Environ. Sci. Technol. 2008, 42, 4764–4770
Identifying Competing Aerobic Nitrobenzene Biodegradation Pathways by Compound-Specific Isotope Analysis T H O M A S B . H O F S T E T T E R , * ,† J I M C . S P A I N , * ,‡ S H I R L E Y F . N I S H I N O , ‡ JAKOV BOLOTIN,† AND ´ P. SCHWARZENBACH† RENE Institute of Biogeochemistry and Pollutant Dynamics, ETH Zurich, 8092 Zurich, Switzerland, and School of Civil & Environmental Engineering, 311 Ferst Drive N.W., Georgia Institute of Technology, Atlanta, Georgia 30332
Received January 11, 2008. Revised manuscript received March 19, 2008. Accepted March 25, 2008.
Nitroaromatic compounds that contaminate soil and groundwater can be biodegraded by different, sometimes competing reaction pathways. We evaluated the combined use of compoundspecific stable C and N isotope analysis to distinguish between enzymatic nitrobenzene oxidation by Comamonas sp. strain JS765 and partial reduction by Pseudomonas pseudoalcaligenes strain JS45 under aerobic conditions. Bulk 13C and 15N enrichment factors for nitrobenzene dioxygenation with JS765 were -3.9‰ ( 0.09‰ ((1σ) and -0.75‰ ( 0.09‰, respectively. The corresponding primary apparent kinetic isotope effects (AKIE) of 1.0241 ( 0.0005 for 13C and a secondary 15N AKIE of 1.0008 ( 0.0001 are in very good agreement with the proposed enzymatic addition of dioxygen to the aromatic ring to form a cis-dihydrodiol in the rate-limiting step of nitrobenzene degradation. For the partial reduction pathway with JS45, εC and εN values were -0.57‰ ( 0.06‰ and -26.6‰ ( 0.7‰. The 13C and 15N AKIEs amount to 1.0034 ( 0.0003 and 1.0273 ( 0.0008, respectively, and are consistent with the two-electron reduction and dehydration of the aromatic NO2 group to nitrosobenzene. The combined evaluation of δ13C and δ15N changes in nitrobenzene, based on the isotope enrichment behavior found in this laboratory study, provide an excellent starting point for assessing of the extent of nitrobenzene biodegradation via competing pathways in contaminated environments.
Introduction Nitroaromatic compounds (NACs) represent a major class of soil and groundwater contaminants owing to their widespread use as pesticides, dyes, explosives, and industrial feedstocks. Depending on the number and position of nitro groups and other aromatic substituents as well as on the prevailing environmental conditions, transformation of NACs occurs along different, sometimes competing enzymatic and abiotic reaction pathways (1, 2). Because some products of * Address correspondence to either author. E-mail:
[email protected] (T.B.H.);
[email protected] (J.C.S.). † ETH Zurich. ‡ Georgia Institute of Technology. 4764
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ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 42, NO. 13, 2008
NAC transformation, such as substituted hydroxylamines and anilines, are equally or even more toxic than the parent compounds, identification of the predominant transformation pathways including estimates of transformation rates are essential. While abiotic reactions of NACs primarily take place under anoxic conditions (3, 4), many NACs are susceptible to microbial degradation in the presence of oxygen (5). Bacteria employ several strategies to transform NACs in the presence of oxygen to key metabolites that are amenable to further degradation (Scheme 1 (5–10)). The nitro group can be released as nitrite following either dioxygenation of the aromatic ring to a substituted catechol (Scheme 1, pathway A) or via monooxygenation to an epoxide, which leads to a phenolic compound (pathway B). An alternative nitro group removal has been observed after nucleophilic addition of hydride to form a hydride-Meisenheimer complex (pathway C). Finally, bacteria can partially reduce some NACs via nitroso- and hydroxylaminobenzene intermediates. The nitro group is later eliminated as ammonia during reactions of substituted o-aminophenols (pathway D). Note that the biodegradation pathways shown in Scheme 1 are not generally applicable to any NAC (e.g., pathway B only works with phenols (7)) because interactions of the substrates with degradative enzymes depend specifically on the contaminant’s molecular structure (11).
SCHEME 1
Thus, identification of degradation pathways and quantitative estimates of the extent of transformation are key elements for assessing the risks caused by soil and groundwater contamination as well as for designing appropriate remediation measures. Because potential degradation products such as aromatic amines can also be primary contaminants (5), they cannot be used to trace degradation processes. Therefore, methods are needed that are able to reveal the predominant biotransformation mechanism based on information obtained from the contaminants found in situ. This information can be provided by compound-specific isotope analysis (CSIA), which has recently become available for δ13C and δ15N analysis of various NACs (12–15). Because the magnitude of stable isotope enrichment is determined by a kinetic isotope effect (KIE) pertinent to each aerobic NAC biodegradation mechanism and type of enzyme involved (e.g., mono- vs dioxygenases, reductases), one can expect an isotopic enrichment in the remaining substrate that is characteristic for each pathway. The bulk isotopic enrichment factors for 13C and 15N (εC and εN, respectively) for each degradation pathway quantify the observed enrichment and relate changes in isotope signature to the extent of biodegradation (16, 17). In addition, product isotope 10.1021/es8001053 CCC: $40.75
2008 American Chemical Society
Published on Web 05/29/2008
signatures from NAC biodegradation follow characteristic trends according to the reaction’s enrichment factors and, therefore, provide information to trace back their formation process(es) (13, 18). Process identification based on isotope fractionation has been applied predominantly in studies on the (bio)transformation of fuel constituents and chlorinated solvents. Examples include methyl tert-butyl ether (MTBE) transformation via oxidation and hydrolysis (16, 19, 20); degradation of chlorinated hydrocarbons involving oxidation, reduction, elimination, or hydrolysis reactions (21–24); and microbial oxidation of aromatic hydrocarbons (25). Analysis of isotope fractionation in NACs, however, has been focused predominantly on 15N fractionation during abiotic reduction under anoxic conditions. Such reactions include reduction of NACs to substituted anilines by surface-bound and structural Fe(II) species in iron-bearing minerals as well as by mercaptojuglone species acting as electron-transfer mediators in homogeneous solutions (13–15). The studies reported large15N enrichment factors, εN, between -30‰ and -40‰, which were independent of the type and position of aromatic substituents. The magnitude of isotope fractionation associated with NAC biodegradation and its variability for structurallydifferentcompoundsduetospecificenzyme-substrate interactions (11), however, has not yet been investigated. It was therefore the objective of this study to determine whether 13C and 15N fractionation during NAC biodegradation could provide the means to distinguish among competing transformation pathways under aerobic conditions. To this end, we investigated the isotope fractionation of nitrobenzene in two biodegradation pathways shown in Scheme 1 (pathways A and D): oxidation to catechol by Comamonas sp. strain JS765 (6) and partial reduction of nitrobenzene to hydroxylaminobenzene by Pseudomonas pseudoalcaligenes strain JS45 (10). Because in enzymatic reactions the actual bond cleavage might not be fully rate-determining due to preceding slow processes such as substrate binding to an enzyme or mass-transfer limitations, observed enrichment factors may not reflect the intrinsic KIE (26, 27). Therefore, bulk 13C and 15N enrichment factors can in some cases be smaller than expected. To deal with effects of such masking on pathway identification with CSIA, one can evaluate the simultaneous changes of δ13C and δ15N in nitrobenzene for the two processes. The strategy has been applied to various element pairs, for example, ∆δ2H/∆δ13C during MTBE transformation (16, 19, 20) and ∆δ18O/∆δ37Cl during perchlorate reduction (28), and provides avenues for the calculation of the extent of biodegradation for competing fractionation pathways by use of an extended formalism of the Rayleigh equation (29).
Experimental Section Biodegradation Experiments. Bacterial cultures were grown in modified nitrogen-free minimal medium (30) containing double the normal amount of MgSO4 · 7H2O (0.16 mM), succinate (3-10 mM), and nitrobenzene (0.5-1.0 mM) provided as the sole nitrogen source. Cultures were incubated at 30 °C, with shaking at 150 rpm. When the cultures became dense and the nitrobenzene was no longer detected, the cells were collected by centrifugation, suspended in fresh minimal medium with nitrobenzene (1 mM) as the sole carbon and nitrogen source, and incubated with shaking as before. Samples (20 mL) were filtered through a sterile aluminum oxide filter (Whatman Anodisc, 0.2 µm, 25 mm) in a stainless steel Swinney filter holder by use of a syringe pump (2 mL min-1). The first milliliter was discarded, the second milliliter was collected in a silanized autosampler vial, and the next 14 mL was collected in a 15 mL serum bottle containing sodium azide (14 µL, 20% azide in H2O). The serum bottles
were sealed with sterile Teflon-lined silicone septa and then stored at 4 °C until they were analyzed. Analytical Procedures. Concentrations of NACs were measured by HPLC on a LC-18 reverse-phase column with UV/vis detection (10, 31). Carbon and nitrogen isotope ratios were measured by the method described previously: solidphase microextraction (SPME) coupled to gas chromatography isotope-ratio mass spectrometry with a combustion interface (GC-C-IRMS (12)). All δ13C and δ15N values were derived from triplicate measurements with good precision (
