Environ. Sci. Technol. 2009, 43, 7205–7211
Sorption to Humic Acids Enhances Polycyclic Aromatic Hydrocarbon Biodegradation KILIAN E.C. SMITH,† MARTIN THULLNER, LUKAS Y. WICK, AND HAUKE HARMS* Department of Environmental Microbiology, UFZ - Centre for Environmental Research, Permoserstrasse 15, 04318, Leipzig, Germany
Received December 23, 2008. Revised manuscript received March 12, 2009. Accepted March 20, 2009.
The hypothesis that humic acids (HA) can act as carriers of polycyclic aromatic hydrocarbon (PAH) compounds and provide bacteria with PAH fluxes beyond those obtained by diffusion of the water-dissolved fraction of these poorly soluble chemicals was tested. Controlled degradation experiments using microcrystalline phenanthrene and Sphingomonas sp. LH162 were performed without HA and in the presence of two HA concentrations. In the absence of HA, phenanthrene was steadily present at the maximum dissolved concentration, indicating that the phenanthrene dissolution flux compensated for the consumption. This was confirmed by dissolution experiments where phenanthrene dissolved roughly an order of magnitude faster than it was consumed in the degradation experiments. Interestingly, the presence of HA further increased the rates of phenanthrene degradation by factors up to 4.8 in an HA concentration-dependent manner. This can onlybeinterpretedbyanHA-mediatedtransportofphenanthrene to the cells, supplementing diffusive uptake from the freely dissolved phase. It is proposed that HA-sorbed phenanthrene is released directly to the cells upon their interaction with HA aggregates, increasing the total phenanthrene flux and also degradation.
Introduction Polycyclic aromatic hydrocarbons (PAHs) are pollutants of many natural habitats, including the marine environment. Here, as a result of their toxic properties (1, 2) and potential for food chain accumulation (3, 4), they raise concerns about human and ecosystem health. Although PAHs are subject to a range of removal processes, including abiotic losses by volatilization or photo-oxidation, biodegradation plays a dominant role. Nevertheless, despite the presence of bacteria possessing the metabolic capacity to degrade PAHs, these contaminants persist at significant levels. In part, this results from their low bioavailability, i.e., they exist in forms or locations that bacteria are unable to access (5, 6). PAH biodegradation depends on the total mass flux to the degrading cells (7). Common to all degrading bacteria is * Corresponding author phone: +49 341 2351260; fax: +49 341 2351351; e-mail:
[email protected]. † Current address: Department of Environmental Chemistry and Microbiology, National Environmental Research Institute - University of Aarhus, Frederiksborgvej 399, P.O. Box 358, 40000 Roskilde, Denmark. 10.1021/es803661s CCC: $40.75
Published on Web 04/10/2009
2009 American Chemical Society
uptake of dissolved PAHs, diffusing from the surrounding aqueous phase toward the cells (8, 9). Here, the freely dissolved concentrations determine the bioavailability, and low freely dissolved concentrations can result in slow degradation (6). The inherent properties of PAHs, primarily low water solubility and high hydrophobicity, cause them to be predominantly sorbed such that freely dissolved concentrations are low. Furthermore, should the freely dissolved reservoir become depleted because of biodegradation, kinetically limited desorption or entrapment in, for example, solid matter micropores, limit replenishment (10). Much of the PAHs thus remain inaccessible and persist over long time periods (5). An increasing body of literature indicates that other transport pathways can provide additional PAHs to the degrading cells. Most evidence comes from degradation kinetics in the presence of artificial and natural surfactants (11, 12). Dependent on the type of surfactant and bacterium, a fraction of the micellar PAH appears to be directly bioavailable to the bacterial cells without needing to first desorb into the freely dissolved phase. This has been attributed to the interaction of the surfactant micelles with the cell surface, followed by the direct release of intercalated PAHs (13, 14). Interestingly, other mobile PAH sorbing matrices such as humic acids (HA) or macromolecular bioemulsifiers also result in faster than expected PAH degradation, again suggesting additional mass transfer routes (15-18). A possible role of HA in the supply of sorbed PAHs is particularly exciting in view of their ubiquity in aquatic and porewater environments (19). While HA can lower biodegradation by reducing the freely dissolved PAH concentrations via sorption, they also may act as PAH carriers. However, uncertainties remain as to which of these two mechanisms dominates under natural conditions. This study was conceived to test the hypothesis that HAsorbed PAHs provide a mass transport pathway acting in parallel to freely dissolved phase diffusion. Phenanthrene was selected as a model PAH because its intermediate aqueous solubility (0.823 mg L-1 (20)) and hydrophobicity (log KOW 4.57 (20)) result in significant sorption to HA, while still leaving a detectable component in the dissolved phase. Phenanthrene-degrading Sphingomonas LH162 was used together with a commercial HA in a saline medium to mimic marine conditions. Solid phase microextraction (SPME) was used to monitor the freely dissolved and sorbed phenanthrene during degradation experiments.
Materials and Methods Reagents and Materials. The composition of the saline mineral medium used is given in Table S1of the Supporting Information. Phenanthrene was obtained from Fluka (purum >97.0% GC, Germany), acenaphthylene-d8 and phenanthrene-d10 from Dr. Ehrenstorfer (99.5% purity, Germany), dimethyl sulfoxide (DMSO) from Merck (p.a., Germany), and sulfuric acid from Merck (95-98%). HA sodium salt was obtained from Sigma-Aldrich (technical grade, Germany) and used without further purification. The measured carbon fraction of the HA of 0.452 g C g-1 (Figure S2 of the Supporting Information) was in the range reported for this particular HA (52.5%, after correcting for ash content (21)). An HA stock solution (concentration 20.78 g L-1) was prepared by dissolving the HA salt in 1 M NaOH (Titrisol, Merck, Germany) and neutralizing this with HCl (25% GR for analysis, Merck) to pH 7, before adding a saline mineral medium to the required volume. All solvents were of analytical grade (Merck). VOL. 43, NO. 19, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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Water was purified using a Milli-Q water purification system (Millipore, U.S.A.). PDMS-coated glass fibers (Polymicro Technologies, Inc., Phoenix, AZ) were obtained with a PDMS coating thickness of 30-31 µm and a PDMS volume of 13.55 ( 0.02 µL m-1. These were cut into 1 cm lengths and cleaned by shaking overnight twice each in MeOH and Milli-Q water before storing in Milli-Q water until use. Kimtech tissue paper (Kimberly-Clark, Germany) was used for harvesting the bacteria. Glassware was baked out at 450 °C for 6 h prior to use and/or solvent rinsed. Bacterium, Cultivation, and Harvesting. Sphingomonas sp. LH162 (22) was grown in a saline mineral medium supplemented with 2 g L-1 of phenanthrene crystals as the sole source of carbon and energy. Prior to use, a sample of the culture was plated on LB agar plates to check colonies for contamination. Cells were harvested after exactly 7 days in a phase of active growth. The culture was filtered through tissue paper to remove remaining crystalline phenanthrene and washed with three cycles of centrifugation at 10000 g for 15 min. Finally, the cells were resuspended in a small volume of saline mineral medium and used as the inoculum for the experiments. The carbon content of an LH162 culture with an OD578 of 0.27 was 0.137 g C L-1 (RSD ) 2.4%, n ) 3). Sphingomonas sp. LH162 has been shown to produce no biosurfactants (23). Analytical Methods. Freely Dissolved and Sorbed Phenanthrene Analysis. Equilibrium nondepletion (nd)-SPME analysis was performed using disposable PDMS-coated glass fibers based on Mayer et al. (24). Ten µg phenanthrene-d10 in methanol was spiked into the sample vial as an internal standard (volume of methanol, 0.3%). A 1 cm length of SPME fiber was added, and the vials were shaken for at least 12 h at 120 rpm on a horizontal shaker at 22 °C and under laboratory lighting. Under these conditions, 5 h was sufficient for partitioning equilibrium. The SPME fiber was removed and placed in 200 µL of toluene, and 1 µg of acenapthylened8 was added as an injection standard before shaking overnight to re-extract the SPME-sorbed phenanthrene into the toluene. A second extraction with 200 µL of fresh toluene yielded concentrations below detection limits. Freely dissolved concentrations were calculated from a seven-point external calibration performed in triplicate. Fifty mL of a saline mineral medium was spiked with increasing amounts of phenanthrene in methanol (range, 10-500 µg L-1; volume of methanol,
