CURRENT MICROBIOLOGY Vol. 43 (2001), pp. 440 – 443 DOI: 10.1007/s002840010335
Current Microbiology An International Journal © Springer-Verlag New York Inc. 2001
Degradation of 2-Methylnaphthalene by Pseudomonas sp. Strain NGK1 U. Sharanagouda, T.B. Karegoudar Department of Biochemistry, Gulbarga University, Gulbarga-585 106, India Received: 20 March 2001 / Accepted: 25 April 2001
Abstract. Pseudomonas sp. strain NGK1, a soil bacterium isolated by naphthalene enrichment from biological waste effluent treatment, capable of utilizing 2-methylnaphthalene as sole source of carbon and energy. To deduce the pathway for biodegradation of 2-methylnaphthalene, metabolites were isolated from the spent medium and identified by thin-layer chromatography and high-performance liquid chromatography. The characterization of purified metabolites, oxygen uptake studies, and enzyme activities revealed that the strain degrades 2-methylnaphthalene through more than one pathway. The growth of the bacterium, utilization of 2-methylnaphthalene, and 4-methylsalicylate accumulation by Pseudomonas sp. strain NGK1 were studied at various incubation periods.
Polycyclic aromatic hydrocarbons (PAHs) are compounds of environmental and human health concern. Some of the PAHs and their biotransformation products have been shown to be toxic, mutagenic, carcinogenic, and teratogenic [9]. Naphthalene and 2-methylnaphthalene are among the most toxic components in the watersoluble fraction of crude oils and have been shown to be concentrated in vertebrate and invertebrate marine organisms [1, 12, 17]. These compounds have also been identified in commercial mosquito repellents [22, 23] and in an aromatic solvent, Aerotex 3470 [19]. Exposure to naphthalene and 2-methylnaphthalne has been reported to cause a decrease in hemoglobin concentration, inhibition of oxygen consumption, and pulmonary damage in various experimental animals [6, 11]. There are several reports available on the degradation of naphthalene by various microorganisms [2, 8, 15, 16, 21]. However, very few reports are available on the degradation of 2-methylnaphthalene by microorganisms. The metabolism of 2-methylnaphthalne has been investigated in rat, rainbow trout [4], fungi [5], and bacteria [14]. Here we report the results on the degradation of 2-methylnaphthalene and accumulation of 4-methylsalicylate by Pseudomonas sp. strain NGK1 at different incubation periods.
Correspondence to: T.B. Karegoudar; email: goudartbk@rediffmail. com
Materials and Methods Chemicals. 2-methylnaphthalene (2-MN), 4-methylsalicylate, and 4-methylcatechol were procured from Lancaster, England. 2-Naphthoic acid was synthesized by oxidizing the methyl side chain of 2-methylnaphthalene in presence of sodium dichromate and sulfuric acid. The other chemicals used were of analytical grade reagents. Microorganism and culture conditions. The Pseudomonas sp. strain NGK1 (NCIM 5120) used in this investigation was isolated from biological wastewater treatment effluent by the naphthalene enrichment culture technique [15]. The culture was maintained on Luria-Bertaini (LB) agar medium. The Pseudomonas sp. strain NGK1 was grown in mineral salts medium with the following composition: 0.38 g K2HPO4, 0.2 g MgSO4 ⫻ 7H20, 1 g KNO3, and 0.05 g FeCl3 in one liter of demineralized water. The pH of the medium was adjusted to 7 and the medium was sterilized. 2-Methylnaphathlene (0.2% wt/vol) was dissolved in a minimum amount of N, N⬘-dimethylformamide-(DMF) and forced into the mineral salt medium containing Tween-80 (approximately 80⫻ critical micelle concentration) with a syringe. This medium was then subjected to ultrasonication (Ultrasonicator Vibra-Cell model 375, USA) for 1 min. The final 2-methylnaphthalene mineral salt medium appeared milky. This medium was inoculated with exponentially growing culture and incubated at room temperature (30⫾ 2° C) on a rotary shaker at 150 rpm. Samples were removed at various time intervals and growth was measured spectrophotometrically at 660 nm. Estimations. The utilization of 2-MN by this bacterium at regular intervals of time was assayed by extraction of residual 2-MN from the media, followed by quantitative UV analysis with UV-Vis Spectrophotometer (Shimadzu model 160A, Japan). The accumulation of 4-methylsalicylate in the culture medium at different time intervals was detected and quantified by the method of Arhana and Brown [2]. Identification and characterization of metabolites. The medium was acidified with 6 M HCl to pH 2.0 and extracted twice with equal volume of ethylacetate. The extracts were dried over anhydrous sodium
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U. Sharanagouda and T.B. Karegoudar: Catabolism of 2-Methylnaphthlene
Fig. 1. Utilization of 2-methylnaphthalene and accumulation of 4-methylsalicylate during the growth of Pseudomonas sp. strain NGK1. (F) growth, (■) residual 2-methylnaphthalene, and (Œ) accumulation of 4-methylsalicylate.
sulfate. The metabolites were isolated by preparative thin layer chromatography with the different solvent systems: (A) cyclohexane-chloroform-acetone (4:1:1), (B) benzene-1, 4-dioxan-acetic acid (45:12:12). The metabolites were visualized with UV lamp (max 254 nm) and detected with detection reagents. The areas carrying the components on TLC were removed from the plates by scraping and the compounds were eluted with methanol and identified by qualitative UV analysis by comparison with standard solution. The reverse phase column HPLC was operated at 28⫾ 2° C with a solvent system, methanol-water (9:1) at a flow rate of 1 ml/min. Metabolites were identified by comparison of retention time with authentic compounds. Oxygen uptake studies. The oxygen uptake by whole cells of Pseudomonas sp. strain NGK1 grown on 2-MN was performed in an oxygraph fitted with Clark type O2 electrode (Hansatech, Germany). The cells were harvested in the early logarithmic phase by centrifugation at 12,000 ⫻ g for 20 min and washed twice with 0.05 M phosphate buffer, pH 7. The final 1 ml reaction mixture contained 50 mmol phosphate buffer (pH 7.5), 100 nmol of substrates in 10l of N,N⬘-dimethylformamide and appropriate amount of cells (4 mg/ml dry weight). Oxygen uptake rates are expressed as nmol of O2 consumed min⫺1 mg⫺1 of dry cells. The values were corrected for endogenous respiration. Enzyme assays. The cell-free extract was prepared from the Pseudomonas sp strain NGK1 grown in the mineral salt medium containing 2-MN [15]. The activity of naphthalene dioxygenase in cell-free extract was measured spectrophotometrically by following the reduction of NAD⫹ at 340 nm [7]. Salicylate hydroxylase activity was also determined by measuring the reduction of NAD⫹ at 340 nm [20]. Catechol2,3-dioxygenase was assayed spectrophotometrically by monitoring the appearance of different semialdehydes from the catechol and 4-methylcatechol which have a max at 375 and 382 nm, respectively [3]. Catechol-1,2-dioxygenase activity was performed by the method of Hegeman [10]. Specific activity of crude extract was calculated as mol of substrate degraded/product formed per min per mg of protein under assay conditions. Protein concentration was determined by the method of Lowry et, al. using bovine serum albumin as standard [13]. Mode of ring cleavage was determined according to the Rothera’s test [18].
Results and Discussion Growth and identification of metabolites. The Pseudomonas sp. strain NGK1 isolated by naphthalene enrichment is also capable of utilizing 2-MN as the sole source of carbon and energy. The maximum growth of the bacterium on 2-MN was observed at 58 – 60 h of incubation (Fig. 1). It is evident that the growth of the bacterium with 2-MN increases with increase in incubation period. The bacterium showed an initial lag phase of 24 h, after which it entered into exponential growth phase that lasted for 48 h. The initial cell population 2 ⫻ 104 cfu ml⫺1 increased to 8 ⫻ 108 cfu ml⫺1 after 24 h, 9 ⫻ 1010 cfu ml⫺1 at 36 h and 26 ⫻ 1011 cfu ml⫺1 at 60 h of incubation. The ethylacetate extracts of the spent medium of this bacterium grown on 2-MN showed the accumulation of 4-methylsalicylate, 4-methylcatechol, and 2-naphthoic acid as intermediates. The metabolites were identified by comparing Rf, Rt, and max values with that of authentic compounds in different solvent systems (Table 1). Oxygen uptake studies. Oxygen uptake studies with washed cells of Pseudomonas sp. strain NGK1, grown on 2-MN, oxidized 2-MN, naphthalene, 4-methylcatechol, and catechol rapidly, whereas the same cells oxidized 4-methylsalicylate, salicylate, and 2-naphthoic acid readily. However, the rates of oxidation were considerably less with 1,2-Dihydroxynaphthalene and salicylaldehyde but failed to oxidize 2-hydroxy-3-naphthoic acid and 2,4-dihydroxybenzoic acid (Table 2) Enzymatic studies. The enzymatic assay of the crude extract of 2-MN grown cells showed high activities of
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CURRENT MICROBIOLOGY Vol. 43 (2001)
Table 1. Rf, Rt, and max values of the isolated and authentic compounds Rf values in solvent system A Compounds 4-methylsalicylate 4-methylcatechol 2-naphthoic acid
max in methanol
Rt values in HPLC
B
a
b
a
b
a
b
a
b
0.087 0.400 0.677
0.090 0.401 0.679
0.838 0.872 0.937
0.839 0.875 0.938
18.46 03.35 03.25
18.57 03.42 03.21
240 236 234
240 236 234
a: isolated. b: authentic. Table 2. Oxygen uptake by whole cells of Pseudomonas sp. strain NGK1 grown on 2-methylnaphthalene
Substrate 2-methylnaphthalene Naphthalene 2-naphthoic acid 1,2-dihydroxy naphthalene Salicylaldehyde 4-methylsalicylate Salicylate 4-methylcatechol Catechol 2-hydroxy-3-naphthoic acid 2,4-dihydroxy benzoic acid
Table 3. Enzyme activities in the cell-free extracts of Pseudomonas sp. strain NGK1 grown on 2-methylnaphthalene.
Oxygen uptake* (nmol min⫺1 mg⫺1 dry cells) 31.66 28.57 21.49 09.99 11.31 25.45 23.94 29.21 27.17 ⬍1.0 ⬍1.0
Enzyme Naphthalene dioxygenase Salicylate hydroxylase Catechol-2,3-dioxygenase Catechol-1,2-dioxygenase
Substrate
Activity (mol min⫺1mg⫺1 protein)
Naphthalene 2-methylnaphthalene Salicylate 4-methylsalicylate Catechol 4-methylcatechol Catechol 4-methylcatechol
0.966 1.020 0.316 0.320 0.406 0.422 ND ND
ND: Not detected. * The values are corrected for endogenous respiration.
naphthalene dioxygenase for both 2-methylnaphthalene and naphthalene as substrates. The cell-free extract showed moderate activity of catechol-2,3-dioxygenase for both 4-methylcatechol and catechol and to a lesser extent of salicylate hydroxylase on 4-methlsalicylate and salicylate. However the cell-free extract did not show the activity of catechol-1,2-dioxygenase (Table 3). The accumulation of 4-methylsalicylate in the spent medium may be because cells grown on 2-MN oxidize 4-methylsalicylate to a lesser extent and the cell-free extract showed less salicylate hydroxylase activity (Fig. 1). The high activity of catechol-2,3-dioxygenase on 4-methylcatechol (0.422) and the absence of catechol1,2-dioxygenase activity revealed that the ring cleavage of 4-methylcatechol is only through meta-pathway. The bacterium degrades 2-MN efficiently by using the same set of enzymes that are involved in the degradation of naphthalene. The accumulation of 4-methylsalicylate and 2-naphthoic acid in the spent medium suggests that 2-MN is catabolized through the involvement of more than one pathways. One pathway accumulated 4-methylsalicylate and 4-methylcatechol, indicating the initial hydroxyla-
tion of aromatic ring of 2-MN in a manner similar to that showed for naphthalene degradation [8, 15]. This bacterium cleaved 4-methylcatechol through meta-pathway as shown by the negative Rothera’s test. This pathway seems to be the major pathway in this bacterium. The accumulation of 2-naphthoic acid in the spent medium indicates the possible alternate pathway for the oxidation of 2-MN, wherein the -CH3 side chain subsequently oxidized to -COOH. Such pathways were proposed in Pseudomonas putida CSV86 [14]. However, the formation of hyrdroxy methylnaphthalene [5] and its subsequent conversion to naphthoic acid by the side chain oxidation has been found exclusively in eukaryotes such as Cunninghamela elegans. ACKNOWLEDGMENTS The authors wish to thank Dr. K. Sreeramulu and Smt. R. M. Vani, Gulbarga University, Gulbarga for their help in carrying out O2 uptake studies. The financial assistance from AICTE, New Delhi, India, is gratefully acknowledged.
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