A method for enhancing the co-metabolic degradation and molecular response of polycyclic aromatic hydrocarbons (PAHs) based on plant aromatic organic acids (AOAs)

Abstract

The present invention discloses a method for enhancing the co-metabolic degradation and molecular response of polycyclic aromatic hydrocarbons (PAHs) based on plant aromatic organic acids (AOAs). Studies have found that AOAs can significantly increase the abundance of PAHs-degrading functional bacteria such as methylophilus and nitrobacterium by providing an easily available carbon source for plant rhizosphere microorganisms, thereby promoting the biodegradation of PAHs. Among them, monophenolic acids are particularly effective in promoting microbial degradation pathways, while diphenolic acids enhance the adsorption and absorption capacity of plants for PAHs by enhancing plant growth activity, thereby promoting the synergistic removal of PAHs. This method uses precise control of the microorganism-plant combined action method to target and enhance the biodegradation of aromatic pollutants such as PAHs using AOAs, while avoiding the risk of secondary pollution, and has the dual advantages of high efficiency and environmental friendliness.

Description

[0001] The invention relates to the technical field of root exudates, and in particular to a method for enhancing the co-metabolic degradation and molecular response of polycyclic aromatic hydrocarbons (PAHs) by plant aromatic organic acids (AOAs). Background Art

[0002] PAHs are aromatic compounds containing two or more fused benzene ring structures in their molecules, which have strong "three-hazard" toxicity. With the rapid development of urbanization and industry, the use of various mineral fuels such as coal, oil and natural gas has increased, and the organic matter in them generates PAHs under incomplete combustion or other reducing conditions. Such pollutants mainly enter the natural environment through three pathways: soil, water and air, thus directly or indirectly causing harm to microbial communities, animals, plants and humans.

[0003] Common PAHs remediation technologies include physical remediation technology, chemical remediation technology, biological remediation technology and cometabolism remediation technology. In view of the problem that pollution caused by PAHs is difficult to be effectively remediated, traditional cometabolism substrates have problems such as poor specificity and easy to cause secondary pollution. This study intends to use AOAs as the cometabolism substrate of PAHs. The root exudates produced by plants contain AOAs, which are natural carbon sources that can be directly used by microorganisms and have strong specificity of benzene ring structure similar to the target pollutants. On the one hand, AOAs promote the growth and metabolism of microorganisms, enhance the activity of microorganisms, accelerate the growth, and thus increase the accumulation of microbial biomass; on the other hand, AOAs root exudates with specific functional structures can enhance the degradation and mineralization of PAHs in the environment by inducing the growth of specific degradation enzymes.

[0004] This study aims to construct an AOAs-PAHs cometabolism system and study the co-metabolism degradation rules of PAHs in the system and the method of microbial response. Based on this, it is planned to use the main active ingredients of plant natural secretion units, p-hydroxybenzoic acid (PHA), p-coumaric acid (PCA), caffeic acid (CA), and ferulic acid (FA) as representative AOAs, and phenanthrene, which is common in the environment, as the target PAHs pollutant, to carry out the following two aspects of research:

[0005] (1) Degradation rules of PAHs under the synergistic effect of AOAs and microorganisms:

[0006] The experiment took PAHs-contaminated water as the research environment, and four AOAs with different chemical structures as the research objects, and compared the degradation effects of AOAs with different functional structures in water. By analyzing the degradation and removal rate of PAHs and the status of related microbial communities, the degradation law of PAHs under the synergistic effect of AOAs and microorganisms was clarified.

[0007] (2) Response methods of microorganisms under the interaction of AOAs and PAHs:

[0008] Combining high-throughput sequencing and bioinformatics analysis techniques, we analyzed the formation characteristics of microbial biofilms at different growth stages, as well as the community structure and abundance of dominant microbial bacteria and PAHs-degrading functional bacteria. We also integrated the mapping relationship among the functional structure of AOAs, PAHs degradation pathway, and microbial community structure to reveal how microorganisms respond to the AOAs-PAHs co-metabolic process. Summary of the invention

[0009] To achieve the above object, the present invention provides the following technical solution: a method for enhancing the co-metabolic degradation and molecular response of polycyclic aromatic hydrocarbons (PAHs) by plant aromatic organic acids (AOAs), comprising the following steps:

[0010] S1: Experimental design: The reed seedlings used in the experiment were purchased from the local market, and reeds with similar size, fresh weight, and root length were selected as experimental subjects;

[0011] S2: Sample collection and pretreatment: After the experimental device started running, water samples from different treatment groups were collected continuously every day for ten days; 5 mL of water samples were collected each time for COD, NH4-N, and PAHs concentration experimental analysis, and an equal amount of sterilized Hoagland nutrient solution was added immediately after sampling as a supplement;

[0012] S3: Analytical methods:

[0013] 1): Chemical oxygen demand: COD is measured and analyzed using a water quality analyzer. The water quality analyzer needs to be configured with COD reagents 1 and 3 with appropriate ranges according to the actual COD range of the water sample; the configuration method is as follows:

[0014] a: Reagent 1: Add 90mL of distilled water to a 200mL beaker, then add 10mL of concentrated sulfuric acid along the wall of the beaker while stirring continuously, then pour the COD reagent 1 powder bag into it to dissolve, and after cooling, put it into a reagent bottle for later use;

[0015] b: Reagent 3: Place the whole package of COD reagent 3 in a 500mL narrow-necked brown bottle, add 400mL of concentrated sulfuric acid, place in a dark place to dissolve, and set aside;

[0016] Determination and analysis of water samples: dilute the COD concentration in the water sample by 10 times, take 2 ml into a test tube and add 0.5 ml of reagent 1 and 2 ml of reagent 3 in sequence, then test on the machine, and convert the analysis result back to the concentration before dilution, which is the COD concentration in the water sample;

[0017] 2): Ammonia nitrogen: NH4-N in water is also measured and analyzed using a water quality analyzer; according to the range of NH4-N in the water sample, take 0.5mL of water sample and add 4.5mL of distilled water to a test tube, add 0.1mL of NH4-N reagent 1 and 0.15mL of NH4-N reagent 2 in sequence, cover and shake well, let it stand for 10 minutes to allow it to develop color, and then test it on the machine. The test result on the machine is the concentration of NH4-N in the water sample;

[0018] 3): Polycyclic aromatic hydrocarbons:

[0019] The concentration of PAHs in water samples was determined by high performance liquid chromatography;

[0020] S4: Microbial community analysis method: A total of 15 root samples from different treatment groups were collected, and the DNA in the water samples was extracted using the PowerSoilDNA Isolation Kit. The concentration and purity of the DNA were detected using a Nano Drop spectrophotometer.

[0021] S5: Numerical calculation: The calculation formulas for the pollutant removal rate constant (Kk, / d) and removal load (RL, mg / d) used in the experiment are as follows: and RL In the formula, K is the removal rate constant of the pollutant, Ct is the concentration of the pollutant in the water sample at time t (mg / L), RL is the removal load of the pollutant, and V is the volume of the water sample (L);

[0022] S6: Data processing and analysis: After the experiment, the data results were processed and analyzed using EXCEL software. The mean value, standard error, pollutant removal rate constant, etc. of each indicator data were calculated, and the differences between the data were analyzed using the one-way analysis of variance method using SPSS software.

[0023] Preferably, before the experiment begins, the soil attached to the roots of the reed seedlings is cleaned with tap water, then rinsed twice with ultrapure water, and the reed seedlings are transferred to a conical flask containing 300 mL of Hoagland nutrient solution, which contains 1 mg / L phenanthrene, a typical representative of PAHs, and 50 mg / L AOAs. The five experimental groups are placed in an artificial biochemical incubator for culture (air temperature 25±1°C, humidity 80%, light intensity 8000Lx, and light 12h / d), and the body of the conical flask is protected from light with tin foil; three parallels are set up in each group, and the AOAs are heated and dissolved in the nutrient solution using a magnetic heating stirrer (SUNNE SNMS-H).

[0024] Preferably, in step 2, in order for the sample to better reflect the actual situation in the water body, a vortex oscillator is used to mix the water sample before sampling, wherein 1.5 mL of the water sample used for PAHs concentration analysis is filtered through a 0.22 μm syringe filter, and the substance on the filter membrane is eluted with methanol solvent and frozen for analysis.

[0025] Preferably, the chromatographic analysis conditions in step 3 are: XTERRA MS C18 chromatographic column (4.6×250mm, 5μm); injection volume 15μL; flow rate 1 mL / min; DAD detection wavelength 254nm; column temperature 40°C; standard curve preparation: take 7 injection vials, add 0, 0.10, 0.20, 0.25, 0.50, 0.75, and 1.0mL of 1mg / L phenanthrene solution in sequence, dilute to 1m with methanol, shake and evenly shake, use methanol as the reference solution, measure the peak area of ​​phenanthrene on the machine, and draw a standard curve with the peak area as the ordinate and the corresponding phenanthrene content as the abscissa.

[0026] Preferably, in step 4, primers 341F and 806R are used to amplify the V3-V4 region of the bacterial 16S rRNA sequence, the PCR reaction system is 50 μL, 2xPremix Taq 25 μL, 341F / 806R primers 1 μL each, DNA 3 μL, Nuclease-free water 20 μL, and the reaction conditions are: 98°C pre-denaturation for 1 min, 98°C denaturation for 10 s, 50°C annealing for 30 s, 72°C extension for 30 s, a total of 30 cycles, and then extended at 72°C for 5 min, and finally stored at 4°C, and finally sequenced using the Illumina HiSeq 2500 platform.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The present invention has found that AOAs can provide an easily available carbon source for plant rhizosphere microorganisms, significantly increase the abundance of PAHs-related degradation bacteria (such as methylophilic bacteria and nitrobacterium and other PAHs degradation functional bacteria), thereby promoting the biodegradation of PAHs. In particular, the promoting effect of monophenolic acids (such as PCA) on the microbial degradation pathway is particularly significant, and this phenomenon is most obvious in the group, while diphenolic acids (such as CA) can enhance the plant growth activity by enhancing the adsorption and absorption capacity of plants for PAHs, thereby promoting the synergistic overall removal effect of PAHs. This method can be seen from the precise regulation of the microbial-plant joint action method that while using AOAs to target and enhance the biodegradation of aromatic organic pollutants such as PAHs, it avoids the risk of secondary pollution and has the dual advantages of high efficiency and environmental friendliness. It is highly targeted to target pollutants and does not produce secondary pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is the technical circuit diagram of the present invention.

[0030] Figure 2 The COD removal rate (a) and removal load (b) of the present invention.

[0031] Figure 3 NH4-H removal rate (a) and removal load (b) of the present invention.

[0032] Figure 4 The phenanthrene removal rate (a) and removal load (b) of the present invention.

[0033] Figure 5 This is the Alpha diversity analysis of the rhizospheric bacterial community of reed under different AOAs treatments of the present invention.

[0034] Figure 6 This is the PCoA analysis of the reed rhizosphere bacterial community under different AOAs treatments based on the Weighted Unifrac (a) and Unweighted Unifrac (b) distances in the present invention.

[0035] Figure 7 This is the distribution of bacterial communities at the phylum classification level in the rhizosphere samples of the present invention.

[0036] Figure 8 This is the distribution of bacterial communities at the genus classification level in the rhizosphere samples of the present invention.

[0037] Fig. 9 This is the ingredient list of Hoagland nutrient solution used in the experiments of the present invention.

[0038] Fig.10 A grouping table was designed for the experiment of the present invention.

[0039] Fig.11 This is a phenotypic abundance table of the samples of the present invention. DETAILED DESCRIPTION

[0040] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0041] See also Figure 1-11 The present invention provides a technical solution: a method for enhancing the co-metabolic degradation and molecular response of polycyclic aromatic hydrocarbons (PAHs) by plant aromatic organic acids (AOAs), comprising the following steps:

[0042] S1: Experimental design: The reed seedlings used in the experiment were purchased from the local market, and reeds with similar size, fresh weight, and root length were selected as experimental subjects;

[0043] S2: Sample collection and pretreatment: After the experimental device started running, water samples from different treatment groups were collected continuously every day for ten days (n=10); 5mL of water samples were collected each time for COD, NH4-N, and PAHs concentration experimental analysis, and an equal amount of sterilized Hoagland nutrient solution was added immediately after sampling as a supplement;

[0044] S3: Analytical methods:

[0045] 1): Chemical oxygen demand: COD is measured and analyzed using a water quality analyzer (YCNPN-4H, JINZHUANG, Shanghai). The water quality analyzer needs to be configured with COD reagents 1 and 3 with appropriate ranges according to the actual COD range of the water sample; the configuration method is as follows:

[0046] a: Reagent 1: Add 90mL of distilled water to a 200mL beaker, then add 10mL of concentrated sulfuric acid (analytical grade) along the wall of the beaker while stirring continuously, then pour the powder bag of COD reagent 1 into it to dissolve, and after cooling, put it into a reagent bottle for later use;

[0047] b: Reagent 3: Place the whole package of COD reagent 3 in a 500mL narrow-necked brown bottle, add 400mL of concentrated sulfuric acid (analytical grade), place in a dark place to dissolve, and set aside;

[0048] Determination and analysis of water samples: dilute the COD concentration in the water sample by 10 times, take 2 ml into a test tube and add 0.5 ml of reagent 1 and 2 ml of reagent 3 in sequence, then test on the machine, and convert the analysis result back to the concentration before dilution, which is the COD concentration in the water sample;

[0049] 2): Ammonia nitrogen: NH4-N in water is also measured and analyzed using a water quality analyzer; according to the range of NH4-N in the water sample, take 0.5mL of water sample and add 4.5mL of distilled water to a test tube, add 0.1mL of NH4-N reagent 1 and 0.15mL of NH4-N reagent 2 in sequence, cover and shake well, let it stand for 10 minutes to allow it to develop color, and then test it on the machine. The test result on the machine is the concentration of NH4-N in the water sample;

[0050] 3): Polycyclic aromatic hydrocarbons:

[0051] The concentration of PAHs in water samples was determined by high performance liquid chromatography (HPLC; 1260 InfinityⅡ, Agilent, USA);

[0052] S4: Microbial community analysis method: A total of 15 root samples from different treatment groups were collected, and the DNA in the water samples was extracted using the PowerSoilDNA Isolation Kit. The concentration and purity of the DNA were detected using a Nano Drop spectrophotometer.

[0053] S5: Numerical calculation: The calculation formulas for the pollutant removal rate constant (Kk, / d) and removal load (RL, mg / d) used in the experiment are as follows: and RL In the formula, K is the removal rate constant of the pollutant, Ct is the concentration of the pollutant in the water sample at time t (mg / L), RL is the removal load of the pollutant, and V is the volume of the water sample (L);

[0054] S6: Data processing and analysis: After the experiment, the data results were processed and analyzed using EXCEL software. The mean value, standard error, pollutant removal rate constant, etc. of each indicator data were calculated, and the differences between the data were analyzed using the one-way analysis of variance method using SPSS software.

[0055] In the present invention, before the experiment begins, the soil attached to the roots of the reed seedlings is cleaned with tap water, then rinsed twice with ultrapure water, and the reed seedlings are transferred to a conical flask containing 300 mL of Hoagland nutrient solution, which contains 1 mg / L of phenanthrene, a typical representative of PAHs, and 50 mg / L of AOAs. Five experimental groups are placed in an artificial biochemical incubator for culture (air temperature 25±1°C, humidity 80%, light intensity 8000Lx, and light 12h / d), and the body of the conical flask is protected from light with tin foil; three parallel experiments are set up for each group, and the AOAs are dissolved in the nutrient solution by heating with a magnetic heating stirrer (SUNNE SNMS-H).

[0056] In the present invention, in order to make the sample better reflect the real situation in the water body, a vortex oscillator is required to mix the water sample before sampling, wherein 1.5 mL of the water sample used for PAHs concentration analysis needs to be filtered through a 0.22 μm syringe filter, and the substance on the filter membrane is eluted with methanol solvent and frozen for analysis.

[0057] In the present invention, the chromatographic analysis conditions are as follows: an XTERRA MS C18 chromatographic column (4.6×250 mm, 5 μm) is selected; the injection volume is 15 μL; the flow rate is 1 mL / min; the DAD detection wavelength is 254 nm; the column temperature is 40°C; standard curve preparation: 7 injection vials are taken, 0, 0.10, 0.20, 0.25, 0.50, 0.75, and 1.0 mL of 1 mg / L phenanthrene solution are added in sequence, diluted to 1 m with methanol, shaken and evenly mixed, methanol is used as a reference solution, the peak area of ​​phenanthrene is measured on a machine, and a standard curve is drawn with the peak area as the ordinate and the corresponding phenanthrene content as the abscissa.

[0058] In the present invention, primers 341F and 806R are used to amplify the V3-V4 region of the bacterial 16S rRNA sequence. The PCR reaction system is 50 μL, 2xPremix Taq 25 μL, 341F / 806R primers 1 μL each, DNA 3 μL, Nuclease-free water 20 μL, and the reaction conditions are: 98°C pre-denaturation for 1 min, 98°C denaturation for 10 s, 50°C annealing for 30 s, 72°C extension for 30 s, a total of 30 cycles, and then extended at 72°C for 5 min, and finally stored at 4°C. Finally, the Illumina HiSeq 2500 platform was used for sequencing.

[0059] Results and Discussion:

[0060] 1. Co-metabolism of AOAs and PAHs in the rhizosphere of reed;

[0061] a. Removal of COD under the action of AOAs, such as Figure 2 As shown in the figure, COD removal rate constant and removal load under different AOAs treatments are shown. The treatments of the five experimental groups are ①+FA, ②+CA, ③+PCA, ④+PHA, and ⑤CK. In the results of water quality parameter COD, the removal rate constant and removal load of COD in all experimental groups with added AOAs (AOAs groups) are lower than those in the CK group (6.56 / d, 59.70 mg / d), and the order of the removal rate constant and removal load of COD in the AOAs groups is: CA group (6.55 / d, 59.54mg / d)>PHA group (6.54 / d, 58.86mg / d)>FA group (6.52 / d, 56.96mg / d)>PCA group (6.49 / d, 53.33mg / d). From the results, CA and PHA have better effects on removing COD from water than FA and PCA, but according to the results of variance analysis, there is no significant difference among the experimental groups. It is speculated that there is no direct relationship between AOAs and changes in COD content in water bodies.

[0062] b. Removal of NH4-H under the action of AOAs, such as Figure 3The results of hydration parameter NH4-H in the experimental group are shown in Figure 2. The removal rate constants and removal loads of all AOAs groups were significantly higher than those of the CK group (5.01 / d, 2.18 mg / d). Among them, the removal rate constants and removal loads of the CA group (5.42 / d, 8.96 mg / L) were the highest, and the removal effect of NH4-H was the best. Although the FA group (5.09 / d, 2.96 mg / L), PCA group (5.10 / d, 5.80 mg / L), and PHA group (5.10 / d, 4.20 mg / L) The removal rates of NH4-H were similar, but the PCA group and the PHA group had higher removal loads. As a pollution-tolerant wetland plant, reed itself had a good removal effect on NH4-H pollution. This showed that during the experiment, the plants were able to grow normally and were not stressed by NH4-H pollution. In addition, due to the presence of AOAs, the initial contents of COD and NH4-H in the water increased. Microorganisms effectively utilized the added carbon source, thereby promoting the growth of NH4-H-related microbial communities and accelerating the decomposition rate of pollutants by microorganisms in the water.

[0063] c. Removal of phenanthrene under the action of AOAs, such as Figure 4 The results show that different AOAs have great differences in the rate of plant removal of phenanthrene pollution: among all the treatment groups, the best treatment effect was in the CA group (0.19-0.24 / d), the removal effect of the PHA group (0.14-0.18 / d) was similar to that of the CK group (0.15-0.19 / d), while the treatment effects of the FA group (0.13-0.15 / d) and the PCA group (0.09-0.14 / d) were not as good as that of the CK group. It is known that microorganisms in plants can both inhibit and amplify the toxicity of phenolic acids, depending on whether the phenolic acids can become a carbon source for nitrogen-fixing bacteria and be used by them. Through research, it was found that some phenolic acids can weaken the allelopathic effect of reeds, including FA and PCA with strong response characteristics. These substances threaten the activity of plants and the reduction and removal of the pollutant phenanthrene. CA and PHA have weak inhibitory ability on the allelopathic effect of plants. As low-molecular organic acids, they can improve the bioavailability of the pollutant phenanthrene, making phenanthrene easier to remove.

[0064] Figure 4b is the removal load of polluted phenanthrene. The pollution removal load of CK group (34.92 mg / L) was significantly higher than that of AOAs treatment group. It is speculated that this may be because CK group was not stressed by AOAs, resulting in higher adsorption of pollutant phenanthrene by plants than other treatment groups, thereby increasing the removal load of CK group. The removal loads of AOAs treatment groups were as follows: CA group (33.42 mg / L) > PHA group (30.04 mg / L) > PCA group (28.37 mg / L) > FA group (25.34 mg / L). The high removal rate of phenanthrene in CA group matched the higher removal load of this group, while FA group and PCA group had similar removal rates, but PCA group had a higher removal load. By comparing the functional structures of AOAs, CA group with high removal rate and high removal load had a vicinal dihydroxy structure. Aromatic organic acids with this structure can better improve the removal of organic matter compared with monohydroxy structures (PHA, PCA and CA).

[0065] 2. Diversity and community composition of rhizosphere microorganisms in reed under co-metabolism

[0066] a. Analysis of Aplha diversity of reed rhizosphere microbial community:

[0067] Alpha diversity is used to analyze the diversity of microbial communities within a sample (within-community). The diversity analysis of a single sample (Alpha diversity) can reflect the richness and diversity of the microbial community within the sample. The Alpha diversity analysis of this experiment uses the Chao1 index ( Figure 5 a) and Shannon index ( Figure 5 b) The higher the specific value, the higher the richness and diversity of the microbial community. Figure 5 It is obvious that the chao1 index in the PCA group is significantly lower than that in other treatment groups. The presence of AOAs did not promote the growth of the microbial community in this group, but inhibited the normal growth of the microbial community. This corresponds to the reason why the phenanthrene removal rate constant of the PCA group is low. Among all the aromatic organic acid groups, the FA group has a higher chao1 index and Shannon index, but the removal rate constant and removal load of the phenanthrene pollutant in the FA group in the previous article are lower. This may be because the strengthening effect of FA inhibits the growth of plants, resulting in a reduction in the adsorption of phenanthrene by plants, but at the same time, as a carbon source for microorganisms, it promotes the growth of microbial communities. The CK group has the highest average value of each index in each treatment group because there is no stress from AOAs on plants. This shows that the growth of the microbial community in the CK group is the best, but the good growth of the microbial community does not mean that the degradation effect of the pollutant phenanthrene is the best. In order to determine the degradation effect of microorganisms on phenanthrene, subsequent analysis of PAHs degrading bacteria is required.

[0068] b. Beta diversity analysis of reed rhizosphere microbial community:

[0069] Beta diversity is a comparative analysis of the composition of microbial communities in different samples. First, based on the species annotation results and OTUs abundance information of all samples, the OTUs information of the same classification is merged to obtain a species abundance information table. At the same time, the phylogenetic relationship between OTUs is used to further calculate the Unifrac distance. Unifrac distance is a method of calculating the distance between samples using the evolutionary information between microbial sequences in each sample. For more than two samples, a distance matrix is ​​obtained. Then, the OTUs abundance information is used to further construct the Weighted Unifrac distance for the Unifrac distance.

[0070] Depend on Figure 6 It can be seen that the AOAs groups tend to cluster in the Unifrac distance matrix. Different AOAs treatments have a significant effect on the Beta diversity of the reed rhizosphere microbial community, and the community structure between the AOAs groups (PHA group, PCA group, CA group, FA group) and the CK group is quite different.

[0071] c. Structure and composition of reed rhizosphere microbial community:

[0072] The top ten dominant bacterial communities in each group at the phylum level were selected for analysis. The distribution of bacterial communities in each group is as follows: Figure 7 As shown, the dominant bacterial phyla detected were Proteobacteria ( Proteobacteria )、Cyanobacteria( Cyanobacteria ), Bacteroidetes ( Bacteroidetes ), Firmicutes ( Firmicutes ), Chloroflexi ( Chloroflexi ), Actinobacteria ( Actinobacteria ), these dominant phyla account for up to 99% of the microbial community, among which Proteobacteria ( Proteobacteria ) accounted for about 60%-66% of each experimental group, the largest proportion in all microbial communities, which is similar to the distribution of this phylum in wetland plants. From a horizontal comparison, the abundance of Proteobacteria and Bacteroidetes in the experimental group with AOAs added was significantly higher than that in the CK group. As a kind of eutrophic bacteria, Proteobacteria and Bacteroidetes can effectively use aromatic organic acids as carbon sources, which makes these two bacterial communities have an advantage in the competition for bacterial community growth. Figure 7 The growth of these two bacteria in the AOAs group with sufficient carbon source was better than that in the CK group. However, due to the lack of effective carbon source, the CK group promoted the growth of Chloroflexi in order to resist the stress of PAHs, which is consistent with the characteristic of Chloroflexi that it can absorb organic acid substances in the environment.

[0073] In previous studies, cyanobacteria rarely became the dominant phylum for degrading PAHs, but in the results of this experiment, this phylum showed a relatively obvious advantage in all groups. Cyanobacteria grow vigorously in water bodies rich in nitrogen and phosphorus, and are often used as indicator organisms for eutrophication of water bodies. The massive reproduction of cyanobacteria is likely related to the high NH4-N value of the experimental water body. The growth of cyanobacteria has a benign relationship with other microorganisms. The coexistence of cyanobacteria and manganese-oxidizing bacteria can promote the enhanced removal rate of benzo[b]fluoranthene (a PAHs) in artificial wetlands. Therefore, the higher removal rate of the pollutant phenanthrene in the CA and CK groups is likely due to the reproduction and growth of the cyanobacterial community.

[0074] Based on the species annotation and abundance information of all samples at the genus level, the top 10 genera in abundance were selected and clustered at both the species and sample levels according to their abundance information in each sample. The differences in species genus levels of communities treated with different AOAs were analyzed. The results are shown in Figure 2. Figure 8 As shown, Methylophilus spp. Methylophilus ) is the dominant genus in this experiment, which can use methyl-like substances as its own carbon source for growth. Judging from the performance of this genus in each group, the abundance of methylophilic bacteria in the CK group was lower than that in the AOAs group; and the treatment effects in the AOAs group were: PCA group>PHA group>FA group>CA group, which showed that the AOAs group contained more methyl-like substances produced during the degradation of phenanthrene, providing sufficient carbon source for methylophilic bacteria and promoting the growth of bacterial communities, confirming that aromatic organic acids enhance the degradation of PAHs phenanthrene by reed rhizosphere microorganisms.

[0075] d. Correlation analysis of environmental factors in reed rhizosphere microbial communities:

[0076] The correlation analysis of environmental factors in this experiment was carried out using the Spearman coefficient, which was used to study the relationship between environmental factors and species, and to obtain the correlation and significance between the two. At present, the bacterial species that have the function of degrading PAHs include acidophilus ( Acidovorax ), Arthrobacter ( Ar-throbacter ), Brevibacterium ( Brevibacterium ), Burkholderia spp. Burkholderia ), Comamonas spp. Comamonas ), Mycobacterium ( Mycobacterium ), Pseudomonas, Sphingomonas Sphingomonas ) etc. In this experiment, the removal of phenanthrene was similar to that of Nitroflavubacillus ( Diaphorobacter ) showed a strong correlation, and the genus belongs to the Comamonasaceae ( Comamonadaceae) of the genus Nitroflavobacterium, whose main characteristics are: Gram-negative, straight rod-shaped cells, monopolar hairs, and oxidase-positive. Studies have shown that Nitroflavobacterium can remove certain benzene derivatives and plays a major role in the biodegradation of phenanthrene. Asticcacaulis , Acetobacteroides , Azospirillum The bacterial species and the environmental factor COD showed a strong correlation, but the current study did not find that these bacterial species had a degradation effect on PAHs, and it was difficult to determine the effect of COD on phenanthrene removal. The environmental factor NH4-H did not have a significant effect on the microbial community. Thermomonas genus ( Thermomonas ) is strongly correlated with the root length of the plant, indicating that the more developed the rhizosphere of the plant, the better the growth of this genus of bacteria. This genus and Nitroflavobacterium have been proven to be bacteria that can efficiently degrade phenanthrene, which shows that the growth of the rhizosphere has a certain degree of influence on the removal of phenanthrene.

[0077] e. Function prediction of PAHs-degrading bacteria:

[0078] The PAHs degradation functional bacteria in this experiment were predicted and analyzed using BugBase. Fig.11 The abundance of seven microbial phenotypes in each experimental group is shown. The PCA group with the best phenanthrene degradation effect has the highest abundance of Gram-negative bacteria compared with other experimental groups. Methylophilus and Nitroflavubacillus mentioned above also belong to Gram-negative bacteria, which shows that the degradation of phenanthrene is closely related to the abundance of Gram-negative bacteria. The CA group with the best phenanthrene removal effect did not stand out significantly in the abundance of the seven microbial phenotypes, indicating that the high removal rate of the CA group is not caused by microbial action. The abundance of facultative anaerobic bacteria is the highest in the PCA group and the PHA group, and their degradation effect on phenanthrene is also the most obvious. It can be inferred that there is a certain connection between the abundance of facultative anaerobic bacteria and the removal of the pollutant phenanthrene, that is, the phenanthrene-degrading bacteria are mostly facultative anaerobic bacteria. Although the FA group has a certain impact on different microbial phenotypes, there is no obvious regularity.

[0079] in conclusion:

[0080] (1) Through the experiment of reed hydroponics, it was found that the removal effect of PAHs in the CA group was the best. The removal effects of the PHA group and the CK group were similar. The removal effects of the PCA group and the FA group were even lower than that of the CK group. However, this does not reflect the actual degradation effect of AOAs on PAHs. The removal of PAHs includes root surface adsorption and biodegradation. Generally, PAHs are adsorbed by the root surface of plants and then biodegraded. CA, as a diphenolic acid, can promote the growth of the reed rhizosphere. More PAHs are adsorbed on the root surface of the reed, which greatly reduces the PAHs in the water. Therefore, PAHs are not removed from the water body through biodegradation. This can be confirmed by the analysis of microbial phenotypic abundance. The other experimental groups FA, PCA and PHA did not promote the removal of PAHs in the water. It may be because the monohydroxyl group inhibits plant growth, thereby reducing the adsorption of PAHs by plants, resulting in the phenomenon that the removal of PAHs by the diphenolic acid groups (PCA group, PHA group, FA group) is lower than that of the CK group.

[0081] (2) In this experiment, AOAs, as an exogenous carbon source, promoted the secretion of multifunctional degradation enzymes by PAHs-related degrading bacteria with similar structures, and then decomposed PAHs through the cleavage pathway, thereby promoting the co-metabolic degradation of PAHs and AOAs. Proteobacteria and Bacteroidetes were the dominant phyla of rhizosphere microorganisms and also the dominant phyla for PAHs degradation, while Cyanobacteria could promote the growth of PAHs-related degrading bacteria, Methylophilus ( Methylophilus ) is a dominant species in the microbial community and has the function of PAHs degradation. After the addition of exogenous AOAs, the abundance of this species was increased, among which the abundance of methylophilic bacteria in the PCA group was most significantly increased, which indicates that the AOAs-PAHs co-metabolism system promotes the growth of PAHs-related degradation species and enhances the biodegradation of PAHs by microorganisms.

[0082] (3) The diphenolic acid CA can enhance the abundance of rhizosphere microbial communities by promoting plant growth, but it does not increase the abundance of phenanthrene-related degrading bacteria, and therefore cannot promote the biodegradation of the pollutant phenanthrene. Although the removal effect of monophenolic acids (PCA, PHA, FA) is not as good as that of diphenolic acids, in fact, the non-specific enzymes released by monophenolic acids increase the abundance of related PAHs-degrading bacteria such as Methylophilus and Nitroflavobacterium, thereby degrading the pollutant phenanthrene through the pathway of co-metabolism. This shows that the type of AOAs affects the growth of plants and the structure of rhizosphere microorganisms, resulting in different degradation behaviors of polycyclic aromatic hydrocarbons.

[0083] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for enhancing the co-metabolic degradation and molecular response of polycyclic aromatic hydrocarbons (PAHs) by plant aromatic organic acids (AOAs), comprising the following steps, characterized in that: S1: Experimental design: The reed seedlings used in the experiment were purchased from the local market, and reeds with similar size, fresh weight, and root length were selected as experimental subjects; S2: Sample collection and pretreatment: After the experimental device started running, water samples from different treatment groups were collected continuously every day for ten days; 5 mL of water sample was collected each time for chemical oxygen demand COD, ammonia nitrogen NH4-N, and PAHs concentration experimental analysis, and an equal amount of sterilized Hoagland nutrient solution was added immediately after sampling as a supplement; S3: Analytical methods: 1): Chemical oxygen demand: COD is measured and analyzed using a water quality analyzer. The water quality analyzer needs to be configured with COD reagents 1 and 3 with appropriate ranges according to the actual COD range of the water sample; the configuration method is as follows: a: Reagent 1: Add 90mL of distilled water to a 200mL beaker, then add 10mL of concentrated sulfuric acid along the wall of the beaker while stirring continuously, then pour the COD reagent 1 powder bag into it to dissolve, and after cooling, put it into a reagent bottle for later use; b: Reagent 3: Place the whole package of COD reagent 3 in a 500mL narrow-necked brown bottle, add 400mL of concentrated sulfuric acid, place in a dark place to dissolve, and set aside; Determination and analysis of water samples: dilute the COD concentration in the water sample by 10 times, take 2 ml into a test tube and add 0.5 ml of reagent 1 and 2 ml of reagent 3 in sequence, then test on the machine, and convert the analysis result back to the concentration before dilution, which is the COD concentration in the water sample; 2): Ammonia nitrogen: NH4-N in water is also measured and analyzed using a water quality analyzer; according to the range of NH4-N in the water sample, take 0.5mL of water sample and add 4.5mL of distilled water to a test tube, add 0.1mL of NH4-N reagent 1 and 0.15mL of NH4-N reagent 2 in sequence, cover and shake well, let it stand for 10 minutes to allow it to develop color, and then test it on the machine. The test result on the machine is the concentration of NH4-N in the water sample; 3): Polycyclic aromatic hydrocarbons: The concentration of PAHs in water samples was determined by high performance liquid chromatography; S4: Microbial community analysis method: A total of 15 root samples were collected from different treatment groups, and the DNA in the water samples was extracted using the Power Soil DNA Isolation Kit. The concentration and purity of the DNA were detected using a Nano Drop spectrophotometer. S5: Numerical calculation: The calculation formulas for the pollutant removal rate constant and removal load used in the experiment are as follows: and RL In the formula, K is the removal rate constant of the pollutant, Ct is the concentration of the pollutant at time t, RL is the removal load of the pollutant, and V is the volume of the water sample; S6: Data processing and analysis: After the experiment, the data results were processed and analyzed using EXCEL software. The mean value, standard error, and pollutant removal rate constant of each indicator data were calculated. SPSS software was used to perform one-way analysis of variance on the differences between the data.

2. The method for enhancing the co-metabolic degradation and molecular response of polycyclic aromatic hydrocarbons (PAHs) by plant aromatic organic acids (AOAs) according to claim 1, characterized in that: Before the experiment, the soil attached to the roots of the reed seedlings was cleaned with tap water, and then rinsed twice with ultrapure water. The reed seedlings were transferred to a conical flask containing 300 mL of Hoagland's nutrient solution, which contained 1 mg / L phenanthrene, a typical representative of polycyclic aromatic hydrocarbons, and 50 mg / L AOAs. The five experimental groups were cultured in an artificial biochemical incubator, and the body of the conical flask was protected from light with tin foil; three parallel sets were set up in each group, and the AOAs were dissolved in the nutrient solution using a magnetic heating stirrer.

3. The method for enhancing the co-metabolic degradation and molecular response of polycyclic aromatic hydrocarbons (PAHs) by plant aromatic organic acids (AOAs) according to claim 1, characterized in that: In order to make the samples better reflect the actual situation in the water body, a vortex oscillator is needed to mix the water sample before sampling. Among them, 1.5mL of water sample used for PAHs concentration analysis needs to be filtered through a 0.22μm syringe filter, and the substances on the filter membrane are eluted with methanol solvent and frozen for analysis.

4. The method for enhancing the co-metabolic degradation and molecular response of polycyclic aromatic hydrocarbons (PAHs) by plant aromatic organic acids (AOAs) according to claim 1, characterized in that: Chromatographic analysis conditions: C18 chromatographic column; injection volume 15 μL; flow rate 1 mL / min; DAD detection wavelength 254 nm; column temperature 40°C; standard curve preparation: take 7 injection vials, add 0, 0.10, 0.20, 0.25, 0.50, 0.75, and 1.0 mL of 1 mg / L phenanthrene solution in sequence, dilute to 1 m with methanol, shake well, use methanol as the reference solution, measure the peak area of ​​phenanthrene on the machine, and draw a standard curve with the peak area as the ordinate and the corresponding phenanthrene content as the abscissa.

5. The method for enhancing the co-metabolic degradation and molecular response of polycyclic aromatic hydrocarbons (PAHs) by plant aromatic organic acids (AOAs) according to claim 1, characterized in that: Primers 341F and 806R were used to amplify the V3-V4 region of the bacterial 16S rRNA sequence. The PCR reaction system was 50 μL, 25 μL of 2xPremix Taq, 1 μL each of 341F / 806R primers, 3 μL of DNA, and 20 μL of Nuclease-free water. The reaction conditions were: pre-denaturation at 98°C for 1 min, denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and extension at 72°C for 30 s, for a total of 30 cycles, followed by extension at 72°C for 5 min. The DNA was then stored at 4°C and sequenced using the Illumina HiSeq 2500 platform.

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