Environmental Science and Pollution Research

, Volume 25, Issue 30, pp 30031–30043 | Cite as

Effect of pressure and temperature on anaerobic methanotrophic activities of a highly enriched ANME-2a community

  • Susma Bhattarai
  • Yu ZhangEmail author
  • Piet N. L. Lens
Research Article


This study investigated the effect of temperature and methane partial pressure on the anaerobic methane-oxidizing and sulfate-reducing (AOM-SR) activities by a highly enriched ANME-2a community. The ANME-2a-enriched biomass was incubated at different pressures, i.e., 2, 10, 20, and 30 MPa at 15 °C for 80 days. The response of the microbial community with temperature was investigated in incubations at 4, 15, and 25 °C at 10 MPa. Among all tested conditions, the incubation at 10 MPa pressure and 15 °C showed the highest AOM-SR activity of the studied ANME-2a phylotype, whereas activity at 2 MPa pressure and 15 °C was almost comparative to the response at 10 MPa pressure. The finding of the most favorable conditions for AOM-SR activity by the studied AOM-SR community comparable to the in situ pressure and temperature (15 °C at 10 MPa) suggests that the studied ANME-2a phylotype was well adapted to the conditions similar to its origin. The microbial community analysis showed that the bacterial community composition shifted upon changing the incubation temperature and pressure.


Anaerobic oxidation of methane Sulfate reduction Anaerobic methanotrophs Sulfate-reducing bacteria High pressure bioreactor Methane pressure 



We thank He Ying (Institute of Oceanology, Shanghai Jiao Tong University) for providing suggestions during the experiments and Yang Shanshan (Institute of Oceanology, Shanghai Jiao Tong University) for her help during microbial analysis. This research was supported by the Erasmus Mundus Joint Doctorate Program ETeCoS3 (Environmental Technologies for Contaminated Solids, Soils and Sediments) under the grant agreement FPA no. 2010-0009. Y. Zhang acknowledges the National Natural Science Foundation of China (grant No. 41476123).

Supplementary material

11356_2018_2573_MOESM1_ESM.docx (83 kb)
ESM 1 (DOCX 83 kb)


  1. Bhattarai S, Cassarini C, Gonzalez-Gil G, Egger M, Slomp CP, Zhang Y, Esposito G, Lens PN (2017) Anaerobic methane-oxidizing microbial community in a coastal marine sediment: anaerobic methanotrophy dominated by ANME-3. Microb Ecol 74:608–622. CrossRefGoogle Scholar
  2. Boetius A, Ravenschlag K, Schubert CJ, Rickert D, Widdel F, Gieseke A, Amann R, Jorgensen BB, Witte U, Pfannkuche O (2000) A marine microbial consortium apparently mediating anaerobic oxidation of methane. Nature 407:623–626. CrossRefGoogle Scholar
  3. Boetius A, Wenzhöfer F (2013) Seafloor oxygen consumption fuelled by methane from cold seeps. Nat Geosci 6:725–734. CrossRefGoogle Scholar
  4. Cassidy J (2014) Optimization of biological sulfate reduction to treat inorganic wastewaters: process control and potential use of methane as electron donor. Dissertation, Joint PhD Program by University of Paris-Est, University of Cassino and Southern Lazio and UNESCO-IHE Institute for Water EducationGoogle Scholar
  5. DeLong EF (1992) Archaea in coastal marine environments. Proc Natl Acad Sci U S A 89:5685–5689. CrossRefGoogle Scholar
  6. Deusner C, Meyer V, Ferdelman T (2009) High-pressure systems for gas-phase free continuous incubation of enriched marine microbial communities performing anaerobic oxidation of methane. Biotechnol Bioeng 105:524–533. CrossRefGoogle Scholar
  7. Egger M, Rasigraf O, Sapart CJ, Jilbert T, Jetten MSM, Röckmann T, van der Veen C, Bândă N, Kartal B, Ettwig KF, Slomp CP (2015) Iron-mediated anaerobic oxidation of methane in brackish coastal sediments. Environ Sci Technol 49:277–283. CrossRefGoogle Scholar
  8. Fang J, Zhang L, Bazylinski DA (2010) Deep-sea piezosphere and piezophiles: geomicrobiology and biogeochemistry. Curr Trends Microbiol 18:413–422. CrossRefGoogle Scholar
  9. Fichtel K, Logemann J, Fichtel J, Rullkötter J, Cypionka H, Engelen B (2015) Temperature and pressure adaptation of a sulfate reducer from the deep subsurface. Front Microbiol 6:1078. CrossRefGoogle Scholar
  10. Greene AC (2014) The family Desulfuromonadaceae. In: The prokaryotes. Springer, pp., pp 143–155CrossRefGoogle Scholar
  11. Hamdi S, Moyano F, Sall S, Bernoux M, Chevallier T (2013) Synthesis analysis of the temperature sensitivity of soil respiration from laboratory studies in relation to incubation methods and soil conditions. Soil Biol Biochem 58:115–126. CrossRefGoogle Scholar
  12. He Y, Li M, Perumal V, Feng X, Fang J, Xie J, Sievert S, Wang F (2016) Genomic and enzymatic evidence for acetogenesis among multiple lineages of the archaeal phylum Bathyarchaeota widespread in marine sediments. Nat Microbiol 1:16035. CrossRefGoogle Scholar
  13. Hinrichs KU, Hayes JM, Sylva SP, Brewer PG, DeLong EF (1999) Methane-consuming archaebacteria in marine sediments. Nature 398:802–805. CrossRefGoogle Scholar
  14. Holler T, Widdel F, Knittel K, Amann R, Kellermann MY, Hinrichs K-U, Teske A, Boetius A, Wegener G (2011) Thermophilic anaerobic oxidation of methane by marine microbial consortia. ISME J 5:1946–1956. CrossRefGoogle Scholar
  15. Jebbar M, Franzetti B, Girard E, Oger P (2015) Microbial diversity and adaptation to high hydrostatic pressure in deep-sea hydrothermal vents prokaryotes. Extremophiles 19:721–740. CrossRefGoogle Scholar
  16. Kato C, Nogi Y, Arakawa S (2008) Isolation, cultivation, and diversity of deep-sea piezophiles. In: Michiels C, Bartlett DH, Artsen A (eds) High-pressure microbiology. ASM Press, Washington DC, pp 203–217CrossRefGoogle Scholar
  17. Kim S-J, Kato C (2010) Sampling, isolation, cultivation, and characterization of piezophilic microbes. In: Timmis K N (ed) Handbook of hydrocarbon and lipid microbiology, springer, pp. 3869–3881Google Scholar
  18. Knittel K, Lösekann T, Boetius A, Kort R, Amann R (2005) Diversity and distribution of methanotrophic archaea at cold seeps. App Environ Microbiol 71:467–479. CrossRefGoogle Scholar
  19. Knittel K, Boetius A (2009) Anaerobic oxidation of methane: progress with an unknown process. Ann Rev Microbiol 63:311–334. CrossRefGoogle Scholar
  20. Krüger M, Wolters H, Gehre M, Joye SB, Richnow H-H (2008b) Tracing the slow growth of anaerobic methane-oxidizing communities by 15N-labelling techniques. FEMS Microbiol Ecol 63:401–411. CrossRefGoogle Scholar
  21. Krüger M, Blumenberg M, Kasten S, Wieland A, Känel L, Klock J-H, Michaelis W, Seifert R (2008a) A novel, multi-layered methanotrophic microbial mat system growing on the sediment of the Black Sea. Environ Microbiol 10:1934–1947. CrossRefGoogle Scholar
  22. Mann KH, Lazier JR (2013) Dynamics of marine ecosystems: biological-physical interactions in the oceans. Blackwell, LondonGoogle Scholar
  23. Marlow JJ, Steele JA, Ziebis W, Thurber AR, Levin LA, Orphan VJ (2014) Carbonate-hosted methanotrophy represents an unrecognized methane sink in the deep sea. Nat Commun 5:1–16. CrossRefGoogle Scholar
  24. Martens CS, Berner RA (1974) Methane production in the interstitial waters of sulfate-depleted marine sediments. Science 185:1167–1169. CrossRefGoogle Scholar
  25. Mason OU, Case DH, Naehr TH, Lee RW, Thomas RB, Bailey JV, Orphan VJ (2015) Comparison of archaeal and bacterial diversity in methane seep carbonate nodules and host sediments, Eel River basin and hydrate ridge, USA. Microb Ecol 70:766–784. CrossRefGoogle Scholar
  26. Meulepas RJW, Jagersma CG, Gieteling J, Buisman CJN, Stams AJM, Lens PNL (2009) Enrichment of anaerobic methanotrophs in sulfate-reducing membrane bioreactors. Biotechnol Bioeng 104:458–470. CrossRefGoogle Scholar
  27. Milucka J, Ferdelman TG, Polerecky L, Franzke D, Wegener G, Schmid M, Lieberwirth I, Wagner M, Widdel F, Kuypers MMM (2012) Zero-valent Sulphur is a key intermediate in marine methane oxidation. Nature 491:541–546. CrossRefGoogle Scholar
  28. Nauhaus K, Boetius A, Kruger M, Widdel F (2002) In vitro demonstration of anaerobic oxidation of methane coupled to sulphate reduction in sediment from a marine gas hydrate area. Environ Microbiol 4:296–305. CrossRefGoogle Scholar
  29. Niemann H, Losekann T, de Beer D, Elvert M, Nadalig T, Knittel K, Amann R, Sauter EJ, Schluter M, Klages M, Foucher JP, Boetius A (2006) Novel microbial communities of the Haakon Mosby mud volcano and their role as a methane sink. Nature 443:854–858. CrossRefGoogle Scholar
  30. Orphan VJ, Hinrichs KU, Ussler W, Paull CK, Taylor LT, Sylva SP, Hayes JM, Delong EF (2001a) Comparative analysis of methane-oxidizing archaea and sulfate-reducing bacteria in anoxic marine sediments. Appl Environ Microbiol 67:1922–1934. CrossRefGoogle Scholar
  31. Orphan VJ, House CH, Hinrichs KU, McKeegan KD, DeLong EF (2001b) Methane-consuming archaea revealed by directly coupled isotopic and phylogenetic analysis. Science 293:484–487. CrossRefGoogle Scholar
  32. Orphan VJ, House CH, Hinrichs KU, McKeegan KD, DeLong EF (2002) Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments. Proc Natl Acad Sci U S A 99:7663–7668. CrossRefGoogle Scholar
  33. Pikuta EV, Itoh T, Krader P, Tang J, Whitman WB, Hoover RB (2006) Anaerovirgula multivorans gen. Nov., sp. nov., a novel spore-forming, alkaliphilic anaerobe isolated from Owens Lake, California, USA. ISME J 56:2623–2629. CrossRefGoogle Scholar
  34. Pruesse E, Quast C, Knittel K, Fuchs BM, Ludwig W, Peplies J, Glöckner FO (2007) SILVA: a comprehensive online resource for quality checked and aligned ribosomal RNA sequence data compatible with ARB. Nucleic Acids Res 35:7188–7196. CrossRefGoogle Scholar
  35. Reeburgh WS (1976) Methane consumption in Cariaco trench waters and sediments. Earth Planet Sci Lett 28:337–344. CrossRefGoogle Scholar
  36. Roof E, Pikuta E, Otto C, Williams G, Hoover R (2013) Some unique features of alkaliphilic anaerobes. Proc. SPIE 8865, instruments, Methods, and Missions for Astrobiology XVI, 88650F (26 September 2013).
  37. Rooze J, Egger M, Tsandev I, Slomp CP (2016) Iron-dependent anaerobic oxidation of methane in coastal surface sediments: potential controls and impact. Limnol Oceanogr 61:S267–S282. CrossRefGoogle Scholar
  38. Ruff SE, Biddle JF, Teske AP, Knittel K, Boetius A, Ramette A (2015) Global dispersion and local diversification of the methane seep microbiome. Proc Natl Acad Sci U S A 112:4015–4020. CrossRefGoogle Scholar
  39. Schloss PD, Westcott SL (2011) Assessing and improving methods used in operational taxonomic unit-based approaches for 16S rRNA gene sequence analysis. Appl Environ Microbiol 77:3219–3226. CrossRefGoogle Scholar
  40. Song Z-Q, Wang F-P, Zhi X-Y, Chen J-Q, Zhou E-M, Liang F, Xiao X, Tang S-K, Jiang H-C, Zhang CL, Dong H, Li W-J (2013) Bacterial and archaeal diversities in Yunnan and Tibetan hot springs, China. Environ Microbiol 15:1160–1175. CrossRefGoogle Scholar
  41. Sousa JA, Sorokin DY, Bijmans MF, Plugge CM, Stams AJ (2015) Ecology and application of haloalkaliphilic anaerobic microbial communities. Appl Microbiol Biotechnol 99:9331–9336. CrossRefGoogle Scholar
  42. Timmers PH, Gieteling J, Widjaja-Greefkes HA, Plugge CM, Stams AJ, Lens PN, Meulepas RJ (2015) Growth of anaerobic methane-oxidizing archaea and sulfate-reducing bacteria in a high-pressure membrane capsule bioreactor. Appl Environ Microbiol 81:1286–1296. CrossRefGoogle Scholar
  43. Treude T, Krüger M, Boetius A, Jørgensen BB (2005) Environmental control on anaerobic oxidation of methane in the gassy sediments of Eckernförde Bay (German Baltic). Limnol Oceanogr 50:1771–1786. CrossRefGoogle Scholar
  44. Treude T, Krause S, Maltby J, Dale AW, Coffin R, Hamdan LJ (2014) Sulfate reduction and methane oxidation activity below the sulfate-methane transition zone in Alaskan Beaufort Sea continental margin sediments: implications for deep sulfur cycling. Geochim Cosmochim Ac 144:217–237. CrossRefGoogle Scholar
  45. Vigneron A, Cruaud P, Pignet P, Caprais J-C, Cambon-Bonavita M-A, Godfroy A, Toffin L (2013) Archaeal and anaerobic methane oxidizer communities in the Sonora margin cold seeps, Guaymas Basin (gulf of California). ISME J 7:1559–1608. CrossRefGoogle Scholar
  46. Wang F-P, Zhang Y, Chen Y, He Y, Qi J, Hinrichs K-U, Zhang X-X, Xiao X, Boon N (2014) Methanotrophic archaea possessing diverging methane-oxidizing and electron-transporting pathways. ISME J 8:1069–1078. CrossRefGoogle Scholar
  47. Wankel SD, Adams MM, Johnston DT, Hansel CM, Joye SB, Girguis PR (2012) Anaerobic methane oxidation in metalliferous hydrothermal sediments: influence on carbon flux and decoupling from sulfate reduction. Environ Microbiol 14:2726–2740. CrossRefGoogle Scholar
  48. Wegener G, Krukenberg V, Ruff SE, Kellermann MY, Knittel K (2016) Metabolic capabilities of microorganisms involved in and associated with the anaerobic oxidation of methane. Front Microbiol 7:46. CrossRefGoogle Scholar
  49. Widdel F, Bak F (1992) Gram negative mesophilic sulfate reducing bacteria, the prokaryotes. Springer, New York, pp 3352–3378Google Scholar
  50. Xiao X, Zhang Y (2014) Life in extreme environments: approaches to study life-environment co-evolutionary strategies. Sci China Earth Sci 57:869–877. CrossRefGoogle Scholar
  51. Zhang Y, Henriet J-P, Bursens J, Boon N (2010) Stimulation of in vitro anaerobic oxidation of methane rate in a continuous high-pressure bioreactor. Bioresour Technol 101:3132–3138. CrossRefGoogle Scholar
  52. Zhang Y, Maignien L, Zhao X, Wang F, Boon N (2011) Enrichment of a microbial community performing anaerobic oxidation of methane in a continuous high-pressure bioreactor. BMC Microbiol 11:1–8. CrossRefGoogle Scholar
  53. Zhang Y, Li X, Bartlett DH, Xiao X (2015) Current developments in marine microbiology: high-pressure biotechnology and the genetic engineering of piezophiles. Curr Opin Biotechnol 33:157–164. CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.UNESCO-IHEDelftthe Netherlands
  2. 2.State Key Laboratory of Ocean Engineering, Institute of OceanographyShanghai Jiao Tong UniversityShanghaiPeople’s Republic of China
  3. 3.National University of Ireland GalwayGalwayIreland

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