Environ. Sci. Technol. 2008, 42, 8290–8296
Isotopic Evidence Suggests Different Initial Reaction Mechanisms for Anaerobic Benzene Biodegradation S I L V I A A . M A N C I N I , †,| CHERYL E. DEVINE,§ MARTIN ELSNER,‡ MONISHA E. NANDI,§ A N I A C . U L R I C H , §,⊥ E L I Z A B E T H A . E D W A R D S , * ,§ A N D BARBARA SHERWOOD LOLLAR† Stable Isotope Laboratory, Department of Geology, University of Toronto, 22 Russell Street, Toronto, Ontario, Canada, M5S 3B1, Institute of Groundwater Ecology, Helmholtz Zentrum Muenchen-National Research Center for Environmental Health, Neuherberg, Germany, and Department of Chemical Engineering and Applied Chemistry, University of Toronto, 200 College Street, Toronto, Canada M5S 3E5
Received April 22, 2008. Revised manuscript received July 29, 2008. Accepted August 6, 2008.
The initial metabolic reactions for anaerobic benzene biodegradation remain uncharacterized. Isotopic data for carbon and hydrogen fractionation from nitrate-reducing, sulfatereducing, and methanogenic benzene-degrading enrichment cultures and phylogenic information were used to investigate the initial reaction step in anaerobic benzene biodegradation. Dual parameter plots of carbon and hydrogen isotopic data (∆δ2H/ ∆δ13C) from each culture were linear, suggesting a consistent reaction mechanism as degradation proceeded. Methanogenic and sulfate-reducing cultures showed consistently higher slopes (m ) 29 ( 2) compared to nitrate-reducing cultures (m ) 13 ( 2) providing evidence for different initial reaction mechanisms. Phylogenetic analyses confirmed that culture conditions were strictly anaerobic, precluding any involvement of molecular oxygen in the observed differences. Using published kinetic data, we explored the possibility of attributing such slopes to reaction mechanisms. The higher slopes found under methanogenic and sulfate-reducing conditions suggest against an alkylation mechanism for these cultures. Observed differences between the methanogenic and nitrate-reducing cultures may not represent distinct reactions of different bonds, but rather subtle differences in relative reaction kinetics. Additional mechanistic conclusions could not be made because kinetic isotope effect data for carboxylation and other putative mechanisms are not available.
* Corresponding author tel: 416-946-3506; fax: 416-978-8605; e-mail:
[email protected]. † Stable Isotope Laboratory, Department of Geology, University of Toronto. ‡ Institute of Groundwater Ecology, Helmholtz Zentrum MuenchenNational Research Center for Environmental Health. § Department of Chemical Engineering and Applied Chemistry, University of Toronto. | Current address: Golder Associates Ltd. 2390 Argentia Rd. Mississauga, Ontario, Canada, L5N 5Z7. ⊥ Current address: Department of Civil and Environmental Engineering, University of Alberta, Edmonton, Alberta, Canada. 8290
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Introduction Benzene is a known human carcinogen that is found in petroleum products and is widely used in the chemical industry. Groundwater contamination by benzene is a significant concern to human and environmental health. Both aerobic (1) and anaerobic (2) biodegradation processes are important for natural and enhanced attenuation of contaminated plumes, although anaerobic processes are much less well understood. In particular, the initial steps in the metabolic pathway for anaerobic benzene biodegradation remain uncharacterized, despite significant effort to this end (2-5). Knowledge of this pathway is a critical bottleneck to understanding this important transformation and detoxification mechanism. Several mechanisms for the initial attack on benzene have been proposed; these include hydroxylation to phenol, carboxylation to benzoate, fumarate addition to a succinyl derivative, reduction, and methylation to toluene (3). Benzoate and phenol have been consistently detected as intermediates of anaerobic benzene degradation in methanogenic (4, 6, 7), sulfate-reducing, and iron-reducing enrichment cultures (5, 7, 8), suggesting that benzene hydroxylation or carboxylation is one of the initial reaction mechanisms. Under nitrate-reducing conditions, Chakraborty et al. (9) also detected phenol in a pure culture, while Ulrich et al. (4) detected toluene, but not phenol, in a nitrate-reducing enrichment culture. The reaction mechanisms and any possible relationship to different terminal electron accepting processes remain unclear. Compound-specific isotope analysis (CSIA) can be used to measure kinetic isotopic effects (KIEs) for compounds during biodegradation, potentially providing insight into biodegradation reaction mechanisms (10-14). CSIA is based on the principle that molecules with light isotopes in the reacting bond, such as 12C and 1H, are generally degraded faster than those with heavy isotopes such as 13C and 2H (15-19). The result is a shift in the isotopic composition of the remaining contaminant pool (expressed as δ13C or δ2H) as it becomes progressively more enriched in molecules containing the heavy isotopes (15-19). The isotopic data measured during biodegradation of aromatic hydrocarbons have been found to fit a Rayleigh isotopic model, which relates the initial isotopic composition of a substrate relative to the isotopic composition of the substrate at any given time point during biodegradation (20). Isotopic shifts may be linked to reaction mechanisms in two ways. First, by evaluating experimental isotope data according to the Rayleigh isotopic model, enrichment factors (ε), which relate isotopic shifts to the extent of biodegradation (more negative values of ε indicate stronger fractionation), can be derived (20). Enrichment factors determined during biodegradation of a compound generally vary as a function of biochemical pathway (21-23). For example, during aerobic toluene biodegradation, pure cultures using different biodegradation pathways exhibited significantly different carbon and hydrogen enrichment factors (εC and εH) (21). In contrast, for anaerobic toluene biodegradation, εC and εH did not vary significantly for different pure cultures under nitrate-reducing, sulfate-reducing, and iron-reducing conditions (24), reflecting the same initial reaction mechanism in each case (25). In a previous study using enrichment cultures, we determined that carbon and hydrogen isotopic fractionation of benzene under anaerobic conditions varied considerably, possibly as a function of terminal electron acceptor (19). However, at that time no definitive interpretation relating 10.1021/es801107g CCC: $40.75
2008 American Chemical Society
Published on Web 10/14/2008
the observed fractionation to a specific degradation pathway and terminal electron acceptor could be made. Recently, a second method for linking isotopic shifts to reaction mechanisms has been documented in the literature. Previous studies have shown that different initial reaction mechanisms are most readily and reliably identified if shifts in isotope values, δ13C or δ2H, are examined in dual parameter plots (13, 14, 26, 27) since different elements are often contained in reacting bonds. In recent studies, such dual parameter plots were successfully employed to distinguish between mechanisms of aerobic benzene biodegradation (18, 26). Pure strains known to degrade benzene using monooxygenase and dioxygenase enzymes showed different slopes of δ2H versus δ13C values, and these slopes were again characteristically different from results with an unidentified sulfate-reducing benzene-degrading culture (26). The goal of the current study was to extend previous work to relate isotopic observations to particular anaerobic benzene biodegradation pathway(s). This was accomplished by first collecting additional isotopic data from multiple enrichment cultures to increase the size of the data set for analysis. Pure cultures would have been ideal for this type of study; unfortunately, though, anaerobic isolates have only been obtained under nitrate-reducing conditions (28), and attempts to grow these cultures in our laboratory under benzene concentrations or volumes high enough for even low detection limit methods (i.e., purge and trap) of hydrogen isotope analysis were unsuccessful. A comparison of the slopes of the δ13C versus δ2H values of benzene for each enrichment culture analyzed was used to interpret isotopic fractionation in the context of putative reaction mechanisms. In addition, the phylogenetic compositions of the isotopically characterized enrichment cultures were compared to investigate to what extent biodegradation mechanism is connected to microbial phylogeny and physiology. We then attempted for the first time to link differences in dual parameter slopes for anaerobic benzene biodegradation to trends in reported KIEs.
Materials and Methods Cultures. In this study, CSIA was performed on five highly enriched benzene-degrading cultures derived from three different field sites: a decommissioned retail gasoline station in Toronto, Ontario (Cart NO3-cons; Cart CH4-1), an oil refinery in Oklahoma (OR CH4-1b) and an uncontaminated swamp near Perth, Ontario (Swamp NO3-1b; Swamp NO3cons). These enrichment cultures have been maintained under nitrate-reducing or methanogenic conditions for many years prior to this study (Table 1). The OR-CH4 and Cart-NO3 cultures were chosen because cultures from these sites were part of a previous metabolite study where evidence for methylation and hydroxylation was found (4). All cultures were maintained with benzene as the sole carbon and energy source in a defined mineral medium (29), as previously described (30). A summary of all cultures investigated is provided in Table 1. This table includes cultures assayed for this study as well as cultures characterized in a previous study by Mancini et al. (19). Experimental Design. All isotopic experiments conducted on the enrichment cultures were set up in 115 or 250 mL glass bottles sealed with Mininert valves (Supelco, Inc.) after the method of Mancini et al. (19). Two or three replicate bottles were prepared for each culture, and for sterile controls containing only medium or double-distilled water. Benzene concentrations and isotopic compositions were measured throughout biodegradation. Experimental conditions for each culture are detailed in the Supporting Information (Table SI-1). Analytical Procedures. Benzene concentrations in all bottles were measured by gas chromatography, and nitrate
and nitrite concentrations were measured by ion chromatography, as previously described (30). δ13C values of benzene were analyzed by direct headspace analysis on a gas chromatograph-combustion-isotope ratio mass spectrometer (GC-C-IRMS) equipped with a Varian 3400 GC interfaced with a combustion oven in-line with a Finnigan Mat 252 IRMS, as previously described (17, 19). The GC was equipped with a VOCOL column (30 m × 0.25 mm i.d.; Supelco Inc.). Measurements of δ13C are expressed relative to the international standard V-PDB, and total analytical uncertainty for compound-specific carbon isotope analysis incorporating both accuracy and reproducibility is ( 0.5 ‰ (31). δ2H values of benzene were determined by direct headspace analysis on a Delta plus XL gas chromatograph continuous-flow mass spectrometer interfaced with a pyrolysis oven, as previously described (19). Measurements of δ2H are expressed relative to the international standard V-SMOW, and total analytical uncertainty for compound-specific hydrogen isotope analysis incorporating both accuracy and reproducibility is ( 5 ‰. Details of these analytical procedures are provided in the Supporting Information. Quantification of Isotopic Fractionation. Enrichment factors were determined using the Rayleigh isotopic model (20) expressed as: ln[(δt ⁄ 1000 + 1) ⁄ (δo ⁄ 1000 + 1)] ) (R - 1) lnf where δt is the isotopic composition of the substrate at time t, δo is the initial isotopic composition of the substrate, R is the fractionation factor, and f is the fraction of substrate (i.e., benzene) remaining. Fractionation factors (R) are determined by plotting lnf versus ln[(δt/1000 + 1)/(δo/1000 + 1)]. The slope of the linear regression is (R - 1). Fractionation factors are then converted into enrichment factors (ε) using ε ) (R - 1) × 1000.
Results and Discussion Isotopic Fractionation During Anaerobic Benzene Biodegradation. Table 2 summarizes experimental results of this study and previous work (19). For all bottles, regression according to the Rayleigh model provided r2 values that were greater than 0.79, indicating a good fit to the data. Since replicate bottles of each culture condition had calculated ε within 95% confidence intervals of each other, mean enrichment factors for replicates are reported (Table 2). Carbon and hydrogen isotopic enrichment in the remaining benzene was observed in all cultures as biodegradation proceeded. In all but one case (Swamp NO3-cons, εH ) 31 ( 7‰), hydrogen enrichment factors (εH) were significantly greater than all of those reported for aerobic cultures (εH ) 0 ( 5‰ to 17 ( 11‰ (26)), where degradation by mono- or dioxygenases does not involve hydrogen-containing bonds and εH values are therefore small (18, 26). This suggests that cleavage of a C-H bond was involved in the rate-limiting step of anaerobic benzene degradation, consistent with our previous conclusions (19) and those of Fischer et al. (26). For the nitrate-reducing cultures, εH and εC from the current study agree with those determined previously (Table 2) (19). In contrast, for the methanogenic cultures, εH and εC were smaller in the current analyses compared to those determined previously (19). These variations in ε suggest that observable isotopic fractionation is to some extent affected by differences in reaction conditions. Potentially, such variations may occur (i) if a nonfractionating rate-limiting step occurs that affects carbon and hydrogen fractionation to the same extent (32), or (ii) if the actual reaction mechanism in the culture changes, giving rise to substantially different intrinsic KIEs. To distinguish between the two cases, changes in values of δ13C and δ2H were visualized in dual parameter plots. VOL. 42, NO. 22, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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TABLE 1. Description of Cultures growth conditionsa
enrichment culture name
date culture bottle startedb
date isotopes run
data from bacterial clone libraries (% of clones)c
methanogenic
Cart CH4-1
May-99
Nov-00d May-05
Dec-06 Desulfobacterium (42%); Desulfotomaculum (6%); Spirochaetes (6%); unclassified (∼30%); [Supporting Information]
methanogenic
OR CH4-1b
Apr-97
May-05
Oct-02 - Desulfurosporosinus (42%); Desulfobacterium (33%) (30)
sulfate-reducing
Cart SO4-1a
May-98
May-01d
no data
nitrate-reducing
Swamp NO3-2b
Nov-95
Feb-01d
no data
nitrate-reducing
Swamp NO3-1b
Nov-95
May-05
Mar-05 - Azoarcus/ Dechloromonas (49%); Thiobacillus denitrificans (11%); Peptococcaceae (14%) [Supporting Information]
nitrate-reducing
Swamp NO3-cons
Sep-02
Sep-06
Mar-05 - Pelotomaculum (52%); Thiobacillus denitrificans (13%); Planctomycetales: Anammoxe (39) Nov-06 - Pelotomaculum (30%); Azoarcus (9%); Chloribium (22%); Other beta Proteobacteria (9%) [Supporting Information]
nitrate-reducing
Cart NO3-PW1
Aug-00
May-05
Oct-02 - Azoarcus/ Dechloromonas (34%); Chlorobium (52%) (30) Mar-05 - Azoarcus/ Dechloromonas (70%) [Supporting Information]
a All cultures were grown in the same FeS-reduced defined mineral medium supplemented with 10 mM sulfate for sulfate-reducing enrichments or 5 mM nitrate for nitrate-reducing enrichments. b Enrichment cultures were derived from transfers from an original microcosm study (34), and have been maintained since first started by reamending with benzene (∼0.1-1 mM, depending on culture) and electron acceptor as needed since the start date. Occasionally, fresh FeS-reduced mineral medium was also added to the bottles, but the cultures were never diluted by more than 50%. c Phylogenetic affiliation listed in order of abundance in Bacterial 16S rRNA gene clone libraries (% total clones in parentheses). Only clones with more than 2% abundance are shown. Archaeal data not shown. d Cultures used in previous study (19). e Anammox organisms were detected in a separate library with different primers (39).
Isotopic Evidence Supporting More Than One Reaction Mechanism. In a dual parameter plot of δ2H versus δ13C, a nonfractionating rate-limiting degradation step influences both the carbon and hydrogen isotopic fractionation patterns to the same extent and in the same direction. As a result, the ratio of carbon to hydrogen isotope effectssand the slope of the straight line plottedsremains similar, despite any observed variability in enrichment factors from one culture to another. Such an approach was used in previous studies for the investigation of MTBE biodegradation pathways (13, 14). Strong linear correlations between δ13C and δ2H values in different cultures degrading MTBE indicated that both isotopic signatures were influenced by the same reaction mechanism for a given biodegradation pathway (13, 14). In contrast, significant differences between the slopes of the regression lines of the δ13C and δ2H values indicated the existence of different initial reaction mechanisms (14, 26, 27). To correct for differences in the initial isotopic composition (δ13C0 and δ2H0) of the benzene used, ∆δ2H and ∆δ13C values were plotted in a dual parameter plot (Figure 1), rather than the absolute measured δ values. Values of ∆δ were calculated simply by subtracting the measured isotopic value 8292
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at time t (δt) from the initial isotopic value (δo) for each culture and experimental condition, such that ∆δ ) δt - δo; raw data are tabulated in the Supporting Information (Table SI-2). The slopes (∆δ2H/∆δ13C) of the linear regressions of ∆δ13C versus ∆δ2H obtained for each culture are provided in Table 2. Strong linear correlations (all r2 values >0.82) were obtained for each culture. Dual isotope plots provide a way to normalize the data, significantly decreasing the variability between experiments. When the data were plotted this way, similar slopes were observed for all methanogenic and sulfate-reducing cultures, indicating that these cultures likely all use the same initial reaction mechanism (Figure 1). Likewise, similar slopes were observed for all nitrate-reducing cultures, suggesting that these cultures also share a common initial reaction mechanism, but one that is distinct from the mechanism in the methanogenic and sulfate-reducing cultures. The ∆δ2H/ ∆δ13C ratios measured for all cultures grown under methanogenic and sulfate-reducing conditions (average slope, m ) 29 ( 2) are significantly higher than those measured under nitrate-reducing conditions (average slope, m ) 13 ( 2). One further data set recently provided by Fischer et al. (26)
a Mean enrichment factors calculated on the basis of combining data from all replicate bottles for each culture (see text). Ninety-five percent confidence intervals (95% CI) were determined from the slope of the linear regression of ln f versus ln[(δt/1000 + 1)/(δo/1000 + 1)] after Mariotti et al. (20) b Slopes of the linear regressions for ∆δ13C vs ∆δ2H benzene values for all replicate bottles of each culture and experimental condition. CI represents 95% confidence intervals determined on the slope of the linear regression. c Sample size is the number of simultaneous isotope and concentration data points taken throughout the degradation experiment. d Fischer et al. (26) also computed slopes from the same previously reported data; slight differences (within error) result from different calculation methods. This study started from raw data while Fischer et al. relied on reported fractionation factors. In addition, it appears that Fischer forced the linear regression lines through zero, while we did not. e Two different initial concentrations were tested for the Cart CH4-1 culture in this study.
39 ( 5 (0.98) 28 ( 1d (0.95) 31 ( 4 (0.87) 29 ( 3d (0.98) 19 ( 3 (0.95) 16 ( 2 (0.98) 8 ( 2d (0.82) 15 ( 4 (0.92) -34 ( 8 (0.88) -59 ( 4 (0.86) -38 ( 6 (0.80) -79 ( 4 (0.79) -47 ( 11 (0.92) -47 ( 4 (0.99) -35 ( 6 (0.91) -31 ( 7 (0.94) -0.8 ( 0.2 (0.93) -2.1 ( 0.1 (0.98) -1.1 ( 0.1 (0.88) -3.6 ( 0.3 (0.92) -2.6 ( 0.6 (0.96) -2.8 ( 0.6 (0.96) -2.2 ( 0.4 (0.95) -1.9 ( 0.7 (0.87) 9 93 38 12 11 8 42 9 this study ref 19 this study ref 19 this study this study ref 19 this study
OR CH4-1b Cart CH4-1 Cart CH4-1 Cart SO4-1a Swamp NO3-1b Cart NO3-PW1 Swamp NO3-2b Swamp NO3-cons
methanogenic, Co∼70 mg/L methanogenic, Co∼60 mg/L methanogenic, Co∼35 mg/L or 70 mg/Le sulfate-reducing, Co∼18 mg/L nitrate-reducing, Co∼20 mg/L nitrate-reducing Co∼20 mg/L nitrate-reducing, Co∼20 mg/L nitrate-reducing, Co∼20 mg/L
∆δ2H/∆δ13C slopes ( 95%CIb (r2) εH ( 95% CI‰a (r2) εC ( 95% CI‰a (r2) sample size nc experimental conditions culture original data from
TABLE 2. Mean Carbon and Hydrogen Enrichment Factors (In ‰ Units) for Anaerobic Benzene Biodegradation, and Slopes of the Linear Regressions of the δ2H versus δ13C Values
FIGURE 1. Plots of the ∆δ2H versus ∆δ13C benzene values measured during anaerobic biodegradation in various enrichment cultures. Values of ∆δ were calculated by subtracting the measured isotopic value at time t (δt) from the initial isotopic value (δo) for each culture, such that ∆δ ) δt δo. Data are from this and a previous study (19) and include methanogenic and sulfate-reducing cultures (closed symbols) and nitrate-reducing cultures (open symbols). The two solid thin lines represent linear regressions of the δ13C and δ2H values for the two different groups of data. 95% confidence intervals (dashed lines) and prediction intervals (thick lines) are plotted correspondingly. measured a slope of m ) 28 ( 3 for an undefined sulfatereducing culture, which is entirely consistent with the data presented here. Based on these results, we therefore hypothesize that most of the variation in our εH and εC data may be explained by the occurrence of nonfractionating ratelimiting steps (e.g., transport, enzyme-binding, etc.) but that we also obtained evidence for at least two different initial benzenedegradationmechanismsunderanaerobicconditions. Relationship to Microbial Phylogeny and Physiology. The slopes of lines in the two-dimensional plot appear to relate to terminal electron accepting process in anaerobic benzene-degrading enrichment cultures (Figure 1). As summarized in Table 1, all of these enrichment cultures were originally derived from subsurface material, and were maintained anaerobically for many years with periodic amendments of benzene and an electron acceptor (nitrate, sulfate (data shown are from a previous study (19)) or without exogenous electron acceptor (methanogenic cultures)) in a defined prereduced anaerobic mineral medium. Electron balances in culture bottles have repeatedly established that overall, benzene oxidation is coupled to the reduction of the electron acceptor provided (30, 33, 34); however, the process by which donor and acceptor are coupled is not clear in all of the enrichment cultures. Attempts at isolating pure cultures capable of anaerobic benzene degradation under sulfatereducing and methanogenic conditions have to date been unsuccessful. One might expect that in nitrate- and sulfatereducing enrichments, a single organism could be responsible for the complete oxidation of benzene to CO2, thereby facilitating isolation. By contrast, in methanogenic cultures, benzene oxidation is necessarily carried out by a minimum of two groups of organisms: methanogens, and fermenting acetogenic syntrophs that produce acetate and hydrogen necessary for the methanogens (35-37). However, because the sulfate-reducing cultures enriched in our laboratory can VOL. 42, NO. 22, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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also degrade benzene methanogenically if sulfate is omitted (30), we believe that syntrophic relationships are present in these cultures as well. Several 16S rRNA bacterial clone libraries have been prepared at various times during enrichment history (Table 1). The clone libraries from methanogenic cultures indicate the presence of strictly anaerobic fermenting and acetogenic organisms (dominated by delta Proteobacteria and Gram positive organisms) and, of course, methanogens (30). In contrast, the clone libraries from various nitrate-amended enrichment cultures contained no Archaea (methanogens), but revealed the presence of typical denitrifying beta Proteobacteria, such as close relatives to Azoarcus and Dechloromonas species that have also been identified in and isolated from other benzene-degrading nitrate-reducing cultures (28, 38). Interestingly, some of the nitrate-reducing enrichments also include organisms only found where highly anaerobic conditions prevail, such as anaerobic ammonium oxidizers (anammox organisms belonging to the Planctomycetales), fermenting or acetogenic bacteria (for example related to the genus Pelotomaculum), and iron oxidizing nitrate-reducers similar to Thiobacillus denitrificans (39). Such strict anaerobes could grow in these cultures because the medium was prereduced with iron sulfide (FeS) to ensure the absence of residual oxygen. On the basis of both isotopic and phylogenetic analyses, we postulate that the strictly anaerobic cultures dominated by syntrophic interactions (methanogenic and sulfatereducing enrichments) fall into one benzene reaction mechanism type, while the cultures dominated by facultative anaerobes such as Azoarcus and Dechloromonas spp., that typically also can use oxygen as an electron acceptor, fall into another. This idea is consistent with the recent analysis of Musat and Widdel (40) who noted that reactive oxygen species cannot be generated in anaerobic metabolism, and propose the existence of a different mechanism for strictly anaerobic organisms. This result is in stark contrast to anaerobic toluene degradation, where a single mechanism prevails (i.e., fumarate addition) under all anaerobic conditions, including nitrate reduction (25). Moreover, we can be certain that the mechanistic differences detected by dual parameter plot analysis in the nitrate-reducing cultures are not the result of oxygen (O2) leaking into the bottles, since these enrichments also maintain strictly anaerobic organisms that would not survive if oxygen were present. This is an important point, as aerobic and chlorate-reducing cultures, where molecular oxygen is known to be involved in the mechanisms, generally have dual parameter slopes lower than the nitrate-reducing cultures presented herein (26). Hydrogen Isotope Effects in Initial Benzene Transformation Reactions. The distinctive differences in dual parameter slopes raises the question of whether these variations can be linked to reported KIEs for specific reaction mechanisms. In previous work, such analyses have elucidated clearly distinct reaction mechanisms for the transformation of 1,2-dichloroethane (41), MTBE (14), and other compounds (10). Following the same approach, we examined possible reaction mechanisms pertaining to different initial steps postulated for benzene degradation (Figure 2). This examination revealed a fundamental difference compared to previous work with 1,2-dichloroethane and MTBE: that all postulated activation mechanisms for anaerobic benzene degradation involve the same chemical C-H bond in a very similar sequence of addition followed by elimination reactions. The first step in this sequence is the reversible addition of an electrophile which causes destabilization of the aromatic ring and the second is the then energetically favorable elimination of a leaving group (i.e., H+) to allow reestablishment of the aromatic structure (Figure 2). Since for benzene, no C-H bond is broken in the first step (k1 and k-1 in Figure 8294
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FIGURE 2. Proposed benzene reaction mechanisms, including hydroxylation of benzene to phenol by a hydroxyl free radical (48), hydroxylation of benzene to phenol by one electron transfer (45) and methylation of benzene to toluene (3, 4). Rate constants k1, k-1, and k2 are those associated with addition, dissociation, and elimination reaction steps, respectively. 2), only weak or negligible (secondary) hydrogen isotope effects are expected here. In contrast, a primary hydrogen isotope effect can be expected in the second step (k2 in Figure 2), since a C-H bond cleavage occurs (42). Hence, depending on whether k-1 or k2 is greater, hydrogen isotope fractionation can be more or less pronounced. If k2 . k-1, quantitative conversion takes place so that the hydrogen isotope effect in k2 is not observable (KIE ) 1). Conversely, in the case of k-1 . k2, the primary hydrogen isotope effect in k2 (KIE ) 3-8) is directly expressed. If k-1 ≈ k2, the observable KIEH will be considerably reduced, but will still be significantly greater than 1 (42). Indeed, the reported hydrogen kinetic isotope effects given in Figure 2 are smaller than expected for primary KIEH, but greater than typical secondary KIEH and suggest that the reaction rates of both steps are of similar magnitude. According to available mechanistic models, the reported KIEH in benzene transformation reactions therefore do not represent distinct reactions of different bonds, but rather subtle differences in relative reaction kinetics. Implications for Dual Parameter Slopes. Slopes in dual parameter plots do not only depend on hydrogen, but also on carbon isotope effects, since they depict changes in isotope ratios of both elements, ∆δ2H/∆δ13C, (eq 23 in ref 10): m)
∆δ2H 13
∆δ C
≈
(Rrp,H)-1 - 1 (Rrp,C)
-1
-1
)
KIEH - 1 1 + KIEC · (zC - 1) · KIEC - 1 1 + KIEH · (zH - 1)
According to this equation, reactions have a greater slope (m) if they have a larger KIEH for the same KIEC. Unfortunately, we do not know of any KIEC values published for the reactions in question; the only fractionation value reported for hydroxylation reactions (1.008) (43) is not a position-specific kinetic isotope effect, and there are no data for carboxylation or methylation to compare it with. Given that explicit values are missing, KIEC may therefore be considered only in a qualitative way. Due to the destabilization of the aromatic structure, all steps k1, k-1, and k2 are expected to cause primary carbon isotope effects (42). It may therefore be expected that the net carbon isotope effect is much less dependent on the relative rates in Figure 2 than the net hydrogen isotope effect. In such a case differences in slopes may be directly related to reported KIEH values in a qualitative way. The greater slopes observed with methanogenic cultures are therefore rather
consistent with a hydroxylation mechanism, and the smaller slope observed with the nitrate-reducing cultures would be in line with a methylation mechanism. We stress, however, that for the reasons given above these lines of evidence do not have the same conclusiveness as in the case of previous studies with other compounds. In addition, because no KIE data are currently available for other possible mechanisms such as carboxylation, no conclusions could be drawn about the likelihood of this mechanism occurring under different redox conditions. While our isotopic data present strong evidence for more than one different anaerobic benzene degradation mechanism, the evidence for the exact nature of these mechanisms needs to be substantiated by alternative approaches. Relating Mechanisms to Metabolites Detected in Benzene-Degrading Cultures. The detection of phenol as a metabolite in several benzene degradation studies suggests that the degradation process may be initiated by a hydroxylation step (4, 7, 9, 44). A hydroxyl radical attack on benzene was proposed to occur in the nitrate-reducing pure culture, Dechloromonas strain RCB (9) (Figure 2); the source of the hydroxyl radical was suggested to originate from a reaction on the outer membrane or in the periplasm of this microorganism (9). As an alternative hydroxylation pathway, a oneelectron transfer from the aromatic ring to a highly oxidized species such as FeIV)O followed by the addition of water to form phenol (Figure 2) was determined in biological experiments involving the metabolism of benzene by cytochrome c (45). Oxygen for this reaction was postulated to originate from water based on the incorporation of 18O labeled water in phenol by immobilized cytochrome c (45), consistent with the results of 18O labeled water experiments carried out during benzene biodegradation using a methanogenic enrichment culture (44). Hence, this reaction mechanism may provide a model for the addition of water to benzene during anaerobic biodegradation in some cultures. A similar reaction mechanism was postulated for anaerobic ethylbenzene biodegradation by Azoarcus sp. strain EB1 where hydroxylation via a hydroxyl group derived from water possibly involves a transfer of an electrophillic oxygen from a ModO cofactor (46, 47). No proof of the exact mechanism of a benzene methylation or carboxylation reaction has been documented, though a methylation might br expected to occur via a Fried-Crafts-type alkylation reaction (Figure 2) and there is recent evidence that the source of a carboxyl group could be bicarbonate (5). Implications for the Initial Reaction Mechanisms in the Benzene-Degrading Cultures. The ∆δ2H/∆δ13C slopes for the strictly anaerobic methanogenic and sulfate-reducing cultures from this study and that reported by Fischer et al. (26) are higher than those that might be expected for benzene methylation. These slopes are more in the range of those expected for a hydroxylation mechanism, consistent with the detection of phenol in the same cultures (4), however, a new study has found that phenol could be an artifact of the extraction procedure (5). The measured ∆δ2H/∆δ13C slopes for the nitrate-reducing cultures are lower and more consistent with slopes expected for benzene methylation. Biochemical evidence for methylation exists from only traces of toluene detected in these cultures (4); therefore caution must be taken with these interpretations. In conclusion, while one can suggest against certain reaction mechanisms where experimental data are different from predictions based on KIEs, one cannot completely rule out other mechanisms particularly those for which KIEs are not available, for example direct carboxylation mechanisms or reactions with a reactive cellular metabolite such as fumarate. In fact, the observed effect of redox on the ∆δ2H/∆δ13C slope may be the result of differences in enzyme or cofactor structure between organisms with different physiologies (e.g., anaerobes vs
facultative organisms), even considering a similar overall reaction. Experiments incorporating both the investigation of intermediates and KIE measurements in pure cultures or in cell free extracts will be a critical next step in elucidating the underlying mechanisms, particularly for proposed mechanisms where no KIE data are currently available. Potential reactive species present only in strictly anaerobic microorganisms need to be particularly scrutinized in the light of obvious mechanistic differences proposed elsewhere (26, 40) and elucidated herein.
Acknowledgments Authors S.A.M. and C.E.D. contributed equally to this work. We acknowledge Jennifer McKelvie and George LacrampeCouloume for assisting with isotopic measurements. We also thank Sandra Little and Roya Gitiafroz (U. of Toronto) for assistance with maintaining the cultures prior to this study. This research was supported by the Natural Science and Engineering Research Council of Canada through a Strategic Grant awarded to the senior authors and postgraduate scholarships awarded to S. M., C. D., A. U., and M. N. Author S. M. thanks the Geological Society of America and the American Association of Petroleum Geologists for their student research funding support.
Supporting Information Available Experimental design for the setup of cultures analyzed in this study (Table SI-1); analytical procedures for isotope analysis; clone library construction; raw data for Figure 1 (Table SI-2 and SI-3). This material is available free of charge via the Internet at http://pubs.acs.org.
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