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Dual Biomarkers of Anaerobic Hydrocarbon Degradation in Historically Contaminated Groundwater Amita. R. Oka,† Craig. D. Phelps,† Xiangyang Zhu,‡ Diane. L. Saber,‡ and L. Y. Young*,† †
Department of Environmental Sciences, School of Environmental and Biological Sciences, Rutgers, The State University of New Jersey, New Brunswick, New Jersey 08901, United States ‡ Environmental Science and Forensic Chemistry Center, Gas Technology Institute, Des Plaines, Illinois 60018, United States
bS Supporting Information ABSTRACT: This study reports that ongoing in situ anaerobic hydrocarbon biodegradation at a manufactured gas plant impacted site is occurring, 9 years after the initial investigation. Groundwater samples from the site monitoring wells (MW) were analyzed for biomarkers by GC-MS, end-point PCR, and quantitative PCR (qPCR). Metabolic biomarkers included specific intermediates of anaerobic naphthalene and/or 2-methylnaphthalene degradation: 2-naphthoic acid (2-NA); 5,6,7,8-tetrahydro-2-NA (TH-2-NA); hexahydro-2-NA (HH-2-NA); and carboxylated-2-methylnaphthalene (MNA). The analogues of gene bssA, encoding alpha subunit of enzyme benzylsuccinate synthase, were used as a genetic biomarker. Results indicate 12 orders of magnitude higher abundance of total bacteria in the impacted wells than in the unimpacted wells. End-point PCR analysis of bssA gene, with degenerate primers, indicated the presence of hydrocarbon degrading bacteria within the plume. In qPCR analysis, using primers based on toluene-degrading denitrifying or sulfate-reducing/methanogenic bacteria, bssA genes were detected only in MW-24, located downstream from the source. Metabolic biomarkers were detected in multiple wells. The highest abundance of 2-NA (6.7 μg/L), TH-2-NA (2.6 μg/L), HH-2-NA, and MNA was also detected in MW-24. The distribution of two independent biomarkers indicates that the site is enriched for anaerobic hydrocarbon biodegradation and provides strong evidence in support of natural attenuation.
’ INTRODUCTION In situ bioremediation at hydrocarbon-impacted sites can be established by demonstrating that biodegradation is actually taking place in the subsurface.1 Independently, biomarkers or indicators of in situ biodegradation of hydrocarbons such as metabolic intermediates (metabolic biomarkers),27 catabolite genes (genetic biomarkers),8,9 and isotope fractionation10,11 have been used in multiple field studies. Only a few studies have demonstrated the use of multiple biomarkers of anaerobic hydrocarbon degradation,1216 including a study by Beller et. al.17 in which the authors demonstrated the use of three different biomarkers in a field test in which benzene, toluene, and o-xylene were injected in the subsurface with or without ethanol. It was found that analysis of multiple biomarkers, rather use of a single biomarker, provided evidence for in situ biodegradation in this field test. Also, Griebler et al.12 found that metabolic biomarkers of polycyclic aromatic hydrocarbons (PAHs) could be identified at a field site, indicating microbial activity even when compound specific isotope analysis (CSIA) did not provide conclusive data. These studies indicate that analysis of multiple biomarkers is important to reliably establish that in situ biodegradation is taking place. r 2011 American Chemical Society
Choice of metabolic as well as genetic biomarkers is largely driven by controlled laboratory studies. Several metabolic intermediates of anaerobic degradation of PAHs have been identified in laboratory studies, and pathways for degradation have been proposed as noted in Figure 1.1822 Products of anaerobic degradation of PAHs including carboxylated substrates like 2-naphthoic acid (2-NA), carboxylated-2-methylnaphthalene, or methylnaphthoic acid (MNA) and products of ring-reduction of carboxylated substrates including tetrahydro-2-naphthoic acid (TH-2-NA) and hexahydro-2-naphthoic acid (HH-2-NA)1921 have been detected at field sites.3,4 Development of genetic biomarkers has focused around genes encoding Bss like enzymes, which are known to catalyze anaerobic biodegradation of a variety of aromatic substrates, including toluene, o-, m-, and p-xylene and ethylbenezene,2325 are also involved in degradation of PAHs like 2-methylnaphthalene,26 and are likely also involved in anaerobic naphthalene degradation.22 Received: November 16, 2010 Accepted: March 8, 2011 Revised: February 28, 2011 Published: March 25, 2011 3407
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Figure 1. Proposed pathways of naphthalene and 2-methylnaphthalene degradation. (A) Hydroxylation of naphthalene to naphthalenol,18 (B) methylation of naphthalene to 2-methylnaphthalene,22 (C) carboxylation of naphthalene to 2-naphthoic acid followed by ring-reduction,20,21 (D) addition of 2-methylnaphthalene to fumarate,34 and (E) carboxylation of 2-methylnaphthalene.19 Metabolic intermediates used as biomarkers in this study are identified by an asterisk.
Indeed, bss gene analogues have also been identified by our lab in the anaerobic biodegradation of alkanes as well.27 Enzymes other than Bss like enzymes that can anaerobically activate hydrocarbons still remain undiscovered, and therefore, the choice of a genetic biomarker is limited to bssA gene analogues at the time of this study. To advance the use of multiple biomarkers as a tool for routine site assessment and remediation projects, studies documenting their effectiveness need to be performed on representative sites, those contaminated over a range of time periods, short to long, and with multiple contaminants. Such studies can provide comparative analysis of the sensitivity of each biomarker in the field. Considering these mostly unmet needs, we framed the overall goal of our research to provide long-term evaluation of the bioremediation potential of a site chronically impacted with a mixture of hydrocarbons. To meet our research goal, we chose to study the distribution of two different biomarkers, (i) metabolic intermediates of anaerobic PAH degradation and (ii) the gene bssA (as a genetic biomarker of anaerobic hydrocarbon degradation), at a historically contaminated manufactured gas plant (MGP) site in Glassboro, NJ, which was previously studied by our laboratory 9 years ago.4 Together, the analysis of the selected biomarkers would provide assessment of anaerobic biodegradation processes involving different types of hydrocarbons at the site, rather than degradation of only one class of compounds. In addition, a comparison of our data could be done with previous results4 to gain a long-term understanding of the biodegradation processes at the site.
The contamination present at our study site was a result of operation of the MGP over decades, and the site has been monitored over time but without active remediation. Our laboratory had examined the site earlier4 and found at that time that intermediates of anaerobic PAH degradation, as well as 2-methylbenzyl succinate (2-MBS), a product of o-xylene addition to fumarate, were detectable, indicating natural attenuation was taking place at the site. At that time, the bssA gene was not available as a biomarker. The same site was sampled again in 2008, although not all the same monitoring wells (MW) were available for this study (MW-24, MW-25, MW-29, and MW-30 were sampled in both studies). We hypothesized that, at such a site, both biomarkers (metabolic intermediates and analogues of catabolic gene bssA) would be detected, and their relative enrichment would be affected by the distribution of contaminants. Our research, therefore, provides evidence for long-term enrichment of naturally occurring anaerobic hydrocarbon biodegradation processes in the groundwater of a chronically contaminated site, which was monitored, but not actively treated or remediated.
’ MATERIALS AND METHODS Groundwater Sampling. Six monitoring wells were selected for this study (see Figure 2), and groundwater was collected from each in July 2008. MW-40 is located within the source area, while MW-29 is near the edge of the contaminant plume, and MW-24 3408
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Figure 2. Location of the monitoring wells at the MGP site in Glassboro, NJ is shown in the map. Impacted wells are shown in black while unimpacted wells are shown in gray. The scale at the bottom of the map reads: 1.5 cm is 250 ft (0.015 m is 76.2 m). Dissolved oxygen (D.O.), oxidation reduction potential (ORP), and pH at each well are given.
is downstream of the source, within the plume. MW-15, -25, and 30 are outside the plume. Sampling was performed during the routine monitoring of the plume. The wells were purged until the parameters measured (pH, temperature, dissolved oxygen (D. O.), turbidity, oxidation reduction potential (ORP), and conductivity) had stabilized, after which samples for analysis of biomarkers were collected. Field measurements are presented in Figure 2 and in Supporting Information S1. For analysis of bssA genes, 2 to 4 liters of groundwater samples were filtered in the field under vacuum through 0.22 μM pore size sterile filters using a sampling manifold. From each well, at least 2 L of water was filtered, but only 1 L of water was filtered through each filter disk. Filters were placed in sterile 50 mL sterile tubes and were frozen immediately in dry ice. For analysis of metabolic intermediates, 4 L of water sample was directly collected in acid cleaned 4 L amber colored glass bottles. Samples were acidified immediately with 6N HCl to pH 2 and stored in an ice packed cooler for transport to the laboratory. In the laboratory, the water samples were stored at 4 °C, while the filters were frozen at 20 °C until further processing. Extraction and Analysis of Metabolic Intermediates. Groundwater samples were extracted and analyzed as described by Phelps et al.3 Slight modifications in the protocol were made; 1 L of sample was extracted two times, with 60 mL of methylene chloride, and an injection standard was not added to the sample. The solvent extracts and standards were derivatized with bis(trimethyl-silyl)trifluoro-acetamide (Sigma Aldrich) prior to GC-MS analysis. Sample extracts and standards of 2-NA (98%, Sigma Aldrich), TH-2-NA (97%, Sigma Aldrich), and phenanthrene carboxylic acid (PHE-CA) (Sigma Aldrich) (isomer not specified) were analyzed using a GC-MS (Shimadzu GC-2010 coupled with GCMS-QP2010) with a Restek RTX-5SIL MS column (30 m 0.25 mm, I.D. 0.25 μm, Bellefonte, PA) with the following program: injector temperature, 250 °C; initial temperature, 80 °C, for 1 min; ramp rate, 10 °C min1; oven temperature, 300 °C for 2 min. A comparison of the retention time (RT) and the GC-MS mass spectra of the samples to the standards were used for identification. Since standards for HH-2-NA,
decalin-2-carboxylate, and MNA were not available, the identification of these metabolites was done by a comparison with published mass spectra.19,21 The diagnostic fragments m/z 115, 127, 155, 185, 229, and 244 of 2-NA (RT 15.31 min), m/z 159, 189, 233, and 248 of TH-2-NA (RT 15.29 min), m/z 161, 191, 235, and 250 of HH-2-NA, and m/z 115, 141, 169, 183, 199, 243, and 258 of MNA were used for identification. Base peaks m/z 229 of 2-NA and m/z 233 of TH-2-NA, m/z 235 of HH-2-NA, and m/z 243 of MNA were used for quantification. DNA Extraction and PCR Analysis. DNA was extracted from filters using the DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA), using the manufacturer’s protocol with slight modifications. Extract from each filter disk was eluted using eight elution columns, and all extracts were pooled together. The DNA concentrations in the extracts were measured with a UV spectrophotometer at 260 nm, and the extracts were stored at 20 °C until further use. End-point PCR analysis of the 16S rRNA gene and bssA gene was performed on each DNA extract. Eubacteria specific 16S rRNA gene primers28 were used to ensure the presence of eubacteria in the water samples. The PCR program for 16S rRNA genes included denaturation at 95 °C for 5 min followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min 30 s, followed by 10 min extension at 72 °C. The 16S rRNA gene PCR included 1 PCR buffer, with 0.2 mM of dNTPs, 0.2 μM of each primer, and 1.5 units of Red Taq in a 50 μL reaction. The bssA gene PCR primers (7772f/8546r) and the program were as described by Winderl et al.8 All reagents were from Sigma (St. Louis, MO). The quantitative PCR (qPCR) analysis with SYBR green assay was performed on all DNA extracts for quantification of the target genes in the samples. For 16S rRNA gene and bssA gene analysis, primers developed by Suzuki et al.29 and Beller et al.17,30 were used, respectively. Two pairs of primers were used for bssA gene analysis; one was based on toluene-degrading denitrifying bacteria (we defined it as bssA-Denitrif qPCR),30 and the other was based on sulfate-reducing/methanogenic toluene-degrading bacteria (we defined it as bssA-Sulf-Meth qPCR).17 A modification of the qPCR protocols by Beller et al.17,30 was used for the 16S rRNA gene and bssA gene analysis. Primers were obtained from 3409
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Environmental Science & Technology Sigma (St. Louis, MO). For qPCR analysis, 2 PCR buffer, 0.5 mM dNTPs, 3 mM MgCl2, 1.25 units of Taq, and 0.5 units of Amperase UNG, along with 5 μL of diluted or undiluted DNA extract, were used. For analysis of 16S rRNA gene and bssA gene (bssA-Denitrif qPCR), 300 nM of primers were used, while 400 nM of primers were used for bssA-Sulf-Meth qPCR. For each bssA gene qPCR, 10 μg of BSA (New England Biolabs, Ipswich, MA) was also used. All other PCR reagents were obtained from Applied Biosystems (Foster City, CA). The program for qPCR analysis included incubation for 2 min at 50 °C and then denaturation at 94 °C for 10 min followed by 45 cycles of 94 °C for 15 s and 58 °C for 1 min. This was followed by a dissociation stage, 30 s at 94 °C, 1 min at 58 °C, and 30 s at 94 °C. For 16S rRNA gene analysis, only 35 cycles were used. All samples and standards were analyzed in triplicates on R 300 Real time PCR analyzer. Calibration curves for bssA-Denitrif qPCR and 16S rRNA gene qPCR were prepared using dilutions of Thauera aromatica T1 genomic DNA, while the calibration curve for bssA-Sulf-Meth qPCR was obtained using dilutions of Desulfobacterium cetonicum DSM7627 (Desulfosarcina cetonica DSM7267) genomic DNA. The number of gene copies in the genomic DNA extracts were calculated using the equation, Gene copies μL1 ¼ ðμg DNA μL1 =bp genome1 Þ ðbp μg1 DNAÞðgenes genome1 Þ For qPCR analysis, we assumed that (i) the size of the genomic DNA of T1 and D. cetonicum each was 4.6 Mbp; (ii) the genomic DNA of T1 had only one 16S rRNA gene and 1 bssA gene, and genomic DNA of D. cetonicum had only 1 bssA gene; (iii) the calibration curves were representative of the other bacteria in the environment. It was also assumed that both bssA gene primer sets used in this study for qPCR analysis would be able to detect bssA genes in the environmental samples.
’ RESULTS AND DISCUSSION Characteristics of the Water Samples. Field measurements (Figure 2) show that impacted wells have low D.O. (0.3 to 0.4 mg/L) and negative ORP (170 to 9 mV) as compared to the positive ORP (77 and 46 mV) and relatively higher D.O. (2.5 and 4.4 mg/L) in the unimpacted wells (MW-15 and MW-25, respectively). This indicates that the wells within the plume area had depleted levels of dissolved oxygen and had developed reducing conditions. Therefore, microbial activity in the impacted wells is expected to be occurring mainly under anaerobic conditions. MW-30, which is outside the plume area, had a relatively high ORP but low D.O., which suggests the presence of microbial activity or redox reactions in this area. Previous data (Supporting Information 2 and 3) showed that detectable sulfate levels were present in the groundwater, but nitrate was not always detected and suggests that the site was not methanogenic. The specific electron accepting processes, however, were not determined. Contaminants Present in the Water Samples. As shown in Table 1, the wells within the plume contained naphthalene and/ or benzene, toluene, ethylbenzene, and xylenes (BTEX), and two wells (MW-40 and -24) also contained phenanthrene. The wells outside the plume, however, did not have any detectable BTEX or naphthalene, though one well (MW-30) contained very low levels of phenanthrene (0.02 μg/L). The highest concentration of BTEX and naphthalene was present in MW-40, which is within the source area. It can also be observed that BTEX concentration
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Table 1. Concentration of Organic Contaminants in the Samples (μg/L)a monitoring wells outside the plume 15
a
25
30
inside the plume 24
29
40
BTEX
121.9
2-methylnaphthalene
naphthalene phenanthrene
0.02
19 0.05
0.5 0.05
1724
BTEX: benzene, toluene, ethylbenzene, and xylenes. : not detected
in MW-29 was higher than in MW-24 but lower than in MW-40. Since MW-29 is down-gradient of MW-24 and the source, loss of BTEX due to physical processes such as dilution and dispersion are expected to decrease the concentration of BTEX from the source. Thus, the BTEX at MW-29 may represent contamination from a separate source. Alternatively, the heterogeneity in groundwater conditions may have affected the results. Metabolic Intermediates. All six groundwater samples were analyzed for the presence of the metabolic intermediates of anaerobic PAH degradation identified in Figure 1. Of these metabolic intermediates, four were detected in the groundwater samples: 2-NA, TH-2-NA, HH-2-NA, and MNA (Figure 3). Since TH2-NA, HH-2-NA, and MNA are formed only during anaerobic degradation of PAHs (naphthalene1921,31), their presence in the groundwater specifically indicates anaerobic biodegradation processes at the site. 2-NA is a product of carboxylation of naphthalene, which is considered to be an initial step in the anaerobic degradation process.20 Although 2-NA can also be produced aerobically from 2-methylnaphthalene,32,33 its presence in the groundwater along with the ring-reduction intermediates TH-2-NA and HH-2-NA, which are formed only under anaerobic conditions, strongly supports that anaerobic microbial degradation of naphthalene and 2-methylnaphthalene is occurring in the groundwater. MNA is a metabolite of anaerobic 2-methylnaphthalene degradation19 and was detected in MW-24. In July 2008, 2-methylnaphthalene was not detected in the groundwater samples, but naphthalene was detected (Table 1); both were detected in the groundwater at MW-24 in Sept 2007 (see Supporting Information S4). Like other anaerobic metabolites (TH-2-NA and HH2-NA), MNA is metabolized further and is unlikely to have accumulated in the groundwater. Since MNA can be a product of 2-methylnaphthalene or naphthalene degradation (via proposed methylation pathway, Figure 1B), we conclude that detection of MNA indicates recent anaerobic degradation of naphthalene or 2-methylnaphthalene. Concentration or abundance of the metabolites detected was compared across all the wells. As shown in Figure 3A, the highest concentration of 2-NA (6.7 μg/L) and TH-2-NA (2.6 μg/L) was detected in MW-24, although the highest concentration of naphthalene (Table 1) was in MW-40. In addition, the highest abundance of HH-2-NA was detected in MW-24 (Figure 3B), with the relative abundance in the order MW-24 > MW-40 > MW-30. Detection of 2-NA, TH-2-NA, and HH-2-NA, metabolites of anaerobic hydrocarbon degradation in high abundance at MW-24, suggests that the microbial community in the vicinity of this well is enriched for anaerobic PAH degradation. Considering 3410
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Figure 3. Concentration of 2-naphthoic acid (2-NA) and tetrahydro2-naphthoic acid (TH-2-NA) (A), and the abundance of hexahydro2-naphthoic acid (HH-2-NA) and carboxylated methylnaphthalene (MNA) (B) in the groundwater samples. TH-2 NA was also detected in monitoring wells 15, 25, and 29 but was below our quantification limit.
that these metabolites are readily metabolized, it can also be concluded that their presence at the site indicates ongoing anaerobic microbial degradation processes and not accumulation of metabolites over time. Interestingly, 2-NA and HH-2-NA were detected in wells outside the plume, albeit in relatively low abundance (see Figure 3). Compared to their parent compounds (naphthalene or 2-methylnaphthalene), the products of their metabolism are more water-soluble due to the presence of dissociable carboxyl group (e.g., solubility of naphthalene is 0.03 g/L, while solubility of 2-NA, a metabolite of naphthalene, is 0.5 g/L). Thus, the distribution of metabolites to the wells outside the contaminant plume is likely a result of their solubility and the groundwater flow. This may also be the explanation for MW-29, which had no detectable naphthalene or 2-methylnaphthalene, but contained detectable levels of 2-NA. Young and Phelps4 also reported on the in situ biodegradation of the alkylbenzenes (at MW-24 and MW-25) and PAHs (at MW-29 and MW-30) at the site. The data provided by Young and Phelps4 and that obtained by us can be used for an overall comparison of microbial processes at the site. A relevant summary of the earlier study4 for a comparative analysis is provided here. The authors noted that (i) the concentration of 2-MBS in the wells was related to the concentration of toluene, (ii) the highest abundance of 2-MBS was found in MW-24 ( MW-24> MW40> MW-25> MW-15> MW-30. Significantly, the abundance of 16S rRNA genes in all the impacted wells was 12 orders of magnitude higher than in the nonimpacted wells. These results indicate enrichment of bacterial population specifically in the impacted wells and are consistent with the site history. The relative increase in bacterial population density in the impacted area likely reflects growth of hydrocarbon degrading bacteria and may also reflect the growth of bacteria not capable of activating hydrocarbons but capable of the use of metabolic intermediates as a carbon source. In comparison, studies have shown that exposure of a microbial community to hydrocarbons over a period of a few days to months has shown no increase or up to 2 log scale increase in 16S rRNA gene abundance.17,30 BssA Gene Analysis. As shown in Figure 5, the bssA-Denitrif qPCR assay yielded positive results only when DNA extracts from MW-24 were used for analysis, while other samples had no detectable signal. The melt curve analysis showed that the fluorescence in sample MW-24 was due to the presence of only one major peak, similar to that in the standards (Tm = 83.3 °C). The negative control did not have any detectable PCR product. These results indicate that bacteria containing bssA genes detectable by bssA-Denitrif qPCR were enriched at MW-24 (2.7 103 copies/ L of bssA gene analogues were detected), which is downstream from the source. Similar results were obtained in MW-24 for bssA-Sulf-Meth qPCR assay (1.7 104 copies/L of bssA gene analogues were detected). These results show that MW-24 contains a relatively high abundance of the analogues of bssA genes, while bssA gene copies in other monitoring wells were below the detection limit of this assay. Please see Supporting Information S6 for details. A comparison of bssA gene analysis shows that, although putative bssA gene analogues could be detected by end-point PCR in all the impacted wells, bssA genes could be quantified only in MW24 with both qPCR assays. To interpret these results, it is essential to consider the following points. First, the primers used for the bssA gene qPCR assay and those for the bssA end-point PCR were based on different reference sequences. In addition, they are designed to amplify different parts of bssA genes and 3412
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Environmental Science & Technology result in amplicons of different lengths. End-point bssA gene PCR primers (7772f/8546r) were based on bssA gene sequences in 7 toluene degrading betaproteobacterial pure cultures that amplify a fragment ∼794 bp long.8 These primers were tested by Winderl et al.8 and could successfully amplify bssA genes from sulfatereducing bacteria, in addition to those from denitrifying bacteria in pure cultures. In addition, Geobacter like bssA gene sequences as well as unrelated bssA sequences were identified from BTEX and PAH impacted sites with the same primer set.8 The bssADenitrif qPCR primers produce an amplicon of 132 bp. These were based on 4 toluene degrading betaproteobacterial bssA gene sequences and were reported to be specific for denitrifying toluenedegrading bacteria (primarily Thauera and Azoarcus8) and were not sensitive toward the sulfate-reducing toluene degrading bacteria.30 The bssA-Sulf-Meth qPCR primers and assay were based on bssA gene sequences from two sulfate-reducing pure cultures and one sequence from a methanogenic consortium17 and amplify a much shorter region that is only 97 bp long. It was designed for analysis of samples from sulfate-reducing environments, although data on the specificity of the primers is lacking. Thus, the two bssA assays (end-point and qPCR) cannot be compared directly due to differences in amplicon length and specificity. Additionally, it should also be noted that qPCR assay methods used in this study had different detection limits. The detection limit of bssA-Sulf-Meth qPCR was 1.59 10 4 copies/L, and it is similar to observations that were made by Beller et al.17 They reported that the detection limit was 3 103 gene copies/L in filtered groundwater extracts, while it was much higher in the samples showing inhibition. The detection limit of bssA-Denitrif qPCR in our study was 7.93 102 gene copies/L. The detection limit of end-point bssA gene PCR primers has not been reported before and was not determined in our study. Considering above factors, the end-point PCR and qPCR data was used only for sample assessment and a direct comparison between these two types of data was not performed. Overall, these data indicate that the impacted site is enriched for hydrocarbon degrading bacteria and that the hydrocarbon degrading microbial community in the area of MW-24 is enriched for organisms containing bssA genes characteristic of both denitrifying as well as sulfate-reducing bacteria. Earlier, Beller et al.17 reported on the abundance of bssA genes at a field site that was stimulated by the addition of benzene, toluene, and o-xylene (BToX) to the groundwater. Our study reported here, on the other hand, is at a site that is historically contaminated, undergoing monitoring, but has not been altered or manipulated. The abundance of bssA genes in our study is comparable to the lower range detected by Beller et al.17 (background concentration was reported as 2 104 bssA gene copies/ L, but after injection of BToX, 104 to more than 106 bssA copies/ L were detected). In our study, the highest abundance of bssA genes detected by bssA-Sulf-Meth qPCR and bssA-Denitrif qPCR were 2.55 103 and 1.66 104 gene copies/L, respectively, in MW-24. In our study, MW-24 had detectable naphthalene (0.5 μg/L) but BToX was not detected, while Beller et al.17 injected 13 mg/L of each monoaromatic hydrocarbon. The difference in the abundance of bssA gene copies in our study and that reported by Beller et al.17 is not surprising since monoaromatic hydrocarbons, as in BToX, are readily degraded as compared to PAHs like naphthalene and 2-methynaphthalene. The choice of the PCR method (end-point or qPCR) for evaluation of in situ biodegradation depends on the question being asked in the investigation. End-point PCR can be used as a
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tool for screening large quantities of field samples, while quantitative evaluation of potential functional capabilities of microbial population at a site can be done by quantitative PCR analysis. Both methods will evolve as more insight is gained into understanding of function of microbial communities involved in in situ anaerobic biodegradation. Since no analysis method is perfect and is subject to bias, it is important to use multiple methods or multiple biomarkers themselves to unearth the actual in situ microbial potential for remediating the site and to reduce the impact of methods used on the conclusions derived. Our data shows that the decrease in concentration of contaminants at this site over the last 10 years (see Supporting Information S4), and likely longer, can be attributed not only to physical and chemical processes but also certainly to anaerobic biodegradation. We have demonstrated that two independent biomarkers (metabolic intermediates and the catabolite gene bssA), specific to anaerobic hydrocarbon biodegradation, were enriched in the impacted wells, as compared to the unimpacted wells. This underscores our earlier study at the site4 that first reported the presence of metabolic intermediates specific to anaerobic degradation of PAHs and BTEX at this site. These two studies collectively provide evidence that biodegradation is relevant to the contaminant loss at the site and that natural attenuation is taking place. Our study together with the earlier data4 provide a long-term perspective of biodegradation processes at the site, which is rarely addressed in field studies. This approach can be a powerful tool for evaluating bioremediation and natural attenuation at subsurface impacted sites.
’ ASSOCIATED CONTENT
bS
Supporting Information. Characteristics of the groundwater samples (S1), concentration of sulfate and nitrate in the impacted wells (S2) and in the unimpacted wells (S3), concentration of contaminants detected at the impacted monitoring wells before 2008 (S4), calibration curves used in qPCR analysis (S5), and deviations in cycle threshold and melting temperature of bssA-Sulf-Meth qPCR (S6). This material is available free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION Corresponding Author
*E-mail:
[email protected]; phone: 732-932-8165, ext. 312; fax: 732-932-0312.
’ ACKNOWLEDGMENT We thank employees of GZA and Test America for extending their help and cooperation during sample collection and for providing us with data of organic analysis of the samples and chemical variables measured in the field. We also thank undergraduate students Mathew Bruno for assistance in sample collection and Shravan Dave for help with sample processing. This work was funded in part by a student grant in aid from New Jersey Water Research Resource Institute for A.R.O. (2009NJ199B) and in part by Gas Technology Institute, Chicago, IL. ’ REFERENCES (1) OSWER. Use of monitored natural attenuation at superfund, RCRA corrective action, and underground storage tank sites; Office of Solid Waste 3413
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