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A microbial consortium couples anaerobic methane oxidation to denitrification


Vol 440|13 April 2006|doi:10.1038/nature04617

LETTERS
A microbial consortium couples anaerobic methane oxidation to denitri?cation
Ashna A. Raghoebarsing1, Arjan Pol1, Katinka T. van de Pas-Schoonen1, Alfons J. P. Smolders2, ? Katharina F. Ettwig1, W. Irene C. Rijpstra3, Stefan Schouten3, Jaap S. Sinninghe Damste3, 1 1 1 Huub J. M. Op den Camp , Mike S. M. Jetten & Marc Strous
Modern agriculture has accelerated biological methane and nitrogen cycling on a global scale1,2. Freshwater sediments often receive increased downward ?uxes of nitrate from agricultural runoff and upward ?uxes of methane generated by anaerobic decomposition3. In theory, prokaryotes should be capable of using nitrate to oxidize methane anaerobically, but such organisms have neither been observed in nature nor isolated in the laboratory 4–8 . Microbial oxidation of methane is thus believed to proceed only with oxygen or sulphate9,10. Here we show that the direct, anaerobic oxidation of methane coupled to denitri?cation of nitrate is possible. A microbial consortium, enriched from anoxic sediments, oxidized methane to carbon dioxide coupled to denitri?cation in the complete absence of oxygen. This consortium consisted of two microorganisms, a bacterium representing a phylum without any cultured species and an archaeon distantly related to marine methanotrophic Archaea. The detection of relatives of these prokaryotes in different freshwater ecosystems worldwide11–14 indicates that the reaction presented here may make a substantial contribution to biological methane and nitrogen cycles. Global biogeochemical cycles are mainly driven by microorganisms feeding on one-carbon compounds such as methane or carbon dioxide. Each step in the element cycles is catalysed by a speci?c group of microorganisms. These may or may not be evolutionarily related, but they share a similar lifestyle and so form an ‘ecological guild’. Thermodynamic calculations show that most of these guilds have already been discovered (Supplementary Fig. S1), but the microorganisms that couple the anaerobic oxidation of methane (AOM) to denitri?cation, shown in equations (1) and (2), are considered missing in nature4–8: 5CH4 ? 8NO32 ? 8H? ! 5CO2 ? 4N2 ? 14H2 O ?1? ?DG0 ? 2765 kJ mol21 CH4 ? 3CH4 ? 8NO2 ? 8H? ! 3CO2 ? 4N2 ? 10H2 O 2 ?2? ?DG0 ? 2928 kJ mol21 CH4 ? As AOM coupled to denitri?cation is possible in theory, both thermodynamically and biochemically (through reverse methanogenesis15,16), such microorganisms might in fact exist and consequently our understanding of biogeochemical methane cycling may be incomplete. The lack of experimental evidence for the occurrence of AOM coupled to denitri?cation is perhaps not surprising, because this process would be expected to occur close to the oxic/anoxic interface in sediments. This interface is generally characterized by
1
0 0

steep gradients, occurring within millimetres, masking the process from geochemical detection. Furthermore, laboratory enrichment of the responsible microorganisms could be dif?cult because of their potentially very slow growth17. Here we report the successful enrichment of consortia of microorganisms capable of AOM coupled to denitri?cation. Anoxic sediment from the Twentekanaal, a canal in the Netherlands, was used as the inoculum for the enrichment culture. This canal contained nitrate concentrations of up to 1 mM and the sediment was saturated with methane, which is typical for freshwater habitats receiving agricultural runoff. A one-litre sample from the upper layer of the sediment was incubated anoxically in the laboratory. Methane was supplied as the only electron donor, and mineral medium containing nitrate (NO3 2), nitrite (NO2 2), bicarbonate and trace elements was supplied and removed continuously. Over 16 months of incubation, the in?uent nitrite concentration was gradually increased to 6 mM, but the actual concentration in the culture medium remained at about 0.1 mM, indicating the growth of a microbial population consuming nitrite. Some nitrate (,1 mM) was also consumed. Methane consumption could not yet be observed experimentally because it was supplied in large excess and any potential conversion remained within the error margin for the CH4 analysis. To measure methane consumption, the media and methane supply were stopped and excess methane was removed by ?ushing with helium gas. The consumption of methane, nitrite and nitrate was now apparent and dinitrogen gas evolved (Fig. 1). Nitrite and nitrate together accounted for all of the produced dinitrogen gas. However, the total denitri?cation rate (28.8 ^ 2 mmol N2 h21) was not completely accounted for by methane oxidation (13.4 ^ 1 mmol CH4 h21) according to equations (1) and (2). Control experiments indicated that this difference was caused by the oxidation of organic compounds from the inoculum or the mineral medium. In these control experiments, the denitri?cation rate in the absence of methane was 5.5 ^ 0.5 mmol N2 h21, and on methane addition it increased to 21.5 ^ 2 mmol N2 h21. As methane itself was consumed at 22.0 ^ 2 mmol CH4 h21, the stoichiometry of AOM was almost completely consistent with the above equations. The enrichment culture used nitrite in preference to nitrate as the substrate for denitri?cation—for experiments in which nitrite was depleted in the presence of both methane and nitrate, AOM ceased, and resumed only when nitrite was re-introduced 2 h after depletion. However, during longer incubation periods in the absence of nitrite (10–20 h), AOM restarted and was then coupled to nitrate consumption. This suggests that the enrichment culture could adapt to nitrate, and that both nitrite and nitrate were suitable substrates for AOM

Department of Microbiology, and 2Department of Aquatic Ecology and Environmental Biology, Institute for Water and Wetland Research, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands. 3Royal Netherlands Institute for Sea Research (NIOZ), Department of Marine Biogeochemistry and Toxicology, PO Box 59, 1790 AB Den Burg, The Netherlands.

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Figure 1 | AOM is coupled to the denitri?cation of nitrite by the enrichment culture after 16 months of enrichment. a, b, The total amounts of methane, dinitrogen gas, nitrate and nitrite present in the culture vessel are indicated. The initial concentrations of these compounds in the culture liquid were 6.0 mM, 0.30 mM, 3.6 mM and 0.24 mM, respectively. During this experiment, the total amount of protein in the enrichment culture was 100 mg.

(equations (1) and (2)). The apparent af?nity constant for methane was very high (less than 0.6 mM, Fig. 1). The af?nity of sulphatedependent AOM for methane is four orders of magnitude lower18 (af?nity constant .16 mM). The addition of 2 mM sulphate to the

enrichment culture neither stimulated nor inhibited AOM. Together, these experiments show unambiguously that methane can be oxidized anaerobically in this system, and that this oxidation is coupled to denitri?cation. The participation of oxygen in this process can be fully excluded. First, we measured oxygen levels continuously in the culture liquid and periodically in the headspace, but did not detect any (detection limit 80 p.p.m.). Second, all of the detected dinitrogen gas in the headspace of the culture was accounted for by the consumption of nitrite and nitrate. Had air leaked into the culture, more dinitrogen gas would have been detected in the headspace than was predicted by the reaction stoichiometry. Third, the stoichiometry of methane consumption coupled to denitri?cation was in good agreement with equations (1) and (2). Had oxygen been involved, much less nitrite or nitrate would have been consumed per mol of methane. Finally, no methane was consumed as the enrichment culture adapted from nitrite to nitrate (see above). We investigated the incorporation of methane into microbial biomass by analysing the membrane lipids of the enrichment culture. We detected a single archaeal and several bacterial biomarkers (Table 1). The archaeal biomarker sn2-hydroxyarchaeol, found in methanogens19 and ANME-2 methanotrophic Archaea20, was the only biomarker substantially depleted in 13C compared to methane. This change indicates that the carbon in this marker compound originated from methane. When 13C-labelled methane was supplied to the culture, incorporation into sn2-hydroxyarchaeol was observed after six days. Notably, the bacterial biomarkers were labelled more rapidly and substantially than the archaeal biomarker (Table 1). These results are comparable to those obtained for a similar 13C-labelling experiment with a consortium performing sulphate-dependent AOM21, in which rapid and substantial incorporation of 13C-labelled methane was noted in the lipids of sulphate-reducing bacteria, but minor 13C incorporation was observed in archaeal lipids only after prolonged incubation (.300 days). Our biomarker data thus indicate that a consortium consisting of an archaeon and a bacterium is responsible for AOM coupled to denitri?cation. To determine the phylogenetic identity of the members of this consortium, we isolated genomic DNA from the biomass in the enrichment culture and constructed bacterial and archaeal 16S ribosomal RNA gene libraries. Sequence analysis of the bacterial clone library showed one dominant group of sequences clustering inside a subdivision with no other cultivated species (Fig. 2a and Supplementary Fig. 2a). This subdivision was distant from all other bacterial subdivisions (sequence identity less than 85%). Similar

Table 1 | Incorporation of methane into the bacterial and archaeal biomarkers of the enrichment culture
Biomarker compound Amount (%) Origin Enrichment culture (t ? 16 months) After labelling with 13C methane 3 days d13C (‰ versus VPDB) 6 days d13C (‰ versus VPDB)

d13C (‰ versus VPDB)

Methane Bicarbonate C14:0 fatty acid Iso-C15:0 fatty acid C16:1 (D9) fatty acid C16:0 fatty acid 10-me-C16:1 (D7) fatty acid 10-me-C16:0 fatty acid C18:0 fatty acid C18:1 (D11 ? D10) fatty acid C19cycloprop. fatty acid Diplopterol Sn2-hydroxyarchaeol

0.8 2.3 6.8 11.4 5.5 28.9 2.8 14.3 12.7 4.1 2.2

Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Bacteria Archaea

240.7 224.6 226.1 231.0 243.7 230.5 237.9 238.5 231.4 235.8 238.0 246.6 267.0

?2,300 ?125 ?4,300 ?370 2? 236.4 ?310 ?900 ?290 ?380 276

?4,400 ?189 2? ?580 2? 236.5 ?380 2? ?420 2? 249

The table shows the relative abundances and stable carbon isotopic composition of the major lipids from the enrichment culture and the incorporation of 13C into these compounds after 3 or 6 days of incubation with 13C-labelled methane (d13C ? [(13C/12C)sample/(13C/12C)standard] 2 1). For the archaeal compound from the enrichment culture, the large depletion in 13C indicates that its carbon is derived from (13C-depleted) methane. The minor but signi?cant incorporation of 13C into this compound during labelling (Dd13C ? 249 2 (267) ? ?18‰) suggests slow growth. VDPB, Vienna Pee Dee Belemnite. ? d13C after 6 days was not determined for some lipids already substantially enriched in 13C after 3 days. ? d13C could not be determined owing to low abundance of the compound and co-elution.

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NATURE|Vol 440|13 April 2006

sequences from this subdivision have previously been retrieved from the denitrifying zone of sediments from Lake Biwa in Japan12 and from contaminated groundwater in the United States13. The archaeal clone library contained a single sequence, which was only distantly related to the AOM Archaea of group 2 (ANME-2 (refs 9, 22), 86–87% identity) and cultivated methanogens (86–88% identity, Fig. 2b and Supplementary Fig. 2b). The highest similarity was found with archaeal clone sequences obtained from freshwater sediments from Lake Michigan in the United States14 and contaminated soils in Japan11. We used both the bacterial and archaeal 16S rRNA gene sequences to design speci?c probes for ?uorescence in situ hybridization (FISH). Samples collected over the 16-month period of enrichment were now hybridized with these probes. In samples from the ?rst three months, only occasional, single cells tested positive. Over time, both the bacterium and the archaeon became increasingly enriched, until they were the dominant microorganisms in the culture. After 16 months, approximately 10% of 4,6-diamidino-2-phenylindole (DAPI)-stained cells consisted of Archaea, all of which hybridized with the speci?c probe targeting the dominant archaeal sequence. The remainder of the culture consisted of bacteria, of which approximately 80% hybridized with the three speci?c probes targeting the dominant bacterial sequence (Fig. 2). As shown in Fig. 2, the Archaea were generally present as clusters inside a matrix of bacterial cells. The ratio of bacterial to archaeal cells (approximately 8:1) was different from the ratio reported for sulphate-dependent AOM18

(approximately 2:1). This difference might be explained by the higher energy yield of denitri?cation compared to sulphate reduction. a-, band g-proteobacteria together made up ,5% of the community. The sulphate reducers known to be involved in sulphate-dependent AOM10 were not detected, consistent with our observation that sulphate was not converted in the culture. The nitrite-dependent AOM rate was 140 mmol CH4 per g protein per hour (Fig. 1), corresponding to approximately 0.4 fmol CH4 per cell per day for the Archaea in our enrichment culture. For sulphatedependent AOM, a similar rate has been reported for the archaeal partner (0.7 fmol CH4 per cell per day)18. This indicates that for AOM coupled to denitri?cation, the archaeal growth rate could be extremely low, with a doubling time in the order of several weeks, consistent with the long duration of the enrichment procedure. To our knowledge this is the ?rst report of archaeal AOM coupled to bacterial denitri?cation. In the 1970s, Mason8 discredited earlier studies that pure cultures of methanotrophic bacteria were able to denitrify using methane as the sole carbon and energy source. Instead, it was established experimentally that methanotrophs can oxidize methane aerobically to methanol or acetate at low oxygen concentrations, and that the methanol or acetate can subsequently be used to drive denitri?cation23–26. Thus, the reaction presented here de?nes a new microbial guild with a potential contribution to biogeochemical cycling that has so far been overlooked. The recovery of related 16S rRNA gene sequences from different habitats and locations11–14 indicates that this process may contribute signi?cantly

Figure 2 | Phylogeny and ?uorescence in situ detection of the archaeal and bacterial members of the consortium mediating AOM coupled to denitri?cation. a, b, Consensus trees of the dominant bacterial (a) and archaeal (b) 16S rRNA sequences of the enrichment culture. Scale bars indicate 10 base substitutions per 100 bases. See also Supplementary Fig. 2 for more detailed trees, bootstrap values and treeing methods. c, Epi?uorescence micrograph after hybridization with the general bacterial probe EUBmix (blue), the speci?c bacterial probe DBACT-193 (red) and the
920

speci?c archaeal probe DARCH-872 (green). The bacterial partner is pink, as it hybridizes with both the general and speci?c bacterial probes. d, Epi?uorescence micrograph after hybridization with the general archaeal probe ARCH915 (blue), the speci?c archaeal probe DARCH-872 (red) and the general bacterial probe EUBmix (green). The archaeal partner is pink because it hybridizes with both the general and speci?c archaeal probes. Scale bars, 5 mm. See Methods for speci?cation of probes.

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to methane oxidation, and could potentially counteract worldwide increases in methane production associated with intensive agriculture. With biomarkers and probes for the responsible microorganisms now available, this possibility can be addressed.
METHODS
Sampling. Samples for inoculation were taken from the sediment of the Twentekanaal (528 11 0 04 00 N and 68 24 0 40 00 E, The Netherlands). Samples were collected from the top 15 cm of the sediment at 1 m water depth. At the time of sampling, the methane concentration at 15 cm sediment depth was 0.8 mM. The nitrate concentration in the water column was 0.1 mM. Cultivation and analytical methods are described in the Supplementary Information. Calculations. The values of the Gibbs energy changes reported for equations (1) and (2) were calculated for standard conditions (25 8C, pH 7). The af?nity constant of a microbial conversion is the substrate concentration at which the conversion rate is half of the maximum conversion rate. In this case the af?nity constant for methane was estimated from the slope of the methane consumption in Fig. 1. Methane incorporation and lipid analysis. Aliquots (60 ml) of the enrichment culture were anaerobically transferred to 120-ml serum bottles with an atmosphere of 90% argon and 10% 13C-labelled methane. The bottles were incubated on a shaker at 30 8C and then used for lipid analysis after three or six days. Lipids were ultrasonically extracted after freeze-drying and analysed by gas chromatography/ mass spectrometry and isotope ratio gas chromatography/mass spectrometry as described previously27. 13CO2 was measured as the end product of AOM, using a GC-isotope ratio mass spectrometer (ThermoFinnigan Delta Plus). 16S rRNA gene sequence analysis and FISH. Chromosomal DNA from 1-ml reactor biomass was isolated and used as a template for polymerase chain reaction (PCR) ampli?cation of 16S rRNA genes. PCR was performed using general bacterial primers (616F and 630R; ref. 27) and general archaeal primers (AR20F and AR958R; ref. 28). Cloning, sequencing and phylogenetic analyses were performed as described previously27. On the basis of the bacterial and archaeal 16S rRNA gene sequences, new oligonucleotide probes were designed. The bacterial probes were S-*-DBACT-0193-a-A-18 (5 0 -CGCTCGCCCCC TTTGGTC-3 0 ), S-*-DBACT-0447-a-A-18 (5 0 -CGCCGCCAAGTCATTCGT-3 0 ) and S-*-DBACT-1027-a-A-18 (5 0 -TCTCCACGCTCCCTTGCG-3 0 ), and the archaeal probe was S-*-DARCH-0872-a-A-18 (5 0 -GGCTCCACCCGTTG TAGT-3 0 ). We also used the general archaeal probe S-D-ARCH-0915-a-A-20, the general bacterial probe EUBmix and probe DSS658 for sulphate reducers10. FISH was performed as described previously27. Formamide concentrations used in FISH experiments varied between 20% and 40%.
Received 30 November 2005; accepted 2 February 2006.
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petroleum-contaminated soil. Environ. Microbiol. 7, 806– -818 (2005). 12. Koizumi, Y., Kojima, H. & Fukui, M. Characterization of depth-related microbial community structure in lake sediment by denaturing gradient gel electrophoresis of ampli?ed 16S rDNA and reversely transcribed 16S rRNA fragments. FEMS Microbiol. Ecol. 46, 147– -157 (2003). 13. Bakermans, C. & Madsen, E. L. Diversity of 16S rDNA and naphthalene dioxygenase genes from coal-tar-waste-contaminated aquifer waters. Microb. Ecol. 44, 95– -106 (2002). 14. Stein, L. Y., La Duc, M. T., Grundl, T. J. & Nealson, K. H. Bacterial and archaeal populations associated with freshwater ferromanganous micronodules and sediments. Environ. Microbiol. 3, 10– (2001). -18 15. Hallam, S. J. et al. Reverse methanogenesis: testing the hypothesis with environmental genomics. Science 305, 1457– -1462 (2004). 16. Kruger, M. et al. A conspicuous nickel protein in microbial mats that oxidize ¨ methane anaerobically. Nature 426, 878– -881 (2003). 17. Strous, M., Kuenen, J. G., Fuerst, J. A., Wagner, M. & Jetten, M. S. The anammox case—a new experimental manifesto for microbiological ecophysiology. Anton. Leeuw. Int. J. G. 81, 693– -702 (2002). 18. Nauhaus, K., Treude, T., Boetius, A. & Kruger, M. Environmental regulation of ¨ the anaerobic oxidation of methane: a comparison of ANME-I and ANME-II communities. Appl. Environ. Microbiol. 7, 98– -106 (2005). 19. Koga, Y., Morii, H., Akagawa-Matsushita, M. & Ohga, M. Correlation of polar lipid composition with 16S rRNA phylogeny in methanogens. Further analysis of lipid component parts. Biosci. Biotechnol. Biochem. 62, 230– -236 (1998). 20. Blumenberg, M., Seifert, R., Reitner, J., Pape, T. & Michaelis, W. Membrane lipid patterns typify distinct anaerobic methanotrophic consortia. Proc. Natl Acad. Sci. USA 101, 11111– -11116 (2004). 21. Blumenberg, M., Seifert, R., Nauhaus, K., Pape, T. & Michaelis, W. In vitro study of lipid biosynthesis in an anaerobically methane-oxidizing microbial mat. Appl. Environ. Microbiol. 71, 4345– -4351 (2005). ¨ 22. Knittel, K., Losekann, T., Boetius, A., Kort, R. & Amann, R. I. Diversity and distribution of methanotrophic archaea at cold seeps. Appl. Environ. Microbiol. 71, 467– -479 (2005). 23. Islas-Lima, S., Thalasso, F. & Gomez-Hernandez, J. Evidence of anoxic methane oxidation coupled to denitri?cation. Water Res. 38, 13– (2004). -16 24. Waki, M., Tanaka, Y., Osada, T. & Suzuki, K. Effects of nitrite and ammonium on methane-dependent denitri?cation. Appl. Microbiol. Biotechnol. 59, 338– -343 (2002). 25. Eisentraeger, A., Klag, P., Vansbotter, B., Heymann, E. & Dott, W. Denitri?cation of groundwater with methane as sole hydrogen donor. Water Res. 35, 2261– -2267 (2001). 26. Costa, C. et al. Denitri?cation with methane as electron donor in oxygenlimited bioreactors. Appl. Microbiol. Biotechnol. 53, 754– -762 (2000). 27. Raghoebarsing, A. A. et al. Methanotrophic symbionts provide carbon for photosynthesis in peat bogs. Nature 436, 1153– -1156 (2005). 28. Hinrichs, K. U., Hayes, J. M., Sylva, S. P., Brewer, P. G. & DeLong, E. F. Methane-consuming archaeabacteria in marine sediments. Nature 398, 802– -805 (1999).

Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements We thank B. Kartal, J. van de Vossenberg and M. Schmid for discussions, and J. G. Kuenen and M. Wagner for critical reading of the manuscript. We thank J. Eigensteyn and W. Geerts for technical support. M.S. and K.F.E. are supported by a VIDI grant from the Dutch Science Foundation (NWO). Help from G. Boedeltje in choosing the sampling location is also gratefully acknowledged. Author Contributions M.S., A.A.R., A.J.P.S. and K.T.P-S. performed the sampling; A.A.R. the enrichment, A.A.R., A.P. and K.F.E. the batch experiments and labelling; W.I.C.R., S.S. and J.S.S.D. the biomarker analysis; A.A.R. and K.T.P-S. the FISH analysis; A.A.R. the molecular analysis; and A.A.R. and H.J.M.O.C. the phylogeny and probe design. The research was conceived by M.S. and M.S.M.J., and pilot experiments were performed by K.T.P-S. A.A.R., A.P., K.F.E., S.S., J.S.S.D., H.J.M.O.C., M.S.M.J. and M.S. contributed to interpreting the data and writing the paper. Author Information The 16S rRNA gene sequences have been deposited in GenBank under accession numbers DQ369741 (archaeal sequence) and DQ369742 (bacterial sequence). Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing ?nancial interests. Correspondence and requests for materials should be addressed to M.S. (m.strous@science.ru.nl).

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