Different metabolic pathways involved in anthracene biodegradation by Brevibacillus, Pseudomonas and Methylocystis Species
Beni-Suef University Journal of Basic and Applied Sciences volume 11, Article number: 4 (2022)
Polycyclic aromatic hydrocarbons (PAHs) such as anthracene are one of the most toxic contaminants to our environment. Microbial biodegradation of these xenobiotics is a cost-effective technological solution. The present study aimed to recover some bacterial isolates from Beni-Suef Governorate in Egypt with high capabilities of anthracene biodegradation. The selected isolates were molecularly characterized by 16S rRNA gene sequencing, the degree of anthracene biodegradation was monitored using optical density (OD) and high-performance liquid chromatography (HPLC), PCR amplification of some selected genes encoding biodegradation of PAHs was monitored, and gas chromatography–mass spectrometry (GC–MS) analysis was applied for detecting the resulted metabolites.
Three bacterial isolates were studied, the 16s rRNA sequences of the isolates showed homology of the first isolate to Brevibacillus sp. (94.58 %), the second isolates showed homology to Pseudomonas sp. (94.53%) and the third isolate showed homology to Methylocystis sp. (99.61 %), all isolates showed the ability to degrade anthracene. PCR amplification of some selected genes encoding biodegradation of PAHs revealed the presence of many biodegrading genes in the selected strains. Gas chromatography-mass spectrometry (GC–MS) analysis of the metabolites resulted from anthracene biodegradation in the present study suggested that more than one biodegradation pathway was followed by the selected isolates.
The selected strains could represent a potential bioremediation tool in solving the PAHs problem in the Egyptian environment with a clean and cost-effective technique.
Polycyclic aromatic hydrocarbons (PAHs) nowadays gain a significant interest due to their environmental toxicity, biological activity, mutagenicity, and carcinogenicity . They are structurally composed of fused two or more benzene rings, created naturally due to forest fires, burning coal tar, oil leakages, and solid waste incineration . They are solids of different colors, with low water solubility . They spread widely in the environment, especially air , water, and soil [5, 6]. They condense onto the particulate matter in the air that facilitates movement to distant places . They have relative stability in soil and are not easily degraded . They constitute a significant health hazard as medically classified as endocrine-disrupting chemicals due to their structural similarities to the real human and animal hormones that, in turn, affect their reproduction, development, metabolism, and may induce cancer [9, 10]. It was reported that high levels of PAHs in the atmosphere could cause 4–8% premature death .
Anthracene is used as a signature compound in PAHs biodegradation studies [12,13,14]. It is composed of three fused benzene rings . Anthracene has been detected in vehicle exhaust fumes . Anthracene as a type of PAHs is widely distributed in the environments due to many anthropogenic activities, like oil refining and the petro-chemical industry . Exposure to high doses of anthracene has toxic effects on skin and tissues .
Anthracene has toxic effects on the growth of some eukaryotic microorganisms . For example, it was investigated that anthracene toxicity induces DNA damage, intracellular oxidative stress effect and decrease in the mitochondrial membrane potential as well as cell viability to some living creatures as earthworm coelomocyte . Also, accumulation of benz(a)anthracene (BaA) in soil sediments causes poisoning to typical enzymes (α-Amylase) of organisms such as Eisenia fetida in soil. . Additionally, anthracene causes dangerous oxidative effect through the increase in the super oxide radicals in the aquatic organism mussel, Mytilus edulis . Moreover, exposure to anthracene reduces the reproductivity of the zooplankton, Daphnia magna .
The exposure to high doses of PAHs has adverse effects on the skin and tissues . So, the elimination of such toxic compounds becomes a necessity. Several bacterial and fungal species were reported in PAHs biodegradation [24,25,26]. Bioremediation is a clean and cheap technique that eliminates PAHs pollutants from the environment . The microorganisms degrade such pollutant compounds and convert them through mono and dioxygenase enzymes to inert substances, CO2, and water [28,29,30]. GC–MS analysis applied in many previous studies [31,32,33] is a technique used to detect the metabolites and the pathways involved in biodegradation.
The present study focused on exploring potential strains adapted in biodegrading PAHs effectively in Egypt's local areas to provide a stress-free environment. The sample location in Egypt was selected based on a recent report which proclaimed that Beni-Suef governorate area is the highest in air pollution with 10 µm in diameter particulate matter levels reached 20 folds more than the WHO limits, and 6 folds more than permissible Egyptian environmental limits, which help in the transfer of such pollutants to the water and soil .
2.1 Materials and culture media
Liquid minimum salt solution (MSM) was prepared using analytical grade chemicals as follows: 1 g K2HPO4, 1 g NH4SO4, 0.3 g MgCl2·6H2O, 0.1 g CaCl2, and 0.02 g FeSO4 0.7H2O . Anthracene (analytical grade) (Sigma-Aldrich Ltd.) was dissolved in methanol, then syringe-filtered, and added to MSM to a final concentration of 50 µg/mL. Agar at 1.5% was added if solid media was required. Acetone, acetonitrile, chloroform, and water which were used in HPLC were of HPLC grade. Ethidium bromide (Fluka), yeast extract (Difco), and agar (Fisher Scientific) were also used in the study.
Soil and sewage water samples were collected from a drainage in Beni-suef Governorate (a drainage ten kilometers away from Beni-suef), Egypt. Nearly 10 gm of soil and 50–100 ml of each sewage water sample were collected in 250-ml sterile conical flasks that were stored at 4 °C until use. One gram of contaminated soil and 10 mL of sewage water samples were suspended in 50 mL MSM containing anthracene (50 µg/mL) as sole carbon source. Then, the flasks were incubated in a shaker incubator at 160 rpm for at least 20 days at 30 °C.
2.3 Bacterial isolation
A volume of 200 µL of the enrichment suspension was plated on solidified MSM-anthracene plates; control plates without anthracene were used to exclude agar-supported bacteria. The plates were wrapped in aluminum foil to prevent the photocatalytic degradation of anthracene and then incubated at 30 °C for at least 20 days. Anthracene degradation was noticed as clear zone around the bacterial colonies.
2.4 Genotypic identification
The identification of the isolates to the genus level was made using the 16S rRNA gene identification method as follows: the bacterial DNA material was extracted using a ZR Fungal/Bacterial DNA MiniPrep extraction kit (Zymo Research, Orange, CA, USA). The 16S rRNA gene was amplified using the forward primer 16F: 5 AAACTYAAAKGAATTGACGG 3 and reverse primer 4R: 5 ACGGGCGGTGTGTRC 3. The reaction mixture (25 μL) contained 12.5 μL MyTaq green Master Mix (Bioline Reagents Ltd, London, UK), 1 μL of forward primer (8 μM), 1 μL of reverse primer (8 μM), 2 μL of template DNA and 8.5 μl of sterile water. The PCR reaction was carried out using Primus 25 advanced® thermocycler (PEQLAB Biotechnologie GmbH, Erlangen, Germany) under the following conditions 5 min initial denaturation at 95 °C, followed by 36 cycles of 1 min denaturation at 94 °C, 1 min annealing at 55 °C, 1.5 min extension at 72 °C, and a final extension step of 7 min at 72 °C . Gel electrophoresis technique (Labnet's ENDURO™, Edison, NJ, USA) used for amplicon separation by using 1% (w/v) agarose gel (Invitrogen, USA) with an electric current of 90 V for 60 min, stained with ethidium bromide and visualized using UV trans-illuminator (Vilber Lourmat Deutschland GmbH, Eberhardzell, Germany). PCR products were purified from agarose gel using Zymo gel extraction kit (Zymo Research, Orange, CA, USA), and the concentration of the purified amplicons was measured using Nanodrop 2000 (Thermo Fisher Scientific Waltham, MA, USA). The PCR amplicons were sent to Macrogen (Macrogen Inc., Seoul, Republic of Korea) for sequencing. Identification was made using Basic Local Alignment Search Tool (BLAST) of the obtained sequence by the NCBI database  in order to identify the sequences that are homologous to the sequence of interest. The obtained sequences were analyzed using MEGA-X (Molecular Evolutionary Genetics Analysis)  and ClustalW  using 15 sequences obtained from the NCBI database. The phylogenetic tree was built by neighbor-joining  with retrieved sequences from the NCBI database bootstrap consensus way with 100 repetitions .
2.5 Detection of PAHs biodegradation genes
2.6 Enhancement of biomass production and anthracene biodegradation
The biomass production and anthracene biodegradation of the selected isolates were investigated under different conditions. The optical density and anthracene residual concentration were monitored using a spectrophotometer (600 nm) and HPLC, respectively. A single pure colony was cultured in nutrient broth at 30 °C for 24 h. A volume of 100 µL of bacterial suspension was added to different flasks to give the following conditions: (a) MSM-anthracene, static incubation at 30 °C, (b) MSM-anthracene with shaking at 160 rpm at 30 °C; , (c) MSM-anthracene with shaking at 160 rpm at 25 °C, (d) MSM-anthracene, yeast extract 0.1% with shaking at 160 rpm at 30 °C , and (e) MSM-anthracene, glucose 0.1% with shaking at 160 rpm at 30 °C . All flasks contain 100 mL of MSM in a 250-mL flask and incubated for 12 days, while the starting pH was adjusted at 7 (± 0.2) throughout the study. Samples were withdrawn at 24-h intervals to assay the change in the bacterial growth through the change in the optical density (OD). OD was detected using the Shimadzu UV-1280 spectrophotometer at λ600 nm. The residual anthracene was analyzed by HPLC, where the 5 mL was withdrawn at 48-h intervals, extracted with chloroform, and dried using anhydrous sodium sulfate . Chloroform layer was evaporated using Rotavap, and the final residue was dissolved in 5 mL acetonitrile. The residual anthracene was detected by HPLC system equipped with Water 996 Photo Diode Array Detector (Water 2690 Alliance, USA). The HPLC gradient system was composed of acetonitrile/water as follows: 50:50 for 2 min, then, 60:40 for 3 min, followed by 70:30 for 2 min, and finally, 80:20 for 5 min. The mobile phase flow rate was 0.4 mL/min, and the UV detection wavelength was 254 nm. The injection volume was 100 µL.
2.7 GC–MS analysis
The metabolites formed during anthracene biodegradation by the selected isolates were qualitatively analyzed by GC–MS spectroscopic analysis. The bacterial isolates in MSM-anthracene were incubated in a shaking incubator at 160 rpm for 4 days at 30 °C. The samples were withdrawn at 48 h intervals and exposed to liquid–liquid extraction using chloroform (1:1 v/v) followed by addition of anhydrous sodium sulfate for drying . The GC–MS analysis was done using Trace GC Ultra-TSQ mass spectrometer (Thermo Scientific, Austin, TX, USA) with a direct capillary column TG-5MS (30 m × 0.25 mm × 0.25 µm film thickness). The column oven temperature was initially held at 100 °C and then increased by 15 °C /min to 160 °C and then increased to 300 with 5 C/min. The injector and MS transfer line temperatures were kept at 280 °C. Helium was used as a carrier gas at a constant flow rate of 1 mL/min. The solvent delay was 3 min, and diluted samples of 3 µL were injected automatically using Autosampler AS3000 coupled with GC in the split mode. EI mass spectra were collected at 70 eV ionization voltages over the range of m/z 40–500 in full scan mode. The ion source was set at 200 °C. The metabolites were putatively identified by comparing their retention times (Rt) and mass spectra with Wiley registry® and Pesticide’s mass spectral database.
3.1 Anthracene biodegradation on solid media
Observation of bacterial growth and zone of clearance indicated possible anthracene-degrading activity. Twenty isolates (9 soil and 11 sewage) were recovered initially; however, three isolates were selected for further experiments based on the maximum anthracene degradation activity. One isolate was recovered from sewage water named PM1, and the two other isolates were recovered from soil named BM1 and MM1.
3.2 Isolates identification using 16S rRNA sequencing
The sequences of the PCR products of the amplification of 16S rRNA genes of the selected isolates were compared to the Genbank database through the NCBI's nucleotide blast tool. The isolates' 16S rRNA sequences and the closely related sequences from NCBI were aligned, and the phylogenetic trees were built. The isolates genus and the sequences of the most closely similar strains were identified. BM1 isolate was identified as Brevibacillus sp. Its 16S rRNA sequence showed high similarity (99.5%) to Brevibacillus panacihumi strain DCY35 (Fig. 1a). Also, PM1 isolate was molecularly assigned as Pseudomonas sp. showing 89.2% similarity to Pseudomonas canadensis strain 2–92 (Fig. 1b). Additionally, MM1 isolate was identified as Methylocystis sp. It displayed 87.7% similarity to Methylocystis heyeri strain H2 (Fig. 1c).
3.3 Enhancement of biomass production and anthracene biodegradation
The spectrophotometric analysis of the bacterial growth of all selected strains using different culture conditions revealed an increase in the OD by time (Fig. 2). Interestingly, the shaking conditions at either 25 °C or 30 °C incubation were superior to stagnant incubation conditions. The lag phase time for the three isolates was reduced by supplementing the media with 0.1% yeast extract or 0.1% glucose. The highest growth level of Methylocystis isolate was at 30 °C shaking condition near to that of 0.1% yeast extract addition.
Figure 2 also shows the biodegradation of the anthracene by the selected strains by time. Shaking at 30 °C and 25 °C significantly decreased the lag phase in the case of Brevibacillus sp. and Methylocystis sp., respectively. Methylocystis sp. showed a high biodegradation rate, as noticed by the anthracene concentration drop to 7.67% at day 2 after shaking incubation at 25 °C. Notably, on day 4 and day 6 for Methylocystis sp. using MSM-anthracene medium supplemented with 0.1% glucose at 30 °C, the residual anthracene concentration reached 0.28% and 0.09%, respectively.
In addition, the residual anthracene percentage using Brevibacillus sp. upon shaking incubation at 30 °C was 29.95%, 15.9%, and 5.43% on days 2, 4, and 6, respectively. Moreover, for Brevibacillus sp. in MSM-anthracene medium at 30 °C with shaking, anthracene utilization was more than that at 0.1% yeast or 0.1% glucose supplementation.
Although the biodegradation efficacy of Pseudomonas sp. in static condition started with 53.56% on day 2, the degradation rate slowly decreased over time. On the other hand, the residual anthracene decreased to 5.48% by Pseudomonas sp. after shaking incubation at 30 °C for 6 days. Similarly, in 0.1% yeast augmented media, Pseudomonas sp. decreased the residual anthracene at day 4 and day 6 to 5.64% and 4.53%, respectively.
Figure 3 shows the follow-up of the anthracene degradation at three different time points, day 0, day 2, and day 4 for the three selected isolates tested using shaking incubation at 160 rpm at 25 °C. As shown in Fig. 3c, Methylocystis sp. almost degraded all anthracene (Rt 12.4 min) used in the experiment at day 4 sample point. Noteworthy, these results were in line with the OD and HPLC results mentioned earlier.
3.4 PCR amplification of PAHs biodegradation genes
Successfully, specific genes were detected in the three selected isolates. Notably, Methylocystis sp. revealed the presence of tnpA-like gene, tadR-like gene, tadQ-like gene, ps pass 2.3-like gene, GS2-like gene, and acin GS-like gene, while Brevibacillus sp. showed presence of tnpA-like gene, tadQ-like gene, acin GS-like gene, tadR-like gene, and tadQ2-like gene. On the other hand, only acin GS-like gene was screened in Pseudomonas sp. using PCR amplification.
3.5 GC–MS analysis of anthracene biodegradation
The GC–MS analysis of the metabolites detected in the crude extracts of the selected isolates incubated in MSM-anthracene for four days confirmed the anthracene biodegradation by the selected strains. Notably, Brevibacillus sp., Pseudomonas sp. and Methylocystis sp. degraded more than 95% of the starting concentration of anthracene. The phthalic acid (2) (MF; C8H6O4) and its esters were detected as the major intermediates in the anthracene biodegradation by all selected strains suggesting that they may followed similar biodegradation pathway to degrade the anthracene as sole carbon source. Interestingly, benzoic acid (TMS derivative) (3) (MF; C7H6O2) putatively identified an intermediate produced by Brevibacillus sp., proposing that this isolate followed this biodegradation pathway (Fig. 4). On the other hand, benzoic acid was not detected by GC–MS analysis of both Pseudomonas sp. and Methylocystis sp., suggesting that they either followed other biodegradation pathways or benzoic acid was further biodegraded into simpler compounds.
Moreover, six other metabolites were detected by the GC–MS analysis and were putatively identified as compounds (4–9) which are suggested to be intermediates in other anthracene biodegradation pathways (Fig. 5). Indeed, only compounds 4 and 8 were detected in all selected isolates, while compound 9 was identified in both Brevibacillus sp. and Pseudomonas sp. On contrast, both compounds 5 and 6 were exclusively identified by GC–MS analysis in Brevibacillus sp., whereas compound 7 was solely detected in Pseudomonas sp.
PAHs were listed as priority pollutants by the US Environmental Protection Agency and the European Environment Agency [49, 50]. Human exposure to such pollutants may be done through inhalation, ingestion, and skin absorption . The samples were obtained from the province of Beni-Suef, as its air quality is considered one of the highly polluted areas in Egypt .
PAHs removal can be adapted through different methods. However, using the biodegradation approach in PAHs removal is considered a cost-effective and environmentally clean technique [27, 51]. PAHs biodegradation capability of different microbial species was detected in several previous studies using different microorganisms including Pseudomonas sp. [12, 26], Brevibacillus sp. [24, 52, 53] and Methylocystis sp. (on hydrocarbons) [54, 55]. Additionally, Bjerkandera adusta degraded 38% of anthracene during three days of incubation  and 70.5% of anthracene (50 µg/mL) was degraded by Sphingomonas sp. KSU05 at 96 h of cultivation . Surprisingly, our isolates showed a high potential to biodegrade anthracene under aerobic conditions within six days as 94.58%, 94.53%, and 99.61% of the starting anthracene concentration were degraded by Brevibacillus sp., Pseudomonas sp., and Methylocystis sp., respectively.
In comparison with the literature, many pieces of literature mentioned the capabilities of Pseudomonas sp.  and Brevibacillus sp.  to degrade the anthracene. These studies match our results, although in case of Methylocystis sp., there are limited pieces of literature talking about its biodegradation actions on anthracene. As shown in our study, Methylocystis sp. reached 99% of anthracene mineralization in 96 h showing the highest biodegradation level among the selected strains. One explanation of this finding is that the rich availability of gene clusters of degrading anthracene as mentioned earlier may stand behind the great capability of this strain to degrade the anthracene.
Performing HPLC analysis for the residual anthracene at 254 nm revealed the decrease in anthracene concentration by time with the presence of any of the three isolates. Shaking conditions increased the biodegradation activity of the three isolates, in agreement with previous studies that confirmed the benefit of the aeration factor on PAH biodegradation by Pseudomonas sp. . Anthracene degradation of any of the three isolates incubation at 30 °C was more efficient than that at 25 °C at day 4 and may reach the double as in Brevibacillus sp. that case is in line with a study revealing that certain conditions including a temperature of 30.04 °C were the optimal conditions for maximum removal efficiency of phenol (99.10%) by Candida tropicalis Z-04 . Methylocystis sp. at day 2 at 25 °C shaking incubation degraded 92.33% of the starting anthracene concentration, while Aspergillus fumigates degraded only 60% of anthracene after 5 days . Anthracene utilization in Brevibacillus sp. with 0.1% yeast or glucose was lower than that of only anthracene MSM media at 30 °C. Annadurai and his colleagues referred to that in a co-metabolic process, addition of a readily degradable secondary source of carbon (glucose) causes the normally stable compound partially degraded but not used as an energy source .
GC–MS analysis has been widely used in many previous studies to identify the byproducts and metabolic intermediates produced during different anthracene biodegradation pathways by various bacterial species [17, 33, 59, 60]. Therefore, it was selected in the current study to detect the different metabolites produced during the anthracene biodegradation and to suggest the proposed anthracene biodegradation pathways followed by the selected strains. In our study, the GC–MS analysis suggested that Brevibacillus sp. biodegraded anthracene via anthraquinone, phthalic acid and benzoic acid derivatives. Of note, many previous research showed that anthracene was biodegraded via similar pathway to ours. For example, Bacillus thuringiensis AT.ISM.1 degraded anthracene through anthraquinone, phthalic acid derivatives, benzoic acid derivatives, and pyrocatechol and benzaldehyde . Also, another study showed that Polyporus sp. S133 followed similar pathway with production of anthraquinone, phthalic acid, benzoic acid, and catechol (TMS derivatives) as metabolic intermediates . Additionally, Bacillus licheniformis MTCC 5514 and Bacillus cereus S13 followed the same pathway of anthracene biodegradation via anthraquinone, phthalic acid, benzoic acid, and catechol [33, 62].
On the other hand, only phthalic acid or its esters were detected as the major intermediates in anthracene degradation by Methylocystis sp. and Pseudomonas sp., while neither benzoic acid nor its derivatives were identified in both isolates. Absence of these intermediates can be explained by the suggestion that the phthalic acid has been involved in another pathway  or it has undergone conversion into other compounds which were used in the central metabolism by these isolates [33, 62].
Moreover, some other metabolic intermediates (4–9) were identified by GC–MS, suggesting the presence of other biodegradation pathways of anthracene biodegradation by the selected isolates. It is well known that microorganisms can follow more than one degrading pathway for the xenobiotics to get best use of them through various pathways. This group of metabolites (4–9) were presented as suggested intermediates in other anthracene biodegradation pathways due to their structural similarity to other byproducts detected in anthracene biodegradation pathways other than that shown in Fig. 4 or because they were derivatives from anthracene itself which may indicate enzymatic modification of anthracene prior to its biodegradation. Also, one compound or more from this group of metabolites may be involved in other new/undiscovered pathways of anthracene biodegradation.
For example, compound 7 (4-[2-(4-methylphenyl) ethenyl]benzoic acid) was putatively identified as a by-product of anthracene degradation by Pseudomonas sp. This compound is closely related in structure to another metabolite (1-methoxy-4-[2-(4-methylphenyl)ethenyl]benzene) detected by GC–MS analysis as intermediate during another anthracene biodegradation pathway by Bacillus thuringiensis in a previous study . This study showed that Bacillus thuringiensis followed a pathway of anthracene degradation via anthracen-9(10H)-one, 1-methoxy-4-[2-(4-methylphenyl)ethenyl]benzene, benzoic acid, benzaldehyde and pyrocatechol. Additionally, the same study as well as many other previous studies identified different phenolic compounds such as phenol, catechol or pyrocatechol and their derivatives as main end products in different pathways of microbial degradation of anthracene [55,56,57]. Noteworthy, a derivative of para-substituted benzenediol compound (8) was putatively identified as 1,4-benzenediol, 2-(1,1-dimethylethyl)-5-(2-propenyl) which is a phenolic compound detected in all selected isolates suggesting that it was one of the intermediates in anthracene biodegradation pathways by these strains. Interestingly, another old study revealed that the benzenediol itself was one of the intermediates in photocatalytic biodegradation of phthalic acid  which is main by-product of anthracene biodegradation in the present study.
In conclusion, this study addresses a potential solution for an area in Egypt that is considered the number-one air-polluted area that needs to be dealt with using cost-effective methods such as biodegradation. One of the recovered microbial species (Methylocystis sp.) showed a potential of anthracene degradation with the efficacy of 99% in 96-h incubation. Although the in vitro PAHs biodegradation abilities of the bacteria are satisfactory, several factors, either abiotic and/or biotic, should be optimized to assess these isolates' full capacities. It was also concluded from the current study that the GC–MS is a precious tool in detecting the metabolic intermediates in the biodegradation of PAHs and in assigning the proposed pathway of their biodegradation.
Availability of data and materials
Will be available upon request.
High-performance liquid chromatography
Gas chromatography–mass spectrometry
Ribosomal ribonucleic acid
National Centre for Biotechnology Information
Basic Local Alignment Search Tool
- KH2PO4 :
Potassium phosphate monobasic
- (NH4)2SO4 :
Magnesium sulfate heptahydrate
Iron(II) sulfate heptahydrate
- KNO3 :
- NaNO3 :
- CaNO3 :
- NH4NO3 :
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I would like to thank Dr. Fatma Molham for her support in PAH biodegrading genes.
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Magdy, M.M., Gaber, Y., Sebak, M. et al. Different metabolic pathways involved in anthracene biodegradation by Brevibacillus, Pseudomonas and Methylocystis Species. Beni-Suef Univ J Basic Appl Sci 11, 4 (2022). https://doi.org/10.1186/s43088-021-00178-9