Abstract
The majority of environmental microbiomes are not amenable to cultivation under standard laboratory growth conditions and hence remain uncharacterized. For environmental applications, such as bioremediation, it is necessary to isolate microbes performing the desired function, which may not necessarily be the fast growing or the copiotroph microbiota. Toward this end, cultivation and isolation of microbial strains using diffusion chambers (DC) and/or microbial traps (MT) have both been recently demonstrated to be effective strategies because microbial enrichment is facilitated by soil nutrients and not by synthetically defined media, thus simulating their native habitat. In this study, DC/MT chambers were established using soils collected from two US Department of Energy (DOE) sites with long-term history of heavy metal contamination, including mercury (Hg). To characterize the contamination levels and nutrient status, soils were first analyzed for total mercury (THg), methylmercury (MeHg), total carbon (TC), total nitrogen (TN), and total phosphorus (TP). Multivariate statistical analysis on these measurements facilitated binning of soils under high, medium and low levels of contamination. Bacterial and fungal microbiomes that developed within the DC and MT chambers were evaluated using comparative metagenomics, revealing Chthoniobacter, Burkholderia and Bradyrhizobium spp., as the predominant bacteria while Penicillium, Thielavia, and Trichoderma predominated among fungi. Many of these core microbiomes were also retrieved as axenic isolates. Furthermore, canonical correspondence analysis (CCA) of biogeochemical measurements, metal concentrations and bacterial communities revealed a positive correlation of Chthoniobacter/Bradyrhizobium spp., to THg whereas Burkholderia spp., correlated with MeHg. Penicillium spp., correlated with THg whereas Trichoderma spp., and Aspergillus spp., correlated with MeHg, from the MT approach. This is the first metagenomics-based assessment, isolation and characterization of soil-borne bacterial and fungal communities colonizing the diffusion chambers (DC) and microbial traps (MT) established with long-term metal contaminated soils. Overall, this study provides proof-of-concept for the successful application of DC/MT based assessment of mercury resistant (HgR) microbiomes in legacy metal-contaminated soils, having complex contamination issues. Overall, this study brings out the significance of microbial communities and their relevance in context to heavy metal cycling for better stewardship and restoration of such historically contaminated systems.
Highlights
Despite advancements in the area of culturomics, which is the coupled application of omics-based tools, such as metagenomics, to improve cultivation efficiencies of environmental microbiota, the vast majority of soil microorganisms remain uncultivable (Keller and Zengler, 2004; Vartoukian et al, 2010; Locey and Lennon, 2016; Hofer, 2018; Hahn et al, 2019; Bodor et al, 2020), and unavailable for downstream environmentally relevant applications, such as bioremediation
Analysis of Hg, MeHg and biogeochemical measurements in the Savannah River Site (SRS) and Oak Ridge Reservation (ORR) soils revealed that the following trends: concentration of Total carbon (TC) was in the following order S1 > S3 > H-02-1 > R1 > B; total nitrogen (TN) followed a similar trend except for site B which was slightly higher than site R1; total phosphorus (TP) concentrations followed this trend except that the B site was relatively higher
To our knowledge, this is the first metagenomics-based assessment and isolation of bacterial and fungal assemblages that colonized in diffusion chambers and microbial traps established with long-term heavy metal contaminated soils
Summary
Despite advancements in the area of culturomics, which is the coupled application of omics-based tools, such as metagenomics, to improve cultivation efficiencies of environmental microbiota, the vast majority of soil microorganisms remain uncultivable (Keller and Zengler, 2004; Vartoukian et al, 2010; Locey and Lennon, 2016; Hofer, 2018; Hahn et al, 2019; Bodor et al, 2020), and unavailable for downstream environmentally relevant applications, such as bioremediation. Soils are serially diluted, mixed with sterile agar, and inoculated in between two semipermeable membranes of variable pore sizes, depending on the microorganisms being targeted for isolation, i.e., bacteria, actinomycetes or fungi. The microbial trap (MT) method, on the other hand, has been developed to enhance growth and isolation of soil-borne actinomycetes and/or fungi (Gavrish et al, 2008) (Supplementary Figure S1), in that the bottom membrane, which lies in close proximity to the soil, consists of 0.2 μm pore size; permitting the entry of fungal hyphae but not bacteria into the trap
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