A purification process for livestock farm wastewater

Abstract

This invention discloses a purification process for livestock farm wastewater, belonging to the field of functional microbial materials and application technology. By introducing a composite functional bacterial agent and a biomass composite filler based on bacterial bran, this invention achieves highly efficient removal of ammonia nitrogen, COD, and other pollutants from wastewater, while significantly reducing the risk of secondary pollution from traditional chemical treatment methods. The composite functional bacterial agent, containing *Pseudomonas hazelnutans*, *Microbacterium keratolyticum*, and *Bacillus licheniformis*, works synergistically to rapidly degrade organic matter in wastewater, while also exhibiting good flocculation effects on ammonia nitrogen. It possesses excellent environmental adaptability and stability, capable of handling complex and changing water quality conditions. Furthermore, the biomass composite filler based on bacterial bran, through acid washing activation and iron-chitosan loading modification, significantly improves its adsorption performance and nitrogen and phosphorus removal capabilities, further optimizing the deep treatment effect. This process is simple to operate, has low operating costs, and is suitable for the wastewater treatment needs of large-scale livestock farms.

Description

Technical Field

[0001] This invention belongs to the field of functional microbial materials and application technology, specifically relating to a purification and treatment process for livestock breeding wastewater. Background Technology

[0002] In recent years, the rapid development of large-scale and intensive livestock and poultry farming technologies has led to a continuous increase in the discharge of wastewater from these operations. The effective treatment of this wastewater is a pressing issue in agricultural production. Livestock and poultry wastewater is complex in composition, containing various suspended particulate matter and organic pollutants, and is dark in color, making it difficult to treat. Large quantities of untreated livestock and poultry wastewater discharged into the surrounding environment cause serious pollution, particularly impacting aquatic and soil ecosystems. Currently, there are various methods for treating livestock and poultry wastewater, which can be broadly categorized into physical remediation, chemical remediation, and biological remediation.

[0003] Physical treatment is a pretreatment process for livestock and poultry wastewater before it enters the biological treatment process. Its purpose is to better remove larger suspended solids from the wastewater, preventing disruption to subsequent equipment operation and reducing equipment load. This is achieved through physical processes such as flocculation, absorption, and sedimentation to remove insoluble pollutants. While physical treatment is simple and efficient, it cannot completely remove organic matter and pollutants such as nitrogen and phosphorus, and the treated water quality may still fail to meet discharge standards.

[0004] Chemical methods refer to using chemical processes to alter the chemical structure of pollutants in wastewater that exist in soluble or colloidal forms, thereby rendering them harmless or transforming them into easily treatable substances. While chemical methods are highly effective, they are very costly. Furthermore, they are uncontrollable and can easily generate other compounds due to environmental changes, leading to secondary pollution.

[0005] Bioremediation refers to the use of plants, specific organisms such as microorganisms or protozoa, to absorb, transform, remove, or degrade pollutants in the surrounding environment to restore the polluted environment and achieve the goal of completely removing pollutants and restoring ecological effects. It is an effective means of improving environmental quality, composed of an ecosystem. Because livestock and poultry farm wastewater contains high concentrations of ammonia nitrogen and COD, it is generally difficult to remove them using physical and chemical methods. Bioremediation can rapidly reduce ammonia nitrogen and COD. Compared with traditional physicochemical remediation, bioremediation is generally considered a sustainable technology with advantages such as cost-effectiveness, low energy consumption, and no secondary pollution.

[0006] For example, Chinese patent application CN202211390528.5 discloses a method for preparing a microbial agent and organic fertilizer for treating dairy farm wastewater. The microbial agent for treating dairy farm wastewater described in this invention is specifically prepared from components comprising the following parts by weight: 12-18 parts of solid fermentation agent, 10-20 parts of liquid fermentation agent, and 10-20 parts of ammonia nitrogen removal agent.

[0007] However, existing bioremediation technologies still face many challenges in practical applications. For example, microbial agents have poor adaptability and stability, and are greatly affected by factors such as environmental temperature, pH, and dissolved oxygen, making it difficult to maintain high-efficiency degradation capabilities in complex and variable wastewater environments. Furthermore, bioremediation cycles are long, and their ability to cope with sudden, high-concentration pollutant shock loads is weak. They also still rely on subsequent complex chemical flocculation and advanced oxidation processes, resulting in high operating costs and the risk of secondary pollution. Therefore, improving the adaptability and remediation efficiency of bioremediation technologies has become a key research focus. We believe that, on the one hand, we can enhance the activity and stability of functional microorganisms with strong tolerance and high degradation efficiency in complex environments by screening and cultivating them. On the other hand, we can combine physical and chemical methods for synergistic treatment to optimize the overall remediation effect. Therefore, this invention aims to solve the problems existing in current technologies from these perspectives. Summary of the Invention

[0008] This invention addresses the problems existing in existing technologies by screening out a strain of Bacillus licheniformis with high ammonia nitrogen degradation capacity and bioflocculation effect. This strain is then combined with Pseudomonas hazelnutans and Microbes keratolyticus to form a functional microbial composition. This composition enables the simultaneous and efficient removal of ammonia nitrogen and COD from livestock wastewater, purifying the water quality while significantly reducing reliance on subsequent chemical treatments, thereby minimizing the risk of secondary pollution. This functional microbial combination exhibits good environmental adaptability and stability, making it particularly suitable for treating livestock wastewater under complex water quality conditions.

[0009] To achieve the above-mentioned technical objectives, the technical solution adopted by the present invention is as follows:

[0010] A purification and treatment process for livestock breeding wastewater includes the following preparation steps:

[0011] (1) Pretreatment: The livestock wastewater to be treated is removed by a bar screen to remove large suspended solids and impurities, and then enters a grit chamber for preliminary sedimentation to separate heavy particles such as sand and gravel, thereby reducing the load on subsequent treatment.

[0012] (2) Primary treatment: The pretreated wastewater is introduced into the hydrolysis acidification tank, with a hydraulic retention time of 6-8 hours, and the facultative microorganisms in the tank are used for preliminary fermentation and decomposition.

[0013] (3) Core microbial treatment: The effluent from the hydrolysis acidification tank is introduced into the aerobic reaction tank, the pH is adjusted to 6.5-7.5, and compound functional bacterial agent is added at 1-3% of the sewage mass. Intermittent aeration is adopted, aeration is carried out for 15 minutes, and the reaction is stopped for 45 minutes. The hydraulic retention time is 12-18 hours.

[0014] (4) Solid-liquid separation: The effluent from the aerobic reaction tank enters the sedimentation tank and is allowed to settle for 1-2 hours. The mud and water are separated by the bioflocculation of the microorganisms themselves.

[0015] (5) Advanced treatment and disinfection: The supernatant from the sedimentation tank enters a filter filled with biomass composite packing material for final nitrogen and phosphorus removal purification. The hydraulic retention time is controlled at 1-2 hours. Finally, it is disinfected by ultraviolet light or ozone before being discharged. When using ultraviolet light for disinfection, the effective ultraviolet dose should be ≥30 mJ / cm² (millijoules / square centimeter). If ozone is used, the ozone dosage is controlled at 5-10 mg / L.

[0016] Furthermore, in step (3), the compound functional microbial agent contains Pseudomonas hazel, Microbacterium keratolyticum and Bacillus licheniformis, with a volume ratio of 1:1:1.

[0017] Furthermore, the strain number of *Pseudomonas hazelnutans* is CGMCC No. 1.3819, with an original deposit date of March 17, 2005; the strain number of *Microbacterium keratolyticum* is CGMCC No. 1.6312, with an original deposit date of May 12, 2006. Both *Pseudomonas hazelnutans* and *Microbacterium keratolyticum* were purchased from the China General Microbiological Culture Collection Center (CGMCC) and are commercially available, requiring no biological preservation. The preservation number of *Bacillus licheniformis* is CGMCC No. 79241, classified as *Bacillus licheniformis*, and deposited at the CGMCC, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of March 5, 2025.

[0018] Furthermore, the preparation method of the compound functional microbial agent is as follows: *Pseudomonas hazelnutans*, *Microbacterium keratolyticum*, and *Bacillus licheniformis* are inoculated into LB medium and cultured with shaking until the bacterial count reaches OD200. 600 The volume ratio of the bacterial solution to the carrier was approximately 3.0. The mixed bacterial solution was then prepared by mixing the carrier with the carrier at a volume-to-mass ratio of 1L:1.5kg. The carrier consisted of attapulgite clay, starch, and calcium chloride at a mass ratio of 100:5:2. The adsorbed material was granulated and vacuum-dried until the moisture content was ≤8%, thus obtaining the composite functional microbial agent. The effective viable count of the composite functional microorganisms was not less than 1×10⁻⁶. 9 CFU / g.

[0019] Furthermore, the preparation method of the bacterial bran-based biomass composite filler in step (5) is as follows:

[0020] (1) Raw material pretreatment: Fresh mushroom substrate taken from enoki mushroom or king oyster mushroom factories is crushed and screened to obtain 40-60 mesh particles. The substrate is repeatedly rinsed with water until the water is clear to remove soluble impurities. It is then dried at 80℃ to constant weight to obtain pretreated mushroom substrate.

[0021] (2) Acid washing and activation: The pretreated mushroom residue is placed in a 5% citric acid solution and shaken at 60℃ and 150 r / min for 2 hours. After the reaction is completed, it is filtered, washed with deionized water until neutral, and dried. This step is used to dissolve and remove some of the inorganic salts and ash in the mushroom residue and expose more pore structures.

[0022] (3) Iron-chitosan loading modification: Prepare a 1.0 mol / L FeCl3 solution and a chitosan acetic acid solution; immerse the acid-washed mushroom bran in the FeCl3 solution at a solid-liquid ratio of 1g:15mL, slowly add the above chitosan solution to the system, the mass ratio of chitosan to mushroom bran is 1:20, and stir slowly at 45℃ for 4 hours; after the reaction is completed, filter, wash the obtained solid with deionized water 2-3 times, vacuum dry at 60℃, and cool to obtain the mushroom bran-based biomass composite filler.

[0023] Furthermore, in step (2), the solid-liquid ratio of citric acid solution and pretreated bacterial bran is 1g:10mL, and in step (3), the solid-liquid ratio of chitosan and acetic acid solution is 1g:100mL, and the mass concentration of acetic acid solution is 3%.

[0024] Furthermore, in step (5), the amount of bacterial bran-based biomass composite filler added is 10-20g per liter of sewage.

[0025] Beneficial effects:

[0026] The livestock wastewater purification process provided by this invention has significant technical advantages and application value. By introducing a composite functional microbial agent and a biomass composite filler based on bacterial bran, it achieves highly efficient removal of ammonia nitrogen, COD, and other pollutants from wastewater, while significantly reducing the risk of secondary pollution from traditional chemical treatment methods. The synergistic effect of *Pseudomonas hazelnutans*, *Microbacterium keratolyticum*, and *Bacillus licheniformis* in the composite functional microbial agent not only rapidly degrades organic matter in wastewater, but also exhibits good flocculation properties for ammonia nitrogen, demonstrating excellent environmental adaptability and stability, and enabling it to cope with complex and changing water quality conditions. Furthermore, the biomass composite filler based on bacterial bran, through acid washing activation and iron-chitosan loading modification, significantly improves its adsorption performance and nitrogen and phosphorus removal capabilities, further optimizing the deep treatment effect.

[0027] This process demonstrates ease of operation and low operating costs in practical applications, making it particularly suitable for the wastewater treatment needs of large-scale farms. Compared to existing technologies, this process eliminates the need for complex chemical flocculation or advanced oxidation processes to achieve compliant wastewater discharge, effectively alleviating the environmental pressure caused by livestock and poultry farming wastewater. Furthermore, precise control over pretreatment, core microbial treatment, and advanced treatment processes ensures the overall stability and reliability of the process, providing crucial technical support for the sustainable development of the livestock and poultry farming industry. Attached Figure Description

[0028] Figure 1 The colony morphology (a) and staining characteristics (b) of Bacillus licheniformis in this invention are shown.

[0029] Figure 2 The diagram shows the antagonistic interactions between bacterial species, where 1 represents *Pseudomonas hazel*, 2 represents *Microbacterium keratolyticum*, and 3 represents *Bacillus licheniformis*.

[0030] Figure 3 The results show the flocculation effect of the microbial bacterial solutions prepared by strains in Example 1 and Comparative Examples 1-9 of this invention. Detailed Implementation

[0031] The technical solution of the present invention will be further described below with reference to specific embodiments, but it is not limited thereto.

[0032] Example 1

[0033] A purification and treatment process for livestock breeding wastewater includes the following preparation steps:

[0034] (1) Pretreatment: The livestock wastewater to be treated is removed by a bar screen to remove large suspended solids and impurities, and then enters a grit chamber for preliminary sedimentation to separate heavy particles such as sand and gravel, thereby reducing the load on subsequent treatment.

[0035] (2) Primary treatment: The pretreated wastewater is introduced into the hydrolysis acidification tank, with a hydraulic retention time of 6 hours, and the facultative microorganisms in the tank are used for preliminary fermentation and decomposition.

[0036] (3) Core microbial treatment: The effluent from the hydrolysis acidification tank is introduced into the aerobic reaction tank, the pH is adjusted to 6.5-7.5, and a compound functional bacterial agent is added at 1% of the sewage mass. Intermittent aeration is adopted, aeration is carried out for 15 minutes, and the reaction is stopped for 45 minutes. The hydraulic retention time is 12 hours.

[0037] (4) Solid-liquid separation: The effluent from the aerobic reaction tank enters the sedimentation tank and is allowed to settle for 1 hour. The mud and water are separated by the bioflocculation of the microorganisms themselves.

[0038] (5) Advanced treatment and disinfection: The supernatant from the sedimentation tank enters a filter filled with biomass composite packing material containing bacterial bran for final denitrification and phosphorus removal purification. The hydraulic retention time is controlled at 1 hour. Finally, ozone is used for disinfection before discharge. The ozone dosage is controlled at 10 mg / L.

[0039] In step (3), the compound functional microbial agent contains Pseudomonas hazel, Microbacterium keratolyticum and Bacillus licheniformis, with a volume ratio of 1:1:1.

[0040] The strain number of *Pseudomonas hazelnutii* is CGMCC No. 1.3819, and the original deposit date is March 17, 2005. The strain number of *Microbacterium keratolyticum* is CGMCC No. 1.6312, and the original deposit date is May 12, 2006. The preservation number of *Bacillus licheniformis* is CGMCC No. 79241, deposited at the China General Microbiological Culture Collection Center, classified and named *Bacillus licheniformis*, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, with a deposit date of March 5, 2025.

[0041] The *Pseudomonas hazelnutans* and *Microbacterium keratolyticum* were purchased from the China General Microbiological Culture Collection Center and can be purchased through commercial channels without the need for preservation.

[0042] The Bacillus licheniformis was isolated from the bottom sludge of a waterworks in the suburbs of Linyi City, Shandong Province. The isolation method was as follows:

[0043] Take 2 g of sample and inoculate it into sterile water. Incubate at 30 ℃ and 180 rpm with shaking for 3 h. After standing, transfer 5 mL of the bacterial suspension to 100 mL of enrichment medium and incubate under the same conditions for 48 h to obtain the enriched bacterial solution. Prepare a series of dilutions of this culture medium (10... -1 10 -2 10 -3 10 -4 10 -5 10 -6 100 µL of the culture medium was spread onto an inverted culture medium and incubated at 30 °C for 48 h. The bacterial growth was observed and recorded daily. Strains with different colony morphologies were picked and streaked for isolation. After repeated streaking isolation, pure strains were obtained.

[0044] The enrichment medium consisted of 3 g / L beef extract, 10 g / L peptone, 30 g / L NaCl, pH 7.0-7.2, and was sterilized at 121℃ for 30 min.

[0045] The screened and purified strains were inoculated into enrichment medium and activated for 1 day. Then, 5 mL of the culture medium was added to 100 mL of isolation medium and cultured in a shaker at 30 ℃ and 180 rpm for 48 h. Samples were taken at 24 and 48 h, and the NH4+ content in the culture medium was determined by Nessler's colorimetric method. + The concentration of -N was used to calculate the total denitrification rate and ammonia nitrogen degradation rate. Three groups were set up for each strain. Based on the total denitrification rate and ammonia nitrogen degradation rate, the strain with the best denitrification effect and the fastest growth rate was selected as the target strain.

[0046] After the target strain of Bacillus licheniformis was cultured on LB solid medium at 30°C for 36 h, the colonies were milky white, raised, smooth, round, and opaque. Figure 1 (a) Gram staining is negative, as shown in (b).

[0047] Antagonistic Experiment: *Pseudomonas hazelnutans*, *Microbacterium keratolyticum*, and *Bacillus licheniformis* were activated and adjusted to the same concentration. Bacterial suspensions were then streaked onto the same nutrient agar plates using an inoculation loop, with each strain intersecting the others. After incubation at 30 °C for 48 h, antagonistic phenomena were observed. Results showed that *Bacillus licheniformis* did not exhibit significant growth inhibition with *Pseudomonas hazelnutans* or *Microbacterium keratolyticum* on the culture medium, indicating no antagonistic effect among the three strains, suggesting they can be used in combination (e.g., ...). Figure 2 (As shown).

[0048] The preparation method of the compound functional microbial agent is as follows: *Pseudomonas hazelnutans*, *Microbacterium keratolyticum*, and *Bacillus licheniformis* are inoculated into LB medium and cultured with shaking until the bacterial count reaches OD200. 600 The volume ratio of the bacterial solution to the carrier was approximately 3.0. The mixed bacterial solution was then prepared by mixing the carrier with the carrier at a volume-to-mass ratio of 1L:1.5kg. The carrier consisted of attapulgite clay, starch, and calcium chloride at a mass ratio of 100:5:2. The adsorbed material was granulated and vacuum-dried until the moisture content was ≤8%, thus obtaining the composite functional microbial agent. The effective viable count of the composite functional microorganisms was not less than 1×10⁻⁶. 9 CFU / g.

[0049] Step (5) The preparation method of the bacterial bran-based biomass composite filler is as follows:

[0050] (1) Raw material pretreatment: Fresh mushroom substrate taken from enoki mushroom or king oyster mushroom factories is crushed and screened to obtain 40-60 mesh particles. The substrate is repeatedly rinsed with water until the water is clear to remove soluble impurities. It is then dried at 80℃ to constant weight to obtain pretreated mushroom substrate.

[0051] (2) Acid washing and activation: The pretreated mushroom residue is placed in a 5% citric acid solution and shaken at 60℃ and 150 r / min for 2 hours. After the reaction is completed, it is filtered, washed with deionized water until neutral, and dried. This step is used to dissolve and remove some of the inorganic salts and ash in the mushroom residue and expose more pore structures.

[0052] (3) Iron-chitosan loading modification: Prepare a 1.0 mol / L FeCl3 solution and a chitosan acetic acid solution; immerse the acid-washed mushroom bran in the FeCl3 solution at a solid-liquid ratio of 1g:15mL, slowly add the above chitosan solution to the system, the mass ratio of chitosan to mushroom bran is 1:20, and stir slowly at 45℃ for 4 hours; after the reaction is completed, filter, wash the obtained solid with deionized water 2-3 times, vacuum dry at 60℃, and cool to obtain the mushroom bran-based biomass composite filler.

[0053] In step (2), the solid-liquid ratio of citric acid solution and pretreated bacterial bran is 1g:10mL. In step (3), the solid-liquid ratio of chitosan and acetic acid solution is 1g:100mL, and the mass concentration of acetic acid solution is 3%.

[0054] The amount of bacterial bran-based biomass composite filler added in step (5) is 10g per liter of sewage.

[0055] Example 2

[0056] A purification and treatment process for livestock breeding wastewater includes the following preparation steps:

[0057] (1) Pretreatment: The livestock wastewater to be treated is removed by a bar screen to remove large suspended solids and impurities, and then enters a grit chamber for preliminary sedimentation to separate heavy particles such as sand and gravel, thereby reducing the load on subsequent treatment.

[0058] (2) Primary treatment: The pretreated wastewater is introduced into the hydrolysis acidification tank, with a hydraulic retention time of 7 hours, and the facultative microorganisms in the tank are used for preliminary fermentation and decomposition.

[0059] (3) Core microbial treatment: The effluent from the hydrolysis acidification tank is introduced into the aerobic reaction tank, the pH is adjusted to 6.5-7.5, and a compound functional bacterial agent is added at 2% of the sewage mass. Intermittent aeration is adopted, aeration is carried out for 15 minutes, and the reaction is stopped for 45 minutes. The hydraulic retention time is 15 hours.

[0060] (4) Solid-liquid separation: The effluent from the aerobic reaction tank enters the sedimentation tank and is allowed to settle for 1 hour. The mud and water are separated by the bioflocculation of the microorganisms themselves.

[0061] (5) Advanced treatment and disinfection: The supernatant from the sedimentation tank enters a filter filled with biomass composite packing material based on bacterial bran for final denitrification and phosphorus removal purification. The hydraulic retention time is controlled at 1.5 hours. Finally, it is discharged after ultraviolet disinfection. The effective ultraviolet dose used for ultraviolet disinfection is 40 mJ / cm² (millijoules per square centimeter).

[0062] In step (3), the compound functional microbial agent contains Pseudomonas hazel, Microbacterium keratolyticum and Bacillus licheniformis, with a volume ratio of 1:1:1.

[0063] The strain number of *Pseudomonas hazel* is CGMCC No. 1.3819, the strain number of *Microbacterium keratolyticum* is CGMCC No. 1.6312, and the preservation number of *Bacillus licheniformis* is CGMCC No. 79241. They are deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0064] The isolation and screening method for Bacillus licheniformis is the same as in Example 1.

[0065] The preparation method of the compound functional microbial agent is the same as in Example 1.

[0066] Step (5) The preparation method of the bacterial bran-based biomass composite filler is as follows:

[0067] (1) Raw material pretreatment: Fresh mushroom substrate taken from enoki mushroom or king oyster mushroom factories is crushed and screened to obtain 40-60 mesh particles. The substrate is repeatedly rinsed with water until the water is clear to remove soluble impurities. It is then dried at 80℃ to constant weight to obtain pretreated mushroom substrate.

[0068] (2) Acid washing and activation: The pretreated mushroom residue is placed in a 5% citric acid solution and shaken at 60℃ and 150 r / min for 2 hours. After the reaction is completed, it is filtered, washed with deionized water until neutral, and dried. This step is used to dissolve and remove some of the inorganic salts and ash in the mushroom residue and expose more pore structures.

[0069] (3) Iron-chitosan loading modification: Prepare a 1.0 mol / L FeCl3 solution and a chitosan acetic acid solution; immerse the acid-washed mushroom bran in the FeCl3 solution at a solid-liquid ratio of 1g:15mL, slowly add the above chitosan solution to the system, the mass ratio of chitosan to mushroom bran is 1:20, and stir slowly at 45℃ for 4 hours; after the reaction is completed, filter, wash the obtained solid with deionized water 2-3 times, vacuum dry at 60℃, and cool to obtain the mushroom bran-based biomass composite filler.

[0070] In step (2), the solid-liquid ratio of citric acid solution and pretreated bacterial bran is 1g:10mL. In step (3), the solid-liquid ratio of chitosan and acetic acid solution is 1g:100mL, and the mass concentration of acetic acid solution is 3%.

[0071] The amount of bacterial bran-based biomass composite filler added in step (5) is 15g per liter of sewage.

[0072] Example 3

[0073] A purification and treatment process for livestock breeding wastewater includes the following preparation steps:

[0074] (1) Pretreatment: The livestock wastewater to be treated is removed by a bar screen to remove large suspended solids and impurities, and then enters a grit chamber for preliminary sedimentation to separate heavy particles such as sand and gravel, thereby reducing the load on subsequent treatment.

[0075] (2) Primary treatment: The pretreated wastewater is introduced into the hydrolysis acidification tank, with a hydraulic retention time of 8 hours, and the facultative microorganisms in the tank are used for preliminary fermentation and decomposition.

[0076] (3) Core microbial treatment: The effluent from the hydrolysis acidification tank is introduced into the aerobic reaction tank, the pH is adjusted to 6.5-7.5, and a compound functional bacterial agent is added at 3% of the sewage mass. Intermittent aeration is adopted, aeration is carried out for 15 minutes, and the reaction is stopped for 45 minutes. The hydraulic retention time is 18 hours.

[0077] (4) Solid-liquid separation: The effluent from the aerobic reaction tank enters the sedimentation tank and is allowed to settle for 2 hours. The mud and water are separated by the bioflocculation of the microorganisms themselves.

[0078] (5) Advanced treatment and disinfection: The supernatant from the sedimentation tank enters a filter filled with biomass composite packing material containing bacterial bran for final denitrification and phosphorus removal purification. The hydraulic retention time is controlled at 2 hours. Finally, it is discharged after ultraviolet disinfection. The effective ultraviolet dose used for ultraviolet disinfection is 40 mJ / cm² (millijoules per square centimeter).

[0079] In step (3), the compound functional microbial agent contains Pseudomonas hazel, Microbacterium keratolyticum and Bacillus licheniformis, with a volume ratio of 1:1:1.

[0080] The strain number of *Pseudomonas hazel* is CGMCC No. 1.3819, the strain number of *Microbacterium keratolyticum* is CGMCC No. 1.6312, and the preservation number of *Bacillus licheniformis* is CGMCC No. 79241. They are deposited at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing.

[0081] The isolation and screening method for Bacillus licheniformis is the same as in Example 1.

[0082] The preparation method of the compound functional microbial agent is the same as in Example 1.

[0083] Step (5) The preparation method of the bacterial bran-based biomass composite filler is as follows:

[0084] (1) Raw material pretreatment: Fresh mushroom substrate taken from enoki mushroom or king oyster mushroom factories is crushed and screened to obtain 40-60 mesh particles. The substrate is repeatedly rinsed with water until the water is clear to remove soluble impurities. It is then dried at 80℃ to constant weight to obtain pretreated mushroom substrate.

[0085] (2) Acid washing and activation: The pretreated mushroom residue is placed in a 5% citric acid solution and shaken at 60℃ and 150 r / min for 2 hours. After the reaction is completed, it is filtered, washed with deionized water until neutral, and dried. This step is used to dissolve and remove some of the inorganic salts and ash in the mushroom residue and expose more pore structures.

[0086] (3) Iron-chitosan loading modification: Prepare a 1.0 mol / L FeCl3 solution and a chitosan acetic acid solution; immerse the acid-washed mushroom bran in the FeCl3 solution at a solid-liquid ratio of 1g:15mL, slowly add the above chitosan solution to the system, the mass ratio of chitosan to mushroom bran is 1:20, and stir slowly at 45℃ for 4 hours; after the reaction is completed, filter, wash the obtained solid with deionized water 2-3 times, vacuum dry at 60℃, and cool to obtain the mushroom bran-based biomass composite filler.

[0087] In step (2), the solid-liquid ratio of citric acid solution and pretreated bacterial bran is 1g:10mL. In step (3), the solid-liquid ratio of chitosan and acetic acid solution is 1g:100mL, and the mass concentration of acetic acid solution is 3%.

[0088] The amount of bacterial bran-based biomass composite filler added in step (5) is 20g per liter of sewage.

[0089] Comparative Example 1

[0090] The only difference is the strain composition in the compound functional microbial agent, and a comparative ratio is set up. All other raw materials and process steps are the same as in Example 1.

[0091] Right now:

[0092] The preparation method of the compound functional microbial agent is as follows: *Pseudomonas hazelnutans*, *Microbacterium keratolyticum*, and *Bacillus licheniformis* are inoculated separately into LB medium and cultured with shaking until the bacterial count reaches OD₂5. 600The volume ratio is approximately 3.0. Then, the mixed bacterial solution is prepared by mixing the bacterial solution with a carrier at a volume-to-mass ratio of 1L:1.5kg. The carrier is composed of attapulgite clay, starch, and calcium chloride at a mass ratio of 100:5:2. The adsorbed material is granulated and vacuum dried until the moisture content is ≤8%, thus obtaining the composite functional microbial agent. The effective viable count in the composite functional microorganisms is not less than 1×10⁻⁶. 9 CFU / g.

[0093] Table 1. Strains in the comparative examples 1-9 (volume ratio) of the compound functional microbial agents

[0094]

[0095] Comparative Example 10

[0096] In this comparative example, except that in step (5) the bacterial bran-based biomass composite filler is replaced with an equal amount of ordinary ceramsite filler, the other raw materials and process steps are the same as in Example 1. That is:

[0097] A purification and treatment process for livestock breeding wastewater includes the following preparation steps:

[0098] (1) Pretreatment: The livestock wastewater to be treated is removed by a bar screen to remove large suspended solids and impurities, and then enters a grit chamber for preliminary sedimentation to separate heavy particles such as sand and gravel, thereby reducing the load on subsequent treatment.

[0099] (2) Primary treatment: The pretreated wastewater is introduced into the hydrolysis acidification tank, with a hydraulic retention time of 6 hours, and the facultative microorganisms in the tank are used for preliminary fermentation and decomposition.

[0100] (3) Core microbial treatment: The effluent from the hydrolysis acidification tank is introduced into the aerobic reaction tank, the pH is adjusted to 6.5-7.5, and a compound functional bacterial agent is added at 1% of the sewage mass. Intermittent aeration is adopted, aeration is carried out for 15 minutes, and the reaction is stopped for 45 minutes. The hydraulic retention time is 12 hours.

[0101] (4) Solid-liquid separation: The effluent from the aerobic reaction tank enters the sedimentation tank and is allowed to settle for 1 hour. The mud and water are separated by the bioflocculation of the microorganisms themselves.

[0102] (5) Advanced treatment and disinfection: The supernatant from the sedimentation tank enters a filter bed filled with ordinary ceramsite for final denitrification and phosphorus removal purification. The hydraulic retention time is controlled at 1 hour. Finally, ozone is used for disinfection before discharge. The ozone dosage is controlled at 10 mg / L.

[0103] The amount of ordinary ceramsite filler added in step (5) is 10g per liter of sewage.

[0104] Performance testing

[0105] Simulated high-salt wastewater was used to determine the degradation effects of *Pseudomonas hazelnutans*, *Microbacterium keratolyticum*, and *Bacillus licheniformis* on COD and ammonia nitrogen under different compositions.

[0106] Simulated high-salt wastewater culture medium: 8g ammonium chloride, 2g anhydrous sodium acetate, 0.05g magnesium sulfate heptahydrate, 0.2g dipotassium hydrogen phosphate, 33g sodium chloride, 0.01g manganese sulfate tetrahydrate, 0.01g ferrous sulfate, 1L water, adjusted pH to 7.2. Sterilize at 121℃ for 30 min before use (ammonia nitrogen content 1027.3 mg / L, COD content 1545 mg / L).

[0107] Preparation of bacterial suspensions: *Pseudomonas hazelnutans*, *Microbacterium keratolyticum*, and *Bacillus licheniformis* were inoculated into LB liquid medium and cultured for 48 h. After centrifugation at 6000 r / min for 10 min, 10 mL of culture medium was taken and the bacterial cells were washed three times with sterile water. Using sterile water as a reference, 4 mL of bacterial suspension was measured at 600 nm. The bacterial suspension with a result of 1.0 was taken to obtain three bacterial suspensions. The three bacterial suspensions were prepared according to the proportions in Table 1 to obtain bacterial suspensions composed of different strains.

[0108] The obtained bacterial suspension was added to 100 mL of simulated wastewater at a dosage of 1%. Three replicates were set up for each experimental group, and the average result was taken. The cultures were incubated at 180 r / min and 30℃. On day 5, the degradation effect of each bacterial strain on the simulated wastewater was measured. Suspended solids had a certain impact on the experimental results; therefore, when measuring COD, the digested sample was centrifuged at 8000 r / min for 5 min, and the supernatant was collected for COD measurement.

[0109] COD degradation rate (%) = (COD concentration of wastewater before treatment - COD concentration of wastewater after treatment) / COD concentration of wastewater before treatment × 100%;

[0110] NH4 + -N degradation rate (%) = (NH4+ in wastewater before treatment) + - N content - NH4 in treated wastewater + - N content) / NH4 in wastewater before treatment + -N content × 100%;

[0111] The test methods refer to the relevant national standards. Among them, the COD determination adopts the potassium dichromate method GB11914-89, and the ammonia nitrogen determination adopts the Nessler's reagent spectrophotometric method GB7479-87.

[0112] Table 2. Test results of simulated degradation effect

[0113]

[0114] As can be seen from the data in Table 2, the purification process of Example 1 of this invention exhibits significant advantages in both COD degradation rate and ammonia nitrogen degradation rate, reaching 78.9% and 90.5% respectively, which are significantly better than other comparative examples. This fully demonstrates that the compound functional microbial agent has the best synergistic effect when mixed with Pseudomonas hazelnutus, Microbacterium keratolyticum, and Bacillus licheniformis in a 1:1:1 volume ratio.

[0115] Flocculation effect test:

[0116] *Pseudomonas hazelnutans*, *Microbacterium keratolyticum*, and *Bacillus licheniformis* were inoculated into 50 mL Erlenmeyer flasks containing 20 mL of flocculation fermentation medium, respectively, and cultured for 3 days in a shaker at 30 °C and 120 rpm. The fermentation broth was centrifuged at 8000 rpm for 15 min, and the supernatant was used to determine its flocculation rate.

[0117] Flocculation fermentation medium: glucose 10.0 g, magnesium sulfate 0.2 g, urea 0.5 g, potassium dihydrogen phosphate 2.0 g, yeast extract 0.5 g, dipotassium hydrogen phosphate 5.0 g, sodium chloride 33.0 g, pH 7.0-7.3, glucose sterilized at 115℃ for 30 min, other components sterilized at 121℃ for 20 min.

[0118] Different bacterial strains were mixed according to the strain ratios in Example 1 and Comparative Examples 1-9, and the flocculation effect of the mixture was measured. The test method was as follows: 200 mL of 4 g / L kaolin suspension, 2 mL of fermentation supernatant, and 300 µL of 10% calcium chloride solution were added to a 250 mL beaker, and the pH was adjusted to 7.3. A smart magnetic stirrer was used with a flocculation stirring program of 700 rpm for 1 min, 80 rpm for 10 min, and then allowed to stand for 5 min. After standing, the absorbance of the supernatant was measured at 550 nm. The control group used deionized water instead of fermentation supernatant, while other conditions remained the same as the experimental group. The flocculation rate was calculated using the formula shown. Each experimental group was repeated three times, and the average value was taken as the final result.

[0119] Flocculation rate (%) = (B - A) / B × 100% Where A is the absorbance of the supernatant of the experimental group and B is the absorbance of the supernatant of the control group.

[0120] Test results are as follows Figure 3 As shown. From Figure 3The test results show that all three active bacterial strains of this invention have a certain flocculation effect, and the composite functional bacterial agent with the three strains mixed in a volume ratio of 1:1:1 exhibits the best flocculation performance, with a flocculation rate of [percentage missing]. This indicates that the three strains have a good synergistic effect at this ratio. Changing the strain composition to comparative ratios 1-9 disrupts the synergistic balance among the three strains, resulting in a certain degree of weakening in both pollutant removal and flocculation capabilities.

[0121] Actual wastewater treatment in aquaculture:

[0122] Wastewater source: Wastewater discharged from a large pig farm in Lanling County, Linyi City, Shandong Province. The wastewater was treated according to the methods of Examples 1-3 and Comparative Examples 1-10 of this invention.

[0123] Detection methods

[0124] Water quality indicators were tested according to national standard methods, as shown in Table 3:

[0125] Table 3 Water quality testing indicators and methods

[0126]

[0127] The test results are shown in Table 4:

[0128] Table 4 Water purification results

[0129]

[0130] Table 5 Water purification effect

[0131]

[0132] From the data in Tables 4-5, we can see that Examples 1-3 of the present invention exhibit significant purification effects in actual aquaculture wastewater treatment, especially in key indicators such as COD, BOD, total phosphorus, total nitrogen, ammonia nitrogen, suspended solids, and heavy metals, all showing superior treatment capabilities compared to the comparative examples. Taking Example 1 as an example, COD decreased from 6900.2 mg / L before treatment to 162.3 mg / L, with a removal rate of over 90%; ammonia nitrogen decreased from 455.1 mg / L to 20.3 mg / L, with a removal rate of 95.5%; and the number of fecal coliforms also decreased from 8.5 × 10⁻⁶. 7 The concentration of CFU / L decreased to 5000 CFU / L, indicating that the process has a highly efficient sterilization effect. Meanwhile, in terms of heavy metal removal, the concentrations of copper, zinc, manganese, cadmium, hexavalent chromium, and arsenic all decreased significantly, fully complying with national emission standards.

[0133] It is worth noting that although Comparative Examples 1-9 also used different combinations of strains for treatment, their purification effect was significantly weaker than that of the Examples because the strain ratios deviated from the optimal synergistic range. This further verifies the superior performance of *Pseudomonas hazelnutans*, *Microbacterium keratolyticum*, and *Bacillus licheniformis* mixed in a 1:1:1 volume ratio.

[0134] In summary, the composite functional microbial agent and its supporting process provided by this invention can not only effectively degrade organic pollutants, but also have good flocculation ability and heavy metal removal effect. It is suitable for actual aquaculture wastewater treatment scenarios with high salinity and high pollution load, and provides reliable technical support for the sustainable development of the livestock breeding industry.

[0135] It should be noted that the above embodiments are merely some preferred embodiments of the present invention, and not all embodiments. Obviously, based on the above embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

Claims

1. A process for purifying livestock breeding wastewater, characterized by, The preparation steps include: (1) Pretreatment: The livestock breeding wastewater to be treated is subjected to removal of large suspended solids and impurities by a grating machine, and then subjected to preliminary sedimentation in a grit chamber to separate heavy particles and reduce subsequent treatment load; (2) Primary treatment: The pretreated wastewater is introduced into a hydrolysis acidification tank, the hydraulic retention time is 6-8 hours, and preliminary fermentation decomposition is performed by using the original facultative microorganisms in the tank; (3) Core microbial treatment: The effluent from the hydrolysis acidification tank is introduced into an aerobic reaction tank, the pH is adjusted to 6.5-7.5, the composite functional bacterial agent is added at 1-3% of the mass of the wastewater, intermittent aeration is adopted, aeration is performed for 15 minutes, and reaction is performed for 45 minutes, the hydraulic retention time is 12-18 hours; (4) Solid-liquid separation: The effluent from the aerobic reaction tank is introduced into a sedimentation tank, and is allowed to stand for 1-2 hours for sedimentation, and mud-water separation is realized by relying on the biological flocculation of the microorganisms themselves; (5) Advanced treatment and disinfection: The supernatant from the sedimentation tank is introduced into a filter tank provided with straw-based biomass composite filler for final denitrification and phosphorus removal purification, the hydraulic retention time is controlled to be 1-2 hours, and finally ultraviolet or ozone disinfection treatment is performed before discharge; The composite functional bacterial agent in step (3) comprises Pseudomonas monteilii, Microbacterium corchorusii and Bacillus licheniformis, and the volume ratio of the three is 1:1:1; The strain number of the Pseudomonas monteilii is CGMCC No. 1.3819, the strain number of the Microbacterium corchorusii is CGMCC No. 1.6312, and the preservation number of the Bacillus licheniformis is CGMCC No. 79241, which is preserved in the General Microbiological Center of China Microbial Culture Collection Management Committee, located at No. 1, Beichen West Road, Haidian District, Beijing.

2. The livestock breeding wastewater purification treatment process according to claim 1, characterized in that, The preparation method of the complex functional microbial agent is as follows: the Pseudomonas fluorescens, the Microbacterium corchori and the Bacillus licheniformis are respectively inoculated into LB culture medium, and are oscillation cultured until the bacterial content is O.D 600 ≈3.0, then mixed to obtain mixed bacterial liquid according to the volume ratio of 1:1:1, the mixed bacterial liquid is mixed and adsorbed with a carrier according to the volume-mass ratio of 1L:1.5kg, the carrier is composed of attapulgite, starch and calcium chloride according to the mass ratio of 100:5:2; the material after adsorption is granulated and vacuum dried until the water content is ≤8%, and the complex functional microbial agent is obtained, the effective viable bacterial count in the complex functional microorganism is not less than 1×10 9 CFU / g.

3. The process for purification of livestock farming wastewater according to claim 1, characterized in that, The preparation method of the straw-based biomass composite filler in step (5) is as follows: (1) Raw material pretreatment: Fresh straw from a golden needle mushroom or a Pleurotus eryngii factory is crushed and sieved, 40-60 mesh particles are taken, and are repeatedly washed with water until the effluent is clear, soluble impurities are removed, and the pretreated straw is dried at 80°C to constant weight to obtain pretreated straw; (2) Acid pickling activation: The pretreated straw is placed in a 5% citric acid solution, and is oscillated at 60°C and 150 r / min for 2 hours; after the reaction is completed, the straw is filtered, washed with deionized water until neutral, and dried; (3) Iron-chitosan loading modification: A 1.0 mol / L FeCl3 solution and a chitosan acetic acid solution are prepared; the acid-pickled straw is immersed in the FeCl3 solution at a solid-liquid ratio of 1g:15mL, and the chitosan solution is slowly added to the system until the mass ratio of chitosan to straw is 1:20, and the system is slowly stirred at 45°C for 4 hours; after the reaction is completed, the solid is filtered, washed with deionized water for 2-3 times, and dried at 60°C under vacuum, and the straw-based biomass composite filler is obtained after cooling.

4. The purification treatment process of livestock breeding wastewater according to claim 3, characterized in that, In step (2), the solid-liquid ratio of the citric acid solution and the pretreated straw is 1g:10mL, and in step (3), the solid-liquid ratio of chitosan and acetic acid solution in the chitosan acetic acid solution is 1g:100mL, and the mass concentration of the acetic acid solution is 3%.

5. The livestock farming wastewater purification treatment process according to claim 1, characterized by, The amount of the fungus-straw-based biomass composite filler added in step (5) is 10-20 g per liter of sewage.

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