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In vitro degradation of fluoranthene by bacteria isolated from petroleum sludge
Bioresource Technology 102 (2011) 3709–3715
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In vitro degradation of fluoranthene by bacteria isolated from petroleum sludge Sushil Kumar a,1, Santosh Kumar Upadhayay b, Babita Kumari a, Sadhna Tiwari a, S.N. Singh a,⇑, P.K. Singh b a b
Environmental Science Division, National Botanical Research Institute, Lucknow-226001, India Plant Molecular Biology and Genetic Engineering Division, Lucknow-226001, India
a r t i c l e
i n f o
Article history: Received 24 August 2010 Received in revised form 19 November 2010 Accepted 22 November 2010 Available online 28 November 2010 Keywords: Fluoranthene Bacteria Catechol 1,2-dioxygenase Catechol 2,3-dioxygenase Molecular size determination
a b s t r a c t An investigation was carried out for in vitro degradation of fluoranthene by four bacterial strains (PSM6, PSM7, PSM10 and PSM11) isolated from the petroleum sludge. Although all the strains registered their growth in MSM with 100 ppm fluoranthene, PSM11 growth was better than other strains. Growth of bacterial strains invariably corresponded to their degradation potential of fluoranthene. After 168 h of incubation, 61% fluoranthene was degraded by PSM11, followed by PSM10 (48%) and PSM6 (42%) and the least was recorded in PSM7 (41%). Besides, 11% loss in fluoranthene was attributed to abiotic factors. Thirty-eight times more activity of catechol 2,3-dioxygenase than catechol 1,2-dioxygenase showed that it played a significant role in fluoranthene degradation. Molecular weight of catechol 2,3-dioxygenase isolated from PSM11 was determined as 136 kDa by size exclusion chromatography and 34 kDa on denaturing SDS–PAGE, indicating tetrameric nature of the enzyme. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Polyaromatic hydrocarbons (PAHs) are characterized as hazardous organic pollutants consisting of two or more fused rings in linear, angular and cluster arrangements. They are ubiquitous in environment and may be present at high concentrations at industrial sites associated with petroleum, coal tar, gas production and wood preservation industries (Wattiau, 2002). The presence of PAHs in the environment causes serious health hazard because of their mutagenic and carcinogenic properties (Kastner et al., 1998). On the basis of their abundance and toxicity, 16 PAH compounds have been included in the US Soil Protection Agency’s list of priority pollutants (Keith and Telliard, 1979). Hence, there is an increasing interest to remediate the sites contaminated with PAHs to minimize their threats to human health. As compared to physico-chemical treatments, use of microbial technology to clean up PAH-contaminated sites has been found an efficient, economical eco-friendly and adaptable choice. Besides, it has potential advantages over physio-chemical methods such as complete degradation of pollutants, greater safety and less soil disturbance (Habe and Omori, 2003). Liu et al. (2010) have studied bioremediation of oily sludge contaminated soil by stimulating indigenous microbes. Low molecular weight PAH (2–3 aromatic rings) are easily metabolized as compared to high molecular weight ones (more than three rings) which are slowly transformed due to their high hydrophobicity (Kanaly and Harayama, 2000) and ⇑ Corresponding author. Tel.: +91 522 2297823; fax: +91 522 2205836. 1
E-mail address: [email protected] (S.N. Singh). He is no more.
0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.101
decreased bioavailability because of absorbtion into the organic matter, strong binding to soil particles and sequestration inside micropores (Hatzinger and Alexander, 1995). Although PAH compounds are reportedly degraded by a wide range of microorganisms (Muller et al., 1996), only a few bacteria are able to grow on the four ring PAHs, specifically, fluoranthene (Rehmann et al., 1999; Luepromchai et al., 2007) and pyrene (Rehmann et al., 1998; Churchill et al., 1999). Mycobacterium, Rhodococcus, Alcaligenes and Sphingomonas are the genera which are generally encountered in this process. A study on the diversity of PAH degrading bacteria shows that Sphingomonas species are generally found fluoranthene degrading, while Pseudomonas strains were commonly associated with phenantherene degradation (Muller et al., 1997). In addition, a number of low molecular weight PAH degraders are also reported to co-metabolize HMW PAHs (Muller et al., 1997). Interestingly, Pizzul et al. (2007) have studied PAH degradation in soil by Mycobacterium sp. in a two-step sequential treatment. Fluoranthene is a major component of petroleum sludge of the Mathura Oil Refinery located in Northern part of India. Hence, it was selected as the model compound for the microbial degradation. Although metabolic pathways of 3-ring PAHs, such as phenantherene and anthracene have been studied in details, the degradation pathway of fluoranthene has not been well elucidated. However, Ho et al. (2000) have characterized fluoranthene and pyrene degrading bacteria isolated from PAH-contaminated soils and sediments and studied their degradation pathways. Gordon and Dorbon (2001) reported fluoranthene degradation by P. alcaligenes PA-10, while Rahmann et al. (2001) studied fluoranthene metabolism by Mycobacterium sp. and identified intermediates during degradation process. Kweon et al. (2007) used a polyomic approach
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to duciate the fluoranthene-degrading pathway in Mycobacterium vanbaalenae PYR-1. Li et al. (2010) studied the degradation of fluoranthene in anaerobic conditions (low or no oxygen) by indigenous and enrichment of a consortium of PAH degrading bacteria with amendment of Fe(III) in mangrove sediment slurry. Kazunga et al. (2001) reported fluoranthene 2,3 and 1,5 diones as novel products from bacterial transformation of fluoranthene. Peng et al. (2008) have recently reviewed the microbial transformation of polyaromatic hydrocarbons. In the present investigation, in vitro fluoranthene degradation was studied in MSM medium using fluoranthene as a sole source of carbon and energy by four bacterial strains isolated from the petroleum sludge of Mathura Oil Refinery in order to find out the highest fluoranthene degrading bacterial strain to be used further in the consortium for petroleum sludge degradation in the field conditions. Besides, involvement of specific fluoranthene degrading enzyme was also investigated in the highly degrading strain. The enzyme was purified for identification based on molecular size determination by size exclusion chromatography. The bacterial strain was identified on the basis of 16s ribosomal DNA sequencing method. 2. Methods 2.1. Bacteria culture media Modified MSM of Ijah et al. (1998) was used for the growth of bacterial strains with the composition (0.1% KH2PO4, 0.05% MgSO4, 0.05% NH4Cl, 0.05% KNO3, containing 0.1% (v/v) of each trace metal solution; 0.5% MnSO4, 0.01% FeSO4, 0.01% CaCl2, 0.1% CoCl2, 0.01% H3BO3, 0.01% ZnSO4, 0.01% Na2MoO4 and 0.1% (v/v) of each vitamin 0.02% biotin, 0.02% folic acid, 0.1% pyridoxine–HCl, 0.05% thiamine HCl, 0.05% riboflavin, 0.05% nicotinic acid, 0.05% pentothenic acid, 0.01% cyanobalamine). 100 ppm fluoranthene in MSM was used as a sole carbon and energy source for the study of in vitro degradation by the bacterial isolates. The pH of MSM medium was adjusted to 7.2. 2.2. Isolation, screening and identification of bacterial strains One gram of petroleum sludge, collected from Mathura oil refinery, Mathura, U.P. (India) was taken in MSM medium and incubated for 5 days for bacterial enrichment. Subsequently, petroleum hydrocarbon degrading bacteria were isolated following serial dilution method. Out of 12 bacterial isolates, four were screened to be potential degraders of fluoranthene on the basis of their growth in MSM media supplemented with different concentrations (50–200 ppm) of fluoranthene. For identification of fluoranthene degrading bacteria on the basis of 16s ribosomal DNA technology, they were grown in nutrient broth separately for overnight and pellets were formed by centrifugation at 15,000g 15 min for DNA extraction. These bacteria were identified on the basis of their homology (>99%) of DNA sequence with NCBI databases of bacteria following 16s DNA technology (Chromous Biotech, Bangalore).
separately in 100 ml of Erlenmeyer flask containing 10 ml of MSM with 100 ppm fluoranthene as sole carbon and energy source. However, control was maintained without isolates with 100 ppm fluoranthene to check the degradation of fluoranthene by the abiotic factor. All the flasks were placed in an orbital rotatory incubator set at 150 rpm for 168 h incubation period. 2.4. Extraction and analysis of fluoranthene degradation The residual fluoranthene after the bacterial degradation was extracted by liquid–liquid extraction (1:1 water:benzene) for overnight. Extracts were evaporated under a gentle nitrogen hood. Residue was dissolved in acetone and was analyzed by gas chromatograph (Agilent GC model 7890A) with flame ionization detector using capillary BP-5 column (5% phenyl methyl polysiloxane column, 30 m 0.32 mm 0.25 lm). Both injection and detector temperatures were maintained at 280 °C. The initial oven temperature was kept 80 °C for 2 min and increased to 300 °C with 10 °C increase per min. The injection volume (1 ll) of sample was taken for analysis. Retention time for fluoranthene was detected at 26.77 min. The concentration of fluoranthene was estimated by comparison against the fluoranthene standard (Sigma–Aldrich). 2.5. Bacterial growth analysis during fluoranthene degradation Bacterial cell density was measured by UV–Vis spectrophotometer at 600 nm and CFU was calculated based on a graph plotted OD versus CFU. 2.6. Protein determination Protein determination was carried out following the method of Lowry et al. (1951) using BSA (Bovine Serum Albumin) as standard. 2.7. Enzyme assays Activities of ortho cleaving catechol 1,2-dioxygenase (decycling) (EC 1.13.11.1) and meta cleaving enzyme catechol 2,3-dioxygenase (decycling) (EC 1.13.11.2) involved in PAH degradation were determined at 25 °C. The reaction was initiated by the addition of enzyme and formation of cis–cis muconate (k = 260 nm, e = 25.6 mM 1 cm 1) or 2 hydroxymuconic semialdehyde (k = 375 nm, e = 33.4 mM 1 cm 1) as transformed product was measured spectrophotometrically (Perkin Elmer Lambda 35) following the method of Ngai et al. (1990) and Sala-Trepat (1971). One unit of enzyme activity is expressed as the amount of enzyme forming 1 lmol of cis–cis muconate or 2 hydroxymuconic semialdehyde per min under the condition of the enzyme assay. 2.8. SDS–PAGE All the fractions of protein were analyzed on SDS–PAGE. Disulphide linkage was reduced by the treatment with DTT (Di Thio Treitol), followed by heating for subunit separation of protein in 12% polyacrylamide gel based on the molecular mass. A standard molecular weight marker (Sigma–Aldrich) was also run parallelly for determination of molecular weight of each protein band.
2.3. Experimental setup 2.9. Expression and purification of catechol 2-3-dioxygenase Four potential fluoranthene degrading bacterial strains (PSM6, PSM7, PSM10 and PSM11) isolated from petroleum sludge were enriched in nutrient broth containing 100 ppm of fluoranthene for 12 h at 37 °C. One milliliter of each inoculum having 1 OD600 nm was centrifuged at 3000 rpm for 15 min to get pellets of intact bacterial cells. After resuspension of the intact cells in MSM, these bacterial cells were inoculated (in triplicates)
Bacterial strain PSM11, which was found to be the highest degrader of fluoranthene, was grown in LB (Luria Bertani Broth) medium with 100 ppm fluoranthene as a sole source of carbon and energy for high expression of catechol 2,3-dioxygenase. Primary culture was inoculated in 100 ml LB and grown for 24 h at 37 °C in orbital shaker at 150 rpm. Cells were harvested by centrifugation
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(13,000g at 4 °C for 10 min) and medium containing extracellular enzyme was used for further purification. All purification steps were performed at 4 °C. Medium containing the desired protein was further centrifuged at 16,000g at 4 °C, filtered by 0.22 l filter (Milipore, USA) and desalted on G25 desalting column in 20 mM Tris buffer (pH 7.5). The desalted protein was loaded on mono-Q column pre-equilibrated with 20 mM Tris (pH 7.5) at the flow rate of 1 ml m 1 and washed by 20 mM Tris (pH 7.5) till OD280 became stable and eluted by linear gradient of NaCl (0–1 M). Fractions containing the higher activity of catechol 2,3-dioxygenase were further purified by size exclusion chromatography on superdex 200 column in buffer containing 20 mM Tris (pH 7.5) and 150 mM NaCl and concentrated by 10 kDa cut off filter (Millipore). All the steps of purifications were analyzed by SDS–PAGE. 2.10. Molecular size estimation The molecular size of the catechol 2,3-dioxygenase was estimated by size exclusion chromatography on Superdex 200 column equilibrated with 20 mM Tris (pH 7.5) and 150 mM NaCl. The column was calibrated with aldolase (158 kDa), albumin (67 kDa) and ovalbumin (43 kDa). 2.11. Iron estimation in purified enzyme Purified fraction of catechol 2,3-dioxygenase was filtered by 0.22 lm syringe filter and iron content of catechol 2,3-dioxygenase was determined by the atomic absorption spectrophotometer. 3. Results and discussion
Fig. 1. Growth of bacterial strains and degradation of fluoranthene (A) and pH change (B) in MSM containing 100 ppm fluoranthene during 168 h of incubation period.
3.1. Bacterial growth Isolated four bacterial strains (104 CFU ml 1) were inoculated in MSM with 100 ppm fluoranthene and incubated at 37 °C in an orbital shaker (150 rpm) for 7 days (168 h) to study their growth. It was observed that the bacterial growth was initially very slow and then got accelerated after the lag phase was over. In PSM6 and PSM11, the maximum CFU value was recorded after 120 h incubation, while in PSM7 and PSM10, the highest growth was recorded after 144 h incubation (Fig. 1A). Then after, their growth showed a declining trend. Among the four bacterial strains, the maximum CFU was recorded for PSM11 (1.2 107 CFU ml 1), followed by PSM10 (1.38 106 CFU ml 1) and PSM7 (1.18 106 CFU ml 1) and the least was recorded in PSM6 (1 106 CFU ml 1). This showed that PSM11 multiplied 1000 times during the same incubation period while other strains multiplied by 100 times only as compared to initial inoculum. There are several reports available on the utilization of fluoranthene as source of carbon and energy by pure bacterial strains (Weibenfels et al., 1990). Faster growth of PSM11 might have impact on degradation of fluoranthene in MSM. 3.2. Fluoranthene degradation Bacteria differed widely in their ability to degrade fluoranthene in MSM. As evident from Fig. 1A, the rate of fluoranthene degradation was initially slow up to 72 h of incubation and then enhanced in all the strains gradually. However, in the case of PSM11, the rate of fluoranthene degradation was recorded always higher than the other three bacterial strains namely PSM6, PSM7 and PSM10. After seven days of incubation, the maximum degradation was recorded in PSM11 (72%), followed by PSM10 (59%) and PSM6 (53%) and the least was observed in PSM7 (52%). After deduction of 11% loss of
fluoranthene by the abiotic factor as observed in control without bacterial inoculum, the actual degradation attributed to the bacterial strain was found to be 61% by PSM11, 48% by PSM10, 42% by PSM6 and a minimum of 41% by PSM7. Degradation of fluoranthene was initially slow due to its hydrophobic nature which restricts its availability to microbes. However, after initial degradation, the polarity was probably introduced into the fluoranthene which enhanced its availability to bacterial strains for degradation by extracellular enzymes. Karsa and Porta (1995) have reported involvement of monoxygenases and dioxygenases synthesized by bacteria in fluoranthene degradation. Most of PAH degradation reports have been made by gram negative bacteria such as Pseudomonas sp. and Mycobacterium sp. (Gordon and Dobson, 2001; Rehmann et al., 2001). In our investigation also, PSM11, a gram negative bacteria was found to be more efficient than three strains, as it could degrade 100 ppm fluoranthene by 61% in one week time in MSM. Some unidentified microbes were also isolated from soil mixed cultures which were capable of metabolizing both fluoranthene and pyrene together. Okparanma et al. (2009) have reported effectiveness of Bacillus substilis and P. aeruginosa in degradation of fluoranthene, pyrene, chrysene and benzo (a) anthracene. These metabolic differences may be linked to relative difference in PAH degrading enzymes. Gordon and Dobson (2001) studied fluoranthene degradation by P. alcaligenes PA-10 and identified four intermediates formed during fluoranthene degradation i.e. 9-fluorenone-1-carboxylic acid, 9-hydroxy-1-fluorene carboxylic acid, 9-fluorenone and 9-fluorenol based on HPLC analysis. However, Kelley et al. (1993) identified ten intermediates as 8-hydroxy-7-methoxyfluoranthene, 9-hydroxyfluorene, 9-fluorenone, 1-acenaphthenone, 9-hydroxy1-fluorenecarboxylic acid, phthalic acid, 2-carboxybenzaldehyde,
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benzoic acid, phenylacetic acid and adipic acid in fluoranthene degradation by Mycobacterium sp. Strain PYR1 on the basis of comparisons with UV-spectrophotometry, TLC, GC–MS and NMR. As far as toxicity of metabolites of fluoranthene degradation is concerned, Šepicˇ et al. (2003) have reported that with the exception of 9-hydroxyfluorene being four times and 9-fluorenone-1carboxylic acid and 9-fluorenone which are 162 and 37 times less to an alga Scenedesmus subspicatus than fluoranthene based on account of EC50 values, but other metabolites were found less toxic in order of 1000–3000 than fluoranthene. 3.3. Effect of degradation on pH It was observed that pH of MSM media prepared for the bacterial growth adjusted to 7.2 was decreased to 6.5 with the degradation of fluoranthene after 7 days of incubation in case of PSM11 (Fig. 1B). However, pH of the MSM media incubated with other bacterial strains for fluoranthene degradation did not show any significant change. Dibble and Bartha (1979) observed that optimum pH for oil sludge mineralization ranged from 5.0 to 7.8. In fluoranthene degradation, a slight drop in pH of the medium from pH 7.2 to 6.5 was noted which indicated the formation of acidic intermediates. According to Rehmann et al. (1999), degradation of fluoranthene leads to seven intermediates, out of which four are acidic in nature, while other three are neutral. In our experiment, slightly acidic conditions after seven days of incubation period made probably catecholases more active to degrade fluoranthene effectively. 3.4. Activities of extracellular catechol dioxygenases Specific activities of catechol 1,2-dioxygenase and catechol 2,3dioxygenase monitored at different days of incubation periods
Fig. 2. Specific activities of catechol 1,2 dioxygenase (A) and catechol 2,3 dioxygenase (B) in different bacterial strains during 168 h of incubation period.
revealed that they were biomarkers for degradation of fluoranthene. Activity of chromosome translated protein catechol 1,2-dioxygenase was recorded maximum after 72 h of inoculation in the case of PSM6 (68.49 g mole mg 1) and PSM7 (79.62 g mole mg 1) (Fig. 2A), while its highest activity was observed in PSM10 (78.13 g mole mg 1) and PSM11 (68.87 g mole mg 1) at 96 h incubation. On other hand, plasmid translated protein catechol 2,3-dioxygenase showed many fold increased activities in all the bacterial strains as compared to catechol 1,2-dioxygenase activity. Its highest activities were recorded as 661.97 and 2134.02 g mole mg 1 in PSM10 and PSM11, respectively after 120 h of incubation, while, PSM7 and PSM6 exhibited its highest specific activities at 96 h and 48 h, respectively (Fig. 2B). The higher specific activity of catechol 2,3-dioxygenase in all the bacterial strains indicated that meta cleaving was the major pathway for catechol degradation, while ortho cleaving was a weak pathway. As aerobic degradation takes place in MSM medium, pH decrease to a slightly acidic condition (from 7.2 to 6.5) further confirmed that incorporation of one molecular oxygen created acidic intermediates by increasing H+ concentration in medium following a substitution reaction. Slightly acidic condition in the MSM probably favored both bacterial growth and catechol 2,3-dioxygenase activity. However, further drop in medium pH might show a negative impact on bacterial multiplication and also on process of fluoranthene degradation. During PAH degradation, some of the genes up regulated while normal metabolic pathway gets inactiviated due to toxicity of these compounds. In our studies, we found that catechol 2,3-dioxygenase activity was enhanced many fold as compared to catechol 1,2-dioxygenase in all the strains in presence of fluoranthene. This indicates that role of plasmid borne catechol 2,3-dioxygenase is very significant in fluoranthene degradation as compared to
Fig. 3. SDS–PAGE analysis of catechol 2-3 dioxygenase purification steps: lane M; molecular weight marker, one; total protein in medium, two; purified on mono-Q column, three; purified on superdex 200 size exclusion column.
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the pathways as illustrated by Kelley et al. (1993) and Rehmann et al. (1999). Catechols are a group of representative central intermediates in metabolism of PAHs by bacteria. They degrade catechol through two pathways, the ortho-cleavage pathway and the meta-cleavage pathway, in which ring cleavage reactions are the first steps mediated by catechol 1,2-dioxygenase and catechol 2,3-dioxygenase, respectively (Smith, 1994). Higher activity of catechol 2,3-dioxygenase in our study clearly indicates that meta cleavage was a predominant process of fluoranthene degradation in contrast to chrysene degradation (Dhote et al., 2010). Junca and Pieper (2004) also reported over expression of catechol 2,3dioxygenase in BTEX contaminated environment. Since this enzyme played a significant role in fluoranthene degradation, it was extracted from PSM11 – the highest degrading strain for purification and molecular size determination to confirm its involvement in degradation process. 3.5. Expression, purification and molecular size analysis of catechol 2,3-dioxygenase Fig. 4. Molecular size analysis of catechol 2,3-dioxygenase on superdex 200 size exclusion elution volume was compared with standard proteins.
chromosomal encoded enzyme catechol 1,2-dioxygenase. In fact, bacterial degradation of fluoranthene starts with mono/di oxygenation of PAH by mono and dioxygenases (Lopez et al., 2005; Boyd et al., 2007). It is initially degraded to form catechol and substituted compounds by decarboxylation as the end product following
Among four degraders of fluoranthene, PSM11 was found to have the highest specific activity of catechol 2,3-dioxygenase, followed by PSM10, PSM7 and PSM6 sequentially at 120 h of incubation period in MSM medium. For purification of enzymes, PSM 11 was grown in LB medium. A total of 407 mg protein, secreted in medium from the 100 ml overnight grown culture, was loaded on mono-Q anion exchange column. The protein was eluted in several fractions. Fractions
Fig. 5. Gene sequence and phylogenetic relationship of PSM11.
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containing catechol 2,3-dioxygenase activities were collected and then further purified on superdex 200 size exclusion column. The purity of desired protein eluted from mono-Q column (with approximately 30% homogeneity) was increased to 80% after size exclusion chromatography (Fig. 3). The protein purified by size exclusion column was used for molecular size analysis. The elution time was compared with standard proteins and molecular size of the desired protein corresponded to the approximately 136 kDa (Fig. 4). However, the molecular mass of the purified protein observed on SDS–PAGE, was approximately 34 kDa. This showed that the catechol 2,3dioxygenase expressed in PSM 11 was tetrameric in nature. 3.6. Iron estimation in purified catechol 2,-dioxygenase In this enzyme, 0.76 iron atom per subunit was calculated which indicated presence of one iron atom per subunit. Addition of strong oxidizer, like hydrogen peroxide at a concentration 40 mM, reduced enzyme activity significantly. This indicated that iron was present in reduced state as co-factor in the enzyme. High activity of catechol 2,3-dioxygenase especially in PSM11 could enhance degradation of fluoranthene to less toxic intermediates. This enzyme is very specific in catabolizing PAH degradation in the environment. This is a tetrameric enzyme, consisting of four subunits each with one Fe atom. Fe atom facilitates the incorporation of oxygen into the substrate, which leads to rapid degradation by the aerobic bacteria. Firmly bound Fe indicated that the incorporation of Fe2+ atom in enzymes takes place during protein synthesis and not during enzymatic reaction at the time of fluoranthene degradation. 3.7. Stereospecificity and enzyme kinetics Catechol 2,3-dioxygenase purified from PSM11 exhibited high specificity towards catechol as compared to the other dihydroxy substituted aromatics like phenol, quinol, resorcinol and protocatechuatic acid (data not given). Highest apparent Km value for catechol was 2.5 lM (1 ml of reaction cocktail contained 50 mM phosphate buffer, pH 6.8, 0.15 mM catechol and 1 U of purified enzyme). It was also observed that enzyme was stable at room temperature. Storage at 25 °C for 20 days did not cause any significant loss in the activity. A range of temperature between 25 °C and 37 °C was found optimum for the maximum activity of enzyme which, in turn, caused degradation of fluoranthene. 3.8. Identification of bacteria Bacterial strain PSM11 which was found highest degrader of fluoranthene in screening was identified by Chromous Biotech based on 16s ribosomal DNA technology. The DNA sequences of PSM11 resembled very closely (>99%) to uncultured Acinetobacter sp. (A. No. GQ009228). The gene sequence and phylogenetic relationship of PSM11 have been reflected in Fig 5. 4. Conclusion Among four bacterial strains, PSM11 was found to be the highest degrader of 4-ring PAH fluoranthene which is a major constituent of petroleum sludge. In the degradation process, the extracellular enzyme catechol 2,3-dioxygenase played a very significant role as evidenced by 38 fold increase in its activity as compared to catechol 1,2 dioxygenase. Molecular size analysis of catechol 2,3dioxygenase isolated from uncultured Acinetobacter sp. PSM11 indicated tetrameric nature of the enzyme.
Acknowledgements The authors are thankful to Director, National Botanical Research Institute, Lucknow for providing laboratory facilities and to CSIR for providing funds to Network Project (NWP-019).
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
In vitro degradation of fluoranthene by bacteria isolated from petroleum sludge Sushil Kumar a,1, Santosh Kumar Upadhayay b, Babita Kumari a, Sadhna Tiwari a, S.N. Singh a,⇑, P.K. Singh b a b
Environmental Science Division, National Botanical Research Institute, Lucknow-226001, India Plant Molecular Biology and Genetic Engineering Division, Lucknow-226001, India
a r t i c l e
i n f o
Article history: Received 24 August 2010 Received in revised form 19 November 2010 Accepted 22 November 2010 Available online 28 November 2010 Keywords: Fluoranthene Bacteria Catechol 1,2-dioxygenase Catechol 2,3-dioxygenase Molecular size determination
a b s t r a c t An investigation was carried out for in vitro degradation of fluoranthene by four bacterial strains (PSM6, PSM7, PSM10 and PSM11) isolated from the petroleum sludge. Although all the strains registered their growth in MSM with 100 ppm fluoranthene, PSM11 growth was better than other strains. Growth of bacterial strains invariably corresponded to their degradation potential of fluoranthene. After 168 h of incubation, 61% fluoranthene was degraded by PSM11, followed by PSM10 (48%) and PSM6 (42%) and the least was recorded in PSM7 (41%). Besides, 11% loss in fluoranthene was attributed to abiotic factors. Thirty-eight times more activity of catechol 2,3-dioxygenase than catechol 1,2-dioxygenase showed that it played a significant role in fluoranthene degradation. Molecular weight of catechol 2,3-dioxygenase isolated from PSM11 was determined as 136 kDa by size exclusion chromatography and 34 kDa on denaturing SDS–PAGE, indicating tetrameric nature of the enzyme. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Polyaromatic hydrocarbons (PAHs) are characterized as hazardous organic pollutants consisting of two or more fused rings in linear, angular and cluster arrangements. They are ubiquitous in environment and may be present at high concentrations at industrial sites associated with petroleum, coal tar, gas production and wood preservation industries (Wattiau, 2002). The presence of PAHs in the environment causes serious health hazard because of their mutagenic and carcinogenic properties (Kastner et al., 1998). On the basis of their abundance and toxicity, 16 PAH compounds have been included in the US Soil Protection Agency’s list of priority pollutants (Keith and Telliard, 1979). Hence, there is an increasing interest to remediate the sites contaminated with PAHs to minimize their threats to human health. As compared to physico-chemical treatments, use of microbial technology to clean up PAH-contaminated sites has been found an efficient, economical eco-friendly and adaptable choice. Besides, it has potential advantages over physio-chemical methods such as complete degradation of pollutants, greater safety and less soil disturbance (Habe and Omori, 2003). Liu et al. (2010) have studied bioremediation of oily sludge contaminated soil by stimulating indigenous microbes. Low molecular weight PAH (2–3 aromatic rings) are easily metabolized as compared to high molecular weight ones (more than three rings) which are slowly transformed due to their high hydrophobicity (Kanaly and Harayama, 2000) and ⇑ Corresponding author. Tel.: +91 522 2297823; fax: +91 522 2205836. 1
E-mail address: [email protected] (S.N. Singh). He is no more.
0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.11.101
decreased bioavailability because of absorbtion into the organic matter, strong binding to soil particles and sequestration inside micropores (Hatzinger and Alexander, 1995). Although PAH compounds are reportedly degraded by a wide range of microorganisms (Muller et al., 1996), only a few bacteria are able to grow on the four ring PAHs, specifically, fluoranthene (Rehmann et al., 1999; Luepromchai et al., 2007) and pyrene (Rehmann et al., 1998; Churchill et al., 1999). Mycobacterium, Rhodococcus, Alcaligenes and Sphingomonas are the genera which are generally encountered in this process. A study on the diversity of PAH degrading bacteria shows that Sphingomonas species are generally found fluoranthene degrading, while Pseudomonas strains were commonly associated with phenantherene degradation (Muller et al., 1997). In addition, a number of low molecular weight PAH degraders are also reported to co-metabolize HMW PAHs (Muller et al., 1997). Interestingly, Pizzul et al. (2007) have studied PAH degradation in soil by Mycobacterium sp. in a two-step sequential treatment. Fluoranthene is a major component of petroleum sludge of the Mathura Oil Refinery located in Northern part of India. Hence, it was selected as the model compound for the microbial degradation. Although metabolic pathways of 3-ring PAHs, such as phenantherene and anthracene have been studied in details, the degradation pathway of fluoranthene has not been well elucidated. However, Ho et al. (2000) have characterized fluoranthene and pyrene degrading bacteria isolated from PAH-contaminated soils and sediments and studied their degradation pathways. Gordon and Dorbon (2001) reported fluoranthene degradation by P. alcaligenes PA-10, while Rahmann et al. (2001) studied fluoranthene metabolism by Mycobacterium sp. and identified intermediates during degradation process. Kweon et al. (2007) used a polyomic approach
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to duciate the fluoranthene-degrading pathway in Mycobacterium vanbaalenae PYR-1. Li et al. (2010) studied the degradation of fluoranthene in anaerobic conditions (low or no oxygen) by indigenous and enrichment of a consortium of PAH degrading bacteria with amendment of Fe(III) in mangrove sediment slurry. Kazunga et al. (2001) reported fluoranthene 2,3 and 1,5 diones as novel products from bacterial transformation of fluoranthene. Peng et al. (2008) have recently reviewed the microbial transformation of polyaromatic hydrocarbons. In the present investigation, in vitro fluoranthene degradation was studied in MSM medium using fluoranthene as a sole source of carbon and energy by four bacterial strains isolated from the petroleum sludge of Mathura Oil Refinery in order to find out the highest fluoranthene degrading bacterial strain to be used further in the consortium for petroleum sludge degradation in the field conditions. Besides, involvement of specific fluoranthene degrading enzyme was also investigated in the highly degrading strain. The enzyme was purified for identification based on molecular size determination by size exclusion chromatography. The bacterial strain was identified on the basis of 16s ribosomal DNA sequencing method. 2. Methods 2.1. Bacteria culture media Modified MSM of Ijah et al. (1998) was used for the growth of bacterial strains with the composition (0.1% KH2PO4, 0.05% MgSO4, 0.05% NH4Cl, 0.05% KNO3, containing 0.1% (v/v) of each trace metal solution; 0.5% MnSO4, 0.01% FeSO4, 0.01% CaCl2, 0.1% CoCl2, 0.01% H3BO3, 0.01% ZnSO4, 0.01% Na2MoO4 and 0.1% (v/v) of each vitamin 0.02% biotin, 0.02% folic acid, 0.1% pyridoxine–HCl, 0.05% thiamine HCl, 0.05% riboflavin, 0.05% nicotinic acid, 0.05% pentothenic acid, 0.01% cyanobalamine). 100 ppm fluoranthene in MSM was used as a sole carbon and energy source for the study of in vitro degradation by the bacterial isolates. The pH of MSM medium was adjusted to 7.2. 2.2. Isolation, screening and identification of bacterial strains One gram of petroleum sludge, collected from Mathura oil refinery, Mathura, U.P. (India) was taken in MSM medium and incubated for 5 days for bacterial enrichment. Subsequently, petroleum hydrocarbon degrading bacteria were isolated following serial dilution method. Out of 12 bacterial isolates, four were screened to be potential degraders of fluoranthene on the basis of their growth in MSM media supplemented with different concentrations (50–200 ppm) of fluoranthene. For identification of fluoranthene degrading bacteria on the basis of 16s ribosomal DNA technology, they were grown in nutrient broth separately for overnight and pellets were formed by centrifugation at 15,000g 15 min for DNA extraction. These bacteria were identified on the basis of their homology (>99%) of DNA sequence with NCBI databases of bacteria following 16s DNA technology (Chromous Biotech, Bangalore).
separately in 100 ml of Erlenmeyer flask containing 10 ml of MSM with 100 ppm fluoranthene as sole carbon and energy source. However, control was maintained without isolates with 100 ppm fluoranthene to check the degradation of fluoranthene by the abiotic factor. All the flasks were placed in an orbital rotatory incubator set at 150 rpm for 168 h incubation period. 2.4. Extraction and analysis of fluoranthene degradation The residual fluoranthene after the bacterial degradation was extracted by liquid–liquid extraction (1:1 water:benzene) for overnight. Extracts were evaporated under a gentle nitrogen hood. Residue was dissolved in acetone and was analyzed by gas chromatograph (Agilent GC model 7890A) with flame ionization detector using capillary BP-5 column (5% phenyl methyl polysiloxane column, 30 m 0.32 mm 0.25 lm). Both injection and detector temperatures were maintained at 280 °C. The initial oven temperature was kept 80 °C for 2 min and increased to 300 °C with 10 °C increase per min. The injection volume (1 ll) of sample was taken for analysis. Retention time for fluoranthene was detected at 26.77 min. The concentration of fluoranthene was estimated by comparison against the fluoranthene standard (Sigma–Aldrich). 2.5. Bacterial growth analysis during fluoranthene degradation Bacterial cell density was measured by UV–Vis spectrophotometer at 600 nm and CFU was calculated based on a graph plotted OD versus CFU. 2.6. Protein determination Protein determination was carried out following the method of Lowry et al. (1951) using BSA (Bovine Serum Albumin) as standard. 2.7. Enzyme assays Activities of ortho cleaving catechol 1,2-dioxygenase (decycling) (EC 1.13.11.1) and meta cleaving enzyme catechol 2,3-dioxygenase (decycling) (EC 1.13.11.2) involved in PAH degradation were determined at 25 °C. The reaction was initiated by the addition of enzyme and formation of cis–cis muconate (k = 260 nm, e = 25.6 mM 1 cm 1) or 2 hydroxymuconic semialdehyde (k = 375 nm, e = 33.4 mM 1 cm 1) as transformed product was measured spectrophotometrically (Perkin Elmer Lambda 35) following the method of Ngai et al. (1990) and Sala-Trepat (1971). One unit of enzyme activity is expressed as the amount of enzyme forming 1 lmol of cis–cis muconate or 2 hydroxymuconic semialdehyde per min under the condition of the enzyme assay. 2.8. SDS–PAGE All the fractions of protein were analyzed on SDS–PAGE. Disulphide linkage was reduced by the treatment with DTT (Di Thio Treitol), followed by heating for subunit separation of protein in 12% polyacrylamide gel based on the molecular mass. A standard molecular weight marker (Sigma–Aldrich) was also run parallelly for determination of molecular weight of each protein band.
2.3. Experimental setup 2.9. Expression and purification of catechol 2-3-dioxygenase Four potential fluoranthene degrading bacterial strains (PSM6, PSM7, PSM10 and PSM11) isolated from petroleum sludge were enriched in nutrient broth containing 100 ppm of fluoranthene for 12 h at 37 °C. One milliliter of each inoculum having 1 OD600 nm was centrifuged at 3000 rpm for 15 min to get pellets of intact bacterial cells. After resuspension of the intact cells in MSM, these bacterial cells were inoculated (in triplicates)
Bacterial strain PSM11, which was found to be the highest degrader of fluoranthene, was grown in LB (Luria Bertani Broth) medium with 100 ppm fluoranthene as a sole source of carbon and energy for high expression of catechol 2,3-dioxygenase. Primary culture was inoculated in 100 ml LB and grown for 24 h at 37 °C in orbital shaker at 150 rpm. Cells were harvested by centrifugation
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(13,000g at 4 °C for 10 min) and medium containing extracellular enzyme was used for further purification. All purification steps were performed at 4 °C. Medium containing the desired protein was further centrifuged at 16,000g at 4 °C, filtered by 0.22 l filter (Milipore, USA) and desalted on G25 desalting column in 20 mM Tris buffer (pH 7.5). The desalted protein was loaded on mono-Q column pre-equilibrated with 20 mM Tris (pH 7.5) at the flow rate of 1 ml m 1 and washed by 20 mM Tris (pH 7.5) till OD280 became stable and eluted by linear gradient of NaCl (0–1 M). Fractions containing the higher activity of catechol 2,3-dioxygenase were further purified by size exclusion chromatography on superdex 200 column in buffer containing 20 mM Tris (pH 7.5) and 150 mM NaCl and concentrated by 10 kDa cut off filter (Millipore). All the steps of purifications were analyzed by SDS–PAGE. 2.10. Molecular size estimation The molecular size of the catechol 2,3-dioxygenase was estimated by size exclusion chromatography on Superdex 200 column equilibrated with 20 mM Tris (pH 7.5) and 150 mM NaCl. The column was calibrated with aldolase (158 kDa), albumin (67 kDa) and ovalbumin (43 kDa). 2.11. Iron estimation in purified enzyme Purified fraction of catechol 2,3-dioxygenase was filtered by 0.22 lm syringe filter and iron content of catechol 2,3-dioxygenase was determined by the atomic absorption spectrophotometer. 3. Results and discussion
Fig. 1. Growth of bacterial strains and degradation of fluoranthene (A) and pH change (B) in MSM containing 100 ppm fluoranthene during 168 h of incubation period.
3.1. Bacterial growth Isolated four bacterial strains (104 CFU ml 1) were inoculated in MSM with 100 ppm fluoranthene and incubated at 37 °C in an orbital shaker (150 rpm) for 7 days (168 h) to study their growth. It was observed that the bacterial growth was initially very slow and then got accelerated after the lag phase was over. In PSM6 and PSM11, the maximum CFU value was recorded after 120 h incubation, while in PSM7 and PSM10, the highest growth was recorded after 144 h incubation (Fig. 1A). Then after, their growth showed a declining trend. Among the four bacterial strains, the maximum CFU was recorded for PSM11 (1.2 107 CFU ml 1), followed by PSM10 (1.38 106 CFU ml 1) and PSM7 (1.18 106 CFU ml 1) and the least was recorded in PSM6 (1 106 CFU ml 1). This showed that PSM11 multiplied 1000 times during the same incubation period while other strains multiplied by 100 times only as compared to initial inoculum. There are several reports available on the utilization of fluoranthene as source of carbon and energy by pure bacterial strains (Weibenfels et al., 1990). Faster growth of PSM11 might have impact on degradation of fluoranthene in MSM. 3.2. Fluoranthene degradation Bacteria differed widely in their ability to degrade fluoranthene in MSM. As evident from Fig. 1A, the rate of fluoranthene degradation was initially slow up to 72 h of incubation and then enhanced in all the strains gradually. However, in the case of PSM11, the rate of fluoranthene degradation was recorded always higher than the other three bacterial strains namely PSM6, PSM7 and PSM10. After seven days of incubation, the maximum degradation was recorded in PSM11 (72%), followed by PSM10 (59%) and PSM6 (53%) and the least was observed in PSM7 (52%). After deduction of 11% loss of
fluoranthene by the abiotic factor as observed in control without bacterial inoculum, the actual degradation attributed to the bacterial strain was found to be 61% by PSM11, 48% by PSM10, 42% by PSM6 and a minimum of 41% by PSM7. Degradation of fluoranthene was initially slow due to its hydrophobic nature which restricts its availability to microbes. However, after initial degradation, the polarity was probably introduced into the fluoranthene which enhanced its availability to bacterial strains for degradation by extracellular enzymes. Karsa and Porta (1995) have reported involvement of monoxygenases and dioxygenases synthesized by bacteria in fluoranthene degradation. Most of PAH degradation reports have been made by gram negative bacteria such as Pseudomonas sp. and Mycobacterium sp. (Gordon and Dobson, 2001; Rehmann et al., 2001). In our investigation also, PSM11, a gram negative bacteria was found to be more efficient than three strains, as it could degrade 100 ppm fluoranthene by 61% in one week time in MSM. Some unidentified microbes were also isolated from soil mixed cultures which were capable of metabolizing both fluoranthene and pyrene together. Okparanma et al. (2009) have reported effectiveness of Bacillus substilis and P. aeruginosa in degradation of fluoranthene, pyrene, chrysene and benzo (a) anthracene. These metabolic differences may be linked to relative difference in PAH degrading enzymes. Gordon and Dobson (2001) studied fluoranthene degradation by P. alcaligenes PA-10 and identified four intermediates formed during fluoranthene degradation i.e. 9-fluorenone-1-carboxylic acid, 9-hydroxy-1-fluorene carboxylic acid, 9-fluorenone and 9-fluorenol based on HPLC analysis. However, Kelley et al. (1993) identified ten intermediates as 8-hydroxy-7-methoxyfluoranthene, 9-hydroxyfluorene, 9-fluorenone, 1-acenaphthenone, 9-hydroxy1-fluorenecarboxylic acid, phthalic acid, 2-carboxybenzaldehyde,
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benzoic acid, phenylacetic acid and adipic acid in fluoranthene degradation by Mycobacterium sp. Strain PYR1 on the basis of comparisons with UV-spectrophotometry, TLC, GC–MS and NMR. As far as toxicity of metabolites of fluoranthene degradation is concerned, Šepicˇ et al. (2003) have reported that with the exception of 9-hydroxyfluorene being four times and 9-fluorenone-1carboxylic acid and 9-fluorenone which are 162 and 37 times less to an alga Scenedesmus subspicatus than fluoranthene based on account of EC50 values, but other metabolites were found less toxic in order of 1000–3000 than fluoranthene. 3.3. Effect of degradation on pH It was observed that pH of MSM media prepared for the bacterial growth adjusted to 7.2 was decreased to 6.5 with the degradation of fluoranthene after 7 days of incubation in case of PSM11 (Fig. 1B). However, pH of the MSM media incubated with other bacterial strains for fluoranthene degradation did not show any significant change. Dibble and Bartha (1979) observed that optimum pH for oil sludge mineralization ranged from 5.0 to 7.8. In fluoranthene degradation, a slight drop in pH of the medium from pH 7.2 to 6.5 was noted which indicated the formation of acidic intermediates. According to Rehmann et al. (1999), degradation of fluoranthene leads to seven intermediates, out of which four are acidic in nature, while other three are neutral. In our experiment, slightly acidic conditions after seven days of incubation period made probably catecholases more active to degrade fluoranthene effectively. 3.4. Activities of extracellular catechol dioxygenases Specific activities of catechol 1,2-dioxygenase and catechol 2,3dioxygenase monitored at different days of incubation periods
Fig. 2. Specific activities of catechol 1,2 dioxygenase (A) and catechol 2,3 dioxygenase (B) in different bacterial strains during 168 h of incubation period.
revealed that they were biomarkers for degradation of fluoranthene. Activity of chromosome translated protein catechol 1,2-dioxygenase was recorded maximum after 72 h of inoculation in the case of PSM6 (68.49 g mole mg 1) and PSM7 (79.62 g mole mg 1) (Fig. 2A), while its highest activity was observed in PSM10 (78.13 g mole mg 1) and PSM11 (68.87 g mole mg 1) at 96 h incubation. On other hand, plasmid translated protein catechol 2,3-dioxygenase showed many fold increased activities in all the bacterial strains as compared to catechol 1,2-dioxygenase activity. Its highest activities were recorded as 661.97 and 2134.02 g mole mg 1 in PSM10 and PSM11, respectively after 120 h of incubation, while, PSM7 and PSM6 exhibited its highest specific activities at 96 h and 48 h, respectively (Fig. 2B). The higher specific activity of catechol 2,3-dioxygenase in all the bacterial strains indicated that meta cleaving was the major pathway for catechol degradation, while ortho cleaving was a weak pathway. As aerobic degradation takes place in MSM medium, pH decrease to a slightly acidic condition (from 7.2 to 6.5) further confirmed that incorporation of one molecular oxygen created acidic intermediates by increasing H+ concentration in medium following a substitution reaction. Slightly acidic condition in the MSM probably favored both bacterial growth and catechol 2,3-dioxygenase activity. However, further drop in medium pH might show a negative impact on bacterial multiplication and also on process of fluoranthene degradation. During PAH degradation, some of the genes up regulated while normal metabolic pathway gets inactiviated due to toxicity of these compounds. In our studies, we found that catechol 2,3-dioxygenase activity was enhanced many fold as compared to catechol 1,2-dioxygenase in all the strains in presence of fluoranthene. This indicates that role of plasmid borne catechol 2,3-dioxygenase is very significant in fluoranthene degradation as compared to
Fig. 3. SDS–PAGE analysis of catechol 2-3 dioxygenase purification steps: lane M; molecular weight marker, one; total protein in medium, two; purified on mono-Q column, three; purified on superdex 200 size exclusion column.
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the pathways as illustrated by Kelley et al. (1993) and Rehmann et al. (1999). Catechols are a group of representative central intermediates in metabolism of PAHs by bacteria. They degrade catechol through two pathways, the ortho-cleavage pathway and the meta-cleavage pathway, in which ring cleavage reactions are the first steps mediated by catechol 1,2-dioxygenase and catechol 2,3-dioxygenase, respectively (Smith, 1994). Higher activity of catechol 2,3-dioxygenase in our study clearly indicates that meta cleavage was a predominant process of fluoranthene degradation in contrast to chrysene degradation (Dhote et al., 2010). Junca and Pieper (2004) also reported over expression of catechol 2,3dioxygenase in BTEX contaminated environment. Since this enzyme played a significant role in fluoranthene degradation, it was extracted from PSM11 – the highest degrading strain for purification and molecular size determination to confirm its involvement in degradation process. 3.5. Expression, purification and molecular size analysis of catechol 2,3-dioxygenase Fig. 4. Molecular size analysis of catechol 2,3-dioxygenase on superdex 200 size exclusion elution volume was compared with standard proteins.
chromosomal encoded enzyme catechol 1,2-dioxygenase. In fact, bacterial degradation of fluoranthene starts with mono/di oxygenation of PAH by mono and dioxygenases (Lopez et al., 2005; Boyd et al., 2007). It is initially degraded to form catechol and substituted compounds by decarboxylation as the end product following
Among four degraders of fluoranthene, PSM11 was found to have the highest specific activity of catechol 2,3-dioxygenase, followed by PSM10, PSM7 and PSM6 sequentially at 120 h of incubation period in MSM medium. For purification of enzymes, PSM 11 was grown in LB medium. A total of 407 mg protein, secreted in medium from the 100 ml overnight grown culture, was loaded on mono-Q anion exchange column. The protein was eluted in several fractions. Fractions
Fig. 5. Gene sequence and phylogenetic relationship of PSM11.
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containing catechol 2,3-dioxygenase activities were collected and then further purified on superdex 200 size exclusion column. The purity of desired protein eluted from mono-Q column (with approximately 30% homogeneity) was increased to 80% after size exclusion chromatography (Fig. 3). The protein purified by size exclusion column was used for molecular size analysis. The elution time was compared with standard proteins and molecular size of the desired protein corresponded to the approximately 136 kDa (Fig. 4). However, the molecular mass of the purified protein observed on SDS–PAGE, was approximately 34 kDa. This showed that the catechol 2,3dioxygenase expressed in PSM 11 was tetrameric in nature. 3.6. Iron estimation in purified catechol 2,-dioxygenase In this enzyme, 0.76 iron atom per subunit was calculated which indicated presence of one iron atom per subunit. Addition of strong oxidizer, like hydrogen peroxide at a concentration 40 mM, reduced enzyme activity significantly. This indicated that iron was present in reduced state as co-factor in the enzyme. High activity of catechol 2,3-dioxygenase especially in PSM11 could enhance degradation of fluoranthene to less toxic intermediates. This enzyme is very specific in catabolizing PAH degradation in the environment. This is a tetrameric enzyme, consisting of four subunits each with one Fe atom. Fe atom facilitates the incorporation of oxygen into the substrate, which leads to rapid degradation by the aerobic bacteria. Firmly bound Fe indicated that the incorporation of Fe2+ atom in enzymes takes place during protein synthesis and not during enzymatic reaction at the time of fluoranthene degradation. 3.7. Stereospecificity and enzyme kinetics Catechol 2,3-dioxygenase purified from PSM11 exhibited high specificity towards catechol as compared to the other dihydroxy substituted aromatics like phenol, quinol, resorcinol and protocatechuatic acid (data not given). Highest apparent Km value for catechol was 2.5 lM (1 ml of reaction cocktail contained 50 mM phosphate buffer, pH 6.8, 0.15 mM catechol and 1 U of purified enzyme). It was also observed that enzyme was stable at room temperature. Storage at 25 °C for 20 days did not cause any significant loss in the activity. A range of temperature between 25 °C and 37 °C was found optimum for the maximum activity of enzyme which, in turn, caused degradation of fluoranthene. 3.8. Identification of bacteria Bacterial strain PSM11 which was found highest degrader of fluoranthene in screening was identified by Chromous Biotech based on 16s ribosomal DNA technology. The DNA sequences of PSM11 resembled very closely (>99%) to uncultured Acinetobacter sp. (A. No. GQ009228). The gene sequence and phylogenetic relationship of PSM11 have been reflected in Fig 5. 4. Conclusion Among four bacterial strains, PSM11 was found to be the highest degrader of 4-ring PAH fluoranthene which is a major constituent of petroleum sludge. In the degradation process, the extracellular enzyme catechol 2,3-dioxygenase played a very significant role as evidenced by 38 fold increase in its activity as compared to catechol 1,2 dioxygenase. Molecular size analysis of catechol 2,3dioxygenase isolated from uncultured Acinetobacter sp. PSM11 indicated tetrameric nature of the enzyme.
Acknowledgements The authors are thankful to Director, National Botanical Research Institute, Lucknow for providing laboratory facilities and to CSIR for providing funds to Network Project (NWP-019).
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