Application of C8-HSL in promoting the ability of nitrogen and phosphorus removal of bacteria-algae symbiotic system in wastewater treatment

Abstract

The application of C8-HSL in promoting the nitrogen and phosphorus removal capacity of bacteria-algae symbiotic system in sewage treatment is disclosed, and 0.8 μM of C8-HSL improves the removal of ammonia nitrogen, TN and total phosphorus of the symbiotic system by 25.17%, 36.22% and 38.74% respectively, and the formation of extracellular polymeric substances (EPS) is increased by 68.62%. The metagenomic sequencing results show that Halomonas and Sphingobium are the main denitrifying and phosphorus removal microorganisms, and the narGHI and nirKS genes play a key role in the denitrification process, while the phnX and ppa genes play a key role in the phosphorus removal process. The study provides a theoretical basis and practical guidance for optimizing sewage treatment through quorum sensing strategy.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment, specifically to the application of C8-HSL in promoting the nitrogen and phosphorus removal capabilities of bacterial-algae symbiotic systems in wastewater treatment. Background Technology

[0002] Livestock and poultry farming wastewater is characterized by high chemical oxygen demand (COD), high ammonia nitrogen, and high phosphorus. Untreated discharge into the environment can lead to ecological problems such as eutrophication, resulting in the proliferation of algae. Traditional microbial treatment technologies have poor tolerance to nitrogen and phosphorus, leading to practical problems such as complex processes, high energy consumption, and low treatment efficiency.

[0003] Research based on heterotrophic nitrification-aerobic denitrification (HN-AD) strains has revealed that HN-AD strains can simultaneously achieve nitrification and denitrification, exhibiting significant nitrogen removal potential under extreme environments such as high organic matter concentration, high salinity, and high ammonia nitrogen. For example, Zhang et al. used HN-AD bacteria to treat aquaculture wastewater, increasing the ammonia nitrogen tolerance concentration from 200 mg / L to 1000 mg / L and the total nitrogen removal rate from 30.4%. However, the high-oxygen requirements of HN-AD strains increase treatment costs, and most HN-AD strains are ineffective in phosphorus removal. As an emerging biological treatment technology, algal-microbe symbiosis technology shows great potential in wastewater treatment due to its highly efficient nitrogen and phosphorus removal capabilities and low operating costs. Microalgae synthesize organic matter from carbon dioxide (CO2) through photosynthesis, while simultaneously releasing oxygen into the environment. This released oxygen provides heterotrophic bacteria with the oxygen needed for metabolism, thereby promoting the oxidation of organic matter and ammonia and reducing aeration energy consumption. Meanwhile, microalgae can also utilize inorganic carbon, nitrogen, and phosphorus released during bacterial metabolism, further enhancing the system's repair capabilities. Our team previously constructed a symbiotic system using our independently developed HN-AD strain and *Chlorella proteoglycans*, finding that the addition of *Chlorella* significantly increased the abundance of dominant HN-AD bacteria. Existing research indicates that this symbiotic relationship is regulated by quorum sensing signaling molecules. Quorum sensing, through the secretion of self-inducing agents to achieve intercellular communication, is a typical ecological behavior of microorganisms. In wastewater treatment, various QS sensing systems have been discovered, among which the AHL-mediated quorum sensing system has been extensively studied and is considered a major signaling molecule affecting microbial community structure and denitrification. Simultaneously, the addition of AHL can exponentially increase the content of extracellular polymeric substances (EPS) in biofilms. Li et al. found that the addition of exogenous C8-HSL can significantly promote biofilm growth. Shah et al. proposed that C8-HSL is a common "signal" for bacterial communication. These studies have made C8-HSL a focus of signaling molecule research. Zhang et al. found that appropriate concentrations (40 nM) of AHLs promote biofilm formation, while high concentrations (200 nM) inhibit it. Therefore, the concentration of signaling molecules affects their pollutant removal efficiency. Currently, the effects of signaling molecules on algal symbiotic systems mainly focus on symbiotic systems constructed with activated sludge or granular sludge and algae. The effects on symbiotic systems constructed with HN-AD strains and Chlorella have not been reported. The effects of the signaling molecules used in this study on this system, their extracellular polymers, and microbial characteristics require further investigation.

[0004] A symbiotic system was established between HN-AD bacteria and Chlorella proteoglycans. The effect of exogenous C8-HSL concentration on the denitrification and phosphorus removal efficiency of the bacterial-algal symbiotic system was investigated to determine the optimal C8-HSL concentration. EPS was measured to explore the effect of C8-HSL concentration on symbiotic biofilm formation. Metagenomic sequencing combined with the KEGG database was used to study the effects of concentration on microbial community structure and metabolic pathways, providing a theoretical basis for the application of quorum sensing signaling molecules in bacterial-algal symbiotic systems. Summary of the Invention

[0005] In view of this, one objective of the present invention is to provide an application of C8-HSL in promoting the nitrogen and phosphorus removal capacity of a bacterial-algae symbiotic system in wastewater treatment; another objective of the present invention is to provide a method for improving the nitrogen and phosphorus removal capacity of wastewater treatment.

[0006] To achieve the above objectives, the present invention provides the following technical solution: Application of C8-HSL in promoting the nitrogen and phosphorus removal capabilities of algae-bacteria symbiotic systems in wastewater treatment.

[0007] Preferably, the concentration of C8-HSL used in this invention is controlled at 0.8 μM.

[0008] Preferably, the bacterial-algae symbiotic system of the present invention is composed of HN-AD bacteria and Chlorella proteoglycans in a volume ratio of 1:3.

[0009] Preferably, the HN-AD bacteria of this invention are composed of Cupriavidus sp. SWA1, Alcaligenes faecalis, Acinetobacter, and Ochrobactrum sp. TAC-2; and the proteocyst Chlorella is Chlorella pyrenoidosa FACHB-9.

[0010] 2. A method for improving the nitrogen and phosphorus removal capacity of wastewater treatment, comprising inoculating the wastewater to be treated with algae species with an OD680 of 1.2 or higher and HN-AD bacteria, and adding C8-HSL, wherein the inoculation ratio of HN-AD bacteria is 5%, the bacteria-algae ratio is 1:3, and then the treatment is carried out at a temperature of 28~30℃, a rotation speed of 300±50, an illumination intensity of 2000±200 lux, an air intake rate of 0.6L / min, and an HRT of 48h.

[0011] Preferably, the amount of C8-HSL added is such that the final concentration after addition is 0.8 μM.

[0012] Preferably, the HN-AD bacteria of this invention are a combination of Cupriavidus sp. SWA1, Alcaligenes faecalis, Acinetobacter, and Ochrobactrum sp. TAC-2; and the proteocyst Chlorella is Chlorella pyrenoidosa FACHB-9.

[0013] In a preferred embodiment of the present invention, the average removal rates of ammonia nitrogen, TN, TP and COD by the method are 91.72%, 81.62% and 87.72%, respectively.

[0014] The beneficial effects of this invention are as follows: This invention provides the application of C8-HSL in enhancing the nitrogen and phosphorus removal capabilities of algae-bacterial symbiotic systems in wastewater treatment. Studies have shown the impact of C8-HSL concentration on the nitrogen and phosphorus removal efficiency of algae-bacterial symbiotic systems. Experimental results show that a C8-HSL concentration of 0.8 μM significantly improves the nitrogen and phosphorus removal capabilities of the symbiotic system, achieving average removal rates of 91.72%, 81.62%, and 87.72% for ammonia nitrogen, TN, TP, and COD, respectively. It also promotes the formation of extracellular polymeric substances (EPS), which has a positive impact on microbial aggregation and sedimentation performance. Metagenomic sequencing analysis revealed that Halomonas and Sphingobium are the key genera for nitrogen and phosphorus removal in the symbiotic system, and the genes narGHI, nirKS, phnX, and ppa play crucial roles in nitrogen and phosphorus removal. By adjusting the C8-HSL concentration, the biological treatment efficiency of algae-bacterial symbiotic systems can be optimized, providing a new approach for achieving efficient and economical wastewater treatment. Attached Figure Description

[0015] To make the objectives, technical solutions, and beneficial effects of this invention clearer, the following figures are provided for illustration: Figure 1 Pollutant removal for algae-microbe symbiotic systems (a: ammonia nitrogen; c: TN; e: TP; g: COD; and average removal rates: b: ammonia nitrogen; d: TN; f: TP; h: COD). Figure 2 For microbial diversity analysis (a: Shannon index; b: Venn diagram; c: NMDS analysis; d: phylum level; e: class level); Figure 3 The effect of signal molecule concentration on EPS secretion in symbiotic systems (a: EPS synthesis; b: key genes for EPS synthesis). Figure 4 Contribution to species (a: nitrogen metabolism; b: phosphorus metabolism; c: EPS; d: regression analysis; e: collinearity network).

[0016] Figure 5 Analysis of nitrogen metabolism in symbiotic systems (a: gene expression in key pathways; b: gene abundance map; c: enzyme abundance map; d: enzyme abundance in each metabolic pathway). Figure 6 Analysis of phosphorus metabolism (a: gene expression in key pathways; b: gene abundance map; c: enzyme abundance map; d: phosphorus metabolism pathway map); Figure 7 To understand the mechanism by which C8-HSL concentration affects the algal-bacterial symbiotic system. Detailed Implementation

[0017] 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 and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0018] This invention uses artificially synthesized high-concentration aquaculture wastewater as experimental water. Anhydrous sodium acetate is used as the carbon source, ammonium sulfate as the nitrogen source, and dipotassium hydrogen phosphate as the phosphorus source. The simulated influent concentrations are as follows: COD concentration 4500–5500 mg / L, ammonia nitrogen concentration 470–550 mg / L, and total phosphorus concentration 55–95 mg / L. Simultaneously, 1 / 200 of the trace elements are added to promote microbial growth. The composition of the trace elements is: 2 g / L MgSO4·7H2O, 0.1 g / L MnSO4·H2O, 1.5 g / L CaCl2, and 0.1 g / L FeSO4·7H2O.

[0019] Inoculum: *Chlorella pyrenoidosa* FACHB-9 from the Institute of Hydrobiology, Chinese Academy of Sciences (Wuhan, China) was used in this study. Before inoculation, the microalgae were stored in solid TAP medium at 4°C. The medium consisted of 0.4 g NH₄Cl, 0.1 g MgSO₄·7H₂O, 0.05 g calcium chloride 2·2H₂O, 0.108 g K₂HPO₄, 0.05 g calcium chloride 2·2H₂O, 0.056 g KH₂PO₄, and 2.42 g Tris per liter. Single algal colonies from the solid medium were picked and inoculated into 500 mL Erlenmeyer flasks containing a mixture of 1.7 g / L BG11 and 20 g / L glucose medium, and incubated under continuous light (150 μmol / m²) on a shaker. 2 The algae were cultured under white LED lamps at a temperature controlled at 28-30℃ and a shaking speed of 160 r / min. Subculture was performed every 3-5 days. All algal strains used in the experiment were in the logarithmic growth phase under these culture conditions, with an OD680 exceeding 1.2.

[0020] HN-AD bacteria are a complex of Cupriavidus sp. SWA1, Alcaligenes faecalis, Acinetobacter, and Ochrobactrum sp. TAC-2.

[0021] Example 1 Seven 500ml Erlenmeyer flasks were used, each filled with 300ml of artificially synthesized high-concentration aquaculture wastewater. Algae with an OD680 of ≥1.2 and HN-AD bacteria were then inoculated, with an HN-AD bacteria inoculation ratio of 5% and a bacteria-to-algae ratio of 1:3. C8-HSL was added separately, resulting in final concentrations of 0μM, 0.2μM, 0.4μM, 0.6μM, 0.8μM, 1.0μM, and 1.2μM, respectively. The Erlenmeyer flasks were then placed in a water bath with a magnetic stirrer at a speed of 300±50 rpm and a temperature of 28~30℃. A 5W white LED was used as a stable light source, with continuous illumination and an intensity of (2000±200) lux. Aeration was provided by external aeration, with the air intake controlled by a flow meter at 0.6L / min, and the HRT (heat recovery time) was 48h.

[0022] Water samples were collected every 48 hours, and ammonia nitrogen, total nitrogen, COD, and TP were measured according to standard methods. At the end of the experiment, microbial samples were collected for metagenomic sequencing. Based on the concentration of added C8-HSL (0 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, and 1.2 μM), the biological samples were labeled A1, A2, A3, A4, A5, A6, and A7, respectively. DNA extraction and sequencing were performed by Shanghai Meiji Biopharmaceutical Technology Co., Ltd. (Shanghai, China) using the Illumina platform.

[0023] Figure 1The changes in pollutant removal efficiency of the algae-bacterial symbiotic system after adding different concentrations of C8-HSL (0 μM, 0.2 μM, 0.4 μM, 0.6 μM, 0.8 μM, 1 μM, 1.2 μM) are shown. Figures a, c, e, and g represent the changes in ammonia nitrogen, total nitrogen, total phosphorus, and COD over operating time, respectively, while b, d, f, and h show the average removal rates of the four pollutants. Figures a and b show that the system reached a steady state around day seven, and the removal efficiencies of ammonia nitrogen and total nitrogen initially increased and then decreased with varying C8-HSL concentrations. Simultaneously, C8-HSL significantly affected the nitrification and denitrification functions of the symbiotic system, similar to the findings of Wang et al. At a C8-HSL concentration of 0.8 μM, the symbiotic system exhibited the best nitrogen removal performance, with ammonia nitrogen and total nitrogen removal rates increasing by 25.17% and 36.23%, respectively. However, when the C8-HSL concentration was 1 μM, the removal efficiencies of both ammonia nitrogen and total nitrogen were significantly inhibited, decreasing by 12.18% and 9.23%, respectively. Studies have shown that exceeding the optimal concentration of AHL (N-acylhomoserine lactone) significantly inhibits microbial denitrification. Song et al. found that QS (quorum sensing) and QQ (quorum quenching) coexist in bioreactors, and their balance is crucial for bioreactor operation. In this study, high concentrations of AHLs disrupted the balance of QS and QQ in the system, negatively impacting system operation and leading to reduced pollutant removal efficiency.

[0024] Figure 1 Figures e and f show the removal of total phosphorus (TP) by the symbiotic system. The removal of TP by the symbiotic system also showed a significant increasing trend with increasing C8-HSL concentration. Phosphorus is crucial for energy formation in microalgal cells, and organisms consume phosphorus from the environment through cellular assimilation of proteins, nucleic acids, and phospholipids. Numerous studies have confirmed that symbiotic systems can better remove phosphorus, accelerating cellular assimilation of phosphorus and thus promoting its removal. The study also found that under aerobic conditions, AHL (Algal Flow Regulator) facilitates phosphorus uptake by polyphosphate-accumulating bacteria. With increasing C8-HSL concentration, the average removal rate of total phosphorus increased by 12.6%, 23.1%, 29.6%, and 38.7%, respectively. However, when the C8-HSL concentration reached 1.2 μM, the average removal rate decreased by 5.2%, which may be due to the excessively high C8-HSL concentration inhibiting the removal of total phosphorus by the symbiotic system.

[0025] Figure 1The figures (g, h) demonstrate the COD removal efficiency of the symbiotic system. In the algal-microbe symbiotic system, microalgae can utilize inorganic carbon for photosynthesis, releasing oxygen for bacterial growth. Furthermore, in the presence of organic carbon, the synergistic photosynthesis and aerobic respiration of microalgae accelerate their development, thereby increasing carbon fixation. Therefore, the algal-microbe symbiotic system can effectively eliminate both organic and inorganic carbon, reducing greenhouse gas emissions. Studies have shown that adding C6-HSL and C8-HSL can significantly improve nitrification and denitrification, but has no significant effect on COD removal, consistent with this study. With increasing signal molecule concentration, the removal rates of ammonia nitrogen, total nitrogen, and total phosphorus show significant changes, but the COD removal rate remains concentrated between 70-85%, generally showing a trend of first increasing and then decreasing. This may be due to significant differences in the denitrification function of enriched microorganisms, thus requiring further analysis of microbial diversity.

[0026] Overall, the addition of 0.8 μM C8-HSL significantly improved the nitrogen removal performance of the symbiotic system, with average removal rates of 91.72%, 81.62%, 87.72%, and 83.46% for ammonia nitrogen, TN, TP, and COD, respectively. Compared with the system without C8-HSL, the removal rates of ammonia nitrogen, total nitrogen, total phosphorus, and COD increased by 25.17%, 36.22%, 38.74%, and 11.34%, respectively. This demonstrates that 0.8 μM C8-HSL can significantly enhance the nitrogen and phosphorus removal efficiency of the symbiotic system.

[0027] Example 2 The effect of signal molecule concentration on the composition and structure of symbiotic microbial communities Microbial composition analysis can provide a better understanding of the denitrification and phosphorus removal processes in symbiotic systems. The Shannon index is an important indicator for measuring community diversity, comprehensively reflecting the richness and evenness of the community. A higher Shannon index indicates greater community diversity and a more even and abundant distribution of species. Figure 2 The results in a show that the Shannon index of A1 and A7 was significantly higher than that of other samples, and the species richness changed significantly with the change of C8-HSL concentration, with A2 significantly decreasing and A5 significantly higher than the other four samples. Figure 2 Figure b shows the Venn diagrams for seven samples. These seven samples share 106 species. A1 and A7 have the most unique species, but since their pollutant removal efficiency is significantly lower than that of other samples, it is inferred that the unique species did not play a key role in nitrogen and phosphorus removal. A5 has two unique species and its Shannon index is higher than that of the other four samples, which may be the reason for its good nitrogen and phosphorus removal efficiency. Figure 2Figure c illustrates the non-metric multidimensional scaling analysis of NMDS. Stress indicates the strength of the test results; when Stress < 0.05, the data is considered highly representative. In this case, Stress = 0.000, indicating significant representativeness. The figure shows that A7 is significantly different from other samples, which may explain its significantly reduced pollutant removal. A4 and A6 show great similarity, and their Shannon indices, unique species, and pollutant removal effects are also very similar, but significantly different from A5. This may be one reason why A5 exhibits the best nitrogen and phosphorus removal efficiency.

[0028] Figure 2 Figures d and e show the species composition at the phylum and genus levels for the seven samples, respectively. It is clear from the figure that *Pseudomonadota* is the most abundant phylum among the seven samples. *Pseudomonadota* includes some Gram-negative bacteria with denitrification functions. Its relative abundance is highest in A1 (76.48%), and with increasing C8-HSL concentration, the relative abundance in A5 and A6 is 99.74% and 99.87%, respectively, while the relative abundance in A7 decreases significantly by 56.42%. Based on the above analysis, A7 has the highest abundance but the worst pollutant removal effect, possibly because most microorganisms do not play a crucial role in nitrogen and phosphorus removal. The significant decrease in the relative abundance of *Pseudomonadota* also indicates that it plays a key pollutant removal role in the symbiotic system.

[0029] Figure 2The results clearly show that Halomonas and Alcaligenes had the highest relative abundance across the seven samples, exhibiting significant differences with varying C8-HSL concentrations. Halomonas has been confirmed as a functional bacterial genus that denitrifies via the HN-AD pathway and is also a typical phosphorus-accumulating organism, considered to play a crucial role in phosphorus removal and also capable of producing extracellular polysaccharides. Studies have demonstrated that Alcaligenes functional bacteria possess highly efficient denitrification capabilities. Research has found that Alcaligenes functional bacteria have the ability to reduce inorganic phosphate and also possess the function of secreting EPS. The relative abundance of Halomonas was greater than 78% in samples A1-A3, decreasing to 3.72% in A4, rising again to 82.43% in A5, decreasing to 1.23% in A6, and increasing to 74.21% in A7. The significant decrease in A4 and A6 is consistent with the previous Alpha diversity analysis. A4 and A6 showed great similarity, with Alcaligenes being the dominant genus, with relative abundances of 93.17% and 94.38%, respectively. The relative abundance of Alcaligenes in other samples was less than 10%. It is speculated that Halomonas plays a key role in pollutant removal in A1-A3 and A5, while Alcaligenes plays a key role in nitrogen and phosphorus removal in A4 and A6. In A7, the relative abundance of both of these functional genera was lower than in other samples, which is why C8-HSL inhibited the nitrogen and phosphorus removal function of the symbiotic system. Zhang et al. found that Sphingobium is crucial in the biosynthesis of polysaccharides and proteins. Its relative abundance in A5 was higher than in other samples (0.84%), which may be due to its higher relative EPS content. Alishewanella, a functional genus with denitrification capabilities, showed a significant correlation between its relative abundance and nitrogen removal in all seven samples, and is therefore considered to play a key role in nitrogen removal in A5.

[0030] Example 3 Effect of signaling molecule concentration on EPS secretion in symbiotic systems Extracellular polysaccharides (EPS) are polymers synthesized by bacterial cells during growth and metabolism. They protect cells from external toxins and provide energy for microorganisms under adverse conditions. EPS also contains abundant functional groups that can adsorb various chemical substances. Currently recognized EPS compositions include S-EPS, LB-EPS, and TB-EPS. EPS determination involves extraction via a low-temperature water bath. The biofilm sample is centrifuged at 8000g, 4℃ for 15min, and filtered through a 0.45μm filter; the supernatant is S-EPS. 0.05% NaCl solution is added to dilute to its original volume (the NaCl solution needs to be preheated to 70℃ to ensure the sludge suspension immediately reaches 50℃). The sludge suspension is immediately vortexed for 1min, then centrifuged at 8000g for 10min at 4℃, and filtered through a 0.45μm filter; the resulting supernatant is LB-EPS (loose type). Add 0.05% NaCl solution again, place in a 60℃ water bath for 30 min, then centrifuge at 8000g for 15 min at 4℃. The collected supernatant is TB-EPS (compact type). Extracellular polysaccharides were determined using the phenol-sulfuric acid method, and extracellular proteins were determined using the BCA protein kit (Shanghai Sangon Biotech Co., Ltd., China). Species and functional annotations of the sequencing results were based on non-redundant protein sequences and the Kyoto Encyclopedia of Genetics and Genomes (KEGG) database. The total sequence length of the non-redundant gene set was obtained as 12,676,050 bp, with an average sequence length of 658.6 bp.

[0031] Figure 3 Figure a shows the different distributions of the three EPS types in seven samples, clearly demonstrating that the concentration of C8-HSL significantly affects EPS secretion. The study indicates that the addition of AHLs exponentially increases the EPS content in biofilms. Compared to the absence of C8-HSL, all three EPS types in A2-A6 increased, while LB-EPS and TB-EPS in A7 were inhibited, which is positively correlated with the aforementioned pollutant removal. It is speculated that low EPS content weakens the symbiotic system's resistance to external pollutants, reduces nutrient utilization, and consequently weakens denitrification and phosphorus removal capabilities. Proteins possess strong flocculation and bioaggregation abilities, with their adhesion ability far exceeding that of polysaccharides. The PN / PS ratios in A2-A5 are all greater than 1, which may be related to increased pollutant removal; the increasing adhesion of proteins enhances pollutant adhesion, thereby improving the sample's denitrification and phosphorus removal capabilities. An EPS gene set was constructed using metagenomics, and 10 functional genes related to EPS were screened. Figure 3 Figure b shows the relative abundance of 10 functional genes. Combined with the analysis of EPS secretion and pollutant removal, it was found that VpsCG is the key functional gene in EPS secretion in A1-A3 and A5, while IcaA and PgaD play a role in A4 and A6.

[0032] Example 4: Analysis of the functional contribution of microorganisms To explore the relationship between metabolic function and dominant species more deeply, a new gene set was established from the KEGG database, consisting of nitrogen metabolism (pathway number: Ko00910), phosphorus metabolism (pathway number: Ko00440), and EPS (pathway number: Ko00543). Figure 4 Figures a, b, and c represent the contributions of the top eight genera in relative abundance to nitrogen metabolism, phosphorus metabolism, and EPS function, respectively, and cluster analysis was performed. The figures show that C8-HSL significantly impacts species function. Different cluster analyses were generated for the seven samples across the three functions, but A4 and A6 showed high consistency. The contributions to all three functions were provided by *Alcaligenes*. Previous analysis indicated that *Alcaligenes* possesses denitrification, phosphorus removal, and EPS generation functions, which explains its high pollutant removal efficiency. For the other five samples, nitrogen metabolism, phosphorus metabolism, and EPS functions were primarily contributed by *Halomonas* and *Alcaligenes*, with a combined contribution exceeding 90%. In nitrogen metabolism, A5 and A7 were grouped together, while in phosphorus metabolism, A5 and A1-3 were grouped together, and in EPS, A5, A3, and A7 were grouped together. This demonstrates that while A5's contribution to the three functions is similar to the other groups, it also exhibits differences. Sphingobium contributed 0.70%, 0.30%, and 5.22% to the three functions of A5, which were significantly higher than those of other samples. This analysis suggests that A5 had higher nitrogen and phosphorus removal and EPS production than other samples, and this type of bacteria played a key role.

[0033] Figure 4 Figure d shows the regression analysis of three functions and species based on the KEGG database. The figure shows that the species in A5 and A6 are significantly positively correlated with these three functions, revealing the consistency between species and functions at the diversity level. The consistency between species and functions in other samples is similar. Figure 4 The first part of the graph (e) is a univariate correlation network analysis of the top 20 genera in relative abundance. The size of the nodes in the graph represents the species abundance. It can be seen that Halomonas and Alcaligenes are the genera with the highest abundance. The graph shows that there is no connection between the two species, which proves that they are relatively independent. The connection between these two genera and other species is mainly green, which indicates that they compete with other species. Their relatively high abundance means that they compete with other species for nutrients and inhibit the growth of other microorganisms, thus making them the dominant species.

[0034] Example 5: Nitrogen metabolism analysis of the symbiotic system Microorganisms play a crucial role in biological nitrogen and phosphorus cycling. In aquatic environments, microorganisms participate in nitrogen degradation through nitrification and denitrification. To investigate the effects of different concentrations of C8-HSL on nitrogen removal performance in a symbiotic system, functional genes and enzymes related to nitrogen metabolism were labeled using the KEGG database. Twenty-two nitrogen metabolism-related genes and twelve functional enzymes were screened from a large pool of genes. Figure 5 Table a shows five nitrogen metabolism pathways: catabolism / assimilation of nitrate fixation, denitrification, nitrification, and nitrogen fixation. Denitrification exhibits the highest gene expression abundance, with A4 and A6 showing high consistency. However, only the gene expression abundance for nitrite reduction to nitrogen is high; other processes are negligible. This may be due to its singular gene expression, resulting in lower nitrogen removal efficiency compared to A5. Tables b and c show the abundance of various functional genes and enzymes, while table d shows the abundance expression of functional enzymes in each nitrogen metabolism pathway. This indicates that nitrate is converted to nitrite via narGHI, napAB, and narB, and to ammonia via nirBD, nrfAH, and nasCD. However, the relative abundance of napAB, narB, and nrfAH, as well as the abundance of their associated enzymes, were low in all seven samples. Therefore, it is believed that nitrate is converted to nitrite via narGHI and to ammonia via nirBD and nasCD. The relative abundance of these enzymes gradually increases in samples A1-A3, decreases in A4 and A6, increases in A5, and decreases in A7, suggesting that A1-A3 and A5 have better nitrate metabolism capabilities. Furthermore, the growth trend of these genes is proportional to that of Halomonas and Alishewanella, thus inferring that these genera are the primary organisms producing these genes. Figure 5 The relative abundance of EC:1.7.2.1 in samples A4 and A6 was significantly higher than in other samples, at 71.13% and 72.40% respectively. This indicates that the denitrification process is mainly driven by the expression of the gene nirKS encoding nitrite reductase. Among the other five samples, A5 had the highest relative abundance of nitrite reductase (17.58%), which is also the key functional gene contributing to A5's strong denitrification capacity. A4-A6 showed the best nitrogen and phosphorus removal efficiency among the seven samples. However, the high expression of denitrification genes and enzymes in A4 and A6 indicated that denitrification was dominant. Conversely, nitrogen genes and enzymes were relatively abundant in A5. Therefore, it is inferred that the nitrogen metabolism pathway is NH4+. + -N→NH2OH→NO3 - -N→NO2 - -N→NO→N2O→N2.

[0035] In aquatic environments, microorganisms participate in the degradation of nitrogen and phosphorus by engaging in nitrification and denitrification processes and releasing enzymes that promote the mineralization of inorganic and organic phosphorus. Figure 6Tables a, b, c, and d show the abundance of functional genes and enzymes related to phosphorus metabolism, as well as phosphorus metabolism pathways, in seven samples. phnX, phnA, and phnY encode acetaldehyde phosphate hydrolase, acetaldehyde phosphate hydrolase, and acetaldehyde phosphate dehydrogenase, respectively. The relative abundance of these three genes decreases progressively across the seven samples, with phnX having the highest relative abundance in A5. Its relative abundance change is proportional to that of *Sphingobium*, suggesting that this bacterium is the ontogeny of this gene. pepM and fom1 encode phosphoenolpyruvate phosphate mutase. Microorganisms transport inorganic phosphates via the PST system regulated by pstB, pstC, pstA, pstS, and phoU, phoR, and phoB. When the external phosphorus concentration is low, the pstBCAS complex separates from the phoR protein, and phoR is phosphorylated by histidine, initiating the expression of genes related to phosphate and inorganic phosphate transport. When the external phosphorus concentration is high, pstBCAS and phoR proteins bind to form a complex. This complex can prevent phosphorylation of the phoB protein and inhibit the expression of genes related to phosphate transport, thus preventing the transport of inorganic phosphate. The enzyme with the highest relative abundance among the seven samples is the one encoded by phoBR, indicating that the seven samples have good phosphorus regulation capabilities. The relative abundance of the pstBCAS gene is proportional to that of Halomonas, therefore it is inferred that this genus is the entity that secretes the pstBCAS gene. The phnC gene is related to phosphorus uptake, increasing in A1-A3 and then decreasing, with a relatively high abundance in A5, demonstrating its role in phosphate uptake. The genes phnG, phnH, phnI, phnJ, phnL, and phnM encode CP lyases, while phoD and phoA encode alkaline phosphatases. These genes are involved in the mineralization of organophosphonates, converting them into inorganic phosphates. Their relative abundance is higher in samples A1 and A7, while it is lower in other samples, which may explain the poor phosphorus removal efficiency in these two samples, leading to phosphorus accumulation. In A5, ppa > ppx > ppk, indicating that inorganic pyrophosphatases rapidly hydrolyze Poly-P into inorganic phosphorus, with the release of inorganic phosphorus being stronger than the formation of Poly-P. Phosphorus removal is achieved through polyphosphate-accumulating organisms (PAOs) converting organic phosphorus (org-Pi) into inorganic phosphorus (Pi), while simultaneously absorbing inorganic phosphorus from the environment and storing it in cells for ATP synthesis and various life activities.

[0036] Example 6: The Mechanism of the Influence of Signal Molecule Concentration on Algal Symbiotic Systems The mechanisms by which signal molecule concentrations affect algal symbiotic systems are multifaceted. Figure 7In these systems, signaling molecules such as N-acylhomoserine lactones (AHLs) regulate microbial behavior and metabolic activities through quorum sensing mechanisms, thereby affecting the system's nitrogen and phosphorus removal efficiency. Specifically, these molecules can activate or inhibit gene expression in specific functional bacteria, such as affecting the activity of key enzymes in nitrification and denitrification processes, thus regulating nitrogen conversion efficiency. Simultaneously, signaling molecules can promote the synthesis of extracellular polymers (EPS), which are crucial for microbial aggregation and sedimentation performance, directly impacting the system's pollutant removal capacity. Furthermore, changes in signaling molecule concentrations can alter the structure and diversity of the microbial community, selectively promoting or inhibiting the growth of certain genera, such as Halomonas and Sphingobium, which play key roles in nitrogen and phosphorus metabolism. By finely controlling the concentration of signaling molecules, the biological treatment efficiency of algal-microbe symbiotic systems can be optimized, providing new insights for efficient and economical wastewater treatment.

[0037] Exogenous C8-HSL was added to an algal symbiotic system to investigate the effects of different concentrations of C8-HSL on the denitrification and phosphorus removal efficiency of the system. C8-HSL is an acyl homoserine lactone signaling molecule, a commonly used signaling pathway in Gram-negative bacteria, controlled by the LuxI / LuxR system. The LuxI protein is responsible for acylating the terminal ligand-binding domain and the hydroxyl-terminal DNA-binding domain of the homoserine lactone signaling molecule. Algae provide oxygen to bacteria, and bacteria produce carbon dioxide and organic matter for algae to use. Exogenous C8-HSL acts on the symbiotic system, promoting symbiosis and the formation of symbiotic biofilms, and significantly promoting denitrification and phosphorus removal. The AHL signaling molecule released by bacteria affects the algal cells' ability to absorb ions, thereby affecting their attachment ability. (10.1111 / j.1365-3040.2005.01440.x)

[0038] The effect of C8-HSL concentration on the nitrogen and phosphorus removal efficiency of a bacterial-algal symbiotic system was investigated. Experimental results showed that a C8-HSL concentration of 0.8 μM significantly improved the nitrogen and phosphorus removal capacity of the symbiotic system, achieving average removal rates of 91.72%, 81.62%, and 87.72% for ammonia nitrogen, TN, TP, and COD, respectively. It also promoted the formation of extracellular polymeric substances (EPS), which positively impacted microbial aggregation and sedimentation performance. Metagenomic sequencing analysis revealed that *Halomonas* and *Sphingobium* are the key genera for nitrogen and phosphorus removal in the symbiotic system, with the genes *narGHI*, *nirKS*, *phnX*, and *ppa* playing crucial roles in nitrogen and phosphorus removal. Adjusting the C8-HSL concentration can optimize the biological treatment efficiency of the bacterial-algal symbiotic system, providing a new approach for achieving efficient and economical wastewater treatment.

[0039] The embodiments described above are merely preferred embodiments for fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are all within the scope of protection of the present invention. The scope of protection of the present invention is defined by the claims.

Claims

1. The application of C8-HSL in promoting the nitrogen and phosphorus removal capacity of bacterial-algae symbiotic systems in wastewater treatment, characterized by: The bacterial-algal symbiotic system consists of HN-AD bacteria and Chlorella pyrenoidosa in a volume ratio of 1:

3. The concentration of C8-HSL used is controlled at 0.8 μM. The HN-AD bacteria are a complex of Cupriavidus sp. SWA1, Alcaligenes faecalis, Acinetobacter, and Ochrobactrum sp. TAC-2. The Chlorella pyrenoidosa is Chlorella pyrenoidosa FACHB-9.

2. A method for improving the nitrogen and phosphorus removal capacity of wastewater treatment, characterized in that: The wastewater to be treated was inoculated with algae species with an OD680 of 1.2 or higher and HN-AD bacteria, and C8-HSL was added. The inoculation ratio of HN-AD bacteria was 5%, and the volume ratio of bacteria to algae was 1:

3. Then, the temperature was 28~30℃, the rotation speed was 300±50, the lighting intensity was set at 2000±200 lux, the air intake was controlled at 0.6L / min, and the HRT was 48h. The C8-HSL was added at a final concentration of 0.8μM. The algae species was Chlorella pyrenoidosa. The HN-AD bacteria were a compound of Cupriavidus sp. SWA1, Alcaligenes faecalis, Acinetobacter, and Ochrobactrum sp. TAC-2. The Chlorella pyrenoidosa was Chlorella pyrenoidosa FACHB-9.

3. The method for improving the nitrogen and phosphorus removal capacity of wastewater treatment according to claim 2, characterized in that: The average removal rates of ammonia nitrogen, TN, TP and COD by this method were 91.72%, 81.62% and 87.72%, respectively.

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