogen consumption, whereas methanogenesis played only a minor role. Only negligible amounts of H(2) were lost by emission, documenting an overall equilibrium between hydrogen production and consumption within the gut. Mathematical modeling revealed that production dominates in the gut lumen and consumption in the gut periphery for R. santonensis and Z. nevadensis, explaining the large accumulation of H(2) in these termites, whereas the moderate hydrogen accumulation in C. secundus indicated a more balanced radial distribution of the two processes. Daily hydrogen turnover rates were 9-33 m(3) H(2) per m(3) hindgut volume, corresponding to 22-26% of the respiratory activity of the termites. This makes H(2) the central free intermediate during lignocellulose degradation and the termite gut-with its high rates of reductive acetogenesis-the smallest and most efficient natural bioreactor currently known.

In situ hydrogen consumption kinetics as an indicator of subsurface microbial activity

There are few methods available for broadly assessing microbial community metabolism directly within a groundwater environment. In this study, hydrogen consumption rates were estimated from in situ injection/withdrawal tests conducted in two geochemically varying, contaminated aquifers as an approach towards developing such a method. The hydrogen consumption first-order rates varied from 0.002 nM h(-1) for an uncontaminated, aerobic site to 2.5 nM h(-1) for a contaminated site where sulfate reduction was a predominant process. The method could accommodate the over three orders of magnitude range in rates that existed between subsurface sites. In a denitrifying zone, the hydrogen consumption rate (0.02 nM h(-1)) was immediately abolished in the presence of air or an antibiotic mixture, suggesting that such measurements may also be sensitive to the effects of environmental perturbations on field microbial activities. Comparable laboratory determinations with sediment slurries exhibited hydrogen consumption kinetics that differed substantially from the field estimates. Because anaerobic degradation of organic matter relies on the rapid consumption of hydrogen and subsequent maintenance at low levels, such in situ measures of hydrogen turnover can serve as a key indicator of the functioning of microbial food webs and may be more reliable than laboratory determinations.

Hydrogen Turnover and Subcellular Compartmentation of Hepatic [2-13C]Glutamate and [3-13C]Aspartate as Detected by 13C NMR

(13)C NMR monitored the dynamics of exchange from specific hydrogens of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate with deuterons from intracellular heavy water providing information on alpha-ketoglutarate/glutamate exchange and subcellular compartmentation. Mouse livers were perfused with [3-(13)C]alanine in buffer containing or not 50% (2)H(2)O for increasing periods of time (1 min < t < 30 min). Liver extracts prepared at the end of the perfusions were analyzed by high resolution (13)C NMR (150.13 MHz) with (1)H decoupling only and with simultaneous (1)H and (2)H decoupling. (13)C-(2)H couplings and (2)H-induced isotopic shifts observed in the glutamate C2 resonance, allowed to estimate the apparent rate constants (forward, reverse; min(-1)) for (i) the reversible exchange of [2-(13)C]glutamate H2 as catalyzed mainly by aspartate aminotransferase (0.32, 0.56), (ii) the reversible exchange of [2-(13)C]glutamate H3(proS) as catalyzed by NAD(P) isocitrate dehydrogenase (0.1, 0.05), and (iii) the irreversible exchanges of glutamate H3(proR) and H3(proS) as catalyzed by the sequential activities of mitochondrial aconitase and NAD isocitrate dehydrogenase of the tricarboxylic acid cycle (0.035), respectively. A similar approach allowed to determine the rates of (1)H-(2)H exchange for the H2 (0.4, 0.5) or H3(proR) (0.3, 0.2) or the H2 and H3(proS) hydrogens (0.20, 0.23) of [3-(13)C]aspartate isotopomers. The ubiquitous subcellular localization of (1)H-(2)H exchange enzymes and the exclusive mitochondrial localization of pyruvate carboxylase and the tricarboxylic acid cycle resulted in distinctive kinetics of deuteration in the H2 and either or both H3 hydrogens of [2-(13)C]glutamate and [3-(13)C]aspartate, allowing to follow glutamate and aspartate trafficking through cytosol and mitochondria.

Homogeneous catalysis of water-gas shift reaction in emulsions

The homogeneous catalysis of the water-gas shift reaction, H 2O + COH 2 + CO 2,has been demonstrated for a number of metal carbonyl complexes under alkaline conditions in toluene-water emulsions. Mo(CO) 6, W(CO) 6 and Ru 3(CO) 12 were found to be effective catalysts at temperatures below 200 °C. Activity of Ru 3(CO) 12 is about 20 times higher than Mo(CO) 6 and W(CO) 6. The effect of process parameters on hydrogen production catalysed by Mo(CO) 6 was studied in some detail. Hydrogen production was found to increase with increasing KOH concentration. Also, hydrogen production increased with increasing Mo(CO) 6 concentration up to 0.04 M and then decreased. Hydrogen turnover number increased with increasing CO loading pressure in the range of 1–3.1 MPa, remained practically constant in the range of 3.1–4.1 MPa and decreased with increasing CO loading pressure from 4.1 to 6.2 MPa. The effect of CO on hydrogen production could be explained by a postulated mechanism involving equilibrium formation of formate ion which reacts with Mo(CO) 5 in a rate determining step to generate a metalloformate, Mo(CO) 5HCOO −. Hydrogen production increased with increasing solvent polarity. Solvent effect is attributed to the stabilization of charged intermediate species.

1,1″,1‴-Trialkyl[4,2′;4′,4″;6′,4‴] quaterpyridinium trichlorides as electron transfer agents

Photo- and electrochemical properties of a novel class of electron transfer substances (1,1″,1‴-trialkyl[4,2′;4′,4″;6′,4‴] quaterpyridinium trichlorides) have been studied for hydrogen producing systems containing sensitizerS, electron transfer agentR, electron donorD, and platinum catalyst. 1,1″,1‴-trimethyl [4,2′;4′,4″;6′,4‴] quaterpyridinium trichloride (1) and 1,1″,1‴-tribenzyl [4,2′;4′,4″;6′,4‴] quaterpyridinium trichloride (2) quench the excited state of Ru(bipy)3Cl2. Irradiation (λ>400 nm) of solutions containing Ru(bipy)3Cl2,1 or2 and EDTA lead to the formation of radicals of1 or2. A solution of radicals of compound1 can change its colour reversibly with change of pH-values (colourless in acidic, blue in alkaline solutions). Also, compounds1 and2 are able to produce H2 without additional sensitizer (irradiation light: λ>300 nm). Hydrogen production rates, quantum yields, and turnover numbers have been determined in two different systems (S, R, D, Pt, λ>400 nm; andR, D, Pt without additional sensitizer, λ>300 nm). Hydrogen production rates with compounds1 and2 are of the same order of magnitude as those with methyl viologen. The radicals of1 and2 are much more stable against oxygen, than those of methyl viologen. Cyclovoltammetry showed that the redox potentials of compounds1 and2 are dependent on pH: increasing pH values result in more negative redox potentials. Coulometric analysis showed that e.g. compound1 could accept 5 electrons at pH=4·8 and 3·4 electrons at pH=12·6 compared to only one electron for methyl viologen (independent of pH). This behaviour is explained with reference to protonated radicals.

Hydrogen turnover by psychrotrophic homoacetogenic and mesophilic methanogenic bacteria in anoxic paddy soil and lake sediment

Hydrogen turnover by psychrotrophic homoacetogenic and mesophilic methanogenic bacteria in anoxic paddy soil and lake sediment

Hydrogen turnover by psychrotrophic homoacetogenic and mesophilic methanogenic bacteria in anoxic paddy soil and lake sediment

The effect of temperature on CH4 production, turnover of dissolved H2, and enrichment of H2-utilizing anaerobic bacteria was studied in anoxic paddy soil and sediment of Lake Constance. When anoxic paddy soil was incubated under an atmosphere of H2/CO2, rates of CH4 production increased 25°C, but decreased at temperatures lower than 20°C. Chloroform completely inhibited methano-genesis in anoxic paddy soil and lake sediment, but did not or only partially inhibit the turnover of dissolved H2, especially at low incubation temperatures. Cultures with H2 as energy source resulted in the enrichment of chemolithotrophic homoacetogenic bacteria whenever incubation temperatures were lower than 20°C. Hydrogenotrophic methanogens could only be enriched at 30°C from anoxic paddy soil. A homoacetogen

Effect of dilution on methanogenesis, hydrogen turnover and interspecies hydrogen transfer in anoxic paddy soil

Dilution of anoxic slurries of paddy soil resulted in a proportional decrease of the rates of total methanogenesis and the rate constants of H2 turnover per gram soil. Dilution did not affect the fraction of H2/CO2-dependent methanogenesis which made up 22% of total CH4 production. However, dilution resulted in a ten fold decrease of the H2 steady state partial pressure from approximately 4 to 0.4 Pa indicating that H2/CO2-dependent methanogenesis was more or less independent of the H2 pool. The rates of H2 production calculated from the H2 turnover rate constants and the H2 steady state partial pressures accounted for only < 5% of H2/CO2-dependent methanogenesis in undiluted soil slurries and for even less after dilution. Upon dilution, the Gibbs free energy available for H2/CO2-dependent methanogenesis decreased from −28.4 to only −5.6 kJ per mol. The results indicate that methane was mainly produced from interspecies H2 transfer within syntrophic bacterial associations and was not significantly affected by the outside H2 pool.

Effect of dilution on methanogenesis, hydrogen turnover and interspecies hydrogen transfer in anoxic paddy soil

Effect of dilution on methanogenesis, hydrogen turnover and interspecies hydrogen transfer in anoxic paddy soil

Influence of pH on microbial hydrogen metabolism in diverse sedimentary ecosystems.

Hydrogen transformation kinetic parameters were measured in sediments from anaerobic systems covering a wide range of environmental pH values to assess the influence of pH on hydrogen metabolism. The concentrations of dissolved hydrogen were measured and hydrogen transformation kinetics of the sediments were monitored in the laboratory by monitoring hydrogen consumption progress curves. The hydrogen turnover rate constants (kt) decreased directly as a function of decreasing sediment pH, and the maximum hydrogen uptake velocities (Vmax) varied as a function of pH within each of the trophic states. Conversely, the half-saturation concentrations (Km) were independent of pH. The steady-state hydrogen concentrations were at least 2 orders of magnitude lower than the half-saturation constants for hydrogen uptake. Dissolved hydrogen concentrations were at least fivefold higher in sediments from eutrophic systems than from oligotrophic and dystrophic systems. The rates of hydrogen production determined from the assumption of steady state decreased with sediment pH. These data indicate that progressively lower pH values inhibit microbial hydrogen-producing and -consuming processes within sedimentary ecosystems.

Photosynthetic Hydrogen and Oxygen Production: Kinetic Studies

Steady-state turnover times for simultaneous photosynthetic production of hydrogen and oxygen have been measured for two systems: the in vitro system comprised of isolated chloroplasts, ferredoxin, and hydrogenase, and the anaerobically adapted green alga Chlamydomonas reinhardtii [137c(+) mating type]. In both systems, the simultaneous photoproduction of hydrogen and oxygen was measured by driving the systems into the steady state with repetitive, single-turnover, flash illumination. The turnover times for production of both oxygen and hydrogen in photosynthetic water splitting are in milliseconds and are equal to or less than the turnover time for carbon dioxide reduction in intact algal cells. The oxygen and hydrogen turnover times are therefore compatible with each other and partially compatible with the excitation rate of the photosynthetic reaction centers under conditions of solar irradiation.

Theoretical radiation dose to the human system from assimilated tritium.

Abstract A theoretical model for tritium behavior in man shows that, for tritium uptake, the radiation energy deposition per unit weight of body hydrogen is essentially invariant for all components of the human system. The model is based on the assumption that tritium enters the body in a manner typical of hydrogen. If assimilated tritium behaves the same as ordinary hydrogen, the amount of tritium passing through any body subsystem varies directly with the hydrogen turnover rate, while the residence time varies inversely with turnover rate. The specific energy deposition in any subsystem is, therefore, the same as that in any other subsystem for tritium assimilated in a manner typical of hydrogen. Pathway effects associated with preferential entry of tritium in atypical hydrogen streams can be significant. An extension of the base model also illustrates that assimilated tritium which becomes permanently bound will not contribute significantly to the total dose. The analysis of a previously reported experiment is shown to conform with the predictions of the theoretical model.