Evaluation of Toluene Degradation Pathways by Two-Dimensional

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Environ. Sci. Technol. 2008, 42, 7793–7800

Evaluation of Toluene Degradation Pathways by Two-Dimensional Stable Isotope Fractionation †

†,‡

CARSTEN VOGT, ESTHER CYRUS, I L K A H E R K L O T Z , †,§ DIETMAR SCHLOSSER,| ARNE BAHR,⊥ STEFFI HERRMANN,† H A N S - H E R M A N N R I C H N O W , * ,† A N D A N K O F I S C H E R †,# Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research, Permoserstrasse 15, D-04318 Leipzig, Germany, Department of Bioremediation, Helmholtz Centre for Environmental Research, Permoserstrasse 15, D-04318 Leipzig, Germany, Center for Applied Geoscience, Working Group Hydrogeochemistry, Eberhard-Karls-University of Tuebingen, Sigwartstrasse 10, D-72076 Tuebingen, Germany, Department of Environmental Microbiology, Helmholtz Centre for Environmental Research, Permoserstrasse 15, D-04318 Leipzig, Germany, Institute of Microbiology, Department of Applied Microbiology, Ernst Moritz Arndt University of Greifswald, F.-L.-Jahn-Str. 15, D-17487 Greifswald, Germany, and Isodetect-Company for Isotope Monitoring, Ingolstaedter Landstrasse 1, D-85764 Neuherberg/Oberschleissheim, Germany

Received February 4, 2008. Revised manuscript received May 26, 2008. Accepted June 9, 2008.

Toluene degradation by several pure and mixed microbial cultures was investigated by two-dimensional compound specific isotope analysis (2D-CSIA). For most of the cultures, the respective toluene degradation pathway and toluene attacking enzymatic step was known. The slope of the linear regression for hydrogen (∆δ2H) vs carbon (∆δ13C) discrimination (Λ ) ∆δ2H/ ∆δ13C ≈ εHbulk/εCbulk) was determined in order to characterize aerobic and anaerobic toluene degradation pathways. The highest Λ value was estimated for the monohydroxylation of the methyl group by Pseudomonas putida (Λ ) 53 ( 5). The lowest value was observed for Rhodococcus opacus (Λ ) 2 ( 2) due to its insignificant hydrogen fractionation, which indicates that a ring dioxygenase was responsible for the initial attack of toluene. The fungus Cladosprium sphaerospermum containing a cytochrome P450-dependent methyl monooxygenase grouped within these extreme values (Λ ) 16 ( 6). Λ values for organisms attacking toluene under anoxic conditions by benzylsuccinate synthase were significantly different and ranged from Λ ) 4 ( 3 (Blastochloris sulfoviridis) to 31 ( 11 (strain TRM1). Values were in the same range for organisms using nitrate (Λ ) 11-14) or sulfate (Λ ) 28-31) * Corresponding author phone: ++49 341 235 1212; fax: ++49 341 235 1443; e-mail: [email protected]. † Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research. ‡ Department of Bioremediation, Helmholtz Centre for Environmental Research. § Eberhard-Karls-University of Tuebingen. | Department of Environmental Microbiology, Helmholtz Centre for Environmental Research. ⊥ Ernst Moritz Arndt University of Greifswald. # Isodetect-Company for Isotope Monitoring. 10.1021/es8003415 CCC: $40.75

Published on Web 08/02/2008

 2008 American Chemical Society

as electron acceptor, indicating that it might be possible to distinguish toluene degradation under different electron acceptor conditions by 2D-CSIA.

Introduction Monitored Natural Attenuation (MNA) has become an important remediation strategy for contaminated sites. Biodegradation processes have been recognized as driving forces for natural attenuation (NA) of pollutants since many studies have shown in the last decades that almost all of the most abundant contaminants, e.g., aromatic hydrocarbons such as benzene and alkylated analogues, can be mineralized under oxic or anoxic conditions by indigenous microorganisms. MNA depends on reliable methods for identifying and quantifying a contaminant’s removal in the environment. A promising and already successfully applied monitoring method for achieving these goals is compound specific stable isotope analysis (CSIA; for an overview see ref 1). CSIA is based on the fact that, in most chemical reactions, lighter isotopomers react faster than heavier ones, leading to a kinetic isotope effect (KIE). KIEs are caused by different bonding strengths of lighter and heavier isotopes and allow characterizing (bio)chemical mechanisms (2). They were used in biochemical studies to elucidate the mechanism of enzymatic catalyzed reactions, since the extent of stable isotope fractionation depends on the first irreversible step of the respective reaction cascade (3). A normal KIE leads to an isotope fractionation between reactant and product: heavier isotopes are enriched in the residual fraction of the reactant. Abiotic, nondestructive NA processes such as dispersion, sorption or volatilization are generally not coupled to significant isotope fractionation (4–7). Thus, the simple observation that decreasing compound concentrations at a polluted site are correlated with an enrichment of heavier isotopes is already a strong hint that the compound is biodegraded (qualitative assessment of biodegradation). The major goal is, however, to quantify biodegradation processes by CSIA. Isotope fractionation factors for known degradation pathways first have to be determined in laboratory experiments using pure or mixed cultures (1, 2). Using these laboratory-derived fractionation factors, it was possible to determine the extent of biodegradation of pollutants in environmental studies (1). However, complications arise by the fact that isotope fractionation factors can be highly variable for a compound, depending on the first irreversible step of the transformation reaction, since many compounds can be degraded by different degradation pathways (1, 2). Moreover, the extent of isotope fractionation can also vary for the same reaction mechanism, if the bond conversion is preceded by non- or slightly isotope fractionating processes (e.g., uptake and transport of a substrate to the reactive site of enzyme, binding of the substrate to form enzyme-substrate complexes) (2, 3). Thus, analysis of isotope fractionation does not only reflect the intrinsic kinetic isotope effect, but also exhibits information on rate-limiting steps, in particular, enzymatic reactions (3). These processes, summarized as “commitment to catalysis” (2, 3), can mask KIE’s significantly, leading to much lower apparent kinetic isotope effects (AKIE’s). To overcome these difficulties, two-dimensional isotope fractionation analysis has been suggested as a tool for determining biodegradation pathways (2, 8–13); here, the isotope fractionation of two elements within a molecule is analyzed and compared with each other. Since commitment to catalysis affects the isotope composition of different elements similarly (2), the correlation of isotope fractionation VOL. 42, NO. 21, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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of these elements may be used to characterize the reaction mechanism more precisely than by the isotope fractionation analysis of only one element. Toluene is a widespread contaminant and can be degraded via several pathways. Under aerobic conditions, different initial steps can be distinguished: (i) hydroxylation of the methyl moiety catalyzed by xylene monooxygenase (14) or toluene monooxygenase (15–17), (ii) monohydroxylation of the aromatic nucleus in ortho-, meta-, or para-position by different toluene monooxygenases (18–21), or (iii) dihydroxylation of the aromatic nucleus by toluene dioxygenase, forming cis-toluene dihydrodiol (22, 23). All these enzymes use molecular oxygen as substrate for hydroxylation. Under anoxic conditions, toluene degradation is initiated by the addition of fumarate to the methyl moiety, yielding (R)benzylsuccinate, catalyzed by the enzyme benzylsuccinate synthase. This reaction is used by nitrate-reducing, sulfatereducing, iron-reducing, and phototrophic bacteria for toluene activation (for an overview see ref 24). Carbon and hydrogen isotope fractionation factors for toluene were determined in laboratory experiments with reference strains using different degradation pathways. The hydroxylation reactions at the aromatic nucleus generally led to only small fractionation factors, whereas the sidechain hydroxylation and the fumarate addition led to higher fractionation factors, especially for hydrogen fractionation (25–30). Hence, the goal of our study was to determine both natural carbon and hydrogen isotope fractionation factors for several toluene activation reactions, and to characterize each reaction by the two-dimensional isotope fractionation approach. The majority of strains examined in our study have not been investigated before in isotopic studies.

Materials and Methods Microorganisms and Cultivation Conditions. The bacterial strains Pseudomonas putida mt-2 (DSM 3931), Thauera aromatica (DSM 6984), Azoarcus sp. strain T (DSM 9506), Desulfosarcina cetonica (DSM 7267), and Blastochloris sulfoviridis (DSM 13255) were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Other strains used in this study were kindly provided by the following persons: Azoarcus sp. EbN1 by Matthias Boll (University of Leipzig, Germany), Rhodococcus opacus B-4 by Junichi Kato (Hiroshima University, Hiroshima, Japan), TRM1 by Christian Griebler and Rainer Meckenstock (Helmholtz Zentrum Mu ¨ nchen, Germany), and the filamentous fungus Cladosporium sphaerospermum T0 by Timm Anke (University of Kaiserslautern, Germany). The toluenedegrading sulfate-reducing mixed culture Zz 53-56 was enriched from groundwater of a BTEX contaminated aquifer near Zeitz, Germany. For the isotope fractionation experiments, the strains were cultivated in mineral salt media spiked with toluene as sole source of carbon and energy. Experiments were either performed in a single bottle of 1 or 2 L culture volume which was periodically sampled, or in several parallel bottles each filled with 50 or 100 mL culture volume which were periodically sacrificed. For Azoarcus EbN1, Azoarcus T, D. cetonica and the mixed culture Zz 53-56, similar data were obtained using both experimental designs (Table 1), showing that they were fully comparable. Detailed information on the composition of the media and the design of the degradation experiments is given in the Supporting Information. Analytical Methods. Toluene was analyzed by headspace gas chromatography, whereas the carbon and hydrogen isotope ratios of toluene were analyzed by gas chromatograph-isotope ratio mass spectroscopy (GC–IRMS). Both analytical methods are described in detail in the Supporting Information. 7794

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Quantification of Isotope Fractionation. The derivations of enrichment factors, AKIE’s and Λ values are explained in detail in the Supporting Information. Position specific enrichment factors (εreactive position) and apparent kinetic isotope effects (AKIE’s) were calculated according to a derivation scheme introduced by Elsner et al. (2). By using those parameters, different toluene transforming biochemical reactions can be compared and mechanistically interpreted.

Results and Discussion Carbon Isotope Fractionation. All investigated strains of this study showed a significant carbon isotope fractionation in the course of toluene degradation (Table 1). The correlation factors (R2) between toluene concentrations and isotope signatures were high (between 0.84 and 0.99, see Figure 1) in each individual degradation experiment, demonstrating the usefulness of the Rayleigh equation for calculating the respective enrichment factors (εC’s) and apparent kinetic isotope effects (AKIEC’s). In sterile controls, no significant loss of toluene and no significant change of toluene isotope signatures could be observed (data not shown). εC’s and AKIEC’s ranged between -0.4 ( 0.2‰ and 1.004 ( 0.002 for C. sphaerospermum and -5.7 ( 0.2‰ and 1.039 ( 0.002 for Azoarcus T, respectively (Table 1, Figure 1; errors are always indicated as 95% confidence intervals). No enzyme-dependent trend could be observed. On the one hand, organisms using a different mechanism for the initial attack of toluene showed roughly similar εC’s and AKIEC’s: P. putida (εC ) -2.8 ( 0.2‰, AKIEC ) 1.02 ( 0.002), using a methyl monooxygenase, and the benzylsuccinate synthase containing strains Azoarcus EbN1, T. aromatica, and D. cetonica as well as the consortium Zz 53-56 (εC’s between -2.4 and -2.9‰, AKIEC’s between 1.017 and 1.020). On the other hand, similar reaction mechanisms produced significantly different εC’s and AKIEC’s, observed for C. sphaerospermum and P. putida, both attacking toluene by a methyl monooxygenase. Also B. sulfoviridis (εC ) -4 ( 0.5, AKIEC ) 1.029 ( 0.004) and Azoarcus T (εC ) -5.7 ( 0.2‰, AKIEC ) 1.039 ( 0.002) showed significantly higher εC’s and AKIEC’s than the other examined anaerobic cultures, although they use the same enzyme, benzylsuccinate synthase, for the initial toluene attack. Remarkably, B. sulfoviridis accumulated high amounts of benzylsuccinate (approximately 30% of the added toluene), while degrading toluene (data not shown). εC’s and AKIEC’s for strain TRM1 and for anaerobic toluene degrading pure and mixed cultures described by others are generally slightly lower than those observed in our study, ranging from -0.5 to -2.2‰ and 1.004 to 1.016, respectively (Table 1). In the literature, most carbon enrichment factors are available for P. putida mt-2. The determined value for strain mt-2 (εC ) -2.8 ( 0.2‰) is similar to the values reported before by Mancini et al. (30) and Meckenstock et al. (29) for low iron conditions. εC and AKIEc of strain mt-2 were significantly higher than the values for R. opacus (εC ) -1.8 ( 0.4, AKIEc ) 1.006 ( 0.001), which initiates toluene degradation presumably by a ring dioxygenase (see below). Hydrogen Isotope Fractionation. Except for R. opacus, toluene concentrations and hydrogen isotope signatures correlated well in the case of fractionation (R2 between 0.81 and 0.99; Figure 1), showing that the Rayleigh equation could be used for calculating the hydrogen enrichment factors (εH’s) and corresponding apparent kinetic isotope effects (AKIEH’s). Those ranged from -8.6 ( 4‰ and 1.026 ( 0.012 for C. sphaerospermum to -140 ( 8‰ and 6.5 ( 2.6 for P. putida mt-2. R. opacus showed an insignificant hydrogen fractionation (εH ) -2 ( 5‰). The εH of P. putida mt-2 determined in our study was nearly in the same range determined by Mancini et al. (30) for this strain under low iron conditions, as already observed for the εC (see above).

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oxic oxic oxic

oxic nitrate-reducing

nitrate-reducing

nitrate-reducing

Ralstonia picketi PKO1 C. sphaerospermum P. putida mt-2

Consortium T. aromatica

Azoarcus T

Azoarcus EbN1

benzylsuccinate synthase (48)

benzylsuccinate synthase (47)

unknown benzylsuccinate synthase (46)

ring monooxygenase (19) methyl monooxygenase (17) methyl monooxygenase (14)

putative ring dioxygenase (32) ring dioxygenase (43)

enzyme of initial toluene attack

sulfate-reducing

sulfate-reducing

sulfate-reducing methanogenic

Strain TRM1

Zz 53-56 (consortium)

consortium consortium

benzylsuccinate synthase

-18 ( 2 -19 ( 2 -19 ( 1 -5.6a -3.5a

-2.0 ( 0.2h -14 ( 1 -1.7 ( 0.2 -12 ( 1.4a -12a -1.7k

1.019 ( 0.002 1.020 ( 0.002 1.019 ( 0.001 1.006 1.004

1.014 ( 0.001 1.012 ( 0.001 1.012

1.018 ( 0.001 1.016 ( 0.001 1.017 ( 0.001 1.016

-18 ( 1 -16 ( 1 -17 ( 1 -15a

-2.6 ( 0.2h -2.3 ( 0.2i -2.4 ( 0.1j -2.2k

-2.7 ( 0.2h -2.8 ( 0.2i -2.8 ( 0.1j putative benzylsuccinate synthase -0.8k benzylsuccinate synthase (53, 54) -0.5k

benzylsuccinate synthase (28)

benzylsuccinate synthase (51)

1.038 ( 0.001 1.044 ( 0.008 1.039 ( 0.002 1.020 ( 0.001 1.027 ( 0.003 1.020 ( 0.001 1.029 ( 0.004 1.013 ( 0.002

-37 ( 1 -43 ( 7 -38 ( 2 -20 ( 1 -26 ( 3 -20 ( 1 -28 ( 3 -13 ( 2a

-5.6 ( 0.3h -6.2 ( 1.1i -5.7 ( 0.2j -3.0 ( 0.1h -3.8 ( 0.5i -2.9 ( 0.2j -4.0 ( 0.5h -1.8 ( 0.3

AKIECl

1.019 ( 0.001 NS 1.019 ( 0.001 1.012 ( 0.001

εC reactive l position [‰]

-18 ( 1a NS -19 ( 1 -12 ( 1a

[‰]

-2.6 ( 0.2 NS -2.7 ( 0.1i -1.7 ( 0.1

l

1.006 ( 0.001 1.001 ( 0.001 1.004 ( 0.001 to 1.008 ( 0.002 1.0004 ( 0.0002 1.02 ( 0.002 1.018 ( 0.002e 1.013 ( 0.001 1.013 ( 0.001 1.024 ( 0.002

bulk

-1.8 ( 0.3h -3.2 ( 0.6 -0.4 ( 0.3 -0.7 ( 0.5a -1.5 ( 0.3a to -1.9 ( 0.4a -1.1 ( 0.2 h -0.4 ( 0.2 -3.0 ( 1.2 -2.8 ( 0.2h -19 ( 1 -2.5 ( 0.3e -18 ( 2a -1.9 ( 0.2g -13 ( 1a -1.8 ( 0.2g -13 ( 1a -3.3 ( 0.3e -23 ( 2a

εC

1.016 ( 0.005c 1.026 ( 0.012 6.5 ( 2.6 NDf NDf NDf

-608 ( 29 -189 ( 32 -157 ( 14 -172 ( 17

2.5 ( 0.1 2.3 ( 0.5 1.9 ( 0.1 2.1 ( 0.2

c

-12 to -65

k

-726k -517 ( 54b to -705 ( 15b -88 ( 5h -81 ( 7i -87 ( 6j

ND

f

NDf -517 ( 54c to -705 ( 15c -211 ( 15 -197 ( 21 -224 ( 23

ND

f

NDf 2.1 ( 0.5c to 3.4 ( 0.03c 2.7 ( 0.3 2.4 ( 0.4 3.1 ( 0.6

NDf NDf -198k -514 ( 21b to -514 ( 21c to 2.1 ( 0.2c to b c -735 ( 76 -735 ( 76 3.8 ( 0.2c -66 ( 19h -152 ( 48 1.8 ( 0.5

-608 ( 29 -80 ( 14h -68 ( 5i -74 ( 7j

c

-607 ( 88c -187 ( 24 -195 ( 46 -187 ( 27 -110 ( 41 -145 ( 23 -122 ( 19 -58 ( 14 -607 ( 88b -79 ( 7h -79 ( 21i -78 ( 12j -45 ( 15h -58 ( 10i -50 ( 7j -23 ( 6h b

1.4 ( 0.2 2.5 ( 0.4c 2.2 ( 0.4 2.4 ( 0.8 2.3 ( 0.4 1.5 ( 0.3 1.8 ( 0.2 1.6 ( 0.1 1.2 ( 0.1

-88 ( 35

-35 ( 14h

NDd

27 ( 4 33 ( 4 28 ( 2

NDd

31 ( 11

28 ( 4 29 ( 3 30 ( 2 NDd

15 ( 2 11 ( 2 11 ( 3 14 ( 4 14 ( 1 14 ( 1 4(3

11 ( 5

NDd 16 ( 6 53 ( 5e 68 ( 5d,e 62 ( 4d,g 54 ( 4d,g NDd

52 52 25

2, 28, 44 this work

2, 28, 44 this work 29 28

28

this work 29 2, 28, 44 this work

this work

2, 28, 44 29 45 this work 29 2, 28, 44 this work

27 this work this work 30 30 30 27

this work 27

Λ ) ∆δ2H/∆δ13C ≈ εHbulk/εCbulk l reference

1.011 ( 0.032 1 ( 2 1.029 ( 0.01c NDd

AKIEHl

-905 ( 71c 10.5 ( 0.1c -905 ( 71b -927 ( 13b to -927 ( 13c to 13.7 ( 0.01c to -956 ( 8b -956 ( 8c 27.7 ( 0.01c

-16 ( 5c -22 ( 10c -282 ( 21 NDf NDf NDf

-16 ( 5b -8.6 ( 3.7h -140 ( 8h -159 ( 11e -115 ( 6g -97 ( 5g

εH reactive l position [‰] -5 ( 15 -28 ( 10c

bulk[‰]

-2 ( 5h -28 ( 10b

εH

a

Approximated by εCreactive position ≈ n/x · εbulk, where n is the number of carbon atoms and x is the number of carbon atoms which are located at the reactive site, respectively. b Hydrogen isotope fractionation determined with mixtures (50:50) of labeled and nonlabeled toluene. c Determined according to (2); for experiments with labeled toluene, εHbulk, εHreactive position, and AKIEH does not need to be corrected for intramolecular competition. d Λ values approximated by εHbulk/εCbulk. If hydrogen and carbon enrichment factors were obtained in independent experiments, Λ values were not estimated since those could be affected by different commitment to catalysis. e Low iron conditions. f εHreactive position approximated by εHreactive reaction ≈ n/x · εbulk can have large systematic errors as well as AKIEH’s derived from these εHreactive position values (2) and were, therefore, not determined. g High iron conditions. h Single bottle experiment. i Sacrifice bottle experiment. j Combined data from single bottle and sacrifice bottle experiments. k Errors for εHbulk and/or εCbulk were not mentioned in the literature. l ND ) not determined; NS ) not significant.

sulfate-reducing

D. cetonica

B. sulfoviridis anoxic phototrophic benzylsuccinate synthase (49) Geobacter metallireducens ferric iron-reducing benzylsuccinate synthase (50)

oxic oxic

growth conditions

R. opacus B-4 P. putida F1

organism

TABLE 1. Bulk Carbon and Hydrogen Enrichment Factors (εCbulk, εHbulk), Position Specific Enrichment Factors (εCreactive position, εHreactive position), Apparent Kinetic Isotope Effects (AKIEC, AKIEH) and Λ Values (Λ = ∆δ2H/∆δ13C ≈ εHbulk/εCbulk) for Toluene-Degrading Microorganisms Examined in This Study and Reported in the Literature

FIGURE 1. Double logarithmic plot according to the Rayleigh equation of the isotopic composition versus the residual concentration of toluene. The lines correspond to a linear regression model. (A) Carbon and (B) hydrogen isotope fractionation by aerobic strains, (C) carbon and (D) hydrogen isotope fractionation by nitrate-reducing strains, (E) carbon and (F) hydrogen isotope fractionation by sulfate-reducing and phototrophic strains and an enrichment culture, respectively. Filled symbols were derived from one bottle experiments and open symbols from experiments with sacrifice batch cultures. The εH’s and AKIEH’s caused by fumarate addition to the methyl moiety, catalyzed by benzylsuccinate synthase were variable, ranging from -23 ( 6‰ and 1.2 ( 0.1 for B. sulfoviridis to -87 ( 6‰ and 3.1 ( 0.6 for the mixed culture Zz 53-56 (Table 1). The three examined nitrate reducers produced different εH’s and AKIEH’s, ranging from -35 ( 14‰ and 1.4 ( 0.2 for T. aromatica to -78 ( 12‰ and 2.3 ( 0.4 for Azoarcus T. Generally, AKIEH’s determined in studies using deuterium-labeled toluene were similar or slightly higher than those determined in our study, as can be expected according to Elsner et al. (2). For strain TRM1, the observed εH (-66 ( 19‰) was an order of magnitude lower than previously reported for experiments with natural abundance toluene (εH ) -726‰ (28)). Analogously, the εH for D. cetonica observed in our study (-74 ( 7‰) was more than 7796

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2-fold lower than previously reported (28). The lower values from our experiments are within the range of εH’s observed with natural abundance toluene for all other anoxic toluene degrading cultures so far (Table 1), indicating its correctness. Probably, the higher εH’s presented in the previous study (28) resulted from incorrect hydrogen isotope analyses. Two-Dimensional Isotope Fractionation Analysis. Apart from R. opacus, a linear regression was observed for the carbon and hydrogen discrimination (∆δ2H, ∆δ13C) in each degradation experiment (Figure 2). The factor Λ given in Table 1 and Figure 2 expresses the slope of the linear regression for ∆δ2H/∆δ13C. Λ values for organisms using benzylsuccinate synthase for toluene activation ranged from 4 ( 3 for the phototrophic B. sulfoviridis over between 11 ( 5 and 14 ( 1 for the three facultative anaerobic nitrate

FIGURE 2. Plot of ∆δ2H vs ∆δ13C for each strain examined in this study, subdivided in (A) aerobic and (B) anaerobic toluene degradation pathways. The slope of regression curve gives the Λ values. reducing strains to between 28 ( 2 and 31 ( 11 for the sulfatereducing, strict anaerobic D. cetonica, strain TRM1 and mixed culture Zz 53-56. Λ values for methyl monohydroxylation by P. putida mt-2 observed in this study and by Mancini et al. (30) were almost similar (53 ( 5 to 68 ( 5) and significantly higher than for fungal monohydroxylation by C. sphaerospermum (16 ( 6) and for putative dihydroxylation by R. opacus (1 ( 2) (Table 1). Toluene Activation by Benzylsuccinate Synthase. Benzylsuccinate synthase adds the cosubstrate fumarate to the methyl moiety of toluene, yielding (R)-benzylsuccinate; this synthesis is thought to be initiated by an abstraction of a hydrogen atom from the methyl group generating a benzyl radical (24). Thus, the cleavage of a C-H bond is hypothesized to be the isotopically sensitive step (28). Λ values for the three nitrate reducers were similar in spite of the significantly higher εCbulk and εHbulk values of Azoarcus T compared to Azoarcus EbN1 and T. aromatica, indicating that isotope fractionation was partially masked by rate limitations others than the isotope sensitive bond cleavage during the biochemical reaction in Azoarcus EbN1 and T. aromatica. Nonetheless, the similar Λ values indicate that the mechanism of the bond cleavage is probably similar in all three investigated nitrate reducers. Remarkably, Λ values for B.

sulfoviridis, the three nitrate reducers, and the three sulfatereducing cultures differed significantly, indicating that the reaction mechanism by which toluene is transformed to benzylsuccinate is not exactly the same in these organisms. Different transitions states might occur during the reaction catalyzed by benzylsuccinate synthase according to the transition state theory (31), leading to different isotope fractionation pattern. Toluene Activation by Ring Di- or Monooxygenation. The putative first step of toluene degradation in R. opacus is the addition of both atoms of dioxygen to the aromatic nucleus to form cis-toluene dihydrodiol catalyzed by a dioxygenase which also catalyzes the dihydroxylation of benzene (32). This reaction would not be linked to the cleavage of a C-H bond. Therefore, small or even negligible carbon and hydrogen isotopic effects can be expected in the course of toluene activation by dioxygenases. The low AKIEc and especially the insignificant AKIEH value determined for R. opacus fit to this theory and suggest that toluene is attacked by means of a dioxygenase in this organism. Correspondingly, Morasch et al. determined only a low carbon isotope fractionation (εC -0.4 ( 0.3‰) for P. putida F1, which dihydroxylates toluene as well by means of a dioxygenase. Small or not measurable enrichment factors for carbon have VOL. 42, NO. 21, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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also been observed for different organisms using dioxygenases for the activation of benzene and chlorobenzene (10, 33, 34). Especially, negligible hydrogen isotope fractionation might be characteristic for aromatic ring dioxygenases, as observed for the dihydroxylation of toluene and benzene (10, 27). No ring monooxygenase using toluene degrading culture was examined in our study. Toluene hydroxylation by the ring monooxygenase of R. picketti was shown to produce relatively low AKIEc (1.004-1.008) and AKIEH (1.016) in a study of Morasch et al. (27). Due to different aromatic hydroxylation mechanisms associated with varying hydrogen isotope effects (35–37), toluene ring monohydroxylation might result in variable Λ values (10). Toluene Activation by Methyl Monooxygenase. The investigated methyl monooxygenases of P. putida mt-2 and C. sphaerospermum produced completely different carbon and hydrogen enrichment factors. The enzyme of strain mt2, xylene monooxygenase, shows NADH: cytochrome c reductase activity and contains FAD as redox centers (14). The high hydrogen isotope effect observed in this study and by others before (27, 28, 30) indicate that the rate-limiting step of the reaction involves a hydrogen atom transfer, a one-step process known for a long time (38). Moreover, AKIEC and AKIEH of our study fit into the range of kinetic isotope effects expected for oxidation of C-H bonds (2). The toluene monooxygenase of the filamentous fungus C. sphaerospermum needs NADPH as electron donor and is thought to be a membrane bound cytochrome P450 (15–17). For toluene transformation by this enzyme, the Λ value (16 ( 6) and also the AKIE’s for carbon and hydrogen were significantly lower than observed for the methyl monooygenase of P. putida, indicating that both enzymes follow different reaction mechanisms. Cytochrome P450 catalyzed hydroxylation reactions of alkylbenzenes can proceed by a two step process, involving first an electron transfer step resulting in the formation of an aromatic radical cation, which is then deprotonated and subsequently hydroxylated in the second step (38). Such a mechanism was proposed for the cytochrome P450 catalyzed side chain hydroxylation of aromatic hydrocarbons of several fungi (39), since the reaction involved only a small hydrogen isotope effect. Thus, our results suggest that also the enzyme of C. sphaerospermum follows the two step electron transfer mechanism. Environmental Implications. Most cultures tested here degrade toluene under anoxic conditions, using benzylsuccinate synthase for toluene activation. This reaction is of high environmental relevance, since no other toluene activation mechanisms under anoxic conditions is known; hence, toluene degradation in the environment (e.g., contaminated aquifers) proceeds particularly by this mechanism due to the low oxygen solubility and rapid consumption of molecular oxygen in aqueous subsurface systems. All nitrate reducers examined in our study produced roughly similar Λ values, demonstrating the potential of 2D-CSIA to identify toluene degradation under nitrate-reducing conditions in environmental samples. Λ values varied between different ecophysiological groups of toluene degraders, since all investigated sulfate-reducing cultures showed significantly higher Λ values than the examined nitrate-reducing cultures; in contrast, the phototrophic toluene degrader produced the lowest value of all tested anaerobic cultures. The variability of isotope fractionation might allow distinguishing toluene degradation under nitrate-reducing and sulfate-reducing conditions in field samples by means of 2D-CSIA. Additionally, our results support the suitability of carbon isotope enrichment factors for quantifying toluene degradation in sulfidic aquifers, as proposed and successfully done by others before (4, 40–42). However, our data also show that carbon enrichment factors can be significantly different even in 7798

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closely related organisms, as shown for the nitrate reducers Azoarcus T, Azoarcus EbN1, and T. aromatica in this study. Hence, at least in aquifers in which toluene is predominantly degraded under nitrate reducing conditions, the amount of biodegradation might be significantly overestimated if using εC’s of around -2.5‰ in the case of a predominant growth of Azoarcus T-like organisms. Our data also imply that Λ values can be taken to differentiate between ring dioxygenase and methyl monooxygenase initiated degradation pathways for toluene in not characterized enrichment cultures or pure strains. Ring dioxygenase initiated degradation pathways for aromatics may generally lead to Λ values around 1 or lower; thus, such reactions can not be quantified or even detected in contaminated aquifers. On the other hand, high Λ values may generally indicate toluene degradation by methyl group oxidation in aerobic contaminated aquifers or enrichment cultures due to the large hydrogen isotope fractionation of this reaction. Ring monooxygenase initiated toluene degradation pathways may produce Λ values in between the values produced by ring dioxygenases and methyl monooxygenases. Summarizing, our results show that 2D-CSIA is a fast and simple method for distinguishing initial transformation reactions of toluene degradation pathways.

Acknowledgments This work was supported by the Helmholtz Centre for Environmental ResearchsUFZ as part of the project “SAFIRA II” and by IsodetectsCompany for Isotope Monitoring. S.H. was supported by the German Federal Ministry of Education and Research (BEOQUE, grant 02 WN 0651). We acknowledge Matthias Gehre and Ursula Gu ¨ nther for their support in the isotope laboratory of the UFZ, Stephanie Hinke and Mario Lange for help during cultivation, and Stefanie Weber for help regarding the analysis of benzylsuccinate.

Supporting Information Available Information on degradation experiments, analytical methods and calculation of enrichment factors, AKIEs and Λ values. This material is available free of charge via the Internet at http://pubs.acs.org.

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