Characterization of anaerobic biotransformation of hexachlorocyclohexanes by novel microbial consortia enriched from channel and river sediments.

The microbial biotransformation of hexachlorocyclohexane (HCH) by novel anaerobic microbial consortia enriched from sediments of an industrial effluent channel and the river Ravi in Pakistan was examined. The anaerobic consortia were capable of biotransforming α-, ß-, γ-, and δ-HCH through reductive dichloroelimination, resulting in the formation of benzene and monochlorobenzene. Concerning γ-HCH biotransformation by the channel and river cultures, isotopic fractionations for carbon (εC) were - 5.3 ± 0.4 (‰) and - 10.6 ± 1.2 (‰), while isotopic fractionations for chlorine (εCl) were - 4.4 ± 0.4 (‰) and - 7.8 ± 0.9 (‰), respectively. Furthermore, lambda values (Λ), representing the correlation of δ13C and δ37Cl fractionation, were determined to be 1.1 ± 0.1 and 1.3 ± 0.1 for γ-HCH biotransformation, suggesting a reductive dichloroelimination as the initial step of HCH biotransformation in both cultures. Amplicon sequencing targeting the 16S rRNA genes revealed that Desulfomicrobium populations were considerably increased in both cultures, indicating their possible involvement in the degradation process. These findings suggest that Desulfomicrobium-like populations may have an important role in biotransformation of HCH and novel anaerobic HCH-degrading microbial consortia could be useful bioaugmentation agents for the bioremediation of HCH-contaminated sites in Pakistan.

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.

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.

Stable isotopes and nanoSIMS single-cell imaging reveals soil plastisphere colonizers able to assimilate sulfamethoxazole.

The presence and accumulation of both, plastics and antibiotics in soils may lead to the colonization, selection, and propagation of soil bacteria with certain metabolic traits, e.g., antibiotic resistance, in the plastisphere. However, the impact of plastic-antibiotic tandem on the soil ecosystem functioning, particularly on microbial function and metabolism remains currently unexplored. Herein, we investigated the competence of soil bacteria to colonize plastics and degrade 13C-labeled sulfamethoxazole (SMX). Using single-cell imaging, isotope tracers, soil respiration and SMX mineralization bulk measurements we show that microbial colonization of polyethylene (PE) and polystyrene (PS) surfaces takes place within the first 30 days of incubation. Morphologically diverse microorganisms were colonizing both plastic types, with a slight preference for PE substrate. CARD-FISH bacterial cell counts on PE and PS surfaces formed under SMX amendment ranged from 5.36 × 103 to 2.06 × 104, and 2.06 × 103 to 3.43 × 103 hybridized cells mm-2, respectively. Nano-scale Secondary Ion Mass Spectrometry measurements show that 13C enrichment was highest at 130 days with values up to 1.29 atom%, similar to those of the 13CO2 pool (up to 1.26 atom%, or 22.55 ‰). Independent Mann-Whitney U test showed a significant difference between the control plastisphere samples incubated without SMX and those in 13C-SMX incubations (P < 0.001). Our results provide direct evidence demonstrating, at single-cell level, the capacity of bacterial colonizers of plastics to assimilate 13C-SMX from contaminated soils. These findings expand our knowledge on the role of soil-seeded plastisphere microbiota in the ecological functioning of soils impacted by anthropogenic stressors.

Continuous-Flow Stable Sulfur Isotope Analysis of Organic and Inorganic Compounds by EA-MC-ICPMS.

Elemental analysis (EA) coupled to isotope ratio mass spectrometry (IRMS) is a well-established method to derive stable isotope ratios of sulfur (34S/32S). Conversion of sulfur to SO2 by EA and measurement of SO2 isotopologues by IRMS represents the simplest and most versatile method to accomplish isotope measurement of sulfur even in bulk samples. Yet, interferences by oxygen isotopes in SO2 often impair the precision and trueness of measured results. In the current study, we coupled EA to multicollector inductively coupled plasma mass spectrometry (MC-ICPMS) to establish a method that avoids such interferences due to direct measurement of S+ ions. In addition, measurement of the 33S/32S isotope ratios is possible, thus representing the first bulk method that is suitable to study mass-independent isotope fractionation (MIF). Analytical precision (σ) of available Ag2S and BaSO4 reference materials (RMs) was, on average, 0.2 mUr for δ33S and δ34S, never exceeding 0.3 mUr within this study (1 mUr = 1‰ = 0.001). Measured δ34S values of reference materials agreed within ±0.2 mUr of officially reported values. Measurement of wood samples yielded good precision (0.2 mUr) for sulfur amounts as low as 3.5 µg, but precision deteriorated for samples at lower sulfur contents due to poor peak shape. Finally, we explored cross-calibration of organic liquids separated via gas chromatography (GC) against solid RMs combusted via EA that avoids challenging offline conversion of RMs. Results indicate good precision (≤0.08 mUr) and acceptable trueness (≤0.34 mUr) for determination of δ34S, demonstrating the future potential of such an approach.

Stable Isotope Probing-nanoFTIR for Quantitation of Cellular Metabolism and Observation of Growth-Dependent Spectral Features.

This study utilizes nanoscale Fourier transform infrared spectroscopy (nanoFTIR) to perform stable isotope probing (SIP) on individual bacteria cells cultured in the presence of 13C-labelled glucose. SIP-nanoFTIR simultaneously quantifies single-cell metabolism through infrared spectroscopy and acquires cellular morphological information via atomic force microscopy. The redshift of the amide I peak corresponds to the isotopic enrichment of newly synthesized proteins. These observations of single-cell translational activity are comparable to those of conventional methods, examining bulk cell numbers. Observing cells cultured under conditions of limited carbon, SIP- nanoFTIR is used to identify environmentally-induced changes in metabolic heterogeneity and cellular morphology. Individuals outcompeting their neighboring cells will likely play a disproportionately large role in shaping population dynamics during adverse conditions or environmental fluctuations. Additionally, SIP-nanoFTIR enables the spectroscopic differentiation of specific cellular growth phases. During cellular replication, subcellular isotope distribution becomes more homogenous, which is reflected in the spectroscopic features dependent on the extent of 13C-13C mode coupling or to specific isotopic symmetries within protein secondary structures. As SIP-nanoFTIR captures single-cell metabolism, environmentally-induced cellular processes, and subcellular isotope localization, this technique offers widespread applications across a variety of disciplines including microbial ecology, biophysics, biopharmaceuticals, medicinal science, and cancer research.

Novel extraction methods and compound-specific isotope analysis of methoxychlor in environmental water and aquifer slurry samples.

Multi-element compound-specific stable isotope analysis (ME-CSIA) allows monitoring the environmental behavior and transformation of most common and persistent contaminants. Recent advancements in analytical techniques have extended the applicability of ME-CSIA to organic micropollutants, including pesticides. Nevertheless, the application of this methodology remains unexplored concerning harmful insecticides such as methoxychlor, a polar organochlorine pesticide usually detected in soil and groundwater. This study introduces methods for dual carbon and chlorine compound-specific stable isotope analysis (δ13C-CSIA and δ37Cl-CSIA) of both methoxychlor and its metabolite, methoxychlor olefin, with a sensitivity down to 10 and 100 mg/L, and a precision lower than 0.3 and 0.5 ‰ for carbon and chlorine CSIA, respectively. Additionally, three extraction and preconcentration techniques suitable for ME-CSIA of the target pesticides at environmentally relevant concentrations were also developed. Solid-phase extraction (SPE) and liquid-solid extraction (LSE) effectively extracted methoxychlor (107 ± 27 % and 87 ± 13 %, respectively) and its metabolite (91 ± 27 % and 106 ± 14 %, respectively) from water and aquifer slurry samples, respectively, with high accuracy (Δδ13C and Δδ37Cl ≤ ± 1 ‰). Combining CSIA with polar organic chemical integrative samplers (POCISs) for the extraction of methoxychlor and methoxychlor olefin from water samples resulted in insignificant fractionation for POCIS-CSIA (Δδ13C ≤ ± 1 ‰). A relevant sorption of methoxychlor was detected within the polyethersulfones membranes of the POCISs resulting in temporary carbon isotope fractionation depending on the sorbed mass fraction during the first deployment days. This highlights the critical role of the interactions of polar analytes with POCIS sorbents and membranes in the performance of this method. Altogether, this study proposes a proof of concept for ME-CSIA of methoxychlor and its metabolites, opening the door for future investigations of their sources and transformation processes in contaminated sites.

Natural attenuation of sulfonamides and metabolites in contaminated groundwater - Review, advantages and challenges of current documentation techniques.

Sulfonamides are applied worldwide as antibiotics. They are emerging contaminants of concern, as their presence in the environment may lead to the spread of antibiotic resistance genes. Sulfonamides are present in groundwater systems, which suggest their persistence under certain conditions, highlighting the importance of understanding natural attenuation processes in groundwater. Biodegradation is an essential process, as degradation of sulfonamides reduces the risk of antibiotic resistance spreading. In this review, natural attenuation, and in particular assessment of biodegradation, is evaluated for sulfonamides in groundwater systems. The current knowledge level on biodegradation is reviewed, and a scientific foundation is built based on sulfonamide degradation processes, pathways, metabolites and toxicity. An overview of bacterial species and related metabolites is provided. The main research effort has focused on aerobic conditions while investigations under anaerobic conditions are lacking. The level of implementation in research is laboratory scale; here we strived to bridge towards field application and assessment, by assessing approaches commonly used in monitored natural attenuation. Methods to document contaminant mass loss are assessed to be applicable for sulfonamides, while the approach is limited by a lack of reference standards for metabolites. Furthermore, additional information is required on relevant metabolites in order to improve risk assessments. Based on the current knowledge on biodegradation, it is suggested to use the presence of substituent-containing metabolites from breakage of the sulfonamide bridge as specific indicators of degradation. Microbial approaches are currently available for assessment of microbial community's capacities, however, more knowledge is required on indigenous bacteria capable of degrading sulfonamides and on the impact of environmental conditions on biodegradation. Compound specific stable isotope analysis shows great potential as an additional in situ method, but further developments are required to analyse for sulfonamides at environmentally relevant levels. Finally, in a monitored natural attenuation scheme it is assessed that approaches are available that can uncover some processes related to the fate of sulfonamides in groundwater systems. Nevertheless, there are still unknowns related to relevant bacteria and metabolites for risk assessment as well as the effect of environmental settings such as redox conditions. Alongside, uncovering the fate of sulfonamides in future research, the applicability of the natural attenuation documentation approaches will advance, and provide a step towards in situ remedial concepts for the frequently detected sulfonamides.

Anaerobic biotransformation of hexachlorocyclohexane isomers in aqueous condition: Dual CCl isotope fractionation and impact on microbial community compositions.

Hexachlorocyclohexane (HCH) isomers are persistent organic pollutants (POPs) with high toxicity, lipid solubility, chemical stability. Despite the current ban on usage of Lindane, residual contamination cannot be ignored, and HCH are frequently detected in groundwater and threaten human health. Cultures capable of degrading α-HCH, ß-HCH, γ-HCH, and δ-HCH individually have been enriched in anoxic aqueous conditions. Compound-Specific Isotope Analysis (CSIA) was applied to examine the transformation mechanisms of different HCH isomers by the four enrichment cultures. 16S rRNA sequencing techniques were employed to examine the community composition of the enrichment cultures and detect changes in these communities resulting from adding individual HCH isomers. The results indicated that the ability of the enrichment cultures for dichloroelimination of HCH isomers was inconsistent. During dichloroelimination, different bond cleavage mode of ß- and δ-HCH led to distinct isotopic effects. HCH isomers had significant impact on the microbial community, while different microbial communities showed comparable isotopic effects during the transformation of a specific HCH isomer. In addition, bacteria in the phyla Proteobacteria and Firmicutes were proposed as the dominant dechlorinators. This study provides a novel perspective on the mode of bond cleavage during HCH dichloroelimination and the effect of HCH on microbial communities, which could potentially support the evaluation of HCH transformation by CSIA and their effects on the microecosystems of groundwater.

Tracking the transformation of persistent organic pollutants in food webs using multi element isotope and enantiomer fractionation.

In order to track the transformation of persistent organic pollutants (POPs) in food webs, field experiments were conducted at two sites using stable isotope and enantiomer fractionation concepts. The enantiomers of α-hexachlorocyclohexane (α-HCH) were selected as representative compounds for POPs. Isotope and enantiomer fractionation allowed the characterization of α-HCH enantiomer biotransformation processes along trophic levels of the food web - from soil and plants to animal livers, fat tissues and milk. The enrichment of heavy isotopes in soils, plants and sediments as well as the changes of enantiomer fractionation indicate that the biotransformation of α-HCH occurred in these compartments. Moreover, the increase of carbon and chlorine isotopic compositions as well as the changes of enantiomer fractionation of liver, fat tissues and milk demonstrated that the overall HCH exposure was much higher than estimates based on concentration levels, while the isotope and enantiomer fractionation revealed the enantiomer specific enantiomer uptake across the blood-brain barriers. Dual element isotope analysis suggested that complex transformation processes have occurred along the potential food web from the HCH sources over different environmental compartments to animal livers, fat tissues and milk. The results imply that the analyses of stable isotope compositions and concentrations has potential to reconstruct the exposure of higher organisms to POPs.

Quantifying energy expenditure in Göttingen Minipigs with the 13C-bicarbonate method under basal and drug-treated conditions.

Effective treatments of obesity focusing on energy expenditure (EE) are needed. To evaluate future EE-modulating drug candidates, appropriate animal models and methods to assess EE are needed. This study aimed to evaluate the stable isotope 13C-bicarbonate method (13C-BM) for estimating EE in Göttingen minipigs under basal and drug-treated conditions. Four experiments (Expt.1-4) were conducted to assess: 1) the 13C-BM reproducibility using breath sample collection (n = 8), on two consecutive days, 2) the effect of two dose levels (5 and 10 mg/kg body weight (BW)) of the mitochondrial uncoupler dinitrophenol (DNP) in a crossover design (n = 8), 3) sampling method agreement; blood vs. exhaled air (n = 6) and 4) 13C-BM using constant isotope infusion compared with indirect calorimetry (IC) (n = 3). Results correlated significantly (p < 0.001) between days (Expt.1), with an average coefficient of variance of 5.4 ± 2.3%. Administration of 10 mg DNP/kg BW increased (p < 0.01) EE by 33.2 ± 6.4% (Expt.2). Results based on different sampling methods correlated significantly (p < 0.001) and EE increased after 10 mg DNP/kg BW (p < 0.05) in Expt.3. However, results based on blood sampling were significantly higher (p < 0.01) than those of exhaled air. No effect of DNP and significantly different EE results (p < 0.05) was observed in a limited number of animals, when constant isotope infusion and blood sampling was compared with IC (Expt.4). In conclusion, the 13C-BM is useful for investigating treatment effects on EE in minipigs. However, further validation under standardized conditions is needed to provide accurate estimates of the 13C recovery factor and respiratory quotient, both of decisive importance when using the 13C-BM.

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.

Tracking deuterium uptake in hydroponically grown maize roots using correlative helium ion microscopy and Raman micro-spectroscopy.

BACKGROUND: Investigations into the growth and self-organization of plant roots is subject to fundamental and applied research in various areas such as botany, agriculture, and soil science. The growth activity of the plant tissue can be investigated by isotope labeling experiments with heavy water and subsequent detection of the deuterium in non-exchangeable positions incorporated into the plant biomass. Commonly used analytical methods to detect deuterium in plants are based on mass-spectrometry or neutron-scattering and they either suffer from elaborated sample preparation, destruction of the sample during analysis, or low spatial resolution. Confocal Raman micro-spectroscopy (CRM) can be considered a promising method to overcome the aforementioned challenges. The substitution of hydrogen with deuterium results in the measurable shift of the CH-related Raman bands. By employing correlative approaches with a high-resolution technique, such as helium ion microscopy (HIM), additional structural information can be added to CRM isotope maps and spatial resolution can be further increased. For that, it is necessary to develop a comprehensive workflow from sample preparation to data processing. RESULTS: A workflow to prepare and analyze roots of hydroponically grown and deuterium labeled Zea mays by correlative HIM-CRM micro-analysis was developed. The accuracy and linearity of deuterium detection by CRM were tested and confirmed with samples of deuterated glucose. A set of root samples taken from deuterated Zea mays in a time-series experiment was used to test the entire workflow. The deuterium content in the roots measured by CRM was close to the values obtained by isotope-ratio mass spectrometry. As expected, root tips being the most actively growing root zone had incorporated the highest amount of deuterium which increased with increasing time of labeling. Furthermore, correlative HIM-CRM analysis allowed for obtaining the spatial distribution pattern of deuterium and lignin in root cross-sections. Here, more active root zones with higher deuterium incorporation showed less lignification. CONCLUSIONS: We demonstrated that CRM in combination with deuterium labeling can be an alternative and reliable tool for the analysis of plant growth. This approach together with the developed workflow has the potential to be extended to complex systems such as plant roots grown in soil.

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.

Carbon and hydrogen stable isotope fractionation of sulfamethoxazole during anaerobic transformation catalyzed by Desulfovibrio vulgaris Hildenborough.

The fate of antibiotics in aquatic environments is of high concern and approaches are needed to assess the transformation of antibiotics in wastewater treatment plants. Here we used the model organism Desulfovibrio vulgaris Hildenborough to analyze compound specific isotope fractionation associated with anaerobic transformation of the antibiotic sulfamethoxazole (SMX). The results show that the rearrangement of the isoxazole ring in SMX is leading to significant carbon and hydrogen isotopic fractionation (εC = -5.8 ± 0.7‰, εH = -34 ± 9‰) during anaerobic transformation. The observed carbon isotopic fractionation is significantly higher than the values reported for aerobic degradation (εC = -0.6 ± 0.1‰) or abiotic reactions (εC = -0.8 to -4.8‰ for photolysis, εC = -0.8 to -2.2‰ for advanced oxidation). This indicates that carbon isotope fractionation can be used as a parameter to differentiate reaction mechanisms of SMX transformation. The corresponding apparent kinetic isotope effect (AKIEC) for anaerobic transformation of SMX was 1.029 ± 0.003, suggesting that the mechanism for anaerobic transformation is distinct from the mechanism reported for microbial aerobic degradation (AKIEC = 1.006 ± 0.001). In addition, dual-element (C-H) isotope analysis of SMX was performed in the present study, which was achieved by utilizing gas chromatography (GC) as the separation method instead of routine liquid chromatography. This dual-element isotope analysis resulted in a Λ value of 4.5 ± 2.2. Overall, compound specific isotope analysis can be a feasible tool to monitor the mitigation of SMX in wastewater treatment plants.

Dehalococcoides mccartyi strain CBDB1 takes up protons from the cytoplasm to reductively dehalogenate organohalides indicating a new modus of proton motive force generation.

Proton translocation across the cytoplasmic membrane is a vital process for all organisms. Dehalococcoides strains are strictly anaerobic organohalide respiring bacteria that lack quinones and cytochromes but express a large membrane-bound protein complex (OHR complex) proposed to generate a proton gradient. However, its functioning is unclear. By using a dehalogenase-based enzyme activity assay with deuterium-labelled water in various experimental designs, we obtained evidence that the halogen atom of the halogenated electron acceptor is substituted with a proton from the cytoplasm. This suggests that the protein complex couples exergonic electron flux through the periplasmic subunits of the OHR complex to the endergonic transport of protons from the cytoplasm across the cytoplasmic membrane against the proton gradient to the halogenated electron acceptor. Using computational tools, we located two proton-conducting half-channels in the AlphaFold2-predicted structure of the OmeB subunit of the OHR complex, converging in a highly conserved arginine residue that could play a proton gatekeeper role. The cytoplasmic proton half-channel in OmeB is connected to a putative proton-conducting path within the reductive dehalogenase subunit. Our results indicate that the reductive dehalogenase and its halogenated substrate serve as both electron and proton acceptors, providing insights into the proton translocation mechanism within the OHR complex and contributing to a better understanding of energy conservation in D. mccartyi strains. Our results reveal a very simple mode of energy conservation in anaerobic bacteria, showing that proton translocation coupled to periplasmic electron flow might have importance also in other microbial processes and biotechnological applications.

Characterization of Hexachlorocyclohexane Isomer Dehydrochlorination by LinA1 and LinA2 Using Multi-element Compound-Specific Stable Isotope Analysis.

Dehydrochlorination is one of the main (thus far discovered) processes for aerobic microbial transformation of hexachlorocyclohexane (HCH) which is mainly catalyzed by LinA enzymes. In order to gain a better understanding of the reaction mechanisms, multi-element compound-specific stable isotope analysis was applied for evaluating α- and γ-HCH transformations catalyzed by LinA1 and LinA2 enzymes. The isotopic fractionation (εE) values for particular elements of (+)α-HCH (εC = -10.8 ± 1.0‰, εCl = -4.2 ± 0.5‰, εH = -154 ± 16‰) were distinct from the values for (-)α-HCH (εC = -4.1 ± 0.7‰, εCl = -1.6 ± 0.2‰, εH = -68 ± 10‰), whereas the dual-isotope fractionation patterns were almost identical for both enantiomers (ΛC-Cl = 2.4 ± 0.4 and 2.5 ± 0.2, ΛH-C = 12.9 ± 2.4 and 14.9 ± 1.1). The εE of γ-HCH transformation by LinA1 and LinA2 were -7.8 ± 1.0‰ and -7.5 ± 0.8‰ (εC), -2.7 ± 0.3‰ and -2.5 ± 0.4‰ (εCl), -170 ± 25‰ and -150 ± 13‰ (εH), respectively. Similar ΛC-Cl values (2.7 ± 0.2 and 2.9 ± 0.2) were observed as well as similar ΛH-C values (20.1 ± 2.0 and 18.4 ± 1.9), indicating a similar reaction mechanism by both enzymes during γ-HCH transformation. This is the first data set on 3D isotope fractionation of α- and γ-HCH enzymatic dehydrochlorination, which gave a more precise characterization of the bond cleavages, highlighting the potential of multi-element compound-specific stable isotope analysis to characterize different transformation processes (e.g., dehydrochlorination and reductive dehalogenation).

Expanding the calibration range of compound-specific chlorine isotope analysis by the preparation of a 37 Cl-enriched tetrachloroethylene.

RATIONALE: The recent development of reliable GC/qMS methods for δ37 Cl compound-specific stable isotope analysis (CSIA) paves the way for dual carbon-chlorine isotope analysis of chlorinated ethenes and thus allows deeper insights into underlying transformation processes/mechanisms. A two-point calibration is indispensable for the precise and correct conversion of raw data to the international δ37 ClSMOC scale. The currently available calibration standards for tetrachloroethylene (PCE) span only a very narrow range from -2.52‰ (EIL2) to +0.29‰ (EIL1), which is considerably smaller than observed δ37 Cl isotope enrichment in (bio-)transformation studies (up to 12‰). METHODS: We describe the preparation and evaluation of a new 37 Cl-enriched PCE standard to avoid bias in δ37 Cl CSIA arising from extrapolation beyond the calibration range. The preparation comprised: (i) partial PCE reduction by zero-valent zinc in a system of PCE, ethanol (initial volume ratio 3/5) and trace amounts of water followed by (ii) liquid-liquid extraction and (iii) a subsequent fractional distillation to purify the 37 Cl-enriched PCE. RESULTS: The obtained PCE (PCEenriched ) showed a purity of 98.8% (mole fraction) and a δ37 ClSMOC value of +10.8 ± 0.5‰. The evaluation of an experimental dataset with and without extrapolation showed no significant variation. CONCLUSIONS: The new PCE standard (PCEenriched ) expands the calibration range to 13.3‰ (previously 2.8‰) and thus prevents potential bias introduced by extrapolation beyond the calibration range.

Uptake and Metabolization of HCH Isomers in Trees Examined over an Annual Growth Period by Compound-Specific Isotope Analysis and Enantiomer Fractionation.

To understand the role of plants for natural attenuation, a field study was conducted to characterize the fate of HCH in trees over an annual growth period using compound-specific isotope analysis and enantiomer fractionation. Stable and slightly higher δ13C and δ37Cl values of HCH of host soil samples compared to the muck (consisting nearly exclusively of HCH) revealed that masking isotope effects caused by the limited bioavailability may underestimate the real extent of HCH transformation in soil. In contrast, an increase of δ13C and δ37Cl values in trees indicated the transformation of HCH. A large variability of δ13C and δ37Cl values in trees over the growth period was observed, representing different transformation extents among different growth times, which is further supported by the shift of the enantiomer fraction (EF), indicating the preferential transformation of enantiomers also varied over the different growth periods. Based on dual-element isotope analysis, different predominant transformation mechanisms were observed during the growing seasons. Our observation implies that plants are acting as biological pumps driving a cycle of uptake and metabolization of HCH and refeed during littering to soil catalyzing their transformation. The changes of the transformation mechanism in different seasons have implications for phytoscreening and shed new light on phytoremediation of HCH at field sites.