No abstract

Primary features of hydrogen and carbon isotope fractionation during toluene degradation were studied to evaluate if analysis of isotope signatures can be used as a tool to monitor biodegradation in contaminated aquifers. D/H hydrogen isotope fractionation during microbial degradation of toluene was measured by gas chromatography. Per-deuterated toluene-d 8 and nonlabeled toluene were supplied in equal amounts as growth substrates, and kinetic isotope fractionation was calculated from the shift of the molar ratios of toluene-d ؊1 . The D/H isotope fractionation (b ‫؍‬ ؊1.580) was 30% greater than that in growth experiments with D. cetonicum. Mass spectroscopic analysis of the product benzylsuccinate showed that H atoms abstracted from the toluene molecules by the enzyme were retained in the same molecules after the product was released. Our findings revealed that the use of deuterium-labeled toluene was appropriate for studying basic features of D/H isotope fractionation. Similar D/H fractionation factors for toluene degradation by anaerobic bacteria, the lack of significant temperature dependence, and the strong fractionation suggest that analysis of D/H fractionation can be used as a sensitive tool to assess degradation activities. Identification of the first enzyme reaction in the pathway as the major fractionating step provides a basis for linking observed isotope fractionation to biochemical reactions.

The influence of microbial degradation on the 13C/12C isotope composition of aromatic hydrocarbons is presented using toluene as a model compound. Four different toluene-degrading bacterial strains grown in batch culture with oxygen, nitrate, ferric iron or sulphate as electron acceptors were studied as representatives of different environmental redox conditions potentially prevailing in contaminated aquifers. The biological degradation induced isotope shifts in the residual, non-degraded toluene fraction and the kinetic isotope fractionation factors alphaC for toluene degradation by Pseudomonas putida (1.0026 +/- 0.00017), Thauera aromatica (1.0017 +/- 0.00015), Geobacter metallireducens (1.0018 +/- 0.00029) and the sulphate-reducing strain TRM1 (1.0017 +/- 0.00016) were in the same range for all four species, although they use at least two different degradation pathways. A similar 13C/12C isotope fractionation factor (alphaC = 1.0015 +/- 0.00015) was observed in situ in a non-sterile soil column in which toluene was degraded under sulphate-reducing conditions. No carbon isotope shifts resulting from soil-hydrocarbon interactions were observed in a non-degrading soil column control with aquifer material under the same conditions. The results imply that microbial degradation of toluene can produce a 13C/12C isotope fractionation in the residual hydrocarbon fraction under different environmental conditions.

13 C/ 12 C and D/H stable isotope fractionation during aerobic degradation was determined for Pseudomonas putida strain mt-2, Pseudomonas putida strain F1, Ralstonia pickettii strain PKO1, and Pseudomonas putida strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.Intrinsic microbial degradation is an important process in elimination of contaminants in polluted aquifers, which can be used for the sustainable cleanup of contaminated sites. However, cost-effective remediation strategies such as natural attenuation require a profound knowledge of the microbial degradation processes in the subsurface. Although biodegradation of aromatic hydrocarbons by aerobic and anaerobic bacteria has been investigated in detail in laboratory systems (11,32), assessment at field sites remains difficult. Stable carbon isotope analysis is one approach to quantify microbial activities in situ. For laboratory cultures, isotope fractionation has been shown to occur during degradation of aromatic hydrocarbons, such as toluene (1,19,20), or chlorinated hydrocarbons, such as trichloroethene (3,7,26). In addition, in contaminated field sites, carbon isotope fractionation could be observed and was interpreted to be indicative of microbial degradation in situ (15,24). For toluene as a model compound, it has been demonstrated that isotope fractionation is caused mainly by the first enzyme reaction in the degradation pathway, whereas transport to and into the cell appears not to be relevant for fractionation. The extent of isotope fractionation is considered to be independent of differences in the growth kinetics of the bacteria (20). Isotope fractionation during anaerobic degradation of toluene was on the same order of magnitude for denitrifying, iron (III)-reducing, sulfate-reducing, and fermenting bacteria (1, 19), probably because, in these cases, degradation was initiated by benzylsuccinate synthase. This finding suggests that, in anoxic environments, isotope fractionation could be applied to assess biological degradation quantitatively, as has been worked out recently for several aquifers (23, 25).The objective of this study was to examine whether carbon and hydrogen isotope fractionation could be used to quantify intrinsic biodegradation as well in oxic environments. Previous studies with the aerobic bacterium Pseudomonas putida strain mt-2 showed an extent of isotope fractionation similar to that of anaerobic toluene-degrading strains (19), whereas isotope fractionation during toluene degradation by undefined aerobic microbial communities was not detected (26). Therefore, we started a systematic investigation of the effects of different oxygenase enzymes and stable isotope fractionation.P. putida strain mt-2 (20), Ralstonia pickettii strain PKO1 (J. J. Kukor, Rutgers University, New Brunswick, N.J.), and P. putida strain F1 (A. M. Cook, Konstanz, German...

A strictly anaerobic bacterium, strain OX39, was isolated with o-xylene as organic substrate and sulfate as electron acceptor from an aquifer at a former gasworks plant contaminated with aromatic hydrocarbons. Apart from o-xylene, strain OX39 grew on m-xylene and toluene and all three substrates were oxidized completely to CO 2 . Induction experiments indicated that o-xylene, m-xylene, and toluene degradation were initiated by different specific enzymes. Methylbenzylsuccinate was identified in supernatants of cultures grown on o-xylene and m-xylene, and benzylsuccinate was detected in supernatants of toluene-grown cells, thus indicating that degradation was initiated in all three cases by fumarate addition to the methyl group. Strain OX39 was sensitive towards sulfide and depended on Fe(II) in the medium as a scavenger of the produced sulfide. Analysis of the PCR-amplified 16S rRNA gene revealed that strain OX39 affiliates with the gram-positive endospore-forming sulfate reducers of the genus Desulfotomaculum and is the first hydrocarbonoxidizing bacterium in this genus. Keywords Sulfate reduction · BTEX · Benzylsuccinate synthase · Desulfotomaculum IntroductionPetroleum hydrocarbons are among the most abundant groundwater contaminants and can be found in gasworks plants, landfill leachates, and accidental fuel spills (US-EPA 1999). The mono-aromatic hydrocarbons benzene, toluene, ethylbenzene, and xylene isomers (BTEX) are putatively mutagenic or carcinogenic substances and make-up more than 50% by weight of the water-soluble gasoline fraction (Coleman et al. 1984). Due to their relatively high solubility, they are mobile with the groundwater flow and form contaminant plumes in aquifers. The degradative potential of anaerobic bacteria towards aromatic hydrocarbons in situ was investigated in several studies and a decrease of BTEX compounds could be demonstrated under denitrifying, Fe(III)-reducing, sulfate-reducing, and methanogenic conditions (Dolfing et al. 1990;Kazumi et al. 1997;Reinhard et al. 1997;Gieg et al. 1999;Phelps and Young 2001). Numerous anaerobic bacterial cultures have been enriched in the past, and pure strains have been isolated. Of all BTEX compounds, toluene has been studied most extensively with respect to its anaerobic degradation. Pure cultures of toluene-degrading bacteria that use NO 3 -, Fe(III), or SO 4 2-as electron acceptors have been isolated (Lovley and Lonergan 1990;Rabus et al. 1993;Seyfried et al. 1994;Zhou et al. 1995;Beller et al. 1996). Under anoxic conditions, degradation of toluene proceeds via addition of fumarate to the methyl group (Biegert et al. 1996;Beller and Spormann 1997b;Leuthner et al. 1998;Rabus and Heider 1998;Kane et al. 2002).Consumption of o-xylene was discovered simultaneously with toluene degradation in methanogenic consortia (Edwards and Grbic-Galic 1994). Later, transformation of o-xylene to o-methyl homologs of benzylsuccinate by toluene-degrading strains was reported; however, no further oxidation steps could be observed (Beller and Spormann...

The occurrence and removal of 58 pharmaceuticals, endocrine disruptors, corrosion inhibitors, biocides, and pesticides, were assessed in the wastewater treatment plant (WWTP) of the city of Lausanne, Switzerland, as well as in the effluent-receiving water body, the Vidy Bay of Lake Geneva. An analytical screening method to simultaneously measure all of the 58 micropollutants was developed based on ultra performance liquid chromatography coupled to a tandem mass spectrometer (UPLC-MS/MS). The selection of pharmaceuticals was primarily based on a prioritization study, which designated them as environmentally relevant for the Lake Geneva region. Except for the endocrine disruptor 17alpha-ethinylestradiol, all substances were detected in 24-h composite samples of wastewater entering the WWTP or in the treated effluent. Of these compounds, 40% were also detected in raw drinking water, pumped from the lake 3 km downstream of the WWTP. The contributions of dilution and degradation to micropollutant elimination between the WWTP outlet and the raw drinking water intake were established in different model scenarios using hypothetical residence times of the wastewater in Vidy Bay of 1, 4, or 90 d. Concentration decrease due to processes other than dilution was observed for diclofenac, beta-blockers, several antibiotics, corrosion inhibitors, and pesticides. Measured environmental concentrations (MECs) of pharmaceuticals were compared to the predicted environmental concentrations (PECs) determined in the prioritization study and agreed within one order of magnitude, but MECs were typically greater than the corresponding PECs. Predicted no-effect concentrations of the analgesic paracetamol, and the two antibiotics ciprofloxacin and sulfamethoxazole, were exceeded in raw drinking water samples and therefore present a potential risk to the ecosystem.

Pharmaceuticals are substances designed to have a biological effect in humans. Their presence in the environment, especially in surface waters, is of increasing concern because of their potential risk to non-target species. A large number of pharmaceuticals are on the market; for example, approximately 2,000 active ingredients are approved in Europe, and many of them have already been detected in surface water. It is therefore crucial to select the substances that may do the most harm to the environment prior to performing measurements and extensive risk assessment. In the present study, a method to determine a list of pharmaceuticals to survey in surface water is proposed. Inclusion of substances on the list was based on a screening procedure, the analytical feasibility, and previous knowledge of pharmaceuticals detected in water. The screening procedure proposed here is an improvement on the standard procedure of the European Medicine Evaluation Agency (EMEA). It is designed to decrease the number of pharmaceuticals to be evaluated in a stepwise manner, thus decreasing the number of data necessary for the evaluation. We applied our approach to determine a list of 37 pharmaceuticals and four hormones to survey in a specific region of Switzerland, the Lake Geneva area, and discussed the advantages and weak points of the method.

No abstract