Benzo(a)pyrene degradation pathway in Bacillus subtilis BMT4i (MTCC 9447)

Chemicals and reagents

The BaP (99.9%) was purchased from Sigma-Aldrich Pvt. Ltd. Missouri, MO, USA. Metabolite standards for benzo(a)pyrene-cis-4,5-dihydrodiol, benzo(a)pyrene-cis-7,8-dihydrodiol were acquired from the NCI Chemical Carcinogen Reference Standards Repository (Kansas, Missouri, MO, USA). HPLC-grade methanol, acetonitrile, dimethylformamide, formic acid were purchased from Merck Pvt. Ltd., Maharashtra, India. Nutrient Agar media and broth were purchased from Himedia Mumbai, Maharashtra, India. The general chemicals including constituents of basal salt mineral media (BSM) and solvents of analytical grade were purchased from Glaxo SmithKline Pvt. Ltd., Mumbai, Maharashtra, India and Merck Life Science Pvt. Ltd., Bengaluru, Karnataka, India.

Identification of metabolites of BaP degradation in Bacillus subtilis BMT4i

For the identification of BaP degradation metabolites, 100 mL BSM culture of B. subtilis BMT4i (10⁷ cells/mL) containing BaP (50 μg/mL) in amber bottle was incubated at 30°C for 40 days in triplicate. At various time intervals (7, 15, and 40 days), 25 mL culture broth was withdrawn and 100 μL of the same was checked for BMT4i growth by CFU method [19]. Remaining withdrawn culture was acidified (pH 2.5) and processed for the recovery of products by ethyl acetate extraction [21]. The extracts were dried using rotary evaporator and dissolved in 5 mL of methanol. The organic extracts were analyzed using UHPLC-MS analysis on commercial basis from Sophisticated Analytical Instrument Facility (SAIF), India Institute of Technology, Mumbai, Maharashtra, India. Identification of metabolites was done by comparing the mass spectral data of metabolites obtained with that of commercially available BaP metabolite standards and those reported in the literature [18], [21].

UHPLC-MS Analysis

Stock solutions of BaP and metabolite standards were prepared at concentration of 5 mg/mL in dimethylformamide since the compounds were readily soluble in dimethylformamide. In addition, working stock solution of the same was prepared in methanol at concentration of 0.2 mg/mL [19]. Furthermore, the standards and samples filtered through glass syringe using 0.2 μm filter and then analyzed in UHPLC (1290 Infinity Binary pump, Agilent Technologies, Santa Clara, CA, USA) coupled with 6550 i-Funnel Quadrupole time-of-flight (QTOF) mass spectrometer. The mobile phase was a gradient of 0.1% formic acid in water and 0.1% formic acid in acetonitrile at a flow rate of 0.3 mL/min for 30 min; the injection volume was 3 μL. The column used was Zorbax SB C18 Rapid Resolution HD, 2.1×100 mm, 1.8 μm, (Agilent Technologies, Santa Clara, CA, USA). The nitrogen gas flow was 13 mL/min at 25°C, and the sheath gas flow was 11 mL/min at 30°C with a nebulizer pressure at 35 psi. The capillary voltage was 3500 V with a nozzle voltage of 1000 V. Fragmentation energy was kept at 175 V. The analysis was done in both positive and negative ESI mode, the data was acquired in Agilent Masshunter Data Acquisition software (Version B.05.00) (Agilent Technologies, Santa Clara, CA, USA) and the data were analyzed in Agilent Masshunter Qualitative Software (Version B.06.00) (Agilent Technologies, Santa Clara, CA, USA).

Identification of metabolites

Metabolic intermediates formed as a result of BaP degradation were recovered and subjected to UHPLC-MS analysis for identification. Firstly, the standards of commercially available metabolite were analyzed to identify the corresponding fragments. Each standard was injected separately, and its mass spectra and m/z values were obtained. The fragmentation pattern of individual metabolite standards and of a mixture of metabolite standards was obtained. Comparison of fragmentation pattern led to the confirmation of the presence of a particular metabolite. MS data (mass of recovered metabolites and their fragmentation pattern) of individual peaks were also compared with the MS data of metabolites of BaP biodegradation reported in the literature [18], [21].

UHPLC-MS analysis of 15-day sample revealed the presence of several peaks out of which eight peaks were identified (Figures 1A–C and 2; Table 1). Peaks II–VIII were observed in 7-day sample in addition to 15-day. However, peak I was exclusively found in 15-day sample. Peaks III–VI were also observed in 7 and 40 day samples in addition to 15 day samples, for peak I, (retention time 3.982 min) the characteristic mass fragment was m/z of 289.124 [M+Na]. The m/z of 289.124 [M+Na] of peak I compound was similar to the benzo(a)pyrene-11,12-epoxide (mol. wt. 266.29), an unstable intermediate formed due to action of cytochrome P450 on BaP which after subsequent hydrolysis converted into benzo(a)pyrene-trans-11,12-dihydrodiol in M. vanbaalenii PYR-1 [18]. Peak II eluted at 6.41 min. Mass fragment of peak II was m/z of 321.129 [M+H] which corresponded to compound 7,8,9,10-tetrahydrobenzo[pqr]tetraphene-7,8,9,10-tetraol (mol. wt. 320.33) shown to be generated in filamentous fungi Cunninghamella elegans by cytochrome-P450 monooxygenase during BaP degradation [24]. Although this kind of compound has not yet been reported in any bacterial species, however, Bacillus megaterium cytochrome-P450 BM3 monooxygenase has been reported to be a unique enzyme which can catalyze a wide range of similar reactions [25]. Peak III eluted at 9.396 min demonstrated mass fragment of m/z of 573.249 (dimer+H). The elution time and mass fragment of peak III was found to be identical with the elution time and mass fragment of the standard metabolite benzo(a)pyrene-cis-7,8-dihydrodiol (mol. wt. 286.32) dimer. Therefore, peak III was identified as benzo(a)pyrene-cis-7,8-dihydrodiol. Peak IV eluted at 10.768 showed mass fragment of m/z of 288.288 [M+CH3CN+H]. Its base peak mass (mol. wt. 246.26) was identical to the mass of 8-carboxy-7-hydroxy pyrene (mol. wt. 246.26) which was shown to be a ring cleavage intermediate in BaP degradation pathway in S. yanoikuyae JAR02 [21]. Peak V eluted at 10.990 min showed mass peak fragment of m/z of 288.28 [M] which corresponded to compound chrysene-4 or 5-carboxylic acid (mol. wt. 288.11) as reported in BaP degradation in M. vanbaalenii PYR-1 [18]. Peak VI was eluted at 12.239 min and its m/z ratio was found to be 316.319 (M). On the basis of its m/z value, it was identified as cis-4-(8-hydroxypyrene-7yl)-2-oxobut-3-enoic acid (mol. wt. 316.31) which was earlier found to be a ring cleavage product of benzo(a)pyrene-cis-9,10-dihydrodiol in M. vanbaalenii PYR-1 [18]. Peak VII with elution time 12.733 showed mass fragment of m/z of 299.105 [M+H] corresponding to the hydroxymethoxybenzo(a)pyrene (mol. wt. 298.33) which was previously reported in B. cereus, B. vireti and M. vanbaalenii PYR-1 [18], [23]. Peak VIII illustrated presence of two compounds on mass fragmentation. Mass fragment of m/z of 313.271 [M+H] with elution time of 17.615 min was found to be parallel to mass of the dimethoxy-benzo(a)pyrene (mol. wt. 312.36), and mass fragment having m/z of 252.099 [M] with elution time of 17.704 min corresponded to the mass peak of benzo(a)pyrene standard itself (mol. wt. 252.32). The finding was in accordance with earlier reports on M. vanbaalenii PYR-1 mediated BaP degradation [18].

Figure 1:

Out of eight peaks, peak I was exclusively found in 15-day sample.

UHPLC chromatograms showing benzo(a)pyrene and its metabolites after (A) 7 day (B) 15 day (C) 40 day.

Figure 2:

Three metabolites namely 8-carboxy-7-hydroxy pyrene, chrysene-4 or 5-carboxylic acid and cis-4-(8-hydroxypyrene-7yl)-2-oxobut-3-enoic acid are the ring cleavage metabolites.

Full scan mass spectra of BaP metabolites showing the abundance of (A) BaP standard as mass peak, (B) peak I (benzo(a)pyrene-11, 12 epoxide as M+Na adduct), (C) peak II (7,8,9,10-tetrahydrobenzo[pqr]tetraphene-7,8,9,10-tetraol as M+H), (D) peak III (benzo(a)pyrene-cis-7,8-dihydrodiol as dimer M+H), (E) peak IV (8-carboxy-7-hydroxy pyrene as M+CH3CN+H), (F) peak V (chrysene-4 or 5-carboxylic acid as mass peak), (G) peak VI (cis-4-(8-hydroxypyrene-7yl)-2-oxobut-3-enoic acid as mass peak), (H) peak VII (hydroxymethoxybenzo(a)pyrene as M+H) and (I) peak VIII (dimethoxy-benzo(a)pyrene as M+H and benzo(a)pyrene as mass peak).

Elucidation of BaP degradation pathway in Bacillus subtilis BMT4i

The identification of total eight metabolites produced during BaP degradation led to elucidation of BaP degradation pathway for the first time in B. subtilis BMT4i (Figure 3). Out of eight, three metabolites namely 8-carboxy-7-hydroxy pyrene, chrysene-4 or 5-carboxylic acid and cis-4-(8-hydroxypyrene-7yl)-2-oxobut-3-enoic acid are the ring cleavage metabolites while others are the intermediates of the degradation pathway prior to ring cleavage. On the basis of identified metabolites, we have proposed BaP biodegradation pathway in B. subtilis BMT4i, which is in agreement with the pathways documented in the previous studies [14], [18], [21], [23]. The listed metabolites are known to be generated by various bacterial species such as Mycobacterium sp. Strain RJGII-135 [14], M. vanbaalenii PYR-1 [18], and S. yanoikuyae JAR02 [21], which suggested functioning of multiple pathways involving dioxygenases and monoxygenase as the initial attacking enzymes in B. subtilis BMT4i. Bacteria initially oxidize aromatic hydrocarbons to cis-dihydrodiols [16]. The oxidation of these compounds involves the enzymatic incorporation of atmospheric oxygen into the substrate. Characteristically, bacteria produce dioxygenases and mono-oxygenases, which incorporate two or one oxygen atoms into the aromatic nucleus forming cis-dihydrodiols and trans-dihydrodiols, respectively [16], [26]. The initial ring oxidation is usually the rate-limiting step in the biodegradation reaction of PAHs. Further oxidation of the cis-dihydrodiols leads to the formation of catechols which are substrates for other dioxygenases that bring about enzymatic cleavage of the aromatic ring.

Figure 3:

Proposed pathways for degradation of BaP by B. subtilis BMT4i (MTCC 9447) which is in consonance to BaP degradation pathways proposed by Moody et al. [18]; Rentz et al. [21].

Peak I (benzo(a)pyrene-11, 12 epoxide), peak II (7,8,9,10-tetrahydrobenzo[pqr]tetraphene-7,8,9,10-tetraol), peak III (benzo(a)pyrene-cis-7,8-dihydrodiol, peak IV (8-carboxy-7-hydroxy pyrene), peak V (chrysene-4 or 5-carboxylic acid), peak VI (cis-4-(8-hydroxypyrene-7yl)-2-oxobut-3-enoic acid, peak VII (hydroxymethoxybenzo(a)pyrene, peak VIII (dimethoxy-benzo(a)pyrene). Metabolites within the box are hypothetical intermediates and have not been measured in this study.

As shown in proposed pathway of BaP degradation in Figure 3, benzo(a)pyrene-11, 12-epoxide is the precursor of benzo(a)pyrene-trans-11,12-dihydrodiol which is further converted to hydroxymethoxybenzo(a)pyrene subsequently leading to formation of dimethoxybenzo(a)pyrene. The dioxygenase mediated hydroxylation at 4, 5 or 11, 12 positions in BaP alternatively may generate benzo(a)pyrene-cis-4,5-dihydrodiol or benzo(a)pyrene-cis-11,12-dihydrodiol, respectively leading to hydroxymethoxybenzo(a)pyrene which is further metabolized to corresponding dimethoxy benzo(a)pyrene [18]. However, at this moment we cannot ascertain the positions of substitution due to lack of substantial data. Formation of tetrahydrobenzo[pqr]tetraphene-7,8,9,10-tetraol might be result of epoxidation of BaP by cytochrome–P450 monooxygenase [24].

Two other accumulated intermediates namely cis-4-(8-hydroxypyrene-7yl)-2-oxobut-3-enoic acid and 8-carboxy-7-hydroxypyrene were considered as products of benzo(a)pyrene-cis-9,10-dihydrodiol and benzo(a)pyrene-cis-7,8-dihydrodiol generated by the action of dioxygenases at 9,10 and 7,8 positions, respectively in BaP [14], [18], [21]. Rentz et al. [21] demonstrated the presence of pyrene-7-hydroxy-8-carboxylic acid or pyrene-8-hydroxy-7-carboxylic acid which was hypothesized to be produced from transformation of 8-carboxy-7-hydroxypyrene. Nevertheless, in our study, presence of 8-carboxy-7-hydroxypyrene is detected, instead of pyrene-7-hydroxy-8-carboxylic acid or pyrene-8-hydroxy-7-carboxylic acid. Formation of chrysene-4 or 5-carboxylic acid is presumed to be the end product of ortho cleavage ring fission product 4,5-chrysene dicarboxylic acid whose precursor is benzo(a)pyrene-cis-4,5- dihyrodiol produced by reaction of dioxygenation of BaP in the K-region (4,5 positions) as reported in Mycobacterium vaanbellenii PYR-1 [18].

To the best of our knowledge, the present study is the first one to report many metabolites of BaP degradation in genus Bacillus. In addition, a metabolic pathway of BaP has been elucidated in much greater detail for the first time in genus Bacillus. The results of this study on B. subtilis BMT4i (MTCC 9447), which is a potent BaP degrader (84.66% BaP degradation in 28 days) [19], could facilitate the elucidation of the biodegradation mechanism in Bacillus, which is currently not well explored. However, further studies are essential to find out whether the entire biodegradation process in B. subtilis results into the metabolic detoxification of BaP or not.