Application of signal molecule C8-HSL in promoting wastewater treatment by bacteria-algae symbiotic granular sludge

Abstract

The present application relates to sewage treatment technology field, especially signal molecule C8-HSL in the application of strengthening bacteria-algae symbiotic granular sludge sewage treatment, the present application finds that signal molecule C8-HSL can improve system TN removal rate through in-depth study;Signal molecule changes the composition of bacterial flora, promotes the enrichment of denitrification functional bacteria, such as Hydrogenophaga, Pseudoxanthomonas, etc., and also enhances the abundance of complex organic matter degrading bacteria Desulfomicrobium;At the same time, the increase of denitrifying bacteria such as Hydrogenophaga, Thauera and Zoogloea optimizes the species diversity, improves the nitrogen removal and organic matter degradation efficiency;Based on PICRUSt2 analysis, signal molecule C8-HSL can increase the abundance of napAB gene, promote aerobic denitrification;nirK, nirS gene expression is enhanced, promotes nitrite to be converted into nitrogen;At the same time, signal molecule C8-HSL can effectively improve the system denitrification performance by optimizing nitrogen metabolism pathway.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment technology, and in particular to the application of the signaling molecule C8-HSL in promoting the treatment of wastewater by granular sludge with bacterial-algae symbiosis. Background Technology

[0002] Rural domestic sewage discharge is generally characterized by extensive and inefficient processes, with relatively small but highly volatile flow rates and widespread and dispersed distribution. Existing treatment methods often employ a combination of biological and ecological processes, but this multi-process system is complex to operate and maintain, has low treatment efficiency, high costs, and struggles to effectively remove nitrogen and phosphorus, resulting in treated water quality that fails to meet Class I discharge standards.

[0003] Existing aerobic granular sludge (AGS) is a wastewater treatment technology. AGS is a self-immobilizing microbial aggregate with a compact structure and layered morphology. Compared to traditional activated sludge, its regular shape significantly improves its nitrogen and phosphorus removal efficiency. Furthermore, the AGS system requires approximately 33% less floor space than traditional activated sludge systems, thus reducing construction costs. However, its high aeration energy consumption and operational stability still require further investigation. Building on this, we further investigated Algal-bacterial granular sludge (ABGS) technology based on HN-AD bacteria. ABGS, as an innovative wastewater treatment technology, combines the advantages of aerobic granular sludge (AGS) and bacteria / algae, making it a resource-friendly, energy-saving, and environmentally friendly treatment solution. We have also applied for and been granted a Chinese patent (publication number CN114835269B).

[0004] Based on the HN-AD bacteria-based symbiotic granular sludge, we conducted further research and found that although the symbiotic granular sludge has made progress compared with aerobic granular sludge (AGS), its microorganisms and algae have poor tolerance to ammonia nitrogen, the treatment cycle is long and the system is unstable. The granular sludge structure is easy to disintegrate during operation, and the total nitrogen removal rate (about 50%) is low, which still needs to be further improved.

[0005] To address this technical problem, we further enhanced the HN-AD bacteria in the HN-AD-based algae-bacterial symbiotic granular sludge (ABGS) system by using signaling molecules, thereby improving the wastewater treatment capacity of the ABGS system with added HN-AD bacteria. Therefore, we propose the application of the signaling molecule C8-HSL in enhancing the wastewater treatment capacity of algae-bacterial symbiotic granular sludge. Summary of the Invention

[0006] This invention provides a symbiotic system of bacteria and algae for wastewater treatment, the system comprising: aerobic granular sludge, Chlorella proteoglycans, HN-AD bacteria, and the signaling molecule C8-HSL.

[0007] This invention provides the application of the signaling molecule C8-HSL in enhancing the wastewater treatment capacity of granular sludge containing HN-AD bacteria in a bacterial-algae symbiotic relationship.

[0008] This invention provides any of the following applications of the signaling molecule C8-HSL:

[0009] (1) Improve the denitrification stability of granular sludge containing HN-AD bacteria and improve the denitrification efficiency of granular sludge containing HN-AD bacteria.

[0010] (2) Promote the removal of organic matter from granular sludge containing HN-AD bacteria;

[0011] (3) Promote the enrichment of HN-AD bacteria in granular sludge containing HN-AD bacteria;

[0012] (4) Promote the release of nutrients from algae in granular sludge containing HN-AD bacteria;

[0013] (5) Inducing changes in the growth pattern of microorganisms in granular sludge containing HN-AD bacteria;

[0014] (6) Improve the surface hydrophobicity of aerobic granular sludge in symbiotic granular sludge containing HN-AD bacteria;

[0015] (7) Modify the granular sludge structure of granular sludge containing HN-AD bacteria and algae symbiotic granular sludge;

[0016] (8) Promotes the secretion of EPS by Chlorella;

[0017] (9) Promote the dominant expression of denitrification functional genes;

[0018] (10) Promotes the increase of napAB gene abundance and promotes aerobic denitrification;

[0019] (11) Promotes the expression of nirK and nirS genes and promotes the conversion of nitrite into nitrogen gas.

[0020] This process promotes the release of nutrients from the algae, specifically nitrogen and phosphorus.

[0021] Among them, inducing changes in the growth pattern of microorganisms means: changing from suspension growth to attached growth.

[0022] Among them, changing the granular sludge structure of the algae-bacterial symbiotic AGS system mainly includes:

[0023] This allows more algae and bacteria to adhere to and entangle on the surface of the granular sludge;

[0024] This makes the granular sludge structure more compact.

[0025] The present invention has at least the following beneficial effects:

[0026] This invention, through in-depth research, discovered that HN-AD bacteria can enhance the wastewater treatment capacity of Chlorella vulgaris and aerobic granular sludge. The constructed symbiotic system improves the enrichment and species richness of denitrifying bacteria, promotes the settling properties of granular sludge, and increases nutrient removal efficiency; it also improves TN removal rate and NH4+ removal rate. + -N removal rate and COD removal rate; helps to improve pollutant removal efficiency, especially the degradation of nitrogen sources and macromolecular organic matter, and helps to improve denitrification and organic matter degradation efficiency; promotes the enrichment of aerobic bacteria, helps to reduce the growth of potential pathogenic microorganisms, and reduces the harm of system operation to the human body; the bacterial-algae symbiotic AGS system promotes good denitrification effect by improving denitrification activity and enriching nitrogen-metabolizing bacteria.

[0027] This invention, through in-depth research, discovered that the signaling molecule C8-HSL can improve the TN removal rate of the algae-microbe symbiotic system. The signaling molecule alters the microbial community composition, promoting the enrichment of denitrifying functional bacteria such as *Hydrogenophaga* and *Pseudoxanthomonas*, while also enhancing the abundance of complex organic matter degrading bacteria *Desulfomicrobium*. Simultaneously, the increase in denitrifying bacteria such as *Hydrogenophaga*, *Thauera*, and *Zoogloea* optimizes species diversity and improves nitrogen removal and organic matter degradation efficiency. Based on PICRUSt2 analysis, this invention shows that the signaling molecule C8-HSL can increase the abundance of the *napAB* gene, promoting aerobic denitrification; and enhance the expression of the *nirK* and *nirS* genes, promoting the conversion of nitrite to nitrogen. Furthermore, the signaling molecule C8-HSL effectively improves the system's nitrogen removal performance by optimizing nitrogen metabolism pathways.

[0028] Furthermore, this invention can provide new theoretical and practical references for the treatment of rural domestic sewage. Attached Figure Description

[0029] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the following description of the embodiments will be briefly introduced. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 The experimental setup is an SBR reactor;

[0031] Figure 2 Comparison of pollutant removal performance of different systems; A: Comparison of algae-bacterial symbiotic AGS system; B: Comparison of algae-granular sludge system; C: Comparison of granular sludge system: (a) TN; (b) COD; (c) NH4 + -N;

[0032] Figure 3 Schematic diagram of granular sludge morphology for each system;

[0033] Figure 4 SEM characterization of aerobic granular sludge in each system; (S1-a, S1-b, S2-c represent S1 aerobic granular sludge images at 120x, 1500x, and 10000x magnification; S2-a, S2-b, S2-c represent S2 aerobic granular sludge images at 120x, 10000x, and 20000x magnification; S3-a, S3-b, S3-c represent S3 aerobic granular sludge images at 120x, 1000x, and 10000x magnification). (10,000x images; X1-a, X1-b, X1-c represent X1 aerobic granular sludge magnified at 120x, 2000x, and 20000x; X2-a, X2-b, X2-c represent X2 aerobic granular sludge magnified at 120x, 10000x, and 20000x; X3-a, X3-b, X3-c represent X3 aerobic granular sludge magnified at 120x, 10000x, and 20000x.)

[0034] Figure 5 For microbial diversity: (a) Dilution curve; (b) Rank-abundance curve; (c) Venn diagram;

[0035] Figure 6 For microbial communities: (a) phylum level; (b) class level; (c) genus level;

[0036] Figure 7 The contribution of key functional bacteria to nitrogen metabolism (a) and carbon metabolism (b), and the species differences between the two groups (c);

[0037] Figure 8 A predicted diagram of denitrification functional genes;

[0038] Figure 9 Abundance diagrams of enzymes involved in the nitrogen cycle: (a) X-SBR; (b) S-SBR. Detailed Implementation

[0039] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0040] This invention primarily proposes to further enhance the HN-AD bacteria through signaling molecules, thereby improving the wastewater treatment capacity of granular sludge containing HN-AD bacteria. Specifically, regarding granular sludge containing HN-AD bacteria, please refer to patent CN114835269B, which represents a more in-depth study of the technology in this invention.

[0041] Example 1: This example proposes to add exogenous quorum sensing signal molecules (C8-HSL) to the above-mentioned algae-bacterial symbiotic granular sludge (ABGS) system with added HN-AD bacteria in order to enhance the synergistic effect of bacteria and algae, improve denitrification efficiency and system stability.

[0042] 1. Materials and Methods

[0043] 1.1. Test Apparatus

[0044] Experimental setup: SBR reactor, etc. Figure 1 As shown, the SBR reactor is made of double-layered plexiglass columns, with an outer water bath insulation layer. A heating rod is used to maintain the reactor temperature at 25±2℃. The effective volume is 5L. A disc-type microporous aerator is installed at the bottom of the reactor, supplied with air via an air pump. A gas flow meter controls the aeration rate at 1L / h. Lamp tubes are attached to the outer wall to provide light for the microalgae, which is beneficial for Chlorella photosynthesis. The SBR reactor operates on a 6-hour cycle, including influent, aeration, settling, drainage, and idle. Each stage is controlled by a timer. Three SBR reactors were operated, numbered 1, 2, and 3 after the anaerobic granular sludge was acclimated to aerobic granular sludge.

[0045] 1.2. Test water

[0046] During the acclimation stage of granular sludge, water was fed into an SBR system, and artificial water distribution was used to simulate rural domestic sewage conditions. The carbon source was anhydrous sodium acetate, the nitrogen source was ammonium sulfate, and the phosphorus source was dipotassium hydrogen phosphate. In the experiment, the COD and ammonia nitrogen concentrations were approximately 300±10 mg / L and 50±5 mg / L, respectively, with a C / N ratio of 6. At the same time, 5 ml of trace elements were added per liter of water to ensure the growth and activity of microorganisms. The composition of the trace elements was 1.00 g / L of MgSO4·7H2O, 0.10 g / L of MnSO4·H2O, 0.50 g / L of CaCl2, and 0.10 g / L of FeSO4·7H2O.

[0047] 1.3. Inoculum

[0048] The activated sludge used in this study was obtained from a domestic wastewater treatment plant in Chongqing. The activated sludge was black and turbid, with an inoculum concentration of 3000 mg / L. Acclimation was completed after approximately 20 days of cultivation. Anaerobic granular sludge was purchased from Liboyuan Environmental Materials Co., Ltd. The inoculum concentration of the granular sludge was 4.45 mg / L. Acclimation was carried out using a sequencing batch reactor (SBR). After approximately 25 days of cultivation, acclimation was completed, and the anaerobic granular sludge was converted into aerobic granular sludge. *Chlorella pyrenoidosa* (FACHB-9) was purchased from a freshwater algae culture bank of a research institute.

[0049] Chlorella was purified before inoculation. The purified microalgae were then inoculated into 500mL Erlenmeyer flasks containing mixed culture medium (1.7g / L BG11 and 20g / L glucose mixed medium) that had been sterilized at 115℃. The flasks were placed under continuous light and shaken at a stable temperature of 26±2℃ and a shaker speed of 160r / min. Subculturing was performed every 3–5 days using an algal culture reactor. Chlorella was inoculated at 15% of the reaction volume.

[0050] The bacterial agent used in the experiment was HN-AD bacteria (a compound bacteria, patent application number: 201810209983.8), a novel bacterial agent obtained by our research group through screening bacteria from extreme environments and subsequent cultivation and domestication, exhibiting excellent denitrification characteristics. HN-AD bacteria were inoculated at 5% of the SBR reactor volume.

[0051] The experiment used N-acylhomoserine lactone (AHL) signaling molecules, which were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. and stored in 250 mL brown reagent bottles containing 99.9% anhydrous ethanol. Our research group previously screened the types and concentrations of AHL signaling molecules, ultimately selecting N-octanoyl-DL-homoserine lactone (C8-HSL) at an inoculum concentration of 87.98 μmol / L.

[0052] 1.4. Analytical Methods

[0053] All water quality indicators in this study were measured using national standard methods. The measured indicators included chemical oxygen demand (COD) and ammonia nitrogen (NH4+). + The influent COD concentration was 300±10 mg / L, and the total nitrogen (TN) was NH4+. +The -N concentration was 50±5 mg / L. The exogenous signaling molecule C8-HSL was introduced, and the results were compared before and after enhancement. In the algae-bacterial symbiotic AGS system, the system before enhancement was S1, and the system after enhancement was X1. In the algae-granular sludge system, the system without C8-HSL was S2, and the system with C8-HSL was X2. In the granular sludge system, the system without C8-HSL was S3, and the system with C8-HSL was X3. Pollutant removal performance was compared. A removal efficiency error within 5% for five consecutive days was considered stable. After approximately 50 days of operation in the six reactors, the effluent indicators stabilized. Biological samples were extracted from the six systems for comparative analysis of pollutant removal performance and biological characteristics. The morphological characteristics of the granular sludge were observed using scanning electron microscopy (SEM). The specific operating steps were as follows: First, granular sludge was extracted from the SBR system and fixed in a 2.5% glutaraldehyde solution. The fixative was placed overnight at 4°C. The granular sludge was rinsed three times with 1 mol / L phosphate buffer (pH 7.0), 15 minutes each time. The samples were then dehydrated sequentially using 30%, 50%, 70%, 80%, 90%, and 95% ethanol solutions, each for 15–30 minutes, followed by two treatments with 100% ethanol solution. Finally, after freeze-drying, the samples were sent to Shanghai Fuda Testing Technology Co., Ltd. for gold sputtering and SEM analysis.

[0054] 1.5. High-throughput sequencing

[0055] DNA was extracted from the biofilm mixture using the manufacturer's standard method and the MobioPowerSoil DNA Isolation Kit. The extracted genomic DNA was then detected by 1% agarose gel electrophoresis. PCR amplification and high-throughput sequencing were performed by Shanghai MajorbioBio-pharm Technology Co., Ltd. PCR amplification was performed using an ABI GeneAmp 9700 instrument and bacterial 16S rRNA gene primers 338F and 806R. The PCR reaction mixture contained 4 μL of 5×FastPfu buffer, 2 μL of dNTPs (2.5 mM), 0.8 μL of forward primer (5 μM), 0.8 μL of reverse primer (5 μM), 0.4 μL of FastPfu polymerase, 0.2 μL of LBSA, 10 ng of template DNA, and was brought to a final volume of 20 μL with ddH2O. High-throughput sequencing results were displayed on the MiSeq platform. Biodiversity analysis of the sequenced 16S rRNA gene variable region was performed using the Majorbio Cloud platform (http: / / www.majorbio.com / ). The results were visualized for further interpretation. Furthermore, PICRUSt2 analysis was used to predict changes in functional genes and metabolic pathways related to nitrogen removal in biological samples from different reactors and at different stages of treatment.

[0056] 1.6. Metagenomic Sequencing

[0057] Metagenomic sequencing is mainly based on high-throughput sequencing platforms such as Illumina / Nanopore. It involves Shotgun sequencing of the genomic DNA of all microorganisms in a biological sample to analyze the species and functional diversity and community structure variation of the microbial community, explore the relationship between microorganisms and the environment and host, and discover and analyze information such as new genes or species with specific functions.

[0058] 2. Results

[0059] 2.1. Comparison of pollutant removal performance

[0060] Figure 2 The study showed the carbon and nitrogen removal effects of the algae-bacterial symbiotic AGS system before and after signal molecule enhancement. In the early stage, when the exogenous signal molecule was added and the system was running for 8-10 days, the TN removal rate was not stable. As the running time increased, the TN removal rate of the X1 system tended to stabilize, while the TN removal rate of the S1 system fluctuated slightly in the early stage of operation until it began to stabilize after 35 days of operation. It is speculated that the added signal molecule C8-HSL can stabilize the denitrification stability of the algae-bacterial symbiotic AGS system and significantly improve the denitrification efficiency. Figure 2As shown in Figure A(a), the TN removal rate of the S1 system was 70.56%, while the TN removal rate of X1 was as high as 95.07%. The total nitrogen removal rate was significantly improved after the introduction of the exogenous signaling molecule C8-HSL. Compared with the S1 system before enhancement, the TN removal rate increased by 25.78%. It is further speculated that the introduction of the exogenous signaling molecule C8-HSL promoted the removal process of organic matter, promoted the enrichment of nitrifying and denitrifying bacteria, improved the activity of functional bacteria, and optimized the microbial community. Figure 2 A(b) and Figure 2 As shown in Figure A(c), the COD removal rates of systems S1 and X1 exhibit similar trends. With reactor operation, the COD removal rate remains stable, with S1 achieving a COD removal rate of 99.16% and X1 achieving 99.46%. The NH4+ removal rates of both systems... + The trend of NH4 removal rate is similar to that of COD, and the NH4 removal rate of S1 is... + -N removal rate was 98.89%, X1 NH4 + -N removal rate was 99.35%. For example... Figure 2 As shown in Figure B, comparing the algae-granular sludge system before and after signal molecule enhancement, the TN removal rate of the X2 system was 87.01%, while that of the S2 system was 61.62%. The TN removal rate of the X2 system after enhancement was 29.17% higher than that of the S2 system before enhancement. It is speculated that the signal molecules can act on the algae, causing nutrients such as nitrogen and phosphorus in the algae to be released from the algae to meet the needs of further growth and reproduction of microorganisms in the reactor, which can promote the metabolism of algae and thus improve the denitrification efficiency. Figure 2 B(b) and Figure 2 The COD removal rate and NH42 content shown in Figure B(c) are as follows. + -N removal rates showed little difference, COD removal rates were all above 95%, and NH4 removal rates were also low. + -N removal rates were all above 95%. Figure 2 As shown in Figure C, comparing the granular sludge systems before and after signal molecule enhancement, the TN removal rate of the X3 system was 81.46%, while that of the S2 system was 54.42%. The TN removal rate of the X3 system after enhancement was 33% higher than that of the S3 system before enhancement. Figure 2 C(b) and Figure 2 The COD removal rate and NH4 in the two systems shown in C(c) are... + The removal rates of -N were not significantly different; both systems achieved Class A standards for COD and ammonia nitrogen removal, with COD removal rates exceeding 95%. NH4 removal... +-N removal rates were all above 95%. Experiments revealed that after the formation of aerobic granular sludge, signaling molecules could induce a shift in microbial growth patterns from suspension to attached growth. Increased EPS content in the exogenous signaling molecule system improved the surface hydrophobicity of the aerobic granular sludge, effectively removing metal ions and organic matter from pollutants. Furthermore, it improved particle stability and addressed issues such as the long operating time and easy disintegration of granular structures in AGS systems.

[0061] 2.2. SEM of different systems

[0062] On day 55, the morphology of aerobic granular sludge in each system was as follows: Figure 3 As shown, the granular sludge in all systems exhibits a full ellipsoidal or spherical shape. Specifically, reactor S3 is an aerobic granular sludge system, with yellow granular sludge. S2 is an algae-granular sludge system, and S1 is a bacterial-algae symbiotic AGS system; both systems have yellow granular sludge with some yellowish-green filamentous material on the surface. X3 is a signal molecule-granular sludge system, with yellow granular sludge and a layer of white filamentous material on the sludge surface. X2 is a signal molecule-algae granular sludge system, and X1 is a signal molecule-bacterial-algae symbiotic AGS system; both systems have green granular sludge, indicating that algae grow on the surface of the aerobic granular sludge. This also demonstrates that the addition of exogenous signal molecules affects the structure of the granular sludge, causing more algae and bacteria to adhere and entangle on its surface.

[0063] To observe the morphology and structure of AGS in each system more clearly, scanning electron microscopy was used to observe the morphology of AGS on day 55 at different magnifications. The results are as follows: Figure 4 As shown in the image, the granular sludge in the X3 system, magnified at 120x magnification, exhibits an ellipsoidal shape. It is clearly observed that the X3 system has more depressions and filamentous entanglement on its surface compared to the S3 system, while the S3 surface is more saturated. In the other systems, the aerobic granular sludge is irregularly ellipsoidal. The granular sludge structures in the S1 and S2 systems are significantly looser, while the X1 and X2 systems with added signaling molecules exhibit a more dense granular sludge structure. Furthermore, the surface of the granular sludge in the X1 and X2 systems shows a large number of filamentous organisms and Chlorella entanglement. The added signaling molecules can stimulate Chlorella to secrete EPS (extracellular polymeric substances), and algal EPS can act as a natural self-flocculating agent, binding to the cell surface and promoting the formation of large aggregates in certain microalgal species. This is why the algae covering the surface of the granular sludge in the X1 and X2 systems can be directly observed. In addition to the filamentous microorganisms and spherical algae acting as a scaffold, many rod-shaped bacteria and Scenedesmus obliqueis can also be seen on the surface of the granular sludge in the image.

[0064] 2.3. Microbial Analysis

[0065] 2.3.1. Microbial diversity analysis

[0066] Microbial diversity analysis primarily assesses the richness and diversity of microbial communities in environmental samples by analyzing richness, coverage, evenness, and diversity. The microbial diversity of the X-SBR and S-SBR systems is shown in Table 2. The coverage of all biological samples was above 99.7%, indicating that the sequencing depth was sufficient to cover most microbial information. Simpson's index, representing species diversity, was significantly higher in the S3 system (0.062) than in other samples, indicating rich diversity in this system. The indices of other systems were lower than those of S3, presumably due to an increased proportion of dominant bacterial groups, leading to reduced diversity. Figure 5 The fact that the dilution curve in (a) gradually flattens out indicates that most of the microorganisms in the biological sample were detected, which is a reasonable result that the reaction system is stable. Figure 5 Figure (b) shows the Rank-abundance curves for the X-SBR and S-SBR systems. Rank-abundance curves can be used to explain two aspects of diversity: species abundance and community evenness. Horizontally, species richness is reflected by the width of the curve; the X-SBR system has a larger range on the horizontal axis than the S-SBR system, indicating that the addition of C8-HSL resulted in higher species richness. The smoothness of the curve reflects the evenness of species in the sample; the X-SBR system's curve is flatter than the S-SBR system's, indicating that the species distribution in the X-SBR system community is more even. Figure 5 Figure (c) shows the OTU distribution in the two systems. The shared OTUs in the X-SBR and S-SBR are 228 and 513, respectively, indicating significant differences in the core OTUs. This observation is consistent with the unique characteristics of the X-SBR system, namely that the signaling molecule C8-HSL promotes the enrichment of the dominant community, enhances the bioactivity of both nitrifying and denitrifying bacteria, and improves the stability of AGS.

[0067] Table 2 Analysis of Microbial Biodiversity Index

[0068]

[0069] 2.3.2. Microbial Community Composition Analysis

[0070] Figure 6 To investigate the microbial community composition of three different systems before and after signal molecule enhancement, the microbial community of the sludge system was analyzed at the phylum, class, and genus levels to explore the influence of the signal molecule C8-HSL on the symbiotic relationships of the three different systems. Figure 6At the phylum level shown in (a), in the granular sludge system, the dominant phyla in both the pre-enhanced S3 system and the post-enhanced X3 system were Proteobacteria, Bacteroidetes, and Chloroflexi, but their relative abundances changed. After enhancement with the signaling molecule C8-HSL, the relative abundance of Proteobacteria increased from 42.26% to 61.94%, while the relative abundances of Bacteridetes and Chloroflexi decreased. This indicates that the addition of the signaling molecule C8-HSL promoted the enrichment of Proteobacteria while also inhibiting the growth of some bacteria. Proteobacteria plays a dominant role in denitrification, and the increase in its relative abundance leads to an increase in the nitrogen removal rate, which is consistent with the performance of pollutant removal. In the algae-granular sludge system, the dominant bacterial phyla changed before and after the introduction of signaling molecules. Before enhancement, the dominant phyla in the S2 system were Proteobacteria (41.84%), Chloroflexi (21.31%), and Bacteroidota (7.40%). After enhancement, the dominant phyla in the X2 system were Proteobacteria (54.57%), Bacteroidota (9.87%), and Cyanobacteria (13.94%). This indicates that signaling molecule enhancement promoted the enrichment of Cyanobacteria, while Cyanobacteria showed increased activity against NH4+. +-N exhibits strong selective absorption, which is beneficial to algal growth and thus improves the removal efficiency of pollutants. In the algal-microbe symbiotic AGS system, the dominant bacterial phyla changed after the addition of the signaling molecule C8-HSL. Before enhancement, the dominant phyla in the S1 system were Proteobacteria (49.79%), Chloroflexi (12.99%), and Actinobacteriota (12.14%). After enhancement, the dominant phyla in the X1 system were Proteobacteria (60.95%), Desulfobacterota (13.40%), and Bacteroidota (13.12%). Desulfobacterota can skillfully degrade recalcitrant organic matter. It can be seen that the changes in the algal-microbe symbiotic AGS system are more obvious after the addition of C8-HSL. It is speculated that C8-HSL can enhance the algal-microbe symbiotic relationship and thus maintain a high denitrification rate. It is noteworthy that Proteobacteria remained the dominant phylum in all six systems. The introduction of the signaling molecule C8-HSL promoted the enrichment of Proteobacteria, and the relative abundance of Firmicutes also increased. Firmicutes had a total relative abundance of 8.44% in the X-SBR system (the three systems after signaling molecule enhancement) and 3.84% in the S-SBR system (the three systems before signaling molecule enhancement), indicating an increase in the relative abundance of Firmicutes. Proteobacteria, Bacteroidota, and Firmicutes belong to HN-AD bacteria. The increased relative abundance of these three in the C8-HSL-enhanced system suggests that C8-HSL can promote the enrichment of HN-AD bacteria, thereby improving denitrification efficiency.

[0071] Figure 6(b) At the class level, a detailed analysis of the relative abundance of each bacterial phylum revealed significant changes in the relative abundance ratios of different bacterial classes in the systems before and after the addition of C8-HSL. Gammaproteobacteria and Alphaproteobacteria were the dominant classes in the biological samples of the six systems. Gammaproteobacteria was abundant in the X-SBR system, with a total relative abundance of 36.58%, higher than that in the S-SBR system, which had a total relative abundance of 21.71%. This indicates that the addition of C8-HSL promoted the growth of Gammaproteobacteria, a result consistent with the pollutant removal performance. The total nitrogen removal rate was significantly improved after the addition of C8-HSL. Both Gammaproteobacteria and Alphaproteobacteria participate in the denitrification process and can secrete EPS, thereby binding cells together and maintaining the stability of the granular sludge structure. In microbial communities at both the phylum and class levels, the addition of the signaling molecule C8-HSL increased the proportion of dominant bacterial groups in the X-SBR system compared to the S-SBR system, indicating that C8-HSL promoted the enrichment of functional bacteria and thus improved denitrification efficiency.

[0072] like Figure 6At the genus level, there are significant differences in the genus composition among the systems, suggesting that the addition of C8-HSL may selectively screen and enrich the bacterial community. In the granular sludge system, the top three dominant bacterial genera in S3 were norank_o__SBR1031 (8.23%), norank_o__Kapabacteriales (8.93%), and Pseudofulvimonas (7.58%). However, the dominant bacterial genera changed after the addition of C8-HSL. The relative abundance of Hydrogenophaga increased from 2.55% to 12.88%, unclassified_f__Comamonadaceae from 1.85% to 12.30%, and Acidovorax from 0.97% to 5.98%, becoming the dominant genera in the X3 system. Unclassified_f__Comamonadaceae possesses aerobic denitrification capabilities, while Acidovorax is a heterotrophic denitrifying bacterium with denitrification activity. It can be seen that the relative abundance of denitrifying bacterial genera significantly increased in the C8-HSL-enhanced system. In the algae-granular sludge system, the dominant bacterial genera in the S2 system were Rhodobacter (10.62%), norank_f__A4b (7.11%), and norank_f__Caldilineaceae (6.95%). In the X2 system enhanced with C8-HSL, the relative abundance of Rhodobacter increased significantly to 17.79%, and new dominant bacterial genera, norank_o__Chloroplast (13.58%) and Pseudoxanthomonas (5.88%), appeared. Pseudoxanthomonas is a well-known denitrifying bacterium. The top three dominant bacterial genera in both systems participated in the denitrification process. It can be seen that the total abundance of the top three dominant bacterial genera in the X2 system was higher than that in the S2 system. It is speculated that the large-scale enrichment of these denitrifying bacterial genera is the reason why the total nitrogen removal efficiency of the X2 system is much higher than that of the S2 system.In the algal symbiotic AGS system, the top three dominant bacterial genera in S1 were Brooklawnia (10.98%), Rhodobacter (9.07%), and Hydrogenophaga (9.61%). These dominant genera can decompose macromolecular organic matter and participate in the denitrification process. In the X1 system with added C8-HSL, the top three dominant bacterial genera were Hydrogenophaga (12.48%), Desulfomicrobium (9.67%), and Pseudoxanthomonas (6.83%). Among them, Hydrogenophaga and Pseudoxanthomonas... S plays a crucial role in denitrification. Desulfomicrobium can degrade some complex, recalcitrant organic matter. Comparing the two systems, the total relative abundance of S1 was 66.18%, while that of X1 was 72.70%. The addition of C8-HSL promoted the enrichment and increased the activity of some bacterial genera, including Thaurea, Flavobacterium, Chlorobium, Sphingorhabdus, and Aquimonas. Among them, Thaurea and Flavobacterium belong to HN-AD bacteria and possess both heterotrophic nitrification and aerobic denitrification functions. It is speculated that the addition of C8-HSL promoted bacterial enrichment and growth. Through respiration, bacteria release CO2, which can be absorbed and utilized by Chlorella as nutrients. Chlorella then produces O2 through photosynthesis. 2供给 Bacteria form a cycle, strengthening the symbiotic relationship between bacteria and algae, thereby improving the degradation efficiency of organic pollutants.

[0073] 2.3.3. Relationship between function and microorganisms

[0074] To further explore the relationship between functional and dominant microorganisms, metagenomic analysis was conducted. A new combinatorial gene catalog was established by selecting nitrogen metabolism (pathway number: Ko00910) and carbon metabolism (pathway number: Ko01200) from the KEGG database, and the contributions of the top 25 abundant genera in nitrogen and carbon metabolism were analyzed.

[0075] according to Figure 7As shown in (a) and (b), the contributing structures of different nitrogen and carbon metabolic functions in the system differ. A comparison of granular sludge S3 (without signaling molecules) and X3 (with signaling molecules) revealed that Methanothrix accounted for the largest proportion in both systems (21.22% and 24.67%, respectively), consumed COD the fastest, and contributed the most to the carbon and nitrogen metabolism degradation of the system. Methanothrix held a dominant position in the complete autotrophic denitrification system for treating biogas digester effluent. Methanothrix is ​​closely related to denitrification and ammonia removal. The granular sludge system with added signaling molecules also enriched some bacterial genera, such as Alicycliphilus (7.06%), Lysobacter (8.29%), and Hydrogenophaga (5.88%). Among them, Hydrogenophaga has denitrification capabilities and plays an important role in the denitrification process. Alicycliphilus is related to the degradation of organic compounds and can provide organic matter for bacteria with denitrification functions, thereby improving denitrification capabilities. Lysobacter can secrete EPS, which is related to nutrient removal and can improve AGS stability. In the algae-granular sludge system, compared to S2, X2 showed a significant enrichment of the relative abundance of Hydrogenophaga, Rhodobacter, Thaurea, and Aquimonas in carbon and nitrogen metabolism. Thaurea, in particular, is an HN-AD bacterium exhibiting heterotrophic nitrification and aerobic denitrification, both of which significantly contribute to carbon and nitrogen metabolism. In the algae-bacterial symbiotic AGS system, Methanothrix, Hydrogenophaga, unclassified_f__Methanobacteriaceae, and Brooklawnia were found to be the most abundant in sample S1. The addition of signaling molecules altered the dominant bacterial genera, with X1 showing enrichment of more genera, greater species diversity, and a smaller difference in relative abundance between genera. This led to the enrichment of genera involved in denitrification, such as Hydrogenophaga, Thaurea, Zoogloea, Flavobacterium, and Anaerolinea.

[0076] Meanwhile, analysis of the abundance changes of the top 20 bacterial genera in the system under the influence of signaling molecules showed that signaling molecules have a significant impact on the composition of the microbial community (e.g., Figure 7(As shown in c). Among them, the genus *Thauera* plays a crucial role in denitrification and COD removal in biological wastewater treatment systems, and the addition of signaling molecules significantly promoted the enrichment of this genus. The genus *Zoogloea*, as a typical aerobic bacterium, is often used for biological nitrogen and phosphorus removal in anaerobic-aerobic wastewater treatment systems; its enrichment under the influence of signaling molecules helps improve the removal efficiency of pollutants such as COD and ammonia nitrogen. Furthermore, the genera *Rhodobacter* and *Hydrogenophaga*, which participate in the degradation and denitrification of macromolecular pollutants, also showed a significant enrichment trend under the regulation of signaling molecules. These findings indicate that signaling molecules can effectively improve the pollutant removal performance of wastewater treatment systems by regulating the abundance of key functional bacterial communities.

[0077] 2.3.4. Predictive Analysis of Functional Genes and Enzymes

[0078] Based on the Kyoto Encyclopedia of Genetics and Genomes (KEGG) database (http: / / www.genome.jp / kegg / ), the migration pathways of nitrogen in six biological samples were studied using PICRUST2. For example... Figure 8As shown, a total of 27 functional genes related to nitrogen removal were found in the six biological samples, including four nitrification-related functional genes (amoA, amoB, amoC, and hao) and 23 denitrification-related functional genes: genes related to processes such as nitrate reduction (narG, narB, narH, narI, napA, napB, nasB, nasC, NR), nitric oxide reduction (norC, norB), nitrite reduction (nirB, nirD, nirA, nirK, nrfA, nirS, nrfH), nitrous oxide reduction (nosZ), and nitrogen fixation (nifD, nifH, anfG, nifK). The presence of the NAP gene in the six biological samples proves that aerobic denitrification occurs in the system. The coexistence of the nirK, nirS, norBC, and nosZ functional genes also indicates that the nitrogen source can be converted into nitrogen gas under aerobic conditions, achieving nitrogen removal. It was found that the relative abundance of the napAB functional gene increased after the addition of the signaling molecule C8-HSL, reaching a maximum of 0.062% in the X1 system, representing a 36.50% increase compared to the unenhanced S1 system. The genes napA and napB, encoding periplasmic nitrate reductase (NAP), are essential functional genes for HN-AD. This confirms that in the X1 system, HN-AD bacterial populations such as Thaurea and Flavobacterium gradually achieved positive enrichment and dominant expression of denitrification functional genes, thus improving denitrification efficiency. This is consistent with the 95.07% increase in total nitrogen removal rate in the X1 system. The changes in the relative abundance of genes napA and napB reflect changes in the system's aerobic denitrification capacity. The total relative abundance of napAB was 0.13% in the S-SBR system and 0.16% in the X-SBR system. The addition of the signaling molecule increased the nap content by 22.69%, suggesting that the addition of C8-HSL promoted the expression of functional genes related to the denitrification process. Although the relative abundance of the napAB functional gene was 0.038% in the X3 system, the relative abundance of the narGHI functional gene increased from 0.17% to 0.30% after the addition of the signaling molecule. This indicates that the reduction of most nitrates to nitrites during the denitrification process in the AGS system with the addition of the signaling molecule mainly relies on the narG, narH, and narI genes. This is one of the reasons why the total nitrogen removal rate of the X3 system was 33% higher than that of the S3 system. In addition, a large number of nitrite reductase genes nirK and nirS are expressed in X1, among which the product encoded by nirK can catalyze NO2. -Nitrogen is oxidized to NO. The presence of nosZ confirms that the final form of nitrogen is nitrogen gas, and it is also highly expressed in X1. The norB and nosZ genes encode nitric oxide reductase (NOR) and nitric oxide reductase (NOS), respectively. In the final stage of denitrification, NOR catalyzes the oxidation of NO to N2O, while NOS catalyzes the oxidation of N2O to N2.

[0079] The abundance of enzymes involved in the nitrogen cycle at different stages, such as Figure 9 As shown, there are three nitrogen metabolism pathways: nitrification, denitrification, and dissimilatory nitrate reduction to ammonia (DNRA). The nitrification process involves NH4+. + -N→NH2OH→NO2 - -N, ammonia nitrogen, is converted to nitrite under the action of enzymes 1.14.99.39 and 1.7.2.6. The relative abundance of related enzymes in the nitrification process was observed to be similar in both systems, indicating that the addition of C8-HSL has a relatively small impact on nitrification. The denitrification pathway, i.e., NO3- － -N→NO2 － -N→NO→N2O→N2, nitrate and nitrite nitrogen are ultimately converted into nitrogen gas during denitrification. Compared with the S-SBR system, the relative abundance of enzymes 1.7.2.1 and 1.7.2.5 is increased in the X-SBR system, especially in the X1 and X3 systems. This results in the conversion of more nitrite into nitrous oxide, thus generating nitrogen gas and achieving efficient nitrogen removal. This is one of the reasons why the X1 system has the best denitrification effect, and why the X3 system has the greatest improvement in total nitrogen removal efficiency compared to the S3 system. The addition of C8-HSL promotes the enrichment of some bacterial genera and increases their activity, including Thaurea and Flavobacterium, which belong to the HN-AD bacteria. These bacteria work synergistically with other bacteria to achieve complete nitrogen removal. It can be observed that, in both S-SBR and X-SBR systems, changes in enzyme abundance are closely related to changes in denitrification efficiency within the system. DNRA pathway strains first use nitrate reductases (1.7.11, 1.7.1.2, 1.7.1.3, 1.7.7.2) to reduce nitrate nitrogen to nitrite nitrogen. Then, the nitrite nitrogen is reduced to ammonium, i.e., NO3, by assimilating nitrite reductases (1.7.7.1, 1.7.2.2, 1.7.1.15). － -N→NO2 － -N→NH4 +-N, compared with the algal symbiotic AGS system, the relative abundance of enzyme 1.7.1.15 was significantly reduced after the addition of the signaling molecule, suggesting that nitrite in the X1 system is more involved in the denitrification process. Notably, the abundance of dissimilatory nitrate reductase (1.7.1.15, 1.7.2.2) in S-SBR and X-SBR was 13.35 times and 10.34 times that of assimilatory nitrate (1.7.7.1) enzymes, respectively, indicating that dissimilatory nitrate reduction is the main pathway of nitrogen metabolism.

[0080] 3. Conclusion

[0081] The TN removal rate of the X1 system enhanced with the signaling molecule C8-HSL reached 95.07%, an increase of 24.51% compared to the unenhanced S1 system (70.56%). The signaling molecule altered the bacterial community composition, promoting the enrichment of denitrifying bacteria such as *Hydrogenophaga* and *Pseudoxanthomonas*, while also increasing the abundance of complex organic matter degrading bacteria *Desulfomicrobium*. After enhancement, the increase in denitrifying bacteria such as *Hydrogenophaga*, *Thauera*, and *Zoogloea* optimized species diversity and improved nitrogen removal and organic matter degradation efficiency. Based on PICRUSt2 analysis, the increased abundance of the *napAB* gene in the X1 system promoted aerobic denitrification; enhanced expression of the *nirK* and *nirS* genes promoted the conversion of nitrite to nitrogen. The signaling molecule effectively improved the system's nitrogen removal performance by optimizing nitrogen metabolism pathways. This invention can provide new theoretical and practical references for the treatment of rural domestic sewage.

[0082] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the claimed invention. The scope of protection claimed by the appended claims and their equivalents is defined.

Claims

1. A symbiotic system of bacteria and algae for wastewater treatment, characterized in that, The system includes: aerobic granular sludge, Chlorella proteoglycans, HN-AD bacteria, and the signaling molecule C8-HSL; The C8-HSL signaling molecule promotes an increase in napAB gene abundance, thus promoting aerobic denitrification. The signaling molecule C8-HSL promotes the expression of nirK and nirS genes, thereby promoting the conversion of nitrite into nitrogen gas.

2. The signaling molecule C8-HSL promotes the application of the HN-AD bacteria, aerobic granular sludge, and Chlorella proteoglycans symbiotic granular sludge contained in the system as described in claim 1 in wastewater treatment capacity.

3. The application according to claim 2, characterized in that, The application is manifested in any of the following aspects: (1) Improve the denitrification stability of granular sludge containing HN-AD bacteria, and improve denitrification efficiency, TN removal rate, NH4+-N removal rate and COD removal rate; (2) Promote the removal of organic matter from granular sludge containing HN-AD bacteria; (3) Promote the enrichment of HN-AD bacteria in granular sludge containing HN-AD bacteria; (4) Promote the release of nutrients from algae in granular sludge containing HN-AD bacteria; (5) Inducing changes in the growth pattern of microorganisms in granular sludge containing HN-AD bacteria; (6) Improve the surface hydrophobicity of aerobic granular sludge in symbiotic granular sludge containing HN-AD bacteria; (7) Modify the granular sludge structure of granular sludge containing HN-AD bacteria; (8) Promotes the secretion of EPS by Chlorella; (9) Promote the dominant expression of denitrification functional genes; (10) Promotes the increase of napAB gene abundance and promotes aerobic denitrification; (11) Promotes the expression of nirK and nirS genes and promotes the conversion of nitrite into nitrogen gas.

4. The application according to claim 3, characterized in that, in, It promotes the release of nutrients from algae, namely nitrogen and phosphorus.

5. The application according to claim 4, characterized in that, in, The growth pattern of microorganisms was induced to change from suspension to attached growth.

6. The application according to claim 4, characterized in that, in, Modifying the granular sludge structure of the algae-microbe symbiotic AGS system mainly includes: This allows more algae and bacteria to adhere to and entangle on the surface of the granular sludge; This makes the granular sludge structure more compact.

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