Environ. Sci. Technol. 2008, 42, 4790–4796
Isomer-Specific Biodegradation of Methylphenanthrenes by Soil Bacteria R A S M U S F . L A M B E R T S , †,‡ JAN H. CHRISTENSEN,§ PHILIPP MAYER,| OLE ANDERSEN,‡ AND A N D E R S R . J O H N S E N * ,† Department of Geochemistry, Geological Survey of Denmark and Greenland, Øster Voldgade 10, DK-1350 Copenhagen K, Denmark, Department of Life Sciences and Chemistry, Roskilde University, Postboks 260, DK-4000 Roskilde, Denmark, Department of Natural Sciences, Faculty of Life Sciences, Copenhagen University, Thorvaldsensvej 40, 1871 Frederiksberg C, Denmark, and Department of Environmental Chemistry and Microbiology, National Environmental Research Institute, Aarhus University, Frederiksborgvej 399, PO Box 358, DK-4000 Roskilde, Denmark
Received January 8, 2008. Revised manuscript received April 7, 2008. Accepted April 14, 2008.
It is assumed that bacteria generally degrade 2-methylphenanthrene (2MPhe) in preference to 1-methylphenanthrene (1MPhe), and that environmental biodegradation of methylated PAHs therefore can be described qualitatively by changes in relative concentrations of these isomers. Our objective was to investigate whether microbial phenanthrene degraders (Sphingomonas and Mycobacterium) show such isomerspecific PAH degradation. Eleven out of twenty-nine phenanthrene degraderscouldgrowonmethylphenanthrene.Themycobacteria grew only on 2MPhe, the sphingomonads grew mostly on 1MPhe, and one sphingomonad could utilize both substrates. Seven strains were tested in a two-phase system where 1MPhe and 2MPhe were supplied in heptamethylnonane. For these strains, a consistent description of biodegradation based on the 2MPhe/1MPhe diagnostic ratio would not be possible because three Mycobacterium and one Sphingomonas degraded 2MPhe faster than 1MPhe, another Sphingomonas degraded 1MPhe and 2MPhe at almost equal rates, and two Sphingomonas degraded 1MPhe faster than 2MPhe. Thus, environmental biodegradation of phenanthrenes may theoretically proceed with only minor changes in 2MPhe/1MPhe ratios if individual members of the degrader community have different isomer preferences. However, two soil microcosms polluted with bunker oil confirmed the general decline in 2MPhe/1MPhe ratio during oil biodegradation.
Introduction Polycyclic aromatic hydrocarbons (PAHs) in the environment originate from two major sources: pyrolytic PAHs generated by incomplete combustion of organic matter, and petrogenic * Corresponding author. Phone: +45 3814 2328, e-mail: arj@ geus.dk. † Geological Survey of Denmark and Greenland. ‡ Roskilde University. § Copenhagen University. | Aarhus University. 4790
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PAHs from mineral oil. Alkyl-PAHs, the subject of this study, dominate the petrogenic PAHs. In spite of their widespread occurrence, the alkyl-PAHs have been given little attention in the biodegradation literature compared to the nonsubstituted PAHs. The fate of environmental PAHs is affected by numerous processes including evaporation, photo-oxidation, chemical oxidation, and biodegradation, processes commonly known as weathering. It is, however, generally accepted that biodegradation is the major process that decontaminates PAH-polluted sediments and soils. It was early recognized that biodegradation of methyl-PAHs in general is slower than biodegradation of the corresponding nonsubstituted PAHs (1), but biodegradation may also be affected by the specific position of the methyl group (2), i.e., different PAH-isomers may be degraded at different rates. This means that changes in the relative concentrations of the individual isomers, socalled diagnostic isomer ratios, may reflect biodegradation. The isomers within a group of alkyl-PAHs, where the only difference is the position of the alkyl group, have similar physicochemical properties. It has therefore been suggested that changes in the ratios of isomer concentration of specific oil PAHs may diagnose the degradation mechanism, i.e., separate microbial oil degradation from physicochemical oil weathering (3, 4). Biodegradation of oils has been demonstrated using such changes in diagnostic ratios of methylnaphthalenes, methylfluorenes, methyldibenzothiophenes, and methylphenanthrenes (3–6). 2- and 3-methylphenanthrene in oil may for instance be degraded faster than 1-, 4and 9-methylphenanthrene (4, 7, 8). Thus, biodegraded oil may show relative enrichments in 1-, 4-, and 9-methylphenanthrene over time when compared to 2- and 3-methylphenanthrene, whereas physically weathered oil may show rather constant ratios of methylphenanthrenes. The concept of diagnostic isomer ratios is based on the assumption that bacteria preferentially degrade some isomers. However, biodegradation of monomethylated phenanthrenes and dibenzothiophenes was not isomer-specific in an in vitro study where an environmental enrichment culture was exposed to oil aromatics (9). Also, different PAH-isomer degradation patterns were seen in a study where bunker oil was degraded in vitro by different microbial consortia (8). This leads to the question whether isomer-specific PAH degradation is a general trait of bacterial PAH-degrading communities, or whether it depends on the actual composition of the bacterial community. We chose 1-methylphenanthrene (1MPhe) and 2-methylphenanthrene (2MPhe) as model compounds because they are present in most crude oils and refined petroleum products, because they were commercially available, and because they have been found to exhibit isomer-specific removal during biodegradation of oil by bacterial pure cultures (7) and consortia (4, 8) and in the environment (5, 6, 10). The objective of our study was to identify new, environmentally relevant 1MPhe- and 2MPhe-degrading bacteria and to characterize them with respect to degradation of 1MPhe and 2MPhe to test the diagnostic isomer ratio hypothesis. On the basis of decreasing 2MPhe/1MPhe ratios reported in the literature, we expected the isolates to preferentially degrade 2MPhe. Diagnostic ratios have mostly been used to describe biodegradation of PAHs in aquatic systems. We therefore tested oil-polluted soil microcosms for isomer-specific biodegradation of 1MPhe and 2MPhe. 10.1021/es800063s CCC: $40.75
2008 American Chemical Society
Published on Web 06/03/2008
TABLE 1. Test of Phenanthrene-Degrading Bacteria for Growth on 1MPhe and 2MPhe as Sole Sources of Carbona,b respiratory WST-1 reduction (∆Abs450) strain Ri456a Ri462b PYR-1 1VB-c Ri464 Ri465b Ri452b VM450 VM451 Ri463b Fan 9 LB501T LB307T Ri457 RL1 Ri455 VF1 VM504 Ri458a Ri466 Ri460 OUS82 LH128 Arj19 EPA505 3VB-ba 10-1 Arj81 Arj13 Arj45
identification c
Arthrobacter oxidans Mycobacterium sp.c Mycobacterium spc M. aurumc M. aurumc M. austroafricanumc M. austroafricanumc M. austroafricanumc M. austroafricanumc. M. frederiksbergensec M. frederiksbergense M. frederiksbergense M. gilvum M. gilvumc M. gilvumc M. gilvumc M. gilvum M. gilvumc M. vaccaec M. vaccaec M. vaccaec Pseudomonas putida Sphingomonas sp.c Sphingomonas sp.c Sphingomonas sp. Sphingomonas sp.c Sphingomonas sp. Sphingomonas sp.c S. subarcticac unclassified β-proteobacteriumc
reference
Phe
1MPhe
2MPhe
No PAH
20 20 DSM7251 22 A. R. Johnsen, unpublished 20 20 20 19 19 20 23 17 17 20 R. F. Lamberts, unpublished 20 DSM11096 24 19 20 20 20 25 17 18 DSM7526 (26) A. R. Johnsen, unpublished DSM12247 27 18 18 18
0.86 ( 0.59 1.79 ( 0.04 1.11 ( 0.15 0.30 ( 0.02 1.50 ( 0.33 0.81 ( 0.46 1.66 ( 0.10 1.59 ( 0.18 1.17 ( 0.08 1.76 ( 0.07 1.54 ( 0.15 0.99 ( 0.07 0.24 ( 0.04 0.49 ( 0.05 1.45 ( 0.20 2.04 ( 0.03 0.50 ( 0.27 0.94 ( 0.14 0.57 ( 0.03 0.93 ( 0.31 0.32 ( 0.05 0.47 ( 0.13 1.88 ( 0.13 1.76 ( 0.28 1.01 ( 0.21 0.22 ( 0.07 0.24 ( 0.21 1.03 ( 0.09 0.52 ( 0.07 0.37 ( 0.06
0.00 ( 0.00 0.02 ( 0.00 0.19 ( 0.05 0.03 ( 0.01 0.07 ( 0.01 0.02 ( 0.01 0.06 ( 0.01 0.15 ( 0.02 0.11 ( 0.02 0.02 ( 0.01 0.04 ( 0.00 0.01 ( 0.00 0.01 ( 0.00 0.12 ( 0.13 0.20 ( 0.01 0.02 ( 0.01 0.02 ( 0.00 0.00 ( 0.00 0.01 ( 0.01 0.00 ( 0.00 0.00 ( 0.00 0.00 ( 0.00 2.29 ( 0.03 1.77 ( 0.05 0.72 ( 0.05 0.42 ( 0.31 0.00 ( 0.01 1.34 ( 0.10 0.78 ( 0.06 0.00 ( 0.00
0.04 ( 0.02 0.00 ( 0.00 0.16 ( 0.02 0.02 ( 0.01 0.26 ( 0.01 0.02 ( 0.00 0.14 ( 0.02 0.36 ( 0.10 0.35 ( 0.03 0.09 ( 0.02 0.08 ( 0.01 0.02 ( 0.00 0.03 ( 0.00 0.09 ( 0.01 0.54 ( 0.05 0.06 ( 0.00 0.06 ( 0.01 0.01 ( 0.01 0.03 ( 0.01 0.02 ( 0.01 0.01 ( 0.00 0.00 ( 0.00 0.00 ( 0.00 1.23 ( 0.10 0.06 ( 0.01 0.00 ( 0.00 0.55 ( 0.05 0.00 ( 0.01 0.03 ( 0.02 0.00 ( 0.00
0.01 ( 0.01 0.03 ( 0.01 0.19 ( 0.03 0.02 ( 0.01 0.07 ( 0.01 0.04 ( 0.01 0.07 ( 0.01 0.15 ( 0.02 0.18 ( 0.05 0.04 ( 0.02 0.07 ( 0.00 0.01 ( 0.00 0.01 ( 0.00 0.11 ( 0.02 0.21 ( 0.03 0.07 ( 0.03 0.03 ( 0.01 0.01 ( 0.00 0.02 ( 0.00 0.02 ( 0.01 0.02 ( 0.01 0.00 ( 0.00 0.00 ( 0.00 0.00 ( 0.00 0.00 ( 0.00 0.00 ( 0.00 0.00 ( 0.00 0.00 ( 0.00 0.00 ( 0.00 0.00 ( 0.00
a Growth was detected as respiratory reduction of the tetrazolium indicator WST-1. b Numbers in bold are significantly larger than those from the negative controls (t-test). c Tentatively identified by partial 16S rRNA gene sequencing.
Materials and Methods Chemicals and Reagents. 1MPhe and 2MPhe were purchased from Ultra Scientific (North Kingstown, RI, 99% purity), phenanthrene from Merck (Glostrup, Denmark, 98% purity), and phenanthrene-d10 from ISOTEC (Champaign, IL). 2,2,4,4,6,8,8-Heptamethylnonane (HMN, 98% purity) was purchased from Sigma-Aldrich (Vallensbæk, Denmark), WST-1 cell proliferation reagent from Roche Molecular Biochemicals (Mannheim, Germany), and sterile, 96-well, flat-bottom microtiter plates from Sarstedt (Nu ¨ mbrecht, Germany, product no. 82.1581.001). Test of Phenanthrene Degrading Bacteria for Growth on 1MPhe and 2MPhe. Thirty isolates (Table 1) capable of growing on phenanthrene were tested for growth on 1MPhe and 2MPhe as sole carbon and energy sources. One isolate (M. gilvum RL1) was already known to grow on 2MPhe and thus was used as a positive control strain. All strains were tested in triplicate for each PAH in 96-well microplates as described previously (11). In short, microplate wells were coated with the phenanthrenes by adding them individually in hexane solution followed by evaporation of the solvent. Hexane without phenanthrene was added to negative control wells. The wells were inoculated with dilute suspensions of degrader cells (OD540 ≈ 0.005). Biomass was assayed for each microplate well after 12-14 days incubation by adding the tetrazolium compound WST-1 together with electron donors. The respiratory reduction of WST-1 in growthpositive wells was determined photometrically (450 nm with a reference wavelength of 650 nm) after 150 min except strains LB501T and LB307T that were read after 90 min, and LH128 that was read after 240 min. The absorbance reads at time zero were subtracted from the subsequent readings to obtain the change in absorbance. Growth was determined by comparison to negative controls (hexane only) using one-
sided t-test at the five-percent significance level. Wells containing phenanthrene as the carbon source were used as positive controls. Kinetics of 1MPhe and 2MPhe Degradation in Pure Cultures. Inocula were grown in closed 500-mL Blue Cap bottles containing 100 mL of phosphate minimal medium (12) supplemented with phenanthrene (200 mg mL-1) as sole carbon source. The phenanthrene was added to the bottles in acetone solution, the acetone was evaporated, and the medium was added. The cultures were incubated for up to 14 days in the dark at 20 °C on a horizontal shaker (150 rev min-1). When turbid, the cultures were harvested by settling phenanthrene crystals for 1 h, followed by filtration into 50mL centrifuge tubes through glasswool-packed funnels. The inocula were washed three times in phosphate-buffered saline (PBS, per liter: NaCl, 8 g; KCl, 0.2 g; Na2HPO4, 1.44 g; NaH2PO4, 0.2 g; pH 7.0) and diluted in PBS to an OD600nm between 0.2 and 0.3. The diluted inocula (1.6 mL) were transferred to Pyrex centrifuge glass vials (10 mL) with Teflon screw caps. A 0.4 mL amount of HMN containing a mixture of 1MPhe and 2MPhe (100 µg mL-1 each) was added to the vials. The vials were incubated on a vertical rotation board (30 rev min-1) at 25 °C for 0, 5, 9, 13, 18, 24, 31, 40, 55, or 72 h. At each time point, vials were sacrificed by adding four drops of HCl (6 M), followed by shaking, to stop microbial activity (final pH < 2). Vials for M. aurum Ri464 at times 9 and 31 h were discarded because of insufficient acidification. The vials were stored at 5 °C until analyzed. Time-zero vials were acidified prior to the addition of HMN to ensure no activity. Vials with PBS and phenanthrenes in HMN were used as negative controls. Vials with HMN containing 100 µg mL-1 of phenanthrene were used as positive controls. The efficiency of HCl-inactivation was initially verified by plating HClVOL. 42, NO. 13, 2008 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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treated inocula (Sphingomonas sp. Arj81 and M. gilvum RL1) on R2A-agar. For PAH quantification, 50 µL of a 40 µg mL-1 solution of phenanthrene-d10 was added to each vial as internal standard. Then NaCl (0.5 g) was added to reduce the heavy HMN emulsification observed by some strains. The vials were vortexed, and the water and HMN phases were separated by centrifugation (10 min, 1500g). The HMN phase was transferred to GC vials with 100-µL inserts and analyzed on a Finnigan TRACE DSQ Single Quadrupole GC-MS (Thermo Electron Corporation) operating in EI mode and equipped with a 60 m HP-5MS capillary column (0.25 mm ID × 0.25 µm film) with 10 m 0.25 mm guard column. One-microliter aliquots were injected in PTV (programmable temperature vaporization) splitless mode starting at 60 °C and increased at 14.5 °C/s to 300 °C and held for 1 min during transfer. The oven temperature program was as follows: initial temperature 80 °C held for 2 min, 60 °C min-1 to 240 °C then followed by an increase of 30 °C min-1 to 315 °C (held for 5 min). The mass spectrometer was operated in selective ion monitoring mode measuring Phe (m/z: 178), Phe-d10 (m/z: 188), and 1MPhe and 2MPhe (m/z: 192). The peak areas of the three phenanthrenes were standardized with respect to the peak area of Phe-d10 in each sample. Five-point calibration curves were used to quantify Phe, while the relative response factors were used for quantification of 1MPhe and 2MPhe. Finally, the concentrations of Phe, 1MPhe, and 2MPhe were normalized to the initial concentration, and data were modeled using three-parameter, one-phase exponential decay curves according to eq 1 Ct)Co+ae-bt
(1)
where Ct was the normalized concentration at time t. The constants a, b, and C0 vary for each decay curve; a is the estimated concentration at time zero, b is the decay constant, and C0 is the final concentration when t approaches infinity. Initial degradation rates were calculated from the first derivative function for t ) 0 (eq 2). dC/dt0) -ba
(2)
For the soil microcosms, day 10 was used as time-zero because of a lag-phase in Phe-degradation. Linear regression was applied when the degradation of phenanthrenes did not follow exponential decay. Diffusive Mass Transfer of 1MPhe and 2MPhe. A previously published, microscale technique (13) was used to determine if there were any differences in the diffusion of 1MPhe and 2MPhe in water. The method is based on transfer from a contaminated silicone disk (source) through a 100µm stagnant water layer to a clean silicone disk (sink). The source discs were contaminated by placing them for 12 h in methanol/water (80:20, v:v) containing 1MPhe (0.126 mM) and 2MPhe (0.134 mM). Diffussive transfer of 1MPhe and 2MPhe was followed for 20 h at room temperature (23 ( 3 °C) by determining the ratio of 2MPhe/1MPhe in the source and sink discs using GCMS as described above for the NAPL phase. Degradation of Methylphenanthrenes in Soil Microcosms. In May 2004, a natural A-horizon soil (the upper 10 cm) was collected at Dronning Mølle (North Sealand, Denmark). Bioremediated subsoil from a tar and asphalt production plant was obtained from a previous study (14). The soils were passed through a 4-mm sieve and stored at 5 °C for 5 days. Portions of 370 g of natural soil were polluted by adding 15 g of heavy bunker oil dissolved in 20 mL of dichloromethane. The dichloromethane was evaporated for 20 h. Microcosms were set up in 4-L stainless steel containers. Microcosm 1 contained 370 g of oil-polluted soil and 1130 g 4792
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of natural soil. Microcosm 2 contained 370 g of oil polluted soil, 1100 g of natural soil, and 30 g of bioremediated soil. The bioremediated soil was added to introduce a PAHdegrading bacterial community. Microcosm 3 was a negative control similar to microcosm 2 but killed by the addition of sodium azide to a final pore water concentration of 5 g L-1. This control accounted for abiotic processes. The reason for polluting only one-fourth of the microcosm soil and then mixing it with fresh soil was to minimize the solvent effect on the microorganisms. The soil organic matter contents were 7.7% (nonprimed) and 7.5% (primed), and the clay+silt contents (
