Fig. 1.
Phylogenetic tree constructed for present study bacteria using MEGA11 software.
Fig. 2.
Growth curve analysis of present study benzene degrading bacteria.
Fig. 3.
Comparison of FTIR spectra of control and present study benzene degrading bacteria to analyze the variation of the peaks contributed by bacterial benzene degradation.
A) Control, B) Paracoccus aestuarii PUB1, C) Bacillus tropicus PUB2, D) Bacillus albus PUB3, E) Bacillus subtilis PUB4, F) Bacillus cereus PUB6.
Table I
Metabolites identified in the present study bacteria, through comparison of m/z ratios of GC-MS spectra with molecular weights of earlier reported benzene degradation metabolites.
| Metabolite identified | Metabolites unidentified | PUB4 | PUB1 | Fragmentation ion m/z | Molecular weight (M/w) | FTlR spectral peaks (cm−1) |
|---|---|---|---|---|---|---|
| Benzene methylation pathway | ||||||
| Toluene | cis-benzoate dihydrodiol, cis-benzene dihydrodiol | I | I | 39,51,65, 77, 91,92 | 92.14 | 1452 (aromatic deformation), 2851 and 2920 (=C–H and –C–H) |
| Benzyl alcohol | I | 65, 77, 79,91, 107, 108 | 108 | 3427 (OH), 3010 (unsaturated CH), 2900 (saturated CH) | ||
| Benzaldehyde | I | I | 29, 39,51,52, 77, 78, 105, 106 | 106 | 1637–1733 (C=O vibs) | |
| Benzoate | I | I | 176, 121,93, 77, 65 | 121 | 3427 (OH), 1271 (C-O-C stretching) | |
| Catechol | I | I | 81,66, 53, 110 | 110 | 3427 (OH), 1658 (C=C), 1250 and 1281 (C-O), 746 and 850 out of plane bending of =C–H bond of aromatic ring | |
| Benzene degradation via benzaldehyde | ||||||
| Benzyl alcohol | benzoate, bcnzoyl-CoA, acetyl CoA | I | I | 65, 77, 79,91, 107,108 | 108 | 3427 (OH), 3010 (unsaturated CH), 2900 (saturated CH) |
| Benzaldehyde | I | I | 29, 39,51,52, 77, 78, 105, 106 | 106 | 1637-1733 (C=O vibs) | |
| Benzene degradation via carboxylation | ||||||
| Benzoate | I | I | 176, 121,93, 77, 65 | 121 | 3427 (OH), 1271 (C–O–C stretching) | |
| cis-1,6-dihydroxy-2,4-Cyelohexadiene-1-Carboxylie acid | I | I | 44, 73, 149 | 156 | 2800-3300 (C–H stretch) | |
| Catechol | I | I | 81,66, 53 | 110 | Same as above | |
| Benzene degradation via phenol | ||||||
| Phenol | I | I | 17, 38, 39, 40, 63, 65, 77, 93, 94 | 94 | 3427 (OH), 1650 (C=C stretch) | |
| Catechol | I | 1 | 81,66, 53, 110 | 110 | Same as above | |
| β-ketoadipate pathway | ||||||
| Catechol | 3-oxoadiphyl CoA, succinyl CoA, succinate, fumarate | I | I | 81,66, 53, 110 | 110 | Same as above |
| cis,cis-muconate | I | --- | 77, 106, 122, 126, 138, 171, 195, 228 | 126 | 1540–1650 (asymmetric stretch of CO2-), 1360–1450 (symmetric stretch of CO2-) | |
| 3-oxoadipatc enol lactone | I | --- | 1637–1733 (C=O vibs), 3427 (OH), | |||
| 3-oxoadipate | I | --- | 40.44, 73, 84, 87, 101, 114, 115, 160 | 160 | 1637–1733 (C=O vibs) | |
| Palmitate | I | I | 41,43, 57, 73, 89, 99, 117, 127, 141, 169, 183, 201,215, 229, 239, 257, 271,285,299, 313 | 255 | 2848 and 2913 (–CH3 and –CH2 vibs), 1695 (C=O), 1464 (–CH2 and –CH3), 939 (–OH out plane vibs) and 1299 (–OH in-plane vibs), 723 and 685 (–OH swinging vibs) | |
Fig. 4.
Four pathways of benzene metabolism suggested in benzene-degrading bacteria in the present study based on GC-MS based identification of metabolites.
A1 – benzyl alcohol dehydrogenase, B1 – benzaldehyde dehydrogenase, C1 – benzoate CoA-ligase, D1 – benzoyl-CoA 2,3-dioxygenase, A2 – methyl monooxygenase, B2 – benzoyl alcohol dehydrogenase, C2 – benzaldehyde dehydrogenase, D2 – benzoate 1,2-dioxygenase, E2 – dihydrocyclohexadiene carbohydrate dehydrogenase, A3 – benzoate dioxygenase, B3 – benzoate cis-diol dehydrogenase, A4 – benzene phenol monooxygenase
Fig. 5.
The β-ketoadipate pathway suggested in the present study benzene degrading bacteria based on GC-MS based identification of metabolites involving this pathway.
a – catechol 1,2-dioxygenase, b – muconate cycloisomerase, c-d – beta-ketoadipate:succinyl CoA transferase, e – succinyl CoA:acetyl CoA C-succinyl transferase, f – succinyl-CoA synthetase, g – succinic dehydrogenase, h – fumarase, i – malate dehydrogenase, j – citrate synthase, k – fatty acid synthase