Correspondence: Tapan K. Dutta, Department of Microbiology, Bose Institute, P-1/12 CIT Scheme VIIM, Kolkata 700054, India. Tel.: +91 33 2569 3241; fax: +91 33 2355 3886; e-mail: email@example.com
The present study describes the assimilation of phenanthrene by an aerobic bacterium, Ochrobactrum sp. strain PWTJD, isolated from municipal waste-contaminated soil sample utilizing phenanthrene as a sole source of carbon and energy. The isolate was identified as Ochrobactrum sp. based on the morphological, nutritional and biochemical characteristics as well as 16S rRNA gene sequence analysis. A combination of chromatographic analyses, oxygen uptake assay and enzymatic studies confirmed the degradation of phenanthrene by the strain PWTJD via 2-hydroxy-1-naphthoic acid, salicylic acid and catechol. The strain PWTJD could also utilize 2-hydroxy-1-naphthoic acid and salicylic acid, while the former was metabolized by a ferric-dependent meta-cleavage dioxygenase. In the lower pathway, salicylic acid was metabolized to catechol and was further degraded by catechol 2,3-dioxygenase to 2-hydroxymuconoaldehyde acid, ultimately leading to tricarboxylic acid cycle intermediates. This is the first report of the complete degradation of a polycyclic aromatic hydrocarbon molecule by Gram-negative Ochrobactrum sp. describing the involvement of the meta-cleavage pathway of 2-hydroxy-1-naphthoic acid in phenanthrene assimilation.
Polycyclic aromatic hydrocarbons (PAHs) comprise a large and diverse group of priority environmental pollutants, which are ubiquitous contaminants derived from both natural and anthropogenic activities. Their abundance in the environment is of great concern, because many of them have been shown to be toxic, mutagenic and/or carcinogenic in nature (Mastrangelo et al., 1996; Marston et al., 2001; Xue & Warshawsky, 2005). The stability, persistency and carcinogenic index of PAHs increase with an increase in the number of aromatic rings, structural angularity and hydrophobicity (Marston et al., 2001).
Phenanthrene has often been used as a model compound to study the microbial metabolism of bay- and K-region-containing PAHs because its structural skeletons are found in many carcinogenic PAHs. Phenanthrene was reported to be degraded by diverse bacterial species documenting several metabolic pathways (Cerniglia, 1984; Peng et al., 2008; Seo et al., 2009 and references therein). In the degradation of phenanthrene, 1-hydroxy-2-naphthoic acid has largely been shown to be one of the intermediates, which can be further degraded either via the phthalate pathway or by the salicylate pathway. However, in the last decade, several studies documented the formation of 2-hydroxy-1-naphthoic acid along with 1-hydroxy-2-naphthoic acid in the degradation of phenanthrene (Balashova et al., 1999; Pinyakong et al., 2000; Kim et al., 2005; Keum et al., 2006; Seo et al., 2006, 2007). In one of the routes, hydroxynaphthoic acids were reported to be transformed to 1,2-dihydroxynaphthalene, which was then metabolized by the classical naphthalene degradation pathway via salicylic acid, while in the other route, 1-hydroxy-2-naphthoic acid was metabolized by ortho-cleavage dioxygenase, leading to the formation tricarboxylic acid cycle intermediates via phthalic acid and protocatechuic acid. However, Mallick et al. (2007) reported for the first time the meta-cleavage of 2-hydroxy-1-naphthoic acid leading to the formation of salicylic acid in the degradation of phenanthrene by a Gram-positive bacterium. Although ortho-cleavage of 1-hydroxy-2-naphthoic acid has been reported from both Gram-positive and Gram-negative bacteria (Kiyohara et al., 1976; Adachi et al., 1999; Zeinali et al., 2008), until now, there has been no report on the meta-cleavage activity of either of the hydroxyl-naphthoic acids from Gram-negative species, which are widely reported to be involved in the degradation of phenanthrene.
Among Gram-negative bacteria, the biodegradative potential of the genus Ochrobactrum has been revealed only recently (El-Sayed et al., 2003; Katsivela et al., 2003; Qiu et al., 2006; Zhong et al., 2007; Yamada et al., 2008). Although Ochrobactrum species are found to be distributed in a wide variety of environmental sources including sewage, soil rhizosphere, animal and human, there is no comprehensive biochemical report on the degradation of PAHs. The present communication describes the isolation and characterization of Gram-negative Ochrobactrum sp. strain PWTJD involved in the assimilation of phenanthrene via meta-cleavage of 2-hydroxy-1-naphthoic acid.
Materials and methods
Enrichment, isolation and characterization of bacterial strain
The test organism used in this study (strain PWTJD) was isolated from municipal waste-contaminated soil (Dhapa, Kolkata, India) using the enrichment culture technique with phenanthrene as the sole source of carbon and energy. The morphological features of the isolate capable of utilizing phenanthrene were studied using a phase-contrast microscope (Olympus CX40, Olympus, Japan). Conventional biochemical tests were performed using standard methods (Kloos & Schleifer, 1986; Smibert & Krieg, 1994). The 16S rRNA gene was amplified using universal bacterial-specific primers f27 and r1492 (Goodwin et al., 2005) and was sequenced according to the manufacturer's specifications (Perkin-Elmer Applied Biosystems). The 16S rRNA gene sequence of the isolate was compared against those in the GenBank database using blast version 2.2.12 of National Center for Biotechnology Information (Altschul et al., 1990).
Media and culture conditions
Cells were grown at 28 °C on a rotary shaker (180 r.p.m.) in 100-mL Erlenmeyer flasks containing 25 mL mineral salt medium (MSM, pH 7.2) and 1 g L−1 of either phenanthrene or succinate as the sole carbon source as described earlier (Mallick et al., 2007). To determine the optimal conditions for phenanthrene degradation by the test organism, different pH values in the range of 5.0–8.0 of the medium, different cultivation temperatures in the range of 15–40 °C and different phenanthrene concentrations in the range of 0.1–2.0 g L−1 were tested individually for growth in MSM. For resting cell transformations, cells were harvested in the late exponential phase by centrifugation (8000 g, 10 min), washed twice with an equal volume of potassium phosphate buffer (50 mM, pH 7.2) and finally resuspended in the same buffer to yield an OD660 nm of 1.0. Phenanthrene and pathway intermediates, viz, 2-hydroxy-1-naphthoic acid, 1-hydroxy-naphtoic acid, 1-naphthol, 2-naphthol, naphthalene-1,2-diol, salicylic acid, o-phthalic acid, protocatechuic acid and catechol in the range of 0.1–1 g L−1 were added individually to washed cell suspensions, and incubated at 28 °C for different periods of time up to 48 h. Unless stated otherwise, each experimental set was performed in triplicate.
Isolation of metabolites
To isolate phenanthrene-degraded metabolites and unutilized phenanthrene, the spent broth and resting cell culture were centrifuged (8000 g, 10 min) and the supernatants were acidified to pH 1.5–2.0 by 6 N hydrochloric acid and extracted three times with equal volumes of ethyl acetate. The combined organic layer was re-extracted with aqueous sodium hydroxide (10 mM). The organic phase was evaporated under reduced pressure (neutral fraction). The aqueous NaOH extracts were acidified as above and then extracted with ethyl acetate (acidic fraction). The combined extracts were dried over anhydrous sodium sulfate and evaporated under reduced pressure. The residues were methylated with a boron trifluoride/methanol solution (Merck) as needed before analysis.
Oxygen uptake assay
Measurements were performed at 25 °C using a YSI model 5300A biological oxygen monitor (Yellow Springs Instrument Co., Yellow Springs, OH) equipped with a Clark-type polarographic oxygen electrodes (YSI model 5331A oxygen probes) and a sample chamber fitted within a YSI model 5301B standard bath. The sample size was 2.0 mL, and the reaction mixture contained 0.5 mL cell suspension (25 mg cells, wet weight), substrate (0.5 mL) and 1 mL phosphate buffer (50 mM, pH 7.0). The reaction was initiated by injecting a suitable amount of the assay substrate and oxygen uptake was monitored for 5 min. Phenanthrene (0.5 mL) was added as a saturated solution (∼1.2 mg L−1), and the possible phenanthrene degradation pathway intermediates were added so as to yield a final concentration of 0.1 mM. The O2 uptake rate was expressed as nanomoles per minute per milligram of protein. The rates were corrected for endogenous oxygen consumption.
Preparation of cell-free extract and enzyme assays
Cells grown in MSM in the presence of phenanthrene (1 g L−1) were harvested at the mid-exponential phase by centrifugation at 8000 g for 10 min at 4 °C. The pellet was washed twice with 10 volumes of 50 mM potassium phosphate buffer (pH 7.2) and resuspended in two volumes of the same buffer. The cell suspension was ultrasonicated (Labsonic-L, Braun Biotech International) for 10 min at 4 °C in 10 pulses and then centrifuged at 20 000 g for 20 min at 4 °C. The supernatant was used as cell-free enzymes for further studies. Protein was measured using the Bradford method (1976) with bovine serum albumin as the standard.
The enzymatic transformations of various substrates were carried out by recording cell-free-extract-catalyzed changes in UV-visible spectra on a Cary 100 Bio UV-visible spectrophotometer (Varian Australia Pty Ltd) using 1 cm path-length quartz cuvettes. The sample and reference cuvettes contained 50 mM potassium phosphate buffer (pH 7.0) in 1-mL volume. The sample cuvette also contained either 2-hydroxy-1-naphthoic acid (50 nmol), salicylaldehyde (50 nmol) or catechol (30 nmol). Data were analyzed using the Varian Cary win uv Scan application software.
The metabolites were resolved by HPLC using a Shimadzu model LC20-AT pump system (Shimadzu Corp., Kyoto, Japan) equipped with a diode array model SIL-M20A detector and an analytical Phenomenex C18 reverse-phase column (Phenomenex Inc., Torrance, CA) attached to a model SIL-20A autosampler. Metabolites were eluted at a flow rate of 1 mL min−1 and detected at 254 nm. UV-visible absorbance spectra were obtained online. The biodegraded products of phenanthrene were eluted with a methanol–water gradient as follows: an initial gradient from 50 : 50 to 95 : 5 (v/v) in 45 min, isocratic for the next 10 min and then back to 50 : 50 (v/v) in 5 min, followed by isocratic for further 3 min. Metabolites were identified by comparing their retention times with those of the authentic compounds analyzed under the same set of conditions.
GC-MS analysis of phenanthrene and its degradation products was performed using a Thermo Scientific model TraceGC Ultra column (Thermo Fischer Scientific Inc., NYSE: TMO) with a model PolarisQ mass spectrometer equipped with a 30 m × 0.25 mm (0.25 μm film thickness) DB-5MS capillary column. The ion source was maintained at 230 °C and both the inlet temperature as well as the transfer line temperature were maintained at 280 °C. The temperature program gave a 2-min hold at 70 °C, an increase to 200 °C at 10 °C min−1, followed by hold for 1 min at 200 °C, further increase to 325 °C at 5 °C min−1 and a 15-min hold at 325 °C. The injection volume was 1 μL, and the carrier gas was helium (1 mL min−1). The mass spectrometer was operated at an electron ionization energy of 70 eV.
Results and discussions
Isolation and characterization of strain PWTJD
Using an enrichment culture technique, a phenanthrene-utilizing bacterium, designated as strain PWTJD, was isolated from a municipal waste-contaminated soil sample. The strain was found to be a Gram-negative rod, nonmotile and nonspore forming. The strain could utilize arabinose, citrate, glucose, lactose, maltose, mannitol and xylose individually as sole carbon sources and was found to be catalase-positive, oxidase-positive, coagulase-positive, nitrate reductase-positive, urease-negative and sensitive to chloramphenicol. On the basis of the above characteristics and other morphological, nutritional and biochemical features of these characteristics (Kloos & Schleifer, 1986; Smibert & Krieg, 1994), strain PWTJD was presumed to be an Ochrobactrum species. To confirm this identification, the partial 16S rRNA gene sequence (1374 bp) of the isolate was determined and deposited in the DDBJ/EMBL/GenBank with the accession no. HM056231. Analysis of that sequence using the blast search revealed 99.9% sequence similarity to Ochrobactrum anthropi LMG 3331T, Ochrobactrum cytisi ESC1T and Ochrobactrum lupini Lup21T. Although the combined analyses indicated a strong correlation at the genus level, a few differential biochemical properties of strain PWTJD were observed when compared with its closest members of the genus Ochrobactrum and as such these data were not sufficient to identify the strain to the species level. Thus, the bacterium has been identified as Ochrobactrum sp. strain PWTJD.
Utilization of phenanthrene by strain PWTJD
Figure 1 shows the growth of strain PWTJD vis-à-vis degradation of phenanthrene under optimal conditions. The strain PWTJD could grow well in MSM at a pH range of 7.2–8.0 and at a temperature range of 25–30 °C. However, both the growth rate and the rate of phenanthrene (1 g L−1) utilization became slower when the pH of the medium was slightly acidic, but favored under a slightly alkaline condition with the optimum pH of 7.2 at 28 °C under shake culture conditions (180 r.p.m.). Although there was a short lag period during the initial incubation, the rate of degradation of phenanthrene rapidly increased after 24 h of incubation and more than 99% of phenanthrene was found to be degraded within 7 days of incubation (Fig. 1). However, during growth on phenanthrene, the pH of the medium declined to as low as 6.8 from 7.2, indicating the possible accumulation of various transient acidic metabolites with time. Apart from phenanthrene, the strain PWTJD could also utilize 2-hydroxy-1-naphthoic acid, although at a much slower rate than that of phenanthrene and salicylic acid individually as sole sources of carbon and energy, but failed to utilize 1-hydroxy-2-naphthoic acid, o-phthalic acid, protoctechuic acid, gentisic acid or catechol.
Oxygen uptake assay
The oxidation of metabolic intermediates of phenanthrene by cells grown on phenanthrene, 2-hydroxy-1-naphthoic acid, salicylic acid or succinate as the sole carbon source was examined with a polarographic oxygen electrode. Cells grown on phenanthrene oxidized phenanthrene, 2-hydroxy-1-naphthoic acid, salicylic acid and catechol efficiently, but failed to respire on 1-hydroxy-2-naphthoic acid, 1,2-dihydroxynaphthalene, naphthalene-1,2-dicarboxylic acid, o-phthalic acid and protocatechic acid. However, cells grown on 2-hydroxy-1-naphthoic acid failed to oxidize phenanthrene, but did oxidize salicylic acid and catechol. On the other hand, cells grown on salicylic acid failed to oxidize both phenanthrene and 2-hydroxy-1-naphthoic acid apart from catechol. Oxygen uptake rates were found to be in the range of 23–40 nmol of oxygen consumed per minute per milligram of protein. Moreover, the immediate oxygen-incorporating activity of the enzymes involved in phenanthrene degradation was not observed with any of the above substrates with succinate-grown cells. It is therefore believed that the oxygen-incorporating enzymes involved in the phenanthrene degradation pathway in strain PWTJD are inducible.
Identification of phenanthrene metabolites
HPLC analysis of a resting cell incubated (48 h) phenanthrene-degraded sample showed a number of well-resolved peaks (Fig. 2), of which, peaks I–V and VII were identified as salicylic acid, catechol, 2-hydroxy-1-naphthoic acid, salicylaldehyde, 2-naphthol and the unutilized phenanthrene, respectively, on comparing their retention times, coelution profiles and UV-visible spectra (Fig. 2, inset) obtained from diode array analysis with those of the authentic compounds analyzed under identical conditions. Identification of 2-naphthol may be due to abiotic decarboxylation of 2-hydroxy-1-naphthoic acid under the experimental conditions used. In addition, the UV-visible spectrum of peak VI eluted at 17.6 min was found to be relatively similar to that of 2-hydroxy-1-naphthoic acid (III), eluted at 5.9 min. Other peaks of Fig. 2 showed neither any match with the UV-visible spectral pattern nor retention behavior of the available authentic compounds that are reported as phenanthrene pathway metabolites in the literature. Compounds corresponding to peaks I, II, IV–VI were also obtained from resting cell incubated 2-hydroxy-1-naphthoic acid-degraded samples by the strain PWTJD grown either on phenanthrene or on 2-hydroxy-1-naphthoic acid.
GC-MS analysis of biodegraded products obtained from the organic extracts (neutral as well as acidic) of the spent culture (96 h) and resting cell incubation (48 h) with phenanthrene are summarized in Table 1. GC-MS data correlate well with those obtained from HPLC analysis, although 2-hydroxy-1-naphthoic acid was not detected as such because this compound was decarboxylated under the GC-MS conditions and furnished the typical spectrum of 2-naphthol (product V, Table 1). This has been verified using authentic 2-hydroxy-1-naphthoic acid under the GC conditions used. However, a methylated derivative of an acidic extract of resting cell incubation with phenanthrene indicated the presence of 2-hydroxy-1-naphthoic acid (metabolite III). Apart from this, 1,2-dihydroxy-1,2-dihydrophenanthrene (metabolite VIII) was detected in the neutral extract while metabolite IX was identified in both neutral and acidic extracts as 5,6-benzocoumarin, similar to the mass spectral data as reported earlier (Pinyakong et al., 2000; Mallick et al., 2007).
Table 1. GC-MS data for the metabolites of phenanthrene obtained from the organic extracts of the culture and resting cell incubation of the strain PWTJD
The ion abundance percentages are shown in parentheses.
Identification was based on the match of mass spectra (fragmentation and peak intensity) and GC retention times with data for authentic samples other than cis-1,2-phenanthrenedihydrodiol and 5,6-benzocoumarin, which were tentatively identified based on their mass spectral data.
Analyses were performed after methylation of organic extract with boron trifluoride/methanol.
The metabolism of 2-hydroxy-1-naphthoic acid by the cell-free extract of strain PWTJD grown on phenanthrene was evidenced by the change in color of the reaction mixture to slightly yellowish and an increase in absorbance at 297 and 334 nm with time (Fig. 3a). An almost similar spectrum was obtained in the HPLC analysis for peak VI (Fig. 2), indicating the possible presence of a ring cleavage product of 2-hydroxy-1-naphthoic acid in the resting cell transformation analysis. However, no change was observed in the spectral pattern when 1,2-dihydroxynaphthalene was incubated with the cell-free extract of phenanthrene-grown cells. All this information indicated the direct ring cleavage of 2-hydroxy-1-naphthoic acid by a ring cleavage dioxygenase present in the strain PWTJD similar to the earlier report from Gram-positive Staphylococcus sp. (Mallick et al., 2007).
Like the previous report on the meta-cleavage of 2-hydroxy-1-naphthoic acid (Mallick et al., 2007), it was also observed that the ring-cleavage dioxygenase possessed dissociable ferric iron at the catalytic center because an increase in the ring-cleavage activity was noticed when the cell-free extract was supplemented with 1 mM FeCl3. In addition, on treatment of the cell-free extract with deferroxamine mesylate, a ferric chelating reagent, the resultant cell-free extract preparation did not show 2-hydroxy-1-naphthoic acid ring-cleavage activity. However, the ring-cleavage activity could be restored on further treatment with FeCl3 solution, verifying the role of ferric iron in catalysis. On the other hand, EDTA, a ferrous chelating reagent, had no impact on the enzyme activity.
In the lower pathway of the degradation of phenanthrene, the metabolism of salicylaldehyde to salicylic acid has been demonstrated in the spectrophotometric analyses (Fig. 3b) by the cell-free extract of both phenanthrene and 2-hydroxy-1-naphthoic acid-grown cells of strain PWTJD. An increase in the absorbance at 296 nm and a simultaneous decrease in absorbance at 254 and 330 nm was observed, indicating the formation of salicylic acid (Fig. 3b) when salicylaldehyde was incubated with a crude cell-free extract (Eaton & Chapman, 1992). Because salicylaldehyde itself has absorbance around 340 nm, the formation of NADH (λmax at 340 nm) from NAD+ during this transformation could not be observed during the early stage of transformation, but became apparent in the later stages of incubation (Fig. 3b). On the other hand, catechol was found to be metabolized by the cell-free extracts of either phenanthrene, 2-hydroxy-1-naphthoic acid or salicylic acid-grown cells of strain PWTJD with the formation of a yellow-colored product, 2-hydroxymuconaldehyde acid (Kojima et al., 1961; Nozaki, 1970), having a λmax at 374 nm (Fig. 3c), indicating the presence of catechol 2,3-dioxygenase activity. This was also confirmed by the immediate appearance of a yellow-colored product when catechol was sprayed on colonies in a Luria–Bertani agar plate (Stillwell et al., 1995) induced with phenanthrene, 2-hydroxy-1-naphthoic acid or salicylic acid. However, none of these activities could be detected in the cell-free extract obtained from succinate-grown cells.
Based on the HPLC, mass, UV-visible spectral data, along with the other observations as stated above, the metabolic pathways involved in the degradation of phenanthrene are proposed (Fig. 4). In the present study, the metabolism of phenanthrene appears to be similar to that reported for Staphylococcus sp. strain PN/Y (Mallick et al., 2007), but the strain PWTJD could not transform indole to indigo (Ensley et al., 1983) as observed in strain PN/Y, indicating structural differences of phenanthrene ring-hydroxylating dioxygenase in these two strains. Interestingly, the ring-hydroxylating dioxygenases from strain PWTJD could not be amplified using the most commonly used primers reported in the literature (Ni Chadhain et al., 2006; Cébron et al., 2008), signifying the possible presence of a structurally unique ring-hydroxylating dioxygenase in Ochrobactrum sp. strain PWTJD. Although the degradative abilities of the genus Ochrobactrum were primarily reported on methyl parathion (Qiu et al., 2006), phenol (El-Sayed et al., 2003), 2,4,6-tribromophenol (Yamada et al., 2008) and 4-nitrocatechol (Zhong et al., 2007), there are few preliminary reports on the degradation of a couple of PAHs by Ochrobactrum sp. (Zhang & Peng, 2008; Arulazhagan & Vasudevan, 2009; Wu et al., 2009). However, neither of the studies describes the structural nature of ring-hydroxylating dioxygenase or the metabolic pathways involved in PAH assimilation.
To the best of our knowledge, this is the first report on the detailed metabolic study of a PAH molecule by an Ochrobactrum species describing the degradation of phenanthrene via meta-cleavage of 2-hydroxy-1-naphthoic acid. Moreover, in this study, the 2-hydroxy-1-naphthoic acid meta-cleavage pathway is reported for the first time from a Gram-negative bacterial species. Further experiments in evaluating the structural nature of phenanthrene ring-hydroxylating dioxygenase and 2-hydroxy-1-naphthoic acid meta-cleavage dioxygenase present in Ochrobactrum sp. strain PWTJD may provide a new insight into the microbial degradation PAHs in general.
The authors gratefully acknowledge Professor P. Sil for reviewing the manuscript. This work was supported in part by a Grant-in aid from Ministry of Environment & Forests, Government of India (#19/34/2005-RE to T.K.D.), and Bose Institute, Kolkata, India.