12C Fractionation Contrasts with Large Enantiomer

Article pubs.acs.org/est

Small 13C/12C Fractionation Contrasts with Large Enantiomer Fractionation in Aerobic Biodegradation of Phenoxy Acids Shiran Qiu,† Erkin Gözdereliler,†,§ Philip Weyrauch,† Eva C. Magana Lopez,† Hans-Peter E. Kohler,‡ Sebastian R. Sørensen,§ Rainer U. Meckenstock,† and Martin Elsner*,† †

Institute of Groundwater Ecology, Helmholtz Zentrum München, Ingolstadter Landstr. 1, 85764 Neuherberg, Germany Swiss Federal Institute of Aquatic Science and Technology (Eawag), Ü berlandstrasse 133, CH-8600 Dübendorf, Switzerland § Department of Geochemistry, Geological Survey of Denmark and Greenland (GEUS), Øster Voldgade 10, 1350 Copenhagen K, Denmark ‡

S Supporting Information *

ABSTRACT: Phenoxy acid herbicides are important groundwater contaminants. Stable isotope analysis and enantiomer analysis are well-recognized approaches for assessing in situ biodegradation in the field. In an aerobic degradation survey with six phenoxyacetic acid and three phenoxypropionic acid-degrading bacteria we measured (a) enantiomer-specific carbon isotope fractionation of MCPP ((R,S)-2-(4-chloro-2-methylphenoxy)propionic acid), DCPP ((R,S)-2-(2,4-dichlorophenoxy)-propionic acid), and 4-CPP ((R,S)-2-(4-chlorophenoxy)-propionic acid); (b) compound-specific isotope fractionation of MCPA (4-chloro2-methylphenoxyacetic acid) and 2,4-D (2,4-dichlorophenoxyacetic acid); and (c) enantiomer fractionation of MCPP, DCPP, and 4-CPP. Insignificant or very slight (ε = −1.3‰ to −2.0‰) carbon isotope fractionation was observed. Equally small values in an RdpA enzyme assay (εea = −1.0 ± 0.1‰) and even smaller fractionation in whole cell experiments of the host organism Sphingobium herbicidovorans MH (εwc = −0.3 ± 0.1‰) suggest that (i) enzyme-associated isotope effects were already small, yet (ii) further masked by active transport through the cell membrane. In contrast, enantiomer fractionation in MCPP, DCPP, and 4-CPP was pronounced, with enantioselectivities (ES) of −0.65 to −0.98 with Sphingomonas sp. PM2, −0.63 to −0.89 with Sphingobium herbicidovorans MH, and 0.74 to 0.97 with Delf tia acidovorans MC1. To detect aerobic biodegradation of phenoxypropionic acids in the field, enantiomer fractionation seems, therefore, a stronger indicator than carbon isotope fractionation.

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INTRODUCTION Phenoxy acids are herbicides that have been widely used in agriculture to control broad-leaved weeds since the mid-20th century. Among them, MCPA (4-chloro-2-methylphenoxyacetic acid), 2,4-D (2,4-dichlorophenoxyacetic acid), MCPP (2-(4-chloro-2-methylphenoxy)-propionic acid), and DCPP (2-(2,4-dichlorophenoxy)-propionic acid) are the most common herbicides (for structures, see Scheme 1). Together with 4-CPP (2-(4-chlorophenoxy)-propionic acid), a byproduct and impurity in commercial herbicide formulations, they are frequently detected in groundwater systems.1 It is therefore important to understand their fate and degradation in the environment. Phenoxy acid molecules are negatively charged and therefore highly mobile at circumneutral pH. In groundwater systems, they are nonvolatile and persistent to hydrolysis2 but can be biodegraded under both oxic3−6 and anoxic conditions (in the latter case usually less efficient).7,8 In particular for aerobic biodegradation, a considerable number of microorganisms have been isolated.9−12 The existence of a degradation potential in the environment could be demonstrated,13,14 and field © 2014 American Chemical Society

studies have given evidence of phenoxy acid degradation in groundwater.2,15 Nonetheless, it is still a challenge to unequivocally detect in situ biodegradation of phenoxy acids because compound concentrations alone are often not conclusive: they may decrease due to dilution and in groundwater mass balances are difficult to close. Hence, alternative approaches are needed for assessing biodegradation of phenoxy acids in the aquatic environment. One such independent approach for quantifying biodegradation is enantiomer analysis. This approach relies on the fact that phenoxypropionic acids such as MCPP and DCPP have a chiral center and consequently exist as two different enantiomers (R and S) (see Scheme 1). Some microorganisms show enantioselective microbial degradation.16 This means that one enantiomer is degraded faster than the other,17−21 either due to preferential microbial uptake or preferential enzyme activity (Figure 1).22 For example, the bacterium Received: Revised: Accepted: Published: 5501

November 18, 2013 April 2, 2014 April 7, 2014 April 7, 2014 dx.doi.org/10.1021/es405103g | Environ. Sci. Technol. 2014, 48, 5501−5511

Environmental Science & Technology

Article

Scheme 1. Chemical Structures and pKa Values56 of Important Phenoxy Acid Compounds, with a Reaction Scheme of the Aerobic Transformation Pathway by Fe(II)- and α-Ketoglutarate (αKG)-Dependent Dioxygenases23a

a

n.r.: not reported.

enantiomer [R] and the sum of both enantiomers [R + S], with values between 0 and 1:

Sphingobium herbicidovorans MH possesses a unique membrane transport system when consuming phenoxy acids as an energy source.18,23 A transporter protein first actively pumps phenoxy acid molecules across the cell membrane via a proton-driven gradient. Then, the substrate is enzymatically cleaved by an α-ketoglutarate-dependent dioxygenase into a phenol derivative and pyruvate (in the case of phenoxypropionic acids) or glyoxylate (in the case of phenoxyacetic acids). Both the transporter protein and the enzyme are enantiospecific (Figure 1).23,24 Selective enrichment of either enantiomer can, therefore, indicate the occurrence of biodegradation, even in the absence of mass balances.20 Two kinds of information are important in this context: (i) By how much is one enantiomer enriched relative to the other in a given sample? This information is typically given by the enantiomer fraction (EF),25 which is the ratio between one

EF =

[R ] [R + S]

(1)

The same information may be expressed by the enantiomer ratio (ER):25,26 ER =

[R ] EF = [S] 1 − EF

(2)

with values between 0 and infinity. (ii) What is the underlying enantiomer fractionation (equal to the preference of microorganisms to degrade one enantiomer compared to the other)? This information is expressed as 5502

dx.doi.org/10.1021/es405103g | Environ. Sci. Technol. 2014, 48, 5501−5511


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Figure 1. Enantiomer fractionation may be caused by either preferential uptake (Step 1) or preferential degradation (Step 2). To this end, enantiospecific transporters and enzymes are expressed in Sphingobium herbicidovorans MH.18,23 Pronounced isotope fractionation, in contrast, is primarily expected in the enzyme reaction, not during uptake.

enantioselectivity (ES) according to the following equation:27

ES =

kR − kS kR + kS

In such cases compound specific isotope analysis (CSIA) is an alternative for assessing biotransformation of organic contaminants in aquifers. The approach relies on the fact that all organic compounds are composed of different stable isotopes (e.g., 13C/12C) inside their structure. During biotransformation, molecules containing only light isotopes (e.g., 12C) usually react faster than those containing a heavy isotope (13C) in the reacting position.29−31 As a result, heavy isotopes are enriched in the remaining fraction of the compound, providing an independent line of evidence for biodegradation. Again, two kinds of information are of interest: (i) By how much is 13C enriched relative to 12C in a given sample? This information is obtained by measuring changes in compound-specific stable isotope ratios (e.g., 13C/12C) at natural abundance.32−35 Measured isotope ratios are generally expressed in the delta (δ) notation, for example, for carbon isotopes (13C, 12C):

(3)

where kR and kS are first-order rate constants for degradation of the R- and S-enantiomers, respectively. Very recent publications26,28 alternatively suggest the use of enantiomeric fractionation and enrichment factors: αER =

kR kS

εER = αER − 1

(4) (5)

They are related to the “traditional” definition of ES according to ES =

αER − 1 εER = αER + 1 εER + 2

13

(6)

13

( ) C C

−

12

δ 13C =

and may experimentally be determined according to ⎛ ER ⎞ ⎡ C (1 + ER 0) ⎤αER − 1 ⎡ C (1 + ER 0) ⎤εER =⎢ · ⎥ ⎥ ⎜ ⎟=⎢ · ⎣ C0 (1 + ER) ⎦ ⎝ ER 0 ⎠ ⎣ C0 (1 + ER) ⎦

sample

( ) C C

13

reference

13

( ) C 13 C

reference

(8)

where (13C/12C)sample and (13C/12C)reference are isotope ratios of the organic compound and the international reference material (V-PDB, Vienna Pee Dee Belemnite for carbon), respectively. (ii) What is the underlying isotope fractionation (equal to the preference of microorganisms to degrade molecules with one isotope compared to the other)? This information is given by the isotope enrichment factor (ε) of a specific compound, which may be determined in laboratory experiments based on the Rayleigh equation (the equivalent of eq 7 for isotopes):36,37

(7)

where C is the total concentration of both enantiomers ([R] + [S]) and subscripts “0” indicate initial values. Values of αER range between 0 to infinity, and εER between −1 and infinity. In contrast, ES values range from −1 to 1, where negative values correspond to the preferential degradation of the S-enantiomer and positive values correspond to the preferential degradation of the R-enantiomer. While definitions of ES, αER, and εER are equivalent and rely on the same physicochemical assumption (i.e., kR/kS is constant throughout the transformation), the ES definition appears most informative in practical terms and will therefore be used in this study. Limitations of enantiomer analysis are, however, (a) that the approach works only if microbial degradation is enantiospecific and (b) that it is not applicable to compounds such as phenoxyacetic acids like MCPA or 2,4-D that do not possess a chiral center.

⎛ δ13C + 1 ⎞ ⎜ 13 t ⎟ = fε ⎝ δ C0 + 1 ⎠

(7a)

where δ Ct and δ C0 are the carbon isotope values measured at time t and time 0, respectively; f is the fraction of the compound (substrate) remaining at time t; and ε is the isotope enrichment factor of biodegradation. 13

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Bacterial Strains and Biodegradation Studies. Sphingobium herbicidovorans MH (DSMZ, Braunschweig, Germany; DSM11019), Delftia acidovorans MC1, Sphingomonas sp. PM2, Cupriavidus basilensis sp. ERG4, and Sphingomonas sp. ERG5 were from our laboratory collection. Cupriavidus sp. ERG4 and Sphingomonas sp. ERG5 have recently been isolated from the MCPA-degrading bacterial communities described by Gözdereliler et al.41 Cupriavidus pinatubonensis JMP134 (DSMZ, Braunschweig, Germany; DSM 4058; formerly Ralstonia eutropha) was kindly provided by Dr. Mette H. Nicolaisen from University of Copenhagen. The strains were chosen because they (i) originate from herbicide-exposed environments,41,42 (ii) are all capable of growth-linked mineralization as found in environmental samples,42−44 and (iii) represent two important groups of phenoxy acid degraders (Groups I and III).45−47 Group I consists of β- and γ-subdivions of proteobacteria, which harbor tfdA-like genes,45 while group III consists of α-proteobacteria closely related to Sphingomonas-related organisms.48,49 The second group, which was not represented in this study, includes the cluster of α-proteobacteria belonging to the Bradyrhizobium-related organisms49,50 Prior to degradation experiments, all strains were grown in 200 mL of R2A-based broth51 overnight at 30 °C and 120 rpm. The strains were harvested during the late exponential phase and washed three times in sterile MSN medium41 before initiation of the experiments. Then, 250 mL sterile glass bottles equipped with screw caps and containing 200 mL of MSN medium were inoculated with 2 mL of washed cell suspensions having an optical density of 0.4 at 600 nm. They were supplemented with phenoxy acid stock solutions (as a sole source of carbon and energy) to a concentration of 0.2 mM and subsequently incubated at 20 °C in the dark. Each degradation experiment was conducted in duplicate where an additional uninoculated bottle served as control for abiotic loss of PAs and an additional bottle without PAs was included. For sampling, 8 mL samples were taken over time and transferred to amber flasks (10 mL) with metal screw caps, followed by the addition of 400 μL of HCl (0.75 M, pH < 2.5) for sample preservation. All samples were stored at 4 °C for analysis by HPLC and isotope measurements. Enzyme Purification and Assay. The enzyme α-ketoglutarate-dependent (R)-dichlorprop dioxygenase (RdpA) was purified from strain E. coli BL21 (DE3)(pLysS) [pET-15b::rdpA],24 which was pregrown at 30 or 37 °C in LB medium (10 g/L tryptone, 5 g/L yeast extract, 10 g/L NaCl, pH 7.4) containing 100 μg/mL ampicillin and 34 μg/mL chloramphenicol. Subsequently, E. coli BL21 (DE3)(pLysS) [pET-15b::rdpA] was grown on liquid LB medium with 100 μg/mL ampicillin and 34 μg/mL chloramphenicol. Incubation was conducted in Erlenmeyer flasks without baffles at 37 °C and 200 rpm until an optical density at 600 nm of 0.5 was reached. Then heterologous gene expression was induced by addition of 1 mM isopropyl-β-D-thiogalactopyranoside (IPTG), and incubation was continued at 20 °C, 200 rpm for 12 h. The cells were then harvested by centrifugation (4,000 × g, 15 min), and the cell pellets were stored at −20 °C. For the preparation of cell-free extracts, the pellets were resuspended in 3 mL of lysis buffer (50 mM Na-phosphate buffer pH 8.0 with 300 mM NaCl and 10 mM imidazole) per 1 g of biomass and sonicated for 3 × 3 min (cycle = 60, amplitude = 0.6). Subsequently, cell debris was pelleted by centrifugation (4,000 × g, 15 min), and the supernatant was applied to a His GraviTrap

Recently, enantiospecific isotope analysis (ESIA) has been brought forward as yet a further instrumental advancement. The method combines the advantages of both approaches, gas chromatographic enantiomer separation and online isotope measurement, and thereby enables the individual isotope analysis of enantiomers separately (in conventional GC separations, they would co-elute).38−40 First laboratory studies demonstrated the simultaneous occurrence of isotope and enantiomer fractionation.26,28 A first application to field samples from a phenoxy acid contaminated site indeed demonstrated pronounced enantiomeric and small but significant isotope fractionation indicating the occurrence of phenoxypropionic acid biodegradation in groundwater.39 However, such field data are difficult to interpret in quantitative terms when laboratory reference studies on isotope fractionation in phenoxy acid degradation are missing. Another prominent research gap that can be tackled with ESIA concerns the underlying drivers of enantiomer versus isotope fractionation. Figure 1 illustrates that (i) enantiomer fractionation can occur either through transporter-driven cell uptake or through enzymatic transformation, whereas (ii) isotope fractionation is primarily expected if chemical bonds are changed in the enzymatic transformation but not during cell uptake. Therefore, if the uptake were the rate-limiting step, enantiomer fractionation would be expected but not isotope fractionation; in contrast, if the enzyme reaction were the rate-limiting step, changes in both enantiomeric fraction and isotope ratios would be expected. Experiments with purified enzyme may allow observing the “intrinsic” isotope fractionation in the (enantioselective) enzyme reaction and assessing if the enzyme reaction or if uptake is rate-limiting in whole-cell biodegradation. The objectives of this study were therefore (i) to systematically investigate (enantiomer-specific) isotope fractionation in aerobic degradation of five different phenoxy acids (two phenoxy acetic, three phenoxy propionic acids) including a comprehensive number of phenoxy acid-degrading microorganisms that have been isolated so far: Sphingobium herbicidovorans MH, Sphingomonas sp. ERG5, Sphingomonas sp. PM2, Delftia acidovorans MC1, Cupriavidus pinatubonensis JMP134, and Cupriavidus basilensis sp. ERG4; (ii) to evaluate the enantioselectivity in degradation of the three phenoxy propionic acids; (iii) to investigate the intrinsic isotope effect in the enantioselective enzyme reaction (RdpA assay), and finally (iv) to assess the respective potential of assessing degradation of phenoxy acids in the field with enantiomer and stable isotope analysis.

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MATERIALS AND METHODS Chemicals. MCPA (4-chloro-2-methylphenoxyacetic acid) (99.8%, CAS No. 94-74-6), 2,4-D (2,4-dichlorophenoxyacetic acid) (99.8%, CAS No. 94-75-7), and MCPP ((R,S)-2-(4-chloro2-methylphenoxy)-propionic acid) (99.6%, CAS No. 7085-19-0) were supplied by Sigma-Aldrich (Germany). DCPP ((R,S)-2(2,4-dichlorophenoxy)-propionic acid) (99.0%, CAS No. 12036-5) was supplied by Dr. Ehrenstorfer GmbH (Germany) and Sigma-Aldrich (Germany), and 4-CPP (2-(4-chlorophenoxy)propionic acid) (CAS No. 307-39-9) was supplied by Aldrich Chemistry (Germany). Milli-Q water was generated with a Millipore Advantage A10 system (Millipore, Molsheim, France). Acetonitrile and n-hexane (>99.9%) were purchased from Carl Roth (Karlruhe, Germany). Acetic acid was purchased from Merck (Darmstadt, Germany). BF3 (10% in methanol) was purchased from Sigma-Aldrich (St. Louis, USA). 5504



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n/a n/a 1 n/a n/a ∞ n/a n/a ∞ n/a −1.3 ± 0.2g n/a n/a n/a n/a n/a +g n/a n/a

Errors given are standard deviations. Determined according to eq 8, where errors represent 95% confidence intervals. cDetermined according to eq 7. dDetermined according to eq 4. eDetermined according to eq 5; in all cases errors represent standard deviations. fSphingomonas sp. PM2 showed significant isotope fractionation in one batch whereas it was not significant in the replicate. gThe values given are for (R)-DCPP. n/s: not significant, changes in isotope values were below twice the uncertainty of the analysis (2‰). n/a: not available. ∞: infinitively large.

n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/s n/s − + +

−

−

n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/s −1.3 ± 0.2 − + +

−

−

n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/a n/s −1.3 ± 0.2 − + +

−

−

0.74 ± 0.12 0.97 ± 0.04 0.90 ± 0.02 6.6 ± 2.3 57 ± 32 20 ± 3 5.6 ± 2.3 56 ± 32 18.8 ± 3.3 n/s n/s n/s n/s n/s + + + +

+

n/s −1.2 ± 0.4 −0.83 ± 0.01 −0.94 ± 0.04 −0.77 ± 0.06 0.17 ± 0.01 0.06 ± 0.04 0.23 ± 0.06 −0.72 ± 0.02 −0.89 ± 0.08 −0.63 ± 0.08 n/s n/s n/s + + + + +

4-CPP MCPP DCPP 4-CPP MCPP DCPP 4-CPP MCPP DCPP 4-CPP MCPP DCPP

n/s −2.0 ± 0.3f

2,4D MCPA

n/s + + + + +

enantiomer fractionation factor (αER)d enantiomer enrichment factor (εER)c isotope enrichment factor ε (‰)b degradation

b a

The CO2 monitoring gas was calibrated in a Finnigan MAT Delta S isotope ratio mass spectrometer with dual inlet system (Thermo Fisher Scientific) against V-PDB by use of international reference materials (RM 8562, RM 8563, RM 8564, provided by the International Atomic Energy Agency). The monitoring

MCPA 2,4D DCPP MCPP 4-CPP

(13C/12C)standard

bacterial strain

(13C/12C)sample − (13C/12C)standard

Sphingomonas sp. PM2 Sphingomonas herbicidovorans MH Delf tia acidovorans MC1 Cupriavidus basilensis sp. ERG4 Cupriavidus necator JMP143 Sphingomonas sanxanigens sp. ERG5 pure enzyme (RdpA)

δ C=

Table 1. Summary of Compound Degradation and Associated Isotope and Enantiomer Fractionation for Targeted Compoundsa

13

enantioselectivity (ES)e

TALON gravity flow column (GE Healthcare, Buckinghamshire, U.K.) equilibrated in lysis buffer. Unbound protein was removed by three wash steps with 5 mL of wash buffer (50 mM Na-phosphate buffer pH 8.0, 300 mM NaCl, 20−100 mM imidazole). The concentration of imidazole was stepwise increased from 20 to 50 mM and finally to 100 mM. Target proteins were then eluted with 3 mL of elution buffer (50 mM Na-phosphate buffer pH 8.0, 300 mM NaCl, 250 mM imidazole) and desalted with 20 mM Tris-HCl (pH 7.5) with VIVASPIN columns (Sartorius Sedim Biotech, Gö ttingen, Germany). The purified RdpA solution was stored at 4 °C. Enzyme assays were conducted in 20 mL of 1 mM α-ketoglutarate, 0.2 mM RS-DCPP, 1 mM ascorbic acid, 0.1 mM (NH4)2Fe(SO4)2, 3.3 μg of purified RdpA, and 20 mM imidazole buffer (pH 6.75). Assays were done at 37 °C and 1.5 mL of aqueous samples was taken at each sampling time for concentration and isotope analysis. Samples were preserved by adding 40 μL of HCl (0.75 M) to stop the enzyme activity (pH < 2). Quantification with HPLC. Concentrations of MCPA, 2,4D, MCPP, DCPP, 4-CPP, and phenol were determined with a Shimadzu LC-10A series high-performance liquid chromatograph (HPLC) equipped with an Allure C18 column, 150 × 4.6 mm, 5 μm particle size (Restek, USA), and a UV−vis detector. The eluting solvents were acetonitrile with 1.25% acetic acid (solvent A) and deionized water with 1.25% acetic acid (solvent B). The gradient was composed of 30% solvent B (1 min) and was increased to 80% (2−10 min) and decreased to 30% (11−12 min). The flow rate was 0.7 mL min−1, and the oven temperature was 45 °C. The sample injection volume was 20 μL, and absorbance of all compounds was measured at 280 nm. Enantiomer concentrations of phenoxypropionic acids were determined from compound concentrations measured by HPLC in combination with enantiomer ratios obtained from GC-IRMS peak areas (see below). Carbon Isotope Analysis. Phenoxy acids in all aqueous samples were derivatized with BF3 and methanol prior to isotope analysis based on the procedure of Maier et al.38 Liquid samples (1−6 μL depending on the concentration of extracts) were then injected (splitless, at 230 °C) via a GC Pal autosampler (CTC, Zwingen, Switzerland) into a gas chromatograph connected to an isotope ratio mass spectrometer (GC−IRMS). The GC−IRMS consisted of a gas chromatograph (Thermo Fisher scientific, Milan, Italy) equipped with a DB5 column (60 m × 0.25 mm, 1 μm film; RESTEK, USA) when measuring phenoxyacetic acids and with a β-6TBDM column (50 m × 0.25 mm, 0.25 μm film; Macherey & Nagel, Düren, Germany) when measuring chiral phenoxypropionic acids. The GC program was as follows: 80 °C (1 min), ramped to 140 °C with a rate of 10 °C min−1, then with 1.5 °C min−1 to 185 °C, and then with 30 °C min−1 to 230 °C (held for 4 min). All analytes were then combusted online in a Finnigan GC combustion interface (Thermo Fisher Scientific, Bremen, Germany) to CO2 with a NiO tube/CuO−NiO reactor operated at 1000 °C (Thermo Fisher Scientific, Bremen, Germany). δ13C is reported relative to Vienna PeeDee Belemnite (VPDB):

n/s −1.0 ± 0.1 −0.99 ± 0.00 −0.98 ± 0.01 −0.79 ± 0.07 0.01 ± 0.00 0.02 ± 0.01 0.21 ± 0.07 −0.98 ± 0.00 −0.97 ± 0.01 −0.65 ± 0.10

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Figure 2. Degradation (a,b) and carbon isotope signatures (c−f) during aerobic degradation of MCPA (left panels) and 2,4D (right panels) using six bacterial strains: Cupriavidus basilensis sp. ERG4 (▼), Sphingomonas sp. PM2 replicate 1 (⧫), replicate 2 (◊), Sphingomonas herbicidovorans MH (●), Delftia acidovorans MC1 (▲), Cupriavidus necator JMP143 (■), Sphingomonas sanxanigens sp. ERG5 (⬢), and uninoculated control (×). The carbon isotope data were grouped such that the middle panel contains the cases of significant isotope fractionation and the bottom panel contains the cases where isotope fractionation was not significant. The data were fitted only if the changes in the isotope values were significant. Enrichment factors were extracted from curve fittings according to the Rayleigh equation and uncertainties for given ε are 95% CI. Error bars represent the standard deviations of triplicate measurements.

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gas was introduced at the beginning and the end of each run. Bias of derivatization was then corrected according to Maier et al.38 by comparison with reference values of phenoxy acid in-house standards (−28.7 ± 0.2‰ for MCPA, −28.6 ± 0.2‰ for MCPP, −27.3 ± 0.2‰ for DCPP, and −26.5 ± 0.2‰ for 4-CPP) determined in measurements by elemental analyzer (EURO EA, EuroVector Instruments, Milan, Italy) coupled to a Finnigan MAT 253 isotope ratio mass spectrometer (Thermo Fisher Scientific, Bremen, Germany). Calibration of EA-IRMS measurements was done by the following reference materials provided by the IAEA Vienna: IAEA 600, IAEA CH3 (Cellulose), IAEA CH6 (sucrose), and IAEA CH7 (polyethylene). Analytical uncertainty of carbon isotope measurements is typically ±0.5‰ and a shift of 2‰ is usually considered to be significant for field applications.35

RESULTS AND DISCUSSION

Microbial Degradation of Phenoxyacetic Acid versus Phenoxypropionic Acid Herbicides. Results of all experiments are summarized in Table 1. Whereas all six bacterial strains were able to grow on the phenoxy acetic acids MCPA and 2,4D (Figure 2, Supplementary Figure S1), only Sphingomonas sp. PM2, Sphingobium herbicidovorans MH, and Delf tia acidovorans MC1 could degrade and grow on the phenoxypropionic acids DCPP, MCPP, and 4-CPP (Figure 3, Supplementary Figure S2). Control batches showed no growth and no degradation, indicating that the degradation process was biotic. The following sections discuss the extent of enantiomer versus isotope fractionation during aerobic biodegradation. Phenoxyacetic acid herbicides, phenoxypropionic acid herbicides, and the herbicide impurity 4-CPP are discussed separately. 5506



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Figure 3. Enantiomeric analysis (a−c), enantiomeric fraction (d−f), and carbon isotope signatures (g−i) of DCPP (left panel), MCPP (middle panel), and 4-CPP (right panel) using three bacterial strains: Sphingomonas sp. PM2 (◆), Sphingomonas herbicidovorans MH (●), and Delftia acidovorans MC1 (▲). Filled symbols are the R-entiomers, and open symbols are the S-entiomers. For isotope ratios the data are the mean (n = 2). Error bars represent estimates of the standard deviation. For enantiomeric analysis, the data are shown individually for duplicate samples, and error bars reflect an estimated relative standard deviation of concentration measurements of 10%.

Enantiomer Fractionation and Enantiomer-Specific Isotope Ratios of the Phenoxypropionic Acid Herbicides MCPP and DCPP. Phenoxy propionic acids contain a chiral center so that both enantiomer as well as isotope fractionation may occur. With all three strains, however, significant carbon isotope fractionation of MCPP and DCPP was observed neither for the S-enantiomer nor for the R-enantiomer (Figure 3). In contrast, enantiomer fractionation occurred, where the S-enantiomers of DCPP and MCPP were preferentially degraded by the strains Sphingomonas sp. PM2 (ESPM2 = −0.98 ± 0.00 and −0.97 ± 0.01, respectively) and Sphingobium herbicidovorans MH (ESMH = −0.72 ± 0.02 and −0.89 ± 0.08, respectively), whereas the R-enantiomers were preferred by the strain Delftia acidovorans MC1 (ESMCl = 0.90 ± 0.02 and 0.97 ± 0.04, respectively). While the enantioselectivity for all compounds with all strains was significant, strain MH expressed slightly weaker enantiomeric preference compared to strains PM2 and MC1. The preferential degradation of R-enantiomers in Sphingobium herbicidovorans MH is consistent with reports that the S-specific catabolic enzyme SdpA seems to be constitutively expressed in this

Compound-Specific Isotope Fractionation of the Phenoxyacetic Acid Herbicides MCPA and 2,4-D. In contrast to phenoxypropionic acids, the acetic acids do not have a chiral center and enantiomer fractionation cannot take place. In contrast, stable isotope fractionation may occur. 13C/12C isotope values of MCPA and 2,4-D during degradation of a selection of bacterial strains are shown in Figure 2 (c and d). Isotopic fractionation of MCPA and 2,4-D was not significant in most experiments (Figure 2, e and f) meaning that changes in isotope values were below twice the uncertainty of the analysis (2‰).37,38 Exceptions were degradation of MCPA with ERG4 and PM2 where small but significant isotope fractionation was observed (changes by overall 2−3‰, enrichment factors of −1.3 ± 0.2‰) (Figure 2, c and d). 2,4-D degradation by Sphingomonas sp. PM2 showed significant isotope fractionation in one batch (εPM2‑rep1 = −2.0 ± 0.3‰), whereas fractionation was not significant in the replicate. (A tentative explanation is given below in the section Implications for Mass Transfer into Cells.) Overall, isotope fractionation in phenoxy acetic acids was, therefore, small, if significant at all. 5507



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which is released into the environment as impurity (up to 7%)15 of commercial propionic acid herbicides. In analogy to MCPP and DCPP, it was again (S)-4-CPP that was preferentially degraded by strains PM2 and MH, whereas (R)-4-CPP was preferred by strain MC1 (Figure 3, panel f). ES values (ESPM2 = −0.65 ± 0.10, ESMH = −0.63 ± 0.08, ESMCl = 0.74 ± 0.12) of 4-CPP degradation, however, were found to be generally smaller than observed for the other two compounds. In contrast to MCPP and DCPP, small carbon isotope fractionation was observed in both enantiomers of 4-CPP during degradation by the strains Sphingomonas sp. PM2 (ε = −1.0 ± 0.4‰) and Sphingobium herbicidovorans MH (ε = −1.2 ± 0.1‰), but there was still no observable isotope effect in 4-CPP during degradation by Delftia acidovorans MC1. Comparison of Enantiomer-Specific Isotope Fractionation of (R)-DCPP in an Enzyme Assay versus Whole Cell Experiments with Sphingobium herbicidovorans MH. An enzyme assay was conducted with RdpA from Sphingobium herbicidovorans MH, which specifically degrades (R)-DCPP, producing phenol and pyruvate. Figure 4a shows that a decrease of (R)-DCPP and a corresponding increase of phenol concentrations were observed, whereas (S)-DCPP concentrations did not change. Figure 4b further demonstrates that this degradation was accompanied by small isotope changes (about 2.5‰) in (R)-DCPP, whereas isotope values of (S)-DCPP, which was not transformed, remained constant. Notably, the values of (R,S)-DCPP illustrate that isotope fractionation in (R)-DCPP would not have been observed with conventional compoundspecific isotope analysis illustrating the necessity of enantiomerspecific isotope analysis. Figure 4c, finally, compares isotope fractionation of (R)-DCPP by RdpA enzyme degradation to whole cell degradation of the host organism Sphingobium herbicidovorans MH. In the enzyme reaction small but significant isotope fractionation was observed (ε = 1.0 ± 0.1‰). In contrast, carbon isotope fractionation associated with degradation by whole cells was too small to be significant. If a Rayleigh regression is conducted nevertheless, a significantly smaller fractionation was determined (ε = 0.3 ± 0.1‰). The greater observable changes in isotope ratios indicate that a masking effect may exist on the observed fractionation of DCPP degradation by strain MH due to an active transport mechanism into the cell over the membrane (see Figure 1). The apparent kinetic isotope effect (AKIE) of (R)-DCPP during enzymatic degradation can be calculated based on the formula53,54 Figure 4. Enantiospecific enzyme (RdpA) assay of DCPP. Concentrations of (R)-DCPP (■), (S)-DCPP (□), phenol produced (▲), and the sum of all species (◆). (b) Carbon isotope values of (R)-DCPP (■), (S)-DCPP (□), and the weighted average of both, (R,S)-DCPP (▲), as the enantiomer fraction EF (⧫) decreases. (c) Changes in carbon isotope values of (R)-DCPP during pure enzyme (RdpA) degradation (▼) compared to degradation by whole cells of the host organism Sphingobium herbicidovorans MH (●). Error bars represent estimated standard deviations of carbon isotope analysis, and dashed lines represent 95% confidence intervals of a regression according to the Rayleigh equation (eq 8).

AKIE ≈

1 n × (εaverage) + 1

(5)

where n is the number of atoms of one element in the molecule. The resultant AKIE (1.009 ± 0.0009) was small, which coincides with values frequently observed for C−H bond oxidation in biodegradation.42 Such a C−H bond oxidation is expected in the α-ketoglutarate-dependent dioxygenase leading to a hemiacetal that is known to readily decompose to 2,4-dichlorophenol and glyoxylate.55 Implications for Mass Transfer into Cells. The significant difference between isotope fractionation in the enzyme assay in comparison to whole cell experiments indicates that active transport across the cell membrane is to a significant extent ratedetermining and becomes the reason for the absence of isotope fractionation. Figure 1 illustrates the underlying mechanism. If the enzyme reaction is rate-determining, molecules can get out of the cell by passive diffusion. In contrast, if transport is slow,

strain,52 whereas RdpA needs to be induced. Preferential degradation of S-enantiomers in Delf tia acidovorans MC1, on the other hand, agrees with the high activity of the R-enantiomer-specific enzyme reported in this strain.53 Enantiomer Fractionation and Enantiomer-Specific Isotope Fractionation of the Herbicide Impurity 4-CPP. In contrast to the herbicides MCPP and DCPP, whose degradation has been subject of previous investigations,5,6,13,20 this study is the first to investigate also degradation of 4-CPP, 5508



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negligible carbon isotope fractionation was observed in aerobic biodegradation, more significant isotope changes in anaerobic degradation would provide a particularly strong evidence for this route of degradation. First indications for such isotope fractionation under anaerobic conditions are given by a recent field study.39 Further insight on the applicability of isotope versus enantiomer fractionation can, therefore, be expected from future research on anaerobic degradation of phenoxy acids.

but the enzyme reaction is comparatively fast, hardly any molecules escape from inside of the cell (where the isotope effect of the enzyme reaction occurs) to outside of the cell (where we measure) to make the (small) isotope effect of the enzyme reaction visible.54 Consequently, the isotope fractionation of the enzyme is masked in the whole cell experiments. These findings also allow considering the results from whole cell experiments with other strains from a new angle. A similar extent of isotope fractionation compared to the enzyme assay was observed in degradation of 4-CPP by strains Sphingomonas sp. PM2 and Sphingobium herbicidovorans MH (Figure 3), showing that here the rate-limiting step is likely to be the enzyme reaction. A similar situation is suggested by the observation of significant isotopic fractionation for MCPA degradation by strains ERG4 and JMP143. The observation, finally, that 2,4-D degradation by Sphingomonas sp. PM2 showed significant isotope fractionation in one batch (εPM2‑rep1 = −2.0 ± 0.3‰) but not in the replicate may be an indication of different rate limitations (uptake vs enzyme reaction) in the two replicates. Our findings therefore delineate an innovative approach to experimentally distinguish different bottlenecks of biodegradation: (i) mass transfer from bulk solution to the outside of the cell, (ii) uptake into the cell by transport, and (iii) enzyme reaction inside the cell. In the first scenario, one would expect neither enantiomer nor isotope fractionation since biodegradation is limited by mass transfer, which is practically not fractionating for enantiomers or isotopes. In the second scenario, only enantiomer fractionation is expected, whereas isotopic fractionation is masked by membrane transport. In the third scenario, both enantiomer and isotope fractionation would be expected because the enzyme reaction may show fractionation in both enantiomers and isotopes. Implications for Field Assessment of Phenoxy Acids. A major objective of this study was to explore the extent of isotope versus enantiomer fractionation during phenoxy acid degradation and to investigate which line of evidence is more indicative in the field. Our results showed that aerobic biodegradation of phenoxy acids brought about only very slight or negligible carbon isotopic fractionation with all strains investigated. Enantiomer fractionation, in contrast, was found to be much more pronounced and may, hence, be a stronger indicator to detect aerobic biodegradation of phenoxy propionic acids. If estimates of biodegradation are warranted, one could further use the ES determined in this study to quantitatively estimate biodegradation based on the following equation brought forward by Reitzel:20

■

Figures of growth curves of bacterial strains and regressions to determine enantiomer enrichment factors (and subsequently enanatioselectivities according to eqs 6 and 7). This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +49 (0)89 31872565. Fax: +49 (0)89 31873361. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS S.Q. and E.G. contributed equally to this work. This study was supported by the Seventh Framework Program (2007-2013) of the European Commission within the GOODWATER Marie Curie Initial Training Network (grant no. 212683), by the Helmholtz Research Platform for the Integrated Assessment of Solute Fluxes and Processes in the Regional Water Cycle, and by the DFG (German National Science Foundation) within the priority program SPP 1315. We thank three anonymous reviewers for helpful comments and suggestions that further improved the quality of the manuscript.

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REFERENCES

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B (%) = ⎛ −1/2( 1 + ES )⎞⎞ ES ⎜ EF0 ⎛ EF0(1 − EF) ⎞ ⎟⎟ ×⎜ 1−⎜ ⎟ ⎟⎟⎟⎟ × 100 ⎜ EF ⎝ EF(1 − EF0) ⎠ ⎝ ⎠⎠

ASSOCIATED CONTENT

S Supporting Information *

(6)

where B is the extent of biodegradation, and EF0 and EF are the enantiomeric fraction at time 0 and time t. Situations where isotope fractionation may nonetheless deliver crucial information in the field, however, are (i) the degradation of 2,4-D and MCPA, where no enantiomers exis, and (ii) simultaneous degradation of, for example, 4-CPP by Sphingobium herbicidovorans MH and Delftia acidovorans MC1. Since enantioselectivities of both strains are opposite, the effects may cancel out, in which case isotope fractionation could still remain as conclusive evidence. Finally, the results of our study lay a basis for further investigations of isotope versus enantiomer fractionation in anaerobic biodegradation of phenoxy acids. Since only small or even 5509



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12C Fractionation Contrasts with Large Enantiomer

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