Molecular and metabolic characterization of petroleum hydrocarbons degrading Bacillus cereus

Hussain, Nadia; Muccee, Fatima; Hammad, Muhammad; Mohiuddin, Farhan; Bunny, Saboor Muarij; Shahab, Aansa

doi:10.33073/pjm-2024-012

Figures & Tables

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.

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.

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.

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.

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

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

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	CH3OH→CH2O+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+12O2→CH2O2methanonemethanoic acid \[\begin{matrix} C{{H}_{2}}O & + & \frac{1}{2}{{O}_{2}} & \to & C{{H}_{2}}{{O}_{2}} \\ \text{methanone} & {} & {} & {} & \text{methanoic}\,\text{acid} \\ \end{matrix}\]
Methylcycleohexane
1	C7H12O+12O2→C7H12O2methylcyclohexanecyclohexane carboxylic acid \[\begin{matrix} {{C}_{7}}{{H}_{12}}O & + & \frac{1}{2}{{O}_{2}} & \to & {{C}_{7}}{{H}_{12}}{{O}_{2}} \\ \text{methylcyclohexane} & {} & {} & {} & \text{cyclohexane}\,\text{carboxylic}\,\text{acid} \\ \end{matrix}\]
2	C7H12O2→C6H5COOH+3H2cyclohexane carboxylic acidbenzoic acid \[\begin{matrix} {{C}_{7}}{{H}_{12}}{{O}_{2}} & \to & {{C}_{6}}{{H}_{5}}COOH+3{{H}_{2}} \\ \text{cyclohexane}\,\text{carboxylic}\,\text{acid} & {} & \text{benzoic}\,\text{acid} \\ \end{matrix}\]
3	C7H6O5+2H2O→C6H6O2+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+O2→C6H4O4+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+O2→C7H8Otolueneo-,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)	C7H8O→C7H8O23-methylcatechol \[\begin{matrix} {{C}_{7}}{{H}_{8}}O & \to & {{C}_{7}}{{H}_{8}}{{O}_{2}} \\ {} & {} & 3\text{-methylcatechol} \\ \end{matrix}\]
3 (TMO)	C7H6O2+12O2→C7H64-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_+C21H36N7O16P3S→C28H36N7O17P3S−4+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+12O2→C6H6O2phenolcatechol \[\begin{matrix} {{C}_{6}}{{H}_{5}}OH & + & \frac{1}{2}{{O}_{2}} & \to & {{C}_{6}}{{H}_{6}}{{O}_{2}} \\ \text{phenol} & {} & {} & {} & \text{catechol} \\ \end{matrix}\]
3	C6H6O2+O2→C6H6O4cis,cis-muconic acid \[\begin{matrix} {{C}_{6}}{{H}_{6}}{{O}_{2}} & + & {{O}_{2}} & \to & {{C}_{6}}{{H}_{6}}{{O}_{4}} \\ {} & {} & {} & {} & cis,cis\text{-muconic}\,\text{acid} \\ \end{matrix}\]
4 (pathway C)	C6H5OH+CO2→C7H6O3phenol4-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	C8H10O→C8H8O+2H+m-methylbenzyl alcoholm-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	C7H8O4→C7H8O2+O23-methylcyclohexa-3,5-diene-1,2-diol-1-carboxylic acid3-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

Molecular and metabolic characterization of petroleum hydrocarbons degrading Bacillus cereus

Figures & Tables

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

Earlier reported pathways in petroleum degrading bacteria and the metabolites identified in Bacillus cereus strain sab41_

Petroleum hydrocarbons degrading bacteria reported in the literature_

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