A Bench-Scale Constructed Wetland As a Model to Characterize Benzene Biodegradation Processes in Freshwater Wetlands

In wetlands, a variety of biotic and abiotic processes can contribute to the removal of organic substances. Here, we used compound-specific isotope analysis (CSIA), hydrogeochemical parameters and detection of functional genes to characterize in situ biodegradation of benzene in a model constructed wetland over a period of 370 days. Despite low dissolved oxygen concentrations (<30 μM), the oxidation of ammonium to nitrate and the complete oxidation of ferrous iron pointed to a dominance of aerobic processes, suggesting efficient oxygen transfer into the sediment zone by plants. As benzene removal became highly efficient after day 231 (>98% removal), we applied CSIA to study in situ benzene degradation by indigenous microbes. Combining carbon and hydrogen isotope signatures by two-dimensional stable isotope analysis revealed that benzene was degraded aerobically, mainly via the monohydroxylation pathway. This was additionally supported by the detection of the BTEX monooxygenase gene <i>tmoA</i> in sediment and root samples. Calculating the extent of biodegradation from the isotope signatures demonstrated that at least 85% of benzene was degraded by this pathway and thus, only a small fraction was removed abiotically. This study shows that model wetlands can contribute to an understanding of biodegradation processes in floodplains or natural wetland systems

Coupling of a Headspace Autosampler with a Programmed Temperature Vaporizer for Stable Carbon and Hydrogen Isotope Analysis of Volatile Organic Compounds at Microgram per Liter Concentrations

One major challenge for the environmental application of compound-specific stable isotope analysis (CSIA) is the necessity of efficient sample treatment methods, allowing isolation of a sufficient mass of organic contaminants needed for accurate measurement of the isotope ratios. Here, we present a novel preconcentration techniquethe coupling of a headspace (HS) autosampler with a programmed temperature vaporizer (PTV)for carbon (δ¹³C) and hydrogen (δ²H) isotope analysis of volatile organic compounds in water at concentrations of tens of micrograms per liter. The technique permits large-volume injection of headspace samples, maintaining the principle of simple static HS extraction. We developed the method for multielement isotope analysis (δ¹³C and δ²H) of methyl <i>tert</i>-butyl ether (MTBE), benzene, toluene, ethylbenzene, and <i>o</i>-xylene (BTEX), and analysis of δ¹³C for chlorinated benzenes and ethenes. Extraction and injection conditions were optimized for maximum sensitivity and minimum isotope effects. Injection of up to 5 mL of headspace sample from a 20 mL vial containing 13 mL of aqueous solution and 5 g of NaCl (10 min of incubation at 90 °C) resulted in accurate δ¹³C and δ²H values. The method detection limits (MDLs) for δ¹³C were from 2 to 60 μg/L (MTBE, BTEX, chlorinated ethenes, and benzenes) and 60–97 μg/L for δ²H (MTBE and BTEX). Overall, the HS–PTV technique is faster, simpler, isotope effect-free, and requires fewer treatment steps and less sample volume than other extraction techniques used for CSIA. The environmental applicability was proved by the analysis of groundwater samples containing BTEX and chlorinated contaminants at microgram per liter concentrations

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 <i>Pseudomonas putida</i> NCIB 9816 and <i>Pseudomonas fluorescens</i> ATCC 17483 containing naphthalene dioxygenases was associated with moderate carbon isotope fractionation (ε_C = −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 (ε_C = −0.2 ± 0.2‰ to −0.4 ± 0.3‰). Notably, anaerobic activation of naphthalene by strain NaphS6 exhibited a normal hydrogen isotope fractionation (ε_H = −11 ± 2‰ to −47 ± 4‰), whereas an inverse hydrogen isotope fractionation was observed for the aerobic strains (ε_H = +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

Dual Carbon–Bromine Stable Isotope Analysis Allows Distinguishing Transformation Pathways of Ethylene Dibromide

The present study investigated dual carbon–bromine isotope fractionation of the common groundwater contaminant ethylene dibromide (EDB) during chemical and biological transformations, including aerobic and anaerobic biodegradation, alkaline hydrolysis, Fenton-like degradation, debromination by Zn(0) and reduced corrinoids. Significantly different correlation of carbon and bromine isotope fractionation (Λ_C/Br) was observed not only for the processes following different transformation pathways, but also for abiotic and biotic processes with, the presumed, same formal chemical degradation mechanism. The studied processes resulted in a wide range of Λ_C/Br values: Λ_C/Br = 30.1 was observed for hydrolysis of EDB in alkaline solution; Λ_C/Br between 4.2 and 5.3 were determined for dibromoelimination pathway with reduced corrinoids and Zn(0) particles; EDB biodegradation by <i>Ancylobacter aquaticus</i> and <i>Sulfurospirillum multivorans</i> resulted in Λ_C/Br = 10.7 and 2.4, respectively; Fenton-like degradation resulted in carbon isotope fractionation only, leading to Λ_C/Br ∞. Calculated carbon apparent kinetic isotope effects (¹³C-AKIE) fell with 1.005 to 1.035 within expected ranges according to the theoretical KIE, however, biotic transformations resulted in weaker carbon isotope effects than respective abiotic transformations. Relatively large bromine isotope effects with ⁸¹Br-AKIE of 1.0012–1.002 and 1.0021–1.004 were observed for nucleophilic substitution and dibromoelimination, respectively, and reveal so far underestimated strong bromine isotope effects

Relative Contributions of <i>Dehalobacter</i> and Zerovalent Iron in the Degradation of Chlorinated Methanes

The role of bacteria and zerovalent iron (Fe⁰) in the degradation of chlorinated solvents in subsurface environments is of interest to researchers and remediation practitioners alike. Fe⁰ used in reactive iron barriers for groundwater remediation positively interacted with enrichment cultures containing <i>Dehalobacter</i> strains in the transformation of halogenated methanes. Chloroform transformation and dichloromethane formation was up to 8-fold faster and 14 times higher, respectively, when a <i>Dehalobacter</i>-containing enrichment culture was combined with Fe⁰ compared with Fe⁰ alone. The dichloromethane-fermenting culture transformed dichloromethane up to three times faster with Fe⁰ compared to without. Compound-specific isotope analysis was employed to compare abiotic and biotic chloroform and dichloromethane degradation. The isotope enrichment factor for the abiotic chloroform/Fe⁰ reaction was large at −29.4 ± 2.1‰, while that for chloroform respiration by <i>Dehalobacter</i> was minimal at −4.3 ± 0.45‰. The combined abiotic/biotic dechlorination was −8.3 ± 0.7‰, confirming the predominance of biotic dechlorination. The enrichment factor for dichloromethane fermentation was −15.5 ± 1.5‰; however, in the presence of Fe⁰ the factor increased to −23.5 ± 2.1‰, suggesting multiple mechanisms were contributing to dichloromethane degradation. Together the results show that chlorinated methane-metabolizing organisms introduced into reactive iron barriers can have a significant impact on trichloromethane and dichloromethane degradation and that compound-specific isotope analysis can be employed to distinguish between the biotic and abiotic reactions involved