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.

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.

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.

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.

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.

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.

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.

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.