Enrichment with Carbon-13 and Deuterium during Monooxygenase

Feb 12, 2018 - For example, a data-mining study has shown attenuation of 1,4-dioxane is associated with higher levels of dissolved oxygen in groundwat...
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Letter Cite This: Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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Enrichment with Carbon-13 and Deuterium during MonooxygenaseMediated Biodegradation of 1,4-Dioxane Peter Bennett,*,† Michael Hyman,‡ Christy Smith,‡ Humam El Mugammar,§ Min-Ying Chu,∥ Michael Nickelsen,⊥ and Ramon Aravena# †

Haley & Aldrich, Inc., 1956 Webster Street, Suite 300, Oakland, California 94612, United States Department of Plant and Microbial Biology, North Carolina State University, Raleigh, North Carolina 27695, United States § Environmental Isotope Laboratory, University of Waterloo, Waterloo, ON N2L 3G1, Canada ∥ Haley & Aldrich, Inc., 400 East Van Buren Street, Suite 545, Phoenix, Arizona 85004, United States ⊥ ECT2, Inc., 200 Town Center Drive, Suite 2, Rochester, New York 14263, United States # Department of Earth and Environmental Sciences, University of Waterloo, Waterloo, ON N2L 3G1, Canada ‡

S Supporting Information *

ABSTRACT: Recent technical developments have enabled the application of compound-specific isotope analysis (CSIA) of low (parts per billion) concentrations of 1,4-dioxane that are often found in groundwater at 1,4-dioxane-contaminated sites. However, to quantify 1,4-dioxane biodegradation, isotopic enrichment factors are needed to interpret the CSIA data obtained from field samples. In this study, the carbon and hydrogen isotopic enrichment factors (εC and εH, respectively) for 1,4dioxane biodegradation have been determined for axenic propane- or isobutanegrown cultures of Rhodococcus rhodochrous ATCC 21198 and for tetrahydrofurangrown cultures of Pseudonocardia tetrahydrof uranoxidans K1. The enrichment factors for propane-grown (εC = −2.7 ± 0.3‰, and εH = −21 ± 2‰) and isobutane-grown (εC = −2.5 ± 0.3‰, and εH = −28 ± 6‰) cells of strain 21198 were similar and substantially smaller than those determined for tetrahydrofuran-grown cells of strain K1 (εC = −4.7 ± 0.9‰, and εH = −147 ± 22‰). The presence of 1-butyne consistently inhibited both the biodegradation and isotopic fractionation of 1,4-dioxane, and this effect implicates monooxygenase enzymes in both the biodegradation and isotopic enrichment of 1,4-dioxane. Our results confirm that an increasing level of enrichment of heavier isotopes of carbon and hydrogen can be used to quantify 1,4-dioxane biodegradation and suggest CSIA can discriminate between the activities of monooxygenase-expressing bacteria expected to be prevalent in engineered, gaseous alkane-stimulated 1,4-dioxane treatment systems and those that may involve microbial metabolism of 1,4-dioxane as a natural attenuation process.

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samplers.11 A correlation between 1,4-dioxane concentration and the distribution and abundance of genes that encode a 1,4dioxane-degrading enzyme, tetrahydrofuran (THF) monooxygenase (THFMO), has also been demonstrated in groundwater.12 In this study, we have explored compound-specific isotope analysis (CSIA) as a direct method for characterizing 1,4dioxane biodegradation. The utility of CSIA for studying the fate of environmental contaminants arises because chemical and biological degradation reactions kinetically favor the breaking of bonds between the highly abundant lighter isotopes (e.g., 12 C−1H) over the breaking of bonds involving the rarer heavier isotopes (13C−1H or 12C−2H). The resulting kinetic isotope effect (KIE) of degradation reactions causes an increase in the levels of 13C/12C and 2H/1H in the remaining contaminant.13

INTRODUCTION 1,4-Dioxane is a cyclic ether considered a possible human carcinogen.1 It has become an important contaminant because of its widespread occurrence and high mobility in groundwater,2,3 high frequency of detection in public water supplies,4 and resistance to conventional water treatment technologies such as carbon filtration (due to low sorption) and air stripping (due to low vapor pressure). Although there was initial uncertainty about the potential for 1,4-dioxane biodegradation,5,6 aerobic 1,4-dioxane biodegradation has now been demonstrated in many studies.7−9 While laboratory studies have identified dozens of bacterial strains that can biodegrade 1,4-dioxane via either direct metabolism or co-metabolism, there are currently few tools available that can unequivocally demonstrate 1,4-dioxane biodegradation in the field. For example, a data-mining study has shown attenuation of 1,4-dioxane is associated with higher levels of dissolved oxygen in groundwater.10 Biodegradation of 1,4-dioxane has also been inferred at an aerobic aquifer based on phospholipid fatty acid stable isotope probing analyses of biomass obtained from 13C-1,4-dioxane-baited Bio-Trap © XXXX American Chemical Society

Received: Revised: Accepted: Published: A

December 19, 2017 February 9, 2018 February 12, 2018 February 12, 2018 DOI: 10.1021/acs.estlett.7b00565 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

Letter

Environmental Science & Technology Letters In contrast, processes such as dilution and evaporation may strongly affect contaminant concentrations but typically do not exhibit significant isotopic effects.13 Via measurement of the stable isotope ratio (e.g., 13C/12C and 2H/1H) of a contaminant by CSIA, the extent of degradation can be calculated using the Rayleigh isotopic enrichment model if both the isotopic ratio of the undegraded contaminant and the relevant carbon and hydrogen enrichment factors (εC and εH, respectively) are known.14 Selection of appropriate values for εC and εH is critical, and for biodegradation reactions, εC and εH are typically established with pure cultures of bacteria with known kinetic and enzymatic features. The KIEs for the rate-determining step of a biodegradation reaction can also provide insights into reaction mechanisms.15 The KIE observed from CSIA is termed the “apparent KIE” (AKIE) because it can be smaller than the actual KIE for enzymatic biodegradation reactions when other nonfractionating reaction steps mask the intrinsic KIE.16 The determination of AKIEs allows for a comparison with published KIEs for known reactions to assess potential reaction mechanisms.15 Simultaneous measurement of isotope ratios for two elements in molecules of interest (e.g., 13C/12C and 2H/1H) allows the determination of two enrichment factors for the reaction. The ratio of these enrichment factors can be diagnostic of a specific reaction mechanism.17,18 This dual-isotope approach has provided valuable insights into degradation mechanisms for other important contaminants, including 1,2-dichloroethane19 and fuel-oxygenate ethers such as methyl tert-butyl ether (MTBE).20 Although CSIA has greatly advanced our understanding of the fate of many chemicals in the environment, CSIA has not yet been widely applied to 1,4-dioxane because analytical limitations have prevented its use at environmentally relevant concentrations. Recent developments now allow CSIA to be reliably conducted for 1,4-dioxane at concentrations in the range of 1−10 μg/L that are found at many 1,4-dioxanecontaminated sites.21,22 However, without published εC and εH values for 1,4-dioxane biodegradation, CSIA results can be inconclusive. To date, only one study, conducted with the 1,4dioxane-metabolizing strain Pseudonocardia dioxanivorans CB1190, has reported an εC value for 1,4-dioxane biodegradation (−1.73 ± 0.14‰).23 To the best of our knowledge, no εH values for 1,4-dioxane biodegradation have been previously reported. In this study, we have examined the 1,4-dioxane-degrading activities of two bacteria. The first of these, Rhodococcus rhodochrous ATCC 21198, is an alkane-metabolizing bacterium that is one of the few commercially available strains known to grow on both propane and isobutane.24 This bacterium does not grow on 1,4-dioxane, and its ability to co-metabolically degrade 1,4-dioxane after growth on gaseous alkanes has not been previously described. However, propane-dependent cometabolic degradation of 1,4-dioxane has been reported in both laboratory and field studies.25,26 The second bacterium, Pseudonocardia tetrahydrof uranoxidans K1, also does not grow on 1,4-dioxane but grows rapidly on THF and can cometabolically degrade 1,4-dioxane after growth on THF.27 This bacterium initiates both THF metabolism and 1,4-dioxane cometabolism through the activity of THFMO.28 Strain K1 was selected for analysis in this study not only because THFMO is responsible for initiating 1,4-dioxane biodegradation in the closely related 1,4-dioxane-metabolizing strain, P. dioxanivorans CB11909 but also because a comprehensive set of εC and εH

values have been previously reported for the co-metabolic degradation of other ethers such as MTBE, ethyl tert-butyl ether (ETBE), and tert-amyl methyl ether (TAME) by THFgrown cells of this bacterium.20 Using laboratory scale studies with pure cultures of strains 21198 and K1, we report here three pairs of εC and εH values for 1,4-dioxane biodegradation. Our results suggest that two-dimensional CSIAs can be used not only to determine the extent of 1,4-dioxane biodegradation but also to discriminate between different types of 1,4-dioxaneoxidizing monooxygenases.

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MATERIALS AND METHODS 1,4-Dioxane Biodegradation Experiments. Propanegrown or isobutane-grown cells of strain 21198 and THFgrown resting cells of strain K1 were used for the biodegradation experiments. Preparation methods for the three sources of washed cells used in the biodegradation reactions are provided in the Supporting Information. Duplicate biodegradation reactions for propane-grown and isobutane-grown cells of strain 21198, and THF-grown cells of strain K1, were conducted in glass serum bottles (1 L) sealed with Teflon-lined Mininert stoppers as described in greater detail in the Supporting Information. The sealed bottles contained sodium phosphate buffer (197 mL) and 1,4-dioxane (91 μL, neat) to give an initial dissolved aqueous 1,4-dioxane concentration of ∼5.4 mM (476 mg/L). A biologically active control bottle was prepared for each cell type by adding 1butyne [10% (v/v) gas phase] as a selective inactivator of monooxygenase enzymes.29 Abiotic controls were prepared with 1,4-dioxane and buffer, but without resting cells. The serum bottles were sampled periodically over the course of the reactions for quantification and isotopic characterization of 1,4dioxane as described in the Supporting Information. Compound-Specific Isotope Analysis. Determinations of 13C/12C and 2H/1H were performed at the University of Waterloo Environmental Isotope Laboratory (UWEIL) in Waterloo, Ontario, Canada, using a recently developed method for concentrating dilute aqueous 1,4-dioxane onto a synthetic carbonaceous sorbent. The sorbent is then placed in a thermal desorption tube where 1,4-dioxane is thermally desorbed into a gas chromatograph fitted with a combustion or pyrolysis reactor, followed by an isotope ratio mass spectrometer. The method is described in greater detail in the Supporting Information. All carbon and hydrogen isotope ratios are reported as heavy to light (R = 13C/12C or 2H/1H) and expressed as δ13C and δ2H relative to Vienna Pee Dee Belemnite (δ13CVPDB) and Vienna Standard Mean Ocean Water (δ2HVSMOW), respectively, based on eq 1: ⎛ R sample ⎞ δ(‰) = ⎜ − 1⎟1000 ⎝ R s tandard ⎠

(1)

Values for εC and εH were calculated from the slope of linear regression analysis of natural logarithms of normalized δ13C and δ2H values, respectively, versus the natural logarithm of the fraction remaining (eqs S1 and S2; based on the Rayleigh isotope enrichment model). AKIEs for carbon (AKIEC) and hydrogen (AKIEH) were calculated according to eq S3, and neglecting secondary isotope effects, using the method of Elsner et al.15 B

DOI: 10.1021/acs.estlett.7b00565 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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Table 1. Summary of Carbon and Hydrogen Isotopic Enrichment Factors and Apparent Kinetic Isotope Effects during 1,4Dioxane Degradation in Microcosms strain

growth substrate

εC (‰)

AKIEC

εH (‰)

AKIEH

Δδ2H/Δδ13C

R. rhodochrous ATCC 21198 R. rhodochrous ATCC 21198 P. tetrahydrof uranoxidans K1

propane isobutane THF

−2.7 ± 0.3 −2.5 ± 0.3 −4.7 ± 0.9

1.01 1.01 1.02

−21 ± 2 −28 ± 6 −147 ± 22

1.2 1.3 >10

7.5 ± 1.1 10.9 ± 2.2 37.2 ± 2.6

Figure 1. Dual-isotope plot highlighting the enrichment with 2H relative to 13C during 1,4-dioxane degradation by strain 21198 grown on propane (blue dots) and isobutane (purple diamonds) and for strain K1 grown on THF (green squares). Solid lines represent the linear best fits of the slope and R2: for propane, slope = 7.5 and R2 = 0.94; for isobutane, slope = 10.9 and R2 = 0.91; for THF, slope = 37.2 and R2 = 0.99. Colored shaded areas represent 95% confidence intervals of the slope of the regression line (Grapher 12, Golden Software).

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Supporting Information shows that the εH values of −28 ± 6 and −21 ± 2‰ are different from one another at a 95% confidence level. Bulk εC and εH values derived through regression (Figure S3) are equivalent to reactive position enrichment factors when there are no nonparticipating atoms15 (Supporting Information). Following the method of Elsner et al.,15 the 13C and 2H apparent kinetic isotope effects (AKIEC and AKIEH, respectively) were calculated from εC and εH based on eq S3 for comparison with the expected range of values reported for experimentally determined carbon and hydrogen kinetic isotope effects (KIEC and KIEH, respectively) (Table 1). Plots of Δδ2Η versus Δδ13C for the three sets of reaction data indicate different slopes for each, suggesting the mechanism for hydroxylation could be different for the propane-, isobutane-, and THF-grown cells. R. rhodochrous ATCC 21198. The AKIEC value (1.01) for propane- and isobutane-grown cells of strain 21198 equals the expected value for cleavage of a C−H bond15 and is consistent with experimentally determined AKIEC values for methyl group C−H bond cleavage during MTBE oxidation.20 On the other hand, the AKIEH values determined for propane- and isobutane-grown cells (1.2 and 1.3, respectively) of strain 21198 are smaller than expected for C−H bond cleavage and more consistent with an SN1-type nucleophilic substitution mechanism or masking of KIEH due to slower nonfractionating steps (Table 1). While the Δδ2Η/Δδ13C for isobutane-grown cells (10.9 ± 2.2; Figure 1 and Table 1) is also consistent with that reported for abiotic acid hydrolysis of MTBE (an SN1-type mechanism),31 such a mechanism does not seem probable for 1,4-dioxane because, unlike MTBE, 1,4-dioxane cannot form the requisite tertiary carbocation. An SN2-type mechanism is typically associated with smaller molecules containing carbon− halogen bonds and is therefore not expected to be important

RESULTS AND DISCUSSION The 1,4-dioxane concentration was stable in all control reactions [494 ± 31.4 mg/L (Figure S1a)], indicating that no significant degradation of 1,4-dioxane occurred either in the abiotic control reactions lacking resting cells or in the biologically active control reactions where monooxygenase activity was inactivated by 1-butyne. The isotopic composition of 1,4-dioxane also remained constant in all control reactions [δ13C = −28.8 ± 0.2‰, n = 16; δ2H = −54 ± 4‰, n = 16 (Figure S1b)] with any variability being within the typical range of analytical precision for δ13C and δ2H analysis by GC-IRMS (±0.5‰ for δ13C and ±5‰ for δ2H).30 Concentration and isotopic data for all individual reactions are listed in Table S1. In the absence of 1-butyne, reactions that included resting cells grown on either propane, isobutane, or THF all biodegraded ≥95% of the initial 1,4-dioxane within 80−220 h (Figure S2a), and this biodegradation was accompanied by a concurrent increase in δ13C and δ2H values in the remaining undegraded 1,4-dioxane (Figure S2b−d). The magnitude of 13 C and 2H enrichment was reproducible in each duplicate biodegradation reaction, and the trends closely followed the Rayleigh isotopic enrichment model (Figure S3). THF-grown cells of strain K1 produced a larger 13C enrichment of residual 1,4-dioxane (εC = −4.7 ± 0.9‰) compared to that of the propane- and isobutane-grown cells of strain 21198 [εC = −2.7 ± 0.3‰ and −2.5 ± 0.3‰ (Table 1)]. The 13C enrichment is larger than that reported for the aerobic metabolism of 1,4dioxane by P. dioxanivorans CB1190 (εC = −1.73 ± 0.14‰).23 Large differences were apparent in εH values for the three different experimental conditions. For THF-grown cells of strain K1, εH = −147 ± 22‰, compared to εH values of −28 ± 6 and −21 ± 2‰ for isobutane- and propane-grown cells of strain 21198, respectively. A statistical analysis included in the C

DOI: 10.1021/acs.estlett.7b00565 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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Residual 1,4-dioxane in reactions with THF-grown K1 also displayed a very high 2H enrichment [≥500‰ at ∼95% degradation (Figure 1)]. The AKIEH for strain K1 was not determined because a high position-specific εH value (−147‰) coupled with intramolecular competition from eight H atoms resulted in a negative AKIEH using eq S3. This is also consistent with the high AKIEH observed for MTBE oxidation by permanganate.31 The large εH is consistent with oxidative C− H bond cleavage by monooxygenase as the rate-determining step during degradation of 1,4-dioxane by strain K1 based on the large enrichment in both C and H isotopes (Table 1). McKelvie et al. evaluated stable C and H isotope fractionation expressed by strain K1 during degradation of oxygenate ethers (MTBE, ETBE, and TAME), reporting an AKIEC of 1.01 for degradation of all three ethers, consistent with oxidative C−H bond cleavage.20 Strong enrichment with 2 H relative to 13C was also observed for degradation of these ethers by strain K1 (Δδ2Η/Δδ13C = 48).20 For 1,4-dioxane degradation by THF-grown strain K1, we report 13C and 2H enrichments for 1,4-dioxane larger than those reported by McKelvie et al. for the oxygenate ethers, and a smaller value for Δδ2Η/Δδ13C (37.2 ± 2.6) due to a higher εC [−4.7 ± 0.9‰ (Table 1)]. This demonstrates that isotope fractionation by the same organism and enzyme system can vary between different (but similar) chemicals even when the initial reaction step (e.g., C−H bond oxidation) is similar. The larger Δδ2Η/Δδ13C compared to those observed for strain 21198 again highlights the potential for CSIA to identify which type of monooxygenase(s) may be responsible for 1,4-dioxane degradation at groundwater cleanup sites (Table 1). In natural environments, multiple organisms capable of degrading 1,4-dioxane may coexist, with multiple competing degradation pathways. When coupled with analyses of monooxygenase biomarkers, CSIA could provide a very compelling case for specific 1,4-dioxane degradation mechanisms in these undefined microbial communities. Such knowledge is critical for selecting the appropriate values for ε C and ε H when attempting to quantify 1,4-dioxane biodegradation based on CSIA results.

for 1,4-dioxane. The isotopic evidence more closely aligns with C−H bond oxidation as a mechanism for 1,4-dioxane hydroxylation rather than SN2 considering the AKIEC of 1.01 appears to be too small for SN2. Reported values for AKIECassociated with SN2 mechanisms are fairly large, generally between 1.029 and 1.072, consistent with chemical literature values of 1.03−1.09.15 In addition, the relatively large apparent hydrogen isotope effects (AKIEH = 1.2−1.3) suggest a primary isotope effect on a hydrogen bond, consistent with C− H oxidation. An SN2 mechanism would initially involve cleavage of a C−O bond, consistent with a smaller (secondary) hydrogen isotope effect. The different slopes of Δδ2Η/Δδ13C (Figure 1) for propanegrown versus isobutane-grown cells of strain 21198 may also suggest different enzymes are involved in the initial oxidation of 1,4-dioxane.16 The annotated draft genome of strain 2119832 indicates this bacterium possesses complete genes for two distinct soluble diiron monooxygenases and that these genes are nearly identical to those described in another closely related bacterium, R. rhodochrous BCP1. Transcriptional studies with strain BCP1 have shown the gene encoding the hydroxylase component of a short chain alkane-oxidizing monooxygenase (SCAM) is expressed at consistently high levels in cells exposed to C2−C6 n-alkanes. In contrast, the gene encoding the hydroxylase component of a propane monooxygenase (PrMO) is maximally expressed in cells exposed to propane. Although isobutane was not tested, the only other volatile alkane supporting expression of this gene, albeit at levels much lower than that of propane, is n-butane.33 It is not currently known what the expression patterns are for these genes and their corresponding enzymes during growth of strain 21198 on propane and isobutane. It is also not clear whether only one or both of these monooxygenases can oxidize 1,4-dioxane. However, in view of the genetic similarities between strains 21198 and BCP1, the experimentally determined values of Δδ2Η/Δδ13C (Table 1) may result from differential expression of these genes and their corresponding enzymes in response to different alkane growth substrates. A similar situation involving an enzyme very similar (84−93% amino acid identity) to SCAM has also recently been described for the 1,4-dioxanemetabolizing strain PH-06.34−36 This bacterium co-expresses genes for both a SCAM-like monooxygenase and a separate copper-containing monooxygenase at similar high levels during growth on 1,4-dioxane. It is not yet known whether only one or both of these enzymes are reactive toward 1,4-dioxane. Our current results suggest that measuring both δ2Η and δ13C during 1,4-dioxane biodegradation may be a useful approach for investigating this issue as these values may be diagnostic of the type of monooxygenase(s) responsible for 1,4-dioxane oxidation by the same bacterium grown under different growth conditions. P. tetrahydrof uranoxidans K1. THF-metabolizing organisms, including strain K1, degrade diverse ethers using THFMO to catalyze an initial oxidative cleavage of a C−H bond.9 The AKIEC value (1.02) for THF-grown cells of strain K1 is at the high end of the range expected for cleavage of a C−H bond15 and slightly above experimentally determined AKIEC values for methyl group C−H bond cleavage during MTBE oxidation (1.01).20 While an AKIEC of 1.02 supports THFMO-mediated oxidation as the initial degradation step, this step exhibits a 13C fractionation higher than what has been previously observed for the degradation of ether oxygenates by THF-grown cells of strain K1.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.estlett.7b00565. Description of growth conditions, biodegradation experiments, compound-specific isotope analytical methods, calculations for εC, εH, AKIEC, and AKIEH, and tabulated experimental data, time series plots, Rayleigh plots, and regression statistics (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +1 510 879 4547. ORCID

Peter Bennett: 0000-0002-0714-4184 Min-Ying Chu: 0000-0001-6417-0736 Notes

The authors declare no competing financial interest. D

DOI: 10.1021/acs.estlett.7b00565 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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Environmental Science & Technology Letters

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(17) Kuder, T.; Wilson, J. T.; Kaiser, P.; Kolhatkar, R.; Philp, P.; Allen, J. Enrichment of stable carbon and hydrogen isotopes during anaerobic biodegradation of MTBE: microcosm and field evidence. Environ. Sci. Technol. 2005, 39 (1), 213−220. (18) Zwank, L.; Berg, M.; Elsner, M.; Schmidt, T. C.; Schwarzenbach, R. P.; Haderlein, S. B. New evaluation scheme for two-dimensional isotope analysis to decipher biodegradation processes: Application to groundwater contamination by MTBE. Environ. Sci. Technol. 2005, 39 (4), 1018−1029. (19) Palau, J.; Shouakar-Stash, O.; Hatijah Mortan, S.; Yu, R.; Rosell, M.; Marco-Urrea, E.; Freedman, D. L.; Aravena, R.; Soler, A.; Hunkeler, D. Hydrogen isotope fractionation during the biodegradation of 1,2-dichloroethane: Potential for pathway identification using a multi-element (C, Cl, and H) isotope approach. Environ. Sci. Technol. 2017, 51 (18), 10526−10535. (20) McKelvie, J. R.; Hyman, M. R.; Elsner, M.; Smith, C.; Aslett, D. M.; Lacrampe-Couloume, G.; Sherwood Lollar, B. Isotopic fractionation of methyl tert-butyl ether suggests different initial reaction mechanisms during aerobic biodegradation. Environ. Sci. Technol. 2009, 43 (8), 2793−2799. (21) SERDP Project ER-2535. Extending the Applicability of Compound-Specific Isotope Analysis to Low Concentrations of 1,4D i o x a n e . h t t p s : / / w w w . s e rd p - e s t c p . o r g / P r o g r a m - A r e a s / Environmental-Restoration/Contaminated-Groundwater/EmergingIssues/ER-2535/ER-2535 (accessed January 16, 2018). (22) Wang, Y. Breakthrough in 2D-CSIA technology for 1,4-dioxane. Remediation 2016, 27 (1), 61−70. (23) Pornwongthong, P.; Paradis, G. L.; Mahendra, S. Stable carbon isotope fractionation during 1,4-dioxane biodegradation. In Proceedings of the Water Environment Federation; Water Environment Federation: Los Angeles, 2011; pp 111−116. (24) MacMichael, G. J.; Brown, L. R. Role of carbon dioxide in catabolism of propane by “Nocardia paraf f inicum” (Rhodococcus rhodochrous). Appl. Environ. Microbiol. 1987, 65−69. (25) Chu, M.-Y.; Bennett, P.; Dolan, M.; Hyman, M.; Anderson, R. H.; Bodour, A.; Peacock, A. Successful Bioremediation of 1,4-Dioxane and 1,2-Dichloroethane in a Dilute Plume. In 2017 GRA Conference and 26th Annual Meeting; Groundwater Resources Association of California: Sacramento, CA, 2017. (26) Lippincott, D.; Streger, S. H.; Schaefer, C. E.; Hinkle, J.; Stormo, J.; Steffan, R. J. Bioaugmentation and propane biosparging for in situ biodegradation of 1,4-dioxane. Groundwater Monit. Rem. 2015, 35 (2), 81−92. (27) Kohlweyer, U.; Thiemer, B.; Schräder, T.; Andreesen, J. R. Tetrahydrofuran degradation by a newly isolated culture of Pseudonocardia sp. strain K1. FEMS Microbiol. Lett. 2000, 186 (2), 301−306. (28) Thiemer, B.; Andreesen, J. R.; Schräder, T. Cloning and characterization of a gene cluster involved in tetrahydrofuran degradation in Pseudonocardia sp. strain K1. Arch. Microbiol. 2003, 179 (4), 266−277. (29) Hyman, M. R.; Daniel, A. Acetylene inhibition of metalloenzymes. Anal. Biochem. 1988, 173, 207−220. (30) Hunkeler, D.; Bernasconi, S. Analysis of Stable Isotopes. In Environmental Isotopes in Biodegradation and Bioremediation; Aelion, C. M., Hohener, P., Hunkeler, D., Aravena, R., Eds.; CRC Press: Boca Raton, FL, 2010; pp 23−42. (31) Elsner, M.; McKelvie, J.; Lacrampe Couloume, G.; Sherwood Lollar, B. Lacrampe Couloume, G.; Sherwood Lollar, B. Insight into methyl tert-butyl ether (MTBE) stable isotope fractionation from abiotic reference experiments. Environ. Sci. Technol. 2007, 41 (16), 5693−5700. (32) Shields-Menard, S. A.; Brown, S. D.; Klingeman, D. M.; Indest, K.; Hancock, D.; Wewalwela, J. J.; French, W. T.; Donaldson, J. R. Draft Genome sequence of Rhodococcus rhodochrous strain ATCC 21198. Genome Announc 2014, 2 (1), e00054-14. (33) Cappelletti, M.; Presentato, A.; Milazzo, G.; Turner, R. J.; Fedi, S.; Frascari, D.; Zannoni, D. Growth of Rhodococcus sp. strain BCP1 on gaseous n-alkanes: New metabolic insights and transcriptional analysis

ACKNOWLEDGMENTS Funding for this research was provided by the U.S. Department of Defense, Strategic Environmental Research and Development Program (Grants ER-2303 and ER-2535), and the Natural Sciences and Engineering Research Council of Canada (Discovery Grant).

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DOI: 10.1021/acs.estlett.7b00565 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.estlett.7b00565 Environ. Sci. Technol. Lett. XXXX, XXX, XXX−XXX