Data_Sheet_1_Carbon and hydrogen stable isotope fractionation due to monooxygenation of short-chain alkanes by butane monooxygenase of Thauera butanivorans Bu-B1211.PDF

Multi element compound-specific stable isotope analysis (ME-CSIA) is a tool to assess (bio)chemical reactions of molecules in the environment based on their isotopic fingerprints. To that effect, ME-CSIA concepts are initially developed with laboratory model experiments to determine the isotope fractionation factors specific for distinct (bio)chemical reactions. Here, we determined for the first time the carbon and hydrogen isotope fractionation factors for the monooxygenation of the short-chain alkanes ethane, propane, and butane. As model organism we used Thauera butanivorans strain Bu-B1211 which employs a non-haem iron monooxygenase (butane monooxygenase) to activate alkanes. Monooxygenation of alkanes was associated with strong carbon and hydrogen isotope effects: εbulkC = −2.95 ± 0.5 ‰ for ethane, −2.68 ± 0.1 ‰ for propane, −1.19 ± 0.18 ‰ for butane; εbulkH = −56.3 ± 15 ‰ for ethane, −40.5 ± 2.3 ‰ for propane, −14.6 ± 3.6 ‰ for butane. This resulted in lambda (Λ ≈ εHbulk/εCbulk) values of 16.2 ± 3.7 for ethane, 13.2 ± 0.7 for propane, and 11.4 ± 2.8 for butane. The results show that ME-CSIA can be used to track the occurrence and impact of monooxygenase-dependent aerobic processes converting short-chain alkanes in natural settings like marine and terrestrial seeps, gas reservoirs, and other geological formations impacted by natural gas.</p

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

Enantioselective Carbon Stable Isotope Fractionation of Hexachlorocyclohexane during Aerobic Biodegradation by <i>Sphingobium</i> spp.

Carbon isotope fractionation was investigated for the biotransformation of γ- and α- hexachlorocyclohexane (HCH) as well as enantiomers of α-HCH using two aerobic bacterial strains: <i>Sphingobium indicum</i> strain B90A and <i>Sphingobium japonicum</i> strain UT26. Carbon isotope enrichment factors (ε_c) for γ-HCH (ε_c = −1.5 ± 0.1‰ and −1.7 ± 0.2‰) and α-HCH (ε_c = −1.0 ± 0.2‰ and −1.6 ± 0.3‰) were similar for both aerobic strains, but lower in comparison with previously reported values for anaerobic γ- and α-HCH degradation. Isotope fractionation of α-HCH enantiomers was higher for (+) α-HCH (ε_c = −2.4 ± 0.8 ‰ and −3.3 ± 0.8 ‰) in comparison to (−) α-HCH (ε_c = −0.7 ± 0.2‰ and −1.0 ± 0.6‰). The microbial fractionation between the α-HCH enantiomers was quantified by the Rayleigh equation and enantiomeric fractionation factors (ε_e) for <i>S. indicum</i> strain B90A and <i>S. japonicum</i> strain UT26 were −42 ± 16% and −22 ± 6%, respectively. The extent and range of isomer and enantiomeric carbon isotope fractionation of HCHs with <i>Sphingobium</i> spp. suggests that aerobic biodegradation of HCHs can be monitored in situ by compound-specific stable isotope analysis (CSIA) and enantiomer-specific isotope analysis (ESIA). In addition, enantiomeric fractionation has the potential as a complementary approach to CSIA and ESIA for assessing the biodegradation of α-HCH at contaminated field sites

MOESM1 of Lessons learned from the microbial ecology resulting from different inoculation strategies for biogas production from waste products of the bioethanol/sugar industry

Additional file 1: Figure S1. Duplicate T-RFLP profiles of the methanogenic community dynamics for each reactor in order to show the reproducibility of the T-RFLP approach. Figure S2. Rarefaction curves of the pyrosequencing data of the 16S ribosomal RNA genes from the four co-digestion reactors (R3.5, R3.6, R3.7 and R3.8) at three different sampling points along the experiment. Table S1. Beta diversity index showing the community similarities between samples. Figure S3. 3D PCA diagram of the beta diversity. Figure S4. N-MDS plot showing the Bray–Curtis similarity of the methanogenic communities in parallel reactors

Rayleigh-Based Concept to Tackle Strong Hydrogen Fractionation in Dual Isotope AnalysisThe Example of Ethylbenzene Degradation by <i>Aromatoleum aromaticum</i>

Compound-specific isotope analysis (CSIA) is a state-of-the-art analytical tool that can be used to establish and quantify biodegradation of pollutants such as BTEX compounds at contaminated field sites. Using isotopes of two elements and characteristic Lambda values (Λ) in dual-isotope-plots can provide insight into reaction mechanisms because kinetic isotope effects (KIEs) of both elements are reflected. However, the concept’s validity in the case of reactions that show strong isotope fractionation needs to be examined. The anaerobic ethylbenzene degradation pathway of <i>Aromatoleum aromaticum</i> is initiated by the ethylbenzene dehydrogenase-catalyzed monohydroxylation of the benzylic carbon atom. Measurements of stable isotope ratios revealed highly pronounced hydrogen fractionation, which could not be adequately described by the classical Rayleigh approach. This study demonstrates the nonlinear behavior of hydrogen isotope ratios caused by anaerobic ethylbenzene hydroxylation both mathematically and experimentally, develops alternative dual plots to enable the comparison of reactions by considering the reacting atoms, and illustrates the importance of the stereochemical aspects of substrate and product for the quantification of hydrogen fractionation in an enzymatic reaction. With regard to field application, proposals for an improved CSIA evaluation procedure with respect to pronounced hydrogen enrichment are given

Anaerobic dihydrogen consumption of nutrient-limited aquifer sediment microbial communities examined by stable isotope analysis

The biogeochemical consequences of dihydrogen (H2) underground storage in porous aquifers are poorly understood. Here, the effects of nutrient limitations on anaerobic H2 oxidation of an aquifer microbial community in sediment microcosms were determined in order to evaluate possible responses to high H2 partial pressures. Hydrogen isotope analyses of H2 yielded isotope depletion in all biotic setups indicating microbial H2 consumption. Carbon isotope analyses of carbon dioxide (CO2) showed isotope enrichment in all H2-supplemented biotic setups indicating H2-dependent consumption of CO2 by methanogens or homoacetogens. Homoacetogenesis was indicated by the detection of acetate and formate. Consumption of CO2 and H2 varied along the differently nutrient-amended setups, as did the onset of methane production. Plotting carbon against hydrogen isotope signatures of CH4 indicated that CH4 was produced hydrogenotrophically and fermentatively. The putative hydrogenotrophic Methanobacterium sp. was the dominant methanogen. Most abundant phylotypes belonged to typical ferric iron reducers, indicating that besides CO2, Fe(III) was an important electron acceptor. In summary, our study provides evidence for the adaptability of subsurface microbial communities under different nutrient-deficient conditions to elevated H2 partial pressures.</p

Compound-Specific Isotope Analysis as a Tool To Characterize Biodegradation of Ethylbenzene

This study applied one- and two-dimensional compound-specific isotope analysis (CSIA) for the elements carbon and hydrogen to assess different means of microbial ethylbenzene activation. Cultures incubated under nitrate-reducing conditions showed significant carbon and highly pronounced hydrogen isotope fractionation of comparable magnitudes, leading to nearly identical slopes in dual-isotope plots. The results imply that <i>Georgfuchsia toluolica</i> G5G6 and an enrichment culture dominated by an <i>Azoarcus</i> species activate ethylbenzene by anaerobic hydroxylation catalyzed by ethylbenzene dehydrogenase, similar to <i>Aromatoleum aromaticum</i> EbN1. The isotope enrichment pattern in dual plots from two strictly anaerobic enrichment cultures differed considerably from those for benzylic hydroxylation, indicating an alternative anaerobic activation step, most likely fumarate addition. Large hydrogen fractionation was quantified using a recently developed Rayleigh-based approach considering hydrogen atoms at reactive sites. Data from nine investigated microbial cultures clearly suggest that two-dimensional CSIA in combination with the magnitude of hydrogen isotope fractionation is a valuable tool to distinguish ethylbenzene degradation and may be of practical use for monitoring natural or technological remediation processes at field sites

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-, β-, 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

Monitoring of a Simulated CO₂ Leakage in a Shallow Aquifer Using Stable Carbon Isotopes

Artificial carbon dioxide leakage into a shallow aquifer was monitored using stable carbon isotope measurements at a field site near the town of Wittstock, Brandenburg, Germany. Approximately 400 000 L of CO₂ were injected into a shallow aquifer at 18 m depth over 10 days. The ¹³C/ ¹²C ratios of the CO₂ were measured in both groundwater and soil gas samples to monitor the distribution of the injected CO₂ plume and to evaluate the feasibility and reliability of this approach to detect potential CO₂ leakage, for example from carbon capture and storage (CCS) sites. The isotopic composition of the injected CO₂ (δ¹³C −30.5 ‰) was differentiable from the background CO₂ (δ¹³C −21.9 ‰) and the artificial CO₂ plume was monitored over a period spanning more than 204 days. The results demonstrate that this stable isotope monitoring approach can be used to identify CO₂ sources and detect potential CO₂ migration from CCS sites into overlying shallow aquifers or even into the upper subsurface. A significant difference between the isotope ratios of the natural background and the injected CO₂ is required for this monitoring approach to be effective

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 β-, 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