Anaerobic oxidation of ethane by archaea from a marine hydrocarbon seep.

Ethane is the second most abundant component of natural gas in addition to methane, and-similar to methane-is chemically unreactive. The biological consumption of ethane under anoxic conditions was suggested by geochemical profiles at marine hydrocarbon seeps1-3, and through ethane-dependent sulfate reduction in slurries4-7. Nevertheless, the microorganisms and reactions that catalyse this process have to date remained unknown8. Here we describe ethane-oxidizing archaea that were obtained by specific enrichment over ten years, and analyse these archaea using phylogeny-based fluorescence analyses, proteogenomics and metabolite studies. The co-culture, which oxidized ethane completely while reducing sulfate to sulfide, was dominated by an archaeon that we name 'Candidatus Argoarchaeum ethanivorans'; other members were sulfate-reducing Deltaproteobacteria. The genome of Ca. Argoarchaeum contains all of the genes that are necessary for a functional methyl-coenzyme M reductase, and all subunits were detected in protein extracts. Accordingly, ethyl-coenzyme M (ethyl-CoM) was identified as an intermediate by liquid chromatography-tandem mass spectrometry. This indicated that Ca. Argoarchaeum initiates ethane oxidation by ethyl-CoM formation, analogous to the recently described butane activation by 'Candidatus Syntrophoarchaeum'9. Proteogenomics further suggests that oxidation of intermediary acetyl-CoA to CO2 occurs through the oxidative Wood-Ljungdahl pathway. The identification of an archaeon that uses ethane (C2H6) fills a gap in our knowledge of microorganisms that specifically oxidize members of the homologous alkane series (CnH2n+2) without oxygen. Detection of phylogenetic and functional gene markers related to those of Ca. Argoarchaeum at deep-sea gas seeps10-12 suggests that archaea that are able to oxidize ethane through ethyl-CoM are widespread members of the local communities fostered by venting gaseous alkanes around these seeps.

Direct Phototransformation of Sulfamethoxazole Characterized by Four-Dimensional Element Compound Specific Isotope Analysis.

The antibiotic sulfamethoxazole (SMX) undergoes direct phototransformation by sunlight, constituting a notable dissipation process in the environment. SMX exists in both neutral and anionic forms, depending on the pH conditions. To discern the direct photodegradation of SMX at various pH levels and differentiate it from other transformation processes, we conducted phototransformation of SMX under simulated sunlight at pH 7 and 3, employing both transformation product (TP) and compound-specific stable isotope analyses. At pH 7, the primary TPs were sulfanilic acid and 3A5MI, followed by sulfanilamide and (5-methylisoxazol-3-yl)-sulfamate, whereas at pH 3, a photoisomer was the dominant product, followed by sulfanilic acid and 3A5MI. Isotope fractionation patterns revealed normal 13C, 34S, and inverse 15N isotope fractionation, which exhibited significant differences between pH 7 and 3. This indicates a pH-dependent transformation process in SMX direct phototransformation. The hydrogen isotopic composition of SMX remained stable during direct phototransformation at both pH levels. Moreover, there was no variation observed in 33S between the two pH levels, indicating that the 33S mass-independent process remains unaffected by changes in pH. The analysis of main TPs and single-element isotopic fractionation suggests varying combinations of bond cleavages at different pH values, resulting in distinct patterns of isotopic fractionation. Conversely, dual-element isotope values at different pH levels did not significantly differ, indicating cleavage of several bonds in parallel. Hence, prudent interpretation of dual-element isotope analysis in these systems is warranted. These findings highlight the potential of multielement compound-specific isotope analysis in characterizing pH-dependent direct phototransformation of SMX, thereby facilitating the evaluation of its natural attenuation through sunlight photolysis in the environment.

Viral Communities Contribute More to the Lysis of Antibiotic-Resistant Bacteria than the Transduction of Antibiotic Resistance Genes in Anaerobic Digestion Revealed by Metagenomics.

Ecological role of the viral community on the fate of antibiotic resistance genes (ARGs) (reduction vs proliferation) remains unclear in anaerobic digestion (AD). Metagenomics revealed a dominance of Siphoviridae and Podoviridae among 13,895 identified viral operational taxonomic units (vOTUs) within AD, and only 21 of the vOTUs carried ARGs, which only accounted for 0.57 ± 0.43% of AD antibiotic resistome. Conversely, ARGs locating on plasmids and integrative and conjugative elements accounted for above 61.0%, indicating a substantial potential for conjugation in driving horizontal gene transfer of ARGs within AD. Virus-host prediction based on CRISPR spacer, tRNA, and homology matches indicated that most viruses (80.2%) could not infect across genera. Among 480 high-quality metagenome assembly genomes, 95 carried ARGs and were considered as putative antibiotic-resistant bacteria (pARB). Furthermore, lytic phages of 66 pARBs were identified and devoid of ARGs, and virus/host abundance ratios with an average value of 71.7 indicated extensive viral activity and lysis. The infectivity of lytic phage was also elucidated through laboratory experiments concerning changes of the phage-to-host ratio, pH, and temperature. Although metagenomic evidence for dissemination of ARGs by phage transduction was found, the higher proportion of lytic phages infecting pARBs suggested that the viral community played a greater role in reducing ARB numbers than spreading ARGs in AD.

Source differentiation of BTEX compounds in groundwater contaminated due to refinery activities.

This study aims to identify sources of groundwater contamination in a refinery area using integrated compound-specific stable isotope analysis (CSIA), oil fingerprinting techniques, hydrogeological data, and distillation analysis. The investigations focused on determination of the origin of benzene, toluene, ethylbenzene, and xylenes (BTEX), and aliphatic hydrocarbons as well. Groundwater and floating oil samples were collected from extraction wells for analysis. Results indicate presence of active leaks in both the northern and southern zones. In the northern zone, toluene was found to primarily originate from oil products like aviation turbine kerosene (ATK or aviation fuel), kerosene, regular gasoline, and diesel fuel. Additionally, stable isotope ratios of carbon and hydrogen for ethylbenzene, o-xylene (ortho xylene) and p-xylene (para xylene) in zone A suggested the pollution originated from gasoline within the northern zone. The origin of super gasoline (with higher octane) identified in southern zone using δ13C and δ2H values of toluene in the floating oil and groundwater samples. Further, biodegradation of toluene likely occurred in southern zone according to δ13C and δ2H. The findings underscore the critical importance of integrating CSIA and fingerprinting techniques to effectively address the challenges of source identification and relying solely on each method independently is insufficient. Accordingly, comparing the GC-MS results of floating oil samples with ATK and jet fuel (JP4) standards can be effectively utilized for source differentiation. However, this method showed no practical application to distinguish different types of diesel or gasoline. The accuracy and reliability of source identification of BTEX compounds may significantly improve when hydrogeological data incorporates with stable isotopes analysis. Additionally, the results of this study will elevate the procedures for fuel-related contaminants source identification of the polluted groundwater that is crucial to develop effective remediation strategies.

Novel stable isotope concepts to track antibiotics in wetland systems.

Antibiotics, their transformation products, and the translocation of antibiotic-resistant genes in the environment pose significant health risks to humans, animals, and ecosystems, aligning with the One Health concept. Constructed wetlands hold substantial yet underutilized potential for treating wastewater from agricultural, domestic sewage, or contaminated effluents from wastewater treatment plants, with the goal of eliminating antibiotics. However, the comprehensive understanding of the distribution, persistence, and dissipation processes of antibiotics within constructed wetlands remains largely unexplored. In this context, we provide an overview of the current application of stable isotope analysis at natural abundance to antibiotics. We explore the opportunities of an advanced multiple stable isotope approach, where isotope concepts could be effectively applied to examine the fate of antibiotics in wetlands. The development of a conceptual framework to study antibiotics in wetlands using multi-element stable isotopes introduces a new paradigm, offering enhanced insights into the identification and quantification of natural attenuation of antibiotics within wetland systems. This perspective has the potential to inspire the general public, governmental bodies, and the broader research community, fostering an emphasis on the utilization of stable isotope analysis for studying antibiotics and other emerging micropollutants in wetland systems.

Microbial diversity and oil biodegradation potential of northern Barents Sea sediments.

The Arctic, an essential ecosystem on Earth, is subject to pronounced anthropogenic pressures, most notable being the climate change and risks of crude oil pollution. As crucial elements of Arctic environments, benthic microbiomes are involved in climate-relevant biogeochemical cycles and hold the potential to remediate upcoming contamination. Yet, the Arctic benthic microbiomes are among the least explored biomes on the planet. Here we combined geochemical analyses, incubation experiments, and microbial community profiling to detail the biogeography and biodegradation potential of Arctic sedimentary microbiomes in the northern Barents Sea. The results revealed a predominance of bacterial and archaea phyla typically found in the deep marine biosphere, such as Chloroflexi, Atribacteria, and Bathyarcheaota. The topmost benthic communities were spatially structured by sedimentary organic carbon, lacking a clear distinction among geographic regions. With increasing sediment depth, the community structure exhibited stratigraphic variability that could be correlated to redox geochemistry of sediments. The benthic microbiomes harbored multiple taxa capable of oxidizing hydrocarbons using aerobic and anaerobic pathways. Incubation of surface sediments with crude oil led to proliferation of several genera from the so-called rare biosphere. These include Alkalimarinus and Halioglobus, previously unrecognized as hydrocarbon-degrading genera, both harboring the full genetic potential for aerobic alkane oxidation. These findings increase our understanding of the taxonomic inventory and functional potential of unstudied benthic microbiomes in the Arctic.

Determination of Stable Hydrogen Isotopic Composition and Isotope Enrichment Factor at Low Hydrogen Concentration.

Determination of stable hydrogen isotopic compositions (δ2H) is currently challenged to achieve a high detection limit for reaching the linear range where δ2H values are independent of concentration. Therefore, it is difficult to assess precise δ2H values for calculating the hydrogen isotope enrichment factor (εH) and for field application where the concentrations of contaminants are relatively low. In this study, a data treatment approach was developed to obtain accurate δ2H values below the linear range. The core concept was to use a logarithmic function to fit the δ2H values below the linear range and then adjust the δ2H values below the linear range into the linear range by using the fitted logarithmic equation. Moreover, the adjusted δ2H values were calibrated by using laboratory reference materials, e.g., n-alkanes. Tris(2-chloroethyl) phosphate (TCEP) and hexachlorocyclohexane (HCH) isomers were selected as examples of complex heteroatom-bearing compounds to develop the data treatment approach. This data treatment approach was then tested using δ2H values from a TCEP transformation experiment with OH radicals. Comparable δ2H values and εH between the low-concentration experiment and the reference experiment were obtained using the developed approach. Therefore, the developed data treatment approach enables a possibility of determining the hydrogen isotopic compositions of organic components in low concentrations. It is especially valuable for determining organic contaminants in environmental samples, which are usually present in low concentrations.

Uptake and Transformation of Hexachlorocyclohexane Isomers (HCHs) in Tree Growth Rings at a Contaminated Field Site.

The potential transformation of hexachlorocyclohexane isomers (HCHs) within tree trunks could have a significant impact on the use of phytoscreening. However, the transformation mechanisms of HCH in trunks particularly in growth rings are not yet well understood. Therefore, a field study on an HCH-contaminated field site was conducted to investigate the fate of HCH, particularly α-HCH in tree trunks using multielement compound-specific isotope analysis (ME-CSIA) and enantiomer fractionation. The results indicate that α-HCH was transformed, as evidenced by higher δ13C and δ37Cl values detected across different growth ring sections and in the bark compared to those in muck and soil. Remarkably, in the middle growth ring section, δ13C values of HCH were only marginally higher or comparable to those in muck, whereas δ37Cl values were higher than those of the muck, indicating a different transformation mechanism. Moreover, the δ37Cl values of ß-HCH also increased in the tree trunks compared to those in soil and muck, implying a transformation of ß-HCH. Additionally, dual-element isotope analysis revealed that there are different transformation mechanisms between the middle growth rings and other sections. Our findings suggest that the transformation of HCHs in trunks could bias quantitative phytoscreening approaches; however, ME-CISA offers an option to estimate the degradation extent.

Humic Substance Photosensitized Degradation of Phthalate Esters Characterized by 2H and 13C Isotope Fractionation.

The photosensitized transformation of organic chemicals is an important degradation mechanism in natural surface waters, aerosols, and water films on surfaces. Dissolved organic matter including humic-like substances (HS), acting as photosensitizers that participate in electron transfer reactions, can generate a variety of reactive species, such as OH radicals and excited triplet-state HS (3HS*), which promote the degradation of organic compounds. We use phthalate esters, which are important contaminants found in wastewaters, landfills, soils, rivers, lakes, groundwaters, and mine tailings. We use phthalate esters as probes to study the reactivity of HS irradiated with artificial sunlight. Phthalate esters with different side-chain lengths were used as probes for elucidation of reaction mechanisms using 2H and 13C isotope fractionation. Reference experiments with the artificial photosensitizers 4,5,6,7-tetrachloro-2',4',5',7'-tetraiodofluorescein (Rose Bengal), 3-methoxy-acetophenone (3-MAP), and 4-methoxybenzaldehyde (4-MBA) yielded characteristic fractionation factors (-4 ± 1, -4 ± 2, and -4 ± 1‰ for 2H; 0.7 ± 0.2, 1.0 ± 0.4, and 0.8 ± 0.2‰ for 13C), allowing interpretation of reaction mechanisms of humic substances with phthalate esters. The correlation of 2H and 13C fractions can be used diagnostically to determine photosensitized reactions in the environment and to differentiate among biodegradation, hydrolysis, and photosensitized HS reaction.

Iron corrosion by methanogenic archaea characterized by stable isotope effects and crust mineralogy.

Carbon and hydrogen stable isotope effects associated with methane formation by the corrosive archaeon Methanobacterium strain IM1 were determined during growth with hydrogen and iron. Isotope analyses were complemented by structural, elemental and molecular composition analyses of corrosion crusts. During growth with H2 , strain IM1 formed methane with average δ13 C of -43.5‰ and δ2 H of -370‰. Corrosive growth led to methane more depleted in 13 C, with average δ13 C ranging from -56‰ to -64‰ during the early and the late growth phase respectively. The corresponding δ2 H were less impacted by the growth phase, with average values ranging from -316 to -329‰. The stable isotope fractionation factors, α 13 C CO 2 / CH 4 , were 1.026 and 1.042 for hydrogenotrophic and corrosive growth respectively. Corrosion crusts formed by strain IM1 have a domed structure, appeared electrically conductive and were composed of siderite, calcite and iron sulfide, the latter formed by precipitation of sulfide (from culture medium) with ferrous iron generated during corrosion. Strain IM1 cells were found attached to crust surfaces and encrusted deep inside crust domes. Our results may assist to diagnose methanogens-induced corrosion in the field and suggest that intrusion of sulfide in anoxic settings may stimulate corrosion by methanogenic archaea via formation of semiconductive crusts.

Genome and proteome analyses show the gaseous alkane degrader Desulfosarcina sp. strain BuS5 as an extreme metabolic specialist.

The metabolic potential of the sulfate-reducing bacterium Desulfosarcina sp. strain BuS5, currently the only pure culture able to oxidize the volatile alkanes propane and butane without oxygen, was investigated via genomics, proteomics and physiology assays. Complete genome sequencing revealed that strain BuS5 encodes a single alkyl-succinate synthase, an enzyme which apparently initiates oxidation of both propane and butane. The formed alkyl-succinates are oxidized to CO2 via beta oxidation and the oxidative Wood-Ljungdahl pathways as shown by proteogenomics analyses. Strain BuS5 conserves energy via the canonical sulfate reduction pathway and electron bifurcation. An ability to utilize long-chain fatty acids, mannose and oligopeptides, suggested by automated annotation pipelines, was not supported by physiology assays and in-depth analyses of the corresponding genetic systems. Consistently, comparative genomics revealed a streamlined BuS5 genome with a remarkable paucity of catabolic modules. These results establish strain BuS5 as an exceptional metabolic specialist, able to grow only with propane and butane, for which we propose the name Desulfosarcina aeriophaga BuS5. This highly restrictive lifestyle, most likely the result of habitat-driven evolutionary gene loss, may provide D. aeriophaga BuS5 a competitive edge in sediments impacted by natural gas seeps. Etymology: Desulfosarcina aeriophaga, aério (Greek): gas; phágos (Greek): eater; D. aeriophaga: a gas eating or gas feeding Desulfosarcina.

Thermophilic archaea activate butane via alkyl-coenzyme M formation.

The anaerobic formation and oxidation of methane involve unique enzymatic mechanisms and cofactors, all of which are believed to be specific for C1-compounds. Here we show that an anaerobic thermophilic enrichment culture composed of dense consortia of archaea and bacteria apparently uses partly similar pathways to oxidize the C4 hydrocarbon butane. The archaea, proposed genus 'Candidatus Syntrophoarchaeum', show the characteristic autofluorescence of methanogens, and contain highly expressed genes encoding enzymes similar to methyl-coenzyme M reductase. We detect butyl-coenzyme M, indicating archaeal butane activation analogous to the first step in anaerobic methane oxidation. In addition, Ca. Syntrophoarchaeum expresses the genes encoding ß-oxidation enzymes, carbon monoxide dehydrogenase and reversible C1 methanogenesis enzymes. This allows for the complete oxidation of butane. Reducing equivalents are seemingly channelled to HotSeep-1, a thermophilic sulfate-reducing partner bacterium known from the anaerobic oxidation of methane. Genes encoding 16S rRNA and methyl-coenzyme M reductase similar to those identifying Ca. Syntrophoarchaeum were repeatedly retrieved from marine subsurface sediments, suggesting that the presented activation mechanism is naturally widespread in the anaerobic oxidation of short-chain hydrocarbons.

Anaerobic Transformation and Detoxification of Sulfamethoxazole by Sulfate-Reducing Enrichments and Desulfovibrio vulgaris.

Sulfamethoxazole (SMX) is a veterinary antibiotic that is not efficiently removed from wastewater by routine treatment and therefore can be detected widely in the environment. Here, we investigated whether microbial anaerobic transformation can contribute to the removal of SMX in constructed systems. We enriched SMX-transforming mixed cultures from sediment of a constructed wetland and from digester sludge of a wastewater treatment plant. Transformation of SMX was observed in both sulfate-reducing and methanogenic cultures, whereas nitrate-reducing cultures showed no SMX transformation. In sulfate-reducing cultures, up to 90% of an initial SMX concentration of 100-250 µM was removed within 6 weeks of incubation, and the experiments demonstrated that the transformation was microbially catalyzed. The transformation products in sulfate-reducing cultures were identified as the reduced and isomerized forms of the isoxazole SMX moiety. The transformation products did not spontaneously reoxidize to SMX after oxygen exposure, and their antibacterial activity was significantly decreased compared to SMX. Population analyses in sequential transfers of the sulfate-reducing cultures revealed a community shift toward the genus Desulfovibrio. We therefore tested a deposited strain of Desulfovibrio vulgaris Hildenborough for its capacity to transform SMX and observed the same transformation products and similar transformation rates as in the enrichment cultures. Our work suggests that an initial anaerobic step in wastewater treatment can reduce the concentration of SMX in effluents and could contribute to decreased SMX concentrations in the environment.

Dual C-Cl Isotope Analysis for Characterizing the Reductive Dechlorination of α- and γ-Hexachlorocyclohexane by Two Dehalococcoides mccartyi Strains and an Enrichment Culture.

Hexachlorocyclohexanes (HCHs) are persistent organic contaminants that threaten human health. Microbial reductive dehalogenation is one of the most important attenuation processes in contaminated environments. This study investigated carbon and chlorine isotope fractionation of α- and γ-HCH during the reductive dehalogenation by three anaerobic cultures. The presence of tetrachlorocyclohexene (TeCCH) indicated that reductive dichloroelimination was the first step of bond cleavage. Isotope enrichment factors (εC and εCl) were derived from the transformation of γ-HCH (εC, from -4.0 ± 0.5 to -4.4 ± 0.6 ‰; εCl, from -2.9 ± 0.4 to -3.3 ± 0.4 ‰) and α-HCH (εC, from -2.4 ± 0.2 to -3.0 ± 0.4 ‰; εCl, from -1.4 ± 0.3 to -1.8 ± 0.2 ‰). During α-HCH transformation, no enantioselectivity was observed, and similar εc values were obtained for both enantiomers. The correlation of 13C and 37Cl fractionation (Λ = Δδ13C/Δδ37Cl ≈ εC/εCl) of γ-HCH (from 1.1 ± 0.3 to 1.2 ± 0.1) indicates similar bond cleavage during the reductive dichloroelimination by the three cultures, similar to α-HCH (1.7 ± 0.2 to 2.0 ± 0.3). The different isotope fractionation patterns during reductive dichloroelimination and dehydrochlorination indicates that dual-element stable isotope analysis can potentially be used to evaluate HCH transformation pathways at contaminated field sites.

Isotopic Characterization (2H, 13C, 37Cl, 81Br) of Abiotic Degradation of Methyl Bromide and Methyl Chloride in Water and Implications for Future Studies.

Methyl bromide (CH3Br) and methyl chloride (CH3Cl) significantly contribute to stratospheric ozone depletion. The atmospheric budgets of both compounds are unbalanced with known degradation processes outweighing known emissions. Stable isotope analysis may be capable to identify and quantify emissions and to achieve a balanced budget. Degradation processes do, however, cause isotope fractionation in methyl halides after emission and hence knowledge about these processes is a crucial prerequisite for any isotopic mass balance approach. In the current study, triple-element isotope analysis (2H, 13C, 37Cl/81Br) was applied to investigate the two main abiotic degradation processes of methyl halides (CH3X) in fresh and seawater: hydrolysis and halide exchange. For CH3Br, nucleophilic attack by both H2O and Cl- caused significant primary carbon and bromine isotope effects accompanied by a secondary inverse hydrogen isotope effect. For CH3Cl only nucleophilic substitution by H2O was observed at significant rates causing large primary carbon and chlorine isotope effects and a secondary inverse hydrogen isotope effect. Observed dual-element isotope ratios differed slightly from literature values for microbial degradation in water and hugely from radical reactions in the troposphere. This bodes well for successfully distinguishing and quantifying degradation processes in atmospheric methyl halides using triple-element isotope analysis.

Enantiomer and Carbon Isotope Fractionation of α-Hexachlorocyclohexane by Sphingobium indicum Strain B90A and the Corresponding Enzymes.

Chiral organic contaminants, like α-hexachlorocyclohexane (α-HCH), showed isotope fractionation and enantiomer fractionation during biodegradation. This study aims to understand the correlation between these two processes. Initial tests of α-HCH degradation by six Sphingobium strains (with different LinA variants) were conducted. Results showed variable enantiomer selectivity over the time course. In contrast, constant enantiomer selectivity was observed in experiments employing (i) cell suspensions, (ii) crude extracts, or (iii) LinA1 and LinA2 enzymes of strain B90A for α-HCH degradation in enzyme activity assay buffer. The average value of enantioselectivity (ES) were -0.45 ± 0.03 (cell suspensions), -0.60 ± 0.05 (crude extracts), and 1 (LinA1) or -1 (LinA2). The average carbon isotope enrichment factors (εc) of (+)α- and (-)α-HCH were increased from cells suspensions (-6.3 ± 0.1‰ and -2.3 ± 0.03‰) over crude extracts (-7.7 ± 0.4‰ and -3.4 ± 0.02‰) to purified enzymes (-11.1 ± 0.3‰ and -3.8 ± 0.2‰). The variability of ES and the εc were discussed based on the effect of mass transport and degradation rates. Our study demonstrates that enantiomer and isotope fractionation of α-HCH are two independent processes and both are affected by underlying reactions of individual enzymes and mass transport to a different extent.

A patchwork pathway for oxygenase-independent degradation of side chain containing steroids.

The denitrifying betaproteobacterium Sterolibacterium denitrificans serves as model organism for studying the oxygen-independent degradation of cholesterol. Here, we demonstrate its capability of degrading various globally abundant side chain containing zoo-, phyto- and mycosterols. We provide the complete genome that empowered an integrated genomics/proteomics/metabolomics approach, accompanied by the characterization of a characteristic enzyme of steroid side chain degradation. The results indicate that individual molybdopterin-containing steroid dehydrogenases are involved in C25-hydroxylations of steroids with different isoprenoid side chains, followed by the unusual conversion to C26-oic acids. Side chain degradation to androsta-1,4-diene-3,17-dione (ADD) via aldolytic C-C bond cleavages involves acyl-CoA synthetases/dehydrogenases specific for the respective 26-, 24- and 22-oic acids/-oyl-CoAs and promiscuous MaoC-like enoyl-CoA hydratases, aldolases and aldehyde dehydrogenases. Degradation of rings A and B depends on gene products uniquely found in anaerobic steroid degraders, which after hydrolytic cleavage of ring A, again involves CoA-ester intermediates. The degradation of the remaining CD rings via hydrolytic cleavage appears to be highly similar in aerobic and anaerobic bacteria. Anaerobic cholesterol degradation employs a composite repertoire of more than 40 genes partially known from aerobic degradation in gammaproteobacteria/actinobacteria, supplemented by unique genes that are required to circumvent oxygenase-dependent reactions.

Compound Specific Stable Chlorine Isotopic Analysis of Volatile Aliphatic Compounds Using Gas Chromatography Hyphenated with Multiple Collector Inductively Coupled Plasma Mass Spectrometry.

Stable chlorine isotope analysis is increasingly used to characterize sources, transformation pathways, and sinks of organic aliphatic compounds, many of them being priority pollutants in groundwater and the atmosphere. A wider use of chlorine isotopes in environmental studies is still inhibited by limitations of the different analytical techniques such as high sample needs, offline preparation, confinement to few compounds and mediocre precision, respectively. Here we present a method for the δ37Cl determination in volatile aliphatic compounds using gas chromatography coupled with multiple-collector inductively coupled plasma mass spectrometry (GC-MC-ICPMS), which overcomes these limitations. The method was evaluated by using a suite of five previously offline characterized in-house standards and eight chlorinated methanes, ethanes, and ethenes. Other than in previous approaches using ICP methods for chlorine isotopes, isobaric interference of the 36ArH dimer with 37Cl was minimized by employing dry plasma conditions. Samples containing 2-3 nmol Cl injected on-column were sufficient to achieve a precision (σ) of 0.1 mUr (1 milliurey = 0.001 = 1‰) or better. Long-term reproducibility and accuracy was always better than 0.3 mUr if organics were analyzed in compound mixtures. Standardization is carried out by using a two-point calibration approach. Drift, even though very small in this study, is corrected by referencing versus an internal standard. The presented method offers a direct, universal, and compound-specific procedure to measure the δ37Cl of a wide array of organic compounds overcoming limitations of previous techniques with the benefits of high sensitivity and accuracy comparable to the best existing approaches.

Carbon, Hydrogen and Chlorine Stable Isotope Fingerprinting for Forensic Investigations of Hexachlorocyclohexanes.

Multielemental stable isotope analysis of persistent organic pollutants (POPs) has the potential to characterize sources, sinks, and degradation processes in the environment. To verify the applicability of this approach for source identification of hexachlorocyclohexane (HCHs), we provide a data set of carbon, hydrogen, and chlorine stable isotope ratios (δ13C, δ2H, δ37Cl) of its main stereoisomers (α-, ß-, δ- and γ-HCHs) from a sample collection based on worldwide manufacturing. This sample collection comprises production stocks, agricultural and pharmaceutical products, chemical waste dumps, and analytical-grade material, covering the production time period from the late 1960s until now. Stable isotope ratios of HCHs cover the ranges from -233‰ to +1‰, from -35.9‰ to -22.7‰, and from -6.69‰ to +0.54‰ for δ2H, δ13C, and δ37Cl values, respectively. Four groups of samples with distinct multielemental stable isotope fingerprints were differentiated, most probably as a result of purification and isolation processes. No clear temporal trend in the isotope compositions of HCHs was found at the global scale. The multielemental stable isotope fingerprints facilitate the source identification of HCHs at the regional scale and can be used to assess transformation processes. The data set and methodology reported herein provide basic information for the assessment of environmental field sites contaminated with HCHs.

Compound Specific and Enantioselective Stable Isotope Analysis as Tools To Monitor Transformation of Hexachlorocyclohexane (HCH) in a Complex Aquifer System.

Technical hexachlorocyclohexane (HCH) mixtures and Lindane (γ-HCH) have been produced in Bitterfeld-Wolfen, Germany, for about 30 years until 1982. In the vicinity of the former dump sites and production facilities, large plumes of HCHs persist within two aquifer systems. We studied the natural attenuation of HCH in these groundwater systems through a combination of enantiomeric and carbon isotope fractionation to characterize the degradation of α-HCH in the areas downstream of a former disposal and production site in Bitterfeld-Wolfen. The concentration and isotope composition of α-HCH from the Quaternary and Tertiary aquifers were analyzed. The carbon isotope compositions were compared to the source signal of waste deposits for the dumpsite and highly contaminated areas. The average value of δ13C at dumpsite was -29.7 ± 0.3 ‰ and -29.0 ± 0.1 ‰ for (-) and (+)α-HCH, respectively, while those for the ß-, γ-, δ-HCH isomers were -29.0 ± 0.3 ‰, -29.5 ± 0.4 ‰, and -28.2 ± 0.2 ‰, respectively. In the plume, the enantiomer fraction shifted up to 0.35, from 0.50 at source area to 0.15 (well T1), and was found accompanied by a carbon isotope enrichment of 5 ‰ and 2.9 ‰ for (-) and (+)α-HCH, respectively. The established model for interpreting isotope and enantiomer fractionation patterns showed potential for analyzing the degradation process at a field site with a complex history with respect to contamination and fluctuating geochemical conditions.