Bringing the uncultivated microbial majority of freshwater ecosystems into culture

Michaela M. Salcher (), Paul Layoun, Clafy Fernandes, Maria-Cecilia Chiriac, Paul-Adrian Bulzu, Rohit Ghai, Tanja Shabarova, Vojtech Lanta, Cristiana Callieri, Bettina Sonntag, Thomas Posch, Fabio Lepori, Petr Znachor and Markus Haber
Additional contact information
Michaela M. Salcher: Biology Centre CAS
Paul Layoun: Biology Centre CAS
Clafy Fernandes: Biology Centre CAS
Maria-Cecilia Chiriac: Biology Centre CAS
Paul-Adrian Bulzu: Biology Centre CAS
Rohit Ghai: Biology Centre CAS
Tanja Shabarova: Biology Centre CAS
Vojtech Lanta: Institute of Botany of the Czech Academy of Sciences
Cristiana Callieri: Water Research Institute (IRSA) - National Research Council (CNR)
Bettina Sonntag: University of Innsbruck
Thomas Posch: University of Zurich
Fabio Lepori: University of Applied Sciences and Arts of Southern Switzerland (SUPSI)
Petr Znachor: Biology Centre CAS
Markus Haber: Biology Centre CAS

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract Axenic cultures are essential for studying microbial ecology, evolution, and genomics. Despite the importance of pure cultures, public culture collections are biased towards fast-growing copiotrophs, while many abundant aquatic prokaryotes remain uncultured due to uncharacterized growth requirements and oligotrophic lifestyles. Here, we applied high-throughput dilution-to-extinction cultivation using defined media that mimic natural conditions to samples from 14 Central European lakes, yielding 627 axenic strains. These cultures include 15 genera among the 30 most abundant freshwater bacteria identified via metagenomics, collectively representing up to 72% of genera detected in the original samples (average 40%) and are widespread in freshwater systems globally. Genome-sequenced strains are closely related to metagenome-assembled genomes (MAGs) from the same samples, many of which remain undescribed. We propose a classification of several novel families, genera, and species, including many slowly growing, genome-streamlined oligotrophs that are notoriously underrepresented in public repositories. Our large-scale initiative to cultivate the “uncultivated microbial majority” has yielded a valuable collection of abundant freshwater microbes, characterized by diverse metabolic pathways and lifestyles. This culture collection includes promising candidates for oligotrophic model organisms, suitable for a wide array of ecological studies aimed at advancing our ecological and functional understanding of dominant, yet previously uncultured, taxa.

Date: 2025
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DOI: 10.1038/s41467-025-63266-9

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