To evaluate the intrinsic bioremediation potential in an anoxic tar oil-contaminated aquifer at a former gasworks site, groundwater samples were qualitatively and quantitatively analyzed by compound-specific isotope analysis (CSIA) and signature metabolites analysis (SMA). 13C/12C fractionation data revealed conclusive evidence for in situ biodegradation of benzene, toluene, o-xylene, m/p-xylene, naphthalene, and 1-methylnaphthalene. In laboratory growth studies, 13C/12C isotope enrichment factors for anaerobic degradation of naphthalene (epsilon = -1.1 +/- 0.4) and 2-methylnaphthalene (epsilon = -0.9 +/- 0.1) were determined with the sulfate-reducing enrichment culture N47, which was isolated from the investigated test site. On the basis of these and other laboratory-derived enrichment factors from the literature, in situ biodegradation could be quantified for toluene, o-xylene, m/p-xylene, and naphthalene. Stable carbon isotope fractionation in the field was also observed for ethylbenzene, 2-methylnaphthalene, and benzothiophene but without providing conclusive results. Further evidence for the in situ turnover of individual BTEX compounds was provided by the presence of acetophenone, o-toluic acid, and p-toluic acid, three intermediates in the anaerobic degradation of ethylbenzene, o-xylene, and p-xylene, respectively. A number of groundwater samples also contained naphthyl-2-methylsuccinic acid, a metabolite that is highly specific for the anaerobic degradation of 2-methylnaphthalene. Additional metabolites that provided evidence on the anaerobic in situ degradation of naphthalenes were 1-naphthoic acid, 2-naphthoic acid, 1,2,3,4-tetrahydronaphthoic acid, and 5,6,7,8-tetrahydronaphthoic acid. 2-Carboxybenzothiophene, 5-carboxybenzothiophene, a putative further carboxybenzothiophene isomer, and the reduced derivative dihydrocarboxybenzothiophene indicated the anaerobic conversion of the heterocyclic aromatic hydrocarbon benzothiophene. The combined application of CSIA and SMA, as two reliable and independent tools to collect direct evidence on intrinsic bioremediation, leads to a substantially improved evaluation of natural attenuation in situ.

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...

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The stable hydrogen isotope ratios (d D) of leaf wax n-alkanes record valuable information on plant and ecosystem water relations. It remains, however, unknown if leaf wax n-alkane d D values record only environmental variation during the brief period of time of leaf growth or if leaf wax n-alkane d D values are affected by environmental variability throughout the entire lifespan of a leaf. To resolve these uncertainties, we irrigated Populus trichocarpa trees with a pulse of deuterium-enriched water and used compound-specific stable hydrogen isotope analyses to test if the applied tracer could be recovered from leaf wax n-alkanes of leaves that were at different stages of their development during the tracer application. Our experiment revealed that only leaf wax n-alkanes from leaves that had developed during the time of the tracer application were affected, while leaves that were already fully matured at the time of the tracer application were not. We conclude from our study that under controlled environmental conditions, leaf wax n-alkanes are synthesized only early in the ontogeny of a leaf. Our experiment has implications for the interpretation of leaf wax n-alkane d D values in an environmental context, as it suggests that these compounds record only a brief period of the environmental variability that a leaf experiences throughout its life.

This study reveals the first analyses of the composition and activity of the microbial community of a saline CO 2 storage aquifer. Microbial monitoring during CO 2 injection has been reported. By using Fluorescence in situ Hybridisation (FISH), we have shown that the microbial community was strongly influenced by the CO 2 injection. Before CO 2 arrival, up to 6x10 6 cells ml -1 were detected by DAPI-staining at a depth of 647 m below the surface. The microbial community was dominated by the domain Bacteria that represented approximately 60 to 90 % of the total cell number, with Proteobacteria and Firmicutes as the most abundant phyla comprising up to 47 % and 45 % of the entire population, respectively. Both the total cell counts as well as the counts of the specific physiological groups revealed quantitative and qualitative changes after CO 2 arrival. Our study revealed temporal shifts in the microbial community from chemoorganotrophic to chemolithotrophic populations, as evidenced by the outcompetition of sulphate-reducing bacteria by methanogenic archaea. In addition, an enhanced activity of the microbial population after five months CO 2 storage indicated that the bacterial community was able to adapt to the extreme conditions of the deep biosphere and to the extreme changes of these atypical conditions.