Environ. Sci. Technol. 2006, 40, 7675-7681
Effects of Trace Element Concentration on Enzyme Controlled Stable Isotope Fractionation during Aerobic Biodegradation of Toluene SILVIA A. MANCINI,† SARAH K. HIRSCHORN,† MARTIN ELSNER,‡ GEORGES LACRAMPE-COULOUME,† BRENT E. SLEEP,§ ELIZABETH A. EDWARDS,| AND B A R B A R A S H E R W O O D L O L L A R * ,† Stable Isotope Laboratory, Department of Geology, University of Toronto, 22 Russell Street, Toronto, Canada M5S 3B12, Institute of Groundwater Ecology, GSF-National Research Center of Environment and Health, Neuherberg, Germany, and Department of Civil Engineering and Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Canada
The effects of iron concentration on carbon and hydrogen isotopic fractionation during aerobic biodegradation of toluene by Pseudomonas putida mt-2 were investigated using a low iron medium and two different high iron media. Mean carbon enrichment factors (C) determined using a Rayleigh isotopic model were smaller in culture grown under high iron conditions (C ) -1.7 ( 0.1‰) compared to low iron conditions (C ) -2.5 ( 0.3‰). Mean hydrogen enrichment factors (H) were also significantly smaller for culture grown under high iron conditions (H ) -77 ( 4‰) versus low iron conditions (H ) -159 ( 11‰). A mechanistic model for enzyme kinetics was used to relate differences in the magnitude of isotopic fractionation for low iron versus high iron cultures to the efficiency of the enzymatic transformation. The increase of carbon and hydrogen enrichment factors at low iron concentrations suggests a slower enzyme-catalyzed substrate conversion step (k2) relative to the enzyme-substrate binding step (k-1) at low iron concentration. While the observed differences were subtle and, hence, do not significantly impact the ability to use stable isotope analysis in the field, these results demonstrated that resolvable differences in carbon and hydrogen isotopic fractionation were related to low and high iron conditions. This novel result highlights the need to further investigate the effects of other trace elements known to be key components of biodegradative enzymes.
Introduction Biodegradation of groundwater pollutants, such as benzene, toluene, ethylbenzene, and xylene (BTEX), is an important * Corresponding author phone: (416)978-0770; fax: (416)978-3938; e-mail:
[email protected]. † Stable Isotope Laboratory, Department of Geology, University of Toronto. ‡ GSF-National Research Center of Environment and Health. § Department of Civil Engineering, University of Toronto. | Department of Chemical Engineering and Applied Chemistry, University of Toronto. 10.1021/es061363n CCC: $33.50 Published on Web 11/17/2006
2006 American Chemical Society
mechanism of natural attenuation in contaminated aquifers (1) and is the only attenuation process resulting in benign end products. However, subsurface sediments can be highly heterogeneous (1), and factors such as the availability of nutrients and appropriate electron acceptors may limit biodegradation potential. Recently, compound specific isotope analysis (CSIA) was successfully used to distinguish between the effects of nondegradative processes of mass loss such as sorption, volatilization, and dilution and those of biodegradation for aromatic hydrocarbons in the field (2, 3). CSIA works on the principle that different isotopes of the same element, such as 12C, 13C (carbon) and 1H, 2H (hydrogen), react at slightly dissimilar rates, resulting in a change in the heavy to light isotope ratio of the remaining substrate, known as a kinetic isotope effect. Laboratory studies investigating isotopic fractionation of aromatic hydrocarbons during processes such as volatilization (4, 5), dissolution (5-7), and sorption (4, 8, 9) showed no discernible isotopic fractionation under equilibrium conditions outside of the analytical uncertainty typical for CSIA ((0.5‰ for carbon isotopes; (5‰ for hydrogen). In contrast, during biodegradation of aromatic hydrocarbons, the remaining contaminant becomes significantly enriched in the heavier isotopes (13C and 2H) resulting in a less negative δ13C or δ2H value compared to the original isotopic value (10-13), where δ is the heavy/light isotope ratio measured in a sample (Rs) relative to an international standard (Rr) (δ ) (Rs/Rr-1)×1000). Isotopic fractionation during biodegradation of toluene under aerobic conditions is well documented in the literature for pure cultures and is known to vary as a function of biodegradation pathway. Carbon isotopic enrichment factors (c’s) calculated using a Rayleigh isotopic model (14) ranged from -0.4 to -3.3‰ for three different aerobic toluene degrading pure cultures (11, 12, 15). The largest c of -3.3 ( 0.3‰ was determined for Pseudomonas putida mt-2 which uses a methyl monooxygenase reaction mechanism and involves a C-H bond breakage on the methyl group of toluene in the initial enzymatic step (12). In contrast, aerobic toluene biodegradation by Ralstonia pickettii strain PKO1 produced a c of -1.1 ( 0.2‰. Ralstonia pickettii strain PKO1 degrades toluene by a ring monooxygenase reaction, which hydroxylates a carbon atom within the aromatic ring and does not involve a distinct C-H or C-C bond breakage within the initial degradation step (12). Pseudomonas putida F1 produced a c of -0.4 ( 0.3‰ (12). For this organism, biodegradation occurs by a ring dioxygenase reaction whereby toluene is oxidized to a cis-dihydrodiol and no accompanying C-C or C-H bond breakage occurs (12). Morasch et al. (12) documented a similar pattern for hydrogen isotopic fractionation consistent with the effects of three different biodegradation pathways and initial enzymatic reactions, where P. putida mt-2 exhibited the largest isotopic fractionation and P. putida F1 exhibited the smallest. In a review on isotope effects during enzyme-catalyzed reactions (16), it was theoretically demonstrated that changes in enzymatic activity during the initial degradation step for an enzymatic reaction can also cause variation in the expression of the isotope effect. Factors known to effect enzymatic activity include temperature (11), pH, ionic activity, and trace element concentration (17). A study by Dinkla et al. (17) showed that during aerobic toluene biodegradation by P. putida mt-2, there was a loss of enzymatic activity of methyl monooxygenase under low iron conditions, suggesting that iron is an important parameter in the oxidative breakdown of toluene. The methyl monooxygenase enzyme consists of an oxygenase component, XylM, and an electronVOL. 40, NO. 24, 2006 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Schematic of the experimental design and setup for experiments I and II. Experiments were conducted in 250-mL glass bottles sealed with PTFE mininert valves. Iron concentrations are described in terms of a ratio of moles of iron to toluene (e.g. [Fe]/ [Tol]) according to Dinkla et al. (17). In experiment I, all bottles were inoculated with 1 mL of the parent culture Pseudomonas putida mt-2 grown in medium I (MI). In experiment II, 1 mL of the parent culture was first transferred to bottles containing MI, monitored over two degradation curves, and then transferred to bottles containing medium II (MII). All cultures were amended with 10 µL of neat toluene. transfer component, XylA, which was shown to be irondependent (18). By varying growth conditions from ironrich to iron-limited (
