Groundwater amendment and sampling.

As previously described (62), an acetate-bromide solution was prepared by mixing native groundwater pumped from an up-gradient portion of the aquifer into a storage tank with sodium acetate (Sigma, St. Louis, MO) and sodium bromide (Sigma). This mixture was added to the subsurface via 10 injection wells to achieve target aquifer concentrations of ~5 mM and ~1 mM for the first 14 days. Additions resumed on day 25, and on day 38 the acetate concentration was increased to provide a target concentration of ~15 mM, with continued additions to day 110 (62). However, a diversion in groundwater flow and acetate consumption at the injection wells diminished the delivery of the injectate to D04 after the groundwater flush (62).

Prior to the initiation of the acetate injection reported here, the site had been under natural groundwater flow without amendments for ~11 months, following a previous, short-duration (ca. 21-day) acetate addition study in 2007 (62).

Groundwater samples for geochemical analyses were collected every 2 days after purging 10 liters of groundwater from the well using a peristaltic pump. Sulfide and ferrous iron were measured spectrophotometrically immediately after sampling using the methylene blue method (hydrogen sulfide test; Hach Company, Loveland, CO) for sulfide and the phenanthroline method (AccuVac ampules; Hach Company) for ferrous iron. After filtration through a 0.2-μm-pore-size polytetrafluoroethylene (PTFE [Teflon]) filter (Alltech Associates, Inc., Deerfield, IL), bromide, acetate, and sulfate concentrations were measured using an Dionex ICS-1000 ion chromatograph equipped with a IonPac AS22 column, an ASRS 300 suppressor, and 4.5 mM carbonate-1.4 mM bicarbonate eluent (Dionex Corporation, Sunnyvale, CA), while U(VI) was measured using a kinetic phosphorescence analyzer (46).

Groundwater samples for molecular analyses were obtained after sampling for geochemical analyses by concentrating 10 liters of groundwater on a 0.2-μm-pore-size, 293-mm-diameter Supor-200 membrane filter (Pall Life Sciences, Ann Arbor, MI). Filters were quickly sealed into a sterile whirl pack, flash frozen in an ethanol-dry ice bath, and stored at −80°C until nucleic acid extraction.

Nucleic acid extraction.

Nucleic acids were extracted from portions of the same filter and crushed with liquid nitrogen (34). Equal volumes of homogenized filter fragments were used for parallel DNA and RNA extractions. Genomic DNA (gDNA) was extracted using a FastDNA Spin Kit for soil (MP Biomedicals, Solon, OH). gDNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific, Wilmington, DE) and stored at −80°C until further analyses.

RNA was extracted using a modified phenol-chloroform method (20). RNA cleanup was performed using an RNeasy Mini Kit (Qiagen GmbH, Hilden, Germany), and RNA was treated with DNase (DNA-free Kit, Ambion, Austin, TX). Successful RNA isolation was checked by visualization on a 1% agarose gel. The absence of DNA contamination was confirmed by PCR amplification. RNA was quantified using a NanoDrop spectrophotometer and stored at −80°C until further analyses.

dsrAB clone library construction and phylogenetic analysis.

The primers used in this study are listed in Table 1. PCR amplification of an approximately 1.9-kbp dsrAB fragment was performed using the primers DSR1Fmix (equimolar mixture of DSR1F, DSR1Fa, DSR1Fb, DSR1Fc, and DSR1Fd) and DSR4Rmix (equimolar mixture of DSR4R, DSR4Ra, DSR4Rb, DSR4Rc, DSR4Rd, and DSR4Re) and the following cycling conditions: initial denaturation at 94°C for 1 min, 35 cycles of 40 s of denaturation at 94°C, 40 s of annealing at 48°C, and 1.5 min of elongation at 72°C, with a final elongation at 72°C for 10 min (30). A positive control of purified dsrAB PCR product from Desulfovibrio vulgaris and a negative control without DNA were always included in PCR amplification experiments. The reaction was carried out in a PTC200 Peltier Thermal Cycler (MJ Research, Waltham, MA). The 50-μl reaction mixture contained 100 ng of DNA, 1× Q-Solution (Qiagen), 1× PCR buffer (Qiagen), 1.5 mM MgCl₂ (Qiagen), a 200 μM concentration of each deoxynucleotide (Sigma), a 0.5 μM concentration of each primer, 0.5× bovine serum albumin (BSA; New England BioLabs, Beverly, MA), and 1.25 U of Taq DNA polymerase (Qiagen). The presence and size of the amplification products were determined by agarose (1%, wt/vol) gel electrophoresis. Bands of the expected sizes were purified from the gel by excision with a sterile surgical blade and purified with a QIAquick Gel Extraction Kit as recommended by the manufacturer (Qiagen).

Clone libraries were constructed from nine representative samples (day 0, 3, 10, 13, 26, 34, 45, 47, and 53 following acetate injection). Four microliters of the agarose gel-purified DNA mixture was ligated into the pCR2.1-TOPO vector (TOPO TA Cloning Kit; Invitrogen, Carlsbad, CA). A dsrAB fragment sequence of approximately 1.9-kbp was determined for Escherichia coli recombinant vector-containing colonies with the primers M13F and M13R in an ABI 3730xl DNA Analyzer using the Sanger chain terminator method with fluorescently labeled nucleotides. Chromatograms were visually inspected using the software 4Peaks, version 1.7 (A. Griekspoor and T. Groothuis, Mekentosj, Aalsmeer, The Netherlands).

Recovered dsrAB sequences (100 clones per library) were compared to the GenBank database (4) for preliminary identification using the BLASTX algorithms (http://www.ncbi.nml.nih.gov/BLAST). The alignment and treeing software of the ARB package (31) (http://www.arb-home.de) were used for the phylogenetic analyses. Concatenated partial dsrA and dsrB sequences were added to an ARB alignment of 1.9-kb dsrAB sequences (64) deposited in the GenBank database. The alignment of the corresponding amino acid sequences was carried out manually using the editor GDE, version 2.2 (51), implemented in ARB. A dsrAB tree was constructed from nucleotide sequences using neighbor-joining analysis with a Jukes-Cantor distance correction. The trees constructed with nucleotide and amino acid sequences yielded similar results. Phylogenetic inference was performed with a total of 1,123 nucleotides; filters were used to exclude from the data set regions of insertion and deletions, as well as the third position in each triplet.

Primer design for quantifying dsrB transcripts.

Conserved regions in the alignment of sequence data from the dsrAB clone libraries were targeted for quantitative PCR (qPCR) primer design. The primer pairs DSRq1F-DSRq1R, DSRq2F-DSRq1R, and DSRq4F-DSRq1R (Table 1) were employed to amplify a portion of 105, 110, and 115 bp of the dsrB portion of the dsrAB of sulfate reducers belonging to the Desulfobacteraceae, Desulfobulbaceae, and Syntrophaceae clusters found in the groundwater at Rifle, respectively. The specificity of the primer pairs was tested in silico using the ARB software. In addition, clone libraries were constructed from PCR-amplified DNA fragments from DNA extracted from the sampling filters using each primer pair and the protocol described above. Proper matching with the targeted SRP was confirmed by inserting the partial dsrB sequences one by one into the tree constructed with long dsrAB sequences using the ARB parsimony tool, without distorting the overall tree topology (data not shown).

RT-PCR of dsrB transcripts.

An Enhanced Avian HS reverse transcription-PCR (RT-PCR) kit (Sigma) was used to generate cDNA from extracted dsrAB transcripts. The reverse transcription (RT) reaction was carried out in two steps. First, the RT master mix contained 2 μl of the appropriate reverse primer (2 μM), 2 μl of deoxynucleoside triphosphate (dNTP) mix (1 mM each dNTP), and 1 μl of diethyl pyrocarbonate (DEPC)-treated water; 5 μl of RNA template (0.01 to 5 μg of RNA) was added for a total reaction mixture volume of 10 μl, and the mixture was incubated at 70°C for 10 min. Then, the PCR master mix (10 μl) consisting of 2 μl of avian myeloblastosis virus (AMV) reverse transcriptase buffer (1×), 1 μl of RNase inhibitor (1 U/μl), 1 μl of Enhanced AMV reverse transcriptase (1 U/μl), and 6 μl of DEPC-treated water was added to the RT reaction mixture, and the samples were incubated at 50°C for 50 min. cDNA was quantified using a NanoDrop spectrophotometer and stored at −80°C until further analyses.

Quantification of genes and transcripts.

The 25-μl qPCR mixture contained 12.5 μl of Power SYBR green PCR Master Mix (Applied Biosystems Inc., Foster City, CA), 1.5 μl of a 150 nM concentration of each primer, and 9.5 μl of a 1:10 dilution of gDNA (dsrB) or cDNA (dsrB transcripts) template. qPCR results were normalized to the total amount of gDNA/cDNA in the 9.5 μl of template solution used to set up the qPCRs. Standard curves were constructed with serial dilutions of known amounts of dsrB amplified with the appropriate primers from environmental gDNA, purified, and quantified with a NanoDrop spectrophotometer. Serial dilutions covered a range of 8 orders of magnitude of template copies per assay (10² to 10⁹). R² values ranged from 0.992 to 0.999. The qPCR efficiency (90% to 95%) was calculated based on the slope of the standard curve. All qPCR assays were run in triplicate. PCR amplification was carried out with a 7500 Real-Time PCR System (Applied Biosystems). Thermal cycling parameters consisted of an activation step at 50°C for 2 min, a denaturation step at 95°C for 10 min, and 50 cycles at 95°C for 15 s and 55°C for 1 min. Amplification and correct amplicon size were verified by running aliquots of qPCRs on an ethidium bromide-stained 1% agarose gel. gDNA extracts were tested for PCR-inhibitory substances by a serial dilution of the template gDNA and subsequent qPCR. Templates were normalized to an equal amount of gDNA/cDNA to enable comparison of different time points.

Sulfate reducers present.

To make a comprehensive inventory of the SRP present at the Rifle site, dsrAB clone libraries were constructed from DNA extracted from samples representative of the entire experimental period. Seven groups of dsrAB sequences were retrieved, with 99 to 100% sequence identity within each group. One group was in the Desulfobacteraceae, three were in the Desulfobulbaceae, and three were in the Syntrophaceae (Fig. 3). Analysis of dsrAB sequences revealed three clades of sulfate reducers at the site: Desulfobacteraceae (dsrAB clone Rifle08_01), Desulfobulbaceae (dsrAB clones Rifle08_02 to Rifle08_04), and Syntrophaceae (dsrAB clones Rifle08_05 to Rifle08_07) (Fig. 3). The closest cultured relatives to the Desulfobacteraceae and Syntrophaceae sequences are the acetate-oxidizing sulfate reducers Desulfobacter postgatei (96 to 97% sequence identity) and Desulfobacca acetoxidans (69 to 72% sequence identity), respectively. The Desulfobulbaceae sequences were not closely related to known acetate-oxidizing sulfate reducers. Primer DSR1F and DSR4R mixes used in this study were recently implemented with additional variants to improve dsrAB coverage (45). Hence, we do not exclude the possibility that our survey underestimated the dsrAB diversity in the groundwater at Rifle.

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Fig. 3.

Phylogenetic tree showing the placement of a representative of each group of the dsrAB sequences recovered from the subsurface (in bold) as well as sequences from pure cultures. The tree was constructed using the neighbor-joining algorithm using full dsrAB sequences for cultured SRP (64) and concatenated dsrA and dsrB sequences for clones. Closed circles indicate bootstrap values (1,000 data resamplings) of ≥90%; open circles indicate values of ≥70%. The dsrAB sequence of Thermodesulfovibrio islandicus was used as an outgroup. The scale bar indicates 10% sequence divergence. GenBank accession numbers are indicated for each sequence.

Prior to the addition of acetate, Desulfobulbaceae and Desulfobacteraceae were comparable in abundance (Fig. 4). However, following the addition of acetate, Desulfobacteraceae became predominant, and the proportion of Desulfobulbaceae declined significantly. Syntrophaceae had lower abundance prior to acetate additions and remained present at a comparable abundance throughout. This specific response of Desulfobacter to the acetate additions was corroborated with 16S rRNA sequence analysis performed with microarrays (8).

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Fig. 4.

Relative abundance of the three sulfate-reducing clades in dsrAB clone libraries. Numbers indicate the number of clones belonging to each group from a total of 100 clones analyzed per library.

Expression of dissimilatory (bi)sulfite reductase genes in the three clades.

The activity of the three major clades of sulfate reducers throughout the field study was evaluated by monitoring the abundance of dsrB transcripts in each group. The number of dsrB transcripts in each clade was normalized to the number of copies of dsrB in that clade. SRP that possess multiple dsr operons in their genome have not been reported, and thus this normalization is expected to approximate dsrB transcripts per cell.

The Desulfobacteraceae had a slightly higher abundance of dsrB transcripts than the Desulfobulbaceae or Syntrophaceae prior to the addition of acetate to the subsurface (Fig. 5). Following the resumption of acetate additions on day 25, the abundance of transcripts in the Desulfobacteraceae increased. This coincided with the enhanced rate of sulfate removal noted above, consistent with higher activity of sulfate reducers. When acetate was no longer being delivered to D04, as indicated by diminished bromide and a rebound in sulfate concentrations, dsrB transcript abundance in Desulfobacteraceae declined rapidly, consistent with the expected decline in the activity of sulfate reducers.

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Fig. 5.

Number of dsrB transcripts per copy of dsrB for the three major clusters of SRP found in uranium-contaminated groundwater at the Rifle site. Data are means ± standard deviation of triplicate determinations.

In contrast, the abundance of dsrB transcripts remained relatively constant in the Desulfobulbaceae throughout the field experiment. The abundance of dsrB transcripts in the Syntrophaceae increased slightly in the later phases of sulfate reduction but remained low compared to transcript abundance in the Desulfobacteraceae (Fig. 5).

These results suggest that, on a per cell basis, Desulfobacteraceae were much more responsive to the changes in the availability of acetate than the other two groups of sulfate reducers. This interpretation is consistent with the finding that Desulfobacteraceae became the most dominant group of sulfate reducers following acetate addition.

We thank the city of Rifle, CO, the Colorado Department of Public Health and Environment, and the U.S. Environmental Protection Agency, Region 8, for their cooperation in this study.

The U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, funded the work under grant number DE-SC0004814 (University of Massachusetts) and contract number DE-AC02-05CH11231 (Lawrence Berkeley National Laboratory [LBNL; operated by the University of California], with support derived equally from the Rifle IFRC and LBNL Sustainable System Science Focus Area research programs).