Article | Published:

Domestication of previously uncultivated Candidatus Desulforudis audaxviator from a deep aquifer in Siberia sheds light on its physiology and evolution

The ISME Journal (2019) | Download Citation


An enigmatic uncultured member of Firmicutes, Candidatus Desulforudis audaxviator (CDA), is known by its genome retrieved from the deep gold mine in South Africa, where it formed a single-species ecosystem fuelled by hydrogen from water radiolysis. It was believed that in situ conditions CDA relied on scarce energy supply and did not divide for hundreds to thousand years. We have isolated CDA strain BYF from a 2-km-deep aquifer in Western Siberia and obtained a laboratory culture growing with a doubling time of 28.5 h. BYF uses not only H2 but also various organic electron donors for sulfate respiration. Growth required elemental iron, and ferrous iron did not substitute for it. A complex intracellular organization included gas vesicles, internal membranes, and electron-dense structures enriched in phosphorus, iron, and calcium. Genome comparison of BYF with the South African CDA revealed minimal differences mostly related to mobile elements and prophage insertions. Two genomes harbored <800 single-nucleotide polymorphisms and had nearly identical CRISPR loci. We suggest that spores with the gas vesicles may facilitate global distribution of CDA followed by colonization of suitable subsurface environments. Alternatively, a slow evolution rate in the deep subsurface could result in high genetic similarity of CDA populations at two sites spatially separated for hundreds of millions of years.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Data availability

The annotated genome sequence of CDA strain BYF has been deposited in the NCBI GenBank database under the accession number CP034260. The Supplementary Information includes Supplementary Figures 1–4 and Supplementary Tables 1–4. Other data that support the findings of this study are available from the corresponding author upon request.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Whitman WB, Coleman DC, Wiebe JW. Prokaryotes: the unseen majority. Proc Natl Acad Sci USA. 1998;95:6578–83.

  2. 2.

    Labonté JM, Field EK, Lau M, Chivian D, Van Heerden E, Wommack KE, et al. Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population. Front Microbiol. 2015;22:349.

  3. 3.

    Magnabosco C, Lin LH, Dong H, Bomberg M, Ghiorse W, Stan-Lotter H, et al. The biomass and biodiversity of the continental subsurface. Nat Geosci. 2018;11:707–17.

  4. 4.

    Inagaki F, Hinrichs K-U, Kubo Y, Bowles MW, Heuer VB, HongInagaki W-L, et al. Exploring deep microbial life in coal-bearing sediment down to ~2.5 km below the ocean floor. Science. 2015;349:420–4.

  5. 5.

    Chivian D, Brodie EL, Alm EJ, Culley DE, Dehal PS, DeSantis TZ, et al. Environmental genomics revealed single-species ecosystem deep within Earth. Science. 2008;322:275–8.

  6. 6.

    Davidson MM, Silver BJ, Onstott TC, Moser DP, Gihring TM, Pratt LM, et al. Capture of planktonic microbial diversity in fractures by long-term monitoring of flowing boreholes, Evander Basin, South Africa. Geomicrobiol J. 2011;28:275–300.

  7. 7.

    Magnabosco C, Tekere M, Lau MCY, Linage B, Kuloyo O, Erasmus M, et al. Comparisons of the composition and biogeographic distribution of the bacterial communities occupying South African thermal springs with those inhabiting deep subsurface fracture water. Front Microbiol. 2014;5:679.

  8. 8.

    Tiago I, Veríssimo A. Microbial and functional diversity of a subterrestrial high pH groundwater associated to serpentinization. Environ Microbiol. 2013;15:1687–706.

  9. 9.

    Kjeldsen KU, Kjellerup BV, Egli K, Frølund B, Nielsen PH, Ingvorsen K. Phylogenetic and functional diversity of bacteria in biofilms from metal surfaces of an alkaline district heating system. FEMS Microbiol Ecol. 2007;61:384–97.

  10. 10.

    Kadnikov VV, Mardanov AV, Beletsky AV, Banks D, Pimenov NV, Frank Y, et al. A metagenomic window into the 2-km-deep terrestrial subsurface aquifer revealed multiple pathways of organic matter decomposition. FEMS Microbiol Ecol. 2018;94:fiy152.

  11. 11.

    Banks D, Frank YA, Kadnikov VV, Karnachuk OV, Watts M, Boyce A, et al. Hydrochemical data report from sampling of two deep abandoned hydrocarbon exploration wells: Byelii Yar and Parabel’, Tomsk Oblast’, Western Siberia, Russian Federation. NGU Report, 2014.034. Trondheim: Geological Survey of Norway; 2014.

  12. 12.

    Frank YA, Kadnikov VV, Lukina AP, Banks D, Beletsky AV, Mardanov AV, et al. Characterization and genome analysis of the first facultatively alkaliphilic Thermodesulfovibrio isolated from the deep terrestrial subsurface. Front Microbiol. 2016;7:2000.

  13. 13.

    Cline JD. Spectrophotometric determination of hydrogen sulfide in natural waters. Limnol Oceanogr. 1969;14:454–8.

  14. 14.

    Widdel FF, Bak R. Gram-negative mesophilic sulfate-reducing bacteria. In: Balows A, Truper HG, Dworkin M, Harder W, Schleifer KH, editors. The Prokaryotes: a handbook on the biology of bacteria: ecophysiology, isolation, identification, applications. 2nd ed. Berlin: Springer; 1992. p. 3352–78.

  15. 15.

    Karnachuk OV, Pimenov NV, Yusupov SK, Frank YA, Puhakka JA, Ivanov MV. Distribution, diversity, and activity of sulfate-reducing bacteria in the water column in Gek-Gel lake, Azerbaijan. Mikrobiologiia. 2006;75:101–9.

  16. 16.

    Frank YA, Kadnikov VV, Gavrilov SN, Banks D, Gerasimchuk AL, Podosokorskaya OA, et al. Stable and variable parts of microbial community in Siberian deep subsurface thermal aquifer system revealed in a long-term monitoring study. Front Microbiol. 2016;7:2101.

  17. 17.

    Ikkert OP, Gerasimchuk AL, Bukhtiyarova PA, Tuovinen OH, Karnachuk OV. Characterization of precipitates formed by H2S-producing, Cu-resistant Firmicute isolates of Tissierella from human gut and Desulfosporosinus from mine waste. Antonie Van Leeuwenhoek. 2013;103:1221–34.

  18. 18.

    Wilson K. Preparation of genomic DNA from bacteria. Curr Protoc Mol Biol. 2001;56:2.4. 1–2.4.5.

  19. 19.

    Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet J. 2011;17:10–2.

  20. 20.

    Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455–77.

  21. 21.

    Cao MD, Nguyen SH, Ganesamoorthy D, Elliott AG, Cooper MA, Coin LJ. Scaffolding and completing genome assemblies in real-time with nanopore sequencing. Nat Commun. 2017;8:14515.

  22. 22.

    Li H, Durbin R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics. 2010;26:589–95.

  23. 23.

    Langmead B, Salzberg SL. Fast gapped-read alignment with Bowtie 2. Nat Methods. 2012;9:357–9.

  24. 24.

    Robinson JT, Thorvaldsdóttir H, Winckler W, Guttman M, Lander ES, Getz G, et al. Integrative genomics viewer. Nat Biotechnol. 2011;29:24–6.

  25. 25.

    Walker BJ, Abeel T, Shea T, Priest M, Abouelliel A, Sakthikumar S, et al. Pilon: an integrated tool for comprehensive microbial variant detection and genome assembly improvement. PLoS ONE. 2014;9:e112963.

  26. 26.

    Brettin T, Davis JJ, Disz T, Edwards RA, Gerdes S, Olsen GJ, et al. RASTtk: a modular and extensible implementation of the RAST algorithm for building custom annotation pipelines and annotating batches of genomes. Sci Rep. 2015;5:8365.

  27. 27.

    Grissa I, Vergnaud G, Pourcel C. CRISPRFinder: a web tool to identify clustered regularly interspaced short palindromic repeats. Nucleic Acids Res. 2007;35:W52–W57.

  28. 28.

    Garrison E, Marth G. Haplotype-based variant detection from short-read sequencing. arXiv:1207.3907 [q-bio.GN]; 2012.

  29. 29.

    Darling AE, Mau B, Perna NT. Progressive Mauve: multiple genome alignment with gene gain, loss and rearrangement. PLoS ONE. 2010;5:e11147.

  30. 30.

    Rodriguez-R LM, Konstantinidis KT. The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. Peer J Prepr. 2016;4:e1900v1.

  31. 31.

    Ramel F, Amrani A, Pieulle L, Lamrabet O, Voordouw G, Seddiki N, et al. Membrane-bound oxygen reductases of the anaerobic sulfate-reducing Desulfovibrio vulgaris Hildenborough: roles in oxygen defence and electron link with periplasmic hydrogen oxidation. Microbiology. 2013;159:2663–73.

  32. 32.

    Jungbluth SP, Glavina del Rio T, Tringe SG, Stepanauskas R, Rappé MS. Genomic comparisons of a bacterial lineage that inhabits both marine and terrestrial deep subsurface systems. PeerJ. 2017;5:e3134.

  33. 33.

    Tyson GW, Banfield JF. Rapidly evolving CRISPRs implicated in acquired resistance of microorganisms to viruses. Environ Microbiol. 2008;10:200–7.

  34. 34.

    Kupczok A, Landan G, Dagan T. The contribution of genetic recombination to CRISPR array evolution. Genome Biol Evol. 2015;7:1925–39.

  35. 35.

    Frank Y, Banks D, Avakian M, Antsiferov D, Kadychagov P, Karnachuk O. Firmicutes is an important component of microbial communities in water-injected and pristine oil reservoirs, Western Siberia, Russia. Geomicrobiol J. 2016;33:387–400.

  36. 36.

    Karnachuk OV, Sasaki K, Gerasimchuk AL, Sukhanova O, Ivasenko DA, Kaksonen AH, et al. Precipitation of Cu-sulfides by copper-tolerant Desulfovibrio isolates. Geomicrobiol J. 2008;25:219–27.

  37. 37.

    Mardanov AV, Panova IA, Beletsky AV, Avakyan MR, Kadnikov VV, Antsiferov DV, et al. Genomic insights into a new acidophilic, copper-resistant Desulfosporosinus isolate from the oxidized tailings area of an abandoned gold mine. FEMS Microbiol Ecol. 2016;92:fiw111.

  38. 38.

    Dinh HT, Kuever J, Mussmann M, Hassel AW, Stratmann M, Widdel F. Iron corrosion by novel anaerobic microorganisms. Nature. 2004;427:829–32.

  39. 39.

    Deng X, Okamoto A. Electrode potential dependency of single-cell activity identifies the energetics of slow microbial electron uptake process. Front Microbiol. 2018;9:2744.

  40. 40.

    Pfeifer F. Distribution, formation and regulation of gas vesicles. Nat Rev Microbiol. 2012;10:705–15.

  41. 41.

    Widdel F, Pfennig N. Sporulation and further nutritional characteristics of Desulfotomaculum acetoxidans. Arch Microbiol. 1981;129:401–2.

  42. 42.

    O’Sullivan LA, Roussel EG, Weightman AJ, Webster G, Hubert CRJ, Bell E, et al. Survival of Desulfotomaculum spores from estuarine sediments after serial autoclaving and high-temperature exposure. ISME J. 2015;9:922–33.

  43. 43.

    Lin L-H, Wang P-L, Rumble D, Lippmann-Pipke J, Boice E, Pratt LM, et al. Long-term sustainability of a high-energy, low-diversity crustal biome. Science. 2006;314:479

  44. 44.

    Karnachuk OV, Kurochkina SY, Tuovinen OH. Growth of sulfate-reducing bacteria with solid-phase electron acceptors. Appl Microbiol Biotechnol. 2002;58:482–6.

  45. 45.

    Kontorovich AE, Yan PA, Zamirailova AG, Kostyreva EA, Eder VG. Classification of rocks of the Bazhenov Formation. Russ Geol Geophys. 2016;57:1606–12.

  46. 46.

    Novikov DA, Shvartsev SL. Hydrogeological conditions of the Pre-Enisei petroleum subprovince. Russ Geol Geophys. 2009;50:873–83.

  47. 47.

    Reno ML, Held NL, Fields CJ, Burke PV, Whitaker RJ. Biogeography of the Sulfolobus islandicus pan-genome. Proc Natl Acad Sci USA. 2009;106:8605–10.

  48. 48.

    Ochman H, Elwyn S, Moran NA. Calibrating bacterial evolution. Proc Natl Acad Sci USA. 1999;96:12638–43.

  49. 49.

    Lawrence JG, Ochman H. Molecular archaeology of the Escherichia coli genome. Proc Natl Acad Sci USA. 1998;95:9413–17.

  50. 50.

    Starnawski P, Bataillon T, Ettema TJG, Jochum LM, Schreiber L, Chen X, et al. Microbial community assembly and evolution in subseafloor sediment. Proc Natl Acad Sci USA. 2017;114:2940–5.

  51. 51.

    Lomstein BA, Langerhuus AT, D’Hondt S, Jørgensen BB, Spivack AJ. Endospore abundance, microbial growth and necromass turnover in deep sub-seafloor sediment. Nature. 2012;484:101–14.

  52. 52.

    Jørgensen BB, D’Hondt S. A starving majority deep beneath the seafloor. Science. 2006;314:932–34.

  53. 53.

    Phelps TJ, Murphy EM, Pfiffner SM, White DC. Comparison between geochemical and biological estimates of subsurface microbial activities. Microb Ecol. 1994;28:335–49.

  54. 54.

    Smith DJ, Timonen HJ, Jaffe DA, Griffin DW, Birmele MN, Perry KD, et al. Intercontinental dispersal of bacteria and archaea by transpacific winds. Appl Environ Microbiol. 2013;79:1134–9.

Download references


Strain BYF isolation was supported by the Russian Foundation for Basic Research (grant # 18–04–00181) to O.V.K. group. Studies of strain BYF morphology and physiology were supported by the Russian Science Foundation (grant # 18-14-00130) to O.V.K. group. The work of N.V.R. group on sequencing and analysis of CDA genome was supported by the Russian Science Foundation (grant 14–14–01016) and the Ministry of Science and Higher Education of the Russian Federation. We thank Andrei Miller for his excellent assistance with TEM. We appreciate the three anonymous reviewers’ suggestions that helped to improve the manuscript.

Author information


  1. Laboratory of Biochemistry and Molecular Biology, Tomsk State University, Tomsk, 634050, Russia

    • Olga V. Karnachuk
    • , Yulia A. Frank
    •  & Anastasia P. Lukina
  2. Institute of Bioengineering, Research Center of Biotechnology of the Russian Academy of Sciences, Moscow, 119071, Russia

    • Vitaly V. Kadnikov
    • , Alexey V. Beletsky
    • , Andrey V. Mardanov
    •  & Nikolai V. Ravin


  1. Search for Olga V. Karnachuk in:

  2. Search for Yulia A. Frank in:

  3. Search for Anastasia P. Lukina in:

  4. Search for Vitaly V. Kadnikov in:

  5. Search for Alexey V. Beletsky in:

  6. Search for Andrey V. Mardanov in:

  7. Search for Nikolai V. Ravin in:


O.V.K. and N.V.R. designed the study and wrote the manuscript. Y.A.F. and A.P.L. performed the research work on CDA isolation, cultivation, morphology, and physiology. A.V.M. and V.V.K. sequenced the genome of strain BYF and performed comparative genomics analysis. A.V.B. performed SNP analysis and genome comparisons. All authors commented on and approved the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

Corresponding authors

Correspondence to Olga V. Karnachuk or Nikolai V. Ravin.

Supplementary information

About this article

Publication history