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 an important trace gas in the atmosphere. Soil microorganisms are known to be an important part of the biogeochemical H 2 cycle, contributing 80 to 90% of the annual hydrogen uptake. Different aquatic ecosystems act as either sources or sinks of hydrogen, but the contribution of their microbial communities is unknown.[NiFe]-hydrogenases are the best candidates for hydrogen turnover in these environments since they are able to cope with oxygen. As they lack sufficiently conserved sequence motifs, reliable markers for these enzymes are missing, and consequently, little is known about their environmental distribution. We analyzed the essential maturation genes of A tmospheric hydrogen concentrations result from a number of counteracting processes. H 2 is mainly produced due to photooxidation of methane and other hydrocarbons in the upper atmosphere and due to fossil fuel and biomass burning. By far the largest proportion of hydrogen (80 to 90%) is consumed by microorganisms in the soil, whereas the remaining part reacts with OH radicals (1, 2). Since OH radicals are important for the degradation of methane, hydrogen levels indirectly control the amount of this trace gas. Currently, the atmospheric concentration of H 2 is about 0.5 ppm (2).The processes governing hydrogen concentrations and exchange in marine and freshwater environments are poorly described. In general, it seems that tropical surface waters act as hydrogen sources contributing about 6% to the global hydrogen production (2). Possible causes of H 2 evolution are photochemical processes in the surface layers as well as nitrogen fixation (3-6). Thorough flux analysis is still lacking. On the other hand, investigations of temperate surface waters showed net hydrogen uptake that could be assigned to microorganisms (7).Three classes of hydrogenases, the enzymes involved in hydrogen turnover, are known. Their classification is based on their active site metal ions being either a single [Fe] Although they are phylogenetically related, it is not possible to derive degenerated primers for the amplification of [NiFe]-hydrogenase genes, because their signature motifs are too scattered and too short. However, all [NiFe]-hydrogenases ultimately depend on the presence of six maturation genes, hypABCDEF (10). The most promising candidate of all maturation genes is the highly conserved hypD, encoding a protein with a ferredoxin:thioredoxin reductase-like [4Fe-4S] cluster and additional cysteine residues that probably work as a redox cascade. In concert with HypC, it works as a scaffold protein that allows the insertion of the Fe(CN) 2 (CO) in the correct oxidation state into the active site of the [NiFe]-hydrogenases (11-17).Since hydrogen leakage from anaerobic habitats is negligible and does not contribute to a significant extent to H 2 levels in the atmosphere (18)(19)(20), microbial influence on its atmospheric concentration apart from nitrogen fixation mainly relies on [NiFe]-hydrogenases. A few studies already attempted to analyze the occurrence o...