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Stable isotope probing (SIP) was used to identify the aerobic toluene-degrading microorganism in soil microcosms. Several approaches (terminal restriction fragment length polymorphism, 16S rRNA gene sequencing, and quantitative PCR) provided evidence that the microorganism responsible was a member of the genus Polaromonas and could grow on toluene. This microorganism also transformed benzene, but not m-xylene or cis-dichloroethene.
Sites containing leaking underground storage tanks (LUST sites) are a national problem in the United States, resulting in BTEX (benzene, toluene, ethylbenzene, and xylenes) or oxygenate (methyl tertiary-butyl ether and tertiary-butyl alcohol) contamination. Information on the organisms responsible for contaminant degradation in mixed community samples, however, is often difficult to obtain. Here, stable isotope probing (SIP) is used in addressing this knowledge gap for toluene.
Two soil samples, obtained at different depths (3 to 4 ft and 5 to 6 ft deep) from a BTEX-contaminated site (in Michigan), were utilized and are referred to hereinafter as soils 1 and 2, respectively. Microcosms were constructed with 6 g soil (wet weight) and 20 ml phosphate-buffered mineral medium (10) in 150-ml serum bottles sealed with rubber stoppers and aluminum seals. Three sets of experiments were conducted; experiment 1 involved SIP to identify the dominant toluene degrader in both soils, experiment 2 focused on real-time quantitative PCR (qPCR) to confirm the SIP results and investigate microorganism growth on toluene, and experiment 3 involved biodegradation tests with other contaminants (benzene, m-xylene, and cis-dichloroethene [cDCE]).
The SIP study was conducted on both soils and involved triplicate abiotic controls, triplicate unlabeled-toluene-amended (1 μl, 99%; Chem Service, West Chester, PA), and triplicate labeled-toluene-amended (1 μl [ring-13C6]toluene, 99%; Cambridge Isotope Laboratories, Inc., Andover, MA) samples. The second experiment (only on soil 2) involved 16 sample microcosms. Unlabeled toluene was added to eight of these, whereas the other eight served as controls without toluene. At each sampling time, two samples and two controls were sacrificed for DNA extraction and qPCR (described below). In experiment 3, for each contaminant, three sample microcosms and two autoclaved control microcosms were constructed using the supernatant of toluene-degrading soil microcosms (from experiment 2, soil 2). Following the depletion of toluene, 5 ml of supernatant was transferred to a serum bottle with 20 ml of medium (as above) and then benzene (99.8%; Sigma Aldrich), cis-dichloroethene (Supelco), or m-xylene (99%; Sigma Aldrich) was added to a final solution concentration of approximately 45 mg liter−1. All microcosms were incubated at room temperature (~20°C) with reciprocal shaking. Contaminant concentrations in headspace gas samples (200 μl) were typically determined daily with a gas chromatograph (Perkin Elmer) equipped with a flame ionization detector and a capillary column (0.53-mm-diameter DB-624; J&W Scientific). The injector and detector temperatures were set at 200°C, and the column temperature was 120°C.
A Powersoil DNA extraction kit (Mo Bio Laboratories) was used for DNA extraction according to the manufacturer's recommended procedure. The extraction time, number of samples, and mass of soil extracted varied depending on the experiment. For SIP, DNA was extracted from two labeled and two unlabeled microcosms (entire sample) following toluene depletion. For qPCR, DNA was extracted (0.3 g soil [wet weight]) from microcosms scarified at successive time points both for the toluene-amended-sample and non-toluene-amended-control microcosms. Two sample and two control microcosms were scarified at day 3, 4, 5, or 6 (or 6.5) when approximately 20, 50, 80, and 100% of the added toluene was transformed.
Ultracentrifugation (initial buoyant density [BD] of 1.730 g ml−1), fractionation, BD measurements, CsCl removal, terminal restriction fragment length polymorphism (TRFLP), and heavy fraction (BD value of 1.744 g ml−1) sequencing were performed as previously described (8). The Ribosomal Database Project (RDP) (Center for Microbial Ecology, Michigan State University, East Lansing, MI) analysis tool Classifier was utilized to assign taxonomic identity to cloned sequences.
A qPCR assay was developed targeting the 16S rRNA gene of the Polaromonas sp. identified as the toluene degrader. The assay was conducted in a Chromo 4 real-time PCR cycler (Bio-Rad) using the primer set PO313F (5′-AATGGATGGTACAGAGGGTC-3′) and PO313R (5′-ATTACTAGCGATTCCGACTT-3′) (Operon Biotechnologies) and produced a 114-bp PCR product. The primer pair were designed with NCBI primer-BLAST, and their specificity was confirmed by cloning and sequencing of the soil PCR products. Both density gradient fractions and total DNA at successive time points were subjected to the qPCR assay. Each 20-μl PCR mixture contained 10 μl SYBR green real-time PCR solution (Applied Biosystems), each primer at 0.25 μM, and 1 μl DNA template. The thermal protocol consisted of an initial denaturation step (95°C for 15 min), 40 cycles of amplification (95°C for 15 s, 60°C for 20 s, and 72°C for 20 s), and a terminal extension step (72°C for 2 min). Melting curves were constructed from 55°C to 95°C and read every 0.6°C for 2 s. For each gradient fraction, 1 μl was diluted with 3 μl water as template. For total DNA samples, 1 μl total DNA was used directly as the template. For both, triplicate samples were measured. Cloned plasmid DNA was utilized as a standard for quantification, and gene copy numbers were determined as previously described (12) (plasmid size was 5,441 bp, including a 1,485-bp insert).
Toluene removal occurred rapidly (~100% removal in seven days) in all sample microcosms but was limited in the autoclaved controls. The TRFLP fraction profiles indicated that one fragment (313 bp) was relatively more abundant in the heavy fractions (1.74 g ml−1) of labeled-toluene-amended microcosms than in the controls (unlabeled toluene) (Fig. (Fig.1).1). This trend was observed in replicates of both soils. Other TRFLP fragments were found in the heavy fractions from the labeled-toluene-amended samples; however, as these were also found at similar levels in the heavy fractions of the controls, they were excluded from further analyses. The identity of the 313-bp TRFLP fragment was determined both by partial 16S rRNA gene sequencing and multiple restriction enzyme digestion. Fractions with dominant 313-bp fragments were chosen for TRFLP analyses with four other enzymes (HhaI, MseI, Bsp1286I, and BsrBI). The dominant fragments obtained from these additional TRFLP digests were compared to the clone library sequences to correlate actual cut sites and predicted cut sites (Table (Table1).1). Only slight differences (1 to 2 nucleotides [nt]) between the predicted and actual lengths were seen; such differences have been noted by others (1, 11). The clone sequence (GenBank accession number GQ254296) containing the five appropriate cut sites was classified as a Polaromonas strain within the class Betaproteobacteria.
Two additional lines of inquiry provided evidence that the Polaromonas strain was responsible for toluene transformation. First, the relative distribution of Polaromonas 16S rRNA genes, as determined via the qPCR assay, indicated an increase in DNA BD between the labeled- and unlabeled-toluene-amended microcosms (Fig. (Fig.22 [results shown are for soil 2]). The maximum 16S rRNA gene abundance levels from labeled-toluene-amended microcosms were found at 1.742 g ml−1 (soil 1) and 1.744 g ml−1(soil 2), a clear increase over the maximum abundance values in the unlabeled-toluene-amended samples (1.719 g ml−1 for soil 1 and 1.726 g ml−1 for soil 2). Thus, the BD differences between the peak abundance levels in unlabeled and labeled microcosms were 0.023 g ml−1 and 0.018 g ml−1 for soil 1 and soil 2, respectively. As expected, the increase is less than has been seen with pure cultures exposed to higher concentrations of labeled substrates (e.g., an increase of 0.038 g ml−1 was noted in Escherichia coli cells following exposure to 1.3 g liter−1 13C-lactate ), yet it is a large enough signal to indicate label uptake from toluene by the Polaromonas population. Second, qPCR of samples at successive time points of toluene removal illustrated a clear increase in Polaromonas sp. gene copies with toluene depletion in the replicate live samples but not in the replicate controls without toluene (Fig. (Fig.33).
Supernatant samples transferred from toluene-degrading microcosms were tested for their ability to transform other contaminants (benzene, m-xylene, and cDCE). Benzene transformation occurred rapidly, whereas neither m-xylene nor cDCE concentrations declined in either the samples or the controls (Fig. (Fig.44).
Since the genus was first reported in 1996 (4), only a small number of Polaromonas isolates have been obtained. The type strain, P. vacuolata, was isolated from Antarctic marine waters (4). Other strains include P. aquatica, obtained from tap water (7), and P. hydrogenivorans, a psychrotolerant, hydrogen-oxidizing bacterium isolated from soil over permafrost (13). More relevant to this research are the remaining two Polaromonas strains, as both have been linked to the degradation of environmental contaminants. The first, isolated from a chlorinated-solvent pump-and-treat plant, Polaromonas sp. strain JS666, is the only known microorganism able to grow using cDCE as a sole carbon and energy source (2, 9). The other, isolated from coal tar-contaminated sediment, P. naphthalenivorans strain CJ2, uses naphthalene as a sole carbon and energy source (5). Interestingly, P. naphthalenivorans strain CJ2 was also first identified with the SIP method (6). The 16S rRNA gene of the Polaromonas strain described here was 96.6% (1,447/1,498 nt) and 97.6% (1,459/1,495 nt) similar to P. naphthalenivorans strain CJ2 and Polaromonas sp. strain JS666, respectively.
In conclusion, several lines of evidence indicated that an organism in the Polaromonas genus was responsible for toluene removal and growth on toluene in complex samples obtained from a BTEX-contaminated site. To our knowledge, this is the first report of growth on toluene of an organism in the Polaromonas genus. These results contribute to the growing body of knowledge regarding the abilities of Polaromonas species to degrade and grow on key environmental organic pollutants. Furthermore, evidence that this particular Polaromonas strain can thrive in a mixed culture was obtained, suggesting that these populations will likely compete well at contaminated sites.
The partial 16S rRNA gene sequence of the Polaromonas sp. was deposited with GenBank under accession number GQ254296.
Published ahead of print on 11 December 2009.