Culturing of “Unculturable” Subsurface Microbes: Natural Organic Carbon Source Fuels the Growth of Diverse and Distinct Bacteria From Groundwater

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.

Dynamics of actively dividing prokaryotes in the western Mediterranean Sea

“…The inter- and intra- phylotypes growth rate variations under different conditions explain the large variability of actively dividing communities’ composition, with Alteromonadales identified as key taxa that caused the largest changes in communities’ structure. The enriched BrdU-labelled community composition is consistent with results obtained with other methods to identify active taxa, such as rDNA vs. rRNA gene composition 10 , 72 , 73 , microautoradiography combined with fluorescence in situ hybridization 74 , 75 , proteomics 76 , 77 , BONCAT 78 or other enrichment experiments 79 , indicating that the method is suitable to identify and characterize active members of the prokaryotic community. In this study it is evidenced that some low abundant phylotypes (< 0.5% abundance in total communities) are relatively active, as indicated by their increased contribution to the actively dividing communities.…”

Size-Fractionated Microbiome Structure in Subarctic Rivers and a Coastal Plume Across DOC and Salinity Gradients

“…A species affiliated to Armatimonas was among the most common OTUs (in relative abundance) in the rDNA large fraction of the SAS and KWK rivers, although it was in low relative abundance in the rRNA, suggesting a slow growth rate. This group (Armatimonadota) has been detected in a groundwater culture enriched with a mixture of sediment organic matter and bacterial cell lysate, and may possess the metabolic potential to utilize recalcitrant organic matter ( Wu et al, 2020 ).…”

A Simple Culture Method Enhances the Recovery of Culturable Actinobacteria From Coastal Sediments

“…Marine agar 2216 (BD) or marine broth 2216 (BD) are frequently and widely used in the isolation of marine bacteria from various habitats, including seawater, sediment, or the isolation of marine bacteria associated with animals or plants ( Gram et al, 2010 ; Sanz-Sáez et al, 2020 ). Several studies revealed that adding extracted inorganic compounds from the natural environment as nutrient sources could be an effective method to isolate novel bacteria from soil ( Nguyen et al, 2018 ) and groundwater ( Wu et al, 2020 ). For example, soil nutrients extracted by autoclaving an equal mixture of water and soil were found to give higher numbers of colonies ( Taylor, 1951a ).…”