Methane oxidation and methylotroph population dynamics in groundwater mesocosms

© The Author(s), 2020. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Kuloyo, O., Ruff, S. E., Cahill, A., Connors, L., Zorz, J. K., de Angelis, I. H., Nightingale, M., Mayer, B., & Strous, M. Methane oxidation and methylotroph population dynamics in groundwater mesocosms. Environmental Microbiology. (2020), doi:10.1111/1462-2920.14929.Extraction of natural gas from unconventional hydrocarbon reservoirs by hydraulic fracturing raises concerns about methane migration into groundwater. Microbial methane oxidation can be a significant methane sink. Here, we inoculated replicated, sand‐packed, continuous mesocosms with groundwater from a field methane release experiment. The mesocosms experienced thirty‐five weeks of dynamic methane, oxygen and nitrate concentrations. We determined concentrations and stable isotope signatures of methane, carbon dioxide and nitrate and monitored microbial community composition of suspended and attached biomass. Methane oxidation was strictly dependent on oxygen availability and led to enrichment of 13C in residual methane. Nitrate did not enhance methane oxidation under oxygen limitation. Methylotrophs persisted for weeks in the absence of methane, making them a powerful marker for active as well as past methane leaks. Thirty‐nine distinct populations of methylotrophic bacteria were observed. Methylotrophs mainly occurred attached to sediment particles. Abundances of methanotrophs and other methylotrophs were roughly similar across all samples, pointing at transfer of metabolites from the former to the latter. Two populations of Gracilibacteria (Candidate Phyla Radiation) displayed successive blooms, potentially triggered by a period of methane famine. This study will guide interpretation of future field studies and provides increased understanding of methylotroph ecophysiology.The authors acknowledge funding from the Alberta Innovates Technology Futures (AITF), and University of Calgary Eyes High Doctoral Scholarships (O.O.K., J.K.Z.) and AITF/Eyes High Postdoctoral Fellowships (S.E.R.), as well as the PROMOS Internship Abroad Scholarship by the German Academic Exchange Service (I.H.d.A.). Additional support was provided by the Natural Sciences and Engineering Research Council of Canada (NSERC), Strategic Project Grant no. 463045‐14, the Campus Alberta Innovation Chair Program (M.S.), Alberta Innovates, The Canadian Foundation for Innovation (M.S.), the Alberta Small Equipment Grant Program (M.S.) and an NSERC Discovery Grant (M.S. and B.M.)

New ecosystems in the deep subsurface follow the flow of water driven by geological activity

Eukarya have been discovered in the deep subsurface at several locations in South Africa, but how organisms reach the subsurface remains unknown. We studied river-subsurface fissure water systems and identified Eukarya from a river that are genetically identical for 18S rDNA. To further confirm that these are identical species one metazoan species recovered from the overlying river interbred successfully with specimen recovered from an underlying mine at −1.4 km. In situ seismic simulation experiments were carried out and show seismic activity to be a major force increasing the hydraulic conductivity in faults allowing organisms to create ecosystems in the deep subsurface. As seismic activity is a non-selective force we recovered specimen of algae and Insecta that defy any obvious other explanation at a depth of −3.4 km. Our results show there is a steady flow of surface organisms to the deep subsurface where some survive and adapt and others perish. As seismic activity is also present on other planets and moons in our solar system the mechanism elucidated here may be relevant for future search and selection of landing sites in planetary exploration.publishedVersio

An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers

Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H₂. Methanogens and sulfate reducers, and the respective energy processes, are thought to be the dominant players and have been the research foci. Recent investigations showed that, in some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa, methanogens contribute <5% of the total DNA and appear to produce sufficient CH₄ to support the rest of the diverse community. This paradoxical situation reflects our lack of knowledge about the in situ metabolic diversity and the overall ecological trophic structure of SLiMEs. Here, we show the active metabolic processes and interactions in one of these communities by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Dominating the active community are four autotrophic β-proteobacterial genera that are capable of oxidizing sulfur by denitrification, a process that was previously unnoticed in the deep subsurface. They co-occur with sulfate reducers, anaerobic methane oxidizers, and methanogens, which each comprise <5% of the total community. Syntrophic interactions between these microbial groups remove thermodynamic bottlenecks and enable diverse metabolic reactions to occur under the oligotrophic conditions that dominate in the subsurface. The dominance of sulfur oxidizers is explained by the availability of electron donors and acceptors to these microorganisms and the ability of sulfur-oxidizing denitrifiers to gain energy through concomitant S and H₂ oxidation. We demonstrate that SLiMEs support taxonomically and metabolically diverse microorganisms, which, through developing syntrophic partnerships, overcome thermodynamic barriers imposed by the environmental conditions in the deep subsurface

Microbial community structure and function in Canadian Prairie groundwater ecosystems.

Groundwater ecosystems are globally widespread yet still poorly understood. The goal of the study was to understand the links between the biogeochemistry and microbial ecology of groundwater ecosystems in diverse geological settings on a broad spatial scale. We investigated the age, aqueous geochemistry, and microbiology of >100 groundwater samples from 90 monitoring wells (<250m depth) located in 14 aquifers in the Canadian Prairie. Geochemistry and microbial ecology were tightly linked revealing large-scale aerobic and anaerobic hydrogen, methane, nitrogen, and sulfur cycling carried out by diverse microbial communities. Details concerning methods, results and conclusions can be found in the associated publication by Ruff et al. 2023

Contextual and environmental data characterizing 138 samples from Canadian groundwater ecosystems

Groundwater ecosystems are globally widespread yet still poorly understood. We therefore investigated contextual and environmental data characterizing 138 groundwater samples from Canadian groundwater ecosystems. The chemical, physical, gas, isotopic, and microbiological measurements were taken from aquifers in the Canadian Prairie between 2015 and 2020. The study area comprised 14 major aquifers and a geographic area of ~210.000 km2. The goal of the study was to understand the links between the biogeochemistry and microbial ecology of groundwater ecosystems in diverse geological settings on a broad spatial scale. Details concerning methods, results and conclusions can be found in the associated publication by Ruff et al. 2023

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

Deep continental subsurface fracture water systems, ranging from 1.1 to 3.3 km 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 1 kmbls, isotopic evidence indicated microbial incorporation of biogenic CH4 by the in situ microbial community. At the shallowest site, 1.05 kmbls in Driefontein mine, this process clearly dominated the isotopic signal. At slightly deeper depths, 1.34 kmbls 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 >3 kmbls 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

South African crustal fracture fluids preserve paleometeoric water signatures for up to tens of millions of years

Fracture fluids in Earth's crust may remain isolated for millions to billions of years, and contain information on paleohydrogeology, subsurface microbial life, and conservative components that help elucidate the atmospheric evolution of the early Earth. Examples include fluids in the South African Kaapvaal craton which host chemolithoautotrophic microbial communities that survive independent of the photosphere, and billion-year-old fluids in the Canadian Shield, which preserve the Xe isotopic signature of an evolving early atmosphere. Stable isotope analyses of the aqueous phase combined with isotopic analyses of the dissolved noble gases provide unrivalled insight into the time-alteration history of aqueous fracture fluids. Here we report stable isotope and noble gas data for fracture fluids in the Witwatersrand Basin and Bushveld Igneous Province systems, South Africa. We determine closed-system radiogenic noble gas residence times of 0.77–97 million years (Myr). Open-system residence times range between 6.0 kyr and 10.8 Myr. One sample from Masimong Mine has a mean closed-system residence time of 85 Myr, making it one of oldest paleometeoric waters ever recorded. The δ2H and δ18O of water in this sample, and in previously reported samples from the same mining district that are shown to have similar ages, require an isotopically depleted source of groundwater recharge. This could reflect a recharge regime at a higher paleolatitude, elevation, or with higher rainfall, established up to tens of Myr ago, and perhaps similar to the recharge regime in the modern Lesotho Highlands. These data suggest that groundwater isotopes can provide useful paleoclimatic information for many Myr

Eukaryotic opportunists dominate the deep-subsurface biosphere in South Africa

Following the discovery of the first Eukarya in the deep subsurface, intense interest has developed to understand the diversity of eukaryotes living in these extreme environments. We identified that Platyhelminthes, Rotifera, Annelida and Arthropoda are thriving at 1.4 km depths in palaeometeoric fissure water up to 12,300 yr old in South African mines. Protozoa and Fungi have also been identified; however, they are present in low numbers. Characterization of the different species reveals that many are opportunistic organisms with an origin due to recharge from surface waters rather than soil leaching. This is the first known study to demonstrate the in situ distribution of biofilms on fissure rock faces using video documentation. Calculations suggest that food, not dissolved oxygen is the limiting factor for eukaryal population growth. The discovery of a group of Eukarya underground has important implications for the search for life on other planets in our solar system