Method for ecological treatment of urban sewage pathogenic bacteria

Abstract

The application discloses an ecological treatment method for pathogenic bacteria in municipal sewage, which comprises the following steps: mixing lactic acid bacteria, photosynthetic bacteria and denitrifying bacteria to form a composite bacteria solution; and directly feeding the composite bacteria solution into a municipal sewage system. The method has simple operation steps and can inhibit pathogenic bacteria in municipal sewage through the composite probiotic agent. The three probiotic bacteria are suitable for growing and breeding in municipal sewage, can work in cooperation, can produce beneficial gain to other bacteria, and can inhibit and remove pathogenic bacteria and harmful substances such as nitrogen and phosphorus in municipal sewage.

Description

Technical Field

[0001] This invention belongs to the field of urban sewage treatment technology and relates to an ecological treatment method for pathogenic bacteria in urban sewage. Background Technology

[0002] The development and utilization of wastewater treatment technologies have practical significance for urban water resource protection and recycling, and for improving the quality of the ecological environment. Microbial metabolism can degrade harmful organic matter in water bodies; however, the type of microorganism has a significant impact on wastewater treatment effectiveness, requiring selection and acclimatization to cultivate microorganisms suitable for treating specific types of wastewater. Urban wastewater is generally a low-oxygen anaerobic environment, heavily polluted with nitrogen and phosphorus, and prone to the proliferation of pathogens. Anaerobic biological treatment technology, operating in the absence of free oxygen, utilizes anaerobic and facultative bacteria to treat organic pollutants in wastewater, producing economically viable combustible gases such as methane and carbon monoxide. This technology is widely applicable, requires no aeration equipment, is low-cost, and shows significant effects in treating COD and ammonia nitrogen in water. However, it is highly sensitive to substances in the wastewater, easily leading to moderate toxicity of the microbial community and resulting in a harsh biological reaction environment. Summary of the Invention

[0003] To address the problems existing in the prior art, this invention provides an ecological treatment method for pathogenic bacteria in urban sewage, thereby solving the technical problem that anaerobic and facultative bacteria are prone to poisoning when treating organic pollutants in urban sewage.

[0004] This invention is achieved through the following technical solution:

[0005] An ecological treatment method for pathogenic bacteria in urban sewage involves mixing lactic acid bacteria, photosynthetic bacteria, and denitrifying bacteria to form a composite bacterial solution, which is then directly added to the urban sewage system.

[0006] Preferably, the volume ratio of the lactic acid bacteria, photosynthetic bacteria and denitrifying bacteria is (1-2):1:1.

[0007] Preferably, the concentration of the compound bacterial solution is 1*10 6 ~5*10 6 CFU / mL.

[0008] Preferably, the urban wastewater contains at least two of the following: ammonia nitrogen, phosphate, sulfide, Vibrio, Spirulina, and Monocystic bacteria.

[0009] Preferably, the lactic acid bacteria are cultured by placing the lactic acid bacteria in a liquid culture medium and fermenting at a constant temperature to complete the culture; after culture, the concentration of the lactic acid bacteria is 1*10⁻⁶. 6 ~5*10 6 CFU / mL.

[0010] Preferably, the photosynthetic bacteria are cultured by placing them in HCH medium and incubating them at a constant temperature under light; after culture, the concentration of the photosynthetic bacteria is 1*10⁻⁶. 6 ~5*10 6 CFU / mL.

[0011] Preferably, the denitrifying bacteria are cultured by adding them to a heterotrophic-denitrification medium and culturing at a constant temperature; after culture, the concentration of the denitrifying bacteria is 1*10⁻⁶. 6 ~5*10 6 CFU / mL.

[0012] Preferably, the lactic acid bacteria culture medium is prepared by dissolving a carbon source, a nitrogen source, minerals, diammonium citrate, sodium acetate, magnesium sulfate, manganese sulfate, and Tween-80 in water, and then sterilizing the solution to obtain the lactic acid bacteria culture medium.

[0013] Preferably, the heterotrophic-denitrification medium is prepared by adding NH4Cl and sodium citrate to a mixed solution of K2HPO4, MgSO4, FeSO4 and MnSO4, adjusting the pH of the system after making up the volume, and sterilizing the solution to obtain the heterotrophic-denitrification medium.

[0014] Preferably, the HCH culture medium is prepared by mixing sodium citrate solution, magnesium sulfate solution, ammonium sulfate solution, calcium chloride solution, potassium dihydrogen phosphate solution, dipotassium hydrogen phosphate solution, Na2EDTA solution, yeast extract solution, and trace element stock solution, adjusting the pH value of the system, and sterilizing it to a fixed volume to obtain the HCH culture medium.

[0015] Compared with the prior art, the present invention has the following beneficial technical effects:

[0016] This invention discloses an ecological treatment method for pathogenic bacteria in urban sewage. The method is characterized by mixing lactic acid bacteria, photosynthetic bacteria, and denitrifying bacteria to form a composite bacterial solution, which is then directly introduced into the urban sewage system. Lactic acid bacteria, photosynthetic bacteria, and denitrifying bacteria are all anaerobic bacteria, suitable for proliferation and growth in anaerobic environments, which are well-suited to the anaerobic environment of sewage. Furthermore, the three bacteria can cooperate and divide their functions. Lactic acid bacteria produce lactic acid, lowering the pH of the sewage and inhibiting pathogenic bacteria growth. Photosynthetic bacteria help improve the sewage environment and promote metabolism. However, photosynthetic bacteria cannot remove the large amounts of higher fatty acids present in the sewage; only the symbiotic lactic acid bacteria can decompose these substances. Denitrifying bacteria have a strong proliferation capacity in anaerobic environments and degrade nitrogen and phosphorus in the sewage, synergistically purifying the water and altering the pathogenic bacteria's reproductive environment. Finally, the three bacterial groups can mutually provide positive benefits. The low pH environment of lactic acid bacteria does not inhibit the proliferation and growth of photosynthetic and denitrifying bacteria, and the nutrients produced by the metabolism of photosynthetic bacteria also supply a large amount of nutrients for the proliferation of lactic acid bacteria and denitrifying bacteria.

[0017] Furthermore, the volume ratio of lactic acid bacteria, photosynthetic bacteria, and denitrifying bacteria is (1-2):1:1. The three types of probiotics occupy different ecological niches in the process of treating urban sewage. This volume ratio can maximize the utilization efficiency of environmental natural resources.

[0018] Furthermore, the concentration of the compound bacterial solution is 1–5 × 10⁻⁶. 6 A concentration of CFU / mL allows bacteria to effectively reach the logarithmic growth phase, resulting in higher activity and better reproduction.

[0019] Furthermore, the urban wastewater contains at least two of the following: ammonia nitrogen, phosphate, sulfide, Vibrio, Spirulina, and Monocystic bacteria. This allows the three types of probiotics to be more targeted: denitrifying bacteria target ammonia nitrogen, phosphate, and sulfide, probiotics can inhibit pathogens, and photosynthetic bacteria significantly reduce ammonia nitrogen, phosphate, and sulfide, thus improving water quality. Detailed Implementation

[0020] To enable those skilled in the art to understand the features and effects of the present invention, the terms and expressions used in the specification and claims are explained and defined in general below. Unless otherwise specified, all technical and scientific terms used herein have the ordinary meaning understood by those skilled in the art regarding the present invention, and in case of conflict, the definitions in this specification shall prevail.

[0021] The theories or mechanisms described and disclosed herein, whether right or wrong, should not in any way limit the scope of the invention, that is, the contents of the invention can be implemented without being limited by any particular theory or mechanism.

[0022] In this document, all features defined by numerical ranges or percentage ranges, such as numerical values, quantities, contents, and concentrations, are for the sake of brevity and convenience only. Accordingly, descriptions of numerical ranges or percentage ranges should be considered as covering and specifically disclosing all possible sub-ranges and individual numerical values ​​(including integers and fractions) within those ranges.

[0023] In this article, unless otherwise specified, “contains,” “includes,” “containing,” “has,” or similar terms cover the meanings of “composed of” and “mainly composed of,” for example, “A contains a” covers the meanings of “A contains a and others” and “A contains only a.”

[0024] For the sake of brevity, not all possible combinations of the technical features in each implementation scheme or embodiment are described herein. Therefore, as long as there is no contradiction in the combination of these technical features, the technical features in each implementation scheme or embodiment can be combined arbitrarily, and all possible combinations should be considered within the scope of this specification.

[0025] This invention provides an ecological treatment method for pathogenic bacteria in urban sewage, which involves mixing lactic acid bacteria, photosynthetic bacteria and denitrifying bacteria to form a composite bacterial solution, and then directly adding the composite bacterial solution into the urban sewage system.

[0026] The lactic acid bacteria are one or more combinations of Lactibacillus, Streptococcus, and Leuconostoc; the denitrifying bacteria are one or more combinations of Aerobic Lactobacillus, Aerobic Bacterium, and Aerobic Mycobacterium; and the photosynthetic strains are Streptococcus thermophilus Rhodopseudomonas sp., Streptococcus thermophilus Rhodovulum sp., or Streptococcus thermophilus Rhodobacter sp. These strains were purchased from Beijing Runzekang Biotechnology Co., Ltd.

[0027] In addition, the volume ratio of lactic acid bacteria, photosynthetic bacteria and denitrifying bacteria is (1-2):1:1.

[0028] After compounding, the concentration of the compound bacterial solution is 1*10. 6 ~5*10 6 CFU / mL.

[0029] Urban wastewater contains at least two of the following: ammonia nitrogen, phosphate, sulfides, Vibrio, Spirulina, and Monotrophic bacteria.

[0030] The cultivation process of lactic acid bacteria involves placing lactic acid bacteria in a liquid culture medium and carrying out constant-temperature fermentation to complete the cultivation of the lactic acid bacteria; after cultivation, the concentration of the lactic acid bacteria is 1*10⁻⁶. 6 ~5*10 6 CFU / mL.

[0031] The cultivation process of photosynthetic bacteria involves placing the bacteria in HCH medium and incubating them at a constant temperature under light; after cultivation, the concentration of the photosynthetic bacteria is 1*10⁻⁶. 6 ~5*10 6 CFU / mL.

[0032] The denitrifying bacteria are cultured by adding them to a heterotrophic-denitrification medium and incubating at a constant temperature; after culture, the concentration of the denitrifying bacteria is 1*10⁻⁶. 6 ~5*10 6 CFU / mL.

[0033] The lactic acid bacteria culture medium is prepared by dissolving carbon source, nitrogen source, minerals, diammonium citrate, sodium acetate, magnesium sulfate, manganese sulfate and Tween-80 in water, and then sterilizing the solution to obtain the lactic acid bacteria culture medium.

[0034] In a preferred embodiment, the carbon source is one of glucose, brown sugar, and maltose. The nitrogen source is one of yeast powder or yeast extract, and the mineral is magnesium sulfate or potassium dihydrogen phosphate.

[0035] In a preferred embodiment, the lactic acid bacteria culture medium is prepared by using 10-20g carbon source, 2-6g nitrogen source, 1-3g minerals, 1-3g diammonium citrate, 3-8g sodium acetate, 0.1-0.3g magnesium sulfate, 0.01-0.05g manganese sulfate, and 0.5-1.5g Tween-80, diluted to 1L with distilled water, and then sterilized by autoclaving at 121℃ and 101kPa for 30min to obtain the lactic acid bacteria culture medium.

[0036] The heterotrophic-denitrification medium is prepared by adding NH4Cl and sodium citrate to a mixed solution of K2HPO4, MgSO4, FeSO4 and MnSO4, adjusting the pH of the system after making up the volume, and sterilizing it to obtain the heterotrophic-denitrification medium.

[0037] In a preferred embodiment, the heterotrophic-denitrification medium is prepared by dissolving 5.0 g K₂HPO₄, 2.5 g MgSO₄·7H₂O, 0.05 g FeSO₄·7H₂O, and 0.05 g MnSO₄·4H₂O sequentially in 1 L of pure water to prepare a Vickers salt solution. Then, 0.5 g NH₄Cl and 5.66 g sodium citrate are added to this solution, and the volume is brought to 1 L with distilled water. The pH is adjusted to 7.0, and the solution is then autoclaved for 30 min to prepare the heterotrophic-denitrification medium.

[0038] The HCH culture medium is prepared by mixing sodium citrate solution, magnesium sulfate solution, ammonium sulfate solution, calcium chloride solution, potassium dihydrogen phosphate solution, dipotassium hydrogen phosphate solution, Na2EDTA solution, yeast extract solution, and trace element stock solution, adjusting the pH value of the system, and sterilizing it to a fixed volume to obtain the HCH culture medium.

[0039] In a preferred embodiment, the HCH culture medium is prepared as follows: 350 mg / L sodium citrate, 120 mg / L magnesium sulfate, 1000 mg / L ammonium sulfate, 75 mg / L calcium chloride, 500 mg / L potassium dihydrogen phosphate, 300 mg / L dipotassium hydrogen phosphate, 20 mg / L Na₂EDTA, 100 mg / L yeast extract, and 1 mL of trace element stock solution are added. The pH is adjusted to 6.8, and 1000 mL of distilled water is added. The mixture is then autoclaved for 30 min to obtain the HCH culture medium. After 48 h of incubation under light and microaerophilic conditions, the culture enters the logarithmic growth phase. The bacterial culture is then stored at 4 °C to obtain the HCH culture medium.

[0040] This invention addresses the challenges of anaerobic environments and severe nitrogen and phosphorus pollution in urban wastewater. It selects three types of probiotics that thrive in anaerobic conditions. These three bacterial communities work collaboratively: lactic acid bacteria produce lactic acid, lowering the pH of the wastewater and inhibiting pathogen growth; photosynthetic bacteria improve the wastewater environment and promote metabolism; however, photosynthetic bacteria cannot remove the large amounts of higher fatty acids present in the wastewater, requiring the addition of symbiotic lactic acid bacteria for decomposition; and denitrifying bacteria, with their strong anaerobic proliferation capacity, degrade nitrogen and phosphorus in the wastewater, synergistically purifying the water and altering the pathogen's reproductive environment. The three bacterial communities mutually enhance each other. The low pH environment created by lactic acid bacteria does not inhibit the proliferation and growth of photosynthetic and denitrifying bacteria, and the nutrients produced by photosynthetic bacteria also support their proliferation. Finally, metagenomic sequencing further elucidates the functional structure, biological denitrification genes, metabolic pathways, and mechanisms of action of probiotics in the ecological inhibition of pathogens in urban wastewater.

[0041] This invention uses a simulated ecological tank of urban sewage to explain the technical solution of the invention.

[0042] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the teachings of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0043] The following examples use instruments and equipment conventional in the art. Experimental methods in the following examples, unless otherwise specified, are generally performed under conventional conditions or as recommended by the manufacturer. All raw materials used in the following examples are conventional commercially available products with specifications conventional in the art. In this specification and the following examples, unless otherwise specified, "%" refers to weight percentage, "parts" refers to parts by weight, and "ratio" refers to weight proportion.

[0044] Example 1

[0045] An ecological treatment method for pathogenic bacteria in urban sewage includes:

[0046] I. Microbial Culture:

[0047] Cultivation of lactic acid bacteria: Lactic acid bacteria are placed in a liquid culture medium and fermented at a constant temperature to complete the cultivation of the lactic acid bacteria; after cultivation, the concentration of the lactic acid bacteria is 1*10⁻⁶. 6 ~5*10 6 CFU / mL.

[0048] Cultivation of denitrifying bacteria: Denitrifying bacteria were added to a heterotrophic-denitrification medium and cultured in a 30℃ incubator. Samples were taken after 24 hours of cultivation for later use. The concentration of denitrifying bacteria after cultivation was 1*10⁻⁶. 6 CFU / mL.

[0049] Cultivation of photosynthetic bacteria: Photosynthetic bacteria were placed in HCH medium and incubated at 30°C under light for 48 hours to enter the logarithmic growth phase. The bacterial culture after 48 hours was stored at 4°C for later use. The concentration of photosynthetic bacteria after cultivation was 5 × 10⁻⁶. 6 CFU / mL.

[0050] The preparation process of the lactic acid bacteria culture medium is as follows: Weigh 10g of glucose, 2g of yeast powder as nitrogen source, 1g of magnesium sulfate, 1g of diammonium hydrogen citrate, 8g of sodium acetate, 0.1g of magnesium sulfate, 0.01g of manganese sulfate and 0.5g of Tween-80, and make up to 1L with distilled water. After sterilization by high pressure steam at 121℃ and 101kPa for 30min, the lactic acid bacteria culture medium is obtained.

[0051] The heterotrophic-denitrification medium was prepared as follows: 5.0 g K₂HPO₄, 2.5 g MgSO₄·7H₂O, 0.05 g FeSO₄·7H₂O, and 0.05 g MnSO₄·4H₂O were dissolved sequentially in 1 L of pure water to prepare a Vickers salt solution. Then, 0.5 g NH₄Cl and 5.66 g sodium citrate were added to this solution, and the volume was adjusted to 1 L with distilled water. The pH was adjusted to 7.0, and the solution was sterilized at 121℃ for 30 min to prepare the heterotrophic-denitrification medium.

[0052] The HCH medium was prepared as follows: 350 mg / L sodium citrate, 120 mg / L magnesium sulfate, 1000 mg / L ammonium sulfate, 75 mg / L calcium chloride, 500 mg / L potassium dihydrogen phosphate, 300 mg / L dipotassium hydrogen phosphate, 20 mg / L Na₂EDTA, 100 mg / L yeast extract, and 1 mL of trace element stock solution were added. The pH was adjusted to 6.8, and 1000 mL of distilled water was added. The mixture was then autoclaved for 30 minutes to obtain the HCH medium. After 48 hours of incubation under light, the culture entered the logarithmic growth phase. The culture was then stored at 4°C to obtain the HCH medium.

[0053] In this invention, when treating urban sewage, a 1:1:1 composite of bacteria is directly introduced into the urban sewage system. Simultaneously, metagenomic sequencing is used to analyze the ecological inhibition effect on pathogens.

[0054] Example 2

[0055] An ecological treatment method for pathogenic bacteria in urban sewage includes:

[0056] I. Microbial Culture:

[0057] Cultivation of lactic acid bacteria: Lactic acid bacteria are placed in a liquid culture medium and fermented at a constant temperature to complete the cultivation of the lactic acid bacteria; after cultivation, the concentration of the lactic acid bacteria is 1*10⁻⁶. 6 CFU / mL.

[0058] Cultivation of denitrifying bacteria: Denitrifying bacteria were incubated at 32℃ for 24 hours, and samples were taken for subsequent compounding. The concentration of denitrifying bacteria after cultivation was 3*10⁻⁶. 6 CFU / mL.

[0059] Cultivation of photosynthetic bacteria: Photosynthetic bacteria were placed in HCH medium and incubated at 31°C under light for 48 hours to enter the logarithmic growth phase. The bacterial culture after 48 hours was stored at 4°C for later use. The concentration of photosynthetic bacteria after cultivation was 2 × 10⁻⁶. 6 CFU / mL.

[0060] The lactic acid bacteria culture medium was prepared as follows: 15g of carbon source brown sugar, 4g of nitrogen source yeast extract, 2g of mineral potassium dihydrogen phosphate, 2g of diammonium hydrogen citrate, 5g of sodium acetate, 0.1g of magnesium sulfate, 0.01g of manganese sulfate and 0.5g of Tween-80 were weighed and diluted to 1L with distilled water. The culture medium was then sterilized by high-pressure steam at 121℃ and 101kPa for 30min to obtain the lactic acid bacteria culture medium.

[0061] The heterotrophic-denitrification medium was prepared as follows: 5.0 g K₂HPO₄, 2.5 g MgSO₄·7H₂O, 0.05 g FeSO₄·7H₂O, and 0.05 g MnSO₄·4H₂O were dissolved sequentially in 1 L of pure water to prepare a Vickers salt solution. Then, 0.5 g NH₄Cl and 5.66 g sodium citrate were added to this solution, and the volume was adjusted to 1 L with distilled water. The pH was adjusted to 7.0, and the solution was sterilized at 121℃ for 30 min to prepare the heterotrophic-denitrification medium.

[0062] The HCH medium was prepared as follows: Sodium citrate 350 mg / L, magnesium sulfate 120 mg / L, ammonium sulfate 1000 mg / L, calcium chloride 75 mg / L, potassium dihydrogen phosphate 500 mg / L, dipotassium hydrogen phosphate 300 mg / L, Na₂EDTA 20 mg / L, yeast extract 100 mg / L, and trace element stock solution 1 mL were added. The pH was adjusted to 6.8, and 1000 mL of distilled water was added. The mixture was autoclaved for 30 min to obtain the HCH medium. After 48 h of incubation under light and microaerophilic conditions, the culture entered the logarithmic growth phase. The bacterial culture was then stored at 4 °C to obtain the HCH medium.

[0063] In this invention, a 2:1:1 composite of bacteria is directly introduced into the urban sewage system during urban wastewater treatment. Simultaneously, metagenomic sequencing is used to analyze the ecological inhibition effect on pathogens.

[0064] Example 3

[0065] An ecological treatment method for pathogenic bacteria in urban sewage includes:

[0066] I. Microbial Culture:

[0067] Cultivation of lactic acid bacteria: Lactic acid bacteria are placed in a liquid culture medium and fermented at a constant temperature to complete the cultivation of the lactic acid bacteria; after cultivation, the concentration of the lactic acid bacteria is 3*10⁻⁶. 6 CFU / mL.

[0068] Cultivation of denitrifying bacteria: Denitrifying bacteria were added to a heterotrophic-denitrification medium and incubated at 33℃. Samples were taken after 24 hours of cultivation for subsequent compounding. The concentration of denitrifying bacteria after cultivation was 4*10⁻⁶. 6 CFU / mL.

[0069] Cultivation of photosynthetic bacteria: Photosynthetic bacteria were placed in HCH medium and incubated at 32°C under light and microaerobic conditions for 48 hours to enter the logarithmic growth phase. The bacterial culture after 48 hours was stored at 4°C for later use. The concentration of photosynthetic bacteria after cultivation was 3 × 10⁻⁶. 6 CFU / mL.

[0070] The lactic acid bacteria culture medium is prepared by weighing 20g of carbon source maltose, 6g of nitrogen source yeast extract, 3g of minerals potassium dihydrogen phosphate, 3g of diammonium hydrogen citrate, 8g of sodium acetate, 0.3g of magnesium sulfate, 0.05g of manganese sulfate and 1.5g of Tween-80, making up to 1L with distilled water, and then sterilizing at 121℃ and 101kPa for 30min.

[0071] The heterotrophic-denitrification medium was prepared by dissolving 5.0 g K₂HPO₄, 2.5 g MgSO₄·7H₂O, 0.05 g FeSO₄·7H₂O, and 0.05 g MnSO₄·4H₂O in 1 L of pure water to prepare a Vickers salt solution. Then, 0.5 g NH₄Cl and 5.66 g sodium citrate were added to this solution, and the volume was brought to 1 L with distilled water. The pH was adjusted to 7.0, and the solution was sterilized at 121°C for 30 min to prepare the heterotrophic-denitrification medium.

[0072] The HCH medium was prepared as follows: 350 mg / L sodium citrate, 120 mg / L magnesium sulfate, 1000 mg / L ammonium sulfate, 75 mg / L calcium chloride, 500 mg / L potassium dihydrogen phosphate, 300 mg / L dipotassium hydrogen phosphate, 20 mg / L Na₂EDTA, 100 mg / L yeast extract, and 1 mL of trace element stock solution were added. The pH was adjusted to 6.8, and 1000 mL of distilled water was added. The mixture was autoclaved for 30 min to obtain the HCH medium. After 48 h of incubation under light and microaerophilic conditions, the culture entered the logarithmic growth phase. The bacterial culture was then stored at 4 °C to obtain the HCH medium.

[0073] In this invention, when treating urban sewage, a 1:1:1 composite of bacteria is directly introduced into the urban sewage system. Simultaneously, metagenomic sequencing is used to analyze the ecological inhibition effect on pathogens.

[0074] The lactic acid bacteria, photosynthetic bacteria, and denitrifying bacteria used in Examples 1-3 above are shown in Table 1:

[0075] Table 1. Lactic acid bacteria, photosynthetic bacteria, and denitrifying bacteria used in Examples 1-3.

[0076]

[0077]

[0078] Example 4

[0079] An ecological treatment method for pathogenic bacteria in urban sewage involves mixing lactic acid bacteria, photosynthetic bacteria, and denitrifying bacteria at a volume ratio of (1-2):1:1 to form a concentration of 1*10. 6 ~5*10 6 A CFU / mL compound bacterial solution was directly added to an urban wastewater system containing ammonia nitrogen, phosphate, and sulfides.

[0080] Among them, the lactic acid bacteria are Lactibacillus, the denitrifying bacteria are Aerobic Lactobacillus Species, and the photosynthetic strain is Rhodopseudomonas sp.

[0081] The lactic acid bacteria cultivation process involves placing the lactic acid bacteria in a liquid culture medium and carrying out constant-temperature fermentation to complete the cultivation of the lactic acid bacteria; after cultivation, the concentration of the lactic acid bacteria is 1–5 × 10⁻⁶. 6 CFU / mL.

[0082] The cultivation process of photosynthetic bacteria involves placing the bacteria in HCH medium and incubating them at a constant temperature under light; the concentration of the photosynthetic bacteria is 1–5 × 10⁻⁵. 6 CFU / mL.

[0083] The denitrifying bacteria are cultured by adding them to a heterotrophic-denitrification medium and incubating at a constant temperature; the concentration of the denitrifying bacteria is 1–5 × 10⁻⁶. 6 CFU / mL.

[0084] The lactic acid bacteria culture medium is prepared as follows: 10g glucose, 2g yeast powder, 1g magnesium sulfate, 1g diammonium citrate, 3g sodium acetate, 0.1g magnesium sulfate, 0.01g manganese sulfate and 0.5g Tween-80 are diluted to 1L with distilled water and sterilized by autoclaving at 121℃ and 101kPa for 30min to obtain the lactic acid bacteria culture medium.

[0085] The heterotrophic-denitrification medium was prepared as follows: 5.0 g K₂HPO₄, 2.5 g MgSO₄·7H₂O, 0.05 g FeSO₄·7H₂O, and 0.05 g MnSO₄·4H₂O were dissolved sequentially in 1 L of pure water to prepare a Vickers salt solution. Then, 0.5 g NH₄Cl and 5.66 g sodium citrate were added to the solution, and the volume was adjusted to 1 L with distilled water. The pH was adjusted to 7.0, and the solution was then autoclaved for 30 min to prepare the heterotrophic-denitrification medium.

[0086] The HCH medium was prepared as follows: Sodium citrate 350 mg / L, magnesium sulfate 120 mg / L, ammonium sulfate 1000 mg / L, calcium chloride 75 mg / L, potassium dihydrogen phosphate 500 mg / L, dipotassium hydrogen phosphate 300 mg / L, Na₂EDTA 20 mg / L, yeast extract 100 mg / L, and trace element stock solution 1 mL were added. The pH was adjusted to 6.8, and 1000 mL of distilled water was added. The mixture was autoclaved for 30 min to obtain the HCH medium. After 48 h of incubation under light and microaerophilic conditions, the culture entered the logarithmic growth phase. The bacterial culture was then stored at 4 °C to obtain the HCH medium.

[0087] Example 5

[0088] The difference from Example 4 is that the lactic acid bacteria are *Streptococcus*, the denitrifying bacteria are *Aerobic Bacterium Species*, and the photosynthetic strain is *Rhodopseudomonas* sp. The volume ratio of the three is 2:1:1. After mixing, the concentration of the composite bacterial solution is 1*10⁻⁶. 6 CFU / mL. The treated municipal wastewater contained ammonia nitrogen, phosphate, sulfides, and Vibrio.

[0089] Example 6

[0090] The difference from Example 4 is that the lactic acid bacteria are *Leuconostoc*, the denitrifying bacteria are *Aerobic Mycobacterium Species*, and the photosynthetic strain is *Rhodopseudomonas* sp. The volume ratio of the three is 1:1:1. After mixing, the concentration of the composite bacterial solution is 3*10⁻⁶. 6 CFU / mL. The treated municipal wastewater contains ammonia nitrogen, phosphate, sulfides, Vibrio, Spirulina, and Monotrophic bacteria.

[0091] Example 7

[0092] The difference from Example 4 is that the concentration of the compound bacterial solution after mixing lactic acid bacteria, photosynthetic bacteria, and denitrifying bacteria is 5*10. 6 CFU / mL.

[0093] The lactic acid bacteria culture medium was prepared as follows: 15g brown sugar, 4g yeast extract, 2g potassium dihydrogen phosphate, 2g diammonium citrate, 5g sodium acetate, 0.2g magnesium sulfate, 0.02g manganese sulfate, and 1.0g Tween-80 were diluted with distilled water to a final volume of 1L. The medium was then autoclaved at 121℃ and 101kPa for 30 minutes to obtain the lactic acid bacteria culture medium. After cultivation, the concentration of the lactic acid bacteria was 1*10⁻⁶. 6 CFU / mL.

[0094] Example 8

[0095] The difference from Example 4 is that the concentration of the compound bacterial solution after mixing lactic acid bacteria, photosynthetic bacteria, and denitrifying bacteria is 2*10. 6 CFU / mL.

[0096] The lactic acid bacteria culture medium was prepared as follows: 20g maltose, 6g yeast extract, 3g potassium dihydrogen phosphate, 3g diammonium citrate, 8g sodium acetate, 0.3g magnesium sulfate, 0.05g manganese sulfate, and 1.5g Tween-80 were diluted with distilled water to a final volume of 1L. The medium was then autoclaved at 121℃ and 101kPa for 30 minutes to obtain the lactic acid bacteria culture medium. After cultivation, the concentration of the lactic acid bacteria was 3 × 10⁻⁶. 6 CFU / mL.

[0097] This invention features a simple operation process, utilizing a compound probiotic agent to ecologically inhibit pathogenic bacteria in urban sewage. Three probiotic strains are all suitable for growth and reproduction in urban sewage. They can work synergistically, while also providing beneficial effects to other strains. Their organic combination inhibits and removes pathogenic bacteria and harmful substances such as nitrogen and phosphorus from urban sewage. Metagenomic sequencing further elucidates the functional structure, biological denitrification genes, metabolic pathways, and mechanisms of action of probiotics in ecologically inhibiting pathogenic bacteria in urban sewage. This invention uses a combination of multiple probiotics as the active ingredient, activated and proliferated through specific culture techniques for the ecological treatment of pathogenic bacteria in urban sewage. This urban sewage pathogen treatment technology is ecological, environmentally friendly, and produces no secondary pollution. The process is easy to operate, exhibits a high pathogen inhibition rate, and has a long-lasting effect, enabling large-scale application in various sewage scenarios.

[0098] Metagenomic testing methods:

[0099] (1) DNA Extraction and Quality Control: Thaw the sample at room temperature for 10 min. Add 1.2 mL of inhibitEX Buffer to a 2 mL centrifuge tube. Mix the sample with a cotton swab, then transfer 180-220 mg to a 2 mL centrifuge tube and vortex until the sample is completely mixed. Centrifuge at 70℃ (the lysis temperature can be increased to 95℃) for 5 min, vortex for 15 s, then centrifuge for 1 min. Transfer 550 μL of the supernatant to a new 1.5 mL EP tube and centrifuge for 1 min. Add 30 μL of proteinase K to a new 1.5 mL EP tube. Transfer 400 μL of the supernatant from step 5 to the EP tube from step 6. Add 400 μL of buffer AL, vortex for 15 s. Incubate at 70℃ for 10 min. Add 400 μL of ethanol (96-100%) and mix (vortex). Transfer 600 μL to the adsorption column, centrifuge for 1 min, discard the tube, and replace with a new collection tube. Add 600 μL to the adsorption column, centrifuge for 1 min, discard the tube, and replace with a new collection tube. Add 500 μL of Buffer AW1, centrifuge for 1 min, discard the tube, and replace with a new collection tube. Add 500 μL of Buffer AW2, centrifuge for 3 min, discard the tube, and replace with a new collection tube. Centrifuge the empty tube for 3 min. Transfer the adsorption column to a new 1.5 mL EP tube, add 200 μL (reduce the volume if the concentration is low), incubate at room temperature for 5 min, and centrifuge for 1 min. DNA concentration: ≥10 ng / μL. The specific quality control steps are as follows: DNA purity: A260 / A280 = approximately 1.8-2.0, total DNA: ≥300 ng, DNA integrity: there should be a clear genomic main band. Run a 1% agarose gel at 120 V for 30 min with a loading volume of 400 ng.

[0100] (2) Library construction and sequencing

[0101] The samples were tested by PCR using primers, the PCR products were purified, a second round of PCR was performed, the amplified products were quality checked by electrophoresis, the products were purified by PCR, and the library was quality checked.

[0102] (3) 16S sequencing analysis

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0104] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the technical solutions of the present invention.

Claims

1. A method for ecological treatment of pathogenic bacteria in urban sewage, characterized in that, Lactic acid bacteria, photosynthetic bacteria and denitrifying bacteria are mixed to form a compound bacterial solution, which is then directly added to the urban sewage system. The volume ratio of lactic acid bacteria, photosynthetic bacteria and denitrifying bacteria is (1~2):1:1; The concentration of the complex bacteria solution is 1*10 6 ~5*10 6 CFU / mL; The culture process of the lactic acid bacteria is to place the lactic acid bacteria in a lactic acid bacteria liquid culture medium, perform constant temperature fermentation, and complete the culture of the lactic acid bacteria; after the culture, the concentration of the lactic acid bacteria is 1*10 6 5*10 6 CFU / mL. The culture process of the photosynthetic bacteria is to place the photosynthetic bacteria in HCH culture medium, and to culture the photosynthetic bacteria under constant temperature and light micro-oxygen condition; after the culture, the concentration of the photosynthetic bacteria is 1*10 6 CFU / mL. 6 CFU / mL. The denitrifying bacteria are cultured by adding them to a heterotrophic-denitrification medium and incubating at a constant temperature; after culture, the concentration of the denitrifying bacteria is 1*10⁻⁶. 6 ~5*10 6 CFU / mL; The urban wastewater contains at least two of the following: ammonia nitrogen, phosphate, sulfide, Vibrio, Spirulina, and Monocystic bacteria. The lactic acid bacteria culture medium is prepared by adding 10-20g carbon source, 2-6g nitrogen source, 1-3g minerals, 1-3g diammonium citrate, 3-8g sodium acetate, 0.1-0.3g magnesium sulfate, 0.01-0.05g manganese sulfate, and 0.5-1.5g Tween-80 to distilled water to a final volume of 1L, followed by sterilization. The heterotrophic-denitrification medium was prepared by dissolving 5.0 g K2HPO4, 2.5 g MgSO4·7H2O, 0.05 g FeSO4·7H2O, and 0.05 g MnSO4·4H2O in 1 L of pure water to prepare a Vickers salt solution. Then, 0.5 g NH4Cl and 5.66 g sodium citrate were added, and the pH was adjusted to 7.0 after making up the volume. The medium was then sterilized to obtain the final product.

2. The method for ecological treatment of pathogenic bacteria in urban sewage according to claim 1, characterized in that, The HCH culture medium is prepared by mixing sodium citrate solution, magnesium sulfate solution, ammonium sulfate solution, calcium chloride solution, potassium dihydrogen phosphate solution, dipotassium hydrogen phosphate solution, Na2EDTA solution, yeast extract solution, and trace element stock solution, adjusting the pH value of the system, and sterilizing it to a fixed volume to obtain the HCH culture medium.

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