Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Innovations to culturing the uncultured microbial majority

Nature Reviews Microbiology volume 19, pages 225–240 (2021)Cite this article

Subjects

Abstract

Despite the surge of microbial genome data, experimental testing is important to confirm inferences about the cell biology, ecological roles and evolution of microorganisms. As the majority of archaeal and bacterial diversity remains uncultured and poorly characterized, culturing is a priority. The growing interest in and need for efficient cultivation strategies has led to many rapid methodological and technological advances. In this Review, we discuss common barriers that can hamper the isolation and culturing of novel microorganisms and review emerging, innovative methods for targeted or high-throughput cultivation. We also highlight recent examples of successful cultivation of novel archaea and bacteria, and suggest key microorganisms for future cultivation attempts.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Cultured bacteria are biased towards Bacteroidetes, Proteobacteria, Firmicutes and Actinobacteria.
Fig. 2: Archaeal diversity is dominated by uncultured groups.
Fig. 3: Workflows for isolating novel microorganisms for cultivation using high-throughput or targeted approaches.
Fig. 4: Innovative methods for the isolation and cultivation of novel microorganisms.
Fig. 5: Reverse genomics for targeted isolation and cultivation of novel microorganisms.

Similar content being viewed by others

References

  1. Hug, L. A. et al. A new view of the tree of life. Nat. Microbiol. 1, 16048 (2016).

    CAS  PubMed  Google Scholar 

  2. Castelle, C. J. & Banfield, J. F. Major new microbial groups expand diversity and alter our understanding of the tree of life. Cell 172, 1181–1197 (2018).

    CAS  PubMed  Google Scholar 

  3. Mulder, A., Van de Graaf, A. A., Robertson, L. & Kuenen, J. Anaerobic ammonium oxidation discovered in a denitrifying fluidized bed reactor. FEMS Microbiol. Ecol. 16, 177–183 (1995).

    CAS  Google Scholar 

  4. Nunoura, T. et al. A primordial and reversible TCA cycle in a facultatively chemolithoautotrophic thermophile. Science 359, 559–563 (2018).

    CAS  PubMed  Google Scholar 

  5. Daims, H. et al. Complete nitrification by Nitrospira bacteria. Nature 528, 504–509 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Thrash, J. C. Culturing the uncultured: risk versus reward. mSystems 4, e00130–e00219 (2019).

    PubMed  PubMed Central  Google Scholar 

  7. Stewart, E. J. Growing unculturable bacteria. J. Bacteriol. 194, 4151–4160 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Overmann, J., Abt, B. & Sikorski, J. Present and future of culturing bacteria. Annu. Rev. Microbiol. 71, 711–730 (2017).

    CAS  PubMed  Google Scholar 

  9. Tyson, G. W. & Banfield, J. F. Cultivating the uncultivated: a community genomics perspective. Trends Microbiol. 13, 411–415 (2005).

    CAS  PubMed  Google Scholar 

  10. Carini, P. A. “Cultural” renaissance: genomics breathes new life into an old craft. mSystems https://doi.org/10.1128/mSystems.00092-19 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  11. Imachi, H. et al. Isolation of an archaeon at the prokaryote–eukaryote interface. Nature 577, 519–525 (2020). This study is the first to culture a member of the Asgard archaea group, the closest prokaryotic relatives of the eukaryote nuclear lineage, providing key insights into the evolutionary process of eukaryogenesis.

    CAS  PubMed  PubMed Central  Google Scholar 

  12. Hamm, J. N. et al. Unexpected host dependency of Antarctic Nanohaloarchaeota. Proc. Natl Acad. Sci. USA 116, 14661–14670 (2019). This study uses traditional enrichment techniques and single-cell sorting to isolate a novel Nanohaloarchaeota, belonging to the poorly characterized DPANN archaea group.

    CAS  PubMed  PubMed Central  Google Scholar 

  13. Chen, S.-C. et al. Anaerobic oxidation of ethane by archaea from a marine hydrocarbon seep. Nature 568, 108–111 (2019).

    CAS  PubMed  Google Scholar 

  14. Hahn, C. J. et al. “Candidatus Ethanoperedens,” a thermophilic genus of Archaea mediating the anaerobic oxidation of ethane. mBio 11, e00600–e00620 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Wiegand, S. et al. Cultivation and functional characterization of 79 planctomycetes uncovers their unique biology. Nat. Microbiol. 5, 126–140 (2019).

    PubMed  PubMed Central  Google Scholar 

  16. Henson, M. W., Lanclos, V. C., Faircloth, B. C. & Thrash, J. C. Cultivation and genomics of the first freshwater SAR11 (LD12) isolate. ISME J. 12, 1846–1860 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. Cross, K. L. et al. Targeted isolation and cultivation of uncultivated bacteria by reverse genomics. Nat. Biotechnol. 37, 1314–1321 (2019). This study demonstrates a novel targeted isolation method, using taxa-specific antibody labelling combined with FACS sorting, to culture a bacterium from a poorly characterized bacterial phylum with few cultured representatives.

    CAS  PubMed  PubMed Central  Google Scholar 

  18. Yu, H. & Leadbetter, J. R. Bacterial chemolithoautotrophy via manganese oxidation. Nature 583, 453–458 (2020).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. Song, Y. et al. Casimicrobium huifangae gen. nov., sp. nov., a ubiquitous “most-wanted” core bacterial taxon from municipal wastewater treatment plants. Appl. Environ. Microbiol. 86, 1038 (2019).

    Google Scholar 

  20. Parks, D. H. et al. Recovery of nearly 8,000 metagenome-assembled genomes substantially expands the tree of life. Nat. Microbiol. 2, 1533–1542 (2017).

    CAS  PubMed  Google Scholar 

  21. Parks, D. H. et al. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat. Biotechnol. 36, 996–1004 (2018).

    CAS  PubMed  Google Scholar 

  22. Yarza, P. et al. Uniting the classification of cultured and uncultured bacteria and archaea using 16S rRNA gene sequences. Nat. Rev. Microbiol. 12, 635–645 (2014).

    CAS  PubMed  Google Scholar 

  23. Castelle, C. J. et al. Genomic expansion of domain Archaea highlights roles for organisms from new phyla in anaerobic carbon cycling. Curr. Biol. 25, 690–701 (2015).

    CAS  PubMed  Google Scholar 

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

    CAS  PubMed  Google Scholar 

  25. Brown, C. T. et al. Unusual biology across a group comprising more than 15% of domain Bacteria. Nature 523, 208–211 (2015).

    CAS  PubMed  Google Scholar 

  26. Baker, B. J., Lazar, C. S., Teske, A. P. & Dick, G. J. Genomic resolution of linkages in carbon, nitrogen, and sulfur cycling among widespread estuary sediment bacteria. Microbiome 3, 1–12 (2015).

    Google Scholar 

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

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Lennon, J. T. & Locey, K. J. The underestimation of global microbial diversity. mBio 7, e01298–e01316 (2016).

    PubMed  PubMed Central  Google Scholar 

  29. Locey, K. J. & Lennon, J. T. Scaling laws predict global microbial diversity. Proc. Natl Acad. Sci. USA 113, 5970–5975 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Lennon, J. T. & Locey, K. J. More support for Earth’s massive microbiome. Biol. Direct 15, e3000106 (2020).

    Google Scholar 

  31. Reimer, L. C. et al. BacDive in 2019: bacterial phenotypic data for high-throughput biodiversity analysis. Nucleic Acids Res. 47, D631–D636 (2019).

    CAS  PubMed  Google Scholar 

  32. Garrity, G. M. NamesforLife BrowserTool takes expertise out of the database and puts it right in the browser. Microbiol. Today 37, 9. (2010).

    Google Scholar 

  33. Parte, A. C. LPSN — list of prokaryotic names with standing in nomenclature (bacterio.net), 20 years on. Int. J. Syst. Evol. Microbiol. 68, 1825–1829 (2018).

    PubMed  Google Scholar 

  34. Lloyd, K. G., Steen, A. D., Ladau, J., Yin, J. & Crosby, L. Phylogenetically novel uncultured microbial cells dominate earth microbiomes. mSystems https://doi.org/10.1128/mSystems.00055-18 (2018). This study demonstrates that a substantial fraction of the microorganisms on Earth belong to phylogenetic groups without cultured representatives.

    Article  PubMed  PubMed Central  Google Scholar 

  35. Olsen, G. J., Lane, D. J., Giovannoni, S. J., Pace, N. R. & Stahl, D. A. Microbial ecology and evolution: a ribosomal RNA approach. Annu. Rev. Microbiol. 40, 337–365 (1986).

    CAS  PubMed  Google Scholar 

  36. Staley, J. T. & Konopka, A. Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial habitats. Annu. Rev. Microbiol. 39, 321–346 (1985).

    CAS  PubMed  Google Scholar 

  37. Overmann, J. Principles of enrichment, isolation, cultivation and preservation of prokaryotes. Prokaryotes 1, 80–136 (2006).

    Google Scholar 

  38. Davis, K. E., Joseph, S. J. & Janssen, P. H. Effects of growth medium, inoculum size, and incubation time on culturability and isolation of soil bacteria. Appl. Environ. Microbiol. 71, 826–834 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. Kappler, A. & Bryce, C. Cryptic biogeochemical cycles: unravelling hidden redox reactions. Environ. Microbiol. 19, 842–846 (2017).

    PubMed  Google Scholar 

  40. Spang, A. et al. Proposal of the reverse flow model for the origin of the eukaryotic cell based on comparative analyses of Asgard archaeal metabolism. Nat. Microbiol. 4, 1138–1148 (2019).

    CAS  PubMed  Google Scholar 

  41. Zhou, Z., Pan, J., Wang, F., Gu, J.-D. & Li, M. Bathyarchaeota: globally distributed metabolic generalists in anoxic environments. FEMS Microbiol. Rev. 42, 639–655 (2018).

    CAS  PubMed  Google Scholar 

  42. Lewis, K. Persister cells, dormancy and infectious disease. Nat. Rev. Microbiol. 5, 48–56 (2007).

    CAS  PubMed  Google Scholar 

  43. Lennon, J. T. & Jones, S. E. Microbial seed banks: the ecological and evolutionary implications of dormancy. Nat. Rev. Microbiol. 9, 119–130 (2011).

    CAS  PubMed  Google Scholar 

  44. Hoehler, T. M. & Jørgensen, B. B. Microbial life under extreme energy limitation. Nat. Rev. Microbiol. 11, 83–94 (2013).

    CAS  PubMed  Google Scholar 

  45. Epstein, S. The phenomenon of microbial uncultivability. Curr. Opin. Microbiol. 16, 636–642 (2013).

    CAS  PubMed  Google Scholar 

  46. Nichols, D. et al. Short peptide induces an “uncultivable” microorganism to grow in vitro. Appl. Environ. Microbiol. 74, 4889–4897 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  47. Dworkin, J. & Shah, I. M. Exit from dormancy in microbial organisms. Nat. Rev. Microbiol. 8, 890–896 (2010).

    CAS  PubMed  Google Scholar 

  48. Zengler, K. & Zaramela, L. S. The social network of microorganisms—how auxotrophies shape complex communities. Nat. Rev. Microbiol. 16, 383–390 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  49. Stams, A. J. M. & Plugge, C. M. Electron transfer in syntrophic communities of anaerobic bacteria and archaea. Nat. Rev. Microbiol. 7, 568–577 (2009).

    CAS  PubMed  Google Scholar 

  50. Hidalgo-Ahumada, C. A. P. et al. Novel energy conservation strategies and behaviour of Pelotomaculum schinkii driving syntrophic propionate catabolism. Environ. Microbiol. 20, 4503–4511 (2018).

    CAS  PubMed  Google Scholar 

  51. Lovley, D. R. Syntrophy goes electric: direct interspecies electron transfer. Annu. Rev. Microbiol. 71, 643–664 (2017).

    CAS  PubMed  Google Scholar 

  52. Guzman, J. J. L., Sousa, D. Z. & Angenent, L. T. Development of a bioelectrochemical system as a tool to enrich H2-producing syntrophic bacteria. Front. Microbiol. 10, 110 (2019).

    PubMed  PubMed Central  Google Scholar 

  53. Valentine, D. L., Reeburgh, W. S. & Blanton, D. C. A culture apparatus for maintaining H2 at sub-nanomolar concentrations. J. Microbiol. Methods 39, 243–251 (2000).

    CAS  PubMed  Google Scholar 

  54. Mountfort, D. O. & Kaspar, H. F. Palladium-mediated hydrogenation of unsaturated hydrocarbons with hydrogen gas released during anaerobic cellulose degradation. Appl. Environ. Microbiol. 52, 744–750 (1986).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Stieb, M. & Schink, B. Cultivation of syntrophic anaerobic bacteria in membrane-separated culture devices. FEMS Microbiol. Lett. 45, 71–76 (1987).

    CAS  Google Scholar 

  56. de Bok, F. A. M., Luijten, M. L. G. C. & Stams, A. J. M. Biochemical evidence for formate transfer in syntrophic propionate-oxidizing cocultures of Syntrophobacter fumaroxidans and Methanospirillum hungatei. Appl. Environ. Microbiol. 68, 4247–4252 (2002).

    PubMed  PubMed Central  Google Scholar 

  57. Beaty, P. S., Wofford, N. Q. & McInerney, M. J. Separation of Syntrophomonas wolfei from Methanospirillum hungatii in syntrophic cocultures by using Percoll gradients. Appl. Environ. Microbiol. 53, 1183–1185 (1987).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. Lynch, M. D. J. & Neufeld, J. D. Ecology and exploration of the rare biosphere. Nat. Rev. Microbiol. 13, 217–229 (2015).

    CAS  PubMed  Google Scholar 

  59. Janssen, P. H. Selective enrichment and purification of cultures of Methanosaeta spp. J. Microbiol. Methods 52, 239–244 (2003).

    CAS  PubMed  Google Scholar 

  60. Nichols, D. et al. Use of iChip for high-throughput in situ cultivation of “uncultivable” microbial species. Appl. Environ. Microbiol. 76, 2445–2450 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  61. Aoi, Y. et al. Hollow-fiber membrane chamber as a device for in situ environmental cultivation. Appl. Environ. Microbiol. 75, 3826–3833 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. Ma, L. et al. Individually addressable arrays of replica microbial cultures enabled by splitting SlipChips. Integr. Biol. 6, 796–805 (2014).

    CAS  Google Scholar 

  63. Ge, Z., Girguis, P. R. & Buie, C. R. Nanoporous microscale microbial incubators. Lab Chip 16, 480–488 (2016).

    CAS  PubMed  Google Scholar 

  64. Connon, S. A. & Giovannoni, S. J. High-throughput methods for culturing microorganisms in very-low-nutrient media yield diverse new marine isolates. Appl. Environ. Microbiol. 68, 3878–3885 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  65. Bartelme, R. P. et al. Influence of substrate concentration on the culturability of heterotrophic soil microbes isolated by high-throughput dilution-to-extinction cultivation. mSphere 5, e00024-20 (2020).

    PubMed  PubMed Central  Google Scholar 

  66. Kaeberlein, T., Lewis, K. & Epstein, S. S. Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296, 1127–1129 (2002).

    CAS  PubMed  Google Scholar 

  67. Dorofeev, A. G. et al. Approaches to cultivation of “nonculturable” bacteria: cyclic cultures. Microbiology 83, 450–461 (2014).

    CAS  Google Scholar 

  68. Goordial, J. et al. Field sequencing and life detection in remote (79°26′N) Canadian high Arctic permafrost ice wedge microbial communities. Front. Microbiol. 8, 2594 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  69. Pascual, J., García-López, M., González, I. & Genilloud, O. Luteolibacter gellanilyticus sp. nov., a gellan-gum-degrading bacterium of the phylum Verrucomicrobia isolated from miniaturized diffusion chambers. Int. J. Syst. Evol. Microbiol. 67, 3951–3959 (2017).

    CAS  PubMed  Google Scholar 

  70. Ling, L. L. et al. A new antibiotic kills pathogens without detectable resistance. Nature 517, 455–459 (2015). This study uses an iChip to culture novel environmental microorganisms, leading to the discovery of a new type of antibiotic, demonstrating one of the functional benefits of culturing novel microorganisms.

    CAS  PubMed  PubMed Central  Google Scholar 

  71. Ferrari, B. C., Binnerup, S. J. & Gillings, M. Microcolony cultivation on a soil substrate membrane system selects for previously uncultured soil bacteria. Appl. Environ. Microbiol. 71, 8714–8720 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  72. Svenning, M. M., Wartiainen, I., Hestnes, A. G. & Binnerup, S. J. Isolation of methane oxidising bacteria from soil by use of a soil substrate membrane system. FEMS Microbiol. Ecol. 44, 347–354 (2003).

    CAS  PubMed  Google Scholar 

  73. Zhao, H. et al. Different phenanthrene-degrading bacteria cultured by in situ soil substrate membrane system and traditional cultivation. Int. Biodeterior. Biodegrad. 117, 269–277 (2017).

    CAS  Google Scholar 

  74. Pudasaini, S. et al. Microbial diversity of Browning Peninsula, Eastern Antarctica revealed using molecular and cultivation methods. Front. Microbiol. 8, 591 (2017).

    PubMed  PubMed Central  Google Scholar 

  75. de Bruyn, J. C., Boogerd, F. C., Bos, P. & Kuenen, J. G. Floating filters, a novel technique for isolation and enumeration of fastidious, acidophilic, iron-oxidizing, autotrophic bacteria. Appl. Environ. Microbiol. 56, 2891–2894 (1990).

    PubMed  PubMed Central  Google Scholar 

  76. Sipkema, D. et al. Multiple approaches to enhance the cultivability of bacteria associated with the marine sponge Haliclona (gellius) sp. Appl. Environ. Microbiol. 77, 2130–2140 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  77. Chaudhary, D. K., Khulan, A. & Kim, J. Development of a novel cultivation technique for uncultured soil bacteria. Sci. Rep. 9, 6666 (2019).

    PubMed  PubMed Central  Google Scholar 

  78. Ma, L. et al. Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project’s most wanted taxa. Proc. Natl Acad. Sci. USA 111, 9768–9773 (2014). This study uses the microfluidic SlipChip device to culture the first member of the genus Ruminococcae, which has relevance for better understanding the human microbiome.

    CAS  PubMed  PubMed Central  Google Scholar 

  79. Du, W., Li, L., Nichols, K. P. & Ismagilov, R. F. SlipChip. Lab Chip 9, 2286 (2009).

    CAS  PubMed  PubMed Central  Google Scholar 

  80. Fodor, A. A. et al. The “most wanted” taxa from the human microbiome for whole genome sequencing. PloS ONE 7, e41294 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  81. Jiang, C.-Y. et al. High-throughput single-cell cultivation on microfluidic streak plates. Appl. Environ. Microbiol. 82, 2210–2218 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  82. Hu, B. et al. High-throughput single-cell cultivation reveals the underexplored rare biosphere in deep-sea sediments along the Southwest Indian Ridge. Lab Chip 20, 363–372 (2020).

    PubMed  Google Scholar 

  83. Park, J., Kerner, A., Burns, M. A. & Lin, X. N. Microdroplet-enabled highly parallel co-cultivation of microbial communities. PloS ONE 6, e17019 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  84. Watterson, W. J. et al. Droplet-based high-throughput cultivation for accurate screening of antibiotic resistant gut microbes. eLife 9, e56998 (2020). This study demonstrates a high-throughput droplet cultivation method that can isolate and grow large numbers of single cells from environmental samples anaerobically.

    CAS  PubMed  PubMed Central  Google Scholar 

  85. Zengler, K. et al. Cultivating the uncultured. Proc. Natl Acad. Sci. USA 99, 15681–15686 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Eun, Y.-J., Utada, A. S., Copeland, M. F., Takeuchi, S. & Weibel, D. B. Encapsulating bacteria in agarose microparticles using microfluidics for high-throughput cell analysis and isolation. ACS Chem. Biol. 6, 260–266 (2011).

    CAS  PubMed  Google Scholar 

  87. Kaminski, T. S., Scheler, O. & Garstecki, P. Droplet microfluidics for microbiology: techniques, applications and challenges. Lab Chip 16, 2168–2187 (2016).

    CAS  PubMed  Google Scholar 

  88. Boitard, L., Cottinet, D., Bremond, N., Baudry, J. & Bibette, J. Growing microbes in millifluidic droplets. Eng. Life Sci. 15, 318–326 (2015).

    CAS  Google Scholar 

  89. Najah, M. et al. Droplet-based microfluidics platform for ultra-high-throughput bioprospecting of cellulolytic microorganisms. Chem. Biol. 21, 1722–1732 (2014).

    CAS  PubMed  Google Scholar 

  90. Leung, K. et al. A programmable droplet-based microfluidic device applied to multiparameter analysis of single microbes and microbial communities. Proc. Natl Acad. Sci. USA 109, 7665–7670 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  91. Shields, C. W. IV, Reyes, C. D. & López, G. P. Microfluidic cell sorting: a review of the advances in the separation of cells from debulking to rare cell isolation. Lab Chip 15, 1230–1249 (2015).

    PubMed  PubMed Central  Google Scholar 

  92. Wang, X. et al. Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies. Lab Chip 11, 3656–3662 (2011).

    CAS  PubMed  Google Scholar 

  93. Huber, H. et al. A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417, 63–67 (2002).

    CAS  PubMed  Google Scholar 

  94. Zhang, H. & Liu, K.-K. Optical tweezers for single cells. J. R. Soc. Interface 5, 671–690 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  95. Wagner, M. & Haider, S. New trends in fluorescence in situ hybridization for identification and functional analyses of microbes. Curr. Opin. Biotechnol. 23, 96–102 (2012).

    CAS  PubMed  Google Scholar 

  96. Haroon, M. F. et al. In-solution fluorescence in situ hybridization and fluorescence-activated cell sorting for single cell and population genome recovery Methods Enzymol. 531, 3–19 (2013).

    CAS  PubMed  Google Scholar 

  97. Batani, G., Bayer, K., Böge, J., Hentschel, U. & Thomas, T. Fluorescence in situ hybridization (FISH) and cell sorting of living bacteria. Sci. Rep. 9, 18618 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  98. Lau, A. Y., Lee, L. P. & Chan, J. W. An integrated optofluidic platform for Raman-activated cell sorting. Lab Chip 8, 1116–1120 (2008).

    CAS  PubMed  Google Scholar 

  99. Berry, D. et al. Tracking heavy water (D2O) incorporation for identifying and sorting active microbial cells. Proc. Natl Acad. Sci. USA 112, E194–E203 (2015).

    CAS  PubMed  Google Scholar 

  100. Lee, K. S. et al. An automated Raman-based platform for the sorting of live cells by functional properties. Nat. Microbiol. 4, 1035–1048 (2019).

    CAS  PubMed  Google Scholar 

  101. Müller, S. & Nebe-von-Caron, G. Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities. FEMS Microbiol. Rev. 34, 554–587 (2010).

    PubMed  Google Scholar 

  102. Morono, Y., Terada, T., Kallmeyer, J. & Inagaki, F. An improved cell separation technique for marine subsurface sediments: applications for high–throughput analysis using flow cytometry and cell sorting. Environ. Microbiol. 15, 2841–2849 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  103. Gutleben, J. et al. The multi-omics promise in context: from sequence to microbial isolate. Crit. Rev. Microbiol. 44, 212–229 (2018).

    CAS  PubMed  Google Scholar 

  104. Sauer, D. B. & Wang, D.-N. Predicting the optimal growth temperatures of prokaryotes using only genome derived features. Bioinformatics 35, 3224–3231 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. Su, M., Satola, S. W. & Read, T. D. Genome-based prediction of bacterial antibiotic resistance. J. Clin. Microbiol. 57, e01405–e01418 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  106. Hatzenpichler, R., Krukenberg, V., Spietz, R. L. & Jay, Z. J. Next-generation physiology approaches to study microbiome function at single cell level. Nat. Rev. Microbiol. 18, 241–256 (2020). This review describes a range of methods that can be used to gain a better understanding of the physiological functions that microorganisms have in their natural environments, which can also be used to inform cultivation strategies.

    CAS  PubMed  PubMed Central  Google Scholar 

  107. Tillich, U. M. et al. High-throughput cultivation and screening platform for unicellular phototrophs. BMC Microbiol. 14, 239 (2014).

    PubMed  PubMed Central  Google Scholar 

  108. Bassi, C. A. & Benson, D. R. Growth characteristics of the slow–growing actinobacterium Frankia sp. strain CcI3 on solid media. Physiol. Plant. 130, 391–399 (2007).

    CAS  Google Scholar 

  109. Tahon, G. & Willems, A. Isolation and characterization of aerobic anoxygenic phototrophs from exposed soils from the Sør Rondane Mountains, East Antarctica. Syst. Appl. Microbiol. 40, 357–369 (2017).

    CAS  PubMed  Google Scholar 

  110. Thrash, J. C., Weckhorst, J. L. & Pitre, D. M. in Hydrocarbon and Lipid Microbiology Protocols Vol. 39 Springer Protocols Handbooks (eds McGenity T. J., Timmis K. N. & Nogales B.) 57–78 (Springer, 2017).

  111. Schrader, C., Schielke, A., Ellerbroek, L. & Johne, R. PCR inhibitors — occurrence, properties and removal. J. Appl. Microbiol. 113, 1014–1026 (2012).

    CAS  PubMed  Google Scholar 

  112. Kim, M. & Chun, J. in New Approaches to Prokaryotic Systematics Vol. 41 (eds Goodfellow, M., Sutcliffe, I. & Chu, J.) 61–74 (Elsevier, 2014).

  113. Bahram, M., Anslan, S., Hildebrand, F., Bork, P. & Tedersoo, L. Newly designed 16S rRNA metabarcoding primers amplify diverse and novel archaeal taxa from the environment. Environ. Microbiol. Rep. 11, 487–494 (2019).

    PubMed  Google Scholar 

  114. Parada, A. E., Needham, D. M. & Fuhrman, J. A. Every base matters: assessing small subunit rRNA primers for marine microbiomes with mock communities, time series and global field samples. Environ. Microbiol. 18, 1403–1414 (2016).

    CAS  PubMed  Google Scholar 

  115. Apprill, A., McNally, S., Parsons, R. & Weber, L. Minor revision to V4 region SSU rRNA 806R gene primer greatly increases detection of SAR11 bacterioplankton. Aquat. Microb. Ecol. 75, 129–137 (2015).

    Google Scholar 

  116. Rettedal, E. A., Gumpert, H. & Sommer, M. O. A. Cultivation-based multiplex phenotyping of human gut microbiota allows targeted recovery of previously uncultured bacteria. Nat. Commun. 5, 4714 (2014).

    CAS  PubMed  Google Scholar 

  117. Eloe-Fadrosh, E. A., Ivanova, N. N., Woyke, T. & Kyrpides, N. C. Metagenomics uncovers gaps in amplicon-based detection of microbial diversity. Nat. Microbiol. 1, 1–4 (2016).

    Google Scholar 

  118. Fiedler, C. J. et al. Assessment of microbial community dynamics in river bank filtrate using high-throughput sequencing and flow cytometry. Front. Microbiol. 9, 2887 (2018).

    PubMed  PubMed Central  Google Scholar 

  119. Props, R. et al. Absolute quantification of microbial taxon abundances. ISME J. 11, 584–587 (2016).

    PubMed  PubMed Central  Google Scholar 

  120. Kim, S. et al. High-throughput automated microfluidic sample preparation for accurate microbial genomics. Nat. Commun. 8, 1–10 (2017).

    Google Scholar 

  121. Dumolin, C. et al. Introducing SPeDE: high-throughput dereplication and accurate determination of microbial diversity from matrix-assisted laser desorption–ionization time of flight mass spectrometry data. mSystems https://doi.org/10.1128/mSystems.00437-19 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  122. Santos, I. C., Hildenbrand, Z. L. & Schug, K. A. Applications of MALDI-TOF MS in environmental microbiology. Analyst 141, 2827–2837 (2016).

    CAS  PubMed  Google Scholar 

  123. Kodama, Y., Shumway, M. & Leinonen, R. The Sequence Read Archive: explosive growth of sequencing data. Nucleic Acids Res. 40, D54–D56 (2012).

    CAS  PubMed  Google Scholar 

  124. Zaremba-Niedzwiedzka, K. et al. Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature 541, 353–358 (2017).

    CAS  PubMed  Google Scholar 

  125. Spang, A. et al. Complex archaea that bridge the gap between prokaryotes and eukaryotes. Nature 521, 173–179 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  126. Williams, T. A., Cox, C. J., Foster, P. G., SzöllŐsi, G. J. & Embley, T. M. Phylogenomics provides robust support for a two-domains tree of life. Nat. Ecol. Evol. 4, 138–147 (2020).

    PubMed  Google Scholar 

  127. Bhattarai, S., Cassarini, C. & Lens, P. Physiology and distribution of archaeal methanotrophs that couple anaerobic oxidation of methane with sulfate reduction. Microbiol. Mol. Biol. Rev. 83, e00074–e00118 (2019).

    PubMed  PubMed Central  Google Scholar 

  128. Vanwonterghem, I. et al. Methylotrophic methanogenesis discovered in the archaeal phylum Verstraetearchaeota. Nat. Microbiol. 1, 1–9 (2016).

    Google Scholar 

  129. Dombrowski, N., Lee, J.-H., Williams, T. A., Offre, P. & Spang, A. Genomic diversity, lifestyles and evolutionary origins of DPANN archaea. FEMS Microbiol. Lett. 366, fnz008 (2019).

    CAS  PubMed Central  Google Scholar 

  130. Zhang, C. L., Xie, W., Martin-Cuadrado, A.-B. & Rodriguez-Valera, F. Marine Group II archaea, potentially important players in the global ocean carbon cycle. Front. Microbiol. 6, 1108 (2015).

    PubMed  PubMed Central  Google Scholar 

  131. Haro-Moreno, J. M., Rodriguez-Valera, F., López-García, P., Moreira, D. & Martin-Cuadrado, A.-B. New insights into Marine Group III Euryarchaeota, from dark to light. ISME J. 11, 1102–1117 (2017).

    CAS  PubMed  PubMed Central  Google Scholar 

  132. Reji, L., Tolar, B. B., Smith, J. M., Chavez, F. P. & Francis, C. A. Differential co-occurrence relationships shaping ecotype diversification within Thaumarchaeota populations in the coastal ocean water column. ISME J. 13, 1144–1158 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. Kielak, A. M., Barreto, C. C., Kowalchuk, G. A., van Veen, J. A. & Kuramae, E. E. The ecology of Acidobacteria: moving beyond genes and genomes. Front. Microbiol. 7, 744 (2016).

    PubMed  PubMed Central  Google Scholar 

  134. Becraft, E. D. et al. Rokubacteria: genomic giants among the uncultured bacterial phyla. Front. Microbiol. 8, 2264 (2017).

    PubMed  PubMed Central  Google Scholar 

  135. Ghai, R., Mizuno, C. M., Picazo, A., Camacho, A. & Rodriguez-Valera, F. Metagenomics uncovers a new group of low GC and ultra-small marine Actinobacteria. Sci. Rep. 3, 2471 (2013).

    PubMed  PubMed Central  Google Scholar 

  136. Liu, Y.-F. et al. Anaerobic hydrocarbon degradation in candidate phylum ‘Atribacteria’ (JS1) inferred from genomics. ISME J. 13, 2377–2390 (2019).

    CAS  PubMed  PubMed Central  Google Scholar 

  137. Nobu, M. K. et al. Phylogeny and physiology of candidate phylum ‘Atribacteria’ (OP9/JS1) inferred from cultivation-independent genomics. ISME J. 10, 273–286 (2016).

    CAS  PubMed  Google Scholar 

  138. Ji, M. et al. Atmospheric trace gases support primary production in Antarctic desert surface soil. Nature 552, 400–403 (2017).

    CAS  PubMed  Google Scholar 

  139. Hawley, A. K. et al. Diverse Marinimicrobia bacteria may mediate coupled biogeochemical cycles along eco-thermodynamic gradients. Nat. Commun. 8, 1–10 (2017).

    CAS  Google Scholar 

  140. Brewer, T. E., Handley, K. M., Carini, P., Gilbert, J. A. & Fierer, N. Genome reduction in an abundant and ubiquitous soil bacterium ‘Candidatus Udaeobacter copiosus’. Nat. Microbiol. 2, 1–7 (2016).

    Google Scholar 

  141. Major, D. W. et al. Field demonstration of successful bioaugmentation to achieve dechlorination of tetrachloroethene to ethene. Environ. Sci. Technol. 36, 5106–5116 (2002).

    CAS  PubMed  Google Scholar 

  142. Okazaki, Y., Salcher, M. M., Callieri, C. & Nakano, S.-I. The broad habitat spectrum of the CL500-11 lineage (phylum Chloroflexi), a dominant bacterioplankton in oxygenated hypolimnia of deep freshwater lakes. Front. Microbiol. 9, 2891 (2018).

    PubMed  PubMed Central  Google Scholar 

  143. Mehrshad, M., Rodriguez-Valera, F., Amoozegar, M. A., López-García, P. & Ghai, R. The enigmatic SAR202 cluster up close: shedding light on a globally distributed dark ocean lineage involved in sulfur cycling. ISME J. 12, 655–668 (2018).

    CAS  PubMed  Google Scholar 

  144. Sheik, C. S., Jain, S. & Dick, G. J. Metabolic flexibility of enigmatic SAR324 revealed through metagenomics and metatranscriptomics. Environ. Microbiol. 16, 304–317 (2014).

    CAS  PubMed  Google Scholar 

  145. Dupont, C. L. et al. Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage. ISME J. 6, 1186–1199 (2012).

    CAS  PubMed  Google Scholar 

  146. Delgado-Baquerizo, M. et al. A global atlas of the dominant bacteria found in soil. Science 359, 320–325 (2018).

    CAS  PubMed  Google Scholar 

  147. Wu, L. et al. Global diversity and biogeography of bacterial communities in wastewater treatment plants. Nat. Microbiol. 4, 1183–1195 (2019).

    CAS  PubMed  Google Scholar 

  148. in ‘t Zandt, M. H., de Jong, A. E., Slomp, C. P. & Jetten, M. S. The hunt for the most-wanted chemolithoautotrophic spookmicrobes. FEMS Microbiol. Ecol. 94, fiy064 (2018).

    PubMed Central  Google Scholar 

  149. Yamaguchi, M. et al. Prokaryote or eukaryote? A unique microorganism from the deep sea. J. Electron. Microsc. 61, 423–431 (2012).

    CAS  Google Scholar 

  150. Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  151. Capella-Gutiérrez, S., Silla-Martínez, J. M. & Gabaldón, T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 25, 1972–1973 (2009).

    PubMed  PubMed Central  Google Scholar 

  152. Nguyen, L.-T., Schmidt, H. A., von Haeseler, A. & Minh, B. Q. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol. Biol. Evol. 32, 268–274 (2015).

    CAS  PubMed  Google Scholar 

  153. Hoang, D. T., Chernomor, O., von Haeseler, A., Minh, B. Q. & Vinh, L. S. UFBoot2: improving the ultrafast bootstrap approximation. Mol. Biol. Evol. 35, 518–522 (2018).

    CAS  PubMed  Google Scholar 

  154. Starr, M. P., Stolp, H., Trüper, H. G., Balows, A. & Schlegel, H. G. The Prokaryotes: A Handbook on Habitats, Isolation and Identification of Bacteria (Springer Science & Business Media, 2013).

  155. Tamaki, H. et al. Comparative analysis of bacterial diversity in freshwater sediment of a shallow eutrophic lake by molecular and improved cultivation-based techniques. Appl. Environ. Microbiol. 71, 2162–2169 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  156. Wolfe, R., Thauer, R. & Pfennig, N. A ‘capillary racetrack’ method for isolation of magnetotactic bacteria. FEMS Microbiol. Ecol. 3, 31–35 (1987).

    Google Scholar 

  157. Goossens, H., Wauters, G., De Boeck, M., Janssens, M. & Butzler, J. Semisolid selective-motility enrichment medium for isolation of salmonellae from fecal specimens. J. Clin. Microbiol. 19, 940–941 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  158. Yu, H. S. & Alam, M. An agarose-in-plug bridge method to study chemotaxis in the Archaeon Halobacterium salinarum. FEMS Microbiol. Lett. 156, 265–269 (1997).

    CAS  PubMed  Google Scholar 

  159. Tanaka, T. et al. A hidden pitfall in the preparation of agar media undermines microorganism cultivability. Appl. Environ. Microbiol. 80, 7659–7666 (2014).

    PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors are grateful to H. Smidt for inspiring discussions and to F. Homa for performing the phylogenetic analyses for the trees depicted in Figs 1 and 2. This work was supported by grants from the European Research Council (ERC consolidator grant 817834), the Dutch Research Council (NWO-VICI grant VI.C.192.016) and the Wellcome Trust foundation (Collaborative award 203276/K/16/Z) to T.J.G.E.

Author information

Authors and Affiliations

  1. Laboratory of Microbiology, Wageningen University and Research, Wageningen, The Netherlands

    William H. Lewis, Guillaume Tahon, Patricia Geesink, Diana Z. Sousa & Thijs J. G. Ettema

Authors
  1. William H. Lewis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Guillaume Tahon
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Patricia Geesink
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Diana Z. Sousa
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thijs J. G. Ettema
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

The authors contributed equally to all aspects of the article.

Corresponding author

Correspondence to Thijs J. G. Ettema.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Peer review information

Nature Reviews Microbiology thanks Slava Epstein, Hiroyuki Imachi, Yoichi Kamagata and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Glossary

Enrichments

Assemblages of several strains that evolve from a taxonomically diverse inoculum in response to controlled environmental selection pressures (such as substrates or temperature).

Pure cultures

Cultures containing cells belonging to the same strain, ideally originating from a single cell or colony, that have minimal genetic variation between them. Also often called axenic cultures.

Co-cultures

Defined assemblages of two or more strains, often artificially introduced and grown together in the laboratory, which may establish interspecies metabolic relationships with one another.

Isolation

The physical separation of a single cell, strain or species from others found in the same sample or habitat.

Fluorescence in situ hybridization

(FISH). A method of labelling cells with a fluorescent signal by binding fluorophore-coupled oligonucleotide probes to complementary target molecules (usually 16S rRNA) in biological samples. Probes can be designed to be highly taxon-specific, making it possible to taxonomically identify microorganisms on the single-cell level.

Dilution-to-extinction

A method of serially diluting a mixed community culture with the aim of isolating single cells that will grow and divide to establish monoclonal and axenic cultures. Can also be called limited dilution.

Growth factors

Any substance that can be used by an organism to facilitate growth.

Symbiosis

The association, usually a physical or metabolic interaction, of two or more organisms, which typically has an influence on the fitness of one or more of the partners involved.

Syntrophy

An interspecies relationship in which metabolites produced by one species are used as growth substrates by another species.

Anaerobic

An organism that grows in the absence of molecular O2.

Inocula

Samples of microorganisms introduced to fresh medium for initiating the growth of a new culture.

Optical tweezers

A method for isolating single cells from cellular suspensions by microscopy and laser capture. Many optical tweezer set-ups are now automated and operate in microfluidic chips. Cells are passed through these chips in a suspension, and those with a detectable phenotype are captured, relocated from the main flow to a sterile outlet and collected.

Phenotypes

The observable or detectable traits of an organism influenced by its genes (genotype) and factors of its environment.

Fluorescence-activated cell sorting

(FACS). The dispersion of cells into separate containers, such as test tubes or wells, based on either natural or artificially induced fluorescent properties (for example, by fluorescent stains or labelling techniques).

Genome-resolved metagenomics

The reconstruction of genome sequences from metagenomic data, typically obtained through bioinformatics approaches in which contigs from a single microorganism are grouped (‘binned’) together.

Anoxic

A state of complete absence of molecular O2, for example, in an environment or a culture.

Optical density

A common spectrophotometric method for assessing the cell density of a liquid suspension, typically by measuring the extent at which light at a 600 nm wavelength is scattered by cells as it passes through a sample.

Flow cytometry

A technique used to detect and count cells based on physical or chemical properties.

MALDI-TOF mass spectrometry

MALDI is an ionization technique used in mass spectrometric analysis based on embedding samples in a special matrix from which they are desorbed by laser light. The technique allows the analysis of biomolecules and organic molecules.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Lewis, W.H., Tahon, G., Geesink, P. et al. Innovations to culturing the uncultured microbial majority. Nat Rev Microbiol 19, 225–240 (2021). https://doi.org/10.1038/s41579-020-00458-8

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41579-020-00458-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing Microbiology

Sign up for the Nature Briefing: Microbiology newsletter — what matters in microbiology research, free to your inbox weekly.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing: Microbiology