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Serpentinization-influenced hydrothermal systems, such as the Lost City Hydrothermal Field (LCHF), are considered as potential sites for the origin of life. Despite an abundance of reducing power in this system (H2 and CH4), microbial habitability may be limited by high pH, elevated temperatures, and/or low concentrations of bioavailable carbon. At the LCHF, the relative contribution of biotic and abiotic processes to the vent fluid composition, especially in the lower temperature vents, remain poorly constrained. We present fluid chemistry and isotope data that suggest that all LCHF fluids are derived from a single endmember produced in the hotter, deeper subsurface essentially in the absence of microbial activity. The strontium isotope composition (87Sr/86Sr) of this fluid records the influence of underlying mantle and/or gabbroic rocks, whereas sulfur isotope composition indicates closed-system thermochemical sulfate reduction. Conductive cooling and transport is accompanied by continued sulfate reduction, likely microbial, and mixing with unaltered seawater, which produce second-order vents characterized by higher δ34Ssulfide and lower δ34Ssulfate values. Third-order vent fluids are produced by varying degrees of subsurface mixing between the first- and second-order fluids and a seawater-dominated fluid. Additional biotic and abiotic processes along different flow paths are needed to explain the spatial variability among the vents. Relationships between sulfur geochemistry and hydrogen concentrations dominantly reflect variations in temperature and/or distance from the primary outflow path. Methane concentrations are constant across the field which point to an origin independent of flow path and venting temperature. At Lost City, not all vent fluids appear to have zero Mg concentrations. Thus, we propose an extrapolation to a Sr isotope-endmember composition as an alternative method to estimate endmember fluid compositions at least in similar systems where a two-component mixing with respect to Sr isotopes between seawater and endmember fluids can be established., Geochimica et Cosmochimica Acta, 332, ISSN:0016-7037, ISSN:1872-9533

The Lost City hydrothermal field (LCHF) is considered an analogue of Archean alkaline hydrothermal vents where life on Earth may have spawned. Although Lost City was discovered more than 20 years ago, it remains unclear to what extent microbes are involved in the precipitation of the various minerals constituting the hydrothermal chimneys. Most chimneys preserve flow textures comprised of mineral walls bounding paleo-channels, which are preserved in inactive vent structures to a varying degree. Brucite lines the internal part of these channels, while aragonite dominates the exterior. Calcite is also present locally, mostly associated with brucite. Based on a combination of microscopic and geochemical analyses, we interpret brucite, calcite, and aragonite as primary minerals that precipitate abiotically from mixing seawater and hydrothermal fluids. We observe local brucite precipitation on microbial filaments and, in some cases, microbial filaments may affect the growth direction of brucite crystals. The link between brucite and organic compounds is further confirmed by fluorescence microscopy: brucite shows a strong fluorescence not observed in calcite and aragonite. Our results point to brucite as an important link to microbial life in alkaline fluids and is potentially a key aspect for future research on the origin of life.

Carbonate-brucite chimneys are a characteristic of low- to moderate-temperature, ultramafic-hosted alkaline hydrothermal systems, such as the Lost City hydrothermal field located on the Atlantis Massif at 30°N near the Mid-Atlantic Ridge. These chimneys form as a result of mixing between warm, serpentinization-derived vent fluids and cold seawater. Previous work has documented the evolution in mineralogy and geochemistry associated with the aging of the chimneys as hydrothermal activity wanes. However, little is known about spatial heterogeneities within and among actively venting chimneys. New mineralogical and geochemical data (87Sr/86Sr and stable C, O, and clumped isotope) indicate that brucite and calcite precipitate at elevated temperatures in vent fluid-dominated domains in the interior of chimneys. Exterior zones dominated by seawater are brucite-poor and aragonite is the main carbonate mineral. Carbonates form mostly out of oxygen and clumped isotope equilibrium due to rapid precipitation upon vent fluid-seawater mixing. In contrast, the carbonates precipitate close to carbon isotope equilibrium, with dissolved inorganic carbon in seawater as the dominant carbon source, and have δ13C values within the range of marine carbonates. Our data suggest that calcite is a primary mineral in the active hydrothermal chimneys and does not exclusively form as a replacement of aragonite during later alteration with seawater. Elevated formation temperatures and lower 87Sr/86Sr relative to aragonite in the same sample suggest that calcite may be the first carbonate mineral to precipitate.

Alkaline fluids venting from chimneys of the Lost City hydrothermal field flow from a potentially vast microbial habitat within the seafloor where energy and organic molecules are released by chemical reactions within rocks uplifted from Earth's mantle. In this study, we investigated hydrothermal fluids venting from Lost City chimneys as windows into subseafloor environments where the products of geochemical reactions, such as molecular hydrogen (H-2), formate, and methane, may be the only available sources of energy for biological activity. Our deep sequencing of metagenomes and metatranscriptomes from these hydrothermal fluids revealed a few key species of archaea and bacteria that are likely to play critical roles in the subseafloor microbial ecosystem. We identified a population of Thermodesulfovibrionales (belonging to phylum Nitrospirota) as a prevalent sulfate-reducing bacterium that may be responsible for much of the consumption of H-2 and sulfate in Lost City fluids. Metagenome-assembled genomes (MAGs) classified as Methanosarcinaceae and Candidatus Bipolaricaulota were also recovered from venting fluids and represent potential methanogenic and acetogenic members of the subseafloor ecosystem. These genomes share novel hydrogenases and formate dehydrogenase-like sequences that may be unique to hydrothermal environments where H-2 and formate are much more abundant than carbon dioxide. The results of this study include multiple examples of metabolic strategies that appear to be advantageous in hydrothermal and subsurface alkaline environments where energy and carbon are provided by geochemical reactions. IMPORTANCE The Lost City hydrothermal field is an iconic example of a microbial ecosystem fueled by energy and carbon from Earth's mantle. Uplift of mantle rocks into the seafloor can trigger a process known as serpentinization that releases molecular hydrogen (H-2) and creates unusual environmental conditions where simple organic carbon molecules are more stable than dissolved inorganic carbon. This study provides an initial glimpse into the kinds of microbes that live deep within the seafloor where serpentinization takes place, by sampling hydrothermal fluids exiting from the Lost City chimneys. The metabolic strategies that these microbes appear to be using are also shared by microbes that inhabit other sites of serpentinization, including continental subsurface environments and natural springs. Therefore, the results of this study contribute to a broader, interdisciplinary effort to understand the general principles and mechanisms by which serpentinization-associated processes can support life on Earth and perhaps other worlds., Applied and Environmental Microbiology, 88 (17), ISSN:0099-2240, ISSN:1098-5336

The isotopic composition of lipid biomarkers and biomass in sedimentary environments are widely used to infer microbial metabolisms and constrain carbon cycling processes. It has been observed that metabolic energy availability in the form of H2 impacts the stable carbon isotopes of CH4 during hydrogenotrophic methanogenesis, but it is unknown whether this relationship extends to lipids and amino acids. Since lipids and amino acids are long-lived, they can be used to reconstruct past conditions over geologic timescales. Therefore, the controls on their isotopic signatures are important to constrain to better interpret past environments. In this study, the isotopic distributions of carbon metabolized and reduced by the hyperthermophile Methanocaldococcus jannaschii were quantified following growth at 82 °C in a chemostat with high (80–83 µM) and low (15–27 µM) H2 concentrations. As has been shown previously, the stable carbon isotope fractionation factors for CH4 were >15‰ larger in low H2 experiments than in high H2 experiments. Lipid biomarkers and amino acids were similarly impacted with approximately 10‰ larger fractionation factors under low H2 conditions. The increase in fractionation factors can be related to the lower availability of thermodynamic energy, suggesting that even larger fractionation factors would be observed in methanogens living close to their threshold energy needs, as they do in most environments. The resulting isotopic signatures of long-lived lipid biomarkers synthesized by hydrogenotrophic methanogens may become as ‘superlight’ as those synthesized by archaea carrying out the anaerobic oxidation of methane. These results help to describe the underlying mechanisms that determine the isotopic composition of long-lived biomarkers and provide constraints for interpreting these signatures in the environment.

The response of terrestrial ecosystems to anthropogenic climate change remains poorly understood and constitutes a major source of uncertainty in future climate projections. Stalagmites are a well-established terrestrial climate archive (Wong and Breecker, 2015), and may also serve as a sensitive recorder of surface ecosystem processes, as they are fed by dripping water that percolates through the soil zone. Coupled climate-ecosystem paleorecords from stalagmites could prove invaluable sources of information on the response of terrestrial ecosystems to past climatic shifts and their sensitivity to climate variability exceeding the instrumental range.Here we present results from a method development study carried out at the Laboratory for the Analysis of Radiocarbon with AMS (LARA) at the University of Bern. Our results are based on a previous protocol by Lechleitner et al. (2019), which describes a rapid, low contamination method for carbon isotope analysis (δ13C and 14C) of the speleothem non-purgeable organic carbon (NPOC) fraction for small samples (3). Decarbonation of acid digested carbonate samples is followed by wet chemical oxidation of the NPOC and analysis of the resulting headspace CO2 via mass spectrometry to determine its isotopic composition.We have made significant progress in resolving the main issues that precluded the routine application of the method as presented in Lechleitner et al. (2019), namely incomplete removal of inorganic carbon from the sample solutions, and contamination from extraneous carbon at different method stages. Apart from an updated pre-cleaning protocol for stalagmite samples and extensive blank assessment, a new needle setup was installed, allowing reduction of the sample amount needed, and the CO2 flow is now being monitored during decarbonation to ensure complete removal of inorganic carbon prior to the oxidation step.These results reiterate the great promise of this method to provide accurate, ecosystem-level information on past terrestrial environments at comparatively high temporal resolution. References:Lechleitner, F.A., Lang, S.Q., Haghipour, N., McIntyre, C., Baldini, J.U.L., Prufer, K.M., Eglinton, T.I., 2019. Towards organic carbon isotope records from stalagmites: coupled d13C and 14C analysis using wet chemical oxidation. Radiocarbon 61, 749–764. doi:10.1017/RDC.2019.35Wong, C.I., Breecker, D.O., 2015. Advancements in the use of speleothems as climate archives. Quat. Sci. Rev. 127, 1–18. doi:10.1016/j.quascirev.2015.07.019

Sources of dissolved and particulate carbon to the Fraser River system vary significantly in space and time. Tributaries in the northern interior of the basin consistently deliver higher concentrations of dissolved organic carbon (DOC) to the main stem than other tributaries. Based on samples collected near the Fraser River mouth throughout 2013, the radiocarbon age of DOC exported from the Fraser River does not change significantly across seasons despite a spike in DOC concentration during the freshet, suggesting modulation of heterogeneous upstream chemical and isotopic signals during transit through the river basin. Dissolved inorganic carbon (DIC) concentrations are highest in the Rocky Mountain headwater region where carbonate weathering is evident, but also in tributaries with high DOC concentrations, suggesting that DOC respiration may be responsible for a significant portion of DIC in this basin. Using an isotope and major ion mass balance approach to constrain the contributions of carbonate and silicate weathering and DOC respiration, we estimate that up to 33 +/- 11% of DIC is derived from DOC respiration in some parts of the Fraser River basin. Overall, these results indicate close coupling between the cycling of DOC and DIC, and that carbon is actively processed and transformed during transport through the river network., Biogeochemistry, 164 (1), ISSN:0168-2563, ISSN:1573-515X

Hydrogen is an important energy source for subsurface microbial communities, but its availability beyond the flow focused through hydrothermal chimneys is largely unknown. We report the widespread export of H2 across the Atlantis Massif oceanic core complex (30°N, Mid-Atlantic Ridge; up to 44 nM), which is distinct from the circulation system feeding the Lost City Hydrothermal Field (LCHF) on the massif’s southern wall. Methane (CH4) abundances are generally low to undetectable (< 3 nM) in fluids that are not derived from the LCHF. Reducing fluids exit the seafloor over a wide geographical area and depth range, including the summit of the massif and along steep areas of mass wasting east of the field. The depth of the fluids in the water column and their H22/CH4 ratios indicate that some are sourced separately from the LCHF. We argue that extensive H2 export is the natural consequence of fluid flow pathways strongly influenced by tectonic features and the volume and density changes that occur when ultramafic rocks react to form serpentinites, producing H2 as a by-product. Furthermore, the circulation of H2-rich fluids through uplifted mantle rocks at moderate temperatures provides geographically expansive and stable environmental conditions for the early evolution of biochemical pathways. These results provide insight into the spatial extent of H2- and CH4-bearing fluids associated with serpentinization, independent of the focused flow emanating from the LCHF., Geology, 49 (7), ISSN:0091-7613, ISSN:1943-2682

Increasing inputs of organic matter (OM) are driving declining dissolved oxygen (DO) concentrations in coastal ecosystems worldwide. The quantity, source, and composition of OM transported to coastal ecosystems via stormwater runoff have been altered by land use changes associated with urbanization and subsequent hydrologic flows that accompany urban stormwater management. To elucidate the role of stormwater in the decline of coastal DO, rain event sampling of biochemical oxygen demand (BOD) in samples collected from the outfall of stormwater ponds and wetlands, as well as samples of largely untreated runoff carried by stormwater ditches, was conducted across a range of urban and suburban development densities. Sampling also included measurements of particulate and dissolved carbon and nitrogen, carbon and nitrogen stable isotopes, and chlorophyll-a. Results suggest stormwater may be a significant source of labile OM to receiving waters, especially during the first flush of runoff, even though BOD concentrations vary both among and within sites in response to rain events. BOD variability was best predicted by particulate OM (POM) and chlorophyll-a, rather than the larger pool of dissolved OM. These findings demonstrate the importance of managing episodic stormwater discharge, especially POM, from urbanized areas to mitigate DO impairment in larger downstream systems.

All living organisms synthesize phospholipids as the primary constituent of their cell membranes. Enzymatic synthesis of diacylphospholipids requires preexisting membrane-embedded enzymes. This limitation has led to models of early life in which the first cells used simpler types of membrane building blocks and has hampered integration of phospholipid synthesis into artificial cells. Here we demonstrate an enzyme-free synthesis of natural diacylphospholipids by transacylation in water, which is enabled by a combination of ion pairing and self-assembly between lysophospholipids and acyl donors. A variety of membrane-forming cellular phospholipids have been obtained in high yields. Membrane formation takes place in water from natural alkaline sources such as soda lakes and hydrothermal oceanic vents. When formed vesicles are transferred to more acidic solutions, electrochemical proton gradients are spontaneously established and maintained. This high-yielding non-enzymatic synthesis of natural phospholipids in water opens up new routes for lipid synthesis in artificial cells and sheds light on the origin and evolution of cellular membranes.

When a range-shifting species colonizes an ecosystem it has not previously inhabited, it may experience suboptimal conditions that challenge its continued persistence and expansion. Some impacts may be partially mitigated by artificial habitat analogues: artificial habitats that more closely resemble a species’ historic ecosystem than the surrounding habitat. If conditions provided by such habitats increase reproductive success, they could be vital to the expansion and persistence of range-shifting species. We investigated the reproduction of the mangrove tree crab Aratus pisonii in its historic mangrove habitat, the suboptimal colonized salt marsh ecosystem, and on docks within the marsh, an artificial mangrove analogue. Crabs were assessed for offspring production and quality, as well as measures of maternal investment and egg quality. Aratus pisonii found on docks produced more eggs, more eggs per unit energy investment, and higher quality larvae than conspecifics in the surrounding salt marsh. Yet, crabs in the mangrove produced the highest quality larvae. Egg lipids suggest these different reproductive outcomes result from disparities in the quality of diet-driven maternal investments, particularly key fatty acids. This study suggests habitat analogues may increase the reproductive fitness of range-shifting species allowing more rapid expansion into, and better persistence in, colonized ecosystems.

The Lost City hydrothermal field is a dramatic example of the biological potential of serpentinization. Microbial life is prevalent throughout the Lost City chimneys, powered by the hydrogen gas and organic molecules produced by serpentinization and its associated geochemical reactions. Microbial life in the serpentinite subsurface below the Lost City chimneys, however, is unlikely to be as dense or active. The marine serpentinite subsurface poses serious challenges for microbial activity, including low porosities, the combination of stressors of elevated temperature, high pH and a lack of bioavailable ∑CO 2 . A better understanding of the biological opportunities and challenges in serpentinizing systems would provide important insights into the total habitable volume of Earth's crust and for the potential of the origin and persistence of life in Earth's subsurface environments. Furthermore, the limitations to life in serpentinizing subsurface environments on Earth have significant implications for the habitability of subsurface environments on ocean worlds such as Europa and Enceladus. Here, we review the requirements and limitations of life in serpentinizing systems, informed by our research at the Lost City and the underwater mountain on which it resides, the Atlantis Massif. This article is part of a discussion meeting issue ‘Serpentinite in the Earth System’.

International Ocean Discovery Program Expedition 357 drilled 17 boreholes across the Atlantis Massif with the goals of investigating carbon cycling and the presence of life in a zone of active serpentinization. The expedition recovered multiple lithologies including gabbros, basalts, carbonate sands, and serpentinites. A subset of contrasting lithologies were analyzed for apolar lipid content to determine if non-volatile organic molecules can be detected in the oceanic subsurface. The definitive detection and identification of abiotic and biological lipids in the subsurface of an actively serpentinizing system would be a significant step towards understanding a variety of scientific processes, including the evolution of pre-biotic chemistry, microbial habitability, and the global carbon cycle. Given the high potential for contamination during drilling, a suite of materials used in sample collection and processing were also analyzed to characterize their signatures. An n-alkane series ranging from C18 to C30 with δ13C isotopic values of −30.9‰ to −28.8‰ was present in lithologically diverse samples. Multiple lines of evidence point to the rock saw used to remove core exteriors during sample processing as the source of these compounds. Many of the other sample-handling procedures designed to reduce surface contamination were determined to be effective and could be implemented in future projects. This result highlights the value of careful prevention and characterization of contamination to allow for more accurate interpretations of complex and dynamic subsurface processes, and the importance that future reports of these compounds occurs in conjunction with thorough contamination assessments.

The composition of the oceans is altered by hydrothermal circulation. These chemical factories sustain microbial life, which in turn alters the chemistry of the fluids that enter the ocean. A decade of research details this complex interchange.

Primitive forms of life may have originated around hydrothermal vents at the bottom of the ancient ocean. The Lost City hydrothermal vent field, fueled by just rock and water, provides an analog for not only primitive ecosystems but also potential extraterrestrial rock-powered ecosystems. The microscopic life covering the towering chimney structures at the Lost City has been previously documented, yet little is known about the carbon cycling in this ecosystem. These results provide a better understanding of how carbon from the deep subsurface can fuel rich microbial ecosystems on the seafloor., The Lost City hydrothermal field on the Mid-Atlantic Ridge supports dense microbial life on the lofty calcium carbonate chimney structures. The vent field is fueled by chemical reactions between the ultramafic rock under the chimneys and ambient seawater. These serpentinization reactions provide reducing power (as hydrogen gas) and organic compounds that can serve as microbial food; the most abundant of these are methane and formate. Previous studies have characterized the interior of the chimneys as a single-species biofilm inhabited by the Lost City Methanosarcinales, but they also indicated that this methanogen is unable to metabolize formate. The new metagenomic results presented here indicate that carbon cycling in these Lost City chimney biofilms could depend on the metabolism of formate by Chloroflexi populations. Additionally, we present evidence for metabolically diverse, formate-utilizing Sulfurovum populations and new genomic and phylogenetic insights into the unique Lost City Methanosarcinales. IMPORTANCE Primitive forms of life may have originated around hydrothermal vents at the bottom of the ancient ocean. The Lost City hydrothermal vent field, fueled by just rock and water, provides an analog for not only primitive ecosystems but also potential extraterrestrial rock-powered ecosystems. The microscopic life covering the towering chimney structures at the Lost City has been previously documented, yet little is known about the carbon cycling in this ecosystem. These results provide a better understanding of how carbon from the deep subsurface can fuel rich microbial ecosystems on the seafloor.

The Lost City hydrothermal field on the Mid-Atlantic Ridge supports dense microbial life on the lofty calcium carbonate chimney structures. The vent field is fueled by chemical reactions between the ultramafic rock under the chimneys and ambient seawater. These serpentinization reactions provide reducing power (as hydrogen gas) and organic compounds that can serve as microbial food; the most abundant of these are methane and formate. Previous studies have characterized the interior of the chimneys as a single-species biofilm inhabited by the Lost City Methanosarcinales, but also indicated that this methanogen is unable to metabolize formate. The new metagenomic results presented here indicate that carbon cycling in these Lost City chimney biofilms could depend on the metabolism of formate by low-abundance Chloroflexi species. Additionally, we present evidence that metabolically diverse, formate-utilizing Sulfurovum species are living in the transition zone between the interior and exterior of the chimneys.IMPORTANCEPrimitive forms of life may have originated around hydrothermal vents at the bottom of the ancient ocean. The Lost City hydrothermal vent field, fueled by just rock and water, provides an analog for not only primitive ecosystems but also extraterrestrial ecosystems that might support life. The microscopic life covering towering chimney structures at the Lost City has been well characterized, yet little is known about the carbon cycling in this ecosystem. These results provide a better understanding of how carbon from the deep subsurface can fuel rich microbial ecosystems on the seafloor.

Speleothem organic matter can be a powerful tracer for past environmental conditions and karst processes. Carbon isotope measurements (δ13C and 14C) in particular can provide crucial information on the provenance and age of speleothem organic matter, but are challenging due to low concentrations of organic matter in stalagmites. Here, we present a method development study on extraction and isotopic characterization of speleothem organic matter using a rapid procedure with low laboratory contamination risk. An extensive blank assessment allowed us to quantify possible sources of contamination through the entire method. Although blank contamination is consistently low (1.7 ± 0.34 – 4.3 ± 0.86 μg C for the entire procedure), incomplete sample decarbonation poses a still unresolved problem of the method, but can be detected when considering both δ13C and 14C values. We test the method on five stalagmites, showing reproducible results on samples as small as 7 μg C for δ13C and 20 μg C for 14C. Furthermore, we find consistently lower non-purgeable organic carbon (NPOC) 14C values compared to the carbonate 14C over the bomb spike interval in two stalagmites from Yok Balum Cave, Belize, suggesting overprint of a pre-aged or even fossil source of carbon on the organic fraction incorporated by these stalagmites.

Hyperthermophilic methanogens are often H2 limited in hot subseafloor environments, and their survival may be due in part to physiological adaptations to low H2 conditions and interspecies H2 transfer. The hyperthermophilic methanogen Methanocaldococcus jannaschii was grown in monoculture at high (80 to 83 μM) and low (15 to 27 μM) aqueous H2 concentrations and in coculture with the hyperthermophilic H2 producer Thermococcus paralvinellae. The purpose was to measure changes in growth and CH4 production kinetics, CH4 fractionation, and gene expression in M. jannaschii with changes in H2 flux. Growth and cell-specific CH4 production rates of M. jannaschii decreased with decreasing H2 availability and decreased further in coculture. However, cell yield (cells produced per mole of CH4 produced) increased 6-fold when M. jannaschii was grown in coculture rather than monoculture. Relative to high H2 concentrations, isotopic fractionation of CO2 to CH4 (eCO2-CH4) was 16‰ larger for cultures grown at low H2 concentrations and 45‰ and 56‰ larger for M. jannaschii growth in coculture on maltose and formate, respectively. Gene expression analyses showed H2-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase expression decreased and coenzyme F420-dependent methylene-H4MPT dehydrogenase expression increased with decreasing H2 availability and in coculture growth. In coculture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H2 availability significantly affects the CH4 and biomass production and CH4 fractionation by hyperthermophilic methanogens in their native habitats. IMPORTANCE Hyperthermophilic methanogens and H2-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H2 can be very low in these environments, and there is a gap in our knowledge about the origin of CH4 in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H2 flux, growth rates, and CH4 production rates decreased. The same trend was observed increasingly with interspecies H2 transfer between M. jannaschii and the hyperthermophilic H2 producer Thermococcus paralvinellae. With decreasing H2 availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4 with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2 transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4 in natural environments.

Hyperthermophilic methanogens and H2-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H2 can be very low in these environments, and there is a gap in our knowledge about the origin of CH4 in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H2 flux, growth rates, and CH4 production rates decreased. The same trend was observed increasingly with interspecies H2 transfer between M. jannaschii and the hyperthermophilic H2 producer Thermococcus paralvinellae. With decreasing H2 availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4 with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2 transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4 in natural environments., Hyperthermophilic methanogens are often H2 limited in hot subseafloor environments, and their survival may be due in part to physiological adaptations to low H2 conditions and interspecies H2 transfer. The hyperthermophilic methanogen Methanocaldococcus jannaschii was grown in monoculture at high (80 to 83 μM) and low (15 to 27 μM) aqueous H2 concentrations and in coculture with the hyperthermophilic H2 producer Thermococcus paralvinellae. The purpose was to measure changes in growth and CH4 production kinetics, CH4 fractionation, and gene expression in M. jannaschii with changes in H2 flux. Growth and cell-specific CH4 production rates of M. jannaschii decreased with decreasing H2 availability and decreased further in coculture. However, cell yield (cells produced per mole of CH4 produced) increased 6-fold when M. jannaschii was grown in coculture rather than monoculture. Relative to high H2 concentrations, isotopic fractionation of CO2 to CH4 (εCO2-CH4) was 16‰ larger for cultures grown at low H2 concentrations and 45‰ and 56‰ larger for M. jannaschii growth in coculture on maltose and formate, respectively. Gene expression analyses showed H2-dependent methylene-tetrahydromethanopterin (H4MPT) dehydrogenase expression decreased and coenzyme F420-dependent methylene-H4MPT dehydrogenase expression increased with decreasing H2 availability and in coculture growth. In coculture, gene expression decreased for membrane-bound ATP synthase and hydrogenase. The results suggest that H2 availability significantly affects the CH4 and biomass production and CH4 fractionation by hyperthermophilic methanogens in their native habitats. IMPORTANCE Hyperthermophilic methanogens and H2-producing heterotrophs are collocated in high-temperature subseafloor environments, such as petroleum reservoirs, mid-ocean ridge flanks, and hydrothermal vents. Abiotic flux of H2 can be very low in these environments, and there is a gap in our knowledge about the origin of CH4 in these habitats. In the hyperthermophile Methanocaldococcus jannaschii, growth yields increased as H2 flux, growth rates, and CH4 production rates decreased. The same trend was observed increasingly with interspecies H2 transfer between M. jannaschii and the hyperthermophilic H2 producer Thermococcus paralvinellae. With decreasing H2 availability, isotopic fractionation of carbon during methanogenesis increased, resulting in isotopically more negative CH4 with a concomitant decrease in H2-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression and increase in F420-dependent methylene-tetrahydromethanopterin dehydrogenase gene expression. The significance of our research is in understanding the nature of hyperthermophilic interspecies H2 transfer and identifying biogeochemical and molecular markers for assessing the physiological state of methanogens and possible source of CH4 in natural environments.

Substances enriched with radiocarbon can easily contaminate samples and laboratories used for natural abundance measurements. We have developed a new method using wet chemical oxidation for swabbing laboratories and equipment to test for 14C contamination. Here, we report the findings of 18 months’ work and more than 800 tests covering studies at multiple locations. Evidence of past and current use of enriched 14C was found at all but one location and a program of testing and communication was used to mitigate its effects. Remediation was attempted with mixed success and depended on the complexity and level of the contamination. We describe four cases from different situations.

Shallow submarine hydrothermal systems are extreme environments with strong redox gradients at the interface of hot, reduced fluids and cold, oxygenated seawater. Hydrothermal fluids are often depleted in sulfate when compared to surrounding seawater and can contain high concentrations of hydrogen sulfide (H2S). It is well known that sulfur in its various oxidation states plays an important role in processing and transformation of organic matter. However, the formation and the reactivity of dissolved organic sulfur (DOS) in the water column at hydrothermal systems are so far not well understood. We investigated DOS dynamics and its relation to the physicochemical environment by studying the molecular composition of dissolved organic matter (DOM) in three contrasting shallow hydrothermal systems off Milos (Eastern Mediterranean), Dominica (Caribbean Sea) and Iceland (North Atlantic). We used ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) to characterize the DOM on a molecular level. The molecular information was complemented with general geochemical data, quantitative dissolved organic carbon (DOC) and DOS analyses as well as isotopic measurements (δ2H, δ18O and F14C). In contrast to the predominantly meteoric fluids from Dominica and Iceland, hydrothermal fluids from Milos were mainly fed by recirculating seawater. The hydrothermal fluids from Milos were enriched in H2S and DOS, as indicated by high DOS/DOC ratios and by the fact that >90% of all assigned DOM formulas that were exclusively present in the fluids contained sulfur. In all three systems, DOS from hydrothermal fluids had on average lower O/C ratios (0.26–0.34) than surrounding surface seawater DOS (0.45–0.52), suggesting shallow hydrothermal systems as a source of reduced DOS, which will likely get oxidized upon contact with oxygenated seawater. Evaluation of hypothetical sulfurization reactions suggests DOM reduction and sulfurization during seawater recirculation in Milos seafloor. The four most effective potential sulfurization reactions were those exchanging an O atom by one S atom in the formula or the equivalent + H2S reaction, correspondingly exchanging H2O, H2 and/or O2 by a H2S molecule. Our study reveals novel insights into DOS dynamics in marine hydrothermal environments and provides a conceptual framework for molecular-scale mechanisms in organic sulfur geochemistry.

The radiocarbon content of dissolved organic carbon (DOC) in rivers, lakes, and other non-saline waters can provide valuable information on carbon cycling dynamics in the environment. DOC is typically prepared for 14C analysis by accelerator mass spectrometry (AMS) either by ultraviolet (UV) oxidation or by freeze-drying and sealed tube combustion. We present here a new method for the rapid analysis of 14C of DOC using wet chemical oxidation (WCO) and automated headspace sampling of CO2. The approach is an adaption of recently developed methods using aqueous persulfate oxidant to determine the δ13C of DOC in non-saline water samples and the 14C content of volatile organic acids. One advantage of the current method over UV oxidation is higher throughput: 22 samples and 10 processing standards can be prepared in one day and analyzed in a second day, allowing a full suite of 14C processing standards and blanks to be run in conjunction with samples. A second advantage is that there is less potential for cross-contamination between samples.

Present-day serpentinization generates groundwaters with conditions (pH > 11, Eh

The stable carbon isotopic composition of dissolved organic matter (δ13C-DOC) reveals information about its source and extent of biological processing. Here we report the lowest δ13C-DOC values (−43.8‰) measured to date in surface waters. The streams were located in the High Arctic, a region currently experiencing rapid changes in climate and carbon cycling. Based on the widespread occurrence of methane cycling in permafrost regions and the detection of the pmoA gene, a proxy for aerobic methanotrophs, we conclude that the low δ13C-DOC values are due to organic matter partially derived from methanotrophs consuming biologically produced, 13C-depleted methane. These findings demonstrate the significant impact that biological activity has on the stream water chemistry exported from permafrost and glaciated environments in the Arctic. Given that the catchments studied here are representative of larger areas of the Arctic, occurrences of low δ13C-DOC values may be more widespread than previously recognized, with implications for understanding C cycling in these environments.

Organic carbon in marine sediments is a critical component of the global carbon cycle, and its degradation influences a wide range of phenomena, including the magnitude of carbon sequestration over geologic timescales, the recycling of inorganic carbon and nutrients, the dissolution and precipitation of carbonates, the production of methane and the nature of the seafloor biosphere. Although much has been learned about the factors that promote and hinder rates of organic carbon degradation in natural systems, the controls on the distribution of organic carbon in modern and ancient sediments are still not fully understood. In this review, we summarize how recent findings are changing entrenched perspectives on organic matter degradation in marine sediments: a shift from a structurally-based chemical reactivity viewpoint towards an emerging acceptance of the role of the ecosystem in organic matter degradation rates. That is, organic carbon has a range of reactivities determined by not only the nature of the organic compounds, but by the biological, geochemical, and physical attributes of its environment. This shift in mindset has gradually come about due to a greater diversity of sample sites, the molecular revolution in biology, discoveries concerning the extent and limits of life, advances in quantitative modeling, investigations of ocean carbon cycling under a variety of extreme paleo-conditions (e.g. greenhouse environments, euxinic/anoxic oceans), the application of novel analytical techniques and interdisciplinary efforts. Adopting this view across scientific disciplines will enable additional progress in understanding how marine sediments influence the global carbon cycle.

Exposure to microbial pathogens is the primary concern of sanitary sewer overflows; however, sewage spills may also be a significant source of toxic metals, including methylmercury (MeHg). Between November 2015 and January 2017, following Hurricane Joaquin, surface water samples were collected routinely from three creeks in Columbia, South Carolina, USA. Routine sampling coincided with six sewage spills. Total mercury (THg) and MeHg (unfiltered and filtered), and 32 other metals (filtered) were measured. Compared to surface water samples, THg (unfiltered and filtered), MeHg (unfiltered), and 19 other metals were significantly higher in sewage spills (all log(10)-transformed) (two-tailed t-test, p

Rock–water–carbon interactions germane to serpentinization in hydrothermal vents have occurred for over 4 billion years, ever since there was liquid water on Earth. Serpentinization converts iron(II) containing minerals and water to magnetite (Fe3O4) plus H2. The hydrogen can generate native metals such as awaruite (Ni3Fe), a common serpentinization product. Awaruite catalyzes the synthesis of methane from H2 and CO2 under hydrothermal conditions. Native iron and nickel catalyze the synthesis of formate, methanol, acetate, and pyruvate—intermediates of the acetyl-CoA pathway, the most ancient pathway of CO2 fixation. Carbon monoxide dehydrogenase (CODH) is central to the pathway and employs Ni0 in its catalytic mechanism. CODH has been conserved during 4 billion years of evolution as a relic of the natural CO2-reducing catalyst at the onset of biochemistry. The carbide-containing active site of nitrogenase—the only enzyme on Earth that reduces N2—is probably also a relic, a biological reconstruction of the naturally occurring inorganic catalyst that generated primordial organic nitrogen. Serpentinization generates Fe3O4 and H2, the catalyst and reductant for industrial CO2 hydrogenation and for N2 reduction via the Haber–Bosch process. In both industrial processes, an Fe3O4 catalyst is matured via H2-dependent reduction to generate Fe5C2 and Fe2N respectively. Whether serpentinization entails similar catalyst maturation is not known. We suggest that at the onset of life, essential reactions leading to reduced carbon and reduced nitrogen occurred with catalysts that were synthesized during the serpentinization process, connecting the chemistry of life and Earth to industrial chemistry in unexpected ways.

Hydrogen produced during water-rock serpentinization reactions can drive the synthesis of organic compounds both biotically and abiotically. We investigated abiotic carbon production and microbial metabolic pathways at the high energy but low diversity serpentinite-hosted Lost City hydrothermal field. Compound-specific 14C data demonstrates that formate is mantle-derived and abiotic in some locations and has an additional, seawater-derived component in others. Lipids produced by the dominant member of the archaeal community, the Lost City Methanosarcinales, largely lack 14C, but metagenomic evidence suggests they cannot use formate for methanogenesis. Instead, sulfate-reducing bacteria may be the primary consumers of formate in Lost City chimneys. Paradoxically, the archaeal phylotype that numerically dominates the chimney microbial communities appears ill suited to live in pure hydrothermal fluids without the co-occurrence of organisms that can liberate CO2. Considering the lack of dissolved inorganic carbon in such systems, the ability to utilize formate may be a key trait for survival in pristine serpentinite-hosted environments.

Chemical input to the deep sea from hydrothermal systems is a globally distributed phenomenon. Hydrothermal discharge is one of the primary mechanisms by which the Earth’s interior processes manifest themselves at the Earth’s surface, and it provides a source of energy for autotrophic processes by microbes that are too deep to capitalize on sunlight. Much is known about the water-column signature of this discharge from high-temperature mid-ocean Ridge (MOR) environments and their neighboring low-temperature counterparts. Hydrothermal discharge farther away from the ridge, however, has garnered less attention, owing in part to the difficulty in finding this style of venting, which eludes methods of detection that work well for high-temperature ‘black smoker’-type venting. Here we present a case study of the plume from one such ‘invisible’ off-axis environment, The Lost City, with an emphasis on the dissolved volatile content of the hydrothermal plume. Serpentinization and abiotic organic synthesis generate significant concentrations of H2 and CH4 in vent fluid, but these species are unevenly transported to the overlying plume, which itself appears to be a composite of two different sources. A concentrated vent cluster on the talus slope channels fluid through at least eight chimneys, producing a water-column plume with the highest observed concentrations of CH4 in the field. In contrast, a saddle in the topography leading up to a carbonate cap hosts broadly distributed, nearly invisible venting apparent only in its water-column signals of redox potential and dissolved gas content, including the highest observed plume H2. After normalizing H2 and CH4 to the 3He background-corrected anomaly (3HeΔ) to account for mixing and relative amount of mantle input, it appears that, while a minimum of 60% of CH4 is transported out of the system, greater than 90% of the H2 is consumed in the subsurface prior to venting. The exception to this pattern occurs in the plume originating from the area dubbed Chaff Beach, in which somewhat more than 10% of the original H2 remains, indicating that this otherwise unremarkable plume, and others like it, may represent a significant source of H2 to the deep sea.

Despite their potential importance as analogs of primitive microbial metabolisms, the knowledge of the structure and functioning of the deep ecosystems associated with serpentinizing environments is hampered by the lack of accessibility to relevant systems. These hyperalkaline environments are depleted in dissolved inorganic carbon (DIC), making the carbon sources and assimilation pathways in the associated ecosystems highly enigmatic. The Prony Bay Hydrothermal Field (PHF) is an active serpentinization site where, similar to Lost City (Mid-Atlantic Ridge), high-pH fluids rich in H-2 and CH4 are discharged from carbonate chimneys at the seafloor, but in a shallower lagoonal environment. This study aimed to characterize the subsurface microbial ecology of this environment by focusing on the earliest stages of chimney construction, dominated by the discharge of hydrothermal fluids of subseafloor origin. By jointly examining the mineralogy and the microbial diversity of the conduits of juvenile edifices at the micrometric scale, we find a central role of uncultivated bacteria belonging to the Firmicutes in the ecology of the PHF. These bacteria, along with members of the phyla Acetothermia and Omnitrophica, are identified as the first chimneys inhabitants before archaeal Methanosarcinales. They are involved in the construction and early consolidation of the carbonate structures via organomineralization processes. Their predominance in the most juvenile and nascent hydrothermal chimneys, and their affiliation with environmental subsurface microorganisms, indicate that they are likely discharged with hydrothermal fluids from the subseafloor. They may thus be representative of endolithic serpentinization-based ecosystems, in an environment where DIC is limited. In contrast, heterotrophic and fermentative microorganisms may consume organic compounds from the abiotic by-products of serpentinization processes and/or from life in the deeper subsurface. We thus propose that the Firmicutes identified at PHF may have a versatile metabolism with the capability to use diverse organic compounds from biological or abiotic origin. From that perspective, this study sheds new light on the structure of deep microbial communities living at the energetic edge in serpentinites and may provide an alternative model of the earliest metabolisms.

Active serpentinization systems are abundant on present-day Earth and are of increasing interest because water–rock reactions lead to alkaline, Ca–OH fluids that have the potential to sequester CO 2 and form reduced chemical species (e.g. CH 4 and H 2 ) that can support chemolithoautotrophy. We present a study of alkaline (pH 10–12) springs in the Voltri Massif (Italy) that focuses on the sources and cycling of inorganic carbon in the basement rocks, the interacting fluids, and the resulting surface carbonate deposits. Most springs are located in mantle rocks that underwent varying degrees of ocean-floor serpentinization and incorporation of carbon (as carbonate and organic carbon) in the Jurassic, which is preserved during subsequent subduction and uplift onto the continent. The springs are fed by meteoric water that evolves into alkaline, Ca-rich spring waters that have 2–3 times higher Ca concentrations than the adjacent rivers. Our study is consistent with previous reaction path modeling and identifies the formation of clay minerals in serpentinites that have been altered by alkaline fluids. These strongly altered basement rocks contain up to 2 wt.% C in the shallow subsurface, also documented by the presence of late calcite veins. Concentrations of dissolved inorganic carbon (DIC) of the alkaline fluids are generally low ( 13 C DIC is between − 24.2‰ and + 1.3‰. We argue that the concentrations and isotopic composition of the DIC in the alkaline waters provide evidence for 1) the precipitation of calcium carbonate under closed-system conditions with respect to atmospheric CO 2 and 2) removal of DIC by microbial activity in the subsurface. Late-stage uptake of atmospheric CO 2 in the shallow subsurface or at the exit sites subsequent to water–rock–microbe interactions in the basement result in fluids with lower pH and Ca, and enrichment in DIC and 13 C. At the surface, interaction of the high pH, Ca–OH fluids with atmospheric CO 2 causes precipitation of carbonates as travertines or crusts on the basement rocks, storing CO 2 as calcium carbonate. Carbonate precipitation at the exit sites is strongly dominated by kinetic processes leading to carbon and oxygen isotope compositions as low as − 27.2‰ and − 18.7‰, respectively. With increasing distance from the springs, the 13 C and 18 O content of the carbonate increases and the fluid pH changes towards neutral. Our study shows that surficial carbonate precipitation plays a subordinate role in carbon sequestration, but that the evolution from Mg–HCO 3 spring waters to Ca–OH waters removes significant amounts of carbon, presumably in the subsurface. We calculate that the serpentinites have the capacity to sequester up to 0.50 to 2.05 × 10 9 mol carbon per year and conclude that they can take up significantly more CO 2 than they currently contain.

We present a method for the isolation and off-line isotope analysis of formate and acetate in marine samples. Organic acids are separated by high performance liquid chromatography and collected in glass Exetainer® screw-capped vials that have been prespiked with an oxidant and flushed with helium. The vials are subsequently heated to convert the organic compounds to CO2 for radiocarbon and δ13C analysis. Small aliquots are sampled for measurement of δ13C by isotope ratio mass spectrometry, whereas the majority of the CO2 is saved for radiocarbon analysis by accelerator mass spectrometry using a gas ion source. Accurate δ13C and radiocarbon values were obtained for formate and acetate standards spiked into deep seawater and saline Milli-Q water at concentrations of 25 to 1000 µM C. The process blank associated with the isolation of formate for radiocarbon analysis was ~1.5 µg C and stable over time. Accurate results could be obtained for marine samples with only 25 µM formate. The radiocarbon analysis of acetate showed significantly higher and more variable extraneous carbon contributions, particularly for samples spiked into seawater. Potential improvements that may make the method appropriate for the radiocarbon analysis of acetate in seawater are discussed. The blanks associated with the wet chemical oxidation were assessed independently and found to be small and consistent (

Samples of young, outer surfaces of brucite-carbonate deposits from the ultramafic-hosted Lost City hydrothermal field were analyzed for DNA and lipid biomarker distributions and for carbon and hydrogen stable isotope compositions of the lipids. Methane-cycling archaeal communities, notably the Lost City Methanosarcinales (LCMS) phylotype, are specifically addressed. Lost City is unlike all other hydrothermal systems known to date and is characterized by metal- and CO2 -poor, high pH fluids with high H2 and CH4 contents resulting from serpentinization processes at depth. The archaeal fraction of the microbial community varies widely within the Lost City chimneys, from 1-81% and covaries with concentrations of hydrogen within the fluids. Archaeal lipids include isoprenoid glycerol di- and tetraethers and C25 and C30 isoprenoid hydrocarbons (pentamethylicosane derivatives - PMIs - and squalenoids). In particular, unsaturated PMIs and squalenoids, attributed to the LCMS archaea, were identified for the first time in the carbonate deposits at Lost City and probably record processes exclusively occurring at the surface of the chimneys. The carbon isotope compositions of PMIs and squalenoids are remarkably heterogeneous across samples and show highly (13) C-enriched signatures reaching δ(13) C values of up to +24.6‰. Unlike other environments in which similar structural and isotopic lipid heterogeneity has been observed and attributed to diversity in the archaeal assemblage, the lipids here appear to be synthesized solely by the LCMS. Some of the variations in lipid isotope signatures may, in part, be due to unusual isotopic fractionation during biosynthesis under extreme conditions. However, we argue that the diversity in archaeal abundances, lipid structure and carbon isotope composition rather reflects the ability of the LCMS archaeal biofilms to adapt to chemical gradients in the hydrothermal chimneys and possibly to perform either methanotrophy or methanogenesis using dissolved inorganic carbon, methane or formate as a function of the prevailing environmental conditions.

The reaction of ultramafic rocks with water during serpentinization at moderate temperatures results in alkaline fluids with high concentrations of reduced chemical compounds such as hydrogen and methane. Such environments provide unique habitats for microbial communities capable of utilizing these reduced compounds in present-day and, possibly, early Earth environments. However, these systems present challenges to microbial communities as well, particularly due to high fluid pH and possibly the availability of essential nutrients such as nitrogen. Here we investigate the source and cycling of organic nitrogen at an oceanic serpentinizing environment, the Lost City hydrothermal field (30°N, Mid-Atlantic Ridge). Total hydrolizable amino acid (THAA) concentrations in the fluids range from 736 to 2300 nm and constitute a large fraction of the dissolved organic carbon (2.5-15.1%). The amino acid distributions, and the relative concentrations of these compounds across the hydrothermal field, indicate they most likely derived from chemolithoautotrophic production. Previous studies have identified the presence of numerous nitrogen fixation genes in the fluids and the chimneys. Organic nitrogen in actively venting chimneys has δ(15) N values as low as 0.1‰ which is compatible with biological nitrogen fixation. Total hydrolizable amino acids in the chimneys are enriched in (13) C by 2-7‰ compared to bulk organic matter. The distribution and absolute δ(13) C(THAA) values are compatible with a chemolithoautotrophic source, an attribution also supported by molar organic C/N ratios in most active chimneys (4.1-5.5) which are similar to those expected for microbial communities. In total, these data indicate nitrogen is readily available to microbial communities at Lost City.

The aqueous alteration of ultramafic rocks through serpentinization liberates mantle carbon and reducing power. Serpentinization occurs in numerous settings on present day Earth, including subduction zones, mid-ocean ridges, and ophiolites and has extended far into Earth’s history, potentially contributing to the origins and early evolution of life. Serpentinization can provide the energy and raw materials to support chemosynthetic microbial communities that may penetrate deep into Earth’s subsurface. Microorganisms may also influence the composition and quantity of carbon-bearing compounds in the deep subsurface. However, conditions created by serpentinization challenge the known limits of microbial physiology in terms of extreme pH, access to electron acceptors, and availability of nutrients. Furthermore, the downward transport of surface carbon and subsequent mixing with calcium-rich fluids at high pH contributes to the precipitation and immobilization of carbonate minerals. The following chapter will explore the physiological challenges presented by the serpentinite environment, data from studies of serpentinite-hosted microbial ecosystems, and areas in need of further investigation. ### Physical and chemical consequences of serpentinization Serpentinization is an alteration process of low-silica ultramafic rocks, characteristic of the lower oceanic crust and upper mantle. These rocks are rich in the minerals olivine and pyroxene. Water-rock reactions result in the oxidation of ferrous iron from olivine and pyroxene, resulting in the precipitation of ferric iron in magnetite (Fe3O4) and other minerals, and in the release of diatomic hydrogen (H2). At low temperatures (< ~150 °C) the reaction results in extremely high pH, commonly above 10. The combination of H2 and CO2 or CO under highly reducing conditions leads to formation of methane and other hydrocarbons through Fischer-Tropsch Type (FTT) synthesis (McCollom and Seewald 2001; Charlou et al. 2002; Proskurowski et al. 2008; McCollom 2013). Serpentinization also results in volume changes in the altered materials, making serpentinites less dense than …

Fire-derived, pyrogenic carbon (PyC), sometimes called black carbon (BC), is the carbonaceous solid residue of biomass and fossil fuel combustion, such as char and soot. PyC is ubiquitous in the environment due to its long persistence, and its abundance might even increase with the projected increase in global wildfire activity and the continued burning of fossil fuel. PyC is also increasingly produced from the industrial pyrolysis of organic wastes, which yields charred soil amendments (biochar). Moreover, the emergence of nanotechnology may also result in the release of PyC-like compounds to the environment. It is thus a high priority to reliably detect, characterize and quantify these charred materials in order to investigate their environmental properties and to understand their role in the carbon cycle.\ud \ud Here, we present the benzene polycarboxylic acid (BPCA) method, which allows the simultaneous assessment of PyC's characteristics, quantity and isotopic composition (13C and 14C) on a molecular level. The method is applicable to a very wide range of environmental sample materials and detects PyC over a broad range of the combustion continuum, i.e., it is sensitive to slightly charred biomass as well as high temperature chars and soot. The BPCA protocol presented here is simple to employ, highly reproducible, as well as easily extendable and modifiable to specific requirements. It thus provides a versatile tool for the investigation of PyC in various disciplines, ranging from archeology and environmental forensics to biochar and carbon cycling research.

Mantle rocks exposed on the seafloor constitute a highly reactive chemical and thermal system, in which interaction with seawater to produce serpentinite has major consequences for lithospheric cooling, global geochemical cycles, and microbial activity. Serpentinite-hosted hydrothermal activity is exemplified by the Lost City Hydrothermal Field (30°N, Mid-Atlantic Ridge) where fluid–rock reactions in the underlying ultramafic rocks result in high concentrations of abiotic hydrogen, methane, C 2 + alkanes, and formate . Such systems have been proposed as possible analogs to the Early Earth environments that gave rise to the first biochemical pathways. Thus, characterizing the local microbial communities and their potential link with abiogenic compounds is of particular significance. Here we demonstrate that in active carbonate chimneys where microbial sulfate reduction is important, up to 50% of the microbial biomass is synthesized from mantle carbon. Conversely, mantle carbon contributes only ∼10% of the biomass in areas with minimal sulfate reduction. We attribute this difference to greater incorporation of formate or methane by the dominant microbial species, the Lost City Methanosarcinales , in locations where sulfate reducers are able to facilitate this assimilation. The ability of autotrophic communities at Lost City to capitalize on the steady stream of chemical products resulting from serpentinization reactions and to utilize abiogenic mantle carbon lend credence to the hypothesis that early biosynthetic pathways could have developed in similar environments.

The stable isotopes of organic matter can provide valuable information on carbon cycling dynamics, microbial metabolisms, and past climates. Since bulk measurements may mask dynamic changes to critical portions of the organic pool, researchers are increasingly isolating individual compounds for isotopic analysis. The amount of carbon isolated is frequently small, requiring specialized equipment for its analysis. We present a simple and accurate method to measure the δ(13)C values of µg-amounts of organic compounds and dissolved organic matter in freshwaters using wet oxidation and a GasBench II preparation device. Samples containing 3 µg C can be analyzed with a precision of

Fluids from the ultramafic-hosted Lost City hydrothermal field were analyzed for total dissolved organic carbon and dissolved organic acids. Formate (36–158 μmol/kg) and acetate (1–35 μmol/kg) concentrations are higher than in other fluids from unsedimented hydrothermal vents, and are a higher ratio of the total dissolved organic carbon than has been found in most marine geothermal systems. Isotopic evidence is consistent with an abiotic formation mechanism for formate, perhaps during serpentinization processes in the sub-surface. Further support comes from previous studies where the abiological formation of low molecular weight organic acids has been shown to be thermodynamically favorable during hydrothermal alteration of olivine, and laboratory studies in which the reduction of carbon dioxide to formate has been confirmed. As the second most prevalent carbon species after methane, formate may be an important substrate to microbial communities in an environment where dissolved inorganic carbon is limited. Acetate is found in locations where sulfate reduction is believed to be important and is likely to be a microbial by-product, formed either directly by autotrophic metabolic activity or indirectly during the fermentative degradation of larger organic molecules. Given the common occurrence of exposed ultramafic rocks and active serpentinization within the worlds ocean basins, the abiotic formation of formate may be an important process supporting life in these high pH environments and may have critical implications to understanding the organic precursors from which life evolved.

The stable isotopes of dissolved organic carbon (DOC) are a powerful tool for distinguishing sources and inputs of organic matter in aquatic systems. While several methods exist to perform these analyses, no labs routinely utilize a high temperature combustion (HTC) instrument. Advantages of HTC instruments include rapid analysis, small sample volumes and minimal sample preparation, making them the favored devices for most routine oceanic DOC concentration measurements. We developed a stable carbon DOC method based around an HTC system. This method has the benefit of a simple setup, requiring neither vacuum nor high pressures. The main drawback of the method is a significant blank, requiring careful accounting of all blank sources for accurate isotopic and concentration values. We present here a series of experiments to determine the magnitude, source and isotopic composition of the HTC blank. Over time, the blank is very stable at ∼ 20 ng of carbon with a δ 13 C of − 18.1‰ vs. VPDB. The similarity of the isotopic composition of the blank and seawater samples makes corrections relatively minor. The precision of the method was determined by oxidizing organic standards with a wide isotopic and concentration range (− 9‰ to − 39‰; 18 μM to 124 μM). Analysis of seawater samples demonstrates the accuracy for low concentration, high salinity samples. The overall error on the measurement is approximately ± 0.8‰.

The circulation of hydrothermal fluid through the upper oceanic crustal reservoir has a large impact on the chemistry of seawater, yet the impact on dissolved organic carbon (DOC) in the ocean has received almost no attention. To determine whether hydrothermal circulation is a source or a sink for DOC in the oceans, we measured DOC concentrations in hydrothermal fluids from several environments. Hydrothermal fluids were collected from high-temperature vents and diffuse, low-temperature vents on the basalt-hosted Juan de Fuca Ridge axis and also from low-temperature vents on the sedimented eastern flanks. High-temperature fluids from Main Endeavour Field (MEF) and Axial Volcano (AV) contain very low DOC concentrations (average = 15 and 17 μM, respectively) compared to background seawater (36 μM). At MEF and AV, average DOC concentrations in diffuse fluids (47 and 48 μM, respectively) were elevated over background seawater, and high DOC is correlated with high microbial cell counts in diffuse fluids. Fluids from off-axis hydrothermal systems located on 3.5-Ma-old crust at Baby Bare Seamount and Ocean Drilling Program (ODP) Hole 1026B had average DOC concentrations of 11 and 13 μM, respectively, and lowered DOC was correlated with low cell counts. The relative importance of heterotrophic uptake, abiotic sorption to mineral surfaces, thermal decomposition, and microbial production in fixing the DOC concentration in vent fluids remains uncertain. We calculated the potential effect of hydrothermal circulation on the deep-sea DOC cycle using our concentration data and published water flux estimates. Maximum calculated fluxes of DOC are minor compared to most oceanic DOC source and sink terms.