Environ. Sci. Technol. 1999, 33, 2571-2578
Kinetics of Degradation of Surfactant-Solubilized Fluoranthene by a Sphingomonas paucimobilis P I A A . W I L L U M S E N * ,† A N D E R I K A R V I N ‡ Department of Marine Ecology and Microbiology, National Environmental Research Institute, P.O. Box 358, Frederiksborgvej 399, DK-4000 Roskilde, Denmark, and Department of Environmental Science and Engineering, The Technical University of Denmark, Lyngby, Denmark
To achieve a better quantitative understanding of the stimulating or inhibiting effect of surfactants on the metabolism of polycyclic aromatic hydrocarbons (PAHs), a biodegradation model describing solubilization, bioavailability, and biodegradation of crystalline fluoranthene is proposed and used to model experimental data. The degradation was investigated in batch systems containing the PAH-degrading bacterium Sphingomonas paucimobilis strain EPA505, the nonionic surfactant Triton X-100, and a fluoranthene-amended liquid mineral salts medium. Surfactant-enhanced biodegradation is complex; however, the biodegradation model predicted fluoranthene disappearance and the initial mineralization well. Surfactant-amendment did increase fluoranthene mineralization rates by strain EPA505; however, the increases were not proportional to the rates of fluoranthene solubilization. The surfactant clearly influenced the microbial PAH metabolism as indicated by a rapid accumulation of colored products and by a surfactantrelated decreased in the overall extent of fluoranthene mineralization. Model estimations of the bioavailability of micelle-solubilized fluoranthene, the relatively fast fluoranthene disappearance, and the accumulation of extracellular compounds in the degradation system suggest that low availability of micellar fluoranthene is not the only factor controlling surfactant-enhanced biodegradation. Also factors such as the extent of accumulation and bioavailability of the PAH metabolites and the crystalline solubilization rate in the presence of surfactants may determine the overall effect of surfactant-enhanced biodegradation of high molecular weight PAHs.
Introduction The molecular structure of two or more fused benzene rings (polycyclic aromatic hydrocarbons, PAHs) are formed whenever organic substances are exposed to high temperatures (pyrolysis) (1). The most significant sources of PAHs in soil and groundwater are generally recognized as contamination from anthropogenic activities, such as disposal of hazardous waste, spillage of oil and coal tar products, or combustion of fossil fuels. The PAHs are considered hazardous because * Corresponding author phone: +45 46301200; fax: +45 46301114; e-mail:
[email protected]. † National Environmental Research Institute. ‡ The Technical University of Denmark. 10.1021/es981022c CCC: $18.00 Published on Web 06/18/1999
1999 American Chemical Society
of cytotoxic, mutagenic, and carcinogenic effects (2, 3). The fate of these compounds in the environment and the remediation of contaminated sites are therefore of high public interest. The aromatic structure and other chemical characteristics of the PAH molecules make these compounds highly hydrophobic, with relatively low aqueous solubilities and with a strong tendency to sorb to particulate organic surfaces or to be dissolved in oily phases at contaminated sites (4, 5). A wide variety of bacteria, fungi, and algae can metabolize the PAHs (3, 4, 6, 7). However, one of the major problems currently limiting technology development for bioremediation is the issue of bioavailability (7). Chemically or biologically produced surface-active compounds (surfactants) have been reported to enhance solubilization of PAHs (8, 9). Introduction of surfactants to a contaminated environment may thus improve biological removal techniques (10, 11). However, inhibitory as well as stimulating effects of surfactants in PAH degradation processes have been reported (12). Possible explanations for surfactant-related inhibition of PAH degradation include decreased bioavailability of micelle-solubilized PAH (1315), surfactant-mediated cell toxic effects, e.g., partly or complete permeabilization of cell membranes (16-18), or surfactant interference with cellular processes, e.g., inhibition of enzymatic dependent processes in biodegradation pathways (19, 20). Despite the numerous reports on surfactantenhanced biodegradation, the mechanisms behind surfactantenhanced biodegradation and the interactions between the microorganisms, the PAHs, and the surfactants are to date not well understood. Before surfactants can be used routinely to promote bioavailability and biodegradation, it is crucial to establish knowledge on how the surfactants influence the survival and activity of the microbial degraders present in the contaminated system. Recently, mathematical models have been proposed to describe bioavailability and kinetics of surfactant-enhanced mineralization of low molecular weight PAHs, naphthalene (13) and phenanthrene (13-15, 21). However, none of these reports have discussed the effect of surfactant-enhanced degradation of high molecular weight (HMW) PAHs, which are known as the more recalcitrant and principal compounds at PAH-contaminated sites (7). In addition, the significance of surfactant-enhanced metabolite accumulation and the possibility of decreased bioavailability of not only the parent compound but also of its metabolite(s) have to our knowledge not been discussed previously. The aim of this study was to achieve a better quantitative understanding of the effects of surfactant on the metabolisms of HMW PAHs, using the HMW four-ring aromatic compound fluoranthene as a model compound. The degradation was investigated in continuously mixed batch systems consisting of a pure PAH-degrading bacterial culture, the nonionic surfactant Triton X-100, and fluoranthene-amended liquid mineral salts medium. The PAH-degrading bacterium Sphingomonas paucimobilis strain EPA505 isolated from a creosote-contaminated soil (22) was chosen because surfactant-enhanced fluoranthene mineralization was obtained previously at up to 34 mM Triton X-100 (18, 23). The experimental data were analyzed in relation to a mathematical model describing solubilization of crystalline substrate, micellar uptake of aqueous-phase substrate, and biodegradation of the substrate and its metabolite(s) in the presence and absence of surfactant. To the best of our knowledge, this is the first report of a model describing the kinetics of surfactant-enhanced degVOL. 33, NO. 15, 1999 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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radation of a HMW PAH present at concentrations greater than its aqueous solubility. The presence of surfactant clearly influenced the microbial PAH metabolism as indicated by a rapid accumulation of colored product(s) following degradation of micelle-solubilized fluoranthene. Few, if any, have discussed the significance of surfactant-induced accumulation of PAH intermediate(s).
Materials and Methods Chemicals. All chemicals were of analytical grade or better. Fluoranthene (98% pure) and [3-14C]fluoranthene with a specific activity of 45 mCi/mmol and a radiochemical purity >95% were obtained from Sigma. A stock solution (160 mM) of the nonionic surfactants Triton X-100 (tert-octylphenoxyethanol, MW ) 625) (Sigma) was prepared in BushnellHaas (BH) medium (see below) and sterilized by autoclaving. Bacterial Strain. Inoculum for batch cultures of the PAHdegrading Sphingomonas paucimobilis strain EPA505 (22), was pregrown in LBg medium (per liter: 10 g of tryptone, 5 g of yeast extract, 10 g of glucose, and 5 g of NaCl at a final pH of 7.2) at 25 °C, harvested in late exponential growth phase, and further prepared as described in ref 24. The number of culturable cells was determined by plating of serial dilutions on LBg plates (LBg medium supplied with 15 g of Bacto agar (Difco)). Fluoranthene Degradation Assays. Biodegradation of fluoranthene in the presence of 0, 0.48, and 8 mM Triton X-100 was quantified at 23 °C using two different assays. 14C-labeled fluoranthene was used to measure the amount of 14CO2 produced during mineralization of fluoranthene, and the disappearance of fluoranthene was determined analytically by HPLC (see below). The same batch of inoculum was used, and similar initial fluoranthene and biomass concentrations were applied in the two separate but parallel experiments (Table 4). The mineral salts medium BushnellHaas (BH) (Difco) was used in both types of assays (per liter: 1 g of Na2HPO4‚H2O, 0.20 g of MgSO4‚7H2O, 0.02 g of CaCl2, 1 g of (NH4)2PO4, 1 g of KNO3, and 0.05 g of FeCl3 at a final pH of 7.2). The fluoranthene-containing medium was prepared as previously described in ref 24 and shaken for 24 h before inoculation. The mineralization assay based on radioactivity was performed as described in ref 24. The fluoranthene concentrations were 20-30 mg/L fluoranthene (Table 4, final radioactivity 1130 Bq/mg fluoranthene). After inoculation, flasks were incubated at 23 °C for 200 rpm. The amount of 14CO collected was quantified using a model LS 1801 2 scintillation counter (Beckman). Counts in controls were never above the background level. At the end of each 14Clabeled carbon mineralization experiment, the recovery of [14C]carbon was determined measuring the radioactivity remaining in the filtrate and retained on a filter as explained in ref 24. The recoveries in the fluoranthene mineralization assays were calculated at 99 ( 7%. The fluoranthene degradation assay was performed in 30-mL glass centrifuge tubes containing 10 mL of BH medium and 20-30 mg/L fluoranthene (Table 4, final radioactivity 1130 Bq/mg fluoranthene). After inoculation, the tubes were sealed with Teflon-lined screw caps and incubated oblique on a shaker table at 23 °C. The tubes were harvested at periodic intervals. The aqueous phase was separated from cells and crystalline fluoranthene by centrifugation (IEC, Centra MP4R, Danmetric, Denmark) at 10 °C and 2240g for 10 min. The total amount of dissolved fluoranthene (aqueous and micellar fluoranthene) was extracted from the culture fluid by passing the supernatant through a 3-mL Supelco Supelclean LC-18 column (B&B, Denmark) under vacuum (1.5 × 104 Pa). The retained substrate was eluted with two times 1000 µL of methanol. The fluoranthene in the pellets was quantified by the addition of 3 mL of methanol to the 2572
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pellets, which were sonicated for 5 min at 140 W in a water bath (Metason 200 (5210E-MT), Struers, Denmark) before the fluoranthene was extracted from the pellet at level 1 on a vertical rotating shaker table (Heidolph REAX2, Struers, Denmark) for 1 h. The cells were removed from the methanol by centrifugation at 10 °C and 2240g for 10 min. Analytical Procedures. The fluoranthene content in the supernatant and pellet samples, respectively, was analyzed by injection of 100 µL of methanol extract into a HPLC system equipped with a Water autosampler (710B WISP), a Shimatzu pump (LC-10AT), and a Supelco C18-column (15 cm by 4.6 mm, 5 µm particle size, octadecylsilane). Peaks were detected by use of a fluorescence detector (model 1046A Hewlett Packard). The wavelengths of excitation and emission were 245 and 350 nm, respectively. Sensitivity of detection was
