Aqueous breakdown of aspartate and glutamate to n-ω-amino acids on the parent bodies of carbonaceous chondrites and asteroid Ryugu.

Amino acids in carbonaceous chondrites may have seeded the origin of life on Earth and possibly elsewhere. Recently, the return samples from a C-type asteroid Ryugu were found to contain amino acids with a similar distribution to Ivuna-type CI chondrites, suggesting the potential of amino acid abundances as molecular descriptors of parent body geochemistry. However, the chemical mechanisms responsible for the amino acid distributions remain to be elucidated particularly at low temperatures (<50°C). Here, we report that two representative proteinogenic amino acids, aspartic acid and glutamic acid, decompose to ß-alanine and γ-aminobutyric acid, respectively, under simulated geoelectrochemical conditions at 25°C. This low-temperature conversion provides a plausible explanation for the enrichment of these two n-ω-amino acids compared to their precursors in heavily aqueously altered CI chondrites and Ryugu's return samples. The results suggest that these heavily aqueously altered samples originated from the water-rich mantle of their water/rock differentiated parent planetesimals where protein α-amino acids were decomposed.

Development of small fluorescent probes for the analysis of autophagy kinetics.

Autophagy is a dynamic process that degrades subcellular constituents, and its activity is measured by autophagic flux. The tandem proteins RFP-GFP-LC3 and GFP-LC3-RFP-LC3ΔG, which enable the visualization of autophagic vacuoles of different stages by differences in their fluorescent color, are useful tools to monitor autophagic flux, but they require plasmid transfection. In this study, we hence aimed to develop a new method to monitor autophagic flux using small cell-permeable fluorescent probes. We previously developed two green-fluorescent probes, DALGreen and DAPGreen, which detect autolysosomes and multistep autophagic vacuoles, respectively. We here developed a red-fluorescent autophagic probe, named DAPRed, which recognizes various autophagic vacuoles. By the combinatorial use of these green- and red-fluorescent probes, we were able to readily detect autophagic flux. Furthermore, these probes were useful not only for the visualization of canonical autophagy but also for alternative autophagy. DAPRed was also applicable for the detection of autophagy in living organisms.

Deep Subseafloor Biogeochemical Processes and Microbial Populations Potentially Associated with the 2011 Tohoku-oki Earthquake at the Japan Trench Accretionary Wedge (IODP Expedition 343).

Post-mega-earthquake geochemical and microbiological properties in subseafloor sediments of the Japan Trench accretionary wedge were investigated using core samples from Hole C0019E, which was drilled down to 851| |m below seafloor (mbsf) at a water depth of 6,890 m. Methane was abundant throughout accretionary prism sediments; however, its concentration decreased close to the plate boundary decollement. Methane isotope systematics indicated a biogenic origin. The content of mole-cular hydrogen (H2) was low throughout core samples, but markedly increased at specific depths that were close to potential faults predicted by logging-while-drilling ana-lyses. Based on isotopic systematics, H2 appeared to have been abundantly produced via a low-temperature interaction between pore water and the fresh surface of crushed rock induced by earthquakes. Subseafloor microbial cell density remained constant at approximately 105| |cells| |mL-1. Amplicon sequences revealed that predominant members at the phylum level were common throughout the units tested, which also included members frequently found in anoxic subseafloor sediments. Metabolic potential assays using radioactive isotopes as tracers revealed homoacetogenic activity in H2-enriched core samples collected near the fault. Furthermore, homoacetogenic bacteria, including Acetobacterium carbinolicum, were isolated from similar samples. Therefore, post-earthquake subseafloor microbial communities in the Japan Trench accretionary prism appear to be episodically dominated by homoacetogenic populations and potentially function due to the earthquake-induced low-temperature generation of H2. These post-earthquake microbial communities may eventually return to the steady-state communities dominated by oligotrophic heterotrophs and hydrogenotrophic and methylotrophic methanogens that are dependent on refractory organic matter in the sediment.

Spectroscopic and Biophysical Methods to Determine Differential Salt-Uptake by Primitive Membraneless Polyester Microdroplets.

α-Hydroxy acids are prebiotic monomers that undergo dehydration synthesis to form polyester gels, which assemble into membraneless microdroplets upon aqueous rehydration. These microdroplets are proposed as protocells that can segregate and compartmentalize primitive molecules/reactions. Different primitive aqueous environments with a variety of salts could have hosted chemistries that formed polyester microdroplets. These salts could be essential cofactors of compartmentalized prebiotic reactions or even directly affect protocell structure. However, fully understanding polyester-salt interactions remains elusive, partially due to technical challenges of quantitative measurements in condensed phases. Here, spectroscopic and biophysical methods are applied to analyze salt uptake by polyester microdroplets. Inductively coupled plasma mass spectrometry is applied to measure the cation concentration within polyester microdroplets after addition of chloride salts. Combined with methods to determine the effects of salt uptake on droplet turbidity, size, surface potential and internal water distribution, it was observed that polyester microdroplets can selectively partition salt cations, leading to differential microdroplet coalescence due to ionic screening effects reducing electrostatic repulsion forces between microdroplets. Through applying existing techniques to novel analyses related to primitive compartment chemistry and biophysics, this study suggests that even minor differences in analyte uptake can lead to significant protocellular structural change.

Low 13C-13C abundances in abiotic ethane.

Distinguishing biotic compounds from abiotic ones is important in resource geology, biogeochemistry, and the search for life in the universe. Stable isotopes have traditionally been used to discriminate the origins of organic materials, with particular focus on hydrocarbons. However, despite extensive efforts, unequivocal distinction of abiotic hydrocarbons remains challenging. Recent development of clumped-isotope analysis provides more robust information because it is independent of the stable isotopic composition of the starting material. Here, we report data from a 13C-13C clumped-isotope analysis of ethane and demonstrate that the abiotically-synthesized ethane shows distinctively low 13C-13C abundances compared to thermogenic ethane. A collision frequency model predicts the observed low 13C-13C abundances (anti-clumping) in ethane produced from methyl radical recombination. In contrast, thermogenic ethane presumably exhibits near stochastic 13C-13C distribution inherited from the biological precursor, which undergoes C-C bond cleavage/recombination during metabolism. Further, we find an exceptionally high 13C-13C signature in ethane remaining after microbial oxidation. In summary, the approach distinguishes between thermogenic, microbially altered, and abiotic hydrocarbons. The 13C-13C signature can provide an important step forward for discrimination of the origin of organic molecules on Earth and in extra-terrestrial environments.

Photochemical Synthesis of Ammonia and Amino Acids from Nitrous Oxide.

Abiotic synthesis of ammonia (NH3) and amino acids is important for the origin of life and early evolution. Ammonia and organic nitrogen species may be produced from nitrous oxide (N2O), which is a second abundant nitrogen species in the atmosphere. Here, we report a new photochemical experiment and evaluate whether N2O can be used as a nitrogen source for prebiotic synthesis in the atmosphere. We conducted a series of experiments by using a gas mixture of N2O+CO, N2O+CO2, or N2O + H2 in the presence of liquid water. The results demonstrate that NH3, methylamine (CH3NH2), and some amino acids such as glycine, alanine, and serine can be synthesized through photochemistry from N2O even without metal catalysts. NH3 can be produced not only from CO + N2O, but also from H2+N2O. Glycine can be synthesized from CH3NH2 and CO2, which can be produced from N2O and CO under ultraviolet irradiation. Our work demonstrates, for the first time, that N2O could be an important nitrogen source and provide a new process for synthesizing ammonia and organic nitrogen species, which has not been previously considered. The contribution of organic synthesis from N2O should, therefore, be considered when discussing the prebiotic chemistry on primitive Earth.

De Novo Design of A Membrane-Anchored Probe for Multidimensional Quantification of Endocytic Dynamics.

As a process of cellular uptake, endocytosis, with gradient acidity in different endocytic vesicles, is vital for the homeostasis of intracellular nutrients and other functions. To study the dynamics of endocytic pathway, a membrane-anchored pH probe, ECGreen, is synthesized to visualize endocytic vesicles under structured illumination microscopy (SIM), a super-resolution technology. Being sensitive to acidity with increasing fluorescence at low pH, ECGreen can differentiate early and late endosomes as well as endolysosomes. Meanwhile, membrane anchoring not only improves the durability of ECGreen, but also provides an excellent anti-photobleaching property for long-time imaging with SIM. Moreover, by taking these advantages of ECGreen, a multidimensional analysis model containing spatial, temporal, and pH information is successfully developed for elucidating the dynamics of endocytic vesicles and their interactions with mitochondria during autophagy, and reveals a fast conversion of endosomes near the plasma membrane.

Scalable and Practical Natural Gradient for Large-Scale Deep Learning.

Large-scale distributed training of deep neural networks results in models with worse generalization performance as a result of the increase in the effective mini-batch size. Previous approaches attempt to address this problem by varying the learning rate and batch size over epochs and layers, or ad hoc modifications of batch normalization. We propose scalable and practical natural gradient descent (SP-NGD), a principled approach for training models that allows them to attain similar generalization performance to models trained with first-order optimization methods, but with accelerated convergence. Furthermore, SP-NGD scales to large mini-batch sizes with a negligible computational overhead as compared to first-order methods. We evaluated SP-NGD on a benchmark task where highly optimized first-order methods are available as references: training a ResNet-50 model for image classification on ImageNet. We demonstrate convergence to a top-1 validation accuracy of 75.4 percent in 5.5 minutes using a mini-batch size of 32,768 with 1,024 GPUs, as well as an accuracy of 74.9 percent with an extremely large mini-batch size of 131,072 in 873 steps of SP-NGD.

Simple Fluorescence Assay for Cystine Uptake via the xCT in Cells Using Selenocystine and a Fluorescent Probe.

The cystine/glutamate antiporter (xCT) is a crucial transporter that maintains cellular redox balance by regulating intracellular glutathione synthesis via cystine uptake. However, no robust and simple method to determine the cystine uptake activity of xCT is currently available. We have developed a method to measure the xCT activity via the reaction of selenocysteine and fluorescein O,O'-diacrylate (FOdA). Selenocystine, a cystine analogue, is transported into cells through xCT on the cell membrane. The amount of the transported selenocystine was then determined by a reaction using tris(2-carboxyethyl)phosphine (TCEP) and FOdA in a weak acidic buffer at pH 6. Using this method, the cystine uptake activity of xCT in various cells and the inhibitory efficiency of xCT inhibitors, were evaluated.

Standardization for 13 C-13 C clumped isotope analysis by the fluorination method.

RATIONALE: The 13 C-13 C isotopologues of C2 molecules have recently been measured using a fluorination method. The C2 compound is first fluorinated into hexafluoroethane (C2 F6 ), and its 13 C-isotopologues are subsequently measured using a conventional isotope ratio mass spectrometer. Here, we present an approach for standardizing the fluorination method on an absolute reference scale by using isotopically enriched C2 F6 . METHODS: We prepared physical mixtures of 13 C-13 C-labeled ethanol and natural ethanol. The enriched ethanol samples were measured using the recently developed fluorination method. Based on the difference between the calculated and measured ∆13 C13 C values, we quantified the extent to which isotopologues were scrambled during dehydration, fluorination, and ionization in the ion source. RESULTS: The measured ∆13 C13 C value was approximately 20% lower than that expected from the amount of 13 C-13 C ethanol. The potential scrambling in the ion source was estimated to be 0.5-2%, which is lower than the observed isotopic reordering. Therefore, isotopic reordering may have occurred during either dehydration or fluorination. CONCLUSIONS: For typical analysis of natural samples, scrambling in the ion source can only change the ∆13 C13 C value by less than 0.04‰, which is lower than the current analytical precision (±0.07‰). Therefore, the observed isotopic reordering may have occurred during the fluorination of ethene through the scrambling of isotopologues of ethene but not in the ion source of the mass spectrometer or during the dehydration of ethanol, given the small amount of C1 and C3+ molecules. Thus, we obtained the empirical transfer function ∆13 C13 CCSC = λ × ∆13 C13 C with a λ value of 1.25 ± 0.01 for ethanol/ethene and 1.00 for ethane. Using the empirical transfer function, the developed fluorination method can provide actual differences in ∆ values.

The Great Oxidation Event preceded a Paleoproterozoic "snowball Earth".

The inability to resolve the exact temporal relationship between two pivotal events in Earth history, the Paleoproterozoic Great Oxidation Event (GOE) and the first "snowball Earth" global glaciation, has precluded assessing causality between changing atmospheric composition and ancient climate change. Here we present temporally resolved quadruple sulfur isotope measurements (δ34S, ∆33S, and ∆36S) from the Paleoproterozoic Seidorechka and Polisarka Sedimentary Formations on the Fennoscandian Shield, northwest Russia, that address this issue. Sulfides in the former preserve evidence of mass-independent fractionation of sulfur isotopes (S-MIF) falling within uncertainty of the Archean reference array with a ∆36S/∆33S slope of -1.8 and have small negative ∆33S values, whereas in the latter mass-dependent fractionation of sulfur isotopes (S-MDF) is evident, with a ∆36S/∆33S slope of -8.8. These trends, combined with geochronological constraints, place the S-MIF/S-MDF transition, the key indicator of the GOE, between 2,501.5 ± 1.7 Ma and 2,434 ± 6.6 Ma. These are the tightest temporal and stratigraphic constraints yet for the S-MIF/S-MDF transition and show that its timing in Fennoscandia is consistent with the S-MIF/S-MDF transition in North America and South Africa. Further, the glacigenic part of the Polisarka Formation occurs 60 m above the sedimentary succession containing S-MDF signals. Hence, our findings confirm unambiguously that the S-MIF/S-MDF transition preceded the Paleoproterozoic snowball Earth. Resolution of this temporal relationship constrains cause-and-effect drivers of Earth's oxygenation, specifically ruling out conceptual models in which global glaciation precedes or causes the evolution of oxygenic photosynthesis.

ß-Galactosidase-Catalyzed Fluorescent Reporter Labeling of Living Cells for Sensitive Detection of Cell Surface Antigens.

The ability to detect cell surface proteins using fluorescent-dye-labeled antibodies is crucial for the reliable identification of many cell types. However, the different types of cell surface proteins used to identify cells are currently limited in number because they need to be expressed at high levels to exceed background cellular autofluorescence, especially in the shorter-wavelength region. Herein we report on a new method, quinone methide-based catalyzed labeling for signal amplification (CLAMP), in which the fluorescence signal is amplified by an enzymatic reaction that strongly facilitates the detection of cell surface proteins on living cells. We used ß-galactosidase as an amplification enzyme and designed a substrate for it, called MUGF, that contains a fluoromethyl group. Upon removal of the galactosyl group in MUGF by ß-galactosidase labeling of the target cell surface proteins, the resulting product containing the quinone methide group was found to be both cell-membrane-permeable and reactive with intracellular nucleophiles, thereby providing fluorescent adducts. Using this method, we successfully detected several cell surface proteins, including programmed death ligand 1 protein, which is difficult to detect using conventional fluorescent-dye-labeled antibodies.

A fluorination method for measuring the 13 C-13 C isotopologue of C2 molecules.

RATIONALE: Doubly substituted isotope species ("clumped" isotopes) can provide insights into the biogeochemical history of a molecule, including its temperature of formation and/or its (bio)synthetic pathway. Here, we propose a new fluorination method for the measurement of 13 C-13 C species in C2 molecules using a conventional isotope ratio mass spectrometer. Target molecules include ethane, ethene and ethanol. METHODS: 13 C-13 C isotope species in C2 molecules were measured as C2 F6 using a conventional isotope ratio mass spectrometer. Ethane and ethene are directly fluorinated to C2 F6 . Ethanol is measured after dehydration to ethene and subsequent fluorination of the latter. The method enables the measurement of the Δ13 C13 C values normalized against a reference working standard. RESULTS: The reproducibility of the whole protocol, including chemical modification steps and measurement of C2 F6 isotopologues, is better than ±0.14‰ for all the compounds. Ethane from natural gas samples and biologically derived ethanol show a narrow range of Δ13 C13 C values, varying from 0.72‰ to 0.90‰. In contrast, synthetic ethanol as well as putative abiotic ethane show Δ13 C13 C values significantly different from this range with values of 1.14‰ and 0.25‰, respectively. CONCLUSIONS: The method presented here provides alternative means of measuring 13 C-13 C species to that using high-resolution mass spectrometry. Preliminary data from natural and synthetic molecules re-emphasizes the potential of 13 C clumped isotope species as a (bio)marker.

Geochemical and Metagenomic Characterization of Jinata Onsen, a Proterozoic-Analog Hot Spring, Reveals Novel Microbial Diversity including Iron-Tolerant Phototrophs and Thermophilic Lithotrophs.

Hydrothermal systems, including terrestrial hot springs, contain diverse geochemical conditions that vary over short spatial scales due to progressive interactions between reducing hydrothermal fluids, the oxygenated atmosphere, and, in some cases, seawater. At Jinata Onsen on Shikinejima Island, Japan, an intertidal, anoxic, iron-rich hot spring mixes with the oxygenated atmosphere and seawater over short spatial scales, creating diverse chemical potentials and redox pairs over a distance of ~10 m. We characterized geochemical conditions along the outflow of Jinata Onsen as well as the microbial communities present in biofilms, mats, and mineral crusts along its traverse using 16S rRNA gene amplicon and genome-resolved shotgun metagenomic sequencing. Microbial communities significantly changed downstream as temperatures and dissolved iron concentrations decreased and dissolved oxygen increased. Biomass was more limited near the spring source than downstream, and primary productivity appeared to be fueled by the oxidation of ferrous iron and molecular hydrogen by members of Zetaproteobacteria and Aquificae. The microbial community downstream was dominated by oxygenic Cyanobacteria. Cyanobacteria are abundant and active even at ferrous iron concentrations of ~150 µM, which challenges the idea that iron toxicity limited cyanobacterial expansion in Precambrian oceans. Several novel lineages of Bacteria are also present at Jinata Onsen, including previously uncharacterized members of the phyla Chloroflexi and Calditrichaeota, positioning Jinata Onsen as a valuable site for the future characterization of these clades.

Intramolecular isotopic evidence for bacterial oxidation of propane in subsurface natural gas reservoirs.

Microbial anaerobic oxidation of hydrocarbons is a key process potentially involved in a myriad of geological and biochemical environments yet has remained notoriously difficult to identify and quantify in natural environments. We performed position-specific carbon isotope analysis of propane from cracking and incubation experiments. Anaerobic bacterial oxidation of propane leads to a pronounced and previously unidentified 13C enrichment in the central position of propane, which contrasts with the isotope signature associated with the thermogenic process. This distinctive signature allows the detection and quantification of anaerobic oxidation of hydrocarbons in diverse natural gas reservoirs and suggests that this process may be more widespread than previously thought. Position-specific isotope analysis can elucidate the fate of natural gas hydrocarbons and provide insight into a major but previously cryptic process controlling the biogeochemical cycling of globally significant greenhouse gases.

Monitoring Lipid Droplet Dynamics in Living Cells by Using Fluorescent Probes.

We have developed three types of lipid droplet (LD)-specific fluorescent probes for live-cell imaging, Lipi-Blue, Lipi-Green, and Lipi-Red, which exhibit fluorescence upon being incorporated into LDs both of living and of fixed cells. These Lipi-probes are LD-specific probes that contain a pyrene or perylene group as a fluorescent scaffold and can be used to observe dynamics of LD in live cells and also interrelations with other organelles by simultaneous staining with multiple organelle-specific probes. Additionally, Lipi-Blue and Lipi-Green allow monitoring LDs in live cells even for 48 h after the staining. Here we show that newly formed LDs and previously existed LDs can be separately monitored in a single cell by using these probes and that intercellular transfer of whole LDs is observed in KB cells, but not in HepG2 cells under the same culturing condition. These findings indicate that newly developed LD-specific probes are useful to analyze the dynamics of LDs in live cells.

Changes of aliphatic C-H bonds in cyanobacteria during experimental thermal maturation in the presence or absence of silica as evaluated by FTIR microspectroscopy.

Aliphatic C-H bonds are one of the major organic signatures detected in Proterozoic organic microfossils, and their origin is a topic of interest. To investigate the influence of the presence of silica on the thermal alteration of aliphatic C-H bonds in prokaryotic cells during diagenesis, cyanobacteria Synechocystis sp. PCC6803 were heated at temperatures of 250-450°C. Changes in the infrared (IR) signals were monitored by micro-Fourier transform infrared (FTIR) spectroscopy. Micro-FTIR shows that absorbances at 2,925 cm-1 band (aliphatic CH2 ) and 2,960 cm-1 band (aliphatic CH3 ) decrease during heating, indicating loss of the C-H bonds, which was delayed by the presence of silica. A theoretical approach using solid-state kinetics indicates that the most probable process for the aliphatic C-H decrease is three-dimensional diffusion of alteration products under both non-embedded and silica-embedded conditions. The extrapolation of the experimental results obtained at 250-450°C to lower temperatures implies that the rate constant for CH3 (kCH3 ) is similar to or lower than that for CH2 (kCH2 ; i.e., CH3 decreases at a similar rate or more slowly than CH2 ). The peak height ratio of 2,960 cm-1 band (CH3 )/2,925 cm-1 band (CH2 ; R3/2 values) either increased or remained constant during the heating. These results reveal that the presence of silica does affect the decreasing rate of the aliphatic C-H bonds in cyanobacteria during thermal maturation, but that it does not significantly decrease the R3/2 values. Meanwhile, studies of microfossils suggest that the R3/2 values of Proterozoic prokaryotic fossils from the Bitter Springs Group and Gunflint Formation have decreased during fossilization, which is inconsistent with the prediction from our experimental results that R3/2 values did not decrease after silicification. Some process other than thermal degradation, possibly preservation of specific classes of biomolecules with low R3/2 values, might have occurred during fossilization.

Geoelectrochemical CO production: Implications for the autotrophic origin of life.

Wächtershäuser's proposal of the autotrophic origin of life theory and subsequent laboratory demonstrations of relevant organic reactions have opened a new gate for the exploration of the origin of life. However, this scenario remains controversial because, at present, it requires a high pressure of CO as a source of carbon and reducing energy, although CO must have been a trace C species on the Hadean Earth. We show that, simulating a geoelectrochemical environment in deep-sea hydrothermal fields, CO production with up to ~40% Faraday efficiency was attainable on CdS in CO2-saturated NaCl solution at ≤-1 V (versus the standard hydrogen electrode). The threshold potential is readily generated in the H2-rich, high-temperature, and alkaline hydrothermal vents that were probably widespread on the early komatiitic and basaltic ocean crust. Thus, Wächtershäuser's scenario starting from CO2 was likely to be realized in the Hadean ocean hydrothermal systems.

Small fluorescent molecules for monitoring autophagic flux.

We have developed two types of fluorescent probes, DALGreen and DAPGreen, for monitoring autophagy, that exhibit fluorescence upon being incorporated into autophagosomes. DALGreen enhances its fluorescence at acidic pH, which is favorable for monitoring late-phase autophagy, whereas DAPGreen remains fluorescent with almost constant brightness during the autophagic process. With these probes that stain autophagosomes as they are being formed, the real-time change of autophagic phenomena of live cells may be traced, which is an advantage over conventional approaches with small molecules that stain mature autophagosomes. The use of both dyes allows monitoring of the membrane dynamics of autophagy in any type of cell without the need for genetic engineering, and therefore, will be useful as a tool to study autophagic phenomena.

Multiple sulfur isotope constraints on microbial sulfate reduction below an Archean seafloor hydrothermal system.

Microbial sulfate reduction is among the most ubiquitous metabolic processes on earth. The oldest evidence of microbial sulfate reduction appears in the ca. 3.5 Ga Dresser Formation in the North Pole area of Pilbara Craton in Western Australia. That evidence was found through analysis of quadruple sulfur isotopes of sulfate and sulfide minerals deposited on the seafloor. However, the activity of microbial sulfate reduction below the Archean seafloor remains poorly understood. Here, we report the quadruple sulfur isotopic compositions of sulfide minerals within hydrothermally altered seafloor basalt and less altered basaltic komatiite collected from the North Pole Dome area. The Δ33 S values of the sulfide minerals were nonzero negative, suggesting that sulfate reduction occurred below the Archean seafloor. To constrain the substrate sulfate sources and sulfate reduction processes, we constructed a numerical model. Comparing the modeled and observed sulfur isotopes, we show that the substrate sulfate comprises seawater sulfate with a negative Δ33 S anomaly and 34 S-enriched sulfate with no anomalous Δ33 S. The latter component probably represents sulfate produced by local hydrothermal processes. The maximum sulfur isotopic fractionation between the putative substrate sulfate and the observed sulfide minerals within the altered basalt and basaltic komatiite is 35‰, which is consistent with a microbial origin. Alternatively, thermochemical sulfate reduction may also produce sulfide. However, considering the hydrothermal temperature inferred from the metamorphic grade of the altered basalt, the sulfur isotopic fractionation produced by inorganic sulfate reduction is probably below 20‰. Collectively, larger fractionations imply the involvement of biological sulfate reduction processes, both in the hydrothermal system below the seafloor and in less altered subsurface settings.