Abstract
Recovery and cultivation of diverse environmentally-relevant microorganisms from the terrestrial subsurface remain a challenge despite recent advances in modern molecular technology. Here, we applied complex carbon (C) sources, i.e., sediment dissolved organic matter (DOM) and bacterial cell lysate, to enrich groundwater microbial communities for 30 days. As comparisons, we also included enrichments amended with simple C sources including glucose, acetate, benzoate, oleic acid, cellulose, and mixed vitamins. Our results demonstrate that complex C is far more effective in enriching diverse and distinct microorganisms from groundwater than simple C. Simple C enrichments yield significantly lower biodiversity, and are dominated by few phyla (e.g., Proteobacteria and Bacteroidetes), while microcosms enriched with complex C demonstrate significantly higher biodiversity including phyla that are poorly represented in published culture collections (e.g., Verrucomicrobia, Planctomycetes, and Armatimonadetes). Subsequent isolation from complex C enrichments yielded 228 bacterial isolates representing five phyla, 17 orders, and 56 distinct species, including candidate novel, rarely cultivated, and undescribed organisms. Results from this study will substantially advance cultivation and isolation strategies for recovering diverse and novel subsurface microorganisms. Obtaining axenic representatives of “once-unculturable” microorganisms will enhance our understanding of microbial physiology and function in different biogeochemical niches of terrestrial subsurface ecosystems.
Highlights
Compared to animal and plant hosts, other non-human environments on Earth such as marine sediment, seawater, soil, and the terrestrial subsurface host prodigious and undescribed microbial populations, as most of them have never been cultured and characterized in the laboratory (Lloyd et al, 2018)
We aim to develop an effective cultivation strategy using naturally occurring complex C to recover diverse, rarely cultivated, and novel bacteria from groundwater collected at the Field Research Center (FRC) in Oak Ridge, TN, United States
Our results show that complex C such as sediment dissolved organic matter (DOM) and bacterial cell lysate are much more effective than conventional simple C sources in encouraging the growth of diverse and distinct bacteria from groundwater, providing a platform for the recovery of undiscovered bacteria that constitute “microbial dark matter” in the subsurface
Summary
Compared to animal and plant hosts, other non-human environments on Earth such as marine sediment, seawater, soil, and the terrestrial subsurface host prodigious and undescribed microbial populations, as most of them have never been cultured and characterized in the laboratory (Lloyd et al, 2018). Yeast extract and simple organic compounds such as glucose, acetate, lactate, pyruvate, and casamino acids are amended routinely, either as an individual carbon (C) source or as a mixture with the understanding that most microbes utilize these C substrates (Eilers et al, 2000) These labile C compounds commonly lead to selective and biased growth of microorganisms with faster growth rates, generally considered as the “weeds” (Wawrik et al, 2005; Cui et al, 2016), and have rarely recovered slow growing yet metabolically active and relevant microbes from the environment (Lok, 2015). For this reason, despite the rapid advances in “omics” technologies, we have still only been able to cultivate less than 2% of microbes on Earth in the laboratory (Overmann et al, 2017; Steen et al, 2019; Martiny, 2020)
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