Deep Continental Subsurface Research Articles

Hydrogeological controls on microbial activity and habitability in the Precambrian continental crust.

Earth's deep continental subsurface is a prime setting to study the limits of life's relationship with environmental conditions and habitability. In Precambrian crystalline rocks worldwide, deep ancient groundwaters in fracture networks are typically oligotrophic, highly saline, and locally inhabited by low-biomass communities in which chemolithotrophic microorganisms may dominate. Periodic opening of new fractures can lead to penetration of surface water and/or migration of fracture fluids, both of which may trigger changes in subsurface microbial composition and activity. These hydrogeological processes and their impacts on subsurface communities may play a significant role in global cycles of key elements in the crust. However, to date, considerable uncertainty remains on how subsurface microbial communities may respond to these changes in hydrogeochemical conditions. To address this uncertainty, the biogeochemistry of Thompson mine (Manitoba, Canada) was investigated. Compositional and isotopic analyses of fracture waters collected here at ~1 km below land surface revealed different extents of mixing between subsurface brine and (paleo)meteoric waters. To investigate the effects this mixing may have had on microbial communities, the Most Probable Number technique was applied to test community response for a total of 13 different metabolisms. The results showed that all fracture waters were dominated by viable heterotrophic microorganisms which can utilize organic materials associated with aerobic/facultative anaerobic processes, sulfate reduction, or fermentation. Where mixing between subsurface brines and (paleo)meteoric waters occurs, the communities demonstrate higher cell densities and increased viable functional potentials, compared to the most saline sample. This study therefore highlights the connection between hydrogeologic heterogeneity and the heterogeneity of subsurface ecosystems in the crystalline rocks, and suggests that hydrogeology can have a considerable impact on the scope and scale of subsurface microbial communities on Earth and potentially beyond.

Global distribution and diversity of antimicrobial genes across subsurface bacterial and archaeal metagenome assembled genomes

Microorganisms have the capability to produce antimicrobial compounds through secondary metabolism, which are not essential within their natural environments, but have been found to have many effects on the ecosystem. Antimicrobial production genes have been identified in a wide range of microorganisms; however, research into natural ecosystems has historically been limited to continental soil environments. Antimicrobial production research has been limited in the deep continental subsurface and marine environments, especially deeply buried marine sediments. We analyzed 466 high-quality metagenome assembled genomes (MAGs) collected from continental and marine subsurface environments through the Deep Carbon Observatory’s Census of Deep Life. A total of 383 MAGs contained biosynthetic gene clusters, namely Type I and Type III polyketide synthase genes, non-ribosomal peptide synthetase genes, and other unspecified ribosomally synthesized and post-translationally modified peptide products. All of these genes were found across continental mines, subglacial lakes, hotsprings, and serpentinizing environments. These environments have previously not been investigated via metagenomics for antimicrobial gene diversity, which may be produced for competition or communication purposes. All other biosynthetic genes identified in this study were less than 50% similar to reference biosynthesis genes indicating the novelty of secondary metabolism in subsurface microorganisms. The majority of predicted antimicrobial products were found to be produced exergonically, which could indicate microbial populations use energy-conserving mechanisms to produce compounds that could offer a competitive advantage.

Charakterisierung von mikrobiellen Biofilmen aus Tunnelwässern

AbstractMicroorganisms mainly from the domain of bacteria dominate life in the terrestrial subsurface up to a depth of several kilometers. Microbial life in that habitat is dependent on the availability of water and nutrients of biotic or abiotic origin. To produce biomass, bacterial primary producers are capable to use simple building blocks such as CO2, H2, CH4, reduced iron and sulfur, nitrate, phosphate as well as other salts and trace elements. Here, we describe the microbial content of several biofilm samples taken during the construction phase of a railway tunnel between the provinces of Styria and Carinthia (Koralm Tunnel). We could identify bacterial cells using light microscopy and used 16S rDNA based microbial profiling to characterize the bacterial community structure. Our results clearly show that the massive biofilms in tunnel drainage water were produced by a bacterial consortium typical for the deep continental subsurface biosphere. In many cases the majority of the consortium could be assigned to autotrophs, methanotrophs and methylotrophs.

Deep mining decreases the microbial taxonomic and functional diversity of subsurface oil reservoirs

Microbes in subsurface oil reservoirs play important roles in elemental cycles and biogeochemical processes. However, the community assembly pattern of indigenous microbiome and their succession under long-term human activity remain poorly understood. Here we studied the microbial community assembly in underground sandstone cores from 190 to 2050 m in northeast China and their response to long-term oil recovery (10–50 years). Indigenous microbiome in subsurface petroleum reservoirs were dominated by Gammaproteobacteria, Firmicutes, Alphaproteobacteria, Bacteroidetes, and Actinobacteria, which exhibited a higher contribution of homogenizing dispersal assembly and different taxonomy distinct ecological modules when compared with perturbed samples. Specifically, the long-term oil recovery reduced the bacterial taxonomic- and functional-diversity, and increased the community co-occurrence associations in subsurface oil reservoirs. Moreover, distinguished from the perturbed samples, both variation partition analysis and structural equation model revealed that the contents of quartz, NO3− and Cl− significantly structured the α- and β-diversity in indigenous subsurface bacterial communities. These findings first provide the holistic picture of microbiome in the deep oil reservoirs, which demonstrate the significant impact of human activity on microbiome in deep continental subsurface.

Earth-like Habitable Environments in the Subsurface of Mars.

In Earth's deep continental subsurface, where groundwaters are often isolated for >106 to 109 years, energy released by radionuclides within rock produces oxidants and reductants that drive metabolisms of non-photosynthetic microorganisms. Similar processes could support past and present life in the martian subsurface. Sulfate-reducing microorganisms are common in Earth's deep subsurface, often using hydrogen derived directly from radiolysis of pore water and sulfate derived from oxidation of rock-matrix-hosted sulfides by radiolytically derived oxidants. Radiolysis thus produces redox energy to support a deep biosphere in groundwaters isolated from surface substrate input for millions to billions of years on Earth. Here, we demonstrate that radiolysis by itself could produce sufficient redox energy to sustain a habitable environment in the subsurface of present-day Mars, one in which Earth-like microorganisms could survive wherever groundwater exists. We show that the source localities for many martian meteorites are capable of producing sufficient redox nutrients to sustain up to millions of sulfate-reducing microbial cells per kilogram rock via radiolysis alone, comparable to cell densities observed in many regions of Earth's deep subsurface. Additionally, we calculate variability in supportable sulfate-reducing cell densities between the martian meteorite source regions. Our results demonstrate that martian subsurface groundwaters, where present, would largely be habitable for sulfate-reducing bacteria from a redox energy perspective via radiolysis alone. We present evidence for crustal regions that could support especially high cell densities, including zones with high sulfide concentrations, which could be targeted by future subsurface exploration missions.

Rock Surface Fungi in Deep Continental Biosphere-Exploration of Microbial Community Formation with Subsurface In Situ Biofilm Trap.

Fungi have an important role in nutrient cycling in most ecosystems on Earth, yet their ecology and functionality in deep continental subsurface remain unknown. Here, we report the first observations of active fungal colonization of mica schist in the deep continental biosphere and the ability of deep subsurface fungi to attach to rock surfaces under in situ conditions in groundwater at 500 and 967 m depth in Precambrian bedrock. We present an in situ subsurface biofilm trap, designed to reveal sessile microbial communities on rock surface in deep continental groundwater, using Outokumpu Deep Drill Hole, in eastern Finland, as a test site. The observed fungal phyla in Outokumpu subsurface were Basidiomycota, Ascomycota, and Mortierellomycota. In addition, significant proportion of the community represented unclassified Fungi. Sessile fungal communities on mica schist surfaces differed from the planktic fungal communities. The main bacterial phyla were Firmicutes, Proteobacteria, and Actinobacteriota. Biofilm formation on rock surfaces is a slow process and our results indicate that fungal and bacterial communities dominate the early surface attachment process, when pristine mineral surfaces are exposed to deep subsurface ecosystems. Various fungi showed statistically significant cross-kingdom correlation with both thiosulfate and sulfate reducing bacteria, e.g., SRB2 with fungi Debaryomyces hansenii.

A window into the abiotic carbon cycle – Acetate and formate in fracture waters in 2.7 billion year-old host rocks of the Canadian Shield

The recent expansion of studies at hydrothermal submarine vents from investigation of abiotic methane formation to include abiotic production of organics such acetate and formate, and rising interest in processes of abiotic organic synthesis on the ocean-world moons of Saturn and Jupiter, have raised interest in potential Earth analogs for investigation of prebiotic/abiotic processes to an unprecedented level. The deep continental subsurface provides an attractive target to identify analog environments where the influence of abiotic carbon cycling may be investigated, particularly in hydrogeological isolated fracture fluids where the products of chemical water-rock reactions have been less overprinted by the biogeochemical signatures of the planet’s surficial water and carbon cycles. Here we report, for the first time, a comprehensive set of concentration measurements and isotopic signatures for acetate and formate, as well as the dissolved inorganic and organic carbon pools, for saline fracture waters naturally flowing 2.4 km below surface in 2.7 billion year-old rocks on the Canadian Shield. These geologically ancient fluids at the Kidd Creek Observatory were the focus of previous investigations of fracture fluid geochemistry, microbiology and noble gas-derived residence times. Here we show the fracture waters of Kidd Creek contain high concentrations of both acetate and formate with concentrations from 1200 to 1900 µmol/L, and 480 to 1000 µmol/L, respectively. Acetate and formate alone account for more than 50–90% of the total DOC – providing a very simple “organic soup”. The unusually elevated concentrations and profoundly 13C-enriched nature of the acetate and formate suggest an important role for abiotic organic synthesis in the deep carbon cycle at this hydrogeologically isolated site. A variety of potential abiotic production reactions are discussed, including a radiolytically driven H, S and C deep cycle that could provide a mechanism for sustaining deep subsurface habitability. Scientific discoveries are beginning to reveal that organic-producing reactions that would have prevailed on Earth before the rise of life, and that may persist today on planets and moons such as Enceladus, Europa and Titan, can be accessed in some specialized geologic settings on Earth that provide valuable natural analog environments for the investigation of abiotic organic chemistry outside the laboratory.

Patterns of in situ Mineral Colonization by Microorganisms in a ~60°C Deep Continental Subsurface Aquifer.

The microbial ecology of the deep biosphere is difficult to characterize, owing in part to sampling challenges and poorly understood response mechanisms to environmental change. Pre-drilled wells, including oil wells or boreholes, offer convenient access, but sampling is frequently limited to the water alone, which may provide only a partial view of the native diversity. Mineral heterogeneity demonstrably affects colonization by deep biosphere microorganisms, but the connections between the mineral-associated and planktonic communities remain unclear. To understand the substrate effects on microbial colonization and the community response to changes in organic carbon, we conducted an 18-month series of in situ experiments in a warm (57°C), anoxic, fractured carbonate aquifer at 752 m depth using replicate open, screened cartridges containing different solid substrates, with a proteinaceous organic matter perturbation halfway through this series. Samples from these cartridges were analyzed microscopically and by Illumina (iTag) 16S rRNA gene libraries to characterize changes in mineralogy and the diversity of the colonizing microbial community. The substrate-attached and planktonic communities were significantly different in our data, with some taxa (e.g., Candidate Division KB-1) rare or undetectable in the first fraction and abundant in the other. The substrate-attached community composition also varied significantly with mineralogy, such as with two Rhodocyclaceae OTUs, one of which was abundant on carbonate minerals and the other on silicic substrates. Secondary sulfide mineral formation, including iron sulfide framboids, was observed on two sets of incubated carbonates. Notably, microorganisms were attached to the framboids, which were correlated with abundant Sulfurovum and Desulfotomaculum sp. sequences in our analysis. Upon organic matter perturbation, mineral-associated microbial diversity differences were temporarily masked by the dominance of putative heterotrophic taxa in all samples, including OTUs identified as Caulobacter, Methyloversatilis, and Pseudomonas. Subsequent experimental deployments included a methanogen-dominated stage (Methanobacteriales and Methanomicrobiales) 6 months after the perturbation and a return to an assemblage similar to the pre-perturbation community after 9 months. Substrate-associated community differences were again significant within these subsequent phases, however, demonstrating the value of in situ time course experiments to capture a fraction of the microbial assemblage that is frequently difficult to observe in pre-drilled wells.

Rectinema subterraneum sp. nov, a chemotrophic spirochaete isolated from the deep terrestrial subsurface.

A novel, obligately anaerobic bacterium (strain SURF-ANA1T) was isolated from deep continental subsurface fluids at a depth of 1500 m below surface in the former Homestake Gold Mine (now Sanford Underground Research Facility, in Lead, South Dakota, USA). Cells of strain SURF-ANA1T were Gram-negative, helical, non-spore-forming and were 0.25-0.55×5.0-75.0 µm with a wavelength of 0.5-0.62 µm. Strain SURF-ANA1T grew at 15-50 °C (optimally at 40 °C), at pH 4.8-9.0 (pH 7.2) and in 1.0-40.0 g l-1 NaCl (10 g l-1 NaCl). The strain grew chemoheterotrophically with hydrogen or mono-, di- and polysaccharides as electron donors. The major cellular fatty acids in order of decreasing abundance (comprising >5% of total) were 10-methyl C16:0, iso-C15:0, C18:2 and C18:0 dimethyl acetal (DMA) and C20:0 methylene-nonadecanoic acid. Phylogenetic analysis based on the 16S rRNA gene sequence of strain SURF-ANA1T indicated a closest relationship with the recently characterized Rectinema cohabitans (99%). Despite high sequence identity, because of its distinct physiology, morphology and fatty acid profile, strain SURF-ANA1T is considered to represent a novel species within the genus Rectinema, for which the name Rectinema subterraneum sp. nov. is proposed. To our knowledge, this is the first report of an isolate within the phylum Spirochaetes from the deep (>100 m) terrestrial subsurface. The GenBank/EMBL/DDBJ accession numbers for the 16S rRNA gene and genomic sequences of strain SURF-ANA1T are KU359248 and GCF 009768935.1, respectively. The type strain of Rectinema subterraneum is SURF-ANA1T (=ATCC TSD-67=JCM 32656).

Bacteria and archaea on Earth and their abundance in biofilms.

Biofilms are a form of collective life with emergent properties that confer many advantages on their inhabitants, and they represent a much higher level of organization than single cells do. However, to date, no global analysis on biofilm abundance exists. We offer a critical discussion of the definition of biofilms and compile current estimates of global cell numbers in major microbial habitats, mindful of the associated uncertainty. Most bacteria and archaea on Earth (1.2 × 1030 cells) exist in the 'big five' habitats: deep oceanic subsurface (4 × 1029), upper oceanic sediment (5 × 1028), deep continental subsurface (3 × 1029), soil (3 × 1029) and oceans (1 × 1029). The remaining habitats, including groundwater, the atmosphere, the ocean surface microlayer, humans, animals and the phyllosphere, account for fewer cells by orders of magnitude. Biofilms dominate in all habitats on the surface of the Earth, except in the oceans, accounting for ~80% of bacterial and archaeal cells. In the deep subsurface, however, they cannot always be distinguished from single sessile cells; we estimate that 20-80% of cells in the subsurface exist as biofilms. Hence, overall, 40-80% of cells on Earth reside in biofilms. We conclude that biofilms drive all biogeochemical processes and represent the main way of active bacterial and archaeal life.

Viable cyanobacteria in the deep continental subsurface

Cyanobacteria are ecologically versatile microorganisms inhabiting most environments, ranging from marine systems to arid deserts. Although they possess several pathways for light-independent energy generation, until now their ecological range appeared to be restricted to environments with at least occasional exposure to sunlight. Here we present molecular, microscopic, and metagenomic evidence that cyanobacteria predominate in deep subsurface rock samples from the Iberian Pyrite Belt Mars analog (southwestern Spain). Metagenomics showed the potential for a hydrogen-based lithoautotrophic cyanobacterial metabolism. Collectively, our results suggest that they may play an important role as primary producers within the deep-Earth biosphere. Our description of this previously unknown ecological niche for cyanobacteria paves the way for models on their origin and evolution, as well as on their potential presence in current or primitive biospheres in other planetary bodies, and on the extant, primitive, and putative extraterrestrial biospheres.

The deep continental subsurface: the dark biosphere.

Although information from devoted geomicrobiological drilling studies is limited, it is clear that the results obtained so far call for a systematic exploration of the deep continental subsurface, similar to what has been accomplished in recent years by the Ocean Drilling Initiatives. In addition to devoted drillings from the surface, much of the continental subsurface data has been obtained using different subterranean "windows," each with their correspondent limitations. In general, the number and diversity of microorganisms decrease with depth, and the abundance of Bacteria is superior to Archaea. Within Bacteria, the most commonly detected phyla correspond to Proteobacteria, Actinobacteria, Bacteroidetes, and Firmicutes. Within Archaea, methanogens are recurrently detected in most analyzed subsurface samples. One of the most controversial topics in the study of subsurface environments is whether the available energy source is endogenous or partly dependent on products photosynthetically generated in the subsurface. More information, at better depth resolution, is needed to build up the catalog of deep subsurface microbiota and the biologically available electron acceptors and donors.

Active microbial biofilms in deep poor porous continental subsurface rocks

Deep continental subsurface is defined as oligotrophic environments where microorganisms present a very low metabolic rate. To date, due to the energetic cost of production and maintenance of biofilms, their existence has not been considered in poor porous subsurface rocks. We applied fluorescence in situ hybridization techniques and confocal laser scanning microscopy in samples from a continental deep drilling project to analyze the prokaryotic diversity and distribution and the possible existence of biofilms. Our results show the existence of natural microbial biofilms at all checked depths of the Iberian Pyrite Belt (IPB) subsurface and the co-occurrence of bacteria and archaea in this environment. This observation suggests that multi-species biofilms may be a common and widespread lifestyle in subsurface environments.

Variations in microbial carbon sources and cycling in the deep continental subsurface

Deep continental subsurface fracture water systems, ranging from 1.1 to 3.3km below land surface (kmbls), were investigated to characterize the indigenous microorganisms and elucidate microbial carbon sources and their cycling. Analysis of phospholipid fatty acid (PLFA) abundances and direct cell counts detected varying biomass that was not correlated with depth. Compound-specific carbon isotope analyses (δ13C and Δ14C) of the phospholipid fatty acids (PLFAs) and carbon substrates combined with genomic analyses did identify, however, distinct carbon sources and cycles between the two depth ranges studied.In the shallower boreholes at circa 1kmbls, isotopic evidence indicated microbial incorporation of biogenic CH4 by the in situ microbial community. At the shallowest site, 1.05kmbls in Driefontein mine, this process clearly dominated the isotopic signal. At slightly deeper depths, 1.34kmbls in Beatrix mine, the isotopic data indicated the incorporation of both biogenic CH4 and dissolved inorganic carbon (DIC) derived from CH4 oxidation. In both of these cases, molecular genetic analysis indicated that methanogenic and methanotrophic organisms together comprised a small component (<5%) of the microbial community. Thus, it appears that a relatively minor component of the prokaryotic community is supporting a much larger overall bacterial community in these samples.In the samples collected from >3kmbls in Tau Tona mine (TT107, TT109 Bh2), the CH4 had an isotopic signature suggesting a predominantly abiogenic origin with minor inputs from microbial methanogenesis. In these samples, the isotopic enrichments (δ13C and Δ14C) of the PLFAs relative to CH4 were consistent with little incorporation of CH4 into the biomass. The most 13C-enriched PLFAs were observed in TT107 where the dominant CO2-fixation pathway was the acetyl-CoA pathway by non-acetogenic bacteria. The differences in the δ13C of the PLFAs and the DIC and DOC for TT109 Bh2 were ∼−24‰ and 0‰, respectively. The dominant CO2-fixation pathways were 3-HP/4-HB cycle>acetyl-CoA pathway>reductive pentose phosphate cycle.

Expanding frontiers in deep subsurface microbiology

The subsurface biosphere on Earth appears to be far more expansive and physiologically and phylogenetically complex than previously thought. Here, several aspects of subsurface microbiology are discussed. Molecular and biogeochemical data, as well as characteristics from new isolates, suggest that ecosystems below deep-sea hydrothermal vents are inhabited primarily by thermophilic archaea and bacteria. The void spaces and conduits in basalt at mid-ocean ridges, and, even more so, at sediment-covered hydrothermal vent sites represent promising hunting grounds for novel chemosynthetic archaea and bacteria. As examples, we highlight the subsurface microbial communities within the basalt flanks of the Juan de Fuca Ridge off British Columbia, and the hydrothermal sediments at the Guaymas Basin in the Gulf of California and at Middle Valley on the Juan de Fuca Ridge. In the deep continental subsurface, microbial studies have primarily targeted aquifers in basaltic and granitic rock, with an almost exclusive emphasis on H 2-driven chemolithoautotrophy. For example, in the subsurface water from Lidy Hot Springs, Idaho (USA), H 2-consuming methanogenic archaea represent the most populous organisms, comprising >95% of all cells. Besides H 2, other potential sources of chemical energy (reducing power) are considered, including abiotically synthesized organic matter and reduced sulfur compounds. In addition, the well-established sequences of redox couples in microbial metabolism are investigated at elevated temperatures using a thermodynamic approach. Values of pε o for 11 common reduction half-reactions are tabulated at 25, 50, 100, and 150 °C. Although many modes of metabolism may be employed by subsurface biota, sulfate reduction appears to be one of the more ubiquitous strategies. Recent studies of sulfate-reducing archaea and bacteria in marine and continental systems are reviewed.