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
Open Access articles citing this article.
Nature Communications Open Access 22 December 2022
Vulcanimicrobium alpinus gen. nov. sp. nov., the first cultivated representative of the candidate phylum “Eremiobacterota”, is a metabolically versatile aerobic anoxygenic phototroph
ISME Communications Open Access 16 December 2022
Investigating the impact of database choice on the accuracy of metagenomic read classification for the rumen microbiome
Animal Microbiome Open Access 18 November 2022
Subscribe to Nature+
Get immediate online access to Nature and 55 other Nature journal
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Hug, L. A. et al. A new view of the tree of life. Nat. Microbiol. 1, 16048 (2016).
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).
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).
Nunoura, T. et al. A primordial and reversible TCA cycle in a facultatively chemolithoautotrophic thermophile. Science 359, 559–563 (2018).
Daims, H. et al. Complete nitrification by Nitrospira bacteria. Nature 528, 504–509 (2015).
Thrash, J. C. Culturing the uncultured: risk versus reward. mSystems 4, e00130–e00219 (2019).
Stewart, E. J. Growing unculturable bacteria. J. Bacteriol. 194, 4151–4160 (2012).
Overmann, J., Abt, B. & Sikorski, J. Present and future of culturing bacteria. Annu. Rev. Microbiol. 71, 711–730 (2017).
Tyson, G. W. & Banfield, J. F. Cultivating the uncultivated: a community genomics perspective. Trends Microbiol. 13, 411–415 (2005).
Carini, P. A. “Cultural” renaissance: genomics breathes new life into an old craft. mSystems https://doi.org/10.1128/mSystems.00092-19 (2019).
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.
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.
Chen, S.-C. et al. Anaerobic oxidation of ethane by archaea from a marine hydrocarbon seep. Nature 568, 108–111 (2019).
Hahn, C. J. et al. “Candidatus Ethanoperedens,” a thermophilic genus of Archaea mediating the anaerobic oxidation of ethane. mBio 11, e00600–e00620 (2020).
Wiegand, S. et al. Cultivation and functional characterization of 79 planctomycetes uncovers their unique biology. Nat. Microbiol. 5, 126–140 (2019).
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).
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.
Yu, H. & Leadbetter, J. R. Bacterial chemolithoautotrophy via manganese oxidation. Nature 583, 453–458 (2020).
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).
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).
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).
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).
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).
Rinke, C. et al. Insights into the phylogeny and coding potential of microbial dark matter. Nature 499, 431–437 (2013).
Brown, C. T. et al. Unusual biology across a group comprising more than 15% of domain Bacteria. Nature 523, 208–211 (2015).
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).
Curtis, T. P., Sloan, W. T. & Scannell, J. W. Estimating prokaryotic diversity and its limits. Proc. Natl Acad. Sci. USA 99, 10494–10499 (2002).
Lennon, J. T. & Locey, K. J. The underestimation of global microbial diversity. mBio 7, e01298–e01316 (2016).
Locey, K. J. & Lennon, J. T. Scaling laws predict global microbial diversity. Proc. Natl Acad. Sci. USA 113, 5970–5975 (2016).
Lennon, J. T. & Locey, K. J. More support for Earth’s massive microbiome. Biol. Direct 15, e3000106 (2020).
Reimer, L. C. et al. BacDive in 2019: bacterial phenotypic data for high-throughput biodiversity analysis. Nucleic Acids Res. 47, D631–D636 (2019).
Garrity, G. M. NamesforLife BrowserTool takes expertise out of the database and puts it right in the browser. Microbiol. Today 37, 9. (2010).
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).
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.
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).
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).
Overmann, J. Principles of enrichment, isolation, cultivation and preservation of prokaryotes. Prokaryotes 1, 80–136 (2006).
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).
Kappler, A. & Bryce, C. Cryptic biogeochemical cycles: unravelling hidden redox reactions. Environ. Microbiol. 19, 842–846 (2017).
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).
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).
Lewis, K. Persister cells, dormancy and infectious disease. Nat. Rev. Microbiol. 5, 48–56 (2007).
Lennon, J. T. & Jones, S. E. Microbial seed banks: the ecological and evolutionary implications of dormancy. Nat. Rev. Microbiol. 9, 119–130 (2011).
Hoehler, T. M. & Jørgensen, B. B. Microbial life under extreme energy limitation. Nat. Rev. Microbiol. 11, 83–94 (2013).
Epstein, S. The phenomenon of microbial uncultivability. Curr. Opin. Microbiol. 16, 636–642 (2013).
Nichols, D. et al. Short peptide induces an “uncultivable” microorganism to grow in vitro. Appl. Environ. Microbiol. 74, 4889–4897 (2008).
Dworkin, J. & Shah, I. M. Exit from dormancy in microbial organisms. Nat. Rev. Microbiol. 8, 890–896 (2010).
Zengler, K. & Zaramela, L. S. The social network of microorganisms—how auxotrophies shape complex communities. Nat. Rev. Microbiol. 16, 383–390 (2018).
Stams, A. J. M. & Plugge, C. M. Electron transfer in syntrophic communities of anaerobic bacteria and archaea. Nat. Rev. Microbiol. 7, 568–577 (2009).
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).
Lovley, D. R. Syntrophy goes electric: direct interspecies electron transfer. Annu. Rev. Microbiol. 71, 643–664 (2017).
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).
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).
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).
Stieb, M. & Schink, B. Cultivation of syntrophic anaerobic bacteria in membrane-separated culture devices. FEMS Microbiol. Lett. 45, 71–76 (1987).
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).
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).
Lynch, M. D. J. & Neufeld, J. D. Ecology and exploration of the rare biosphere. Nat. Rev. Microbiol. 13, 217–229 (2015).
Janssen, P. H. Selective enrichment and purification of cultures of Methanosaeta spp. J. Microbiol. Methods 52, 239–244 (2003).
Nichols, D. et al. Use of iChip for high-throughput in situ cultivation of “uncultivable” microbial species. Appl. Environ. Microbiol. 76, 2445–2450 (2010).
Aoi, Y. et al. Hollow-fiber membrane chamber as a device for in situ environmental cultivation. Appl. Environ. Microbiol. 75, 3826–3833 (2009).
Ma, L. et al. Individually addressable arrays of replica microbial cultures enabled by splitting SlipChips. Integr. Biol. 6, 796–805 (2014).
Ge, Z., Girguis, P. R. & Buie, C. R. Nanoporous microscale microbial incubators. Lab Chip 16, 480–488 (2016).
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).
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).
Kaeberlein, T., Lewis, K. & Epstein, S. S. Isolating “uncultivable” microorganisms in pure culture in a simulated natural environment. Science 296, 1127–1129 (2002).
Dorofeev, A. G. et al. Approaches to cultivation of “nonculturable” bacteria: cyclic cultures. Microbiology 83, 450–461 (2014).
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).
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).
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.
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).
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).
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).
Pudasaini, S. et al. Microbial diversity of Browning Peninsula, Eastern Antarctica revealed using molecular and cultivation methods. Front. Microbiol. 8, 591 (2017).
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).
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).
Chaudhary, D. K., Khulan, A. & Kim, J. Development of a novel cultivation technique for uncultured soil bacteria. Sci. Rep. 9, 6666 (2019).
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.
Du, W., Li, L., Nichols, K. P. & Ismagilov, R. F. SlipChip. Lab Chip 9, 2286 (2009).
Fodor, A. A. et al. The “most wanted” taxa from the human microbiome for whole genome sequencing. PloS ONE 7, e41294 (2012).
Jiang, C.-Y. et al. High-throughput single-cell cultivation on microfluidic streak plates. Appl. Environ. Microbiol. 82, 2210–2218 (2016).
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).
Park, J., Kerner, A., Burns, M. A. & Lin, X. N. Microdroplet-enabled highly parallel co-cultivation of microbial communities. PloS ONE 6, e17019 (2011).
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.
Zengler, K. et al. Cultivating the uncultured. Proc. Natl Acad. Sci. USA 99, 15681–15686 (2002).
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).
Kaminski, T. S., Scheler, O. & Garstecki, P. Droplet microfluidics for microbiology: techniques, applications and challenges. Lab Chip 16, 2168–2187 (2016).
Boitard, L., Cottinet, D., Bremond, N., Baudry, J. & Bibette, J. Growing microbes in millifluidic droplets. Eng. Life Sci. 15, 318–326 (2015).
Najah, M. et al. Droplet-based microfluidics platform for ultra-high-throughput bioprospecting of cellulolytic microorganisms. Chem. Biol. 21, 1722–1732 (2014).
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).
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).
Wang, X. et al. Enhanced cell sorting and manipulation with combined optical tweezer and microfluidic chip technologies. Lab Chip 11, 3656–3662 (2011).
Huber, H. et al. A new phylum of Archaea represented by a nanosized hyperthermophilic symbiont. Nature 417, 63–67 (2002).
Zhang, H. & Liu, K.-K. Optical tweezers for single cells. J. R. Soc. Interface 5, 671–690 (2008).
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).
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).
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).
Lau, A. Y., Lee, L. P. & Chan, J. W. An integrated optofluidic platform for Raman-activated cell sorting. Lab Chip 8, 1116–1120 (2008).
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).
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).
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).
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).
Gutleben, J. et al. The multi-omics promise in context: from sequence to microbial isolate. Crit. Rev. Microbiol. 44, 212–229 (2018).
Sauer, D. B. & Wang, D.-N. Predicting the optimal growth temperatures of prokaryotes using only genome derived features. Bioinformatics 35, 3224–3231 (2019).
Su, M., Satola, S. W. & Read, T. D. Genome-based prediction of bacterial antibiotic resistance. J. Clin. Microbiol. 57, e01405–e01418 (2019).
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.
Tillich, U. M. et al. High-throughput cultivation and screening platform for unicellular phototrophs. BMC Microbiol. 14, 239 (2014).
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).
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).
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).
Schrader, C., Schielke, A., Ellerbroek, L. & Johne, R. PCR inhibitors — occurrence, properties and removal. J. Appl. Microbiol. 113, 1014–1026 (2012).
Kim, M. & Chun, J. in New Approaches to Prokaryotic Systematics Vol. 41 (eds Goodfellow, M., Sutcliffe, I. & Chu, J.) 61–74 (Elsevier, 2014).
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).
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).
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).
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).
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).
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).
Props, R. et al. Absolute quantification of microbial taxon abundances. ISME J. 11, 584–587 (2016).
Kim, S. et al. High-throughput automated microfluidic sample preparation for accurate microbial genomics. Nat. Commun. 8, 1–10 (2017).
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).
Santos, I. C., Hildenbrand, Z. L. & Schug, K. A. Applications of MALDI-TOF MS in environmental microbiology. Analyst 141, 2827–2837 (2016).
Kodama, Y., Shumway, M. & Leinonen, R. The Sequence Read Archive: explosive growth of sequencing data. Nucleic Acids Res. 40, D54–D56 (2012).
Zaremba-Niedzwiedzka, K. et al. Asgard archaea illuminate the origin of eukaryotic cellular complexity. Nature 541, 353–358 (2017).
Spang, A. et al. Complex archaea that bridge the gap between prokaryotes and eukaryotes. Nature 521, 173–179 (2015).
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).
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).
Vanwonterghem, I. et al. Methylotrophic methanogenesis discovered in the archaeal phylum Verstraetearchaeota. Nat. Microbiol. 1, 1–9 (2016).
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).
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).
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).
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).
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).
Becraft, E. D. et al. Rokubacteria: genomic giants among the uncultured bacterial phyla. Front. Microbiol. 8, 2264 (2017).
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).
Liu, Y.-F. et al. Anaerobic hydrocarbon degradation in candidate phylum ‘Atribacteria’ (JS1) inferred from genomics. ISME J. 13, 2377–2390 (2019).
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).
Ji, M. et al. Atmospheric trace gases support primary production in Antarctic desert surface soil. Nature 552, 400–403 (2017).
Hawley, A. K. et al. Diverse Marinimicrobia bacteria may mediate coupled biogeochemical cycles along eco-thermodynamic gradients. Nat. Commun. 8, 1–10 (2017).
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).
Major, D. W. et al. Field demonstration of successful bioaugmentation to achieve dechlorination of tetrachloroethene to ethene. Environ. Sci. Technol. 36, 5106–5116 (2002).
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).
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).
Sheik, C. S., Jain, S. & Dick, G. J. Metabolic flexibility of enigmatic SAR324 revealed through metagenomics and metatranscriptomics. Environ. Microbiol. 16, 304–317 (2014).
Dupont, C. L. et al. Genomic insights to SAR86, an abundant and uncultivated marine bacterial lineage. ISME J. 6, 1186–1199 (2012).
Delgado-Baquerizo, M. et al. A global atlas of the dominant bacteria found in soil. Science 359, 320–325 (2018).
Wu, L. et al. Global diversity and biogeography of bacterial communities in wastewater treatment plants. Nat. Microbiol. 4, 1183–1195 (2019).
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).
Yamaguchi, M. et al. Prokaryote or eukaryote? A unique microorganism from the deep sea. J. Electron. Microsc. 61, 423–431 (2012).
Katoh, K. & Standley, D. M. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol. Biol. Evol. 30, 772–780 (2013).
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).
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).
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).
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).
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).
Wolfe, R., Thauer, R. & Pfennig, N. A ‘capillary racetrack’ method for isolation of magnetotactic bacteria. FEMS Microbiol. Ecol. 3, 31–35 (1987).
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).
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).
Tanaka, T. et al. A hidden pitfall in the preparation of agar media undermines microorganism cultivability. Appl. Environ. Microbiol. 80, 7659–7666 (2014).
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.
The authors declare no competing interests.
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.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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.
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.
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.
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.
An interspecies relationship in which metabolites produced by one species are used as growth substrates by another species.
An organism that grows in the absence of molecular O2.
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.
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.
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.
About this article
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
This article is cited by
Nature Methods (2023)
Discovering untapped microbial communities through metagenomics for microplastic remediation: recent advances, challenges, and way forward
Environmental Science and Pollution Research (2023)
Functional differentiation determines the molecular basis of the symbiotic lifestyle of Ca. Nanohaloarchaeota
Strain-level profiling with picodroplet microfluidic cultivation reveals host-specific adaption of honeybee gut symbionts
Investigating the impact of database choice on the accuracy of metagenomic read classification for the rumen microbiome
Animal Microbiome (2022)