Removal of benzo[a]pyrene by a highly degradable microbial community immobilized by modified wheat straw biochar

Removal of benzo[a]pyrene by a highly degradable microbial community immobilized by modified wheat straw biochar | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Removal of benzo[a]pyrene by a highly degradable microbial community immobilized by modified wheat straw biochar Xiaoxiao Chen, Rong Meng, Meihui Geng, Jiahui Zhou, Yuewu Pu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4052065/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Dec, 2024 Read the published version in Environmental Science and Pollution Research → Version 1 posted 6 You are reading this latest preprint version Abstract Benzo[a]pyrene, a high-molecular-weight polycyclic aromatic hydrocarbon, is a prevalent organic pollutant. In this study, a microbial community with high efficiency of BaP degradation was enriched in oil-contaminated soil, and the microbial community was immobilized using modified wheat straw biochar. The highest removal efficiency of 5–20 mg/L BaP was 75.18% in 12 days. Through 16SrRNA sequencing, Pseudomonas , Stenotrophomonas and Bacillus were found to be the dominant bacteria in the community. Additionally, metagenomic annotation revealed the gene function and metabolic pathway of the microbial community during BaP degradation. Degradation of benzo[a]pyrene Biochar immobilized microorganisms Macrogenomic analysis Microbial community Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction Benzo[a]pyrene (BaP) is a kind of polycyclic aromatic hydrocarbons (PAH) organic pollutant characterized by its five-benzene-ring structure, classifying it as a high molecular weight PAH (HMW-PAH). Featuring stable chemical properties(Nzila and Musa 2021 ) and hydrophobic nature(Guo and Wen 2021 ), BaP readily adheres to solid particles. It is commonly found in the atmosphere, water bodies, vegetation, and soil. Notably toxic and profoundly carcinogenic(Bukowska et al. 2022 ), BaP poses significant health risks to humans(Kim et al. 2013 ). The Council of Ministers of the Environment of Canada (CCME) ever proposed BaP as one of the indicators for the classification of soil pollution. Meanwhile, BaP is also the earliest and most important carcinogen found in PAHs(Wang et al. 2023 ). To this end, BaP was hereby selected as the representative pollutant of HMW-PAHs for corresponding remediation technology research. This endeavor holds immense importance in the realm of treating soil contaminated with HMW-PAHs. Microbial remediation is one of the most important remediation methods for BaP-contaminated soil at present(Bezza and Chirwa 2017 ; Ostrem Loss and Yu 2018 ). While employing PAH-degrading bacteria directly might prove more efficient in laboratory settings, their viability and degradation efficacy in contaminated soil are often hampered by environmental factors and competition from indigenous microbial populations, making it imperative to address the challenge of maintaining microbial activity and enhancing degradation efficiency(Hou et al. 2019 ; Punetha et al. 2022 ). Based on the above, immobilized microtechnology becomes an important method. This technology can maintain the high biomass content of microorganisms, enhance the tolerance to harmful environmental conditions of strains, and prevent their loss. At the same time, BaP migrates from pollutants to the carrier surface through hydrophobic interaction, adsorption between donors and receptors or specific interactions, which promotes the degradation of BaP by carrier immobilized microorganisms and improves the removal efficiency of BaP(Bianco et al. 2021 ). Among numerous carrier materials, biochar made from agricultural and forestry wastes is a preferred choice. Biochar immobilized microorganisms are reported to have the shortest half-life and the highest biodegradation efficiency in the process of biodegradation of pollutants, and release enough nutrients for microbial metabolism(Zhang et al. 2019 ). The present study was conducted to screen the efficient BaP-degrading microbial community and improve the BaP removal efficiency by immobilizing BaP-degrading microbial community on biochar. At the same time, the differences of microbial community succession during the removal of different concentrations of BaP before and after biochar immobilization were studied by means of amplification sequencing and macrogenomic detection, and the functional genes playing an important role in BaP degradation were also identified. 2 Materials and Methods 2.1 Soil sampling and enrichment Soil samples (0 ~ 10cm from the surface) were collected from the oil field in Karamay (Xinjiang, China). With BaP as carbon source, the leaching solution of soil samples was added to MSM medium to enrich BaP-degrading microbial community. The enrichment process is as follows: 1g soil samples were added to 100mL aseptic water and cultured in shaker (200rpm) at 30 ℃ for 2 days. BaP (Macklin, Shanghai, China) was dissolved in acetone (Macklin) to prepare BaP- acetone concentrate (2g/L). The MSM medium was prepared by adding the BaP concentrate to the sterilized MSM-BaP medium (Supplementary Table S1 ) and waiting for acetone to volatilize. At the same time, the MSM-BaP medium with 1g/L glucose (Damao, Tianjin, China) was added under the same conditions. The experiments of two kinds of media were carried out respectively: the leaching solution of 2mL soil sample was transferred into 20mL medium with BaP concentration of 5mg/L. After being cultured in 200rpm at 30 ℃ for 7 days, the 2mL culture material was transferred into 20mL medium with BaP concentration of 10mg/L for further culture. By taking 7 days as a round, the concentration of BaP carbon source was gradually increased to 15, 20 and 25mg/L. The screened microorganisms degraded the BaP added in MSM medium within 7 days, and the bacterial community with high removal efficiency was finally selected. 2.2 Preparation and characterization of biochar The pretreated wheat straw was sealed in a porcelain crucible and put into a muffle furnace. Subsequently, the wheat straw underwent pyrolysis and carbonization for a duration of 2 hours, with a heating rate maintained at 10 ℃/min under N 2 atmosphere. After the complete reaction, the biochar of wheat straw (WBC) was obtained by being naturally cooled down to room temperature. WBC and sodium hydroxide solid (Guangzhou Chemical Reagent Factory, China) were added to 150mL aseptic water at 1:2(v/v) and transferred to a shaker (180rpm) at 30 ℃ for 24 hours, which were dried in an oven at 105 ℃ after centrifugation, and then pyrolyzed again under the same conditions. After cooling, the solution was soaked in deionized water, and its pH was adjusted to neutral. After washing and drying, the modified wheat straw biochar (MWBC) was obtained through 40 mesh sieve and encapsulation. The specific surface area and pore size of MWBC samples were measured by BET, and the structure and morphology were observed using SEM scanning electron microscope. 2.3 Preparation of microbial community immobilized by biochar Preparation of biochar-immobilized microbial community was conducted as follows: 0.02gMBC was added to 18mL inorganic salt medium, and then placed in a conical bottle for 20 minutes of sterilization in a sterilizer at 121C. After cooling to room temperature, 2mL of heavy suspension (OD 600 = 1) was added. After mixing, the bacteria were placed in a shaker (30 ℃, 180rpm) and fully oscillated to be loaded on the biochar as much as possible. After 24 hours of adsorption and fixation, the bacteria were removed and transferred to a high-speed centrifuge and centrifuged for 5 minutes under the condition of 4500rpm. Then, the supernatant was removed and washed with sterile water. The MWBC immobilized bacterial community was obtained by repeated centrifugation for 2 times. The fixed time was 4, 8, 12, 16, 20 and 24 hours, respectively. After each group was fixed, MWBC immobilization was transferred to 5min in 20mL aseptic water, then 10min was placed statically, the supernatant was taken, and the supernatant was diluted 10000 times. The number of microorganisms adsorbed was determined by dilution coating plate method to determine the best fixed time. On the basis of the optimal fixed time, the pH values of MSM were 5, 6, 7, 8 and 9, respectively. After each group was fixed, the best pH value was determined by the same method mentioned above. Under the conditions of the best fixed time and the best pH value, the MWBC-immobilized microbial community were prepared for later experiments. 2.4 Experiments on BaP removal of microbial community and MWBC-immobilized microbial community Experiment on BaP removal of microbial community (Experiment 1): The above bacterial community was inoculated into SOB broth medium (Supplementary Table S2 ) and transferred to a shaker at 30 ℃ for 15 hours. After 5 minutes of centrifugation under the condition of 8000rpm, the culture was washed with inorganic salt medium (MSM) for 3 times, and then re-suspended in MSM. In the BaP removal experiment of microbial community, the initial BaP concentration was 5, 10 and 20mg/L, respectively. The experiments were carried out at 15 ℃, 20 ℃, 25 ℃, 30 ℃ and 35 ℃. The grouping of specific experimental conditions is shown in Table 1 , and it is named T1-T15 according to different temperatures and different BaP concentrations. Besides, 2ml resuspension inoculant (OD 600 = 1) was inoculated in 20mLMSM-BaP medium. A blank control group was set up to evaluate abiotic loss, all experiments were designed with 3 parallels, and destructive sampling was carried out after 2, 4, 6, 8, 10 and 12 days to detect the removal of BaP. Experiment on BaP removal of MWBC-immobilized microbial community (Experiment 2): The experiment was carried out under the same conditions by using MBC-immobilized bacterial community instead of bacteria in free state in Experiment 1. A blank control group was set up to evaluate abiotic loss, and all experiments were designed with 3 parallels. The grouping of specific experimental conditions is shown in Table 1 , and it is named W1-W15 according to different temperatures and different BaP concentrations. After 2, 4, 6, 8, 10 and 12 days, destructive sampling was carried out to detect the removal of BaP.. Table 1 Grouping conditions for experiments on BaP removal of microbial community and MWBC-immobilized microbial community Temperature (℃) BaP concentration (mg/L) Group name of Experiment 1 Group name of Experiment 2 15 5 T1 W1 10 T2 W2 20 T3 W3 20 5 T4 W4 10 T5 W5 20 T6 W6 25 5 T7 W7 10 T8 W8 20 T9 W9 30 5 T10 W0 10 T11 W11 20 T12 W12 35 5 T13 W13 10 T14 W14 20 T15 W15 2.5 Extraction and detection of BaP By taking dichloromethane (Macklin) with the same volume as the culture medium, the samples were extracted and repeated twice. The concentration of BaP was determined using high performance liquid chromatography. The mobile phase was acetonitrile (Macklin) and water, with a ratio of 9:1 (v/v). A C18 column (250 × 4.6 mm) was adopted, the injection volume was 10 µL, and the detection wavelength was 290nm. In order to calculate the concentration of BaP, the standard curve of the external standard detection of BaP was drawn, and the correlation coefficient was 0.99. 2.6 Detection and analysis of microbial community amplifier and macro genome In the two BaP removal experiments described in Section 2.2 and 2.4 , when T10, T11, T12, W10, W11 and W12 were cultured at 30 ℃ to the 6th and 12th day, the cultures were taken for amplification and macrogenomic detection. The samples of T10, T11 and T12 were recorded as B groups, while those of W10, W11 and W12 were recorded as M groups. The groups were named according to the concentration of BaP: B5, B10, B20, M5, M10 and M20, respectively. The forward primer for 16srRNA analysis is Bakt_341F (CCTAYGGGRBGCASCAG), and the reverse primer is Bakt_806R (GGACTACNNGGGTATCTAAT).The amplifiers were sequenced and analyzed by Novogene Company (Beijing, China). The process involved DNA and PCR extraction, DNA amplification, library construction, NovaSeq6000 analysis, sequencing and data processing. The DADA2 method (Callahan BJ et al., 2016) was used to reduce the noise of each de-weight sequence to generate ASVs (Amplicon Sequence Variants), and QIIME2's classify-sklearn algorithm (Bokulich NA et al., 2018; Bolyen E et al.,2019) was adopted to annotate the species of each ASV using the pre-trained NaiveBayes classifier. According to the results of ASVs annotation and the characteristic table of each sample, the species abundance table was obtained, and the Alpha diversity index was calculated. The functional genes of the community were annotated and mapped to the Kyoto Encyclopedia of genes and Genomes (KEGG, Version: 2018.01), the function of the homologous group was annotated to the evolutionary pedigree of genes, the unsupervised homologous group (eggNOG, Version: 4.5), and the function and relative abundance of carbohydrate enzymes were annotated to the carbohydrate active enzyme database (CAZy, Version: 2018.01). 3 Results and Discussion 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 m 2 /g, and the average pore diameter was 87.373cm 3 /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. 2 a. 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. 2 b. 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. 3 a), 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. 3 b), 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. 3 c), 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. 4 a), 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. 4 b), 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. 4 c), 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 5 a 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 5 b 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 5 c 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 6 a 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. 2 a 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 6 b 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. 7 a, 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 7 b 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). 4 Conclusion In this study, a highly efficient microbial community capable of removing benzo[a]pyrene (BaP) was enriched from oil-contaminated soil. Upon immobilization with modified wheat straw biochar, this microbial community exhibited a remarkable BaP removal efficiency of up to 75.18% within 12 days in an inorganic salt medium containing 5–20 mg/L BaP. Meanwhile, the microbial community was observed and studied by 16SrRNA sequencing and metagenomic analysis, and the results demonstrated Pseudomonas , Stenotrophomonas and Bacillus as the main genus in the microbial community. Additionally, the function and relative abundance of genes were annotated by eggNOG, CAZy and KEGG databases to provide insights into why the bacterial community had the ability to degrade BaP, and to study the metabolic pathway of bacterial community to BaP. Declarations Data Availability Statement: All data analyzed are available from the corresponding author on reasonable request. Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Ethics approval: Not applicable. Consent to participate: The authors agree to participate. Consent for publication: The authors agree to publish. Author Contributions: Conceptualization, X.C.; methodology, X.C., M.G and J.Z.; validation, X.C. and R.M.; formal analysis, X.C.; writing—original draft preparation, X.C.; writing—review and editing, R.M., M.G and J.Z.; project administration, Y.P.; funding acquisition, Y.P. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the National Key R&D Program of China (No. 2020YFC1808802). Competing Interests: The authors have no relevant financial or non-financial interests to disclose. 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05:49:35","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4052065/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4052065/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11356-024-35717-1","type":"published","date":"2024-12-06T15:57:28+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53870192,"identity":"c23cfbb8-ba05-491f-bdf4-db3feec2f495","added_by":"auto","created_at":"2024-04-01 15:13:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":774412,"visible":true,"origin":"","legend":"\u003cp\u003eSEM scanning images of modified wheat straw biochar (MWBC)\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4052065/v1/eee848d497bfb025270484d7.png"},{"id":53870186,"identity":"8c1d7ccf-5064-4939-9606-eacab9d7d091","added_by":"auto","created_at":"2024-04-01 15:13:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":89651,"visible":true,"origin":"","legend":"\u003cp\u003eRelationship between the number of fixed microorganisms in MWBC and fixed time (a) and pH value (b) of MSM\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4052065/v1/fe4dfc0803c2193ac536e4fa.png"},{"id":53871246,"identity":"c6bfcb1c-1ca0-4af6-b40f-e70c78f55c88","added_by":"auto","created_at":"2024-04-01 15:21:28","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":159129,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of removal efficiency of 5 mg/L (a), 10 mg/L (b) and 20 mg/L (c) BaP by microbial community\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4052065/v1/feb5be02f1ae3381688bde47.png"},{"id":53870180,"identity":"368b2c95-0ffd-4b61-9c25-d44cba08f7e9","added_by":"auto","created_at":"2024-04-01 15:13:25","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":48400,"visible":true,"origin":"","legend":"\u003cp\u003eChanges of removal efficiency of 5 mg/L (a), 10 mg/L (b) and 20 mg/L (c) BaP by MWBC -immobilized microbial community\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4052065/v1/04d499051ce7b39cc3c64d9c.png"},{"id":53870181,"identity":"0864a4c7-412b-4300-8fa9-6e21ed365767","added_by":"auto","created_at":"2024-04-01 15:13:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":177311,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of bacterial community structure at phylum (a), family (b) and genus (c) level\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4052065/v1/4742533b0762b4938cea8e84.png"},{"id":53870191,"identity":"0749e25a-3e39-4a7e-adf2-8b16ea0fe69d","added_by":"auto","created_at":"2024-04-01 15:13:29","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":234168,"visible":true,"origin":"","legend":"\u003cp\u003eThe relative abundance of eggNOG at level 1 (a) and level 2 (b)\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4052065/v1/125620b3f4ad822bfca8e925.png"},{"id":53870189,"identity":"87b60fad-1fb0-4324-9c50-826fd24df551","added_by":"auto","created_at":"2024-04-01 15:13:28","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":194102,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundance of the CAZy at the L1 (a) and family (b)\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4052065/v1/8b0f51094b0ab22f4df1271b.png"},{"id":53870184,"identity":"621fca86-fed2-4c84-91b2-f318d00daf99","added_by":"auto","created_at":"2024-04-01 15:13:27","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":527096,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of genes of KEGG pathway annotation at level 2\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4052065/v1/3354190c1c55241567dab9c5.png"},{"id":70964694,"identity":"f940f5a6-4585-4eab-be6c-aeec252bf82e","added_by":"auto","created_at":"2024-12-09 16:14:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3338139,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4052065/v1/2c1287e7-fa61-45c4-9d10-6de2fe4a6bb3.pdf"},{"id":53870176,"identity":"aea7c591-44c8-4fde-98df-9ecce7da07a8","added_by":"auto","created_at":"2024-04-01 15:13:24","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":82192,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-4052065/v1/71ee9aaa061be7ef806a791c.docx"}],"financialInterests":"","formattedTitle":"Removal of benzo[a]pyrene by a highly degradable microbial community immobilized by modified wheat straw biochar","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eBenzo[a]pyrene (BaP) is a kind of polycyclic aromatic hydrocarbons (PAH) organic pollutant characterized by its five-benzene-ring structure, classifying it as a high molecular weight PAH (HMW-PAH). Featuring stable chemical properties(Nzila and Musa \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and hydrophobic nature(Guo and Wen \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), BaP readily adheres to solid particles. It is commonly found in the atmosphere, water bodies, vegetation, and soil. Notably toxic and profoundly carcinogenic(Bukowska et al. \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), BaP poses significant health risks to humans(Kim et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The Council of Ministers of the Environment of Canada (CCME) ever proposed BaP as one of the indicators for the classification of soil pollution. Meanwhile, BaP is also the earliest and most important carcinogen found in PAHs(Wang et al. \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). To this end, BaP was hereby selected as the representative pollutant of HMW-PAHs for corresponding remediation technology research. This endeavor holds immense importance in the realm of treating soil contaminated with HMW-PAHs.\u003c/p\u003e \u003cp\u003eMicrobial remediation is one of the most important remediation methods for BaP-contaminated soil at present(Bezza and Chirwa \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Ostrem Loss and Yu \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). While employing PAH-degrading bacteria directly might prove more efficient in laboratory settings, their viability and degradation efficacy in contaminated soil are often hampered by environmental factors and competition from indigenous microbial populations, making it imperative to address the challenge of maintaining microbial activity and enhancing degradation efficiency(Hou et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Punetha et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eBased on the above, immobilized microtechnology becomes an important method. This technology can maintain the high biomass content of microorganisms, enhance the tolerance to harmful environmental conditions of strains, and prevent their loss. At the same time, BaP migrates from pollutants to the carrier surface through hydrophobic interaction, adsorption between donors and receptors or specific interactions, which promotes the degradation of BaP by carrier immobilized microorganisms and improves the removal efficiency of BaP(Bianco et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Among numerous carrier materials, biochar made from agricultural and forestry wastes is a preferred choice. Biochar immobilized microorganisms are reported to have the shortest half-life and the highest biodegradation efficiency in the process of biodegradation of pollutants, and release enough nutrients for microbial metabolism(Zhang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe present study was conducted to screen the efficient BaP-degrading microbial community and improve the BaP removal efficiency by immobilizing BaP-degrading microbial community on biochar. At the same time, the differences of microbial community succession during the removal of different concentrations of BaP before and after biochar immobilization were studied by means of amplification sequencing and macrogenomic detection, and the functional genes playing an important role in BaP degradation were also identified.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Soil sampling and enrichment\u003c/h2\u003e \u003cp\u003eSoil samples (0\u0026thinsp;~\u0026thinsp;10cm from the surface) were collected from the oil field in Karamay (Xinjiang, China). With BaP as carbon source, the leaching solution of soil samples was added to MSM medium to enrich BaP-degrading microbial community.\u003c/p\u003e \u003cp\u003eThe enrichment process is as follows: 1g soil samples were added to 100mL aseptic water and cultured in shaker (200rpm) at 30 ℃ for 2 days. BaP (Macklin, Shanghai, China) was dissolved in acetone (Macklin) to prepare BaP- acetone concentrate (2g/L). The MSM medium was prepared by adding the BaP concentrate to the sterilized MSM-BaP medium (Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) and waiting for acetone to volatilize. At the same time, the MSM-BaP medium with 1g/L glucose (Damao, Tianjin, China) was added under the same conditions. The experiments of two kinds of media were carried out respectively: the leaching solution of 2mL soil sample was transferred into 20mL medium with BaP concentration of 5mg/L. After being cultured in 200rpm at 30 ℃ for 7 days, the 2mL culture material was transferred into 20mL medium with BaP concentration of 10mg/L for further culture. By taking 7 days as a round, the concentration of BaP carbon source was gradually increased to 15, 20 and 25mg/L. The screened microorganisms degraded the BaP added in MSM medium within 7 days, and the bacterial community with high removal efficiency was finally selected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Preparation and characterization of biochar\u003c/h2\u003e \u003cp\u003eThe pretreated wheat straw was sealed in a porcelain crucible and put into a muffle furnace. Subsequently, the wheat straw underwent pyrolysis and carbonization for a duration of 2 hours, with a heating rate maintained at 10 ℃/min under N\u003csub\u003e2\u003c/sub\u003e atmosphere. After the complete reaction, the biochar of wheat straw (WBC) was obtained by being naturally cooled down to room temperature. WBC and sodium hydroxide solid (Guangzhou Chemical Reagent Factory, China) were added to 150mL aseptic water at 1:2(v/v) and transferred to a shaker (180rpm) at 30 ℃ for 24 hours, which were dried in an oven at 105 ℃ after centrifugation, and then pyrolyzed again under the same conditions. After cooling, the solution was soaked in deionized water, and its pH was adjusted to neutral. After washing and drying, the modified wheat straw biochar (MWBC) was obtained through 40 mesh sieve and encapsulation.\u003c/p\u003e \u003cp\u003eThe specific surface area and pore size of MWBC samples were measured by BET, and the structure and morphology were observed using SEM scanning electron microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Preparation of microbial community immobilized by biochar\u003c/h2\u003e \u003cp\u003ePreparation of biochar-immobilized microbial community was conducted as follows: 0.02gMBC was added to 18mL inorganic salt medium, and then placed in a conical bottle for 20 minutes of sterilization in a sterilizer at 121C. After cooling to room temperature, 2mL of heavy suspension (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1) was added. After mixing, the bacteria were placed in a shaker (30 ℃, 180rpm) and fully oscillated to be loaded on the biochar as much as possible. After 24 hours of adsorption and fixation, the bacteria were removed and transferred to a high-speed centrifuge and centrifuged for 5 minutes under the condition of 4500rpm. Then, the supernatant was removed and washed with sterile water. The MWBC immobilized bacterial community was obtained by repeated centrifugation for 2 times. The fixed time was 4, 8, 12, 16, 20 and 24 hours, respectively. After each group was fixed, MWBC immobilization was transferred to 5min in 20mL aseptic water, then 10min was placed statically, the supernatant was taken, and the supernatant was diluted 10000 times. The number of microorganisms adsorbed was determined by dilution coating plate method to determine the best fixed time. On the basis of the optimal fixed time, the pH values of MSM were 5, 6, 7, 8 and 9, respectively. After each group was fixed, the best pH value was determined by the same method mentioned above. Under the conditions of the best fixed time and the best pH value, the MWBC-immobilized microbial community were prepared for later experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Experiments on BaP removal of microbial community and MWBC-immobilized microbial community\u003c/h2\u003e \u003cp\u003eExperiment on BaP removal of microbial community (Experiment 1): The above bacterial community was inoculated into SOB broth medium (Supplementary Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e) and transferred to a shaker at 30 ℃ for 15 hours. After 5 minutes of centrifugation under the condition of 8000rpm, the culture was washed with inorganic salt medium (MSM) for 3 times, and then re-suspended in MSM. In the BaP removal experiment of microbial community, the initial BaP concentration was 5, 10 and 20mg/L, respectively. The experiments were carried out at 15 ℃, 20 ℃, 25 ℃, 30 ℃ and 35 ℃. The grouping of specific experimental conditions is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and it is named T1-T15 according to different temperatures and different BaP concentrations. Besides, 2ml resuspension inoculant (OD\u003csub\u003e600\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1) was inoculated in 20mLMSM-BaP medium. A blank control group was set up to evaluate abiotic loss, all experiments were designed with 3 parallels, and destructive sampling was carried out after 2, 4, 6, 8, 10 and 12 days to detect the removal of BaP.\u003c/p\u003e \u003cp\u003eExperiment on BaP removal of MWBC-immobilized microbial community (Experiment 2): The experiment was carried out under the same conditions by using MBC-immobilized bacterial community instead of bacteria in free state in Experiment 1. A blank control group was set up to evaluate abiotic loss, and all experiments were designed with 3 parallels. The grouping of specific experimental conditions is shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, and it is named W1-W15 according to different temperatures and different BaP concentrations. After 2, 4, 6, 8, 10 and 12 days, destructive sampling was carried out to detect the removal of BaP..\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGrouping conditions for experiments on BaP removal of microbial community and MWBC-immobilized microbial community\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTemperature (℃)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eBaP concentration (mg/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup name of Experiment 1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup name of Experiment 2\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT7\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT11\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW11\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT13\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW13\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e10\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003eT15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eW15\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Extraction and detection of BaP\u003c/h2\u003e \u003cp\u003eBy taking dichloromethane (Macklin) with the same volume as the culture medium, the samples were extracted and repeated twice. The concentration of BaP was determined using high performance liquid chromatography. The mobile phase was acetonitrile (Macklin) and water, with a ratio of 9:1 (v/v). A C18 column (250 \u0026times; 4.6 mm) was adopted, the injection volume was 10 \u0026micro;L, and the detection wavelength was 290nm. In order to calculate the concentration of BaP, the standard curve of the external standard detection of BaP was drawn, and the correlation coefficient was 0.99.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Detection and analysis of microbial community amplifier and macro genome\u003c/h2\u003e \u003cp\u003eIn the two BaP removal experiments described in Section \u003cspan refid=\"Sec4\" class=\"InternalRef\"\u003e2.2\u003c/span\u003e and \u003cspan refid=\"Sec6\" class=\"InternalRef\"\u003e2.4\u003c/span\u003e, when T10, T11, T12, W10, W11 and W12 were cultured at 30 ℃ to the 6th and 12th day, the cultures were taken for amplification and macrogenomic detection. The samples of T10, T11 and T12 were recorded as B groups, while those of W10, W11 and W12 were recorded as M groups. The groups were named according to the concentration of BaP: B5, B10, B20, M5, M10 and M20, respectively.\u003c/p\u003e \u003cp\u003eThe forward primer for 16srRNA analysis is Bakt_341F (CCTAYGGGRBGCASCAG), and the reverse primer is Bakt_806R (GGACTACNNGGGTATCTAAT).The amplifiers were sequenced and analyzed by Novogene Company (Beijing, China). The process involved DNA and PCR extraction, DNA amplification, library construction, NovaSeq6000 analysis, sequencing and data processing. The DADA2 method (Callahan BJ et al., 2016) was used to reduce the noise of each de-weight sequence to generate ASVs (Amplicon Sequence Variants), and QIIME2's classify-sklearn algorithm (Bokulich NA et al., 2018; Bolyen E et al.,2019) was adopted to annotate the species of each ASV using the pre-trained NaiveBayes classifier. According to the results of ASVs annotation and the characteristic table of each sample, the species abundance table was obtained, and the Alpha diversity index was calculated. The functional genes of the community were annotated and mapped to the Kyoto Encyclopedia of genes and Genomes (KEGG, Version: 2018.01), the function of the homologous group was annotated to the evolutionary pedigree of genes, the unsupervised homologous group (eggNOG, Version: 4.5), and the function and relative abundance of carbohydrate enzymes were annotated to the carbohydrate active enzyme database (CAZy, Version: 2018.01).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and Discussion","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Characterization of biochar\u003c/h2\u003e \u003cp\u003eThe SEM scanning images of MWBC are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. 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 m\u003csup\u003e2\u003c/sup\u003e/g, and the average pore diameter was 87.373cm\u003csup\u003e3\u003c/sup\u003e/g.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Determination of optimal fixed time and pH value\u003c/h2\u003e \u003cp\u003eThe relationship between the number of fixed microorganisms in MWBC and fixed time is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb. 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.3 BaP removal efficiency of microbial community\u003c/h2\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e presents the removal efficiency of different concentrations of BaP by microbial community at different temperatures. For the BaP of 5 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea), 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\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb), 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\u0026ndash;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\u0026ndash;46%. For the BaP of 20 mg/L (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), 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\u0026ndash;30 ℃, the BaP removal efficiency of bacterial community was similar, and finally reached the range of 35\u0026ndash;40%.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAccording to Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, 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\u0026ndash;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\u0026ndash;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\u0026ndash;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\u0026ndash;20 mg/L BaP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.4 BaP removal efficiency of MWBC-immobilized microbial community\u003c/h2\u003e \u003cp\u003eThe removal efficiency of different concentrations of BaP by MWBC-immobilized microbial community at different temperatures is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. For 5 mg/L BaP (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), 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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb), 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\u0026ndash;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.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec), 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\u0026ndash;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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe 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\u0026ndash;35 ℃, MWBC immobilized bacteria performed well in removing 5\u0026ndash;20 mg/L BaP. Especially, the removal effect of 5 mg/L BaP was the best at 30 ℃, and that of 10\u0026ndash;20 mg/L BaP was the best at 35 ℃.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Analysis of microbial community structure\u003c/h2\u003e \u003cp\u003eThe 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\u0026thinsp;\u0026ge;\u0026thinsp;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. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). 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. \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 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. \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Efficient microbial alliance was an important reason to improve the degradation efficiency of BaP(Zhang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea 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. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). 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. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Sun et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). 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. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 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\u0026ndash;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. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2020\u003c/span\u003e; Lee et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). 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. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb depicts the relative abundance of six groups of samples at the family level (the richness was among the top 10). In B groups, \u003cem\u003ePseudomonadaceae\u003c/em\u003e and \u003cem\u003eXanthomonadaceae\u003c/em\u003e were the two kinds of families with the highest relative abundance. The relative abundance of \u003cem\u003ePseudomonadaceae\u003c/em\u003e increased with the increase of BaP concentration, which was 31.40% (B5), 41.30% (B10) and 65.21% (B20), while that of \u003cem\u003eXanthomonadaceae\u003c/em\u003e 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 \u003cem\u003eBacillaceae\u003c/em\u003e, which was about 70%, followed by \u003cem\u003ePseudomonadaceae\u003c/em\u003e, accounting for close to a quarter. In addition to these advantages of the family, there were also \u003cem\u003eAlcaligenaceae\u003c/em\u003e and \u003cem\u003eSphingobacteriaceae\u003c/em\u003e. Both of these two kinds of families have been mentioned in studies and reports on the degradation of PAHs(Mangwani et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Yang et al. \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), having a certain ability to degrade BaP.\u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec 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 \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eStenotrophomonas\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e was similar to that of the family. \u003cem\u003ePseudomonas\u003c/em\u003e is a common genus of PAHs-degrading bacteria(Premnath et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), which can degrade not only LMW-PAHs, but also HMW-PAHs. According to the report, \u003cem\u003ePseudomonas\u003c/em\u003e can effectively biodegrade polycyclic aromatic hydrocarbons and heterocyclic derivatives through the transverse hydrogen peroxide pathway(Liu et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). In addition, \u003cem\u003ePseudomonas\u003c/em\u003e aeruginosa can use biosurfactant(Zang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) and has an anti-heavy metal effect(Safahieh et al. \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which may be the basis of BaP degradation ability. \u003cem\u003eStenotrophomonas\u003c/em\u003e, a genus within the \u003cem\u003eXanthomonadaceae\u003c/em\u003e 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 \u003cem\u003eStenotrophomonas\u003c/em\u003e species possess the capability to utilize PAHs as their sole carbon source for growth. As part of a microbial alliance, \u003cem\u003eStenotrophomonas\u003c/em\u003e 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. \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). 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, \u003cem\u003eStenotrophomonas\u003c/em\u003e 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, \u003cem\u003ePseudomonas\u003c/em\u003e still possessed a high degradation ability to a higher concentration of BaP and occupied a dominant position. \u003cem\u003eBacillus\u003c/em\u003e 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. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), as well as tolerance to heavy metals(Rabani et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). \u003cem\u003eBacillus\u003c/em\u003e exerts a good degradation effect on PAHs such as BaP, BaA, Pyr and heavy crude oil(Kong et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). In the case of microbial alliance, whether the combination of \u003cem\u003eBacillus\u003c/em\u003e licheniformis and \u003cem\u003eBacillus\u003c/em\u003e mojavensis of the same genus(Eskandary et al. \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), or the interaction with other genus microorganisms(Jacques et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), PAHs removal becomes more efficient. Herein, in M groups, \u003cem\u003eBacillus\u003c/em\u003e was always the dominant genus, indicating that the environment and nutrition provided by biochar could be preferentially utilized by \u003cem\u003eBacillus\u003c/em\u003e, and then cooperate with \u003cem\u003ePseudomonas\u003c/em\u003e to effectively degrade BaP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Metagenomic analysis\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003e3.6.1 Metagenomic analysis of eggNOG\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea 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.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea is given in Supplementary Table S3). The results demonstrated Amino acid transport and metabolism (7.28\u0026ndash;7.47%) and Signal transduction mechanisms (6.04\u0026ndash;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. \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), the relative abundance of Replication, recombination and repair (4.67\u0026ndash;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\u0026ndash;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 \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Zang et al. \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). They are generally produced by secondary metabolites of organisms(Nie et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2012\u003c/span\u003e), which can improve the solubility, bioavailability and biodegradability of BaP, i.e., a hydrophobic compound, thereby causing emulsification in the process of BaP extraction.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb 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\u0026thinsp;\u0026gt;\u0026thinsp;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 \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), 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 \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). 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. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), which might promote bacterial utilization of BaP.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003e3.6.2 Metagenomic analysis of CAZy\u003c/h2\u003e \u003cp\u003eCAZys 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. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). These enzymes also play an important role in the degradation of BaP. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, 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. \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; van der Maarel and Leemhuis \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). 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. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). 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.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFigure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb shows the level 2 data analysis of CAZy at the family. The two enzyme types with the highest relative abundance were GT2 (\u0026gt;\u0026thinsp;8%) and GT4 (\u0026gt;\u0026thinsp;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. \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). 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. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). 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. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), 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. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). 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. \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), a laccase from \u003cem\u003eBacillus\u003c/em\u003e subtilis could efficiently oxidize BaP and anthracene. To sum up, the synergism between different CAZys was beneficial to the improvement of BaP removal efficiency.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e \u003ch2\u003e3.6.3 Metagenomic analysis of KEGG\u003c/h2\u003e \u003cp\u003eKEGG 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. \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2022\u003c/span\u003e). 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. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Bezza and Chirwa \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; 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. \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Some microorganisms such as \u003cem\u003ePseudomonas\u003c/em\u003e 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.\u003c/p\u003e \u003cp\u003eAccording 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.\u0026nbsp;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.\u003c/p\u003e \u003cp\u003eAccording 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\u003csup\u003e+\u003c/sup\u003e 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. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2022\u003c/span\u003e), 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).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eIn this study, a highly efficient microbial community capable of removing benzo[a]pyrene (BaP) was enriched from oil-contaminated soil. Upon immobilization with modified wheat straw biochar, this microbial community exhibited a remarkable BaP removal efficiency of up to 75.18% within 12 days in an inorganic salt medium containing 5\u0026ndash;20 mg/L BaP. Meanwhile, the microbial community was observed and studied by 16SrRNA sequencing and metagenomic analysis, and the results demonstrated \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eStenotrophomonas\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e as the main genus in the microbial community. Additionally, the function and relative abundance of genes were annotated by eggNOG, CAZy and KEGG databases to provide insights into why the bacterial community had the ability to degrade BaP, and to study the metabolic pathway of bacterial community to BaP.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData Availability Statement:\u003c/strong\u003e All data analyzed are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInstitutional Review Board Statement:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eInformed Consent Statement:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e The authors agree to participate.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e The authors agree to publish.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u0026nbsp;\u003c/strong\u003eConceptualization, X.C.; methodology, X.C., M.G and J.Z.; validation, X.C. and R.M.; formal\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eanalysis, X.C.; writing\u0026mdash;original draft preparation, X.C.; writing\u0026mdash;review and editing, R.M., M.G and J.Z.; project administration, Y.P.; funding acquisition, Y.P. All authors have read and agreed to the\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003epublished version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research was funded by the National Key R\u0026amp;D Program of China (No. 2020YFC1808802).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting Interests:\u003c/strong\u003e The authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAbdel-Mawgoud AM, Lepine F, Deziel E (2010) Rhamnolipids: diversity of structures, microbial origins and roles. Appl Microbiol Biotechnol. 86(5): 1323-1336. 10.1007/s00253-010-2498-2\u003c/li\u003e\n\u003cli\u003eAkash S, Sivaprakash B, Rajamohan N, Selvankumar T (2023) Biotransformation as a tool for remediation of polycyclic aromatic hydrocarbons from polluted environment - review on toxicity and treatment technologies. 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Journal of Hazardous Materials. 419: 126524. 10.1016/j.jhazmat.2021.126524\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Degradation of benzo[a]pyrene, Biochar immobilized microorganisms, Macrogenomic analysis, Microbial community","lastPublishedDoi":"10.21203/rs.3.rs-4052065/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4052065/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBenzo[a]pyrene, a high-molecular-weight polycyclic aromatic hydrocarbon, is a prevalent organic pollutant. In this study, a microbial community with high efficiency of BaP degradation was enriched in oil-contaminated soil, and the microbial community was immobilized using modified wheat straw biochar. The highest removal efficiency of 5\u0026ndash;20 mg/L BaP was 75.18% in 12 days. Through 16SrRNA sequencing, \u003cem\u003ePseudomonas\u003c/em\u003e, \u003cem\u003eStenotrophomonas\u003c/em\u003e and \u003cem\u003eBacillus\u003c/em\u003e were found to be the dominant bacteria in the community. Additionally, metagenomic annotation revealed the gene function and metabolic pathway of the microbial community during BaP degradation.\u003c/p\u003e","manuscriptTitle":"Removal of benzo[a]pyrene by a highly degradable microbial community immobilized by modified wheat straw biochar","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-01 15:11:54","doi":"10.21203/rs.3.rs-4052065/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major Revision","date":"2024-04-26T02:54:49+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-28T09:29:44+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-27T17:07:28+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Environmental Science and Pollution Research","date":"2024-03-25T13:30:01+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-18T05:07:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Science and Pollution Research","date":"2024-03-14T01:54:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-science-and-pollution-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"espr","sideBox":"Learn more about [Environmental Science and Pollution Research](https://www.springer.com/journal/11356)","snPcode":"11356","submissionUrl":"https://submission.nature.com/new-submission/11356/3","title":"Environmental Science and Pollution Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"45387fda-eb5d-4d8b-a76f-d075e61aeb4c","owner":[],"postedDate":"April 1st, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-12-09T16:02:00+00:00","versionOfRecord":{"articleIdentity":"rs-4052065","link":"https://doi.org/10.1007/s11356-024-35717-1","journal":{"identity":"environmental-science-and-pollution-research","isVorOnly":false,"title":"Environmental Science and Pollution Research"},"publishedOn":"2024-12-06 15:57:28","publishedOnDateReadable":"December 6th, 2024"},"versionCreatedAt":"2024-04-01 15:11:54","video":"","vorDoi":"10.1007/s11356-024-35717-1","vorDoiUrl":"https://doi.org/10.1007/s11356-024-35717-1","workflowStages":[]},"version":"v1","identity":"rs-4052065","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4052065","identity":"rs-4052065","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}