A sustained-release bacterial agent and its preparation method and application

Abstract

The present invention relates to a slow-release bacterial agent, a preparation method thereof and an application thereof. The present invention realizes sewage denitrification treatment through the symbiotic coordination between Bacillus megaterium, Bacillus sphingosine and Bacillus chestnut brown, provides a large specific surface area for microorganisms through a special skeleton carrier, ensures the stability of the microorganisms in the carrier, provides a stable and low-viscosity film-wrapped microorganism through an embedding agent formed by a composite of polyvinyl alcohol, sodium alginate, agar and gelatin, delays its release rate, and can also improve the loading success rate of the microorganism. Some skeleton carrier components can absorb moisture and expand to help the microorganism release. The long-term activity of the microorganism is guaranteed by a suitable slow-release carbon source. Through the interaction between the components, the overall mechanical properties of the slow-release bacterial agent are improved, the carrier crushing speed is slowed down, and when used in wetlands, wetland blockage can be alleviated, and the sewage treatment effect is guaranteed to be long-term and stable. The slow-release bacterial agent preparation process of the present invention is simple and can be industrialized.

Description

Technical Field

[0001] The present invention belongs to the technical field of sustained-release bacterial agents, and in particular relates to a sustained-release bacterial agent, a preparation method thereof and an application thereof. Background Art

[0002] With the improvement of urbanization, water pollution is becoming more and more serious. Common sewage treatment methods include chemical precipitation, crystallization and adsorption. Chemical precipitation and crystallization will produce a lot of sludge, resulting in increased sewage treatment costs, which is not conducive to large-scale promotion. The adsorption method has stable conditions, high removal efficiency, relatively simple equipment and low cost. It is more suitable for small and medium-sized, decentralized water treatment facilities when combined with biofilters, artificial wetlands, and bioretention tanks. Artificial wetlands are an ecosystem that simulates natural wetlands and treats sewage. They have the characteristics of low construction and operation costs, high efficiency and easy operation. Artificial wetlands treat sewage mainly by utilizing the synergistic effects of matrix adsorption, plant absorption and microbial assimilation to remove pollutants such as organic matter and nitrogen in sewage. Among them, artificial wetland matrices, such as volcanic rocks and zeolites, provide a large number of attachment sites for microorganisms due to their large specific surface area and high porosity, which can effectively improve the denitrification efficiency through nitrification / denitrification.

[0003] During the operation of artificial wetlands, microbial agents need to be added to quickly start the effect. On the one hand, conventional powdered agents are added to artificial wetlands, which can easily cause blockage of artificial wetlands; on the other hand, additional carbon sources need to be added during operation to increase the efficiency of denitrification, while slow-release granular agents are not easy to cause wetland blockage and can alleviate wetland blockage. At the same time, they can slowly release functional microorganisms and carbon sources, improve denitrification efficiency, and do not require additional carbon sources, thereby reducing maintenance costs. The current slow-release microbial agents themselves have low mechanical strength, unstable slow-release effects, are easily broken when exposed to water erosion, and the stability of long-term treatment effects needs to be improved. In addition, the preparation process of existing slow-release agents is complicated and involves many processes, resulting in a high composition, which is not conducive to promotion. Therefore, there is an urgent need to develop a slow-release agent with high mechanical strength, stable slow-release effect and sewage treatment effect, erosion resistance, and simple preparation process. Summary of the invention

[0004] The purpose of the present invention is to provide a slow-release bacterial agent with high mechanical strength, stable slow-release effect and good long-term sewage treatment effect.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned sustained-release bacterial agent.

[0006] Another object of the present invention is to provide application of the above-mentioned slow-release bacterial agent in sewage treatment.

[0007] In order to achieve the above object, the technical solution adopted by the present invention is:

[0008] The first aspect of the present invention provides a sustained-release bacterial agent, which is in granular form. The raw materials thereof include a composite microbial fermentation broth, a skeleton carrier, an embedding agent and a sustained-release carbon source. The mass ratio of the composite microbial fermentation broth, the skeleton carrier, the embedding agent and the sustained-release carbon source is 1:(6-8):(1-3):(1-3). The composite microbial fermentation broth is composed of Bacillus megaterium fermentation broth, Bacillus sphingosine fermentation broth and Bacillus chestnut brown fermentation broth. The Bacillus megaterium is deposited in China Center for Type Culture Collection (CCTCC). The address of the collection center is Wuhan University, Wuhan, China. The collection date is May 27, 2022. The collection number is CCTCC NO: M2022744. The classification name is Bacillus megaterium Q-11 Bacillus megaterium Q-11, the Sphingobacillus is deposited in China Center for Type Culture Collection (CCTCC), the address of the collection center is Wuhan University, Wuhan, China, the collection date is May 27, 2022, the collection number is CCTCC NO: M2022746, and the classification name is Sphingobium sp Q-13, the chestnut brown Bacillus is deposited in the China Center for Type Culture Collection - CCTCC, the address of the collection center is Wuhan University, Wuhan, China, the collection date is May 27, 2022, the collection number is CCTCC NO: M2022748, and the classification name is Bacillus sp. Q-15, the mass ratio of the Bacillus megaterium fermentation broth, the Bacillus sphingosine fermentation broth and the Bacillus chestnut brown fermentation broth is (3~8):(1~3):1, the skeleton carrier is a mixture of one or more of rice husk powder, diatomaceous earth, zeolite powder, kaolin, and medical stone, the embedding agent is composed of polyvinyl alcohol, gelatin, agar and sodium alginate, and the slow-release carbon source is a mixture of one or more of starch, polycaprolactone, and polylactic acid, and the slow-release carbon source at least includes the polycaprolactone.

[0009] Preferably, the particle size of the sustained-release bacterial agent is 0.2-2 cm and the density is 0.5×10 3 ~3×10 3 kg / m 3 , moisture content ≤5%.

[0010] More preferably, the particle size of the sustained-release bacterial agent is 0.5-1 cm, and the density is 1×10 3 ~2×10 3 kg / m 3 , moisture content ≤5%, no adhesion.

[0011] Preferably, the mass ratio of the Bacillus megaterium fermentation broth, the Bacillus sphingosine fermentation broth and the Bacillus chestnut brown fermentation broth is (3-5):(1-3):1.

[0012] Further preferably, the mass ratio of the Bacillus megaterium fermentation broth, the Bacillus sphingosine fermentation broth and the Bacillus chestnut brown fermentation broth is (3-4):1:1.

[0013] Preferably, the concentration of live bacteria in the Bacillus megaterium fermentation broth, the Bacillus sphingosine fermentation broth and the Bacillus chestnut fermentation broth is ≥ 1×10 10 CFU / mL.

[0014] Preferably, the skeleton carrier is composed of rice husk powder and diatomaceous earth or zeolite powder, and the mass of the rice husk powder accounts for 20% to 30% of the total mass of the skeleton carrier, and more preferably 22% to 28%.

[0015] Preferably, the mass of the sodium alginate accounts for 50% to 60% of the total mass of the embedding agent, and more preferably 55% to 60%.

[0016] Preferably, the mass ratio of polyvinyl alcohol, gelatin, agar and sodium alginate is 1:(1-2):(1-2):(3-5).

[0017] Preferably, the mass of the polycaprolactone accounts for 50% to 70% of the total mass of the slow-release carbon source, and more preferably 55% to 65%.

[0018] Further preferably, the slow-release carbon source consists of starch and polycaprolactone.

[0019] The second aspect of the present invention also provides a method for preparing the above-mentioned sustained-release bacterial agent, the preparation method comprising:

[0020] (1) mixing a Bacillus megaterium fermentation liquid, a Bacillus sphingosine fermentation liquid and a Bacillus chestnut fermentation liquid to obtain a composite microbial fermentation liquid, and concentrating the composite microbial fermentation liquid under vacuum and then mixing it with a skeleton carrier to obtain an adsorption mixture;

[0021] (2) heating and dissolving polyvinyl alcohol, and then mixing it with gelatin, agar and sodium alginate to obtain an embedding agent;

[0022] (3) uniformly mixing the adsorption mixture of step (1) with the embedding agent and the slow-release carbon source of step (2), and granulating;

[0023] (4) adding the particles obtained in step (3) into a cross-linking agent for cross-linking reaction, washing the particles after the reaction with physiological saline, and then drying to obtain the sustained-release bacterial agent,

[0024] There is no particular order in which steps (1) and (2) are performed.

[0025] Preferably, in the step (1), before the vacuum concentration, 0.5 wt% to 1.5 wt% of glycerol is added to the composite microbial fermentation broth as a protective agent.

[0026] Preferably, the cross-linking agent in step (4) contains 2wt% to 5wt% CaCl 2 of saturated boric acid solution.

[0027] Preferably, the cross-linking reaction in step (4) is carried out at room temperature, and the cross-linking reaction time is 36 to 48 hours.

[0028] Preferably, the drying temperature is 25°C to 40°C, and the water content of the sustained-release bacterial agent after drying is ≤5%.

[0029] The third aspect of the present invention also provides the use of the above-mentioned slow-release bacterial agent in sewage treatment.

[0030] Preferably, the above-mentioned slow-release bacterial agent is added to a wetland for sewage treatment, and the addition amount is preferably 1% to 10% of the total mass of the water body in the wetland.

[0031] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0032] The present invention realizes sewage denitrification treatment through the symbiotic coordination between Bacillus megaterium, Bacillus sphingosine and Bacillus chestnut brown, provides a large specific surface area for microorganisms through a special skeleton carrier, ensures the stability of microorganisms in the carrier, provides a stable and low-viscosity film-wrapped microorganism through an embedding agent formed by a composite of polyvinyl alcohol, sodium alginate, agar and gelatin, delays its release rate, and can also improve the loading success rate of microorganisms, and some skeleton carrier components can absorb moisture and expand to help microorganisms release, and ensure the long-term activity of microorganisms through a suitable slow-release carbon source, and through the interaction between the components, the overall mechanical properties of the slow-release microorganism are improved, the carrier crushing speed is slowed down, and when used in wetlands, wetland blockage can be alleviated, and the sewage treatment effect is ensured to be long-term and stable. The slow-release microorganism preparation process of the present invention is simple and can be industrialized. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a morphological diagram of the sustained-release bacterial agent of Example 1;

[0034] Figure 2 This is the change chart of nitrate nitrogen value and COD value of effluent in Experiment 2;

[0035] Figure 3 This is the change diagram of nitrate nitrogen removal rate in the effluent of Experiment 2;

[0036] Figure 4 This is the change diagram of nitrate nitrogen value in influent and effluent in Experiment 3;

[0037] Figure 5 This is the change diagram of COD value of influent and effluent in Experiment 3; DETAILED DESCRIPTION

[0038] In the present invention, Bacillus megaterium is deposited in China Center for Type Culture Collection-CCTCC, the address of the collection center is Wuhan University, Wuhan, China, the collection date is May 27, 2022, the collection number is CCTCC NO: M2022744, and the classification name is Bacillus megaterium Q-11 Bacillus megaterium Q-11.

[0039] In the present invention, Sphingobacillus is deposited in China Center for Type Culture Collection (CCTCC), the address of the collection center is Wuhan University, Wuhan, China, the deposit date is May 27, 2022, the deposit number is CCTCC NO: M2022746, and the classification name is Sphingobium sp .Q-13.

[0040] In the present invention, the chestnut brown Bacillus is deposited in the China Center for Type Culture Collection-CCTCC, the address of the collection center is Wuhan University, Wuhan, China, the collection date is May 27, 2022, the collection number is CCTCC NO: M2022748, and the classification name is Bacillus sp. Q-15.

[0041] In the following, only some exemplary embodiments are briefly described. As those skilled in the art will appreciate, the described embodiments may be modified in various ways without departing from the spirit or scope of the embodiments of the present invention. Therefore, the drawings and descriptions are considered to be exemplary and non-restrictive in nature.

[0042] In the present invention, all the raw materials can be purchased commercially or / and prepared by known means. Unless otherwise specified, they all meet the requirements of standard chemical products. Among them, polyvinyl alcohol comes from Merck Life Sciences, product number 341584, Mw 89000-98000; polycaprolactone comes from Jiangsu Bosite Chemical Technology Co., Ltd., CAS 36890-68-3; polylactic acid comes from Merck Life Sciences, product number, 719943, Mw 18000-24000.

[0043] The experimental methods used in the present invention are all conventional methods unless otherwise specified.

[0044] In the present invention, room temperature refers to 25±5°C.

[0045] In the present invention, the COD detection method refers to the dichromate method for determination of chemical oxygen demand in water (HJ 828-2017); nitrate nitrogen (NO 3- -N) Detection method reference standard HJ / T 346-2007 Water quality - Determination of nitrate nitrogen - UV spectrophotometry.

[0046] In the present invention, the fermentation broth of each strain is prepared by the following method:

[0047] (1) Strain activation: The functional strain was streaked onto the slant of a test tube or onto a plate culture medium (culture medium formula: 5 g / L glucose, 5 g / L peptone, 0.5 g / L beef extract, 3 g / L sodium chloride, 20 g / L agar, pH = 7.2) under sterile conditions and cultured in a constant temperature incubator at 32°C for 24 h.

[0048] (2) Preparation of strain seed solution: Pick one loop of functional strain from the test tube slant or plate and inoculate it into a 1000mL Erlenmeyer flask containing 200mL of culture medium (culture medium composition: 5g / L glucose, 5g / L peptone, 0.5g / L beef extract, 3g / L sodium chloride, pH=7.2). Culture at 30℃ for 12h~18h. Microscopic examination showed that the strain was in the logarithmic growth phase and free of foreign bacteria, which was the first-level seed solution. Transfer it to a 50L fermenter under sterile conditions and culture it for 18h~24h. Microscopic examination showed that the strain was in the logarithmic growth phase and free of foreign bacteria, which was the second-level seed solution.

[0049] (3) Preparation of culture materials: Weigh the components of the culture medium (glucose 5 g / L, peptone 5 g / L, beef extract 0.5 g / L, sodium chloride 3 g / L, pH = 7.2) and soybean oil defoamer and add them to the fermenter.

[0050] (4) Fermentation culture: The fermentation system was sterilized by high temperature and high pressure, inoculated, and cultured for 36 h to 48 h at 30-32 °C, pH = 6.0-7.5, and DO ≥ 25%. Microscopic examination was performed to check the growth status of the functional strain and the amount of fermented bacteria (> 10 9 / mL) and / or spore rate level (>90%).

[0051] (5) Fermentation broth concentration: After the fermentation culture is completed, the fermentation broth is concentrated and the bacterial count is determined by the plate colony count method. If the bacterial count is greater than 1×10 11 CFU / mL, and obtain high-density strain fermentation broth.

[0052] For ease of understanding of the present invention, the present invention lists the following examples. Those skilled in the art should understand that the examples are only to help understand the present invention and should not be regarded as specific limitations of the present invention. The following examples and comparative examples are only some typical experimental examples in the development process of the present invention. Example 1

[0053] This embodiment provides a sustained-release bacterial agent, the raw materials of which include composite microbial fermentation broth, a skeleton carrier, an embedding agent and a sustained-release carbon source, and the mass ratio of the composite microbial fermentation broth, the skeleton carrier, the embedding agent and the sustained-release carbon source is 1:8:2:2. The specific preparation method is as follows:

[0054] The Bacillus megaterium fermentation liquid, the Bacillus sphingosine fermentation liquid and the Bacillus chestnut brown fermentation liquid were mixed in a mass ratio of 3.5:1:1 to obtain a composite microbial fermentation liquid;

[0055] The skeleton carrier is prepared by mixing rice husk powder and diatomaceous earth in a mass ratio of 1:4;

[0056] The composite microbial fermentation liquid and the skeleton carrier were weighed in a ratio of 1:8, 1 wt% glycerol was added to the composite microbial fermentation liquid as a protective agent, and the composite microbial fermentation liquid was mixed with the skeleton carrier after vacuum concentration, and the composite microbial fermentation concentrate was adsorbed into the skeleton carrier to obtain an adsorption mixture.

[0057] (2) Weigh polyvinyl alcohol, gelatin, agar and sodium alginate in a mass ratio of 1:1:1:4, heat the polyvinyl alcohol until it is dissolved (90-95°C), add gelatin, agar and sodium alginate, stir, place in an ultrasonic device for ultrasonic mixing, and let stand to cool to 37°C to obtain an embedding agent.

[0058] (3) Starch and polycaprolactone are mixed in a mass ratio of 1:1.5 to prepare a slow-release carbon source, and the adsorption mixture of step (1) is evenly mixed with the embedding agent and the slow-release carbon source of step (2) in a mass ratio of 4:1:1 among the skeleton carrier, the embedding agent and the slow-release carbon source in the adsorption mixture, and granulation is performed using a granulator to control the particle size range to be 1-2 cm.

[0059] (4) Add the particles obtained in step (3) to a mixture containing 2.5 wt% CaCl 2 The particles were placed in a saturated boric acid solution at room temperature for a cross-linking reaction for 36 hours. The particles were washed 1 to 3 times with saline and then dried (35 to 45°C) to obtain a sustained-release bacterial agent with a water content of <5%. Example 2

[0060] This embodiment provides a sustained-release bacterial agent, which is basically the same as that in Embodiment 1, except that the mass ratio of Bacillus megaterium fermentation broth, Bacillus sphingosine fermentation broth and Bacillus chestnut brown fermentation broth in the composite microbial fermentation broth is 8:1:1, and the preparation method refers to that in Embodiment 1. Example 3

[0061] The embodiment provides a sustained-release bacterial agent, which is basically the same as the embodiment 1, except that: the skeleton carrier is prepared by mixing rice husk powder and zeolite powder in a mass ratio of 1:4, and the preparation method is referred to the embodiment 1. Example 4

[0062] This embodiment provides a sustained-release bacterial agent, which is basically the same as that in Embodiment 1, except that the skeleton carrier is prepared by mixing rice husk powder and kaolin in a mass ratio of 1:4, and the preparation method is referred to that in Embodiment 1. Example 5

[0063] This embodiment provides a sustained-release bacterial agent, which is basically the same as that in Embodiment 1, except that: the skeleton carrier is all rice husk powder, and the preparation method is referred to that in Embodiment 1. Example 6

[0064] This embodiment provides a sustained-release bacterial agent, which is basically the same as that of Embodiment 1, except that the skeleton carriers are all medical stones, and the preparation method is referred to that of Embodiment 1. Example 7

[0065] This embodiment provides a sustained-release bacterial agent, which is basically the same as that in Embodiment 1, except that the sustained-release carbon source is prepared by mixing starch and polylactic acid in a mass ratio of 1:1.5, and the preparation method is referred to that in Embodiment 1. Example 8

[0066] This embodiment provides a sustained-release bacterial agent, which is basically the same as that in Embodiment 1, except that the sustained-release carbon sources are all polycaprolactone, and the preparation method is as in Embodiment 1. Example 9

[0067] This embodiment provides a sustained-release bacterial agent, which is basically the same as that in Embodiment 1, except that the mass ratio of Bacillus megaterium fermentation broth, Bacillus sphingosine fermentation broth and Bacillus chestnut brown fermentation broth in the composite microbial fermentation broth is 6:1:2, and the preparation method is referred to that in Embodiment 1. Example 10

[0068] This embodiment provides a sustained-release bacterial agent, which is basically the same as that in Embodiment 1, except that the mass ratio of the composite microbial fermentation broth, the skeleton carrier, the embedding agent and the sustained-release carbon source is 1:7:1:3, and the preparation method refers to that in Embodiment 1. Embodiment 11

[0069] This embodiment provides a sustained-release bacterial agent, which is basically the same as that in Embodiment 1, except that the mass ratio of the composite microbial fermentation broth, the skeleton carrier, the embedding agent and the sustained-release carbon source is 1:6:3:3, and the preparation method refers to that in Embodiment 1. Example 12

[0070] This embodiment provides a sustained-release bacterial agent, which is basically the same as Example 1, except that: the embedding agent is composed of polyvinyl alcohol, gelatin, agar, and sodium alginate in a mass ratio of 3:1:1:8, and the preparation method refers to Example 1.

[0071] Comparative Example 1

[0072] This comparative example provides a sustained-release bacterial agent, which is basically the same as Example 1, except that: the mass ratio of Bacillus megaterium fermentation broth, Bacillus sphingosine fermentation broth and Bacillus chestnut brown fermentation broth in the composite bacterial fermentation broth is 1.5:1:1, the embedding agent is composed of polyvinyl alcohol, gelatin, agar and sodium alginate in a mass ratio of 2:1:1:8, and the preparation method refers to Example 1.

[0073] Comparative Example 2

[0074] This comparative example provides a sustained-release bacterial agent, which is basically the same as Example 1, except that: the mass ratio of Bacillus megaterium fermentation broth, Bacillus sphingosine fermentation broth and Bacillus chestnut brown fermentation broth in the composite bacterial fermentation broth is 11:1:2, the embedding agent is composed of polyvinyl alcohol, gelatin, agar and sodium alginate in a mass ratio of 2:1:1:8, and the preparation method refers to Example 1.

[0075] Comparative Example 3

[0076] This comparative example provides a sustained-release bacterial agent, which is basically the same as Example 1, except that: the composite bacterial fermentation broth does not contain chestnut brown Bacillus fermentation broth, and the mass ratio of Bacillus megaterium fermentation broth to Sphingobacillus fermentation broth is 7:2, the embedding agent is composed of polyvinyl alcohol, gelatin, agar, and sodium alginate in a mass ratio of 2:1:1:8, and the preparation method refers to Example 1.

[0077] Comparative Example 4

[0078] This comparative example provides a sustained-release bacterial agent, which is basically the same as Example 1, except that: the composite bacterial fermentation broth does not contain Sphingobacillus fermentation broth, and the mass ratio of Bacillus megaterium fermentation broth to Bacillus chestnut brown fermentation broth is 7:2, the embedding agent is composed of polyvinyl alcohol, gelatin, agar, and sodium alginate in a mass ratio of 2:1:1:8, and the preparation method refers to Example 1.

[0079] Comparative Example 5

[0080] This comparative example provides a sustained-release bacterial agent, which is basically the same as Example 1, except that the mass ratio of Bacillus megaterium fermentation broth, Bacillus sphingosine fermentation broth and Bacillus chestnut brown fermentation broth in the composite bacterial fermentation broth is 7:2:5, and the preparation method refers to Example 1.

[0081] Comparative Example 6

[0082] This comparative example provides a sustained-release bacterial agent, which is basically the same as Example 1, except that the mass ratio of Bacillus megaterium fermentation broth, Bacillus sphingosine fermentation broth and Bacillus chestnut brown fermentation broth in the composite bacterial fermentation broth is 7:5:2, and the preparation method refers to Example 1.

[0083] Comparative Example 7

[0084] This comparative example provides a sustained-release bacterial agent, which is basically the same as Example 1, except that: the embedding agent is composed of polyvinyl alcohol, gelatin, agar, and sodium alginate in a mass ratio of 1:1:1:1.5, and the preparation method refers to Example 1.

[0085] Comparative Example 8

[0086] This comparative example provides a sustained-release bacterial agent, which is basically the same as Example 1, except that the embedding agent is composed of polyvinyl alcohol, gelatin, agar, and sodium alginate in a mass ratio of 5:1:2:5, and the preparation method refers to Example 1.

[0087] Comparative Example 9

[0088] This comparative example provides a sustained-release bacterial agent, which is basically the same as Example 1, except that the mass ratio of the composite microbial fermentation broth, the skeleton carrier, the embedding agent and the sustained-release carbon source is 1:7:2:5, and the preparation method refers to Example 1.

[0089] Comparative Example 10

[0090] This comparative example provides a sustained-release bacterial agent, which is basically the same as Example 1, except that the mass ratio of the composite microbial fermentation broth, the skeleton carrier, the embedding agent and the sustained-release carbon source is 1:7:5:2, and the preparation method refers to Example 1.

[0091] Comparative Example 11

[0092] This comparative example provides a sustained-release bacterial agent, which is basically the same as Example 1, except that the mass ratio of the composite microbial fermentation broth, the skeleton carrier, the embedding agent and the sustained-release carbon source is 1:4:2:2, and the preparation method refers to Example 1.

[0093] Comparative Example 12

[0094] This comparative example provides a sustained-release bacterial agent, which differs from Example 1 mainly in that no embedding agent is used for cross-linking, and its raw materials include composite microbial fermentation broth, skeleton carrier and sustained-release carbon source, and the mass ratio of composite microbial fermentation broth, skeleton carrier and sustained-release carbon source is 1:7:3. The preparation method is as follows:

[0095] (1) mixing the fermentation liquid of Bacillus megaterium, the fermentation liquid of Bacillus sphingosine and the fermentation liquid of Bacillus chestnut brown in a mass ratio of 3.5:1:1 to obtain a composite microbial fermentation liquid;

[0096] The skeleton carrier is prepared by mixing rice husk powder and diatomaceous earth in a mass ratio of 1:4;

[0097] The composite microbial fermentation liquid and the skeleton carrier were weighed in a ratio of 1:7, 1 wt% glycerol was added to the composite microbial fermentation liquid as a protective agent, and the composite microbial fermentation liquid was mixed with the skeleton carrier after vacuum concentration, and the composite microbial fermentation concentrate was adsorbed into the skeleton carrier to obtain an adsorption mixture.

[0098] (2) Starch and polycaprolactone are mixed in a mass ratio of 1:1.5 to prepare a slow-release carbon source.

[0099] (3) The adsorption mixture of step (1) and the slow-release carbon source of step (2) are uniformly mixed in a mass ratio of 7:3 between the skeleton carrier and the slow-release carbon source in the adsorption mixture, granulated using a granulator, the particle size range of which is controlled to be 1-2 cm, and dried (35-45° C.) to a moisture content of less than 5%.

[0100] Comparative Example 13

[0101] This comparative example provides a sustained-release bacterial agent, which differs from Example 1 mainly in that no sustained-release carbon source is used, and its raw materials include composite microbial fermentation broth, skeleton carrier and embedding agent, and the mass ratio of composite microbial fermentation broth, skeleton carrier and embedding agent is 1:7:3. The specific preparation method is as follows:

[0102] (1) mixing the fermentation liquid of Bacillus megaterium, the fermentation liquid of Bacillus sphingosine and the fermentation liquid of Bacillus chestnut brown in a mass ratio of 3.5:1:1 to obtain a composite microbial fermentation liquid;

[0103] The skeleton carrier is prepared by mixing rice husk powder and diatomaceous earth in a mass ratio of 1:4;

[0104] The composite microbial fermentation liquid and the skeleton carrier were weighed in a ratio of 1:7, 1 wt% glycerol was added to the composite microbial fermentation liquid as a protective agent, and the composite microbial fermentation liquid was mixed with the skeleton carrier after vacuum concentration, and the composite microbial fermentation concentrate was adsorbed into the skeleton carrier to obtain an adsorption mixture.

[0105] (2) Weigh polyvinyl alcohol, gelatin, agar and sodium alginate in a mass ratio of 1:1:1:4, heat the polyvinyl alcohol until it is dissolved (90-95°C), add gelatin, agar and sodium alginate, stir, place in an ultrasonic device for ultrasonic mixing, and let stand to cool to 37°C to obtain an embedding agent.

[0106] (3) The adsorption mixture of step (1) and the embedding agent of step (2) are uniformly mixed in a ratio of 7:3 in which the mass ratio of the skeleton carrier to the embedding agent in the adsorption mixture is 7:3, and granulated using a granulator to control the particle size range to be 1-2 cm.

[0107] (4) Add the particles obtained in step (3) to a mixture containing 2.5 wt% CaCl 2 The particles were placed in a saturated boric acid solution at room temperature for a cross-linking reaction for 36 hours. The particles were washed 1 to 3 times with saline and then dried (35 to 45°C) to obtain a sustained-release bacterial agent with a water content of <5%.

[0108] Experiment 1:

[0109] Test sample: the sustained-release bacterial agent prepared in each embodiment and each comparative example.

[0110] Test water sample: sewage with nitrate nitrogen value of 35mg / L and COD value of 78.0mg / L.

[0111] Test method: A columnar plexiglass reactor was selected as the test container, where the inner diameter of the reactor was 6 cm, the height was 30 cm, and the effective volume was 350 mL. The sustained-release bacteria agent was added into the reactor at a dosage of 5% of the total mass of the sewage in the reactor. The sewage was continuously introduced into the reactor from the bottom and discharged from the top through the water inlet pump. The residence time was set to 6 h. After running for 7 days and stabilizing, the nitrate nitrogen value and COD value of the effluent water sample were detected, and the state of the sustained-release bacteria agent in the water body was observed. The results are shown in Table 1.

[0112]

[0113] Table 1 shows that the slow-release bacteria agents of Examples 1 to 12 can not only greatly reduce the effluent COD and nitrate nitrogen content, but also are stable in state without adhesion and dissolution. The sewage treatment effect of the slow-release bacteria agents of Examples 1 to 12 is significantly better than that of the slow-release bacteria agents of Comparative Examples 1 to 13. Some of the slow-release bacteria agents of the comparative examples also have adhesion and dissolution phenomena, which will easily cause wetland blockage when used in wetlands.

[0114] Experiment 2:

[0115] Test sample: the sustained-release bacterial agent of Example 1.

[0116] Test water sample: Water from the pilot wetland.

[0117] Testing time: May 2 to June 29, 2024.

[0118] Test method: A laboratory-scale simulated artificial wetland was selected, which was constructed with 15 mm thick plexiglass. The interior of the wetland included a water distribution area, a treatment area and a water collection area from the front end to the back end. A water inlet was provided at the top of the water distribution area, and the upper parts of the water distribution area and the treatment area were connected by overflow. The lower end of the treatment area was connected to the lower part of the water collection area, and a water outlet was provided at the top of the water collection area. The water distribution area and the water collection area were filled with gravel with a particle size range of 0.5~2 cm respectively. The specifications of the treatment area were: side length 0.5m×side length 0.5m×height 0.80m. The filler of the treatment area was 5~30 mm gravel with a paving height of 60cm. The plants planted in the wetland were 4 reeds. Slow-release bacteria were added to the treatment area at a dosage of 3% of the total mass of the water in the treatment area. The water from the pilot wetland is continuously introduced into the water distribution area through a water pipe equipped with an inlet pump. The water overflows from the upper part of the water distribution area into the treatment area, flows through the treatment area, and enters the water collection area from the lower rear end of the treatment area. Finally, it flows out continuously from the outlet at the upper part of the water collection area. The water retention time in the treatment area is controlled by the inlet pump to be 6 hours. After one day of operation, the nitrate nitrogen value and COD value of the effluent are continuously tested at intervals of 2 days, with no addition of slow-release bacteria as the control. The monitoring results are shown in Figure 2 and Figure 3 .

[0119] Figure 1 It shows that after adding the slow-release bacteria agent of Example 1, the COD and nitrate nitrogen contents are significantly lower than those of the control group. Even under the condition of low carbon-nitrogen ratio, the removal rate of nitrate nitrogen by the slow-release bacteria agent of Example 1 is always maintained above 50% ( Figure 3 , nitrate nitrogen removal rate (%) = (control NO 3 - -N - Process NO 3 - -N) / control NO 3 - -N×100%), it can be seen that the slow-release bacterial agent of Example 1 has a good removal ability for nitrate nitrogen and COD in sewage, and the long-term operation effect is stable.

[0120] Experiment 3:

[0121] Test sample: the sustained-release bacterial agent of Example 3.

[0122] Test water sample: sewage with nitrate nitrogen value of 25~35mg / L and COD value of 25~45mg / L.

[0123] Testing time: July 14 to August 15, 2024.

[0124] Test method: A columnar organic glass reactor was used to simulate artificial wetlands, where the reactor had an inner diameter of 20 cm, a height of 30 cm, and an effective volume of 9400 mL. The slow-release bacteria agent and filler volcanic rock were filled into the columnar organic glass reactor at a volume ratio of 1:9, and the filling height was 25 cm. The influent entered the reactor from the bottom through the inlet pump and was discharged from the top through the filler layer. The residence time was set to 6 hours. After running for 5 days and stabilizing, the nitrate nitrogen value and COD value of the effluent were continuously tested at intervals. The results are shown in the figure. Figure 4 and Figure 5 The slow-release bacterial agent of Example 3 can stabilize the effluent COD concentration below 20 mg / L and the nitrate nitrogen concentration below 10 mg / L for a long time.

[0125] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent changes or modifications made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. A sustained-release bacterial agent, characterized in that: The sustained-release bacterial agent is in granular form, and its raw materials include composite microbial fermentation broth, skeleton carrier, embedding agent and sustained-release carbon source, and the mass ratio of the composite microbial fermentation broth, skeleton carrier, embedding agent and sustained-release carbon source is 1:(6-8):(1-3):(1-3), The composite microbial fermentation liquid is composed of Bacillus megaterium fermentation liquid, Bacillus sphingosine fermentation liquid and Bacillus chestnut fermentation liquid. The Bacillus megaterium deposit number is CCTCC NO: M2022744, The Sphingobacterium sphingosineum deposit number is CCTCC NO: M2022746, The deposit number of Bacillus chestnut is CCTCC NO: M2022748. The mass ratio of the Bacillus megaterium fermentation liquid, the Bacillus sphingosine fermentation liquid and the Bacillus chestnut fermentation liquid is (3-8): (1-3): 1, The skeleton carrier is composed of rice husk powder and diatomaceous earth or zeolite powder, and the mass of the rice husk powder accounts for 20% to 30% of the total mass of the skeleton carrier. The embedding agent is composed of polyvinyl alcohol, gelatin, agar and sodium alginate, and the mass ratio of the polyvinyl alcohol, gelatin, agar and sodium alginate is 1:(1-2):(1-2):(3-5). The slow-release carbon source consists of starch and polycaprolactone, and the mass of the polycaprolactone accounts for 50% to 70% of the total mass of the slow-release carbon source.

2. The sustained-release bacterial agent according to claim 1, characterized in that The concentration of live bacteria in the Bacillus megaterium fermentation liquid, the Bacillus sphingosine fermentation liquid and the Bacillus chestnut fermentation liquid is ≥1×10 10 CFU / mL; And / or, the particle size of the sustained-release bacterial agent is 0.5-2 cm, and the density is 0.5×10 3 ~3×10 3 kg / m 3 , moisture content ≤5%.

3. The sustained-release bacterial agent according to claim 1, characterized in that The mass of the sodium alginate accounts for 50% to 60% of the total mass of the embedding agent.

4. The method for preparing a sustained-release bacterial agent according to any one of claims 1 to 3, characterized in that: The preparation method comprises: (1) mixing a Bacillus megaterium fermentation liquid, a Bacillus sphingosine fermentation liquid and a Bacillus chestnut fermentation liquid to obtain a composite microbial fermentation liquid, and concentrating the composite microbial fermentation liquid under vacuum and then mixing it with a skeleton carrier to obtain an adsorption mixture; (2) heating and dissolving polyvinyl alcohol, and then mixing it with gelatin, agar and sodium alginate to obtain an embedding agent; (3) uniformly mixing the adsorption mixture of step (1) and the embedding agent of step (2), and granulating; (4) adding the particles obtained in step (3) into a cross-linking agent for cross-linking reaction, washing the particles after the reaction with physiological saline, and then drying to obtain the sustained-release bacterial agent, There is no particular order in which steps (1) and (2) are performed.

5. The method for preparing the sustained-release bacterial agent according to claim 4, characterized in that: In the step (1), before the vacuum concentration, 0.5 wt% to 1.5 wt% of glycerol is added to the composite microbial fermentation broth. And / or, the cross-linking agent in step (4) is a saturated boric acid solution containing 2wt% to 5wt% CaCl2, And / or, the drying temperature is 25° C. to 40° C., and the water content of the sustained-release bacterial agent after drying is ≤5%.

6. Use of the sustained-release bacterial agent according to any one of claims 1 to 5 in sewage treatment.

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