Marcus Elvert

Novel microbial communities of the Haakon Mosby mud volcano and their role as a methane sink

Mud volcanism is an important natural source of the greenhouse gas methane to the hydrosphere and atmosphere. Recent investigations show that the number of active submarine mud volcanoes might be much higher than anticipated (for example, see refs 3–5), and that gas emitted from deep-sea seeps might reach the upper mixed ocean. Unfortunately, global methane emission from active submarine mud volcanoes cannot be quantified because their number and gas release are unknown. It is also unclear how efficiently methane-oxidizing microorganisms remove methane. Here we investigate the methane-emitting Haakon Mosby Mud Volcano (HMMV, Barents Sea, 72° N, 14° 44′ E; 1,250 m water depth) to provide quantitative estimates of the in situ composition, distribution and activity of methanotrophs in relation to gas emission. The HMMV hosts three key communities: aerobic methanotrophic bacteria (Methylococcales), anaerobic methanotrophic archaea (ANME-2) thriving below siboglinid tubeworms, and a previously undescribed clade of archaea (ANME-3) associated with bacterial mats. We found that the upward flow of sulphate- and oxygen-free mud volcano fluids restricts the availability of these electron acceptors for methane oxidation, and hence the habitat range of methanotrophs. This mechanism limits the capacity of the microbial methane filter at active marine mud volcanoes to <40% of the total flux.

Characterization of Specific Membrane Fatty Acids as Chemotaxonomic Markers for Sulfate-Reducing Bacteria Involved in Anaerobic Oxidation of Methane

Membrane fatty acids were extracted from a sediment core above marine gas hydrates at Hydrate Ridge, NE Pacific. Anaerobic sediments from this environment are characterized by high sulfate reduction rates driven by the anaerobic oxidation of methane (AOM). The assimilation of methane carbon into bacterial biomass is indicated by carbon isotope values of specific fatty acids as low as m 103. Specific fatty acids released from bacterial membranes include C 16:1 y 5c , C 17:1 y 6c , and cyC 17:0 y 5,6 , all of which have been fully characterized by mass spectrometry. These unusual fatty acids continuously display the lowest i 13 C values in all sediment horizons and two of them are detected in high abundance (i.e., C 16:1 y 5c and cyC 17:0 y 5,6 ). Combined with microscopic examination by fluorescence in situ hybridization specifically targeting sulfate-reducing bacteria (SRB) of the Desulfosarcina/Desulfococcus group, which are present in the aggregates of AOM consortia in extremely high numbers, these specific fatty acids appear to provide a phenotypic fingerprint indicative for SRB of this group. Correlating depth profiles of specific fatty acid content and aggregate number in combination with pore water sulfate data provide further evidence of this finding. Using mass balance calculations we present a cell-specific fatty acid pattern most likely displaying a very close resemblance to the still uncultured Desulfosarcina/Desulfococcus species involved in AOM.

Archaea mediating anaerobic methane oxidation in deep-sea sediments at cold seeps of the eastern Aleutian subduction zone

Cold seeps in the Aleutian deep-sea trench support prolific benthic communities and generate carbonate precipitates which are dependent on carbon dioxide delivered from anaerobic methane oxidation. This process is active in the anaerobic sediments at the sulfate reduction-methane production boundary and is probably performed by archaea working in syntrophic co-operation with sulfate-reducing bacteria. Diagnostic lipid biomarkers of archaeal origin include irregular isoprenoids such as 2,6,11,15-tetramethylhexadecane (crocetane) and 2,6,10,15,19-pentamethylicosane (PMI) as well as the glycerol ether lipid archaeol (2,3-di-O-phytanyl-sn-glycerol). These biomarkers are prominent lipid constituents in the anaerobic sediments as well as in the carbonate precipitates. Carbon isotopic compositions of the biomarkers are strongly depleted in 13C with values of δ13C as low as −130.3‰ PDB. The process of anaerobic methane oxidation is also reflected in the carbon isotope composition of organic matter with δ13C-values of −39.2 and −41.8‰ and of the carbonate precipitates with values of −45.4 and −48.7‰. This suggests that methane-oxidizing archaea have accumulated within the microbial community, which is active at the cold seep sites. The dominance of crocetane in sediments at one station indicates that, probably due to decreased methane venting, archaea might no longer be growing, whereas high amounts of crocetenes found at other more active stations may indicate recent fluid venting and active archaea. Comparison with other biomarker studies suggests that various archaeal assemblages might be involved in the anaerobic consumption of methane. The assemblages are apparently dependent on specific conditions found at each cold seep environment. Selective conditions probably include water depth, temperature, degree of anoxia, and supply of free methane.

Evidence for Microbial Carbon and Sulfur Cycling in Deeply Buried Ridge Flank Basalt

Under the Sea Floor Microorganisms living in basaltic sea floor buried beneath sediments derive energy from inorganic components from the host rocks that interact with infiltrating seawater, which brings dissolved oxygen and other trace nutrients with it. Lever et al. (p. 1305) directly sampled the subseafloor community off the eastern flank of the Juan de Fuca Ridge in the Pacific Ocean and found evidence for ongoing microbial sulfate reduction and methanogenesis. Multiyear incubation experiments with samples of host rock confirmed the microbial activities measured in situ. Active methane- and sulfur-cycling microbial communities exist in deep basaltic ocean crust. Sediment-covered basalt on the flanks of mid-ocean ridges constitutes most of Earths oceanic crust, but the composition and metabolic function of its microbial ecosystem are largely unknown. By drilling into 3.5-million-year-old subseafloor basalt, we demonstrated the presence of methane- and sulfur-cycling microbes on the eastern flank of the Juan de Fuca Ridge. Depth horizons with functional genes indicative of methane-cycling and sulfate-reducing microorganisms are enriched in solid-phase sulfur and total organic carbon, host δ13C- and δ34S-isotopic values with a biological imprint, and show clear signs of microbial activity when incubated in the laboratory. Downcore changes in carbon and sulfur cycling show discrete geochemical intervals with chemoautotrophic δ13C signatures locally attenuated by heterotrophic metabolism.

Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor

A deep sleep in coal beds Deep below the ocean floor, microorganisms from forest soils continue to thrive. Inagaki et al. analyzed the microbial communities in several drill cores off the coast of Japan, some sampling more than 2 km below the seafloor (see the Perspective by Huber). Although cell counts decreased with depth, deep coal beds harbored active communities of methanogenic bacteria. These communities were more similar to those found in forest soils than in other deep marine sediments. Science, this issue p. 420; see also p. 376 Coal beds more than 2 kilometers below the seafloor host methanogenic bacteria related to those found in forest soils. [Also see Perspective by Huber] Microbial life inhabits deeply buried marine sediments, but the extent of this vast ecosystem remains poorly constrained. Here we provide evidence for the existence of microbial communities in ~40° to 60°C sediment associated with lignite coal beds at ~1.5 to 2.5 km below the seafloor in the Pacific Ocean off Japan. Microbial methanogenesis was indicated by the isotopic compositions of methane and carbon dioxide, biomarkers, cultivation data, and gas compositions. Concentrations of indigenous microbial cells below 1.5 km ranged from <10 to ~104 cells cm−3. Peak concentrations occurred in lignite layers, where communities differed markedly from shallower subseafloor communities and instead resembled organotrophic communities in forest soils. This suggests that terrigenous sediments retain indigenous community members tens of millions of years after burial in the seabed.

Assimilation of methane and inorganic carbon by microbial communities mediating the anaerobic oxidation of methane

The anaerobic oxidation of methane (AOM) is a major sink for methane on Earth and is performed by consortia of methanotrophic archaea (ANME) and sulfate-reducing bacteria (SRB). Here we present a comparative study using in vitro stable isotope probing to examine methane and carbon dioxide assimilation into microbial biomass. Three sediment types comprising different methane-oxidizing communities (ANME-1 and -2 mixture from the Black Sea, ANME-2a from Hydrate Ridge and ANME-2c from the Gullfaks oil field) were incubated in replicate flow-through systems with methane-enriched anaerobic seawater medium for 5-6 months amended with either (13)CH(4) or H(13)CO(3)(-). In all three sediment types methane was anaerobically oxidized in a 1:1 stoichiometric ratio compared with sulfate reduction. Similar amounts of (13)CH(4) or (13)CO(2) were assimilated into characteristic archaeal lipids, indicating a direct assimilation of both carbon sources into ANME biomass. Specific bacterial fatty acids assigned to the partner SRB were almost exclusively labelled by (13)CO(2), but only in the presence of methane as energy source and not during control incubations without methane. This indicates an autotrophic growth of the ANME-associated SRB and supports previous hypotheses of an electron shuttle between the consortium partners. Carbon assimilation efficiencies of the methanotrophic consortia were low, with only 0.25-1.3 mol% of the methane oxidized.

Intact phospholipids—microbial “life markers” in marine deep subsurface sediments

AbstractDeepsubsurfacesedimentsfromtheNankaiTrough,JapanSea,ODPLeg190,sites1173,1174,1177,andnear-surfacesedimentsfromHydrateRidge,NE-Pacifichavebeenanalysedbyhighperformanceliquidchromatography(HPLC)–electrosprayionisation(ESI)-massspectrometry(MS).Themainobjectivewastoutilizethepresenceofintactphospholipidsasadirectindicatorofviablemicroorganisms.TheextractsofNankaiTroughsedimentswerefoundtocontainavarietyofphospholipid(PL)structures,well-knowntostemfrommicroorganisms,todepthsasgreatas745mbsfandinsitutemperaturesashighas85C.Inaddition,highrelativeamountsoflysophospholipids(e.g.lysopho-sphatidylcholines)exceedingthoseoftheregularphospholipidsweredetected.DiglyceridemassfragmentsofvariousPLshavebeenassignedtofattyacylside-chainsoftypicalchainlength(C 14 ,C 16 ,C 18 ,C 20 )anddegreeofunsaturation(zero,oneortwodoublebonds).Similarresultswereobtainedforthephospholipiddistributioninextractsoforganicmatter-richHydrateRidgesediments.Todate,theenhancedoccurrenceoflysophospholipidscannotbeexplainedcompletelybutaresponsetoincreasingthermalandecologicalstressseemsprobable.# 2003ElsevierScienceLtd.Allrightsreserved.1.IntroductionBiologicalmarkersprovideinformationonthepre-cursorbiotaandpost-depositionalhistoryoforganicmatterpreservedinsediments,therebyenablingbiolo-gicalevolution,palaeoclimateandpetroleum-formingprocessestobedocumentedindetail(Brassell,1993;PetersandMoldowan,1993).Thisinformation,whetherbasedonhydrocarbonsorfunctionalisedcompounds,haslargelybeengatheredusinggaschromatography-massspectrometry,andwasinitiallymadepossiblebyrapidtechnicaladvancesinthemid-1960s.Inthelatterpartofthelastcentury,anewandcompellingchallengebegantotakeshape,thisbeingtodefinethetempera-ture-anddepthlimitstowhichlifecanoccurwithintheEarth.Thiswasmotivatedbythediscoveryofmicro-bially-basedecosystemsfuelledbygeothermalenergyathydrothermalvents,andtheoccurrenceofdiversemicrobialpopulationstohundredsofmetresdepthinclasticsediments(Craggetal.,1992;Parkesetal.,1994,2000;FredricksonandOnstott,1996;Wellsburyetal.,1997;Boetiusetal.,2000).Carbonatemoundswhoseformationmaybeinitiatedbymethanotrophicbacteria,gashydrates,andbiodegradedpetroleumreservoirsalsobearwitnesstothefactthatthisso-calleddeepbio-spheremaybeubiquitousandinterceptfluxesfromthegeosphere.Bacterialcommunitiesinsubsurfaceenvironmentsaremostcommonlyquantifiedbycellcounting(Cragget

Autotrophy as a predominant mode of carbon fixation in anaerobic methane-oxidizing microbial communities.

The methane-rich, hydrothermally heated sediments of the Guaymas Basin are inhabited by thermophilic microorganisms, including anaerobic methane-oxidizing archaea (mainly ANME-1) and sulfate-reducing bacteria (e.g., HotSeep-1 cluster). We studied the microbial carbon flow in ANME-1/ HotSeep-1 enrichments in stable-isotope–probing experiments with and without methane. The relative incorporation of 13C from either dissolved inorganic carbon or methane into lipids revealed that methane-oxidizing archaea assimilated primarily inorganic carbon. This assimilation is strongly accelerated in the presence of methane. Experiments with simultaneous amendments of both 13C-labeled dissolved inorganic carbon and deuterated water provided further insights into production rates of individual lipids derived from members of the methane-oxidizing community as well as their carbon sources used for lipid biosynthesis. In the presence of methane, all prominent lipids carried a dual isotopic signal indicative of their origin from primarily autotrophic microbes. In the absence of methane, archaeal lipid production ceased and bacterial lipid production dropped by 90%; the lipids produced by the residual fraction of the metabolically active bacterial community predominantly carried a heterotrophic signal. Collectively our results strongly suggest that the studied ANME-1 archaea oxidize methane but assimilate inorganic carbon and should thus be classified as methane-oxidizing chemoorganoautotrophs.

Diagenetic transformation of dissolved organic nitrogen compounds under contrasting sedimentary redox conditions in the Black Sea

Remineralization of organic matter in reactive marine sediments releases nutrients and dissolved organic matter (DOM) into the ocean. Here we focused on the molecular-level characterization of DOM by high-resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) in sediment pore waters and bottom waters from contrasting redox regimes in the northern Black Sea with particular emphasis on nitrogen-bearing compounds to derive an improved understanding of the molecular transformations involved in nitrogen release. The number of nitrogen-bearing molecules is generally higher in pore waters than in bottom waters. This suggests intensified degradation of nitrogen-bearing precursor molecules such as proteins in anoxic sediments: No significant difference was observed between sediments deposited under oxic vs anoxic conditions (average O/C ratios of 0.55) suggesting that the different organic matter quality induced by contrasting redox conditions does not impact protein diagenesis in the subseafloor. Compounds in the pore waters were on average larger, less oxygenated, and had a higher number of unsaturations. Applying a mathematical model, we could show that the assemblages of nitrogen-bearing molecular formulas are potential products of proteinaceous material that was transformed by the following reactions: (a) hydrolysis and deamination, both reducing the molecular size and nitrogen content of the products and intermediates; (b) oxidation and hydration of the intermediates; and (c) methylation and dehydration.

Assessing production of the ubiquitous archaeal diglycosyl tetraether lipids in marine subsurface sediment using intramolecular stable isotope probing

The membrane lipids diglycosyl-glycerol dibiphytanyl glycerol tetraethers (2G-GDGTs) in marine subsurface sediments are believed to originate from uncultivated benthic archaea, yet the production of 2G-GDGTs from subseafloor samples has not been demonstrated in vitro. In order to validate sedimentary biosynthesis of 2G-GDGTs, we performed a stable carbon isotope probing experiment on a subseafloor sample with six different (13) C-labelled substrates (bicarbonate, methane, acetate, leucine, glucose and Spirulina platensis biomass). After 468 days of anoxic incubation, only glucose and S. platensis resulted in label uptake in lipid moieties of 2G-GDGTs, indicating incorporation of carbon from these organic substrates. The hydrophobic moieties of 2G-GDGTs showed minimal label incorporation, with up to 4‰ (13) C enrichment detected in crenarchaeol-derived tricyclic biphytane from the S. platensis-supplemented slurries. The 2G-GDGT-derived glucose or glycerol moieties also showed (13) C incorporation (Δδ(13) C = 18-38‰) in the incubations with glucose or S. platensis, consistent with a lipid salvage mechanism utilized by marine benthic archaea to produce new 2G-GDGTs. The production rates were nevertheless rather slow, even when labile organic matter was supplied. The 2G-GDGT turnover times of 1700-20,500 years were much longer than those estimated for subseafloor microbial communities, implying that sedimentary 2G-GDGTs as biomarkers of benthic archaea are cumulative records of past and present generations.