Development of an advanced on-line position-specific stable carbon isotope system and application to methyl tert-butyl ether

We present an advanced system for on-line position-specific carbon isotope analysis. The main limitation of on-line intramolecular isotope ratio measurements has been that optimal pyrolytic fragments are obtained mostly at temperatures where the analyte has not completely reacted. As a result of undetermined isotopic fractionation, the isotopic signatures of the pyrolysis products are not strictly equal to these of the equivalent moieties in the parent molecule. We designed a pyrolytic unit in which both temperature and reaction time are variable parameters, enabling determination of the enrichment factor of the pyrolysis at optimal temperature by construction of a Rayleigh plot. In the case of methyl tert-butyl ether (MTBE) presented here, a 'pre-pyrolysis' fractionation of MTBE leading to a depletion of 0.9 parts per thousand was discovered and the enrichment factor of the optimal pyrolysis reaction was determined at -1.7 parts per thousand. Absolute delta C-13 values of two functional groups of MTBE - the methoxy group and the 2-methylpropane group - could be determined with 95% confidence intervals of 0.4 parts per thousand and 0.5 parts per thousand, respectively

Physico-chemical and biological characterization of an aquifer polluted with ETBE

International audiencePetroleum compounds and among them, gasoline, is the most massively used chemicals worldwide and, as a consequence gasoline derives compounds are the most frequently found contaminants in groundwate

Isotopic Characterization ( 2 H, 13 C, 37 Cl, 81 Br) of Abiotic Degradation of Methyl Bromide and Methyl Chloride in Water and Implications for Future Studies

International audienceMethyl bromide (CH 3 Br) and methyl chloride (CH 3 Cl) significantly contribute to stratospheric ozone depletion. The atmospheric budgets of both compounds are unbalanced with known degradation processes outweighing known emissions. Stable isotope analysis may be capable to identify and quantify emissions and to achieve a balanced budget. Degradation processes do, however, cause isotope fractionation in methyl halides after emission and hence knowledge about these processes is a crucial prerequisite for any isotopic mass balance approach. In the current study, triple-element isotope analysis (2 H, 13 C, 37 Cl/ 81 Br) was applied to investigate the two main abiotic degradation processes of methyl halides (CH 3 X) in fresh and seawater: hydrolysis and halide exchange. For CH 3 Br, nucleophilic attack by both H 2 O and Cl − caused significant primary carbon and bromine isotope effects accompanied by a secondary inverse hydrogen isotope effect. For CH 3 Cl only nucleophilic substitution by H 2 O was observed at significant rates causing large primary carbon and chlorine isotope effects and a secondary inverse hydrogen isotope effect. Observed dual-element isotope ratios differed slightly from literature values for microbial degradation in water and hugely from radical reactions in the troposphere. This bodes well for successfully distinguishing and quantifying degradation processes in atmospheric methyl halides using triple-element isotope analysis. ■ INTRODUCTION Methyl chloride (CH 3 Cl, chloromethane) and methyl bromide (CH 3 Br, bromomethane) together contribute about 30% to halogen induced ozone loss even though atmospheric concentrations are very low: 540 pptv and 7 pptv, respectively. 1 CH 3 Cl and CH 3 Br are emitted by both anthropogenic and natural sources such as fumigation for quarantine and preshipment treatment (for CH 3 Br), 2 marine macroalgae, 3 salt marshes, 4 soils, 5 biomass burning, 6 and tropical plants. 7 Main degradation processes for both of these compounds are reaction with OH and Cl radicals in the troposphere, 8 degradation in oceans 9 and soils. 10 The atmospheric budgets of both compounds are unbalanced with known degradation processes exceeding the best estimates of known emissions by approximately 20% for CH 3 Cl and 30% for CH 3 Br. 1,11 A better understanding of emission and degradation processes will be necessary in order to better quantify emission and degradation of CH 3 X and to improve budget estimates. Previous studies suggested that degradation in oceans is primarily driven by the abiotic processes hydrolysis and halide exchange as well as microbial degradation. 9,12,13 To a minor extent, hydrolysis may also contribute to degradation of CH 3 Br in soils. 14 Hydrolysis and halide exchange of CH 3 X (CH 3 Cl and CH 3 Br) are both nucleophilic substitution reactions (S N 2) following second order reaction kinetics. The attacking nucleophiles are either water (H 2 O), hydroxide ions (OH −), or halide ions such as Cl − and Br − (Y −): 15−1

Anaerobic Microbial Degradation of Hydrocarbons: From Enzymatic Reactions to the Environment

Hydrocarbons are abundant in anoxic environments and pose biochemical challenges to their anaerobic degradation by microorganisms. Within the framework of the Priority Program 1319, investigations funded by the Deutsche Forschungsgemeinschaft on the anaerobic microbial degradation of hydrocarbons ranged from isolation and enrichment of hitherto unknown hydrocarbon-degrading anaerobic microorganisms, discovery of novel reactions, detailed studies of enzyme mechanisms and structures to process-oriented in situ studies. Selected highlights from this program are collected in this synopsis, with more detailed information provided by theme-focused reviews of the special topic issue on 'Anaerobic biodegradation of hydrocarbons' [this issue, pp. 1-244]. The interdisciplinary character of the program, involving microbiologists, biochemists, organic chemists and environmental scientists, is best exemplified by the studies on alkyl-/arylalkylsuccinate synthases. Here, research topics ranged from in-depth mechanistic studies of archetypical toluene-activating benzylsuccinate synthase, substrate-specific phylogenetic clustering of alkyl-/arylalkylsuccinate synthases (toluene plus xylenes, p-cymene, p-cresol, 2-methylnaphthalene, n-alkanes), stereochemical and co-metabolic insights into n-alkane-activating (methylalkyl) succinate synthases to the discovery of bacterial groups previously unknown to possess alkyl-/arylalkylsuccinate synthases by means of functional gene markers and in situ field studies enabled by state-of-the-art stable isotope probing and fractionation approaches. Other topics are Mo-cofactor-dependent dehydrogenases performing O-2-independent hydroxylation of hydrocarbons and alkyl side chains (ethylbenzene, p-cymene, cholesterol, n-hexadecane), degradation of p-alkylated benzoates and toluenes, glycyl radical-bearing 4-hydroxyphenylacetate decarboxylase, novel types of carboxylation reactions (for acetophenone, acetone, and potentially also benzene and naphthalene), W-cofactor-containing enzymes for reductive dearomatization of benzoyl-CoA (class II benzoyl-CoA reductase) in obligate anaerobes and addition of water to acetylene, fermentative formation of cyclohexanecarboxylate from benzoate, and methanogenic degradation of hydrocarbons

Antibiotic resistance indicator genes in biofilm and planktonic microbial communities after wastewater discharge

The spread of bacteria with antibiotic resistance genes (ARGs) in aquatic ecosystems is of growing concern as this can pose a risk of transmission to humans and animals. While the impact of wastewater treatment plant (WWTP) effluent on ARG abundance in surface waters has been studied extensively, less is known about the fate of ARGs in biofilms. The proximity and dense growth of microorganisms in combination with the accumulation of higher antibiotic concentrations in biofilms might render biofilms a reservoir for ARGs. Seasonal parameters such as water temperature, precipitation, and antibiotic concentrations should be considered as well, as they may further influence the fate of ARGs in aquatic ecosystems. Here we investigated the effect of WWTP effluent on the abundance of the sulfonamide resistance genes sul1 and sul2, and the integrase gene intI1 in biofilm and surface water compartments of a river in Germany with a gradient of anthropogenic impact using quantitative PCR. Furthermore, we analyzed the bacterial community structure in both compartments via 16S rRNA gene amplicon sequencing, following the river downstream. Additionally, conventional water parameters and sulfonamide concentrations were measured, and seasonal aspects were considered by comparing the fate of ARGs and bacterial community diversity in the surface water compartment between the summer and winter season. Our results show that biofilm compartments near the WWTP had a higher relative abundance of ARGs (up to 4.7%) than surface waters (10 km) of the WWTP in the hot and dry summer season than in winter. This finding is likely a consequence of the higher proportion of wastewater and thus wastewater-derived microorganisms in the river during summer periods. We observed distinct bacterial communities and ARG abundance between the biofilm and surface water compartment, but even greater variations when considering seasonal and spatiotemporal parameters. This underscores the need to consider seasonal aspects when studying the fate of ARGs in aquatic ecosystems

Evaluation of ethyl tert-butyl ether biodegradation in a contaminated aquifer by compound specific isotope analysis and in situ microcosms

Ethyl tert-butyl ether (ETBE) is an upcoming groundwater pollutant in Europe whose environmental fate has been less investigated thus far. In the present study, we investigated the in situ biodegradation of ETBE in a fuel-contaminated aquifer using compound-specific stable isotope analysis (CSIA) and in situ microcosms in combination with total lipid fatty acid (TLFA)-stable isotope probing (SIP). In a first field investigation, CSIA revealed no significant carbon isotope fractionation but low hydrogen isotope fractionation of up to +14 ¿ along the prevailing anoxic ETBE plume suggesting biodegradation of ETBE. Ten months later, oxygen injection was conducted to enhance the biodegradation of petroleum hydrocarbons (PH) at the field site. Within the framework of this remediation measure, in situ microcosms loaded with [13C6]-ETBE (BACTRAP®s) were exposed for 119 days in selected groundwater wells to assess the biodegradation of ETBE by TLFA-SIP under the following conditions: (i) ETBE as main contaminant; (ii) ETBE as main contaminant subjected to oxygen injection; (iii) ETBE plus other PH; (iv) ETBE plus other PH subjected to oxygen injection. Under all conditions investigated, significant 13C-incorporation into microbial total lipid fatty acids extracted from the in situ microcosms was found, providing clear evidence of ETBE biodegradation

Microbial methane formation in deep aquifers of a coal-bearing sedimentary basin, Germany

Published version. Also available at http://dx.doi.org/10.3389/fmicb.2015.00200Coal-bearing sediments are major reservoirs of organic matter potentially available for methanogenic subsurface microbial communities. In this study the specific microbial community inside lignite-bearing sedimentary basin in Germany and its contribution to methanogenic hydrocarbon degradation processes was investigated. The stable isotope signature of methane measured in groundwater and coal-rich sediment samples indicated methanogenic activity. Analysis of 16S rRNA gene sequences showed the presence of methanogenic Archaea, predominantly belonging to the orders Methanosarcinales and Methanomicrobiales, capable of acetoclastic or hydrogenotrophic methanogenesis. Furthermore, we identified fermenting, sulfate-, nitrate-, and metal-reducing, or acetogenic Bacteria clustering within the phyla Proteobacteria, complemented by members of the classes Actinobacteria, and Clostridia. The indigenous microbial communities found in the groundwater as well as in the coal-rich sediments are able to degrade coal-derived organic components and to produce methane as the final product. Lignite-bearing sediments may be an important nutrient and energy source influencing larger compartments via groundwater transport

Critical evaluation of the 2D-CSIA scheme for distinguishing fuel oxygenate degradation reaction mechanisms

Although the uniform initial hydroxylation of methyl tert-butyl ether (MTBE) and other oxygenates during aerobic biodegradation has already been proven by molecular tools, variations in carbon and hydrogen enrichment factors (εC and εH) have still been associated with different reaction mechanisms (McKelvie et al. Environ. Sci. Technol. 2009, 43, 2793-2799). Here, we present new laboratory-derived εC and εH data on the initial degradation mechanisms of MTBE, ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) by chemical oxidation (permanganate, Fenton reagents), acid hydrolysis and aerobic bacteria cultures (species of Aquincola, Methylibium, Gordonia, Mycobacterium, Pseudomonas and Rhodococcus). Plotting of Δδ2H/ Δδ13C data from chemical oxidation and hydrolysis of ethers resulted in slopes (Λ values) of 22 ± 4 and between 6 and 12, respectively. With A. tertiaricarbonis L108, R. zopfii IFP 2005 and Gordonia sp. IFP 2009, εC was low (<|-1|¿) and εH insignificant. Fractionation obtained with P. putida GPo1 was similar to acid hydrolysis and M. austroafricanum JOB5 and R. ruber DSM 7511 displayed Λ values previously only ascribed to anaerobic attack. The fractionation patterns rather correlate with the employment of different P450, AlkB and other monooxygenases, likely catalyzing ether hydroxylation via different transition states. Our data questions the value of 2D-CSIA for a simple distinguishing of oxygenate biotransformation mechanisms, therefore caution and complementary tools are needed for proper interpretation of groundwater plumes at field sites

Compartment-specific effect of sulfamethoxazole at low μg/L concentrations on microbial nitrogen assimilation in a river system

Sulfamethoxazole (SMX) is one of the most frequently detected antibiotics in rivers, with concentrations occasionally exceeding the predicted no-effect concentration (PNEC). The impact of such concentrations on the microbial activity of riverine microbial communities remains poorly studied. Here, we investigated the effect of SMX concentrations at the upper end of reported PNEC values (12.5 µg/L) on microbial communities in flume systems with either near-pristine or wastewater-impacted river water. Using a combination of microbiological and chemical methods, we found that SMX was persistent in both near-pristine and wastewater-impacted river water over a time course of 63 days, and had no significant impact on the planktonic bacterial community composition. However, there was an increase in microbial activity after SMX addition. Tracking 15N incorporation in both sample types using Nanoscale Secondary Ion Mass Spectrometry (NanoSIMS) and an Elemental Analyser - Isotope Ratio Mass Spectrometer (EA-IRMS) revealed that SMX concentrations in the test range (10, 100 and 1000 µg/L) enhanced nitrogen assimilation from ammonium up to 64 %. The highest increase was found almost always at 10 µg/L SMX. The response was stronger in samples from the near-pristine site compared to the wastewater-impacted site, and in planktonic biomass compared to biofilms. Overall, our findings reveal a transient increase in microbial nitrogen assimilation with environmentally relevant concentrations of SMX in a habitat-specific manner, but not of SMX degradation, which could be of significance for nutrient dynamics and primary productivity in impacted rivers