Carbonate minerals are considered a metastable carbon reservoir under alkaline conditions. Here we demonstrate calcium carbonate as a sole inorganic carbon source at moderately alkaline pH for a methanogenic microbial community enriched from alkaline, saline wetland soil. In reactors amended with calcium carbonate as the sole source of inorganic carbon, concentrations of methane and aqueous calcium increased concurrent with headspace hydrogen depletion. Cells were observed in association with the carbonate mineral matrix via coherent anti-Stokes Raman scattering and two-photon excitation fluorescence microscopy. Genome-resolved metagenomics of the enrichment community confirmed the presence of a methanogen (Methanobacterium sp.) with the metabolic potential for hydrogenotrophic methanogenesis in addition to five bacterial community members. These results demonstrate that carbonate minerals can serve as an inorganic carbon source for hydrogenotrophic methanogenesis, leading to mineral dissolution under stable alkaline conditions. As such, hydrogenotrophic methanogens may impact carbon biogeochemistry and carbonate mineral stability in environmental systems. Carbonate minerals can be used as inorganic carbon source for microbial methanogenesis resulting in mineral dissolution under stable alkaline conditions, according to soil enrichment culture experiments supported by metagenomic sequencing.

Lignin degradation is a major process in the global carbon cycle across both terrestrial and marine ecosystems. Bathyarchaeia, which are among the most abundant microorganisms in marine sediment, have been proposed to mediate anaerobic lignin degradation. However, the mechanism of bathyarchaeial lignin degradation remains unclear. Here, we report an enrichment culture of Bathyarchaeia, named Candidatus Baizosediminiarchaeum ligniniphilus DL1YTT001 (Ca. B. ligniniphilus), from coastal sediments that can grow with lignin as the sole organic carbon source under mesophilic anoxic conditions. Ca. B. ligniniphilus possesses and highly expresses novel methyltransferase 1 (MT1, mtgB) for transferring methoxyl groups from lignin monomers to cob(I)alamin. MtgBs have no homology with known microbial methyltransferases and are present only in bathyarchaeial lineages. Heterologous expression of the mtgB gene confirmed O-demethylation activity. The mtgB genes were identified in metagenomic data sets from a wide range of coastal sediments, and they were highly expressed in coastal sediments from the East China Sea. These findings suggest that Bathyarchaeia, capable of O-demethylation via their novel and specific methyltransferases, are ubiquitous in coastal sediments.

Deep sea cold seep sediments have been discovered to harbor novel, abundant, and diverse bacterial and archaeal viruses. However, little is known about viral genetic features and evolutionary patterns in these environments. Here, we examined the evolutionary ecology of viruses across active and extinct seep stages in the area of Haima cold seeps in the South China Sea. A total of 338 viral operational taxonomic units are identified and linked to 36 bacterial and archaeal phyla. The dynamics of host-virus interactions are informed by diverse antiviral defense systems across 43 families found in 487 microbial genomes. Cold seep viruses are predicted to harbor diverse adaptive strategies to persist in this environment, including counter-defense systems, auxiliary metabolic genes, reverse transcriptases, and alternative genetic code assignments. Extremely low nucleotide diversity is observed in cold seep viral populations, being influenced by factors including microbial host, sediment depth, and cold seep stage. Most cold seep viral genes are under strong purifying selection with trajectories that differ depending on whether cold seeps are active or extinct. This work sheds light on the understanding of environmental adaptation mechanisms and evolutionary patterns of viruses in the sub-seafloor biosphere.

Groundwater uranium (U) concentrations have been measured above the U.S. EPA maximum contaminant level (30 μg/L) in many U.S. aquifers, including in areas not associated with anthropogenic contamination by milling or mining. In addition to carbonate, nitrate has been correlated to uranium groundwater concentrations in two major U.S. aquifers. However, to date, direct evidence that nitrate mobilizes naturally occurring U from aquifer sediments has not been presented. Here, we demonstrate that the influx of high-nitrate porewater through High Plains alluvial aquifer silt sediments bearing naturally occurring U(IV) can stimulate a nitrate-reducing microbial community capable of catalyzing the oxidation and mobilization of U into the porewater. Microbial reduction of nitrate yielded nitrite, a reactive intermediate, which was further demonstrated to abiotically mobilize U from the reduced alluvial aquifer sediments. These results indicate that microbial activity, specifically nitrate reduction to nitrite, is one mechanism driving U mobilization from aquifer sediments in addition to previously described bicarbonate-driven desorption from mineral surfaces, such as Fe(III) oxides.

Horizontal gene transfer is an important driver of adaptation and evolution in microorganisms. Transducing biological nanoparticles such as viral particles are believed to be key facilitators of horizontal gene transfer. In deep subseafloor sediments, energy can be highly limiting, supporting only extremely slow metabolisms. In such low-energy, isolated environments where communities may subsist for millions of years, the mechanisms of subsurface microbial adaptation and evolution remain a mystery. Virus particles have been found everywhere that life has been found, including deep subsurface environments. Although microorganisms are abundant and active in the Earth's subsurface, the role of viruses in shaping and influencing these slow-growing communities is only recently starting to be explored. Here, we analyzed the deeply buried microbial community from a lignite coalbed layer 2km below the seafloor offshore Shimokita, Japan (IODP Expedition 337) that had been buried for 20 million years. We harvested cells (>0.2µm) and biological nanoparticles (<0.2µm) from a bioreactor enrichment seeded by lignite core samples. We sequenced DNA from the cells and nanoparticles and subsequently analyzed the metagenomes. Within the nanoparticle metagenome, numerous complete novel virus genomes were reconstructed. Comparison of the virus genomes to the prokaryotic MAGs (metagenome assembled genomes) revealed that many of the virus genomes had been integrated prophage within bacterial genomes, suggesting the potential for virus-host interactions to occur in the deep subseafloor. Additionally, lysogeny may be an important survival mechanism for viruses in deeply buried, low-energy environments. Host genes were found to be packaged by viral particles, demonstrating the potential for specialized and general transduction by viruses. Not only viral particles, but there was also evidence that membrane vesicles and gene transfer agents may participate in transduction in this deep subsurface community. Horizontal gene transfer mediated by biological nanoparticles may be an important mechanism of adaptation for deep subsurface microbial communities and may provide insight into possible evolutionary processes shaping microbial communities in the deep subsurface. These results may also shed some light onto the nature of viral infection in the subsurface, potentially revealing insights about the long-term persistence of life under extreme energy limitation and how viruses may survive this over geological timescales.

Here, we report the draft genome sequence of the siderophilic cyanobacterium Fischerella thermalis JSC-11, which was isolated from an iron-depositing hot spring. JSC-11 has bioremediation potential because it is capable of both extracellular absorption and intracellular mineralization of colloidal iron. This genomic information will facilitate the exploration of JSC-11 for bioremediation.

Anaerobic lignin degradation is a major process in the global carbon cycle that would significantly influence estimates of carbon flux in both terrestrial and marine ecosystems. The ubiquitous Bathyarchaeia , one of the most abundant taxa in marine sediments, have been proposed to be key players in this process. However, the mechanism of Bathyarchaeial lignin degradation is unclear due to the lack of cultured strains. Here we report the cultivation of Candidatus Marisediminiarchaeum ligniniphilus DL1YTT001, a Bathyarchaeial representative from nearshore marine sediments that can grow with lignin as the sole organic carbon source under mesophilic and anaerobic conditions. Strain DL1YTT001 possesses and highly expresses a novel and specific methyltransferase system for O-demethylation of lignin-derived methoxylated aromatic compounds (ArOCH 3 ). The key gene, methyltransferase 1 (MtgB), is not homologous to any other lineages. Enzymatic activity was confirmed through the heterologous expression of the MtgB gene, showing O-demethylation activity with guaiacol as the substrate. Considering that Bathyarchaeial lineages carrying this specific methyltransferase system are widely distributed in diverse anoxic environments, especially lignin-rich nearshore sediments, Bathyarchaeia -mediated O-demethylation is likely a key step in global anaerobic lignin remineralization.

Uranium minerals are commonly found in soils and sediment across the United States at an average concentration of 2-4 mg/kg. Uranium occurs in the environment primarily in two forms, the oxidized, mostly soluble uranium(VI) form, or the reduced, sparingly soluble reduced uranium(IV) form. Here we describe subsurface geochemical conditions that result in low uranium concentrations in an alluvial aquifer with naturally occurring uranium in soils and sediments in the presence of complexing ligands under oxidizing conditions. Groundwater was saturated with respect to calcite and contained calcium (78-90 mg/L) with elevated levels of carbonate alkalinity (291-416 mg/L as HCO3-). X-ray adsorption near edge structure (XANES) spectroscopy identified that sediment-associated uranium was oxidized as a uranium(VI) form (85%). Calcite was the predominant mineral by mass in the ultrafine fraction in uranium-bearing sediments (>16 mg/kg). Groundwater geochemical modeling indicated calcite and/or a calcium-uranyl-carbonate mineral such as liebigite in equilibrium with groundwater. The δ13C (0.57‰ ± 0.15‰) was indicative of abiotic carbonate deposition. Thus, solid-phase uranium(VI) associated with carbonate is likely maintaining uranium(VI) groundwater levels below the maximum contaminant level (MCL; 30 µg/L), presenting a deposition mechanism for uranium attenuation rather than solely a means of mobilization.

The siderophilic, thermophilic Leptolyngbyaceae cyanobacterium JSC-12 was isolated from a microbial mat in an iron-depositing hot spring. Here, we report the high-quality draft genome sequence of JSC-12, which may help elucidate the mechanisms of resistance to extreme iron concentrations in siderophilic cyanobacteria and lead to new remediation biotechnologies.

A moderately acidophilic Geobacter sp. strain, strain FeAm09, was isolated from forest soil. The complete genome sequence is 4,099,068 bp, with an average GC content of 61.1%. No plasmids were detected. The genome contains a total of 3,843 genes and 3,608 protein-coding genes, including genes supporting iron and nitrogen biogeochemical cycling.

Viruses are enigmatic, with scientists debating, often heatedly, about the nature of their existence, such as whether viruses should be categorized as living or non-living. They are not even represented in most conventional trees of life. However, viruses have played a driving role in the co-evolution of life on Earth since its origins. Viruses can serve as molecular fossils, providing a possible glimpse at a pool of genes that may have been present from a time before the Last Universal Common Ancestor (LUCA). The study of virology and the origin of life have often been intertwined. Since the earliest studies of viruses, viruses have been hypothesized to be ancient, possibly anteceding cell-based life. Other hypotheses propose that cell-based life preceded viruses, with viruses “escaping” from cellular life forms. This chapter will introduce viruses and their relationship with the origin of life, including the various proposed origin scenarios involving viruses and virus-like elements.

In marine ecosystems, viruses exert control on the composition and metabolism of microbial communities, influencing overall biogeochemical cycling. Deep sea sediments associated with cold seeps are known to host taxonomically diverse microbial communities, but little is known about viruses infecting these microorganisms. Here, we probed metagenomes from seven geographically diverse cold seeps across global oceans to assess viral diversity, virus-host interaction, and virus-encoded auxiliary metabolic genes (AMGs). Gene-sharing network comparisons with viruses inhabiting other ecosystems reveal that cold seep sediments harbour considerable unexplored viral diversity. Most cold seep viruses display high degrees of endemism with seep fluid flux being one of the main drivers of viral community composition. In silico predictions linked 14.2% of the viruses to microbial host populations with many belonging to poorly understood candidate bacterial and archaeal phyla. Lysis was predicted to be a predominant viral lifestyle based on lineage-specific virus/host abundance ratios. Metabolic predictions of prokaryotic host genomes and viral AMGs suggest that viruses influence microbial hydrocarbon biodegradation at cold seeps, as well as other carbon, sulfur and nitrogen cycling via virus-induced mortality and/or metabolic augmentation. Overall, these findings reveal the global diversity and biogeography of cold seep viruses and indicate how viruses may manipulate seep microbial ecology and biogeochemistry.

Viruses are highly abundant, diverse, and active components of marine environments. Flow cytometry has helped to increase the understanding of their impact on shaping microbial communities and biogeochemical cycles in the pelagic zone. However, to date, flow cytometric quantification of sediment viruses is still hindered by interference from the sediment matrix. Here, we developed a protocol for the enumeration of marine sediment viruses by flow cytometry based on separation of viruses from sediment particles using a Nycodenz density gradient. Results indicated that there was sufficient removal of background interference to allow for flow cytometric quantification. Applying this new protocol to deep-sea and tidal-flat samples, viral abundances enumerated by flow cytometry correlated well (R2 = 0.899) with counts assessed by epifluorescence microscopy over several orders of magnitude from marine sediments of various compositions. Further optimization may be needed for sediments with low biomass or high organic content. Overall, the new protocol enables fast and accurate quantification of marine sediment viruses, and opens up the options for virus sorting, targeted viromics, and single-virus sequencing.

Microorganisms in marine subsurface sediments substantially contribute to global biomass. Sediments warmer than 40°C account for roughly half the marine sediment volume, but the processes mediated by microbial populations in these hard-to-access environments are poorly understood. We investigated microbial life in up to 1.2-kilometer-deep and up to 120°C hot sediments in the Nankai Trough subduction zone. Above 45°C, concentrations of vegetative cells drop two orders of magnitude and endospores become more than 6000 times more abundant than vegetative cells. Methane is biologically produced and oxidized until sediments reach 80° to 85°C. In 100° to 120°C sediments, isotopic evidence and increased cell concentrations demonstrate the activity of acetate-degrading hyperthermophiles. Above 45°C, populated zones alternate with zones up to 192 meters thick where microbes were undetectable.

X-ray computed tomography (XCT) can be used to identify lithologies and deformation structures within geological core, with the potential for the identification processes to be applied automatically. However, because of drilling disturbance and other artifacts, the use of large XCT-datasets in automated processes requires methods of quality control that can be applied systematically. We propose a new systematic method for quality control of XCT data that applies numerical measures to CT slices, and from this obtains data reflective of core quality. Because the measures are numerical they can be applied quickly and consistently between different sections and cores. This quality control processing protocol produces downhole radiodensity profiles from mean CT-values that can be used for geological interpretation. The application of this quality control protocols was applied to XCT data from International Ocean Discovery Program (IODP) Expedition 370 Site C0023 located at the toe of the Nankai accretionary complex. The evaluation of core quality based on this protocol was found to be a good fit to standard-evaluations based on the visual description of core, and could be used to select samples free from drilling disturbance or contamination. The quality-controlled downhole mean CT-value profile has features that can be used to identify lithologies within a formation, the presence and type of deformation structures and to distinguish formations.

Viruses are the most abundant biological entities on Earth and perform essential ecological functions in aquatic environments by mediating biogeochemical cycling and lateral gene transfer. Cellular life as well as viruses have been found in deep subseafloor sediment. However, the study of deep sediment viruses has been hampered by the complexities involved in efficiently extracting viruses from a sediment matrix. Here, we developed a new method for the extraction of viruses from sediment based on density separation using a Nycodenz density step gradient. The density separation method resulted in up to 2 orders of magnitude greater recovery of viruses from diverse subseafloor sediments compared to conventional methods. The density separation method also showed more consistent performance between samples of different sediment lithology, whereas conventional virus extraction methods were highly inconsistent. Using this new method, we show that previously published virus counts have underestimated viral abundances by up to 2 orders of magnitude. These improvements suggest that the carbon contained within viral biomass in the subseafloor environment may potentially be revised upward to 0.8-3.7 Gt from current estimates of 0.2 Gt. The vastly improved recovery of viruses indicate that viruses represent a far larger pool of organic carbon in subseafloor environments than previously estimated.

The study investigates the in-situ strength of sediments across a plate boundary décollement using drilling parameters recorded when a 1180-m-deep borehole was established during International Ocean Discovery Program (IODP) Expedition 370, Temperature - Limit of the Deep Biosphere off Muroto ( T - Limit ). Information of the in-situ strength of the shallow portion in/around a plate boundary fault zone is critical for understanding the development of accretionary prisms and of the décollement itself. Studies using seismic reflection surveys and scientific ocean drillings have recently revealed the existence of high pore pressure zones around frontal accretionary prisms, which may reduce the effective strength of the sediments. A direct measurement of in-situ strength by experiments, however, has not been executed due to the difficulty in estimating in-situ stress conditions. In this study, we derived a depth profile for the in-situ strength of a frontal accretionary prism across a décollement from drilling parameters using the recently established equivalent strength (EST) method. At site C0023, the toe of the accretionary prism area off Cape Muroto, Japan, the EST gradually increases with depth but undergoes a sudden change at ~ 800 mbsf, corresponding to the top of the subducting sediment. At this depth, directly below the décollement zone, the EST decreases from ~ 10 to 2 MPa, with a change in the baseline. This mechanically weak zone in the subducting sediments extends over 250 m (~ 800–1050 mbsf), corresponding to the zone where the fluid influx was discovered, and high-fluid pressure was suggested by previous seismic imaging observations. Although the origin of the fluids or absolute values of the strength remain unclear, our investigations support previous studies suggesting that elevated pore pressure beneath the décollement weakens the subducting sediments.

The growing complexity and interdependence of water management processes requires the involvement of multiple stakeholders in water governance. Multi-party collaboration is increasingly vital at both the strategy development and implementation levels. Multi-party collaboration involves a process of joint decision-making among key stakeholders in a problem domain directed towards the future of that domain. However, the common goal is not present from the beginning; rather, the common goal emerges during the process of collaboration. Unfortunately, when the conflicting interests of different actors are at stake, the large majority of environmental multi-party efforts often do not reliably deliver sustainable improvements to policy and/or practice. One of the reasons for this, which has been long established by many case studies, is that social learning with a focus on relational practices is missing. The purpose of this paper is to present the design and initial results of a pilot study that utilized a game-based approach to explore the effects of relational practices on the effectiveness of water governance. This paper verifies the methods used by addressing the following question: are game mechanisms, protocols for facilitation and observation, the recording of decisions and results, and participant surveys adequate to reliably test hypotheses about behavioral decisions related to water governance? We used the "Lords of the Valley" (LOV) game, which focuses on the local-level management of a hypothetical river valley involving many stakeholders. We used an observation protocol to collect data on the quality of relational practices and compared this data with the quantitative outcomes achieved by participants in the game. In this pilot study, we ran the game three times with different groups of participants, and here we provide the outcomes within the context of verifying and improving the methods.

Reduced zones in the subsurface represent biogeochemically active hotspots enriched in buried organic matter and reduced metals. Within a shallow alluvial aquifer located near Rifle, CO, reduced zones control the fate and transport of uranium (U). Though an influx of dissolved oxygen (DO) would be expected to mobilize U, we report U immobilization. Groundwater U concentrations decreased following delivery of DO (21.6 mg O2/well/h). After 23 days of DO delivery, injection of oxygenated groundwater was paused and resulted in the rebound of groundwater U concentrations to preinjection levels. When DO delivery resumed (day 51), groundwater U concentrations again decreased. The injection was halted on day 82 again and resulted in a rebound of groundwater U concentrations. DO delivery rate was increased to 54 mg O2/well/h (day 95) whereby groundwater U concentrations increased. Planktonic cell abundance remained stable throughout the experiment, but virus-to-microbial cell ratio increased 1.8-3.4-fold with initial DO delivery, indicative of microbial activity in response to DO injection. Together, these results indicate that the redox-buffering capacity of reduced sediments can prevent U mobilization, but could be overcome as delivery rate or oxidant concentration increases, mobilizing U.

International Ocean Discovery Program (IODP) Expedition 370 aimed to explore the limits of life in the deep subseafloor biosphere at a location where temperature increases with depth at an intermediate rate and exceeds the known temperature maximum of microbial life (~120°C) at the sediment/basement interface ~1.2 km below the seafloor. Drilling Site C0023 is located in the vicinity of Ocean Drilling Program (ODP) Sites 808 and 1174 at the protothrust zone in the Nankai Trough off Cape Muroto at a water depth of 4776 m. ODP Leg 190 in 2000, revealed the presence of microbial cells at Site 1174 to a depth of ~600 meters below seafloor (mbsf), which corresponds to an estimated temperature of ~70°C, and reliably identified a single zone of higher cell concentrations just above the decollement at around 800 mbsf, where temperature presumably reached 90°C; no cell count data was reported for other sediment layers in the 70°–120°C range, because the limit of manual cell count for low-biomass samples was not high enough. With the establishment of Site C0023, we aimed to detect and investigate the presence or absence of life and biological processes at the biotic–abiotic transition with unprecedented analytical sensitivity and precision. Expedition 370 was the first expedition dedicated to subseafloor microbiology that achieved time-critical processing and analyses of deep biosphere samples by simultaneous shipboard and shore-based investigations. Our primary objectives during Expedition 370 were to study the relationship between the deep subseafloor biosphere and temperature. We aimed to comprehensively study the factors that control biomass, activity, and diversity of microbial communities in a subseafloor environment where temperatures increase from ~2°C at the seafloor to ~120°C at the sediment/basement interface and thus likely encompasses the biotic–abiotic transition zone. We also aimed to determine geochemical, geophysical, and hydrogeological characteristics in sediment and the underlying basaltic basement and elucidate if the supply of fluids containing thermogenic and/or geogenic nutrient and energy substrates may support subseafloor microbial communities in the Nankai accretionary complex. To address these primary scientific objectives and questions, we penetrated 1180 m and recovered 112 cores across the sediment/basalt interface. More than 13,000 samples were collected, and selected samples were transferred to the Kochi Core Center by helicopter for simultaneous microbiological sampling and analysis in laboratories with a super-clean environment. Following the coring operations, a temperature observatory with 13 thermistor sensors was installed in the borehole to 863 mbsf.