Microbial hydrogen sinks in the sand-bentonite backfill material for the deep geological disposal of radioactive waste.

The activity of subsurface microorganisms can be harnessed for engineering projects. For instance, the Swiss radioactive waste repository design can take advantage of indigenous microorganisms to tackle the issue of a hydrogen gas (H2) phase pressure build-up. After repository closure, it is expected that anoxic steel corrosion of waste canisters will lead to an H2 accumulation. This occurrence should be avoided to preclude damage to the structural integrity of the host rock. In the Swiss design, the repository access galleries will be back-filled, and the choice of this material provides an opportunity to select conditions for the microbially-mediated removal of excess gas. Here, we investigate the microbial sinks for H2. Four reactors containing an 80/20 (w/w) mixture of quartz sand and Wyoming bentonite were supplied with natural sulfate-rich Opalinus Clay rock porewater and with pure H2 gas for up to 108 days. Within 14 days, a decrease in the sulfate concentration was observed, indicating the activity of the sulfate-reducing bacteria detected in the reactor, e.g., from Desulfocurvibacter genus. Additionally, starting at day 28, methane was detected in the gas phase, suggesting the activity of methanogens present in the solid phase, such as the Methanosarcina genus. This work evidences the development, under in-situ relevant conditions, of a backfill microbiome capable of consuming H2 and demonstrates its potential to contribute positively to the long-term safety of a radioactive waste repository.

Growth and Persistence of an Aerobic Microbial Community in Wyoming Bentonite MX-80 Despite Anoxic in situ Conditions.

Microbial activity has the potential to enhance the corrosion of high-level radioactive waste disposal canisters, which, in the proposed Swiss deep geological repository, will be embedded in bentonite and placed in the Opalinus Clay (OPA) rock formation. A total of 12 stainless steel cylindrical vessels (referred to as modules) containing bentonite were deployed in an anoxic borehole in OPA for up to 5.5 years. Carbon steel coupons were embedded in the bentonite. Individual modules were retrieved after 1, 1.5, 2.5, and 5.5 years. Enumeration of aerobic and anaerobic heterotrophs and sulfate-reducing bacteria (SRB) revealed microbial growth for 1.5 years followed by a decline or stagnation in microbial viability. It was surprising to observe the growth of aerobic heterotrophs followed by their persistent viability in bentonite, despite the nominally anoxic conditions. In contrast, SRB numbers remained at very low levels. DNA-based amplicon sequencing confirmed the persistence of aerobes and the relatively low contribution of anaerobes to the bentonite microbiome. Bentonite dry density, in situ exposure time, and bioavailable trapped oxygen are observed to shape the bentonite microbial community in the clay.

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.

Active sulfur cycling in the terrestrial deep subsurface.

The deep terrestrial subsurface remains an environment where there is limited understanding of the extant microbial metabolisms. At Olkiluoto, Finland, a deep geological repository is under construction for the final storage of spent nuclear fuel. It is therefore critical to evaluate the potential impact microbial metabolism, including sulfide generation, could have upon the safety of the repository. We investigated a deep groundwater where sulfate is present, but groundwater geochemistry suggests limited microbial sulfate-reducing activity. Examination of the microbial community at the genome-level revealed microorganisms with the metabolic capacity for both oxidative and reductive sulfur transformations. Deltaproteobacteria are shown to have the genetic capacity for sulfate reduction and possibly sulfur disproportionation, while Rhizobiaceae, Rhodocyclaceae, Sideroxydans, and Sulfurimonas oxidize reduced sulfur compounds. Further examination of the proteome confirmed an active sulfur cycle, serving for microbial energy generation and growth. Our results reveal that this sulfide-poor groundwater harbors an active microbial community of sulfate-reducing and sulfide-oxidizing bacteria, together mediating a sulfur cycle that remained undetected by geochemical monitoring alone. The ability of sulfide-oxidizing bacteria to limit the accumulation of sulfide was further demonstrated in groundwater incubations and highlights a potential sink for sulfide that could be beneficial for geological repository safety.

Microbially Mediated Release of As from Mekong Delta Peat Sediments.

Peat layers within alluvial sediments are considered effective arsenic (As) sinks under reducing conditions due to the binding of As(III) to thiol groups in natural organic matter (NOM) and the formation of As-bearing sulfide phases. However, their possible role as sources of As for anoxic groundwaters remains unexplored. Here, we perform laboratory experiments to provide evidence for the role of a sediment peat layer in releasing As. Our results show that the peat layer, deposited about 8,000 years ago in a paleomangrove environment in the nascent Mekong Delta, could be a source of As to porewater under reducing conditions. X-ray absorption spectroscopy (XAS) analysis of the peat confirmed that As was bound to NOM thiol groups and incorporated into pyrite. Nitrate was detected in peat layer porewater, and flow-through and batch experiments evidenced the release of As from NOM and pyrite in the presence of nitrate. Based on poisoning experiments, we propose that the microbially mediated oxidation of arsenic-rich pyrite and organic matter coupled to nitrate reduction releases arsenic from this peat. Although peat layers have been proposed as As sinks in earlier studies, we show here their potential to release depositional- and/or diagenetically-accumulated As.

Colloidal Size and Redox State of Uranium Species in the Porewater of a Pristine Mountain Wetland.

Uranium (U) speciation was investigated in anoxically preserved porewater samples of a natural mountain wetland in Gola di Lago, Ticino, Switzerland. U porewater concentrations ranged from less than 1 µg/L to tens of µg/L, challenging the available analytical approaches for U speciation in natural samples. Asymmetrical flow field-flow fractionation coupled with inductively coupled plasma mass spectrometry allowed the characterization of colloid populations and the determination of the size distribution of U species in the porewater. Most of the U was associated with three fractions: <0.3 kDa, likely including dissolved U and very small U colloids; a 1-3 kDa fraction containing humic-like organic compounds, dispersed Fe, and, to a small extent, Fe nanoparticles; and a third fraction (5-50 nm), containing a higher amount of Fe and a lower amount of organic matter and U relative to the 1-3 kDa fraction. The proportion of U associated with the 1-3 kDa colloids varied spatially and seasonally. Using anion exchange resins, we also found that a significant proportion of U occurs in its reduced form, U(IV). Tetravalent U was interpreted as occurring within the colloidal pool of U. This study suggests that U(IV) can occur as small (1-3 kDa), organic-rich, and thus potentially mobile colloidal species in naturally reducing wetland environments.

As release under the microbial sulfate reduction during redox oscillations in the upper Mekong delta aquifers, Vietnam: A mechanistic study.

The impact of seasonal fluctuations linked to monsoon and irrigation generates redox oscillations in the subsurface, influencing the release of arsenic (As) in aquifers. Here, the biogeochemical control on As mobility was investigated in batch experiments using redox cycling bioreactors and As- and SO42--amended sediment. Redox potential (Eh) oscillations between anoxic (-300-0 mV) and oxic condition (0-500 mV) were implemented by automatically modulating an admixture of N2/CO2 or compressed air. A carbon source (cellobiose, a monomer of cellulose) was added at the beginning of each reducing cycle to stimulate the metabolism of the native microbial community. Results show that successive redox cycles can decrease arsenic mobility by up to 92% during reducing conditions. Anoxic conditions drive mainly the conversion of soluble As(V) to As(III) in contrast to oxic conditions. Phylogenetic analyses of 16S rRNA amplified from the sediments revealed the presence of sulfate and iron - reducing bacteria, confirming that sulfate and iron reduction are key factors for As immobilization from the aqueous phase. As and S K-edge X-ray absorption spectroscopy suggested the association of Fe-(oxyhydr)oxides and the importance of pyrite (FeS2(s)), rather than poorly ordered mackinawite (FeS(s)), for As sequestration under oxidizing and reducing conditions, respectively. Finally, these findings suggest a role for elemental sulfur in mediating aqueous thioarsenates formation in As-contaminated groundwater of the Mekong delta.

Biogeochemical Cycling by a Low-Diversity Microbial Community in Deep Groundwater.

Olkiluoto, an island on the south-west coast of Finland, will host a deep geological repository for the storage of spent nuclear fuel. Microbially induced corrosion from the generation of sulphide is therefore a concern as it could potentially compromise the longevity of the copper waste canisters. Groundwater at Olkiluoto is geochemically stratified with depth and elevated concentrations of sulphide are observed when sulphate-rich and methane-rich groundwaters mix. Particularly high sulphide is observed in methane-rich groundwater from a fracture at 530.6 mbsl, where mixing with sulphate-rich groundwater occurred as the result of an open drill hole connecting two different fractures at different depths. To determine the electron donors fuelling sulphidogenesis, we combined geochemical, isotopic, metagenomic and metaproteomic analyses. This revealed a low diversity microbial community fuelled by hydrogen and organic carbon. Sulphur and carbon isotopes of sulphate and dissolved inorganic carbon, respectively, confirmed that sulphate reduction was ongoing and that CO2 came from the degradation of organic matter. The results demonstrate the impact of introducing sulphate to a methane-rich groundwater with limited electron acceptors and provide insight into extant metabolisms in the terrestrial subsurface.

Arsenic Speciation in Mekong Delta Sediments Depends on Their Depositional Environment.

Arsenic contamination in groundwater is pervasive throughout deltaic regions of Southeast Asia and threatens the health of millions. The speciation of As in sediments overlying contaminated aquifers is poorly constrained. Here, we investigate the chemical and mineralogical compositions of sediment cores collected from the Mekong Delta in Vietnam, elucidate the speciation of iron and arsenic, and relate them to the sediment depositional environment. Gradual dissolution of ferric (oxyhydr)oxides with depth is observed down to 7 m, corresponding to the establishment of reducing conditions. Within the reduced sediment, layers originating from marine, coastal or alluvial depositional environments are identified and their age is consistent with a late Holocene transgression in the Mekong Delta. In the organic matter- and sulfur-rich layers, arsenic is present in association with organic matter through thiol-bonding and in the form of arsenian pyrite. The highest arsenic concentration (34-69 ppm) is found in the peat layer at 16 m and suggests the accumulation of arsenic due to the formation of thiol-bound trivalent arsenic (40-55%) and arsenian pyrite (15-30%) in a paleo-mangrove depositional environment (∼8079 yr BP). Where sulfur is limited, siderite is identified, and oxygen- and thiol-bound trivalent arsenic are the predominant forms. It is also worth noting that pentavalent arsenic coordinated to oxygen is ubiquitous in the sediment profile, even in reduced sediment layers. But the identity of the oxygen-bound arsenic species remains unknown. This work shows direct evidence of thiol-bound trivalent arsenic in the Mekong Delta sediments and provides insight to refine the current model of the origin, deposition, and release of arsenic in the alluvial aquifers of the Mekong Delta.

Phylogenetic comparison of Desulfotomaculum species of subgroup 1a and description of Desulfotomaculum reducens sp. nov.

A genome and physiological comparison was made of the type strains of Desulfotomaculum species belonging to subgroup 1a and of 'Desulfotomaculum reducens' strain MI-1. Phenotypically, 'Desulfotomaculum reducens' strain MI-1 can be distinguished from the other described Desulfotomaculum species of subgroup 1a by its ability to grow with propionate and butyrate. In addition, the strain is able to use a variety of metals as electron acceptors. Metal reduction has not been tested in the other species, but seems likely based on our genome analysis. Phylogenetic 16S rRNA gene sequence analysis and the average nucleotide identity between the genomes of the species of subgroup 1a show that strain MI-1 represents a novel species within the Desulfotomaculum 1a subgroup, Desulfotomaculum reducens sp. nov. The type strain is MI-1T.

Mobile uranium(IV)-bearing colloids in a mining-impacted wetland.

Tetravalent uranium is commonly assumed to form insoluble species, resulting in the immobilization of uranium under reducing conditions. Here we present the first report of mobile U(IV)-bearing colloids in the environment, bringing into question this common assumption. We investigate the mobility of uranium in a mining-impacted wetland in France harbouring uranium concentrations of up to 14,000 p.p.m. As an apparent release of uranium into the stream passing through the wetland was observable, we examine soil and porewater composition as a function of depth to assess the geochemical conditions leading to this release. The analyses show the presence of U(IV) in soil as a non-crystalline species bound to amorphous Al-P-Fe-Si aggregates, and in porewater, as a distinct species associated with Fe and organic matter colloids. These results demonstrate the lability of U(IV) in these soils and its association with mobile porewater colloids that are ultimately released into surface water.

Metal reduction by spores of Desulfotomaculum reducens.

The bioremediation of uranium-contaminated sites is designed to stimulate the activity of microorganisms able to catalyze the reduction of soluble U(VI) to the less soluble mineral UO(2). U(VI) reduction does not necessarily support growth in previously studied bacteria, but it typically involves viable vegetative cells and the presence of an appropriate electron donor. We characterized U(VI) reduction by the sulfate-reducing bacterium Desulfotomaculum reducens strain MI-1 grown fermentatively on pyruvate and observed that spores were capable of U(VI) reduction. Hydrogen gas - a product of pyruvate fermentation - rather than pyruvate, served as the electron donor. The presence of spent growth medium was required for the process, suggesting that an unknown factor produced by the cells was necessary for reduction. Ultrafiltration of the spent medium followed by U(VI) reduction assays revealed that the factor's molecular size was below 3 kDa. Pre-reduced spent medium displayed short-term U(VI) reduction activity, suggesting that the missing factor may be an electron shuttle, but neither anthraquinone-2,6-disulfonic acid nor riboflavin rescued spore activity in fresh medium. Spores of D. reducens also reduced Fe(III)-citrate under experimental conditions similar to those for U(VI) reduction. This is the first report of a bacterium able to reduce metals while in a sporulated state and underscores the novel nature of the mechanism of metal reduction by strain MI-1.

Metastasizing melanoma formation caused by expression of activated N-RasQ61K on an INK4a-deficient background.

In human cutaneous malignant melanoma, a predominance of activated mutations in the N-ras gene has been documented. To obtain a mouse model most closely mimicking the human disease, a transgenic mouse line was generated by targeting expression of dominant-active human N-ras (N-RasQ61K) to the melanocyte lineage by tyrosinase regulatory sequences (Tyr::N-RasQ61K). Transgenic mice show hyperpigmented skin and develop cutaneous metastasizing melanoma. Consistent with the tumor suppressor function of the INK4a locus that encodes p16INK4A and p19(ARF), >90% of Tyr::N-RasQ61K INK4a-/- transgenic mice develop melanoma at 6 months. Primary melanoma tumors are melanotic, multifocal, microinvade the epidermis or epithelium of hair follicles, and disseminate as metastases to lymph nodes, lung, and liver. Primary melanoma can be transplanted s.c. in nude mice, and if injected i.v. into NOD/SCID mice colonize the lung. In addition, primary melanomas and metastases contain cells expressing the stem cell marker nestin suggesting a hierarchical structure of the tumors comprised of primitive nestin-expressing precursors and differentiated cells. In conclusion, a novel mouse model with melanotic and metastasizing melanoma was obtained by recapitulating genetic lesions frequently found in human melanoma.