Whole Genome Shotgun Sequencing-Based Insights into the Benzene and Xylene Degrading Potentials of Bacteria

Benzene is declared carcinogenic by the International Agency for Research on Cancer (IARC) and the Environmental Protection Agency (EPA) of the USA (Li et al. 2021; Matheson et al. 2023). Xylene is non-carcinogenic with risk class 3 (moderate risk), but when it is found in the form of benzene, toluene, ethylbenzene, and xylene (BTEX), it proves hazardous (Li et al. 2025). These two pollutants adversely affect nervous, respiratory, reproductive, endocrine, urinary and cardiovascular systems (Caron-Beaudoin et al. 2022).

Given the ubiquitous nature, persistence, carcinogenicity, and other associated health hazards, extended range of transport, highly volatile nature, high emission rate, and internal resistance to degradation, benzene and xylene are the significant concerns today. Multiple approaches, including catalytic oxidation, soil vapor extraction, photocatalysis, and thermal incineration, have been employed (Tabakova 2024). However, as per the previously documented research, these conventional methods are not eco-friendly due to high carbon footprint and are highly expensive (Unegg et al. 2023; Sornaly et al. 2024; Kaur and Sood 2025). In comparison, bioremediation using microbes is found to be more significant due to being economically profitable, eco-friendly, capable of degrading the pollutants in dilute amounts, simple operation, least energy consumption, low carbon footprint, and reduced formation of secondary pollutants (Kuppan et al. 2024). Literature documents various case studies that successfully bio-remediated the water and soil contaminated with oil, polycyclic aromatic hydrocarbons (PHCs) and polychlorinated biphenyls (PCBs) (You et al. 2014; Schad 2016). Hence, bioremediation is capable of diluting the pollutants in environmental resources even without the use of conventional techniques. However, this will require optimizing the bioremediation process, selecting the right microorganism, adaptive management, and sufficient ongoing monitoring. In contrast, physicochemical and mechanical methods cannot completely remove pollutants when employed without bioremediation (Sales da Silva et al. 2020). However, a strategic combination of conventional and biological methods improves the outcome synergistically (Aparicio et al. 2022; Kumar et al. 2022; Nagda et al. 2022; Sornaly et al. 2024; Ha and Tan 2025).

Bacteria, adaptable to various environmental conditions and nutritionally versatile, are the most suitable candidates for bioremediation. The biodegradable potential of bacteria is reflected in their genomes. Whole Genome Sequencing (WGS) could provide a holistic view of the genes of these environmentally competent bacteria concerning bioremediation. In addition, it is also helpful in tracking the changes that occur in microbial communities during the biodegradation process, thus providing insights into its effectiveness and possible ways of optimizing the technique (Chettri et al. 2024).

Ortega-González et al. (2013) documented the genes toluene 1,2-dioxygenase (todC1), and xylene monooxygenase (xylM) in a bacterial sample comprising Cellulomonas hominis, Ralstonia insidiosa, Serratia marcescens, and Burkholderia kururiensis from a contaminated area in Southern Mexico. In another investigation, bacteria from m-, o- and p-xylene degrading enrichments, from BTEX contaminated soil in Hungary, were found to exhibit catechol 2,3-dioxygenase (C23O) encoding genes. Prominent genera of the bacterial sample were Pseudomonas, Acidovorax, and Hydrogenophaga (Banerjee et al. 2022a). Another study also documented the same C23O gene in related bacteria (Táncsics et al. 2020). The same gene was also identified via Illumina sequencing in microaerobic enrichment dominated by Rhodoferax, Pseudomonas, and Acidovorax (Bedics et al. 2022). A bacterial sample from petrochemical contaminated water, predominated by Proteobacteria, was found to exhibit toluene 2-monooxygenase (dmpK), glutaryl-CoA dehydrogenase (gcd), and benzoyl-CoA reductase (bamB) genes. These genes were detected using metagenome-assembled genome (MAG) and contig-based genome (Zhang et al. 2024). Other bacteria explored so far include Pseudomonas aromaticivorans sp. nov., Hydrogenophaga, (Banerjee et al. 2022b). Metagenome analysis based functional annotation of a bacterial sample unraveled the presence of enzymes toluene methyl monooxygenase, benzoate/toluate 1,2-dioxygenase, aryl alcohol dehydrogenase, benzaldehyde dehydrogenase, dihydroxycyclohexadiene carboxylate dehydrogenase, catechol 2,3-dioxygenase, catechol 1,2-dioxygenase, muconolactone D-isomerase, muconate cycloisomerase, 3-oxoadipate enol-lactonase, 2-hydroxymuconate-6-semialdehyde dehydrogenase, 2-hydroxymuconate semialdehyde hydrolase, 4-oxalocrotonate tautomerase, 2-keto-4-pentenoate hydratase, 2-oxo-3-hexenedioate decarboxylase, toluene 4-monooxygenase (T4MO), 4-hydroxy-2-oxovalerate aldolase, 3-methylcatechol 2,3-dioxygenase, acetaldehyde dehydrogenase, 2-hydroxy-6-oxo-octa-2,4-dienoate hydrolase and 2,3-dihydroxyethylbenzene 1,2-dioxygenase in addition to toluene, benzene, ethylbenzene, and xylene mono- and dioxygenases (Eze 2021).

The current study has targeted the genomes of eleven bacteria, which were identified in two separate projects. One project isolated benzene, and another project targeted the xylene-degrading bacteria. Ten of these eleven isolates belonged to the genus Bacillus and were Gram-positive.

Although we can find many benzene and xylene-metabolizing bacteria in previously published data, among these, only the pathways of Gram-negative bacteria are well characterized, while Gram-positive, particularly genus Bacillus, is the least understood in this regard (Wongbunmak et al. 2020). In addition, Bacillus species explored so far have not been optimized for factors affecting bioremediation like temperature, pH, and concentration of contaminants (Mohammadpour et al. 2020). This is uncommon among Gram-positive bacteria reported so far to metabolize benzene and xylene under anaerobic conditions as compared to Gram-negative microbes (Weelink et al. 2010; Banerjee et al. 2022a). Previously reported Gram-positive bacteria associated with degradation of these two pollutants have been documented as more susceptible to environmental constraints like the presence of toxins, including hydrogen sulfide, low temperature, and electron acceptors like sulfate and nitrate (Kaur et al. 2023; 2025).

Bacillus species explored so far exhibited a narrow substrate range and poor degradation efficiencies (Handayani et al. 2019). Lastly, degradation of these pollutants has never been explored in the native area of Multan, Pakistan. These limitations have been a big hurdle in optimizing the degradation potentials of bacteria isolated and characterized.

So, considering these constraints of earlier reported bacteria, we initiated a current research project hypothesizing that we might identify unusual and novel pathways and associated genes in benzene and xylene metabolizing bacteria isolated from the tannery industry of Multan.

WGS strategies are proving a source of insights into the biodegradation capabilities of bacteria (Oyewusi et al. 2021). It decodes both the uncultivable and cultivable microbes. The current study attempts to decode the underlying benzene and xylene-metabolizing genes and pathways of cultivable bacterial species. Ten out of eleven bacteria analyzed belonged to the genus Bacillus.

Taxonomic profiling revealed 49% B. cereus species and 20% B. thuringiensis. This is a validation of our previously performed 16S rRNA sequencing according to which most of the bacterial community was comprised of Bacillus species. This finding is also consistent with earlier literature reporting different species of the genus Bacillus as associated with benzene and xylene metabolism. i.e., B. thuringiensis, B. cereus, Bacillus amyloliquefaciens, Bacillus infantis, Bacillus pumilus (Dou et al. 2010; Undugoda et al. 2018; Wongbunmak et al. 2020; Kesavan et al. 2021; Kaur et al. 2023; 2025; Wu et al. 2023; Jin et al. 2025;). Our findings are not in line with some of the literature because species belonging to Pseudomonas, Sphingobium, Azoarcus, Cupriavidus, Hydrogenophaga, and Rhodococcus, have also been documented as benzene and xylene metabolizing bacteria (Lee et al. 2012; You et al. 2018; Devanadera et al. 2019; Lopez et al. 2022; Sathesh-Prabu et al. 2023). All of these bacteria except Rhodococcus are Gram-negative while those documented in current study are Gram-positive. According to literature, as compared to Gram-positive bacteria, Gram-negative bacteria exhibit greater efficiency of benzene and xylene metabolism (Mazzeo et al. 2010; Chicca et al. 2020; Bacosa et al. 2021). Dominance of Gram-positive isolates in the current project is justified as per the literature finding that Gram-positive bacteria dominate tannery industry soils due to their high adaptability to the heavy metals and organic compounds found in tannery waste (Albokari et al. 2018; Oruko et al. 2019; Abate et al. 2021). This adaptability is contributed by thick cell walls containing peptidoglycan.

WGS analysis revealed the enzymes involved in o-, m- and p-xylene catabolism, 3-aminobenzenesulfonate, toluene and 2-, 3- and 4-fluorobenzoate. Based on the identification of enzymes, the xylene degradation pathways suggested in a bacterial sample of the current study are shown in Fig. 4.

Fig. 4.

Reconstruction of ortho, meta and para-xylene degradation pathways based on whole genome shotgun functional annotation.

The o-xylene degradation pathway starts with its oxidation into 2-methylbenzyl alcohol in the presence of toluene methylmonooxygenase, which is oxidized by arylalcohol dehydrogenase into 2-methylbenzaldehyde. After this, 2-methylbenzaldehyde is oxidized in the presence of benzaldehyde dehydrogenase into o-methylbenzoate, which is transformed into 1,2-dihydroxy-6-methylcyclohexa-3,5-dienecarboxylate by benzoate 1,2-dioxygenase subunit alpha.

Following this, 3-methylcatechol is produced, which is oxidized into cis, cis-2-hydroxyl-6-oxohept-2,4-dienoate in the presence of catechol 2,3-dioxygenase. The cis-2-hydroxyl-6-oxohept-2,4-dienoate is then converted into cis-2-hydroxypenta-2,4-dienoate, which is acted upon by enzyme 2-oxopent-4-enoate hydratase, and 4-hydroxy-2-oxopentanoate is produced. This compound is then transformed into pyruvate and acetaldehyde in 4-hydroxyl-2-oxovalerate aldolase. Pyruvate enters glycolysis, and acetaldehyde is oxidized into acetyl-CoA by acetaldehyde dehydrogenase. Acetyl-CoA then enters the Krebs cycle. Enzymes involved in all these steps were identified in the current study.

The m-xylene degradation pathway comprises the initial oxidation of m-xylene into 3-methylbenzyl alcohol by the enzyme toluene methyl monooxygenase. It is followed by forming 3-methyl benzaldehyde in the presence of aryl-alcohol dehydrogenase, benzoate 1,2-dioxygenase subunit alpha. Then, benzaldehyde dehydrogenase oxidizes 3-methyl benzaldehyde into m-methyl benzoate, which is further oxidized into 1,2-dihydroxy-3-methylcyclohexa-3,5-dienecarboxylate by benzoate 1,2-dioxygenase subunit alpha, which is then transformed into 3-methylcatechol in the presence of dihydroxycyclohexadiene carboxylate dehydrogenase. Further degradation of 3-methylcatechol follows the exact mechanism as documented in o-xylene. All enzymes of this pathway were identified in the current study bacterial sample.

During degradation of p-xylene, it is first oxidized into 4-methylbenzyl alcohol by toluene methyl monooxygenase.

Following this, benzaldehyde dehydrogenase performs oxidation and forms p-methylbenzoate, which is transformed into cis-1,2-dihydroxy-4-methylcyclohexa-3,5-diene-1-carboxylate in the presence of benzoate/toluate 1,2-dioxygenase subunit alpha. Afterwards, dihydroxy cyclohexadiene carboxylate dehydrogenase catalyzes the formation of 4-methylcatechol, which is acted upon by catechol 2,3-dioxygenase and 2-hydroxyl-5-methyl-cis, cis-muconic semialdehyde is formed. In a subsequent step, 2-hydroxyl-5-methyl-cis, cis-muconic semialdehyde is transformed into 2-hydroxyl-5-methyl-cis, cis-muconate in the presence of hydroxymuconate 6-semialdehyde dehydrogenase. Following this, tautomerization by 4-oxalocrotonate tautomerase results in the formation of 2-oxo-5-methyl cis-muconate. This is decarboxylated by 2-oxo-3-hexenedioate decarboxylase into 2-hydroxyl-cis-hexa-2,4-dienoate. Following this, cis-2-oxohexa-4-enoate hydratase catalyzes the formation of 4-hydroxyl-2-oxohexanoate, which is then converted into pyruvate and propanol in the presence of enzyme 4-hydroxyl-2-oxohexanoate aldolase. Pyruvate enters the Krebs cycle, and propanol enters propanoate metabolism through conversion into propanoyl-CoA in the presence of propanol dehydrogenase. All the enzymes of this pathway except hydroxymuconate-6-semialdehyde dehydrogenase, 4-oxalocrotonate tautomerase, cis-2-oxohexa-4-enoate hydratase, and 4-hydroxyl-2-oxohexanoate aldolase were identified in the current study bacterial DNA sample. These pathways of xylene degradation are consistent with earlier reported pathways in BTEX-degrading B. amyloliquefaciens subsp. plantarum W1 (Lee et al. 2019; Wongbunmak et al. 2020).

Pathways identified in the current bacteria involved in benzene, 3-aminobenzene sulfonate, toluene, and 2-, 3-, and 4-fluorobenzoate are shown in Fig. 5. In benzene degradation pathways, benzene is first converted into benzyl alcohol by benzyl alcohol dehydrogenase. Then, benzyl alcohol is converted into benzaldehyde, which is transformed into benzoate by benzaldehyde dehydrogenase. Benzoate is oxidized by benzoate/toluate 1,2-dioxygenase subunit alpha into cis-1,2-dihydroxycyclohexa-3,5-diene-1-carboxylate, which is transformed into catechol in the presence of dihydroxycyclohexadiene carboxylate dehydrogenase. Catechol is oxidized into cis, cis-muconate by catechol 1,2-dioxygenase. The cis, cis-muconate is isomerized into muconolactone by muconate cycloisomerase. This is followed by forming 3-oxoadipate-enol lactone in the presence of muconolactone D-isomerase. Afterwards, 3-oxoadipate enol lactonase catalyzes the formation of 3-oxoadipate, which is then transformed into 3-oxoadipyl-CoA in the presence of enzyme 3-oxoadipate CoA-transferase alpha subunit. The 3-oxoadipyl-CoA is then converted into acetyl-CoA and succinyl-CoA by acetyl-CoA acyltransferase and 3-oxoadipyl-CoA thiolase, respectively. Succinyl-CoA then enters the Krebs cycle. All the enzymes of this pathway, except benzylalcohol dehydrogenase and benzaldehyde dehydrogenase, were identified in the current study. This pathway has been documented in literature in Burkholderia sp. strain NK8 (Francisco et al. 2001).

Fig. 5.

Reconstruction of benzene, 3-aminobenzene sulfonate, toluene and 2-, 3- and 4-fluorobenzoate degradation pathways based on whole genome shotgun functional annotation.

During degradation of 3-aminobenzene sulfonate, it is first converted into 3-sulfocatechol in the presence of 2-aminobenzene sulfonate 2,3-dioxygenase. Following this, oxidation occurs by catechol 2,3-dioxygenase and 2-hydroxymuconate is formed. Then, 2-hydroxymuconate tautomerizes into gamma-oxalocrotonate by 2-hydroxymuconate tautomerase. Afterwards, decarboxylation occurs in the presence of 2-oxo-3-hexenedioate decarboxylase, and 2-oxopent-4-enoate is formed, which undergoes hydration to form 4-hydroxy-2-oxopentanoate. The hydration reaction is catalyzed by 2-keto-4-pentenoate hydratase. The 4-hydroxy-2-oxopentanoate is further cleaved into acetaldehyde and pyruvate in 4-hydroxyl-2-oxovalerate aldolase. All the enzymes involved in this pathway, except 2-aminobenzene sulfonate 2,3-dioxygenase, 2-hydroxymuconate tautomerase, and 2-oxo-3-hexenedioate decarboxylase, were identified in the current bacteria.

During toluene degradation, it is initially oxidized by toluene methyl monooxygenase into benzyl alcohol, which is then oxidized into benzaldehyde in the presence of arylalcohol dehydrogenase. Following this, benzaldehyde is converted into benzoate by benzaldehyde dehydrogenase. All three enzymes involved in these steps were identified in the bacteria in the current study. This pathway is consistent with earlier reported toluene degradation pathways in BTEX-degrading B. amyloliquefaciens subsp. plantarum W1 (Lee et al. 2019; Wongbunmak et al. 2020).

Enzyme benzoate 1,2-dioxygenase subunit alpha that catalyzes the transformation of 2-, 3-, and 4-fluorobenzoate into 6-, 3-, 5-, and 4-fluorocyclohexadiene-cis, cis-1,2-diol-1-carboxylate, respectively, has been identified in the current study bacteria. Among these compounds, 6- and 3-fluorocyclohexadiene-cis, cis-1,2-diol-1-carboxylate are converted into 3-fluorocatechol by enoate reductase and dihydroxycyclohexadiene carboxylate dehydrogenase, respectively. On the other hand, 5- and 4-fluorocyclohexadiene-cis, cis-1,2-diol-1-carboxylate are transformed into 4-fluorocatechol in the presence of dihydroxycyclohexadiene carboxylate dehydrogenase. Then, 3- and 4-fluorocatechols are converted into 2- and 3-fluoro-cis, cis-muconate by enzyme catechol 1,2-dioxygenase. All the enzymes of these pathways except enoate reductase were identified in the current study. These pathways are consistent with the literature and have been documented in Sphingomonas sp. HB-1 and in a microbial sample comprising Marinobacter, Vibrio natriegens, Gramella sp., and Methyloceanibacter (Hidde Boersma et al. 2004; Kannan et al. 2023).

In addition to enzymes associated with the above pathways, some additional enzymes were also identified in bacteria, including amidase, 3-hydroxybenzoate monooxygenase, 4-hydroxybenzoate decarboxylase subunit c, 3-carboxy-cis, cis-muconate cycloisomerase, procatechuate-3,4-dioxygenase alpha subunit, 2-hydroxymuconate-6-semialdehyde dehydrogenase, 2-keto-4-pentenoate hydratase, and 4-carboxymuconolactone decarboxylase. Reactions catalyzed by these bacteria are shown in Fig. 6.

Fig. 6.

Reactions of benzene and xylene metabolism catalyzed by bacterial enzymes identified in the current study using whole genome shotgun functional annotation.

Enzymes identified in the current study are in accordance with previous literature, as a study focused on WGS of twenty-six different strains of bacteria reported the enzymes associated with benzene, xylene, benzoate, naphthalene, and toluene (Révész et al. 2020; Eze 2021; Bedics et al. 2022; Hossain et al. 2024). Another study reported xylene degrading enzyme catechol 2,3-dioxygenase in a Pseudomonas strain (Miri et al. 2022).

The current project documenting the Gram-positive hydrocarbonoclastic Bacillus bacteria is a novel addition to the current state of knowledge because the benzene degradation pathway identified in current study have been reported in Gram-negative bacteria in literature but not in any Gram-positive Bacilli except B. cereus which was isolated from petroleum contaminated soil (Hussain et al. 2025). Fluorobenzoate degradation pathways identified in the current bacteria have never been documented in any Gram-positive bacteria. However, they are reported in Gram-negative isolates. Toluene and xylene degrading bacteria documented in the current study have been identified earlier either in Gram-negative bacteria or in B. amyloliquefaciens subsp. plantarum W1 (Wongbunmak et al. 2020). No other Bacillus species have been reported to exhibit this xylene degradation pathway. However, the toluene degradation pathway is also reported in another Bacillus. i.e., B. pumilus MVSV3 (Surendra et al. 2017).

In addition, these isolates exhibit the coexistence of metabolic pathways for diverse substrates. i.e., benzene, toluene, o-, m- and p-xylene, 3-aminobenzene-sulfonate and 2-, 3- and 4-fluorobenzoates.

Despite these facts, current data have significance because they document Gram-positive bacteria, which can be more potent bioremediating agents than previously reported Gram-negative bacteria. Due to the unique characteristics of Gram-positive bacterial cell walls, including the absence of an outer membrane, they have a greater tendency to uptake organic pollutants and bind with and accumulate heavy metals from external sources (Liaqat and Sabri 2008). These features render them more potent bioremediation candidates than Gram-negative bacteria (Ganesh and Lin 2009; Nanda et al. 2019). In Gram-negative bacteria, efflux pumps are present, which confer them tolerance against various pollutants like organic solvents, heavy metals, dyes, and antibiotics. Hence, they contribute to the survivability of bacteria in the highly polluted environment. According to the literature, the efflux pumps contribute to the bioremediation potentials of bacteria by allowing them to persist in contaminated environments (Blair et al. 2014).

However, bioremediation is considered an eco-friendly mode of pollutant degradation. A bacterium possessing pumps that confer antibiotics and other multi-drug resistance can be a source of spread of multi-drug resistance (MDR) (Cunningham et al. 2020). According to this fact, we cannot use such bacteria as whole cells for eco-friendly bioremediation. Hence, Gram-positive bacteria can be more potent and sustainable bioremediating agents because they can accumulate large concentrations of pollutants in their cells and have the least chance of developing MDR due to the absence of efflux pumps (Biswas et al. 2021).