Culturing of ‘Unculturable’ Subsurface Microbes: Natural Organic Carbon Source Fuels the Growth of Diverse and Distinct Bacteria from Groundwater

Abstract

22The recovery and cultivation of diverse field-related microorganisms from the terrestrial 23 subsurface environment remains a challenge despite recent advances in modern 24 molecular technology. Here we applied natural organic carbon (C), i.e., sediment-derived 25 natural organic matter (NOM) and bacterial cell lysate, to groundwater microbial 26 communities for a 30-day enrichment incubation, followed by conventional direct-plating 27 for isolation. The groundwater was collected from a background well at the Oak Ridge 28Reservation Field Research Center, Tennessee. As a comparison, we also included 29 enrichments amended with simple organic C sources, including glucose, acetate, 30 benzoate, oleic acid, cellulose, and mixed vitamins. Our results demonstrate that complex 31 natural organic C sources are more effective in enriching diverse bacterial species from 32 groundwater than simple organic C sources. Microcosms amended with simple organic C 33 (glucose, acetate, benzoate, or oleic acid) show significantly lower biodiversity than 34 unamended control and are dominated by only few phyla such as Proteobacteria and 35Bacteroidetes. In contrast, microcosms amended with complex natural organic C 36 (sediment NOM or bacterial cell lysate) display significantly higher biodiversity, and 37 enrich distinct species from the phyla that are poorly represented in published culture 38 collections (e.g., Verrucomicrobia, Planctomycetes, and Armatimonadetes). Our 39 subsequent isolation efforts from natural organic C-amended enrichments led to 222 40 purified bacterial isolates representing 5 phyla, 16 orders, and 54 distinct species 41 including candidate novel, rarely cultivated, and undescribed organisms. 42 43 Importance 44Innovative strategies for recovering bacterial strains representing the true diversity of 45 microbial communities in the terrestrial subsurface would significantly advance the 46 understanding of ecologically critical taxa residing in these ecosystems. In this study, we 47 demonstrate that complex natural organic C that mimic the naturally available resources 48 for microbes encourages the growth of diverse bacteria much more robustly than 49 traditional simplistic organic C sources. Results from this study will substantially 50 advance and improve the design of strategies to effectively cultivate and isolate diverse 51 and novel subsurface microorganisms in the laboratory. Obtaining axenic cultures of the 52 'once-unculturable' microorganisms will greatly enhance our understanding of microbial 53 physiology, function, and roles in different biogeochemical niches in terrestrial 54 subsurface ecosystems. 55 56 57Compared to animal and plant hosts, other non-human environments on Earth 58 such as marine sediment, seawater, soil, and the terrestrial subsurface host prodigious and 59 undiscovered microbial populations, as most of them have never been cultured and 60 characterized in the laboratory 1 . In the terrestrial subsurface, it is estimated that there are 61 2,500 × 10 26 microbial cells, of whi...

Developing stable, simplified, functional consortia from Brachypodium rhizosphere for microbial application in sustainable agriculture

“…For carbon sources, single carbon sources dominant at Brachypodium distachyon root exudates were used: citrate, malate, glucose, asparagine, glutamine, glucuronic acid, and a mixture of all these carbon sources (also at 2 mM). Lastly, 1/10th dilution of commercially available media, R2A (hereafter referred to as 1/10 R2A) was used as an additional media for reference since R2A has been widely adopted for enrichment and isolation of soil and subsurface microbes (Jenkins et al, 2017;Zhalnina et al, 2018;Wu et al, 2020;de Raad et al, 2022;Gushgari-Doyle et al, 2022). However, in keeping with the carbon concentrations in the other enrichment media, it was diluted 10-fold to mimic similar carbon concentrations as other carbon sources (2 mM) and adjusted to pH 6.…”

“…Genomic DNA of these samples were extracted at UC Berkeley DNA Sequencing Facility and submitted to Novogene Corporation Inc. for 16S amplicon sequencing of the V4 region using the universal bacterial primers 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT). The method previously published (Wu et al, 2020) was employed to analyze the amplicon sequence variants (ASVs). In summary, analysis of microbial community sequences was conducted using QIIME 2020.8 (Bolyen et al, 2019).…”

“…However, this method could inadvertently exclude microbes that resist cultivation under routine cultivation and incubation conditions including some rare and dormant taxa (de Souza et al, 2020;McClure et al, 2020;Choi et al, 2021). In addition, for subsequent consortia stability, knowledge about individual microbes and their interactions with partner microbes is imperative (Kaeberlein et al, 2002;Gilmore et al, 2019;Wu et al, 2020). Conversely, the top-down approach employs enrichments to generate consortia of reduced complexity after multiple passages (Gilmore et al, 2019;Díaz-García et al, 2021).…”

“…Conversely, the top-down approach employs enrichments to generate consortia of reduced complexity after multiple passages (Gilmore et al, 2019;Díaz-García et al, 2021). However, this approach may only yield fast-growing generalists of low diversity if routine carbon sources are used, overlooking the true rich phylogenetic and functional diversity of rhizosphere (Kaeberlein et al, 2002;Wu et al, 2020). Consequently, vital to construction of useful RCC is the design framework, that demonstrates stability, complexity, reproducibility, scalability, and genetic tractability-which appears to be generally lacking (Wu et al, 2020;Liang et al, 2022).…”

Reaching unreachables: Obstacles and successes of microbial cultivation and their reasons

“…Another decisive factor that enhances cultivation success is the choice of substrates and notably their concentration. Differing carbon concentrations create niches that are occupied by different bacteria (Eichorst et al, 2011;Wu et al, 2020). In environments prone to drastic environmental changes such as soil or water, selective pressure favors cells with a low metabolic cost existence (Mukamolova et al, 2003).…”