Protocols for Measuring Methanogenesis

  • Oleg KotsyurbenkoEmail author
  • Mikhail Glagolev
Part of the Springer Protocols Handbooks book series (SPH)


Methanogenesis is one of the most important terminal processes in the microbial degradation of organic matter in many anoxic environments. Since ancient times, methane was known as a combustion gas, but its microbiological origin was proved only in the nineteenth century. The contribution of methane to the global warming and its beneficial importance in ecological biotechnology and bioenergetics dictate the need in proper estimations of its fluxes and measurements of its production rates in different microbiological processes.

Measuring methanogenesis is mostly conducted in laboratory experiments with different types of methanogenic samples, in fields or in ruminants. The samples used for such measurements are either liquid methanogenic cultures and slurries prepared by homogenization and dilution or intact soil cores. All types of methanogenic samples are incubated, and accumulated CH4 is analyzed in order to calculate methanogenesis rate. The samples as slurries incubated under laboratory conditions are referred to as potential production rates, whereas rates measured in intact samples or in fields are referred to as actual (in situ) production rates.

To initiate methanogenesis, characteristic substrates of methanogens are used as additions to the samples. Radiotracers are also used to measure rates of certain methanogenesis pathways in samples.

Classification of methods of measuring methanogenesis is based on the mass balance equation relating the rate of change in concentration of methane with its source and flux. The two major methods are described in detail.


Chamber method Methanogenesis Methanogens Potential methane production Radiotracers 


  1. 1.
    Wolfe RS (1993) A historical overview of methanogenesis. In: Ferry JG (ed) Methanogenesis, Chapman & Hall microbiology series. Chapman & Hall, New YorkGoogle Scholar
  2. 2.
    Barker HA (ed) (1956) Bacterial fermentation. Wiley, New YorkGoogle Scholar
  3. 3.
    Ehhalt DH, Schmidt U (1978) Sources and sinks of atmospheric methane. Pageoph 116:452–464CrossRefGoogle Scholar
  4. 4.
    Großkopf R, Janssen PH, Liesack W (1998) Diversity and structure of the methanogenic community in anoxic rice paddy soil microcosms as examined by cultivation and direct 16S rRNA gene sequence retrieval. Appl Environ Microbiol 64:960–969PubMedPubMedCentralGoogle Scholar
  5. 5.
    Garcia JL (1990) Taxonomy and ecology of methanogens. FEMS Microbiol Rev 87:297–308. doi: 10.1111/j.1574-6968.1990.tb04928.x CrossRefGoogle Scholar
  6. 6.
    Chan OC, Claus P, Casper P et al (2005) Vertical distribution of methanogenic archaeal community in Lake Dagow sediment. Environ Microbiol 7:1139–1149. doi: 10.1111/j.1462-2920.2005.00790.x CrossRefPubMedGoogle Scholar
  7. 7.
    Jeanthon C, L’Haridon S, Reysenbach AL et al (1999) Methanococcus vulcanius sp. nov., a novel hyperthermophilic methanogen isolated from East Pacific Rise, and identification of Methanococcus sp. DSM 4213T as Methanococcus fervens sp. nov. Int J Syst Evol Microbiol 49:583–589Google Scholar
  8. 8.
    Ganzert L, Jurgens G, Münster U et al (2007) Methanogenic communities in permafrost-affected soils of the Laptev Sea coast, Siberian Arctic, characterized by 16S rRNA gene fingerprints. FEMS Microbiol Ecol 59:476–488CrossRefPubMedGoogle Scholar
  9. 9.
    Kobabe S, Wagner D, Pfeiffer EM (2004) Characterisation of microbial community composition of a Siberian tundra soil by fluorescence in situ hybridization. FEMS Microbiol Ecol 50:13–23. doi: 10.1016/j.femsec.2004.05.003 CrossRefPubMedGoogle Scholar
  10. 10.
    Lin C, Raskin L, Stahl DA (1997) Microbial community structure in gastrointestinal tracts of domestic animals: comparative analyzes using rRNA-targeted oligonucleotide probes. FEMS Microbiol Ecol 22:281–294. doi: 10.1111/j.1574-6941.1997.tb00380.x CrossRefGoogle Scholar
  11. 11.
    Brune A (2010) Methanogenesis in the digestive tract of termites. In: Hackstein JHP (ed) (Endo)symbiotic methanogenic archaea, vol 19, Microbiology monographs. Springer, Berlin. doi: 10.1007/978-3-642-13615-3_1 CrossRefGoogle Scholar
  12. 12.
    Hackstein JHP (2010) Anaerobic ciliates and their methanogenic endosymbionts. In: Hackstein JHP (ed) (Endo)symbiotic methanogenic archaea, vol 19, Microbiology monographs. Springer, Berlin. doi: 10.1007/978-3-642-13615-3_1 CrossRefGoogle Scholar
  13. 13.
    Bapteste E, Brochier C, Boucher Y (2005) Higher-level classification of the Archaea: evolution of methanogenesis and methanogens. Archaea 1:353–363. doi: 10.1155/2005/859728 CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Lefèvre F, Forget F (2009) Observed variations of methane on Mars unexplained by known atmospheric chemistry and physics. Nature 460(7256):720–723. doi: 10.1038/nature08228 CrossRefPubMedGoogle Scholar
  15. 15.
    Börjesson P, Mattiasson B (2008) Biogas as a resource-efficient vehicle fuel. Trends Biotechnol 26:7–13CrossRefPubMedGoogle Scholar
  16. 16.
    Beliaev SS, Ivanov MV (1975) The rate of methane formation by bacteria determined by isotopic labeling technique. Microbiology 44:166–168 (in Russian)Google Scholar
  17. 17.
    Lein AY, Ivanov MV (eds) (2009) Biogeochemical cycle of methane in the ocean. Nauka, Moscow (in Russian)Google Scholar
  18. 18.
    Min’ko OI, Kasparov SV, Amosova YM (1987) Gaseous compounds metabolic products of microbial coenoses of waterlogged soils. Biol Bull Rev 48:182–193Google Scholar
  19. 19.
    Orlov DS, Minko OI, Ammosova Ya M et al (1987) Research methods for soil gas function. In: Voroin AD, Orlov DS (eds) Modern physical and chemical methods of soil studies. MGU, Moscow (in Russian)Google Scholar
  20. 20.
    Alperin MJ, Reeburg WS, Whiticar MJ (1988) Carbon and hydrogen isotope fractionation resulting from anaerobic methane oxidation. Glob Biogeochem Cycles 2:279–288CrossRefGoogle Scholar
  21. 21.
    Glagolev MV (1998) Modeling of production, oxidation and transportation processes of methane. In: Global Environment Research Fund: Eco-Frontier Fellowship (EFF) in 1997, Environment Agency. Global Environment Department. Research & Information Office, TokyoGoogle Scholar
  22. 22.
    Panikov NS, Dedysh SN, Kolesnikov OM et al (2001) Metabolic and environmental control on methane emission from soils: mechanistic studies of mesotrophic fen in West Siberia. Water Air Soil Pollut Focus 1:415–428CrossRefGoogle Scholar
  23. 23.
    Martin JL, McCutcheon SC (eds) (1999) Hydrodynamics and transport for water quality modeling. Lewis, Boca RatonGoogle Scholar
  24. 24.
    Arsenin VY (1984) Methods of mathematical physics and higher functions. Nauka, Moscow (in Russian)Google Scholar
  25. 25.
    Bridgham SD, Richardson CJ (1992) Mechanisms controlling soil respiration (CO2 and CH4) in southern peatlands. Soil Biol Biochem 24:1089–1099CrossRefGoogle Scholar
  26. 26.
    Bodelier PLE, Hahn AP, Arth IR et al (2000) Effects of ammonium-based fertilisation on microbial processes involved in methane emission from soils planted with rice. Biogeochemistry 51:225–257CrossRefGoogle Scholar
  27. 27.
    Blodau C, Basiliko N, Moore TR (2004) Carbon turnover in peatland mesocosms exposed to different water table levels. Biogeochemistry 67:331–351CrossRefGoogle Scholar
  28. 28.
    Baldwin DS, Rees GN, Mitchell AM et al (2006) The short-term effects of salinization on anaerobic nutrient cycling and microbial community structure in sediment from a freshwater wetland. Wetlands 26:455–464CrossRefGoogle Scholar
  29. 29.
    Conrad R, Klose M (1999) Anaerobic conversion of carbon dioxide to methane, acetate and propionate on washed rice roots. FEMS Microbiol Ecol 30:147–155CrossRefPubMedGoogle Scholar
  30. 30.
    Schulz S, Conrad R (1996) Influence of temperature on pathways to methane production in the permanently cold profundal sediment of Lake Constance. FEMS Microbiol Ecol 20:1–14CrossRefGoogle Scholar
  31. 31.
    Thebrath B, Mayer H-P, Conrad R (1992) Bicarbonate-dependent production and methanogenic consumption of acetate in anoxic paddy soil. FEMS Microbiol Ecol 86:295–302CrossRefGoogle Scholar
  32. 32.
    Conrad R, Klose M (2000) Selective inhibition of reactions involved in methanogenesis and fatty acid production on rice roots. FEMS Microbiol Ecol 34:27–34CrossRefPubMedGoogle Scholar
  33. 33.
    Bartlett KB, Harriss RC, Sebacher DI (1985) Methane flux from coastal salt marshes. J Geophys Res 90:5710–5720CrossRefGoogle Scholar
  34. 34.
    Galy-Lacaux C, Delmas R, Jambert C et al (1997) Gaseous emissions and oxygen consumption in hydroelectric dams: a case study in French Guyana. Glob Biogeochem Cycles 11:471–483CrossRefGoogle Scholar
  35. 35.
    Nakano T, Sawamoto T, Morishita T et al (2004) A comparison of regression methods for estimating soil-atmosphere diffusion gas fluxes by a closed-chamber technique. Soil Biol Biochem 36:107–113CrossRefGoogle Scholar
  36. 36.
    Sabrekov AF, Runkle BRK, Glagolev MV et al (2014) Seasonal variability as a source of uncertainty in the West Siberian regional CH4 flux upscaling. Environ Res Lett 9(4): 045008. doi:10.1088/1748-9326/9/4/045008
  37. 37.
    Pape L, Ammann C, Nyfeler-Brunner A et al (2009) An automated dynamic chamber system for surface exchange measurement of non-reactive and reactive trace gases of grassland ecosystems. Biogeosciences 6:405–429CrossRefGoogle Scholar
  38. 38.
    Stefanik KC, Mitsch WJ (2013) Metabolism and methane flux of dominant macrophyte communities in created riverine wetlands using open system flow through chambers. Ecol Eng. doi: 10.1016/j.ecoleng.2013.10.036 Google Scholar
  39. 39.
    Bedard C, Knowles R (1989) Physiology, biochemistry, and specific inhibitors of CH4, NH4 +, and CO oxidation by methanotrophs and nitrifiers. Microbiol Rev 53:68–84PubMedPubMedCentralGoogle Scholar
  40. 40.
    Novikov VV, Stepanov AL, Pozdnyakov AI et al (2004) Seasonal dynamics of CO2, CH4, N2O, and NO emissions from peat soils of the Yakhroma river floodplain. Eurasian Soil Sci 37:755–761Google Scholar
  41. 41.
    Dise NB (1992) Winter fluxes of methane from Minnesota peatlands. Biogeochemistry 17:71–83CrossRefGoogle Scholar
  42. 42.
    Baldocchi DD, Hicks BB, Meyers TP (1988) Measuring biosphere-atmosphere exchanges of biologically related gases with micrometeorological methods. Ecology 69:1331–1340CrossRefGoogle Scholar
  43. 43.
    Brouček J (2014) Methods of methane measurement in ruminants. Slovak J Anim Sci 47:51–60Google Scholar
  44. 44.
    Harper LA, Denmead OT, Flesch TK (2011) Micrometeorological techniques for measurement of enteric greenhouse gas emissions. Anim Feed Sci Technol 166–167:227–239CrossRefGoogle Scholar
  45. 45.
    Garnsworthy PC, Craigon J, Hernandez-Medrano JH et al (2012) On-farm methane measurements during milking correlate with total methane production by individual dairy cows. J Dairy Sci 95:3166–3180CrossRefPubMedGoogle Scholar
  46. 46.
    Derno M, Elsner HG, Paetow EA et al (2009) Technical note: a new facility for continuous respiration measurements in lactating cows. J Dairy Sci 92:2804–2808CrossRefPubMedGoogle Scholar
  47. 47.
    Lassey K, Walker C, McMillan A et al (2001) On the performance of SF6 permeation tubes used in determining methane emission from grazing livestock. Chemosphere Global Change Sci 3:367–376CrossRefGoogle Scholar
  48. 48.
    Martin C, Rouel J, Jouany JP et al (2008) Methane output and diet digestibility in response to feeding dairy cows crude linseed, extruded linseed, or linseed oil. J Anim Sci 86:2642–2650CrossRefPubMedGoogle Scholar
  49. 49.
    Storm IMLD, Hellwing ALF, Nielsen NI et al (2012) Methods for measuring and estimating methane emission from ruminants. Animals 2:160–183CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Hegarty RS (2013) Applicability of short-term emission measurements for on-farm quantification of enteric methane. Animal 7:401–408CrossRefPubMedGoogle Scholar
  51. 51.
    Lü F, Ji J, Shao L et al (2013) Bacterial bioaugmentation for improving methane and hydrogen production from microalgae. Biotechnol Biofuels 6:92CrossRefPubMedPubMedCentralGoogle Scholar
  52. 52.
    Esposito G, Frunzo L, Liotta F et al (2012) Bio-methane potential tests to measure the biogas production from the digestion and co-digestion of complex organic substrates. TOENVIEJ 5:1–8CrossRefGoogle Scholar
  53. 53.
    Wolin EA, Wolin MG, Wolfe RS (1963) Formation of methane by bacterial extracts. J Biol Chem 238:2882–2886PubMedGoogle Scholar
  54. 54.
    Nakano T, Kuniyoshi S, Fukuda M (2000) Temporal variation in methane emission from tundra wetlands in a permafrost area, northeastern Siberia. Atmos Environ 34:1205–1213CrossRefGoogle Scholar
  55. 55.
    Silvola J, Saarnio S, Foot J et al (2003) Effects of elevated CO2 and N deposition on CH4 emissions from European mires. Glob Biogeochem Cycles 17(2):1068. doi: 10.1029/2002GB001886 CrossRefGoogle Scholar
  56. 56.
    Augustin J, Merbach W, Rogasik J (1998) Factors influencing nitrous oxide and methane emissions from minerotrophic fens in northeast Germany. Biol Fertil Soils 28:1–4CrossRefGoogle Scholar
  57. 57.
    Poth M, Anderson IC, Miranda HS et al (1995) The magnitude and persistence of soil NO, N2O, CH4, and CO2 fluxes from burned tropical savanna in Brazil. Glob Biogeochem Cycles 9:503–513CrossRefGoogle Scholar
  58. 58.
    Crill PM (1991) Seasonal patterns of methane uptake and carbon dioxide release by a temperate woodland soil. Glob Biogeochem Cycles 5:319–334CrossRefGoogle Scholar
  59. 59.
    Frenzel P, Karofeld E (2000) CH4 emission from a hollow-ridge complex in a raised bog: the role of CH4 production and oxidation. Biogeochemistry 51:91–112CrossRefGoogle Scholar
  60. 60.
    Glagolev MV, Sabrekov AF, Kleptsova IE et al (2012) Methane emission from bogs in the subtaiga of Western Siberia: the development of standard model. Eurasian Soil Sci 45:947–957. doi: 10.1134/S106422931210002X CrossRefGoogle Scholar
  61. 61.
    Granberg G, Mikkela C, Sundh I et al (1997) Sources of spatial variation in methane emission from mires in northern Sweden: a mechanistic approach in statistical modeling. Glob Biogeochem Cycles 11:135–150CrossRefGoogle Scholar
  62. 62.
    Panikov NS, Dedysh SN (2000) Cold season CH4 and CO2 emission from boreal peat bogs (West Siberia): winter fluxes and thaw activation dynamics. Glob Biogeochem Cycles 14:1071–1080CrossRefGoogle Scholar
  63. 63.
    Chanton JP, Whiting GJ, Showers WJ et al (1992) Methane flux from Peltandra virginica: stable isotope tracing and chamber effects. Glob Biogeochem Cycles 6:15–31CrossRefGoogle Scholar
  64. 64.
    Press WH, Teukolsky SA, Vetterling WT et al (1995) Numerical recipes in FORTRAN. The art of scientific computing. Cambridge University Press, CambridgeGoogle Scholar
  65. 65.
    Kays WM (1966) Convective heat and mass transfer. McGraw-Hill, New YorkGoogle Scholar
  66. 66.
    Ertekin T, Abou-Kassem JH, King GR (2001) Basic applied reservoir simulation. Society of Petroleum Engineers, RichardsonGoogle Scholar
  67. 67.
    Odum EP (1983) Basic ecology. Saunders College, PhiladelphiaGoogle Scholar
  68. 68.
    Christensen TR, Michelsen A, Jonasson S et al (1997) Carbon dioxide and methane exchange of a subarctic heath in response to climate change related environmental manipulations. OIKOS 79:34–44CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2015

Authors and Affiliations

  1. 1.Lomonosov Moscow State UniversityMoscowRussia
  2. 2.Yugra State UniversityKhanty-MansiyskRussia
  3. 3.Institute of Microbiology, Russian Academy of SciencesMoscowRussia
  4. 4.Institute of Forest Science, Russian Academy of SciencesMoscowRussia
  5. 5.Tomsk State UniversityTomskRussia

Personalised recommendations