Degradation of polycyclic aromatic hydrocarbons dissolved in Tween 80 surfactant solutions by Sphingomonas paucimobilis EPA 505 Dianne J. Luning Prak and Parmely H. Pritchard
Abstract: The degradation rates of mixtures of pyrene (PYR). fluoranthene (FLA), and phenanthrene (PHE) by Sphingornonas paucfmo/Jilis EPA 505 were measured in the presence of the nonionic surfactant Tween 80. For strain EPA 505, FLA and PIlE are grO\vth substrates, while PYR is not. Linear degradation rates ranging from 0.05 to 2.2 mg·t·l·h· 1 were observed for F.LA, PYR, and PRE at approximately 107 colony-forming units (CFU)!mL At lower biomass, PYR degradation exhibited lognormal degradation. The degradation rates of PYR, FLA, and PUE increased with increasing biomass and substrate concentration. At highFLA concentrati.ons, FLA degradation rates were £1ster in the presence of surfactant than in the absence of surfactant, suggesting that some of the FLA was transp0l1ed directly into the cell from the micellar phase. In mixtures, PHE was the preferred substrate and was utilized first, followed by FLA and then PYR. Once the competing substrates were degraded, the remaining substrate was degraded at the same rate or Hlster than the rate found in the single-substrate system. Based on the results with Tween 80, it appears that PHE, PYR, andFLA are competing for the same enzymatic sites.
Key words: PAH mixtures, microbial degradation, surfactant, solubilization, EPA 505.
Resume : Nous avons mesure les taux de degradation de melanges de pyrene (PYR), de fluoranthene (FLA) et de phenanthrene (PHE) par la souche EPA 505 de Sphingomonas paucimobilis en presence de Tween 80, un agent tensio actif non ionique. Dans Ie cas de cettc souche EPA 505, FLA et pnE sont des substrats de croissance mais pas Ie PYR. Des taux de degradation lineaire variant de 0.05 a 2.2 ont ete observes pour FLA, PYR et PHE en presence d'environ 107 UFC/ml... Avec une biomasse plus faible, PYR etait degrade de fayon lognormale. Les tau x de degradation de PYR, FLA et de PHE etaient plus eleves selon l'augmentation de la biomasse et de la concentration des substrats. En presence de fortes concentrations de FLi\. les taux de degradation de ce produit etaient plus rapides en presence qu'en absence de l'agent tensio-actif, laissant croire qu'ul1e partie du FLA etait transportee directement dans la cellule lors de la phase micellaire. Dans les melanges. Ie PHE etait Ie substrat prefere et il etait utilise en premier, suiv! du FLA et finalement du PYR. Suite ,\ Ia degradation des substrats competitifs, Ie substrat restant etait degrade a la meme vitesse ou plus rapidement que Ie taux de degradation observe dans un systeme a un seu) substrat. D'apres les resultats obtenus avec Ie Tween 80, it semble que Ie PHE, le PYR et Ie .1"L/\ competitionnent pour les memes s.ites enzymatiques.
Tt.10ts eMs: melanges de PHA, degradation microbienne, agent tensio-actif, solubilisation, EPA 505. [Traduit par la Redaction]
Introduction One limitation to degradation of polycyclic aromatic hy drocarbons (PAHs) is their low solubility in water and their consequent high affinity for surfaces. Since most microor ganisms take in PAHs from the aqueous phase, mass transfer from the sorbed or nonaqueous liquid phase into the aqueous phase often limits PAR degradation rates. One way to en hance the rate of PAH transfer into the aqueous phase is to
add surfactants that fOIm micelles and solubil ize the PAH. Surfactants have been found to enhance the degradation rates of individual PAHs by pure and mixed cultures (Tiehm 1994; Volkering et al. 1995; Liu ct al. 1995; Guha and Jaffe 1996; Guha et aL 1998; Willumsen et al. 1998; Grimberg et al. 1996; Madsen and Kristensen 1997). Only a few stud ies, however, have examined the effectiveness of surfac tants in enhancing the degradation of P AH mixtures (Tichm 1994; Guha et al. 1998), especially when it involves
Received 8 August 2001. Revision received 19 December 2001. Accepted 20 December 2001. Published on the NRC Research Press Web site at ht1p:/lcjm.nrc.ca on 2 March 2002. D.J. Luning Prak1,2 and P.H. Pritchard. 3 Naval Research Laboratory, Washington, DC 20357, U.S.A. 1Corresponding author ( e-mail: [email protected]
2Present address: The USNA Chemistry Depat1ment, 572 Hollaway Road, Annapolis, MD 21402, U.S.A.
3Present address: Department of Microb.ial Ecology and Biotechnology, National Environmental Research Institute,
Frederiksborgvej 399, 4000 Roskilde, Denmark.
Can . .1. Microbiol. 48: 15]-158 (2002)
DOl: 10.1 139!W()2-004
2()02 NRC Canada
co-metabolism (Boonchan et a1. 1998). For example, Boonchan et a1. (1998) found that Stenotrophomonas rnaltophi/ia VUN 1,001 Os degraded mixtures of P AHs faster in the presence of surfactant than in the absence of surfac tant, and the maximum specific degradation rate o:f each component from the mixture was smaller than when it was degraded alone. Further work in this area is necessary to provide a greater understanding of how surfactants influence the microbial degradation of mixtures and to enable engi neers to design appropriate methods to clean up contami nated sites. To this end, we have chosen to study the metabolism and co-metabolism of PAH mixtures in the presence of surfac tants. Of the various PAH-degrading cultures, Sphingomona.'i' strains have been shown to degrade a large variety of PAHs (up to five-ring PAHs) (e.g., Ye et a1. 1996; Muel.ler et a1. 1990; Ho et al. 2000) and are now being studied to under stand cell biochemistry (e.g., Story et al. 2000). In this work, we sought to investigate the degradation of mixtures of PAHs by Sphingomonas paucimobilis EPA 505 in the pres ence of a surfactant, Tween 80. This microorganism has been shown to metabolize phenanthrene (PIlE) and fluoranthene (FLA) and co-metabolize (transfonn but not grow on) pyrene (PYR) (Ho et al. 2000).
Materials and methods The PAHs used in this study were pyrene, fluoranthene, and phenanthrene (Aldrich, St. Louis, Mo.; 99+% purity). The reported aqueous solubilities of PYR, FLA, and PHE are 0.15, 0.25, and 1.3 mg/L, respectively (Pearlman et al. 1984). Bushnell Haas (BH) medium contained the following (giL): 1.0 NH4N0 3 , 0.2 MgS0 4 '7H2 0, 1.0 K2HP0 4 , 1.0 KJI 2P04 , 0.05 FeClj'7H zO, and 0.02 CaCli4H20. Miller Luria-Bertani (LB) broth was purchased from Fisher Scien tific (Chicago, Ill.). PYGT agar contained the following (giL): 0.6 Bacto peptone, 1.0 yeast extract, 1.0 dextrose, 0.5 tryptone peptone, and 15 Bacto agar, all purchased from Difco LaboratOlies (Detroit, Mich.). Tween 80 (Sigma Aldrich, St. Louis, Mo.) was used as supplied and contained a critical micelle concentration of 33··A5 mglL (Pennell et a1. 2000). All solutions were prepared in NANOpure water and autoclaved. Sphingomonas paucimobilis EPA 505 was originally iso lated from soil containing creosote (Mueller et a1. 1990). Cultures of strain EPA 505 were maintained in BH broth in the presence of solid FLA (10·.. .20 mg). To develop inocula for experiments in BH medium containing PAHs, sub samples of the maintenance culture were grown in LB broth, in the absence of PARs. This procedure, therefore, avoided any confounding induction effects from pre-exposure to PARs. Inocula prepared in LB were not washed because centrifuging often partially inactivated the microbes. It is possible that the small amount of carry-over of LB medium helped to initiate PAR degradation, possibJy through the supply of energy resources. Viable cell counts were deter mined by serial dHution and plating on PYGT agar. Optical densities were measured at 600 nm (OD 600) using a SPEC 20 spectrophotometer (VWR, Bridgeport, N.J.). All biodegradation experiments were run with PAHs dis solved in the surfactant solution; no solid was present. Initial
Can. J. Microbial. Vol. 48, 2002
PAH concentrations ranging from 1 to 18 mg/L (less than the maximum solubility in Tween solution) were selected to ensure that crystals did not fonn and to allow some flexibil ity in testing different P AH concentrations. Concentrations of Tween 80 ranging from 0.6 to 1.8 giL were selected to give high enough concentrations of PAHs for direct high pressure liquid c.hromatography (HPLC) analyses but not to be toxic to strain EPA 505. Because of the analytical inten sity of each experiment, duplicate samples were not taken at each time point; however, all experiments were run at least twice. LB-!,'Town cultures were added to 250-mL flasks con taining 130 mL BH, Tween 80, and dissolved PAHs to ob tain an initial biomass of approximately 107 cells/mL. Flasks were shaken on a temperature-controlled orbital shaker (Lab Line Instruments, Dubuque, Iowa) in the dark at approxi mately 150 rpm and 25°C. Periodically, l-mL aqueous sam ples were taken f1- 0.9SC
0.73±0.04 2.1:r:O.1 1.9±0.2 2.0±0.2
2.0±0.2 1.6±0.2 1.8±O.l
0.28±0.07 1.2±0.6 0.56±0.1
NA 2.0e 2.1±0.8
Note: NA, not enough data points to calculate an accurate slope; FLA, fluoranthcne: PYR, pyrene; PHE, phenanthrene; PAH, polycyclic aromatic hydrocarbons. Errors are thc 95% confidence intervals for tllC fitted slope. °Pyrene degradation rates are based on a linear fit of concentration···time data for concentTation values that are greater than 10% of the initial concentration. "Only three data points were available for slope determination, 1