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Keywords:

  • Bioaugmentation;
  • VC-assimilation;
  • Mycobacterium sp.

Abstract

  1. Top of page
  2. Abstract
  3. 1Introduction and background
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

An aerobic bacterium, Mycobacterium sp. strain TRW-2 that assimilated vinyl chloride (VC) or ethene (ETH) as the sole carbon source was isolated from a chloroethene-degrading enrichment culture. The strain TRW-2 also degraded cis-dichloroethene (cis-DCE) in mineral salts medium, but only when VC was present as the primary carbon source. However, no degradation of tran s-dichloroethene or trichloroethene occurred in either the presence or absence of added VC. The measured growth yield values were 6.53 and 14.1 g protein/mol of VC and ETH utilized, respectively. Inoculation by strain TRW-2 in microcosms prepared with aquifer samples resulted in rapid degradation of VC, whereas native bacteria degraded negligible amounts of VC within the same time period, thus suggesting bioaugmentation potential of the isolate. Phylogenetic analysis of the 16S rDNA sequence of the isolate revealed 98% sequence similarity to the members of the genus Mycobacterium. In summary, the isolate's ability to degrade VC, cis-DCE, and ETH and also its ability to survive and degrade VC in the presence of other microorganisms is relevant to the remediation of VC-impacted aquifers.


1Introduction and background

  1. Top of page
  2. Abstract
  3. 1Introduction and background
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

Accumulation of cis-dichloroethene (cis-DCE) and vinyl chloride (VC) under anaerobic conditions is commonly seen at most sites due to incomplete dechlorination of higher chloroethenes, including perchloroethene (PCE), trichloroethene (TCE), 1,1,2,2-tetrachloroethane, and 1,1,2-trichloroethane [1–3]. Other major sources of VC contamination include the industrial use of VC for the production of chlorinated solvents and polyvinyl chloride [4]. The accumulation of cis-DCE and VC can be attributed mainly to the lack of bacteria such as Dehalococcoides spp. [5,6] which are implicated in catalyzing complete dechlorination of chloroethenes to ethene (ETH) under anaerobic conditions.

Many reports have shown aerobic biodegradation of cis-DCE and VC through metabolic [7–11] and co-metabolic processes (see [12]). Only a handful of organisms belonging to different genera that assimilate VC as the growth substrate have been isolated. These include members of the genus Mycobacterium, Pseudomonas, Ochrobactrum, and Nocardioides[7–11]. Only one isolate, a β-Proteobacterium capable of degrading cis-DCE as the sole source carbon has been reported [13]. Among the isolated strains, only members of the genus Mycobacterium have been widely detected at chloroethene-contaminated sites, thus suggesting their importance in the natural attenuation of VC.

While aerobic VC degradation is frequently reported, it is by no means a ubiquitous process. Recent studies [7] have documented biodegradation of VC in only 11 of 33 samples tested. Similarly, studies in our laboratory with aquifer material obtained from 11 different sites showed biodegradation of cis-DCE and VC at only 4 sites (data not shown). In the present study we report the isolation of a Mycobacterium sp. strain TRW-2 that assimilates VC as a sole growth substrate. We also tested its ability to degrade VC in the presence of other bacteria by inoculating microcosms containing aquifer material obtained from different chloroethene-contaminated sites. These results are relevant to bioaugmentation strategies because fast-growing Mycobacteria have been isolated from diverse habitats and shown to biotransform a wide range of petroleum and chlorinated hydrocarbons [7,9,14–17], thus attesting to their versatility and plasticity for survival under different growth conditions.

2Materials and methods

  1. Top of page
  2. Abstract
  3. 1Introduction and background
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

2.1Chemicals and media

VC, cis-DCE, trans-dichloroethene (trans-DCE), TCE, and ETH were 98% pure and obtained from Aldrich Chemical Co., Milwaukee, WI. All other chemicals used were of reagent grade. The mineral salts medium (MSM) amended with no yeast-extract used in the present study was described previously [12].

2.2Analytical methods

Biodegradation of VC, cis-DCE, trans-DCE, TCE, and ETH was monitored using an Agilent 6890 gas chromatograph (GC) equipped with a flame ionization detector (FID) and an electron capture detector (ECD) as described before [12]. For monitoring the concentrations of these volatile compounds, 20 or 30 μl of head-space gases were injected into the GC. The GC response for each compound tested was calibrated to give the total mass in that bottle.

Assuming the headspace and aqueous phase concentrations were in equilibrium, the total mass present in the bottle was determined using standards prepared similarly. Standards were prepared in 160-ml serum bottles filled with 50-ml of MSM and closed with Teflon-faced septa and aluminum caps. After equilibration (approximately 1.5 h) at room temperature, the GC response for a range of mass (μmol/bottle) of each compound tested was plotted and the slopes were used to quantify the unknown. The GC detection limit for VC was <0.05 μmol/bottle.

Total protein was estimated by withdrawing 1 ml of the culture samples from serum bottles and cells were pelleted by centrifugation at 8000 rpm for 15 min. at room temperature and the supernatant was discarded. The cell pellet was mixed with 1 ml of 1 N NaOH and heated at 90°C for 10 min. The samples were analyzing for total protein using Lowry's method [18,19]. The standards were prepared using bovine albumin treated with 1 N NaOH and heating at 90°C. The resulting plot was used to estimate the concentration of cell protein.

2.3Isolation and identification of a VC-assimilating organism

A VC-assimilating pure culture was isolated from a highly enriched chloroethene degrading enrichment culture [12]. The isolation was accomplished by performing a 10-fold serial dilution of the enrichment and streaking 0.1 ml of diluted aliquots onto plates prepared with 1/10-strength nutrient agar (NA). The plates were incubated at 30°C. Yellow colonies appeared within 5–7 days of incubation. Individual colonies were aseptically transferred to bottles containing liquid MSM with VC as the sole carbon source. The bottles that showed VC degradation were further plated and colonies were tested for purity by culturing the isolate on MSM-VC agar, MSM-glucose agar, Tris Soy Agar (TSA), Luria–Bertani agar (LB) and NA plates. The MSM-VC plates were prepared by spreading 0.1 ml of the culture onto agar plates prepared with MSM. The plates were incubated in a dessiccator filled with air and VC.

Phylogenetic identification of the isolate was performed by comparative 16S rRNA gene sequence analysis. A large fragment of the DNA corresponding to positions 8-27f and 1492–1513r of the Escherichia coli 16S rRNA-gene was amplified by PCR using a Perkin–Elmer DNA Thermal Cycler (Perkin–Elmer Corp, Foster City, CA). The 100 μl PCR mix contained 50 ng of DNA, 0.6 μM of each primer, 200 μM of PCR nucleotide mix (Fisher Bioreagents, Fisher Scientific, Pittsburgh, PA), 1.75 mM MgCl2, and 2.5 units Taq polymerase in Buffer A (M1865, Promega Chemicals, Madison, WI). The amplification was performed with the program which consists of an initial denaturation at 94°C for 2 min, 30 cycles of denaturation at 94°C for 1 min, annealing at 54°C for 2 min, and extension at 72°C for 3 min, and another 8 min at 72°C for the final extension. The PCR product (5 μl) was purified using 1 μl of Exonulease I (1 units/μl) (USB, Cleveland, OH) and 2 μl of Shrimp Alkaline Phosphatase (1 units/μl) (USB, Cleveland, OH) as described else where [20]. The mixture was placed in the Thermal Cycler for 30 min at 37°C then 15 min at 85°C. The purified PCR product was quantified using gel electrophoresis with low DNA mass ladder (Invitrogen, Inc). The purified PCR product was directly sequenced using the ABI PRISM 3700 DNA analyzer (Applied Biosystems).

The 16S rDNA sequence obtained from the strain TRW-2 was compared to the most similar (>98% sequence similarity) sequences from the GenBank as well as with the 16S rDNA sequences of the other known VC-assimilating organisms. These sequences were aligned using the integrated Clustal-W (MEGA 3.0), and the phylogenetic tree was constructed using neighbor-joining algorithm and by p-distance estimation method implemented in MEGA, version 3.0 [21]. The confidence for individual branches of the resulting tree was estimated by performing 1000 bootstrap replicates. Pseudomanas aruginosa DL1 was used as the outgroup.

2.4Batch studies

Batch studies were carried out using 160 ml serum bottles (Wheaton) closed with 20-mm Teflon-coated rubber septa and aluminum caps (West Pharmaceutical, Lionville, PA). In order to provide a consistent source of inoculum for experiments, a stock culture (500 ml) of the isolated Mycobacterium sp. was maintained on VC in 1 L bottle. VC and ETH were injected into bottles using 1 and 5 ml sterile disposable syringes and sterile filters (0.2 μM pore size, Nalgene), respectively. Addition of TCE, cis-DCE, and trans-DCE was performed with dedicated 10-μl glass syringes (Hamilton Co., Reno, NV). For each treatment, triplicate inoculated active and duplicate autoclaved control bottles were used. Bottles were incubated in an inverted position without shaking in the dark at 30°C. Prior to analysis, the bottles were kept at room temperature for 30 min (23 ± 2°C).

2.5Starvation experiment

The ability of Mycobacterium sp. strain TRW-2 to resume VC degradation after various periods of starvation was assessed. For each starvation regimen, sets of three active bottles were established as described above with VC as the sole carbon and energy source. Initially, bottles were amended with 25 μmol/bottle VC twice and when all the added VC was degraded, the bottles were incubated without added VC for various time periods including 1, 3, 7, 14, and 28 days. At the end of each starvation period, the bottles were spiked with 38 μmol/bottle VC and the degradation was monitored with GC.

2.6Growth yield on VC or ETH utilization

Bottles containing 50-ml of MSM amended with 64 μmol/bottle VC were inoculated with 2.5 ml of VC-grown culture. Both headspace (30 μl) and liquid phase (1 ml) samples were withdrawn periodically for the determination of VC and protein, respectively. Additional VC (64 μmol/bottle) was added to the bottles as required. Similarly, bottles were prepared with ETH (190 μmol/bottle) and inoculated with ETH-grown culture for the determination of growth yield on ETH. Growth yield (grams of protein produced/mol of substrate consumed) was calculated from a linear regression of protein versus VC or ETH consumed.

2.7Bioaugmentation test

Microcosms were prepared with samples of aquifer solids or groundwater obtained from three geographically different chloroethene-contaminated sites. Aquifer slurry microcosms were prepared with 10 g of wet aquifer solids and 40 ml of MSM. Groundwater microcosms were prepared with 50 ml of groundwater samples. The pH was adjusted to neutral using 1 N HCl or NaOH. Microcosms were inoculated with the Mycobacterium sp. at various initial cell densities. To assess the bioaugmentation potential of the isolate in the absence of native organisms, autoclaved microcosms were prepared as above for each aquifer solid or groundwater sample tested. The initial cell densities of the inoculated TRW-2 cells were determined by performing a 10-fold serial dilution of the inoculum and plating an aliquot of 0.1 ml of the diluted culture onto 1/10-strength LB-plates. The plates were incubated at 30°C and colonies were counted after 7 days of incubation.

2.8Nucleotide sequence Accession Number

Sequence obtained in this study was deposited in GenBank under Accession No. DQ054531.

3Results and discussion

  1. Top of page
  2. Abstract
  3. 1Introduction and background
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

In this article, we briefly describe the physiology, phylogeny, and bioaugmentation potential of the Mycobacterium sp. strain TRW-2 isolated from a chloroethene degrading enrichment culture. The isolate is a Gram positive rod. The phylogenetic analysis of the nearly full-length 16S rDNA sequence (1392 bases) of the isolate showed close sequence similarity with the members of the genus Mycobacterium (98% similar to Mycobacterium rhodesiae). Tentatively we will refer this organism as Mycobacterium sp. strain TRW-2. As shown in Fig. 1, the strain TRW-2 formed a close cluster with other members of the genus Mycobacterium including M. rhodesiae JS60, M. aurum, and M. strain RJGll-135. Both M. rhodesiae JS60 and M. aurum are capable of assimilating VC, while strain RJGll-135 was shown to degrade polyaromatic hydrocarbons [7,9,22]. Also, the strain TRW-2 loosely clustered with a VC-assimilating Nocardioides sp [7]. On the other hand, no close grouping was found with other VC-degrading bacteria including Ralstonia pickettii (unpublished results), Pseudomonas putida and β-Proteobacterium[8,13]. These groupings clearly show that VC and cis-DCE degrading activity is not confined to one phylogenetic group.

image

Figure 1. Phylogenetic tree resulting from the analysis of 16S rDNA gene from Mycobacterium sp. strain TRW-2 and the other known VC and cis-DCE-assimilating bacteria. The tree was constructed using the neighbor-joining algorithm and p-distance estimation method of the software MEGA 3.O. Bootstrap values (>50%) generated from 1000 replicates are shown as percent values at the nodes. The scale bar represents nucleotide substitution per site. Pseudomonas aeruginosa DL1 was used as the outgroup.

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The pure culture grew on VC or ETH as the sole carbon source (Fig. 2) as evidenced by the concomitant formation of protein. The organism grew on VC with a doubling time of 35.4 h at 30°C calculated from protein production from VC-assimilation under our experimental conditions. In addition to VC and ETH, the Mycobacterium sp. also degraded cis-DCE, but only in the presence of VC or ETH as a primary substrate. No degradation of trans-DCE or TCE occurred either in the presence or absence of VC. These observations are important because cis-DCE and VC are often detected as co-contaminants at most chloroethene-contaminated aquifers.

image

Figure 2. Biodegradation of (a) VC coupled to growth of Mycobacterium sp. strain TRW-2. Bottles containing 50 ml of MSM inoculated with 2.5 ml of VC-grown culture were spiked with VC as the sole source of carbon and energy. (b) Biodegradation of ETH coupled to growth of Mycobacterium sp strain TRW-2. Bottles containing 50 ml of MSM inoculated with 2.5 ml of VC-grown culture were spiked with ETH as the sole source of carbon and energy. Headspace and liquid samples were withdrawn aseptically from bottles at time intervals and analyzed for the degradation of VC or ETH and corresponding accumulation of total protein. Symbols: (•) VC or ETH -assimilation; (▄) protein production. Autoclaved controls showed no degradation (data not shown). Error bars indicate the standard deviation of triplicate bottles.

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The assimilation of VC at different temperatures ranging from 5 to 50°C was assessed (data not shown). VC was degraded at maximum rates between 30 and 37°C. Degradation proceeded at much slower rates at 5, 10, 15, 20, 25, 45, and 50°C. However, VC degradation rate increased from 0.65 μmol/bottle/day to 2.24 μmol/bottle/day in bottles incubated at 25°C after they have been fed VC once more. These observations suggest that strain TRW-2 can easily adapt to a lower temperature range found in field conditions. The ability of the strain TRW-2 to resume VC degradation after various periods of starvation was assessed (data not shown). Results show that VC degradation resumed with no lag time after 1 or 3 days of starvation. However, 7 or 14 days of VC starvation resulted in slow degradation requiring more than 4 weeks to completely degrade 25 μmol/bottle VC. Recently isolated VC-assimilating strains including Mycobacterium sp. strain JS60 and strain Pseudomonas aeruginosa MF1 display similar capabilities [7,10]. The strain MF1 degraded VC after 15 days of lag when grown without VC for 24 days. Similarly, the strain JS60 readily degraded VC following at least 1 week of VC starvation. On the other hand, M. aurum L1 and Nocardioides sp. lost their ability to degrade VC after a brief interruption of VC in the growth media. This loss of activity was attributed to the accumulation of highly toxic chlorooxirane or chloroacetaldehyde [7,9,10]. Growth yield on VC and ETH were 6.53 g protein/mol VC and 14.1 g protein/mol ETH. These values are similar to the values reported for other Mycobacterium strains [7]. The growth yield on VC was almost 50% lower than for ETH-grown cells. Lower yield on VC could be because VC is more oxidized than ETH and also VC-epoxide is a toxic intermediate generated during VC metabolism [7,9,10].

To assess if the same or different enzyme(s) are involved in the degradation of VC and ETH, the culture was fed VC and ETH at various proportions (Fig. 3). Results show that depletion of VC and ETH occurred concurrently (data not shown) and the removal rates were dependent on the relative concentrations of each compound present. VC was degraded at a maximum rate of 2.85 μmol/mg protein/day at a total mole percent VC:ETH ratio of 100:0. The rate of VC degradation decreased proportionally with decreasing concentration of VC. Similarly, ETH was degraded at a maximum rate of 2.33 μmol/mg protein/day at a total mole percent VC:ETH ratio of 0:100, and the rate deceased proportionally with declining ETH concentrations. These results suggest that VC and ETH are degraded by the same enzymatic system. These observations are important because VC and ETH are common co-contaminants in many chloroethene-contaminated aquifers. Addition of acetylene at 5% or 10% of the headspace volume resulted in complete inhibition of VC degradation, implying the involvement of a monooxygenase enzyme in the degradation process [10,23].

image

Figure 3. Biodegradation of VC and ETH when fed together at different ratios (total mole percent). Bottles were spiked with VC and ETH at varied total mole percent VC: ETH ratios of 100:0 (32 μmol VC: 0 μmol ETH), 47:53 (17.33 μmol VC: 19.48 μmol ETH), 21:79 (11.55 μmol VC: 42.27 μmol ETH), 6:95 (3.94 μmol VC: 65.63 μmol ETH), or 0:100 (0 μmol VC: 82 μmol ETH). Results are averages of three bottles.

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3.1Bioaugmentation potential of Mycobacterium sp. strain TRW-2

Although VC is readily degraded under aerobic conditions and the different bacteria responsible for its degradation are widely distributed in a variety of habitats, VC still persists at many contaminated sites [24]. Biostimulation may not be sufficient to ensure rapid and complete degradation of VC in the field if the aquifer does not contain specific degraders in sufficient numbers to exert measurable degradation rates [25]. This may be particularly true for plumes that migrate from anaerobic to aerobic zones. In a recent field study, the anaerobic biodegradation of chloroethenes using bioaugmentation and biostimulation was compared [26]. Results showed that complete conversion of both sorbed and dissolved phase chloroethene to ETH occurred within 43 days through bioaugmenation, while it took more than 121 days through biostimulation. The shorter time and more complete dechlorination suggests the potential for significant cost savings using a bioaugmentation strategy. In addition, many other studies have demonstrated the benefits of bioaugmentation in the degradation of chlorinated solvents [27–30]. We believe that members of the genus Mycobacterium may have advantages over other VC-degraders for in situ transformation by virtue of their ability to grow and metabolize a number of environmental pollutants under a wide range of environmental conditions [7,9,14–17]. Therefore, one of the goals of this study was to assess the ability of the strain TRW-2 to degrade VC in the presence or absence of native bacteria in aquifer samples obtained from different sites.

The bioaugmentation potential of the strain TRW-2 was tested by injecting varied concentrations of the cells in live and sterile microcosms. Complete degradation of VC occurred in live as well as sterile bottles within 6–10 days in most microcosms established with different aquifer samples. On the other hand, approximately 10–15% the initially added VC was removed in most of the un-inoculated microcosms even after incubating >30 days. This could be due to the absence of VC-degrading bacteria and/or absence of substrate (for cometabolic degradation) in aquifer materials used in this study (Table 1). Results showed that the rate of VC degradation was dependent on the population density of the Mycobacterium added to the microcosms. In general, addition of higher cell numbers (107–108 CFU/bottle) resulted in more rapid degradation of VC. This is the first study to obtain preliminary data showing bioaugmentation potential of a VC-assimilating Mycobacterium sp. in aquifer samples under aerobic condition.

Table 1.  Bioaugmentation potential of VC by Mycobacterium sp. strain TRW-2
Sample locationMediaSample typeInoculum CFU/bottleVC degradation (%)
    AutoclavedNon-autoclaved days
  1. Microcosms were inoculated with 0-ml, 2-ml, 4-ml, or 6-ml of the isolate and spiked with initial concentration of 30–38 μmol/bottle VC.

  2. a2-ml inoculum added.

  3. b4-ml inoculum added.

  4. c6-ml inoculum added.

  5. dMicrocosms incubated for more than 30 days.

  6. eHydrite MP-71 sample was taken from a highly contaminated location in the plume. Groundwater concentrations of PCE, TCE, cis-DCE, and VC were 10, 44, 47, and 1.3 mg/L, respectively.

  7. f15 days for autoclaved samples; 43 days for non-autoclaved samples.

Hydrite, WISoilAnoxic0.00E+0d13.213.330
 SoilAnoxic1.60E+0898.298.36
 SoilAnoxic3.20E+0899.899.66
 SoilAnoxic4.80E+08100.0100.06
       
Wichita, KSSoilOxic0.00E+0d10.3−1.230
 SoilOxic1.68E+0699.199.77
 SoilOxic3.36E+06100.0100.07
 SoilOxic5.04E+06100.0100.07
       
B&J Industrial, MISoilOxic0.00E+0d10.3−1.230
 SoilOxic7.17E+0799.199.77
 SoilOxic1.43E+08100.0100.07
 SoilOxic2.15E+08100.0100.07
       
HydriteP-71, WIeGroundwaterAnoxic0.00E+0d21.032.615/43f
 GroundwaterAnoxicunknowna97.542.215/43f
 GroundwaterAnoxicunknownb100.068.715/43f
 GroundwaterAnoxicunknownc100.033.915/43f
       
Hydrite MP-2–2, WIGroundwaterAnoxic0.00E+0d15.515.930
 GroundwaterAnoxic2.90E+07100.098.110
 GroundwaterAnoxic5.80E+07100.0100.010
 GroundwaterAnoxic8.70E+07100.0100.010

Degradation proceeded relatively slowly in microcosms established with groundwater samples obtained from the Hydrite MP-71 site. Complete removal of VC occurred in sterile microcosms in 2 weeks after inoculation with strain TRW-2 and rates increased with increasing number of cells injected (Fig. 4(a)). However, little degradation occurred in live microcosms even after incubating for more than 43 days (Fig. 4(b)). The exact reason for this slow or negligible degradation of VC in the Hydrite MP-71 sample is not known. It could be that this sample had unusually high levels of chloroethenes as determined at the time of sample collection. The field concentrations of PCE, TCE, cis-DCE and VC in the sample were 10,000, 44,000, 47,000, and 1300 μg/L, respectively. Therefore, we speculate that perhaps this sample was toxic for the bioaugmented TRW-2 cells [31–33]. Autoclaving might have reduced the concentrations of these compounds in sterile microcosms. Alternatively, the inhibition of VC degradation in live microcosms could be due to competition by the native microflora.

image

Figure 4. (a) Biodegradation of VC in microcosms established with sterile groundwater obtained from the Hydrite P-71 site. The microcosms were inoculated with Mycobacterium sp strain TRW-2 at varied cell densities. Symbol: (•) 0-ml of inoculum; (∘) 2-ml of inoculum; (▴) 4-ml of inoculum; (▵) 6-ml of inoculum. Error bars indicate the standard deviation of triplicate bottles. (b) Biodegradation of VC in microcosms established with non-autoclaved groundwater obtained from the Hydrite P-71 site. The microcosms were inoculated with Mycobacterium sp. strain TRW-2 at varied cell densities. Symbol: (•) 0-ml of inoculum; (∘) 2-ml of inoculum; (▴) 4-ml of inoculum; (▵) 6-ml of inoculum. Error bars indicate the standard deviation of triplicate bottles.

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Overall these observations suggest that strain TRW-2 is capable of rapid degradation of VC in the presence of native population present in aquifer materials obtained from different chloroethene-contaminated sites. However, it should be noted that the bioaugmented TRW-2 cells in our studies may have had relatively less competition from the native bacteria because the aquifer samples used in this study were mostly taken from anoxic zones and thus may not represent typical population densities found in aerobic zones. The organism's capacity to degrade a variety of compounds such as VC, ETH and cis-DCE, combined with its ability to survive >2 weeks of starvation makes this organism a better candidate for field bioremediation applications, especially for sites lacking VC degradation activity.

Acknowledgements

  1. Top of page
  2. Abstract
  3. 1Introduction and background
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References

This work was supported by a grant from the Department of Defense's Strategic Environmental Research and Development Program (SERDP) and by the Environmental Institute's Water Research Center at Oklahoma State University. We are grateful to Al Bourquin, Camp Dresser & McKee for providing the TRW aquifer material. We also thank Collin Talbot, Maya Kitaoka, Udaya DeSilva, and Charaka Fernando for help with maintenance of the culture, GC analysis, and phylogenetic evaluation.

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  1. Top of page
  2. Abstract
  3. 1Introduction and background
  4. 2Materials and methods
  5. 3Results and discussion
  6. Acknowledgements
  7. References
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