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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 conducted to characterize metabolic pathway of BaP in the bacterium B. subtilis BMT4i. The metabolites were separated 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 benzo(a)pyrene-11,12-epoxide, 7,8,9,10-tetrahydrobenzo[pqr]tetraphene-7,8,9,10-tetraol, benzo(a)pyrene-cis-7,8-dihydrodiol, 8-carboxy-7-hydroxy pyrene, chrysene-4 or 5-carboxylic acid, cis-4-(8-hydroxypyrene-7yl)-2-oxobut-3-enoic acid, hydroxymethoxybenzo(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.

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The present study demonstrated the growth kinetics of a potent high molecular weight polycyclic aromatic hydrocarbon (HMW-PAH) degrader Bacillus subtilis BMT4i (MTCC 9447) using mobil oil or engine oil as the sole carbon and energy source. The study was performed to demonstrate the variations in morphology and growth kinetics in benzo-a-pyrene (BaP) degrading BMT4i due to the mobil oil induced stress conditions. The morphological variations were evaluated using Gram staining and spore staining (Schaeffer-Fulton method) followed by assessment of viability and growth using colony forming units (CFU)/ml method by means of growing Bacillus subtilis BMT4i in basal salt medium (BSM) with mobil oil (2%: BSMM) as sole carbon and energy source at different time intervals. The findings represented that mobil oil has noticeable effect on the shape and size of BMT4i cells. After 24 h of exposure to mobil oil, maximum BMT4i cells entered in to endospore development and several exospores were released after 24 h. The growth kinetics depicted an exponential increase in BMT4i cells in BSMM with increase in incubation time up to 3 days accomplishing maxima of 2.1 x 1019 demonstrating approx. 2x1011-fold enhancements in cell number and afterwards cell number declined. Increase in CFU number (approx. 2x1011 fold) was directly linked with the BMT4i potential to use mobil oil as the sole carbon and energy source leading to elevation in cell number within just 3 days. Thus, it could be concluded that Bacillus subtilis BMT4i (MTCC 9447) is very competent in using mobil oil as the sole source of carbon and energy and hence it could be employed for bioremediation of mobil oil and PAH contaminated sites.

The present study demonstrated the growth kinetics of a potent high molecular weight polycyclic aromatic hydrocarbon (HMW-PAH) degrader Bacillus subtilis BMT4i (MTCC 9447) using mobil oil or engine oil as the sole carbon and energy source. The study was performed to demonstrate the variations in morphology and growth kinetics in benzo-a-pyrene (BaP) degrading BMT4i due to the mobil oil induced stress conditions. The morphological variations were evaluated using Gram staining and spore staining (Schaeffer-Fulton method) followed by assessment of viability and growth using colony forming units (CFU)/ml method by means of growing Bacillus subtilis BMT4i in basal salt medium (BSM) with mobil oil (2%: BSMM) as sole carbon and energy source at different time intervals. The findings represented that mobil oil has noticeable effect on the shape and size of BMT4i cells. After 24 h of exposure to mobil oil, maximum BMT4i cells entered in to endospore development and several exospores were released after 24 h. The growth kinetics depicted an exponential increase in BMT4i cells in BSMM with increase in incubation time up to 3 days accomplishing maxima of 2.1 x 1019 demonstrating approx. 2x1011-fold enhancements in cell number and afterwards cell number declined. Increase in CFU number (approx. 2x1011 fold) was directly linked with the BMT4i potential to use mobil oil as the sole carbon and energy source leading to elevation in cell number within just 3 days. Thus, it could be concluded that Bacillus subtilis BMT4i (MTCC 9447) is very competent in using mobil oil as the sole source of carbon and energy and hence it could be employed for bioremediation of mobil oil and PAH contaminated sites.