Environ. Sci. Technol. 2008, 42, 6065–6072
Comparative Assessments of Benzene, Toluene, and Xylene Natural Attenuation by Quantitative Polymerase Chain Reaction Analysis of a Catabolic Gene, Signature Metabolites, and Compound-Specific Isotope Analysis H A R R Y R . B E L L E R , * ,† S T A C I R . K A N E , † TINA C. LEGLER,† JENNIFER R. MCKELVIE,‡ BARBARA SHERWOOD LOLLAR,‡ FRANCESCA PEARSON,† LIANNA BALSER,† AND DOUGLAS M. MACKAY§ Lawrence Livermore National Laboratory, Livermore, California, Department of Geology, University of Toronto, Toronto, ON, Canada, and Department of Land, Air & Water Resources, University of California, Davis, California
Received April 7, 2008. Revised manuscript received May 31, 2008. Accepted June 9, 2008.
A controlled-release study conducted at Vandenberg Air Force Base involved the injection of anaerobic groundwater amended with benzene, toluene, and o-xylene (BToX; 1-3 mg/L each) in two parallel lanes: lane A injectate contained no ethanol, whereas lane B injectate contained ∼500 mg/L ethanol. As reported previously by Mackay and co-workers, ethanol led to slower BToX disappearance in lane B. Here, we report on assessments of BToX natural attenuation by three independent and specific monitoring approaches: signature metabolites diagnostic of anaerobic TX metabolism (benzysuccinates), compound-specific isotope analysis (CSIA), and quantitative polymerase chain reaction (qPCR) analysis of a catabolic gene involved in anaerobic TX degradation (bssA). In combination, the three monitoring methods provided strong evidence of in situ TX biodegradation in both lanes A and B; however, no single method provided strong evidence for TX biodegradation in both lanes. Benzylsuccinates were detected almost exclusively in lane B, where slower TX degradation and higher residual TX concentrations led to higher metabolite concentrations. In contrast, CSIA provided evidence of TX biodegradation almost exclusively in lane A, as greater degradation rates led to more pronounced isotopic enrichment. qPCR analyses of bssA were more complex. Evidence of increases in bssA copy number (up to 200-fold) after the release started was stronger in lane A, but higher absolute bssA copy number (and bacterial abundance, based on 16S rRNA * Corresponding author (current address): Lawrence Berkeley National Laboratory, 1 Cyclotron Rd., Mail Stop 70A-3317, Berkeley, CA 94720. E-mail
[email protected]. Phone (510) 486-7321. Fax (510) 486-7152. † Lawrence Livermore National Laboratory. ‡ University of Toronto. § University of California. 10.1021/es8009666 CCC: $40.75
Published on Web 07/16/2008
2008 American Chemical Society
genes) was observed in lane B, where bacteria genetically capable of anaerobic TX degradation may have been growing primarily on ethanol or its metabolites rather than TX.
Introduction Advances in monitoring in situ BTEX (benzene, toluene, ethylbenzene, and xylene) biodegradation have been driven by dramatic improvements in understanding of the biochemistry and genetics underlying anaerobic alkylbenzene metabolism (1, 2) as well as advances in mass spectrometric and molecular techniques over the past 10 to 15 years. Some of these monitoring methods have focused on metabolites (benzylsuccinates) or genes (bssA) associated with benzylsuccinate synthase (BSS), an enzyme that catalyzes the first step of anaerobic toluene and xylene degradation (1). Advantages of the BSS reaction with regard to in situ monitoring include the unique and diagnostic nature of its cognate metabolite (3) as well as its relevance to physiologically and phylogenetically diverse bacteria. The presence of BSS has been observed in cultures that degrade toluene under a wide range of environmentally relevant electron-accepting conditions, including denitrifying, sulfate-reducing, ferric iron-reducing, and methanogenic conditions (1, 2, 4, 5). BSS has also been shown to catalyze the first step in anaerobic xylene degradation (6, 7), and a homologous enzyme apparently catalyzes ethylbenzene degradation under sulfatereducing conditions (8). To date, no enzyme other than BSS has been described that catalyzes the first step of anaerobic toluene or xylene degradation. Specific monitoring techniques for in situ alkylbenzene metabolism in field studies of contaminated aquifers have included: (a)mass spectrometric analysis of benzylsuccinates (9–13), after this class of compounds was first identified as signature metabolites in 1995 (9), (b) compound-specific isotope analysis (CSIA) (12, 14–17), and (c) quantitative polymerase chain reaction (qPCR) analysis of a catabolic gene essential to anaerobic toluene and xylene degradation, bssA. The latter method was first developed in a 2002 laboratory study (18) and was recently used in a field study (19). In some cases, the first two approaches (signature metabolites and CSIA) have been used together in the same study (12, 16, 17), but, to our knowledge, there have been no field studies in which all three were used together. In this article, we report the use of all three specific and credible monitoring approaches (signature metabolites, CSIA, and qPCR of bssA) in a controlled-release field study designed to examine the effects of ethanol on the natural attenuation of benzene, toluene, and o-xylene (BToX). This study was funded by the State of California to address the substitution of methyl tert-butyl ether with ethanol as a fuel oxygenate, and the inevitable release of ethanol along with gasoline hydrocarbons from leaking underground fuel storage tanks. Mackay et al. (20) presented geochemical data (including concentrations of BToX and electron acceptors) from this study previously and showed that ethanol led to the depletion of sulfate in the aquifer, resulting in the development of methanogenic/acetogenic conditions and slower disappearance of BToX. The geochemical data and assessment of the effect of ethanol on BToX disappearance rates will not be repeated here. Instead, our objective is to evaluate the performance of three independent monitoring methods in assessing (i.e., providing definitive evidence for) in situ, anaerobic BToX metabolism during this controlled-release study. VOL. 42, NO. 16, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Groundwater monitoring wells sampled for this study. Injection wells for continuous controlled release of BToX and ethanol are indicated with an X. The wide arrows indicate the approximate paths of the injected water and solutes in lanes A and B.
Experimental Section Controlled-Release Site and Experimental Design. The field site and experimental design were described in detail by Mackay and co-workers (20) and will only be briefly summarized here. The study was conducted between May 2004 and February 2005 at Site 60, Vandenberg Air Force Base (VAFB) in California. The release area was the site of a leaking fuel storage tank in 1994; however, the tanks and piping were excavated the following year, and the pre-release concentrations of BTX in this area in 2004 were low (typically
