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Metabolic pathway for degradation of anthracene by halophilic Martelella sp. AD-3
International Biodeterioration & Biodegradation 89 (2014) 67e73
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Metabolic pathway for degradation of anthracene by halophilic Martelella sp. AD-3 Changzheng Cui a, Lei Ma b, Jie Shi a, Kuangfei Lin a, Qishi Luo c, Yongdi Liu a, * a State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China b Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China c State Environmental Protection Engineering Center for Urban Soil Contamination Control and Remediation, Institute of Wastes and Soil Environment, Shanghai Academy of Environmental Science, Shanghai 200233, China
a r t i c l e i n f o
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Article history: Received 11 October 2013 Received in revised form 17 January 2014 Accepted 21 January 2014 Available online 11 February 2014
Anthracene (40 mg l1) was completely depleted by Martelella sp. strain AD-3 under 3% salinity and a pH of 9.0 after 6 days of incubation. The metabolites were extracted and identified by high-performance liquid chromatography (HPLC) retention times, mass spectrometry, 1H and 13C nuclear magnetic resonance spectrometry, and comparison to authentic compounds or literature data. On the basis of the identified metabolites, enzyme activities and the utilization of probable intermediates, anthracene degradation by strain AD-3 is proposed via two distinct routes. In route I, metabolism of anthracene is initiated by the dioxygenation at C-1,2, then proceeds through 6,7-benzocoumarin, 3-hydroxy-2naphthoic acid, salicylic acid and gentisic acid. In route II, anthracene is metabolized to 9,10anthraquinone. The results suggest that strain AD-3 possesses efficient anthracene biodegradability in high salinity. To our knowledge, this work presents the first report of anthracene degradation by a halophilic PAH-degrading strain via two routes. The strain AD-3 may be a useful candidate for PAHcontaminated saline-alkali soil bioremediation. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Anthracene Biodegradation Moderate halophilic bacteria Metabolic pathway 3-Hydroxy-2-naphthoic acid
1. Introduction The petroleum industry generates a huge amount of oily and saline waste water (oily brines, production waters) after separation of crude oil from reservoir water, which has to be disposed. The main contaminants in this production water are aromatic and polycyclic aromatic hydrocarbons (PAHs) (Borgne et al., 2008; Zhuang et al., 2010). In addition, some marine and coastal sites are polluted by PAHs due to occasional accidents, such as oil spills during production, transportation or refining processes of the oil (Borgne et al., 2008). It is crucial to develop an efficient remedial process for both saline water and soil where natural attenuation is very slow. Anthracene (ANT), a tricyclic PAH, is found in high concentrations in PAH-contaminated sediments, surfaces soils, and waste sites (Wilson and Jones, 1993). It has been registered as a persistent compound that is toxic to marine life (Choi and Oris, 2003). In addition, ANT is considered a benchmark PAHs and serves as a * Corresponding author. Tel./fax: þ86 21 64253988. E-mail addresses: [email protected] (C. Cui), [email protected] (Y. Liu). 0964-8305/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2014.01.012
signature compound to detect PAHs contamination, since its chemical structure is found in carcinogenic PAHs, such as benz[a] anthracene and benzo[a]pyrene. According to previous reports, biodegradation of ANT by Pseudomonas (Evans et al., 1965; Menn et al., 1993), Mycobacterium (Moody et al., 2001; Herwijnen et al., 2003a), Rhodococcus (Tongpim and Pickard, 1996; Dean-Ross et al., 2001), Sphingomonas (Herwijnen et al., 2003b), Nocardia (Zeinali et al., 2008), and Bacillus (Ahmed et al., 2012) under non-halophilic conditions has been demonstrated to include two major types of metabolic pathways. One is via dioxgenation at the C-1 and C-2 ring positions, to form 1,2-dihydroxy-anthracene. This dihydroxylated intermediate is further metabolized by cleavage of the ring at the meta position to yield 4-(2-hydroxynaphth-3-yl)-2-oxobut-3-enoic acid. This compound is unstable and will spontaneously rearrange to form 6,7Benzocoumarin, or can be further metabolized to 3-hydroxy-2naphthoic acid. However, to our knowledge, all studies of this pathway since Evans et al. (1965), have been unable to detect 3hydroxy-2-naphthoic acid from biodegradation of anthracene by wild-type strains. Subsequent metabolism proceeds via a pathway analogous to that of naphthalene biodegradation to yield salicylate or phthalic acid (Dean-Ross et al., 2001; Zeinali et al., 2008). The
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second pathway involves dioxygenation at the C-9 and C-10 positions to form 9,10-dihydrodiol, which converts non-enzymaticallly to the dead-end product 9,10-anthraquinone. In addition, Ye’s research group showed that fungi Aspergillus fumigates metabolizes ANT to form 9,10-anthraquinone, followed by conversion via an extracellular peroxidase enzyme (manganese peroxidase and lignin peroxidase) to yield phthalic acid under high salt conditions (Ye et al., 2001). Conventional microorganisms are unable to operate efficiently at salinities above that of seawater and their adaptation to salinity is easily lost after exposure to low salinity conditions. Moderately halophilic bacteria are potential candidates for use in the degradation of pollutants at high salt concentrations, since they can survive under high salinity (Ceylan et al., 2011). Either high or fluctuating salinity promotes the loss of cell wall integrity, protein denaturalization and changes in osmotic pressure (Borgne et al., 2008). Within halophiles, moderately halophilic microorganisms constitute an important group that grows optimally between 0.5 mol l1 (3%) and 2.5 mol l1 (15%) NaCl (Kushner, 1978). The biological treatment of industrial hyper-saline wastewaters and the bioremediation of polluted hyper-saline environments are important factors in selecting appropriate halophilic or halotolerant PAH-degrading microorganism (Betancur-Galvis et al., 2006; Arulazhagan and Vasudevan, 2011a; Haddadi and Shavandi, 2013). To our knowledge, the catabolic pathways of ANT biodegradation by halophilic bacteria have not been investigated. Martelella sp. strain AD-3 (CCTCC M 2011218), a moderate halophilic bacterium which was originally isolated in our laboratory from a petroleumcontaminated soil with high salinity (Cui et al., 2012), is capable of degrading naphthalene, phenanthrene, ANT and pyrene as the sole carbon source (Feng et al., 2012). The biodegradation pathway has been elucidated for the metabolism of phenanthrene by Martelella sp. strain AD-3 (Feng et al., 2012). We describe in the present study the possible metabolic pathways for degradation of ANT by this Martelella species under halophilic conditions, based on the metabolites identified from initial ring oxidation and ring cleavage products. 2. Materials and methods 2.1. Chemicals ANT, phenanthrene, 3-hydroxy-2-naphthoic acid, 1-hydroxy-2naphthoic acid, 2-hydroxy-1-naphthoic acid, 2,3dihydroxynaphthalene, salicylic acid and gentisic acid were purchased from SigmaeAldrich (St Louis, MO, USA) and were of the highest purity available. Nuclear magnetic resonance (NMR) solvents were purchased from Isotec, Inc. (Miamisburg, Ohio). Other organic solvents were purchased from (Rathburn, Walkerburn, Scotland) and were high-performance liquid chromatography grade. 2.2. Culture conditions Cultures of Martelella sp. AD-3 were grown in 500-ml Erlenmeyer flasks containing 200 ml of 3% minimal basal salt medium (pH 9.0) supplemented with 2.5 g l1 and 5 g l1 concentrations of yeast extract and peptone, respectively (Feng et al., 2012). A 20 ml aliquot of ANT (10 mg ml1) in N,N-dimethylformamide was added to flasks for enzyme induction. The cells from the flasks were harvested 2 days after inoculation by centrifugation at 8500 rpm for 10 min at 4 C. The cultures (OD600, 1) were incubated in 50 ml of 3% minimal basal medium (pH 9.0) in 250 ml Erlenmeyer flasks. ANT was dissolved in N,N-dimethylformamide and added to the
cultures to a final concentrations 40 mg l1. All cultures were incubated in the dark at 30 C with shaking at 150 rpm. 2.3. Analytical methods To identify the metabolites, contents of the flask were extracted four times with 50 ml of ethyl acetate, twice at neutral pH and twice after acidification to pH 2 with 5 M HCl. The neutral and acidic extracts were pooled, and the solvents were evaporated completely under gentle sparging with high-purity nitrogen. The residues were dissolved in 2 ml of acetonitrile and concentrated for analysis by reverse-phase HPLC and Gas chromatography/mass spectrometry (GCeMS). Flasks were extracted at 0, 2, 4, 6 and 8 days, respectively. ANT and its metabolites were analyzed by reverse-phase HPLC (Waters) equipped with a diode array UVevisible light (Vis) detector and a Waters XBridgeTM C18 analytical column (150 4.6 mm, i.d., 3.5 mm particle-size). A guard column was used to protect the analytical column. Initial HPLC conditions were 95% buffer A (water-0.01% TFA) and 5% buffer B (acetonitrile-0.01% TFA) with the following linear gradient: From 0 to 5 min chromatography was maintained in 95% buffer A. From 5 to 20 min chromatography was in a linear gradient from 95% buffer A to 95% buffer B; from 20 to 25 min at constant 95% buffer B; 25e25.1 min switched sharply to 95% buffer A; and from 25.1 to 32 min maintained in 95% A. The injection volume was 20 ml. For collection of sufficient quantities of metabolites to perform nuclear magnetic resonance (NMR) analysis, Waters reverse-phase HPLC was preformed with a diode array UVevisible light (Vis) detector and a Waters XBridgeTM C18 Prep column (50 19 mm, i.d., particle-size is 5 mm). Purification was performed in a gradient of buffer A to buffer B at a flow rate of 17 ml min1 using the following method. From 0 to 9 min, a linear gradient from 95% buffer A to 14% buffer A; from 9 to 9.1 min sharply switched to 95% buffer B; from 9.1 to 11 min in 95% buffer B; from 11 to 11.1 min a sharp switch from 95% buffer B to 95% buffer A; and from 11.1 to 13 min in 95% buffer A. The 8.44 min fraction was chosen to purify the compound. The injection volume was 150 ml. Proton (1H) NMR spectra were obtained on a Bruker AM500 spectrometer (Bruker Instruments, Billerica, MA, USA). Each metabolite was dissolved in 0.5 ml deuterated methanol (99.96 atom% 2H) for analysis. Chemical shifts are reported on the d scale (ppm) by assigning the residual solvent peak to 2.49 or 3.30 ppm, respectively. Typical 1H data acquisition parameters were: data size, 3200; sweep width, 7042 Hz; filter width, 8900 Hz; acquisition time, 2.33 s; flip angle, 900; relaxation delay, 0 s; temperature, 298.5 K. For measurement of coupling constants, the free induction decay was zero-filled to 64,000 resulting in a final data point resolution of 0.215 Hz per point. Coupling constants reported are first order. Those that were non-first order and those of overlapping resonances were omitted. Assignments were made from integration, analysis of substituent effects, and comparison to spectra of pure, known standards. A 13C NMR spectrum was obtained for one metabolite. EI-MS: Finnigan MAT-90/95 sector-field mass spectrometer; in m/z. GC/MS was performed using Agilent Technologies 7890A GC System coupled with an Agilent 5975C VL MSD with Triple-axis mass detector with an Agilent HP5-MS Ultra Inert (60 m 0.25 mm, 0.25 mm film thickness) column. The column temperature program followed the method as described by Feng et al. (2012). Derivatization prior to GC/EI-MS analysis was performed by silylation with N,O-Bis(trimethylsilyl)trifluoroacetamide 1% with trimethylchlorosilane (Sigma). 100 ml of sample was dissolved in acetonitrile, then mixed with 150 ml of silylation reagent and allowed to react for 1 h at 60 C. The injection volume was 1 ml.
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2.4. Utilization of other organic compounds The ability of the strain to grow on alternative organic compounds, including 3-hydroxy-2-naphthoic acid, 2,3dihydroxynaphthalene, salicylic acid, o-phthalic acid, catechol, gentisic acid and succinic acid, was evaluated. The test was performed using MSM (100 ml, pH 7.5) containing a given organic compound with a concentration of 100 mg l1 in a sealed 250 ml flask. In order to prevent decomposition of 2,3-dihydroxynaphthalene, the test was performed using MSM (100 ml, pH 5.5). The un-inoculated control and duplicate samples were prepared for each treatment. After three days of incubation at 30 C and agitation speed of 150 rpm, the reaction solutions and controls were sampled and examined for their OD at 600 nm (OD600). 2.5. Enzyme assays Enzyme activities in selected cell-free extracts were applied to confirm the route of metabolism of ANT by strain AD-3. Reported spectrophotometric methods were used to monitor the activities of 3-hydroxy-2-naphthoic acid hydroxylase, 1-hydroxy-2-naphthoic acid hydroxylase (Deveryshetty and Phale, 2010), 1-hydroxy-2naphthoic acid dioxygenase (Deveryshetty and Phale, 2009), salicylate 5-hydroxylase (S5H) (Zhou et al., 2002), gentisate 1,2dioxygenase (GDO) (Fu and Oriel, 1998), catechol 1,2-dioxygenase (Doddamani and Ninnekar, 2000) and catechol 2,3-dioxygenase (Doddamani and Ninnekar, 2000). Cell-free extracts included ANT, phenanthrene, salicylic acid and glucose. The preparation of the cell-free extracts was carried out using a previously described procedure (Fu and Oriel, 1998). A published Bradford assay method (1976), with bovine serum albumin as the standard, was used to determine protein concentrations. Enzyme activity was defined as units (nmol of the product formed or substrate disappeared, NADH formed) min1 ml1. The specific activities were expressed as units: mg1 protein.
Fig. 1. HPLC elution profile of metabolites produced during the growth of Martelella sp. strain AD-3 by different incubation time in the presence of anthracene. The UV absorbance wavelength was used at 254 nm. (B) 4 days, (C) 6 days, (D) 8 days.
To our knowledge, ANT degradation has been little studied in halophilic microorganisms. According to previous reports, Ochrobactrum sp. VA1 utilized 87% of anthracene (3 mg l1) in 4 days at 30 g l1 of NaCl concentration along with glucose as additional carbons source (Arulazhagan and Vasudevan, 2011a). Ochrobactrum sp. VA1 utilized 65% of anthracene (3 mg l1) in 4 days at 60 g l1 of NaCl concentration along with yeast extract (Arulazhagan and Vasudevan, 2011b). However, in present research, anthracene (40 mg l1) was completely depleted by Martelella sp. strain AD-3 under 3% salinity and a pH of 9.0 after 6 days of incubation. The results suggest that strain AD-3 possesses efficient anthracene biodegradation ability in high salinity. 3.2. ANT metabolite identification
2.6. Statistical analysis Statistical analysis was carried out using SPSS version 17.0 software. Multiple comparison analysis was performed using the least significant difference (LSD) test at the significant level of P > 0.05. The results were expressed as the mean values and corresponding standard errors. 3. Results and discussion 3.1. Characterization of ANT degradation and metabolite formation The characterization of ANT biodegradation and the formation of specific ANT metabolites by Martelella sp. strain AD-3 cultures was measured over time. The degradation of ANT was monitored, by HPLC, for up to 8 days. After 4 days of incubation, most ANT was degraded to metabolites by AD-3 cultures; a visible yellow color in the medium indicated the accumulation of some metabolite. The was not a significant color change in any of the controls. After 4 days incubation, four metabolite fractions of ANT, designated ANT-I, ANT-II, ANT-III, and ANT-IV, with retention times of 15.2, 15.3, 17.0 and 17.3 min, respectively, were observed after HPLC analysis. The parent compound, ANT, was eluted at 21.1 min (Fig. 1). The metabolites found were not present in any of the controls after HPLC analysis. We observed the ANT peak gradually disappear; while the ANT-III peak areas gradually increased, followed by ANT-III peak areas gradually decreasing. These results showed Martelella sp. strain AD-3 is not only capable of degrading ANT, but also capable of continuing to degrade metabolic intermediates ANT-III (Fig. 1).
HPLC collection of the extract from ANT incubations produced one big peak of metabolite ANT-III as shown in Fig. 1. Compound ANT-III was obtained as an amorphous yellow powder after drying the eluent. The molecular formula was determined as C11H8O3 by EI mass spectroscopy, from the molecular ion signal at an m/z of 188.0. The 1H NMR assignments and coupling constants are as follows: d 8.50 (s, H1), 7.80 (dd, J ¼ 8.1, 1.3, H8), 7.66 (dd, J ¼ 8.3, 1.2 Hz, H5), 7.47 (ddd, J ¼ 8.3, 6.8, 1.3 Hz, H6), 7.29 (ddd, J ¼ 8.1, 6.8, 1.2 Hz, H7), 7.22 (s, H4). The 1H NMR spectrum showed six aromatic resonances with a coupling pattern (singlet peaks at d 8.50 and 7.22) indicative of substitution at the C-2 (carboxyl group) and C-3 (hydroxyl group) positions. From the above EI mass and 1H NMR data, the structure of ANT-III was identified as 3-hydroxy-2-naphthoic acid. The 13C NMR (125 MHz, CD3OD) data are as follows: d 173.2, 157.9, 139.2, 133.8, 130.2, 130.0, 128.4, 127.1, 124.8, 115.8, 112.0 (Fig. 2), which further confirmed that the structure of ANT-III as 3-hydroxy-2naphthoic acid. Other metabolites (ANT-I, ANT-II, and ANT-IV) were not present in sufficient quantities to perform EI mass or NMR analyses. A variety of bacterial species have been isolated that have the ability to utilize ANT as the sole source of carbon and energy. The initial reaction in the degradation of ANT is catalyzed by multicomponent dioxygenases that incorporate both atoms of molecular oxygen into the PAHs nucleus to produce cis-dihydrodiols. The original papers propose that ANT -degrading strains oxidize ANT in the 1,2 position to form (þ)-(1R,2S)-cis-1,2-dihydroxy-1,2dihydroanthraene, which is subsequently converted to 1,2dihydroanthracene, which is further metabolized to 3-hydroxy-2-
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Fig. 2. 1H NMR spectrum (A) and hydroxy-2-naphthoic acid.
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C NMR spectrum of metabolite II identified as 3-
naphthoic acid, salicylate and catechol by enzymes of the pathway (Evans et al., 1965). However, to our knowledge, the metabolic products of ANT biodegradation by wild-type strains (Moody et al., 2001; Dean-Ross et al., 2001; Herwijnen et al., 2003a; Zeinali et al., 2008; Ahmed et al., 2012), specifically, 3-hydroxy-2-naphthoic acid, have not been isolated. The metabolite 2-hydroxy-3-naphthoic acid from biodegradation of anthracene was identified by Pseudomonas fluorescens 5R mutant 5RL (Menn et al., 1993). Similarly, the mutant strains VM843 and VM844 of Mycobacterium sp. strain PYR-1 from these works contained a very small peak which was identified with the use of a derivatized sample as derivatized 3-hydroxy-2naphthoic acid (Moody et al., 2001). However, in the present investigation, we find 3-hydroxy-2-naphthoic acid is formed from ANT. Its very large HPLC peak suggests degradation of ANT through 3-hydroxy-2-naphthoic acid as the main pathway, and that 3hydroxy-2-naphthoic acid is not a dead-end product, but could be further degraded (Fig. 1). A cleavage reaction similar to that of 1hydroxy-2-naphthoic acid in the Martelella AD-3 degradation pathway of phenanthrene has been shown to form the cleavage product (Feng et al., 2012). GCeMS chromatograms of derivatized samples from cultures contained three potential metabolite peaks, designated as metabolite I, metabolite II, and metabolite III. Metabolite I, eluting at 22.7 min, had a mass spectra with a molecular ion (M)þ at m/z 208 and fragment ions at m/z 180, 152, 126 and 76 indicating that the compound could be 9,10-anthraquinone on the basis of its m/z values and comparison of the mass spectra with the NIST library available in GCeMS (Fig. 3a), which was consistent with those of
9,10-anthraquinone described by Dean-Ross et al. (2001). An alternate route of enzymatic attack by Martelella sp. strain AD-3 is at the C-9 and C-10 positions of ANT. Initial attack on ANT at C-9,10 positions is consistent with previous reports on Mycobacterium (Moody et al., 2001) and Bacillus (Ahmed et al., 2012) growing on ANT. The presence of the dead-end product 9,10-anthraquinone could be explained by the formation and non-enzymatic oxidation of 9,10-dihydroxyanthracene (Moody et al., 2001). Metabolite II, eluting at 23.3 min, had a mass spectra with a molecular ion (M)þ at m/z 332 and major fragment ions at m/z 317, 244, 185, 141 and 73 (Fig. 3b), which was identified as derivatized, indicating that the compound could be 3-hydroxy-2-naphthoic acid on the basis of its m/z values and comparison of the mass spectra with the NIST library available in GCeMS. Therefore, Metabolite II was identified as derivatized 3-hydroxy-2-naphthoic acid. Metabolite III, eluting at 24.1 min, had a mass spectra with a molecular ion (Mþ) at m/z 196 and fragment ions at m/z 168, 139, 98, 84, 70 and 63 (Fig. 3c), which was consistent with those of 6,7benzocoumarin described by Dean-Ross et al. (2001) and Herwijnen et al. (2003a). The formation of 6,7-benzocoumarin indicates that dioxygenase occurred at the C-1 and C-2 position of ANT to form 1,2-dihydroxyanthracene as the initial oxidation product followed by its meta-cleavage (Herwijnen et al., 2003a). However, 1,2-dihydroxyanthracene was not detected in the present study. The 6,7-benzocoumarin has also been found to occur in Mycobacterium (Moody et al., 2001), Rhodococcus (Dean-Ross et al., 2001), Bacillus (Ahmed et al., 2012), and Pseudomonas (Evans et al., 1965) species, suggesting that it may be a common pathway for the initial degradation of ANT in both non-halophilic and halophilic organisms. The original papers propose 6,7-benzocoumarin to be a dead-end product (Evans et al., 1965), but recent literature (Moody et al., 2001; Herwijnen et al., 2003a) propose 6,7-benzocoumarin as an intermediate in the degradation pathway. The results show that Martelella sp. strain AD-3 efficiently metabolized ANT. The isolation and characterization of the major oxidation and ring fission products indicated two routes of enzymatic attack. The degradation pathways of ANT by Martelella sp. strain AD-3 is proposed in Fig. 4. Present research and previous studies of the biodegradation of ANT by Martelella sp. strain AD-3 suggest that both dioxygenases and monooxygenases catalyze the initial attack on the aromatic ring. Since positional isomers of cis-dihydrodiols are formed, it may also be speculated that several dioxygenase are present in Martelella sp. strain AD-3. The broad range of PAHs that are degraded by Martelella sp. strain AD-3 may also be due to a relaxed specificity of the same dioxygenase for initial attack on PAHs. The identification of ortho-ring cleavage intermediates from the degradation of dihydroxylated metabolites of phenanthrene (Feng et al., 2012) indicate alternative enzymatic routes in the degradation of PAHs by Martelella sp. strain AD-3. Bioremediation is an economically and environmentally attractive solution for cleaning PAHs in saline-alkaline soils. In order to achieve an efficient bioremediation process, it is important that the bacteria involved perform a complete degradation pathway so that potentially toxic metabolites do not accumulate in the soil (Herwijnen et al., 2003a). In this study, we examined the pathway for ANT degradation by Martelella sp. strain AD-3 to determine whether metabolites are expected to accumulate. 3.3. Transformation and formation of intermediates We tested growth of Martelella sp. strain AD-3 on several possible intermediates. 3-hydroxy-2-naphthoic acid, 1-hydroxy-2naphthoic acid, 2-hydroxy-1-naphthoic acid, 2,3-
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Fig. 3. EI mass spectrum of metabolite I (a), II (b) and III (c) identified as, 9,10-anthraquinone, 3-hydroxy-2-naphthoic acid and 6,7-benzocoumarin, respectively.
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not be observed. This observation shows that enzymes responsible for degradation of 3-hydroxy-2-naphthoic acid are induced during growth on ANT and this may explain the poor growth on 3hydroxy-2-naphthoic acid. Similarly, Mycobacterium sp. strain PYR-1 could degrade ANT through o-phthalic acid, however, no growth was observed on 3-hydroxy-2-naphthoic acid (Herwijnen et al., 2003a). 3.4. Enzyme activities in cell-free extracts
Fig. 4. Proposed pathway for the degradation of anthracene by the halophilic strain Martelella sp. AD-3 on the basis of identified metabolites, enzyme activity and utilization profile of different substrates. Compounds in brackets are hypothetical intermediates, but not detected.
dihydroxynaphthalene, salicylic acid, benzoic acid, catechol, and gentisic acid and succinic acid were tested to see if they could serve as growth substrates. Anthracene, salicylic acid, gentisic acid, and succinic acid as the sole carbon showed good growth. While culture with 1-hydroxy-2-naphthoic acid, 3-hydroxy-2-naphthoic acid and 2,3-dihydroxynaphthalene as the sole carbon showed poor growth. This was evident by measuring significant cell density increases after 3 days of incubation. Moreover, no growth was observed on 2hydroxy-1-naphthoic acid, o-phthalic acid and catechol. The detailed results are shown in Table 1. To examine the transformation of 3-hydroxy-2-naphthoic acid by this bacterium, we preformed experiments with cell extracts in which we compared bacteria grown on ANT and yeast extract. Transformation of 3-hydroxy-2-naphthoic acid could only be observed in cell extracts of cultures grown on the concentration of ANT 10 mg l1. In assays with cell extracts from cultures grown on yeast extract, transformation of 3-hydroxy-2-naphthoic acid could Table 1 Profile of utilization of different substrates by strain AD-3. Substrates
Concentration (mg l
Anthracene 3-hydroxy-2-naphthoic acid 1-hydroxy-2-naphthoic acid 2-hydroxy-1-naphthoic acid 2,3-dihydroxynaphthalene Salicylic acid o-phthalic acid Succinic acid Gentisic acid Catechol
40 100 100 100 100 100 100 100 100 100
1
)
An enzyme extract prepared from ANT-grown cells showed 3hydroxy-2-naphthoic acid hydroxylase activity, but failed to showed the activity of 1-hydroxy-2-naphthoic acid hydroxylase, catechol-1,2-dioxygeanse, catechol-2,3-dioxygenase, or gentisate 1,2-dioxygenase. An enzyme extract prepared from phenanthrenegrown cells showed 1-hydroxy-2-naphthoic acid hydroxylase activity, but failed to show the activity of 3-hydroxy-2-naphthoic acid hydroxylase activity. In this preliminary study, we assume 3hydroxy-2-naphthoic acid hydroxylase is different from 1hydroxy-2-naphthoic acid hydroxylase. Salicylic acid-grown cells showed comparatively reduced activities for 1-hydroxy-2naphthoic acid hydroxylase and significantly higher activities of salicylate 5-hydroxylase, gentisate 1,2-dioxygenase (Feng et al., 2012). Similarly, comparatively little activity for 3-hydroxy-2naphthoic acid hydroxylase was detected. The lack of activity of these enzymes in glucose-grown cell-free extracts indicated that the enzymes responsible for anthracene metabolism were inducible (Table 2). The specific activity versus growth profile indicated the presence of three hydroxylases in strain AD-3, salicylate-5hydroxylase, 1-hydroxy-2-naphthoic acid hydroxylase, and 3hydroxy-2-naphthoic acid hydroxylase. Salicylate 5-hydroxylase and 1-hydroxy-2-naphthoic acid hydroxylase from various bacterial sources have been characterized and reported (Adachi et al., 1999; Zhou et al., 2002; Deveryshetty and Phale, 2010). However, to our knowledge, the enzyme characterization for 3-hydroxy-2naphthoic acid hydroxylase has not been reported so far and is worthy of further study. 4. Conclusion Martelella sp. AD-3 extends our knowledge of moderate halophilic bacteria that can initiate an attack on ANT through two different pathways: via the C-1,2 or C-9,10 positions. The observation of the degradation of ANT through 3-hydroxy-2-naphthoic acid, 2,3-dihydroxyanthracene and gentisate acid indicate the presence of a complete ANT degradation pathway in the ANTTable 2 Specific activity of various enzymes in the cell-free extract of halophilic strain AD-3 grown on different substrates to the mid-exponential phase. Enzymes
Specific activitya (nmol min1 mg1 protein) Glucose
Salicylic acid
Anthracene
1-Hydroxy-2-naphthoic acid hydroxylase 1-Hydroxy-2-naphthoic acid dioxygenase 3-hydroxy-2-naphthoic acid hydroxylase Salicylate 5-hydroxylase (S5H) Catechol-1,2-dioxygenase Catechol-2,3-dioxygenase Gentisate 1,2-dioxygenase (GDO)
NDb
34.1
30.2
ND
ND
ND
ND
38.6
120.4
ND
185.1
106.3
ND ND ND
ND ND 80.5
ND ND ND
Growth þþ þ þ e þ þþ e þþ þþ e
(þþ) Good growth: OD600 > 0.2; (þ) growth: 0.05 < OD600 < 0.2; () no growth: OD600 < 0.05. Cells were cultivated in MSM (salinity 3%, pH 7.5) containing each substrate individually at specific concentration at 30 C and 150 rpm in the dark for 3 days incubation except 2,3-dihydroxynaphthalene in MSM (salinity 3%, pH 5.5).
a b
The values are the average values measured in triplicate. Activity could not be detected.
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utilizing bacteria. This finding warrants further extensive investigations of degradation enzymes in moderate halophilic PAHdegrading bacteria. The strain AD-3 may be a useful candidate for PAH-contaminated saline-alkali soil bioremediation experiments. Acknowledgments This research was supported by the National Natural Science Foundation of China (41301329, 51378208), the Shanghai Municipal Natural Science Foundation (13ZR1410900), the Specialized Research Fund for the Doctoral Program of Higher Education (20110074130002), and the Fundamental Research Funds for the Central Universities (WB1114031). References Adachi, K., Iwabuchi, T., Sano, H., Harayama, S., 1999. Structure of the ring cleavage product of 1-hydroxy-2-naphthoate, and intermediate of the phenanthrenedegradative pathway of Nocardioides sp. strain KP7. J. Bacteriol. 181, 757e763. Ahmed, A.T., Othman, M.A., Sarwade, V.D., Gawai, K.R., 2012. Degradation of anthracene by Alkaliphilic bacteria Bacillus badius. Environ. Pollut. 2, 97e104. Arulazhagan, P., Vasudevan, N., 2011a. Role of nutrients in the utilization of polycyclic aromatic hydrocarbons by halotolerant bacterial strain. J. Environ. Sci. 23, 282e287. Arulazhagan, P., Vasudevan, N., 2011b. Biodegradation of polycyclic aromatic hydrocarbons by a halotolerant bacterial strain Ochrobactrum sp. VA1. Mar. Pollut. Bull. 62, 388e394. Betancur-Galvis, L.A., Alvarez-Bernal, D., Ramos-Valdivia, A.C., Dendooven, L., 2006. Bioremediation of polycyclic aromatic hydrocarbon-contaminated saline-alkaline soils of the former Lake Texcoco. Chemosphere 62, 1749e1760. Borgne, S.L., Paniagua, D., Rafael, V.D., 2008. Biodegradation of organic pollutants by halophilic bacteria and archaea. J. Mol. Microbiol. Biotechnol. 15, 74e92. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Ceylan, S., Akbulut, B.S., Denizci, A.A., Kazan, D., 2011. Proteomic insight into phenolic adaptation of a moderately halophilic Halomonas sp. strain AAD12. Can. J. Microbiol. 57, 295e302. Choi, J., Oris, J.T., 2003. Assessment of the toxicity of anthracene photo-modification products using the topminnow (Poeciliopsis lucida) hepatoma cell line (PLHC-1). Aquat. Toxicol. 65, 243e251. Cui, C.Z., Feng, T.C., Yu, Y.Q., Dong, F., Yang, X.M., Feng, Y.Y., Liu, Y.D., Lin, H.P., 2012. Isolation, characterization of an anthracene degrading bacterium Martelella sp. AD-3 and cloning of dioxygenase gene. Environ. Sci. Chin. 33, 4062e4068. Dean-Ross, D., Moody, J.D., Freeman, J.P., Doerge, D.R., Cerniglia, C.E., 2001. Metabolism of anthracene by a Rhodococcus species. FEMS Microbiol. Lett. 204, 205e211.
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Deveryshetty, J., Phale, P.S., 2009. Biodegradation of phenanthrene by Pseudomonas sp. strain PPD: purification and characterization of 1-hydroxy-2-naphthoic acid dioxygenase. Microbiology 155, 3083e3091. Deveryshetty, J., Phale, P.S., 2010. Biodegradation of phenanthrene by Alcaligenes sp. strain PPH: partial purification and characterization of 1-hydroxy-2-naphthoic acid hydroxylase. FEMS Microbiol. Lett. 311, 93e101. Doddamani, H.P., Ninnekar, H.Z., 2000. Biodegradation of phenanthrene by a Bacillus Species. Curr. Microbiol. 41, 11e14. Evans, W.C., Fernley, H.N., Griffiths, E., 1965. Oxidative metabolism of phenanthrene and anthracene by soil Pseudomonads. Biochem. J. 95, 819e831. Feng, T.C., Cui, C.Z., Dong, F., Feng, Y.Y., Liu, Y.D., Yang, X.M., 2012. Phenanthrene biodegradation by halophilic Martelella sp. AD-3. J. Appl. Microbiol. 113, 779e 789. Fu, W., Oriel, P., 1998. Gentisate 1,2-dioxygenase from Haloferax sp. D1227. Extremophiles 2, 439e446. Haddadi, A., Shavandi, M., 2013. Biodegradation of phenol in hypersaline conditions by Halomonas sp. strain PH2-2 isolated from saline soil. Int. Biodeterior. Biodegrad. 85, 29e34. Herwijnen, R.V., Springael, D., Slot, P., Govers, H.A.J., Parsons, J.R., 2003a. Degradation of anthracene by Mycobacterium sp. strain LB501T proceeds via a novel pathway, though o-Phthalic acid. Appl. Environ. Microbiol. 69, 186e190. Herwijnen, R.V., Wattiau, P., Bastiaens, L., Daal, L., Jonker, L., Springael, D., Govers, H.A.J., Parsons, J.R., 2003b. Elucidation of the metabolic pathway of fluorene and cometabolic pathways of phenanthrene, fluoranthene, anthracene and dibenzothiophene by Sphingomonas sp. LB126. Res. Microbiol. 154, 199e206. Kushner, D.J., 1978. Life in high salt and solute concentrations: halophilic bacteria. In: Kushner, D.J. (Ed.), Microbial Life in Extreme Environments. Academic Press, London, pp. 317e368. Menn, F.M., Applegate, B.M., Sayler, G.S., 1993. NAH plasmid-mediated catabolism of anthracene and phenanthrene to naphthoic acids. Appl. Environ. Microbiol. 59, 1938e1942. Moody, J.D., Freeman, J.P., Doerge, D.R., Cerniglia, C.E., 2001. Degradation of phenanthrene and anthracene by cell suspensions of Mycobacterium sp. strain PYR-1. Appl. Environ. Microbiol. 67, 1476e1483. Tongpim, S., Pickard, M.A., 1996. Growth of Rhodococcus S1 on anthracene. Can. J. Microbiol. 42, 289e294. Wilson, S.C., Jones, K.C., 1993. Bioremediation of soil contaminated with polynuclear aromatic hydrocarbons (PAHs): a review. Environ. Pollut. 81, 229e249. Ye, J.S., Yin, H., Qiang, J., Peng, H., Qin, H.M., Zhang, N., He, B.Y., 2001. Biodegradation of anthracene by Aspergillus fumigates. J. Hazard. Mater. 185, 174e181. Zeinali, M., Vossoughi, M., Ardestani, S.K., 2008. Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium. J. Appl. Microbiol. 105, 398e406. Zhou, N.Y., Al-Dulayymi, J., Baird, M.S., Williams, P.A., 2002. Salicylate 5-hydroxylase from Ralstonia sp. strain U2: a monooxygenase with close relationships to and shared electron transport proteins with naphthalene dioxygenase. J. Bacteriol. 184, 1547e1555. Zhuang, X.L., Han, Z., Bai, Z.H., Zhuang, G.Q., Shim, H., 2010. Progress in decontamination by halophilic microorganisms in saline wastewater and soil. Environ. Pollut. 158, 1119e1126.
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Metabolic pathway for degradation of anthracene by halophilic Martelella sp. AD-3 Changzheng Cui a, Lei Ma b, Jie Shi a, Kuangfei Lin a, Qishi Luo c, Yongdi Liu a, * a State Environmental Protection Key Laboratory of Environmental Risk Assessment and Control on Chemical Process, School of Resources and Environmental Engineering, East China University of Science and Technology, Shanghai 200237, China b Shanghai Key Laboratory of New Drug Design, School of Pharmacy, East China University of Science and Technology, Shanghai 200237, China c State Environmental Protection Engineering Center for Urban Soil Contamination Control and Remediation, Institute of Wastes and Soil Environment, Shanghai Academy of Environmental Science, Shanghai 200233, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 October 2013 Received in revised form 17 January 2014 Accepted 21 January 2014 Available online 11 February 2014
Anthracene (40 mg l1) was completely depleted by Martelella sp. strain AD-3 under 3% salinity and a pH of 9.0 after 6 days of incubation. The metabolites were extracted and identified by high-performance liquid chromatography (HPLC) retention times, mass spectrometry, 1H and 13C nuclear magnetic resonance spectrometry, and comparison to authentic compounds or literature data. On the basis of the identified metabolites, enzyme activities and the utilization of probable intermediates, anthracene degradation by strain AD-3 is proposed via two distinct routes. In route I, metabolism of anthracene is initiated by the dioxygenation at C-1,2, then proceeds through 6,7-benzocoumarin, 3-hydroxy-2naphthoic acid, salicylic acid and gentisic acid. In route II, anthracene is metabolized to 9,10anthraquinone. The results suggest that strain AD-3 possesses efficient anthracene biodegradability in high salinity. To our knowledge, this work presents the first report of anthracene degradation by a halophilic PAH-degrading strain via two routes. The strain AD-3 may be a useful candidate for PAHcontaminated saline-alkali soil bioremediation. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Anthracene Biodegradation Moderate halophilic bacteria Metabolic pathway 3-Hydroxy-2-naphthoic acid
1. Introduction The petroleum industry generates a huge amount of oily and saline waste water (oily brines, production waters) after separation of crude oil from reservoir water, which has to be disposed. The main contaminants in this production water are aromatic and polycyclic aromatic hydrocarbons (PAHs) (Borgne et al., 2008; Zhuang et al., 2010). In addition, some marine and coastal sites are polluted by PAHs due to occasional accidents, such as oil spills during production, transportation or refining processes of the oil (Borgne et al., 2008). It is crucial to develop an efficient remedial process for both saline water and soil where natural attenuation is very slow. Anthracene (ANT), a tricyclic PAH, is found in high concentrations in PAH-contaminated sediments, surfaces soils, and waste sites (Wilson and Jones, 1993). It has been registered as a persistent compound that is toxic to marine life (Choi and Oris, 2003). In addition, ANT is considered a benchmark PAHs and serves as a * Corresponding author. Tel./fax: þ86 21 64253988. E-mail addresses: [email protected] (C. Cui), [email protected] (Y. Liu). 0964-8305/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.ibiod.2014.01.012
signature compound to detect PAHs contamination, since its chemical structure is found in carcinogenic PAHs, such as benz[a] anthracene and benzo[a]pyrene. According to previous reports, biodegradation of ANT by Pseudomonas (Evans et al., 1965; Menn et al., 1993), Mycobacterium (Moody et al., 2001; Herwijnen et al., 2003a), Rhodococcus (Tongpim and Pickard, 1996; Dean-Ross et al., 2001), Sphingomonas (Herwijnen et al., 2003b), Nocardia (Zeinali et al., 2008), and Bacillus (Ahmed et al., 2012) under non-halophilic conditions has been demonstrated to include two major types of metabolic pathways. One is via dioxgenation at the C-1 and C-2 ring positions, to form 1,2-dihydroxy-anthracene. This dihydroxylated intermediate is further metabolized by cleavage of the ring at the meta position to yield 4-(2-hydroxynaphth-3-yl)-2-oxobut-3-enoic acid. This compound is unstable and will spontaneously rearrange to form 6,7Benzocoumarin, or can be further metabolized to 3-hydroxy-2naphthoic acid. However, to our knowledge, all studies of this pathway since Evans et al. (1965), have been unable to detect 3hydroxy-2-naphthoic acid from biodegradation of anthracene by wild-type strains. Subsequent metabolism proceeds via a pathway analogous to that of naphthalene biodegradation to yield salicylate or phthalic acid (Dean-Ross et al., 2001; Zeinali et al., 2008). The
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second pathway involves dioxygenation at the C-9 and C-10 positions to form 9,10-dihydrodiol, which converts non-enzymaticallly to the dead-end product 9,10-anthraquinone. In addition, Ye’s research group showed that fungi Aspergillus fumigates metabolizes ANT to form 9,10-anthraquinone, followed by conversion via an extracellular peroxidase enzyme (manganese peroxidase and lignin peroxidase) to yield phthalic acid under high salt conditions (Ye et al., 2001). Conventional microorganisms are unable to operate efficiently at salinities above that of seawater and their adaptation to salinity is easily lost after exposure to low salinity conditions. Moderately halophilic bacteria are potential candidates for use in the degradation of pollutants at high salt concentrations, since they can survive under high salinity (Ceylan et al., 2011). Either high or fluctuating salinity promotes the loss of cell wall integrity, protein denaturalization and changes in osmotic pressure (Borgne et al., 2008). Within halophiles, moderately halophilic microorganisms constitute an important group that grows optimally between 0.5 mol l1 (3%) and 2.5 mol l1 (15%) NaCl (Kushner, 1978). The biological treatment of industrial hyper-saline wastewaters and the bioremediation of polluted hyper-saline environments are important factors in selecting appropriate halophilic or halotolerant PAH-degrading microorganism (Betancur-Galvis et al., 2006; Arulazhagan and Vasudevan, 2011a; Haddadi and Shavandi, 2013). To our knowledge, the catabolic pathways of ANT biodegradation by halophilic bacteria have not been investigated. Martelella sp. strain AD-3 (CCTCC M 2011218), a moderate halophilic bacterium which was originally isolated in our laboratory from a petroleumcontaminated soil with high salinity (Cui et al., 2012), is capable of degrading naphthalene, phenanthrene, ANT and pyrene as the sole carbon source (Feng et al., 2012). The biodegradation pathway has been elucidated for the metabolism of phenanthrene by Martelella sp. strain AD-3 (Feng et al., 2012). We describe in the present study the possible metabolic pathways for degradation of ANT by this Martelella species under halophilic conditions, based on the metabolites identified from initial ring oxidation and ring cleavage products. 2. Materials and methods 2.1. Chemicals ANT, phenanthrene, 3-hydroxy-2-naphthoic acid, 1-hydroxy-2naphthoic acid, 2-hydroxy-1-naphthoic acid, 2,3dihydroxynaphthalene, salicylic acid and gentisic acid were purchased from SigmaeAldrich (St Louis, MO, USA) and were of the highest purity available. Nuclear magnetic resonance (NMR) solvents were purchased from Isotec, Inc. (Miamisburg, Ohio). Other organic solvents were purchased from (Rathburn, Walkerburn, Scotland) and were high-performance liquid chromatography grade. 2.2. Culture conditions Cultures of Martelella sp. AD-3 were grown in 500-ml Erlenmeyer flasks containing 200 ml of 3% minimal basal salt medium (pH 9.0) supplemented with 2.5 g l1 and 5 g l1 concentrations of yeast extract and peptone, respectively (Feng et al., 2012). A 20 ml aliquot of ANT (10 mg ml1) in N,N-dimethylformamide was added to flasks for enzyme induction. The cells from the flasks were harvested 2 days after inoculation by centrifugation at 8500 rpm for 10 min at 4 C. The cultures (OD600, 1) were incubated in 50 ml of 3% minimal basal medium (pH 9.0) in 250 ml Erlenmeyer flasks. ANT was dissolved in N,N-dimethylformamide and added to the
cultures to a final concentrations 40 mg l1. All cultures were incubated in the dark at 30 C with shaking at 150 rpm. 2.3. Analytical methods To identify the metabolites, contents of the flask were extracted four times with 50 ml of ethyl acetate, twice at neutral pH and twice after acidification to pH 2 with 5 M HCl. The neutral and acidic extracts were pooled, and the solvents were evaporated completely under gentle sparging with high-purity nitrogen. The residues were dissolved in 2 ml of acetonitrile and concentrated for analysis by reverse-phase HPLC and Gas chromatography/mass spectrometry (GCeMS). Flasks were extracted at 0, 2, 4, 6 and 8 days, respectively. ANT and its metabolites were analyzed by reverse-phase HPLC (Waters) equipped with a diode array UVevisible light (Vis) detector and a Waters XBridgeTM C18 analytical column (150 4.6 mm, i.d., 3.5 mm particle-size). A guard column was used to protect the analytical column. Initial HPLC conditions were 95% buffer A (water-0.01% TFA) and 5% buffer B (acetonitrile-0.01% TFA) with the following linear gradient: From 0 to 5 min chromatography was maintained in 95% buffer A. From 5 to 20 min chromatography was in a linear gradient from 95% buffer A to 95% buffer B; from 20 to 25 min at constant 95% buffer B; 25e25.1 min switched sharply to 95% buffer A; and from 25.1 to 32 min maintained in 95% A. The injection volume was 20 ml. For collection of sufficient quantities of metabolites to perform nuclear magnetic resonance (NMR) analysis, Waters reverse-phase HPLC was preformed with a diode array UVevisible light (Vis) detector and a Waters XBridgeTM C18 Prep column (50 19 mm, i.d., particle-size is 5 mm). Purification was performed in a gradient of buffer A to buffer B at a flow rate of 17 ml min1 using the following method. From 0 to 9 min, a linear gradient from 95% buffer A to 14% buffer A; from 9 to 9.1 min sharply switched to 95% buffer B; from 9.1 to 11 min in 95% buffer B; from 11 to 11.1 min a sharp switch from 95% buffer B to 95% buffer A; and from 11.1 to 13 min in 95% buffer A. The 8.44 min fraction was chosen to purify the compound. The injection volume was 150 ml. Proton (1H) NMR spectra were obtained on a Bruker AM500 spectrometer (Bruker Instruments, Billerica, MA, USA). Each metabolite was dissolved in 0.5 ml deuterated methanol (99.96 atom% 2H) for analysis. Chemical shifts are reported on the d scale (ppm) by assigning the residual solvent peak to 2.49 or 3.30 ppm, respectively. Typical 1H data acquisition parameters were: data size, 3200; sweep width, 7042 Hz; filter width, 8900 Hz; acquisition time, 2.33 s; flip angle, 900; relaxation delay, 0 s; temperature, 298.5 K. For measurement of coupling constants, the free induction decay was zero-filled to 64,000 resulting in a final data point resolution of 0.215 Hz per point. Coupling constants reported are first order. Those that were non-first order and those of overlapping resonances were omitted. Assignments were made from integration, analysis of substituent effects, and comparison to spectra of pure, known standards. A 13C NMR spectrum was obtained for one metabolite. EI-MS: Finnigan MAT-90/95 sector-field mass spectrometer; in m/z. GC/MS was performed using Agilent Technologies 7890A GC System coupled with an Agilent 5975C VL MSD with Triple-axis mass detector with an Agilent HP5-MS Ultra Inert (60 m 0.25 mm, 0.25 mm film thickness) column. The column temperature program followed the method as described by Feng et al. (2012). Derivatization prior to GC/EI-MS analysis was performed by silylation with N,O-Bis(trimethylsilyl)trifluoroacetamide 1% with trimethylchlorosilane (Sigma). 100 ml of sample was dissolved in acetonitrile, then mixed with 150 ml of silylation reagent and allowed to react for 1 h at 60 C. The injection volume was 1 ml.
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2.4. Utilization of other organic compounds The ability of the strain to grow on alternative organic compounds, including 3-hydroxy-2-naphthoic acid, 2,3dihydroxynaphthalene, salicylic acid, o-phthalic acid, catechol, gentisic acid and succinic acid, was evaluated. The test was performed using MSM (100 ml, pH 7.5) containing a given organic compound with a concentration of 100 mg l1 in a sealed 250 ml flask. In order to prevent decomposition of 2,3-dihydroxynaphthalene, the test was performed using MSM (100 ml, pH 5.5). The un-inoculated control and duplicate samples were prepared for each treatment. After three days of incubation at 30 C and agitation speed of 150 rpm, the reaction solutions and controls were sampled and examined for their OD at 600 nm (OD600). 2.5. Enzyme assays Enzyme activities in selected cell-free extracts were applied to confirm the route of metabolism of ANT by strain AD-3. Reported spectrophotometric methods were used to monitor the activities of 3-hydroxy-2-naphthoic acid hydroxylase, 1-hydroxy-2-naphthoic acid hydroxylase (Deveryshetty and Phale, 2010), 1-hydroxy-2naphthoic acid dioxygenase (Deveryshetty and Phale, 2009), salicylate 5-hydroxylase (S5H) (Zhou et al., 2002), gentisate 1,2dioxygenase (GDO) (Fu and Oriel, 1998), catechol 1,2-dioxygenase (Doddamani and Ninnekar, 2000) and catechol 2,3-dioxygenase (Doddamani and Ninnekar, 2000). Cell-free extracts included ANT, phenanthrene, salicylic acid and glucose. The preparation of the cell-free extracts was carried out using a previously described procedure (Fu and Oriel, 1998). A published Bradford assay method (1976), with bovine serum albumin as the standard, was used to determine protein concentrations. Enzyme activity was defined as units (nmol of the product formed or substrate disappeared, NADH formed) min1 ml1. The specific activities were expressed as units: mg1 protein.
Fig. 1. HPLC elution profile of metabolites produced during the growth of Martelella sp. strain AD-3 by different incubation time in the presence of anthracene. The UV absorbance wavelength was used at 254 nm. (B) 4 days, (C) 6 days, (D) 8 days.
To our knowledge, ANT degradation has been little studied in halophilic microorganisms. According to previous reports, Ochrobactrum sp. VA1 utilized 87% of anthracene (3 mg l1) in 4 days at 30 g l1 of NaCl concentration along with glucose as additional carbons source (Arulazhagan and Vasudevan, 2011a). Ochrobactrum sp. VA1 utilized 65% of anthracene (3 mg l1) in 4 days at 60 g l1 of NaCl concentration along with yeast extract (Arulazhagan and Vasudevan, 2011b). However, in present research, anthracene (40 mg l1) was completely depleted by Martelella sp. strain AD-3 under 3% salinity and a pH of 9.0 after 6 days of incubation. The results suggest that strain AD-3 possesses efficient anthracene biodegradation ability in high salinity. 3.2. ANT metabolite identification
2.6. Statistical analysis Statistical analysis was carried out using SPSS version 17.0 software. Multiple comparison analysis was performed using the least significant difference (LSD) test at the significant level of P > 0.05. The results were expressed as the mean values and corresponding standard errors. 3. Results and discussion 3.1. Characterization of ANT degradation and metabolite formation The characterization of ANT biodegradation and the formation of specific ANT metabolites by Martelella sp. strain AD-3 cultures was measured over time. The degradation of ANT was monitored, by HPLC, for up to 8 days. After 4 days of incubation, most ANT was degraded to metabolites by AD-3 cultures; a visible yellow color in the medium indicated the accumulation of some metabolite. The was not a significant color change in any of the controls. After 4 days incubation, four metabolite fractions of ANT, designated ANT-I, ANT-II, ANT-III, and ANT-IV, with retention times of 15.2, 15.3, 17.0 and 17.3 min, respectively, were observed after HPLC analysis. The parent compound, ANT, was eluted at 21.1 min (Fig. 1). The metabolites found were not present in any of the controls after HPLC analysis. We observed the ANT peak gradually disappear; while the ANT-III peak areas gradually increased, followed by ANT-III peak areas gradually decreasing. These results showed Martelella sp. strain AD-3 is not only capable of degrading ANT, but also capable of continuing to degrade metabolic intermediates ANT-III (Fig. 1).
HPLC collection of the extract from ANT incubations produced one big peak of metabolite ANT-III as shown in Fig. 1. Compound ANT-III was obtained as an amorphous yellow powder after drying the eluent. The molecular formula was determined as C11H8O3 by EI mass spectroscopy, from the molecular ion signal at an m/z of 188.0. The 1H NMR assignments and coupling constants are as follows: d 8.50 (s, H1), 7.80 (dd, J ¼ 8.1, 1.3, H8), 7.66 (dd, J ¼ 8.3, 1.2 Hz, H5), 7.47 (ddd, J ¼ 8.3, 6.8, 1.3 Hz, H6), 7.29 (ddd, J ¼ 8.1, 6.8, 1.2 Hz, H7), 7.22 (s, H4). The 1H NMR spectrum showed six aromatic resonances with a coupling pattern (singlet peaks at d 8.50 and 7.22) indicative of substitution at the C-2 (carboxyl group) and C-3 (hydroxyl group) positions. From the above EI mass and 1H NMR data, the structure of ANT-III was identified as 3-hydroxy-2-naphthoic acid. The 13C NMR (125 MHz, CD3OD) data are as follows: d 173.2, 157.9, 139.2, 133.8, 130.2, 130.0, 128.4, 127.1, 124.8, 115.8, 112.0 (Fig. 2), which further confirmed that the structure of ANT-III as 3-hydroxy-2naphthoic acid. Other metabolites (ANT-I, ANT-II, and ANT-IV) were not present in sufficient quantities to perform EI mass or NMR analyses. A variety of bacterial species have been isolated that have the ability to utilize ANT as the sole source of carbon and energy. The initial reaction in the degradation of ANT is catalyzed by multicomponent dioxygenases that incorporate both atoms of molecular oxygen into the PAHs nucleus to produce cis-dihydrodiols. The original papers propose that ANT -degrading strains oxidize ANT in the 1,2 position to form (þ)-(1R,2S)-cis-1,2-dihydroxy-1,2dihydroanthraene, which is subsequently converted to 1,2dihydroanthracene, which is further metabolized to 3-hydroxy-2-
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Fig. 2. 1H NMR spectrum (A) and hydroxy-2-naphthoic acid.
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C NMR spectrum of metabolite II identified as 3-
naphthoic acid, salicylate and catechol by enzymes of the pathway (Evans et al., 1965). However, to our knowledge, the metabolic products of ANT biodegradation by wild-type strains (Moody et al., 2001; Dean-Ross et al., 2001; Herwijnen et al., 2003a; Zeinali et al., 2008; Ahmed et al., 2012), specifically, 3-hydroxy-2-naphthoic acid, have not been isolated. The metabolite 2-hydroxy-3-naphthoic acid from biodegradation of anthracene was identified by Pseudomonas fluorescens 5R mutant 5RL (Menn et al., 1993). Similarly, the mutant strains VM843 and VM844 of Mycobacterium sp. strain PYR-1 from these works contained a very small peak which was identified with the use of a derivatized sample as derivatized 3-hydroxy-2naphthoic acid (Moody et al., 2001). However, in the present investigation, we find 3-hydroxy-2-naphthoic acid is formed from ANT. Its very large HPLC peak suggests degradation of ANT through 3-hydroxy-2-naphthoic acid as the main pathway, and that 3hydroxy-2-naphthoic acid is not a dead-end product, but could be further degraded (Fig. 1). A cleavage reaction similar to that of 1hydroxy-2-naphthoic acid in the Martelella AD-3 degradation pathway of phenanthrene has been shown to form the cleavage product (Feng et al., 2012). GCeMS chromatograms of derivatized samples from cultures contained three potential metabolite peaks, designated as metabolite I, metabolite II, and metabolite III. Metabolite I, eluting at 22.7 min, had a mass spectra with a molecular ion (M)þ at m/z 208 and fragment ions at m/z 180, 152, 126 and 76 indicating that the compound could be 9,10-anthraquinone on the basis of its m/z values and comparison of the mass spectra with the NIST library available in GCeMS (Fig. 3a), which was consistent with those of
9,10-anthraquinone described by Dean-Ross et al. (2001). An alternate route of enzymatic attack by Martelella sp. strain AD-3 is at the C-9 and C-10 positions of ANT. Initial attack on ANT at C-9,10 positions is consistent with previous reports on Mycobacterium (Moody et al., 2001) and Bacillus (Ahmed et al., 2012) growing on ANT. The presence of the dead-end product 9,10-anthraquinone could be explained by the formation and non-enzymatic oxidation of 9,10-dihydroxyanthracene (Moody et al., 2001). Metabolite II, eluting at 23.3 min, had a mass spectra with a molecular ion (M)þ at m/z 332 and major fragment ions at m/z 317, 244, 185, 141 and 73 (Fig. 3b), which was identified as derivatized, indicating that the compound could be 3-hydroxy-2-naphthoic acid on the basis of its m/z values and comparison of the mass spectra with the NIST library available in GCeMS. Therefore, Metabolite II was identified as derivatized 3-hydroxy-2-naphthoic acid. Metabolite III, eluting at 24.1 min, had a mass spectra with a molecular ion (Mþ) at m/z 196 and fragment ions at m/z 168, 139, 98, 84, 70 and 63 (Fig. 3c), which was consistent with those of 6,7benzocoumarin described by Dean-Ross et al. (2001) and Herwijnen et al. (2003a). The formation of 6,7-benzocoumarin indicates that dioxygenase occurred at the C-1 and C-2 position of ANT to form 1,2-dihydroxyanthracene as the initial oxidation product followed by its meta-cleavage (Herwijnen et al., 2003a). However, 1,2-dihydroxyanthracene was not detected in the present study. The 6,7-benzocoumarin has also been found to occur in Mycobacterium (Moody et al., 2001), Rhodococcus (Dean-Ross et al., 2001), Bacillus (Ahmed et al., 2012), and Pseudomonas (Evans et al., 1965) species, suggesting that it may be a common pathway for the initial degradation of ANT in both non-halophilic and halophilic organisms. The original papers propose 6,7-benzocoumarin to be a dead-end product (Evans et al., 1965), but recent literature (Moody et al., 2001; Herwijnen et al., 2003a) propose 6,7-benzocoumarin as an intermediate in the degradation pathway. The results show that Martelella sp. strain AD-3 efficiently metabolized ANT. The isolation and characterization of the major oxidation and ring fission products indicated two routes of enzymatic attack. The degradation pathways of ANT by Martelella sp. strain AD-3 is proposed in Fig. 4. Present research and previous studies of the biodegradation of ANT by Martelella sp. strain AD-3 suggest that both dioxygenases and monooxygenases catalyze the initial attack on the aromatic ring. Since positional isomers of cis-dihydrodiols are formed, it may also be speculated that several dioxygenase are present in Martelella sp. strain AD-3. The broad range of PAHs that are degraded by Martelella sp. strain AD-3 may also be due to a relaxed specificity of the same dioxygenase for initial attack on PAHs. The identification of ortho-ring cleavage intermediates from the degradation of dihydroxylated metabolites of phenanthrene (Feng et al., 2012) indicate alternative enzymatic routes in the degradation of PAHs by Martelella sp. strain AD-3. Bioremediation is an economically and environmentally attractive solution for cleaning PAHs in saline-alkaline soils. In order to achieve an efficient bioremediation process, it is important that the bacteria involved perform a complete degradation pathway so that potentially toxic metabolites do not accumulate in the soil (Herwijnen et al., 2003a). In this study, we examined the pathway for ANT degradation by Martelella sp. strain AD-3 to determine whether metabolites are expected to accumulate. 3.3. Transformation and formation of intermediates We tested growth of Martelella sp. strain AD-3 on several possible intermediates. 3-hydroxy-2-naphthoic acid, 1-hydroxy-2naphthoic acid, 2-hydroxy-1-naphthoic acid, 2,3-
C. Cui et al. / International Biodeterioration & Biodegradation 89 (2014) 67e73
Fig. 3. EI mass spectrum of metabolite I (a), II (b) and III (c) identified as, 9,10-anthraquinone, 3-hydroxy-2-naphthoic acid and 6,7-benzocoumarin, respectively.
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not be observed. This observation shows that enzymes responsible for degradation of 3-hydroxy-2-naphthoic acid are induced during growth on ANT and this may explain the poor growth on 3hydroxy-2-naphthoic acid. Similarly, Mycobacterium sp. strain PYR-1 could degrade ANT through o-phthalic acid, however, no growth was observed on 3-hydroxy-2-naphthoic acid (Herwijnen et al., 2003a). 3.4. Enzyme activities in cell-free extracts
Fig. 4. Proposed pathway for the degradation of anthracene by the halophilic strain Martelella sp. AD-3 on the basis of identified metabolites, enzyme activity and utilization profile of different substrates. Compounds in brackets are hypothetical intermediates, but not detected.
dihydroxynaphthalene, salicylic acid, benzoic acid, catechol, and gentisic acid and succinic acid were tested to see if they could serve as growth substrates. Anthracene, salicylic acid, gentisic acid, and succinic acid as the sole carbon showed good growth. While culture with 1-hydroxy-2-naphthoic acid, 3-hydroxy-2-naphthoic acid and 2,3-dihydroxynaphthalene as the sole carbon showed poor growth. This was evident by measuring significant cell density increases after 3 days of incubation. Moreover, no growth was observed on 2hydroxy-1-naphthoic acid, o-phthalic acid and catechol. The detailed results are shown in Table 1. To examine the transformation of 3-hydroxy-2-naphthoic acid by this bacterium, we preformed experiments with cell extracts in which we compared bacteria grown on ANT and yeast extract. Transformation of 3-hydroxy-2-naphthoic acid could only be observed in cell extracts of cultures grown on the concentration of ANT 10 mg l1. In assays with cell extracts from cultures grown on yeast extract, transformation of 3-hydroxy-2-naphthoic acid could Table 1 Profile of utilization of different substrates by strain AD-3. Substrates
Concentration (mg l
Anthracene 3-hydroxy-2-naphthoic acid 1-hydroxy-2-naphthoic acid 2-hydroxy-1-naphthoic acid 2,3-dihydroxynaphthalene Salicylic acid o-phthalic acid Succinic acid Gentisic acid Catechol
40 100 100 100 100 100 100 100 100 100
1
)
An enzyme extract prepared from ANT-grown cells showed 3hydroxy-2-naphthoic acid hydroxylase activity, but failed to showed the activity of 1-hydroxy-2-naphthoic acid hydroxylase, catechol-1,2-dioxygeanse, catechol-2,3-dioxygenase, or gentisate 1,2-dioxygenase. An enzyme extract prepared from phenanthrenegrown cells showed 1-hydroxy-2-naphthoic acid hydroxylase activity, but failed to show the activity of 3-hydroxy-2-naphthoic acid hydroxylase activity. In this preliminary study, we assume 3hydroxy-2-naphthoic acid hydroxylase is different from 1hydroxy-2-naphthoic acid hydroxylase. Salicylic acid-grown cells showed comparatively reduced activities for 1-hydroxy-2naphthoic acid hydroxylase and significantly higher activities of salicylate 5-hydroxylase, gentisate 1,2-dioxygenase (Feng et al., 2012). Similarly, comparatively little activity for 3-hydroxy-2naphthoic acid hydroxylase was detected. The lack of activity of these enzymes in glucose-grown cell-free extracts indicated that the enzymes responsible for anthracene metabolism were inducible (Table 2). The specific activity versus growth profile indicated the presence of three hydroxylases in strain AD-3, salicylate-5hydroxylase, 1-hydroxy-2-naphthoic acid hydroxylase, and 3hydroxy-2-naphthoic acid hydroxylase. Salicylate 5-hydroxylase and 1-hydroxy-2-naphthoic acid hydroxylase from various bacterial sources have been characterized and reported (Adachi et al., 1999; Zhou et al., 2002; Deveryshetty and Phale, 2010). However, to our knowledge, the enzyme characterization for 3-hydroxy-2naphthoic acid hydroxylase has not been reported so far and is worthy of further study. 4. Conclusion Martelella sp. AD-3 extends our knowledge of moderate halophilic bacteria that can initiate an attack on ANT through two different pathways: via the C-1,2 or C-9,10 positions. The observation of the degradation of ANT through 3-hydroxy-2-naphthoic acid, 2,3-dihydroxyanthracene and gentisate acid indicate the presence of a complete ANT degradation pathway in the ANTTable 2 Specific activity of various enzymes in the cell-free extract of halophilic strain AD-3 grown on different substrates to the mid-exponential phase. Enzymes
Specific activitya (nmol min1 mg1 protein) Glucose
Salicylic acid
Anthracene
1-Hydroxy-2-naphthoic acid hydroxylase 1-Hydroxy-2-naphthoic acid dioxygenase 3-hydroxy-2-naphthoic acid hydroxylase Salicylate 5-hydroxylase (S5H) Catechol-1,2-dioxygenase Catechol-2,3-dioxygenase Gentisate 1,2-dioxygenase (GDO)
NDb
34.1
30.2
ND
ND
ND
ND
38.6
120.4
ND
185.1
106.3
ND ND ND
ND ND 80.5
ND ND ND
Growth þþ þ þ e þ þþ e þþ þþ e
(þþ) Good growth: OD600 > 0.2; (þ) growth: 0.05 < OD600 < 0.2; () no growth: OD600 < 0.05. Cells were cultivated in MSM (salinity 3%, pH 7.5) containing each substrate individually at specific concentration at 30 C and 150 rpm in the dark for 3 days incubation except 2,3-dihydroxynaphthalene in MSM (salinity 3%, pH 5.5).
a b
The values are the average values measured in triplicate. Activity could not be detected.
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utilizing bacteria. This finding warrants further extensive investigations of degradation enzymes in moderate halophilic PAHdegrading bacteria. The strain AD-3 may be a useful candidate for PAH-contaminated saline-alkali soil bioremediation experiments. Acknowledgments This research was supported by the National Natural Science Foundation of China (41301329, 51378208), the Shanghai Municipal Natural Science Foundation (13ZR1410900), the Specialized Research Fund for the Doctoral Program of Higher Education (20110074130002), and the Fundamental Research Funds for the Central Universities (WB1114031). References Adachi, K., Iwabuchi, T., Sano, H., Harayama, S., 1999. Structure of the ring cleavage product of 1-hydroxy-2-naphthoate, and intermediate of the phenanthrenedegradative pathway of Nocardioides sp. strain KP7. J. Bacteriol. 181, 757e763. Ahmed, A.T., Othman, M.A., Sarwade, V.D., Gawai, K.R., 2012. Degradation of anthracene by Alkaliphilic bacteria Bacillus badius. Environ. Pollut. 2, 97e104. Arulazhagan, P., Vasudevan, N., 2011a. Role of nutrients in the utilization of polycyclic aromatic hydrocarbons by halotolerant bacterial strain. J. Environ. Sci. 23, 282e287. Arulazhagan, P., Vasudevan, N., 2011b. Biodegradation of polycyclic aromatic hydrocarbons by a halotolerant bacterial strain Ochrobactrum sp. VA1. Mar. Pollut. Bull. 62, 388e394. Betancur-Galvis, L.A., Alvarez-Bernal, D., Ramos-Valdivia, A.C., Dendooven, L., 2006. Bioremediation of polycyclic aromatic hydrocarbon-contaminated saline-alkaline soils of the former Lake Texcoco. Chemosphere 62, 1749e1760. Borgne, S.L., Paniagua, D., Rafael, V.D., 2008. Biodegradation of organic pollutants by halophilic bacteria and archaea. J. Mol. Microbiol. Biotechnol. 15, 74e92. Bradford, M.M., 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248e254. Ceylan, S., Akbulut, B.S., Denizci, A.A., Kazan, D., 2011. Proteomic insight into phenolic adaptation of a moderately halophilic Halomonas sp. strain AAD12. Can. J. Microbiol. 57, 295e302. Choi, J., Oris, J.T., 2003. Assessment of the toxicity of anthracene photo-modification products using the topminnow (Poeciliopsis lucida) hepatoma cell line (PLHC-1). Aquat. Toxicol. 65, 243e251. Cui, C.Z., Feng, T.C., Yu, Y.Q., Dong, F., Yang, X.M., Feng, Y.Y., Liu, Y.D., Lin, H.P., 2012. Isolation, characterization of an anthracene degrading bacterium Martelella sp. AD-3 and cloning of dioxygenase gene. Environ. Sci. Chin. 33, 4062e4068. Dean-Ross, D., Moody, J.D., Freeman, J.P., Doerge, D.R., Cerniglia, C.E., 2001. Metabolism of anthracene by a Rhodococcus species. FEMS Microbiol. Lett. 204, 205e211.
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