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

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

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

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

Genome and proteome analyses show the gaseous alkane degrader Desulfosarcina sp. strain BuS5 as an extreme metabolic specialist

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Chen, S.-C., Ji, J., Popp, D., Jaekel, U., Richnow, H.-H., Sievert, S. M., & Musat, F. Genome and proteome analyses show the gaseous alkane degrader Desulfosarcina sp. strain BuS5 as an extreme metabolic specialist. Environmental Microbiology, 24, (2022): 1964-1976, https://doi.org/10.1111/1462-2920.15956.The metabolic potential of the sulfate-reducing bacterium Desulfosarcina sp. strain BuS5, currently the only pure culture able to oxidize the volatile alkanes propane and butane without oxygen, was investigated via genomics, proteomics and physiology assays. Complete genome sequencing revealed that strain BuS5 encodes a single alkyl-succinate synthase, an enzyme which apparently initiates oxidation of both propane and butane. The formed alkyl-succinates are oxidized to CO2 via beta oxidation and the oxidative Wood–Ljungdahl pathways as shown by proteogenomics analyses. Strain BuS5 conserves energy via the canonical sulfate reduction pathway and electron bifurcation. An ability to utilize long-chain fatty acids, mannose and oligopeptides, suggested by automated annotation pipelines, was not supported by physiology assays and in-depth analyses of the corresponding genetic systems. Consistently, comparative genomics revealed a streamlined BuS5 genome with a remarkable paucity of catabolic modules. These results establish strain BuS5 as an exceptional metabolic specialist, able to grow only with propane and butane, for which we propose the name Desulfosarcina aeriophaga BuS5. This highly restrictive lifestyle, most likely the result of habitat-driven evolutionary gene loss, may provide D. aeriophaga BuS5 a competitive edge in sediments impacted by natural gas seeps.This study was financed by the Max Planck Society and by the Helmholtz Association of German Research Centres. The draft genome was sequenced as part of the U.S. Department of Energy Joint Genome Institute (DOE-JGI) Community Science Program project 1078203 awarded to S. M. Sievert and F. Musat. The work conducted by the DOE-JGI, a DOE Office of Science User Facility, is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. Lynne Goodwin (Los Alamos National Laboratory) is acknowledged for project management support of the draft genome sequencing. Further support was provided by the U.S. National Science Foundation grant MCB-0702677 (to SMS), and by the Helmholtz Association grant ERC-RA-0020 (to FM). We acknowledge the Centre for Chemical Microscopy (ProVIS) platform at the Helmholtz Centre for Environmental Research – UFZ, for using their analytical facilities. ProVIS is supported by European Regional Development Funds (EFRE – Europe funds Saxony). We acknowledge the Bundesministerium für Bildung und Forschung (BMBF)-funded German Network for Bioinformatics Infrastructure de.NBI (031A537B, 031A533A, 031A538A, 031A533B, 031A535A, 031A537C, 031A534A, 031A532B) for providing computational resources

Improving protein extraction and separation methods for investigating the metaproteome of anaerobic benzene communities within sediments

BTEX compounds such as benzene are frequent soil and groundwater contaminants that are easily biodegraded under oxic conditions by bacteria. In contrast, benzene is rather recalcitrant under anaerobic conditions. The analysis of anoxic degradation is often hampered by difficult sampling conditions, limited amounts of biomass and interference of matrix compounds with proteomic approaches. In order to improve the procedure for protein extraction we established a scheme consisting of the following steps: dissociation of cells from lava granules, cell lysis by ultrasonication and purification of proteins by phenol extraction. The 2D-gels revealed a resolution of about 240 proteins spots and the spot patterns showed strong matrix dependence, but still differences were detectable between the metaproteomes obtained after growth on benzene and benzoate. Using direct data base search as well as de novo sequencing approaches we were able to identify several proteins. An enoyl-CoA hydratase with cross species homology to Azoarcus evansii, is known to be involved in the anoxic degradation of xenobiotics. Thereby the identification confirmed that this procedure has the capacity to analyse the metaproteome of an anoxic living microbial community

The fate of sulfonamide resistance genes and anthropogenic pollution marker intI1 after discharge of wastewater into a pristine river

Introduction: Currently there are sparse regulations regarding the discharge of antibiotics from wastewater treatment plants (WWTP) into river systems, making surface waters a latent reservoir for antibiotics and antibiotic resistance genes (ARGs). To better understand factors that influence the fate of ARGs in the environment and to foster surveillance of antibiotic resistance spreading in such habitats, several indicator genes have been proposed, including the integrase gene intI1 and the sulfonamide resistance genes sul1 and sul2. Methods: Here we used quantitative PCR and long-read nanopore sequencing to monitor the abundance of these indicator genes and ARGs present as class 1 integron gene cassettes in a river system from pristine source to WWTP-impacted water. ARG abundance was compared with the dynamics of the microbial communities determined via 16S rRNA gene amplicon sequencing, conventional water parameters and the concentration of sulfamethoxazole (SMX), sulfamethazine (SMZ) and sulfadiazine (SDZ). Results: Our results show that WWTP effluent was the principal source of all three sulfonamides with highest concentrations for SMX (median 8.6 ng/l), and of the indicator genes sul1, sul2 and intI1 with median relative abundance to 16S rRNA gene of 0.55, 0.77 and 0.65%, respectively. Downstream from the WWTP, water quality improved constantly, including lower sulfonamide concentrations, decreasing abundances of sul1 and sul2 and lower numbers and diversity of ARGs in the class 1 integron. The riverine microbial community partially recovered after receiving WWTP effluent, which was consolidated by a microbiome recovery model. Surprisingly, the relative abundance of intI1 increased 3-fold over 13 km of the river stretch, suggesting an internal gene multiplication. Discussion: We found no evidence that low amounts of sulfonamides in the aquatic environment stimulate the maintenance or even spread of corresponding ARGs. Nevertheless, class 1 integrons carrying various ARGs were still present 13 km downstream from the WWTP. Therefore, limiting the release of ARG-harboring microorganisms may be more crucial for restricting the environmental spread of antimicrobial resistance than attenuating ng/L concentrations of antibiotics