A method for evaluating the stress response and resistance mechanism of extracellular polymers to UV / peracetic acid disinfection

Abstract

The present invention relates to a method for evaluating the stress response and resistance mechanism of extracellular polymers to ultraviolet / peracetic acid disinfection, and belongs to the technical field of sewage treatment. The method comprises the following steps: extracting the total extracellular polymers (EPS) from the secondary effluent of a sewage treatment plant, and setting different experimental groups and control groups; performing an ultraviolet / peracetic acid disinfection experiment on the obtained experimental groups using ultraviolet light and peracetic acid to obtain sludge samples to be tested; filtering the obtained sludge samples to be tested from the control group and the experimental group to obtain solid microorganism samples to be tested; and performing multi-omics association analysis on the obtained solid microorganism samples to be tested. The present invention is suitable for evaluating the stress response and resistance mechanism of extracellular polymers to ultraviolet / peracetic acid disinfection from different perspectives. Peracetic acid is widely available and inexpensive, and its use in sewage treatment can effectively reduce costs. Due to the short reaction time and the low ultraviolet dose used in the process, the construction cost and operating cost are low.

Description

Technical Field

[0001] The present invention relates to the technical field of sewage treatment, and in particular to a method for evaluating the pressure response and resistance mechanism of extracellular polymers to ultraviolet / peracetic acid disinfection. Background Art

[0002] With the increasingly severe global water resource situation, wastewater regeneration has become a key strategic response. Disinfection, as the final barrier, plays a crucial role in ensuring the safety of wastewater regeneration. Furthermore, traditional disinfection processes, such as chlorine disinfection, produce toxic and harmful disinfection byproducts and incompletely inactivate pathogens. Therefore, further research is needed to develop new wastewater disinfection processes based on traditional wastewater disinfection techniques that can both efficiently inactivate pathogens and effectively control the generation of toxic and harmful disinfection byproducts.

[0003] Compared to traditional disinfection processes, peracetic acid (PAA), a peroxy organic acid, has gained widespread application in Europe and North America due to its high pathogen inactivation efficiency and minimal risk of byproduct formation. Furthermore, combining PAA disinfection with UV disinfection, which also produces fewer disinfection byproducts (i.e., UV / PAA disinfection, or UV / PAA disinfection), leverages their synergistic effects, achieving highly effective pathogen inactivation while reducing PAA dosage, making it a promising new wastewater disinfection process. Its mechanism relies on the efficient generation of reactive oxygen species. Pathogens in wastewater are encapsulated by extracellular polymeric substances (EPS), which serve as a crucial interface for the exchange of substances and information between them and the external environment. During the disinfection process, the EPS encapsulating the pathogens is converted or degraded by oxidative species generated by the disinfection system. Furthermore, the pathogens produce more EPS as the disinfection process progresses to resist the process. Therefore, EPS exhibits dynamic changes during UV / PAA disinfection, and the microbial responses and resistance mechanisms it mediates significantly influence the effectiveness of UV / PAA disinfection.

[0004] In recent years, with the development of omics technology, multi-omics methods such as metagenomics, metatranscriptomics and non-targeted metabolomics have provided important technical support for in-depth revelation of the molecular mechanisms of microbial-environment interactions. However, in the field of sewage disinfection research, single omics technology often has obvious limitations. For example, metagenomics can only reveal changes in microbial community structure and functional genes, but cannot reflect the true expression level of genes. Although non-targeted metabolomics can capture the phenotypic output of metabolic pathways, it is difficult to trace their upstream regulatory genes. Therefore, the combination of multi-omics has become an important means to explain the internal mechanisms of complex biological processes. However, there is currently no special study on the comprehensive evaluation method of EPS stress response and resistance mechanism to UV / PAA disinfection process. Summary of the Invention

[0005] To address the above technical issues, the present invention provides a method for evaluating the stress response and resistance mechanism of extracellular polymers to UV / peracetic acid disinfection. The method of the present invention utilizes a heating-sodium carbonate combined method to extract total EPS from actual secondary effluent from a sewage treatment plant. After retaining the microorganisms, the groups are divided into EPS-containing and EPS-free groups. 10 mg / L PAA solution is added to each group and UV irradiation is performed (UV light radiation dose is 108 mJ / cm 2 ), and a multi-omics association analysis was used to comprehensively evaluate the stress response and resistance mechanism of EPS. First, the metagenome was used to study the structural succession characteristics of the microbial community during EPS-mediated UV / PAA disinfection. Further, the stress response mechanism of EPS to the UV / PAA disinfection process was systematically analyzed through metagenomic and metatranscriptomic association analysis, including oxidative stress, material transport and signal transduction, and quorum sensing. Finally, the response mechanism of EPS to metabolites and key metabolic pathways during UV / PAA disinfection was analyzed through non-targeted metabolomics. The results showed that EPS significantly affected the succession of microbial communities in the UV / PAA disinfection system. Compared with the EPS_60 group, the nEPS_60 group had a significantly higher succession rate than the EPS_60 group. Nitrosomonas 、 Sulfuritalea and Thauera The relative abundance of dominant bacterial genera such as _____ were significantly reduced, indicating that the presence of EPS can provide a protective barrier to prevent some dominant bacterial genera from being inactivated by disinfection. Nitrospira The nEPS_60 group maintained a high abundance (9.53%), indicating that this genus possesses strong resistance to UV / PAA disinfection and is minimally affected by the absence of EPS. Furthermore, EPS can influence the expression of transport and signaling genes, with the presence of EPS upregulating the abundance of related genes. The nEPS_60 sample also showed upregulation of DNA repair and oxidative stress genes. Furthermore, EPS mediates the quorum sensing system, with the nEPS_60 sample showing a significant increase in the abundance of genes involved in the QS pathway. Finally, the presence of EPS enriches metabolites in nucleic acid metabolism and the ATP-Binding Cassette Transport Pathway, conferring a degree of disinfection resistance. In this disclosure, " / " represents a combined mechanism.

[0006] In order to achieve the technical effects of the present invention, the following technical solutions are used:

[0007] The present invention aims to provide a method for evaluating the stress response and resistance mechanism of extracellular polymers to ultraviolet / peracetic acid disinfection, comprising the following steps:

[0008] S1. Extract the total extracellular polymers (EPS) from the secondary effluent of a sewage treatment plant and set up different experimental and control groups;

[0009] S2. The experimental group obtained in step S1 was subjected to a UV / peracetic acid disinfection experiment using UV and peracetic acid to obtain a sludge sample to be tested;

[0010] S3. The sludge sample to be tested in the control group obtained in step S1 and the sludge sample to be tested in the experimental group obtained in step S2 are filtered to obtain a solid microbial sample to be tested;

[0011] S4. Perform multi-omics association analysis on the solid microbial sample obtained in step S3.

[0012] In some embodiments of the present invention, in step S1, the extraction method includes the following steps:

[0013] Providing a sludge mixed liquid, and centrifuging and washing the sludge mixed liquid;

[0014] resuspending the washed sludge mixed solution in a NaCl solution, and adding sodium carbonate to obtain a mixed solution;

[0015] The obtained mixed solution is heated and then centrifuged to obtain a solid microbial sample and a supernatant. The supernatant is further filtered through a filter membrane to obtain a filtrate, which is then dialyzed in deionized water using a dialysis membrane to remove residual chemical reagents and obtain total EPS.

[0016] In some embodiments of the present invention, the centrifugal washing conditions are: centrifugation at 8000-12000 rpm for 5-6 min at 1-4° C., and washing with 0.9% NaCl solution at least 3 times.

[0017] In some embodiments of the present invention, the concentration of sodium carbonate in the mixed solution is 0.5 wt %. The present invention mainly uses sodium carbonate to promote the dissociation of EPS from the cell wall and stabilize the enzyme protein structure.

[0018] The temperature of the heating treatment is 60-80°C and the time is 25-30 min; the molecular cutoff value of the dialysis membrane is 3500 Da.

[0019] In some embodiments of the present invention, the experimental group and the control group are specifically as follows:

[0020] EPS_0 control group (i.e., EPS-containing group): EPS was not extracted and the sample group was before UV / peracetic acid disinfection;

[0021] EPS_60 experimental group (i.e., EPS-containing group): EPS was not extracted and the samples were disinfected with UV / peracetic acid for 60 minutes;

[0022] nEPS_0 control group (i.e., EPS-free group): EPS extraction, sample group before UV / peracetic acid disinfection;

[0023] The nEPS_60 experimental group is the EPS-free group: the sample group was extracted and disinfected with UV / peracetic acid for 60 minutes.

[0024] In some embodiments of the present invention, in step S2, the radiation dose of ultraviolet disinfection in the ultraviolet / peracetic acid disinfection experiment is 108 mJ / cm 2 The addition concentration of peracetic acid is 10 mg / L.

[0025] In some embodiments of the present invention, in step S3, the multi-omics association analysis includes one or more of metagenomics analysis, macrotranscriptomics analysis, and non-targeted metabolomics analysis.

[0026] In some embodiments of the present invention, the metagenomic analysis specifically comprises the following steps:

[0027] DNA was extracted from the solid microbial sample to be tested, and the DNA concentration and purity were tested. The 350 bp fragment was screened for library construction;

[0028] After matching the libraries, genome sequencing is performed, and then the DNA gene sequences are clustered by retaining the valid DNA data and performing splicing and assembly to construct a non-redundant gene set;

[0029] The non-redundant gene set was compared with the non-redundant protein database NR and the Kyoto Encyclopedia of Genes and Genomes KEGG database, and the abundance of each species was counted.

[0030] In some embodiments of the invention, the macrotranscriptomic analysis comprises the following steps:

[0031] Agarose gel electrophoresis was used to evaluate the degree of RNA degradation in the solid microbial samples to be tested, and the RNA purity was determined. RNA was enriched from qualified samples (OD260 / 280 of 1.8-2.4).

[0032] The enriched RNA is purified, end-repaired, and then PCR amplified to obtain a library;

[0033] After library construction, the end-repaired PCR was initially quantified using Qubit2.0, then diluted to 1 ng / μL. The insert size was then detected using Agilent 2100. If it met expectations, accurate quantification was performed using qPCR.

[0034] When the effective concentration of PCR is >2 nM, the library is determined to have passed the quality inspection. After sequencing the qualified library, the raw data is obtained. The effective data obtained after quality control of the raw data is compared with the macrotranscriptome gene set that has been disclosed in the existing technology, and gene abundance is calculated. Gene expression analysis is performed in combination with species / function annotations.

[0035] In some embodiments of the present invention, the non-targeted metabolomics analysis process comprises the following steps:

[0036] The solid microbial sample to be tested is dried and ground, and then metabolites are extracted. An equal volume of the extracted metabolite sample is mixed to prepare a quality control sample. Metabolite detection is performed using ultra-performance liquid chromatography-Fourier transform mass spectrometry.

[0037] Mass spectrometry information was compared and identified using the HMDB database;

[0038] Subsequently, principal component analysis was performed using R language to screen differential metabolites, pathway annotation was performed using the KEGG database, and finally pathway enrichment analysis was performed using Python.

[0039] The above technical solution of the present invention has the following advantages over the prior art:

[0040] 1) The evaluation method of this invention utilizes multi-omics technology, which can overcome the limitations of single-omics technology and help to further analyze the stress response and resistance mechanism of EPS to UV / PAA disinfection.

[0041] 2) The present invention uses metagenomics and metatranscriptomics association analysis to jointly reveal the gene expression and community structure characteristics of EPS in UV / PAA disinfection. Among them, EPS significantly affected the microbial community succession in the UV / PAA disinfection system. Compared with the EPS_60 group, the nEPS_60 group Nitrosomonas 、 Sulfuritalea and Thauera The relative abundance of dominant bacterial genera such as _____ were significantly reduced, indicating that the presence of EPS can provide a protective barrier to prevent some dominant bacterial genera from being inactivated by disinfection. Nitrospira The nEPS_60 group maintained a high abundance (9.53%), indicating that this genus is highly resistant to UV / PAA disinfection and that EPS deficiency has little effect on it. Furthermore, EPS regulates oxidative stress genes, DNA repair genes, transport and signaling genes, and quorum sensing genes.

[0042] 3) The present invention's non-targeted metabolomics analysis further explored EPS-mediated differential metabolic information. The results showed that EPS can regulate metabolic pathways and significantly affect the disinfection efficacy of UV / PAA. In the presence of EPS, metabolic pathways related to substance transport and DNA replication were enriched, while in the absence of EPS, toxic substance metabolism dominated. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0044] Figure 1 This is the significant difference analysis of the microbial community of the present invention (genus level) (the significance of the differences between the groups was assessed using Fisher's exact test (*P<0.05; **P<0.01; ***P<0.001).

[0045] Figure 2 Fisher's exact test was used to evaluate the significance of differences between groups (*P<0.05; **P<0.01; ***P<0.001).

[0046] Figure 3 Genes related to ABC transport, two-component system, DNA replication and oxidative stress (ROS) were differentially expressed.

[0047] Figure 4 These are the significantly different functional genes related to the two-component system (TCS) among the EPS_60 vs EPS_0, nEPS_60 vs nEPS_0, and nEPS_60 vs EPS_60 groups in the present invention.

[0048] Figure 5 This is a scatter plot of the differences in functional genes related to quorum sensing (QS) in the present invention.

[0049] Figure 6 This is the principal component analysis (PCA) of metabolites containing / excluding EPS in the present invention.

[0050] Figure 7 This is the partial least squares discriminant analysis (PLS-DA) of metabolites containing / not containing EPS in the present invention.

[0051] Figure 8 This is the enrichment analysis of differential metabolic pathways in the groups containing / excluding EPS in the present invention (top 20 in abundance).

[0052] Figure 9 It is a collinear network between the significantly different metabolites and the significantly enriched metabolic pathways in the EPS-containing / non-EPS-containing groups in the present invention. DETAILED DESCRIPTION

[0053] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0054] Example 1

[0055] This embodiment provides a method for evaluating the stress response and resistance mechanism of EPS to UV / PAA disinfection, comprising the following steps:

[0056] Step S1. Total EPS was extracted using a method that caused less damage to microorganisms and had a better extraction effect, without UV / PAA disinfection. The two groups were marked as EPS extraction group and EPS non-extraction group, denoted as EPS_0 and nEPS_0, respectively.

[0057] Step S2: The EPS extraction group and the non-EPS extraction group obtained in step S1 were irradiated with a dose of 108 mJ / cm 2 The sludge samples were irradiated with ultraviolet light (the light source was an ultraviolet lamp with a wavelength of 254 nm) and disinfected with 10 mg / L PAA for 60 min to obtain the disinfected sludge samples of the two experimental groups, which were recorded as EPS_60 and nEPS_60.

[0058] Step S3. Filter the sludge samples to be tested of the control group EPS_0 and nEPS_0 obtained in step S1, and the sludge samples to be tested of the experimental group EPS_60 and nEPS_60 obtained in step S2 to obtain solid microbial samples to be tested.

[0059] In step S1, the total EPS is extracted from the secondary effluent of the sewage treatment plant by using a heat-sodium carbonate combined method, and the extraction method comprises the following steps:

[0060] Step a. providing a uniformly mixed sludge mixture, centrifuging the sludge mixture at 8000 rpm for 5 min at 4 ° C, and then washing three times with 0.9% NaCl solution to obtain a first mixed solution;

[0061] Step b. The first mixed solution after washing in step a was resuspended in an equal volume of 0.9% NaCl solution, and sodium carbonate powder was added to a concentration of 0.5wt% to obtain a second mixed solution;

[0062] Step c. The second mixed solution obtained in step b was placed in an 80°C water bath for 30 minutes, and then centrifuged at 11,000 g for 15 minutes to obtain the supernatant; the supernatant and the solid microbial sample were retained at the same time; the supernatant was then filtered through a 0.22 μm filter membrane to obtain a total EPS sample, and further dialyzed in deionized water using a dialysis membrane with a molecular cutoff value of 3500 Da to remove residual chemical reagents.

[0063] In addition, the sludge sample to be tested of nEPS_0 in step S3 mainly comes from the solid microorganism sample retained in step c, which is dispersed in the secondary effluent to obtain the sludge sample to be tested; and the sludge sample to be tested of EPS_0 is obtained by dispersing an equal amount of sludge sample as the solid microorganism sample retained in step c in an equal volume of secondary effluent.

[0064] Example 2

[0065] The solid microbial samples to be tested corresponding to the different control groups EPS_0 and nEPS_0, and the different experimental groups EPS_60 and nEPS_60 obtained in Example 1 were subjected to metagenomic sequencing analysis.

[0066] Each group underwent metagenomic analysis, which included the following steps:

[0067] (1) DNA was extracted from the solid microbial samples of each experimental group and the control group using the FastPure Stool DNA Isolation Kit (Magnetic bead) (MJYH, Shanghai, China).

[0068] (2) After DNA extraction, the DNA concentration and purity were tested, and DNA integrity was checked by 1% agarose gel electrophoresis. The DNA was fragmented using a Covaris M220, and fragments of approximately 350 bp were selected for the construction of a PE library.

[0069] (3) Paired-end libraries were constructed using NEXTFLEX Rapid DNA-Seq (Bio Scientific, USA), and metagenomic sequencing was performed using the Illumina NovaSeq™ X Plus (Illumina, USA) platform.

[0070] (4) Valid DNA data were then screened and retained through data quality control. The optimized sequences were assembled using MEGAHIT software, and the gene sequences predicted from all samples were clustered using CD-HIT to construct a non-redundant gene set.

[0071] (5) Diamond was used to compare the non-redundant gene sets in the samples with the non-redundant protein database (NR) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the gene and species abundances were calculated and analyzed.

[0072] The experimental results are shown in Figure 1 .

[0073] Example 3

[0074] The samples from each group obtained in Example 1 were subjected to macrotranscriptomics analysis, which specifically included the following steps:

[0075] (1) Agarose gel electrophoresis was used to evaluate the RNA degradation degree of the solid microbial samples in the experimental and control groups. The RNA purity was determined by Nanodrop. RNA samples with OD260 / 280 values ​​in the range of 1.8–2.4 were identified as qualified samples.

[0076] (2) For qualified samples, eukaryotic mRNA was enriched using Oligo(dT) magnetic beads, while prokaryotic rRNA was removed using a kit to enrich mRNA. Subsequently, double-stranded cDNA was purified using PBS buffer and AMPure XP beads. After end repair and other steps, PCR amplification was performed, and the library was finally purified using AMPure XP beads.

[0077] (3) After sequencing the libraries that passed the quality control (libraries with an effective concentration >2 nM were determined to be qualified), the raw data were obtained. The valid data obtained after the quality control of the raw data were aligned to the macrotranscriptome gene set that has been published in the existing technology. The gene abundance was calculated based on the number of reads, and subsequent gene expression analysis was carried out in combination with species / function annotations.

[0078] (4) The expression of genes related to oxidative stress, DNA replication, material transport, signal transduction and quorum sensing was analyzed in detail. The experimental results are shown in Tables 1 and Figures 2 to 5 .

[0079] Example 4

[0080] The solid microbial samples obtained in Example 1 were subjected to non-targeted metabolomics analysis, comprising the following steps:

[0081] (1) The solid microbial samples obtained from each group in Example 1 were dried to obtain solid samples. The solid samples were then ground and metabolites were extracted. Equal volumes of the extracted metabolite samples were mixed to prepare quality control samples. Metabolite detection was performed using ultra-performance liquid chromatography-Fourier transform mass spectrometry. The raw data were processed using Progenesis QI (Waters Corporation, Milford, USA), and the MS / MS spectra were compared with the HMDB database for identification.

[0082] (2) The R language ropls package (Version 1.6.2) was used to perform principal component analysis to screen differential metabolites. The screening criterion was that the variable weight value VIP>1 of the OPLS-DA model was combined with the t-test (p<0.05).

[0083] (3) Metabolic pathway annotation was performed using the KEGG database, and pathway enrichment analysis was performed using Python. The experimental results are shown in Figures 6-9 .

[0084] Table 1

[0085]

[0086] The analysis results of Examples 2-4 are as follows Figure 1-9 From the results in Table 1, we can conclude that:

[0087] Figure 1 The following table shows the structural succession characteristics and significant difference analysis of the microbial community in different groups of Example 2. It can be seen from the figure that EPS significantly affected the succession of the microbial community in the UV / PAA disinfection system. The dominant genera in the four groups of samples are mainly Hyphomicrobium , Nitrosomonas , Nitrospira , unclassified_c__Caudoviricetes , Sulfuritalea and Thauera Compared with the EPS_60 group, the nEPS_60 group Nitrosomonas 、 Sulfuritalea and Thauera The relative abundance of dominant bacterial genera such as _____ were significantly reduced, indicating that the presence of EPS can provide a protective barrier to prevent some dominant bacterial genera from being inactivated by disinfection. Nitrospira The abundance of the bacteria in the nEPS_60 group was still high (9.53%), indicating that the bacteria genus had strong resistance to UV / PAA disinfection and the absence of EPS had little effect on it.

[0088] Figure 2 Table 1 shows the enrichment pathways of functional genes identified in different groups. It was found that the differential metabolic pathways between groups mainly involved key metabolic pathways such as secondary metabolite biosynthesis, environmental adaptability metabolism, two-component signal transduction system, cofactor synthesis, carbon metabolism, amino acid synthesis, quorum sensing (QS), ABC transport system, purine metabolism, pyruvate metabolism, and DNA replication. Notably, the relative abundance of these pathways was significantly higher in the nEPS_60 group than in the EPS_60 group.

[0089] The results of metagenomic and metatranscriptomic analyses, such as Figure 3 、 Figure 4 and Figure 5 As shown, Figure 3Differential expression of genes related to ABC transport, two-component transport systems, DNA replication, and oxidative stress was demonstrated, revealing significant differences in the expression of these genes among the different groups after UV / PAA disinfection. Analysis of oxidative stress and DNA repair gene expression revealed that the expression of the oxidative stress-related gene sodA and the DNA repair-related gene ahpC increased by 1.71-fold and 9.71-fold, respectively, in the EPS-free group, reflecting the key role of EPS in protecting against oxidative damage caused by disinfection. Analysis of transporter and signaling genes revealed that EPS effectively mitigated intracellular oxidative damage induced by UV / PAA disinfection through the expression of genes related to the TCS and ABC transport systems. Compared to the EPS-free group (nEPS_60), the expression of the membrane integrity-related gene lolD and the signaling-related gene atoC increased by 1.67-fold and 0.34-fold, respectively, in the EPS-containing group. Furthermore, differential expression analysis of quorum sensing system genes revealed that EPS regulates QS by upregulating the expression of biofilm integrity genes, acting as both a protective barrier and an information mediator to synergistically enhance microbial disinfection resistance. In the BDSF / DSF-mediated QS pathway, the abundance of crp genes in the EPS_60 and nEPS_60 groups increased by 22.2% and 368.4%, respectively, and the expression levels of biofilm formation-related genes (RpfF, RpfB, etc.) increased by 27.8% and 324.5%, respectively.

[0090] The results of non-targeted metabolomics analysis are as follows Figure 6 、 Figure 7 、 Figure 8 and Figure 9 As shown. Figure 6 and Figure 7 The PCA analysis and PLS-DA analysis of metabolites in the EPS group and the non-EPS group respectively show that a total of 490 differential metabolites were identified in the two groups of samples. Under the two classification conditions, the metabolites were well dispersed and significantly separated from each other. Among them, the R 2 The X value is 0.805, indicating that the model fit is good. The differential metabolites of the two groups of samples were further analyzed by partial least squares discriminant analysis (PLS-DA). All grouped samples showed a good degree of separation, with an R 2 X value and R 2 The Y values ​​were 0.841 and 0.997, respectively, which indicated the stability and reliability of the PLS-DA model and that it could successfully predict the differential metabolites among different groups. Figure 8 and Figure 9"" represent differential metabolic pathway enrichment analysis and collinear network analysis, respectively. Enrichment analysis results showed that in the EPS_60 group, nucleic acid metabolism and ABC transport pathways were significantly enriched, with enrichment ratios of 0.26 and 0.12, respectively, confirming that EPS can enhance microbial antioxidant defense. In contrast, in the nEPS_60 group, histidine metabolism and α-linolenic acid metabolism dominated, indicating that the lack of EPS protection leads to the accumulation of toxic substances and accelerated cell death. Collinear network analysis revealed that differential metabolites in the EPS-containing group were significantly correlated with pathways such as nucleotide metabolism, ABC transporters, and central carbon metabolism in cancer (p < 0.001). In contrast, metabolites in the EPS-free group were primarily enriched in stress response pathways such as histidine metabolism and α-linolenic acid metabolism. This difference in metabolic pathway distribution confirms that EPS not only serves as a physical protective barrier but also plays a crucial role in regulating microbial metabolism.

[0091] The above embodiment has the following beneficial effects compared to the prior art:

[0092] The present invention has developed an evaluation method for the stress response and resistance mechanism of extracellular polymers to UV / peracetic acid disinfection, and adopted multi-omics association analysis to jointly reveal its mechanism from different angles. First, the structural succession characteristics of the microbial community and the differential expression of functional genes in different groups were analyzed by metagenomics and metatranscriptomics. It was found that EPS significantly affected the succession of the microbial community in the UV / PAA disinfection system. Compared with the EPS_60 group, the relative abundance of dominant bacterial genera such as Nitrosomonas, Sulfuritalea and Thauera in the nEPS_60 group were significantly reduced after disinfection. In addition, Nitrospira The abundance of the bacteria in the nEPS_60 group was still high (9.53%), indicating that the bacteria had strong resistance to UV / PAA disinfection and that the absence of EPS had little effect on it. Gene expression analysis found that EPS could effectively alleviate the intracellular oxidative damage caused by UV / PAA disinfection. Compared with the group without EPS (nEPS_60), the genes related to membrane integrity in the group with EPS were significantly increased. lol The expression of D gene and atoC gene related to signal transduction were up-regulated by 1.67 times and 0.34 times respectively. sod A and DNA repair related ahpThe expression levels of the C gene increased by 1.71 times and 9.71 times, respectively, reflecting the key role of EPS in resisting oxidative damage caused by disinfection. On this basis, the present invention further explained the metabolic differences of EPS in response to UV / PAA disinfection through non-targeted metabolomics. The results showed that in the EPS_60 group, nucleic acid metabolism and ABC transport pathways were significantly enriched, with enrichment rates of 0.26 and 0.12, respectively, confirming that EPS can enhance microbial antioxidant defense. In contrast, the nEPS_60 group was dominated by histidine metabolism and α-linolenic acid metabolism, indicating that the lack of EPS protection will lead to the accumulation of toxic substances and accelerate cell death.

[0093] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications can be made based on the above description. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A method for evaluating the stress response and resistance mechanism of extracellular polymers to UV / peracetic acid disinfection, characterized in that: The following steps are involved: S1. Extract the total extracellular polymers (EPS) from the secondary effluent of a sewage treatment plant and set up different experimental and control groups; S2. The experimental group obtained in step S1 was subjected to a UV / peracetic acid disinfection experiment using UV and peracetic acid to obtain a sludge sample to be tested; S3. The sludge sample to be tested in the control group obtained in step S1 and the sludge sample to be tested in the experimental group obtained in step S2 are filtered to obtain a solid microbial sample to be tested; S4. Perform multi-omics association analysis on the solid microbial sample obtained in step S3; The experimental and control groups are as follows: EPS_0 control group (i.e., EPS-containing group): EPS was not extracted and the sample group was before UV / peracetic acid disinfection; EPS_60 experimental group (i.e., EPS-containing group): EPS was not extracted and the samples were disinfected with UV / peracetic acid for 60 min; nEPS_0 control group (i.e., EPS-free group): EPS extraction, sample group before UV / peracetic acid disinfection; nEPS_60 experimental group (i.e., EPS-free group): EPS was extracted and the samples were disinfected with UV / peracetic acid for 60 min; In step S3, the multi-omics association analysis includes metagenomics analysis, macrotranscriptomics analysis and non-targeted metabolomics analysis; The metagenomic analysis specifically includes the following steps: DNA was extracted from the solid microbial sample to be tested, and the DNA concentration and purity were tested. The 350 bp fragment was screened for library construction; After matching the libraries, genome sequencing is performed, and then the DNA gene sequences are clustered by retaining the valid DNA data and performing splicing and assembly to construct a non-redundant gene set; The non-redundant gene set was compared with the non-redundant protein database NR and the Kyoto Encyclopedia of Genes and Genomes KEGG database, and the abundance of each species was counted; The macrotranscriptomic analysis includes the following steps: Agarose gel electrophoresis was used to evaluate the degree of RNA degradation in the solid microbial samples to be tested, and the RNA purity was determined. RNA was enriched in qualified samples. The enriched RNA is purified, end-repaired, and then PCR amplified to obtain a library; After library construction is complete, Qubit2.0 is used to perform preliminary quantification of the end-repaired PCR, and then diluted to 1 ng / μL. The insert fragment size is then detected by Agilent2100. If it meets expectations, the qPCR method is used for accurate quantification; When the effective concentration of PCR is >2 nM, the library is determined to have passed the quality inspection. The library that has passed the quality inspection is sequenced to obtain the raw data. The valid data obtained after the raw data quality control is compared with the macrotranscriptome gene set that has been published in the existing technology, and the gene abundance is calculated. The gene expression analysis is then carried out in combination with the species / function annotation. The non-targeted metabolomics analysis process includes the following steps: The solid sample obtained after drying the solid microbial sample to be tested is ground, and metabolites are extracted after grinding. An equal volume of the extracted metabolite sample is taken and mixed to prepare a quality control sample; metabolite detection is performed using ultra-performance liquid chromatography-Fourier transform mass spectrometry; The mass spectrometry information was compared and identified using the human metabolome database (HMDB); Then, principal component analysis was performed using R language to screen differential metabolites, pathway annotation was performed using the KEGG database, and finally pathway enrichment analysis was performed using Python; The specific contents of the multi-omics association analysis include: species abundance, species gene abundance, and expression of genes related to oxidative stress, DNA replication, material transport, signal transduction, and quorum sensing, KEGG metabolic pathway analysis, and differential metabolic pathways between groups; the differential metabolic pathways between groups include secondary metabolite biosynthesis, environmental adaptability metabolism, two-component signal transduction system, cofactor synthesis, carbon metabolism, amino acid synthesis, quorum sensing QS, ABC transport system, purine metabolism, pyruvate metabolism and DNA replication.

2. The method for evaluating the stress response and resistance mechanism of extracellular polymers to UV / peracetic acid disinfection according to claim 1, wherein: In step S1, the extraction method includes the following steps: Providing a sludge mixed liquid, and centrifuging and washing the sludge mixed liquid; resuspending the washed sludge mixed solution in a NaCl solution, and adding sodium carbonate to obtain a mixed solution; The obtained mixed solution is heated and then centrifuged to obtain a solid microbial sample and a supernatant. The supernatant is further filtered through a filter membrane to obtain a filtrate, which is then dialyzed in deionized water using a dialysis membrane to remove residual chemical reagents and obtain total extracellular polymers.

3. The method for evaluating the stress response and resistance mechanism of extracellular polymers to UV / peracetic acid disinfection according to claim 2, wherein: The centrifugal washing conditions are as follows: centrifugation at 8000-12000 rpm for 5-6 minutes at 1-4°C, and washing with 0.9% NaCl solution for at least 3 times.

4. The method for evaluating the stress response and resistance mechanism of extracellular polymers to UV / peracetic acid disinfection according to claim 2, wherein: The concentration of the NaCl solution is 0.01 mol / L; The concentration of sodium carbonate in the mixed solution is 0.5wt%; The temperature of the heating treatment is 60-80° C., and the time is 25-30 min. The molecular cutoff value of the dialysis membrane is 3500 Da.

5. The method for evaluating the stress response and resistance mechanism of extracellular polymers to UV / peracetic acid disinfection according to claim 1, wherein: In step S2, the radiation dose of ultraviolet disinfection in the ultraviolet / peracetic acid disinfection experiment is 108mJ / cm 2 The addition concentration of peracetic acid is 10 mg / L.

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