Primary N2-He gas field formation in intracratonic sedimentary basins.

Helium, nitrogen and hydrogen are continually generated within the deep continental crust1-9. Conceptual degassing models for quiescent continental crust are dominated by an assumption that these gases are dissolved in water, and that vertical transport in shallower sedimentary systems is by diffusion within water-filled pore space (for example, refs. 7,8). Gas-phase exsolution is crucial for concentrating helium and forming a societal resource. Here we show that crustal nitrogen from the crystalline basement alone-degassing at a steady state in proportion to crustal helium-4 generation-can reach sufficient concentrations at the base of some sedimentary basins to form a free gas phase. Using a gas diffusion model coupled with sedimentary basin evolution, we demonstrate, using a classic intracratonic sedimentary basin (Williston Basin, North America), that crustal nitrogen reaches saturation and forms a gas phase; in this basin, as early as about 140 million years ago. Helium partitions into this gas phase. This gas formation mechanism accounts for the observed primary nitrogen-helium gas discovered in the basal sedimentary lithology of this and other basins, predicts co-occurrence of crustal gas-phase hydrogen, and reduces the flux of helium into overlying strata by about 30 per cent because of phase solubility buffering. Identification of this gas phase formation mechanism provides a quantitative insight to assess the helium and hydrogen resource potential in similar intracontinental sedimentary basins found worldwide.

Greenland melt drives continuous export of methane from the ice-sheet bed.

Ice sheets are currently ignored in global methane budgets1,2. Although ice sheets have been proposed to contain large reserves of methane that may contribute to a rise in atmospheric methane concentration if released during periods of rapid ice retreat3,4, no data exist on the current methane footprint of ice sheets. Here we find that subglacially produced methane is rapidly driven to the ice margin by the efficient drainage system of a subglacial catchment of the Greenland ice sheet. We report the continuous export of methane-supersaturated waters (CH4(aq)) from the ice-sheet bed during the melt season. Pulses of high CH4(aq) concentration coincide with supraglacially forced subglacial flushing events, confirming a subglacial source and highlighting the influence of melt on methane export. Sustained methane fluxes over the melt season are indicative of subglacial methane reserves that exceed methane export, with an estimated 6.3 tonnes (discharge-weighted mean; range from 2.4 to 11 tonnes) of CH4(aq) transported laterally from the ice-sheet bed. Stable-isotope analyses reveal a microbial origin for methane, probably from a mixture of inorganic and ancient organic carbon buried beneath the ice. We show that subglacial hydrology is crucial for controlling methane fluxes from the ice sheet, with efficient drainage limiting the extent of methane oxidation5 to about 17 per cent of methane exported. Atmospheric evasion is the main methane sink once runoff reaches the ice margin, with estimated diffusive fluxes (4.4 to 28 millimoles of CH4 per square metre per day) rivalling that of major world rivers6. Overall, our results indicate that ice sheets overlie extensive, biologically active methanogenic wetlands and that high rates of methane export to the atmosphere can occur via efficient subglacial drainage pathways. Our findings suggest that such environments have been previously underappreciated and should be considered in Earth's methane budget.

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.

Advances in carbon isotope analysis of trapped methane and volatile hydrocarbons in crystalline rock cores.

RATIONALE: The isotopic composition of hydrocarbons trapped in rocks on the microscale (fluid inclusions, mineral grain boundaries, microfractures) can provide powerful information on geological and biological processes but are an analytical challenge due to low concentrations. We present a new approach for the extraction and carbon isotopic analysis of methane (CH4 ) and hydrocarbons in trapped volatiles in crystalline rocks. METHODS: An off-line crusher with cryogenic trapping and a custom-made silica glass U-trap were attached to an external injector port on a continuous flow gas chromatograph/combustion/isotope ratio mass spectrometer to demonstrate the accuracy, reproducibility, and sensitivity of δ13 C measurements for CH4 . RESULTS: The method can isotopically characterize CH4 in crushed rock samples with concentrations as low as 3.5 × 10-9 mol/g of rock, and both sample and isotopic standards are analyzed with an accuracy and reproducibility of ±0.5‰. High H2 O/CH4 ratios of 98 to 500 have no effect on measured δ13 CCH4 values. The method is successfully applied to natural samples from the north range of Sudbury Basin, Ontario, Canada. The δ13 C isotopic signatures of CH4 trapped microscopically in rock from the north range overlap significantly with that of CH4 contained in larger scale flowing fracture fluids from the same part of the Sudbury Basin, indicating a potential genetic link. CONCLUSIONS: A novel method for δ13 CCH4 analysis was developed for the extraction of nanomole quantities of CH4 trapped microscopically in rocks. The technique has an accuracy and reproducibility comparable to that of on-line crushing techniques but importantly provides the capability of crushing larger rock quantities (up to 100 g). The benefit is improved detection levels for trace hydrocarbon species. Such a capability will be important for future extension of such crushing techniques for measurement of 2 H/1 H for CH4 , clumped isotopologues of CH4 and other trapped volatiles species, such as C2 H6 , C3 H8 , C4 H10 , CO2 and N2 .

Requirements for Chromium Reactors for Use in the Determination of H Isotopes in Compound-Specific Stable Isotope Analysis of Chlorinated Compounds.

There is a strong need for careful quality control in hydrogen compound-specific stable isotope analysis (CSIA) of halogenated compounds. This arises in part due to the lack of universal design of the chromium (Cr) reactors. In this study, factors that optimize the critical performance parameter, linearity, for the Cr reduction method for hydrogen isotope analysis were identified and evaluated. These include the effects of short and long vertically mounted reactors and temperature profiles on trapping of Cl to ensure accurate and precise hydrogen isotope measurements. This paper demonstrates the critical parameters that need consideration to optimize any Cr reactor applications to ensure the accuracy of δ2H analysis for organic compounds and to enhance intercomparability for both international standards and reference materials run by continuous flow versus an elemental analyzer.

Reductive Dehalogenation of Trichloromethane by Two Different Dehalobacter restrictus Strains Reveal Opposing Dual Element Isotope Effects.

Trichloromethane (TCM) is a frequently detected and persistent groundwater contaminant. Recent studies have reported that two closely related Dehalobacter strains (UNSWDHB and CF) transform TCM to dichloromethane, with inconsistent carbon isotope effects (ε13CUNSWDHB = -4.3 ± 0.45‰; ε13CCF = -27.5 ± 0.9‰). This study uses dual element compound specific isotope analysis (C; Cl) to explore the underlying differences. TCM transformation experiments using strain CF revealed pronounced normal carbon and chlorine isotope effects (ε13CCF = -27.9 ± 1.7‰; ε37ClCF = -4.2 ± 0.2‰). In contrast, small carbon and unprecedented inverse chlorine isotope effects were observed for strain UNSWDHB (ε13CUNSWDHB = -3.1 ± 0.5‰; ε37ClUNSWDHB = 2.5 ± 0.3‰) leading to opposing dual element isotope slopes (λCF = 6.64 ± 0.14 vs λUNSWDHB = -1.20 ± 0.18). Isotope effects of strain CF were identical to experiments with TCM and Vitamin B12 (ε13CVitamin B12 = -26.0 ± 0.9‰, ε37ClVitamin B12 = -4.0 ± 0.2‰, λVitamin B12 = 6.46 ± 0.20). Comparison to previously reported isotope effects suggests outer-sphere-single-electron transfer or SN2 as possible underlying mechanisms. Cell suspension and cell free extract experiments with strain UNSWDHB were both unable to unmask the intrinsic KIE of the reductive dehalogenase (TmrA) suggesting that enzyme binding and/or mass-transfer into the periplasm were rate-limiting. Nondirected intermolecular interactions of TCM with cellular material were ruled out as reason for the inverse isotope effect by gas/water and gas/hexadecane partitioning experiments indicating specific, yet uncharacterized interactions must be operating prior to catalysis.

Mechanistic Dichotomy in Bacterial Trichloroethene Dechlorination Revealed by Carbon and Chlorine Isotope Effects.

Tetrachloroethene (PCE) and trichloroethene (TCE) are significant groundwater contaminants. Microbial reductive dehalogenation at contaminated sites can produce nontoxic ethene but often stops at toxic cis-1,2-dichloroethene ( cis-DCE) or vinyl chloride (VC). The magnitude of carbon relative to chlorine isotope effects (as expressed by ΛC/Cl, the slope of δ13C versus δ37Cl regressions) was recently recognized to reveal different reduction mechanisms with vitamin B12 as a model reactant for reductive dehalogenase activity. Large ΛC/Cl values for cis-DCE reflected cob(I)alamin addition followed by protonation, whereas smaller ΛC/Cl values for PCE evidenced cob(I)alamin addition followed by Cl- elimination. This study addressed dehalogenation in actual microorganisms and observed identical large ΛC/Cl values for cis-DCE (ΛC/Cl = 10.0 to 17.8) that contrasted with identical smaller ΛC/Cl for TCE and PCE (ΛC/Cl = 2.3 to 3.8). For TCE, the trend of small ΛC/Cl could even be reversed when mixed cultures were precultivated on VC or DCEs and subsequently confronted with TCE (ΛC/Cl = 9.0 to 18.2). This observation provides explicit evidence that substrate adaptation must have selected for reductive dehalogenases with different mechanistic motifs. The patterns of ΛC/Cl are consistent with practically all studies published to date, while the difference in reaction mechanisms offers a potential answer to the long-standing question of why bioremediation frequently stalls at cis-DCE.

An oligotrophic deep-subsurface community dependent on syntrophy is dominated by sulfur-driven autotrophic denitrifiers.

Subsurface lithoautotrophic microbial ecosystems (SLiMEs) under oligotrophic conditions are typically supported by H2 Methanogens and sulfate reducers, and the respective energy processes, are thought to be the dominant players and have been the research foci. Recent investigations showed that, in some deep, fluid-filled fractures in the Witwatersrand Basin, South Africa, methanogens contribute <5% of the total DNA and appear to produce sufficient CH4 to support the rest of the diverse community. This paradoxical situation reflects our lack of knowledge about the in situ metabolic diversity and the overall ecological trophic structure of SLiMEs. Here, we show the active metabolic processes and interactions in one of these communities by combining metatranscriptomic assemblies, metaproteomic and stable isotopic data, and thermodynamic modeling. Dominating the active community are four autotrophic ß-proteobacterial genera that are capable of oxidizing sulfur by denitrification, a process that was previously unnoticed in the deep subsurface. They co-occur with sulfate reducers, anaerobic methane oxidizers, and methanogens, which each comprise <5% of the total community. Syntrophic interactions between these microbial groups remove thermodynamic bottlenecks and enable diverse metabolic reactions to occur under the oligotrophic conditions that dominate in the subsurface. The dominance of sulfur oxidizers is explained by the availability of electron donors and acceptors to these microorganisms and the ability of sulfur-oxidizing denitrifiers to gain energy through concomitant S and H2 oxidation. We demonstrate that SLiMEs support taxonomically and metabolically diverse microorganisms, which, through developing syntrophic partnerships, overcome thermodynamic barriers imposed by the environmental conditions in the deep subsurface.

Dual Carbon-Chlorine Isotope Analysis Indicates Distinct Anaerobic Dichloromethane Degradation Pathways in Two Members of Peptococcaceae.

Dichloromethane (DCM) is a probable human carcinogen and frequent groundwater contaminant and contributes to stratospheric ozone layer depletion. DCM is degraded by aerobes harboring glutathione-dependent DCM dehalogenases; however, DCM contamination occurs in oxygen-deprived environments, and much less is known about anaerobic DCM metabolism. Some members of the Peptococcaceae family convert DCM to environmentally benign products including acetate, formate, hydrogen (H2), and inorganic chloride under strictly anoxic conditions. The current study applied stable carbon and chlorine isotope fractionation measurements to the axenic culture Dehalobacterium formicoaceticum and to the consortium RM comprising DCM degrader Candidatus Dichloromethanomonas elyunquensis. Degradation-associated carbon and chlorine isotope enrichment factors (εC and εCl) of -42.4 ± 0.7‰ and -5.3 ± 0.1‰, respectively, were measured in D. formicoaceticum cultures. A similar εCl of -5.2 ± 0.1‰, but a substantially lower εC of -18.3 ± 0.2‰, were determined for Ca. Dichloromethanomonas elyunquensis. The εC and εCl values resulted in distinctly different dual element C-Cl isotope correlations (ΛC/Cl = Δδ13C/Δδ37Cl) of 7.89 ± 0.12 and 3.40 ± 0.03 for D. formicoaceticum and Ca. Dichloromethanomonas elyunquensis, respectively. The distinct ΛC/Cl values obtained for the two cultures imply mechanistically distinct C-Cl bond cleavage reactions, suggesting that members of Peptococcaceae employ different pathways to metabolize DCM. These findings emphasize the utility of dual carbon-chlorine isotope analysis to pinpoint DCM degradation mechanisms and to provide an additional line of evidence that detoxification is occurring at DCM-contaminated sites.

Carbon Kinetic Isotope Effects and the Mechanisms of Acid-Catalyzed Decarboxylation of 2,4-Dimethoxybenzoic Acid and CO2 Incorporation into 1,3-Dimethoxybenzene.

The rate of decarboxylation of 2,4-dimethoxybenzoic acid (1) is accelerated in parallel to the extent that the carboxyl group acquires a second proton (1H+). However, the conjugate acid would resist C-C bond breaking as that would lead to formation of doubly protonated CO2. An alternative via formation of a higher-energy protonated phenyl tautomer (2H+) prior to C-C bond breaking would produce protonated CO2, an energetically inaccessible species that can be avoided by transfer of the carboxyl proton to water in the same step. Headspace sampling of CO2 that evolves in the acid-catalyzed process and analysis by GC-IRMS gives a smaller than expected value of 1.022 for the carbon kinetic isotope (CKIE), k12/k13. While this value establishes that C-C cleavage is part of the rate-determining process, intrinsic CKIEs for decarboxylation reactions are typically greater than 1.03. Computational analysis of the C-C bond cleavage from 2H+ gives an intrinsic CKIE of 1.051 and suggests two partially rate-determining steps in the decarboxylation of 1: transfer of the second carboxyl proton to the adjacent phenyl carbon and C-C cleavage in which the carboxyl proton is also transferred to water. Applying the principle of microscopic reversibility to fixation of CO2 in acidic solutions reveals the importance of proton transfers to both carbon and oxygen in the overall fixation process.

Vapor Pressure Isotope Effects in Halogenated Organic Compounds and Alcohols Dissolved in Water.

Volatilization causes changes in the isotopic composition of organic compounds as a result of different vapor pressures of molecules containing heavy and light isotopes. Both normal and inverse vapor pressure isotope effects (VPIE) have been observed, depending on molecular interactions in the liquid phase and the investigated element. Previous studies have focused mostly on pure compound volatilization or on compounds dissolved in organic liquids. Environmentally relevant scenarios, such as isotope fractionation during volatilization of organics from open water surfaces, have largely been neglected. In the current study, open-system volatilization experiments (focusing thereby on kinetic/-nonequilibrium effects) were carried out at ambient temperatures for trichloromethane, trichloroethene, trichlorofluoromethane, trichlorotrifluoroethane, methanol, and ethanol dissolved in water and, if not previously reported in the literature for these compounds, for volatilization from pure liquids. Stable carbon isotopic signatures were measured using continuous flow isotope ratio mass spectrometry. The results demonstrate that volatilization of the four halogenated compounds from water does not cause a measurable change in the carbon isotopic composition, whereas for pure-phase evaporation, significant inverse isotope effects are consistently observed (+0.3 ‰< ε < + 1.7 ‰). In contrast, methanol and ethanol showed normal isotope effects for evaporation of pure organic liquids (-3.9 ‰ and -1.9 ‰) and for volatilization of compounds dissolved in water (-4.4 ‰ and -2.9 ‰), respectively. This absence of measurable carbon isotope fractionation considerably facilitates the application of isotopic techniques for extraction of field samples and preconcentration of organohalogens-known to be important pollutants in groundwater and in the atmosphere.

Sediment Monitored Natural Recovery Evidenced by Compound Specific Isotope Analysis and High-Resolution Pore Water Sampling.

Monitoring natural recovery of contaminated sediments requires the use of techniques that can provide definitive evidence of in situ contaminant degradation. In this study, a passive diffusion sampler, called "peeper", was combined with Compound Specific Isotope Analysis to determine benzene and monochlorobenzene (MCB) stable carbon isotope values at a fine vertical resolution (3 cm) across the sediment water interface at a contaminated site. Results indicated significant decrease in concentrations of MCB from the bottom to the top layers of the sediment over 25 cm, and a 3.5 ‰ enrichment in δ13C values of MCB over that distance. Benzene was always at lower concentrations than MCB, with consistently more depleted δ13C values than MCB. The redox conditions were dominated by iron reduction along most of the sediment profile. These results provide multiple lines of evidence for in situ reductive dechlorination of MCB to benzene. Stable isotope analysis of contaminants in pore water is a valuable method to demonstrate in situ natural recovery of contaminated sediments. This novel high-resolution approach is critical to deciphering the combined effects of parent contaminant (e.g., MCB) degradation and both production and simultaneous degradation of daughter products, especially benzene.

Compound-Specific Stable Carbon Isotope Analysis of Chlorofluorocarbons in Groundwater.

Chlorofluorocarbons (CFCs) and hydrochlorofluorocarbons (HCFCs), controlled substances due to their role in stratospheric ozone loss, also occur as dissolved contaminants in groundwaters. Stable carbon isotopic signatures may provide valuable new information on the fate of these compounds as has been seen for other priority hydrocarbon contaminants, but to date no method for extraction and isotopic analysis of dissolved CFCs from groundwaters has been developed. Here we describe a cryogenic purge and trap system coupled to continuous flow compound-specific stable carbon isotope analysis mass spectrometry for concentrations as low as 35 µg/L. The method is validated by comparing isotopic signatures from water extracted CFCs against a new suite of isotopic CFC standards. Fractionation of CFCs in volatilization experiments from pure-phase CFC-11 and CFC-113 resulted in enrichment factors (ε) of +1.7 ± 0.1‰ and +1.1 ± 0.1‰, respectively, indicating that such volatile loss, if significant, would produce a more (13)C depleted signature in the remaining CFCs. Importantly, no significant fractionation was observed during volatile extraction of dissolved CFCs from aqueous solutions. δ(13)C values for groundwaters from a CFC-contaminated site were, on average, more enriched than δ(13)C values for pure compounds. Such enriched δ(13)C values have been seen in other hydrocarbon contaminants such as chlorinated ethenes and ethanes due to in situ degradation, but definitive interpretation of such enriched signatures in field samples requires additional experiments to characterize fractionation of CFCs during biodegradation. The establishment of a robust and sensitive method of extraction and analysis, as described here, provides the foundation for such future directions.

Compound specific isotope analysis of hexachlorocyclohexane isomers: a method for source fingerprinting and field investigation of in situ biodegradation.

RATIONALE: The manufacturing and uses of hexachlorocyclohexane (HCH) have resulted in a serious environmental challenge and legacy. This study highlights the ability of compound specific isotope analysis (CSIA) to distinguish among various HCH sources and to support the evaluation of the potential for in situ biodegradation in contaminated groundwater. METHODS: Tests were conducted to verify the absence of significant isotope fractionation during HCH sample pre-concentration including dichloromethane extraction, solvent exchange into iso-octane, and H2SO4 clean-up, and analysis by gas chromatography/combustion-isotope ratio mass spectrometry (GC/C-IRMS). The method was then applied to four Technical Grade (TG) HCH mixtures procured from different sources and to groundwater samples from a contaminated site. RESULTS: The pre-concentration method enabled determination of carbon isotope ratios (δ(13)C values) of HCH isomers with no significant isotopic fractionation. The TG-HCH mixtures had significantly different δ(13)C values. Moreover, for any given TG-HCH, all isomers had δ(13)C values within 1.1‰ of each other - a distinctly uniform fingerprint. At the HCH-contaminated field site, compared with source wells, downgradient wells showed significant (up to 5.1‰) enrichment in (13)C and the δ(13)C values of the HCH isomers were significantly different from each other. CONCLUSIONS: A method was successfully developed for the CSIA of HCH isomers that showed potential for HCH source differentiation and identification of HCH in situ biodegradation. At the HCH-contaminated site, the observed preferential isotopic enrichment of certain isomers relative to others for a given source allows differentiation between biodegraded and non-biodegraded HCH.

Measurement of a doubly substituted methane isotopologue, ¹³CH3D, by tunable infrared laser direct absorption spectroscopy.

Methane is an important energy resource and significant long-lived greenhouse gas. Carbon and hydrogen isotope ratios have been used to better constrain the sources of methane but interpretations based on these two parameters alone can often be inconclusive. The precise measurement of a doubly substituted methane isotopologue, (13)CH3D, is expected to add a critical new dimension to source signatures by providing the apparent temperature at which methane was formed or thermally equilibrated. We have developed a new method to precisely determine the relative abundance of (13)CH3D by using tunable infrared laser direct absorption spectroscopy (TILDAS). The TILDAS instrument houses two continuous wave quantum cascade lasers; one tuned at 8.6 µm to measure (13)CH3D, (12)CH3D, and (12)CH4, and the other at 7.5 µm to measure (13)CH4. With the use of an astigmatic Herriott cell with an effective path length of 76 m, a precision of 0.2‰ (2σ) was achieved for the measurement of (13)CH3D abundance in ca. 10 mL STP (i.e., 0.42 mmol) pure methane samples. Smaller quantity samples (ca. 0.5 mL STP) can be measured at lower precision. The accuracy of the Δ(13)CH3D measurement is 0.7‰ (2σ), evaluated by thermally equilibrating methane with a range of δD values. The precision of ±0.2‰ corresponds to uncertainties of ±7 °C at 25 °C and ±20 °C at 200 °C for estimates of apparent equilibrium temperatures. The TILDAS instrument offers a simple and precise method to determine (13)CH3D in natural methane samples to distinguish geological and biological sources of methane in the atmosphere, hydrosphere, and lithosphere.

Distinct carbon isotope fractionation during anaerobic degradation of dichlorobenzene isomers.

Chlorinated benzenes are ubiquitous organic contaminants found in groundwater and soils. Compound specific isotope analysis (CSIA) has been increasingly used to assess natural attenuation of chlorinated contaminants, in which anaerobic reductive dechlorination plays an essential role. In this work, carbon isotope fractionation of the three dichlorobenzene (DCB) isomers was investigated during anaerobic reductive dehalogenation in methanogenic laboratory microcosms. Large isotope fractionation of 1,3-DCB and 1,4-DCB was observed while only a small isotope effect occurred for 1,2-DCB. Bulk enrichment factors (εbulk) were determined from a Rayleigh model: -0.8 ± 0.1 ‰ for 1,2-DCB, -5.4 ± 0.4 ‰ for 1,3-DCB, and -6.3 ± 0.2 ‰ for 1,4-DCB. εbulk values were converted to apparent kinetic isotope effects for carbon (AKIE) in order to characterize the carbon isotope effect at the reactive positions for the DCB isomers. AKIE values are 1.005 ± 0.001, 1.034 ± 0.003, and 1.039 ± 0.001 for 1,2-DCB, 1,3-DCB, and 1,4-DCB, respectively. The large difference in AKIE values between 1,2-DCB and 1,3-DCB (or 1,4-DCB) suggests distinct reaction pathways may be involved for different DCB isomers during microbial reductive dechlorination by the methanogenic cultures.

Hydrogeological controls on microbial activity and habitability in the Precambrian continental crust.

Earth's deep continental subsurface is a prime setting to study the limits of life's relationship with environmental conditions and habitability. In Precambrian crystalline rocks worldwide, deep ancient groundwaters in fracture networks are typically oligotrophic, highly saline, and locally inhabited by low-biomass communities in which chemolithotrophic microorganisms may dominate. Periodic opening of new fractures can lead to penetration of surface water and/or migration of fracture fluids, both of which may trigger changes in subsurface microbial composition and activity. These hydrogeological processes and their impacts on subsurface communities may play a significant role in global cycles of key elements in the crust. However, to date, considerable uncertainty remains on how subsurface microbial communities may respond to these changes in hydrogeochemical conditions. To address this uncertainty, the biogeochemistry of Thompson mine (Manitoba, Canada) was investigated. Compositional and isotopic analyses of fracture waters collected here at ~1 km below land surface revealed different extents of mixing between subsurface brine and (paleo)meteoric waters. To investigate the effects this mixing may have had on microbial communities, the Most Probable Number technique was applied to test community response for a total of 13 different metabolisms. The results showed that all fracture waters were dominated by viable heterotrophic microorganisms which can utilize organic materials associated with aerobic/facultative anaerobic processes, sulfate reduction, or fermentation. Where mixing between subsurface brines and (paleo)meteoric waters occurs, the communities demonstrate higher cell densities and increased viable functional potentials, compared to the most saline sample. This study therefore highlights the connection between hydrogeologic heterogeneity and the heterogeneity of subsurface ecosystems in the crystalline rocks, and suggests that hydrogeology can have a considerable impact on the scope and scale of subsurface microbial communities on Earth and potentially beyond.

Carbon kinetic isotope effects reveal variations in reactivity of intermediates in the formation of protonated carbonic acid.

Kinetic evidence suggests that acid-catalyzed decarboxylation reactions of aromatic carboxylic acids can occur by a hydrolytic process that generates protonated carbonic acid (PCA) as the precursor of CO2. Measurements of reaction rates and carbon kinetic isotope effects (CKIE) for decarboxylation of isomeric sets of heterocyclic carboxylic acids in acidic solutions reveal that C-C cleavage to form PCA is rate-determining with significant variation in the magnitude of the observed CKIE (1.018-1.043). Larger values are associated with the more reactive member in each isomeric pair. This variation is consistent with stepwise mechanisms in which C-C cleavage is competitive with C-O cleavage, leading to reversion to the protonated reactant to varying degrees with an invariant intrinsic CKIE for C-C cleavage. Thus, the relative barriers to reversion and formation of PCA control the magnitude of the observed CKIE in a predictable manner that correlates with reactivity. Application of the proposed overall mechanism reveals that carboxylation reactions in acidic solutions will proceed by way of initial formation of PCA.

Pressure-monitored headspace analysis combined with compound-specific isotope analysis to measure isotope fractionation in gas-producing reactions.

RATIONALE: Processes that lead to pressure changes in closed experimental systems can dramatically increase the total uncertainty in enrichment factors (ε) based on headspace analysis and compound-specific isotope analysis (CSIA). We report: (1) A new technique to determine ε values for non-isobaric processes, and (2) a general approach to evaluate the experimental error in calculated ε values. METHODS: ε values were determined by monitoring the change in headspace pressure from the production of CO2 in a decarboxylation reaction using a pressure gauge and measuring the δ(13) C values using CSIA. The statistical error was assessed over shorter reaction progress intervals to evaluate the impact of experimental error on the total uncertainty associated with calculated ε values. RESULTS: As an alternative to conventional compositional analysis, calculation of CO2 produced during the reaction monitored with a pressure gauge resulted in rate constants and ε values with improved correlation coefficients and confidence intervals for a non-isobaric process in a closed system. Further, statistical evaluation of the ε values as a function of reaction progress showed that uncertainty in data points for reaction progress (f) at late stages of the reaction can have a significant impact on the reported ε value. CONCLUSIONS: Pressure-monitored headspace analysis reduces the uncertainty associated with monitoring the reaction progress (f) based on estimating substrate removal and headspace dilution during sampling. Statistical calculations over shorter intervals should be used to evaluate the total error for reported ε values.

Anaerobic conversion of chlorobenzene and benzene to CH4 and CO2 in bioaugmented microcosms.

Chlorobenzene is a widespread groundwater contaminant found at many industrial sites. Reductive dechlorination of chlorobenzene requires input of electron donor and results in problematic accumulation of benzene, which is more toxic than chlorobenzene. We hypothesized that coupling a culture capable of reductive dechlorination of chlorobenzene to benzene with a second benzene-degrading methanogenic culture would completely detoxify chlorobenzene. To this end, active chlorobenzene-dechlorinating microcosms that were producing benzene were inoculated with a previously described enriched methanogenic benzene-degrading consortium. The combination resulted in the transformation of chlorobenzene via benzene to the nontoxic degradation products, CO2 and CH4. Sustainable degradation of chlorobenzene and benzene was observed in the microcosms and was further confirmed by shifts in the carbon isotopic ratios of chlorobenzene and benzene during degradation. Moreover, we could show that benzene derived electrons fueled chlorobenzene dechlorination removing the need to provide exogenous electron donor. The results have promising implications for sustainable bioremediation of sites contaminated with chlorinated benzenes and benzene.