Innovations to culturing the uncultured microbial majority

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.

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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.

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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.

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Correspondence to Thijs J. G. Ettema.

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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.

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Lewis, W.H., Tahon, G., Geesink, P. et al. Innovations to culturing the uncultured microbial majority. Nat Rev Microbiol (2020). https://doi.org/10.1038/s41579-020-00458-8

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