Compound-specific Stable Isotope Analysis Research Articles

Trophic interaction among organisms in a seagrass meadow ecosystem as revealed by bulk δ13C and amino acid δ15N analyses

AbstractNumerous studies have used stable isotope analysis (SIA) of carbon and nitrogen within the bulk tissues of organisms to determine the trophic structure among organisms in a food web. Recently, SIA has evolved to compound‐specific stable isotope analysis (CSIA) of nitrogen within amino acids to significantly reduce the uncertainty in the estimated trophic position (TP) of organisms based on the isotopic difference between glutamic acid and phenylalanine within a single organism. However, because the initial offset (β) between glutamic acid and phenylalanine differs between aquatic algae (ca. +3.4‰) and vascular plants (ca. −8.4‰) in food webs that rely on both resources, β should be replaced by a value adapted to the admixture of primary producers for each specimen. In this study, we established a new method involving the β value (βmix) of each consumer specimen determined based on its bulk tissue δ13C value and successfully obtained realistic TPs (TPmix) for organisms in a complex seagrass meadow food web. Remarkable differences between the TPmix and traditional TPalgal values were found in deposit feeders due to the large contribution of seagrass to their basal resources. The estimated TPs of organisms increased by up to 1.5 units (from TPalgal to TPmix), in terms of trophic transfer, when their diets included substantial seagrass‐derived contributions. Thus, combinatorial analysis of the amino acid δ15N and specimen‐specific βmix values provides better understanding of the trophic interactions in food webs, even in complex seagrass meadow ecosystems.

Strong linkage of polar cod (Boreogadus saida) to sea ice algae-produced carbon: Evidence from stomach content, fatty acid and stable isotope analyses

The polar cod (Boreogadus saida) is considered an ecological key species, because it reaches high stock biomasses and constitutes an important carbon source for seabirds and marine mammals in high-Arctic ecosystems. Young polar cod (1–2years) are often associated with the underside of sea ice. To evaluate the impact of changing Arctic sea ice habitats on polar cod, we examined the diet composition and quantified the contribution of ice algae-produced carbon (αIce) to the carbon budget of polar cod. Young polar cod were sampled in the ice-water interface layer in the central Arctic Ocean during late summer 2012. Diets and carbon sources of these fish were examined using 4 approaches: (1) stomach content analysis, (2) fatty acid (FA) analysis, (3) bulk nitrogen and carbon stable isotope analysis (BSIA) and (4) compound-specific stable isotope analysis (CSIA) of FAs.The ice-associated (sympagic) amphipod Apherusa glacialis dominated the stomach contents by mass, indicating a high importance of sympagic fauna in young polar cod diets. The biomass of food measured in stomachs implied constant feeding at daily rates of ∼1.2% body mass per fish, indicating the potential for positive growth. FA profiles of polar cod indicated that diatoms were the primary carbon source, indirectly obtained via amphipods and copepods. The αIce using bulk isotope data from muscle was estimated to be >90%. In comparison, αIce based on CSIA ranged from 34 to 65%, with the highest estimates from muscle and the lowest from liver tissue. Overall, our results indicate a strong dependency of polar cod on ice-algae produced carbon. This suggests that young polar cod may be particularly vulnerable to changes in the distribution and structure of sea ice habitats. Due to the ecological key role of polar cod, changes at the base of the sea ice-associated food web are likely to affect the higher trophic levels of high-Arctic ecosystems.

Transformation of chlorpyrifos in integrated recirculating constructed wetlands (IRCWs) as revealed by compound-specific stable isotope (CSIA) and microbial community structure analysis

Carbon isotope analysis and 454 pyrosequencing methods were used to investigate in situ biodegradation of chlorpyrifos during its transport through three model integrated recirculating constructed wetlands (IRCWs). Results show that plant and Fe-impregnated biochar promoted degradation of chlorpyrifos and its metabolite 3,5,6-trichloro-2-pyridinol (TCP). Carbon isotope ratios in the IRCWs shifted to −31.24±0.58‰ (IRCW1, plant free), −26.82±0.60‰ (IRCW2, with plant) and −24.76±0.94‰ (IRCW3, with plant and Fe-biochar). The enrichment factors (Ɛbulk,c) were determined as −0.69±0.06‰ (IRCW1), −0.91±0.07‰ (IRCW2) and −1.03±0.09‰ (IRCW3). Microbial community analysis showed that IRCW3 was dominated by members of Bacillus, which can utilize and degrade chlorpyrifos. These results reveal that plant and Fe-biochar can induce carbon isotope fractionation and have a positive impact on the chlorpyrifos degradation efficiency by influencing the development of beneficial microbial communities.

Validation of GC-IRMS techniques for δ13C and δ2H CSIA of organophosphorus compounds and their potential for studying the mode of hydrolysis in the environment.

Compound-specific stable isotope analysis (CSIA) is among the most promising tools for studying the fate of organic pollutants in the environment. However, the feasibility of multidimensional CSIA was limited by the availability of a robust method for precise isotope analysis of heteroatom-bearing organic compounds. We developed a method for δ 13C and δ 2H analysis of eight organophosphorus compounds (OPs) with different chemical properties. In particular, we aimed to compare high-temperature conversion (HTC) and chromium-based HTC (Cr/HTC) units to explore the limitations of hydrogen isotope analysis of heteroatom-bearing compounds. Analysis of the amount dependency of the isotope values (linearity analysis) of OPs indicated that the formation of HCl was a significant isotope fractionation process leading to inaccurate δ 2H analysis in HTC. In the case of nonchlorinated OPs, by-product formation of HCN, H2S, or PH3 in HTC was observed but did not affect the dynamic range of reproducible isotope values above the limit of detection. No hydrogen-containing by-products were found in the Cr/HTC process by use of ion trap mass spectrometry analysis. The accuracy of gas chromatography-isotope ratio mass spectrometry was validated in comparison with elemental analyzer-isotope ratio mass spectrometry. Dual-isotope fractionation yielded Λ values of 0 ± 0 at pH 7, 7 ± 1 at pH 9, and 30 ± 6 at pH 12, indicating the potential of 2D CSIA to characterize the hydrolysis mechanisms of OPs. This is the first report on the combination of δ 2H and δ 13C isotope analysis of OPs, and this is the first study providing a systematic evaluation of HTC and Cr/HTC for hydrogen isotope analysis using OPs as target compounds. Graphical Abstract Comparison of δ2H measurement of non-chlorinated and chlorinated OPs via GC-Cr/HTC-IRMS and GC-HTC-IRMS system.

多環芳香族炭化水素類(PAHs)の安定炭素同位体分析を用いた環境化学的研究の現状と展望

多環芳香族炭化水素類(PAHs)の安定炭素同位体分析を用いた環境化学的研究の現状と展望

Development of a novel method for the enrichment of unsubstituted naphthalene and phenanthrene from crude oils for compound-specific stable isotope analysis

A novel, effective and easy-to-operate method to enrich unsubstituted naphthalene and phenanthrene directly from crude oils was developed based on closed-system pyrolysis.

Geochemically distinct carbon isotope distributions in Allochromatium vinosum DSM 180T grown photoautotrophically and photoheterotrophically.

Anoxygenic, photosynthetic bacteria are common at redox boundaries. They are of interest in microbial ecology and geosciences through their role in linking the carbon, sulfur, and iron cycles, yet much remains unknown about how their flexible carbon metabolism-permitting either autotrophic or heterotrophic growth-is recorded in the bulk sedimentary and lipid biomarker records. Here, we investigated patterns of carbon isotope fractionation in a model photosynthetic sulfur-oxidizing bacterium, Allochromatium vinosum DSM180T . In one treatment, A.vinosum was grown with CO2 as the sole carbon source, while in a second treatment, it was grown on acetate. Different intracellular isotope patterns were observed for fatty acids, phytol, individual amino acids, intact proteins, and total RNA between the two experiments. Photoautotrophic CO2 fixation yielded typical isotopic ordering for the lipid biomarkers: δ13 C values of phytol>n-alkyl lipids. In contrast, growth on acetate greatly suppressed intracellular isotopic heterogeneity across all molecular classes, except for a marked 13 C-depletion in phytol. This caused isotopic "inversion" in the lipids (δ13 C values of phytol<n-alkyl lipids). The finding suggests that inverse δ13 C patterns of n-alkanes and pristane/phytane in the geologic record may be at least in part a signal for photoheterotrophy. In both experimental scenarios, the relative isotope distributions could be predicted from an isotope flux-balance model, demonstrating that microbial carbon metabolisms can be interrogated by combining compound-specific stable isotope analysis with metabolic modeling. Isotopic differences among molecular classes may be a means of fingerprinting microbial carbon metabolism, both in the modern environment and the geologic record.

Mercury levels in largemouth bass (Micropterus salmoides) from regulated and unregulated rivers

Within areas of comparable atmospheric mercury deposition rates methylmercury burden in largemouth bass populations vary significantly between regulated and unregulated rivers. To investigate if trophic dynamics strongly influenced pollutant body load, we sampled largemouth bass from two adjacent rivers, one regulated and one unregulated, and applied a suite of biochemical and stable isotope assays to compare their trophic dynamics. Total mercury burden in the bass from the unregulated Sipsey River (Elrod, AL, USA) and the regulated Black Warrior River (Demopolis, AL, USA) averaged 0.87 mg kg−1 and 0.19 mg kg−1 wet weight, respectively. For both populations, age, weight, and length were positively correlated with muscle mercury concentration. Compound specific stable isotope analysis of amino acids showed the trophic position of both populations was just under four. Quantitative and isotopic analysis of neutral lipid fatty acid of Sipsey River bass indicated a greater reliance upon the detrital component of the food web compared to Demopolis Reservoir bass which fed within the autochthonous, pelagic component of the food web. Since the close proximity of the rivers makes differences in atmospheric deposition unlikely and both populations had similar trophic position, our findings indicate that food web dynamics should be included among the factors that can strongly influence mercury concentration in fish.

Compound-Specific Stable Isotope Analysis: Implications in Hexachlorocyclohexane in-vitro and Field Assessment.

Assessment of biotic and abiotic degradation reactions by studying the variation in stable isotopic compositions of organic contaminants in contaminated soil and aquifers is being increasingly considered during the last two decades with development of Compound specific stable isotope analysis (CSIA) technique. CSIA has been recognized as a potential tool for evaluating both qualitative and quantitative degradation with measurement of shifts in isotope ratios of contaminants and their degradation products as its basis. Amongst a wide variety of environmental pollutants including monoaromatics, chlorinated ethenes and benzenes etc., it is only recently that its efficacy is being tested for assessing biodegradation of a noxious pollutant namely hexachlorocyclohexane (HCH), by pure microbial cultures as well as directly at the field site. Anticipating the increase in demand of this technique for monitoring the microbial degradation along with natural attenuation, this review highlights the basic problems associated with HCH contamination emphasizing the applicability of emerging CSIA technique to absolve the major bottlenecks in assessment of HCH. To this end, the review also provides a brief overview of this technique with summarizing the recent revelations put forward by both in vitro and in situ studies by CSIA in monitoring HCH biodegradation.

Fatty acid stable isotope signatures of molluscs exposed to finfish farming outputs

Tracing the flow of nutrients from aquaculture operations to the surrounding biota is important for environmental monitoring and developing integrated aquaculture practices. A novel approach in this context, compound specific stable isotope analysis (CSIA) was used to trace fatty acid (FA) carbon in benthic invertebrates from multiple aquaculture sites in the Broughton Archi- pelago, British Columbia, Canada, up to 2500 m away from each site. We found that the carbon isotope ratios (δ 13 C) of certain FA in mussels and limpets were gradually 13 C-enriched with increasing distance away from the site, suggesting that the farm contributed to more depleted iso- topic signatures among organisms within the vicinity of the site. In mussels, the δ 13 C of 16:2n-4, 16:3n-4, 20:1n-9, 22:6n-3, saturated FA, the bacterial FA marker, and the overall weighted mean of FA became significantly more 13 C-enriched with increasing distance from the farm (r 2 > 0.28, p 0.37, p < 0.05). The δ 13 C of mussels was also influenced by location of the farm, regardless of distance from the site. Non-indigenous isotopic signatures, as a result of feed inputs, physical properties of the farm location, and other anthro- pogenic influences, likely contributed to the depleted signature of mussels and limpets within the vicinity of the farm. This study was the first to use CSIA to determine the uptake and movement of organic nutrients from aquaculture outputs to the surrounding ecosystem.

Feeding ecologies of key bivalve and polychaete species in the Bering Sea as elucidated by fatty acid and compound-specific stable isotope analyses

Feeding ecologies of key bivalve and polychaete species in the Bering Sea as elucidated by fatty acid and compound-specific stable isotope analyses

Labelled microbial culture as a calibration medium for (13) C-isotope measurement of derivatized compounds: application to tert-butyldimethylsilyl amino acids.

Compound-specific stable carbon isotope analysis by gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) is widely used in studies of environmental or biological functioning. In the case of derivatized molecules, a calibration might be required due to added non-analyte carbon and in some cases non-stoichiometric recovery by the mass spectrometer. Two biological materials of known isotopic composition were produced by microbial cell cultures on either (13) C-labelled glucose or non-labelled glucose as sole source of carbon. Subsequent hydrolyzed amino acids were derivatized as tert-butyldimethylsilyl (tBDMSi) derivatives and analyzed by GC/C/IRMS. The (13) C-enrichment measurements were used as a direct calibration to calculate the original (13) C/(12) C ratios of individual amino acids. We tested this calibration on both known and unknown samples. For the main proteinogenic amino acids we could determine the number of non-analyte added carbon atoms and assess the non-stoichiometrical recovery of tBDMSi carbon atoms, due to their incomplete oxidation in the combustion step of GC/C/IRMS. The calibration enabled the determination of the natural abundances (δ(13) C values) of amino acids with an average accuracy of ±1.1 ‰. We illustrate the application of the calibration to determine the (13) C/(12) C ratios of amino acids, and the associated uncertainty, in biological and plant materials. The analysis of a labelled microbial cell culture offers a straightforward, rapid and reliable estimate of non-analyte carbon contribution to stable isotope composition. We recommend this method as a calibration or a control in artificial or natural (13) C-tracing experiments. Copyright © 2016 John Wiley & Sons, Ltd.

The importance of ice algae-produced carbon in the central Arctic Ocean ecosystem: Food web relationships revealed by lipid and stable isotope analyses

To better predict ecological consequences of changing Arctic sea ice environments, we aimed to quantify the contribution of ice algae-produced carbon (αIce) to pelagic food webs in the central Arctic Ocean. Eight abundant under-ice fauna species were submitted to fatty acid (FA) analysis, bulk stable isotope analysis (BSIA) of nitrogen (δ15N) and carbon (δ13C) isotopic ratios, and compound-specific stable isotope analysis (CSIA) of δ13C in trophic marker FAs. A high mean contribution αIce was found in Apherusa glacialis and other sympagic (ice-associated) amphipods (BSIA: 87% to 91%, CSIA: 58% to 92%). The pelagic copepods Calanus glacialis and C. hyperboreus, and the pelagic amphipod Themisto libellula showed substantial, but varying αIce values (BSIA: 39% to 55%, CSIA: 23% to 48%). Lowest αIce mean values were found in the pteropod Clione limacina (BSIA: 30%, CSIA: 14% to 18%). Intra-specific differences in FA compositions related to two different environmental regimes were more pronounced in pelagic than in sympagic species. A comparison of mixing models using different isotopic approaches indicated that a model using δ13C signatures from both diatom-specific and dinoflagellate-specific marker FAs provided the most conservative estimate of αIce. Our results imply that ecological key species of the central Arctic Ocean thrive significantly on carbon synthesized by ice algae. Due to the close connectivity between sea ice and the pelagic food web, changes in sea ice coverage and ice algal production will likely have important consequences for food web functioning and carbon dynamics of the pelagic system.

Tracing the biotransformation of polychlorinated biphenyls (PCBs) in common carp (Cryprinus carpio): Enantiomeric fraction and compound-specific stable carbon isotope analyses

Metabolites of polychlorinated biphenyls (PCBs) in fish are difficult to detect in vivo due to the complexity of biometabolism. In the present study, atropisomeric fraction analysis of chiral PCB congeners and compound-specific isotopic analysis (CSIA) were applied to trace the biotransformation of PCBs in fish by exposure of common carp (Cryprinus carpio) to the commercial PCB mixture Aroclor 1242. Stereoselective elimination of the chiral PCB congeners 91, 95, and 136 was observed, indicating a stereoselective biotransformation process. The δ13C values of PCBs 5/8, 18, and 20/33 in fish were increased compared with those in the spiked food, while PCBs 47/48 and 49 showed significant heavy isotope depletion. These results suggested a significant biotransformation of the corresponding individual PCB congeners although the potential PCB metabolites, hydroxylated PCBs (OH-PCBs) and methylsulfone PCBs (MeSO2-PCBs), were not detected in the fish tissue samples throughout this experiment. The results of the present study demonstrated that a combination of chiral analysis and CSIA is a promising new approach for investigating the biotransformation of PCBs in biota.

Compound-specific δ13C isotopes and Bayesian inference for erosion estimates under different land use in Vietnam

Recent studies have pointed out the potential of the compound specific stable isotope (CSSI) technique based on long-chain fatty acids methylester (FAME) to identify hot spots of soil erosion by means of land use types. We tested the applicability of the CSSI technique on the basis of soil and sediment samples derived from a small agriculturally used catchment in Vietnam which is exemplary for many mountainous areas in Southeast Asia. Following CSSI analysis we set up a statistical decision sequence to identify hot spots of soil erosion by i) testing for significant differences between δ13C values of fatty acids (FA) of different contributing land use types and thereafter ii) examining the data using a Monte Carlo simulation of mixing polygons to provide a quantitative basis for model rejection and exclusion for sediment samples which violate the point-in-polygon assumption and iii) applying a Bayesian model with a Markov chain Monte Carlo (MCMC) model fitting using “SIAR‟ (Stable Isotope Analysis in R), which produces simulations of plausible values and therefore representing a true probability density for the proportional contribution of source soils. Our results confirmed that there were significantly different δ13C values for identical FAMEs extracted from soils under different land uses. Most fatty acids with significantly different δ13C values were found between soils under C3 (protected and secondary forest, teak and fruit plantations) and C4 (maize) plants but also within different soils of land use types which consisted only of C3 plants (e.g. protected forest, fruit plantation and teak). The resulting soil proportions were plausible for the six investigated sedimentation areas and suggested that fields under crop production such as maize and cassava, but also teak plantations were the main sources of eroding soil in the upland area surrounding the Chieng Khoi reservoir. Based on our data, we can conclude that the developed integrated Bayesian SIAR-CSSI approach represents a unique tool to identify and apportion soil sources to major land use types in small heterogeneous catchments by linking biomarkers of land use types to the sediment in deposition zones.

Characterization of a site contaminated by chlorinated ethenes and ethanes using multi-analysis

Chlorinated ethenes and ethanes are the most commonly detected groundwater contaminants in industrial areas. Compound-specific stable isotope analysis combined with hydrogeological, and geochemical analyses was applied to identify contaminant sources and to monitor the natural degradation processes of chlorinated ethenes and ethanes in an industrial area of Asan, Korea. The concentrations of the detected trichloroethene (TCE) and 1,1,1-trichloroethane (1,1,1-TCA) ranged from 0.004 to 5.8 mg/L and from non-detected to 1.8 mg/L in August 2013. TCE concentrations correspond to 65–87 % of the molar fraction of the volatile organic compounds. Most of the flow system in the study area is aerobic with dissolved oxygen (DO) ranging 0.1–8.3 mg/L. The highest concentration of TCE (5.8 mg/L) and the most depleted δ13C and δ37Cl values (−27.1 and −1.7 ‰, respectively) were observed in the well near an electrical parts company, indicating a source area. The slight enrichments in both the carbon and chlorine isotopic values of contaminants in some wells can be explained by isotopic shifts caused by degradation processes in the study area. 1,1-dichloroethene (1,1-DCE) was present in higher concentrations than cis-DCE and trans-DCE. The concentrations of 1,1-DCA, the biotic degradation product of 1,1,1-TCA, were much lower than those of 1,1-DCE due to the limited anaerobic conditions of the study area. High correlations of 1,1-DCE and 1,1,1-TCA in the study area indicated that some 1,1-DCE might be the dehydrohalogenation product of 1,1,1-TCA. Further isotopic analysis of 1,1,1-TCA are recommended to identify the degradation pathway of 1,1,1-TCA. Hydrogeochemical, VOCs, and dual isotope data showed that dilution with an insignificant rate of biodegradation is the main process attenuating VOCs in the groundwater system. Thus, an active remediation plan is necessary to reduce contaminant concentration in the study area.

A novel high-temperature combustion interface for compound-specific stable isotope analysis of carbon and nitrogen via high-performance liquid chromatography/isotope ratio mass spectrometry.

In aqueous samples compound-specific stable isotope analysis (CSIA) plays an important role. No direct method (without sample preparation) for stable nitrogen isotope analysis (δ(15) N SIA) of non-volatile compounds is known yet. The development of a novel HPLC/IRMS interface based on high-temperature combustion (HTC) for both δ(13) C and δ(15) N CSIA and its proof of principle are described in this study. To hyphenate high-performance liquid chromatography (HPLC) with isotope ratio mass spectrometry (IRMS) a modified high-temperature combustion total organic carbon analyzer (HTC TOC) was used. A system to handle a continuously large amount of water (three-step drying system), favorable carrier and reaction gas mix and flow, an efficient high-temperature-based oxidation and subsequent reduction system and a collimated beam transfer system were the main requirements to achieve the necessary performance. The proof of principle with caffeine solutions of the system succeeded. In this initial testing, both δ(13) C and δ(15) N values of tested compounds were determined with precision and trueness of ≤0.5‰. Further tests resulted in lower working limit values of 3.5 μgC for δ(13) C SIA and 20 μgN for δ(15) N SIA, considering an accuracy of ±0.5‰ as acceptable. The development of a novel HPLC/IRMS interface resulted in the first system reported to be suitable for both δ(13) C and δ(15) N direct CSIA of non-volatile compounds. This highly efficient system will probably open up new possibilities in SIA-based research fields. Copyright © 2016 John Wiley & Sons, Ltd.

Hydrogen Isotope Fractionation As a Tool to Identify Aerobic and Anaerobic PAH Biodegradation.

Aerobic and anaerobic polycyclic aromatic hydrocarbon (PAH) biodegradation was characterized by compound specific stable isotope analysis (CSIA) of the carbon and hydrogen isotope effects of the enzymatic reactions initiating specific degradation pathways, using naphthalene and 2-methylnaphtalene as model compounds. Aerobic activation of naphthalene and 2-methylnaphthalene by Pseudomonas putida NCIB 9816 and Pseudomonas fluorescens ATCC 17483 containing naphthalene dioxygenases was associated with moderate carbon isotope fractionation (εC = -0.8 ± 0.1‰ to -1.6 ± 0.2‰). In contrast, anaerobic activation of naphthalene by a carboxylation-like mechanism by strain NaphS6 was linked to negligible carbon isotope fractionation (εC = -0.2 ± 0.2‰ to -0.4 ± 0.3‰). Notably, anaerobic activation of naphthalene by strain NaphS6 exhibited a normal hydrogen isotope fractionation (εH = -11 ± 2‰ to -47 ± 4‰), whereas an inverse hydrogen isotope fractionation was observed for the aerobic strains (εH = +15 ± 2‰ to +71 ± 6‰). Additionally, isotope fractionation of NaphS6 was determined in an overlaying hydrophobic carrier phase, resulting in more reliable enrichment factors compared to immobilizing the PAHs on the bottle walls without carrier phase. The observed differences especially in hydrogen fractionation might be used to differentiate between aerobic and anaerobic naphthalene and 2-methylnaphthalene biodegradation pathways at PAH-contaminated field sites.

Characterization of toluene and ethylbenzene biodegradation under nitrate-, iron(III)- and manganese(IV)-reducing conditions by compound-specific isotope analysis

Ethylbenzene and toluene degradation under nitrate-, Mn(IV)-, or Fe(III)-reducing conditions was investigated by compound specific stable isotope analysis (CSIA) using three model cultures (Aromatoleum aromaticum EbN1, Georgfuchsia toluolica G5G6, and a Azoarcus-dominated mixed culture). Systematically lower isotope enrichment factors for carbon and hydrogen were observed for particulate Mn(IV). The increasing diffusion distances of toluene or ethylbenzene to the solid Mn(IV) most likely caused limited bioavailability and hence resulted in the observed masking effect. The data suggests further ethylbenzene hydroxylation by ethylbenzene dehydrogenase (EBDH) and toluene activation by benzylsuccinate synthase (BSS) as initial activation steps. Notably, significantly different values in dual isotope analysis were detected for toluene degradation by G. toluolica under the three studied redox conditions, suggesting variations in the enzymatic transition state depending on the available TEA. The results indicate that two-dimensional CSIA has significant potential to assess anaerobic biodegradation of ethylbenzene and toluene at contaminated sites.

Characterization of phenol and cresol biodegradation by compound-specific stable isotope analysis

Microbial degradation of phenol and cresols can occur under oxic and anoxic conditions by different degradation pathways. One recent technique to take insight into reaction mechanisms is compound-specific isotope analysis (CSIA). While enzymes and reaction mechanisms of several degradation pathways have been characterized in (bio)chemical studies, associated isotope fractionation patterns have been rarely reported, possibly due to constraints in current analytical methods. In this study, carbon enrichment factors and apparent kinetic isotope effects (AKIEc) of the initial steps of different aerobic and anaerobic phenol and cresols degradation pathways were analyzed by isotope ratio mass spectrometry connected with liquid chromatography (LC-IRMS). Significant isotope fractionation was detected for aerobic ring hydroxylation, anoxic side chain hydroxylation, and anoxic fumarate addition, while anoxic carboxylation reactions produced small and inconsistent fractionation. The results suggest that several microbial degradation pathways of phenol and cresols are detectable in the environment by CSIA.