Microbial Ecology

, Volume 69, Issue 2, pp 319–332 | Cite as

Heterotrophic Communities Supplied by Ancient Organic Carbon Predominate in Deep Fennoscandian Bedrock Fluids

  • Lotta Purkamo
  • Malin Bomberg
  • Mari Nyyssönen
  • Ilmo Kukkonen
  • Lasse Ahonen
  • Merja Itävaara


The deep subsurface hosts diverse life, but the mechanisms that sustain this diversity remain elusive. Here, we studied microbial communities involved in carbon cycling in deep, dark biosphere and identified anaerobic microbial energy production mechanisms from groundwater of Fennoscandian crystalline bedrock sampled from a deep drill hole in Outokumpu, Finland, by using molecular biological analyses. Carbon cycling pathways, such as carbon assimilation, methane production and methane consumption, were studied with cbbM, rbcL, acsB, accC, mcrA and pmoA marker genes, respectively. Energy sources, i.e. the terminal electron accepting processes of sulphate-reducing and nitrate-reducing communities, were assessed with detection of marker genes dsrB and narG, respectively. While organic carbon is scarce in deep subsurface, the main carbon source for microbes has been hypothesized to be inorganic carbon dioxide. However, our results demonstrate that carbon assimilation is performed throughout the Outokumpu deep scientific drill hole water column by mainly heterotrophic microorganisms such as Clostridia. The source of carbon for the heterotrophic microbial metabolism is likely the Outokumpu bedrock, mainly composed of serpentinites and metasediments with black schist interlayers. In addition to organotrophic metabolism, nitrate and sulphate are other possible energy sources. Methanogenic and methanotrophic microorganisms are scarce, but our analyses suggest that the Outokumpu deep biosphere provides niches for these organisms; however, they are not very abundant.


Carbon cycling Nitrogen cycling Deep subsurface Functional microbial communities Heterotrophy 



This research was funded by the Academy of Finland, The Kone Foundation and Finnish Research Program on Nuclear Waste Management (KYT). Mirva Pyrhönen, Aura Nousiainen and Marjo Öster are acknowledged for their work with sampling at Outokumpu drill hole as well as in laboratory. Riikka Kietäväinen is thanked for commenting on the manuscript and Dr. David Thomas for critical language editing.


  1. 1.
    Ahonen L, Kietäväinen R, Kortelainen N, Kukkonen IT, Pullinen A, Toppi T, Bomberg M, Itävaara M, Nousiainen A, Nyyssönen M (2011) Hydrogeological characteristics of the Outokumpu deep drill hole. In: Kukkonen IT (ed) Outokumpu Deep Drilling Project 2003–2010, Special paper 51. Geological Survey of Finland, Espoo, pp 151–168Google Scholar
  2. 2.
    Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ (1990) Basic local alignment search tool. J Mol Biol 215:403PubMedCrossRefGoogle Scholar
  3. 3.
    Auclair J, Lépine F, Parent S, Villemur R (2010) Dissimilatory reduction of nitrate in seawater by a Methylophaga strain containing two highly divergent narG sequences. ISME J 4:1302PubMedCrossRefGoogle Scholar
  4. 4.
    Auguet J, Borrego CM, Bañeras L, Casamayor EO (2008) Fingerprinting the genetic diversity of the biotin carboxylase gene (accC) in aquatic ecosystems as a potential marker for studies of carbon dioxide assimilation in the dark. Environ Microbiol 10:2527PubMedCrossRefGoogle Scholar
  5. 5.
    Baker BJ, Moser DP, MacGregor BJ, Fishbain S, Wagner M, Fry NK, Jackson B, Speolstra N, Loos S, Takai K (2003) Related assemblages of sulphate-reducing bacteria associated with ultradeep gold mines of South Africa and deep basalt aquifers of Washington State. Environ Microbiol 5:267PubMedCrossRefGoogle Scholar
  6. 6.
    Berg IA, Kockelkorn D, Ramos-Vera WH, Say RF, Zarzycki J, Hügler M, Alber BE, Fuchs G (2010) Autotrophic carbon fixation in archaea. Nat Rev Microbiol 8:447PubMedCrossRefGoogle Scholar
  7. 7.
    Berg IA (2011) Ecological aspects of the distribution of different autotrophic CO2 fixation pathways. Appl Environ Microbiol 77:1925PubMedCentralPubMedCrossRefGoogle Scholar
  8. 8.
    Boden R, Cunliffe M, Scanlan J, Moussard H, Kits KD, Klotz MG, Jetten MS, Vuilleumier S, Han J, Peters L, Mikhailova N, Teshima H, Tapia R, Kyrpides N, Ivanova N, Pagani I, Cheng JF, Goodwin L, Han C, Hauser L, Land ML, Lapidus A, Lucas S, Pitluck S, Woyke T, Stein L, Murrell JC (2011) Complete genome sequence of the aerobic marine methanotroph Methylomonas methanica MC09. J Bacteriol 193:7001PubMedCentralPubMedCrossRefGoogle Scholar
  9. 9.
    Brazelton WJ, Morrill PL, Szponar N, Schrenk MO (2013) Bacterial communities associated with subsurface geochemical processes in continental serpentinite springs. Appl Environ Microbiol 79:3906PubMedCentralPubMedCrossRefGoogle Scholar
  10. 10.
    Casamayor EO, García-Cantizano J, Pedrós-Alió C (2008) Carbon dioxide fixation in the dark by photo-synthetic bacteria in sulfide-rich stratified lakes with oxic-anoxic interfaces. Limnol Oceanogr 53:1193CrossRefGoogle Scholar
  11. 11.
    Cheng YS, Halsey JL, Fode KA, Remsen CC, Collins ML (1999) Detection of methanotrophs in groundwater by PCR. Appl Environ Microbiol 65:648PubMedCentralPubMedGoogle Scholar
  12. 12.
    Daumas S, Cord-Ruwisch R, Garcia J (1988) Desulfotomaculum geothermicum sp. nov., a thermophilic, fatty acid-degrading, sulfate-reducing bacterium isolated with H2 from geothermal ground water. Antonie Van Leeuwenhoek 54:165PubMedCrossRefGoogle Scholar
  13. 13.
    Doerfert SN, Reichlen M, Iyer P, Wang M, Ferry JG (2009) Methanolobus zinderi sp. nov., a methylotrophic methanogen isolated from a deep subsurface coal seam. Int J Syst Evol Microbiol 59:1064PubMedCrossRefGoogle Scholar
  14. 14.
    Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792PubMedCentralPubMedCrossRefGoogle Scholar
  15. 15.
    Ettwig KF, Shima S, De Pas‐Schoonen V, Katinka T, Kahnt J, Medema MH, Op Den C, Huub JM, Jetten MS, Strous M (2008) Denitrifying bacteria anaerobically oxidize methane in the absence of Archaea. Environ Microbiol 10:3164PubMedCrossRefGoogle Scholar
  16. 16.
    Fardeau ML, Ollivier B, Patel BK, Dwivedi P, Ragot M, Garcia JL (1995) Isolation and characterization of a thermophilic sulfate-reducing bacterium, Desulfotomaculum thermosapovorans sp. nov. Int J Syst Bacteriol 45:218PubMedCrossRefGoogle Scholar
  17. 17.
    Fisher E, Dawson AM, Polshyna G, Lisak J, Crable B, Perera E, Ranganathan M, Thangavelu M, Basu P, Stolz JF (2008) Transformation of inorganic and organic arsenic by Alkaliphilus oremlandii sp. nov. strain OhILAs. Ann N Y Acad Sci 1125:230PubMedCrossRefGoogle Scholar
  18. 18.
    Gagen EJ, Denman SE, Padmanabha J, Zadbuke S, Al Jassim R, Morrison M, McSweeney CS (2010) Functional gene analysis suggests different acetogen populations in the bovine rumen and tammar wallaby forestomach. Appl Environ Microbiol 76:7785PubMedCentralPubMedCrossRefGoogle Scholar
  19. 19.
    Geets J, Borremans B, Diels L, Springael D, Vangronsveld J, van der Lelie D, Vanbroekhoven K (2006) DsrB gene-based DGGE for community and diversity surveys of sulfate-reducing bacteria. J Microbiol Methods 66:194PubMedCrossRefGoogle Scholar
  20. 20.
    Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52:696PubMedCrossRefGoogle Scholar
  21. 21.
    Hales BA, Edwards C, Ritchie DA, Hall G, Pickup RW, Saunders JR (1996) Isolation and identification of methanogen-specific DNA from blanket bog peat by PCR amplification and sequence analysis. Appl Environ Microbiol 62:668PubMedCentralPubMedGoogle Scholar
  22. 22.
    Hallbeck L, Pedersen K (2012) Culture-dependent comparison of microbial diversity in deep granitic groundwater from two sites considered for a Swedish final repository of spent nuclear fuel. FEMS Microbiol Ecol 81:66PubMedCrossRefGoogle Scholar
  23. 23.
    Hallbeck L, Pedersen K (2008) Characterization of microbial processes in deep aquifers of the Fennoscandian Shield. Appl Geochem 23:1796CrossRefGoogle Scholar
  24. 24.
    Hammer Ø, Harper D, Ryan P (2001) Past: paleontological statistics software package for education and data analysis. Paleontología Electrónica 4: 1–9. URL Http://
  25. 25.
    Hoehler TM, Jørgensen BB (2013) Microbial life under extreme energy limitation. Nat Rev Microbiol 11:83PubMedCrossRefGoogle Scholar
  26. 26.
    Itävaara M, Nyyssönen M, Kapanen A, Nousiainen A, Ahonen L, Kukkonen I (2011) Characterization of bacterial diversity to a depth of 1500 m in the Outokumpu deep borehole, Fennoscandian Shield. FEMS Microbiol Ecol 77:295PubMedCrossRefGoogle Scholar
  27. 27.
    Jetten MS, Strous M, Pas‐Schoonen KT, Schalk J, Dongen UG, Graaf AA, Logemann S, Muyzer G, Loosdrecht M, Kuenen JG (1998) The anaerobic oxidation of ammonium. FEMS Microbiol Rev 22:421PubMedCrossRefGoogle Scholar
  28. 28.
    Katoh K, Misawa K, Kuma K, Miyata T (2002) MAFFT: a novel method for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res 30:3059PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Kietäväinen R, Ahonen L, Kukkonen IT, Hendriksson N, Nyyssönen M, Itävaara M (2013) Characterisation and isotopic evolution of saline waters of the Outokumpu Deep Drill Hole, Finland—implications for water origin and deep terrestrial biosphere. Appl Geochem 32:37CrossRefGoogle Scholar
  30. 30.
    Kukkonen IT, Rath V, Kivekäs L, Šafanda J, Čermak V (2011) Geothermal studies of the Outokumpu Deep Drill Hole, Finland: vertical variation in heat flow and palaeoclimatic implications. Phys Earth Planet Inter 188:9CrossRefGoogle Scholar
  31. 31.
    Lalucat J, Bennasar A, Bosch R, Garcia-Valdes E, Palleroni NJ (2006) Biology of Pseudomonas stutzeri. Microbiol Mol Biol Rev 70:510PubMedCentralPubMedCrossRefGoogle Scholar
  32. 32.
    Lever MA (2013) Functional gene surveys from ocean drilling expeditions—a review and perspective. FEMS Microbiol Ecol 84:1PubMedCrossRefGoogle Scholar
  33. 33.
    Lever MA (2012) Acetogenesis in the energy-starved deep biosphere—a paradox? Front Microbiol 2:284PubMedCentralPubMedCrossRefGoogle Scholar
  34. 34.
    Lin L, Hall J, Onstott T, Gihring T, Lollar BS, Boice E, Pratt L, Lippmann-Pipke J, Bellamy RE (2006) Planktonic microbial communities associated with fracture-derived groundwater in a deep gold mine of South Africa. Geomicrobiol J 23:475CrossRefGoogle Scholar
  35. 35.
    Llirós M, Alonso‐Sáez L, Gich F, Plasencia A, Auguet O, Casamayor EO, Borrego CM (2011) Active bacteria and archaea cells fixing bicarbonate in the dark along the water column of a stratified eutrophic lagoon. FEMS Microbiol Ecol 77:370PubMedCrossRefGoogle Scholar
  36. 36.
    López-Gutiérrez JC, Henry S, Hallet S, Martin-Laurent F, Catroux G, Philippot L (2004) Quantification of a novel group of nitrate-reducing bacteria in the environment by real-time PCR. J Microbiol Methods 57:399PubMedCrossRefGoogle Scholar
  37. 37.
    Nakatsu CH, Hristova K, Hanada S, Meng XY, Hanson JR, Scow KM, Kamagata Y (2006) Methylibium petroleiphilum gen. nov., sp. nov., a novel methyl tert-butyl ether-degrading methylotroph of the Betaproteobacteria. Int J Syst Evol Microbiol 56:983PubMedCrossRefGoogle Scholar
  38. 38.
    Nanba K, King GM, Dunfield K (2004) Analysis of facultative lithotroph distribution and diversity on volcanic deposits by use of the large subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase. Appl Environ Microbiol 70:2245PubMedCentralPubMedCrossRefGoogle Scholar
  39. 39.
    Nealson KH, Inagaki F, Takai K (2005) Hydrogen-driven subsurface lithoautotrophic microbial ecosystems (SLiMEs): do they exist and why should we care? Trends Microbiol 13:405PubMedCrossRefGoogle Scholar
  40. 40.
    Nicolaisen MH, Ramsing NB (2002) Denaturing gradient gel electrophoresis (DGGE) approaches to study the diversity of ammonia-oxidizing bacteria. J Microbiol Methods 50:189PubMedCrossRefGoogle Scholar
  41. 41.
    Nurmi PA, Kukkonen IT (1986) A new technique for sampling water and gas from deep drill holes. Can J Earth Sci 23:1450CrossRefGoogle Scholar
  42. 42.
    Nyyssönen M, Hultman J, Ahonen L, Kukkonen I, Paulin L, Laine P, Itävaara M, Auvinen P (2014) Taxonomically and functionally diverse microbial communities in deep crystalline rocks of the Fennoscandian shield. ISME J 8:126–138PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Nyyssönen M, Bomberg M, Kapanen A, Nousiainen A, Pitkänen P, Itävaara M (2012) Methanogenic and sulphate-reducing microbial communities in deep groundwater of crystalline rock fractures in Olkiluoto, Finland. Geomicrobiol J 29:863CrossRefGoogle Scholar
  44. 44.
    Okland I, Huang S, Dahle H, Thorseth IH, Pedersen RB (2012) Low temperature alteration of serpentinized ultramafic rock and implications for microbial life. Chem Geol 318:75CrossRefGoogle Scholar
  45. 45.
    Orsi WD, Edgcomb VP, Christman GD, Biddle JF (2013) Gene expression in the deep biosphere. Nature 499:205PubMedCrossRefGoogle Scholar
  46. 46.
    Osaka T, Yoshie S, Tsuneda S, Hirata A, Iwami N, Inamori Y (2006) Identification of acetate- or methanol-assimilating bacteria under nitrate-reducing conditions by stable-isotope probing. Microb Ecol 52:253PubMedCrossRefGoogle Scholar
  47. 47.
    Pedersen K, Arlinger J, Eriksson S, Hallbeck A, Hallbeck L, Johansson J (2008) Numbers, biomass and cultivable diversity of microbial populations relate to depth and borehole-specific conditions in groundwater from depths of 4–450 m in Olkiluoto, Finland. ISME J 2:760PubMedCrossRefGoogle Scholar
  48. 48.
    Pedersen K (2000) Exploration of deep intraterrestrial microbial life: current perspectives. FEMS Microbiol Lett 185:9PubMedCrossRefGoogle Scholar
  49. 49.
    Pedersen K (1997) Microbial life in deep granitic rock. FEMS Microbiol Rev 20:399CrossRefGoogle Scholar
  50. 50.
    Pedersen K, Ekendahl S (1992) Assimilation of CO2 and introduced organic compounds by bacterial communities in groundwater from southeastern Sweden deep crystalline bedrock. Microb Ecol 23:1PubMedCrossRefGoogle Scholar
  51. 51.
    Petsch S, Edwards K, Eglinton T (2005) Microbial transformations of organic matter in black shales and implications for global biogeochemical cycles. Palaeogeogr Palaeoclimatol Palaeoecol 219:157CrossRefGoogle Scholar
  52. 52.
    Petsch ST, Eglington TI, Edwards KJ (2001) 14C-dead living biomass: evidence for microbial assimilation of ancient organic carbon during shale weathering. Science 292:1127PubMedCrossRefGoogle Scholar
  53. 53.
    Proskurowski G, Lilley MD, Seewald JS, Fruh-Green GL, Olson EJ, Lupton JE, Sylva SP, Kelley DS (2008) Abiogenic hydrocarbon production at lost city hydrothermal field. Science 319:604PubMedCrossRefGoogle Scholar
  54. 54.
    Purkamo L, Bomberg M, Nyyssönen M, Kukkonen I, Ahonen L, Kietäväinen R, Itävaara M (2013) Dissecting the deep biosphere: retrieving authentic microbial communities from packer-isolated deep crystalline bedrock fracture zones. FEMS Microbiol Ecol 85:324PubMedCrossRefGoogle Scholar
  55. 55.
    Rosewarne CP, Greenfield P, Li D, Tran-Dinh N, Midgley DJ, Hendry P (2013) Draft genome sequence of Methanobacterium sp. Maddingley, reconstructed from metagenomic sequencing of a methanogenic microbial consortium enriched from coal-seam gas formation water. Genome Announc 1:e00081–12. doi: 10.1128/genomeA.00081-12 PubMedCentralPubMedGoogle Scholar
  56. 56.
    Roslev P, Larsen MB, Jørgensen D, Hesselsoe M (2004) Use of heterotrophic CO2 assimilation as a measure of metabolic activity in planktonic and sessile bacteria. J Microbiol Methods 59:381PubMedCrossRefGoogle Scholar
  57. 57.
    Russell MJ, Martin W (2004) The rocky roots of the acetyl-CoA pathway. Trends Biochem Sci 29:358PubMedCrossRefGoogle Scholar
  58. 58.
    Sahl JW, Schmidt R, Swanner ED, Mandernack KW, Templeton AS, Kieft TL, Smith RL, Sanford WE, Callaghan RL, Mitton JB, Spear JR (2008) Subsurface microbial diversity in deep-granitic-fracture water in Colorado. Appl Environ Microbiol 74:143PubMedCentralPubMedCrossRefGoogle Scholar
  59. 59.
    Schrenk MO, Brazelton WJ, Lang SQ (2013) Serpentinization, carbon, and deep life. Rev Mineral Geochem 75:575CrossRefGoogle Scholar
  60. 60.
    Shlimon AG, Friedrich MW, Niemann H, Ramsing NB, Finster K (2004) Methanobacterium aarhusense sp. nov., a novel methanogen isolated from a marine sediment (Aarhus Bay, Denmark). Int J Syst Evol Microbiol 54:759PubMedCrossRefGoogle Scholar
  61. 61.
    Spiridonova E, Berg I, Kolganova T, Ivanovsky R, Kuznetsov B, Tourova T (2004) An oligonucleotide primer system for amplification of the ribulose-1, 5-bisphosphate carboxylase/oxygenase genes of bacteria of various taxonomic groups. Microbiology 73:316CrossRefGoogle Scholar
  62. 62.
    Swanner E, Templeton A (2011) Potential for nitrogen fixation and nitrification in the granite-hosted subsurface at Henderson Mine, CO. Front Microbiol 2:254PubMedCentralPubMedCrossRefGoogle Scholar
  63. 63.
    Takai K, Campbell BJ, Cary SC, Suzuki M, Oida H, Nunoura T, Hirayama H, Nakagawa S, Suzuki Y, Inagaki F, Horikoshi K (2005) Enzymatic and genetic characterization of carbon and energy metabolisms by deep-sea hydrothermal chemolithoautotrophic isolates of Epsilonproteobacteria. Appl Environ Microbiol 71:7310PubMedCentralPubMedCrossRefGoogle Scholar
  64. 64.
    Taran LN, Onoshko MP, Mikhailov ND (2011) Structure and composition of organic matter and isotope geochemistry of the Palaeoproterozoic graphite and sulphide-rich metasedimentary rocks from the Outokumpu deep drill hole, eastern Finland. In: Kukkonen IT (ed) Outokumpu Deep Drilling Project 2003–2010, Special paper 51. Geological Survey of Finland, Espoo, pp 219–228Google Scholar
  65. 65.
    Teske A, Biddle JF (2008) Analysis of deep subsurface microbial communities by functional genes and genomics. In Dilek et al. (ed) Links between geological processes, microbial activities & evolution of life, Springer, pp 159–176Google Scholar
  66. 66.
    Tiago I, Veríssimo A (2013) Microbial and functional diversity of a subterrestrial high pH groundwater associated to serpentinization. Environ Microbiol 15:1687PubMedCrossRefGoogle Scholar
  67. 67.
    Västi K (2011) Petrology of the drill hole R2500 at Outokumpu, eastern Finland—the deepest drill hole ever drilled in Finland. In: Kukkonen IT (ed) Outokumpu Deep Drilling Project 2003-2010, Special paper 51. Geological Survey of Finland, Espoo, pp 17–46Google Scholar
  68. 68.
    Wagner M, Roger AJ, Flax JL, Brusseau GA, Stahl DA (1998) Phylogeny of dissimilatory sulfite reductases supports an early origin of sulfate respiration. J Bacteriol 180:2975PubMedCentralPubMedGoogle Scholar
  69. 69.
    Welander PV, Metcalf WW (2005) Loss of the mtr operon in Methanosarcina blocks growth on methanol, but not methanogenesis, and reveals an unknown methanogenic pathway. Proc Natl Acad Sci U S A 102:10664PubMedCentralPubMedCrossRefGoogle Scholar
  70. 70.
    Whelan S, Goldman N (2001) A general empirical model of protein evolution derived from multiple protein families using a maximum-likelihood approach. Mol Biol Evol 18:691PubMedCrossRefGoogle Scholar
  71. 71.
    Zhu J, Liu X, Dong X (2011) Methanobacterium movens sp. nov. and Methanobacterium flexile sp. nov., isolated from lake sediment. Int J Syst Evol Microbiol 61:2974PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Lotta Purkamo
    • 1
  • Malin Bomberg
    • 1
  • Mari Nyyssönen
    • 1
  • Ilmo Kukkonen
    • 2
    • 3
  • Lasse Ahonen
    • 2
  • Merja Itävaara
    • 1
  1. 1.VTT Technical Research Centre of FinlandEspooFinland
  2. 2.Geological Survey of Finland (GTK)EspooFinland
  3. 3.Department of PhysicsUniversity of HelsinkiHelsinkiFinland

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