Article pubs.acs.org/est
Insight on RDX Degradation Mechanism by Rhodococcus Strains Using 13C and 15N Kinetic Isotope Effects Anat Bernstein,†,§ Zeev Ronen,†,* and Faina Gelman‡ †
Zuckerberg Institute for Water Research, Department of Environmental Hydrology and Microbiology, Ben-Gurion University of the Negev, Sede Boqer Campus, 84990, Israel ‡ Geological Survey of Israel, 30 Malkhey Israel St., Jerusalem, 95501, Israel ABSTRACT: The explosive Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) is known to be degraded aerobically by various isolates of the Rhodococcus species, with denitration being the key step, mediated by Cytochrome P450. Our study aimed at gaining insight into the RDX degradation mechanism by Rhodococcus species and comparing isotope effects associated with RDX degradation by distinct Rhodococcus strains. For these purposes, enrichment in 13C and 15N isotopes throughout RDX denitration was studied for three distinct Rhodococcus strains, isolated from soil and groundwater in an RDX-contaminated site. The observable 15N enrichment throughout the reaction, together with minor 13C enrichment, suggests that N−N bond cleavage is likely to be the key rate-limiting step in the reaction. The similarity in the kinetic 15N isotope effect between the three tested strains suggests that either isotope-masking effects are negligible, or are of a similar extent for all tested strains. The lack of variability in the kinetic 15N isotope effect allows the interpretation of environmental studies with greater confidence.
■
The denitration mechanism was first studied by Bhushan et al.,14 who carried out experiments with rabbit liver cytochrome P450 and Rhodococcus sp. strain DN22. These authors suggested that cytochrome P450 catalyzes two sequential transfers of single electrons from NADPH, each resulting in denitration. The remaining RDX product, having only one nitro group, was identified as being unstable and as spontaneously decomposing to the ring-cleavage product 4-nitro-2,4-diazabutanal (NDAB; Figure 1). Halasz et al.,15 on the other hand, postulated that the very first step in the aerobic degradation of RDX by cytochrome P450 is a C−H bond cleavage rather than a direct attack of the nitro group. Specifically, they suggested that the C−H bond cleavage is initiated by superoxide radicals that are formed from O2 by cytochrome P450, resulting in an H• abstraction as the first catalytic step, which is followed by denitration. Then, the reaction normally proceeds to the second denitration step; although in some cases only one denitration step occurs. As documented also by Bhushan et al.,14 the denitration is followed by ring cleavage and the formation of NDAB (and, to a slight extent, methylenedinitramine, MEDINA; Figure 1). The mechanism suggested by Halasz et al.15 can be seen as analogous to the denitration of RDX by alkaline hydrolysis, in which a nucleophilic attack results in the C−H bond cleavage accompanied by denitration and the spontaneous formation of NDAB16 (Figure 1). Although this latter mechanism does not involve the formation of a radical intermediate, it shows that when degradation is initiated by a C−H bond cleavage, subsequent denitration and the formation of NDAB is indeed possible.
INTRODUCTION The explosive compound hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) frequently pollutes soils and groundwater adjacent to production plants and military training areas. Microbial biodegradation of RDX has been extensively studied in recent years.1 It was shown that RDX serves as a substrate, mostly a nitrogen source, for various Rhodococcus species under aerobic conditions (e.g., refs 2−4), thus promoting its removal from the environment.5Considerable effort has been made to elucidate the aerobic biochemical pathway by which RDX is degraded by these species. Studies have shown that denitration is a key step in the process and that it is followed by ring cleavage.2 The denitration process was found to be catalyzed by a unique form of the enzyme cytochrome P4506 encoded by the gene XplA,3,5,7−9 with NADPH as an electron donor. Activity of cytochrome P450 in RDX denitration was demonstrated in vivo for various Rhodococcus strains isolated from contaminated habitats worldwideamong them, strain DN22 (isolated from contaminated soils in Australia 6); 11Y (isolated from contaminated soils in England10); YH1, T7, and T9N (isolated from contaminated soils11 and groundwater12 in Israel). This global distribution may indicate that the denitration mechanism is a fundamental degradation pathway for the compound under aerobic conditions. Although the denitration pathway has been thoroughly studied, the exact mechanism by which this process takes place is still questioned. Specifically, there is no consensus on which bond is actually initially cleaved during the enzymatic reaction. Since the triazinic RDX ring lacks stability, in contradiction to the aromatic ring of the explosive compound TNT for example, it was suggested that any theoretical attack of either an N−NO2 or C−H bond will result in ring cleavage.13 The observation of denitration alone does not necessarily provide sufficient knowledge about the first mechanistic step. © 2012 American Chemical Society
Received: Revised: Accepted: Published: 479
July 6, 2012 December 6, 2012 December 6, 2012 December 6, 2012 dx.doi.org/10.1021/es302691g | Environ. Sci. Technol. 2013, 47, 479−484
Environmental Science & Technology
Article
Figure 1. Postulated denitration pathways of RDX.
Compound-specific isotope analysis (CSIA) may be applied as a tool for distinguishing between the different postulated degradation mechanisms: Atoms within bonds that are cleaved during the reaction’s rate limiting step frequently lead to observable isotope enrichment (“primary isotope effect”), whereas adjacent atoms typically show only slight, or undistinguished, enrichment (“secondary isotope effect”).17 Thus, the knowledge gaps regarding the RDX denitration mechanism may be discovered by monitoring the isotope composition of a postulated target element within the molecule. Previous experiments with Rhodococcus strain YH1 have already shown that denitration is accompanied by primary nitrogen isotope effects,18 whereas carbon isotope effects were not studied. Thus, a complementary CSIA of carbon in RDX throughout the microbial degradation reaction may assist in better understanding of the denitration mechanism. Monitoring the nitrogen and carbon isotope effects during RDX degradation by different Rhodococcus species is not only of mechanistic importance, but of further interest for environmental applications, for the following reasons: 1. Dual-isotope analysis may assist in distinguishing between different potential degradation pathways in the complex environment (e.g., refs 17 and 19), and 2. In recent years, increasing lines of evidence show that the magnitude of the isotope effect may differ between different strains, even if they catalyze the same reaction, and even if they share the same enzymes, as shown experimentally, for example, for atrazine,20 or tetrachloroethylene.21 This phenomenon is explained by differences in slow transport steps prior to the enzymatic reaction, such as diffusion in the growth medium to the cell, diffusion through the cell membrane, or transport to the enzyme’s active site.22,23 These processes mask the intrinsic kinetic isotope effects to different extents, independently from the actual enzymatic reaction. In mechanistic studies, important understanding can be derived, even if differences in the masking effects are evident, since the magnitude of the primary isotope effect is of less importance. In environmental studies, on the other hand, when the goal is to assess the extent of biodegradation in the field, it
is desirable to reveal the variability in isotope effects for a range of degrading strains. The present study aimed to investigate the RDX degradation mechanism of Rhodococcus species by monitoring isotope fractionation of both 13C and 15N throughout the process. To this end, biodegradation experiments were carried out by three distinct RDX-degrading Rhodococcus strains: strain YH1, isolated from contaminated soils,11 and strains T7 and T9N, isolated from a contaminated aquifer.12 The three different strains are known to catalyze the same reaction with the same enzyme and the same mechanism.12 Therefore, comparing the kinetic isotope effects associated with the degradation enabled us to investigate how representative the laboratory-derived isotope enrichment factors are for distinct Rhodococcus strains.
■
EXPERIMENTAL SECTION Biodegradation Experiments. Biodegradation experiments were carried out for three distinct isolates of Rhodococcus sp. strains YH1, T7, and T9N, thoroughly studied in previous works.4,12 Incubation (up to 10 days) experiments were carried out in 3 L Erlenmeyer flasks, filled with 2 L of a liquid growth medium comprised of phosphate buffer (1.0 g L−1 K2HPO4 and 0.5 g L−1 KH2PO4), 0.2 g L−1 MgSO4, trace elements (2.5 mg L−1 FeSO4, 5 mg L−1 CaCl2, 1.25 mg L−1 MnSO4, 0.25 mg L−1 CuSO4, and 0.25 mg L−1 Na2MoO4) and glucose as the carbon source (1 g L−1). RDX was added to the growth medium at an initial concentration of ca. 20 mg L−1, and was the only nitrogen source. Flasks were kept in the dark at 25 °C and were continuously shaken on an orbital shaker at 150 rpm. Aliquots of the samples for isotope analysis were collected throughout the incubation periods. The volumes of the aliquots were increased during the incubation period in order to ensure a sufficient mass of RDX required for isotope analysis. For isotope analysis, RDX was extracted into dichloromethane (DCM). The DCM extracts were reduced to a final volume of 0.5−1.0 mL under gentle nitrogen flow at room temperature. Analytical Methods. Concentration of RDX in the samples during the incubation period was analyzed by high-performance 480
dx.doi.org/10.1021/es302691g | Environ. Sci. Technol. 2013, 47, 479−484
Environmental Science & Technology
Article
The uncertainty of ε (± in the following sections) is the 95% confidence interval, calculated by multiplying the standard error of the curve’s slope by the student t value for this uncertainty for the given degrees of freedom.
liquid chromatography (HPLC) (Agilent 1100 series, Palo Alto, CA) equipped with a SUPELCOSIL LC-18 column (Supelco, Bellefonte, PA) according to EPA method 8330.24 The detection was carried out at 254 nm using a mobile phase consisting of methanol (30%) and water (70%) . The method enables to detect nitroso derivates of RDX as well. Isotope analysis of RDX extracted from the incubation experiments was performed using Trace GC Ultra (Thermo Electron Corporation, Milan, Italy) interfaced to a DeltaV Plus (Thermo Fisher Scientific, Bremen, Germany) via GC Combustion III interface. The combustion reactor was operated at 980 °C, and reduction oven was held at 660 °C. The analytical method followed that previously reported by Gelman et al.25 Briefly, 2 μL of the solution was injected into a PTV injector operated in a split mode with a split ratio of 1:10. The injector followed a temperature program of 75 °C for 0.05 min, a ramp of 14.5 °C/sec to 260 °C (hold for 15 min), and a final cleaning ramp of 14 °C/sec to 270 °C (hold for 2 min) under a vent flow of 50 mL/min. DB-5 capillary column (30 m × 0.25 mm, film thickness 0.25 μm) was used for the GC separation following a temperature program of 60 °C for 1 min, 15 °C/min to 180 °C and finally 90 °C/min to 290 °C (hold for 5 min). Helium was used as a carrier gas in a constant flow mode with a flow rate of 3 mL/min. To correct for instrumental drifts, each sample analysis was bracketed by a nonreacted RDX standard having an isotope composition of δ13C = −37.8‰ and a δ15N = −9.8‰. Each sample was analyzed at least in duplicates. Calculations. The isotope ration mass spectrometry (IRMS) yielded nitrogen and carbon isotopic ratios (R) of RDX, relative to isotope composition of international standards (atmospheric nitrogen and Vienna PeeDee Belemnite (VPDB) respectively) and expressed in δ values in per mil units according to eq 1: ⎛R − R std ⎞ δ RDX[‰] = ⎜ RDX ⎟ × 1000 R std ⎝ ⎠
■
RESULTS AND DISCUSSION Kinetic Nitrogen Isotope Effects for Distinct Rhodococcus Strains. In this study δ15N and δ13C isotope effects were studied for three distinct RDX degrading Rhodococcus strains. Previous work has shown that these three Rhodococcus strains share the same cytochrome P450 enzymatic system for catalyzing RDX degradation.12 However, the three strains are clearly not identical. Previous phylogenetic analysis based on 16S rRNA gene sequence revealed 94% identity between strains T7 and YH1, and 92% identity between T9N and YH1. These variations are reflected, for example, by differences in the influence of nitrate and ammonium on the RDX degradation rate,12 by the fact that strain YH1 can grow on cyclohexanone as a sole carbon source,4 whereas the other two cannot (unpublished data), and by the observation that the degradation of RDX by strain T9N is accompanied by a distinct pinkish color. Nevertheless, it could be assumed that differences in measured isotope effects, if observed, should not have been the result of the enzymatic reaction itself but rather of other distinct, strain-specific rate limiting steps, such as diffusion through the cell membrane, etc: The similar 15N isotope effects for RDX denitration by the three strains imply that either rate limiting steps that mask the kinetic isotope effects are negligible for all strains, or that these steps mask the isotope effect to the same extent. It should be noted that while some previous studies presented considerable differences in masking effects for strains belonging to a different genus (e.g., atrazine20 or tetrachloroethylene21), others have documented considerable differences even within the same genus (e.g., differences in kinetic 13C isotope effects during tetrachloroethylene dechlorination by Desulf itobacterium sp. strain PCE-S and by Desulf itobacterium sp. strain Viet1;26). From our current study, it seems that the similarity between distinct RDX degraders within the Rhodococcus genus is high, making differences in the masking effect negligible. Since members of the Rhodococcus species are found as players in the degradation of RDX in aerobic environments worldwide, it would be of great interest to reveal the variability in kinetic isotope effects within this genus by exploring other isolates as well. The results obtained for strain YH1 in this study perfectly agree with those observed in a previous study (Table 1) in which a very different analytical concept was applied that included crude offline purification using thin-layer chromatographic plates.18 Our current study provides validation for the kinetic isotope effects obtained in the previous study, as well as validation for the offline analytical technique. Although the offline purification technique is labor-intensive and likely not suitable for δ13C analysis, it may be still a method of choice when only EA-IRMS rather GC-IRMS is available. Mechanistic Aspects. The studied bacteria degraded RDX following the denitration pathway, as shown previously for the tested strains.12 Nitroso derivates were not detected along the incubation period, excluding the possibility for the occurrence of the typical anaerobic reductive pathway. Moreover, Fuller et al.27 indicated that even at low oxygen concentrations (that may have existed in our incubation), 4-nitro-2,4-diazabutanal was the only product of RDX degradation by Rhodococcus strains
(1)
where RRDX and Rstd are the ratios between the heavy and light isotopes (either 13C/12C or 15N/14N) in the investigated compound and in the international standard, respectively. The mathematical description of the relation between RDX degradation extent and its isotopic composition is described by the Rayleigh distillation equation ⎛ RRDX, t ⎞ ⎟⎟ = (α − 1) ·ln f ln⎜⎜ ⎝ RRDX,0 ⎠
(2)
with f=
Ct C0
(3)
where C0 and Ct are RDX concentrations at times 0 and t, respectively. The stable isotope fractionation factor α was obtained by plotting the natural logarithm of the isotopic enrichment, (RRDX,t/RRDX,0), against the natural logarithm of the extent of degradation, f., where the linear slope of the obtained curve is (α − 1). Enrichment factors (ε) were determined for the different degradation experiments which are expressed as ε = (α − 1) × 1000
(4) 481
dx.doi.org/10.1021/es302691g | Environ. Sci. Technol. 2013, 47, 479−484
Environmental Science & Technology
Article
Table 1. Isotope Enrichment of RDX during Enzymatic and Abiotic Denitration Processes isotope enrichment, ε [‰] δ15N
δ13C
T7
−2.3 ± 0.8
T9N
−2.3 ± 0.8
YH1 YH1a
−1.9 ± 0.4 −1.9 ± 0.3 (−2.1 ± 0.1) −5.3 ± 0.8
in the range of analytical uncertainty in the range of analytical uncertainty 0.86 ± 0.84‰ not studied
strain
alkaline hydrolysis25
−7.8 ± 0.5
a
Results of former study18 were reevaluated without forcing the previously published data points through the origin. In brackets is the original data.
(strains DN22 and 11Y). Thus, the isotope shifts recorded in our study are expected to reflect denitration solely. Dual-isotope analysis of 13C and 15N in RDX may potentially shed light on the denitration mechanism, distinguishing between: (1) N−N bond cleavage, (2) C−H cleavage, and (3) simultaneous loosening of C−H and N−N bonds, as different hypothetical rate limiting steps. If the enzymatic attack focuses on the nitro group and the N−N bond cleavage occurs directly without any involvement of a C−H bond, nitrogen is expected to present primary isotope effects, whereas carbon is expected to present secondary isotope effects only. Alternatively, if hydrogen abstraction by a radical reaction takes place, and cleavage of a C−H bond occurs during the reaction’s rate limiting step, one would expect to observe a primary carbon isotope effect, as shown to occur in radical reactions during C−H bond cleavage of aromatic compounds,28,29 whereas nitrogen is expected to present secondary isotope effects only. A primary carbon isotope effect was shown to occur in RDX during abiotic alkaline hydrolysis;25 however, during alkaline hydrolysis, the reaction is also accompanied by a primary nitrogen isotope effect, suggesting that both loosening of the C−H bond, as well as of the N−NO2 bond, probably occurs simultaneously with the reaction’s transition state.25 If the microbial enzymatic reaction proceeds in a similar manner as alkaline hydrolysis, with the simultaneous loosening of the C−H and N−NO2 bonds at the transition state, both primary carbon and nitrogen isotope effects can be expected. A primary 15N enrichment was detected during the enzymatic denitration by all three Rhodococcus strains (Figure 2), whereas a13C enrichment was almost in the range of the analytical method uncertainty (ε = −0.86 ± 0.84‰ for strain YH1; Figure 3). This low, almost undistinguishable carbon isotope enrichment reflects secondary isotope effects. Combining both elements from biodegraded samples by the tested strains in a dual-isotope plot (δ13C vs δ15N; Figure 4), shows a slope of 0.35 ± 0.31, which is significantly different from the slope of the alkaline hydrolysis (which was 1.49 ± 0.31). This leads us to suggest that denitration of RDX by Rhodococcus species is initiated by N−N bond cleavage, rather than by a C− H bond cleavage or by a simultaneous loosening of both C−H and N−N bonds. This typical enrichment is shown for all three strains tested (Figure 4). Environmental Significance. When variability in isotope effects is observed for the biodegradation of target compounds by different strains, the uncertainty in extrapolating the laboratory-derived factors to the field increases (e.g., ref 26).
Figure 2. δ15N enrichment during denitration by strain YH1 (upper panel), T7 (middle panel), and T9N (lower panel).
Figure 3. δ13C enrichment during denitration by strain YH1.
The fact that all three strains here tested share the same kinetic 15 N isotope effect is encouraging from an environmental point of view. Two of the three strains that were tested in this study were isolated from groundwater within the same contamination plume, and the third strain was isolated from unsaturated sediments in the same site. Although only three strains were isolated from the site and tested in this current work, it is likely that other distinct Rhodococcus strains may exist within the plume as well, catalyzing the same reaction under the same mechanism. For example, Seth-Smith et al.3 isolated 19 distinct Rhodococcus strains that are capable of utilizing RDX as a sole N source from the sediments of a contaminated site in the UK. In 482
dx.doi.org/10.1021/es302691g | Environ. Sci. Technol. 2013, 47, 479−484
Environmental Science & Technology
Article
Present Address §
Anat Bernstein: Institute for Soil, Water and Environmental Sciences, Agricultural Research Organization (ARO), Volcani Center, Bet Dagan, Israel. Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
■
REFERENCES
This work was supported, in part, by grant 167/2008 from the Israel Science Foundation. The work of A.B. was supported by a generous contribution from Vera Barcza, Toronto, Canada, the Rosinger-Barcza Family Fund, in support of Young Researchers at the Zuckerberg Institute for Water Research. We thank the anonymous reviewers for their important comments and suggestions.
(1) Bernstein, A.; Ronen, Z., Biodegradation of the Explosives TNT, RDX and HMX. Microbial Degradation of Xenobiotics; Singh, S. N., Ed.; Springer: Berlin Heidelberg, 2012; pp 135−176. (2) Fournier, D.; Halasz, A.; Spain, J.; Fiurasek, P.; Hawari, J. Determination of key metabolites during biodegradation of hexahydro1,3,5-Trinitro-1,3,5-triazine with Rhodococcus sp. strain DN22. Appl. Environ. Microbiol. 2002, 68 (1), 166−172. (3) Seth-Smith, H. M. B.; Edwards, J.; Rosser, S. J.; Rathbone, D. A.; Bruce, N. C. The explosive-degrading cytochrome P450 system is highly conserved among strains of Rhodococcus spp. Appl. Environ. Microbiol. 2008, 74 (14), 4550−4552. (4) Nejidat, A.; Kafka, L.; Tekoah, Y.; Ronen, Z. Effect of organic and inorganic nitrogenous compounds on RDX degradation and cytochrome P-450 expression in Rhodococcus strain YH1. Biodegradation 2008, 19 (3), 313−320. (5) Rylott, E. L.; Jackson, R. G.; Sabbadin, F.; Seth-Smith, H. M. B.; Edwards, J.; Chong, C. S.; Strand, S. E.; Grogan, G.; Bruce, N. C. The explosive-degrading cytochrome P450 XplA: Biochemistry, structural features and prospects for bioremediation. BBA - Proteins & Proteomics 2011, 1814 (1), 230−236. (6) Coleman, N. V.; Spain, J. C.; Duxbury, T. Evidence that RDX biodegradation by Rhodococcus strain DN22 is plasmid-borne and involves a cytochrome P450. J. Appl. Microbiol. 2002, 93 (3), 463−472. (7) Indest, K. J.; Crocker, F. H.; Athow, R. A TaqMan polymerase chain reaction method for monitoring RDX-degrading bacteria based on the xplA functional gene. J. Microbiol. Meth. 2007, 68 (2), 267−274. (8) Jackson, R. G.; Rylott, E. L.; Fournier, D.; Hawari, J.; Bruce, N. C. Exploring the biochemical properties and remediation applications of the unusual explosive-degrading P450 system XplA/B. Proc. Natl. Acad. Sci. U. S. A. 2007, 104 (43), 16822−16827. (9) Roh, H.; Yu, C.-P.; Fuller, M. E.; Chu, K.-H. Identification of hexahydro-1,3,5-trinitro-1,3,5-triazine-degrading microorganisms via 15 N-stable isotope probing. Environ. Sci. Technol. 2009, 43 (7), 2505−2511. (10) Seth-Smith, H. M. B.; Rosser, S. J.; Basran, A.; Travis, E. R.; Dabbs, E. R.; Nicklin, S.; Bruce, N. C. Cloning, sequencing, and characterization of the hexahydro-1,3,5-trinitro-1,3,5-triazine degradation gene cluster from Rhodococcus rhodochrous. Appl. Environ. Microbiol. 2002, 68 (10), 4764−4771. (11) Brenner, A.; Ronen, Z.; Harel, Y.; Abeliovich, A. Use of hexahydro-1,3,5-trinitro-1,3,5-triazine as a nitrogen source in biological treatment of munitions wastes. Water Environ. Res. 2000, 72 (4), 469− 475. (12) Bernstein, A.; Adar, E.; Nejidat, A.; Ronen, Z. Isolation and characterization of RDX-degrading Rhodococcus species from a contaminated aquifer. Biodegradation 2011, 22 (5), 997−1005. (13) Hawari, J.; Beaudet, S.; Halasz, A.; Thiboutot, S.; Ampleman, G. Microbial degradation of explosives: Biotransformation versus mineralization. Appl. Microbiol. Biotechnol. 2000, 54 (5), 605−618.
Figure 4. Dual isotope reflection for selected samples of denitrated RDX by strain YH1 (●), T7 (□), and T9N (Δ). Error bars are of 0.3 and 1.3‰ for δ15N and δ13C, respectively (1σ). Dashed lines represent postulated curves for N−N bond attack (horizontal line), and postulated curve for C−H bond attack (vertical line). Solid line is the measured curve for alkaline hydrolysis (diagonal line; based on 25). The intersection between the three lines is the measured isotope composition of RDX prior to degradation, which is −37.8‰ and −9.8‰ for carbon and nitrogen, respectively.
18 of these strains, the XplA-XplB system encoding RDXdegrading cytochrome P450 was present. The fact that the three strains tested in our current work presented a similar kinetic 15N isotope effect suggests that this value may be highly representative for the aerobic reaction and that the laboratoryderived isotope enrichment factor may be further extrapolated to the field with somewhat greater confidence. The gene XplA, encoding for the cytochrome P450 from RDX-degrading strains, is highly conserved. Therefore, it is suggested that our results may be also representative even for RDX-degrading bacteria that harbor this RDX degrading gene isolated from different geographical locations. Nevertheless, other factors that may mask the isotope effects in the environment and are not observable in the experimental laboratory system (such as bioavailability restrictions;22) may still come into play in the field. Distinguishing between different degradation mechanisms of RDX cannot always be achieved in contaminated groundwater by tracing degradation products. For example, tracing the relatively stable denitration product NDAB in groundwater30 cannot give conclusive evidence regarding whether denitration is promoted biotically by Rhodococcus strains or abiotically followed alkaline hydrolysis, since this product may be detected in both cases (Figure 1). Tracing RDX 13C and 15N isotope signatures, on the other hand, may be successfully implemented for field studies to distinguish between these two specific mechanisms: whereas denitration by Rhodococcus species should record 15N enrichment only, abiotic alkaline hydrolysis, if it occurs in situ, should record 13C enrichment as well.
■
AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. 483
dx.doi.org/10.1021/es302691g | Environ. Sci. Technol. 2013, 47, 479−484
Environmental Science & Technology
Article
(14) Bhushan, B.; Trott, S.; Spain, J. C.; Halasz, A.; Paquet, L.; Hawari, J. Biotransformation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by a rabbit liver cytochrome P450: Insight into the mechanism of RDX biodegradation by Rhodococcus sp. strain DN22. Appl. Environ. Microbiol. 2003, 69 (3), 1347−1351. (15) Halasz, A.; Manno, D.; Strand, S. E.; Bruce, N. C.; Hawari, J. Biodegradation of RDX and MNX with Rhodococcus sp. Strain DN22: New insights into the degradation pathway. Environ. Sci. Technol. 2010, 44 (24), 9330−9336. (16) Balakrishnan, V. K.; Halasz, A.; Hawari, J. Alkaline hydrolysis of the cyclic nitramine explosives RDX, HMX, and CL-20: New insights into degradation pathways obtained by the observation of novel intermediates. Environ. Sci. Technol. 2003, 37 (9), 1838−1843. (17) Elsner, M.; Zwank, L.; Hunkeler, D.; Schwarzenbach, R. P. A new concept linking observable stable isotope fractionation to transformation pathways of organic pollutants. Environ. Sci. Technol. 2005, 39 (18), 6896−6916. (18) Bernstein, A.; Ronen, Z.; Adar, E.; Nativ, R.; Lowag, H.; Stichler, W.; Meckenstock, R. U. Compound-specific isotope analysis of RDX and stable isotope fractionation during aerobic and anaerobic biodegradation. Environ. Sci. Technol. 2008, 42 (21), 7772−7777. (19) 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. (20) Meyer, A. H.; Penning, H.; Elsner, M. C and N isotope fractionation suggests similar mechanisms of microbial atrazine transformation despite involvement of different enzymes (AtzA and TrzN). Environ. Sci. Technol. 2009, 43 (21), 8079−8085. (21) Nijenhuis, I.; Andert, J.; Beck, K.; Kästner, M.; Diekert, G.; Richnow, H.-H. Stable isotope fractionation of tetrachloroethene during reductive dechlorination by Sulf urospirillum multivorans and Desulf itobacterium sp. strain PCE-S and abiotic reactions with cyanocobalamin. Appl. Environ. Microbiol. 2005, 71 (7), 3413−3419. (22) Kampara, M.; Thullner, M.; Richnow, H. H.; Harms, H.; Wick, L. Y. Impact of bioavailability restrictions on microbially induced stable isotope fractionation. 2. Experimental evidence. Environ. Sci. Technol. 2008, 42 (17), 6552−6558. (23) Northrop, D. B. The expression of isotope effects on enzymecatalyzed reactions. Annu. Rev. Biochem. 1981, 50, 103−131. (24) U.S. EPA, Nitroaromatics and Nitramines by High Performance Liquid Chromatography (HPLC), Second update SW-846 Method 8330. U.S. Environmental Protection Agency: Washington, DC, 1994. (25) Gelman, F.; Kotlyar, A.; Chiguala, D.; Ronen, Z. Precise and accurate compound-specific carbon and nitrogen isotope analysis of RDX by GC-IRMS. Int. J. Environ. Anal. Chem. 2011, 91 (14), 1392− 1400. (26) Cichocka, D.; Imfeld, G.; Richnow, H.-H.; Nijenhuis, I. Variability in microbial carbon isotope fractionation of tetra- and trichloroethene upon reductive dechlorination. Chemosphere 2008, 71 (4), 639−648. (27) Fuller, M. E.; Perreault, N.; Hawari, J. Microaerophilic degradation of hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) by three Rhodococcus strains. Lett. Appl. Microbiol. 2010, 51 (3), 313−318. (28) Morasch, B.; Richnow, H. H.; Schink, B.; Meckenstock, R. U. Stable hydrogen and carbon isotope fractionation during microbial toluene degradation: Mechanistic and environmental aspects. Appl. Environ. Microbiol. 2001, 67 (10), 4842−4849. (29) Morasch, B.; Richnow, H. H.; Vieth, A.; Schink, B.; Meckenstock, R. U. Stable isotope fractionation caused by glycyl radical enzymes during bacterial degradation of aromatic compounds. Appl. Environ. Microbiol. 2004, 70 (5), 2935−2940. (30) Paquet, L.; Monteil-Rivera, F.; Hatzinger, P. B.; Fuller, M. E.; Hawari, J. Analysis of the key intermediates of RDX (hexahydro-1,3,5trinitro-1,3,5-triazine) in groundwater: Occurrence, stability and preservation. J. Environ. Monit.. 2011, 13 (8), 2304−2311.
484
dx.doi.org/10.1021/es302691g | Environ. Sci. Technol. 2013, 47, 479−484
