INTRODUCTION

Top Abstract Introduction Materials and Methods References

Aromatic hydrocarbons are pervasive contaminants of terrestrial environments, particularly shallow aquifers. There are a variety of anthropogenic sources for these chemicals, but the most environmentally significant are hydrocarbon fuel mixtures leaking from buried storage tanks. In situ bioremediation is often considered for cleanup or containment of hydrocarbon-contaminated groundwater, and these applications have generated increasing interest in anaerobic processes. Electron acceptor demands typically created in aquifers following fuel contamination greatly exceed dissolved oxygen supplies (9, 28). Thus, the systems become anaerobic, and biodegradation processes operative under these conditions are important for supporting intrinsic bioremediation (9, 28). Anaerobic processes have also been considered for potential use in enhanced bioremediation to obviate difficulties associated with introducing, dispersing, and maintaining oxygen at levels adequate to sustain aerobic biodegradation (33). These applications for electron acceptor supplementation with nitrate have attracted attention because of its high water solubility and general lack of noxious by-products (23, 24).

Elucidation of the microbiological and hydrogeochemical influences on anaerobic biodegradation of hydrocarbons in aquifers is needed to gain an improved understanding of both intrinsic and enhanced bioremediation processes (33). Microbiological investigations focusing on laboratory microcosm and selective enrichment experiments have demonstrated anaerobic degradation of aromatic hydrocarbons under redox conditions ranging from nitrate-reducing to methanogenic (18, 24, 29) and allowed identification of axenic cultures (14, 15, 26, 27, 37) or defined consortia (7) of anaerobic, aromatic hydrocarbon degraders. Hydrogeochemical studies have demonstrated the impacts of hydrocarbon contamination on altering temporal and spatial zonation of dominant terminal electron-accepting processes (7, 25). Collectively, these findings indicate that in contaminated aquifers, anaerobic degradation of hydrocarbons is mediated by a diversity of organisms, the nature and variety of which change in time and space.

While community successions appear to be a fundamental aspect of the microbial ecology underlying anaerobic hydrocarbon biodegradation, there is relatively little information on this process, partly because the diversity of the microbial trophic groups that comprise these communities has rendered culture-based techniques alone inadequate to comprehensively evaluate community structure. However, the recent development of phylogenetic probes, which may be used to detect and quantify specific taxonomically defined groups of microorganisms without culturing, offers microbial ecologists the possibility of gaining new insights into microbial population dynamics. For analysis of microbial community structure in complex environments, a suite of phylogenetically nested probes may be used to conduct analysis of the population in hybridization assays from the top down (5). In these experiments, amounts of broad-spectrum (e.g., domain-level) probes hybridizing to nucleic acids extracted from the environment are quantified, and the community structure is further defined in subsequent hybridization assays by using probes with progressively greater specificity. However, because phylogenetic probes do not directly assay function, their utility for deciphering potential activities of the targeted organisms is strengthened when they are integrated with physical-chemical analysis of the environment that facilitates the relation of phylotype to ecotype.

Phylogenetic probe analysis has been applied to study microbial communities in a variety of environments (12, 35, 39, 42), but there have been relatively few examinations of aquifers. Fry et al. (16) used this approach to analyze groundwater from two deep (>300 m below ground) aquifers and showed that the populations were similar in that they were dominated by Bacteria, with Eucarya comprising a smaller but significant fraction (5 to 14%). Archaea were detected at low levels (<2%) and were more abundant in groundwater that was methanogenic. Hess et al. (21) applied in situ hybridization in a laboratory study of fuel-contaminated (FC) aquifer material incubated under denitrifying conditions. These experiments demonstrated that the microbial community structure varied with electron acceptor gradients and that the -Proteobacteria appeared to predominate under denitrifying conditions. Detection of Azoarcus spp. with specific probes perhaps strengthened this hypothesis, as this organism is classified under -Proteobacteria and has members that are known aromatic hydrocarbon-degrading denitrifiers (15). Dojka et al. (13) conducted a phylogenetic survey of an anaerobic contaminated aquifer. They examined partial 16S rRNA gene sequences from 104 clones and identified representatives of established phylogenetic groups as well as several that were apparently of unknown lineage. In addition, some gene sequences were encountered more frequently in areas with a particular redox condition. This study provided evidence of the vast diversity of microorganisms inhabiting contaminated aquifers but did not develop correlations between changes in groundwater chemistry and changes in microbial populations, which might help to identify organisms involved in biodegradation processes.

The objectives of this study were to evaluate the use of phylogenetic probes to identify alterations in aquifer microbial community structure elicited by anthropogenic perturbances. Analyses were done on FC and noncontaminated (NC) aquifer material taken directly from the field and on aquifer materials used to establish anaerobic microcosms supplemented with toluene and/or nitrate. The latter experiments were done to gain information on the effects of specific, isolated variables relevant to the bioremediation of anaerobic aquifers and to facilitate the establishment of phylotype-ecotype linkages.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods References

Aquifer characterization and sampling. Aquifer samples were obtained from a military installation (Fort McCoy; Sparta, Wis.) where fuel hydrocarbons leaking from buried tanks and transfer lines had contaminated a shallow (1.4 m below ground surface) sand aquifer. Groundwater samples were collected from miniature multilevel sampling devices (37) installed to sample the aquifer near the water table surface at 0.6-m intervals to a depth of 11 m. A peristaltic pump was attached to the sampling lines, and after approximately 3 well volumes were withdrawn, water samples were collected for analysis of redox indicators (dissolved oxygen, NO₃, Fe²⁺, SO₄², HS) and volatile organic compounds (VOC). All redox indicators except HS were analyzed immediately in the field by using colorimetric techniques with CHEMets kits (CHEMetrics, Inc., Claverton, Va.). Sulfide analysis was done by ion chromatography at the University of Wisconsin-Madison, Soil and Plant Analysis Laboratory, and VOC determinations were done by the U.S. Army Analytical Laboratory (Sauk City, Wis.).

Microcosm experiments. Microcosms were established in 100-ml (nominal volume) serum bottles under an N₂ atmosphere in an anaerobic glovebox (model HE-493; Vacuums Atmosphere Co., Hawthorn, Calif.). Each microcosm contained 50 g of homogenized, sediment material from FC and NC regions of the aquifer and 50 ml of base medium. The base medium was composed of 330 µM KH₂PO₄ and 70 µM NH₄Cl and was adjusted to pH 7 with NaOH (24). Duplicate microcosms were then amended to a final concentration of 2 mM KNO₃ and/or 250 µM toluene. Duplicate microcosms containing the sediment and base medium alone were used as nonamended controls. The serum bottles were sealed with Teflon-coated butyl rubber stoppers affixed with aluminum crimp caps and then incubated inverted in the dark with continuous, gentle agitation on an orbital shaker (150 rpm) at 24°C. Agitation was applied to facilitate diffusion and did not generate a turbulent, water sediment suspension or disturb the sediment in any way.

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Statistical analysis. Data were analyzed with repeated-measures analysis of variance. Means were compared with pairwise comparisons of the estimated population marginal means, by using the statistical procedure PROC MIXED (SAS Institute Inc., Cary, N.C.).

Nucleic acid extraction techniques. A variety of nucleic acid extraction procedures were evaluated to determine the optimal technique for extracting the most DNA from the aquifer materials used in this study. The nucleic acid extraction protocol ultimately adopted for routine use was a lysozyme-freeze-thaw cell lysis technique for DNA extraction modified from that reported by Tsai and Olsen (41). Briefly, aquifer material (50 g) was suspended in 50 ml of TE buffer containing 0.3% Tween 20 and mixed for 2 h on a rotary shaker (150 rpm, 26°C). The supernatant was transferred to a 35-ml centrifuge tube and spun at 8,200 × g (20 min, 4°C). The pellets were resuspended in 5 ml of lysis solution (15 mg of lysozyme in TE ml¹) and incubated at 37°C for 1.5 h. After incubation, sodium dodecyl sulfate was added to a final concentration of 2% (wt/vol) and the sample was subjected to three freeze-thaw cycles (70°C, 65°C). The solution was then extracted twice (1:1 [vol/vol]) with TE-buffered phenol and then twice (1:1 [vol/vol]) with chloroform. DNA was precipitated with ethanol and sodium acetate as described above with 2 volumes of 100% ethanol and 0.1 volume of 3 M sodium acetate overnight at 20°C. The solutions were then centrifuged (8,200 × g, 20 min), and the resulting DNA pellets were washed once with 75% ethanol and then dissolved in double-distilled water (ddH₂O).

Nucleic acid purification and quantification. A two-step chromatographic purification system was adopted for routine application to the extracts, consisting of sequential filtration over Sepharose 2B (Sigma, St. Louis, Mo.) and Wizard Plus Minipreps (Promega, Madison, Wis.). For the first step, Sepharose gels were hydrated in 40 ml of TE buffer overnight and loaded into 5-ml syringes plugged with glasswool, which were then spun at 1,200 × g (10 min). Next, crude DNA extracts (200 to 400 µl) were loaded, and the tubes were spun at 1,200 × g (10 min). The effluents were collected, and the columns were washed twice with 100 µl of TE buffer. The effluents were then combined, and DNA was precipitated as described above and dissolved in ddH₂O. In the second step, the samples were further purified by using Wizard spin-columns according to the manufacturers' instructions.

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Hybridization analysis. Nucleic acid samples were transferred to Hybond-N⁺ nylon membranes (Amersham, Arlington Heights, Ill.), by using a minifold II 72-well slot-blot manifold (Schleicher and Schuell, Keene, N.H.), and cross-linked to the membranes by exposure to 120 mJ of UV light energy with a UV Stratalinker 1800 (Stratagene, Torrey Pines, Calif.). All oligonucleotides used as probes (Table 1) were conjugated at the 5' ends to digoxigenin by the supplier (NBI, Plymouth, Minn.). The probes were tested and calibrated with reference genomic DNAs prepared from 19 organisms representing the -, -, and -Proteobacteria, sulfate-reducing bacteria, high-G+C gram positives, low-G+C gram positives, Eucarya, and Archaea (Table 2). Hybridization and wash conditions were empirically optimized for each probe. Probe-target hybrids were detected following a 45-min exposure of the membranes to X-ray film by using the Genius chemiluminescence system (Boehringer Mannheim, Indianapolis, Ind.). Rapid-Hyb buffer (Amersham) was used instead of the Easi-Hyb buffer supplied with the Genius system, because in comparative tests the former yielded much stronger hybridization signals.

	RESULTS AND DISCUSSION

Chemical and molecular characterization of field samples. In the FC region of the aquifer, total BTEX (benzene, toluene, ethylbenzene, and xylene) levels ranged from 4,000 to >5,000 µg liter¹, reflecting significant spatial and temporal variation. Natural organic matter content ranged from 18 to 53 g kg¹, reflecting vertical heterogeneity of the aquifer material resulting from shifting patterns of fluvial deposition and development of peat in a riparian wetland. Samples collected from the NC portion of the aquifer were lower in natural organic matter (2 to 31 g kg¹), and groundwater from this location contained no detectable fuel hydrocarbons. Although peat layers also existed in the NC portion of the aquifer, these were not recovered in the samples examined in these experiments. The NC and FC areas also differed in that the groundwater chemistry of the former indicated predominantly aerobic conditions (pH 7.0 to 7.5) with 1 to 5 mg of dissolved oxygen (DO) liter¹ and no detectable Fe²⁺. In the FC region, DO concentrations across the 0.6-m sampling interval ranged from 0 (nondetectable) to 0.1 mg liter¹, while Fe²⁺ levels were 10 to 100 mg liter¹ (pH 5.6 to 6.2). Additional evidence for the occurrence of anaerobic processes in the FC area was a depletion of other dissolved electron acceptors compared to the concentrations measured in the NC section. Specifically, in the NC area NO₃ ranged from 6 to 10 mg liter¹ and SO₄² ranged from 10 to 20 mg liter¹, while in the FC region NO₃ was nondetectable and SO₄² concentrations varied from 0 to 10 mg liter¹, respectively.

View larger version (99K): [in this window] [in a new window]   FIG. 1.   (A) Agarose gel analysis of DNA extracted from aquifer field samples and standards. Lanes 1 to 3, Pseudomonas genomic DNA loaded at 0.59, 1.18, and 2.47 µg; lanes 4 and 5, aliquots (10 µl) of purified DNAs extracted from the NC or FC sediment (total volumes of the DNA extracts were 100 and 600 µl for the NC and FC extracts, respectively). (B) Relative abundance (universal probe-normalized signals) of domains and subgroups (phylum and subclass levels) in the NC and FC aquifer samples. The sums of domain and subgroup hybridization signals (± standard deviations) are given in the box above the graph. Legend for probe target groups: , universal; , Bacteria; , Eucarya; , ALF; , B+G; , SRB; , HGC. Data are means of duplicate measurements (± standard deviations). An asterisk indicates that the hybridization signal from a given probe differed significantly (P  0.05) from that for the nonamended control.

Chemical and molecular analyses of NC sediment microcosms. Toluene supplementation stimulated microbial growth as indicated by levels of CO₂ production and DNA yields compared to those of nonamended controls (Table 3). Toluene degradation was detectable between days 10 and 12 and coincided with the period of enhanced CO₂ production. The total amount of CO₂ was not significantly different from the 32-µmol amount expected based on equation 1. The DNA extracted from the nonamended and toluene-amended microcosms totaled 6 and 9 µg, respectively. The amount of toluene added should have supported the production of ca. 88 µg of bacterial biomass containing 3.5 µg of DNA. Thus, the 3 µg of additional DNA produced in the toluene-amended treatment agreed well with the theoretical yield and indicated effective recovery of DNA from the microcosms. Domain probe analysis showed that in both the toluene-amended and nonamended flasks, Bacteria accounted for 85 to 95% of the community DNA, Eucarya accounted for 2 to 8%, and Archaea were nondetectable (Fig. 2). Shifts in microbial community structure were detectable at the phylum and subclass levels. In the nonamended microcosm, the relative abundance was 50% B+G, 20% ALF, 15% SRB, and 2% HGC (Fig. 2). In the toluene-supplemented microcosms there was reversal of ALF versus B+G as the dominant subclass and significant increases in the SRB and HGC groups (Fig. 2).

View larger version (37K): [in this window] [in a new window]   FIG. 2.   Molecular analysis of DNAs extracted from microcosms established with the NC aquifer material. Relative abundance of the domain or subgroups was determined by hybridization to the indicated probe (see the legend to Fig. 1 for the key to probe abbreviations). Bars marked with an asterisk were significantly different (P  0.05) from those for the nonamended microcosms. Probes for which data are not plotted gave hybridization signals that were below background.

Chemical and molecular analyses of FC aquifer microcosms. Toluene supplementation increased total CO₂ production to ca. 40 µmol in the nonamended treatment compared to ca. 25 µmol in the nitrate-amended treatments (Table 4). Since the theoretical CO₂ production should be about 32 µmol, this result may have indicated that microbial activity was supported in part by oxidation of native organic materials. While CO₂ analysis indicated that toluene supplementation stimulated microbial activity, there was no significant effect on DNA yield. This result may have been attributed to the high organic matter content of the FC sediment interfering with DNA recovery from these materials. Toluene degradation was detectable between days 6 and 10 and coincided with the period of enhanced CO₂ production. Toluene degradation was more rapid than that observed in the FC sediment and may have reflected the effect of prior fuel exposure on acclimation of the microbial population. Results of domain probe analysis were similar to those of the NC sediment in abundance patterns and the lack of significant treatment effects on domain abundance (Fig. 3). However, significant effects of toluene supplementation on community structure were detected at the lower taxonomic levels. The FC sediment showed a 40% decrease in B+G and an 8% increase in the ALF. Most striking, however, was the fact that the majority of the DNA (67%) was not accounted for by the phylum or subclass probe (Fig. 3).

View larger version (39K): [in this window] [in a new window]   FIG. 3.   Molecular analysis of DNA extracted from microcosms established with the FC aquifer material. Relative abundance of the domain or subgroups was determined by hybridization to the indicated probe (see the legend to Fig. 1 for the key to probe abbreviations). Bars marked with an asterisk were significantly different (P  0.05) from those for the nonamended microcosms. Probes for which data are not plotted gave hybridization signals that were below background.

Comparison of field sample analysis and microcosm experiments: insights into effects of hydrocarbon contaminants and/or nitrate supplementation on microbial community structure. At the domain level, the microbial community structure showed no significant differences regardless of the aquifer material type or microcosm amendment. The detection of Eucarya in the microcosms was in agreement with the hybridization analysis of Fry et al. (16), who reported that Eucarya comprised 6 to 14% of the microbial community in anaerobic groundwater from deep aquifers. While the nature and function of Eucarya observed in the microcosms are unknown, the detection of a consistent fraction suggests that these organisms were not directly affected by the treatments applied. These Eucarya could be anaerobic protozoa grazing on the microbial populations that were stimulated in the microcosms.