A compound bacterial agent for rural domestic sewage treatment and its application

Abstract

This invention belongs to the field of wastewater treatment, specifically relating to a compound microbial agent for rural domestic wastewater treatment and its application. The compound microbial agent contains at least two of the following strains: Rhodococcus sp. 24、Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The aforementioned compound microbial agent can efficiently remove ammonia nitrogen and organic pollutants in rural black and gray wastewater treatment, and it exhibits strong adaptability and stable treatment effects, showing broad application prospects. This invention provides a new solution for rural wastewater treatment, with significant environmental and economic benefits.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment, specifically relating to a compound bacterial agent for rural domestic wastewater treatment and its application. Background Technology

[0002] With the advancement of urban-rural integration, rural domestic sewage treatment has become a critical issue urgently needing to be addressed in the environmental protection field. The high concentrations of ammonia nitrogen, phosphorus, and organic pollutants in black and grey water from rural domestic sewage have severely impacted the aquatic environment. Traditional sewage treatment methods, such as chemical precipitation, physical adsorption, and activated sludge processes, while effective in reducing pollutants in some cases, suffer from problems such as complex operation, high cost, and unstable treatment effects in rural sewage and black and grey water treatment. Furthermore, these methods often cannot simultaneously address multiple pollutants, especially given the demand for low-cost and simple treatment equipment, making them unsuitable for the actual needs of rural areas. Therefore, developing efficient and adaptable black and grey water treatment technologies has become an important research topic in environmental science and technology. In recent years, biological treatment technologies, particularly utilizing the pollutant degradation capabilities of microorganisms, have become important means of black and grey water treatment. Microorganisms can degrade organic matter through natural metabolism and possess strong nitrogen removal capabilities. However, the treatment effect of a single strain is often limited by various factors; the synergistic effect between strains and the rational configuration of the microbial community are crucial for improving treatment efficiency. Especially in the treatment of black and grey water, the synergistic effect of microorganisms and their ability to treat multiple organic substances are particularly important. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention proposes a composite microbial agent for rural domestic sewage, based on the construction of such an agent. This composite agent utilizes the synergistic effect of multiple strains with excellent denitrification and organic matter degradation capabilities. Specifically, this composite agent not only improves nitrogen removal efficiency but also exhibits a carbon-nitrogen synergistic effect; that is, during sewage treatment, microorganisms can simultaneously and efficiently treat both nitrogen and carbon sources in the sewage. This carbon-nitrogen synergistic effect optimizes the metabolic pathways of the microbial community, promoting the mutual degradation of organic matter and the removal of ammonia nitrogen, thereby significantly improving treatment efficiency. Studies have shown that this composite agent exhibits significant degradation effects in black and grey water treatment, effectively removing nitrogen and chemical oxygen demand (COD), and possesses good adaptability and stability, capable of handling different types of domestic sewage. This invention not only provides a new solution for the efficient treatment of rural domestic sewage but also provides theoretical support and technical guarantees for the development and utilization of microbial resources and the development of sewage resource-based treatment.

[0004] Therefore, the present invention relates to a compound microbial agent for rural domestic sewage treatment and its application, especially the application of the compound microbial agent in ammonia nitrogen removal and organic matter degradation.

[0005] Therefore, one object of the present invention is to provide a compound microbial agent for rural domestic sewage treatment, said compound microbial agent comprising at least two of the following strains: Rhodococcus sp. 24、Rhodococcus sp. YB , Pseudomonas tianjinensis sp. .

[0006] The strain Rhodococcus sp. 24 The strain CPZ24 involved in the published patent document CN103525734B ( Rhodococuus pyridinivorans ).

[0007] The strain Rhodococcus sp. YB It is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 35172.

[0008] The strain Pseudomonas tianjinensis sp. It is deposited at the China Industrial Microbial Culture Collection Center (CICC), accession number: CICC 24204; it is available for purchase from the China Industrial Microbial Culture Collection Center.

[0009] All three strains have denitrification capabilities and can be used alone to remove ammonia nitrogen or total nitrogen from domestic sewage. They have good adaptability and denitrification efficiency, and can be used alone or in combination with other strains depending on the sewage quality to achieve flexible treatment results under different denitrification requirements.

[0010] When any two of the three strains are used in combination (i.e., a two-component compound bacterial agent), the two strains produce a synergistic effect, which can more effectively remove ammonia nitrogen from rural domestic sewage and black and gray water, and degrade the organic matter therein.

[0011] Specifically, the compound microbial agent contains the following strains:

[0012] Rhodococcus sp. 24 , Rhodococcus sp. YB and Pseudomonas tianjinensis sp. ;or,

[0013] Rhodococcus sp. 24 and Rhodococcus sp. YB ;or,

[0014] Rhodococcus sp. YB and Pseudomonas tianjinensis sp. ;or,

[0015] Rhodococcus sp. 24 and Pseudomonas tianjinensis sp. .

[0016] Preferably, the compound microbial agent contains the following strains:

[0017] When the compound microbial agent contains strains Rhodococcus sp. 24 , Rhodococcus sp. YB and Pseudomonas tianjinensis sp. At that time, the strain Rhodococcus sp. 24、Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The volume ratio is 18.8%~53.7%:10%~40.7%:8.7%~46.3%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase; or

[0018] When the compound microbial agent contains strains Rhodococcus sp. 24 and Rhodococcus sp. YB At that time, the strain Rhodococcus sp. 24 and Rhodococcus sp. YB The volume ratio is 10%~90%:10%~90%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase; or,

[0019] When the compound microbial agent contains strains Rhodococcus sp. YB and Pseudomonas tianjinensis sp. At that time, the strain Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The volume ratio is 10%~90%:10%~90%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase; or,

[0020] When the compound microbial agent contains strains Rhodococcus sp. 24 and Pseudomonas tianjinensis sp. At that time, the strain Rhodococcus sp. 24 and Pseudomonas tianjinensis sp. The volume ratio is 10%~90%:10%~90%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase.

[0021] More preferably, the compound microbial agent contains the following strains:

[0022] When the compound microbial agent contains strains Rhodococcus sp. 24 , Rhodococcus sp. YB and Pseudomonas tianjinensis sp. At that time, the strain Rhodococcus sp. 24、Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The volume ratio is 20%~40%:20%~40%:20%~45%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase; or

[0023] When the compound microbial agent contains strains Rhodococcus sp. 24 and Rhodococcus sp. YB At that time, the strain Rhodococcus sp. 24 and Rhodococcus sp. YB The volume ratio is 40%~60%:40%~60%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase; or,

[0024] When the compound microbial agent contains strains Rhodococcus sp. YB and Pseudomonas tianjinensis sp. At that time, the strain Rhodococcus sp. YB and Pseudomonas tianjinensis sp.The volume ratio is 40%~60%:40%~60%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase; or,

[0025] When the compound microbial agent contains strains Rhodococcus sp. 24 and Pseudomonas tianjinensis sp. At that time, the strain Rhodococcus sp. 24 and Pseudomonas tianjinensis sp. The volume ratio is 40%~60%:40%~60%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase.

[0026] More preferably, the compound microbial agent contains the following strains:

[0027] When the compound microbial agent contains strains Rhodococcus sp. 24 , Rhodococcus sp. YB and Pseudomonas tianjinensis sp. At that time, the strain Rhodococcus sp. 24, Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The volume ratio was 36.5%:22.7%:40.8%, where the volume ratio was the volume ratio of each strain during the logarithmic growth phase; or

[0028] When the compound microbial agent contains strains Rhodococcus sp. 24 and Rhodococcus sp. YB At that time, the strain Rhodococcus sp. 24 and Rhodococcus sp. YB The volume ratio is 50%:50%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase; or,

[0029] When the compound microbial agent contains strains Rhodococcus sp. YB and Pseudomonas tianjinensis sp. At that time, the strain Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The volume ratio is 50%:50%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase; or,

[0030] When the compound microbial agent contains strains Rhodococcus sp. 24 and Pseudomonas tianjinensis sp. At that time, the strain Rhodococcus sp. 24 and Pseudomonas tianjinensis sp. The volume ratio is 50%:50%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase.

[0031] Preferably, the compound microbial agent contains the following three strains: Rhodococcus sp. 24, Rhodococcus sp. YB and Pseudomonas tianjinensis sp. .

[0032] The compound microbial agent containing three strains (i.e., the three-component compound microbial agent) can produce a better synergistic effect, and can more effectively remove ammonia nitrogen from rural domestic sewage and black and gray water, and degrade the organic matter therein. In other words, the compound microbial agent (i.e., the three-component compound microbial agent) can synergistically improve the ammonia nitrogen removal rate and organic matter degradation capacity.

[0033] The compound microbial agent (especially the three-component compound microbial agent) is suitable for denitrification treatment of black water, grey water or mixed domestic sewage. By adding the compound microbial agent, the removal of total nitrogen or ammonia nitrogen and nitrate nitrogen in sewage can be achieved.

[0034] The method for constructing the compound microbial agent mainly involves first conducting antagonistic tests, and then optimizing the interaction ratio to maximize the removal rate.

[0035] The optimal ratio of bacterial strains in the compound microbial agent was obtained using a simplex centroid design model with Design Expert 13 software.

[0036] The optimal ratio was optimized using the simplex centroid method, which can achieve the widest range of ammonia nitrogen removal and organic matter degradation effects.

[0037] Preferably, the cell activity of the compound microbial agent is 1×10⁻⁶. 8 ~100×10 10 CFU / mL.

[0038] Preferably, the compound microbial agent achieves an ammonia nitrogen removal rate of over 90% in both heterotrophic nitrification and aerobic denitrification media, more preferably 92% to 98% (e.g., 92.67% to 97.82%). The compound microbial agent exhibits significant ammonia nitrogen removal capability.

[0039] Preferably, the composite microbial agent has a COD removal rate of over 60% in wastewater, more preferably 61% to 74% (e.g., 61.21% to 73.31%). The composite microbial agent exhibits significant organic matter degradation capabilities.

[0040] Preferably, the compound bacterial agent has a TOC removal rate of over 90% in wastewater, and more preferably 94.65%.

[0041] Preferably, the compound microbial agent significantly increases the proportion of protein substances in DOM, enhances its autogenic characteristics, and reduces the level of humification.

[0042] Another objective of this invention is to provide the application of the compound microbial agent in actual rural domestic sewage treatment.

[0043] The compound microbial agent is suitable for various wastewater samples, including traditional rural domestic sewage, black water, grey water, and their mixed wastewater. In different types of wastewater, the compound microbial agent can synergistically improve ammonia nitrogen and COD removal capabilities, exhibiting excellent denitrification and organic matter degradation abilities.

[0044] Preferably, the wastewater is selected from: black water, grey water, and a mixture of black and grey water.

[0045] Preferably, the ammonia nitrogen concentration of the wastewater is 0~200 mg / L, more preferably 20 mg / L~200 mg / L, and even more preferably 20 mg / L~100 mg / L.

[0046] Another object of the present invention is to provide a method for treating actual rural domestic sewage, the method comprising: inoculating the compound bacterial agent into the sewage, and performing aerobic shaking culture for a reaction time of 48-120 hours.

[0047] The method described above can achieve efficient removal of ammonia nitrogen and degradation of organic matter. Depending on the type of wastewater and the concentration of ammonia nitrogen, the removal effect of the compound microbial agent is stable and highly efficient.

[0048] The composite microbial agent has demonstrated significant effectiveness in actual wastewater treatment. When treating different types of wastewater (including unsterilized and sterilized wastewater), the composite microbial agent can efficiently remove ammonia nitrogen and total nitrogen, and effectively reduce COD concentration. Furthermore, the composite microbial agent can significantly reduce the concentration of dissolved organic carbon (DOC) in actual wastewater, further validating its powerful ability to degrade organic pollutants.

[0049] Preferably, the ammonia nitrogen removal rate of the method is above 88%, preferably 89% to 98% (e.g., 89.68% to 97.28%); the total nitrogen removal rate is above 80%, preferably 81% to 93% (e.g., 81.23% to 92.04%), and it can significantly reduce dissolved organic carbon (DOC) in wastewater.

[0050] Furthermore, this invention provides a bacterium, which is classified and named as: Rhodococcus. Rhodococcus sp. YB It was deposited on July 10, 2025 at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35172.

[0051] Preferably, the 16S rRNA of the bacteria is as shown in SEQ ID NO: 1. SEQ ID NO: 1 is:

[0052] .

[0053] The bacteria YB has denitrification function and can be used to remove nitrogen or organic matter from domestic sewage. It has good adaptability and can be used alone or in combination with other strains depending on the sewage quality to achieve flexible treatment effects under different denitrification requirements.

[0054] In the process of degrading dissolved organic matter (DOM) in actual wastewater, the composite microbial agent not only effectively removed organic pollutants from the water but also significantly altered the compositional characteristics of DOM. The addition of the composite microbial agent enhanced the proportion of organic matter generated by microbial autobiology and reduced the degree of humification, demonstrating its potential in aquatic ecological restoration.

[0055] This invention's compound microbial agent can efficiently remove nitrogen and organic pollutants in rural domestic sewage treatment, exhibiting strong adaptability and stable treatment effects, and has broad application prospects. This invention provides a new solution for rural sewage treatment, with significant environmental and economic benefits.

[0056] Preservation Notes: Strain Name: YB, Classification: Rhodococcus ( Rhodococcus sp. Depository Institution: China General Microbiological Culture Collection Center (CGMCC). Depository Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. Depository Number: CGMCC No. 35172. Deposit Date: July 10, 2025. Attached Figure Description

[0057] Figure 1 This is a schematic diagram of an antagonism test.

[0058] Figure 2 This is an antagonistic phenomenon.

[0059] Figure 3 The average ammonia nitrogen removal rate of each strain in synthetic wastewater with an ammonia nitrogen concentration of 20 mg / L is given.

[0060] Figure 4 This study investigates the nitrogen removal and variation trends of CMI-5 in heterotrophic nitrification and aerobic denitrification media. Specifically, in the heterotrophic nitrification medium, (a) changes in nitrogen content, and (b) changes in TN and NH4+. + -N removal rate changes; (c) nitrogen content changes in aerobic denitrification medium, (d) TN and NO3- changes. - -N removal rate change.

[0061] Figure 5 The effect of CMI-5 on nitrogen content in sterilized black water (MA) and unsterilized black water (YA). Among them: (a) TN; (b) NH4. + -N; (c)NO3 - -N; (d)NO2-N.

[0062] Figure 6 The effect of CMI-5 on nitrogen content in sterilized ash water (MB) and unsterilized ash water (YB) was investigated. Among them: (a) TN; (b) NH4+. + -N; (c)NO3 --N; (d)NO2-N.

[0063] Figure 7 The effect of CMI-5 on nitrogen content in sterilized black ash (MC) and unsterilized black ash (YC) was investigated. Among the nitrogen content: (a) TN; (b) NH4+. + -N; c)NO3 - -N; (d)NO2-N.

[0064] Figure 8 The effects of CMI-5 on COD content in actual domestic sewage are shown. Among them: (a) MA and YA; (b) MB and YB; (c) MC and YC.

[0065] Figure 9 This refers to the DOC content in wastewater.

[0066] Figure 10 The fluorescence intensity of each fluorescent component is represented by .

[0067] Figure 11 This represents the percentage of fluorescence intensity for each fluorescent component.

[0068] Figure 12 The three-dimensional fluorescence spectral index of DOM is defined as follows: (a) fluorescence index; (b) biogenic index; and (c) humification index. Detailed Implementation

[0069] I. Experimental Methods

[0070] 1. Construction of compound denitrifying bacterial agent

[0071] (1) Strain activation and bacterial culture preparation

[0072] Using an inoculation loop, a loopful of the bacterial strain preserved on a test tube slant was transferred to a 250 mL Erlenmeyer flask containing 100 mL of sterile tryptone soybean broth (TSB). The flask was then wrapped with a sterile breathable membrane and incubated at 37 °C and 160 r / min for 48 h for activation. After 48 h of aerobic incubation, 10 mL of the bacterial solution was transferred to another 250 mL Erlenmeyer flask containing 100 mL of sterile TSB liquid medium, and the same process was repeated for another 48 h to rejuvenate the strain. Finally, 10 mL of the rejuvenated bacterial solution was inoculated into 100 mL of sterile 20 mg / L heterotrophic nitrification liquid medium and incubated on a shaker for 48 h to complete the bacterial culture preparation.

[0073] (2) Verification of functional bacterial antagonism

[0074] Dilute all bacterial solutions by 10 4Prepare bacterial suspensions by dilution, and use the suspension of one strain as the background culture. Take 150 μL of this suspension and spread it onto sterile tryptone soybean agar (TSA) medium. Figure 1 As shown, the plate was divided into 4-5 regions. Sterile stainless steel tubes were used to punch holes in each region of the plate. Residual culture medium in the holes was removed with a sterile needle, resulting in 4-5 round holes of the same diameter. An equal volume of a suspension of other bacterial strains was injected into 3-4 wells as verification bacteria. Sterile water was injected into one well as a blank control. Each experiment was repeated twice. The cells were incubated at 37 ℃ for 48 h. The presence of inhibition zones around the wells was observed. The absence of inhibition zones indicated no antagonistic effect between the background bacteria and the inoculated strain; conversely, the presence of inhibition zones indicated antagonistic activity between the two bacteria. The groups of background bacteria and verification bacteria are shown in Table 1.

[0075] H1 is Rhodococcus sp. 24 The strain CPZ24 involved in the published patent document CN103525734B ( Rhodococuus pyridinivorans );

[0076] H2 is Rhodococcussp. YB Accession number: CGMCC No. 35172;

[0077] H3 is Pseudomonas tianjinensis sp. Accession number: CICC 24204; purchased from China Industrial Microbial Culture Collection Center.

[0078] H4 is Pseudomonas plecoglossicida Accession number: CGMCC No.1.16111; purchased from the China General Microbiological Culture Collection Center.

[0079] H5 is Pseudomonas parafulva Accession number: CGMCC No.1.15634; purchased from the China General Microbiological Culture Collection Center.

[0080] H6 is Pseudomonas psychrotolerans Accession number: CGMCC No.1.15631; purchased from the China General Microbiological Culture Collection Center.

[0081] H7 is Hydrogenophaga atypica Accession number: CGMCC No.1.12740; purchased from the China General Microbiological Culture Collection Center.

[0082] H8 is Hydrogenophaga flavaAccession number: CGMCC No.1.8793; purchased from the China General Microbiological Culture Collection Center.

[0083] H9 is Enterobacter cloacae Accession number: CGMCC No.1.8726; purchased from the China General Microbiological Culture Collection Center.

[0084] H10 is Enterobacter hormaechei Accession number: CGMCC No.1.10608; purchased from the China General Microbiological Culture Collection Center.

[0085] H11 is Virgibacillus halophilus Accession number: CGMCC No.1.15481; purchased from the China General Microbiological Culture Collection Center.

[0086] H12 is Virgibacillus halodenitrificans Accession number: CGMCC No.1.8915; purchased from the China General Microbiological Culture Collection Center.

[0087] H13 is Virgibacillus kekensis Accession number: CGMCC No.1.6298; purchased from the China General Microbiological Culture Collection Center.

[0088] Table 1. Grouping of background and verification bacteria in the antagonistic test

[0089]

[0090] (3) Denitrification rate determination of single bacteria

[0091] The prepared 10 mL single-strain bacterial suspension was inoculated into a 250 mL Erlenmeyer flask containing 100 mL of sterile 20 mg / L heterotrophic nitrification liquid medium, and reacted in a shaker at 37 ℃ and 160 r / min for 48 h. After filtration through a disposable 0.45 μm filter membrane, NH4 was measured using a continuous flow meter. + -N concentration.

[0092] (4) Construction of denitrifying bacterial agents using the simple morphology centroid method

[0093] Based on the results of functional bacterial antagonism verification and single-strain denitrification rate, a comprehensive evaluation was conducted, and strains with high ammonia nitrogen removal rates and no antagonistic interactions among the 13 strains were selected for the construction of denitrifying bacterial agents. The volume of the bacterial suspension obtained after culturing the finally selected strains in 20 mg / L heterotrophic nitrification medium for 24 h was used as the independent variable, and the ammonia nitrogen removal rate after 48 h of shaking reaction in synthetic wastewater environments with ammonia nitrogen levels of 20, 50, 100, and 200 mg / L, respectively, was denoted as η. 20N η 50N η 100N and η 200N The boundary values ​​for the proportion of each strain were set to 0 to 1. Using Design Expert 13 software, simplex centroid design was employed to obtain different combinations of simplex points and replication points, which were recorded as a simplex centroid design table.

[0094] Bacterial suspensions of various strains were inoculated into synthetic wastewater with different ammonia nitrogen concentrations according to the proportions specified in the simplex centroid design table. The total inoculation volume was 2 mL, and the culture medium volume was 20 mL. The inoculation was carried out at 37℃ and 160 r·min. -1 After 48 hours of incubation in a shaker under specific conditions, ammonia nitrogen concentration was measured, and the ammonia nitrogen removal rate was calculated. The obtained experimental data were tested against linear, quadratic, specific cubic, and cubic models to obtain their respective regression coefficients. The recommended model was selected, and its adequacy was tested using analysis of variance (ANOVA). The optimal compound microbial agent ratio under different ammonia nitrogen levels was determined through three-dimensional response surface methodology, thus obtaining the compound microbial agent.

[0095] 2. Analysis of the denitrification performance of the bacterial agent

[0096] The compound microbial agent was inoculated into heterotrophic nitrification medium and aerobic denitrification medium, respectively, and cultured for 72 h. Samples were taken at 0 h, 3 h, 6 h, 9 h, 12 h, 15 h, 18 h, 24 h, 36 h, and 72 h. After filtration through a 0.45 μm filter membrane, TN and NO2 were measured using a continuous flow analyzer. - -N, NO3 - -N,NH4 + -N.

[0097] Bacterial suspensions were collected at 0 h and 72 h and ultrasonically disrupted using an ultrasonic cell disruptor. Each sample underwent 80 ultrasonic cycles using a 6 mm ultrasonic probe, with each cycle consisting of a 5-second pulse and a 6-second pause, at a power of 400 W. The ultrasonically disrupted samples were then centrifuged at 10,000 rpm for 10 min in a high-speed refrigerated centrifuge, filtered through a 0.45 μm filter, and their TN and NO2 content was measured using a continuous flow analyzer. - -N, NO3- -N,NH4 + -N was analyzed for nitrogen balance using calculations. All experiments were repeated three times.

[0098] (1) Analysis of the carbon source utilization capacity of the microbial agent

[0099] Cell proliferation and decarbonization performance

[0100] The compound bacterial agent was inoculated into heterotrophic nitrification medium and cultured for 72 h. Samples were taken at 0 h, 3 h, 6 h, 9 h, 12 h, 15 h, 18 h, 24 h, 36 h, and 72 h. After filtration through a 0.45 μm filter membrane, the cell optical density (OD) was measured using a UV spectrophotometer. 600 TOC concentration was measured using a TOC-L analyzer. All experiments were repeated three times.

[0101] Carbon source utilization capacity

[0102] To evaluate the carbon metabolism activities of the compound microbial agent and the individual bacterial strains to different carbon sources, the carbon source metabolism activities were determined using ecological microplate technology. The compound microbial agent and each individual bacterial strain were cultured in a shaker for 24 h on heterotrophic nitrification medium. Five mL of enriched bacterial solution was collected and placed in a 50 mL sterile centrifuge tube. The OD values ​​of the bacterial solutions from the compound microbial agent and the individual bacterial strains were then analyzed using sterile physiological saline. 600 Adjust the concentration to approximately 0.05. Transfer the diluted bacterial culture to a 10 mm sterile petri dish, then use an 8-line pipette to add 100 μL to each well of a 96-well ECO plate. Incubate the ECO plates at 37 ℃ for 5 days. Measure the optical density (OD) of each well at 590 nm using a microplate reader at 11 time points: 0 h, 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 48 h, 72 h, 96 h, and 120 h. 590 And record it.

[0103] OD was used in this study 590 To measure carbon utilization efficiency (CUE), the CUE value is given by the formula Calculate. Where C i CUE represents the absorbance value of the i-th carbon source well at 590 nm in the ECO ecological board, and R is the absorbance value of the blank well at 590 nm. If CUE < 0, it is recorded as 0 in the calculation. The metabolic activity of the carbon source is studied using the average color change of the color well (AWCD). The AWCD value is given by the formula... Calculation, where CUE i This represents the CUE value of the i-th carbon source pore, where n refers to the number of carbon sources.

[0104] 3. Application of microbial agents in actual wastewater treatment

[0105] (1) The denitrification effect and COD removal capacity of compound bacterial agents on actual wastewater

[0106] When using compound microbial agents to treat actual wastewater (black water A from toilets), grey water B from domestic bathing and kitchens, and mixed black and grey wastewater C, the presence of the original microbial community in the actual wastewater may, to some extent, compete with or synergize with the compound microbial agent. To investigate the treatment capacity of the compound microbial agent on wastewater under two conditions: one affected by the original microorganisms in the wastewater and the other unaffected, the three types of water samples were divided into two parts. One part was left untreated, and the other part was sterilized at 121 °C for 30 min before use. The compound microbial agent was used to treat both the unsterilized and sterilized actual wastewater. The unsterilized treatment groups were named YA, YB, and YC, and the sterilized treatment groups were named MA, MB, and MC. In addition, considering the volatilization of nitrogen in the actual wastewater and the degradation of nitrogen by the original microbial community, control groups YA-CK, YB-CK, YC-CK, MA-CK, MB-CK, and MC-CK were also set up in the experiment.

[0107] In the treatment groups, the compound microbial agent was inoculated at a 10% inoculum into the unsterilized treatment groups YA, YB, and YC, and the sterilized treatment groups MA, MB, and MC, respectively. The mixture was aerobically cultured in a shaker, and samples were taken at 0 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, 60 h, 72 h, 96 h, and 120 h, and filtered through a 0.45 μm filter membrane. The control group was not inoculated with the compound microbial agent but was cultured, sampled, and treated in the same manner as the treatment groups. Each treatment group consisted of three replicates, while each control group had no replicates.

[0108] A portion of the filtered sample was analyzed using a continuous flow analyzer to determine NH4. + -N, NO3 - -N, NO2 - -N and TN, and another part is digested by a COD rapid digester, and the COD concentration is measured using a multi-parameter water quality analyzer.

[0109] (2) The effect of compound microbial agents on the composition of DOM in actual wastewater

[0110] To investigate the effects of compound microbial agents on the degradation of soluble organic carbon (DOC) and the specific composition and changes of dissolved organic matter (DOM) during the actual rural domestic sewage purification process, untreated and unreacted raw water (without compound microbial agents) A_0d, B_0d, C_0d, and unsterilized and sterilized treatment groups (with compound microbial agents) were collected. Samples YA_5d, YB_5d, YC_5d, MA_5d, MB_5d, and MC_5d were collected after 5 days of reaction using a high-speed refrigerated centrifuge at 10000 ×g and 4 ℃ for 5 min. The supernatant was filtered through a 0.45 μm filter membrane. A portion was used to determine the DOC using a TOC-L analyzer, and the other portion was used to obtain the fluorescence excitation-emission matrix (EEM) using an F-7000 fluorescence spectrophotometer (Hitachi, Japan). Three replicates were performed for each group.

[0111] Test methods for denitrification of rural domestic sewage using different combinations of compound bacterial agents

[0112] To further verify the nitrogen removal efficiency of the three strains of this invention in actual rural domestic sewage when used alone, in pairs, and in triplicate, a comparative experiment was designed. Simulated mixed rural domestic sewage was used as the treatment object, with an influent total nitrogen concentration of approximately 45 mg / L. The experiment consisted of seven groups, including three single-strain treatment groups (inoculated separately). Rhodococcus sp. 24 , Rhodococcussp. YB , Pseudomonas tianjinensis sp. ), three groups of two-by-two treatment groups ( Rhodococcus sp. 24 and Rhodococcus sp. YB The volume ratio of the strains was 50%:50%; Rhodococcus sp. 24 and Pseudomonas tianjinensis sp. The volume ratio of the strains was 50%:50%; Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The strain volume ratio was 50%:50%, and a three-strain treatment group was also included. Rhodococcus sp. 24 , Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The bacterial strain volume ratio was 36.5%:22.7%:40.8%. 10 mL of bacterial solution was added to 100 mL of wastewater. Each group of experiments was carried out at 30℃ under aerobic conditions for 48 hours. After the reaction, water samples were collected, the total nitrogen concentration was measured, and the removal rate was calculated to evaluate the denitrification effect of each group of bacterial agents.

[0113] II. Results and Analysis

[0114] 1. Construction of denitrifying bacterial agents

[0115] (1) Antagonism test and single-strain denitrification rate results

[0116] Thirteen bacterial strains, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, and H13, were used in a hole-punch inhibition zone experiment. The results are as follows: Figure 2 As shown, strain H10 showed obvious antagonistic reactions with H1, H2, H6, and H7, strain H9 showed obvious antagonistic reactions with H8, and no antagonistic phenomena were found among other strains.

[0117] The ammonia nitrogen removal rates of these 13 strains after reacting in heterotrophic nitrification liquid medium with an ammonia nitrogen concentration of 20 mg / L for 48 h are as follows: Figure 3 As shown, the strains with an average ammonia nitrogen removal rate greater than 80% are: H10 (90.86%), H1 (88.93%), H3 (88.56%), H4 (86.16%), H2 (85.02%), H11 (84.52%), H8 (83.57%), and H9 (81.09%).

[0118] Based on the results of antagonistic reactions between strains, strains H1, H2, H3, H4 and H11, which had good ammonia nitrogen removal effects and no obvious antagonistic effects among each other, were finally selected for compounding of bacterial agents.

[0119] (2) Construction of compound microbial agents

[0120] Using the bacterial suspension volumes of five strains (H1, H2, H3, H4, and H11) as independent variables, and their ammonia nitrogen removal rates after 48 h of reaction in synthetic wastewater environments with ammonia nitrogen levels of 20, 50, 100, and 200 mg / L as response values, a simplex centroid design was used with Design Expert 13 software to obtain 41 simplex points and 11 replication points (Table 2). Each point represents the proportion of each strain, with a total of 1. The ammonia nitrogen removal rates of the five strains under different combinations at four ammonia nitrogen loading levels were determined, expressed as η. 20N η 50N η 100N and η 200N The results are shown in Table 3. The results were fitted using Design Expert 13 software, and regression analysis showed that the most suitable approach for η... 20N η 50N η 100N and η 200N All models are special cubic models (Table 4), and are extremely significant ( p <0.001). Based on this, improvements are made by selecting model terms with higher coefficient importance ( p <0.1) were included in the final model. As shown in Table 5, the positive and negative values ​​of some regression coefficients in the nonlinear term of the final model represent the synergistic and antagonistic effects in the microbial agent compound, respectively. η 20N η 50N η100N and η 200N The final model's p-values ​​were all less than 0.001, and the F-values ​​were generally at a medium-high level, at 37.67, 200.82, 70.28, and 21.68 respectively, indicating the feasibility of the model in predicting response factors. η 20N R 2 94.96%, η 50N R 2 99.17%, η 100N R 2 It is 97.46%, η 200N R 2 The accuracy rate was 90.83%, indicating that the final model basically matched the experimental data. To avoid the added independent variables causing R... 2 Increasing the R-squared value can lead to model overfitting; in statistics, an adjusted R-squared value should be used. 2 (Adjusted coefficient of determination R) 2 ). η 20N η 50N η 100N and η 200N The coefficient of determination R of the adjustment 2 The percentages were 92.44%, 98.67%, 96.07%, and 86.65%, respectively, with predicted R... 2 (Prediction Determination Coefficient R) 2 The percentages were 88.90%, 96.75%, 93.29%, and 80.63%, respectively, indicating that the fitted model had high significance. Sufficient accuracy was used to measure the signal-to-noise ratio; generally, a ratio greater than 4 is desirable. In this study, η... 20N η 50N η 100N and η 200N The appropriate precision ratios were 32.029, 57.383, 32.882, and 19.193, reflecting appropriate signal-to-noise ratio relationships. The coefficient of variation (CV) is typically used as a statistical measure of variability among repeated measures; a larger CV value indicates greater variability among repeated measures, and vice versa. Therefore, a smaller CV value indicates more reliable measurement results. The results show that η 20N The CV is 0.48%, η 50N The CV is 1.08%, η 100N The CV is 1.21%, η 200N The CV was 3.14%, and none of them exceeded 4%, which indicates that the strain combination model has high reproducibility and effectiveness.

[0121] Table 2 Five-component experimental design table

[0122]

[0123] Table 3. Response values ​​of ammonia nitrogen removal rate under different ammonia nitrogen loading levels

[0124]

[0125] Table 4. Summary of Fitting Different Models

[0126]

[0127] Table 5. Model regression equations and analysis of variance results

[0128]

[0129] To evaluate the fitting effect of the strain combination model, the model was tested based on the residual properties of the model corresponding to each response value, i.e., the difference between the observed value and the fitted value. The results show that η 20N η 50N η 100N and η 200N The residuals follow a normal distribution, and the model shows a sufficient fit, indicating that the basic assumptions about typicality in the test are correct, and that the developed model is approved based on these straight lines. This means that the developed model is suitable for predicting ammonia nitrogen removal rates obtained with strain combinations.

[0130] In this experiment, the inventors evaluated the denitrification performance of compound bacterial strains under different ammonia nitrogen loading levels. The optimization objective was to obtain the maximum denitrification rate, but the optimal formulations obtained to meet the corresponding response variables differed significantly under different ammonia nitrogen loading levels. Contour analysis of the superimposed response values ​​showed that at ammonia nitrogen loading levels of 20 mg / L and 50 mg / L, strains H1, H2, and H3 played a dominant role in ammonia nitrogen removal. At an ammonia nitrogen loading level of 100 mg / L, in addition to the three strains, strain H11 also played a certain role in denitrification. At an ammonia nitrogen loading level of 200 mg / L, only H1 and H3 had a dominant effect on ammonia nitrogen removal. The software provides the optimal compound ratio and optimized predicted values ​​for each ammonia nitrogen load level: the proportions of H1, H2, H3, H4, and H11 are 18.8%~53.7%, 0%~40.7%, 8.7%~46.3%, 0%, and 0%~19.8%, respectively.

[0131] To obtain a microbial agent formulation that maintains high denitrification performance under different ammonia nitrogen loading levels, it is necessary to comprehensively consider the interactions between different independent factors. Therefore, based on the optimization objective of achieving the maximum denitrification rate, the inventors assigned equal weight values ​​to all optimization items and used software to derive a microbial agent formulation that satisfies four ammonia nitrogen loading levels. In this formulation, strains H1, H2, and H3 are allocated in proportions of 36.5%, 22.7%, and 40.8%, respectively, while strains H4 and H5 were removed from the formulation due to their potential competitive and antagonistic relationships with other strains in the mixed agent. In this case, η 20N η 50N η 100N and η 200N The predicted values ​​were 91.44%, 94.86%, 86.34%, and 67.66%, respectively. A composite ideality of 0.828 was observed, thus determining the optimal combination of microbial agents.

[0132] Strains H1, H2, and H3 were inoculated into sterilized synthetic wastewater with ammonia nitrogen concentrations of 20 mg / L, 50 mg / L, 100 mg / L, and 200 mg / L, respectively, according to the optimal compound ratio obtained above. The mixture was reacted in a shaker for 48 h, with three replicates per group. The ammonia nitrogen removal rate was measured for each group. The results showed that η 20N η 50N η 100N and η 200N The values ​​were 93.38±1.1%, 95.55±0.63%, 87.41±1.26%, and 68.6±1.2%, respectively, which were 0.69%-1.94% higher than the predicted values, indicating that the model is effective and the optimal bacterial agent formulation has a high efficiency in removing ammonia nitrogen. Therefore, the optimal compound ratio of bacterial agents is 36.5%, 22.7%, and 40.8% for strains H1, H2, and H3, respectively. The compound bacterial agent obtained from this formulation is named CMI-5.

[0133] (3) Study on the denitrification performance and mechanism of compound bacterial agents

[0134] ① Names of the bacterial strains that make up the compound bacterial agent

[0135] The bacterial strains H1, H2, and H3 that make up the compound bacterial agent CMI-5 are respectively Rhodococcus sp. 24 , Rhodococcus sp. YB and Pseudomonas tianjinensis sp. .

[0136] ② The heterotrophic nitrification and aerobic denitrification capabilities of compound microbial agents

[0137] like Figure 4As shown in (a) and (b), under aerobic conditions, the compound bacterial agent CMI-5 reacted for 72 h in a heterotrophic nitrification medium with an initial ammonia nitrogen concentration of 50 mg / L. NH4 + The nitrogen (TN) concentration decreased from 49.47 ± 0.29 mg / L to 0.46 ± 0.13 mg / L, achieving a removal rate of 99.07%, corresponding to an average removal rate of 0.68 mg / (L·h). Furthermore, within the first 24 hours of the reaction, CMI-5 achieved a TN removal rate of 90.66%, while the TN concentration slightly increased after 24 hours. This may be because, with the consumption of exogenous organic carbon, some bacteria entered a period of death or decline, and organic nitrogen released during cell lysis re-entered the water, leading to a slight increase in total nitrogen levels. Notably, no NO2 was detected throughout the entire reaction process. - -N and NO3 - The accumulation of intermediates such as -N. This phenomenon suggests that CMI-5 may have the ability to convert NH4+ into NH4+. + The ability of NO2- to be directly converted into gaseous nitrogen such as N2O and N2. However, it cannot be ruled out that CMI-5 has a faster denitrification rate, and that the relevant conversion process is mainly completed intracellularly, leading to NO2... - -N and NO3 - -N did not accumulate in the culture medium. This hypothesis needs further verification using metagenomics and metatranscriptomics. Comprehensive analysis suggests that CMI-5 may primarily convert ammonia nitrogen to gaseous nitrogen via a heterotrophic nitrification-aerobic denitrification pathway, or it may assimilate some ammonia nitrogen for cellular biosynthesis, thereby achieving effective ammonia nitrogen removal.

[0138] like Figure 4 As shown in (c) and (d), CMI-5 also exhibited a certain nitrate removal capacity in aerobic denitrification medium with an initial nitrate nitrogen concentration of 50 mg / L. After 72 h of reaction, NO3... - The NO3- concentration decreased from 49.73±0.15 mg / L to 29.07±0.2 mg / L, with an overall removal rate of 41.53%. Specifically, within the first 12 hours, NO3- concentration decreased significantly. - -N decreased rapidly, with a removal rate of 57.63%, corresponding to an average rate of 2.39 mg / (L·h), demonstrating the strong early denitrification activity of CMI-5. However, NO3 decreased from 18 h onwards. - The -N concentration gradually increased, and NO2 was detected in the system. - The continuous accumulation of -N, reaching a concentration of 11.95 ± 1.21 mg / L after 72 hours, indicates that CMI-5 plays a role in reducing NO3-. - -N is reduced to NO2 - After -N, NO2 --N could not be effectively reduced further to gaseous nitrogen, leading to its gradual accumulation in the system. Due to NO2... - -N exhibits certain biotoxicity, and its continuous accumulation may inhibit bacterial cell growth, thereby weakening its denitrification activity and overall nitrogen conversion capacity. Furthermore, a small amount of NH4 was detected in the system. + The fluctuation of -N may suggest that CMI-5 participates in nitrogen transformation through multiple pathways besides the classic denitrification pathway, including assimilation and dissimilation of nitrate reduction. These results indicate that under these experimental conditions, the denitrification process of CMI-5 is limited by the intermediate product NO2. - With the accumulation of -N, nitrogen transformation mainly occurs at the NO3 stage. - -N and NO2 - The dynamic conversion between -N and -N means that the overall denitrification process did not fully progress to the final stage of gaseous nitrogen as a product in the later stages. The specific nitrogen metabolism mechanism within this process requires further investigation.

[0139] ③ Nitrogen balance analysis

[0140] Nitrogen balance analysis further revealed the main nitrogen transformation pathways and metabolic fate of the inoculant CMI-5 in different reaction systems. As shown in Table 6, in heterotrophic nitrification media, NH4+... + -N is the initial nitrogen source in the system, and the initial total nitrogen (TN) mass is 50.46 ± 0.24 mg. After 72 h of reaction, the residual NH4 in the system... + -N was 0.46±0.13 mg, organic nitrogen content was 10.99±0.56 mg, and nitrogen used for microbial biomass synthesis reached 30.76±0.46 mg, accounting for approximately 61% of the total nitrogen. An additional 8.24±0.95 mg of nitrogen was lost in undetectable form, presumably through heterotrophic nitrification-aerobic denitrification processes, where it was converted into gaseous products such as NO, N2O, and N2 and discharged from the system. It is noteworthy that during heterotrophic nitrification, a large amount of NH4+ was released. + -N was converted for microbial biomass synthesis, indicating that CMI-5 has a strong ammonia assimilation capacity. In contrast, in aerobic denitrification media, NO3- was converted for microbial biomass synthesis. - -N was the initial nitrogen source, and the initial total nitrogen was 50.44 ± 0.20 mg. After 72 h, 29.07 ± 0.20 mg of NO3 remained in the system. - -N, 11.95±0.41 mg of NO2 - -N, 0.37±0.02 mg of NH4 +The CMI-5 system produced 2.56 ± 0.63 mg of organic nitrogen and 2.56 ± 0.63 mg of nitrogen, while only 5.16 ± 0.51 mg of nitrogen was used for microbial biomass synthesis. The estimated gaseous nitrogen loss was 1.33 ± 0.10 mg, significantly lower than that of heterotrophic nitrification systems. This result indicates that the denitrification efficiency and nitrogen assimilation efficiency of CMI-5 under aerobic denitrification conditions are both limited, with nitrogen primarily produced as NO3-. - -N and NO2 - The -N form is retained in the liquid phase. It can be seen that CMI-5 is retained in the form of NH4+. + Heterotrophic nitrification systems with -N as substrates exhibit higher ammonia assimilation capacity and gaseous nitrogen release potential, while those with NO3- as substrates show higher potential. - In aerobic denitrification systems with N as the substrate, nitrogen metabolism pathways are relatively limited. This difference reflects the regulatory strategy of CMI-5 on assimilation and denitrification mechanisms under different nitrogen source forms.

[0141] Table 6. Nitrogen balance analysis of CMI-5 in heterotrophic nitrifying and aerobic denitrifying media (unit: mg)

[0142]

[0143] (4) Research on the application of compound microbial agents in actual wastewater

[0144] ① The denitrification effect of compound bacterial agents on actual wastewater

[0145] To systematically evaluate the stability and adaptability of the compound microbial agent CMI-5 in actual wastewater treatment, this study introduced the raw water microbial community as an influencing variable. Three types of actual wastewater (A, B, C) from different sources were designated as unsterilized groups (YA, YB, YC) and sterilized groups (MA, MB, MC), respectively. The denitrification performance of CMI-5 was investigated under conditions of both inoculation and non-inoculation, with and without raw water microbial interference. A control group was set up to eliminate the influence caused by nitrogen volatilization and spontaneous metabolism of the native microbial community.

[0146] like Figure 5As shown, due to the loss of ammonia and organic nitrogen caused by high-temperature sterilization, there were certain differences in the initial total nitrogen and ammonia nitrogen levels between the unsterilized and sterilized wastewater control groups. The initial total nitrogen concentrations of YA-CK and MA-CK were 69.72 mg / L and 56.16 mg / L, respectively, and the initial ammonia nitrogen concentrations were 60.85 mg / L and 48.05 mg / L, respectively. After 120 h of reaction, due to the action of the raw water microbial community or the influence of nitrogen volatilization, the total nitrogen and ammonia nitrogen concentrations of both control groups decreased, but the decrease was relatively limited. Throughout the process, only trace levels of nitrate nitrogen (<0.09 mg / L) were detected in YA-CK and MA-CK, and no accumulation of nitrite nitrogen was detected, indicating that the spontaneous nitrogen conversion process within the system was weak and the self-purification and denitrification effect of the water body was limited. In contrast, the treatment group inoculated with the compound bacterial agent CMI-5 showed a significant denitrification capacity. After 120 h, the total nitrogen concentrations in the YA and MA systems decreased from 66.19±1.71 mg / L and 52.56±0.95 mg / L to 6.83±0.43 mg / L and 5.67±0.35 mg / L, respectively, with removal rates as high as 89.68% and 89.21%. Ammonia nitrogen concentrations also decreased from 58.76±1.71 mg / L and 44.05±0.18 mg / L to 4.31±1.28 mg / L and 1.20±0.23 mg / L, respectively, with removal rates reaching 92.67% and 97.28%, demonstrating that CMI-5 has good ammonia and total nitrogen removal effects in actual wastewater environments. During the denitrification process, nitrate nitrogen was briefly detected in the YA system within the first 24 h, but disappeared rapidly after 36 h. The nitrate nitrogen concentration in the MA system remained at a low level overall, with the highest fluctuation range not exceeding 0.23±0.37 mg / L. Similar to the control group, no accumulation of nitrite nitrogen was detected in either treatment system, indicating that CMI-5 can achieve efficient removal of ammonia nitrogen in actual wastewater environments and may rapidly convert it into gaseous nitrogen and intracellular substances through heterotrophic nitrification-aerobic denitrification pathway, avoiding the accumulation of intermediate products.

[0147] like Figure 6As shown, the total nitrogen and ammonia nitrogen concentrations in the control groups YB-CK and MB-CK decreased to some extent during the 120-h reaction, but the decrease was relatively limited, similar to the control groups YA-CK and MA-CK. However, a significant increase in nitrate nitrogen (4.03 mg / L) and accumulation of nitrite nitrogen (0.34 mg / L) were detected in the YB-CK system at the end of the reaction, suggesting a risk of intermediate product accumulation in its nitrogen transformation pathway. In contrast, no nitrate or nitrite nitrogen was detected in the MB-CK system, indicating that the activity of raw water microorganisms in YB-CK was the cause of nitrate and nitrite accumulation in the system. After inoculation with CMI-5, both the YB and MB treatment groups showed significant denitrification effects. Within 120 hours, the total nitrogen concentrations in both groups decreased from 59.64±0.45 mg / L and 44.12±0.82 mg / L to 11.19±0.89 mg / L and 3.51±0.33 mg / L, respectively, with removal rates as high as 81.23% and 92.04%. Ammonia nitrogen concentrations also decreased to 3.10±1.04 mg / L and 1.11±0.31 mg / L, with removal rates of 93.27% and 96.99%, respectively, significantly better than the corresponding control groups. This indicates that CMI-5 can rapidly remove ammonia nitrogen and achieve a high degree of total nitrogen reduction in both types of raw water. It is noteworthy that nitrate nitrogen levels (5.39±0.5 mg / L) and nitrite nitrogen accumulation (1.48±0.4 mg / L) were also detected in the YB system, with trends largely consistent with the control group. This suggests that the activity of microorganisms in the raw water or differences in substrate within the system may have affected the normal progress of the CMI-5 denitrification process, leading to incomplete conversion of intermediate products. In contrast, the MB system only showed trace fluctuations in nitrate nitrogen in the first 60 hours, followed by rapid degradation, and no nitrite nitrogen was detected throughout the process. This further demonstrates the adverse effects of substrate differences or environmental limitations on the nitrogen removal efficiency of CMI-5.

[0148] like Figure 7As shown, the nitrogen removal and conversion in the control groups YC-CK and MC-CK exhibited similar trends to those in YB-CK and MB-CK. On the one hand, the concentrations of total nitrogen and ammonia nitrogen decreased to some extent with increasing time. On the other hand, significant accumulation of nitrate nitrogen and nitrite nitrogen was observed in the YC-CK system, rising to 5.23 mg / L and 1.58 mg / L, respectively, while no accumulation of nitrate nitrogen or nitrite nitrogen was detected in MC-CK. This suggests that although the raw water microorganisms in the YC-CK system possess a certain nitrification capacity, the subsequent denitrification process may not have been effectively carried out, leading to the accumulation of intermediate products in the system. In the treatment groups after inoculation with CMI-5, both YC and MC showed significant nitrogen removal advantages. Within 120 h, the total nitrogen removal rates of the two groups were 87.74% and 89.54%, respectively, and the ammonia nitrogen removal rates were 97.15% and 97.82%, respectively, demonstrating that CMI-5 has a strong ability to remove both ammonia nitrogen and total nitrogen in both sterilized and non-sterilized raw water. In the YC system, nitrate nitrogen and nitrite nitrogen still accumulated to some extent, but significantly less than in the corresponding control group. This indicates that the denitrification process of CMI-5 by the original water microorganisms in this system was less restricted, or that there was a co-metabolic metabolism of substrates by CMI-5 and the original water microorganisms. The MC system, consistent with its control group, showed no detectable nitrate nitrogen during the reaction, indicating a relatively complete nitrogen conversion process without significant accumulation of intermediate products.

[0149] In summary, CMI-5 achieved high-efficiency denitrification in three different types of wastewater. Its metabolic pathway may be somewhat disturbed in the unsterilized system, showing the phased accumulation of intermediate products. However, in the sterilized system, the metabolic pathway is complete and the product transformation is thorough, indicating that it has strong autonomous denitrification potential.

[0150] ② The ability of compound bacterial agents to remove COD from actual wastewater

[0151] To further evaluate the comprehensive purification capacity of the compound microbial agent CMI-5 in actual wastewater treatment, this study not only examined its denitrification performance but also monitored the changes in chemical oxygen demand (COD) during the treatment process to reflect its ability to degrade organic matter.

[0152] like Figure 8As shown, both the treatment groups (YA, YB, YC, MA, MB, MC) inoculated with the compound microbial agent CMI-5 and the uninoculated control groups (YA-CK, YB-CK, YC-CK, MA-CK, MB-CK, MC-CK) showed varying degrees of COD concentration reduction within 120 h. Due to the pyrolysis of organic matter during high-temperature sterilization, the initial COD concentrations of MA-CK, MB-CK, and MC-CK after sterilization were significantly lower than those of the corresponding unsterilized control groups YA-CK, YB-CK, and YC-CK, decreasing by approximately 9.78 mg / L to 18.68 mg / L, respectively. This indicates that sterilization treatment has a certain weakening effect on the content of degradable organic matter in wastewater. In the control system without inoculation of microbial agents, the COD concentration decreased to some extent within 120 h of reaction. The decrease in the unsterilized groups (YA-CK, YB-CK, YC-CK) was generally greater than that in the sterilized groups (MA-CK, MB-CK, MC-CK). Specifically, YA-CK decreased by 28.97 mg / L, YB-CK by 24.84 mg / L, and YC-CK by 24.81 mg / L, significantly higher than the decreases in MA-CK, MB-CK, and MC-CK (16.3 mg / L, 11.02 mg / L, and 19.66 mg / L, respectively). This result indicates that autotrophic microorganisms in the raw water still possess a certain degree of activity during the degradation of organic matter and can partially participate in the COD conversion process, while the COD removal capacity is significantly limited in the sterilized system.

[0153] In the CMI-5 inoculated treatment group, the COD concentration decreased significantly more than that in the corresponding control group, reflecting that the compound microbial agent has a strong ability to degrade organic matter. Specifically, the COD concentrations of YA and MA decreased from 120.80±0.53 mg / L and 111.38±1.01 mg / L to 44.86±1.41 mg / L and 38.82±1.44 mg / L, respectively, corresponding to COD removal rates of 62.86% and 65.13%. YB and MB decreased from 110.84±0.88 mg / L and 92.67±0.8 mg / L to 43.00±1.36 mg / L and 34.60±1.35 mg / L, respectively, with removal rates of 61.21% and 62.67%. YC and MC decreased from 115.36±0.39 mg / L and 96.55±1.00 mg / L to 44.19±0.49 mg / L and 33.81±1.89 mg / L. The removal rates were 61.69% and 64.97%, respectively, for mg / L. Compared with the natural degradation and physical volatilization effect of raw water by microorganisms in the control group, CMI-5 effectively accelerated the oxidation and mineralization of organic matter by enhancing heterotrophic metabolism, demonstrating good COD removal performance. This result further confirms that CMI-5 not only has nitrogen removal function in wastewater treatment, but also has the potential to synergistically degrade organic pollutants, and is expected to play a greater role in the treatment of wastewater with high overall pollution load.

[0154] ③ The effect of compound microbial agents on the composition of DOM in actual wastewater

[0155] Effect of compound microbial agents on DOC concentration

[0156] To further evaluate the degradation capacity of the compound microbial agent CMI-5 for soluble organic matter during the treatment of actual wastewater, this study monitored the changes in dissolved organic carbon (DOC) before and after the reaction in each group. Figure 9As shown, the DOC concentrations in untreated actual wastewater samples A_0d, B_0d, and C_0d were 72.68±0.45 mg / L, 66.55±1.12 mg / L, and 68.75±0.43 mg / L, respectively. After 5 days of reaction, the DOC concentrations in the unsterilized treatment groups YA, YB, and YC, inoculated with the compound bacterial agent CMI-5, decreased significantly, to 21.64±0.62 mg / L, 17.76±1.52 mg / L, and 20.57±1.06 mg / L, respectively. In contrast, the final DOC concentrations in the sterilized treatment groups MA, MB, and MC were 25.04±0.72 mg / L, 22.09±1.58 mg / L, and 24.89±0.83 mg / L, respectively, showing a relatively smaller decrease. In terms of DOC removal efficiency, the degradation rate of the unsterilized group was between 70.08% and 73.31%, generally higher than that of the sterilized group (63.80%–66.81%). This indicates that the presence of microorganisms in the raw water may have promoted the degradation of dissolved organic carbon by the compound microbial agent to some extent. This promoting effect may stem from the synergistic metabolism between the microbial agent and native microorganisms, such as through primary substrate degradation, complementary metabolites, or synergistic expression of extracellular enzyme systems, jointly accelerating the conversion and mineralization of DOC. Furthermore, regardless of sterilization, CMI-5 significantly reduced the DOC content in the water, further validating its ability to degrade complex organic matter in actual wastewater and demonstrating strong heterotrophic metabolic potential. This result is consistent with the aforementioned COD trend, indicating that CMI-5 can not only remove recalcitrant macromolecular organic matter but also effectively treat soluble organic carbon.

[0157] DOM three-dimensional fluorescence spectroscopy and parallel factorial analysis

[0158] To deeply analyze the compositional characteristics and structural changes of dissolved organic matter (DOM) in water during treatment with the compound microbial agent CMI-5, this study employed a parallel factor analysis (PARAFAC) model to deconstruct and analyze the three-dimensional fluorescence spectra (3D-EEM) of all samples. Through model fitting, four main fluorescent components were identified, denoted as C1 to C4. The peak positions of the excitation wavelength (Ex) and emission wavelength (Em) for each component were: C1 (Ex: 314 / <250 nm; Em: 393 nm), C2 (Ex: 272 nm; Em: 345 nm), C3 (Ex: <250 / 382 nm; Em: 483 nm), and C4 (Ex: 352 / <250 nm; Em: 426 nm). C1, C3, and C4 can be classified as humic-like fluorescent substances, mainly originating from high-molecular-weight organic matter formed during humification, while C2 is a protein-like substance, often produced by microbial metabolism or the release of cellular components.

[0159] Analysis of component intensity and proportion in three-dimensional fluorescence spectra of DOM

[0160] To further investigate the effect of the compound bacterial agent CMI-5 on the fluorescence characteristics of DOM in actual wastewater, this study, based on the four fluorescent components (C1-C4) extracted from the aforementioned PARAFAC model, analyzed the maximum fluorescence intensity (F) of DOM in each treated sample. max The relative proportions of the microbial agents were quantitatively analyzed to reveal the regulatory effect of microbial agents on the degradation characteristics and structural composition of DOM.

[0161] From the change in fluorescence intensity ( Figure 10 From the perspective of CMI-5, the addition of CMI-5 significantly reduced the F of each DOM component. max Compared to the untreated raw water samples (A_0d, B_0d, C_0d), the fluorescence intensities of C1, C2, C3, and C4 in the six treatment groups after 5 days of reaction decreased by 39.21%–64.86%, 21.22%–53.19%, 44.67%–61.35%, and 42.38%–67.15%, respectively, indicating that the bacterial agent CMI-5 has good degradation ability in removing DOM. Notably, the total fluorescence intensity of the unsterilized treatment groups was generally lower than that of the corresponding sterilized treatment groups, suggesting a possible synergistic metabolic relationship between the microorganisms in the raw water and CMI-5. This trend is consistent with the aforementioned DOC change results.

[0162] Further analysis of the relative abundance of each fluorescent component ( Figure 11 The results showed that the addition of CMI-5 not only reduced the overall fluorescence intensity of DOM but also altered its composition. In the untreated raw water A_0d, the proportions of each component were C1 (32.72%) > C2 (24.36%) > C4 (21.51%) > C3 (21.41%). After CMI-5 treatment, the proportions of C2 and C3 in YA_5d increased significantly to 27.96% and 23.95%, respectively, while the proportion of C4 decreased to 18.81%. In the corresponding sterilized group MA_5d, the proportion of C2 further increased to 41.31%, becoming the dominant component. Similar changes were also observed in the B and C group samples. In B_0d, C2 was the dominant component (31.80%), but in YB_5d, its proportion significantly increased to 43.45%, while the proportions of C1 and C4 decreased significantly. In contrast, C1 levels in sterilized MB_5d showed a slight increase, reflecting the difference in CMI-5 degradation patterns under conditions without native microbial interference. In group C, regardless of sterilization treatment, C2 consistently dominated the reaction (YC_5d: 35.00%, MC_5d: 33.89%), indicating that the effect of the microbial agent on protein-like components is consistent across different water qualities.

[0163] In summary, the results show that while CMI-5 degraded the total amount of DOM (degradable organic matter), it also significantly increased the relative abundance of the protein-like fluorescent component (C2) in DOM. C2 is generally considered to be a protein or cellular metabolic organic matter of microbial origin; its increased proportion may indicate that the active microbial metabolic behavior during CMI-5 treatment significantly enhanced the turnover rate of endogenous proteins in wastewater, reflecting characteristics such as increased microbial activity and accelerated metabolic cycles. This trend also supports the potential role of CMI-5 in promoting microbial structure reconstruction and niche regulation in wastewater systems.

[0164] DOM three-dimensional fluorescence spectral index analysis

[0165] To further reveal the effects of compound microbial agent CMI-5 on the source and structural characteristics of DOM in wastewater, this study quantitatively characterized the source attributes and humification degree of DOM in each sample using three commonly used parameters: fluorescence index (FI), biogenic index (BIX), and humification index (HIX).

[0166] The fluorescence index (FI) reflects the source of fluorescence in DOM (Distinct DOM), defined as the ratio of the intensity at emission wavelengths of 470 nm to 520 nm at an excitation wavelength of 370 nm. FI values ​​are significantly negatively correlated with aromatic substances in DOM. Generally, when FI < 1.4, DOM exhibits heterogeneous terrestrial fluorescence characteristics, containing a higher content of non-aromatic substances; when FI > 1.8, DOM exhibits local microbial fluorescence characteristics, with a high content of aromatic amino acids and strong microbial activity. For example... Figure 12 As shown in (a), the mean FI value of all samples in this study was greater than 2, indicating that the DOM in each water sample was mainly autogenous and the microbial activity was strong. Further comparison showed that the FI values ​​of the treatment groups after inoculation with CMI-5 were generally higher than those of the corresponding control groups. Among them, the FI values ​​of YA_5d (2.08±0.08) and MA_5d (2.08±0.06) were higher than those of the original sample A_0d (2.03±0.03), YB_5d (2.09±0.12) was also higher than B_0d (2.06±0.02), and MC_5d (2.05±0.07) was higher than C_0d (2.03±0.05). This indicates that the addition of CMI-5 enhanced the microbial metabolic activity in the system to a certain extent and strengthened the autogenous fluorescence characteristics of the DOM in the water.

[0167] The Biogenicity Index (BIX) measures the relative contribution of newly generated organic matter from microorganisms in the DOM (Disseminated Organic Matter). It is defined as the ratio of the intensity at emission wavelengths of 380 nm and 430 nm at an excitation wavelength of 310 nm. Generally, when BIX < 0.6, the autogenic contribution of DOM in the water body is considered to be small, and the main source is terrestrial input; when BIX > 0.8, the main source of DOM in the water body is algae or bacteria, and the autogenic source is strong. Figure 12As shown in (b), the mean BIX value of all samples was higher than 1.1, further confirming the high proportion of autogenic organic matter in the water and significant microbial activity. Compared with the control group, the BIX values ​​of all groups were generally higher after CMI-5 treatment, indicating that it effectively enhanced the accumulation of newly generated autogenic substances in the system. For example, MA_5d (1.19±0.09) was higher than A_0d (1.16±0.03), YB_5d (1.20±0.1) and MB_5d (1.17±0.05) were both higher than B_0d (1.13±0.02), and YC_5d (1.18±0.06) and MC_5d (1.20±0.07) were also higher than C_0d (1.14±0.05), further reflecting that the application of CMI-5 helps to improve the microbial productivity in the system.

[0168] The Humus Index (HIX) is used to assess the degree of humification of DOM (Dissolved Morphological Material). It is calculated as the ratio of the sum of fluorescence intensities at 435 nm–480 nm to the sum of fluorescence intensities at 300 nm–345 nm. A higher HIX indicates a stronger degree of humification in the DOM. Generally, an HIX < 0.8 indicates a relatively fresh DOM with low humification, while an HIX > 0.85 indicates abundant humus content. Figure 12 As shown in (c), the mean HIX value of all samples in this study was below 0.8, indicating that the overall humification level of DOM was low, mainly consisting of low-humification organic matter such as microbial metabolites. Furthermore, the HIX value was lower in most treatment groups than in the corresponding control group, suggesting that the addition of CMI-5 may have reduced the accumulation of highly humic organic matter in the system by promoting the fresh renewal and molecular remodeling of DOM. For example, MA_5d (0.6±0.12) was significantly lower than A_0d (0.72±0.03), YB_5d (0.61±0.14) was lower than B_0d (0.67±0.08), and YC_5d (0.66±0.01) and MC_5d (0.64±0.15) were both lower than C_0d (0.68±0.01).

[0169] The changes in the above indices show that the introduction of CMI-5 not only effectively removed organic pollutants from the water, but also significantly changed the composition and source characteristics of DOM, enhanced the autogenic input of microorganisms, and reduced the degree of humification, thus playing a positive role in improving the content of active substances in the water and the metabolic efficiency of the system.

[0170] (5) Results of nitrogen removal tests of different combinations of compound bacterial agents for rural domestic sewage

[0171] Experimental results showed that all three single strains had a certain ability to treat ammonia nitrogen in actual rural domestic sewage, with an average removal rate of 67%. The ammonia removal effect of any two strains combined was significantly better than that of single strains, with total nitrogen removal rates between 70% and 80%. When all three strains were used in combination, the total nitrogen removal rate significantly increased to 90%, which was significantly better than single strains or combinations of two strains. These results indicate that the three strains have good complementary and synergistic effects in the nitrogen removal process. The above-mentioned compound bacterial agent is suitable for nitrogen pollution control in various types of domestic sewage in rural areas, including black water, grey water, and mixed sewage. In practical applications, single strains, dual-strain combinations, or triple-strain combinations can be flexibly selected according to the sewage composition and treatment objectives to optimize resource utilization and cost control while ensuring treatment effectiveness.

[0172] Based on the above description of the invention, those skilled in the art can fully apply the present invention, and all similar modifications and principles used should be considered within the scope of the present invention.

Claims

1. A compound bacterial agent with synergistic carbon and nitrogen removal function, characterized in that, It consists of the following three strains: Rhodococcus sp. 24, Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The compound microbial agent simultaneously possesses heterotrophic nitrification-aerobic denitrification denitrification capabilities, chemical oxygen demand (COD) removal capabilities, dissolved organic carbon (DOC) reduction capabilities, dissolved organic matter (DOM) degradation capabilities, and the ability to increase the proportion of proteinaceous substances to enhance autotrophic characteristics and reduce humification levels; wherein: The strain Rhodococcus sp. 24 The strain CPZ24 is mentioned in patent document CN103525734B; The strain Rhodococcussp. YB It is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number: CGMCC No. 35172; The strain Pseudomonas tianjinensis sp. It is deposited at the China Industrial Microbial Culture Collection Center (CICC), accession number: CICC 24204.

2. The compound microbial agent according to claim 1, characterized in that, The strain Rhodococcus sp. 24 Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The volume ratio was 18.8%~53.7%:10%~40.7%:8.7%~46.3%, where the volume ratio was the volume ratio of each strain during the logarithmic growth phase; wherein, the volume ratio was obtained using the simplex centroid design model with Design Expert 13 software.

3. The compound microbial agent according to claim 1, characterized in that, The strain Rhodococcus sp. 24 Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The volume ratio is 20%~40%:20%~40%:20%~45%, where the volume ratio is the volume ratio of each strain during the logarithmic growth phase.

4. The compound microbial agent according to claim 1, characterized in that, The strain Rhodococcus sp. 24 Rhodococcus sp. YB and Pseudomonas tianjinensis sp. The volume ratio was 36.5%:22.7%:40.8%, which is the volume ratio of each strain during the logarithmic growth phase.

5. The compound microbial agent according to claim 1, characterized in that, The cell activity of the compound microbial agent is 1×10⁻⁶. 8 ~100×10 10 CFU / mL.

6. The application of the compound microbial agent according to any one of claims 1 to 5 in the treatment of rural domestic sewage.

7. The application according to claim 6, characterized in that, The wastewater is selected from: black water, grey water and mixed black and grey water; the ammonia nitrogen concentration of the wastewater is 20~200 mg / L.

8. The application according to claim 7, characterized in that, The ammonia nitrogen concentration in the wastewater is 20~100 mg / L.

9. The application according to claim 6, characterized in that, The compound microbial agent enhances the fluorescence index (FI) and biogenic index (BIX) of wastewater, and reduces the humification index (HIX).

10. A method for treating actual rural domestic sewage, characterized in that, The method includes: inoculating the composite bacterial agent according to any one of claims 1 to 5 into the wastewater, and carrying out aerobic culture for a reaction time of 48 to 120 hours.

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