3.1 Characterization of biochar
The SEM scanning images of MWBC are shown in Fig. 1. Remarkably, the carbonization process retained the intricate pore structure of the original biomass, resulting in a highly porous biochar with increased specific surface area. Consequently, this enhanced its adsorption capacity significantly. Such biochar could serve as an excellent immobilized carrier for preparing microorganisms capable of both adsorption and degradation, thus facilitating the remediation of contaminants. By BET detection, the specific surface area of MWBC was 172.407 m2/g, and the average pore diameter was 87.373cm3/g.
3.2 Determination of optimal fixed time and pH value
The relationship between the number of fixed microorganisms in MWBC and fixed time is shown in Fig. 2a. The best fixed time was 20 hours. Under this adsorption time, the relationship between the number of microorganisms fixed by MBC and the pH value of MSM is shown in Fig. 2b. The optimum pH value was 8, close to the original pH value of MSM. The follow-up experiments were carried out with the MWBC-immobilized microbial community prepared under the condition of a fixed time of 20 h and a pH of 8.
3.3 BaP removal efficiency of microbial community
Figure 3 presents the removal efficiency of different concentrations of BaP by microbial community at different temperatures. For the BaP of 5 mg/L (Fig. 3a), the BaP removal efficiency of T1, T4, T7, T10 and T13 reached 41.56%, 48.20%, 57.88%, 56.50% and 53.69%, respectively on the 12th day. At 25–30 ℃, the removal efficiency of 5 mg/L BaP reached 38.78% and 34.44% from the 2nd day, and was higher than that at other temperatures throughout the experiment. Besides, the final removal efficiency reached more than 56%. Secondly, the removal efficiency was only about 20% on the 2nd-4th day at 35 ℃ and 20 ℃, and the removal efficiency of the former increased rapidly to 41.83% on the 6th day, while that of the latter increased significantly on the 10th-12th day. However, at 15 ℃, the removal efficiency of only about 40% was the lowest. For the BaP of 10 mg/L (Fig. 3b), the removal efficiency of T2, T5, T8, T11 and T14 reached 39.79%, 46.52%, 44.83%, 45.59% and 55.34%. At 35 ℃, although the BaP removal efficiency of microbial community was the lowest in 4 days, the growth rate of removal efficiency increased significantly after the 4th day, and surpassed that of other experimental groups after the 10th day. Finally, it reached the highest BaP removal efficiency. At 15–30 ℃, although the BaP removal efficiency of bacteria could be improved either quickly or slowly, the final BaP removal efficiency was approximately within the range of 39–46%. For the BaP of 20 mg/L (Fig. 3c), the removal efficiency of T3, T6, T9, T12 and T15 reached 35.18%, 34.36%, 39.07%, 35.59% and 46.88%, respectively. On the 6th day, the BaP removal efficiency of bacterial community in each experimental group was rather similar, reaching 26.29%. After that, the removal efficiency at 35 ℃ was much higher than that at other temperatures, reaching 46.70% on the 10th day, and the improvement of the removal efficiency was basically stagnant in the following 2 days. At 15–30 ℃, the BaP removal efficiency of bacterial community was similar, and finally reached the range of 35–40%.
According to Fig. 3, the microbial community presented obvious differences in the removal efficiency of different concentrations of BaP. From the final BaP removal efficiency, for 5mg/L, the highest value was obtained at 25–30 ℃, and for 10 and 20 mg/L, the highest at 35 ℃. On the other hand, comparison could be made from the curve trend. Except for B7 and B10, the BaP removal efficiency of other experimental groups was relatively low in 4 days, which might be attributed to the nature of BaP. BaP features a large octanol-water partition coefficient related to highly oxidized carbon, and is thus not preferred by microorganisms as an energy source, and the initial degradation effect was not ideal. At 35 ℃, the removal efficiency of BaP increased significantly after the 4th-6th day, and the final removal efficiency of 5 or 20 mg/L BaP was higher. At 25–30 ℃, the removal efficiency generally maintained a steady increase in 8 days, and tended to stagnate after the 8th day. The BaP removal efficiency of microbial community increased slowly at 15–20 ℃, usually in a very slow stage during the 4th-8th day, and the curve of this stage tended to be flat. It generally started to ascend again after the 8th-10th day, which might be related to the lower growth and metabolic ability of microbial cells than that of other temperatures in the experimental group caused by the lower temperature conditions. At the same time, the experimental results showed that the flora had high ability to degrade 5–20 mg/L BaP.
3.4 BaP removal efficiency of MWBC-immobilized microbial community
The removal efficiency of different concentrations of BaP by MWBC-immobilized microbial community at different temperatures is shown in Fig. 4. For 5 mg/L BaP (Fig. 4a), the removal efficiency of W1, W4, W7, W10 and W13 reached 50.58%, 65.24%, 69.04%, 75.18% and 68.20% on the 12th day. Within 4 days, the BaP removal efficiency of W7, W10 and W13 was similar. At 25 ℃, the removal efficiency of 5 mg/L BaP was significantly higher than that of other experimental groups after the 4th day. Secondly, at 35 ℃ and 20 ℃, the removal efficiency increased following a similar trend after 4th day. At 20 ℃, the removal efficiency of BaP was highly improved after the 10th day, and the final removal efficiency reached more than 65%. However, the removal efficiency was the lowest at 15 ℃, which was only about 50%. For the BaP of 10mg/L (Fig. 4b), the BaP removal rates of W2, W5, W8, W11 and W14 reached 47.40%, 56.56%, 65.55%, 66.63% and 72.85%, respectively on the 12th day. While the BaP removal efficiency was the lowest in 2 days at 35 ℃, the growth rate of the removal efficiency increased significantly after the 2nd day, remaining same as the promotion of the other two groups on the 6th-10th day, and exceeding that at the other temperatures after the 10th day. Finally, it achieved the highest BaP removal efficiency. At 20–30 ℃, the BaP removal efficiency of W11 was significantly higher than that of the other two groups. The removal efficiency of W5 and W8 maintained the same improvement in 10 days, but the final removal efficiency of W8 and W11 was similar after the 10th day, while that of W5 was lower than that of the former two. At 15 ℃, the removal efficiency of BaP was significantly lower than that of other experimental groups after the 2nd day. For the BaP of 20mg/L (Fig. 4c), the BaP removal rates of W3, W6, W9, W12 and W15 reached 43.71%, 42.29%, 48.36%, 55.72% and 65.27%, respectively on the 12th day. At 35 ℃, although the BaP removal efficiency was low in 2 days, it maintained a high trend in the whole experiment, and was significantly higher than that of other experimental groups after the 8th day. At 30 ℃, the BaP removal efficiency was higher than that of other experimental groups on the 2nd-6th day, and was similar to that of W15 on the 6th-8th day. However, the increase tended to be flat after the 8th day. At 15–25 ℃, although the BaP removal efficiency of W3 was the highest in the 2 days (probably caused by the difference of biochar adsorption), the improvement of W3 removal efficiency became rather slow after the 2nd day. After the 6th day, the removal efficiency of W3 was similar to that of W6, while that of W9 was slightly higher than that of the former two groups.
The experimental results showed that under the same temperature and BaP concentration, the BaP removal efficiency of MWBC-immobilized microbial community was significantly higher than that of bacteria in free state. At 25–35 ℃, MWBC immobilized bacteria performed well in removing 5–20 mg/L BaP. Especially, the removal effect of 5 mg/L BaP was the best at 30 ℃, and that of 10–20 mg/L BaP was the best at 35 ℃.
3.5 Analysis of microbial community structure
The microbial community diversity in the samples was analyzed using Alpha diversity, and Shannon, Simpson, chao1, ACE and coverage were used to measure the diversity and richness of microbial community (supplementary table S3). The coverage index ≥ 0.999, indicating the efficiency of the sequencing results in representing the real situation of microorganisms in the sample. According to the chao1 index, the species abundance in the same concentration was similar in the group with only free flora, and the higher the concentration of BaP, the greater the species abundance. According to some reports, higher PAHs concentrations should have led to lower species diversity(Quero et al. 2015). However, this might be due to the fact that BaP itself was difficult to degrade and its bioavailability was much lower than that of LMW-PAHs, resulting in little change in species richness at the same concentration even if the concentration of BaP was low. At the same time, the higher the concentration of BaP, the more kinds of microorganisms should be degraded together, so the microbial richness was greater. In M groups with the addition of WMBC-immobilized microbial community, the situation was quite different. The species abundance of M groups was much higher than that of B groups, suggesting that the biochar carrier optimized the growth environment of microorganisms, so that more microorganisms could grow and join in the process of BaP degradation. Among them, possibly due to the dominant microorganisms in the process of BaP degradation, the species abundance of M5.12 and M10.12 decreased compared with M5.6 and M10.6. On this basis, the higher the concentration of BaP substrate in the range of 5-10mg/L, the stronger the dominant role of dominant microorganisms, and the lower the species richness, metabolic diversity and phylogenetic diversity. However, possibly given that the concentration of BaP exceeded the degradation ability of the original dominant microorganisms and required more microorganisms to cooperate, the species abundance of M20.12 was higher than that of M20.6. The micropore structure of biochar provided a suitable growth environment for microorganisms, which could release carbon sources and nutrients, and accumulate foreign carbon sources through adsorption to accelerate the growth of microorganisms(Mukherjee et al. 2022). In addition, according to reports, biochar could induce the transfer of microbial community, greatly improve species abundance, and was considered more conducive to the cooperation between microorganisms(Zhang et al. 2018). Efficient microbial alliance was an important reason to improve the degradation efficiency of BaP(Zhang et al. 2021).
Figure 5a shows the relative abundance of six groups of samples at the phylum level (the richness was among the top 10). In B groups, Proteobacteria was the phylum with the highest relative abundance, with relative abundance of 96.95% (B5), 96.11% (B10) and 91.95% (B20), followed by Firmicutes and Bacteroidota. The relative abundance of the three phylums added together was more than 99%. In M groups, the phylum with the highest relative abundance was Firmicutes, which was 72.46% (M5), 69.72% (M10) and 68.56% (M20), followed by Proteobacteria, accounting for more than 25%. Proteobacteria is a common PAHs degrading bacteria(Akash et al. 2023). According to the study, in most mixed microorganisms, γ- Proteobacteria, α- Proteobacteria and β- Proteobacteria can all occupy a dominant position at the class level and the order level(Gosai et al. 2022; Sun et al. 2010). Therefore, the high abundance of Proteobacteria might explain its high BaP removal efficiency. Herein, under the condition that only this microbial community was added in B groups, Proteobacteria occupied an absolutely dominant position as the dominant phylum. On the other hand, Firmicutes is also a common phylum isolated from PAHs enrichment culture(Akash 2023). Some studies have confirmed its better efficiency in adapting to higher temperature than Proteobacteria, and its degradation rate of total PAHs generally increases significantly with the increase of the temperature(Sun et al. 2022). This may be one of the reasons that the BaP removal efficiency of microbial community at 35 ℃ is not significantly lower than or even higher than that at 25–30 ℃. In M groups, the relative abundance of Firmicutes was much larger than that of Proteobacteria, indicating that the environment and nutrients provided by WMBC contributed to the accumulation of Firmicutes, became the dominant bacteria in the degradation of BaP, and significantly improved the removal efficiency of BaP. At the same time, Proteobacteria also accounted for more than 1/4, which still played its own role in the removal of BaP in M groups. In many reports, many PAHs-degrading microbial communities isolated from highly polluted aged PAHs contaminated soil or heavy crude oil contaminated soil contain high abundance of Proteobacteria and Firmicutes, and they could still maintain high growth and metabolic ability after different treatments of soil(Gou et al. 2020; Lee et al. 2018; Yang et al. 2018). This demonstrated that the favorable synergism between them could improve the activity of microbial community and the degradation effect of PAHs. In addition, Bacteroidota is good at degrading various polymers(Huang et al. 2023), and is also a PAHs-degrading bacteria(Akash 2023). Therefore, although relatively abundant in the flora, it could also contribute to the removal of BaP and metabolic intermediates.
Figure 5b depicts the relative abundance of six groups of samples at the family level (the richness was among the top 10). In B groups, Pseudomonadaceae and Xanthomonadaceae were the two kinds of families with the highest relative abundance. The relative abundance of Pseudomonadaceae increased with the increase of BaP concentration, which was 31.40% (B5), 41.30% (B10) and 65.21% (B20), while that of Xanthomonadaceae decreased with the increase of BaP concentration, which was 61.70% (B5), 49.26% (B10) and 24.65% (B20). In M groups, the family with the highest relative abundance was Bacillaceae, which was about 70%, followed by Pseudomonadaceae, accounting for close to a quarter. In addition to these advantages of the family, there were also Alcaligenaceae and Sphingobacteriaceae. Both of these two kinds of families have been mentioned in studies and reports on the degradation of PAHs(Mangwani et al. 2017; Yang et al. 2015), having a certain ability to degrade BaP.
Figure 5c shows the relative abundance of six groups of samples at the genus level (the richness ranked in the top 30). On the whole, the relative abundance of Pseudomonas, Stenotrophomonas and Bacillus was similar to that of the family. Pseudomonas is a common genus of PAHs-degrading bacteria(Premnath et al. 2021), which can degrade not only LMW-PAHs, but also HMW-PAHs. According to the report, Pseudomonas can effectively biodegrade polycyclic aromatic hydrocarbons and heterocyclic derivatives through the transverse hydrogen peroxide pathway(Liu et al. 2021). In addition, Pseudomonas aeruginosa can use biosurfactant(Zang et al. 2021) and has an anti-heavy metal effect(Safahieh et al. 2012), which may be the basis of BaP degradation ability. Stenotrophomonas, a genus within the Xanthomonadaceae family, is traditionally recognized as a type of plant pathogenic bacteria. However, it has also been discovered in studies of microbial communities inhabiting oil-contaminated soils. Various research findings indicate that certain Stenotrophomonas species possess the capability to utilize PAHs as their sole carbon source for growth. As part of a microbial alliance, Stenotrophomonas not only efficiently degrades pollutants such as Phe and Pyr(Gosai 2022), but also improves the tolerance to HMW-PAHs such as BaP and its ability as a source of energy(Zafra et al. 2014). This may be one of the reasons explaining the high BaP degradation ability of the microbial community. Herein, in B groups, when the concentration of BaP was low, Stenotrophomonas demonstrated a higher capability to utilize BaP compared to other microorganisms, making it the dominant genus initially. However, when the BaP concentration rose, its utilization ability diminished. This decrease in BaP utilization ability might be attributed to the inhibitory effect of higher BaP concentrations on microorganisms within the community. However, at this time, Pseudomonas still possessed a high degradation ability to a higher concentration of BaP and occupied a dominant position. Bacillus is another common genus of PAHs-degrading bacteria, which plays a pioneering role in microbial recovery by consuming mobilized organic matter under adverse environmental conditions(Medina et al. 2020), as well as tolerance to heavy metals(Rabani et al. 2022). Bacillus exerts a good degradation effect on PAHs such as BaP, BaA, Pyr and heavy crude oil(Kong et al. 2022). In the case of microbial alliance, whether the combination of Bacillus licheniformis and Bacillus mojavensis of the same genus(Eskandary et al. 2017), or the interaction with other genus microorganisms(Jacques et al. 2008), PAHs removal becomes more efficient. Herein, in M groups, Bacillus was always the dominant genus, indicating that the environment and nutrition provided by biochar could be preferentially utilized by Bacillus, and then cooperate with Pseudomonas to effectively degrade BaP.
3.6 Metagenomic analysis
3.6.1 Metagenomic analysis of eggNOG
Figure 6a shows the functional annotations of genes and the relative abundance of the number of genes in the eggNOG level 1 database (the specific meaning of the abbreviations in Fig. 2a is given in Supplementary Table S3). The results demonstrated Amino acid transport and metabolism (7.28–7.47%) and Signal transduction mechanisms (6.04–7.14%) as the two genes with the highest abundance, followed by Transcription, Cell wall/membrane/envelope biogenesis, and Energy production and conversion. Their relative abundance was all more than 5%, indicating the exuberant growth and metabolism of microorganisms. In addition, considering the genotoxicity and mutagenicity of BaP(Ghosal et al. 2016), the relative abundance of Replication, recombination and repair (4.67–5.66%) was also high. This gene enabled the microbial community to repair its own variation, improve microbial survival, and utilize and degrade BaP. By comparison, Signal transduction mechanisms, Energy production and conversion in M groups were significantly higher than those in B groups. Due to the obvious difference of dominant microorganisms before and after MWBC fixation, the microbial community of M groups was more active in these two aspects. These two genes were also closely related to the improvement of BaP removal efficiency. In addition, the eggNOG database also annotated Secondary metabolites biosynthesis, transport and catabolism (2.62–2.70%), which might be related to the emulsification observed in the remaining BaP extraction process of the sample in the experiment. Glycolipids and lipopeptides are biosurfactants often secreted by microorganisms in the presence of aromatic compounds(Bezza and Chirwa 2017; Zang et al. 2021). They are generally produced by secondary metabolites of organisms(Nie et al. 2012), which can improve the solubility, bioavailability and biodegradability of BaP, i.e., a hydrophobic compound, thereby causing emulsification in the process of BaP extraction.
Figure 6b shows the further level 2 data analysis of eggNOG. Transcriptional regulator and Protein conserved in bacteria were the two functions with the highest relative content (relative abundance > 1%). The LysR family of transcriptional regulatory factors was reported to be involved in the catabolism, cell movement and quorum sensing of aromatic compounds(Maddocks and Oyston 2008), making microorganisms move to better sites, contact and degrade BaP more fully. It was also hereby revealed that the relative abundance of Transcriptional regulator in B groups was significantly higher than that in M groups, which might be attributed to the lower utilization efficiency of BaP in B groups than that in M groups. The microorganisms in B groups were more involved in transcriptional activities and failed to carry out further metabolic activities. On the other hand, proteins were conserved, and residues important to maintain protein function were also highly conserved(Jing-Fei and Blundell 1999). The phenomenon of high relative content of Protein conserved in bacteria might be the response of bacteria to reduce the aberration rate and improve the survival rate in BaP environment. In addition, the relative abundance of Histidine kinase in M groups was significantly higher than that in B groups. According to reports, histidine kinases could mediate signal transduction of bacterial chemotaxis(Oshkin et al. 2020), which might promote bacterial utilization of BaP.
3.6.2 Metagenomic analysis of CAZy
CAZys are indeed crucial enzymes for catalyzing the utilization of hydrocarbons. Their function is to degrade, modify and form glycosidic bonds, further transform glycosidic bonds, and obtain energy. CAZys are classified as Carbohydrate-Binding Modules (CBMs), Carbohydrate Esterases (CEs), Glycoside Hydrolases (GHs), Glycosyl Transferases (GTs), Polysaccharide Lyases (PLs) and Auxiliary Activities (AAs). Some CAZys are reported to be capable of effectively oxidizing polycyclic aromatic hydrocarbons, phenolic and non-phenolic aromatic compounds(Imam et al. 2022). These enzymes also play an important role in the degradation of BaP. As shown in Fig. 7a, GTs and GHs were dominant enzymes in all samples. Some of these two types of enzymes could play a role in the cleavage and cleavage of polymers(Janecek et al. 2014; van der Maarel and Leemhuis 2013). The relative abundance of CBMs was more than 7.8%. CBMs could promote the interaction between a given enzyme and its substrate, thus improving the catalytic efficiency(Sidar et al. 2020). To this end, it was speculated that CBMs might be more conducive to targeting BaP and intermediate metabolites in the process of degradation, or combine the active sites of the enzyme with BaP or intermediate metabolites, which is more likely to be caused by the degradation of BaP.
Figure 7b shows the level 2 data analysis of CAZy at the family. The two enzyme types with the highest relative abundance were GT2 (> 8%) and GT4 (> 7%), followed by GH13, CMB50, etc. GT2 and GT4 contained many kinds of glucosyltransferases and galactosyltransferases, which, together with CBM50, promoted the formation and further decomposition of oligosaccharides, disaccharides, polysaccharides and cellulose(Thompson et al. 2013). Their high content indicated that the microbial community could use BaP more efficiently and had transformed it into sugars that could be used more easily. There were many kinds of GH13 family, which could further hydrolyze the sugars obtained from the previous conversion, and monosaccharides and other metabolic end products to be directly used could be finally obtained. Other enzymes, such as CBM48, were associated with amylase, causing amylase to target starch binding and promoting its degradation(Wilkens et al. 2018). This could also support the above conjecture. At the same time, GH13 subfamily enzymes contained α-amylase and other enzymes that could directly degrade alkanes(Pinto et al. 2020), which had strong adaptability to extreme environment. Notably, significant differences in GT2 and GH13 could be observed between B groups and M groups. The relative abundance of them in M groups was higher than that in B groups. This also suggested that the metabolism of MWBC-immobilized microbial community was more active, and that the degradation effect of BaP was better. Many studies have shown that the degradation process of lignin, cellulose and other polysaccharides can greatly promote the biodegradation of PAHs, such as crop residue, compost, etc(Molina-Barahona et al. 2004). In addition to the enzymes given in the figure, it was also hereby found that enzymes such as AA1 and AA5 were also in the annotation ranks. AA1 was a polycopper oxidase, involving subfamily including laccase and laccase-like polycopper oxidase. These enzymes introduced a hydrogen peroxide molecule into two water molecules to oxidize aromatics. According to reports, laccase could significantly promote the conversion of polycyclic aromatic hydrocarbons, phenols and other aromatic compounds(Imam 2022). On the other hand, according to the study of Zeng et al.(Zeng et al. 2016), a laccase from Bacillus subtilis could efficiently oxidize BaP and anthracene. To sum up, the synergism between different CAZys was beneficial to the improvement of BaP removal efficiency.
3.6.3 Metagenomic analysis of KEGG
KEGG comments generally fall into seven broad categories, i.e., Metabolism, Genetic Information Processing, Environmental Information Processing, Cellular Processes, Organismal Systems, Human Diseases, and Drug Development. Each large category has several small classes. In all samples, most of the genes (a total of 67589) were annotated to Metabolism. At the level 2, Carbohydrate metabolism and Amino acid metabolism were the most annotated ways. Carbohydrate metabolism and amino acid metabolism were related to the degradation of polycyclic aromatic hydrocarbons. The enrichment regulation of carbohydrate metabolism genes can aid in enriching genes responsible for polycyclic aromatic hydrocarbon degradation. Amino acid metabolism holds a central role in cell metabolism. For example, many amino acids are involved in the TCA cycle, and the TCA cycle is associated with reactions such as glycolysis and improves environmental stress(Xiao et al. 2022). Such a network connects the metabolism of nitrogen and carbon, and is closely related to the degradation of polycyclic aromatic hydrocarbons. Numerous genes are also annotated to lipid metabolism. Certain aromatic compounds and polycyclic aromatic hydrocarbons can induce bacteria to produce biosurfactants, including lipids, fatty acids, glycolipids, lipopeptides, and phospholipids(Abdel-Mawgoud et al. 2010; Bezza and Chirwa 2017, 2017; Zang 2021). These biosurfactants enhance the solubility and accessibility of polycyclic aromatic hydrocarbons, thereby facilitating their degradation. Other studies have shown that the presence of some fatty acids can improve the removal efficiency of HMW-PAHs(Wang et al. 2020). Some microorganisms such as Pseudomonas capable of producing this kind of biosurfactant possess significant potential and practicability for the degradation of HMW-PAHs(Zang 2021). The genes annotated to Glycan biosynthesis and metabolism support each other with the annotated results of GT2 and GT4 in CAZy. Similar to the results of eggNOG comments, replication and repair, as well as folding, sorting, and degradation in Genetic Information Processing, are also annotated in the KEGG database. They maintain the stability of microbial life by removing defective proteins. More importantly, the microbial community annotated to the Xenobiotics biodegradation and metabolism gene, indicating that the flora will focus a large part of its efforts on the degradation of exogenous organisms such as BaP.
According to the annotation of KEGG, it was hereby inferred that there were two main metabolic pathways for the BaP of this microbial community to convert to low molecular weight PAHs (Supplementary Fig. 1). One was the conversion of benzo[a]pyrene to pyrene and then to phenanthrene, while the other was the conversion of benzo[a]pyrene to benzo[a]anthracene, and then to anthracene. Then, through the metabolic pathways such as naphthalene degradation and benzoic acid degradation, the resulting metabolites entered the TCA cycle and glycolysis/gluconeogenesis.
According to the annotation results, the sample detected benzo[a]pyrene dioxygenase (EC.1.14.12), including benzo[a]pyren1,2-dioxygenase (EC.1.14.12.12). The benzo[a]pyrene-cis-9,10-dihydrodiol: NAD+ 9,10-oxidoreductase (EC.1.3.1.29) on the pathway of conversion to pyrene was also annotated. Under the action of these two enzymes and 9,10-dihydroxybenzoate[a]pyrene dioxygenase (EC1.13.11) and other catalysts, BaP was converted to pyrene, then to phenanthrene, and then into the phthalic acid degradation pathway(Imam 2022). In another experiment conducted by the present research group(Xu et al. 2022), the microbial community obtained the results of efficient degradation of low molecular weight polycyclic aromatic hydrocarbons such as phenanthrene and pyrene. On the other hand, due to the annotating of the degradation process and related enzymes of 7,12-dimethylbenz[a]anthracene, the existence of benzo[a]anthracene and its derivatives in the upper reaches of the pathway was further confirmed. BaP was converted to 11,12-Dihydroxybenzo[a]pyrene under the action of dioxygenase and dehydrogenase, or BaP was synthesized by cyclooxygenase, epoxide hydrolase and dehydrogenase to produce 11,12-dihydroxybenzo[a]pyrene. At the same time, two catechol dioxygenases were detected. The substance could also act on the same structure in addition to catechol. Therefore, 11,12-dihydroxybenzo[a]pyrene was further converted to benzo[a]anthracene and anthracene under the action of dioxygenase. Anthracene entered the metabolic pathway towards phthalic acid under the action of dioxygenase (EC.1.14.12.12) and oxidoreductase (EC.1.3.1.29).