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

Background: Benzo(a)pyrene (BaP), a high molecular weight pentacyclic aromatic hydrocarbon, is a priority pollutant of extreme concern. Bacillus subtilis BMT4i (MTCC 9447) degrades BaP through chromosomally encoded pathway. Nevertheless, inadequate information is available on BaP degradation pathway in genus Bacillus despite of its species being shown as potent BaP degrader. The objective of this study was to elucidate BaP degradation pathway in B. subtilis strain BMT4i by identifying metabolites through UHPLC-MS. Materials and methods: Batch experiments were con-ducted to characterize metabolic pathway of BaP in the bacterium B. subtilis BMT4i. The metabolites were sepa-rated and characterized by UHPLC-MS. Results: The major intermediates of BaP metabolism that had accumulated in the culture media after 15 days of incubation were cis-4-(8-hydroxypyr-ene-7yl)-2-oxobut-3-enoic acid, hydroxy methoxybenzo(a) pyrene and dimethoxybenzo(a)pyrene. Among above, 8-carboxy-7-hydroxy pyrene, chrysene-4 or 5-carboxylic acid, and cis-4-(8-hydroxypyrene-7yl)-2-oxobut-3-enoic acid are ring cleavage products of BaP. Conclusion: The identified metabolites indicated that BMT4i initially oxidized BaP with monooxygenases and dioxygenases at C-11,12 or and C-7,8 and C-9,10 positions, suggesting operation of multiple pathways for BaP degradation in B. subtilis . Further studies are essential to find out whether the entire biodegradation process in B. subtilis results into metabolic detoxification of BaP or not.

As an extension of previous study, the present work is performed to elucidate BaP degradation pathway in B. subtilis strain BMT4i by identifying metabolites through Ultra High Performance Liquid Chromatography-Mass Spectroscopy (UHPLC-MS). The present study reports the presence of eight different metabolites including ring cleavage products suggesting functioning of multiple degradation pathways involving dioxygenases and monoxygenase as the initial attacking enzymes.

Identification of metabolites of BaP degradation in Bacillus subtilis BMT4i
For the identification of BaP degradation metabolites, 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 At various time intervals (7,15, and 40 days), 25 mL culture broth was withdrawn and 100 μL of the same was checked for BMT4i growth by CFU method [19]. Remaining withdrawn culture was acidified (pH 2.5) and processed for the recovery of products by ethyl acetate extraction [21].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 [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].
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.