1. Methanogenes in permafrost Prepared by Anatoli Brouchkov 2005

2. Area of permafrost

3. Ancient ice, about 22000 years old; Arctic coast

4. Aldan river exposure, about 40 m above water level (left) and Sirdah lake exposure, about 10 m above water level (right). Ice wedges do not contain as much methane as frozen grounds, but the average content is high. Icewedges are different: Sirdah site dosn’t contain methane, but carbon dioxide; Neleger site contain much methane. Ice wedges, about 40000 years old

5. Microorganisms in Arctic permafrost Microorganisms from marine ice saturated deposits of Ugorsky peninsula, Cara sea coast. Age of permafrost of about 20000 years. 3 μm

6. Diversity of permafrost microorganisms

7. Methane content in permafrost: first section Methane content, ppmv ForestSlopeAlas Distance, m

8. Methane content and depth

9. Methane content in permafrost on Alaska (Fairbanks) 778.8 485.9 4890.5 1244.9 45023.7 122244.0

10. Methane content in permafrost on Alaska (Barrow) 1381.7772.4 2113.0 362.9 84.2213.1 soilice

11. High greenhouse gas load observed in debris-rich basal ice from the GRIP ice cores from the Greenland ice sheet: 130,000 ppmv of CO2 and 6,000 ppmv of CH4 (Souchez et al., 1995)

12. Methane content in permafrost It is suggested that permafrost contains 40 ml/kg CH 4 in average (Pearce, 1989). The estimation of gashydrate content in permafrost has not been done yet. Methane in ice of Arctic coast of Siberia (Moriizumi, Iida and Fukuda, 1994) Vertical profiles of total bacteria and methanogens in West Siberia wetland (Utsumi et al., 2000)

13. Utsumi et al., 2000

14. All organisms in the sample are marked in blue by using DAPI (4',6- Diamidino-2- phenylindole), a fluorescent dye which bounds to all DNA. Methane producing microorganisms of the order Methanomicrobia les are marked in red by hybridisation with a specific oligonucleotide probe (MG1200) By overlaying the pictures the fraction of Methanomicrobia les cells per DAPI-detected cells can be visualised. Methanogen’s detection by DAPI (Eastern Siberia soils, Alfred Wegener Institute, Potsdam, Germany)

15. Concentrations of headspace gases generated during 12 months of incubation at 4°C by 1997 thawed ice samples amended with 0.25× R2A medium Sample CO 2 CH 4 Basal ice (debris rich) 58,000 ± 2,70016,000 ± 100 Glacier ice (debris poor) 5,500 ± 1501.7 ± 0.1 Uninoculated medium 8,600 ± 3001.4 ± 0.2 a Values are means ± standard deviations based on the results of duplicate analyses. Arctic glacier, eastern Ellesmere Island, Canada

16. Interannual variations in Alaskan tundra methane emission (Whalen and Reeburg, 1992) Methane emission in frozen soils

17. Methane producing in permafrost? Methanogenic bacteria and metane both present in permafrost (Rivkina et al., 1993; Jansen, Bock, 1994). The most ancient bacteria (up to 3 mln years) found in permafrost is a methane producing archeobacteria. Methane could be more easy lost in common deposits, but not in ice-saturated permafrost. Origin of known fields of gashydrates in permafrost (Pradho Bay and others) is not explained yet. It has shown that upper layer of permafrost (about 10 m) of eastern Siberia is enriched by methane (up to 1%) in the air bubbles and the methane is younger then 5000 years (Fukuda, 1994). Methane content in active layer is much less at the beginning of winter than at the end. Methane collection in active layer in the winter period is about 100 ml/kg, and could be from 4 to 9 times more then in summer (Glotov, 1991). Relationship between soil temperature at 5 cm depth and the cumulative methane flux, g/sq.m a day in Alaska (Ota et al., 2000)

18. Experimental methane emission below freezing point (Mountfort et al., 2003) Plots of decrease in the log specific radioactivity of acetate after addition of [2-14C]acetate to sediments which were incubated at 4°C (a) and below freezing (b). Plots of log ratio of methane production rate: sulfate reduction rate versus temperature for low-sulfate pond systems.

19. Days of experimentSamples/Methane content, ppmv Tomakomai Alaska Yakutsk 11.430.90 1.23 21.18 1.54 - 232.06 1.98 - 702.05 2.12 1.98 2092.07 2.02 2.29*) 2302.01 2.02 - *)148 days Methane emission in frozen soils: experiments

20. Methane producing in frozen soils: experiments

21. Methane producing at low temperatures (-5 deg. C): effect of thawing

22. Technique of sampling is important

23. Technique of permafrost sampling Sample washing in 70% ethanol

24. Phylogenic analysis of archaeal clones retrieved from a Siberian permafrost sample, showing unknown methanogenic communities may be present in the environment.

25. Anaerobic incubation

26. Incubation of local (Tomakomai), Yakutsk and Siberian soil Marked in red cells - increase of methane Calculated values of CH4 in head space, ppmv SoilMediumTemperatureSampleComment11.09.0404.10.04 YakutskH220120817880 YakutskH202210502 YakutskButyrate20532951493 YakutskButyrate063511757 YakutskButyrate-57turn red once192399 YakutskButyrate-58170215 YakutskAcetate2092335585 Local - TomakomaiH22013587113274 Local - TomakomaiH20149041523 Local - TomakomaiH2-51593415261 Local - TomakomaiH2-516was frozen702753 Siberian soilAcetate201738518 Siberian soilAcetate-52032136 Siberian soilAcetate-521342681 Siberian soilGlucose202233106 Siberian soilGlucose0231534971

27. Tomakomai soil, methane emission experiment at -5°C CloneLengthHomologyOrganismEnvironment LocH-55285100Methanobacterium bryantii LocH-5247899 Oryza sativarice roots LocH-52044099 LocH-51251598Methanobacterium bryantii LocH-51535198Uncultured archaeon clone LocH-52246898 Methanobacterium bryantii LocH-53141298 LocH-5446398 LocH-52134397 LocH-52539497 LocH-52947797

28. Yakutsk soil, from active layer, methane emission experiment at 20°C CloneLengthHomologyOrganismEnvironment YakH201539098 Uncultured Methanomicrobiaceae archaeonsoil YakH20255798 YakH20347098Methanobacterium palustretundra soil YakH20643198 Uncultured Methanomicrobiaceae archaeonsoil YakH20841098 YakH203140297Methanobacterium palustretundra soil YakH201123396Uncultured Methanomicrobiaceae archaeonsoil YakH201643296 Methanobacterium palustretundra soil YakH202643296 YakH20542896 YakH202541795

29. Yakutsk soil, from active layer, methane emission experiment at -5°C CloneLengthHomologyOrganismEnvironment YakH-51140999Uncultured methanogencontaminated aquifer YakH-52640299Uncultured archaeonriparian flooding YakH-52840999 YakH-53049999 YakH-54140699 YakH-54640599Methanobacteriaceae archaeonacidic peat YakH-5643499Uncultured archaeonriparian flooding YakH-53147298 YakH-53336898Methanobacteriaceae archaeonacidic peat YakH-5439098Uncultured methanogencontaminated aquifer YakH-54239498Methanobacteriaceae archaeonacidic peat YakH-54754797Ruminococcus palustrisswamp YakH-53839196Methanobacteriaceae archaeonacidic peat

30. Siberian permafrost soil, methane emission experiment at 20°C CloneLengthHomologyOrganismEnvironment SibH20143299 Uncultured crenarchaeotecold soil SibH201250499 SibH201650399 SibH202048299 SibH202250899 SibH202735899 SibH202950499 SibH203253099Uncultured bacterium clonecontaminated soil SibH20851199Uncultured crenarchaeotecold soil SibH201340693Uncultured Chloroflexi bacterium clonepasture soil

31. Siberian permafrost soil, methane emission experiment at -5°C CloneLengthHomologyOrganismEnvironment SibH-51647599 Archaeon soil SibH-51849099 SibH-5138998 SibH-52449898 SibH-52753598 SibH-52941891 Uncultured bacterium SibH-52851288 SibH-5733686 Uncultured archaeon rice soil SibH-51136385rice soil SibH-5536385rice soil SibH-5651885 Uncultured bacteriumsurface coal mining SibH-5850885 SibH-51428484 Uncultured archaeonrice soil SibH-52126984 SibH-52540584 SibH-51946682 SibH-5350082 SibH-52653981Uncultured archaeondeep sea SibH-52346880 Uncultured archaeoncold sulfidic springs SibH-5446780 SibH-52051079 SibH-52250679

32. Methane producing and content in permafrost: consequences Latitude, degree N Area of northern wetland, 10 in 9 degree m2 Measure- ments, methane flux, mg/m2 a day Measured methane emission, Tg a year Methan e produ- cing in active layer, Tg/year Addition as a result of 0,5 m increase of active layer, Tg a year Addition as a result of thawing permafrost (Tg), taking average 1000 ppmv methane content and 5% air content Methane produced and stored in permafrost for 10000 years 75-72.530.2450.1 0.80.2320.7 72.5-7080.21000.70.82.01.21703.2 70-67.591.91701.41.62.32.02927.4 Total202.32.22.55.13.44951.3

33. Conclusion Methane concentration is high in permafrost soils and ice in Siberia. Experiments show a production of methane in soils at - 5 °C. A large amount of produced methane was trapped in frozen soil and emitted due to thawing only. Methanogenes, which were possibly responsible for the production, were identified in the samples. Finding of a number of methanogenes in permafrost present another evidence of the opportunity. Therefore permafrost might be a source of methane, which could be emitted in case of thawing.