Combined Application of Stable Carbon Isotope Analysis and Specific Metabolites Determination for Assessing In Situ Degradation of Aromatic Hydrocarbons in a Tar Oil-Contaminated Aquifer

Abstract

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.

DNA-SIP identifies sulfate-reducing Clostridia as important toluene degraders in tar-oil-contaminated aquifer sediment

“…To ensure more or less constant substrate supply over a long period at low concentrations, XAD7 adsorber resin was used, which lowered initial toluene concentrations in the cosms from B1 mM to an average of B66 mM over the first 50 days of incubation. This was only twofold of the maximal toluene concentrations of B33 mM reported close to the investigated well (Griebler et al, 2004). In addition, the added 10 mM of sulfate were in a similar relation to reported in situ concentrations of up to B5 mM (Griebler et al, 2004).…”

“…This was only twofold of the maximal toluene concentrations of B33 mM reported close to the investigated well (Griebler et al, 2004). In addition, the added 10 mM of sulfate were in a similar relation to reported in situ concentrations of up to B5 mM (Griebler et al, 2004). Most relevantly, we used freshly sampled aquifer sediment material containing a complex in situ microbial community for our experiment.…”

“…Sampling site and sample acquisition Sampling was carried out at a former gasworks site (Testfeld Sü d), which has been intensively studied and described elsewhere (Herfort et al, 1998;Bockelmann et al, 2001;Zamfirescu and Grathwohl, 2001;Griebler et al, 2004). Aquifer sediment from the bottom of monitoring well B49 (at B7.50 m depth, groundwater table at B3.25 m) was retrieved in December 2006 with an aqua-sampler (Bü rkle, Bad Bellingen, Germany).…”

“…Especially for hydrocarbon-contaminated groundwater systems, our present understanding of the diversity and ecology of relevant degraders is still in its infancy. At a former coal gasification plant in Germany with an underlying tar-oil-contaminated aquifer, the anaerobic degradation of benzene, toluene, ethylbenzene and xylenes (BTEX) has been reported to occur mostly under conditions of sulfate reduction (Griebler et al, 2004). One of the few available Gram-positive BTEX-degrading sulfate reducers, Desulfotomaculum sp.…”

Carbon isotope fractionation during aerobic biodegradation of n-alkanes and aromatic compounds in unsaturated sand

“…Usually intrinsic biodegradation by indigenous microbial populations is the key process for contaminant elimination and methods are required to demonstrate its efficacy. In groundwater studies, compound-specific isotope analysis (CSIA) has been used increasingly to demonstrate in situ biodegradation of various types of organic contaminants (Kelley et al, 1997;Sturchio et al, 1998;Hunkeler et al, 1999;Sherwood Lollar et al, 2001;Kolhatkar et al, 2002;Mancini et al, 2002;Meckenstock et al, 2002;Song et al, 2002;Kirtland et al, 2003;Richnow et al, 2003a,b;Chu et al, 2004;Griebler et al, 2004;Steinbach et al, 2004;Hunkeler et al, 2005;Morrill et al, 2005). In contrast, only a few studies have investigated the use of CSIA in the unsaturated zone (Stehmeier et al, 1999;Kirtland et al, 2005).…”