Microbial Ecology

, Volume 71, Issue 3, pp 686–699 | Cite as

Archaeal Distribution in Moonmilk Deposits from Alpine Caves and Their Ecophysiological Potential

  • Christoph ReitschulerEmail author
  • Christoph Spötl
  • Katrin Hofmann
  • Andreas O. Wagner
  • Paul Illmer
Environmental Microbiology


(Alpine) caves are, in general, windows into the Earth’s subsurface. Frequently occurring structures in caves such as moonmilk (secondary calcite deposits) offer the opportunity to study intraterrestrial microbial communities, adapted to oligotrophic and cold conditions. This is an important research field regarding the dimensions of subsurface systems and cold regions on Earth. On a methodological level, moonmilk deposits from 11 caves in the Austrian Alps were collected aseptically and investigated using a molecular (qPCR and DGGE sequencing-based) methodology in order to study the occurrence, abundance, and diversity of the prevailing native Archaea community. Furthermore, these Archaea were enriched in complex media and studied regarding their physiology, with a media selection targeting different physiological requirements, e.g. methanogenesis and ammonia oxidation. The investigation of the environmental samples showed that all moonmilk deposits were characterized by the presence of the same few habitat-specific archaeal species, showing high abundances and constituting about 50 % of the total microbial communities. The largest fraction of these Archaea was ammonia-oxidizing Thaumarchaeota, while another abundant group was very distantly related to extremophilic Euryarchaeota (Moonmilk Archaea). The archaeal community showed a depth- and oxygen-dependent stratification. Archaea were much more abundant (around 80 %), compared to bacteria, in the actively forming surface part of moonmilk deposits, decreasing to about 5 % down to the bedrock. Via extensive cultivation efforts, it was possible to enrich the enigmatic Moonmilk Archaea and also AOA significantly above the level of bacteria. The most expedient prerequisites for cultivating Moonmilk Archaea were a cold temperature, oligotrophic conditions, short incubation times, a moonmilk surface inoculum, the application of erythromycin, and anaerobic (microaerophilic) conditions. On a physiological level, it seems that methanogenesis is of marginal importance, while ammonia oxidation and a still undiscovered metabolic pathway are vital elements in the (archaeal) moonmilk biome.


Moonmilk Methanogens Ammonia-Oxidizing Archaea Non-extremophilic Archaea Uncultured Archaea Microbial Cave communities 



We are grateful to the Tiroler Wissenschaftsfonds (TWF) for funding a significant part of this study and to A. Rettenbacher, R. Erlmoser and R. Pavuza for providing moonmilk samples.

Supplementary material

248_2015_727_MOESM1_ESM.pdf (88 kb)
Fig. S1 (PDF 88 kb)
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Fig. S2 (PDF 185 kb)
248_2015_727_MOESM3_ESM.pdf (166 kb)
Fig. S3 (PDF 165 kb)
248_2015_727_MOESM4_ESM.pdf (42 kb)
Table S1 (PDF 42 kb)


  1. 1.
    Castelle CJ, Wrighton KC, Thomas BC, Hug LA, Brown CT, Wilkins MJ, Frischkorn KR, Tringe SG, Singh A, Markillie LM, Taylor RC, Williams KH, Banfield JF (2015) Genomic expansion of domain Archaea highlights roles for organisms from new phyla in anaerobic carbon cycling. Curr Biol 25:690–701CrossRefPubMedGoogle Scholar
  2. 2.
    Zuo G, Xu Z, Hao B (2015) Phylogeny and taxonomy of Archaea: a comparison of the whole-genome-based CVTree approach with 16S rRNA sequence analysis. Life 5:949–968CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Petitjean C, Deschamps P, Lopez-Garcia P, Moreira D, Brochier-Armanet (2015) Extending the conserved phylogenetic core of Archaea disentangles the evolution of the third domain of life. Mol Biol Evol 32:1242–1254CrossRefPubMedGoogle Scholar
  4. 4.
    Cavicchioli R (2011) Archaea—timeline of the third domain. Nature 9:51–61Google Scholar
  5. 5.
    Rinke C, Schwientek P, Sczyrba A, Ivanova NN, Anderson IJ, Cheng JF, Darling A, Malfatti S, Swan BK, Gies EA, Dodsworth JA, Hedlund BP, Tsiamis G, Sievert SM, Liu WT, Eisen JA, Hallam SJ, Kyrpides NC, Stepanauskas R, Rubin EM, Hugenholtz P, Woyke T (2013) Insights into the phylogeny and coding potential of microbial dark matter. Nature 499:431–437CrossRefPubMedGoogle Scholar
  6. 6.
    Petitjean C, Deschamps P, Lopez-Garcia P, Moreira D (2014) Rooting the domain Archaea by phylogenetic analysis supports the foundation of the new kingdom Proteoarchaeota. Genome Biol Evol 7:191–204CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Woese CR, Fox GE (1977) Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc Natl Acad Sci U S A 74:5088–5090CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Woese CR, Kandler O, Wheelis ML (1990) Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci U S A 87:4576–4579CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Brochier-Armanet C, Boussau B, Gribaldo S, Forterre P (2008) Mesophilic crenarchaeota: proposal for a third archaeal phylum, the Thaumarchaeota. Nature 6:245–252Google Scholar
  10. 10.
    Epstein SS (2013) The phenomenon of microbial uncultivability. Curr Opin Microbiol 16:636–642CrossRefPubMedGoogle Scholar
  11. 11.
    Alain K, Querellou J (2009) Cultivating the uncultured: limits, advances and future challenges. Extremophiles 13:583–594CrossRefPubMedGoogle Scholar
  12. 12.
    Vartoukian SR, Palmer RM, Wade WG (2010) Strategies for culture of ‘unculturable’ bacteria. FEMS Microbiol Lett 309:1–7PubMedGoogle Scholar
  13. 13.
    Leadbetter JR (2003) Cultivation of recalcitrant microbes: cells are alive, well and revealing their secrets in the 21st century laboratory. Curr Opin Microbiol 6:274–281CrossRefPubMedGoogle Scholar
  14. 14.
    Knittel K, Boetius A (2009) Anaerobic oxidation of methane: progress with an unknown process. Annu Rev Microbiol 63:311–334CrossRefPubMedGoogle Scholar
  15. 15.
    Rooney DC, Hutchens E, Clipson N, Baldini J, McDermott F (2010) Microbial community diversity of moonmilk deposits at Ballynamintra cave, Co. Waterford, Ireland. Microb Ecol 60:753–761CrossRefPubMedGoogle Scholar
  16. 16.
    Canaveras JC, Cuezva S, Sanchez-Moral S, Lario J, Laiz L, Gonzalez JM, Saiz-Jimenez C (2006) On the origin of fiber calcite crystals in moonmilk deposits. Naturwissenschaften 93:27–32CrossRefPubMedGoogle Scholar
  17. 17.
    Portillo MC, Gonzalez JM (2011) Moonmilk deposits originate from specific bacterial communities in Altamira cave (Spain). Microb Ecol 61:182–189CrossRefPubMedGoogle Scholar
  18. 18.
    Probst AJ, Moissl-Eichinger C (2015) “Altiarchaeales”: uncultivated Archaea from the subsurface. Life 5:1381–1395CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Ramanan R, Kannan K, Sivanesan SD, Mudliar S, Kaur S, Tripathi AK, Chakrabarti T (2009) Bio-sequestration of carbon dioxide using carbonic anhydrase enzyme purified from Citrobacter freundii. World J Microbiol Biotechnol 25:981–987CrossRefGoogle Scholar
  20. 20.
    Emerson JB, Thomas BC, Alvarez W, Banfield JF (2015) Metagenomic analysis of a high carbon dioxide subsurface microbial community populated by chemolithoautotrophs and bacteria and Archaea from candidate phyla. Environ Microbiol. doi: 10.1111/1462-2920.12817 PubMedGoogle Scholar
  21. 21.
    Dubey G, Kollah B, Ahirwar U, Tiwari S, Mohanty SR (2015) Significance of Archaea in terrestrial biogeochemical cycles and global climate change. Afr J Microbiol Res 9:201–208CrossRefGoogle Scholar
  22. 22.
    Banks ED, Taylor NM, Gulley J, Lubbers BR, Giarrizo JG, Bullen HA, Hoehler TM, Barton HA (2010) Bacterial calcium carbonate precipitation in cave environments: a function of calcium homeostasis. Geomicrobiol J 27:444–454CrossRefGoogle Scholar
  23. 23.
    Gonzalez JM, Portillo MC, Saiz-Jimenez C (2006) Metabolically active Crenarchaeota in Altamira cave. Naturwissenschaften 93:42–45CrossRefPubMedGoogle Scholar
  24. 24.
    Snider JR, Goin C, Miller RV, Boston PJ, Northup DE (2009) Ultraviolet radiation sensitivity in cave bacteria: evidence of adaption to the subsurface? Int J Speleol 38:11–22CrossRefGoogle Scholar
  25. 25.
    Reitschuler C, Lins P, Schwarzenauer T, Spötl C, Wagner AO, Illmer P (2015) New undescribed lineages of non-extremophilic Archaea form a constant and dominant element within alpine moonmilk microbiomes. Geomicrobiol J 32:890–902CrossRefGoogle Scholar
  26. 26.
    Reitschuler C, Lins P, Wagner AO, Illmer P (2014) Cultivation of moonmilk-born non-extremophilic Thaum and Euryarchaeota in mixed culture. Anaerobe 29:73–79CrossRefPubMedGoogle Scholar
  27. 27.
    Spötl C, Reimer PJ, Luetscher M (2014) Long-term mass balance of perennial firn and ice in an Alpine cave (Austria): constraints from radiocarbon-dated wood fragments. The Holocene 24:165–175CrossRefGoogle Scholar
  28. 28.
    Yu Y, Lee C, Hwang S (2005) Group-specific primer and probe sets to detect methanogenic communities using quantitative real-time polymerase chain reaction. Biotechnol Bioeng 89:670–679CrossRefPubMedGoogle Scholar
  29. 29.
    Reitschuler C, Lins P, Illmer P (2014) Primer evaluation and adaption for cost-efficient SYBR Green-based qPCR and its applicability for specific quantification of methanogens. World J Microbiol Biotechnol 30:293–304CrossRefPubMedGoogle Scholar
  30. 30.
    Rooney-Varga JN, Anderson RT, Fraga JL, Ringelberg D, Lovley DR (1999) Microbial communities associated with anaerobic benzene degradation in a petroleum-contaminated aquifer. Appl Environ Microbiol 65:3056–3063PubMedPubMedCentralGoogle Scholar
  31. 31.
    Schabereiter-Gurtner C, Maca S, Rölleke S, Nigl K, Lukas J, Hirschl A (2001) 16S rDNA-based identification of bacteria from conjunctival swabs by PCR and DGGE fingerprinting. IOVS 42:1164–1171Google Scholar
  32. 32.
    Pereyra LP, Hiibel SR, Prieto Riquelme MV, Reardon KF, Pruden A (2010) Detection and quantification of functional genes of cellulose-degrading, fermentative, and sulfate-reducing bacteria and methanogenic archaea. Appl Environ Microbiol 76:2192–2202CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Martineau C, Whyte LG, Greer CW (2010) Stable isotope probing analysis of the diversity and activity of methanotrophic bacteria in soils from the Canadian High Arctic. Appl Environ Microbiol 76:5773–5784CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Auguet JC, Nomokonova N, Camarero L, Casamayor EO (2011) Seasonal changes of freshwater ammonia-oxidizing archaeal assemblages and nitrogen species in oligotrophic alpine lakes. Appl Environ Microbiol 6:1937–1945CrossRefGoogle Scholar
  35. 35.
    Harms G, Layton AC, Dionisi HM, Gregory IR, Garrett VM, Hawkins SA, Robinson KG, Sayler GS (2003) Real-time PCR quantification of nitrifying bacteria in a municipal wastewater treatment plant. Environ Sci Technol 37:343–351CrossRefPubMedGoogle Scholar
  36. 36.
    Dedysh SN, Panikov NS, Tiedje JM (1998) Acidophilic methanotrophic communities from sphagnum peat bogs. Appl Environ Microbiol 62:922–929Google Scholar
  37. 37.
    Onstott TC, Moser DP, Pfiffner SM, Fredrickson JK, Brockman FJ, Phelps TJ, White DC, Peacock A, Balkwill D, Hoover R, Krumholz LR, Borscik M, Kieft TL, Wilson R (2003) Indigenous and contaminant microbes in ultradeep mines. 5:1168-1191Google Scholar
  38. 38.
    Könneke M, Bernhard AE, de la Torre JR, Walker CB, Waterbury JB, Stahl DA (2005) Isolation of an autotrophic ammonia-oxidizing marine archaeon. Nature 437:543–546CrossRefPubMedGoogle Scholar
  39. 39.
    Weber KA, Achenbach LA, Coates JD (2006) Microorganisms pumping iron: anaerobic microbial iron oxidation and reduction. Nature 752:752–764Google Scholar
  40. 40.
    Berg IA, Kockelkorn D, Ramos-Vera WH, Say RF, Zarzycki J, Hügler M, Alber BE, Fucks G (2010) Autotrophic carbon fixation in Archaea. Nature 8:447–460Google Scholar
  41. 41.
    Khelaifia S, Drancourt M (2012) Susceptibility of Archaea to antimicrobial agents: applications to clinical microbiology. Clin Microbiol Infect 18:841–848CrossRefPubMedGoogle Scholar
  42. 42.
    Lins P, Reitschuler C, Illmer P (2012) Development and evaluation of inocula combating high acetate concentrations during the start-up of an anaerobic digestion. Bioresour Technol 110:167–173CrossRefPubMedGoogle Scholar
  43. 43.
    Wagner AO, Lins P, Illmer P (2012) A simple method for the enumeration of methanogens by most probable number counting. Biomass Bioenerg 45:311–314CrossRefGoogle Scholar
  44. 44.
    Kandeler E, Gerber H (1988) Short-term assay of soil urease activity using colorimetric determination of ammonium. Biol Fertil Soils 6:68–72CrossRefGoogle Scholar
  45. 45.
    Walker CB, de la Torre JR, Klotz MG, Urakawa H, Pinel N, ArpDJ B-AC, Chain PSG, Chan PP, Gollabgir A, Hemp J, Hügler M, Karr EA, Könneke M, Shin M, Lawton TJ, Lowe T, Martens-Habbena W, Sayavedra-Soto LA, Lang D, Sievert SM, Rosenzweig AC, Manning G, Stahl DA (2010) Nitrosopumilus maritimus genome reveals unique mechanisms for nitrification and autotrophy in globally distributed marine crenarchaea. PNAS 107:8818–8823CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Pester M, Schleper C, Wagner M (2011) The Thaumarchaeota: an emerging view of their phylogeny and ecophysiology. Curr Opin Microbiol 14:300–306CrossRefPubMedPubMedCentralGoogle Scholar
  47. 47.
    Itoh T, Yoshikawa N, Takashina T (2007) Thermogymnomonas acidicola gen. nov., sp. nov., a novel thermoacidophilic, cell wall-less archaeon in the order Thermoplasmatales, isolated from a solfataric soil in Hakone, Japan. Int J Syst Evol Microbiol 57:2557–2561CrossRefPubMedGoogle Scholar
  48. 48.
    Reysenbach AL, Liu Y, Banta AB, Beveridge TJ, Kirshtein JD, Schouten S, Tivey MK, von Damm KL, Voytek MA (2006) A ubiquitous thermoacidophilic archaeon from deep-sea hydrothermal vents. Nature 442:444–447CrossRefPubMedGoogle Scholar
  49. 49.
    Hanson RS, Hanson TE (1996) Methanotrophic bacteria. Microbiol Rev 60:439–471PubMedPubMedCentralGoogle Scholar
  50. 50.
    Ercolini D (2004) PCR-DGGE fingerprinting: novel strategies for detection of microbes in food. J Microbiol Methods 56:297–314CrossRefPubMedGoogle Scholar
  51. 51.
    Portillo MC, Gonzalez JM (2008) Statistical differences between relative quantitative molecular fingerprints from microbial communities. Antonie Van Leeuwenhoek 94:157–163CrossRefPubMedGoogle Scholar
  52. 52.
    Lin X, Kennedy D, Fredrickson J, Bjornstad B, Konopka A (2011) Vertical stratification of subsurface microbial community composition across geological formations at the Hanford Site. Environ Microbiol 14:414–425CrossRefPubMedGoogle Scholar
  53. 53.
    Margesin R, Miteva V (2011) Diversity and ecology of psychrophilic microorganisms. Res Microbiol 162:346–361CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  • Christoph Reitschuler
    • 1
    Email author
  • Christoph Spötl
    • 2
  • Katrin Hofmann
    • 1
  • Andreas O. Wagner
    • 1
  • Paul Illmer
    • 1
  1. 1.Institute of MicrobiologyUniversity of InnsbruckInnsbruckAustria
  2. 2.Institute of GeologyUniversity of InnsbruckInnsbruckAustria

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