Microbial carbon cycling in oligotrophic regional aquifers near the Tono Uranium Mine, Japan as inferred from δC and ΔC values of in situ phospholipid fatty acids and carbon sources

Microorganisms are ubiquitous in deep subsurface environments, but their role in the global carbon cycle is not well-understood. The natural abundance δ¹³C and Δ¹⁴C values of microbial membrane phospholipid fatty acids (PLFAs) were measured and used to assess the carbon sources of bacteria in sedimentary and granitic groundwaters sampled from three boreholes in the vicinity of the Tono Uranium Mine, Gifu, Japan. Sample storage experiments were performed and drill waters analyzed to characterize potential sources of microbial contamination. The most abundant PLFA structures in all waters sampled were 16:0, 16:1ω7c, cy17:0, and 18:1ω7c. A PLFA biomarker for type II methanotrophs, 18:1ω8c, comprised 3% and 18% of total PLFAs in anoxic sedimentary and granitic waters, respectively, sampled from the KNA-6 borehole. The presence of this biomarker was unexpected given that type II methanotrophs are considered obligate aerobes. However, a bacterium that grows aerobically with CH₄ as the sole energy source and which also produces 56% of its total PLFAs as 18:1ω8c was isolated from both waters, providing additional evidence for the presence of type II methanotrophs. The Δ¹⁴C values determined for type II methanotroph PLFAs in the sedimentary (−861‰) and granite (−867‰) waters were very similar to the Δ¹⁴C values of dissolved inorganic carbon (DIC) in each water (∼−850‰). This suggests that type II methanotrophs ultimately derive all their carbon from inorganic sources, whether directly from DIC and/or from CH₄ produced by the reduction of DIC. In contrast, δ¹³C values of type II PLFAs in the sedimentary (−93‰) and granite (−60‰) waters indicate that these organisms use different carbon assimilation schemes in each environment despite very similar δ¹³C_CH4 values (∼−95‰) for each water. The δ¹³C_PLFA values (−28‰ to −45‰) of non-methanotrophic bacteria in the KNA-6 LTL water do not clearly distinguish between heterotrophic and autotrophic metabolisms, but Δ¹⁴C_PLFA values indicate that >65% of total bacteria filtered from the KNA-6 LTL water are heterotrophs. Ancient Δ¹⁴C values (∼−1000‰) of some PLFAs suggest that many heterotrophs utilize ancient organic matter, perhaps from lignite seams within the sedimentary rocks. The more negative range of δ¹³C_PLFA values determined for the KNA-6 granitic water (−42‰ to −66‰) are likely the result of a microbial ecosystem dominated by chemolithoautotrophy, perhaps fuelled by abiogenic H₂. Results of sample storage experiments showed substantial shifts in microbial community composition and δ¹³C_PLFA values (as much as 5‰) during 2-4 days of dark, refrigerated, aseptic storage. However, water samples collected and immediately filtered back in the lab from freshly drilled MSB-2 borehole appeared to maintain the same relative relationships between δ¹³C_PLFA values for sedimentary and granitic host rocks as observed for samples directly filtered under artesian flow from the KNA-6 borehole of the Tono Uranium Mine.     

Distributions of phospholipid and glycolipid fatty acids in two strains of different functional Erythrobacter sp. isolated from South China Sea

The comparison of the fatty acids between aerobic anoxygenic phototrophic bacteria (AAPB) and their phylogenetic relatives has been a fascinating but yet enigmatic topic, enhancing our understanding of physiological variations between these evolutionarily related microorganisms. Two strains of marine bacteria, both phylogenetically falling into Erythrobacter sp., were isolated from the South China Sea, and demonstrated, respectively, to be an aerobic anoxygenic phototrophic bacteria (AAPB) (JL475) which is capable of anoxygenic photosynthesis via BChl a, and an obligate heterotroph (JL316) with a lack of BChl a, on the basis of phylogenetic analysis and pure culture cultivation. Phospholipid fatty acids (PLFA) and glycolipid fatty acids (GLFA) of the two strains were extracted and analyzed by gas chromatographymass spectrometry. The PLFA in JL475 AAPB are characterized by C_18:1 C_18:2ω7,13 and C_18:1, with the C_18:2ω7,13 being a specific compound for AAPB and in particular for Erythrobacter longus and some of its phylogenetically closely related relatives. The JL316 strain is characterized in PLFA by the presence of C_18:1, C_16:1 and C_16:0, and in particular C_17:1. GLFA do not show any discrimination between the two strains. Four α,ω-dicarboxylic acids, including 1,8-octanedioic acid, 1,9-nonanedioic acid, 1,10-decanedioic acid and 1,11-undecanedioic acid, are present only in JL316 GLFA, presumably derived from metabolic products. C₁₄-C₁₆ 2-hydroxy fatty acids were found in the two strains, probably assuming a similar function of their LPS in outer membranes.     

Stable isotopic evidence in support of active microbial methane cycling in low-temperature diffuse flow vents at 9°50′N East Pacific Rise

A unique dataset from paired low- and high-temperature vents at 9°50′N East Pacific Rise provides insight into the microbiological activity in low-temperature diffuse fluids. The stable carbon isotopic composition of CH₄ and CO₂ in 9°50′N hydrothermal fluids indicates microbial methane production, perhaps coupled with microbial methane consumption. Diffuse fluids are depleted in ¹³C by ∼10‰ in values of δ¹³C of CH₄, and by ∼0.55‰ in values of δ¹³C of CO₂, relative to the values of the high-temperature source fluid (δ¹³C of CH₄ =−20.1 ± 1.2‰, δ¹³C of CO₂ =−4.08 ± 0.15‰). Mixing of seawater or thermogenic sources cannot account for the depletions in ¹³C of both CH₄ and CO₂ at diffuse vents relative to adjacent high-temperature vents. The substrate utilization and ¹³C fractionation associated with the microbiological processes of methanogenesis and methane oxidation can explain observed steady-state CH₄ and CO₂ concentrations and carbon isotopic compositions. A mass-isotope numerical box model of these paired vent systems is consistent with the hypothesis that microbial methane cycling is active at diffuse vents at 9°50′N. The detectable ¹³C modification of fluid geochemistry by microbial metabolisms may provide a useful tool for detecting active methanogenesis.     

Bacterial incorporation of relict carbon in the hydrothermal environment of Guaymas Basin

Radiocarbon analyses of bulk carbon and individual organic compounds are presented for the hydrothermal environment of the Rebecca’s Roost vent in the southern trough of the Guaymas Basin hydrothermal field. The Δ¹⁴C values of CO₂ and CH₄in the hottest hydrothermal fluids (317°C) are nearly “radiocarbon dead” (−944‰ and −923‰, respectively). In contrast, the Δ¹⁴C values of sediments and individual fatty acids (−418‰ to −227‰) obtained from a bacterial mat located south of the vent site are similar to values previously reported for hydrothermal petroleum in this environment and are more depleted in ¹⁴C than overlying waters. Hydrothermal fluids moving through the sediments appear to supply ¹⁴C of intermediate age to the bacteria. This carbon may take the form of, or may be supplied by processes similar to, the generation of hydrothermal petroleum. Although the bacterial mat visibly was dominated by Beggiatoa spp., such mats are known to include numerous other species. Individual compound data show that preaged carbon is being consumed by the integrated bacterial assemblage. Values of δ¹³C and Δ¹⁴C indicate that petroleum-derived carbon is incorporated directly into fresh bacterial biomass. Subsequently, some of this newly synthesized material also is consumed by heterotrophs, as eukaryotic sterols from the same sample also have ¹⁴C-depleted values (Δ¹⁴C = −136‰ to −110‰). Therefore, the entire system may operate as a complex consortium to transform relict carbon back into biomass. Bacterial consumption of relict carbon occurs despite the ample supply of fresh carbon delivered from the productive, overlying water column.     

Fractionation of carbon isotopes in biosynthesis of fatty acids by a piezophilic bacterium Moritella japonica strain DSK1

We examined stable carbon isotope fractionation in biosynthesis of fatty acids of a piezophilic bacterium Moritella japonica strain DSK1. The bacterium was grown to stationary phase at pressures of 0.1, 10, 20, and 50 MPa in media prepared using sterile-filtered natural seawater supplied with glucose as the sole carbon source. Strain DSK1 synthesized typical bacterial fatty acids (C_14-19 saturated, monounsaturated, and cyclopropane fatty acids) as well as long-chain polyunsaturated fatty acids (PUFA) (20:6ω3). Bacterial cell biomass and individual fatty acids exhibited consistent pressure-dependent carbon isotope fractionations relative to glucose. The observed Δδ_FA-glucose (−1.0‰ to −11.9‰) at 0.1 MPa was comparable to or slightly higher than fractionations reported in surface bacteria. However, bulk biomass and fatty acids became more depleted in ¹³C with pressure. Average carbon isotope fractionation (Δδ_FA-glucose) at high pressures was much higher than that for surface bacteria: −15.7‰, −15.3‰, and −18.3‰ at 10, 20, and 50 MPa, respectively. PUFA were more ¹³C depleted than saturated and monounsaturated fatty acids at all pressures. The observed isotope effects may be ascribed to the kinetics of enzymatic reactions that are affected by hydrostatic pressure and to biosynthetic pathways that are different for short-chain and long-chain fatty acids. A simple quantitative calculation suggests that in situ piezophilic bacterial contribution of polyunsaturated fatty acids to marine sediments is nearly two orders of magnitude higher than that of marine phytoplankton and that the carbon isotope imprint of piezophilic bacteria can override that of surface phytoplankton. Our results have important implications for marine biogeochemistry. Depleted fatty acids reported in marine sediments and the water column may be derived simply from piezophilic bacteria resynthesis of organic matter, not from bacterial utilization of a ¹³C-depleted carbon source (i.e., methane). The interpretation of carbon isotope signatures of marine lipids must be based on principles derived from piezophilic bacteria.     

Paleogene paleoclimate reconstruction using oxygen isotopes from land and freshwater organisms: the use of multiple paleoproxies

Understanding past climate change is critical to the interpretation of earth history. Even though relative temperature change has been readily assessed in the marine record, it has been more difficult in the terrestrial record due to restricted taxonomic distribution and isotopic fractionation. This problem could be overcome by the use of multiple paleoproxies. Therefore, the δ¹⁸O isotopic composition of five paleoproxies (rodent tooth enamel, δ¹⁸O_Phosphate = +17.7 ± 2.0‰ n = 74 (VSMOW); fish scale ganoine δ¹⁸O_Phosphate = +19.7 ± 0.7‰ n = 20 (VSMOW); gastropod shell δ¹⁸O_Calcite = −1.7 ± 1.3‰ n = 50 (VPDB); charophyte gyrogonite δ¹⁸O_Calcite = −2.4 ± 0.5‰ n = 20 (VPDB); fish otolith δ¹⁸O_Aragonite = δ¹⁸O = −3.6 ± 0.6‰ n = 20 (VPDB)) from the Late Eocene (Priabonian) Osborne Member (Headon Hill Formation, Solent Group, Hampshire Basin, UK) were determined. Because diagenetic alteration was shown to be minimal the phosphate oxygen component of rodent tooth enamel (as opposed to enamel carbonate oxygen) was used to calculate an initial δ¹⁸O_{Local water} value of 0.0 ± 3.4‰. However, a skewed distribution, most likely as a result of the ingestion of evaporating water, necessitated the calculation of a corrected δ¹⁸O_{Local water} value of −1.3 ± 1.7‰ (n = 62). This δ¹⁸O_{Local water} value corresponds to an approximate mean annual temperature of 18 ± 1°C. Four other mean paleotemperatures can also be calculated by combining the δ¹⁸O_{Local water} value with four independent freshwater paleoproxies. The calculated paleotemperature using the fish scale thermometry equations most likely represents the mean temperature (21 ± 2°C) of the entire length of the growing season. This should be concordant with the paleotemperature calculated using the Lymnaea shell thermometry equation (23 ± 2°C). The lack of concordance is interpreted to be the result of diagenetic alteration of the originally aragonitic Lymnaea shell to calcite. The mean paleotemperature calculated using the charophyte gyrogonite thermometry equation (21 ± 2°C), on the other hand, most likely represents the mean temperature of a single month toward the end of the growing season. The fish otolith mean paleotemperature (28 ± 2°C) most likely represents the mean temperature of the warmest months of the growing season. An approximate mean annual temperature of 18 ± 1°C, in addition to a mean growing season paleotemperature of 21 ± 2°C (using fish scale only) with a warmest month temperature of 28 ± 2°C, and high associated standard deviations suggest that a subtropical to warm temperate seasonal climate existed during the deposition of the Late Eocene Osborne Member.     

中国南方灯影峡期海洋碳酸盐岩原始δ13C和δ18O组成及海水温度

中国南方灯影峡期(晚前寒武纪)是白云岩广泛发育的海洋碳酸盐沉积时期,在灯影组中部发育从海水直接沉积沉淀的原生白云岩,目前仍保留其原始组构特征。从40个原生白云石(岩)中测得:泥晶白云石的δ¹³C值为3.64‰,δ¹⁸O值为-1.17‰(n=6);白云岩的13C值为3.52‰,δ¹⁸O值为-1.86‰(n=15);海水纤状白云石胶结物δ¹³C值为2.90‰,δ¹⁸O值2.65‰(n=8);海水刃状白云石胶结物的δ¹³C值为2.96‰,δ¹⁸O值为-2.41‰(n=8);晶纹层和海水纤状白云石胶结物的δ¹³C值为2.79‰,δ¹⁸O值为-3.13‰。40个岩样的δ¹³C平均值为3.25‰±0.44‰,δ¹⁸O平均值为-2.12‰±0.98‰(均以PDB标准)。对于灯影峡期海相云岩的原始δ¹³C和δ¹⁸O值,不采用所有样品的平均值,而是采用原生白云石沉积物与海水白云石结物δ¹³C值和δ¹⁸O值两个图示分布区重叠部分的最重同位素值,即：δ¹C值为4.43‰(PDB标准),δ¹⁸O值为-0.62‰(PDB标准),将其作为灯影峡期海洋碳酸盐岩的原始同位素组成。对海水原生白云石胶结物包裹体盐度进行了测定,海水δ¹⁸O计算值为2.90(SMOW标准),用原始δ¹⁸O值计算的原生白云石形成时的海水温度为40.8 ℃。这说明中国南方灯影峡的海洋为炎热的较高的海水温度环境。     

The implications for paleodietary and paleoclimatic reconstructions of intrapopulation variability in the oxygen and carbon isotopes of teeth from modern feral horses

We analyzed the isotopic patterns found in the tooth enamel of modern feral horses from Shackleford Banks, North Carolina (USA), which has a temperate climate and supports primarily C₄ grasslands. Enamel δ¹³C values averaged −4.1‰ with a standard deviation (1σ) of 1.7‰, which corresponds to an average diet of 66 ± 12% C₄ plants. Our results differ from dietary reconstructions from 1978 to 1981, which found that horses consumed 91% C₄ plants. This suggests that horses have increased their consumption of C₃ forbs, likely as a result of the removal of cattle, sheep, and goats from the island. Shackleford surface waters had δ¹⁸O values that averaged −3.3 ± 0.5‰ and −1.3 ± 1.8‰ on the western and eastern ends of the island, respectively. Tooth enamel samples averaged 27.3 ± 1.5‰ and displayed the same range of δ¹⁸O values as surface waters. The variability of both δ¹⁸O and the δ¹³C values among individuals within this population demonstrates that horses from relatively homogenous temperate environments can display a wide range of isotopic values. Given the observed range of isotopic values for modern horses, we suggest that researchers use the mean values of multiple (≥9) equids when attempting to reconstruct average paleodiets and/or paleoenvironmental conditions.     

Stable carbon and nitrogen isotopic compositions of high molecular weight dissolved organic matter from four U.S. estuaries

High molecular weight dissolved organic matter (HMW-DOM) represents an important component of dissolved organic carbon (DOC) in seawater and fresh-waters. In this paper, we report measurements of stable carbon (δ¹³C) isotopic compositions in total lipid, total hydrolyzable amino acid (THAA), total carbohydrate (TCHO) and acid-insoluble “uncharacterized” organic fractions separated from fourteen HMW-DOM samples collected from four U.S. estuaries. In addition, C/N ratio, δ¹³C and stable nitrogen (δ¹⁵N) isotopic compositions were also measured for the bulk HMW-DOM samples. Our results indicate that TCHO and THAA are the dominant organic compound classes, contributing 33-46% and 13-20% of the organic carbon in HMW-DOM while total lipid accounts for only <2% of the organic carbon in the samples. In all samples, a significant fraction (35-49%) of HMW-DOM was included in the acid-insoluble fraction. Distinct differences in isotopic compositions exist among bulk samples, the compound classes and the acid-insoluble fractions. Values of δ¹³C and δ¹⁵N measured for bulk HMW-DOM varied from −22.1 to −30.1‰ and 2.8 to 8.9‰, respectively and varied among the four estuaries studied as well. Among the compound classes, TCHO was more enriched in ¹³C (δ¹³C = −18.5 to −22.8‰) compared with THAA (δ¹³C = −20.0 to −29.6‰) and total lipid (δ¹³C = −25.7 to −30.7‰). The acid-insoluble organic fractions, in general, had depleted ¹³C values (δ¹³C = −23.0 to −34.4‰). Our results indicate that the observed differences in both δ¹³C and δ¹⁵N were mainly due to the differences in sources of organic matter and nitrogen inputs to these estuaries in addition to the microbial processes responsible for isotopic fractionation among the compound classes. Both terrestrial sources and local sewage inputs contribute significantly to the HMW-DOM pool in the estuaries studied and thus had a strong influence on its isotopic signatures.     

Geochemical characterization of secondary microbial gas occurrence in the Songliao Basin, NE China

A suite of natural gases from the northern Songliao Basin in NE China were characterized for their molecular and carbon isotopic composition. Gases from shallow reservoirs display clear geochemical evidence of alteration by biodegradation, with very high dryness (C₁/C₂₊ > 100), high C₂/C₃ and i-C₄/n-C₄ ratios, high nitrogen content and variable carbon dioxide content. Isotopic values show wide range variations (δ¹³C_CH4 from −79.5‰ to −45.0‰, δ¹³C_C2H6 from −53.7‰ to −32.2‰, δ¹³C_C3H8 from −36.5‰ to −20.1‰, δ¹³C_nC4H10 from −32.7‰ to −24.5‰, and δ¹³C_CO2 from −21.6‰ to +10.5‰). A variety of genetic types can be recognized on the basis of chemical and isotopic composition together with their geological occurrence. Secondary microbial gas generation was masked by primary microbial gas and the mixing of newly generated methane with thermogenic methane already in place in the reservoir can cause very complicated isotopic signatures. System openness also was considered for shallow biodegraded gas accumulations. Gases from the Daqing Anticline are relatively wet with ¹³C enriched methane and ¹³C depleted CO₂, representing typically thermogenic origin. Gases within the Longhupao-Da’an Terrace have variable dryness, ¹³C enriched methane and variable δ¹³C of CO₂, suggesting dominant thermogenic origin and minor secondary microbial methane augment. The Puqian-Ao’nan Uplift contains relatively dry gas with ¹³C depleted methane and ¹³C enriched CO₂, typical for secondary microbial gas with a minor part of thermogenic methane. Gas accumulations in the Western Slope are very dry with low carbon dioxide concentrations. Some gases contain ¹³C depleted methane, ethane and propane, indicating low maturity/primary microbial origin. Recognition of varying genetic gas types in the Songliao Basin helps explain the observed dominance of gas in the shallow reservoir and could serve as an analogue for other similar shallow gas systems.     

Carbon isotopes and petrography of kerogens in ∼3.5-Ga hydrothermal silica dikes in the North Pole area, Western Australia

More than 600 specimens of ∼3.5 Ga-old hydrothermal silica dikes from the North Pole area, Pilbara craton, Western Australia, have been studied petrographically. The kerogens in 44 samples have been analyzed isotopically (C and N) and chemically (C, N, and H). The silica dikes are composed mainly of fine-grained silica (modal abundance: >97%) and are classified into two types by minor mineral assemblages: B(black)-type and G(gray)-type. The B-type silica dikes contain kerogen (0.37 to 6.72 mgC/g; average 2.44 mgC/g, n = 21) and disseminated sulfides, dominantly pyrite and Fe-poor sphalerite. In some cases, carbonate and apatite are also present. Their silica-dominated and sulfide-poor mineral assemblages suggest precipitation from low-temperature reducing hydrothermal fluid (likely 100-200°C). On the other hand, the G-type silica dikes are sulfide-free and concentrations of kerogen are relatively low (0.05 to 0.41 mgC/g; average 0.17 mgC/g, n = 13). They typically contain Fe-oxide (mainly hematite) which commonly replaces cubic pyrite and rhombic carbonate. Some G-types occur along secondary quartz veins. These textures indicate that the G-type silica dikes were formed by postdepositional metasomatism (oxidation) of the B-types, and that the B-types probably possess premetasomatic signatures. The δ¹³C values of kerogen in the B-types are −38.1 to −33.1‰ (average −35.9‰, n = 21), which are ∼4‰ lower than those of the G-types (−34.5 to −30.0‰; average −32.2‰, n = 19), and ∼6‰ lower than bedded chert (−31.2 to −29.4‰; average −30.5‰, n = 4). This indicates the preferential loss of ¹²C during the metasomatism (estimated fractionation factor: 0.9985). Considering the metasomatic effect on carbon isotopes with probably minor diagenetic and metamorphic overprints, we conclude that the original δ¹³C values of the kerogen in the silica dikes would have been heterogeneous (∼5‰) and at least some material had initial δ¹³C values of ≤ −38‰. The inferred ¹³C-depletions of organic carbon could have been produced by anaerobic chemoautotrophs such as methanogen, but not by aerobic photoautotrophs. This is consistent with the estimated physical and chemical condition of the hydrothermal fluid, which was probably habitable for anaerobic and thermophilic/hyperthermophilic chemoautotrophs. Alternatively, the organic matter may have been possibly produced by abiological reaction such as Fischer-Tropsch Type (FTT) synthesis under the hydrothermal condition. However, the estimated condition is inconsistent with the presence of the effective catalysts for the FTT reaction (i.e., Fe-Ni alloy, magnetite, and hematite). These lines of evidence suggest the possible existence of biosphere in the ∼3.5 Ga sub-seafloor hydrothermal system.     

Carbon and hydrogen isotope fractionation associated with the aerobic microbial oxidation of methane, ethane, propane and butane

Carbon isotope fractionation factors associated with the aerobic consumption of methane (C1), ethane (C2), propane (C3), and n-butane (C4) were determined from incubations of marine sediment collected from the Coal Oil Point hydrocarbon seep field, located offshore Santa Barbara, CA. Hydrogen isotope fractionation factors for C1, C2 and C3 were determined concurrently. Fresh sediment samples from two seep areas were each slurried with sea water and treated with C1, C2, C3 or C4, or with mixtures of all four gases. Triplicate samples were incubated aerobically at 15 °C, and the stable isotope composition and headspace levels of C1-C4 were monitored over the course of the experiment. Oxidation was observed for all C1-C4 gases, with an apparent preference for C3 and C4 over C1 and C2 in the mixed-gas treatments. Fractionation factors were calculated using a Rayleigh model by comparing the δ¹³C and δD of the residual C1-C4 gases to their headspace levels. Carbon isotope fractionation factors (reported in ε or (α-1) × 1000 notation) were consistent between seep areas and were −26.5‰ ± 3.9 for C1, −8.0‰ ± 1.7 for C2, −4.8‰ ± 0.9 for C3 and −2.9‰ ± 0.9 for C4. Fractionation factors determined from mixed gas incubations were similar to those determined from individual gas incubations, though greater variability was observed during C1 consumption. In the case of C1 and C3 consumption, carbon isotope fractionation appears to decrease as substrate becomes limiting. Hydrogen isotope fractionation factors determined from the two seep areas differed for C1 oxidation but were similar for C2 and C3. Hydrogen isotope fractionation factors ranged from −319.9‰ to −156.4‰ for C1 incubations, and averaged −61.9‰ ± 8.3 for C2 incubations and −15.1‰ ± 1.9 for C3 incubations. The fractionation factors presented here may be applied to estimate the extent of C1-C4 oxidation in natural gas samples, and should prove useful in further studying the microbial oxidation of these compounds in the natural environment.     

Low temperature volatile production at the Lost City Hydrothermal Field, evidence from a hydrogen stable isotope geothermometer

Although commonly utilized in continental geothermal work, the water-hydrogen and methane-hydrogen isotope geothermometers have been neglected in hydrothermal studies. Here we report δD-CH₄ and δD-H₂ values from high-temperature, black smoker-type hydrothermal vents and low-temperature carbonate-hosted samples from the recently discovered Lost City Hydrothermal Field. Methane deuterium content is uniform across the dataset at − 120 ± 12‰. Hydrogen δD values vary from − 420‰ to − 330‰ at high-temperature vents to − 700‰ to − 600‰ at Lost City. The application of several geothermometer equations to a suite of hydrothermal vent volatile samples reveals that predicted temperatures are similar to measured vent temperatures at high-temperature vents, and 20-60 °C higher than those measured at the Lost City vents. We conclude that the overestimation of temperature at Lost City reflects 1) that methane and hydrogen are produced by serpentinization at > 110 °C, and 2) that isotopic equilibrium at temperatures < 70 °C is mediated by microbial sulfate reduction. The successful application of hydrogen isotope geothermometers to low-temperature Lost City hydrothermal samples encourages its employment with low-temperature diffuse hydrothermal fluids.     

The stable carbon isotope biogeochemistry of acetate and other dissolved carbon species in deep subseafloor sediments at the northern Cascadia Margin

Ocean drilling has revealed the existence of vast microbial populations in the deep subseafloor, but to date little is known about their metabolic activities. To better understand the biogeochemical processes in the deep biosphere, we investigate the stable carbon isotope chemistry of acetate and other carbon-bearing metabolites in sediment pore-waters. Acetate is a key metabolite in the cycling of carbon in anoxic sediments. Its stable carbon isotopic composition provides information on the metabolic processes dominating acetate turnover in situ. This study reports our findings for a methane-rich site at the northern Cascadia Margin (NE Pacific) where Expedition 311 of the Integrated Ocean Drilling Program (IODP) sampled the upper 190 m of sediment. At Site U1329, δ¹³C values of acetate span a wide range from −46.0‰ to −11.0‰ vs. VPDB and change systematically with sediment depth. In contrast, δ¹³C values of both the bulk dissolved organic carbon (DOC) (−21.6 ± 1.3‰ vs. VPDB) and the low-molecular-weight compound lactate (−20.9 ± 1.8‰ vs. VPDB) show little variability. These species are interpreted to represent the carbon isotopic composition of fermentation products. Relative to DOC, acetate is up to 23.1‰ depleted and up to 9.1‰ enriched in ¹³C. Broadly, ¹³C-depletions of acetate relative to DOC indicate flux of carbon from acetogenesis into the acetate pool while ¹³C-enrichments of pore-water acetate relative to DOC suggest consumption of acetate by acetoclastic methanogenesis. Isotopic relationships between acetate and lactate or DOC provide new information on the carbon flow and the presence and activity of specific functional microbial communities in distinct biogeochemical horizons of the sediment. In particular, they suggest that acetogenic CO₂-reduction can coexist with methanogenic CO₂-reduction, a notion contrary to the hypothesis that hydrogen levels are controlled by the thermodynamically most favorable electron-accepting process. Further, the isotopic relationship suggests a relative increase in acetate flow to acetoclastic methanogenesis with depth although its contribution to total methanogenesis is probably small. Our study demonstrates how the stable carbon isotope biogeochemistry of acetate can be used to identify pathways of microbial carbon turnover in subsurface environments. Our observations also raise new questions regarding the factors controlling acetate turnover in marine sediments.     

Fate of bacterial biomass derived fatty acids in soil and their contribution to soil organic matter

Soil organic matter (SOM) is a major pool of the global C cycle and determines soil fertility. The stability of SOM strongly depends on the molecular precursors and structures. Plant residues have been regarded as the dominant precursors, but recent results showed a major contribution of microbial biomass. The fate of microbial biomass constituents has not yet been explored; therefore, we investigated the fate of fatty acids (FA) from ¹³C labeled Gram-negative bacteria (Escherichia coli) in a model soil study [Kindler, R., Miltner, A., Richnow, H.H., Kästner, M., 2006. Fate of gram negative bacterial biomass in soil—mineralization and contribution to SOM. Soil Biology & Biochemistry 38, 2860–2870]. After 224 days of incubation, the label in the total fatty acids (t-FA) in the soil decreased to 24% and in the phospholipid fatty acids (PLFA) of living microbes to 11% of the initially added amount. Since the bulk C decreased only to 44% in this period, the turnover of FA is clearly higher indicating that other compounds must have a lower turnover. The ¹³C label in the t-FA reached a stable level after 50 days but the label of the PLFA of the living microbial biomass declined until the end of the experiment. The isotopic enrichment of individual PLFA shows that the biomass derived C was spread across the microbial food web. Modelling of the C fluxes in this experiment indicated that microbial biomass is continuously mineralized after cell death and recycled by other organisms down to the 10% level, whereas the majority of biomass derived residual bulk C (～33%) was stabilized in the non-living SOM pool.     

Archaeal lipids in Neogene dolomites (Monterey and Sisquoc Formations, California) - Planktic versus benthic archaeal sources

The distribution of archaeal lipids, including archaeol and glycerol dibiphytanyl glycerol tetraethers (GDGTs), in dolomite concretions and surrounding sediment from the Monterey Formation (Miocene) and the Sisquoc Formation (Miocene-Pliocene) were examined to distinguish planktic from benthic contributions. For this purpose, dolomites with positive δ¹³C values (+7‰ to +13‰) were chosen; such highly positive values point to pronounced methanogenesis of benthic archaea in the sedimentary column. At first glance, distributions and relative abundances of GDGTs in both dolomites and background sediment were similar, resembling patterns of marine planktic crenarchaea. A contribution of benthic euryarchaea to the GDGT pool became evident only from variations in the δ¹³C values of different biphytanes obtained after ether cleavage of GDGTs. Whereas bi- and tricyclic biphytanes had an isotopic signal typical of planktic archaea (δ¹³C −23.6‰ to −20.5‰ and −23.4‰ to −21.2‰, respectively) for both dolomite and background sediment, acyclic and monocyclic biphytanes showed lower values for dolomite samples (−25.1‰ to −22.6‰ and −27.6‰ to −24.7‰, respectively), indicating a contribution of lipids from benthic archaea. The isoprenoid diether archaeol (δ¹³C −23.9‰ to −22.9‰), assigned to euryarchaea, was only detected in dolomite samples, also reflecting additional input from sedimentary archaea, probably autotrophic methanogens. The occurrence of lipids derived from methanogenic archaea agrees with the strong ¹³C-enrichment of dolomites and with mineral formation taking place in the zone of archaeal methanogenesis. This implies that the lipid biomarker inventory of sedimentary strata needs to be interpreted carefully, as it is often not straightforward to discriminate between input from the water column and sedimentary microbial activity.     

A menagerie of graphite morphologies in the Acapulco meteorite with diverse carbon and nitrogen isotopic signatures: Implications for the evolution history of acapulcoite meteorites

Morphologies, petrographic settings and carbon and nitrogen isotopic compositions of graphites in the Acapulco meteorite, the latter determined by secondary ionization mass spectrometry, are reported. Seven different graphite morphologies were recognized, the majority of which occur enclosed exclusively in kamacite. Individual graphite grains also rarely occur in the silicate matrix. Kamacite rims surrounding taenite cores of metal grains are separated from the Ni-rich metal cores by graphite veneers. These graphite veneers impeded or prevented Ni-Fe interdiffusion during cooling. In addition, matrix FeNi metal contains considerable amounts of phosphorous (≈ 700 ppm) and silicon (≈ 300 ppm) (Pack et al., 2005 in preparation) thus indicating that results of laboratory cooling experiments in the Fe-Ni binary system are inapplicable to Acapulco metals. Graphites of different morphologies display a range of carbon and nitrogen isotopic compositions, indicating a diversity of source regions before accretion in the Acapulco parent body. The isotopic compositions point to at least three isotopic reservoirs from which the graphites originated: (1) A reservoir with heavy carbon, represented by graphite in silicates (δ¹³C = 14.3 ± 2.4 ‰ and δ¹⁵N = −103.4 ± 10.9 ‰), (2) A reservoir with isotopically light carbon and nitrogen, characteristic for the metals. Its C- and N-isotopic compositions are probably preserved in the graphite exsolutions that are isotopically light in carbon and lightest in nitrogen (δ¹³C = −17 to −23 ‰ δ¹⁵N = −141 to −159 ‰). (3) A reservoir with an assumed isotopic composition (δ¹³C ∼ −5 ‰; δ¹⁵N ∼ −50 ‰). A detailed three-dimensional tomography in reflected light microscopy of the decorations of metal-troilite spherules in the cores of orthopyroxenes and olivines and metal-troilite veins was conducted to clarify their origin. Metal and troilite veins are present only near the fusion crust. Hence, these veins are not pristine to Acapulco parent body but resulted during passage of Acapulco in Earth’s atmosphere. A thorough search for symplectite-type silicate-troilite liquid quench textures was conducted to determine the extent of closed-system partial silicate melting in Acapulco.Metal-troilite spherules in orthopyroxenes and olivines are not randomly distributed but decorate ferromagnesian silicate restite cores, indicating that the metal-spherule decoration around restite silicates took place in a silicate partial melt. Graphite inclusions in these spherules have C- and N- isotopic compositions (δ¹³C = −2.9 ± 2.5 ‰ and δ¹⁵N = −101.2 ± 32 ‰) close to the average values of graphite in metals and in the silicate matrix, thus strongly suggesting that they originated from a mixture of graphite inclusions in metals and silicate matrix graphite during a closed system crystallization process subsequent to silicate-metal-sulfide partial melting. Troilite-orthopyroxene quench symplectite textures in orthopyroxene rims are clear evidence that silicate-sulfide partial melting took place in Acapulco. Due to petrographic heterogeneity on a centimeter scale, bulk REE abundances of individual samples or of individual minerals provide only limited information and the REE abundances alone are not entirely adequate to unravel the formational processes that prevailed in the acapulcoite-lodranite parent body. The present investigations demonstrate the complexity of the evolutionary stages of acapulcoites from accretion to parent body processes.     

Extraordinary C enrichment of diether lipids at the Lost City Hydrothermal Field indicates a carbon-limited ecosystem

Active and inactive carbonate chimneys from the Lost City Hydrothermal Field contain up to 0.6% organic carbon with diverse lipid assemblages. The δ¹³C values of total organic carbon range from −21.5‰ vs. VPDB at an extinct carbonate chimney to −2.8‰ at a 70 °C, actively venting carbonate chimney. Samples collected at locations with total organic carbon with δ¹³C > −15‰ also contained high abundances of isoprenoidal and nonisoprenoidal diether lipids. Samples with TOC more depleted in ¹³C lacked or contained lower amounts of these diethers.Isoprenoidal diethers, including sn-2 hydroxyarchaeol, sn-3 hydroxyarchaeol, and putative dihydroxyarchaeol, are likely to derive from methanogenic archaea. These compounds have δ¹³C values ranging from −2.9 to +6.7‰ vs. VPDB. Nonisoprenoidal diethers and monoethers are presumably derived from bacteria, and have structures similar to those produced by sulfate-reducing bacteria in culture and at cold seeps. In samples that also contained abundant hydroxyarchaeols, these diethers have δ¹³C values between −11.8 and +3.6‰. In samples without abundant hydroxyarchaeols, the nonisoprenoidal diethers were typically more depleted in ¹³C, with δ¹³C as low as −28.7‰ in chimneys and −45‰ in fissures.The diethers at Lost City are probably derived from hydrogen-consuming methanogens and bacteria. High hydrogen concentrations favor methanogenesis over methanotrophy and allow the concurrent growth of methanogens and sulfate-reducing bacteria. The unusual enrichment of ¹³C in lipids can be attributed to nearly complete consumption of bioavailable carbon in vent fluids. Under carbon-limited conditions, the isotope effects that usually lead to ¹³C-depletion in organic material cannot be expressed. Consequently, metabolic products such as lipids and methane have δ¹³C values typical of abiotic carbon.     

Sulfur isotope fractionation during growth of sulfate-reducing bacteria on various carbon sources

Stable sulfur isotope fractionation during microbial sulfate reduction is a potential tool to estimate sulfate reduction rates at field sites. However, little is known about the influence of the utilized carbon source on the magnitude of sulfur isotope fractionation. To investigate this effect, both a pure culture (strain PRTOL1) and enrichment cultures from a petroleum hydrocarbon (PHC)-contaminated aquifer were used and grown in batch cultures on various carbon sources with an initial sulfate concentration of 1 mmol/L. As sole carbon sources the PHC components naphthalene, 1,3,5-trimethylbenzene, and heating oil (enrichment culture) and the organic acids acetate, pyruvate, benzoate, and 3-phenylpropionate (enrichment culture and PRTOL1) were used. Sulfate reduction rates of all cultures ranged from 6 ± 1 nmol cm⁻³ d⁻¹ (enrichment culture grown on 1,3,5-trimethylbenzene) to 280 ± 6 nmol cm⁻³ d⁻¹ (enrichment culture grown on pyruvate). Cell-specific sulfate reduction rates ranged from 1.1 × 10⁻¹⁴ mol cell⁻¹ d⁻¹ (PRTOL1 grown on pyruvate) to 1.5 × 10⁻¹³ mol cell⁻¹ d⁻¹ (PRTOL1 grown on acetate). Sulfur isotope enrichment factors (ε) for the enrichment culture ranged from 16.1‰ (3-phenylpropionate) to 34.5‰ (1,3,5-trimethylbenzene) and for PRTOL1 from 30.0‰ (benzoate) to 36.0‰ (pyruvate). Cultures of PRTOL1 always showed higher ε values than the enrichment culture when grown on the same carbon source due to culture-specific properties. Higher ε values were obtained when the enrichment culture was grown on PHC components than on organic acids. No relationship between ε values and cell-specific sulfate reduction rate existed when all data were combined. When comparing the magnitude of ε values determined in this laboratory study with ε values measured at contaminated and uncontaminated field sites, it becomes evident that a multitude of factors influences ε values at field sites and complicates their interpretation. The results of this study help us assess some of the general parameters that govern the magnitude of ε in sulfate-reducing environments.     

Isotopic Fingerprints of Paleoclimates during the Last 30,000 Years in Deep Confined Groundwaters of Southern India

Isotopic and geochemical evidence of paleoclimates, especially for the last glaciation, has been obtained from deep confined groundwaters of southern India. The δ¹³C, δ¹⁸O, chloride, and deuterium analyses of groundwaters show distinct excursions inferred to be related to climatic variations. The arid climatic episode associated with the last glaciation (18,000 ± 2000 yr B.P.) is conspicuously identified by signatures of relatively enriched δ¹³C (−10 to −12‰ PDB) and δ¹⁸O (−5.3 to −4.8‰ SMOW) values, and high chloride concentration (80 to 160 mg/l). The transition from an arid to humid period ca. 12,000–8000 yr B.P. is shown by a decreasing trend in the δ¹³C (−9.5 to −17‰) and δ¹⁸O (−4.5 to −6.3‰) contents of groundwaters. The late Holocene (since 4000 yr B.P.), marked by a more humid but unstable climate, is identified by further depletion of δ¹³C (−13 to −20‰) and δ¹⁸O (−5.2 to −6.3‰). Similar variation between δ¹⁸O and chloride values in confined groundwaters further demonstrates two distinct climatic excursions (arid and humid) governed by the “amount effect.” This is the first time that isotopic and geochemical signatures related to changing paleoclimates have been identified in the confined groundwaters of the southern Indian landmass.