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Optimization of an inducible, chromosomally encoded benzo [a] pyrene (BaP) degradation pathway in Bacillus subtilis BMT4i (MTCC 9447)

Optimization of an inducible, chromosomally encoded benzo [a] pyrene (BaP) degradation pathway in... Ann Microbiol (2010) 60:51–58 DOI 10.1007/s13213-009-0010-y ORIGINAL ARTICLE Optimization of an inducible, chromosomally encoded benzo [a] pyrene (BaP) degradation pathway in Bacillus subtilis BMT4i (MTCC 9447) Madhuri Kaushish Lily & Ashutosh Bahuguna & Koushalya Dangwal & Veena Garg Received: 9 June 2009 /Accepted: 4 December 2009 /Published online: 27 January 2010 Springer-Verlag and the University of Milan 2010 Abstract Benzo [a] pyrene (BaP), a pentacyclic polyaro- standardization of effective bioremediation protocols matic hydrocarbon, is 1 of the 12 target compounds defined in using B. subtilis BMT4i. the new US Environmental Protection Agency strategy for . . controlling persistent, bioaccumulative, and toxic pollutants. Keywords Bacillus subtilis BMT4i Benzo [a] pyrene . . We previously isolated a novel strain Bacillus subtilis BMT4i Biodegradation High performance liquid chromatography capable of utilizing BaP as sole source of carbon and energy. Plasmid curing The present study investigated (1) whether the BaP degradation pathway is inducible, and (2) whether it is plasmid-encoded. Furthermore, physical (temperature, pH, Introduction and UV-induced photolysis of BaP) and chemical (BaP concentration, surfactant, and ionic strength) parameters for Benzo [a] pyrene (BaP), a high molecular weight polycyclic BaP degradation were determined. Our findings revealed a aromatic hydrocarbon (HMW-PAH) containing five ben- ten-fold enhanced degradation rate in induced vs non- zene rings, is commonly found as pollutant of air, water, induced culture in the presence of chloramphenicol, suggest- and soil (California Environmental Protection Agency ing that the BaP degradation pathway is inducible. Physical 1997). Due to its multiple ring structure and low aqueous methods demonstrated the lack of plasmid in BMT4i—a solubility, BaP is thermodynamically stable and recalcitrant result further complimented by plasmid curing, which had no to microbial degradation (Heitkamp and Cerniglia 1987; effect on BaP degradability thus a chromosomal localization Shuttleworth and Cerniglia 1995; Kanaly and Harayama can be inferred. Maximum BaP degradation in BMT4i 2000). BaP has been shown to possess potent carcinogenic, was observed under the following physical and chemical genotoxic and cytotoxic properties (Miller et al. 1988; Kalf conditions: 30°C, pH 8.0, UV-induced photolysis of BaP- and Crommentuijn 1997; NTP 2002; Hsu et al. 2005). On basal salt mineral medium (BSM), 150 μg/ml BaP, the basis of its abundance in the environment and its potent 0.01% Tween-20, and 400–1,800 μMMgSO .These toxicity, BaP is included in the list of 12 target compounds conditions could be beneficial in the development and defined in the new US Environmental Protection Agency strategy for controlling persistent, bioaccumulative, and toxic pollutants (ATSDR 1990; Renner 1999). The natural : : M. K. Lily A. Bahuguna K. Dangwal (*) environmental sources of BaP include forest fires, volcanic Department of Biotechnology, eruptions, peat fires, burning of crude oil and shale oil, Modern Institute of Technology (MIT), while anthropogenic sources include the incomplete com- Dhalwala, bustion of fossil fuel, coke oven emissions, aluminum Rishikesh 249201 Uttarakhand, India e-mail: kdangwal1@yahoo.co.in smelters, coal combustion and conversion industries, incinerators, vehicle exhausts, cigarette, and cigar and V. Garg marijuana smoke (Blumer 1976; California Environmental Department of Biosciences and Biotechnology, Protection Agency 1997). The removal of BaP from Banasthali University, contaminated soil during remediation is essential in order Rajasthan, India 52 Ann Microbiol (2010) 60:51–58 to meet current “clean up” standards. Microbial degradation and solvents of analytical grade were purchased from Glaxo of BaP may play a major role in the decontamination of (Mumbai, India) and Merck (Mumbai, India). sediment and surface soils (Sims and Overcash 1983; Habe and Omori 2003). Only a few bacterial species, including Preparation of media Mycobacterium sp. (Heitkamp and Cerniglia 1989; Schneider et al. 1996; Grosser et al. 1991), Sphingomonas Basal salt medium, pH 7.0, was prepared by dissolving paucimobilis (Ye et al. 1996), Strenotrophomonas malto- 0.38 g KH PO ,0.6 g K HPO , 0.2 g MgSO ·7H O, 1.0 g 2 4 2 4 4 2 philia (Juhasz and Naidu 2000; Juhasz et al. 2002) and NH Cl and 0.05 g FeCl in 1 l double distilled water and 4 3 Mycobacterium vanbaalenii Pyr-1; (Moody et al. 2004), are autoclaving. All solid media, BSM and nutrient agar (5 g capable of degrading BaP co-metabolically. However, no peptone, 5 g NaCl, 3 g beef extract and 3 g yeast extract) study has yet demonstrated utilization of BaP as the sole contained 1.5% agar. Stock solutions of BaP (10 mg/ml in source of carbon and energy (Kanaly and Haryama 2000; N, N-dimethylformamide), dextrose (5% in double distilled Peng et al. 2008; Seo et al. 2009). water), chloramphenicol (34 mg/ml in absolute ethanol) and Although bioremediation of BaP-contaminated soils is a acridine orange (1 mg/ml in double distilled water) were promising alternative remedial strategy (Cerniglia 1992), prepared and sterilized using a Millipore micro syringe the bioremediation rate of BaP in the environment is limited filter assembly (0.45 µm pore size). by various factors (Providenti et al. 1993). Considering the above problems, approaches should be taken to isolate BaP degradation by induced and non-induced cultures microbes that can degrade BaP and its byproducts, and also of Bacillus subtilis strain BMT4i (MTCC 9447) to optimize various physical and chemical parameters for optimum BaP degradation. In this context, we have All operations were carried out under dim yellow light in reported recently for the first time, utilization of BaP as a order to avoid photodegradation of BaP, and all experi- sole source of carbon and energy by a novel strain Bacillus ments were set up in triplicate. To determine the inducible/ subtilis BMT4i (MTCC 9447), a potential degrader of BaP non-inducible nature of the BaP degradation pathway, a and various PAHs, which showed a 10 -fold enhancement single colony of B. subtilis BMT4i maintained on a BSM in cell number within 7 days and more than 80% BaP agar plate supplemented with BaP (50 μg/ml; BaP-BSM) degradation after 28 days growth (Lily et al. 2009). As an was used to inoculate a 50 ml conical flask containing extension of this previous study, the present work was 10 ml nutrient broth and grown for 24 h at 37°C in a performed to find out whether the BaP degradation pathway shaking incubator. The cells were harvested by centrifuga- in B. subtilis strain BMT4i is inducible in nature, and tion at 3,000 g for 10 min and the cell pellet was washed whether it is contributed by the plasmid or located on three times with BSM to remove traces of nutrient broth. chromosomal DNA. This study further aimed to optimize The pellet was re-suspended in BSM to adjust the cell the various physical (temperature, pH, and UV-induced density to 10 cells/ml. For induction, two separate conical photolysis of BaP) and chemical (BaP concentration, flasks, having 50 ml BSM containing BaP (50 μg/ml; presence of surfactant and ionic strength) parameters for induced starter culture) and dextrose (1%; BSMD, non- maximum BaP biodegradation, which could be useful in the induced starter culture), respectively, as the sole source of development of an effective bioremediation protocol for the carbon and energy, were inoculated with 1 ml cell removal of HMW-PAH including BaP from contaminated suspension. Cultures were grown for 2 weeks at 37°C in a soil. shaking incubator (Heitkamp et al. 1988; Khan et al. 2002). Culture purity was determined by Gram staining and microscopic examination. After thorough washing with Materials and methods BSM, 10 cells/ml each of BMT4i induced and non- induced starter cultures were inoculated into 50 ml BSM Chemicals and reagents containing BaP (50 μg/ml) as the sole source of carbon and energy and chloramphenicol (25 μg/ml) as an inhibitor of Benzo (a) pyrene (99.9%) was purchased from Supelco, protein synthesis. The respective cultures were grown for 0 (Bellefonte, PA). Tryptone, peptone, beef extracts, bacto-agar, and 7 days at 37°C with constant stirring (Heitkamp et al. yeast extract, beef extract, dextrose, tris-base, ethylene 1988). The number of colony forming units (CFU/ml) in diamine tetraacetic acid (EDTA), sodium dodecyl sulphate the induced and non-induced cultures was determined after (SDS), boric acid, lysozyme, agarose, chloramphenicol, 0 and 7 days of growth as described previously (Lily et al. acridine orange, and staining reagents were obtained from 2009). The extent of BaP degradation in induced and non- HiMedia Laboratories (Mumbai, India). General chemicals, induced cultures was determined by ethyl acetate extraction including constituents of basal salt mineral medium (BSM) of the corresponding cultures followed by HPLC analysis Ann Microbiol (2010) 60:51–58 53 −1 −10 (Heitkamp et al. 1988; Lily et al. 2009). To study the effect 10 –10 and spread on BSMD agar plates followed by of induction on the growth kinetics of BMT4i, 10 cells/ml incubation at 37°C for 24 h. The plate with distinct colonies −4 of induced and non-induced starter cultures were inoculated (10 dilution) was selected as a master plate and exact into two different conical flask containing 50 ml BaP-BSM imprints of the BMT4i colonies from the master plate broth and grown for 38 days at 37°C with constant stirring. were transferred onto two different BaP-BSM (50 μg/ml) At various time points (0, 3, 6, 12 h, and 1 day to 38 days at agar plates by replica plating (Huys et al. 2006). The 1-day intervals), CFU/ml of the respective cultures was replica plates were incubated at 37°C for 48 h, while the determined and plotted against incubation time. The extent master plate was stored at 4°C for post incubation of BaP degradation in induced and non-induced cultures comparison. To isolate plasmid-cured BMT4i colonies, after 38 days of growth was determined by HPLC analysis loss of BaP degradation ability was used as the selectable of leftover BaP and its by-products as described earlier marker for the presence of plasmid in B. subtilis BMT4i. (Lily et al. 2009). Colonies present on the master plates but absent on the replica plates were selected as cured colonies from the Isolation of plasmid and plasmid curing master plates. Curing was also checked by growing cured B. subtilis BMT4i colonies in BaP-BSM broth for 7 days. For isolation of plasmid, a single colony each of B. subtilis After 7 days, the BaP-BSM broth culture of cured BMT4i BMT4i and Agrobacterium tumefaciens (ATCC No. 7953) was extracted with ethyl acetate, dried, dissolved in harboring the ~250 kb Ti plasmid as a positive control were methanol and processed for HPLC analysis to determine inoculated into nutrient broth and grown for 24 h at 37°C whether the ability of BaP degradation still existed in and 28°C, respectively. The presence of plasmids was cured B. subtilis BMT4i and to what extent. The success analyzed by pulse field gel electrophoresis (PFGE) on 1% of the curing method was demonstrated by the loss of the PFGE-certified agarose in 0.5X TBE buffer at 14°C for Ti plasmid from cured A. tumefaciens as assessed by in- 26 h, 40 min at 6.0 V/cm (200 V) using a 120° included well cell lysis electrophoresis. angle with a 2.77–26.32 second linear switch time ramp (Smith et al. 1988; Gunderson and Chu 1991). The PFGE Optimization of physical and chemical parameters facility was provided by the National Centre for Cell for degradation of BaP Science (NCCS), Pune, India. Plasmids were also extracted using an in-well cell lysis To optimize the various physical and chemical parameters technique as described by Pedraza and Ricci (2002). In for optimum BaP biodegradation, an overnight culture of brief, 0.1 ml of each culture was harvested by centrifuga- BMT4i grown in nutrient broth was washed three times tion at 10,000 rpm for 10 min and pellets were suspended with BSM to remove traces of nutrient broth. A 1 ml in 0.02 ml lysis solution [sucrose 10%, RNase 10 μg/ml aliquot of BSM suspension culture of BMT4i (10 cell/ml) and lysozyme 1 μg/ml in Tris-Borate EDTA (TBE) buffer; was inoculated in various 50 ml flasks, each carrying 10 ml TBE contains (per liter) Tris 10.8 g, EDTA 0.93 g, boric BSM supplemented with BaP (50 μg/ml) as a sole source acid 5.5 g, pH 8.0). The lysed cell pellets were mixed with of carbon and energy. Triplicate flasks were grown at 0.03 ml loading buffer and electrophoresed on a 0.7% different physical (varied temperature, pH and UV-treated agarose gel prepared in TBE-SDS (1%) buffer. The gel was BaP-BSM) and chemical (varied BaP concentrations, ionic run at 0.7 V/cm for 1 h, followed by 1.40 V/cm for 2 h and strength and presence of surfactants) parameters for 7 days 2.81 V/cm for a further 3 h as described (Pedraza and Ricci at a constant rotation speed of 120 rpm in a shaking 2002). DNA bands were stained for 30 min with ethidium incubator. The effects of temperature and pH on BaP bromide (0.5 μg/ml), washed in distilled water for 50 min degradation were assessed by growing BMT4i at various and photographed. temperatures ranging from 10°C to 50°C, and at various BSM To check whether BaP degradation ability depends on pH values (2.0–14.0). The pH was adjusted using 0.1 N HCl the presence of a plasmid present at low copy number in and 1 N NaOH solution. BMT4i that our physical methods are unable to detect, a To assess the effect of UV-mediated photolysis of BaP plasmid curing experiment was performed using acridine on BaP degradation, BaP-BSM broth in a Petri-dish was orange as the curing agent (Rasool et al. 2003; Huys et al. exposed to UV irradiation at a wavelength of 254 nm for 2006; Mojgani et al. 2006). A. tumefaciens was used as a 15 min at a distance of 5 cm in a UV irradiation chamber positive control in the plasmid curing experiment. An (Vikrant Equipment, Ahmedabad , India). Thereafter, the overnight culture of B. subtilis BMT4i (10 cells/ml) was UV-treated BaP-BSM was transferred to 50 ml conical inoculated in 10 ml BSMD broth containing acridine flasks and inoculated with BMT4i and grown as described orange (40 μg/ml) and grown at 37°C for 72 h. After above. A control experiment without UV treatment was incubation, 100 μl cured BMT4i culture was diluted up to also set up simultaneously. 54 Ann Microbiol (2010) 60:51–58 The effect of BaP concentration on BaP biodegradation induced culture as new protein synthesis was inhibited by was studied by growing BMT4i in BSM containing various chloramphenicol (Table 1). concentrations of BaP ranging from 10 to 250 μg/ml. The To check the effect of induction on the growth kinetics effect of ionic strength was studied in BaP-BSM containing of BMT4i, the induced and non-induced starter culture various MgSO concentrations, ranging from 100 to were grown in BaP-BSM broth and CFU/ml was deter- 2,400 μM. The effect of surfactants was assessed by mined at different time points (0 h–38 days). As seen in growing BMT4i in BaP-BSM containing 0.01% each of Fig. 1, the induced culture showed a lag phase of only 3 h, Tween-20, Triton-X-100 and SDS in separate flasks. afterwards the log CFU/ml of BMT4i increased exponen- Negative controls without BMT4i were also set up for tially up to day 7, declining rapidly thereafter to reach zero each experiment for all physical and chemical parameters. on day 18. In contrast to the induced culture, the non- After 7 days incubation, 100 μl grown culture was removed induced culture showed a prolonged lag phase of 24 h, and the CFU/ml determined. The percent BaP degradation delayed attainment of maximum growth on day 10 and in the respective cultures was quantified by HPLC analysis extended viability up to day 37. HPLC analysis of extracts as described earlier (Lily et al. 2009). The physical and of induced and non-induced cultures at the end of chemical parameters showing maximum BaP degradation experiment revealed >90% and ~84% BaP degradation, were considered to be optimum for BaP degradation by respectively. The above observations clearly indicate the BMT4i. inducible nature of the BaP degradation pathway in B. subtilis BMT4i. To determine whether the BaP degradation pathway in Results BMT4i is a function of the chromosome, PFGE and the in- well cell lysis method were performed to determine the BaP degradation by induced and non-induced cultures presence or absence of plasmid. A. tumefaciens harboring of Bacillus subtilis strain BMT4i (MTCC 9447) the ∼250 kb Ti plasmid was processed in parallel as a positive control. Our observations clearly demonstrated the Since all reported PAH degradation pathways to date have lack of plasmid in BMT4i. We were unable to detect any been shown to be inducible and to require prior new protein plasmid band in the BMT4i lane in agarose gels despite synthesis, we checked the inducibility of the BaP degrada- several attempts, while the high molecular weight Ti tion pathway in BMT4i. For this purpose, the BaP plasmid (250 kb) was successfully isolated and detected degradation (%) was determined by growing induced and in the A. tumefaciens lane (Fig. 2a, b). To finally exclude non-induced starter cultures of BMT4i in BaP-BSM broth the remote possibility of the presence of a plasmid-encoded containing chloramphenicol, an inhibitor of protein synthe- BaP degradation pathway in BMT4i, acridine orange was sis. HPLC analysis of extracts of induced and non-induced used as a curing agent. If a plasmid-borne BaP degradation BMT4i cultures revealed that BaP degradation was en- ability exists in BMT4i, it would be lost after curing; hanced around ten-fold in induced culture compared to non- however, if the BaP degradation activity resides on the induced culture in the presence of chloramphenicol, chromosome it will be retained in BMT4i after plasmid suggesting inducibility of the BaP degradation pathway curing. We observed that all cured BMT4i colonies from (Table 1). This data revealed the inducibility of BaP the BSMD master plate retained BaP degradation ability degradation pathway since induced starter culture were when replica plated on BaP-BSM agar. Moreover, HPLC already possessed the enzyme machinery for BaP degrada- analysis of extracts of cured and uncured BMT4i cultures tion due to the prolonged prior exposure to BaP, whereas after 7 days of growth revealed the retention of the BaP non-induced culture required new protein synthesis, which degradation ability in cured BMT4i culture. The cured is inhibited by chloramphenicol, for BaP degradation. To BMT4i culture was able to degrade BaP up to 54%, which check the effect of chloramphenicol on the viability of is comparable to uncured BMT4i culture (49%). The lack BMT4i culture, the number of CFU/ml in induced and non- of plasmid, together with the maintenance of BaP degrada- induced cultures was determined at 0 h and 7 days of tion activity after curing, indicate that the BaP degradation growth in BaP-BSM broth containing chloramphenicol. In pathway in BMT4i is chromosomally encoded. the induced culture, the log CFU/ml remained static (8.0) from 0 h to 7 days; however, in non-induced culture, log Optimization of physical parameters for degradation of BaP CFU/ml declined from the initial log CFU/ml values of by Bacillus subtilis strain BMT4i (MTCC 9447) 8.0 to 3.6 after 7 days. This indicates that the presence of the enzymatic machinery for BaP degradation due to prior Various physical parameters such as temperature, pH and induction supported the viability of BMT4i in the induced UV-induced photolysis of BaP have profound effects on the BaP degradation activity of BMT4i, as detailed below. BMT4i culture and that this support was lacking in non- Ann Microbiol (2010) 60:51–58 55 Table 1 Showing the log col- Day Non-induced culture Induced culture ony forming units (CFU)/ml and degradation of benzo [a] pyrene log CFU/ml Degradation (%) log CFU/ml Degradation (%) 10 10 (BaP) by non-induced and in- duced starter culture of Bacillus 0 8.0 - 8.0 - subtilis BMT4i in the presence of chloramphenicol 7 3.6 <5 8.0 54 Temperature of BMT4i was inoculated. As shown in Fig. 3, BaP degradation was enhanced approximately 1.5-fold BMT4i-mediated BaP degradation activity was observed to (70.12%) when BaP was pretreated with UV as compared be temperature dependent, with maximum BaP degradation to control values (46.32%). Accordingly, BMT4i showed (62.86%) at 30°C; however, growth at higher temperatures an increase in growth in the UV-treated BaP experiment of (range 35–50°C) resulted in a gradual decline in BaP approximately 1.8-fold compared to that of the control degradation. The growth and viability of BMT4i (log (Fig. 3). This clearly indicated that photolytic degradation CFU/ml) at the above-mentioned temperatures were in of BaP resulted in some less complex source of carbon, accordance with those obtained for % BaP degradation. leading to comparatively higher rates of BaP degradation and growth of BMT4i. pH Optimization of chemical parameters for degradation The pH value had a significant impact on the extent of BaP of BaP by Bacillus subtilis strain BMT4i (MTCC 9447) degradation. A steady increase in BaP degradation activity was observed with increase in pH from 5.0 to 7.0, with the Chemical parameters, viz. BaP concentration, surfactants highest level achieved at pH 8.0 (50.28%). Moreover, and ionic strength, were studied to determine their effect on increasing the pH range still further (9.0–14.0) leads to a BaP biodegradation. The results showed that BaP degrada- slow decline in BaP degradation rate. In contrast, viability tion is affected significantly by BaP concentration and the remained constant in the pH range 7.0–11.0, indicating the presence of surfactant. wide range of pH tolerance of BMT4i. BaP concentration UV treatment The results showed an almost exponential increase in BaP To determine the effect of UV-mediated photolysis of BaP degradation when the BaP concentration was increased on its biodegradation, BaP-BSM (50 μg/ml) was exposed to UV irradiation (254 nm) for 15 min and then a pure culture 0 3 6 9 12 15 18 21 24 27 30 33 36 39 Incubation time (days) Induced Non-induced Fig. 2 a Pulse field gel electrophoresis (PFGE) and b in-well cell Fig. 1 Growth kinetics of non-induced and induced Bacillus subtilis lysis gel electrophoresis showing the absence of plasmid in BMT4i. BMT4i (MTCC 9447) in basal salts medium (BSM) with benzo [a] Lanes: M Marker (range 2–194 Kb), 1 uncured Bacillus subtilis pyrene (BaP) against incubation time (days). Data points represent BMT4i, 2 plasmid cured B. subtilis BMT4i (PC), 3 uncured average values from triplicate flasks Agrobacterium tumefaciens, 4 plasmid cured A. tumefaciens (PC) log CFU/ml of BMT4i in BaP-BSM 10 56 Ann Microbiol (2010) 60:51–58 80 60 60 40 40 40 30 10 0 0 Control Tween 20 Triton X-100 SDS % BaP Degradation log CFU/ml 0 0 UV-treated BSM-BaP Control BSM-BaP Fig. 5 BaP degradation (%) and log CFU/ml in the presence of % BaP Degradation log CFU/ml different surfactants. Data points represent mean values from three independent experiments. Error bars Standard deviations of triplicate Fig. 3 BaP biodegradation (%) and log CFU/ml of UV-treated BaP- independent experiments BSM and control BaP-BSM. Data points represent the mean of three independent experiments. Error bars Standard deviation of triplicate independent experiments had an inhibitory effect on BaP degradation (28.81%). The CFU data was in agreement with that of BaP degradation from 10 μg/ml (26.31%) to 30 μg/ml (40.94%), with a (Fig. 5). Hence, the BaP degradation and viability of BMT4i subsequent steady increase in BaP degradation reaching a is enhanced by the presence of the surfactants Tween-20 and maximum (61.04%) at 150 μg/ml (Fig. 4). Further increase Triton-X-100, while SDS showed adverse effects. in BaP concentration (from 200–250 μg/ml) resulted in a steep decrease in BaP degradation from 55.28% to 32.37%. Ionic strength Likewise, the viability of BMT4i was enhanced as the BaP concentration increased from 10 to 150 μg/ml. Further BMT4i was grown in BaP-BSM at various ionic strengths increase in BaP concentration resulted in a decrease in with respect to MgSO . As shown in Fig. 6,BaP viability. Therefore, the optimum BaP concentration at 37°C, degradation activity increased significantly (from 20.24 to pH 7.0 was found to be 150 μg/ml. 46.4%) when the MgSO concentration in the BaP-BSM was increased from 100 to 400 μM. With further increase in Surfactant treatment MgSO concentration, up to 1800 μM, the BaP degradation activity became stationary (48.12%), and then slowly To assess the impact of surfactant treatment on BaP declined to 45.64% at 2,400 μM. The viability data (log degradation, BMT4i was grown in the presence of 0.01% CFU/ml) was in agreement with that of BaP degradation different surfactants: Tween-20, Triton-X-100 and SDS. (Fig. 6). These findings suggest 1,800 μM MgSO as the The results demonstrated enhanced BaP degradation optimum concentration for the BaP degradation activity of (58.64%) in the presence of Tween-20 followed by Triton- BMT4i. However, higher concentrations of MgSO X-100 (50.12%) as compared to the control (46.32%) as (2,400 μM) had a negligible inhibitory effect on BaP shown in Fig. 5. Conversely, surfactant treatment with SDS degradation. 70 50 60 30 15 20 10 10 5 0 0 0 0 50 100 150 200 250 300 0 500 1000 1500 2000 2500 3000 BaP Concentration (microgram/ml) MgSO Concentration (microgram/ml) % BaP Degradation log CFU/ml % BaP Degradation log CFU/ml Fig. 4 BaP degradation (%) and log CFU/ml at different BaP Fig. 6 BaP degradation (%) and log CFU/ml at different ionic 10 10 concentrations. Data points represent mean values from three strength (MgSO ). Data points represent mean values from three independent experiments. Error bars Standard deviations of triplicate independent experiments. Error bars Standard deviations of triplicate independent experiments independent experiments % BaP Degradation of BMT4i % BaP Degradation of BMT4i log CFU/ml of BMT4i log10 CFU/ml of BMT4i % BaP Degradation of BMT4i % BaP Degradation of BMT4 log10 CFU/ml of BMT4i log10 CFU/ml of BMT4 Ann Microbiol (2010) 60:51–58 57 Discussion Acknowledgements This work was supported in part by the Modern Institute of Technology, Rishikesh, Uttarakhand, India and Uttarakhand Council of Science and Technology, Uttarakhand, India The present study was performed to extend our knowledge (UCOST; grant no. UCS and T/R and D/LS-46/06-07), which are about the BaP degradation activity of Bacillus subtilis gratefully acknowledged. We also thank Prof Aditya Shastri, Vice BMT4i (MTCC 9447). We have shown earlier that B. Chancellor, Banasthali University, Rajasthan, India for providing facilities in the Department of Bioscience and Biotechnology at this subtilis BMT4i (MTCC 9447) is an efficient degrader of university. We gratefully acknowledge Dr. Yogesh Shouche, Scientist BaP and possesses the ability to utilize BaP as a sole source E and Mr. Jay Siddharth, Senior Research Fellow (SRF), Molecular of carbon and energy for growth (Lily et al. 2009). This Biology Laboratory, National Centre for Cell Science (NCCS), Pune, India for providing the pulse field gel electrophoresis (PFGE) facility study reports that the BaP degradation activity of BMT4i is and invaluable help and cooperation during this research work. highly inducible, since induced culture showed more than 50% BaP degradation whereas non-induced culture did not show any significant BaP degradation in the presence of chloramphenicol. 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Med J Islam World Acad Sci 16 polycyclic hydrocarbons on soil surfaces using TiO under UV (1):19–24 light. J Hazard Mater 158:478–484 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Optimization of an inducible, chromosomally encoded benzo [a] pyrene (BaP) degradation pathway in Bacillus subtilis BMT4i (MTCC 9447)

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Springer Journals
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Copyright © 2010 by Springer-Verlag and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Mycology; Medical Microbiology; Applied Microbiology
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1590-4261
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DOI
10.1007/s13213-009-0010-y
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Abstract

Ann Microbiol (2010) 60:51–58 DOI 10.1007/s13213-009-0010-y ORIGINAL ARTICLE Optimization of an inducible, chromosomally encoded benzo [a] pyrene (BaP) degradation pathway in Bacillus subtilis BMT4i (MTCC 9447) Madhuri Kaushish Lily & Ashutosh Bahuguna & Koushalya Dangwal & Veena Garg Received: 9 June 2009 /Accepted: 4 December 2009 /Published online: 27 January 2010 Springer-Verlag and the University of Milan 2010 Abstract Benzo [a] pyrene (BaP), a pentacyclic polyaro- standardization of effective bioremediation protocols matic hydrocarbon, is 1 of the 12 target compounds defined in using B. subtilis BMT4i. the new US Environmental Protection Agency strategy for . . controlling persistent, bioaccumulative, and toxic pollutants. Keywords Bacillus subtilis BMT4i Benzo [a] pyrene . . We previously isolated a novel strain Bacillus subtilis BMT4i Biodegradation High performance liquid chromatography capable of utilizing BaP as sole source of carbon and energy. Plasmid curing The present study investigated (1) whether the BaP degradation pathway is inducible, and (2) whether it is plasmid-encoded. Furthermore, physical (temperature, pH, Introduction and UV-induced photolysis of BaP) and chemical (BaP concentration, surfactant, and ionic strength) parameters for Benzo [a] pyrene (BaP), a high molecular weight polycyclic BaP degradation were determined. Our findings revealed a aromatic hydrocarbon (HMW-PAH) containing five ben- ten-fold enhanced degradation rate in induced vs non- zene rings, is commonly found as pollutant of air, water, induced culture in the presence of chloramphenicol, suggest- and soil (California Environmental Protection Agency ing that the BaP degradation pathway is inducible. Physical 1997). Due to its multiple ring structure and low aqueous methods demonstrated the lack of plasmid in BMT4i—a solubility, BaP is thermodynamically stable and recalcitrant result further complimented by plasmid curing, which had no to microbial degradation (Heitkamp and Cerniglia 1987; effect on BaP degradability thus a chromosomal localization Shuttleworth and Cerniglia 1995; Kanaly and Harayama can be inferred. Maximum BaP degradation in BMT4i 2000). BaP has been shown to possess potent carcinogenic, was observed under the following physical and chemical genotoxic and cytotoxic properties (Miller et al. 1988; Kalf conditions: 30°C, pH 8.0, UV-induced photolysis of BaP- and Crommentuijn 1997; NTP 2002; Hsu et al. 2005). On basal salt mineral medium (BSM), 150 μg/ml BaP, the basis of its abundance in the environment and its potent 0.01% Tween-20, and 400–1,800 μMMgSO .These toxicity, BaP is included in the list of 12 target compounds conditions could be beneficial in the development and defined in the new US Environmental Protection Agency strategy for controlling persistent, bioaccumulative, and toxic pollutants (ATSDR 1990; Renner 1999). The natural : : M. K. Lily A. Bahuguna K. Dangwal (*) environmental sources of BaP include forest fires, volcanic Department of Biotechnology, eruptions, peat fires, burning of crude oil and shale oil, Modern Institute of Technology (MIT), while anthropogenic sources include the incomplete com- Dhalwala, bustion of fossil fuel, coke oven emissions, aluminum Rishikesh 249201 Uttarakhand, India e-mail: kdangwal1@yahoo.co.in smelters, coal combustion and conversion industries, incinerators, vehicle exhausts, cigarette, and cigar and V. Garg marijuana smoke (Blumer 1976; California Environmental Department of Biosciences and Biotechnology, Protection Agency 1997). The removal of BaP from Banasthali University, contaminated soil during remediation is essential in order Rajasthan, India 52 Ann Microbiol (2010) 60:51–58 to meet current “clean up” standards. Microbial degradation and solvents of analytical grade were purchased from Glaxo of BaP may play a major role in the decontamination of (Mumbai, India) and Merck (Mumbai, India). sediment and surface soils (Sims and Overcash 1983; Habe and Omori 2003). Only a few bacterial species, including Preparation of media Mycobacterium sp. (Heitkamp and Cerniglia 1989; Schneider et al. 1996; Grosser et al. 1991), Sphingomonas Basal salt medium, pH 7.0, was prepared by dissolving paucimobilis (Ye et al. 1996), Strenotrophomonas malto- 0.38 g KH PO ,0.6 g K HPO , 0.2 g MgSO ·7H O, 1.0 g 2 4 2 4 4 2 philia (Juhasz and Naidu 2000; Juhasz et al. 2002) and NH Cl and 0.05 g FeCl in 1 l double distilled water and 4 3 Mycobacterium vanbaalenii Pyr-1; (Moody et al. 2004), are autoclaving. All solid media, BSM and nutrient agar (5 g capable of degrading BaP co-metabolically. However, no peptone, 5 g NaCl, 3 g beef extract and 3 g yeast extract) study has yet demonstrated utilization of BaP as the sole contained 1.5% agar. Stock solutions of BaP (10 mg/ml in source of carbon and energy (Kanaly and Haryama 2000; N, N-dimethylformamide), dextrose (5% in double distilled Peng et al. 2008; Seo et al. 2009). water), chloramphenicol (34 mg/ml in absolute ethanol) and Although bioremediation of BaP-contaminated soils is a acridine orange (1 mg/ml in double distilled water) were promising alternative remedial strategy (Cerniglia 1992), prepared and sterilized using a Millipore micro syringe the bioremediation rate of BaP in the environment is limited filter assembly (0.45 µm pore size). by various factors (Providenti et al. 1993). Considering the above problems, approaches should be taken to isolate BaP degradation by induced and non-induced cultures microbes that can degrade BaP and its byproducts, and also of Bacillus subtilis strain BMT4i (MTCC 9447) to optimize various physical and chemical parameters for optimum BaP degradation. In this context, we have All operations were carried out under dim yellow light in reported recently for the first time, utilization of BaP as a order to avoid photodegradation of BaP, and all experi- sole source of carbon and energy by a novel strain Bacillus ments were set up in triplicate. To determine the inducible/ subtilis BMT4i (MTCC 9447), a potential degrader of BaP non-inducible nature of the BaP degradation pathway, a and various PAHs, which showed a 10 -fold enhancement single colony of B. subtilis BMT4i maintained on a BSM in cell number within 7 days and more than 80% BaP agar plate supplemented with BaP (50 μg/ml; BaP-BSM) degradation after 28 days growth (Lily et al. 2009). As an was used to inoculate a 50 ml conical flask containing extension of this previous study, the present work was 10 ml nutrient broth and grown for 24 h at 37°C in a performed to find out whether the BaP degradation pathway shaking incubator. The cells were harvested by centrifuga- in B. subtilis strain BMT4i is inducible in nature, and tion at 3,000 g for 10 min and the cell pellet was washed whether it is contributed by the plasmid or located on three times with BSM to remove traces of nutrient broth. chromosomal DNA. This study further aimed to optimize The pellet was re-suspended in BSM to adjust the cell the various physical (temperature, pH, and UV-induced density to 10 cells/ml. For induction, two separate conical photolysis of BaP) and chemical (BaP concentration, flasks, having 50 ml BSM containing BaP (50 μg/ml; presence of surfactant and ionic strength) parameters for induced starter culture) and dextrose (1%; BSMD, non- maximum BaP biodegradation, which could be useful in the induced starter culture), respectively, as the sole source of development of an effective bioremediation protocol for the carbon and energy, were inoculated with 1 ml cell removal of HMW-PAH including BaP from contaminated suspension. Cultures were grown for 2 weeks at 37°C in a soil. shaking incubator (Heitkamp et al. 1988; Khan et al. 2002). Culture purity was determined by Gram staining and microscopic examination. After thorough washing with Materials and methods BSM, 10 cells/ml each of BMT4i induced and non- induced starter cultures were inoculated into 50 ml BSM Chemicals and reagents containing BaP (50 μg/ml) as the sole source of carbon and energy and chloramphenicol (25 μg/ml) as an inhibitor of Benzo (a) pyrene (99.9%) was purchased from Supelco, protein synthesis. The respective cultures were grown for 0 (Bellefonte, PA). Tryptone, peptone, beef extracts, bacto-agar, and 7 days at 37°C with constant stirring (Heitkamp et al. yeast extract, beef extract, dextrose, tris-base, ethylene 1988). The number of colony forming units (CFU/ml) in diamine tetraacetic acid (EDTA), sodium dodecyl sulphate the induced and non-induced cultures was determined after (SDS), boric acid, lysozyme, agarose, chloramphenicol, 0 and 7 days of growth as described previously (Lily et al. acridine orange, and staining reagents were obtained from 2009). The extent of BaP degradation in induced and non- HiMedia Laboratories (Mumbai, India). General chemicals, induced cultures was determined by ethyl acetate extraction including constituents of basal salt mineral medium (BSM) of the corresponding cultures followed by HPLC analysis Ann Microbiol (2010) 60:51–58 53 −1 −10 (Heitkamp et al. 1988; Lily et al. 2009). To study the effect 10 –10 and spread on BSMD agar plates followed by of induction on the growth kinetics of BMT4i, 10 cells/ml incubation at 37°C for 24 h. The plate with distinct colonies −4 of induced and non-induced starter cultures were inoculated (10 dilution) was selected as a master plate and exact into two different conical flask containing 50 ml BaP-BSM imprints of the BMT4i colonies from the master plate broth and grown for 38 days at 37°C with constant stirring. were transferred onto two different BaP-BSM (50 μg/ml) At various time points (0, 3, 6, 12 h, and 1 day to 38 days at agar plates by replica plating (Huys et al. 2006). The 1-day intervals), CFU/ml of the respective cultures was replica plates were incubated at 37°C for 48 h, while the determined and plotted against incubation time. The extent master plate was stored at 4°C for post incubation of BaP degradation in induced and non-induced cultures comparison. To isolate plasmid-cured BMT4i colonies, after 38 days of growth was determined by HPLC analysis loss of BaP degradation ability was used as the selectable of leftover BaP and its by-products as described earlier marker for the presence of plasmid in B. subtilis BMT4i. (Lily et al. 2009). Colonies present on the master plates but absent on the replica plates were selected as cured colonies from the Isolation of plasmid and plasmid curing master plates. Curing was also checked by growing cured B. subtilis BMT4i colonies in BaP-BSM broth for 7 days. For isolation of plasmid, a single colony each of B. subtilis After 7 days, the BaP-BSM broth culture of cured BMT4i BMT4i and Agrobacterium tumefaciens (ATCC No. 7953) was extracted with ethyl acetate, dried, dissolved in harboring the ~250 kb Ti plasmid as a positive control were methanol and processed for HPLC analysis to determine inoculated into nutrient broth and grown for 24 h at 37°C whether the ability of BaP degradation still existed in and 28°C, respectively. The presence of plasmids was cured B. subtilis BMT4i and to what extent. The success analyzed by pulse field gel electrophoresis (PFGE) on 1% of the curing method was demonstrated by the loss of the PFGE-certified agarose in 0.5X TBE buffer at 14°C for Ti plasmid from cured A. tumefaciens as assessed by in- 26 h, 40 min at 6.0 V/cm (200 V) using a 120° included well cell lysis electrophoresis. angle with a 2.77–26.32 second linear switch time ramp (Smith et al. 1988; Gunderson and Chu 1991). The PFGE Optimization of physical and chemical parameters facility was provided by the National Centre for Cell for degradation of BaP Science (NCCS), Pune, India. Plasmids were also extracted using an in-well cell lysis To optimize the various physical and chemical parameters technique as described by Pedraza and Ricci (2002). In for optimum BaP biodegradation, an overnight culture of brief, 0.1 ml of each culture was harvested by centrifuga- BMT4i grown in nutrient broth was washed three times tion at 10,000 rpm for 10 min and pellets were suspended with BSM to remove traces of nutrient broth. A 1 ml in 0.02 ml lysis solution [sucrose 10%, RNase 10 μg/ml aliquot of BSM suspension culture of BMT4i (10 cell/ml) and lysozyme 1 μg/ml in Tris-Borate EDTA (TBE) buffer; was inoculated in various 50 ml flasks, each carrying 10 ml TBE contains (per liter) Tris 10.8 g, EDTA 0.93 g, boric BSM supplemented with BaP (50 μg/ml) as a sole source acid 5.5 g, pH 8.0). The lysed cell pellets were mixed with of carbon and energy. Triplicate flasks were grown at 0.03 ml loading buffer and electrophoresed on a 0.7% different physical (varied temperature, pH and UV-treated agarose gel prepared in TBE-SDS (1%) buffer. The gel was BaP-BSM) and chemical (varied BaP concentrations, ionic run at 0.7 V/cm for 1 h, followed by 1.40 V/cm for 2 h and strength and presence of surfactants) parameters for 7 days 2.81 V/cm for a further 3 h as described (Pedraza and Ricci at a constant rotation speed of 120 rpm in a shaking 2002). DNA bands were stained for 30 min with ethidium incubator. The effects of temperature and pH on BaP bromide (0.5 μg/ml), washed in distilled water for 50 min degradation were assessed by growing BMT4i at various and photographed. temperatures ranging from 10°C to 50°C, and at various BSM To check whether BaP degradation ability depends on pH values (2.0–14.0). The pH was adjusted using 0.1 N HCl the presence of a plasmid present at low copy number in and 1 N NaOH solution. BMT4i that our physical methods are unable to detect, a To assess the effect of UV-mediated photolysis of BaP plasmid curing experiment was performed using acridine on BaP degradation, BaP-BSM broth in a Petri-dish was orange as the curing agent (Rasool et al. 2003; Huys et al. exposed to UV irradiation at a wavelength of 254 nm for 2006; Mojgani et al. 2006). A. tumefaciens was used as a 15 min at a distance of 5 cm in a UV irradiation chamber positive control in the plasmid curing experiment. An (Vikrant Equipment, Ahmedabad , India). Thereafter, the overnight culture of B. subtilis BMT4i (10 cells/ml) was UV-treated BaP-BSM was transferred to 50 ml conical inoculated in 10 ml BSMD broth containing acridine flasks and inoculated with BMT4i and grown as described orange (40 μg/ml) and grown at 37°C for 72 h. After above. A control experiment without UV treatment was incubation, 100 μl cured BMT4i culture was diluted up to also set up simultaneously. 54 Ann Microbiol (2010) 60:51–58 The effect of BaP concentration on BaP biodegradation induced culture as new protein synthesis was inhibited by was studied by growing BMT4i in BSM containing various chloramphenicol (Table 1). concentrations of BaP ranging from 10 to 250 μg/ml. The To check the effect of induction on the growth kinetics effect of ionic strength was studied in BaP-BSM containing of BMT4i, the induced and non-induced starter culture various MgSO concentrations, ranging from 100 to were grown in BaP-BSM broth and CFU/ml was deter- 2,400 μM. The effect of surfactants was assessed by mined at different time points (0 h–38 days). As seen in growing BMT4i in BaP-BSM containing 0.01% each of Fig. 1, the induced culture showed a lag phase of only 3 h, Tween-20, Triton-X-100 and SDS in separate flasks. afterwards the log CFU/ml of BMT4i increased exponen- Negative controls without BMT4i were also set up for tially up to day 7, declining rapidly thereafter to reach zero each experiment for all physical and chemical parameters. on day 18. In contrast to the induced culture, the non- After 7 days incubation, 100 μl grown culture was removed induced culture showed a prolonged lag phase of 24 h, and the CFU/ml determined. The percent BaP degradation delayed attainment of maximum growth on day 10 and in the respective cultures was quantified by HPLC analysis extended viability up to day 37. HPLC analysis of extracts as described earlier (Lily et al. 2009). The physical and of induced and non-induced cultures at the end of chemical parameters showing maximum BaP degradation experiment revealed >90% and ~84% BaP degradation, were considered to be optimum for BaP degradation by respectively. The above observations clearly indicate the BMT4i. inducible nature of the BaP degradation pathway in B. subtilis BMT4i. To determine whether the BaP degradation pathway in Results BMT4i is a function of the chromosome, PFGE and the in- well cell lysis method were performed to determine the BaP degradation by induced and non-induced cultures presence or absence of plasmid. A. tumefaciens harboring of Bacillus subtilis strain BMT4i (MTCC 9447) the ∼250 kb Ti plasmid was processed in parallel as a positive control. Our observations clearly demonstrated the Since all reported PAH degradation pathways to date have lack of plasmid in BMT4i. We were unable to detect any been shown to be inducible and to require prior new protein plasmid band in the BMT4i lane in agarose gels despite synthesis, we checked the inducibility of the BaP degrada- several attempts, while the high molecular weight Ti tion pathway in BMT4i. For this purpose, the BaP plasmid (250 kb) was successfully isolated and detected degradation (%) was determined by growing induced and in the A. tumefaciens lane (Fig. 2a, b). To finally exclude non-induced starter cultures of BMT4i in BaP-BSM broth the remote possibility of the presence of a plasmid-encoded containing chloramphenicol, an inhibitor of protein synthe- BaP degradation pathway in BMT4i, acridine orange was sis. HPLC analysis of extracts of induced and non-induced used as a curing agent. If a plasmid-borne BaP degradation BMT4i cultures revealed that BaP degradation was en- ability exists in BMT4i, it would be lost after curing; hanced around ten-fold in induced culture compared to non- however, if the BaP degradation activity resides on the induced culture in the presence of chloramphenicol, chromosome it will be retained in BMT4i after plasmid suggesting inducibility of the BaP degradation pathway curing. We observed that all cured BMT4i colonies from (Table 1). This data revealed the inducibility of BaP the BSMD master plate retained BaP degradation ability degradation pathway since induced starter culture were when replica plated on BaP-BSM agar. Moreover, HPLC already possessed the enzyme machinery for BaP degrada- analysis of extracts of cured and uncured BMT4i cultures tion due to the prolonged prior exposure to BaP, whereas after 7 days of growth revealed the retention of the BaP non-induced culture required new protein synthesis, which degradation ability in cured BMT4i culture. The cured is inhibited by chloramphenicol, for BaP degradation. To BMT4i culture was able to degrade BaP up to 54%, which check the effect of chloramphenicol on the viability of is comparable to uncured BMT4i culture (49%). The lack BMT4i culture, the number of CFU/ml in induced and non- of plasmid, together with the maintenance of BaP degrada- induced cultures was determined at 0 h and 7 days of tion activity after curing, indicate that the BaP degradation growth in BaP-BSM broth containing chloramphenicol. In pathway in BMT4i is chromosomally encoded. the induced culture, the log CFU/ml remained static (8.0) from 0 h to 7 days; however, in non-induced culture, log Optimization of physical parameters for degradation of BaP CFU/ml declined from the initial log CFU/ml values of by Bacillus subtilis strain BMT4i (MTCC 9447) 8.0 to 3.6 after 7 days. This indicates that the presence of the enzymatic machinery for BaP degradation due to prior Various physical parameters such as temperature, pH and induction supported the viability of BMT4i in the induced UV-induced photolysis of BaP have profound effects on the BaP degradation activity of BMT4i, as detailed below. BMT4i culture and that this support was lacking in non- Ann Microbiol (2010) 60:51–58 55 Table 1 Showing the log col- Day Non-induced culture Induced culture ony forming units (CFU)/ml and degradation of benzo [a] pyrene log CFU/ml Degradation (%) log CFU/ml Degradation (%) 10 10 (BaP) by non-induced and in- duced starter culture of Bacillus 0 8.0 - 8.0 - subtilis BMT4i in the presence of chloramphenicol 7 3.6 <5 8.0 54 Temperature of BMT4i was inoculated. As shown in Fig. 3, BaP degradation was enhanced approximately 1.5-fold BMT4i-mediated BaP degradation activity was observed to (70.12%) when BaP was pretreated with UV as compared be temperature dependent, with maximum BaP degradation to control values (46.32%). Accordingly, BMT4i showed (62.86%) at 30°C; however, growth at higher temperatures an increase in growth in the UV-treated BaP experiment of (range 35–50°C) resulted in a gradual decline in BaP approximately 1.8-fold compared to that of the control degradation. The growth and viability of BMT4i (log (Fig. 3). This clearly indicated that photolytic degradation CFU/ml) at the above-mentioned temperatures were in of BaP resulted in some less complex source of carbon, accordance with those obtained for % BaP degradation. leading to comparatively higher rates of BaP degradation and growth of BMT4i. pH Optimization of chemical parameters for degradation The pH value had a significant impact on the extent of BaP of BaP by Bacillus subtilis strain BMT4i (MTCC 9447) degradation. A steady increase in BaP degradation activity was observed with increase in pH from 5.0 to 7.0, with the Chemical parameters, viz. BaP concentration, surfactants highest level achieved at pH 8.0 (50.28%). Moreover, and ionic strength, were studied to determine their effect on increasing the pH range still further (9.0–14.0) leads to a BaP biodegradation. The results showed that BaP degrada- slow decline in BaP degradation rate. In contrast, viability tion is affected significantly by BaP concentration and the remained constant in the pH range 7.0–11.0, indicating the presence of surfactant. wide range of pH tolerance of BMT4i. BaP concentration UV treatment The results showed an almost exponential increase in BaP To determine the effect of UV-mediated photolysis of BaP degradation when the BaP concentration was increased on its biodegradation, BaP-BSM (50 μg/ml) was exposed to UV irradiation (254 nm) for 15 min and then a pure culture 0 3 6 9 12 15 18 21 24 27 30 33 36 39 Incubation time (days) Induced Non-induced Fig. 2 a Pulse field gel electrophoresis (PFGE) and b in-well cell Fig. 1 Growth kinetics of non-induced and induced Bacillus subtilis lysis gel electrophoresis showing the absence of plasmid in BMT4i. BMT4i (MTCC 9447) in basal salts medium (BSM) with benzo [a] Lanes: M Marker (range 2–194 Kb), 1 uncured Bacillus subtilis pyrene (BaP) against incubation time (days). Data points represent BMT4i, 2 plasmid cured B. subtilis BMT4i (PC), 3 uncured average values from triplicate flasks Agrobacterium tumefaciens, 4 plasmid cured A. tumefaciens (PC) log CFU/ml of BMT4i in BaP-BSM 10 56 Ann Microbiol (2010) 60:51–58 80 60 60 40 40 40 30 10 0 0 Control Tween 20 Triton X-100 SDS % BaP Degradation log CFU/ml 0 0 UV-treated BSM-BaP Control BSM-BaP Fig. 5 BaP degradation (%) and log CFU/ml in the presence of % BaP Degradation log CFU/ml different surfactants. Data points represent mean values from three independent experiments. Error bars Standard deviations of triplicate Fig. 3 BaP biodegradation (%) and log CFU/ml of UV-treated BaP- independent experiments BSM and control BaP-BSM. Data points represent the mean of three independent experiments. Error bars Standard deviation of triplicate independent experiments had an inhibitory effect on BaP degradation (28.81%). The CFU data was in agreement with that of BaP degradation from 10 μg/ml (26.31%) to 30 μg/ml (40.94%), with a (Fig. 5). Hence, the BaP degradation and viability of BMT4i subsequent steady increase in BaP degradation reaching a is enhanced by the presence of the surfactants Tween-20 and maximum (61.04%) at 150 μg/ml (Fig. 4). Further increase Triton-X-100, while SDS showed adverse effects. in BaP concentration (from 200–250 μg/ml) resulted in a steep decrease in BaP degradation from 55.28% to 32.37%. Ionic strength Likewise, the viability of BMT4i was enhanced as the BaP concentration increased from 10 to 150 μg/ml. Further BMT4i was grown in BaP-BSM at various ionic strengths increase in BaP concentration resulted in a decrease in with respect to MgSO . As shown in Fig. 6,BaP viability. Therefore, the optimum BaP concentration at 37°C, degradation activity increased significantly (from 20.24 to pH 7.0 was found to be 150 μg/ml. 46.4%) when the MgSO concentration in the BaP-BSM was increased from 100 to 400 μM. With further increase in Surfactant treatment MgSO concentration, up to 1800 μM, the BaP degradation activity became stationary (48.12%), and then slowly To assess the impact of surfactant treatment on BaP declined to 45.64% at 2,400 μM. The viability data (log degradation, BMT4i was grown in the presence of 0.01% CFU/ml) was in agreement with that of BaP degradation different surfactants: Tween-20, Triton-X-100 and SDS. (Fig. 6). These findings suggest 1,800 μM MgSO as the The results demonstrated enhanced BaP degradation optimum concentration for the BaP degradation activity of (58.64%) in the presence of Tween-20 followed by Triton- BMT4i. However, higher concentrations of MgSO X-100 (50.12%) as compared to the control (46.32%) as (2,400 μM) had a negligible inhibitory effect on BaP shown in Fig. 5. Conversely, surfactant treatment with SDS degradation. 70 50 60 30 15 20 10 10 5 0 0 0 0 50 100 150 200 250 300 0 500 1000 1500 2000 2500 3000 BaP Concentration (microgram/ml) MgSO Concentration (microgram/ml) % BaP Degradation log CFU/ml % BaP Degradation log CFU/ml Fig. 4 BaP degradation (%) and log CFU/ml at different BaP Fig. 6 BaP degradation (%) and log CFU/ml at different ionic 10 10 concentrations. Data points represent mean values from three strength (MgSO ). Data points represent mean values from three independent experiments. Error bars Standard deviations of triplicate independent experiments. Error bars Standard deviations of triplicate independent experiments independent experiments % BaP Degradation of BMT4i % BaP Degradation of BMT4i log CFU/ml of BMT4i log10 CFU/ml of BMT4i % BaP Degradation of BMT4i % BaP Degradation of BMT4 log10 CFU/ml of BMT4i log10 CFU/ml of BMT4 Ann Microbiol (2010) 60:51–58 57 Discussion Acknowledgements This work was supported in part by the Modern Institute of Technology, Rishikesh, Uttarakhand, India and Uttarakhand Council of Science and Technology, Uttarakhand, India The present study was performed to extend our knowledge (UCOST; grant no. UCS and T/R and D/LS-46/06-07), which are about the BaP degradation activity of Bacillus subtilis gratefully acknowledged. We also thank Prof Aditya Shastri, Vice BMT4i (MTCC 9447). We have shown earlier that B. Chancellor, Banasthali University, Rajasthan, India for providing facilities in the Department of Bioscience and Biotechnology at this subtilis BMT4i (MTCC 9447) is an efficient degrader of university. We gratefully acknowledge Dr. Yogesh Shouche, Scientist BaP and possesses the ability to utilize BaP as a sole source E and Mr. Jay Siddharth, Senior Research Fellow (SRF), Molecular of carbon and energy for growth (Lily et al. 2009). This Biology Laboratory, National Centre for Cell Science (NCCS), Pune, India for providing the pulse field gel electrophoresis (PFGE) facility study reports that the BaP degradation activity of BMT4i is and invaluable help and cooperation during this research work. highly inducible, since induced culture showed more than 50% BaP degradation whereas non-induced culture did not show any significant BaP degradation in the presence of chloramphenicol. 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Annals of Microbiology – Springer Journals

Published: Jan 27, 2010

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