1. Gold, T. The deep, hot biosphere. Proc. Natl Acad. Sci. USA 89, 6045–6049 (1992).

2. Whitman, W. B., Coleman, D. C. & Wiebe, W. J. Prokaryotes: the unseen majority. Proc. Natl Acad. Sci. USA 95, 6578–6583 (1998).

3. Anantharaman, K. et al. Thousands of microbial genomes shed light on interconnected biogeochemical processes in an aquifer system. Nat. Commun. 7, 13219 (2016).

4. Rinke, C. et al. Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, 431–437 (2013).

5. Michalski, J. R. et al. The search for clues to abiogenesis on Mars. Nat. Geosci. 11, 21–26 (2017).

6. Onstott, T. C. Deep Life (Princeton Univ. Press, Princeton, 2016).

7. Kallmeyer, J., Pockalny, R., Adhikari, R. R., Smith, D. C. & D’Hondt, S. Global distribution of microbial abundance and biomass in subseafloor sediment. Proc. Natl Acad. Sci. USA 109, 16213–16216 (2012).

8. Onstott, T. C. et al. in Enigmatic Microorganisms and Life in Extreme Environments (ed. Seckbach, J.) 489–499 (Kluwer Academic Publishers, ​Alphen aan den Rijn, 1998).

9. Fredrickson, J. & Balkwill, D. Geomicrobiological processes and diversity in the deep terrestrial subsurface. Geomicrobiol. J. 23, 345–356 (2006).

10. Parkes, R. J. et al. A review of prokaryotic populations and processes in sub-seafloor sediments, including biosphere:geosphere interactions. Mar. Geol. 352, 409–425 (2014).

11. Parkes, R. J. et al. Deep bacterial biosphere in Pacific Ocean sediments. Nature 371, 410–413 (1994).

12. McMahon, S. & Parnell, J. Weighing the deep continental biosphere. FEMS Microbiol. Ecol. 87, 113–120 (2014).

13. Bar-On, Y. M., Phillips, R. & Milo, R. The biomass distribution on Earth. Proc. Natl Acad. Sci. USA 115, 6506–6511 (2018).

14. Heberling, C., Lowell, R. P., Liu, L. & Fisk, M. R. Extent of the microbial biosphere in the oceanic crust. Geochem. Geophys. Geosyst. 11, Q08003 (2010).

15. Lipp, J. S., Morono, Y., Inagaki, F. & Hinrichs, K.-U. Significant contribution of Archaea to extant biomass in marine subsurface sediments. Nature 454, 991–994 (2008).

16. D’Hondt, S. et al. Subseafloor sedimentary life in the South Pacific Gyre. Proc. Natl Acad. Sci. USA 106, 11651–11656 (2009).

17. Pedersen, K. & Ekendahl, S. Distribution and activity of bacteria in deep granitic groundwaters of southeastern Sweden. Microb. Ecol. 20, 37–52 (1990).

18. Hazen, T. C., Jimenez, L., López de Victoria, G. & Fliermans, C. B. Comparison of bacteria from deep subsurface sediment and adjacent groundwater. Microb. Ecol. 22, 293–304 (1991).

19. Sinclair, J. & Ghiorse, W. Distribution of aerobic bacteria, protozoa, algae, and fungi in deep subsurface sediments. Geomicrobiol. J. 7, 15–31 (1989).

20. Colwell, F. S. Microbiological comparison of a surface soil and unsaturated subsurface soil from a semiarid high desert. Appl. Environ. Microbiol. 55, 2420–2423 (1989).

21. Federle, T. W., Dobbins, D. C., Thornton-Manning, J. R. & Jones, D. D. Microbial biomass, activity, and community structure in subsurface soils. Groundwater 24, 365–374 (1986).

22. Harvey, R. W., Smith, R. L. & George, L. Effect of organic contamination upon microbial distributions and heterotrophic uptake in a Cape Cod, Mass., aquifer. Appl. Environ. Microbiol. 48, 1197–1202 (1984).

23. Balkwill, D., Leach, F., Wilson, J., McNabb, J. & White, D. C. Equivalence of microbial biomass measures based on membrane lipid and cell wall components, adenosine triphosphate, and direct counts in subsurface sediments. Microb. Ecol. 16, 73–84 (1988).

24. Beloin, R. M., Sinclair, J. L. & Ghiorse, W. C. Distribution and activity of microorganisms in subsurface sediments of a pristine study site in Oklahoma. Microb. Ecol. 16, 85–97 (1988).

25. Benjamini, Y. & Hochberg, Y. Controlling the false discovery rate: a practical and powerful approach to multiple testing. J. R. Stat. Soc. Series B Stat. Methodol. 57, 289–300 (1995).

26. Onstott, T. C. et al. Does aspartic acid racemization constrain the depth limit of the subsurface biosphere? Geobiology 12, 1–19 (2014).

27. Head, I. M., Gray, N. D. & Larter, S. R. Life in the slow lane; biogeochemistry of biodegraded petroleum containing reservoirs and implications for energy recovery and carbon management. Front. Microbiol. 5, 566 (2014).

28. Miteva, V. I., Sheridan, P. P. & Brenchley, J. E. Phylogenetic and physiological diversity of microorganisms isolated from a deep Greenland glacier ice core. Appl. Environ. Microbiol. 70, 202–213 (2004).

29. Telling, J. et al. Rock comminution as a source of hydrogen for subglacial ecosystems. Nat. Geosci. 8, 851–855 (2015).

30. Boyd, E. S., Hamilton, T. L., Havig, J. R., Skidmore, M. L. & Shock, E. L. Chemolithotrophic primary production in a subglacial ecosystem. Appl. Environ. Microbiol. 80, 6146–6153 (2014).

31. Chapelle, F. R. et al. A hydrogen-based subsurface microbial community dominated by methanogens. Nature 415, 312–314 (2002).

32. Bomberg, M., Lamminmäki, T. & Itävaara, M. Microbial communities and their predicted metabolic characteristics in deep fracture groundwaters of the crystalline bedrock at Olkiluoto, Finland. Biogeosci. Discuss. 13, 6031–6047 (2016).

33. Simkus, D. N. et al. Variations in microbial carbon sources and cycling in the deep continental subsurface. Geochim. Cosmochim. Acta. 173, 264–283 (2015).

34. Bomberg, M., Nyyssönen, M., Pitkänen, P., Lehtinen, A. & Itävaara, M. Active microbial communities inhabit sulphate-methane interphase in deep bedrock fracture fluids in Olkiluoto, Finland. BioMed Res. Int. 2015, 979530 (2015).

35. Knittel, K. & Boetius, A. Anaerobic oxidation of methane: progress with an unknown process. Annu. Rev. Microbiol. 63, 311–334 (2009).

36. Crespo-Medina, M. et al. Insights into environmental controls on microbial communities in a continental serpentinite aquifer using a microcosm-based approach. Front. Microbiol. 5, 604 (2014).

37. Tiago, I. & Veríssimo, A. Microbial and functional diversity of a subterrestrial high pH groundwater associated to serpentinization. Environ. Microbiol. 15, 1687–1706 (2013).

38. Brazelton, W. J. et al. Metagenomic identification of active methanogens and methanotrophs in serpentinite springs of the Voltri Massif, Italy. PeerJ 5, e2945 (2017).

39. Brazelton, W. J., Morrill, P. L., Szponar, N. & Schrenk, M. O. Bacterial communities associated with subsurface geochemical processes in continental serpentinite springs. Appl. Environ. Microbiol. 79, 3906–3916 (2013).

40. Lavalleur, H. J. & Colwell, F. S. Microbial characterization of basalt formation waters targeted for geological carbon sequestration. FEMS Microbiol. Ecol. 85, 62–73 (2013).

41. Nyyssönen, M. et al. Taxonomically and functionally diverse microbial communities in deep crystalline rocks of the Fennoscandian shield. ISME J. 8, 126–138 (2014).

42. Pedersen, K., Bengtsson, A. F., Edlund, J. S. & Eriksson, L. C. Sulphate-controlled diversity of subterranean microbial communities over depth in deep groundwater with opposing gradients of sulphate and methane. Geomicrobiol. J. 31, 617–631 (2014).

43. Osburn, M. R., LaRowe, D. E., Momper, L. M. & Amend, J. P. Chemolithotrophy in the continental deep subsurface: Sanford Underground Research Facility (SURF), USA. Front. Microbiol. 5, 610 (2014).

44. Magnabosco, C. 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. 5, 679–689 (2014).

45. Magnabosco, C. et al. Fluctuations in populations of subsurface methane oxidizers in coordination with changes in electron acceptor availability. FEMS Microbiol. Ecol. 94, fiy089 (2018).

46. Katayama, T. et al. Physicochemical impacts associated with natural gas development on methanogenesis in deep sand aquifers. ISME J. 9, 436–446 (2015).

47. Dong, Y. et al. Halomonas sulfidaeris-dominated microbial community inhabits a 1.8 km-deep subsurface Cambrian Sandstone reservoir. Environ. Microbiol. 16, 1695–1708 (2014).

48. Cluff, M. A., Hartsock, A., MacRae, J. D., Carter, K. & Mouser, P. J. Temporal changes in microbial ecology and geochemistry in produced water from hydraulically fractured marcellus shale gas wells. Environ. Sci. Technol. 48, 6508–6517 (2014).

49. Kryachko, Y., Dong, X., Sensen, C. W. & Voordouw, G. Compositions of microbial communities associated with oil and water in a mesothermic oil field. Antonie van Leeuwenhoek 101, 493–506 (2012).

50. O’Mullan, G. et al. Microbial stimulation and succession following a test well injection simulating CO 2 leakage into a shallow newark basin aquifer. PLoS ONE 10, e0117812 (2015).

51. Marteinsson, V. T. et al. Microbial communities in the subglacial waters of the Vatnajokull ice cap, Iceland. ISME J. 7, 427–437 (2013).

52. Lerm, S. et al. Thermal effects on microbial composition and microbiologically induced corrosion and mineral precipitation affecting operation of a geothermal plant in a deep saline aquifer. Extremophiles 17, 311–327 (2013).

53. Chivian, D. et al. Environmental genomics reveals a single species ecosystem deep within the Earth. Science 322, 275–278 (2008).

54. Magnabosco, C. et al. A metagenomic window into carbon metabolism at 3 km depth in Precambrian vontinental crust. ISME J. 10, 730–741 (2016).

55. New, M. G., Hulme, M. & Jones, P. D. Representing twentieth-century space–time climate variability. Part I: development of a 1961–90 mean monthly terrestrial climatology. J. Climate 12, 829–856 (1999).

56. Genthon, C. & Braun, A. ECMWF analyses and predictions of the surface climate of Greenland and Antarctica. J. Climate 8, 2324–2332 (1995).

57. Davies, J. H. Global map of solid Earth surface heat flow. Geochem. Geophys. Geosyst. 14, 4608–4622 (2013).

58. UNESCO-IHP-ISARM-Programme (United Nations Educational, Scientific and Cultural Organization, 2009).

59. Laske, G., Masters, G., Ma, Z. & Pasyanos, M. Update on CRUST1. 0 — A 1‐degree global model of Earth’s crust. Geophys. Res. Abstracts 15, 2658 (2013).

60. Norland, S., Heldal, M. & Tumyr, O. On the relation between dry matter and volume of bacteria. Microb. Ecol. 13, 95–101 (1987).

61. Griebler, C., Mindl, B., Slezak, D. & Geiger-Kaiser, M. Distribution patterns of attached and suspended bacteria in pristine and contaminated shallow aquifers studied with an in situ sediment exposure microcosm. Aquat. Microb. Ecol. 28, 117–129 (2002).

62. Marxsen, J. Bacterial biomass and bacterial uptake of glucose in polluted and unpolluted groundwater of sandy and gravelly deposits. Verh. Int. Ver. Limnol. 21, 1371–1375 (1981).

63. Mason, O. U. et al. First investigation of the microbiology of the deepest layer of ocean crust. PLoS ONE 5, e15399 (2010).

64. Locey, K. J. & Lennon, J. T. Scaling laws predict global microbial diversity. Proc. Natl Acad. Sci. USA 113, 5970–5975 (2015).

65. Willis, A. Extrapolating abundance curves has no predictive power for estimating microbial biodiversity. Proc. Natl Acad. Sci. USA 113, E5096 (2016).

66. Locey, K. J. & Lennon, J. T. Reply to Willis: powerful predictions of biodiversity from ecological models and scaling laws. Proc. Natl Acad. Sci. USA 113, E5097 (2016).

67. Curtis, T. P., Sloan, W. T. & Scannell, J. W. Estimating prokaryotic diversity and its limits. Proc. Natl Acad. Sci. USA 99, 10494–10499 (2002).

68. Rosenzweig, M. L. Species Diversity in Space and Time (Cambridge Univ. Press, Cambridge, 1995).

69. Hanson, C. A., Fuhrman, J. A., Horner-Devine, M. C. & Martiny, J. B. H. Beyond biogeographic patterns: processes shaping the microbial landscape. Nat. Rev. Microbiol. 10, 497–506 (2012).

70. Horner-Devine, M. C., Lage, M., Hughes, J. B. & Bohannan, B. J. M. A taxa–area relationship for bacteria. Nature 432, 750–753 (2004).

71. Morlon, H. et al. A general framework for the distance–decay of similarity in ecological communities. Ecol. Lett. 11, 904–917 (2008).

72. Sheik, C. S. et al. Identification and removal of contaminant sequences from ribosomal gene databases: lessons from the census of deep life. Front. Microbiol. 9, 840 (2018).

73. Lau, M. C. Y. et al. Deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers. Proc. Natl Acad. Sci. USA 113, E7927–E7936 (2016).

74. Sohlberg, E. et al. Revealing the unexplored fungal communities in deep groundwater of crystalline bedrock fracture zones in Olkiluoto, Finland. Front. Microbiol. 6, 573 (2015).

75. Pachiadaki, M. G., Rédou, V., Beaudoin, D. J., Burgaud, G. & Edgcomb, V. P. Fungal and prokaryotic activities in the marine subsurface biosphere at Peru Margin and Canterbury Basin inferred from RNA-based analyses and microscopy. Front. Microbiol. 7, 846 (2016).

76. Bengtson, S. et al. Deep-biosphere consortium of fungi and prokaryotes in Eocene sub-seafloor basalts. Geobiology 12, 489–496 (2014).

77. Anderson, R. E., Brazelton, W. J. & Baross, J. A. The deep viriosphere: assessing the viral impact on microbial community dynamics in the deep subsurface. Rev. Mineral. Geochem. 75, 649–675 (2013).

78. Kyle, J. E., Eydal, H. S., Ferris, F. G. & Pedersen, K. Viruses in granitic groundwater from 69 to 450 m depth of the Äspö hard rock laboratory, Sweden. ISME J. 2, 571–574 (2008).

79. Labonté, J. et al. Single cell genomics indicates horizontal gene transfer and viral infections in a deep subsurface Firmicutes population. Front. Microbiol. 6, 349 (2015).

80. Eydal, H., Jägevall, S., Hermansson, M. & Pedersen, K. Bacteriophage lytic to Desulfovibrio aespoeensis isolated from deep groundwater. ISME J. 3, 1139 (2009).

81. Lloyd, K. G., May, M. K., Kevorkian, R. T. & Steen, A. D. Meta-analysis of quantification methods shows that Archaea and Bacteria have similar abundances in the subseafloor. Appl. Environ. Microbiol. 79, 7790–7799 (2013).

82. Arístegui, J., Gasol, J. M., Duarte, C. M. & Herndl, G. J. Microbial oceanography of the dark ocean’s pelagic realm. Limnol. Oceanogr. 54, 1501–1529 (2009).

83. Buitenhuis, E. et al. Global Distribution of Picoheterotrophs (Bacteria and Archaea) Abundance and Biomass-Gridded Data Product (NetCDF)-Contribution to the MAREDAT World Ocean Atlas of Plankton Functional Types (PANGEA, 2012); https://doi.org/10.1594/PANGAEA.779142

84. Buitenhuis, E. T. et al. Picophytoplankton biomass distribution in the global ocean. Earth Syst. Sci. Data 4, 37–46 (2012b).

85. Xu, X., Thornton, P. E. & Post, W. M. A global analysis of soil microbial biomass carbon, nitrogen and phosphorus in terrestrial ecosystems. Global Ecol. Biogeogr. 22, 737–749 (2013).

86. Serna‐Chavez, H. M., Fierer, N. & Van Bodegom, P. M. Global drivers and patterns of microbial abundance in soil. Global Ecol. Biogeogr. 22, 1162–1172 (2013).

87. Joergensen, R. G. & Wichern, F. Quantitative assessment of the fungal contribution to microbial tissue in soil. Soil Biol. Biochem. 40, 2977–2991 (2008).

88. Kieft, T. L. & Simmons, K. A. Allometry of animal–microbe interactions and global census of animal-associated microbes. Proc. R. Soc. B 282, 20150702 (2015).

89. Morisita, M. Measuring of interspecific association and similarity between communities. Mem. Fac. Sci. Kyushu Univ. Ser. 3, 65–80 (1959).

90. Turnbaugh, P. J. et al. The human microbiome project. Nature 449, 804–810 (2007).

91. Dorofeeva, R. P. & Lysak, S. V. Heat Flow of Central Asia (Proc. World Geothermal Congress, 2010); https://www.geothermal-energy.org/pdf/IGAstandard/WGC/2010/1308.pdf

92. Keller, C. B. & Schoene, B. Statistical geochemistry reveals disruption in secular lithospheric evolution about 2.5 Gyr ago. Nature 485, 490–493 (2012).

93. Stevens, T. O., McKinley, J. P. & Fredrickson, J. K. Bacteria associated with deep, alkaline, anaerobic groundwaters in southeast Washington. Microb. Ecol. 25, 35–50 (1993).

94. Takai, K. et al. Shifts in archaeal communities associated with lithological and geochemical variations in subsurface Cretaceous rock. Environ. Microbiol. 5, 309–320 (2003).

95. Lloyd, K. G. in Microbial Life of the Deep Biosphere Vol. 1 (eds Kallmeyer, J. & Wagner, D.) 121–142 (DeGruyter, Berlin, 2014).

96. Rajala, P. & Bomberg, M. Reactivation of deep subsurface microbial community in response to methane or methanol amendment. Front. Microbiol. 8, 431 (2017).

97. Morono, Y. & Inagaki, F. in Advances in Applied Microbiology Vol. 95 (eds Gladd, G.M. & Sariaslani, S.) Ch. 3, 149–178 (Elsevier Inc., New York, 2016).

98. White, D. C. in Microbes in Their Natural Environments: Thirty-fourth Symposium of the Society for General Microbiology (eds Slater, J. H., Whittenbury, R. & Wimpenny, J. W. T.) 37–66 (Cambridge Univ. Press, New York, 1983).

99. Green, C. T. & Scow, K. M. Analysis of phospholipid fatty acids (PLFA) to characterize microbial communities in aquifers. Hydrogeol. J. 8, 126–141 (2000).

100. Stouthamer, A. H. in Microbial Biochemistry Vol. 21 (ed Quayle, J. R.) Ch. 1, 1–47 (Univ. Park Press, 1979).

101. Ringelberg, D., Sutton, S. & White, D. C. Biomass, bioactivity and biodiversity: microbial ecology of the deep subsurface: analysis of ester-linked phospholipid fatty acids. FEMS Microbiol. Rev. 20, 371–377 (1997).

102. Lin, L. H. et al. Long term biosustainability in a high energy, low diversity crustal biome. Science 314, 479–482 (2006b).

103. Byl, T. D. et al. Adaptations of indigenous bacteria to fuel contamination in karst aquifers in south-central Kentucky. J. Cave and Karst Studies 76, 104–113 (2014).

104. Griebler, C., Mindl, B. & Slezak, D. Combining DAPI and SYBR Green II for the enumeration of total bacterial numbers in aquatic sediments. Internat. Rev. Hydrobiol. 86, 453–465 (2001).

105. Webster, J., Hampton, G., Wilson, J., Ghiorose, W. & Leach, F. Determination of microbial cell numbers in subsurface samples. Ground Water 23, 17–25 (1985).

106. Eydal, H. & Pedersen, K. Use of an ATP assay to determine viable microbial biomass in Fennoscandian Shield groundwater from depths of 3–1000 m. J. Microbiol. Methods 70, 363–373 (2007).

107. Neidhardt, F. et al. Escherichia coli and Salmonella typhimurium (American Society for Microbiology, 1996).

108. Lomstein, B. A., Langerhuus, A. T., D’Hondt, S., Jørgensen, B. B. & Spivack, A. J. Endospore abundance, microbial growth and necromass turnover in deep sub-seafloor sediment. Nature 484, 101–104 (2012).

109. Kembe, S. W., Wu, M., Eisen, J. A. & Green, J. L. Incorporating 16S gene copy number information improves estimates of microbial diversity and abundance. PLoS Comput. Biol. 8, e1002743 (2012).

110. Xiao, X., White, E. P., Hooten, M. B. & Durham, S. L. On the use of log-transformation vs. nonlinear regression for analyzing biological power laws. Ecology 92, 1887–1894 (2011).

111. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114–2120 (2014).

112. Eren, A. M., Vineis, J. H., Morrison, H. G. & Sogin, M. L. A Filtering method to generate high quality short reads using Illumina paired-end technology. PLoS ONE 8, e66643 (2013).

113. Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).