From Surface to Subsurface: Diversity, Composition, and Abundance of Sessile and Endolithic Bacterial, Archaeal, and Eukaryotic Communities in Sand, Clay and Rock Substrates in the Laurentians (Quebec, Canada)

Microbial communities play an important role in shallow terrestrial subsurface ecosystems. Most studies of this habitat have focused on planktonic communities that are found in the groundwater of aquifer systems and only target specific microbial groups. Therefore, a systematic understanding of the processes that govern the assembly of endolithic and sessile communities is still missing. This study aims to understand the effect of depth and biotic factors on these communities, to better unravel their origins and to compare their composition with the communities detected in groundwater. To do so, we collected samples from two profiles (~0–50 m) in aquifer sites in the Laurentians (Quebec, Canada), performed DNA extractions and Illumina sequencing. The results suggest that changes in geological material characteristics with depth represent a strong ecological and phylogenetical filter for most archaeal and bacterial communities. Additionally, the vertical movement of water from the surface plays a major role in shallow subsurface microbial assembly processes. Furthermore, biotic interactions between bacteria and eukaryotes were mostly positive which may indicate cooperative or mutualistic potential associations, such as cross-feeding and/or syntrophic relationships in the terrestrial subsurface. Our results also point toward the importance of sampling both the geological formation and groundwater when it comes to studying its overall microbiology.

Rates of primary production in groundwater rival those in oligotrophic marine systems

The terrestrial subsurface contains nearly all of Earth's freshwater reserves and harbors upwards of 60% of our planet's total prokaryotic biomass. While genetic surveys suggest these organisms rely on in situ carbon fixation, rather than the translocation of photosynthetically derived organic carbon, corroborating measurements of carbon fixation in the subsurface are absent. Using a novel ultra-low level 14C-labeling technique, we show that in situ carbon fixation rates in a carbonate aquifer reached 10% of the median rates measured in oligotrophic marine surface waters, and were up to six-fold greater than those observed in lower euphotic zone waters where deep chlorophyll levels peak. Empirical carbon fixation rates were substantiated by both nitrification and anammox rate data. Metagenomic analyses revealed a remarkable abundance of putative chemolithoautotrophic members of an uncharacterized order of Nitrospiria - the first representatives of this class expected to fix carbon via the Wood-Ljungdahl pathway. Based on these fixation rates, we extrapolate global primary production in carbonate groundwaters to be 0.11 Pg of carbon per year.

Metabolic diversity and aero-tolerance in anammox bacteria from geochemically distinct aquifers

Background: Anaerobic ammonium oxidation (anammox) is important for converting bioavailable nitrogen into dinitrogen gas, particularly in carbon poor environments. Yet, the diversity and prevalence of anammox bacteria in the terrestrial subsurface – a typically oligotrophic environment – is little understood across different geochemical conditions. To determine the distribution and activity of anammox bacteria across a range of aquifer lithologies and physicochemistries, we analysed 16S rRNA genes, metagenomes and metatranscriptomes, and quantified hydrazine synthase genes and transcripts sampled from 59 groundwater wells distributed over 1 240 km2. Results: Data indicate that anammox-associated bacteria (class Brocadiae) and the anammox process are prevalent in aquifers (identified in aquifers with sandy-gravel, sandsilt and volcanic lithologies). While Brocadiae diversity decreased with increasing DO, Brocadiae 16S rRNA genes and hydrazine synthase genes and transcripts (hydrazine synthase, hzsB) were detected across a wide range of bulk groundwater dissolved oxygen (DO) concentrations (0 – 10 mg/L). Anammox genes and transcripts (hzsB) correlated significantly with those involved in bacterial and archaeal ammonia oxidation (ammonia monooxygenase, amoA), which could represent a major source of nitrite for anammox. Differences in anammox community composition were strongly associated with DO and bore depth (and to a lesser extent pH and phosphate), revealing niche differentiation among anammox bacteria in groundwater that was largely driven by water oxygen contents, and not ammonium/nitrite. Eight Brocadiae genomes (63-95% estimated completeness) reconstructed from a subset of groundwater sites belong to 2 uncharacterized families and 6 novel species (based on average nucleotide identity). Distinct groups of these genomes dominated the anammox-associated community at dysoxic and oxic sites, further reflecting the influence of DO on Brocadiae composition. Six of the genomes (dominating dysoxic or oxic sites) have genes characteristic of anammox (hydrazine synthase and/or dehydrogenase). These genes, in addition to aerotolerance genes, belonging to four Brocadiae genomes, were transcriptionally active, although transcript numbers clearly highest in dyoxic groundwater. Conclusions: Our findings indicate anammox bacteria contribute to loss of fixed N across diverse anoxic-to-oxic aquifer conditions, and that this is likely supported by nitrite from aerobic ammonia oxidation. Results provide an insight into the distribution and activity of anammox bacteria across distinct aquifer physicochemisties.

Active anaerobic methane oxidation and sulfur disproportionation in the deep terrestrial subsurface

Microbial life is widespread in the terrestrial subsurface and present down to several kilometers depth, but the energy sources that fuel metabolism in deep oligotrophic and anoxic environments remain unclear. In the deep crystalline bedrock of the Fennoscandian Shield at Olkiluoto, Finland, opposing gradients of abiotic methane and ancient seawater-derived sulfate create a terrestrial sulfate-methane transition zone (SMTZ). We used chemical and isotopic data coupled to genome-resolved metaproteogenomics to demonstrate active life and, for the first time, provide direct evidence of active anaerobic oxidation of methane (AOM) in a deep terrestrial bedrock. Proteins from Methanoperedens (formerly ANME-2d) are readily identifiable despite the low abundance (≤1%) of this genus and confirm the occurrence of AOM. This finding is supported by 13C-depleted dissolved inorganic carbon. Proteins from Desulfocapsaceae and Desulfurivibrionaceae, in addition to 34S-enriched sulfate, suggest that these organisms use inorganic sulfur compounds as both electron donor and acceptor. Zerovalent sulfur in the groundwater may derive from abiotic rock interactions, or from a non-obligate syntrophy with Methanoperedens, potentially linking methane and sulfur cycles in Olkiluoto groundwater. Finally, putative episymbionts from the candidate phyla radiation (CPR) and DPANN archaea represented a significant diversity in the groundwater (26/84 genomes) with roles in sulfur and carbon cycling. Our results highlight AOM and sulfur disproportionation as active metabolisms and show that methane and sulfur fuel microbial activity in the deep terrestrial subsurface.Significance StatementThe deep terrestrial subsurface remains an environment in which there is limited understanding of the extant microbial metabolisms, despite its reported large contribution to the overall biomass on Earth. It is much less well studied than deep marine sediments. We show that microorganisms in the subsurface are active, and that methane and sulfur provide fuel in the oligotrophic and anoxic subsurface. We also uncover taxonomically and metabolically diverse ultra-small organisms that interact with larger host cells through surface attachment (episymbiosis). Methane and sulfur are commonly reported in terrestrial crystalline bedrock environments worldwide and the latter cover a significant proportion of the Earth’s surface. Thus, methane- and sulfur-dependent microbial metabolisms have the potential to be widespread in the terrestrial deep biosphere.

A metagenomic view of novel microbial and metabolic diversity found within the deep terrestrial biosphere

The deep terrestrial subsurface is a large and diverse microbial habitat and a vast repository of biomass. However, in relation to its size and physical heterogeneity we have limited understanding of taxonomic and metabolic diversity in this realm. Here we present a detailed metagenomic analysis of samples from the Deep Mine Microbial Observatory (DeMMO) spanning depths from the surface to 1.5 km deep in the crust. From these eight geochemically and spatially distinct fluid samples we reconstructed ~600 metagenome assembled genomes (MAGs), representing 50 distinct phyla and including 18 candidate phyla. These novel clades include many members of the Patescibacteria superphylum and two new MAGs from candidate phylum OLB16, a phylum originally identified in DeMMO fluids and for which only one other MAG is currently available. We find that microbes spanning this expansive phylogenetic diversity and physical space are often capable of numerous dissimilatory energy metabolisms and are poised to take advantage of nutrients as they become available in relatively isolated fracture fluids. This metagenomic dataset is contextualized within a four-year geochemical and 16S rRNA time series, adding another invaluable piece to our knowledge of deep subsurface microbial ecology.

Draft Genome Sequence of Dietzia sp. Strain SYD-A1, Isolated from Coal Seam Formation Water

ABSTRACT Subsurface coal seams harbor an array of diverse microbial species subsisting as a community on the organic matter present in coal. Here, we present the annotated genome sequence of Dietzia sp. strain SYD-A1, a bacterium isolated from a terrestrial subsurface coal seam in New South Wales, Australia.

Initial Investigations into Microbial Dynamics and Biogeochemical Cycling in the Bedretto Tunnel

<p>Over 70% of Earth’s bacteria and archaea live in the subsurface. These rock-dwelling microorganisms are capable of exerting considerable influence on their environment by altering and recycling nutrients, as well as inducing changes to fluid flow paths through bioclogging. Subsurface life, therefore, has considerable implications for both natural and engineered subsurface environments. The Bedretto tunnel, located within the Swiss Alps, is 5,218 meters long and is host to the Bedretto Underground Laboratory for Geosciences and Geoenergies (BULGG), which was built to study the feasibility of large-scale geothermal energy storage and extraction. The tunnel, with a maximum overburden of approximately 1,650m, is embedded within both gneiss and Rotondo granite and offers an ideal location to investigate the biogeochemical feedbacks associated with natural fluids as well as the effect that stimulation has on the biological and chemical properties of subsurface fluids. For these reasons, a multi-year, monthly survey of fracture fluids at over 20 locations across the entire length of the tunnel has been carried out since August 2020 with the goal of performing 16S rRNA sequencing of cells captured by 0.22µm Millipore Sterivex filters and cell enumeration by epifluorescence microscopy of cells fixed with ethanol. By studying the microorganisms inhabiting BULG, we will be able to understand how the physical-chemical heterogeneities of the subsurface influence microbial physiology and community structure. Preliminary results of DNA extractions from the cells concentrated on Sterivex filters show that there is a measurable amount of DNA found in the fluids of the Bedretto tunnel that correlates with pH, indicating the presence of microbial communities which may vary with changes in fluid chemistry. With continued monitoring through 2021, we will determine whether there is significant variability of microbial taxa at different locations within the tunnel and the relationship between the hydrochemical properties of the fluids and the microbial communities. Alongside the profiling survey, whole genome sequencing as well as targeted virome sequencing procedures will be developed and used to learn more about the genetic and metabolic capacity of the microbial communities and to better understand how viruses can influence their hosts in such an environment. These results will be compared to other subsurface environments around the globe to gain a more holistic understanding of microbial dynamics in the terrestrial subsurface. Together, these results provide a new and important tool for tracking subsurface processes.</p>

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

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.