Cultivating the uncultivated majority – can we use genomic strategies to close this gap?

Microbes are the most dominant and diverse lifeform on Earth, yet the vast majority remain uncultivated.Despite advances in isolation methods, less than 0.5% of bacteria and archaea, and under 2% of fungal species, have been successfully cultured. [1][2]2][3] In extreme environments like terrestrial Antarctica, the gap is even wider, as metagenomic surveys continue to uncover diverse, underexplored microbial communities. [4][5]5][6] Most microbial life continues to exist as this microbial dark matter, recalcitrant to laboratory cultivation efforts.Given the role of microbes in climate change, geochemical cycling and their biomining potential, our focus needs to shift back to the isolation of the yet-to-be cultured majority.The advent of single-cell sequencing technologies, metagenomics and sophisticated differential coverage binning approaches now allow for the reconstruction of metagenome assembled genomes (MAGs) from diverse ecosystems. [5][6]6][7][8] The significant increase in genome data has led to ever changing views of the tree of life, and the development of new hypotheses around what metabolic capacities and ecological roles uncultivated taxa perform. 4,6While metagenomics has revolutionised our understanding of microbial diversity and functioning, it cannot replace experimental validation to confirm predictions on the cell biology, metabolism and physiological traits of microbial dark matter through the cultivation of pure cultures, co-cultures or stable enrichments.In the early 2000s, novel cultivation approaches began to close this gap, by simply mimicking the natural environment, extending incubation times and using dilute media, the ubiquitous SAR11 clade that had evaded cultivation for decades were recovered. 9,10e began to understand that not all microbes grow as macrocolonies, instead slow growing oligophiles or k-strategists which prefer nutrient poor-stable environments, and grow as microcolonies only -invisible to the naked eye. 11,12Once isolated, domestication allows microbes to adapt to an r-strategist lifestyle and become a faster growing species.Additionally, new understanding of metabolic cooperation and the requirements for signalling molecules have emerged, such as syntrophic relationships thus 'pure' cultures may not be possible, 13 and in 2018, a reverse-genomics cultivation method was developed that combined the design of antibodies to predicted surface proteins with cell sorting. 8These innovations led to the targeted isolation of the first members of the Saccharibacteria (formerly TM7), an epiparasite requiring an Actinobacterial host. 8,13ut the isolation of such rare, recalcitrant taxa requires patience, thinking outside the box, and in some cases a lot of luck.A new resurgence in targeted and innovative cultivation efforts is needed, and is timely, as genomes have been retrieved from a myriad of extreme environments with metabolic reconstructions of a taxa's functional potential invaluable for informing isolation efforts.For example, we discovered bacteria from novel phyla (formerly AD3 and WPS-2) in Antarctic soils support primary production via the genomic capacity to 'live on air' -using high affinity enzymes to oxidise atmospheric levels of molecular hydrogen and carbon monoxide to provide energy requirements to not only sustain dormancy, but support growth via CO 2 fixation (Fig. 1). 6However, difficulty in laboratory isolation of a trace gas chemotroph means this hypothesis has not been confirmed experimentally.Can we select for, and enrich for these air-eating bacteria through the addition of these gasses in enrichment cultures?A pioneering study by Nobs et al., supports this approach. 14 In this work, culture conditions informed by MAG reconstructions led to the enrichment of an Asgard archaeon alongside a sulfate-reducing bacterium.These enrichment cultures were derived from incubating anoxic layers of modern microbial mats from Hamelin Pool, Shark Bay, and incubation under anerobic and hypersaline conditions, thereby mimicking their natural environment.The successful enrichment and subsequent analysis of these critical putative syntrophic partners have major implications for the origin of eukaryogenesis.