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Y involving 2-hydroxy-1-naphthoic acid in a novel metabolic pathway
Process Biochemistry 43 (2008) 1004–1008
Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Kinetics of phenanthrene degradation by Staphylococcus sp. strain PN/Y involving 2-hydroxy-1-naphthoic acid in a novel metabolic pathway Somnath Mallick, Tapan K. Dutta * Department of Microbiology, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 November 2007 Received in revised form 15 April 2008 Accepted 28 April 2008
Staphylococcus sp. strain PN/Y isolated from creosote-contaminated soil, was previously reported to degrade phenanthrene as sole source of carbon and energy. Unlike other phenanthrene degraders, Staphylococcus sp. PN/Y degraded phenanthrene by a novel pathway involving 2-hydroxy-1-naphthoic acid (2H1NA), which was further metabolized by unique meta-cleavage dioxygenase, ultimately leading to TCA cycle intermediates. In the present study, kinetics of phenanthrene degradation and the dynamic fate of the key intermediates, 2H1NA, salicylic acid and catechol were demonstrated. When cells were grown on 50, 100, 200, 500 and 1000 mg l1 of phenanthrene, the doubling time was 31.55, 30.2, 29.32, 26.84, 25.22 h and the specific growth rate was 0.0314, 0.0331, 0.034, 0.0372 and 0.0396 h1, respectively. At a concentration of 1 g l1 of phenanthrene, the maximum accumulation of 2H1NA was found to be 323 mg l1. In addition, 2H1NA utilized by Staphylococcus sp. PN/Y as sole carbon source, the growth yield at 96 h was 445, 557, 560, 480, 419, 334, 212 and 66 mg of protein (g 2H1NA)1 when cells were grown on 50, 100, 200, 400, 500, 650, 800 and 1000 mg l1 of 2H1NA, respectively. Growth was found to be inhibited at initial higher concentration of 2H1NA, which may attributed due to concentration-dependent toxicity of 2H1NA and/or the toxicity of its decarboxylated metabolite, 2naphthol. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Phenanthrene 2-Hydroxy-1-naphthoic acid 2-Naphthol Biodegradation Staphylococcus sp. Kinetic study
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are an important class of ubiquitous environmental contaminants because of their high potential toxicity, mutagenicity and/or carcinogenicity [1–3]. Among other PAHs, phenanthrene, a polycyclic aromatic hydrocarbon with three condensed rings fused in angular fashion is widely distributed in the environment primarily because of anthropogenic and pyrolytic processes. Phenanthrene has been used in the synthesis of different organic compounds like pesticides, fungicides, detergents, dyes and mothballs [4]. Phenanthrene is known to be a photosensitizer of human skin, a mild allergen and mutagenic to bacterial systems under specific conditions [5]. It is a weak inducer of sister chromatid exchanges and a potent inhibitor of gap junctional intercellular communication [6]. Since phenanthrene is the smallest aromatic hydrocarbon to have a ‘‘bay-region’’ and a ‘‘K-region’’, it is often used as a model substrate for studies on the metabolism of carcinogenic polycyclic aromatic hydrocarbons.
* Corresponding author. Tel.: +91 33 2355 9544; fax: +91 33 2355 3886. E-mail address: [email protected] (T.K. Dutta). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.04.022
Numerous microbial species are known to degrade phenanthrene, and there appear to be several metabolic routes available, depending upon the species, strains, and even the culture conditions [7–19]. In majority of cases reported so far, the degradation pathway proceeds through the well-known central metabolite 1-hydroxy-2-naphthoic acid. In recent years, some investigators predict assimilation of phenanthrene via both 1hydroxy-2-naphthoic acid and 2-hydroxy-1-naphthoic acid, the later being the minor product [20–24]. In our previous study, we reported phenanthrene degradation exclusively via the central metabolite 2-hydroxy-1-naphthoic acid through a novel phenanthrene degradation pathway, with a new phenanthrene degrading bacterium Staphylococcus sp. strain PN/Y [25]. Although, aerobic biodegradation is an important attenuation mechanism for PAHs in the environment but the ability to estimate PAH biodegradation rates is important for predicting environmental fate and for designing remediation efforts. During phenanthrene biodegradation, the degradation kinetics including the fate of the metabolite 1-hydroxy-2-naphthoic acid (1H2NA) was described [26–29], however, no such effort had been made in the alternative phenanthrene degradation pathway that involves 2-hydroxy-1-naphthoic acid. In the present study, the kinetics of phenanthrene degradation and the dynamic fate of the intermediate 2-hydroxy-1-naphthoic acid were investigated in the
S. Mallick, T.K. Dutta / Process Biochemistry 43 (2008) 1004–1008
novel phenanthrene degradation pathway by Staphylococcus sp. strain PN/Y. 2. Materials and methods 2.1. Chemicals Phenanthrene, 2-hydroxy-1-naphthoic acid, salicylic acid, catechol and 2naphthol used in this study were purchased from Sigma–Aldrich (Milwaukee, WI). All other chemicals and reagents used in this study were of analytical grade and used without further purification. 2.2. Organism and culture conditions A new phenanthrene degrading strain Staphylococcus sp. strain PN/Y used in the present study was isolated from a PAH-contaminated soil by enrichment culture technique with phenanthrene as the sole source of carbon and energy [25]. Cells were grown in liquid mineral salt medium (MSM, pH 7.0) containing (l1) 3.34 g of K2HPO4, 0.87 g of NaH2PO4, 2.0 g of NH4Cl, 123 mg of nitrilotriacetic acid, 200 mg of MgSO47H2O, 12 mg of FeSO47H2O, 3 mg of MnSO4H2O, 3 mg of ZnSO47H2O and 1 mg of CoCl26H2O. Solid media contained 2% (w/v) agar (HiMedia, India). 2.3. Degradation experiments Phenanthrene degradation experiments were conducted in 100 ml Erlenmeyer flask containing 25 ml of MSM supplemented with 0.05–4.0 g l1 of phenanthrene as sole carbon source and incubated for different periods of time at 28 8C on a rotary shaker (180 rpm). To investigate the effect of temperature and pH on phenanthrene degradation by Staphylococcus sp. strain PN/Y, replicate bacterial cultures in MSM containing 1 g l1 of phenanthrene were incubated at various temperature and at different pH of the medium. In the degradation of 2H1NA used as sole carbon source in MSM, 2H1NA was supplemented in the range of 0.05–1.0 g l1 while in another experiment, 0.5 g l1 of 2H1NA was supplemented in four installments (125 g l1) at 0, 24, 48 and 72 h. In case of phenathrene degradation, 1% inoculum (OD660 adjusted to 0.5) grown in MSM with phenanthrene (1 g l1) for 72 h while in 2H1NA degradation, 2% inoculum (OD600 adjusted to 0.25) grown in MSM with 2H1NA (0.5 g l1) for 48 h, individually as sole carbon sources were used. Occasionally, the cell growth was estimated as cellular protein amount, which was determined by the method of Lowry with bovine serum albumin as the standard [30]. Unless stated otherwise, each experimental set was performed in triplicate.
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soil and was identified as Staphylococcus sp. strain PN/Y [25]. The organism was able to use phenanthrene as sole source of carbon and energy. Fig. 1A shows the growth profile of Staphylococcus sp. strain PN/Y during degradation at different initial concentrations of phenanthrene, ranging from 0.05 to 1 g l1. When cells were grown on 0.05, 0.1, 0.2, 0.5 and 1.0 g l1 of phenanthrene, the doubling time was 31.55, 30.2, 29.32, 26.84, 25.22 h and the specific growth rate was 0.0314, 0.0331, 0.034, 0.0372 and 0.0396 h1, respectively. However, at higher concentration of phenanthrene (up to 4 g l1), the degradation rate was observed to be slower (Table 1). After a series of phenanthrene degradation experiments at incubation temperatures ranging from 24 to 42 8C and pH values from 5.0 to 9.0 with the optimum concentration of phenanthrene (1 g l1), the optimal conditions for growth of the Staphylococcus sp. strain PN/Y were determined as 28 8C and pH 7.0 under shake culture condition at 180 rpm. Fig. 1B illustrates the biodegradative fate of phenanthrene during incubation with the Staphylococcus sp. strain PN/Y, at different initial concentrations of phenanthrene. During cultivation, biodegradation of phenanthrene experienced a lineardecrease stage concomitant with the log-phase growth of the Staphylococcus sp. strain PN/Y. A good linear relationship between phenanthrene degradation rates (PDR) and the initial concentration of phenanthrene were observed during the log-phase of the growth of the Staphylococcus sp. strain PN/Y (Table 1). A steady increase in PDR was observed with the increase in phenanthrene
2.4. Isolation metabolites and unconverted substrates After incubation, at each sampling point, the spent broth culture were centrifuged (8000 g, 10 min) and the supernatants were acidified to pH 1.5– 2.0 by concentrated hydrochloric acid and extracted thrice with equal volume of ethyl acetate. The combined extracts were dried over anhydrous sodium sulfate and evaporated under reduced pressure at 40 8C. 2.5. Analysis Phenanthrene, 2H1NA and their derived metabolites were resolved and quantified by HPLC system (Waters, USA) on an analytical Inertsil ODS-3 column (5 mm, 4.6 mm 250.0 mm; MetaChem Technologies, Torrance, CA) equipped with a guard column packed with the same stationary phase attached to a Waters 515 solvent delivery system. The biodegraded products were eluted with a programmed methanol–water gradient as solvent system at a flow rate of 1.0 ml min1 and detected by Waters 486 tunable absorbance UV detector at 254 nm. The mobile phase was a 45 min linear gradient from 50% (v/v) to 95% (v/v) aqueous methanol with holding at 95% aqueous methanol for 10 min followed by 95–50% (v/v) aqueous methanol in 5 min. Metabolites were identified by comparing the retention times with that of the authentic compounds analyzed under the same set of conditions [25]. Concentrations of unconverted phenanthrene, remaining and/or accumulated 2-hydroxy-1-naphthoic acid and 2-naphthol were quantified using Millennium Session Manager Software package (version 2.15.01). The Folin-Ciocalteau reaction was performed on the culture supernatants to analyze the presence of phenolic compounds produced during phenanthrene utilization according to the procedure reported earlier [31]. A standard curve was prepared with 2hydroxy-1-naphthoic acid and the concentration of metabolic intermediates was estimated as 2-hydroxy-1-naphthoic acid (2H1NA) equivalents in mg l1.
3. Results and discussion 3.1. Kinetics of phenanthrene degradation and bacterial growth Using an enrichment culture technique, a phenanthrenedegrading bacterium was isolated from petroleum-contaminated
Fig. 1. Growth (A) and degradation of phenanthrene (B) by Staphylococcus sp. strain PN/Y in MSM at various initial phenanthrene concentrations of 50 mg l1 (&), 100 mg l1 (*), 200 mg l1 (~), 500 mg l1 (^) and 1000 mg l1 (*). Bars indicate the errors of three independent samples.
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S. Mallick, T.K. Dutta / Process Biochemistry 43 (2008) 1004–1008
Table 1 The phenanthrene degradation rates (PDR) during the linear-decrease stage at each initial concentration Initial concentration (mg l1)
PDR (mg l1 h1)
R2 for linear relationship a
Incubation period (h)b
100 200 500 1000 2000 4000
0.89 1.69 3.4 7.12 11.85 11.89
0.980 0.982 0.988 0.990 0.982 0.994
24–72 24–72 24–72 24–72 24–72 24–72
R2 represents the regression coefficient. a Linear relationship was determined from five data points within the range of 24–72 h. b The time interval involved in the calculation of PDR.
concentration. However, above 1 g l1of phenanthrene concentration, relative biodegradation rate per gram of phenanthrene falls gradually and at a concentration of 4 g l1, a drastic fall of the same was observed (Table 1). The decreasing biodegradation rate per gram of phenanthrene beyond 1 g l1 of phenanthrene concentration may be due to the increased level of toxic metabolite(s) generated during the degradation process. 3.2. Kinetics of accumulation and utilization of 2H1NA and other metabolites The dynamic fate of the central metabolite 2H1NA in the assimilation of phenanthrene by the Staphylococcus sp. strain PN/Y is shown in Fig. 2A. In the degradation of phenanthrene, highest
accumulation of 2H1NA was noticed during mid-log phase (42– 48 h) of growth irrespective of initial phenanthrene concentration. The accumulation of 2H1NA was found to be 323 mg l1 at 48 h of incubation when grown on 1 g l1 of phenanthrene. However, at higher initial concentration of phenanthrene, amount of accumulated 2H1NA was found to be 487 and 705 mg l1 when grown on 2 and 4 g l1 of phenanthrene, respectively. Since phenanthrene is exclusively assimilated via 2H1NA, it may be inferred that the activity and/or expression of initial set of enzymes including phenanthrene-1,2-dioxygenase transforming phenanthrene to 2H1NA is higher than that of 2H1NA ring-cleavage dioxygenase activity resulting in the accumulation of 2H1NA in the reaction medium. It may be mentioned that 2H1NA is also used as sole carbon source by Staphylococcus sp. strain PN/Y and the oxygen
Fig. 2. Accumulation and utilization of 2H1NA (A), salicylic acid (B) and catechol (C) and Folin-Ciocalteau reactive phenolic compounds (D) by Staphylococcus sp. strain PN/Y during incubation in MSM with phenanthrene as the sole source of carbon and energy at various initial concentrations of phenanthrene. The symbols are the same as in Fig. 1.
S. Mallick, T.K. Dutta / Process Biochemistry 43 (2008) 1004–1008
uptake experiment indicated that the genetic elements responsible for the degradation of phenanthrene to 2H1NA and the metabolism of 2H1NA by a novel meta-cleavage dioxygenase belongs to two different degradative operon [25]. Distinct metabolic regulation of these two operons may possibly lead to the accumulation of 2H1NA until mid-log phase of growth on phenanthrene. However, beyond the mid-log phase, a progressive depletion of 2H1NA was noticed. This may be accounted for reducing accumulation of 2H1NA at this stage due to lower available concentration of its precursor, phenanthrene in the reaction medium and the presence of higher concentration of protein (relative to biomass) involved in the ring-cleavage of 2H1NA. Apart from 2H1NA, dynamic fate of other two metabolites viz., salicylic acid and catechol, in the assimilation of phenanthrene by
Fig. 3. Growth (A) and degradation (B) of 2H1NA by Staphylococcus sp. strain PN/Y in MSM at various initial 2H1NA concentrations of 50 mg l1 (&), 100 mg l1 (*), 200 mg l1 (~), 400 mg l1 (&), 500 mg l1 (^), 650 mg l1 (^), 800 mg l1 (~) and 1000 mg l1 (*). Growth (^) and degradation (&) of 2H1NA (500 mg l1) supplemented in installments, 125 mg l1 at 24 h interval (C).
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the Staphylococcus sp. strain PN/Y were demonstrated (Fig. 2B and C). Very small amounts of both salicylic acid and catechol were found to be accumulated during the degradation process. Maximum accumulation of salicylic acid and catechol were observed to be 2.72 and 1.63 mg l1, respectively during 72 of incubation, when grown on 1 g l1 of phenanthrene. Phenanthrene degraded products were also analyzed to estimate reactive phenolic compounds in the reaction medium. Accumulation of Folin-Ciocalteau reactive phenolic substances (2H1NA equivalents) is presented in Fig. 2D. It has been observed that accumulation pattern of phenolic compounds is similar to that of 2H1NA, which is the major phenolic intermediate in phenanthrene degradation. To understand the growth profile of Staphylococcus sp. strain PN/Y on 2H1NA, various initial concentration of 2H1NA in the range of 0.05–1.0 g l1 was used as sole carbon and energy source (Fig. 3A). While Fig. 3B illustrates the biodegradative fate of 2H1NA during incubation. It was observed that although growth increases with increasing 2H1NA concentration up to 650 mg l1, but a sharp decline in growth was observed at 1 g l1 of 2H1NA. When the cells were grown on 50, 100, 200, 400, 500, 650, 800 and 1000 mg l1 of 2H1NA the growth yield at 96 h was 445, 557, 560, 480, 419, 334, 212 and 66 mg of protein (g 2H1NA)1, respectively. Declining growth at higher initial concentration of 2H1NA may be due to the
Fig. 4. Accumulation of 2-naphthol during the growth of Staphylococcus sp. strain PN/Y in MSM in presence of 1 g l1 of phenanthrene (A) and at various initial concentrations of 2H1NA (B). The symbols are the same as in Fig. 3A.
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possible toxic nature of 2H1NA at higher concentration or toxic metabolite(s) so generated from 2H1NA in the degradation process. To examine the situation, 500 mg l1of 2H1NA was added in installments (125 mg l1 at 24 h interval), and the growth vis-a`vis degradation of 2H1NA was examined (Fig. 3C). It has been observed that when 2H1NA was added in installments, the growth yield was 559 mg protein (g 2H1NA)1, 33.4% higher than the growth yield for the same amount of 2H1NA added at zero hour indicating the possible adverse effect on the growth of the organism at higher initial concentration. It may be mentioned that 2-naphthol, a decarboxylated product of 2H1NA was detected as a minor metabolite in the degradation of phenantherene by Staphylococcus sp. strain PN/Y [25]. Moreover, it has been observed that 2-naphthol was toxic intermediate and the minimum growth inhibitory concentration was found to be 45 mg l1 [25]. Toxicity of 2-naphthol has also been reported earlier in Burkholderia and Pseudomonas spp. [20]. Fig. 4A represents the accumulated 2-naphthol concentration during the growth of Staphylococcus sp. strain PN/Y in presence of phenanthrene (1 g l1) and various initial concentration of 2H1NA. It has been observed that in the degradation of phenanthrene, initially 2-naphthol concentration increases, reaching a peak value of 4.7 mg l1 at 36 h, and then slowly decreases. However, at higher initial concentration of 2H1NA the accumulation of 2naphthol increases, which may be affecting the growth of the organism (Fig. 4B). But when 500 mg l1 of 2H1NA was added in supplements, the concentration of 2-naphthol remained substantially less (data not shown), which again supported the inhibitory effect of 2-naphthol on the growth of the test organism. Since 2naphthol is not the intermediate of phenanthrene assimilatory pathway in Staphylococcus sp. strain PN/Y, the slow decrease in the concentration of accumulated 2-naphthol during incubation may be accounted for the transformation of 2-naphthol to 2-methoxynaphthalene, a dead-end product was detected earlier in the spent culture of Staphylococcus sp. strain PN/Y on phenanthrene and was produced possibly by catechol-O-methyltransferase activity of strain PN/Y against the toxic 2-naphthol [25]. Thus it may be inferred that the degradation kinetics of phenanthrene and the growth of Staphylococcus sp. strain PN/Y depends on the metabolic regulation as well as on the accumulation of growth inhibitory metabolites 2H1NA and/or 2-naphthol. Acknowledgements Financial support for this work was provided by Bose Institute, Kolkata, India. SM was supported with fellowship from the Council of Scientific & Industrial Research, Government of India. References [1] Mastrangela G, Fadda E, Marzia V. Polycyclic aromatic hydrocarbons and cancer in man. Environ Health Perspect 1996;104:1166–70. [2] Marston CP, Pereira ZC, Ferguson J, Fischer L, Hedstrom O, Dashwood WM, et al. Effect of a complex environmental mixture from coal tar containing polycyclic aromatic hydrocarbons (PAH) on tumor initiation, PAH-DNA binding and metabolic activation of carcinogenic PAH in mouse epidermis. Carcinogenesis 2001;22:1077–86. [3] Xue W, Warshawsky D. Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: a review. Toxicol Appl Pharmacol 2005;206:73–93.
[4] Shennan JL. Hydrocarbons as substrates in industrial fermentation. In: Atlas RM, editor. Petroleum microbiology. New York: Macmillan; 1984. p. 643–83. [5] Fawell JK, Hunt S. The polycyclic aromatic hydrocarbons. In: Fawell JK, Hunt S, editors. Environmental toxicology: Organic pollutants. West Sussex: Ellis Horwood; 1988. p. 241–69. [6] Weis LM, Rummel AM, Masten SJ, Trosko JE, Upham BL. Bay and baylike regions of polycyclic aromatic hydrocarbons were potent inhibitors of gap junctional intercellular communication. Environ Health Perspect 1998;106:17–22. [7] Rogoff MH, Wender I. The microbiology of coal. I. Bacterial oxidation of phenanthrene. J Bacteriol 1957;73:264–8. [8] Evans WC, Fernley HN, Griffiths E. Oxidative metabolism of phenanthrene and anthracene by soil pseudomonads: the ring fission mechanism. Biochem J 1965;95:819–31. [9] Kiyohara H, Nagao K, Nomi R. Degradation of phenanthrene through o-phthalate by an Aeromonas sp.. Agric Biol Chem 1976;40:1075–82. [10] Kiyohara H, Nagao K. The catabolism of phenanthrene and naphthalene by bacteria. J Gen Microbiol 1978;105:69–75. [11] Kiyohara H, Nagao K, Kouno K, Yano K. Phenanthrene-degrading phenotype of Alcaligenes faecalis AFK2. Appl Environ Microbiol 1982;43:458–61. [12] Bransley EA. Phthalate pathway of phenanthrene metabolism: formation of 20 carboxybenzalpyruvate. J Bacteriol 1983;154:113–7. [13] Ghosh DK, Mishra AK. Oxidation of phenanthrene by strain of Micrococcus: evidence of protocatechuate pathway. Curr Microbiol 1983;9:219–24. [14] Gibson DT, Subramanian V. Microbial degradation of aromatic hydrocarbons. In: Gibson DT, editor. Microbial degradation of organic compounds. New York: Dekker Inc; 1984. p. 181–252. [15] Houghton JE, Shanley MS. Catabolic potential of Pseudomaonads: a regulatory perspective. In: Chaudhry RG, editor. Biological degradation and bioremediation of toxic chemicals. London: Chapman and Hall; 1994. p. 11–32. [16] Adachi K, Iwabuchi T, Sano H, Harayama S. Structure of the ring cleavage product of 1-hydroxy-2-naphthoate, an intermediate of the phenanthrene-degradative pathway of Nocardioides sp. strain KP7. J Bacteriol 1999;181:757–63. [17] Samanta SK, Chakraborti AK, Jain RK. Degradation of phenanthrene by different bacteria: evidence for novel transformation sequences involving the formation of 1-naphthol. Appl Microbiol Biotechnol 1999;53:98–107. [18] Prabhu Y, Phale PS. Biodegradation of phenanthrene by Pseudomonas sp. Strain PP2: novel metabolic pathway, role of biosurfactant and cell surface hydrophobicity in hydrocarbon assimilation. Appl Microbiol Biotechnol 2003;61:342–51. [19] Kim Y, Freeman JP, Moody JD, Engesser K, Cerniglia CE. Effects of pH on the degradation of phenanthrene and pyrene by Mycobacterium vanbaalenii PYR-1. Appl Microbiol Biotechnol 2005;67:275–85. [20] Balashova NV, Kosheleva IA, Golovchenko NP, Boronin AM. Phenanthrene metabolism by Pseudomonas and Burkholderia strains. Process Biochem 1999;35:291–6. [21] Pinyakong O, Habe H, Supaka N, Pinpanichkarn P, Juntongjin K, Yoshida T, et al. Identification of novel metabolites in the degradation of phenanthrene by Sphingomonas sp. strain P2. FEMS Microbiol Lett 2000;191:115–21. [22] Keum YS, Seo JS, Hu Y, Li QX. Degradation pathways of Phenanthrene by Sinorhizobium sp. C4. Appl Microbiol Biotechnol 2006;71:935–41. [23] Seo JS, Keum YS, Hu Y, Lee SE, Li QX. Phenanthrene degradation in Arthrobacter sp. P1-1: initial 1,2-, 3,4- and 9,10-dioxygenation, and meta- and orthocleavages of naphthalene-1,2-diol after its formation from naphthalene-1,2dicarboxylic acid and hydroxyl naphthoic acids. Chemosphere 2006;65:2388– 94. [24] Seo JS, Keum YS, Hu Y, Lee SE, Li QX. Degradation of phenanthrene by Burkholderia sp. C3: initial 1,2- and 3,4-dioxygenation and meta- and ortho-cleavage of naphthalene-1,2-diol. Biodegradation 2006;18:123–31. [25] Mallick S, Chatterjee S, Dutta TK. A novel degradation pathway in the assimilation of phenanthrene by Staphylococcus sp. strain PN/Y via meta-cleavage of 2-hydroxy-1-naphthoic acid: formation of trans-2,3-dioxo-5-(20 -hydroxyphenyl)-pent-4-enoic acid. Microbiology 2007;153:2104–15. [26] Tiehm A. Degradation of polycyclic aromatic hydrocarbons in the presence of synthetic surfactants. Appl Environ Microbiol 1994;60:258–63. [27] Grifoll M, Selifonov SA, Gatlin CV, Chapman PJ. Actions of a versatile fluorinedegrading bacterial iosolate on polycyclic aromatic compounds. Appl Environ Microbiol 1995;61:3711–23. [28] Tian L, Ma P, Zhong J. Kinetics and key enzyme activities of phenanthrene degradation by Pseudomonas mendocina. Process Biochem 2002;37:1431–7. [29] Tian L, Ma P, Zhong J. Impact of the presence of salicylate or glucose on enzyme activity and phenanthrene degradation by Pseudomonas mendocina. Process Biochem 2003;38:1125–32. [30] Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. 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Contents lists available at ScienceDirect
Process Biochemistry journal homepage: www.elsevier.com/locate/procbio
Short communication
Kinetics of phenanthrene degradation by Staphylococcus sp. strain PN/Y involving 2-hydroxy-1-naphthoic acid in a novel metabolic pathway Somnath Mallick, Tapan K. Dutta * Department of Microbiology, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, India
A R T I C L E I N F O
A B S T R A C T
Article history: Received 7 November 2007 Received in revised form 15 April 2008 Accepted 28 April 2008
Staphylococcus sp. strain PN/Y isolated from creosote-contaminated soil, was previously reported to degrade phenanthrene as sole source of carbon and energy. Unlike other phenanthrene degraders, Staphylococcus sp. PN/Y degraded phenanthrene by a novel pathway involving 2-hydroxy-1-naphthoic acid (2H1NA), which was further metabolized by unique meta-cleavage dioxygenase, ultimately leading to TCA cycle intermediates. In the present study, kinetics of phenanthrene degradation and the dynamic fate of the key intermediates, 2H1NA, salicylic acid and catechol were demonstrated. When cells were grown on 50, 100, 200, 500 and 1000 mg l1 of phenanthrene, the doubling time was 31.55, 30.2, 29.32, 26.84, 25.22 h and the specific growth rate was 0.0314, 0.0331, 0.034, 0.0372 and 0.0396 h1, respectively. At a concentration of 1 g l1 of phenanthrene, the maximum accumulation of 2H1NA was found to be 323 mg l1. In addition, 2H1NA utilized by Staphylococcus sp. PN/Y as sole carbon source, the growth yield at 96 h was 445, 557, 560, 480, 419, 334, 212 and 66 mg of protein (g 2H1NA)1 when cells were grown on 50, 100, 200, 400, 500, 650, 800 and 1000 mg l1 of 2H1NA, respectively. Growth was found to be inhibited at initial higher concentration of 2H1NA, which may attributed due to concentration-dependent toxicity of 2H1NA and/or the toxicity of its decarboxylated metabolite, 2naphthol. ß 2008 Elsevier Ltd. All rights reserved.
Keywords: Phenanthrene 2-Hydroxy-1-naphthoic acid 2-Naphthol Biodegradation Staphylococcus sp. Kinetic study
1. Introduction Polycyclic aromatic hydrocarbons (PAHs) are an important class of ubiquitous environmental contaminants because of their high potential toxicity, mutagenicity and/or carcinogenicity [1–3]. Among other PAHs, phenanthrene, a polycyclic aromatic hydrocarbon with three condensed rings fused in angular fashion is widely distributed in the environment primarily because of anthropogenic and pyrolytic processes. Phenanthrene has been used in the synthesis of different organic compounds like pesticides, fungicides, detergents, dyes and mothballs [4]. Phenanthrene is known to be a photosensitizer of human skin, a mild allergen and mutagenic to bacterial systems under specific conditions [5]. It is a weak inducer of sister chromatid exchanges and a potent inhibitor of gap junctional intercellular communication [6]. Since phenanthrene is the smallest aromatic hydrocarbon to have a ‘‘bay-region’’ and a ‘‘K-region’’, it is often used as a model substrate for studies on the metabolism of carcinogenic polycyclic aromatic hydrocarbons.
* Corresponding author. Tel.: +91 33 2355 9544; fax: +91 33 2355 3886. E-mail address: [email protected] (T.K. Dutta). 1359-5113/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.procbio.2008.04.022
Numerous microbial species are known to degrade phenanthrene, and there appear to be several metabolic routes available, depending upon the species, strains, and even the culture conditions [7–19]. In majority of cases reported so far, the degradation pathway proceeds through the well-known central metabolite 1-hydroxy-2-naphthoic acid. In recent years, some investigators predict assimilation of phenanthrene via both 1hydroxy-2-naphthoic acid and 2-hydroxy-1-naphthoic acid, the later being the minor product [20–24]. In our previous study, we reported phenanthrene degradation exclusively via the central metabolite 2-hydroxy-1-naphthoic acid through a novel phenanthrene degradation pathway, with a new phenanthrene degrading bacterium Staphylococcus sp. strain PN/Y [25]. Although, aerobic biodegradation is an important attenuation mechanism for PAHs in the environment but the ability to estimate PAH biodegradation rates is important for predicting environmental fate and for designing remediation efforts. During phenanthrene biodegradation, the degradation kinetics including the fate of the metabolite 1-hydroxy-2-naphthoic acid (1H2NA) was described [26–29], however, no such effort had been made in the alternative phenanthrene degradation pathway that involves 2-hydroxy-1-naphthoic acid. In the present study, the kinetics of phenanthrene degradation and the dynamic fate of the intermediate 2-hydroxy-1-naphthoic acid were investigated in the
S. Mallick, T.K. Dutta / Process Biochemistry 43 (2008) 1004–1008
novel phenanthrene degradation pathway by Staphylococcus sp. strain PN/Y. 2. Materials and methods 2.1. Chemicals Phenanthrene, 2-hydroxy-1-naphthoic acid, salicylic acid, catechol and 2naphthol used in this study were purchased from Sigma–Aldrich (Milwaukee, WI). All other chemicals and reagents used in this study were of analytical grade and used without further purification. 2.2. Organism and culture conditions A new phenanthrene degrading strain Staphylococcus sp. strain PN/Y used in the present study was isolated from a PAH-contaminated soil by enrichment culture technique with phenanthrene as the sole source of carbon and energy [25]. Cells were grown in liquid mineral salt medium (MSM, pH 7.0) containing (l1) 3.34 g of K2HPO4, 0.87 g of NaH2PO4, 2.0 g of NH4Cl, 123 mg of nitrilotriacetic acid, 200 mg of MgSO47H2O, 12 mg of FeSO47H2O, 3 mg of MnSO4H2O, 3 mg of ZnSO47H2O and 1 mg of CoCl26H2O. Solid media contained 2% (w/v) agar (HiMedia, India). 2.3. Degradation experiments Phenanthrene degradation experiments were conducted in 100 ml Erlenmeyer flask containing 25 ml of MSM supplemented with 0.05–4.0 g l1 of phenanthrene as sole carbon source and incubated for different periods of time at 28 8C on a rotary shaker (180 rpm). To investigate the effect of temperature and pH on phenanthrene degradation by Staphylococcus sp. strain PN/Y, replicate bacterial cultures in MSM containing 1 g l1 of phenanthrene were incubated at various temperature and at different pH of the medium. In the degradation of 2H1NA used as sole carbon source in MSM, 2H1NA was supplemented in the range of 0.05–1.0 g l1 while in another experiment, 0.5 g l1 of 2H1NA was supplemented in four installments (125 g l1) at 0, 24, 48 and 72 h. In case of phenathrene degradation, 1% inoculum (OD660 adjusted to 0.5) grown in MSM with phenanthrene (1 g l1) for 72 h while in 2H1NA degradation, 2% inoculum (OD600 adjusted to 0.25) grown in MSM with 2H1NA (0.5 g l1) for 48 h, individually as sole carbon sources were used. Occasionally, the cell growth was estimated as cellular protein amount, which was determined by the method of Lowry with bovine serum albumin as the standard [30]. Unless stated otherwise, each experimental set was performed in triplicate.
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soil and was identified as Staphylococcus sp. strain PN/Y [25]. The organism was able to use phenanthrene as sole source of carbon and energy. Fig. 1A shows the growth profile of Staphylococcus sp. strain PN/Y during degradation at different initial concentrations of phenanthrene, ranging from 0.05 to 1 g l1. When cells were grown on 0.05, 0.1, 0.2, 0.5 and 1.0 g l1 of phenanthrene, the doubling time was 31.55, 30.2, 29.32, 26.84, 25.22 h and the specific growth rate was 0.0314, 0.0331, 0.034, 0.0372 and 0.0396 h1, respectively. However, at higher concentration of phenanthrene (up to 4 g l1), the degradation rate was observed to be slower (Table 1). After a series of phenanthrene degradation experiments at incubation temperatures ranging from 24 to 42 8C and pH values from 5.0 to 9.0 with the optimum concentration of phenanthrene (1 g l1), the optimal conditions for growth of the Staphylococcus sp. strain PN/Y were determined as 28 8C and pH 7.0 under shake culture condition at 180 rpm. Fig. 1B illustrates the biodegradative fate of phenanthrene during incubation with the Staphylococcus sp. strain PN/Y, at different initial concentrations of phenanthrene. During cultivation, biodegradation of phenanthrene experienced a lineardecrease stage concomitant with the log-phase growth of the Staphylococcus sp. strain PN/Y. A good linear relationship between phenanthrene degradation rates (PDR) and the initial concentration of phenanthrene were observed during the log-phase of the growth of the Staphylococcus sp. strain PN/Y (Table 1). A steady increase in PDR was observed with the increase in phenanthrene
2.4. Isolation metabolites and unconverted substrates After incubation, at each sampling point, the spent broth culture were centrifuged (8000 g, 10 min) and the supernatants were acidified to pH 1.5– 2.0 by concentrated hydrochloric acid and extracted thrice with equal volume of ethyl acetate. The combined extracts were dried over anhydrous sodium sulfate and evaporated under reduced pressure at 40 8C. 2.5. Analysis Phenanthrene, 2H1NA and their derived metabolites were resolved and quantified by HPLC system (Waters, USA) on an analytical Inertsil ODS-3 column (5 mm, 4.6 mm 250.0 mm; MetaChem Technologies, Torrance, CA) equipped with a guard column packed with the same stationary phase attached to a Waters 515 solvent delivery system. The biodegraded products were eluted with a programmed methanol–water gradient as solvent system at a flow rate of 1.0 ml min1 and detected by Waters 486 tunable absorbance UV detector at 254 nm. The mobile phase was a 45 min linear gradient from 50% (v/v) to 95% (v/v) aqueous methanol with holding at 95% aqueous methanol for 10 min followed by 95–50% (v/v) aqueous methanol in 5 min. Metabolites were identified by comparing the retention times with that of the authentic compounds analyzed under the same set of conditions [25]. Concentrations of unconverted phenanthrene, remaining and/or accumulated 2-hydroxy-1-naphthoic acid and 2-naphthol were quantified using Millennium Session Manager Software package (version 2.15.01). The Folin-Ciocalteau reaction was performed on the culture supernatants to analyze the presence of phenolic compounds produced during phenanthrene utilization according to the procedure reported earlier [31]. A standard curve was prepared with 2hydroxy-1-naphthoic acid and the concentration of metabolic intermediates was estimated as 2-hydroxy-1-naphthoic acid (2H1NA) equivalents in mg l1.
3. Results and discussion 3.1. Kinetics of phenanthrene degradation and bacterial growth Using an enrichment culture technique, a phenanthrenedegrading bacterium was isolated from petroleum-contaminated
Fig. 1. Growth (A) and degradation of phenanthrene (B) by Staphylococcus sp. strain PN/Y in MSM at various initial phenanthrene concentrations of 50 mg l1 (&), 100 mg l1 (*), 200 mg l1 (~), 500 mg l1 (^) and 1000 mg l1 (*). Bars indicate the errors of three independent samples.
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Table 1 The phenanthrene degradation rates (PDR) during the linear-decrease stage at each initial concentration Initial concentration (mg l1)
PDR (mg l1 h1)
R2 for linear relationship a
Incubation period (h)b
100 200 500 1000 2000 4000
0.89 1.69 3.4 7.12 11.85 11.89
0.980 0.982 0.988 0.990 0.982 0.994
24–72 24–72 24–72 24–72 24–72 24–72
R2 represents the regression coefficient. a Linear relationship was determined from five data points within the range of 24–72 h. b The time interval involved in the calculation of PDR.
concentration. However, above 1 g l1of phenanthrene concentration, relative biodegradation rate per gram of phenanthrene falls gradually and at a concentration of 4 g l1, a drastic fall of the same was observed (Table 1). The decreasing biodegradation rate per gram of phenanthrene beyond 1 g l1 of phenanthrene concentration may be due to the increased level of toxic metabolite(s) generated during the degradation process. 3.2. Kinetics of accumulation and utilization of 2H1NA and other metabolites The dynamic fate of the central metabolite 2H1NA in the assimilation of phenanthrene by the Staphylococcus sp. strain PN/Y is shown in Fig. 2A. In the degradation of phenanthrene, highest
accumulation of 2H1NA was noticed during mid-log phase (42– 48 h) of growth irrespective of initial phenanthrene concentration. The accumulation of 2H1NA was found to be 323 mg l1 at 48 h of incubation when grown on 1 g l1 of phenanthrene. However, at higher initial concentration of phenanthrene, amount of accumulated 2H1NA was found to be 487 and 705 mg l1 when grown on 2 and 4 g l1 of phenanthrene, respectively. Since phenanthrene is exclusively assimilated via 2H1NA, it may be inferred that the activity and/or expression of initial set of enzymes including phenanthrene-1,2-dioxygenase transforming phenanthrene to 2H1NA is higher than that of 2H1NA ring-cleavage dioxygenase activity resulting in the accumulation of 2H1NA in the reaction medium. It may be mentioned that 2H1NA is also used as sole carbon source by Staphylococcus sp. strain PN/Y and the oxygen
Fig. 2. Accumulation and utilization of 2H1NA (A), salicylic acid (B) and catechol (C) and Folin-Ciocalteau reactive phenolic compounds (D) by Staphylococcus sp. strain PN/Y during incubation in MSM with phenanthrene as the sole source of carbon and energy at various initial concentrations of phenanthrene. The symbols are the same as in Fig. 1.
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uptake experiment indicated that the genetic elements responsible for the degradation of phenanthrene to 2H1NA and the metabolism of 2H1NA by a novel meta-cleavage dioxygenase belongs to two different degradative operon [25]. Distinct metabolic regulation of these two operons may possibly lead to the accumulation of 2H1NA until mid-log phase of growth on phenanthrene. However, beyond the mid-log phase, a progressive depletion of 2H1NA was noticed. This may be accounted for reducing accumulation of 2H1NA at this stage due to lower available concentration of its precursor, phenanthrene in the reaction medium and the presence of higher concentration of protein (relative to biomass) involved in the ring-cleavage of 2H1NA. Apart from 2H1NA, dynamic fate of other two metabolites viz., salicylic acid and catechol, in the assimilation of phenanthrene by
Fig. 3. Growth (A) and degradation (B) of 2H1NA by Staphylococcus sp. strain PN/Y in MSM at various initial 2H1NA concentrations of 50 mg l1 (&), 100 mg l1 (*), 200 mg l1 (~), 400 mg l1 (&), 500 mg l1 (^), 650 mg l1 (^), 800 mg l1 (~) and 1000 mg l1 (*). Growth (^) and degradation (&) of 2H1NA (500 mg l1) supplemented in installments, 125 mg l1 at 24 h interval (C).
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the Staphylococcus sp. strain PN/Y were demonstrated (Fig. 2B and C). Very small amounts of both salicylic acid and catechol were found to be accumulated during the degradation process. Maximum accumulation of salicylic acid and catechol were observed to be 2.72 and 1.63 mg l1, respectively during 72 of incubation, when grown on 1 g l1 of phenanthrene. Phenanthrene degraded products were also analyzed to estimate reactive phenolic compounds in the reaction medium. Accumulation of Folin-Ciocalteau reactive phenolic substances (2H1NA equivalents) is presented in Fig. 2D. It has been observed that accumulation pattern of phenolic compounds is similar to that of 2H1NA, which is the major phenolic intermediate in phenanthrene degradation. To understand the growth profile of Staphylococcus sp. strain PN/Y on 2H1NA, various initial concentration of 2H1NA in the range of 0.05–1.0 g l1 was used as sole carbon and energy source (Fig. 3A). While Fig. 3B illustrates the biodegradative fate of 2H1NA during incubation. It was observed that although growth increases with increasing 2H1NA concentration up to 650 mg l1, but a sharp decline in growth was observed at 1 g l1 of 2H1NA. When the cells were grown on 50, 100, 200, 400, 500, 650, 800 and 1000 mg l1 of 2H1NA the growth yield at 96 h was 445, 557, 560, 480, 419, 334, 212 and 66 mg of protein (g 2H1NA)1, respectively. Declining growth at higher initial concentration of 2H1NA may be due to the
Fig. 4. Accumulation of 2-naphthol during the growth of Staphylococcus sp. strain PN/Y in MSM in presence of 1 g l1 of phenanthrene (A) and at various initial concentrations of 2H1NA (B). The symbols are the same as in Fig. 3A.
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possible toxic nature of 2H1NA at higher concentration or toxic metabolite(s) so generated from 2H1NA in the degradation process. To examine the situation, 500 mg l1of 2H1NA was added in installments (125 mg l1 at 24 h interval), and the growth vis-a`vis degradation of 2H1NA was examined (Fig. 3C). It has been observed that when 2H1NA was added in installments, the growth yield was 559 mg protein (g 2H1NA)1, 33.4% higher than the growth yield for the same amount of 2H1NA added at zero hour indicating the possible adverse effect on the growth of the organism at higher initial concentration. It may be mentioned that 2-naphthol, a decarboxylated product of 2H1NA was detected as a minor metabolite in the degradation of phenantherene by Staphylococcus sp. strain PN/Y [25]. Moreover, it has been observed that 2-naphthol was toxic intermediate and the minimum growth inhibitory concentration was found to be 45 mg l1 [25]. Toxicity of 2-naphthol has also been reported earlier in Burkholderia and Pseudomonas spp. [20]. Fig. 4A represents the accumulated 2-naphthol concentration during the growth of Staphylococcus sp. strain PN/Y in presence of phenanthrene (1 g l1) and various initial concentration of 2H1NA. It has been observed that in the degradation of phenanthrene, initially 2-naphthol concentration increases, reaching a peak value of 4.7 mg l1 at 36 h, and then slowly decreases. However, at higher initial concentration of 2H1NA the accumulation of 2naphthol increases, which may be affecting the growth of the organism (Fig. 4B). But when 500 mg l1 of 2H1NA was added in supplements, the concentration of 2-naphthol remained substantially less (data not shown), which again supported the inhibitory effect of 2-naphthol on the growth of the test organism. Since 2naphthol is not the intermediate of phenanthrene assimilatory pathway in Staphylococcus sp. strain PN/Y, the slow decrease in the concentration of accumulated 2-naphthol during incubation may be accounted for the transformation of 2-naphthol to 2-methoxynaphthalene, a dead-end product was detected earlier in the spent culture of Staphylococcus sp. strain PN/Y on phenanthrene and was produced possibly by catechol-O-methyltransferase activity of strain PN/Y against the toxic 2-naphthol [25]. Thus it may be inferred that the degradation kinetics of phenanthrene and the growth of Staphylococcus sp. strain PN/Y depends on the metabolic regulation as well as on the accumulation of growth inhibitory metabolites 2H1NA and/or 2-naphthol. Acknowledgements Financial support for this work was provided by Bose Institute, Kolkata, India. SM was supported with fellowship from the Council of Scientific & Industrial Research, Government of India. References [1] Mastrangela G, Fadda E, Marzia V. Polycyclic aromatic hydrocarbons and cancer in man. Environ Health Perspect 1996;104:1166–70. [2] Marston CP, Pereira ZC, Ferguson J, Fischer L, Hedstrom O, Dashwood WM, et al. Effect of a complex environmental mixture from coal tar containing polycyclic aromatic hydrocarbons (PAH) on tumor initiation, PAH-DNA binding and metabolic activation of carcinogenic PAH in mouse epidermis. Carcinogenesis 2001;22:1077–86. [3] Xue W, Warshawsky D. Metabolic activation of polycyclic and heterocyclic aromatic hydrocarbons and DNA damage: a review. Toxicol Appl Pharmacol 2005;206:73–93.
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