Compound-specific isotope analysis (CSIA) evaluation of degradation of chlorinated benzenes (CBs) and benzene in a contaminated aquifer.

Compound-specific isotope analysis (CSIA) has become a valuable tool in understanding the fate of organic contaminants at field sites. However, its application to chlorinated benzenes (CBs), a group of toxic and persistent groundwater contaminants, has received less attention. This study employed CSIA to investigate the occurrence of natural degradation of various CBs and benzene in a contaminated aquifer. Despite the complexity of the study area (e.g., installation of a sheet pile barrier and the presence of a complex set of contaminants), the substantial enrichments in δ13C values (i.e., >2‰) for all CBs and benzene across the sampling wells indicate in situ degradation of these compounds. In particular, the 13C enrichments for 1,2,4-trichlorobenzene (1,2,4-TCB) and 1,2-dichlorobenzene (1,2-DCB) display good correlations with decreasing groundwater concentrations, consistent with the effects of in situ biodegradation. Using the Rayleigh model, the extent of degradation (EoD) is estimated to be 47-99% for 1,2-DCB, and 21-73% for 1,2,4-TCB. The enrichments observed for the other CBs (1,4-DCB and chlorobenzene (MCB)) and benzene at the site are also suggestive of in situ biodegradation. Due to simultaneous degradation and production of 1,4-DCB (a major 1,2,4-TCB degradation product), MCB (from DCB degradation), and benzene (from MCB degradation), the estimation of EoD for these intermediate compounds is more complex but a modelling simulation supports in situ biodegradation of these daughter products. In particular, the fact that the δ13C values of MCB and benzene (i.e., daughter products of 1,2,4-TCB) are more enriched than the original δ13C value of their parent 1,2,4-TCB provides definitive evidence for the occurrence of in situ biodegradation of the MCB and benzene.

Exploring Space via Astromycology: A Report on the CIFAR Programs Earth 4D and Fungal Kingdom Inaugural Joint Meeting.

"Fungi on Mars!": a popular news heading that piques public interest and makes scientists' blood boil. While such a statement is laden with misinformation and light on evidence, the search for past and present extraterrestrial life is an ongoing scientific effort. Moreover, it is one that is increasingly gaining momentum with the recent collection of martian rock cores from Jezero Crater by NASA's Perseverance rover. Despite the increasingly sophisticated approaches guiding the search for microbial life on other planets, fungi remain relatively underexplored compared to their bacterial counterparts, highlighting a gap between the astrobiological and fungal research communities. Through a meeting in April 2021, the CIFAR Earth 4D and Fungal Kingdom research programs worked to bridge this divide by uniting experts in each field. CIFAR is a Canadian-based global research organization that convenes researchers across disciplines to address important questions facing science and humanity. The CIFAR Earth 4D: Subsurface Science & Exploration and Fungal Kingdom: Threats & Opportunities research programs were launched by CIFAR in July 2019, each made up of approximately two dozen international researchers who are experts in their fields. The Earth 4D program, led by co-directors John Mustard (Brown University, USA) and Barbara Sherwood Lollar (University of Toronto, Canada), aims to understand the complex chemical, physical, and biological interactions that occur within and between Earth's surface and subsurface to explore questions on the evolution of planets and life. The Fungal Kingdom program, led by co-directors Leah Cowen (University of Toronto, Canada) and Joseph Heitman (Duke University, USA), seeks to tackle the most pressing threats fungi pose to human health, agriculture, and biodiversity and to harness their extraordinary potential. The programs met to explore areas for synergy within four major themes: (1) the origins of life; (2) the evolution and diversification of life; (3) life in diverse and extreme environments; and (4) extinction: lessons learned and threats. This report covers the research discussed during the meeting across these four themes.

Novel Microbial Groups Drive Productivity in an Archean Iron Formation.

Deep subsurface environments are decoupled from Earth's surface processes yet diverse, active, and abundant microbial communities thrive in these isolated environments. Microbes inhabiting the deep biosphere face unique challenges such as electron donor/acceptor limitations, pore space/fracture network limitations, and isolation from other microbes within the formation. Of the few systems that have been characterized, it is apparent that nutrient limitations likely facilitate diverse microbe-microbe interactions (i.e., syntrophic, symbiotic, or parasitic) and that these interactions drive biogeochemical cycling of major elements. Here we describe microbial communities living in low temperature, chemically reduced brines at the Soudan Underground Mine State Park, United States. The Soudan Iron mine intersects a massive hematite formation at the southern extent of the Canadian Shield. Fractured rock aquifer brines continuously flow from exploratory boreholes drilled circa 1960 and are enriched in deuterium compared to the global meteoric values, indicating brines have had little contact with surface derived waters, and continually degas low molecular weight hydrocarbons C1-C4. Microbial enrichments suggest that once brines exit the boreholes, oxidation of the hydrocarbons occur. Amplicon sequencing show these borehole communities are low in diversity and dominated by Firmicute and Proteobacteria phyla. From the metagenome assemblies, we recovered approximately thirty genomes with estimated completion over 50%. Analysis of genome taxonomy generally followed the amplicon data, and highlights that several of the genomes represent novel families and genera. Metabolic reconstruction shows two carbon-fixation pathways were dominant, the Wood-Ljungdahl (acetogenesis) and Calvin-Benson-Bassham (via RuBisCo), indicating that inorganic carbon likely enters into the microbial foodweb with differing carbon fractionation potentials. Interestingly, methanogenesis is likely driven by Methanolobus and suggests cycling of methylated compounds and not H2/CO2 or acetate. Furthermore, the abundance of sulfate in brines suggests cryptic sulfur cycling may occur, as we detect possible sulfate reducing and thiosulfate oxidizing microorganisms. Finally, a majority of the microorganisms identified contain genes that would allow them to participate in several element cycles, highlighting that in these deep isolated systems metabolic flexibility may be an important life history trait.

Transformation of Chlorofluorocarbons Investigated via Stable Carbon Compound-Specific Isotope Analysis.

Compound-specific isotope analysis (CSIA) is a valuable tool in contaminant remediation studies. Chlorofluorocarbons (CFCs) are ozone-depleting substances previously thought to be persistent in groundwater under most geochemical conditions but more recently have been found to (bio)transform in some laboratory experiments. To date, limited applications of CSIA to CFCs have been undertaken. Here, biotransformation-associated carbon isotope enrichment factors, εC,bulk for CFC-113 (εC,bulk = -8.5 ± 0.4‰) and CFC-11 (εC,bulk = -14.5 ± 1.9‰), were determined. δ13C signatures of pure-phase CFCs and hydrochlorofluorocarbons were measured to establish source signatures. These findings were applied to investigate potential in situ CFC transformation in groundwater at a field site, where carbon isotope fractionation of CFC-11 suggests naturally occurring biotransformation by indigenous microorganisms. The maximum extent of CFC-11 transformation is estimated to be up to 86% by an approximate calculation using the Rayleigh concept. CFC-113 δ13C values in contrast were not resolvably different from pure-phase sources measured to date, demonstrating that CSIA can aid in identifying which compounds may, or may not, be undergoing reactive processes at field sites. Science and public attention remains focused on CFCs, as unexplained source inputs to the atmosphere have been recently reported, and the potential for CFC biotransformation in surface and groundwaters remains unclear. This study proposes δ13C CSIA as a novel application to study the fate of CFCs in groundwater.

Thermoanaerosceptrum fracticalcis gen. nov. sp. nov., a Novel Fumarate-Fermenting Microorganism From a Deep Fractured Carbonate Aquifer of the US Great Basin.

Deep fractured rock ecosystems across most of North America have not been studied extensively. However, the US Great Basin, in particular the Nevada National Security Site (NNSS, formerly the Nevada Test Site), has hosted a number of influential subsurface investigations over the years. This investigation focuses on resident microbiota recovered from a hydrogeologically confined aquifer in fractured Paleozoic carbonate rocks at 863 - 923 meters below land surface. Analysis of the microorganisms living in this oligotrophic environment provides a perspective into microbial metabolic strategies required to endure prolonged hydrogeological isolation deep underground. Here we present a microbiological and physicochemical characterization of a deep continental carbonate ecosystem and describe a bacterial genus isolated from the ecosystem. Strain DRI-13T is a strictly anaerobic, moderately thermophilic, fumarate-respiring member of the phylum Firmicutes. This bacterium grows optimally at 55°C and pH 8.0, can tolerate a concentration of 100 mM NaCl, and appears to obligately metabolize fumarate to acetate and succinate. Culture-independent 16S rRNA gene sequencing indicates a global subsurface distribution, while the closest cultured relatives of DRI-13T are Pelotomaculum thermopropionicum (90.0% similarity) and Desulfotomaculum gibsoniae (88.0% similarity). The predominant fatty acid profile is iso-C15 : 0, C15 : 0, C16 : 0 and C14 : 0. The percentage of the straight-chain fatty acid C15 : 0 is a defining characteristic not present in the other closely related species. The genome is estimated to be 3,649,665 bp, composed of 87.3% coding regions with an overall average of 45.1% G + C content. Strain DRI-13T represents a novel genus of subsurface bacterium isolated from a previously uncharacterized rock-hosted geothermal habitat. The characterization of the bacterium combined with the sequenced genome provides insights into metabolism strategies of the deep subsurface biosphere. Based on our characterization analysis we propose the name Thermoanaerosceptrum fracticalcis (DRI-13T = DSM 100382T = ATCC TSD-12T).

Sources of Uncertainty in Biotransformation Mechanistic Interpretations and Remediation Studies using CSIA.

Compound-specific isotope analysis (CSIA) is a powerful tool to understand the fate of organic contaminants. Using CSIA, the isotope ratios of multiple elements (δ13C, δ2H, δ37Cl, δ15N) can be measured for a compound. A dual-isotope plot of the changes in isotope ratios between two elements produces a slope, lambda (Λ), which can be instrumental for practitioners to identify transformation mechanisms. However, practices to calculate and report Λ and related uncertainty are not universal, leading to the potential for misinterpretations. Here, the most common methods are re-evaluated to provide the basis for a more accurate best-practice representation of Λ and its uncertainty. The popular regression technique, ordinary linear regression, can introduce mathematical bias. The York method, which incorporates error in both variables, better adapts to the wide set of data conditions observed for dual-isotope data. Importantly, the existing technique of distinguishing between Λs using the 95% confidence interval alone produces inconsistent results, whereas statistical hypothesis testing provides a more robust method to differentiate Λs. The propensity for Λ to overlap for a variety of conditions and mechanisms highlights the requirement for statistical justification when comparing data sets. Findings from this study emphasize the importance of this evaluation of best practice and provide recommendations for standardizing, calculating, and interpreting dual-isotope data.

Determining Carbon Kinetic Isotope Effects Using Headspace Analysis of Evolved CO2.

Isotope ratio mass spectrometry (IRMS) provides accurate measurements of relative abundance of isotopes of heavy atoms for reactions that are subject to kinetic isotope effects (KIEs). The recent development of compound-specific isotope analysis (CSIA) allows the use of multiple time points that provide data for a rate plot as well as isotope ratios. Utilizing CSIA in enzymology presents opportunities for obtaining heavy atom KIEs in diverse areas.

Diffusion related isotopic fractionation effects with one-dimensional advective-dispersive transport.

Aqueous phase diffusion-related isotope fractionation (DRIF) for carbon isotopes was investigated for common groundwater contaminants in systems in which transport could be considered to be one-dimensional. This paper focuses not only on theoretically observable DRIF effects in these systems but introduces the important concept of constraining "observable" DRIF based on constraints imposed by the scale of measurements in the field, and on standard limits of detection and analytical uncertainty. Specifically, constraints for the detection of DRIF were determined in terms of the diffusive fractionation factor, the initial concentration of contaminants (C0), the method detection limit (MDL) for isotopic analysis, the transport time, and the ratio of the longitudinal mechanical dispersion coefficient to effective molecular diffusion coefficient (Dmech/Deff). The results allow a determination of field conditions under which DRIF may be an important factor in the use of stable carbon isotope measurements for evaluation of contaminant transport and transformation for one-dimensional advective-dispersive transport. This study demonstrates that for diffusion-dominated transport of BTEX, MTBE, and chlorinated ethenes, DRIF effects are only detectable for the smaller molar mass compounds such as vinyl chloride for C0/MDL ratios of 50 or higher. Much larger C0/MDL ratios, corresponding to higher source concentrations or lower detection limits, are necessary for DRIF to be detectable for the higher molar mass compounds. The distance over which DRIF is observable for VC is small (less than 1m) for a relatively young diffusive plume (<100years), and DRIF will not easily be detected by using the conventional sampling approach with "typical" well spacing (at least several meters). With contaminant transport by advection, mechanical dispersion, and molecular diffusion this study suggests that in field sites where Dmech/Deff is larger than 10, DRIF effects will likely not be observable for common groundwater contaminants. Importantly, under most field conditions, Dmech/Deff≥10 is usually satisfied in the longitudinal direction, suggesting that DRIF is not likely to be observable in most groundwater systems in which contaminant transport is predominantly one-dimensional. Given the importance in the MDL it is recommended that MDL should always be explicitly reported in both modeling and field studies.

Methane cycling. Nonequilibrium clumped isotope signals in microbial methane.

Methane is a key component in the global carbon cycle, with a wide range of anthropogenic and natural sources. Although isotopic compositions of methane have traditionally aided source identification, the abundance of its multiply substituted "clumped" isotopologues (for example, (13)CH3D) has recently emerged as a proxy for determining methane-formation temperatures. However, the effect of biological processes on methane's clumped isotopologue signature is poorly constrained. We show that methanogenesis proceeding at relatively high rates in cattle, surface environments, and laboratory cultures exerts kinetic control on (13)CH3D abundances and results in anomalously elevated formation-temperature estimates. We demonstrate quantitatively that H2 availability accounts for this effect. Clumped methane thermometry can therefore provide constraints on the generation of methane in diverse settings, including continental serpentinization sites and ancient, deep groundwaters.

The contribution of the Precambrian continental lithosphere to global H2 production.

Microbial ecosystems can be sustained by hydrogen gas (H2)-producing water-rock interactions in the Earth's subsurface and at deep ocean vents. Current estimates of global H2 production from the marine lithosphere by water-rock reactions (hydration) are in the range of 10(11) moles per year. Recent explorations of saline fracture waters in the Precambrian continental subsurface have identified environments as rich in H2 as hydrothermal vents and seafloor-spreading centres and have suggested a link between dissolved H2 and the radiolytic dissociation of water. However, extrapolation of a regional H2 flux based on the deep gold mines of the Witwatersrand basin in South Africa yields a contribution of the Precambrian lithosphere to global H2 production that was thought to be negligible (0.009 × 10(11) moles per year). Here we present a global compilation of published and new H2 concentration data obtained from Precambrian rocks and find that the H2 production potential of the Precambrian continental lithosphere has been underestimated. We suggest that this can be explained by a lack of consideration of additional H2-producing reactions, such as serpentinization, and the absence of appropriate scaling of H2 measurements from these environments to account for the fact that Precambrian crust represents over 70 per cent of global continental crust surface area. If H2 production via both radiolysis and hydration reactions is taken into account, our estimate of H2 production rates from the Precambrian continental lithosphere of 0.36-2.27 × 10(11) moles per year is comparable to estimates from marine systems.

Diffusion sampler for compound specific carbon isotope analysis of dissolved hydrocarbon contaminants.

Compound Specific Isotope Analysis (CSIA) is widely utilized to study the fate of organic contaminants in groundwater. To date, however, no method is available to obtain CSIA samples at a fine (cm) spatial scale across the sediment-surface water interface (SWI), a key boundary for discharge of contaminated groundwater to surface water. Dissolved contaminants in such discharged zones undergo rapid temporal and spatial changes due to heterogeneity in redox conditions and microbial populations. The compatibility of a passive sediment pore water sampler ("peeper") to collect 40 mL samples for CSIA of benzene, toluene, monochlorobenzene, and 1,2-dichlorobenzene at field-relevant concentrations (0.1-5 mg L(-1)) was evaluated in laboratory experiments. Results demonstrate that physical diffusion across the polysulfone membrane does not alter the carbon isotope values (±0.5‰). Measured δ(13)C values also remain invariant despite significant adsorption of the compounds on the peeper material, an effect which increased with higher numbers of chlorine atoms and sorption coefficient (Koc) values. In addition, isotope equilibrium between the peeper chamber and the sediment pore water occurred in less than a day, indicating the peeper method can be used to provide samples for CSIA analysis at fine spatial and temporal sampling resolutions in contaminated sediments.

Integration of stable carbon isotope, microbial community, dissolved hydrogen gas, and ²HH2O tracer data to assess bioaugmentation for chlorinated ethene degradation in fractured rocks.

An in situ bioaugmentation (BA) experiment was conducted to understand processes controlling microbial dechlorination of trichloroethene (TCE) in groundwater at the Naval Air Warfare Center (NAWC), West Trenton, NJ. In the BA experiment, an electron donor (emulsified vegetable oil and sodium lactate) and a chloro-respiring microbial consortium were injected into a well in fractured mudstone of Triassic age. Water enriched in ²H was also injected as a tracer of the BA solution, to monitor advective transport processes. The changes in concentration and the δ¹³C of TCE, cis-dichloroethene (cis-DCE), and vinyl chloride (VC); the δ²H of water; changes in the abundance of the microbial communities; and the concentration of dissolved H2 gas compared to pre- test conditions, provided multiple lines of evidence that enhanced biodegradation occurred in the injection well and in two downgradient wells. For those wells where the biodegradation was stimulated intensively, the sum of the molar chlorinated ethene (CE) concentrations in post-BA water was higher than that of the sum of the pre-BA background molar CE concentrations. The concentration ratios of TCE/(cis-DCE+VC) indicated that the increase in molar CE concentration may result from additional TCE mobilized from the rock matrix in response to the oil injection or due to desorption/diffusion. The stable carbon isotope mass-balance calculations show that the weighted average ¹³C isotope of the CEs was enriched for around a year compared to the background value in a two year monitoring period, an effective indication that dechlorination of VC was occurring. Insights gained from this study can be applied to efforts to use BA in other fractured rock systems. The study demonstrates that a BA approach can substantially enhance in situ bioremediation not only in fractures connected to the injection well, but also in the rock matrix around the well due to processes such as diffusion and desorption. Because the effect of the BA was intensive only in wells where an amendment was distributed during injection, it is necessary to adequately distribute the amendments throughout the fractured rock to achieve substantial bioremediation. The slowdown in BA effect after a year is due to some extend to the decrease abundant of appropriate microbes, but more likely the decreased concentration of electron donor.

Large carbon isotope fractionation during biodegradation of chloroform by Dehalobacter cultures.

Compound specific isotope analysis (CSIA) has been applied to monitor bioremediation of groundwater contaminants and provide insight into mechanisms of transformation of chlorinated ethanes. To date there is little information on its applicability for chlorinated methanes. Moreover, published enrichment factors (ε) observed during the biotic and abiotic degradation of chlorinated alkanes, such as carbon tetrachloride (CT); 1,1,1-trichloroethane (1,1,1-TCA); and 1,1-dichloroethane (1,1-DCA), range from -26.5‰ to -1.8‰ and illustrate a system where similar C-Cl bonds are cleaved but significantly different isotope enrichment factors are observed. In the current study, biotic degradation of chloroform (CF) to dichloromethane (DCM) was carried out by the Dehalobacter containing culture DHB-CF/MEL also shown to degrade 1,1,1-TCA and 1,1-DCA. The carbon isotope enrichment factor (ε) measured during biodegradation of CF was -27.5‰ ± 0.9‰, consistent with the theoretical maximum kinetic isotope effect for C-Cl bond cleavage. Unlike 1,1,1-TCA and 1,1-DCA, reductive dechlorination of CF by the Dehalobacter-containing culture shows no evidence of suppression of the intrinsic maximum kinetic isotope effect. Such a large fractionation effect, comparable to those published for cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC) suggests CSIA has significant potential to identify and monitor biodegradation of CF, as well as important implications for recent efforts to fingerprint natural versus anthropogenic sources of CF in soils and groundwater.

Insights into enzyme kinetics of chloroethane biodegradation using compound specific stable isotopes.

While compound specific isotope analysis (CSIA) has been used extensively to investigate remediation of chlorinated ethenes, to date considerably less information is available on its applicability to chlorinated ethanes. In this study, biodegradation of 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA) was carried out by a Dehalobacter-containing mixed culture. Carbon isotope fractionation factors (ε) measured during whole cell degradation demonstrated that values for 1,1,1-TCA and 1,1-DCA (-1.8‰ and -10.5‰, respectively) were significantly smaller than values reported for abiotic reductive dechlorination of these same compounds. Similar results were found in experiments degrading these two priority pollutants by cell free extracts (CFE) where values of -0.8‰ and -7.9‰, respectively, were observed. For 1,1,1-TCA in particular, the large kinetic isotope effect expected for cleavage of a C-Cl bond was almost completely masked during biodegradation by both whole cells and CFE. Comparison to previous studies demonstrates that these patterns of isotopic fractionation are not attributable to transport effects across the cell membrane, as had been seen for other compounds such as PCE. In contrast these results reflect significant differences in the kinetics of the enzymes catalyzing chlorinated ethane degradation.

Microbial characterization of a subzero, hypersaline methane seep in the Canadian High Arctic.

We report the first microbiological characterization of a terrestrial methane seep in a cryo-environment in the form of an Arctic hypersaline (∼24% salinity), subzero (-5 °C), perennial spring, arising through thick permafrost in an area with an average annual air temperature of -15 °C. Bacterial and archaeal 16S rRNA gene clone libraries indicated a relatively low diversity of phylotypes within the spring sediment (Shannon index values of 1.65 and 1.39, respectively). Bacterial phylotypes were related to microorganisms such as Loktanella, Gillisia, Halomonas and Marinobacter spp. previously recovered from cold, saline habitats. A proportion of the bacterial phylotypes were cultured, including Marinobacter and Halomonas, with all isolates capable of growth at the in situ temperature (-5 °C). Archaeal phylotypes were related to signatures from hypersaline deep-sea methane-seep sediments and were dominated by the anaerobic methane group 1a (ANME-1a) clade of anaerobic methane oxidizing archaea. CARD-FISH analyses indicated that cells within the spring sediment consisted of ∼84.0% bacterial and 3.8% archaeal cells with ANME-1 cells accounting for most of the archaeal cells. The major gas discharging from the spring was methane (∼50%) with the low CH(4)/C(2+) ratio and hydrogen and carbon isotope signatures consistent with a thermogenic origin of the methane. Overall, this hypersaline, subzero environment supports a viable microbial community capable of activity at in situ temperature and where methane may behave as an energy and carbon source for sustaining anaerobic oxidation of methane-based microbial metabolism. This site also provides a model of how a methane seep can form in a cryo-environment as well as a mechanism for the hypothesized Martian methane plumes.

Hydrolytic decarboxylation of carboxylic acids and the formation of protonated carbonic acid.

Acid-catalyzed decarboxylation reactions of carboxylic acids should avoid formation of protonated carbon dioxide, a very high energy species. A potential alternative route parallels ester hydrolysis, with addition of water to the carboxyl group followed by protonation of the unsaturated leaving group and formation of protonated carbonic acid, a species that had been predicted to be a viable reaction intermediate. The hydrolytic mechanism for the decarboxylation of pyrrole-2-carboxylic acid is consistent with observed (12)C/(13)C kinetic isotope effects (1.010 +/- 0.001 at H(0) = -0.01 and 1.043 +/- 0.001 at H(0) = -2.6), solvent kinetic isotope effects (k(H(2))(O)/k(D(2))(O) = 2 at H(0) = 0.9; k(H(2))(O)/k(D(2))(O) = 1 at H(0) = -2.9), and activation parameters [DeltaH() = 23.5 kcal.mol(-1) and DeltaS() = 5.5 cal.deg(-1).mol(-1) at H(0) = -2.9]. Thus, the specific route for a decarboxylation process is a consequence of the nature of the potential carbanion (or its conjugate acid), the acidity of the medium and avoidance of formation of protonated carbon dioxide.

Isotopic fractionation of methyl tert-butyl ether suggests different initial reaction mechanisms during aerobic biodegradation.

Carbon isotopic enrichment factors (epsilonC) measured during cometabolic biodegradation of methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) by Pseudonocardia tetrahydrofuranoxydans strain K1 were -2.3 +/- 0.2 per thousand, -1.7 +/- 0.2 per thousand, and -1.7 +/- 0.3 per thousand, respectively. The measured carbon apparent kinetic isotope effect was 1.01 for all compounds, consistent with the expected kinetic isotope effects for both oxidation of the methoxy (or ethoxy) group and enzymatic SN1 biodegradation mechanisms. Significantly, delta13C measurements of the tert-butyl alcohol and tert-amyl alcohol products indicated that the tert-butyl and tert-amyl groups do not participate in the reaction and confirmed that ether biodegradation by strain K1 involves oxidation of the methoxy (or ethoxy) group. Measured hydrogen isotopic enrichment factors (epsilonH) were -100 +/- 10 per thousand, -73 +/- 7 per thousand, and -72 +/- 20 per thousand for MTBE, ETBE, and TAME respectively. Previous results reported for aerobic biodegradation of MTBE by Methylibium petroleiphilum PM1 and Methylibium R8 showed smaller epsilonH values (-35 per thousand and -42 per thousand, respectively). Plots of Delta2H/Delta13C show different slopes for strain K1 compared with strains PM1 and R8, suggesting that different mechanisms are utilized by K1 and PM1/R8 during aerobic MTBE biodegradation.

Proteomic and transcriptomic analyses reveal genes upregulated by cis-dichloroethene in Polaromonas sp. strain JS666.

Polaromonas sp. strain JS666 is the only bacterial isolate capable of using cis-dichloroethene (cDCE) as a sole carbon and energy source. Studies of cDCE degradation in this novel organism are of interest because of potential bioremediation and biocatalysis applications. The primary cellular responses of JS666 to growth on cDCE were explored using proteomics and transcriptomics to identify the genes upregulated by cDCE. Two-dimensional gel electrophoresis revealed upregulation of genes annotated as encoding glutathione S-transferase, cyclohexanone monooxygenase, and haloacid dehalogenase. DNA microarray experiments confirmed the proteomics findings that the genes indicated above were among the most highly upregulated by cDCE. The upregulation of genes with antioxidant functions and the inhibition of cDCE degradation by elevated oxygen levels suggest that cDCE induces an oxidative stress response. Furthermore, the upregulation of a predicted ABC transporter and two sodium/solute symporters suggests that transport is important in cDCE degradation. The omics data were integrated with data from compound-specific isotope analysis (CSIA) and biochemical experiments to develop a hypothesis for cDCE degradation pathways in JS666. The CSIA results indicate that the measured isotope enrichment factors for aerobic cDCE degradation ranged from -17.4 to -22.4 per thousand. Evidence suggests that cDCE degradation via monooxygenase-catalyzed epoxidation (C C cleavage) may be only a minor degradation pathway under the conditions of these experiments and that the major degradation pathway involves carbon-chloride cleavage as the initial step, a novel mechanism. The results provide a significant step toward elucidation of cDCE degradation pathways and enhanced understanding of cDCE degradation in JS666.

Solubility trapping in formation water as dominant CO(2) sink in natural gas fields.

Injecting CO(2) into deep geological strata is proposed as a safe and economically favourable means of storing CO(2) captured from industrial point sources. It is difficult, however, to assess the long-term consequences of CO(2) flooding in the subsurface from decadal observations of existing disposal sites. Both the site design and long-term safety modelling critically depend on how and where CO(2) will be stored in the site over its lifetime. Within a geological storage site, the injected CO(2) can dissolve in solution or precipitate as carbonate minerals. Here we identify and quantify the principal mechanism of CO(2) fluid phase removal in nine natural gas fields in North America, China and Europe, using noble gas and carbon isotope tracers. The natural gas fields investigated in our study are dominated by a CO(2) phase and provide a natural analogue for assessing the geological storage of anthropogenic CO(2) over millennial timescales. We find that in seven gas fields with siliciclastic or carbonate-dominated reservoir lithologies, dissolution in formation water at a pH of 5-5.8 is the sole major sink for CO(2). In two fields with siliciclastic reservoir lithologies, some CO(2) loss through precipitation as carbonate minerals cannot be ruled out, but can account for a maximum of 18 per cent of the loss of emplaced CO(2). In view of our findings that geological mineral fixation is a minor CO(2) trapping mechanism in natural gas fields, we suggest that long-term anthropogenic CO(2) storage models in similar geological systems should focus on the potential mobility of CO(2) dissolved in water.

Isotopic evidence suggests different initial reaction mechanisms for anaerobic benzene biodegradation.

The initial metabolic reactions for anaerobic benzene biodegradation remain uncharacterized. Isotopic data for carbon and hydrogen fractionation from nitrate-reducing, sulfate-reducing, and methanogenic benzene-degrading enrichment cultures and phylogenic information were used to investigate the initial reaction step in anaerobic benzene biodegradation. Dual parameter plots of carbon and hydrogen isotopic data (deltadelta2H/ deltadelta13C) from each culture were linear, suggesting a consistent reaction mechanism as degradation proceeded. Methanogenic and sulfate-reducing cultures showed consistently higher slopes (m = 29 +/- 2) compared to nitrate-reducing cultures (m = 13 +/- 2) providing evidence for different initial reaction mechanisms. Phylogenetic analyses confirmed that culture conditions were strictly anaerobic, precluding any involvement of molecular oxygen in the observed differences. Using published kinetic data, we explored the possibility of attributing such slopes to reaction mechanisms. The higher slopes found under methanogenic and sulfate-reducing conditions suggest against an alkylation mechanism for these cultures. Observed differences between the methanogenic and nitrate-reducing cultures may not represent distinct reactions of different bonds, but rather subtle differences in relative reaction kinetics. Additional mechanistic conclusions could not be made because kinetic isotope effect data for carboxylation and other putative mechanisms are not available.