Environmental Microbiology (2000) 2(5), 477±484

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New perspectives on anaerobic methane oxidation

David L. Valentine² and William S. Reeburgh* the net modern atmospheric methane flux (20± Department of Earth System Science, University of 100 Â 1012 g year21). A variety of different environments California, Irvine, 220 Rowland Hall, Irvine, CA 92679- exist in which sulphate-dependent methane oxidation 3100 USA. (SDMO) is thought to occur, including recent marine sediments, methane seeps and vents, anoxic waters, soda lakes and deep continental margin sediments. Summary Despite extensive study, the mechanism of SDMO is not Anaerobic methane oxidation is a globally important understood, and no organisms have been isolated but poorly understood process. Four lines of evidence capable of explaining environmental observations. have recently improved our understanding of this A recent hypothesis postulates that process. First, studies of recent marine sediments () operate in reverse to oxidize methane and indicate that a consortium of methanogens and produce hydrogen. Sulphate reducers then use the sulphate-reducing bacteria are responsible for anae- hydrogen generated from methane, thereby maintaining robic methane oxidation; a mechanism of `reverse conditions that allow methane oxidation to proceed methanogenesis' was proposed, based on the prin- exergonically. Recent advances based on lipid biomarker ciple of interspecies hydrogen transfer. Second, isotope analysis and culture-independent identification studies of known methanogens under low hydrogen also indicate that an association of Archaea and sulphate- and high methane conditions were unable to induce reducing bacteria (SRB) is responsible for the observed methane oxidation, indicating that `reverse methano- oxidation of methane. The present review analyses new genesis' is not a widespread process in methanogens. data that pertain to the mechanism and environmental Third, lipid biomarker studies detected isotopically importance of SDMO, and provides a new perspective on depleted archaeal and bacterial biomarkers from this enigmatic process. In particular, we review evidence marine methane vents, and indicate that Archaea are contrary to the reverse methanogenesis hypothesis and the primary consumers of methane. Finally, phyloge- explore alternative mechanisms to explain environmental netic studies indicate that only specific groups of and experimental observations. Archaea and SRB are involved in methane oxidation. This review integrates results from these recent studies to constrain the responsible mechanisms. Background SDMO has been reviewed previously by Alperin and Introduction Reeburgh (1984), Hoehler et al. (1994) and Hoehler and Alperin (1996). Three lines of evidence have traditionally Methane oxidation in anoxic environments is microbially been used to support the existence of SDMO: (i) diagenetic mediated and of global significance. Methane is an (advection±diffusion±reaction) models of methane con- important trace gas in the atmosphere, and methane centration profiles in anoxic sediments and water columns oxidation in anoxic waters and marine sediments serves (Barnes and Goldberg, 1976; Reeburgh, 1976; Martens as an important control on the flux of methane to the and Berner, 1977; Reeburgh and Heggie, 1977; Alperin and atmosphere. Methane produced in deep sediment dif- Reeburgh, 1984); (ii) tracer measurements using [14C]- fuses upwards and is consumed by a population of CH ,[3H]-CH or [35S]-SO 2± (Reeburgh, 1980; Devol and through an undefined process in which 4 4 4 Ahmed, 1981; Iversen and Blackburn, 1981; Devol, 1983; sulphate acts as the terminal oxidant. This process is Iversen and Jùrgensen, 1985; Iversen et al., 1987; Ward estimated to consume methane equivalent to 5±20% of et al., 1987; 1989; Alperin, 1989; Reeburgh et al., 1991; Received 31 March, 2000; revised 9 June, 2000; accepted 10 July, Joye et al., 1999); and (iii) stable isotope distributions 2000. ²Present address, Marine Biology Research Division, Scripps (Oremland and DesMarais, 1983; Whiticar et al., 1986; Institution of Oceanography, Hubbs Hall Room 4305, 8750 Biological Grade, La Jolla, CA 92037, USA. *For correspondence. E-mail Oremland et al., 1987; Alperin et al., 1988; Blair and Aller, [email protected]; Tel. (11) 949 824 2986; Fax (11) 949 824 3256. 1995; Martens et al., 1999). Methane oxidation has been

Q 2000 Blackwell Science Ltd 478 D. L. Valentine and W. S. Reeburgh

13 21 13 Fig. 1. Sediment depth profiles of CH4 concentration (W,mMpw), [d C]-CH4 (A, ½), CH4 oxidation rate (D,mMpw CH4 year ), [d C]-DIC 2± 2± 2± 21 (dissolved inorganic carbon, L, ½), SO4 concentration (S,mMpw), SO4 reduction rate ((,mMpw SO4 year ) in sediments of Skan Bay, 13 Alaska. Values for CH4 concentration analysis and [d C]-CH4 analysis were taken by D. Valentine during an August 1997 cruise. Values for 13 2± 2± CH4 oxidation rate, [d C]-DIC, SO4 concentration, and SO4 reduction rate were taken from Alperin, 1989). The depth of zero represents the sediment±water interface. The zone of anaerobic methane oxidation is highlighted by hatched rectangles and extends from approximately 25 to 35 cm depth. studied extensively in the anoxic sediments of Skan Bay, environments. Its concentration represents a dynamic Alaska (see Fig. 1). steady state and is indicative of the dominant terminal Despite extensive study of SDMO, the organisms electron-accepting process (Lovley and Phillips, 1987; responsible have not been isolated, and the process remains Lovley and Goodwin, 1988; Hoehler et al., 1998). The a puzzle. Numerous bacterial isolates conserve energy from maintenance of low H2 allows for syntrophic oxidation of methane oxidation, but all use molecular oxygen to activate organic material through the process of interspecies H2 the very stable methane molecule (Hanson and Hanson, transfer (Wolin, 1982; Schink, 1997). Using results from 1996). SDMO occurs in environments completely devoid of field and laboratory studies, Hoehler et al. (1994) oxygen, and all evidence indicates that aerobic methano- hypothesized that, under sufficiently low H2, methano- trophs are not involved. The oxidation of methane by pure gens reverse their metabolism and mediate the net cultures of anaerobes has been reported in both SRB (Davis reversal of methanogenesis (acting as methane oxidizers; and Yarborough, 1966; Iversen, 1984) and methanogens MO, eqn 1), using water as the terminal electron acceptor.

(Zehnder and Brock, 1979; 1980; Harder, 1997). However, The H2 is removed efficiently and maintained at low the observed methane oxidation occurred as only a small concentrations by SRB (eqn 2) operating in a syntrophic fraction of the total metabolism, and such mechanisms are association with the methanogens (Hoehler and Alperin, unable to account for the net methane oxidation observed in 1996). The sulphate reducers are more efficient at using the environment. H2 as an electron donor; thus, they can create conditions that thermodynamically favour the oxidation of methane. The net reaction of the syntrophic association (eqn 3) Reverse methanogenesis and H syntrophy 2 yields approximately 225 kJ mol21 methane oxidized. Hoehler et al. (1994) used field and laboratory studies to The available energy can be shared by the two partners in suggest that SDMO is mediated by a consortium of the association, and the H2 level determines the methanogens and SRB through a process of `reverse partitioning of energy between the two organisms. methanogenesis'. Hoehler et al. (1994) proposed that a CH 1 2H O ! CO 1 4H MO† 1† ±sulphate reducer consortium provided a 4 2 2 2 biochemically feasible mechanism for net anaerobic SO2 2 1 4H 1 H 1 ! HS 2 1 4H O SRB† 2† methane oxidation and was consistent with seasonal 4 2 2 variations observed in Cape Lookout Bight. The hypothesis SO2 2 1 CH ! HCO 2 1 HS 2 1 H O Net† 3† was also attractive for several reasons: it was consistent 4 4 3 2 with all previous field, tracer, stable isotope and modelling Metabolic reversibility like that hypothesized in eqn 1 is studies, with results from inhibition experiments, it required known to occur among anaerobic microbes. Hydrogen acts no new organism, and it was more credible energetically as a thermodynamic trigger for metabolic reversal in the than previously proposed reaction schemes. `Reversibacter', a homoacetogen capable of converting

Hydrogen syntrophy is hypothesized to form the basis acetate to CO2 and H2 under low H2 (Zinder and Koch, of the methanogen±sulphate reducer consortium. Hydro- 1984; Lee and Zinder, 1988). Furthermore, some methano- gen is known to be a competitive substrate in anaerobic gens are capable of oxidizing methane to CO2 during growth,

Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 477±484 Anaerobic methane oxidation 479

Table 1. Selected 13C-depleted lipids from environments associated with anaerobic methane oxidation.

Known Santa Barbara Basina Eel River Basinb Marmorito Carbonatec Napolid Hydrate Ridgee Lipid source (½) (½) (½) (½) (½)

Archaeol Archaea 2119 2100 ± 276.2 ± sn-2-hydroxyarchaeol 2128 2110 ± ± ± Hydroxyarchaeols Archaea ±±±277f ± Crocetane Unknown (Archaea?) 2119 ± 2108.3 ± 2117.9 Phytanol Archaea ± ± 2108.5 ± ± Biphytanediol Archaea ± ± ± 277 ± PMI Archaea 2129 ± 2105.5 264.8 2123.8 PMI:4 Archaea ± ± ± ± 2107.3 Monoalkylglycerolethers Deeply branched bacteria 2104g ±± ±± n-hexadecan-1-ol Bacteria (SRB) ± ± 287.6h ±± i Fatty acids (C14-C18) Bacteria (SRB) 267 ±± ±± Iso/anteiso fatty acids Bacteria (SRB) ± ± ± 268 ± Diplopterol Bacteria ± ± ± 260 ±

±, indicates that values were either not reported or are not included here. a. Seep environment (Hinrichs et al., 2000). b. Seep environment (Hinrichs et al., 1999). c. Ancient seep environment (Thiel et al., 1999). d. Cold seep environment (Pancost et al. 2000). e. Core SO109-1 TVG 41-2 (Elvert et al., 1999). f. Both sn-2 and sn-3-hydroxyarchaeol were present. g. Average d13C of all monoalkylglycerolethers found. h. Additional lipids were found with similar structures and isotope values. i. Average d13C; several fatty acids were identified with isotope compositions as light as 2114½. although the rate of methane oxidation is always a fraction of carbon isotope signatures of the observed lipids serves as the production rate (Zehnder and Brock, 1979; 1980; Harder, compelling evidence that methane-derived carbon is enter- 1997). ing the anabolic pathways of the organisms that produce the We designed experiments aimed at testing the `reverse lipids, and also indicates that these organisms are directly methanogenesis' hypothesis, which involved screening involved in SDMO. Many of the lipids are so isotopically light pure cultures of several methanogens for H2 production that a substantial isotope fractionation (<60½) must still under conditions of low H2 and high CH4.AH2 removal occur even if methane is the initial carbon source. technique was developed based on the principle of gas The majority of 13C-depleted lipids that have been sparging (Valentine et al., 2000), and observed are thought to be unique to Archaea, and are concilii, Methanosaeta thermophila, Methanobacterium commonly used as biomarkers of archaeal metabolism strain Marburg and barkeri were screened (De Rosa et al., 1986; Koga et al., 1993; Table 1). The for H2 production/CH4 oxidation potential. Although most predominance of archaeal lipids indicates that Archaea cultures produced H2 initially, production was not sus- are the primary consumers of methane. However, tained, and methane oxidation was never observed different environments seem to contain different specific (Valentine, 2000). Similar studies have also been per- lipids, indicating that several distinct populations of formed by other researchers using other methanogens with Archaea may be involved in SDMO (Hinrichs et al., similar results (J. Harder, personal communication). 1999; 2000; Thiel et al., 1999; Pancost et al., 2000). Many of the observed lipids are known to be produced by the Methanosarcinales (a genus of mainly methylotrophic New evidence pertaining to anaerobic methane methanogens), although some lipids are produced more oxidation broadly within the Archaea, and the producers of some lipids remain unknown. Some 13C-depleted lipids gener- Isotopically (13C) depleted lipid biomarkers ally associated with Bacteria have also been observed Methane in marine environments is generally isotopically along with archaeal lipids (Thiel et al., 1999; Hinrichs et al., light, with values ranging from 250 to 290½ (Whiticar, 2000; Pancost et al., 2000). The putative bacterial lipids 1999) relative to the Pee Dee Belemnite standard. Pro- are especially prevalent in SRB (Kaneda, 1991) and have cesses that oxidize methane are expected to yield catabolic been found with isotopic signatures intermediate between and anabolic products, which are also depleted in 13C. the source methane and the archaeal lipids. Such lipids Several recent studies have focused on determining the are probably produced by organisms that are indirectly isotopic compositions and molecular structures of specific involved in SDMO, and may possibly be anabolic products lipids associated with SDMO (Table 1). The extremely light of syntrophic SRB.

Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 477±484 480 D. L. Valentine and W. S. Reeburgh Culture-independent evidence archaeal lipids and heavier bacterial lipids (Thiel et al., 1999; Hinrichs et al., 2000; Pancost et al., 2000); without Culture-independent identification techniques have interspecies carbon transfer, it is difficult to explain why recently been applied to the problem of anaerobic some bacterial lipids are found to be isotopically depleted, methane oxidation. Hinrichs et al. (1999) analysed whereas others in the same sample are not. Finally, small-subunit ribosomal RNA (16S rRNA) sequences phylogenetic and microscopic evidence indicates that from a methane seep in the Eel River Basin, a site at Archaea related to the Methanosarcinales (possibly which SDMO is thought to occur. The results of this study closely related to the Methanosaeta) may be responsible found a mixture of Bacteria and Archaea. The Bacteria for methane oxidation in some environments (Pimenov present were mainly related to known anaerobes includ- et al., 1997; Hinrichs et al., 1999; B. Lanoil et al. ing SRB. The Archaea consisted primarily of a novel unpublished data); many Methanosarcinales are unable group (ANME-1, probably representing a new archaeal to use H during methanogenesis, and tend to grow from genus) peripherally related to the Methanosarcinales,as 2 methylated compounds and acetate instead. well as novel species of Methanosarcinales. Isotopic We explored two alternative mechanisms for SDMO to analysis of lipid biomarkers in the same sediments account for the new findings. Mechanisms were sought (Table 1) provides circumstantial evidence linking the that allow for greater thermodynamic yields for the novel archaeal phylotypes to methane oxidation through organisms involved, involve interspecies carbon transfer, both the lipid structures and the isotopic signatures. require no novel biochemical pathways and are consistent More recent 16S rRNA studies of methane hydrates, with all previous observations. One such mechanism that borehole fluid and high-methane sediments have also matches all these requirements involves the formation of found sequences from ANME-1, but Methanosarcinales acetic acid and H from two molecules of methane (eqn 4) are found to predominate over ANME-1 in each of the 2 by methane-oxidizing Archaea (MO) with subsequent environments sampled (B. Lanoil et al., unpublished data). consumption of H and acetic acid (eqns 5 and 6) by SRB: The Methanosarcinales-related clones found by B. Lanoil 2 and colleagues are most closely related to the Methano- 2CH4 1 2H2O ! CH3COOH 1 4H2 MO† 4† saeta, although some are more deeply branching within the 22 1 2 Methanosarcinales. Additional evidence from the Black 4H2 1 SO4 1 H ! HS 1 4H2O SRB† 5† Sea also supports the possibility that Methanosarcinales are involved in anaerobic methane oxidation. Microscopic 22; 2 2 1 CH3COOH 1 SO4 ! 2HCO3 1 HS 1 H SRB† 6† analysis of microbial communities associated with methane seep carbonates (in which SDMO is thought to occur) 22 2 2 2CH4 1 2SO4 ! 2HCO3 1 2HS 1 2H2O Net† 7† indicated that the dominant populations strongly resemble The net reaction (eqn 7) is simply twice the reaction the Methanosaeta (Pimenov et al., 1997). Phylogenetic normally associated with anaerobic methane oxidation (eqn (16S rRNA) analysis of Black Sea anoxic waters also 3). The coupling of two methane molecules to form acetic reveal the presence of Methanosarcinales (Vetriani and acid allows for energy conservation from the net process Kerkhof, 1999); methane oxidation has been observed in (eqn 7), which would normally be reduced (i.e. divided by the anoxic waters of the Black Sea (Reeburgh et al., 1991). two to equal eqn 3) for thermodynamic calculations. The net reaction (eqn 7) allows for twice as much free energy as the mechanism of reverse methanogenesis (eqn 3), although Alternative mechanisms for anaerobic methane the energy must be split three ways instead of two. oxidation A second alternative mechanism has been considered Several recent studies raise questions about reverse previously (Zehnder and Brock, 1980; Hoehler et al., methanogenesis (Hoehler et al., 1994) as the mechanism 1994) and involves the formation of acetate from CO2 and responsible for SDMO. First, repeated attempts to induce CH4 (a reversal of aceticlastic methanogenesis) by a reverse methanogenesis using low H2 and high CH4 methane-oxidizing Archaea (eqn 8). Acetate would then have proved unsuccessful (Valentine, 2000; J. Harder, be consumed by a SRB (eqn 9) acting in a syntrophic personal communication). Secondly, the thermodynamic association with the methane oxidizer: 21 yield of reverse methanogenesis allows only 225 kJ mol 2 2 CH4 1 HCO3 ! CH3COO 1 H2O MO† 8† (CH4) to be shared by an Archaea and a SRB (assuming typical sediment concentrations), a value below the com- CH COO2 1 SO22 ! 2HCO2 1 HS2 SRB† 9† monly accepted biological energy quantum 3 4 3 (< 220 kJ mol21 per organism; Schink, 1997). Thirdly, CH 1 SO22 ! HCO2 1 HS2 1 H O Net† 3† specific 13C-depleted bacterial lipids (probably from SRB) 4 4 3 2 have been found in samples with a mixture of light The net reaction (eqn 3) does not show the fixation of

Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 477±484 Anaerobic methane oxidation 481

Table 2. Thermodynamics of two proposed mechanisms for sulphate-dependent methane oxidation.

Reaction DGo DGo0a (pH, T) DG0ab Eqn

2 1 c CH3COO 1 H ! CH3COOH At equilibrium At equilibrium 0 Mechanism 1 d 2CH4 1 2H2O ! CH3COOH 1 4H2 1166.6 1169.4 214.4 4 22 1 2 d 4H2 1 SO4 1 H ! HS 1 4H2O 2152.2 2154.1 217.8 5 22 2 2 1 CH3COOH 1 SO4 ! 2HCO3 1 HS 1 H 247.6 243.3 218.5 6 22 2 2 2CH4 1 2SO4 ! 2HS 1 2HCO3 1 2H2O 233.2 228.0 250.7 7 Mechanism 2 2 2 CH4 1 HCO3 ! CH3COO 1 H2O 131.0 128.5 15.3 8 2 22 2 2 CH3COO 1 SO4 ! 2HCO3 1 HS 247.6 242.4 230.6 9 22 2 2 CH4 1 SO4 ! HCO3 1 HS 1 H2O 216.6 214.0 225.4 10 a. Calculations corrected for typical summer conditions in sediments of Skan Bay, AK (T ˆ 48C, pH ˆ 7.2). 5 22 2 2 b. Calculations used chemical concentrations of: CH4 (2.2  10 Pa), SO4 (2 mM), HS (1 mM), acetate (3 mM), acetic acid (11 nM), HCO3 22 (32 mM), H2 (3.16  10 Pa) (Shaw et al., 1984; Alperin, 1989). 22 c. H2 levels have not been accurately quantified in Skan Bay sediments; the value of 3.16  10 Pa was chosen as a reasonable estimate based on the work of Hoehler et al., 1998). d. Equilibrium is assumed for the acid±base reaction involving acetate (pKa ˆ 4.75), allowing for calculations of acetic acid concentration.Standard thermodynamic values and equations were used for all calculations (Thauer et al., 1977; Atkins, 1994). All free energy yields are given in kJ reaction21. bicarbonate to form acetate, as that carbon is later SDMO, it is unlikely that metabolic energy is used actively to reoxidized by the SRB. import or export acetate. Instead, it is anticipated that acetic To determine the feasibility of the proposed mechan- acid will pass through the membrane in the undissociated isms, we calculated the free energy change of each form (Thauer et al., 1977). The transfer of acetic acid (not reaction for the conditions present within the methane acetate) does not alter the net thermodynamics of methane oxidation zone in sediments of Skan Bay, Alaska (Table 2). oxidation, but it does alter the partitioning of energy between We feel that the mechanism involving the formation of the various organisms involved as a result of concentration acetic acid from two methane molecules (eqns 4±7) is differences between acetate and acetic acid. If acetate is the advantageous over the other hypotheses, as it allows for species transferred between organisms, the thermodynamic more energy for each organism involved, and better yield of the methane-oxidizing Archaea (according to eqn 8) explains why some acetate-using SRB may contain would be lower, and the yield for acetate-consuming SRB isotopically depleted lipids. Lipids formed by Archaea would increase correspondingly. However, interspecies producing acetate from methane and bicarbonate (eqn 8) acetate transfer could occur in structured microenviron- are likely to have isotopic values intermediate between ments in which SRB maintain acetate concentrations below methane and ambient bicarbonate. Also, the coupling of the level in the surrounding pore water; in such a case, the two methane molecules to form acetate is biochemically energetics of acetate transfer might be similar to those of feasible (Valentine, 2000), requires no new organisms and acetic acid transfer. is consistent with previous field and laboratory studies. The hypotheses presented here can be tested experi- The thermodynamics of acetate production from methane mentally, both in the laboratory and in the field. Laboratory and bicarbonate (eqn 8) are poor under the conditions experiments involving mixed cultures of H2 and acetate- assumed in Table 2, but would be more favourable under using SRB can be used to screen known methanogens by conditions of high methane (i.e. deep methane vents), in feeding the cultures sulphate and methane. Direct methane microenvironments surrounded by SRB, or if acetic acid was consumption could be observed in such cultures. Various the molecule transferred between species (as in eqns 4 and isotopic techniques can also be used to test this hypothesis 6). The production of acetic acid (not acetate) from methane in environmental samples. Given the light isotopic signature and carbon dioxide under 50 atm of methane (otherwise of methane in marine systems, one could analyse the assuming the conditions of Skan Bay sediments) would yield isotopic signature of porewater acetate in a well-defined 212.9 kJ mol21. Such a thermodynamic yield is still below methane-oxidizing sediment. Abnormally isotopically light the biologicalenergy quantum of < 220 kJ mol21 and is well acetate would be an indication of the validity of this below the yield of 228.7 kJ mol21 for the production of mechanism. However, if isotopically heavy sources of acetic acid and H2 from two methane molecules (eqn 4) acetate contribute to the acetate pool, a light signal could under identical conditions. be diluted. This may be the case when methane oxidation The presumed transfer of acetic acid instead of acetate only accounts for a fraction of sulphate reduction. Methane (eqns 4±7) represents a `whole cell' perspective on the vent sites may provide a good location to test this hypothesis, bioenergetics of metabolism. Given the low energy yield of as methane oxidation seems to be the dominant form of

Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 477±484 482 D. L. Valentine and W. S. Reeburgh metabolism, and the isotopic signature of acetate is more (Iversen and Jùrgensen, 1985; Niewohner et al., 1998; likely to reflect the methane source. Isotope tracer incubation Borowski et al., 1999). In such environments, there is no experiments could also be performed to track methane opportunity for co-metabolism, as there is no available through dissolved acetate (or other intermediates). Any organic material, and sulphate (the sole terminal electron isotope of carbon or hydrogen could be used to perform such acceptor available in the sediments) reduction is quantita- experiments by incubating sediments with labelled methane, tively linked to methane oxidation. Further, the high separating or isolating acetate from the porewater and abundance of specific 13C-depleted lipids from different analysing the isotopes of acetate. methane-oxidizing environments indicates that methane is the sole carbon source during growth of these populations, with no other carbon entering the lipid-synthesizing anabolic Problems and prospects pathways. Methane oxidation rate measurements rely on Thermodynamic insights methane conversion to CO2, further indicating that methane oxidation is primarily a dissimilatory process. In addition, One factor frequently overlooked in discussions of SDMO is there may be more energy available for methane oxidation how the thermodynamics and kinetics of the process vary than previously realized, as discussed above. Taken between the different sorts of environments in which it occurs. together, the rate, isotope and thermodynamic evidence Differences in methane and sulphate concentrations tend to strongly indicate that methane oxidation is an energy- drive such variations. Vent sites have methane partial conserving metabolic process for some Archaea. pressures proportional to depth, often greater than 50 atm, Assuming that organisms can grow from SDMO, why whereas methane in recent sediments, such as those of Skan have all attempts at enrichment failed? There are a number Bay, Alaska, generally ranges from 0.2 to 2.0 atm. Methane of possible explanations for this, but one of the most likely is partial pressure in anoxic waters, such as those of the Black that the organisms involved have exceptionally slow growth Sea, are generally , 0.01 atm. Given typical conditions, free rates because of their marginal thermodynamic yields. The energy yields for methane oxidation (according to eqn 3) in probable syntrophic association will further decrease the these environments range from 235 kJ (vents) to 225 kJ growth rate. If the same mechanism is active in recent (sediments) to 222 kJ (anoxic waters). Variations in the sediments and in seeps, then growth may be expected to partial pressure of methane will also significantly influence occur much faster under the methane levels found at the the kinetics of methane uptake. At low methane levels, it is seeps because of the more favourable thermodynamics. difficult to bind and activate the stable methane molecule, Enrichment attempts using proper inoculum and high whereas at high levels, a methane-binding enzyme may not pressures of methane (501 atm) have a greater potential require a high affinity for substrate. Along with other for success than standard roll-tube techniques. interenvironmental differences, including pH, temperature, salinity and pressure, differences in thermodynamics and kinetics may select for different methane-oxidizing commu- Future studies nities at different sites. The variety of different lipid The mechanism behind anaerobic methane oxidation will biomarkers found at different sites (Table 1) seems to be remain a mystery until either representative organisms are consistent with such an assessment. cultured or known organisms are shown to perform methane oxidation in a manner consistent with field observations. Environmental studies have the potential to yield further SDMO is coupled to energy conservation insight into the mechanism and prevalence of the process. Numerous attempts to enrich for methane-oxidizing anae- Highly integrated studies that include concentration, rate, robes have failed. Coupled with the poor thermodynamic phylogenetic, biomarker, microscopy and isotope analyses yield of SDMO, this has led to the idea that methane oxidation can simultaneously demonstrate metabolic activity, popula- is a co-metabolic activity, and that the responsible organ- tion abundance, bioenergetics and pathways of carbon flow ism(s) do not conserve energy from the process (Schink, for a given environment. These studies can be used to test 1997) (i.e. organisms cannot be enriched based on co- the various hypotheses for methane oxidation, and the use of metabolic activity). Evidence from natural settings seems to exogenous isotope tracers can be used to follow the indicate that this is not the case. The strongest evidence immediate fate of methane in samples (i.e. to lipids, acetate comes from comparing rates of methane oxidation with rates or other intermediates). of sulphate reduction for a given setting. In several marine sediments containing little organic material, the depth- Expanding the importance of SDMO integrated rates of methane oxidation are equal to the depth-integrated rates of sulphate reduction, indicating that In addition to the importance of SDMO in present-day all sulphate reduction is coupled to methane oxidation methane cycling, the process may have played an

Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 477±484 Anaerobic methane oxidation 483 important role in the early biogeochemical evolution of the closely related to Desulfosarcina/Desulfocococcus. By demonstrat- earth. During the early Precambrian era, the sun was much ing the close physical association of the consortium and the specific fainter than today, yet the climate was warm. To explain this species involved, this study has added further insight to our understanding of SDMO. `faint young sun paradox', it has been hypothesized that an abundance of greenhouse gases was present in the atmosphere of early earth (Kasting, 1997). Because CO2 References levels have been constrained by geological evidence (Rye Alperin, M.J. (1989) The Carbon Cycle in an Anoxic Marine et al., 1995), methane has been postulated to be the key Sediment: Concentrations, Rates, Isotope Ratios, and Dia- component. If abundant methane and sulphate were genetic Models. PhD Thesis, Institute of Marine Science, present on the early earth, then SDMO may have played University of Alaska, Fairbanks. IMS report; R89-1, pp. 241. a key role in modulating the climate of early earth and may Alperin, M.J., and Reeburgh, W.S. (1984) Geochemical observa- tions supporting anaerobic methane oxidation. In Microbial have been the primary sink for atmospheric methane. Growth on C-1 Compounds. Crawford, R., and Hanson, R. Evidence pertaining to such a hypothesis may be located in (eds). Washington, DC: American Society for Microbiology the geological record (in isotopes and biomarkers), in the Press, pp. 282±289. biochemistry/genetics of the process (through the relation- Alperin, M.J., Reeburgh, W.S., and Whiticar, M.J. (1988) Carbon ships of undiscovered enzymes/genes) and can be further and hydrogen isotope fractionation resulting from anaerobic understood through bioenergetic and kinetic studies of methane oxidation. Global Biogeochem Cycles 2: 279±288. Atkins, P.W. (1994). Physical Chemistry. New York: W.H. Freeman. methane consumption in extant ecosystems. Barnes, R.O., and Goldberg, E.D. (1976) Methane production and consumption in anaerobic marine sediments. Geology 4: 297±300. Concluding remarks Blair, N.E., and Aller, R.C. (1995) Anaerobic methane oxidation on SDMO is a process that occurs widely in anoxic marine the Amazon Shelf. Geochim Cosmochim Acta 59: 3707±3715. Borowski, W.S., Paull, C.K., and Ussler, W. (1999) Global and systems and acts as a barrier for methane release to the local variations of interstitial sulfate gradients in deep-water, water column and the atmosphere. Recent evidence continental margin sediments: Sensitivity to underlying indicates that the mechanism of methane oxidation methane and gas hydrates. Mar Geol 159: 131±154. involves methane-oxidizing Archaea and SRB, acting in Davis, J.B., and Yarborough, H.F. (1966) Anaerobic oxidation of a syntrophic association. Hydrogen appears to be a key hydrocarbons by Desulfovibrio desulfuricans. Chem Geol 1: intermediate in this syntrophic association, and we hypoth- 137±144. De Rosa, M., Gambacorta, A., and Gliozzi, A. (1986) Structure, esize that acetate is also involved. Although methane biosynthesis, and physicochemical properties of Archaebac- oxidation occurs in a variety of different settings and terial lipids. Microbiol Rev 50: 70±80. appears to be performed by several different organisms, Devol, A.H. (1983) Methane oxidation rates in the anaerobic there are indications that similar mechanisms may be sediments of Saanich Inlet. Limnol Oceanogr 28: 738±742. active in all such settings. SDMO appears to be a Devol, A.H., and Ahmed, S.L. (1981) Are high rates of sulfate fundamental metabolic process in some Archaea and is reduction associated with anaerobic oxidation of methane? Nature 291: 407±408. probably coupled to energy conservation and growth. Elvert, M., Suess, E., and Whiticar, M.J. (1999) Anaerobic Future field and laboratory studies will undoubtedly yield methane oxidation associated with marine gas hydrates: super- further insights into this important and enigmatic process. light C-isotopes from saturated and unsaturated C-20 and C-25 irregular isoprenoids. Naturwissenschaften 86: 295±300. Hanson, R.S., and Hanson, T.E. (1996) Methanotrophic bacteria. Acknowledgements Microbiol Rev 60: 439±471. Harder, J. (1997) Anaerobic methane oxidation by bacteria We thank Kai Hinrichs, John Hayes, Brian Lanoil and Marcus employing C-14-methane uncontaminated with C-14-carbon Elvert for kindly supplying us with unpublished data. Support for monoxide. Mar Geol 137: 13±23. this work was provided by the NSF through the Life in Extreme Hinrichs, K.U., Hayes, J.M., Sylva, S.P., Brewer, P.G., and Environments special competition (MCB 97-13967 to W.S.R.) and DeLong, E.F. (1999) Methane-consuming archaebacteria in through a UC Regents Dissertation Quarter Fellowship (to D.L.V.). marine sediments. Nature 398: 802±805. Hinrichs, K.U., Summons, R.E., Orphan, V., Sylva, S.P., and Note added in proof Hayes, J.M. (2000) Molecular and isotopic analysis of anaerobic methane-oxidizing communities in marine sedi- A recent study of hydrate-containing marine sediments conducted by ments. Org Geochem (in press). A. Boetius and colleagues (in press) describes an Archaea-SRB Hoehler, T.M., and Alperin, M.J. (1996) Anaerobic methane consortium apparently responsible for SDMO in this environment. oxidation by a methanogen-sulfate reducer consortium: geo- Using 16S rRNA-targeted oligonucleotide probes and fluorescence chemical evidence and biochemical considerations. In Microbial in situ hybridization, the authors are able to visualize clusters of Growth on C-1 Compounds. Lidstrom, M.E., and Tabita, R.F. archaeal cells surrounded by layers of SRB. The specificity of the (eds). Dordrecht: Kluwer Academic Publishing, pp. 326±333. probes also indicates that the consortium consists of specific Hoehler, T.M., Alperin, M.J., Albert, D.B., and Martens, C.S. Archaea closely related to the Methanosarcinales and specific SRB (1994) Field and laboratory studies of methane oxidation in an

Q 2000 Blackwell Science Ltd, Environmental Microbiology, 2, 477±484 484 D. L. Valentine and W. S. Reeburgh

anoxic marine sediment ± evidence for a methanogen-sulfate on coral-like structures at methane seeps in the Black Sea. reducer consortium. Global Biogeochem Cycles 8: 451±463. Microbiology 66: 354±360. Hoehler, T.M., Alperin, M.J., Albert, D.B., and Martens, C.S. Reeburgh, W.S. (1976) Methane consumption in Cariaco Trench (1998) Thermodynamic control on hydrogen concentrations in waters and sediments. Earth Planet Sci Lett 28: 337±344. anoxic sediments. Geochim Cosmochim Acta 62: 1745±1756. Reeburgh, W.S. (1980) Anaerobic methane oxidation: rate depth Iversen, N. (1984) Interaktioner mellem Fermenterings ± distributions in Skan Bay sediments. Earth Planet Sci Lett 47: Processer de Terminale Processer. PhD thesis. Aarhus, 345±352. Denmark: Aarhus University. 129 pp. Reeburgh, W.S., and Heggie, D.T. (1977) Microbial methane Iversen, N., and Blackburn, H.T. (1981) Seasonal rates of consumption reactions and their effect on methane distributions methane oxidation in anoxic marine sediments. Appl Environ in freshwater and marine environments. Limnol Oceanogr 22:1±9. Microbiol 41: 1295±1300. Reeburgh, W.S., Ward, B.B., Whalen, S.C., Sandbeck, K.A., Iversen, N., and Jùrgensen, B.B. (1985) Anaerobic methane Kilpatrick, K.A., and Kerkhof, L.J. (1991) Black Sea methane oxidation rates at the sulfate±methane transition in marine geochemistry. Deep-Sea Res Part A Oceanogr Res Papers 38: sediments from Kattegat and Skagerrak (Denmark). Limnol S1189±S1210. Oceanogr 30: 944±955. Rye, R., Kuo, P.H., and Holland, H.D. (1995) Atmospheric carbon Iversen, N., Oremland, R.S., and Klug, M.J. (1987) Big Soda dioxide concentrations before 2.2 billion years ago. Nature 378: Lake (Nevada). 3. Pelagic methanogenesis and anaerobic 603±605. methane oxidation. Limnol Oceanogr 32: 804±814. Schink, B. (1997) Energetics of syntrophic cooperation in Joye, S.B., Connell, T.L., Miller, L.G., Oremland, R.S., and methanogenic degradation. Mol Microbiol Rev 61: 262±280. Jellison, R.S. (1999) Oxidation of ammonia and methane in an Shaw, D.G., Alperin, M.J., Reeburgh, W.S., and McIntosh, D.J. alkaline, saline lake. Limnol Oceanogr 44: 178±188. (1984) Biogeochemistry of acetate in anoxic sediments of Skan Kaneda, T. (1991) Iso-fatty and anteiso-fatty acids in bacteria ± Bay, AK. Geochim Cosmochim Acta 48: 1819±1825. biosynthesis, function, and taxonomic significance. Microbiol Thauer, R.K., Jungermann, K., and Decker, K. (1977) Energy Rev 55: 288±302. conservation in chemotrophic anaerobic bacteria. Bacteriol Kasting, J.F. (1997) Planetary atmospheres ± warming early Rev 41: 100±180. Earth and Mars. Science 276: 1213±1215. Thiel, V., Peckmann, J., Seifert, R., Wehrung, P., Reitner, J., and Koga, Y., Nishihara, M., Morii, H., and Akagawamatsushita, M. (1993) Michaelis, W. (1999) Highly isotopically depleted isoprenoids: Ether polar lipids of methanogenic bacteria ± structures, compara- molecular markers for ancient methane venting. Geochim tive aspects, and biosyntheses. Microbiol Rev 57: 164±182. Cosmochim Acta 63: 3959±3966. Lee, M.J., and Zinder, S.H. (1988) Isolation and characterization of Valentine, D.L. (2000) Biogeochemistry of Hydrogen and a thermophilic bacterium which oxidizes acetate in syntrophic Methane in Anoxic Environments: Thermodynamic and Iso- association with a methanogen and which grows acetogenically topic Studies. PhD Thesis, Earth System Science, University of on H -CO . Appl Environ Microbiol 54: 124±129. 2 2 California at Irvine, pp. 173. Lovley, D.R., and Goodwin, S. (1988) Hydrogen concentrations as an Valentine, D.L., Reeburgh, W.S., and Blanton, D.C. (2000) A indicator of the predominant terminal electron-accepting reactions culture apparatus for maintaining H at sub-nanomolar in aquatic sediments. Geochim Cosmochim Acta 52: 2993±3003. 2 concentrations. J Microbiol Methods 39: 243±251. Lovley, D.R., and Phillips, E.J.P. (1987) Competitive mechanisms Vetriani, C., and Kerkhof, L. (1999) Phylogenetic and functional for inhibition of sulfate reduction and methane production in the characterization of microbial assemblages at the oxic/anoxic zone of ferric iron reduction in sediments. Appl Environ interface in the Black Sea. Eos Trans Am Geophys Union 80:288. Microbiol 53: 2636±2641. Martens, C.S., and Berner, R.A. (1977) Interstitial water Ward, B.B., Kilpatrick, K.A., Novelli, P.C., and Scranton, M.I. chemistry of Long Island Sound sediments, I, dissolved (1987) Methane oxidation and methane fluxes in the ocean gases. Limnol Oceanogr 22: 10±25. surface layer and deep anoxic waters. Nature 327: 226±229. Martens, C.S., Albert, D.B., and Alperin, M.J. (1999) Stable isotope Ward, B.B., Kilpatrick, K.A., Wopat, A.E., Minnich, E.C., and tracing of anaerobic methane oxidation in the gassy sediments of Lidstrom, M.E. (1989) Methane oxidation in Saanich Inlet EckernfoÈrde Bay, German Baltic Sea. Am J Sci 299: 589±610. during summer stratification. Cont Shelf Res 9: 65±75. Niewohner, C., Hensen, C., Kasten, S., Zabel, M., and Schulz, Whiticar, M.J. (1999) Carbon and hydrogen isotope systematics H.D. (1998) Deep sulfate reduction completely mediated by of bacterial formation and oxidation of methane. Chem Geol anaerobic methane oxidation in sediments of the upwelling 161: 291±314. area off Namibia. Geochim Cosmochim Acta 62: 455±464. Whiticar, M.J., Faber, E., and Schoell, M. (1986) Biogenic Oremland, R.S., and DesMarais, D.J. (1983) Distribution, methane formation in marine and freshwater environments: abundance and carbon isotope composition of gaseous CO2 reduction vs. acetate fermentation-isotope evidence. hydrocarbons in Big Soda Lake, Nevada: an alkaline mero- Geochim Cosmochim Acta 50: 693±709. mictic lake. Geochim Cosmochim Acta 47: 2107±2114. Wolin, M.J. (1982) Hydrogen transfer in microbial communities. In Oremland, R.S., Miller, L.G., and Whiticar, M.J. (1987) Sources Microbial Interactions and Communities, Vol. 1. Bull, A.T., and and flux of natural gases from Mono Lake, California. Geochim Slater, J.H. (eds). London: Academic Press pp. 323±356. Cosmochim Acta 51: 2915±2929. Zehnder, A.J., and Brock, T.D. (1979) Methane formation and Pancost, R.D., Sinninghe Damste, J.S., de Lint, S., Van Der methane oxidation by methanogenic bacteria. J Bacteriol 137: Maarel, M.J.E.C., and Gottschal, J.C. (2000) Biomarker 420±432. evidence for widespread anaerobic methane oxidation in Zehnder, A.J., and Brock, T.D. (1980) Anaerobic methane oxidation: Mediterranean sediments by a consortium of methanogenic occurrence and ecology. Appl Environ Microbiol 39: 194±204. Archaea and Bacteria. Appl Environ Microbiol 66: 1126±1132. Zinder, S.H., and Koch, M. (1984) Non-aceticlastic methanogen- Pimenov, N.V., Rusanov, I.I., Poglazova, M.N., Mityushina, L.L., esis from acetate: acetate oxidation by a thermophilic Sorokin, D.Y., Khmelenina, V.N., et al. (1997) Bacterial mats syntrophic coculture. Arch Microbiol 138: 263±272.

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