Evidence for in situ degradation of mono-and polyaromatic hydrocarbons in alluvial sediments based on microcosm experiments with 13C-labeled contaminants
Introduction
The assessment of biodegradation in contaminated aquifers has become an increasingly important issue. This is closely related to the acceptance of intrinsic bioremediation (or monitored natural attenuation) as a means to manage contaminated sites (Scow and Hicks, 2005, US-EPA, 1999). In this context, it is necessary to distinguish between mineralization leading to complete removal of pollutants, partial degradation to – possibly harmful – products, and physico-chemical processes such as sorption and dilution which locally reduce concentrations but do not remove pollutants (Norris, 1994, Wiedemeier et al., 1999). The differentiation becomes even more complicated, as in the majority of cases rather undefined mixtures of pollutants are present. Among the main contributors to environmental pollution are aromatic hydrocarbons. Although polyaromatic hydrocarbons (PAHs) are still continuously generated by combustion of organic matter and atmospherically deposited, immense contaminations of the environment typically originate from industrial sites, from coal gasification, landfill leachates, or accidental petroleum and fuel spills (US-EPA, 1999). The microbial degradation of aromatic hydrocarbons is hampered for three reasons: Firstly, because of the high activation energy needed to chemically attack aromatic rings (Boll et al., 2002), secondly by the substances' toxicities towards bacteria (Jonker et al., 2006, Loibner et al., 2004), and thirdly because aromatic hydrocarbons have a tendency to sorb on hydrophobic surfaces. This tendency is increasing with the number of their aromatic rings (Kleineidam et al., 2002, Kubicki, 2006). Sorption and poor solubility in water makes the major fraction of PAHs inaccessible for bacterial degradation (Bosma et al., 1997, Volkering et al., 1992). Although one controversial study reported on bacteria that directly accessed the sorbed substrate (Guerin and Boyd, 1997), presently it is believed that PAHs adsorbed on soil particles, solid PAH crystals, or hydrocarbons dissolved in non-aqueous phase liquids (NAPLs) remain unavailable to bacteria (Johnsen et al., 2005).
Until now, no standard procedure exists that has the potential to demonstrate biodegradation of organic environmental pollutants in contaminated environments. In practice, often two or more independent approaches are combined (Smets and Pritchard, 2003, Watanabe and Hamamuray, 2003), among these are evidence of concentration decrease of contaminants over time and distance (Wiedemeier et al., 1999), enrichment of heavy stable isotopes in the remaining fraction of organic contaminants (Hunkeler et al., 2002, Meckenstock et al., 2002, Sherwood Lollar et al., 1999), radiotracer studies (Bianchin et al., 2006, Conrad et al., 1997), succession of redox zones in the field (Kuhn and Suflita, 1989, Vroblesky and Chapelle, 1994); accumulation of signature metabolites (Beller, 2002, Elshahed et al., 2001), investigation of the intrinsic microbial biodegradation potential in microcosm studies – in parts with 14C-labeled substrates – (Aelion and Bradley, 1991, Ambrosoli et al., 2005, Lovley, 2001); characterization of the bacterial community by molecular techniques (Amann et al., 1995, Bakermans et al., 2002), tracing 13C in fatty acid profiles of bacteria (Geyer et al., 2005), and detection of bacterial enzymes (Hanson et al., 1999, Heinaru et al., 2005, Hendrickx et al., 2006, Löffler et al., 2000). Each of these methods has its advantages and limitations, and therefore it is advisable to base investigations on several approaches, even more when dealing with complex field sites. It must be noted that only isotope-based methods (13C or 14C) targeting CO2 are suitable to prove complete mineralization of single pollutants. Minor amounts of substrate which are incompletely mineralized or used to synthesize cellular building blocks remain unconsidered. Since the use of radioactive markers such as 14C is usually restricted, radio-labeled compounds were almost exclusively-applied in laboratory experiments (Chapelle et al., 1996, Conrad et al., 1997). Recovery of 14C-CO2 in the headspace of the respective test systems is a very sensitive tool for proving complete mineralization of the labeled substrate (Bradley et al., 2002). However, radioactivity causes difficulty in sample treatment, detection, and waste disposal, and as a consequence the interest in 13C-based methods is increasing. Moreover, 13C-labeled substances can be used in lab and field studies.
This study focuses on the evaluation of the intrinsic bacterial degradation potential of mono- and polyaromatic hydrocarbons at the site of a former cokery located in direct proximity to a river. In terms of risk assessment, it is imperative to investigate whether or not biodegradation is sufficiently rapid to prevent dissolved contaminants from reaching the river. Here, we present a new method based on the addition of 13C-labeled contaminants and the subsequent analysis of 13C-CO2 generated during biodegradation. Hence this approach conveys compound-specific information on mineralization and 13C was found to be an ideal marker to test the fate of one particular contaminant out of a mixture. Microcosm experiments with sediments from the former cokery site were performed under conditions resembling in situ conditions using benzene, naphthalene, and acenaphthene as substrates. All three substances were major contaminants at the field site and belong to the 16 priority PAHs designated by the United States Environmental Protection Agency (US-EPA; http://www.epa.gov/) and to the 33 priority substances recently defined in the EU Water Framework Directive (EC, 2006).
Section snippets
Field site
A microcosm study was conducted using alluvial sediments from the premises of a former cokery in the alluvial plain of the river Meuse. The area covering more than 7 ha of land is located in the region of Luik, Belgium, in direct proximity to the river (Fig. 1). The upper 2.5 m are partly made up of backfill material and the sediments below consist of silt, sand, clay, and small gravels. A gravel aquifer is below the depth of 5.5–7 m. The groundwater flow direction is South-East towards the Meuse (
Aerobic intrinsic degradation potential at the field site
The distribution of the intrinsic biodegradation potential was investigated in sediments sampled from the vadose and from the saturated zone of 7 different drilling locations (Fig. 1). Microcosms were set up with non-labeled benzene, naphthalene, or acenaphthene (U13) and with the respective 13C-labeled compounds (all 7 locations). In parallel, controls of all 7 locations were investigated that were either without addition of substrate or that were inhibited by sodium azide but contained 13
Isotope shifts in CO2 in experiments with non-labeled substrates
One condition to be met in order to apply the 13C-based screening method, was to verify whether the 13C-CO2 in the headspace was a degradation product of the individual labeled compounds or of any organic or inorganic substance containing about 1% of naturally abundant 13C. Parallel incubations of sediments from drilling location U13 with 13C-labeled and non-labeled substrates (Fig. 3) were compared to each other and served to quantify the increase that came from labeled compounds. In former
Conclusions
A semi-quantitative screening method based on 13C-CO2 analysis by GC-IRMS has been developed and evaluated. This new approach permitted to investigate the intrinsic biodegradation potential under in situ-like conditions, e.g. low temperature, no light and no shaking, using a high sediment to groundwater ratio and groundwater from the site. In situ biodegradation of benzene, naphthalene, and acenaphthene in alluvial sediments from the site of a former cokery was evident in aerobic and anaerobic
Acknowledgements
This work was supported by the European Union FP6 Integrated Project AquaTerra (Project No. GOCE 505428) under the thematic priority sustainable development, global change and ecosystems. SPAQuE is thanked for authorization and support concerning the field campaign.
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