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Fig. 1.

Growth phases and petroleum hydrocarbons degradation potential analysis of Bacillus cereus strain sab41in present study. a) Growth curve showing lag, log, static, and death phases; b) graph showing degradation efficiency of petroleum by B. cereus strain sab41 estimated by measuring OD600 of supernatant containing DCPIP.
Growth phases and petroleum hydrocarbons degradation potential analysis of Bacillus cereus strain sab41in present study. a) Growth curve showing lag, log, static, and death phases; b) graph showing degradation efficiency of petroleum by B. cereus strain sab41 estimated by measuring OD600 of supernatant containing DCPIP.

Fig. 2.

Biochemical characterization of Bacillus cereus strain sab41 using Remel RapID™ One panel.
a) Remel RapID™ One panel showing the results; b) table illustrating the biochemical tests being analyzed in the present study.
Biochemical characterization of Bacillus cereus strain sab41 using Remel RapID™ One panel. a) Remel RapID™ One panel showing the results; b) table illustrating the biochemical tests being analyzed in the present study.

Fig. 3.

Phylogenetic tree of Bacillus cereus strain sab41 constructed using MEGA 11 software.
A maximum composite likelihood neighbor-joining tree using a bootstrap value 100 was constructed.
Phylogenetic tree of Bacillus cereus strain sab41 constructed using MEGA 11 software. A maximum composite likelihood neighbor-joining tree using a bootstrap value 100 was constructed.

Fig. 4.

GC chromatograms of Bacillus cereus strain sab41 showing the identified metabolites formed by degradation of petroleum hydrocarbons in the present study.
a) Peaks showing methylalcohol, methanoic acid, cyclohexene, cyclohexane, catechol, 4-methylcyclohexanone; b) peak showing benzoate; c) peak showing 3-methyl salicylic acid; d) peak showing acetaldehyde and o-cresol; e) peak showing 2-methylmuconate.
GC chromatograms of Bacillus cereus strain sab41 showing the identified metabolites formed by degradation of petroleum hydrocarbons in the present study. a) Peaks showing methylalcohol, methanoic acid, cyclohexene, cyclohexane, catechol, 4-methylcyclohexanone; b) peak showing benzoate; c) peak showing 3-methyl salicylic acid; d) peak showing acetaldehyde and o-cresol; e) peak showing 2-methylmuconate.

Fig. 5.

Pathways identified in Bacillus cereus strain sab41 associated with the degradation of alkanes and cycloalkanes.
a – alkane 1-monooxygenase; b – alcohol dehydrogenase; c – cyclohexanone monooxygenase; d – 6-hexanolactone hydrolase; a1 – benzoate 1,2-dioxygenase; a2 – dihydroxybenzoate dehydrogenase; a3 – catechol 1,2-dioxygenase; e – methane monooxygenase; f – methanol dehydrogenase; g – formaldehyde dehydrogenase; h – formate dehydrogenase
Pathways identified in Bacillus cereus strain sab41 associated with the degradation of alkanes and cycloalkanes. a – alkane 1-monooxygenase; b – alcohol dehydrogenase; c – cyclohexanone monooxygenase; d – 6-hexanolactone hydrolase; a1 – benzoate 1,2-dioxygenase; a2 – dihydroxybenzoate dehydrogenase; a3 – catechol 1,2-dioxygenase; e – methane monooxygenase; f – methanol dehydrogenase; g – formaldehyde dehydrogenase; h – formate dehydrogenase

Fig. 6.

Pathways identified in Bacillus cereus strain sab41 involved in the degradation of aromatics.
a1, a2 – toluene 3-monooxygenase; b1, b2 – toluene 2-monooxygenase; c1 – toluene 4-monooxygenase; c2 – 4-hydroxymethyl hydroxylase; c3 – 4-hydroxybenzaldehyde dehydrogenase; d1 – xylene oxidase; d2 – alcohol dehydrogenase; d3 – aldehyde dehydrogenase
Pathways identified in Bacillus cereus strain sab41 involved in the degradation of aromatics. a1, a2 – toluene 3-monooxygenase; b1, b2 – toluene 2-monooxygenase; c1 – toluene 4-monooxygenase; c2 – 4-hydroxymethyl hydroxylase; c3 – 4-hydroxybenzaldehyde dehydrogenase; d1 – xylene oxidase; d2 – alcohol dehydrogenase; d3 – aldehyde dehydrogenase

Fig. 7.

Benzene degradation pathways identified in Bacillus cereus strain sab41 in present study via GC-MS analysis.
a – benzylalcohol dehydrogenase; b – benzaldehyde dehydrogenase; c – benzoate CoA-ligase; d – 4-hydroxybenzoate CoA-ligase; e –4-hydroxybenoyl CoA reductase
Benzene degradation pathways identified in Bacillus cereus strain sab41 in present study via GC-MS analysis. a – benzylalcohol dehydrogenase; b – benzaldehyde dehydrogenase; c – benzoate CoA-ligase; d – 4-hydroxybenzoate CoA-ligase; e –4-hydroxybenoyl CoA reductase

Stoichiometric equations of reactions involved in formation of metabolic intermediates of petroleum hydrocarbons degradation identified in Bacillus cereus strain sab41

No. of reactions Stoichiometric equations
Methane
1 CH3OHCH2O+2H+methylalcoholmethanone \[\begin{align} & \\ & \begin{matrix} C{{H}_{3}}OH & \to & C{{H}_{2}}O & + & 2{{H}^{+}} \\ \text{methylalcohol} & {} & \text{methanone} & {} & {} \\ \end{matrix} \\ \end{align}\]
2 CH2O+12O2CH2O2methanonemethanoicacid \[\begin{matrix} C{{H}_{2}}O & + & \frac{1}{2}{{O}_{2}} & \to & C{{H}_{2}}{{O}_{2}} \\ \text{methanone} & {} & {} & {} & \text{methanoic}\,\text{acid} \\ \end{matrix}\]
Methylcycleohexane
1 C7H12O+12O2C7H12O2methylcyclohexanecyclohexanecarboxylicacid \[\begin{matrix} {{C}_{7}}{{H}_{12}}O & + & \frac{1}{2}{{O}_{2}} & \to & {{C}_{7}}{{H}_{12}}{{O}_{2}} \\ \text{methylcyclohexane} & {} & {} & {} & \text{cyclohexane}\,\text{carboxylic}\,\text{acid} \\ \end{matrix}\]
2 C7H12O2C6H5COOH+3H2cyclohexanecarboxylicacidbenzoicacid \[\begin{matrix} {{C}_{7}}{{H}_{12}}{{O}_{2}} & \to & {{C}_{6}}{{H}_{5}}COOH+3{{H}_{2}} \\ \text{cyclohexane}\,\text{carboxylic}\,\text{acid} & {} & \text{benzoic}\,\text{acid} \\ \end{matrix}\]
3 C7H6O5+2H2OC6H6O2+CO2+2H2O2-hydro-1,2-dihydroxybenzoatecatechol \[\begin{matrix} {{C}_{7}}{{H}_{6}}{{O}_{5}} & + & 2{{H}_{2}}O\to {{C}_{6}}{{H}_{6}}{{O}_{2}}+C{{O}_{2}}+2{{H}_{2}}O \\ \text{2-hydro-1,2-dihydroxybenzoate} & {} & \text{catechol} \\ \end{matrix}\]
4 C6H6O2+O2C6H4O4+2Hcis,cis-muconate \[\begin{matrix} {{C}_{6}}{{H}_{6}}{{O}_{2}} & + & {{O}_{2}} & \to & {{C}_{6}}{{H}_{4}}{{O}_{4}} & + & 2H \\ {} & {} & {} & {} & cis,cis\text{-muconate} & {} & {} \\ \end{matrix}\]
Toluene
1 (Tbu, TMO, TOM) C6H5CH3+O2C7H8Otolueneo-,m-,p-cresol \[\begin{matrix} {{C}_{6}}{{H}_{5}}C{{H}_{3}} & + & {{O}_{2}} & \to & {{C}_{7}}{{H}_{8}}O \\ \text{toluene} & {} & {} & {} & o\text{-,}m\text{-},p\text{-cresol} \\ \end{matrix}\]
2 (Tbu, TMO) C7H8OC7H8O23-methylcatechol \[\begin{matrix} {{C}_{7}}{{H}_{8}}O & \to & {{C}_{7}}{{H}_{8}}{{O}_{2}} \\ {} & {} & 3\text{-methylcatechol} \\ \end{matrix}\]
3 (TMO) C7H6O2+12O2C7H64-hydroxybenzaldehyde4-hydroxybenzoate \[\begin{matrix} {{C}_{7}}{{H}_{6}}{{O}_{2}} & + & \frac{1}{2}{{O}_{2}} & \to & {{C}_{7}}{{H}_{6}} \\ \text{4-hydroxybenzaldehyde} & {} & {} & {} & \text{4-hydroxybenzoate} \\ \end{matrix}\]
Benzene
1 (pathway A) C7H5O2_+C21H36N7O16P3SC28H36N7O17P3S4+12O2benzoateCoAbenzoyl-CoA \[\begin{matrix} {{C}_{7}}{{H}_{5}}O_{2}^{\_} & + & {{C}_{21}}{{H}_{36}}{{N}_{7}}{{O}_{16}}{{P}_{3}}S & \to & {{C}_{28}}{{H}_{36}}{{N}_{7}}{{O}_{17}}{{P}_{3}}{{S}^{-4}} & + & \frac{1}{2}{{O}_{2}} \\ \text{benzoate} & {} & \text{CoA} & {} & \text{benzoyl-CoA} & {} & {} \\ \end{matrix}\]
2 (pathway B) C6H5OH+12O2C6H6O2phenolcatechol \[\begin{matrix} {{C}_{6}}{{H}_{5}}OH & + & \frac{1}{2}{{O}_{2}} & \to & {{C}_{6}}{{H}_{6}}{{O}_{2}} \\ \text{phenol} & {} & {} & {} & \text{catechol} \\ \end{matrix}\]
3 C6H6O2+O2C6H6O4cis,cis-muconicacid \[\begin{matrix} {{C}_{6}}{{H}_{6}}{{O}_{2}} & + & {{O}_{2}} & \to & {{C}_{6}}{{H}_{6}}{{O}_{4}} \\ {} & {} & {} & {} & cis,cis\text{-muconic}\,\text{acid} \\ \end{matrix}\]
4 (pathway C) C6H5OH+CO2C7H6O3phenol4-hydroxybenzoate \[\begin{matrix} {{\text{C}}_{6}}{{\text{H}}_{5}}\text{OH} & + & C{{O}_{2}} & \to & {{C}_{7}}{{H}_{6}}{{O}_{3}} \\ \text{phenol} & {} & {} & {} & 4\text{-hydroxybenzoate} \\ \end{matrix}\]
Xylene
1 C8H10OC8H8O+2H+m-methylbenzylalcoholm-tolualdehyde \[\begin{matrix} {{C}_{8}}{{H}_{10}}O & \to & {{C}_{8}}{{H}_{8}}O & + & 2{{H}^{+}} \\ m\text{-methylbenzyl}\,\text{alcohol} & {} & m\text{-tolualdehyde} & {} & {} \\ \end{matrix}\]
2 C7H8O4C7H8O2+O23-methylcyclohexa-3,5-diene-1,2-diol-1-carboxylicacid3-methylcatechol \[\begin{matrix} {{C}_{7}}{{H}_{8}}{{O}_{4}} & \to & {{C}_{7}}{{H}_{8}}{{O}_{2}} & + & {{O}_{2}} \\ 3\text{-methylcyclohexa-3,5-diene-1,2-diol-1-carboxylic}\,\text{acid} & {} & 3\text{-methylcatechol} & {} & {} \\ \end{matrix}\]

Earlier reported pathways in petroleum degrading bacteria and the metabolites identified in Bacillus cereus strain sab41.

Petroleum hydrocarbon component Pathway/bacterium reported in literature Metabolites identified in present study Reference
Methane Methylomicrobium alcaliphilum 20Z methylalcohol, methanone, methanoic acid Kalyuzhnaya et al. 2013
Methylcyclohexane Pseudophaeobacter, Gilvimarinus, Pseudomonas, Cycloclasticus, Roseovarius cyclohexane carboxylic acid, benzoic acid, catechol, cis,cis-muconate Li et al. 2023a
Toluene Thauera sp. strain T1 benzoate, acetaldehyde, cyclohexene, pyruvate, cresol, 3-methyl catechol, 4-hydroxybenzoate Heider et al. 1998; Muccee et al. 2019
Toluene 4-monoxygenase pathway in Pseudomonas mendocina KR1 p-cresol, 4-hydroxybenzoate Whited and Gibson 1991
toluene 3-monooxygenase pathway in Pseudomonas pickettii m-cresol, 3-methylcatechol Olsen et al. 1994
Benzene Acinetobacter calcoaceticus, Rhodopseudomonas palustris, Pseudomonas putida CSV86 benzoate, catechol, cis,cis-muconic acid, 4-hydroxybenzoate Mackintosh and Fewson 1988; Egland et al. 1997; Basu et al. 2003
Xylene m-xylene oxidation in Pseudomonas Pxy m-tolualdehyde, 3-methylcatechol Davey and Gibson 1974

Petroleum hydrocarbons degrading bacteria reported in the literature.

Bacteria References
Acinetobacter XS-4 Zou et al. 2023
Neorhizobium, Allorhizobium, Rhizobium, Pararhizobium, Pseudomonas, Nocardioides, Simplicispira Eziuzor and Vogt 2023
Dehalococcoidia Zehnle et al. 2023
Pinisolibacter aquiterrae Bedics et al. 2022
Enterobacter Hossain et al. 2022
Talaromyces sp. Zhang et al. 2021
Pseudomonas pseudoalcaligenes, Rhodococcus Feng et al. 2021; Chuah et al. 2022
Aquabacterium Xu et al. 2019
Nesiotobacter exalbescens Ganesh Kumar et al. 2019
Bradyrhizobium, Koribacter, Acidimicrobium Jeffries et al. 2018
Sphingomonas Zhou et al. 2016
Exiguobacterium aurantiacum Mohanty and Mukherji 2008
Bacillus subtilis, Alcaligenes sp., Flavobacterium sp., Micrococcus roseus, Corynebacterium sp. Adebusoye et al. 2007
Marinobacter, Alcanivorax, Sphingomonas, Gordonia, Micrococcus, Cellulomonas, Dietzia Brito et al. 2006
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