Low-temperature-resistant sewage treatment agent as well as preparation method and application thereof

Abstract

The invention relates to a low-temperature-resistant sewage treatment agent as well as a preparation method and application thereof, in particular to a low-temperature-resistant sewage treatment agent which comprises an effective amount of bacillus velezensis fungicide, glycerol, xanthan gum, betaine and humic acid. The treatment agent is prepared from the following components in parts by weight: 20 to 30 parts of bacillus velezensis fungicide with an effective dose, 5 to 8 parts of glycerol, 0.5 to 1 part of xanthan gum, 1 to 2 parts of glycine betaine and 0.3 to 0.5 part of humic acid. The effective viable count in the bacillus velezensis microbial inoculum is greater than or equal to 5 * 10 < 9 > CFU / mL. The strain disclosed by the invention can stably play a role at 4-25 DEG C after being domesticated at a low temperature, still keeps efficient activity at an extremely low temperature (4 DEG C), can synchronously remove pollutants such as COD (Chemical Oxygen Demand), ammonia nitrogen and the like in sewage, and is high in storage stability and suitable for various sewage treatment processes.

Description

Technical Field

[0001] This invention relates to the field of wastewater treatment, specifically to a low-temperature resistant wastewater treatment agent, its preparation method, and its application. The low-temperature wastewater referred to in this invention refers to wastewater with a treatment temperature of 4-25°C, including urban domestic sewage, livestock and poultry breeding wastewater, and lightly polluted industrial wastewater. Background Technology

[0002] With the acceleration of urbanization and increasingly stringent environmental standards in my country, the demand for wastewater treatment continues to grow. In cold northern regions and high-altitude areas, the efficiency of biological wastewater treatment is significantly reduced under low-temperature conditions (≤15℃), becoming a technical bottleneck restricting stable compliance with discharge standards. Existing biological nitrogen and phosphorus removal technologies mainly rely on mesophilic microorganisms, whose enzyme activity decreases and metabolic rate slows down under low-temperature environments, resulting in a significant reduction in the removal efficiency of ammonia nitrogen, total phosphorus, and COD, failing to meet the requirements for winter operation.

[0003] To address this challenge, developing low-temperature resistant microbial agents has become an important direction. However, current related products still have significant shortcomings: single strains have limited functionality, making it difficult to simultaneously and efficiently achieve nitrogen removal, phosphorus removal, and COD degradation; antagonistic effects easily occur when using multi-genera strains in combination, and most agents have a narrow low-temperature adaptability range (usually not lower than 6℃), weak adaptability to fluctuations in wastewater pH and salinity, and poor stability in practical applications. In addition, the complex production processes and high costs of some agents also limit their large-scale application. Therefore, there is an urgent need to develop a compound microbial agent that exhibits high activity at low temperatures, synergistic functions, strong adaptability, and reasonable cost. Summary of the Invention

[0004] The present invention aims to solve the complex technical problems existing in the current low temperature (especially 4-15℃) wastewater treatment technology: how to obtain a microbial agent that can efficiently, synergistically and stably remove multiple pollutants such as ammonia nitrogen, total phosphorus and COD at extreme low temperatures, and has no antagonism between strains, wide environmental adaptability and easy industrial production.

[0005] The first inventive point of this invention is a low-temperature resistant wastewater treatment agent, comprising an effective amount of Bacillus belye ( Bacillus velezensis Ingredients: Bacterial agent, glycerin, xanthan gum, betaine, humic acid.

[0006] Further, the treatment agent, by weight, comprises 20-30 parts of an effective amount of Bacillus belyssus (B. belyssus). Bacillus velezensis Ingredients: 5-8 parts glycerin, 0.5-1 part xanthan gum, 1-2 parts betaine, 0.3-0.5 parts humic acid.

[0007] Furthermore, the effective viable count of the *Bacillus vesiculosus* agent is ≥5 × 10⁻⁶. 9 CFU / mL.

[0008] Furthermore, the Bacillus berberis agent is produced by strain Bacillus berberis LTS-AF1 ( Bacillus from Velez LTS-AF1) fermentation broth, Bacillus belye LTS-AF2 ( Bacillus velezensis LTS-AF2) fermentation broth, Bacillus belye LTS-AF3 ( Bacillus velezensis LTS-AF3) fermentation broth, Bacillus vesiculosus LTS-AF4 ( Bacillus velezensis It is a mixture of LTS-AF4 fermentation broth; The Bacillus berberis LTS-AF1 ( Bacillus velezensis LTS-AF1), its accession number is CCTCC NO: M 2025378; The Bacillus berberis LTS-AF2 ( Bacillus velezensis LTS-AF2), its accession number is CCTCC NO: M 2025379; The Bacillus berberis LTS-AF3 ( Bacillus velezensis LTS-AF3), its accession number is CCTCC NO: M 2025380; The Bacillus berberis LTS-AF4 ( Bacillus velezensis LTS-AF4), its accession number is CCTCC NO: M 2025381; All of the above-mentioned Bacillus belyes are deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, on March 5, 2025.

[0009] Furthermore, the viable cell ratio of the Bacillus belyssus LTS-AF1 fermentation broth, LTS-AF2 fermentation broth, LTS-AF3 fermentation broth, and LTS-AF4 fermentation broth is (1~3):(2~4):(2~4):(1~3).

[0010] Further, the treatment agent is in the form of a solid powder or a liquid formulation; the solid powder is composed of a liquid formulation and a carrier, wherein the mass ratio of the liquid formulation to the carrier is 1:(2-4); the carrier includes diatomaceous earth and / or corn cob powder; the number of viable bacteria in the solid powder is ≥5×10⁻⁶. 9 CFU / g, effective viable bacteria count in liquid formulation ≥1×10 10 CFU / mL.

[0011] The second inventive point of this invention is to provide a method for preparing the aforementioned low-temperature resistant wastewater treatment agent, comprising the following steps: S1: Strain screening: The Bacillus beleibacillus LTS-AF1, LTS-AF2, LTS-AF3 and LTS-AF4 were isolated and screened from activated sludge in a low-temperature environment; S2: Low temperature acclimatization: The strains obtained in step S1 were subjected to low temperature gradient acclimatization, with the acclimatization temperature gradient from 15℃→10℃→6℃→4℃, and each temperature gradient was cultured for 3 days. S3: Expansion culture: Each strain after domestication in step S2 is subjected to expansion culture to obtain a high-concentration fermentation broth; S4: Compound formulation: The fermentation broths of each strain obtained in step S3 are mixed according to the ratio of live bacteria, and glycerol, xanthan gum, betaine and humic acid are added according to the mass ratio. After stirring evenly, the mixture is concentrated under low temperature vacuum to obtain the liquid preparation of the low temperature resistant sewage treatment agent.

[0012] In step S2, the culture medium used for low-temperature acclimatization is an acclimatization culture medium that simulates the components of low-temperature wastewater; the culture medium is based on ammonia nitrogen 100 mg / L, nitrate nitrogen 80 mg / L, nitrite nitrogen 20 mg / L, total phosphorus 5 mg / L, and COD 300 mg / L.

[0013] In step S3, the culture medium used for the expanded culture contains 8-12 g / L corn flour, 6-10 g / L soybean meal, 0.3-0.7 g / L KH2PO4, and 0.1-0.3 g / L MgSO4·7H2O. The culture conditions are: temperature 28-37℃, aeration rate 1.0-1.2 vvm, stirring speed 160-200 rpm, and culture time 24-48 hours.

[0014] Furthermore, it also includes step S5: continuing to add a carrier in step S4 to obtain the low-temperature wastewater treatment agent solid powder.

[0015] The third inventive point of this invention is the application of the aforementioned low-temperature resistant wastewater treatment agent in wastewater treatment.

[0016] Furthermore, it includes urban domestic sewage, livestock and poultry breeding wastewater, and lightly polluted industrial wastewater; the low temperature is 4℃~25℃; the initial concentration of ammonia nitrogen in the low-temperature wastewater is 50~150 mg / L, nitrate nitrogen is 30~100 mg / L, nitrite nitrogen is 10~30 mg / L, total phosphorus is 3~10 mg / L, and COD is 200~500 mg / L; the pH value of the low-temperature wastewater is 5.5~9.5.

[0017] Furthermore, the application includes the simultaneous removal of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus and COD from wastewater.

[0018] Furthermore, when the treatment agent is added in liquid form, the dosage is 0.1% to 0.3% of the wastewater volume; when added in solid form, the dosage is 1 to 3 g / m³ of wastewater.

[0019] Compared with the prior art, the advantages of this invention are: The strains of this invention, after gradient low-temperature acclimatization, can function stably at temperatures ranging from 4 to 25°C, maintaining high activity even at extreme low temperatures (4°C), overcoming the technical bottleneck of existing bacterial agents where efficiency drops sharply below 6°C. This invention avoids the antagonistic effects of multi-generic strain combinations through the specific compounding of four homologous Bacillus beryllus strains, and innovatively combines them with a composite excipient system of "glycerol + xanthan gum + betaine + humic acid." Through multiple synergistic mechanisms, it not only extends the storage period at 4°C to 12 months (with viable cell attenuation ≤20%), but also improves the bacterial agent's tolerance to salinity and heavy metal stress. Furthermore, this invention utilizes inexpensive industrial culture media for expansion, resulting in a simple preparation process, low dosage, and easy large-scale application in existing wastewater treatment processes, reducing overall operating costs. Detailed Implementation

[0020] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0022] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0023] Example 1: Composition, formulation, and formulation design of a low-temperature resistant wastewater treatment agent. This embodiment provides a low-temperature resistant wastewater treatment agent, comprising an effective amount of Bacillus berberis (B. berberis). Bacillus from Velez Ingredients: Bacterial agent, glycerin, xanthan gum, betaine, humic acid.

[0024] As a further preferred embodiment, the treatment agent comprises 20-30 parts by weight of an effective amount of Bacillus belyssus (B. belyssus). Bacillus velezensis Ingredients: 5-8 parts glycerin, 0.5-1 part xanthan gum, 1-2 parts betaine, 0.3-0.5 parts humic acid.

[0025] Preferably, the number of viable bacteria in the Bacillus berberis agent is ≥5×10⁻⁶. 9 CFU / mL.

[0026] In the low-temperature resistant wastewater treatment agent provided in this embodiment, Bacillus vesiculosus, as an active substance, provides pollutant degradation function. It works synergistically with glycerol, xanthan gum, betaine, and humic acid to play the roles of low-temperature protection, physical dispersion and stabilization, nutrient supplementation and pollutant adsorption, and together ensure the survival rate and functional activity of the agent under low-temperature stress.

[0027] Adding glycerin as a cryoprotectant and xanthan gum as a dispersant improves the low-temperature storage stability of the bacterial agent (≤15% decrease in viable cell count after 6 months of storage at 4℃). Betaine (as an osmotic pressure protectant) and glycerol (as a cryoprotectant) work synergistically to protect the strain through a dual mechanism of regulating osmotic pressure and stabilizing enzyme structure, preventing cell membrane rupture at low temperatures. At the same time, the functional groups (carboxyl, hydroxyl, etc.) on humic acid can adsorb some of the recalcitrant COD and heavy metals (such as trace amounts of lead and cadmium), reducing the inhibitory effect of pollutants on the strain. In addition, the small molecule organic acids produced by the degradation of humic acid can provide a fast carbon source for Bacillus belyssus, making up for the lack of carbon source at low temperatures.

[0028] As a further preferred embodiment, the Bacillus berberis inoculant is derived from the strain Bacillus berberis LTS-AF1 ( Bacillus velezensis LTS-AF1) fermentation broth, Bacillus belye LTS-AF2 ( Bacillus from Velez LTS-AF2) fermentation broth, Bacillus belye LTS-AF3 ( Bacillus velezensis LTS-AF3) fermentation broth, Bacillus vesiculosus LTS-AF4 ( Bacillus velezensis It is a mixture of LTS-AF4 fermentation broth; The Bacillus berberis LTS-AF1 ( Bacillus velezensis LTS-AF1), its accession number is CCTCC NO: M 2025378; The Bacillus berberis LTS-AF2 ( Bacillus velezensis LTS-AF2), its accession number is CCTCC NO: M 2025379; The Bacillus berberis LTS-AF3 ( Bacillus velezensis LTS-AF3), its accession number is CCTCC NO: M 2025380; The Bacillus berberis LTS-AF4 ( Bacillus velezensis LTS-AF4), its accession number is CCTCC NO: M 2025381; All of the above-mentioned Bacillus belyes are deposited at the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, on March 5, 2025. The four strains of Bacillus belye in this invention have a division of labor and synergy: LTS-AF1 and LTS-AF2 are in charge of ammonia nitrogen transformation and secrete low-temperature adapted ammonia monooxygenase (AMO); LTS-AF3 is in charge of nitrate and nitrite nitrogen reduction and produces denitrifying enzyme system; LTS-AF4 is in charge of phosphorus absorption and COD degradation and secretes phosphatases and extracellular polymers; the strains form a synergistic system of "denitrification-phosphorus removal-COD degradation-low-temperature adaptation".

[0029] Preferably, the viable cell count ratio of the Bacillus belyssus LTS-AF1 fermentation broth, LTS-AF2 fermentation broth, LTS-AF3 fermentation broth, and LTS-AF4 fermentation broth is (1~3):(2~4):(2~4):(1~3).

[0030] Preferably, the dosage form of the treatment agent is a solid powder or a liquid preparation; the solid powder is composed of a liquid preparation and a carrier, wherein the mass ratio of the liquid preparation to the carrier is 1:(2-4); the carrier includes diatomaceous earth and / or corn cob powder.

[0031] Preferably, the number of viable bacteria in the liquid bacterial agent is ≥1×10⁻⁶. 10 CFU / mL, effective viable bacteria count in solid powder ≥5×10 9 CFU / g.

[0032] Liquid formulations are suitable for immediate addition; solid powders use porous carriers (diatomaceous earth, corn cob powder) to adsorb bacteria and excipients, making them easy to store and transport, and providing an initial attachment point after addition to water, allowing for the slow release of bacteria.

[0033] Example 2 Preparation of Low-Temperature Resistant Wastewater Treatment Agent (Strain Screening - Acclimation - Propagation - Compound Formulation) This embodiment provides a method for preparing the low-temperature resistant wastewater treatment agent described in Embodiment 1, comprising the following steps: S1: Strain screening: The Bacillus beleibacillus LTS-AF1, LTS-AF2, LTS-AF3 and LTS-AF4 were isolated and screened from activated sludge in a low-temperature environment; S2: Low temperature acclimatization: The strains obtained in step S1 were subjected to low temperature gradient acclimatization, with the acclimatization temperature gradient from 15℃→10℃→6℃→4℃, and each temperature gradient was cultured for 3 days. S3: Expansion culture: Each strain after domestication in step S2 is subjected to expansion culture to obtain a high-concentration fermentation broth; S4: Compound formulation: The fermentation broths of each strain obtained in step S3 are mixed according to the ratio of live bacteria, and glycerol, xanthan gum, betaine and humic acid are added according to the mass ratio. After stirring evenly, the mixture is concentrated under low temperature vacuum to obtain the liquid preparation of the low temperature resistant sewage treatment agent.

[0034] As a further preferred embodiment, step S5 is also included: continuing to add a carrier in step S4 to obtain the low-temperature wastewater treatment agent solid powder.

[0035] As a further preferred embodiment, in step S2, the culture medium used for low-temperature acclimatization is an acclimatization culture medium that simulates the components of low-temperature wastewater; the culture medium is based on ammonia nitrogen 100 mg / L, nitrate nitrogen 80 mg / L, nitrite nitrogen 20 mg / L, total phosphorus 5 mg / L, and COD 300 mg / L.

[0036] As a further preferred embodiment, in step S3, the culture medium used for the expanded culture includes 8-12 g / L corn flour, 6-10 g / L soybean meal, 0.3-0.7 g / L KH2PO4, and 0.1-0.3 g / L MgSO4·7H2O. The culture conditions are: temperature 28-37℃, aeration rate 1.0-1.2 vvm, stirring speed 160-200 rpm, and culture time 24-48 hours.

[0037] Specifically, Source of strains: Four strains of Bacillus belye were isolated and screened from activated sludge of a wastewater treatment plant in northern my country during winter, and were named as follows: Bacillus velezensis LTS-AF1, LTS-AF2, LTS-AF3, LTS-AF4.

[0038] S1: Filtering method: S1-1: Strain activation: Culture medium: Beef extract peptone basal medium (beef extract 5g / L, peptone 10g / L, NaCl 5g / L, pH 7.0~7.5); Conditions: 30℃, 150rpm shaking culture for 24h, OD of activated strain 600 The value reaches 1.2~1.5; S1-2: Enrichment culture: Enrichment culture was carried out for 7 days at 4°C and 120 rpm with a low-temperature wastewater simulated medium; S2: Low temperature acclimatization: The activated strains were inoculated into a low temperature acclimatization medium (simulating the composition of low temperature wastewater, with a temperature gradient from 15℃→10℃→6℃→4℃, and each gradient cultured for 3 days), with an acclimatization period of 12 days, to enhance the low temperature adaptability of the strains. S2-1: Isolation and purification: Using the gradient dilution plating method, single colonies were picked from the low-temperature screening medium (temperature 4℃) and purified by streak plating three times to obtain pure strains; S2-2: Functional screening: The denitrification (ammonia nitrogen, nitrate nitrogen, nitrite nitrogen), phosphorus removal and COD degradation efficiency of each strain at 4℃, 10℃ and 15℃ were determined to screen out strains with functional advantages. S2-3: Strain identification: Identification was performed by 16S rRNA gene sequencing. After sequence alignment, the 16S rRNA gene sequences of LTS-AF1, LTS-AF2, LTS-AF3, and LTS-AF4 showed ≥99.8% homology with the Bacillus belysii model strain. The strain classification was confirmed by combining physiological and biochemical characteristics.

[0039] S3: Expansion culture: Industrial-grade fermenters were used, with corn flour 10g / L, soybean meal powder 8g / L, KH2PO4 0.5g / L, and MgSO4. Using 0.2 g / L 7H2O as the expansion medium, the culture was carried out at 30℃, with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm for 48 h to obtain a high concentration of Bacillus belyssus fermentation broth. S4: Compound formulation: Mix the fermentation broth of each strain according to the above ratio, and add glycerol, xanthan gum, betaine and humic acid according to the mass ratio. After stirring evenly, concentrate under low temperature vacuum (below 40℃) to prepare a low temperature resistant sewage treatment agent liquid preparation. S5: Continue to add carrier (diatomaceous earth, corn cob powder), the mass ratio of the liquid preparation to the carrier is 1:(2-4), to make a low-temperature resistant sewage treatment agent solid powder.

[0040] The domesticated strains can synthesize low-temperature induced proteins, reduce the cell membrane phase transition temperature, and increase the specific activity of enzymes at low temperatures, ensuring that they can still maintain efficient metabolism at 4°C. The pollutant transformation pathway is as follows: ammonia nitrogen undergoes nitrification (first converting to nitrite nitrogen, then to nitrate nitrogen), and then nitrate nitrogen and nitrite nitrogen undergo denitrification to be converted into N2 and released; phosphorus is absorbed by the strain and converted into intracellular polyphosphate for storage; COD is metabolized by the strain as a carbon source and energy source and decomposed into CO2 and H2O.

[0041] Example 3: Application scenarios, conditions, and core performance advantages of low-temperature resistant wastewater treatment agents This embodiment provides an application of the low-temperature resistant wastewater treatment agent described in Embodiment 1 in wastewater treatment.

[0042] As a further preferred embodiment, the wastewater is low-temperature wastewater, and the low temperature is 4℃~25℃.

[0043] The wastewater is urban domestic sewage, livestock and poultry breeding wastewater, and lightly polluted industrial wastewater; the pH value of the low-temperature wastewater is 5.5~9.5.

[0044] As a further preferred embodiment, the application includes the simultaneous removal of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus and COD from wastewater.

[0045] As a further preferred embodiment, when the treatment agent is added in liquid form, the dosage is 0.1% to 0.3% of the wastewater volume; when added in solid form, the dosage is 1 to 3 g / m³ of wastewater.

[0046] Treatment conditions: Wastewater pH 5.5~9.5, salinity (NaCl) 0.1~35g / L, dissolved oxygen concentration ≥2mg / L, bacterial agent retention time ≥1d (at temperatures above 10℃, 1d retention time is sufficient to achieve ideal results; at 4℃, 1.5~2d retention time is recommended). Process compatibility: Can be directly added to existing wastewater treatment plant A 2 For aeration tanks using processes such as O and MBR, no modification to existing equipment is required. The dosing method can be continuous or intermittent (replenished every 24 hours, with the replenishment amount being 50% of the initial dosing amount).

[0047] This invention can achieve the following: Low-temperature treatment efficiency: At 4℃, after a 1.5-day retention period, the removal rates of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus, and COD are ≥75%, ≥68%, ≥70%, ≥65%, and ≥80%, respectively. At 10℃, after a 1-day retention period, the removal rates of all indicators are ≥85%, which is superior to existing similar microbial agents (the average removal rate is increased by 15%~20% at low temperatures).

[0048] Wide adaptability range: Within the pH range of 5.5~9.5 and salinity range of 0.1~35g / L, the treatment efficiency fluctuates by ≤10%, making it suitable for different regions and types of wastewater in northern China.

[0049] Storage stability: After 6 months of storage at 4℃, the number of viable bacteria decreased by ≤15%; after 3 months of storage at room temperature (25℃), the number of viable bacteria decreased by ≤20%, which is better than the storage performance of existing microbial agents.

[0050] Environmental safety: Bacillus belesii is a GRAS (Generally Recognized As Safe) strain, which is non-pathogenic, its metabolites do not cause secondary pollution, and the addition of compound bacterial agents will not cause sludge bulking.

[0051] Industrialization and application advantages: The raw materials for the basic culture medium are inexpensive and readily available, and the expansion process does not require special equipment. The production cost is reduced by 30% to 40% compared with existing bacterial agents. The dosage is only 1 / 5 to 1 / 3 of some existing bacterial agents, and the retention time is shortened to 1 day, which greatly reduces the application cost of large-scale sewage treatment plants and is suitable for large-scale promotion.

[0052] Example 4 Bacillus belye ( Bacillus velezensisPreparation and ratio optimization verification of compound formulations Preparation method: This implementation of Bacillus belysinus ( Bacillus velezensis The compound microbial agent is made by compounding Bacillus beleibacillus LTS-AF1, LTS-AF2, LTS-AF3, and LTS-AF4 in a certain proportion after primary seed culture, secondary seed culture, fermentation culture, and fermentation culture. The primary and secondary seed cultures of *Bacillus belyssioides* LTS-AF1, LTS-AF2, LTS-AF3, and LTS-AF4 were cultured using beef extract peptone basal medium (5 g / L beef extract, 10 g / L peptone, 5 g / L NaCl, pH 7.0–7.5); culture conditions: 30℃, 150 rpm shaking culture for 24 h; fermentation culture: 10 g / L corn flour, 8 g / L soybean meal, 0.5 g / L KH2PO4, MgSO4 0.2 g / L 7H2O was used as the expansion medium. The culture was carried out at 30℃, with an aeration rate of 1.0 vvm and a stirring speed of 200 rpm for 48 h to obtain a high-concentration fermentation broth. The fermentation broths of the above strains were mixed and compounded in a certain proportion to obtain Bacillus belyssus (B. belyssus). Bacillus from Velez Compound microbial agent.

[0053] Experimental Group 1: Bacillus belysin inoculum: The ratio of viable bacteria in the fermentation broth of strains LTS-AF1, LTS-AF2, LTS-AF3, and LTS-AF4 was 2:3:3:2.

[0054] Experimental Group 2: Bacillus belysin inoculum: The ratio of viable bacteria in the fermentation broth of strains LTS-AF1, LTS-AF2, LTS-AF3, and LTS-AF4 was 1:1:1:1.

[0055] Testing process: ① Prepare the culture medium substrate according to the low temperature acclimatization substrate formula, containing 100 mg / L ammonia nitrogen, 80 mg / L nitrate nitrogen, 20 mg / L nitrite nitrogen, 5 mg / L total phosphorus, and 300 mg / L COD. Adjust the pH to 7.0, control the salinity (NaCl) to 0.1 g / L, and the dissolved oxygen ≥2 mg / L; ② The prepared Bacillus belyssus compound inoculants for experimental group 1 (live bacteria ratio 2:3:3:2) and experimental group 2 (live bacteria ratio 1:1:1:1) were adjusted to have an effective live bacteria count of ≥5×10⁹ CFU / mL and set aside for later use; ③ Add the corresponding compound bacterial agent to the simulated low-temperature wastewater of each experimental group, with the addition amount being 0.1% of the wastewater volume; place the reaction system in a 4℃ constant temperature incubation device, maintain aeration (dissolved oxygen ≥2mg / L), and continuously incubate for 1.5 days, without adding any other nutrients during the period, and control the environmental parameters of the reaction system to be stable.

[0056] ④ After culturing for 1.5 days, take the supernatant of the reaction solution of each experimental group and determine the concentration of ammonia nitrogen, total phosphorus and nitrite nitrogen according to the national standard detection method. Calculate the removal rate of ammonia nitrogen and total phosphorus and count the accumulation of nitrite nitrogen. At the same time, calculate the unit ton water treatment cost of the two groups of bacterial agents.

[0057] Table 1. Wastewater removal efficiency of Bacillus belyssin inoculant (4℃, 1.5d retention time)

[0058] Conclusion: Experimental group 1, which used a specific ratio of Bacillus belye inoculant, showed significantly higher ammonia nitrogen removal rate and total phosphorus removal rate than experimental group 2 (equal ratio of inoculant) at 4℃, and also had lower nitrite nitrogen accumulation. This indicates that the specific ratio of viable bacteria in the compound system can achieve synergistic effect of bacterial strains and improve the effect of low-temperature wastewater treatment.

[0059] Example 5: Comparative Study on the Preparation and Treatment Effects of Different Types of Low-Temperature Resistant Wastewater Treatment Agents (Liquid Formulations / Solid Powders) Experimental Example 1 This experimental example provides a low-temperature resistant wastewater treatment agent (liquid formulation), including Bacillus belye prepared in experimental group 1 of Example 4. Bacillus velezensis 25 g of bacterial agent (effective viable count is 1×10⁻⁶) 10 The ingredients include: CFU / mL, glycerol 6g, xanthan gum 0.8g, betaine 1.5g, and humic acid 0.4g.

[0060] Preparation method: The Bacillus berberis inoculant from Experimental Group 1 in Example 4 was mixed with glycerol, xanthan gum, betaine, and humic acid at a specific mass ratio. After stirring evenly, the mixture was concentrated under low-temperature vacuum (40°C) to 1 / 3 of its original volume to prepare a low-temperature resistant wastewater treatment liquid formulation (effective viable count ≥1×10⁻⁶). 10 (CFU / mL).

[0061] Experimental Example 2 This experimental example provides a low-temperature resistant wastewater treatment agent (solid powder), including Bacillus belye prepared in experimental group 1 of Example 4. Bacillus velezensis 25 g of bacterial agent (effective viable count is 1×10⁻⁶) 10 The ingredients include: CFU / mL, 6g glycerol, 0.8g xanthan gum, 1.5g betaine, 0.4g humic acid, and 101g corn cob powder as carrier.

[0062] Preparation method: The Bacillus berberis agent from Experimental Group 1 in Example 4 was mixed with glycerol, xanthan gum, betaine, and humic acid at a mass ratio. After stirring evenly, the mixture was concentrated under low-temperature vacuum (40°C) to 1 / 3 of its original volume to prepare a liquid preparation intermediate. Then, 101g of corn cob powder (treatment agent to carrier mass ratio 1:3) was added, mixed evenly, dried, and pulverized to obtain a low-temperature resistant wastewater treatment agent solid powder (effective viable count ≥5×10⁻⁶). 9 CFU / g).

[0063] Comparative Example 1 Wastewater treatment agent: Klebsiella pneumoniae compound bacterial agent, specifically referencing Example 2 of the State Intellectual Property Office patent CN119685210 A (this patent's core low-temperature performance verification example specifically discloses the denitrification effect at 6℃, which is highly compatible with the core scenario of "low-temperature wastewater treatment" in this invention, and fully discloses the strain combination, key preparation process, and performance data of the compound bacterial agent, objectively reflecting the inherent low-temperature denitrification performance of this type of bacterial agent). To ensure the fairness of the comparative test, based on Example 2 of the patent and the content disclosed in the specification, the specific formula and preparation method are determined as follows, adapted to the test conditions of this invention: Core ingredients and proportions: The functional strain is Klebsiella pneumoniae ( Klebsiella sp.) TYF-CJJ-P07 (accession number CGMCC NO.29833) and Klebsiella pneumoniae ( Klebsiella sp.) TYF-CJJ-A11 (preservation number CGMCCNO.29831), the fermentation broths of the two strains were mixed at a viable cell ratio of 1:1, and the total effective viable cell count was ≥1×10 9 CFU / mL; no additional excipients were added (the original patent did not disclose the composite excipient system, and only used the basic culture medium to prepare the bacterial solution), and the solvent was sterile water; Preparation method: The process was carried out according to the process disclosed in Examples 1-2 of patent CN 119685210 A, namely, the strain was activated in beef extract peptone medium (30℃, 120rpm shaking culture for 24h, medium composition: beef extract 5g / L, peptone 10g / L, NaCl 5g / L, pH 7.0±0.2), and expanded in denitrified medium (composition: C6H5Na3O7). 2H2O 5.719g / L, (NH4)2SO40.472g / L, KNO30.722g / L, NaNO20.246g / L, K2HPO40.200g / L, MgSO4 7H2O 0.050g / L, MnSO4 After culturing with 0.010 g / L H₂O, 0.010 g / L FeSO₄, 0.120 g / L NaCl, pH 7.0±0.2, at 30℃ and 120 rpm for 48 h, the fermentation broths of the two strains were mixed in a 1:1 ratio to prepare a liquid preparation (consistent with the dosage form of Experiment Example 1 of this invention).

[0064] The basis for selecting this embodiment and formulation is as follows: Example 2 of patent CN 119685210 A is its only performance verification embodiment under low temperature conditions of 6℃, which can directly reflect the low temperature denitrification limit of the bacterial agent; the formulation and process strictly follow the original patent disclosure, without adding any additional modified components, which can truly reflect its original performance shortcomings (such as no low temperature protectant, single function focusing only on denitrification); the dosage form is uniformly a liquid preparation, and the dosage is converted to 0.5% of the sewage volume according to the "5% inoculum amount" recommended by the original patent, ensuring that the comparative test only has a single variable (bacterial agent type), and the data comparison is effective and convincing.

[0065] Comparative Example 2 Wastewater treatment agent: Low-temperature resistant compound denitrifying bacterial agent (containing Pseudomonas strains), specifically referencing Example 2 of the State Intellectual Property Office patent CN 114854641 A (this patent's core low-temperature performance verification example specifically discloses the denitrification and COD degradation effects at 6℃, which is highly compatible with the core scenario of "low-temperature wastewater treatment" in this invention, and fully discloses the strain combination, key preparation process, and low-temperature performance data of the compound bacterial agent, objectively reflecting the inherent performance of this type of Pseudomonas-containing compound bacterial agent). To ensure the fairness of the comparative test, based on Example 2 of this patent and the content disclosed in the specification, the specific formula and preparation method are determined as follows, adapted to the test conditions of this invention: Core ingredients and proportions: The functional strain is *Laenia aquaticis* (…). Rahnella aquatica ) strain WS33 (CGMCC No. 22997), Pseudomonas ( Pseudomonas strain DT04 (CGMCC No. 22998), Pseudomonas (sp.) Pseudomonas strain DT06 (CGMCC No. 22999), fermentation broth of the three strains were mixed at a cell number ratio of 2:2:1, with a total effective viable cell count ≥1×10⁻⁶. 8 CFU / mL; no additional excipients were added (the original patent did not disclose the composite excipient system, and only used the basic culture medium to prepare the bacterial solution), and the solvent was sterile water; Preparation method: The process was carried out according to the procedures disclosed in Examples 1-2 of patent CN 114854641 A, namely, the strain was activated in an enrichment medium (cultured at 30℃ and 180 rpm for 36 h with shaking; medium composition: NaCl 10.0 g / L, peptone 10.0 g / L, yeast extract 5.0 g / L, distilled water 1000 mL, pH 7.2~8.0), and expanded in a denitrified medium (composition: CH3COONa 8.2 g / L, NH4Cl 0.50 g / L, KNO3 0.58 g / L, NaNO2 0.24 g / L, KH2PO4 0.08 g / L, CaCl2). 2H₂O 0.1g / L, MgSO₄ 7H2O 1.0g / L, FeSO4 After culturing with 0.006 g / L H2O, 1 mL of trace element solution, 1 L of distilled water, pH 7.0~7.5, at 10℃ and 180 rpm for 48 h, the fermentation broth of the three strains was mixed in a ratio of 2:2:1 to prepare a liquid preparation (consistent with the dosage form of Experimental Example 1 and Comparative Example 1 of this invention).

[0066] The basis for selecting this embodiment and formulation is as follows: Embodiment 2 of patent CN 114854641 A is its core performance verification embodiment under low temperature conditions of 6℃, which can directly reflect the low temperature treatment limit capability of the Pseudomonas compound bacterial agent; the formulation and process strictly follow the content disclosed in the original patent, without adding additional low temperature protectants or functional modification components, which can truly reflect its original performance shortcomings (such as low denitrification and COD degradation efficiency below 6℃, no phosphorus removal function, and limited salinity adaptability); the dosage form is uniformly a liquid preparation, and the dosage is set according to the "1% inoculum amount" recommended by the original patent (the dosage in Experiment Example 1 of this invention is 0.1%), ensuring that the comparative test only has a single variable (the type of bacterial agent), and the data comparison is effective and convincing.

[0067] Comparative Example 3 Wastewater treatment agent: Compared with Experimental Example 1, it contains only glycerol and xanthan gum. The preparation method is the same as that of Experimental Example 1.

[0068] Comparative Example 4 Wastewater treatment agent: Compared with Test Example 1, it contains only glycerol, xanthan gum and betaine (without humic acid).

[0069] Comparative Example 5 Wastewater treatment agent: Compared with Test Example 1, it contains glycerol, xanthan gum and humic acid (but no betaine).

[0070] Test conditions: Simulated low-temperature wastewater (the culture medium used for the low-temperature acclimatization was an acclimatization culture medium simulating the components of low-temperature wastewater; the culture medium was based on ammonia nitrogen 100 mg / L, nitrate nitrogen 80 mg / L, nitrite nitrogen 20 mg / L, total phosphorus 5 mg / L, and COD 300 mg / L, as in Example 4), 10℃, held for 1 day, and salinity adaptability was tested simultaneously (0.1 g / L, 25 g / L, 35 g / L). The specific results are shown in Table 2.

[0071] Table 2. Wastewater removal efficiency of various wastewater treatment agents (10℃, 1 day retention)

[0072] in conclusion: 1. The removal rates of ammonia nitrogen, total phosphorus, and COD in Experimental Example 1 (liquid) and Experimental Example 2 (solid) of this invention are significantly higher than those in Comparative Examples 1-2 of the prior art, and the dosage is only 1 / 5 of that in Comparative Example 1 and 1 / 10 of that in Comparative Example 2, demonstrating the advantages of high efficiency and low dosage. 2. The treatment effects of Comparative Examples 3-5 were all lower than those of Experimental Example 1, proving that the synergistic effect of the "glycerol + xanthan gum + betaine + humic acid" composite excipient system is crucial and indispensable; 3. The bacterial agent of this invention is adaptable to a salinity range of 0.1~35g / L, which is superior to existing bacterial agents (0.1~30g / L), and has stronger adaptability to high-salt and low-temperature wastewater.

[0073] Example 6 This embodiment verifies the pollutant removal performance of the liquid formulation in Experiment Example 1 at different temperatures.

[0074] Testing process: ①Preparation of simulated low-temperature wastewater: The culture medium used for the low-temperature acclimatization is an acclimatization culture medium that simulates the components of low-temperature wastewater; the culture medium is based on ammonia nitrogen 100mg / L, nitrate nitrogen 80mg / L, nitrite nitrogen 20mg / L, total phosphorus 5mg / L, and COD 300mg / L, the pH of the wastewater is adjusted to 7.0, the salinity (NaCl) is controlled at 0.1g / L, and aeration is carried out until the dissolved oxygen concentration is ≥2mg / L, for later use; ② Preparation of microbial agents: Take the low-temperature resistant wastewater treatment liquid formulation prepared in Example 1 (effective viable bacteria count ≥ 1 × 10⁻⁶). 10 (CFU / mL), no dilution required, ready for immediate use; ③ Experimental grouping: Five temperature treatment groups were set up, namely 4℃ (1d), 4℃ (1.5d), 10℃ (1d), 15℃ (1d), and 25℃ (1d). ④ Add the liquid preparation of Experiment Example 1 to the simulated low-temperature wastewater of each temperature group, with an addition amount of 0.1% of the wastewater volume; place the reaction system of each experimental group in a constant temperature incubation device at the corresponding temperature, maintain aeration throughout the process (dissolved oxygen concentration stable ≥2mg / L), and continuously incubate for the preset time (4℃ group incubated for 1 day and 1.5 days respectively, 10℃, 15℃ and 25℃ groups incubated for 1 day each), without adding any additional nutrients during the incubation period, and control the environmental parameters of the reaction system to be stable; after reaching the preset incubation time, stop aeration, let stand for 30 minutes, and take the supernatant of each reaction system for the detection of pollutant indicators such as ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus, and COD.

[0075] The specific results are shown in Table 3.

[0076] Table 3. Wastewater treatment effect of wastewater treatment agent in Experiment 1 at different temperatures.

[0077] Conclusion: The wastewater treatment agent of this invention can function stably in a wide temperature range of 4~25℃, and the treatment efficiency increases with increasing temperature; after 1.5 days of treatment at extreme low temperature (4℃), the removal rate of various pollutants reached a high level, breaking through the technical bottleneck of the rapid drop in activity of existing bacterial agents below 6℃, and can meet the needs of low-temperature wastewater treatment in northern winters.

[0078] Example 7 pH Adaptability Test (Experiment 1 Liquid Formulation) This embodiment verifies the stability of the liquid formulation in Experiment Example 1 under different pH conditions.

[0079] Test conditions: Simulated low-temperature wastewater (the culture medium used for the low-temperature acclimatization was an acclimatization culture medium simulating the components of low-temperature wastewater; the culture medium was based on ammonia nitrogen 100 mg / L, nitrate nitrogen 80 mg / L, nitrite nitrogen 20 mg / L, total phosphorus 5 mg / L, and COD 300 mg / L, as in Example 6), temperature 10℃, treatment time 1 day, dosage 0.1%, and pH adjusted to 5.5, 7.0, and 9.5 respectively. The specific results are shown in Table 4.

[0080] Table 4. Results of pH adaptability test (10℃, 1 day).

[0081] Conclusion: The wastewater treatment agent of this invention has good adaptability in the pH range of 5.5~9.5, with the best effect under neutral conditions. Under acidic and alkaline conditions, the removal rate of various pollutants is ≥80% with fluctuation ≤10%. It can be adapted to low-temperature wastewater with different pH levels and has a wide range of applications.

[0082] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. A low-temperature resistant wastewater treatment agent, characterized in that, Including effective amount of Bacillus belysinus ( Bacillus velezensis Ingredients: Bacterial agent, glycerin, xanthan gum, betaine, humic acid.

2. The treatment agent according to claim 1, characterized in that, The treatment agent, by weight, comprises 20-30 parts of effective amount of Bacillus belyssus (B. belyssus). Bacillus velezensis The bacterial agent contains 5-8 parts glycerol, 0.5-1 part xanthan gum, 1-2 parts betaine, and 0.3-0.5 parts humic acid; the effective viable count of the *Bacillus belyi* bacterial agent is ≥5 × 10⁻⁶. 9 CFU / mL.

3. The treatment agent according to claim 1, characterized in that, The Bacillus berberis inoculant is derived from strain Bacillus berberis LTS-AF1 ( Bacillus velezensis LTS-AF1) fermentation broth, Bacillus belye LTS-AF2 ( Bacillus velezensis LTS-AF2) fermentation broth, Bacillus belye LTS-AF3 ( Bacillus velezensis LTS-AF3) fermentation broth, Bacillus vesiculosus LTS-AF4 ( Bacillus velezensis It is a mixture of LTS-AF4 fermentation broth.

4. The treatment agent according to claim 3, characterized in that, The viable cell ratio of the Bacillus belyssus LTS-AF1 fermentation broth, LTS-AF2 fermentation broth, LTS-AF3 fermentation broth, and LTS-AF4 fermentation broth is (1~3):(2~4):(2~4):(1~3).

5. The treatment agent according to claim 1, characterized in that, The treatment agent is in the form of a solid powder or a liquid formulation; the solid powder is composed of a liquid formulation and a carrier, wherein the mass ratio of the liquid formulation to the carrier is 1:(2-4); the carrier includes diatomaceous earth and / or corn cob powder; the effective viable count in the solid powder is ≥5×10⁻⁶. 9 CFU / g, effective viable bacteria count in liquid formulation ≥1×10 10 CFU / mL.

6. A method for preparing a low-temperature resistant wastewater treatment agent according to any one of claims 1-5, characterized in that, Includes the following steps: S1: Strain screening: The Bacillus beleibacillus LTS-AF1, LTS-AF2, LTS-AF3 and LTS-AF4 were isolated and screened from activated sludge in a low-temperature environment; S2: Low temperature acclimatization: The strains obtained in step S1 were subjected to low temperature gradient acclimatization, with the acclimatization temperature gradient from 15℃→10℃→6℃→4℃, and each temperature gradient was cultured for 3 days. S3: Expansion culture: Each strain after domestication in step S2 is subjected to expansion culture to obtain a high-concentration fermentation broth; S4: Compound formulation: The fermentation broths of each strain obtained in step S3 are mixed according to the ratio of live bacteria, and glycerol, xanthan gum, betaine and humic acid are added according to the mass ratio. After stirring evenly, the mixture is concentrated under low temperature vacuum to obtain the liquid preparation of the low temperature resistant sewage treatment agent. In step S3, the culture medium used for the expanded culture includes 8-12 g / L corn flour, 6-10 g / L soybean meal, 0.3-0.7 g / L KH2PO4, and 0.1-0.3 g / L MgSO4·7H2O. The culture conditions are: temperature 28-37℃, aeration rate 1.0-1.2 vvm, stirring speed 160-200 rpm, and culture time 24-48 hours.

7. The preparation method according to claim 6, characterized in that, It also includes step S5: In step S4, a carrier is added to obtain the low-temperature wastewater treatment agent solid powder.

8. The application of a low-temperature resistant wastewater treatment agent as described in any one of claims 1-5 in wastewater treatment.

9. The application according to claim 8, characterized in that, The wastewater includes urban domestic sewage, livestock and poultry breeding wastewater, and lightly polluted industrial wastewater; the low temperature is 4℃~25℃; the initial concentration of ammonia nitrogen in the low-temperature wastewater is 50~150 mg / L, nitrate nitrogen is 30~100 mg / L, nitrite nitrogen is 10~30 mg / L, total phosphorus is 3~10 mg / L, and COD is 200~500 mg / L; the pH value of the low-temperature wastewater is 5.5~9.

5. The application includes the simultaneous removal of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen, total phosphorus and COD from wastewater.

10. The application according to claim 8, characterized in that, When the treatment agent is added in liquid form, the dosage is 0.1% to 0.3% of the wastewater volume; when added in solid form, the dosage is 1 to 3 g / m³ of wastewater.

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