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Compound-Specific Isotope Analysis as a Tool To Characterize Biodegradation of Ethylbenzene Conrad Dorer,†,‡ Carsten Vogt,*,† Sabine Kleinsteuber,§ Alfons J. M. Stams,∥ and Hans-Hermann Richnow† †

Department of Isotope Biogeochemistry and §Department of Environmental Microbiology, UFZ−Helmholtz Centre for Environmental Research, Permoserstrasse 15, D-04318 Leipzig, Germany ‡ Faculty of Biology, University of Freiburg, Schänzlestrasse 1, D−79104 Freiburg, Germany ∥ Laboratory of Microbiology, Wageningen University, 6703 HB Wageningen, The Netherlands S Supporting Information *

ABSTRACT: This study applied one- and two-dimensional compound-specific isotope analysis (CSIA) for the elements carbon and hydrogen to assess different means of microbial ethylbenzene activation. Cultures incubated under nitratereducing conditions showed significant carbon and highly pronounced hydrogen isotope fractionation of comparable magnitudes, leading to nearly identical slopes in dual-isotope plots. The results imply that Georgf uchsia toluolica G5G6 and an enrichment culture dominated by an Azoarcus species activate ethylbenzene by anaerobic hydroxylation catalyzed by ethylbenzene dehydrogenase, similar to Aromatoleum aromaticum EbN1. The isotope enrichment pattern in dual plots from two strictly anaerobic enrichment cultures differed considerably from those for benzylic hydroxylation, indicating an alternative anaerobic activation step, most likely fumarate addition. Large hydrogen fractionation was quantified using a recently developed Rayleigh-based approach considering hydrogen atoms at reactive sites. Data from nine investigated microbial cultures clearly suggest that two-dimensional CSIA in combination with the magnitude of hydrogen isotope fractionation is a valuable tool to distinguish ethylbenzene degradation and may be of practical use for monitoring natural or technological remediation processes at field sites.

1. INTRODUCTION Benzene, toluene, ethylbenzene, and xylenes (BTEX) are components of crude oil and produced on a large scale for use as solvents and precursors of other chemicals.1−4 For instance, ethylbenzene serves primarily for the manufacture of styrene, which is the raw material for polystyrene synthesis. However, improper human handling, waste disposal, or accidental fuel spills release aromatic hydrocarbons to the environment.2,5 Due to their relatively high mobility and elevated water solubility in comparison to other hydrocarbons, BTEX compounds easily contribute to groundwater pollution.6 Therefore, the assessment of their degradation potential is of high interest in order to make decisions about remediation measures and to minimize risks to health and environment. Because of the stabilizing resonance energy of the aromatic ring and missing destabilizing functional groups, BTEX compounds are chemically relatively inert compounds. Nevertheless, they can be biochemically activated, and their biodegradation under both aerobic and anaerobic conditions has been demonstrated. In all investigated aerobic organisms, biodegradation of aromatic hydrocarbons proceeds via one or more hydroxylation reactions that are catalyzed by mono- or dioxygenases. In the case of aerobic ethylbenzene degradation, the initial attack can © 2014 American Chemical Society

occur either at the aromatic ring by mono- or dihydroxylation or at the side chain (Figure 1 and Supporting Information Table S2a).7−9 In several cases multiple parallel activation mechanisms have been shown to occur in a single organism. For example, toluene dioxygenases in Pseudomonas putida strains F1 and UV4 catalyze the dihydroxylation of the aromatic ring as the dominating activation step, but (S)-1-phenylethanol is also formed as a minor product that is quickly dihydroxylated at the ring to the corresponding triol.10−12 In P. putida NCIB 9816, ethylbenzene degradation is initiated primarily via benzylic hydroxylation to form (S)-1-phenylethanol but (R)1-phenylethanol and styrene are also produced to a minor extent.13 These hydroxylations are catalyzed by naphthalene dioxygenase (NDO), an enzyme with relaxed substrate specificity.14 However, the catalytic cycles of mono- and dihydroxygenases are still not fully understood.15−17 Anaerobic degradation of ethylbenzene has been shown to occur under denitrifying,18,19 iron-reducing,20 sulfate-reducing,21 or methanogenic conditions.22 Only a limited number of Received: Revised: Accepted: Published: 9122

January 17, 2014 May 27, 2014 June 27, 2014 June 27, 2014 dx.doi.org/10.1021/es500282t | Environ. Sci. Technol. 2014, 48, 9122−9132

Environmental Science & Technology

Article

Figure 1. Initial steps of aerobic and anaerobic microbial ethylbenzene degradation.10,13,33,71−74

is based on slightly different reaction rates of isotopologues in isotopically sensitive rate-limiting steps. In this way, either substrates or products become enriched in heavy isotopes at the reactive position, which is referred to as isotope fractionation. The term kinetic isotope effect (KIE) indicates the ratio of the reaction rates of the corresponding light and heavy isotopologue in unidirectional transformations and is one way to quantify isotope enrichment.39,40 The magnitude of KIEs reflects the geometric structure of the transition state41 and thus may be of help in identifying the isotopically sensitive step in a complex enzymatic reaction. Small observed KIEs as compared to theoretical maximum values indicate that the transition state resembles either the substrate or the product, e.g., for reactions with strong intermolecular interactions such as heterolytic H abstraction, whereas large KIEs indicate symmetric transition states.42−44 In biological systems, isotope enrichment factors for the same type of reaction may vary depending on the presence of other nonfractionating, rate-limiting processes, such as substrate uptake into the cell. These processes may mask intrinsic isotope enrichment upon bond cleavage and lead to apparent kinetic isotope effects (AKIEs) that are smaller than intrinsic KIEs.40,45 Multidimensional isotope analysis allows the correlation of isotope ratios of several elements relative to each other. As masking effects can act on two or more elements in a similar way, multidimensional CSIA enables an evaluation that is independent from bioconversion rates.37 The correlation of

anaerobic ethylbenzene-degrading bacteria have been isolated, e.g., Aromatoleum aromaticum strain EbN1,23 strain EB1,24 and Georgf uchsia toluolica strain G5G6,25 that use nitrate as the terminal electron acceptor. The initial hydroxylation reaction at the benzylic carbon atom leading to (S)-1-phenylethanol (Figure 1) was intensively studied using the model strains EB124,26,27 and A. aromaticum.28−31 Note that these strains employ oxygen from water, whereas obligate aerobic bacteria incorporate molecular oxygen from the air. In A. aromaticum, the rate-limiting step of the initial enzyme ethylbenzene dehydrogenase under physiological conditions is a homolytic C−H bond activation of the substrate forming a radical intermediate.32 By contrast, the sulfate-reducing ethylbenzenedegrading strain EbS7 isolated from marine sediment33 was shown to activate ethylbenzene by fumarate addition33 (Figure 1), a well-known reaction mechanism to activate hydrocarbons under anoxic conditions. For instance, fumarate addition to toluene is catalyzed by the enzyme benzylsuccinate synthase, in which hydrogen abstraction from the methyl group generating a benzyl radical is suggested the rate-limiting step.34,35 Compound-specific isotope analysis (CSIA) is a common analytical tool with broad application in the environmental sciences, such as in the field of contaminant hydrology of organic compounds. Small variations in the isotopic composition of chemical compounds make it possible to identify pollution sources and to assess degradation processes.36−38 Quantification and elucidation of biochemical transformations 9123

dx.doi.org/10.1021/es500282t | Environ. Sci. Technol. 2014, 48, 9122−9132


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same medium was used under oxic conditions with nitrate substituted with 10 mM NH4Cl as the source of nitrogen. From an aquifer sample from the above-mentioned contaminated site, an anaerobic enrichment culture (DDAnox2) was also established under methanogenic conditions. To prepare DD-Anox2, approximately 100 g sediment was transferred into a 1 L bottle which was filled with anoxic mineral medium55 modified by replacing sulfate with 1 mM Na2S. A sulfate-reducing ethylbenzene-degrading culture (Zz3) originated from the coarse sand used as filling material of a column system located in the area of a former coal hydrogenation and benzene production plant near Zeitz (Germany). The bacterial community colonizing this sand has previously been shown to mineralize benzene under sulfatereducing conditions.55 The enrichment culture Zz3 was prepared by transferring 700 mL of coarse sand into 1 L bottles, after which ca. 600 mL of anoxic mineral medium for sulfate-reducing bacteria, described by Vogt et al.,55 was added. For isotope fractionation experiments, cultures were prepared by inoculating the various mineral salt media with ethylbenzene-grown preparatory cultures. The enrichment cultures DD-Anox2 and Zz3 were used for isotope fractionation experiments directly after setup (see below). In both the preparation of anoxic media and the handling of anaerobic cultures, established anaerobic techniques of this research group were used as described elsewhere.49,55 Neat ethylbenzene was used as the sole source of carbon and energy except for experiments with P. putida NCIB 9816, which degraded ethylbenzene only co-metabolically in the presence of naphthalene. In this case, 1 mg of crystalline naphthalene was added to 1 L of medium. All fractionation experiments were run at room temperature. Experiments were performed either in a single bottle of 1 or 2 L culture volume, which was periodically sampled, or in several parallel 118 mL serum bottles each filled with 50 mL of culture volume, which were periodically sacrificed for destructive sampling. For all experiments, control bottles containing medium and ethylbenzene did not display any fractionation for either element and no significant changes in substrate concentration occurred. The Supporting Information gives more detailed information on the design of the degradation experiments. Analytical Methods. Ethylbenzene was analyzed by GCFID with helium or nitrogen as carrier gases as described in Vogt et al., 2008.49 Metabolites of ethylbenzene transformation were identified by GC-MS and quantified by HPLC techniques (see Supporting Information). Nitrite was determined photometrically by a modified Griess reaction according to the German standard DIN EN 26 777 (EPOS Analyzer 5060, Eppendorf AG, Hamburg, Germany). The applied analytical techniques for CSIA were described recently in the Supporting Information of Dorer et al., 2014.51 Phylogenetic Identification of Dominant Organisms in DD-Anox1 and DD-Ox. Cells were harvested from 50 mL each of the aerobic- (DD-Ox) and nitrate-reducing enrichment cultures (DD-Anox1) by centrifugation. The methods for DNA extraction, 16S rRNA gene fragment amplification, phylogenetic assignment of the gained 16S rRNA gene sequences, and T-RFLP analysis of the 16S rRNA amplicons are described in the Supporting Information of this manuscript. Isotope Calculations. One-dimensional plots considering only bulk values of one element were calculated by means of the simplified Rayleigh equation (eq 1)

hydrogen and carbon fractionation in dual-isotope plots has been employed to characterize degradation pathways and to identify enzymatic reaction mechanisms, e.g., for benzene,46−48 toluene,49 or xylene50 biodegradation. However, this approach is challenged when large hydrogen isotope fractionation occurs, as observed recently for ethylbenzene biodegradation in A. aromaticum.51 Nevertheless, even very large hydrogen isotope enrichment can be tackled by a proposed refinement to the two-dimensional CSIA evaluation procedure based on the Rayleigh equation and considering reactive positions51 and by enhanced approaches to model KIE values.52 In this study, carbon and hydrogen stable isotope fractionation of four pure strains and four enrichment cultures were investigated. Together with the previous isotope analyses of ethylbenzene-degrading A. aromaticum, the potential of twodimensional (2D) CSIA to characterize microbial ethylbenzene activation was assessed (see overview in Table S1, Supporting Information). As a variety of aerobic microorganisms can simultaneously attack ethylbenzene in multiple ways, the number of model organisms at lab scale for only one specific initial reaction was limited. The major objectives of this study were to determine (i) whether different pathways under oxic and anoxic conditions could be distinguished by characteristic ranges for enrichment factors and values from dual-isotope plots and (ii) if large hydrogen fractionation plays a role and the refined CSIA evaluation procedure proposed recently51 is applicable for these cultures. Finally, the authors discuss what implications the findings of this study have for application at contaminated sites.

2. MATERIAL AND METHODS Chemicals. Chemicals were purchased as follows from the following companies: ethylbenzene (Merck, ≥99% GC), racemic 1-phenylethanol (Aldrich, 98% GC), (R)-1-phenylethanol (Aldrich, 97% GC), (S)-1-phenylethanol (Aldrich, 97% GC), acetophenone (Sigma-Aldrich, 99% GC), 2-ethylphenol (Aldrich, 99% GC), 3-ethylphenol (Merck-Schuchardt, 98% GC), and 4-ethylphenol (Fluka, >97% GC). Microorganisms and Cultivation Conditions. The aerobic bacterial strains Pseudomonas putida NCIB 9816 (DSM 8368), Pseudomonas putida F1 (DSM 6899), and Pseudomonas f luorescens SK1 (DSM 16274) were purchased from the Leibniz Institute DSMZ - German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany). They were cultivated in Brunner mineral salt medium,53 the medium for the latter strain being supplemented with 0.5% yeast extract. Georgf uchsia toluolica G5G6 (DSM 19032) was taken from the strain collection of the Laboratory of Microbiology of the University of Wageningen, The Netherlands, and grown under nitrate-reducing conditions in a medium described by Holliger et al., 1993,54 with the modifications given in Weelink et al., 2009.25 A nitrate-reducing ethylbenzene-degrading culture dominated by an Azoarcus sp. (DD-Anox1) and an aerobic ethylbenzene-degrading culture dominated by an Acidovoraxrelated phylotype (DD-Ox) were enriched from anoxic aquifer samples taken from a BTEX-contaminated site in Dresden, Germany, by repeated transfer under nitrate-reducing and aerobic conditions, respectively. DD-Anox1 was cultivated under nitrate-reducing conditions in a mineral salt medium as previously described.23 For the enrichment culture DD-Ox, the 9124



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Figure 2. Rayleigh plots and dual-isotope plots for ethylbenzene hydroxylation by G. toluolica and DD-Anox1. Bulk isotopic data resulted in good linearity for carbon isotope enrichment (first column), whereas large hydrogen fractionation led to nonlinear Rayleigh plots that do not allow determination of an εbulk,H representative for a larger concentration interval (second column). Considering hydrogen atoms in the reactive position (n/x = 10) enables linearization and the possibility to deduce robust isotope enrichment factors (εrp,H) over the same concentration interval (third column). Traditional dual-isotope plots characterized by the slope Λ (eq 3) led to nonlinear effects. The increasing deviation of a constant Λ is shown by the extrapolated line for delta values considering δt − δ0 < 100‰ (fourth column). ζ*(rp) plots (eq 4) enable a linear regression (fifth column). Straight lines represent linear regressions and dashed lines the lower and upper 95% confidence interval.

⎛ R t ⎞ ⎛ ct ⎞εbulk = f εbulk ⎜ ⎟=⎜ ⎟ ⎝ R 0 ⎠ ⎝ c0 ⎠

ζ *(rp) = (x /n)H (1)

δt 2 H − δ0 2 H Δδ 2 H = δt 13C − δ013C Δδ13C

⎞

( nx )H ln⎝(R 0 + ( nx )H ΔR)/R 0⎠ ⎜

=

⎟

ln(R t /R 0)c

H

(4)

AKIEs were calculated according to eq 5 when secondary isotope effects were neglected. AKIE =

1 z εrp + 1

(5)

Derivations and a comprehensive discussion of Rayleigh equations, dual plots and AKIE calculations, Λ simulation plots, and an alternative logarithmic regression are described by Dorer et al.51

3. RESULTS AND DISCUSSION Ethylbenzene Degradation under Nitrate-Reducing Conditions. At about 90% ethylbenzene degradation, G. toluolica showed more than 10‰ and 400‰ enrichment of heavy carbon and hydrogen isotopes, respectively, as compared to the initial isotopic composition. Carbon isotope enrichment factors were calculated at εbulk,C = −4.1 ± 0.2‰ and hydrogen isotope factors varied with increasing substrate transformation, exhibiting values below −100‰ (Figure 2 and Supporting Information Table S3). A similar isotope fractionation pattern was reported recently for A. aromaticum degrading ethylbenzene under nitrate-reducing conditions.51 A series of transfers of a BTEX-contaminated aquifer sample resulted in the enrichment culture DD-Anox1 dominated by an Azoarcus species, which showed 99% BLAST identity to A. toluclasticus, A. tolulyticus, and A. denitrificans. T-RFLP analysis confirmed that the culture consisted almost exclusively of this phylotype (Figure S1, Supporting Information). Isotope

(2)

In “classical” dual-isotope plots, Δδ2H versus Δδ13C was plotted. The slope Λ was calculated using eq 3 Λ=

εbulk,C ⎛

where R is the isotope ratio (RC = 13C/12C and RH = 2H/1H), c expresses the ethylbenzene concentration, and the subscripts 0 and t refer to the beginning and a later time point t of the degradation process. The ratio is defined as the reaction progress f, and ε is the enrichment factor, correlating changes in concentration to changes in the isotope composition. Taking into consideration the number x of hydrogen atoms at the reactive position (rp) (given for each culture in Table S1, Supporting Information) and the total number n of hydrogen atoms in the compound (e.g., n = 10 hydrogen atoms for ethylbenzene), eq 2 was applied as derived in Elsner et al., 200537 ⎛ R 0 + n (R t − R 0) ⎞ ⎛ c ⎞εrp x ⎜⎜ ⎟⎟ = ⎜ t ⎟ = f εrp R0 ⎝ ⎠ ⎝ c0 ⎠

εrp,H

(3)

and can be often approximated by Λ ≈ εbulk,H/εbulk,C.47 The dimensionless delta value (δ) expresses the relation δSample = (RSample/RStandard) − 1. The alternative two-dimensional parameter ζ*(rp)51 considers hydrogen isotopes at reactive sites and is defined according to eq 4 9125



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Table 1. Overview of CSIA Parameters for Nine Microbial Cultures Capable of Ethylbenzene Degradationa

a Enrichment factors for reactive positions and AKIEs were calculated with the following factors. (i) Ring-2,3-dihydroxylation (nC = 8, xC = 4, zC = 2); (nH = 10, xH = 4, zH = 2). (ii) Ring-monohydroxylation in the ortho position (nC = 8, xC = 2, zC = 1); (nH = 10, xH = 2, zH = 2). (iii) Side-chainmonohydroxylation at the benzylic carbon (nC = 8, xC = 1, zC = 1); (nH = 10, xH = 1 or 2, zH = 1 or 2). Errors are indicated for a 95% confidence interval. In the case of AKIEs, errors are given in brackets and were calculated using the lowest and highest fractionation factor of the corresponding confidence interval. R2 and the number of considered data points refer to the appropriate parameter.

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for G. toluolica and DD-Anox1, respectively. Equation 4 provides the ability to calculate robust ζ*(rp) values in an alternative dual-isotope plot in order to quantify the carbon− hydrogen bond cleavage at the benzylic carbon atom of an ethylbenzene molecule, leading to ζ*(rp) = 18 ± 1 for G. toluolica and ζ*(rp) = 22 ± 1 for DD-Anox1 (Figure 2). The Λ and ζ*(rp) values of both investigated microbial cultures were similar to the values previously determined for A. aromaticum (Λ = 24 ± 2 and ζ*(rp) = 19 ± 1) (Figure S2, Supporting Information). AKIEHs calculated with zH = 1 and without consideration of secondary isotope effects were of the order of magnitude typical for a C−H bond cleavage:37 4.4 for G. toluolica and 4.2 for DD-Anox1 (Table 1, Table S5, Supporting Information) compared to 3.9 for A. aromaticum.51 The magnitude of this hydrogen AKIE is consistent with a C−H bond cleavage and indicates a relatively symmetrical transition state, as expected for a homolytic bond cleavage. Two conclusions can be drawn from these results: (i) the previously refined evaluation procedure for CSIA52 proved once again to be suitable to address large hydrogen fractionation and deliver robust values, and (ii) the almost identical carbon and hydrogen enrichment factors, Λ and ζ*(rp) factors, hydrogen AKIEs, and metabolites of the initial ethylbenzene degradation pathway for the two nitrate-reducing cultures studied here as well as the previously characterized A. aromaticum strongly indicate that the ethylbenzene attack in G. toluolica and DD-Anox1 is catalyzed by an ethylbenzene dehydrogenase with similar kinetic and mechanistic properties as in A. aromaticum. The occurrence of a gene predicted to encode ethylbenzene dehydrogenase in the genome of G. toluolica57 lends support to the conclusions from CSIA. Keeping in mind that strain EB1 also catalyzes the hydroxylation of ethylbenzene to 1-phenylethanol,24,26,27 it can be inferred that this reaction is a common mechanism for microbial ethylbenzene activation under nitrate-reducing conditions. Ethylbenzene Degradation under Nitrate-Free, Strictly Anoxic Conditions. The two anaerobic enrichment cultures DD-Anox2 and Zz3 showed a very distinct carbon and hydrogen isotope fractionation pattern for ethylbenzene degradation compared to the cultures incubated under nitrate-reducing conditions. Both the sulfate-reducing enrichment culture Zz3 and the enrichment culture DD-Anox2, set up under methanogenic conditions, degraded about 0.25 mM ethylbenzene within 1 year. Isotope samples taken regularly over this period exhibited small carbon fractionation at εbulk,C = −0.6 ± 0.1‰ and εbulk,C = −0.7 ± 0.3‰ and moderate hydrogen fractionation at εbulk,H = −76 ± 16‰ and εbulk,H = −96 ± 7‰ for Zz3 and DD-Anox2, respectively (Table 1, Table S3 and Figure S3, Supporting Information). Decreasing ethylbenzene concentrations were accompanied by increasing sulfide concentrations in Zz3, indicating sulfate as the terminal electron acceptor (data not shown). As no production of methane was measured for DD-Anox2, use of other electron acceptors present in the sediment cannot be ruled out, e.g., ferric iron or remaining sulfate. In addition, sulfide was not detected but could have been precipitated as metal sulfides in the sediment slurry. 1-Phenylethanol and acetophenone, typical metabolites of the ethylbenzene degradation pathway initiated by ethylbenzene dehydrogenase, were not detected in either culture. Notably, enrichments of about 800‰ compared to initial hydrogen isotope composition were detected for Zz3 at

fractionation experiments for ethylbenzene degradation resulted in values similar to those observed for G. toluolica: carbon and hydrogen isotope enrichment was about 7‰ and 360‰, respectively, after 88% ethylbenzene degradation. The strong hydrogen fractionation again led to nonlinear hydrogen isotope enrichment in Rayleigh plots based on bulk values, whereas a stable carbon isotope enrichment factor was determined to be εbulk,C = −3.7 ± 0.1 (Figure 2). In the experiments with G. toluolica and DD-Anox1, ethylbenzene degradation was accompanied by an increase in the amount of nitrite, suggesting that nitrate was used as the terminal electron acceptor (Figure S7, Supporting Information). In order to establish whether the intermediates suggest the same initial activation steps for ethylbenzene degradation as in A. aromaticum, metabolites were identified by GC-MS for G. toluolica and DD-Anox1 and in both cases revealed to be 1phenylethanol and acetophenone (Figure S9, Supporting Information). Additionally, both organisms were able to grow on (R)-1-phenylethanol, (S)-1-phenylethanol, racemic 1phenylethanol, and acetophenone as the sole carbon and energy sources. In this study, complete degradation of these intermediates was shown by HPLC (Figure S8, Supporting Information). The concomitant increasing optical density at 600 nm and increasing nitrite concentrations indicate that the metabolization of ethylbenzene is coupled to growth and that nitrate is used as an electron acceptor. No changes were seen in control bottles containing either autoclaved cultures or medium with the same metabolite concentration (data not shown). For A. aromaticum, (S)-1-phenylethanol is produced as the first intermediate in ethylbenzene degradation. For each of the enantiomers of 1-phenylethanol, a 1-phenylethanol dehydrogenase was recently identified. Interestingly, (R)-1-phenylethanol dehydrogenase was sufficiently induced even when cells grew solely on (S)-1-phenylethanol.56 Thus, degradation of both 1-phenylethanol enantiomers in G. toluolica and DDAnox1 may be explained by the occurrence of two separate enantiomer-specific isoenzymes and does not contradict formation of an enantiopure first intermediate. In summary, these findings suggest that the first two steps of ethylbenzene degradation in G. toluolica and DD-Anox1 are identical with those in A. aromaticum and hence that ethylbenzene hydroxylation at the benzylic carbon atom is catalyzed by an ethylbenzene dehydrogenase. Assuming that the enzymatic reaction produces a pure enantiomer, probably (S)-1-phenylethanol, only (R)-[1-2H1]ethylbenzene contributes to the primary kinetic isotope effect, and in the case of (R)-1-phenylethanol only (S)-[1-2H1]ethylbenzene contributes. Consequently, determination of hydrogen isotope enrichment factors considering the hydrogen atoms at the reactive site (eq 2) with xH = 1 resulted in εrp,H = −772 ± 29‰ and −764 ± 17‰ for G. toluolica and DDAnox1, respectively (Figure 2). These values are virtually identical to the factors determined for A. aromaticum containing ethylbenzene dehydrogenase: εbulk,C = −3.8 ± 0.1‰ and εrp,H = −744 ± 19‰.51 Applying a logarithmic regression as described in a prior study,51 the data from both elements support the assumed stereochemically pure enzymatic reaction with a fitted xH = 1.4 ± 0.3 for G. toluolica and xH = 1.2 ± 0.2 for DD-Anox1 (Table S4 and Figure S2, Supporting Information). Λ values are robust only in the case of small concentrations of degraded ethylbenzene, leading to relatively small changes in hydrogen delta values with Λ = 24 ± 5 and Λ = 32 ± 2 (δt − δ0 < 100‰) 9127



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Figure 3. Isotopic characterization of four microbial cultures capable of aerobic ethylbenzene activation. Regression lines of the double-logarithmic Rayleigh plots for bulk carbon and bulk hydrogen isotope fractionation obtained from four different aerobic ethylbenzene degrading cultures using three different major activation strategies (ring mono- and dihydroxylation as well as benzylic side-chain hydroxylation). Although all cultures showed a similar, very low carbon isotope fractionation, the patterns were different for hydrogen isotope fractionation exhibiting the highest enrichment factor for the activation at the methylene group at the side chain. The same pattern is seen for Λ values (which are for low fractionation identical with ζ*(rp) values).

Aerobic Ethylbenzene Degradation. The four aerobic cultures showed very similar small carbon isotope fractionation with bulk enrichment factors of between −0.4‰ and −0.6‰. By contrast, the hydrogen isotope fractionation differed considerably (Figure 3). P. putida NCIB 9816 using benzylic hydroxylation as the major activation step exhibited moderate fractionation (εbulk,H = −28 ± 3‰). An aerobic culture (DDOx) enriched from a BTEX-contaminated site in Dresden, Germany, consisting almost completely of a single phylotype according to T-RFLP analysis and affiliated to the genus Acidovorax, resulted in small but still significant fractionation (εbulk,H = −6 ± 1‰). For P. f luorescens SK1, no significant fractionation was detected (εbulk,H = −2 ± 3‰), and for P. putida F1 employing a dioxygenase to catalyze the initial degradation step, slight inverse fractionation was observed (εbulk,H = +4 ± 1‰) (Figure 3, Figure S4 and Table S3, Supporting Information). 2-Ethylphenol was detected as a major product of the ethylbenzene activation step for both the enrichment culture DD-Ox and P. f luorescens SK1 (Figure S9, Supporting Information). Metabolite degradation experiments with the culture DD-Ox showed that 2-ethylphenol was used as substrate. However, the culture also degraded a variety of other possible activation products: (S)-1-phenylethanol, (R)-1phenylethanol, acetophenone, 3-ethylphenol, and 4-ethylphenol. Consumption of all mentioned substrates was accompanied by a clear increase in the optical density at 600 nm, indicating growth (Figure S8, Supporting Information). For aerobic cultures with ring hydroxylation as the initial enzymatic step, which in this study consisted of P. putida F1, P. f luorescens SK1, and DD-Ox, Λ values were rather small at −7 ± 3, 5 ± 6, and 9 ± 3, respectively. In contrast, P. putida NCIB 9816, activating ethylbenzene by benzylic hydroxylation as major mechanism, showed a Λ value of 35 ± 9. The characteristic isotope fractionation patterns make CSIA a useful tool to distinguish between mechanistically different arene epoxidation and alkylic side-chain hydroxylations for ethylbenzene activation. Ring dihydroxylations as the initial step for the investigated cultures capable of degradation of

approximately 99% ethylbenzene degradation; thus, the observed range of hydrogen isotope fractionation was similar to those observed for G. toluolica and recently for A. aromaticum. However, due to the relatively small carbon isotope fractionation, dual-isotope plots delivered a distinct pattern compared to the nitrate-reducers involved in ethylbenzene transformation: the anaerobic enrichment cultures Zz3 and DD-Anox2 produced extremely large Λ and ζ*(rp) values (Figure S3 and Table S4, Supporting Information). When using eq 4, regardless of whether xH = 1 assuming an enantiopure product or xH = 2 assuming a racemic product was applied, ζ*(rp) values were at least three times higher than those of the nitrate-reducing cultures (Table 1). Hence, the isotopic and metabolite pattern indicate a second activation mechanism initiating ethylbenzene degradation under anoxic conditions. Thus far, fumarate addition is the only known alternative activation mechanism for anaerobic ethylbenzene degradation, reported for the marine sulfate-reducing strain EbS7.33 Comparing the Λ or ζ*(rp) value of EbS7 to our enrichment cultures Zz3 and DD-Anox2 would have demonstrated whether fumarate addition and anoxic hydroxylation give significantly different isotope fractionation patterns. Unfortunately, EbS7 did not metabolize ethylbenzene in this study. The small carbon isotope fractionation factors, and the large Λ values do not fit with isotope data recently determined for fumarate addition of toluene and xylenes, both of which were associated with higher carbon isotope fractionation leading eventually to smaller Λ values than those observed for ethylbenzene degradation in this study.49,50 Nevertheless, in a few cases very low carbon enrichments factors between = −0.5‰ and −0.8‰ were reported for sulfate-reducing and methanogenic consortia capable of toluene and o-xylene degradation50,58,59 but never reaching hydrogen isotope fractionation of similar magnitude as that observed for Zz3 and DD-Anox2. However, Λ values for anaerobic fumarate addition were shown to be variable depending on the eco-physiology of the tested organisms,49,50 probably reflecting slightly different acting isoenzymes catalyzing the same reaction step.34 9128



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Figure 4. Traditional and alternative dual-isotope plots as a means to distinguish enzymatic ethylbenzene activation mechanisms. Two-dimensional Λ plot (Δδ2H/Δδ13C) shows data for δt − δ0 < 100‰ (note that for the cultures DD-Anox2 and Zz3 the range δt − δ0 < 200‰ was considered because not enough data points were available for the smaller fractionation interval). ζ*(rp) plot considers data over the whole ethylbenzene concentration range during biodegradation. Three characteristic ranges can be recognized for both dual-isotope plots: Λ and ζ*(rp) values with a very low slope (

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