The effects of sediment slurrying on microbial processes,and the role of amino acids as substrates for sulfate reduction in anoxic marine sediments

In sediment slurry experiments with anoxic marine sediments collected in Cape Lookout Bight, NC, and a site in mid-Chesapeake Bay, the rates of sulfate reduction and ammonium production decrease with increasing dilution of sediment with oxygen-free sea-water. The effect of sediment dilution on the rates of these processes can be described by a simple mathematical relationship, and when these rates are corrected for sediment dilution they yield values which agree well with direct measurements of these processes.In sediment slurry studies of amino acid utilization in Cape Lookout Bight sediments, the fermentative decarboxylation of glutamic acid (to -aminobutyric acid) or aspartic acid (to alanine or -alanine) did not occur when either of these amino acids were added to Cape Lookout Bight slurries. The addition of glutamic acid did however lead to a small (1) transient build-up of -aminoglutaric acid. Measured rates of glutamic acid uptake in these slurries also decreased with increasing sediment dilution.Molybdate inhibition experiments demonstrated that dissolved free amino acids represent 1–3% of the carbon sources/electron donors used for sulfate reduction in Cape Lookout Bight sediments. The direct oxidation of amino acids by sulfate reducing bacteria also accounts for 13–20% of the total ammonium produced. Glutamic acid, alanine, -aminoglutaric acid, aspartic acid and asparagine are the major amino acids oxidized by sulfate reducing bacteria in Cape Lookout Bight sediments.     

A thermodynamic analysis of the anaerobic oxidation of methane in marine sediments

Anaerobic oxidation of methane (AOM) in anoxic marine sediments is a significant process in the global methane cycle, yet little is known about the role of bulk composition, temperature and pressure on the overall energetics of this process. To better understand the biogeochemistry of AOM, we have calculated and compared the energetics of a number of candidate reactions that microorganisms catalyse during the anaerobic oxidation of methane in (i) a coastal lagoon (Cape Lookout Bight, USA), (ii) the deep Black Sea, and (iii) a deep-sea hydrothermal system (Guaymas basin, Gulf of California). Depending on the metabolic pathway and the environment considered, the amount of energy available to the microorganisms varies from 0 to 184 kJ mol(-1). At each site, the reactions in which methane is either oxidized to HCO3(-), acetate or formate are generally only favoured under a narrow range of pressure, temperature and solution composition--particularly under low (10(-10 )m) hydrogen concentrations. In contrast, the reactions involving sulfate reduction with H2, formate and acetate as electron donors are nearly always thermodynamically favoured. Furthermore, the energetics of ATP synthesis was quantified per mole of methane oxidized. Depending on depth, between 0.4 and 0.6 mol of ATP (mol CH4(-1) was produced in the Black Sea sediments. The largest potential productivity of 0.7 mol of ATP (mol CH4(-1) was calculated for Guaymas Basin, while the lowest values were predicted at Cape Lookout Bight. The approach used in this study leads to a better understanding of the environmental controls on the energetics of AOM.     

Biogeochemical Processes of Methane Cycle in the Soils,Bogs, and Lakes of Western Siberia

The biogeochemical processes of methane production and oxidation were studied in the upper horizons of tundra and taiga soils and raised bogs and lake bottom sediments near the Tarko-Sale gas field in western Siberia. Both in dry and water-logged soils, the total methane concentration (in soil particles and gaseous phase) was an order of magnitude higher than in the soil gaseous phase alone (22 and 1.1 nl/cm³, respectively). In bogs and lake bottom sediments methane concentration was as high as 11 l/cm³. Acetate was the major precursor of the newly formed methane. The rate of aceticlastic methanogenesis reached 55 ng C/(cm³day), whereas that of autotrophic methanogenesis was an order of magnitude lower. The most active methane production and oxidation were observed in bogs and lake sediments, where the ¹³C values of CO₂were inversely related to the intensity of bacterial methane oxidation. Methane diffusing from bogs and lake bottom sediments showed ¹³C values ranging from –78 to –47, whereas the ¹³C value of carbon dioxide ranged from –18 to –1. In these ecosystems, methane emission comprised from 3 to 206 mg CH₄/(m²day). Conversely, the dry and water-logged soils of the tundra and taiga took up atmospheric methane at a rate varying from 0.3 to 5.3 mg CH₄/(m²day). Methane consumption in soils was of biological nature. This was confirmed by the radioisotopic method and chamber experiments, in which weighting of methane carbon was observed (the ¹³C value changed from –51 to –41).     

Energy coupling and respiration inNitrosomonas europaea

Intact cells ofNitrosomonas europaea grown in an ammonium salts medium will oxidise ammonium ions, hydroxylamine and ascorbate-TMPD; there is no oxidation of carbon monoxide, methane or methanol. TheK _m value for ammonia oxidation is highly pH dependent with a minimum value of 0.5 mM above pH 8.0. This suggests that free ammonia is the species crossing the cytoplasmic membrane(s). The measurement of respiration driven proton translocation indicates that there is probably only one proton translocating loop (loop 3) association with hydroxylamine oxidation. The oxidation of endogenous substrates is sometimes associated with more than one proton-translocating loop. These results indicate that during growth hydroxylamine oxidation is probably associated with a maximum P/O ratio of 1.Abbreviations H⁺/O ratio g equiv. H⁺ translocated/g atom O consumed     

Vertical distribution and diversity of bacteria and archaea in sulfide and methane-rich cold seep sediments located at the base of the Florida Escarpment

The bacterial and archaeal communities of the sediments at the base of the Florida Escarpment (Gulf of Mexico, USA) were investigated using molecular phylogenetic analysis. The total microbial community DNA of each of three vertical zones (top, middle and bottom) of a sediment core was extracted and the 16S rRNA genes were amplified by PCR, cloned and sequenced. Shannon–Weaver Diversity measures of bacteria were high in all three zones. For the archaea, diversity was generally low, but increased with depth. The archaeal clonal libraries were dominated by representatives of four groups of organisms involved in the anaerobic oxidation of methane (ANME groups). Phylogenetic analysis of bacteria suggests the dominance of -proteobacteria in the top zone, the -, - and -proteobacteria in the middle zone and the -proteobacteria in the bottom zone of the core. Members of the Cytophaga–Flexibacter–Bacteroidetes group, the Chloroflexi/green non-sulfur bacteria, the Gram+ (Firmicutes), the Planctomyces, candidate division WS3 and Fusobacterium were also detected. Our data suggest that the community structure and diversity of microorganisms can shift greatly within small vertical distances, possibly in response to changes in the physical and chemical conditions.     

Comparative ecology of H2 cycling in sedimentary and phototrophic ecosystems

The simple biochemistry of H₂ is critical to a large number of microbial processes, affecting the interaction of organisms with each other and with the environment. The sensitivity of each of these processes to H₂ can be described collectively, through the quantitative language of thermodynamics. A necessary prerequisite is to understand the factors that, in turn, control H₂ partial pressures. These factors are assessed for two distinctly different ecosystems. In anoxic sediments from Cape Lookout Bight (North Carolina, USA), H₂ partial pressures are strictly maintained at low, steady-state levels by H₂-consuming organisms, in a fashion that can be quantitatively predicted by simple thermodynamic calculations. In phototrophic microbial mats from Baja California (Mexico), H₂ partial pressures are controlled by the activity of light-sensitive H₂-producing organisms, and consequently fluctuate over orders of magnitude on a daily basis. The differences in H₂ cycling can subsequently impact any of the H₂-sensitive microbial processes in these systems. In one example, methanogenesis in Cape Lookout Bight sediments is completely suppressed through the efficient consumption of H₂ by sulfate-reducing bacteria; in contrast, elevated levels of H₂ prevail in the producer-controlled phototrophic system, and methanogenesis occurs readily in the presence of 40 mM sulfate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Asparagine and glutamine side-chain conformation in solution and crystal: A comparison for hen egg-white lysozyme using residual dipolar ouplings

Experimental ¹⁵N–¹H and ¹H–¹H residual dipolar couplings (RDCs) for the asparagine (Asn) and glutamine (Gln) side chains of hen egg-white lysozyme are measured and analysed in conjunction with ¹N relaxation data, information about ¹ torsion angles in solution and molecular dynamics simulations. The RDCs are compared to values predicted from 16 high-resolution crystal structures. Two distinct groups of Asn and Gln side chains are identified. The first contains residues whose side chains show a fixed, relatively rigid, conformation in solution. For these residues there is good agreement between the experimental and predicted RDCs. This agreement improves when the experimental order parameter, S, is included in the calculation of the RDCs from the crystal structures. The comparison of the experimental RDCs with values calculated from the X-ray structures shows that the similarity between the oxygen and nitrogen electron densities is a limitation to the correct assignment of the Asn and Gln side-chain orientation in X-ray structures. In the majority of X-ray structures a 180° rotation about ² or ³, leading to the swapping of N ² and O ¹, is necessary for at least one Asn or Gln residue in order to achieve good agreement between experimental and predicted RDCs. The second group contains residues whose side chains do not adopt a single, well-defined, conformation in solution. These residues do not show a correlation between the experimental and predicted RDCs. In many cases the family of crystal structures shows a range of orientations for these side chains, but in others the crystal structures show a well-defined side-chain position. In the latter case, this is found to arise from crystallographic contacts and does not represent the behaviour of the side chain in solution.     

Methane emissions from the Orinoco River floodplain, Venezuela

Methane emissions were measured over a 17-monthinterval at 21 locations on the Orinoco fringingfloodplain and upper delta (total area,14,000 km²). Emissions totaled 0.17 Tgyr^–1, or 7.1 mmol d^–1 (114 mg d^–1;standard deviation, ±18%) per m² of watersurface. Ebullition accounted for 65% of emissions. Emission rates were about five times as high forfloodplain forest as for open water or macrophytemats. Emission rates were positively correlated withcarbon content of sediment and amount of methane inthe water column, and negatively correlated withdissolved oxygen, but the correlations were weak. Emission from floodplain soils occurred only when thewater content of soil exceeded 25%, which occurredwithin 20 m of standing water during floodplaindrainage (3 months/yr). Bare soils emitted 60mmol/day per m of shoreline length; soils covered bystranded macrophyte beds emitted five times thisamount. Total emissions were accounted for primarilyby flooded forest (94%); macrophyte mats, open water,and exposed soils made only small contributions. Theflux-weighted mean ¹³C for the floodplainwas –62 ± 8; for D the mean was –271 ± 27. The ¹³C and D were negativelycorrelated. Overall emission rates were notably lowerthan for the Amazon. The depth and duration offlooding are considerably less for the Orinoco thanfor the Amazon floodplain; oxygen over sediments isthe rule for the Orinoco but not for the Amazon. TheOrinoco data illustrate the difficulty of generalizingemission rates. Current information for tropicalAmerica, including revised estimates for inundatedarea along the Amazon, indicate that methane emissionsfrom tropical floodplains have been overestimated.     

Release of fossil methane from mineral soil particles, and its implication for estimation of methane oxidation in a mineral subsoil

In a preliminary experiment we found that methane evolved from a sandy subsoil during aerobic incubation of shaken soil slurries. In the study presented here the methane was found to be released from the sand particles by mechanical weathering, caused by the grinding effect of the shaking. Large amounts of gas (about 0.5 ml gas g^–1 soil) were extracted by intense grinding of the soil in gas tight serum vials. Methane was the main hydrocarbon in the emitted gas, but also a considerable amount of ethane was present, as well as minor amounts of heavier hydrocarbons (up to C₆). The ¹³C-values of the emitted methane and ethane were –33 and –29 , respectively. Together these results demonstrate a thermogenic origin of the gas. This paper also reports the results of an incubation experiment where possible methane oxidation was looked for. If a possible release of methane is not accounted for, methane oxidation may be overlooked, as illustrated in this paper. Methane consumption was detected only in soil from 40 cm, in contrast to soil sampled at 100 cm and deeper where a slight production was measured. When methane oxidation was inhibited by dimethyl-ether, a significant release of methane was seen. The release was probably caused by chemical weathering. When this methane release was taken into account, methane oxidation was found to be present at all measured depths (40 to 200 cm). Fertilization with urea inhibited the methane oxidation at 40 cm but not at deeper layers. It is hypothesized that ammonia oxidizing bacteria were the main methane oxidizers in this mineral subsoil (deeper than 1 m), and that oxidation of methane might be a survival mechanism for ammonia oxidizers in ammonia limited environments.     

Geochemical Peculiarities of the Carbonate Constructions Formed during Microbial Oxidation of Methane under Anaerobic Conditions

The aragonite constructions of the Black Sea are formed in a stable anaerobic zone and are a perfect object to study the natural mechanism of anaerobic methane oxidation. The most probable pathway of methane oxidation is its methanogen-mediated reaction with bicarbonates, dissolved in seawater, with the formation of water and acetate, which is then consumed by other components of the anaerobic community. Comparison of the ¹³C values of carbonate minerals and organic matter once more demonstrated that the formation of the organic matter of biomass is accompanied by intense fractionation of carbon isotopes, as a result of which the total organic matter of biomass acquires an extremely light isotopic composition, characterized by ¹³C values as low as –83.8.     

Detection of inconspicuous epiphytic algae supporting food webs in seagrass meadows

Summary Detritus from common seagrasses and other marine angiosperms may often be a less important basis for estuarine food webs than previously believed. In NW Gulf of Mexico seagrass meadows, epiphytic algae have high productivities, palatability, and a more important trophic role than common large plants have. Interdisciplinary field experiments show (1) intensive night-time ingestion of epiphytes by various invertebrate detritivores, (2) very high productivity of epiphytic algae on seagrasses, and (3) assimilation of epiphytes rather than seagrasses, as measured by ¹³C comparisons. These combined data show that many naturally concentrated and potentially competing invertebrates in Gulf of Mexico seagrass meadows feed largely on the algal overgrowth on seagrass blades, even when such algae appear to be sparse. Primary productivity of these epiphytic algae can equal that of the seagrasses, per blade or per unit biomass. Animal ¹³C values tracked epiphytic values rather than seagrass values when comparisons were made over six sites. These measurements reinforce the view that epiphytic algae can be the primary basis of the food web in seagrass meadows.Contribution No. 608 of The University of Texas Marine Science Institute     

Bacterial Processes of the Methane Cycle in Bottom Sediments of Lake Baikal

The activity of methanogenic and methanotrophic bacteria was evaluated in bottom sediments of Lake Baikal. Methane concentration in Baikal bottom sediments varied from 0.0053 to 81.7 ml/dm³. Bacterial methane was produced at rates of 0.0004–534.7 l CH₄/(dm³ day) and oxidized at rates of 0.005–1180 l CH₄/(dm³ day). Peak methane production and oxidation were observed in Frolikha Bay near a methane vent. Methane was emitted into water at rates of 49.2–4340 l CH₄/(m² day). Rates of bacterial methane oxidation in near-bottom water layers ranged from 0.002 to 1.78 l/(l day). Methanogens and methanotrophs were found to play an important role in the carbon cycle through all layers of sediments, particularly in the areas of methane vent and gas-hydrate occurrence.     

Carbon-13 variation with depth in soils of Brazil and climate change during the Quaternary

Paleoecological and geomorphological studies indicate that, during the middle Holocene, there was a predominance of drier conditions with grassy savannahs replacing forests across the South American continent. Modern savannahs are composed mainly of C4 plants and soils developed under this type of vegetation show enrichment in ¹³C compared to soils under C3 vegetation cover. If soils contain stabilized organic matter formed in the middle Holocene, we hypothesize that former C4 vegetation would be evidenced by a large enrichment of ¹³C in soil organic matter (SOM). We investigate this possibility examining the depth variation of carbon isotopic composition in 21 soil profiles collected by different researchers at 14 different sites in Brazil. Of these, profiles from only three sites showed a marked increase of ¹³C with depth (9–10 enrichment in ¹³C difference between the surface soil and deepest depth); two sites showed intermediate enrichment (4–5), and nine sites showed a small enrichment of approximatelly 2.5. The majority of sites showing all-C3 derived SOM were in the Amazon region. Possible causes for the absence of a large ¹³C enrichment with depth are: (1) dominance of C3 rather than C4 grasses in mid-Holocene savannahas, (2) soil profiles did not preserve organic matter derived from mid-Holocene plants, (3) the retreat of forest areas did not occur on a regional scale, but was a much more localized phenomenon.     

Sources and fluxes of particulate organic matter in shallow coastal waters characterized by summer macroaggregate formation

The origin and temporal variation in composition of sedimented particulate organic matter (POM) in the Gulf of Trieste (northern Adriatic) was studied over the year with special reference to the composition and sedimentation of macroaggregates in summer of 1991 using sediment traps.Suspended and sedimented POM, comprising a minor part of the largely inorganic total particulate matter, was prevalently of marine origin and composed mostly of humic substances followed by carbohydrates and proteins. Seasonal variations of particulate proteins and carbohydrates were correlated with variations of phytoplankton biomass. New production, occurring in late spring as a consequence of massive riverine inputs of N, Si and P nutrients in the surface layer of the Gulf, produced high particulate carbohydrate and protein concentrations. Subsequent depletion of introduced nutrients caused the decrease of particulate protein concentration but not that of particulate carbohydrate. The prolonged plankton biosynthesis of carbohydrates successively produced marine snow and later macroaggregates. The macroaggregates were characterized by ¹³C value of –19.9. and their carbohydrates were mostly composed of glucose followed in decreasing order by mannose, fructose, galactose, arabinose, ribose, xylose and fucose, suggesting a prevalent origin from phytoplankton structural heteropolysaccharides. Sedimentation of particulate organic constituents in the summertime, characterized by the massive presence of macroaggregates in the surface layer above the pycnocline, was the highest at a depth of 10 m at the end of this phenomenon, about six weeks after its first appearance. Sedimented macroaggregates were clearly traced by a characteristic ¹³C signal and higher carbohydrate concentrations. The monosaccharide composition was influenced by selective degradation in the water column. Sedimented POM in the bottom layer was, on the other hand, more affected by sediment resuspension. The mean yearly decrease of particulate protein-C and carbohydrate-C by 40–50% in the water column between the depths of 10 and 20 m indicates the preferential utilization of these constituents by microorganisms. The decrease of particulate humic-C is probably more the result of the export of particulate matter from the Gulf. This study also indicates that the macroaggregate formation has little impact on the annual C and N budget in such coastal areas.     

Stable isotopic signature of El Niño-Southern Oscillation events in eastern tropical Pacific reef corals

In this study we measured stable oxygen and carbon isotope ratios in the skeletons of massive reef-building corals (Porites lobata and Pavona gigantea) at four widespread locations in the tropical eastern Pacific, each with a unique marine climate. Annual variation in sea surface temperature (SST) varied from 5–7°C at upwelling sites (Gulf of Panamá and Galápagos Islands) to 2–3°C where upwelling was absent [Caño Isand (Costa Rica) and Gulf of Chiriquí (Panamá)]. Annual range in salinity was high in the gulfs of Panamá and Chiriquí (up to 15) and low at Cãno Island and Galápagos (2–3). We characterize the isotopic signatures of recent (15–40 y long records) El Niño/Southern Oscillation (ENSO) events, particularly the very severe 1982–1983 event. Subannual sampling at 1–2 month resolution reveals that the ¹⁸O signal at Caño Island records strong to very strong ENSOs. In the Gulf of Chiriquí, this signal is governed mainly (80%) by salinity and thus is a weak recorder of ENSO events: only the 1951–1952, 1957–1958 and 1972–1973 events appear as significant ¹⁸O anomalies over the period 1940–1984. In the Gulf of Panamá, high variation in both SST and salinity contribute to the ¹⁸O signal. ENSO events at this site are poorly recorded, probably due to ameliorating effects of cool upwelled water during the early stage of the ENSO event. The ¹⁸O record in Galápagos, however, shows a strong correlation with SST and accurately records all but the most severe 1982–1983 ENSO event. Thus, ENSOs are most clearly recorded at sites where salinity variation is minimal. At Caño only strong ENSOs are recorded while mild to strong events appear in the Galápagos record. Nowhere did the ¹⁸O signal accurately record the full range of temperatures that occurred during the 1982–1983 ENSO; however, a stress band on the coral skeleton was evident at all sites. By comparing the ¹⁸O records and skeletal features across sites it may be possible to identify the occurrence of strong to very severe ENSOs prior to instrumental records. The relationship between ¹⁸O and ¹³C was examined and found to be significantly positive and in phase (i.e., either depleted or enriched at the same time) at three of the four sites studied. Weak correlations at some sites may be explained by high variability in water column clarity leading to depleted ¹³C when waters are cool and surface isolation high.     

Sulphur isotope ratios in sulphate and oxygen isotopes in water from a small watershed in Central Sweden

During 1988–89 water samples for sulphur and oxygen isotope measurements were collected in the Lake Mjösjön watershed (7.3 km²), central Sweden. Samples included: precipitation, throughfall, lakewater, shallow groundwater and inlet and outlet streams.The ³⁴S of sulphate in precipitation ranged from + 6.41 in winter to + 3.88 in summer, the higher winter values attributed to seasonal differences in the kinetic and equilibrium isotope fractionation during oxidation of atmospheric sulphur dioxide to sulphate. The ³⁴S in rain samples and in pine and spruce throughfall were similar, indicating no gain of sulphur from the trees. In the inflowing stream, the ³⁴S value increased as discharge decreased, from + 5.57 in spring to + 26.21 in summer, indicating bacterial sulphate reduction. The fluctuations in the inlet water were damped by the lake and in the outlet water, only a small decrease in the ³⁴S value during spring discharge was observed.During winter 1988–89, the near surface waters in the lake showed the same ³⁴S as snow indicating that meltwater governs the isotopic composition. During the winter, the ³⁴S in the near bottom waters increased while oxygen decreased due to bacterial sulphate reduction in the sediments. This also caused an increase in the alkalinity in the near bottom waters.Based on the ¹⁸O data the water within the watershed is derived largely from meteoric water. During spring discharge, meltwater governs the inflow and outflow stream while additional groundwater influences occurred during the drier period. Most sulphur is derived from atmospheric deposition and the ³⁴S in sulphate increased during passage through the watershed due to bacterial sulphate reduction.     

Spatial change of reservoir nitrite-dependent methane-oxidizing microorganisms

Nitrite-dependent anaerobic methane oxidation (n-damo), catalyzed by microorganisms affiliated with bacterial phylum NC10, can have an important contribution to the reduction of the methane emission from anoxic freshwater sediment to the atmosphere. However, information on the variation of sediment n-damo organisms in reservoirs is still lacking. The present study monitored the spatial change of sediment n-damo organisms in the oligotrophic freshwater Xinfengjiang Reservoir (South China). Sediment samples were obtained from six different sampling locations and two sediment depths (0–5 cm, 5–10 cm). Sediment n-damo bacterial abundance was found to vary with sampling location and layer depth, which was likely influenced by pH and nitrogen level. The presence of the n-damo pmoA gene was found in all these samples. A remarkable shift occurred in the diversity and composition of sediment n-damo pmoA gene sequences. A variety of distinctively different n-damo pmoA clusters existed in reservoir sediments. The pmoA sequences affiliated with Candidatus Methylomirabilis oxyfera formed the largest group, while a significant proportion of the obtained n-damo pmoA gene sequences showed no close relationship to those from any known NC10 species. In addition, the present n-damo process was found in reservoir sediment, which could be enhanced by nitrite nitrogen amendment.     

Enrichment and Molecular Detection of Denitrifying Methanotrophic Bacteria of the NC10 Phylum

Anaerobic methane oxidation coupled to denitrification was recently assigned to bacteria belonging to the uncultured phylum NC10. In this study, we incubated sediment from a eutrophic ditch harboring a diverse community of NC10 bacteria in a bioreactor with a constant supply of methane and nitrite. After 6 months, fluorescence in situ hybridization showed that NC10 bacteria dominated the resulting population. The enrichment culture oxidized methane and reduced nitrite to dinitrogen gas. We assessed NC10 phylum diversity in the inoculum and the enrichment culture, compiled the sequences currently available for this bacterial phylum, and showed that of the initial diversity, only members of one subgroup had been enriched. The growth of this subgroup was monitored by quantitative PCR and correlated to nitrite-reducing activity and the total biomass of the culture. Together, the results indicate that the enriched subgroup of NC10 bacteria is responsible for anaerobic methane oxidation coupled to nitrite reduction. Due to methodological limitations (a strong bias against NC10 bacteria in 16S rRNA gene clone libraries and inhibition by commonly used stopper material) the environmental distribution and importance of these bacteria could be largely underestimated at present.Atmospheric concentrations of methane have risen 2.6-fold since preindustrial times (10). After several years of stagnation, there was again a clear increase in the methane concentration in 2007 (29). Currently, it is uncertain whether an increase in the number of sources and production or a decrease in the number of sinks and consumption is responsible for this reversal of the trend.Freshwater habitats like natural wetlands and rice fields are a major source (38% [9]) of atmospheric methane. In the absence of other documented electron donors, aerobic methane oxidation is assumed to be the most important sink in these habitats, but the role of alternative electron donors is not well understood (19, 30). Anaerobic methane oxidation coupled to denitrification is energetically favorable, but evidence that it occurs is scarce. In marine, methane-containing sediments, nitrate and nitrite are usually not quantitatively important electron acceptors; in freshwater sediments the denitrifying and aerobic zones are in close proximity (3, 22, 35), possibly masking the process from detection. To our knowledge, concomitant methane and nitrate profiles of sediments have never been published.So far, methane oxidation coupled to denitrification has received the most attention in the field of hydrogeology. In groundwater, contamination with nitrate and nitrite occurs frequently, whereas electron donors are limiting. Methane plumes often form around landfills, and their attenuation has sometimes been attributed to denitrification (2, 37). So far, a single previous study unambiguously demonstrated anaerobic oxidation of methane coupled to denitrification in a contaminated freshwater aquifer (32). The first in vitro observation of anaerobic methane oxidation coupled to denitrification came from a laboratory-scale sludge digestor (11). The use of a laboratory enrichment culture also eventually resulted in identification of the organisms involved; bacteria of the NC10 phylum and archaea of the order Methanosarcinales dominated a mixed culture carrying out anaerobic methane oxidation coupled to denitrification (27). This culture was enriched from a freshwater canal sediment after 1 year of continuous supply of methane and nitrite. Subsequently, the archaea were shown to be dispensable, as they disappeared after prolonged incubation of the same culture (7). Mass balance calculations showed that methane oxidation was coupled to the reduction of nitrite with a 3:8 stoichiometry, in accordance with theoretical expectations. The bacteria that dominated the mixed culture and apparently oxidized methane anaerobically are members of the NC10 phylum, one of the many phyla having no members in pure culture (8, 28). The 16S rRNA gene sequences of such organisms, however, have been found in a number of environmental surveys of aquatic environments; e.g., the most closely related sequences have been found in aquifers (1, 23) and lake sediments (13, 17). Sequence similarity and phylogenetic affiliation may indicate similar metabolic capacities of organisms, but by itself this is not sufficient to infer similar metabolism (5). This is especially true for denitrifying methanotrophs, because only a single enrichment culture has been described so far (7, 27).The objective of the present study was to generalize the previous finding that NC10 bacteria were associated with anaerobic methane oxidation, a necessary step forward in addressing the significance of this poorly understood process as a methane sink in freshwater habitats.     

Microbial Diversity of Hydrothermal Sediments in the Guaymas Basin: Evidence for Anaerobic Methanotrophic Communities

Microbial communities in hydrothermally active sediments of the Guaymas Basin (Gulf of California, Mexico) were studied by using 16S rRNA sequencing and carbon isotopic analysis of archaeal and bacterial lipids. The Guaymas sediments harbored uncultured euryarchaeota of two distinct phylogenetic lineages within the anaerobic methane oxidation 1 (ANME-1) group, ANME-1a and ANME-1b, and of the ANME-2c lineage within the Methanosarcinales, both previously assigned to the methanotrophic archaea. The archaeal lipids in the Guaymas Basin sediments included archaeol, diagnostic for nonthermophilic euryarchaeota, and sn-2-hydroxyarchaeol, with the latter compound being particularly abundant in cultured members of the Methanosarcinales. The concentrations of these compounds were among the highest observed so far in studies of methane seep environments. The δ-¹³C values of these lipids (δ-¹³C = −89 to −58‰) indicate an origin from anaerobic methanotrophic archaea. This molecular-isotopic signature was found not only in samples that yielded predominantly ANME-2 clones but also in samples that yielded exclusively ANME-1 clones. ANME-1 archaea therefore remain strong candidates for mediation of the anaerobic oxidation of methane. Based on 16S rRNA data, the Guaymas sediments harbor phylogenetically diverse bacterial populations, which show considerable overlap with bacterial populations of geothermal habitats and natural or anthropogenic hydrocarbon-rich sites. Consistent with earlier observations, our combined evidence from bacterial phylogeny and molecular-isotopic data indicates an important role of some novel deeply branching bacteria in anaerobic methanotrophy. Anaerobic methane oxidation likely represents a significant and widely occurring process in the trophic ecology of methane-rich hydrothermal vents. This study stresses a high diversity among communities capable of anaerobic oxidation of methane.     

Gene and primary structures of dye-linked <Emphasis Type="SmallCaps">l</Emphasis>-proline dehydrogenase from the hyperthermophilic archaeon<Emphasis Type="Italic"> Thermococcus profundus</Emphasis> show the presence of a novel heterotetrameric amino acid dehydrogenase complex

Dye-linked l-proline dehydrogenase catalyzes the oxidation of l-proline in the presence of artificial electron acceptors such as 2, 6-dichloroindophenol and ferricyanide. The enzyme from the hyperthermophilic archaeon Thermococcus profundus was purified and characterized for the first time in archaea by Sakuraba et al. in 2001. In this study, cloning and sequencing analyses of the gene encoding the enzyme and functional analysis of the subunits were performed. The gene formed an operon that consisted of four genes, pdhA, pdhB, pdhF, and pdhX, which are tandemly arranged in the order of pdhA-F-X-B. SDS-PAGE analysis of the purified recombinant enzyme showed four different bands corresponding to (54 kDa), (43 kDa), (19 kDa), and (8 kDa) subunits encoded by pdhA, pdhB, pdhF, and pdhX, respectively, and the molecular ratio of these subunits was determined to be equal. This indicates that the enzyme consists of a heterotetrameric structure. Functional analysis of each subunit revealed that the subunit catalyzed the dye-linked l-proline dehydrogenase reaction by itself and that, unexpectedly, the subunit exhibited dye-linked NADH dehydrogenase activity. This is the first example showing the existence of a bifunctional dye-linked l-proline/NADH dehydrogenase complex. On the basis of genome analysis, similar gene clusters were observed in the genomes of Pyrococcus horikoshii, Pyrococcus abyssi, Pyrococcus furiosus, and Archaeoglobus fulgidus. These results indicate that the dye-linked l-proline dehydrogenase is a novel type of heterotetrameric amino acid dehydrogenase that might be widely distributed in the hyperthermophilic archaeal strain.Communicated by K. Horikoshi