Mushroom residue immobilized bacillus mucilaginosus embedding inoculant and its application in pharmaceutical wastewater treatment

Abstract

This invention discloses a mushroom residue immobilized Bacillus mucilaginosus encapsulation agent and its application in pharmaceutical wastewater treatment. Specifically, the preparation method of the immobilized microbial strain of this invention includes three steps: mushroom residue carrier pretreatment and modification, Bacillus mucilaginosus activation and cultivation, and immobilization reaction. This invention uses widely available and low-cost mushroom residue as the raw material for nano-biochar. The selected solid waste mushroom residue is pretreated with an inorganic salt culture solution before drying. The carrier performance is further optimized through material modification, and then adsorbed and immobilized with a high-concentration Bacillus mucilaginosus bacterial solution. Specific excipients and processes are selected to improve the survival rate and performance stability of the immobilized agent.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology. Specifically, this invention relates to an immobilized Bacillus mucilaginosus agent from mushroom residue and its application in pharmaceutical wastewater. Background Technology

[0002] With the development of my country's industry and urbanization, the public's attention to environmental pollution is increasing, and methods for treating organic matter in water and soil environments are gradually developing. Compared with physical and chemical methods (coagulation sedimentation, ion exchange, photochemical catalysis, etc.), microbial remediation is favored due to its high efficiency, low energy consumption, and environmental friendliness.

[0003] Pharmaceutical wastewater is complex in composition, containing large amounts of recalcitrant organic matter, antibiotics, heavy metal ions, and residual mycelium. It is characterized by high COD (Chemical Oxygen Demand), low BOD / COD ratio, high toxicity, and large fluctuations in water quality. Direct discharge poses a serious threat to aquatic ecosystems and human health. Currently, the main methods for treating pharmaceutical wastewater include physical, chemical, and biological methods. Among these, biological methods have become one of the mainstream technologies for wastewater treatment due to their advantages such as low treatment cost, environmental friendliness, and no secondary pollution.

[0004] Paenibacillus mucilaginosus spp. K1-1 is a Gram-positive bacterium with strong extracellular polysaccharide production and adsorption capabilities. It can remove pollutants from wastewater through biosorption and biodegradation, showing promising application prospects in wastewater treatment. However, during use, free Paenibacillus mucilaginosus exhibits problems such as easy loss, poor stability, and difficulty in recycling and reuse, limiting its practical application effectiveness.

[0005] Free-floating microbial agents such as Bacillus subtilis are prone to problems in wastewater treatment, including agent loss, poor buffering capacity of biological treatment systems, and poor solid-liquid separation. Therefore, immobilizing microorganisms for wastewater treatment is a more ideal choice. Compared with free-floating microbial agents, immobilized microorganisms have advantages such as stability, strong shock resistance, ease of solid-liquid separation, and higher microbial density per unit space and volume of pharmaceutical wastewater. This ensures stable growth and reproduction of microorganisms and efficient wastewater treatment.

[0006] Immobilized bacterial agents, such as those containing Bacillus subtilis, are an effective way to solve the above problems. By immobilizing microorganisms on a carrier material, their stability and tolerance can be improved, enabling their recycling and reuse. Currently, commonly used immobilization carriers mainly include inorganic carriers (such as zeolite, activated carbon, and ceramics), organic carriers (such as chitosan), and natural polymer carriers. However, existing carriers have drawbacks such as high cost, poor mass transfer performance, and unstable immobilization effects.

[0007] Mushroom residue is a solid waste generated during edible mushroom cultivation, mainly composed of mycelial remains, residual mycelium, and incompletely utilized culture medium. It contains abundant natural macromolecules such as cellulose, hemicellulose, and lignin, and is widely available and inexpensive. Currently, most mushroom residue is discarded or incinerated, resulting in resource waste and environmental pollution. Using mushroom residue as a microbial immobilization carrier can achieve resource utilization of mushroom residue while reducing immobilization costs, aligning with the concept of green and environmentally friendly development. However, there are currently no reports on using mushroom residue for immobilizing Bacillus mucilaginosus and applying it to pharmaceutical wastewater treatment. Summary of the Invention

[0008] The purpose of this invention is to provide a mushroom residue immobilized Bacillus mucilaginosus encapsulation agent and its application in pharmaceutical wastewater treatment.

[0009] In a first aspect of the present invention, a mushroom residue immobilized Bacillus mucilaginosus encapsulation agent is provided, the mushroom residue immobilized Bacillus mucilaginosus encapsulation agent comprising an immobilization carrier made of mushroom residue and Bacillus mucilaginosus encapsulated on the immobilization carrier.

[0010] In another preferred embodiment, the method for preparing the immobilized carrier includes the steps of: (1) Take mushroom residue, dry it to constant weight, crush it to obtain mushroom residue powder, place the mushroom residue powder in a tube furnace, heat it to 400-600℃ at a heating rate of 5-10℃ / min under nitrogen atmosphere, pyrolyze for 2-4 h, cool it to room temperature and take it out to obtain mushroom residue biochar. (2) Add mushroom residue biochar to a 0.5-2 mol / L magnesium sulfate solution with a solid-liquid ratio of 1:10-1:20 (g / mL) and modify it by constant temperature shaking at 30-50℃ for 4-8 h at a shaking rate of 120-180 r / min. After modification, wash with distilled water until neutral and dry to constant weight to obtain the immobilized carrier.

[0011] In another preferred embodiment, the method for preparing the encapsulated bacterial agent includes the following steps: a. Disperse sodium alginate, polyvinyl alcohol and immobilization carrier separately in pure water, then add bacterial suspension and mix thoroughly to obtain mixed suspension; b. Prepare a calcium chloride solution by dissolving calcium chloride in pure water; c. Place the calcium chloride solution on a magnetic stirrer and add the mixed suspension dropwise into the calcium chloride solution. Let it stand at room temperature for 24-36 h to allow cross-linking. d. After cross-linking is completed, the immobilized microspheres are rinsed with physiological saline to remove impurities and free bacteria from the surface of the microspheres, thereby obtaining the encapsulating bacterial agent.

[0012] In another preferred embodiment, in step a, the weight-to-volume ratio of sodium alginate, polyvinyl alcohol, immobilization carrier and bacterial solution is: 2.0-5.0 (w): 8.0-10.0 (w): 6.0-10.0 (w): 5 (v).

[0013] In another preferred embodiment, in step a, 2.0-5.0 g of sodium alginate, 8.0-10.0 g of polyvinyl alcohol and 6.25-10.00 g of immobilized carrier (mushroom residue modified biochar) are dissolved in 30-50 mL of pure water and mixed evenly. Then, 5 mL of bacterial suspension is added, and pure water is added to make up to 100 mL to make the solution fully mixed to obtain a mixed suspension.

[0014] In another preferred embodiment, the concentration of Bacillus mucilaginosus in the bacterial suspension is 10. 8 -10 9 CFU / mL.

[0015] In another preferred embodiment, in step b, the mass concentration of the calcium chloride solution is 4%-5%.

[0016] In another preferred embodiment, the gelatinous Bacillus is Paenibacillus mucilaginosus Kl-1, accession number CCTCC No. M 2021439.

[0017] In another preferred embodiment, the method for preparing the bacterial suspension includes the steps of: a. Take the Bacillus subtilis strain frozen in glycerol and thaw it at room temperature; b. After preparing solid culture medium, sterilize it, pour it into plates, and cool it for later use. c. Take 200 μl of the liquid from the thawed Bacillus mucilaginosus cryopreservation tube and spread it onto the solid culture plate in step b. Incubate the plate upside down in a 30°C incubator for 24 h. d. Prepare liquid culture medium, fill it into an Erlenmeyer flask, seal it, sterilize and cool it before use; e. Take the solid plate culture from c, examine it under a microscope, pick a single colony culture and transfer it to the liquid culture medium in d. Incubate it in a shaker at 30℃ and 150 rpm for 24 h, then centrifuge and filter. Wash the lower bacterial precipitate three times with sterile water to remove cell growth metabolites and residual culture medium. Make up the volume with sterile physiological saline to prepare a bacterial suspension for later use.

[0018] In another preferred embodiment, the liquid culture medium has the following specific composition: The composition of each 1000 mL culture medium is as follows: 15-20 g / L sucrose, 1000-1200 mg Na2SO4, 1000-1200 mg NH4Cl, 80-100 mg CaCl2·2H2O, 1800-2000 mg MgSO4·7H2O, 500-600 mg KH2PO4, 800-1000 mg yeast extract, 800-1000 mg CH3COONa, 400-500 mg FeSO4·7H2O, 5 mL trace element stock solution, and the remainder is water. The pH is 7.0-7.5. The medium is sterilized at 121 °C for 30 min before use.

[0019] In another preferred embodiment, the trace element stock solution comprises KI, FeCl3·6H2O, H3BO3, CuSO4, MnSO4·H2O, and ZnSO4·7H2O.

[0020] In another preferred embodiment, the trace element supplementation culture medium consists of the following per 1000 mL: 0.25-0.30 mg KI, 5.5-6.0 mg FeCl3·6H2O, 0.5-0.6 mg H3BO3, 0.5-0.6 mg CuSO4, 0.75-0.80 mg MnSO4·H2O, 0.375-0.400 mg ZnSO4·7H2O, with the remainder being water, filtered and sterilized before use.

[0021] In a second aspect, the present invention provides the application of the mushroom residue immobilized Bacillus mucilaginosus encapsulation agent described in the first aspect of the present invention in pharmaceutical wastewater treatment.

[0022] In another preferred embodiment, the pharmaceutical wastewater is antibiotic pharmaceutical wastewater, chemically synthesized pharmaceutical wastewater, or traditional Chinese medicine pharmaceutical wastewater.

[0023] In another preferred embodiment, the COD concentration in the pharmaceutical wastewater is 1000-5000 mg / L, the ammonia nitrogen concentration is 50-200 mg / L, and the heavy metal ion concentration is 10-50 mg / L.

[0024] A third aspect of the present invention provides a method for treating pharmaceutical wastewater using the mushroom residue immobilized Bacillus mucilaginosus encapsulation agent described in the first aspect of the present invention, the method comprising the steps of: The mushroom residue immobilized Bacillus mucilaginosus encapsulation agent was added to pharmaceutical wastewater, the pH of the wastewater was adjusted to 6.5-8.0, the reaction temperature was controlled at 25-35℃, the dissolved oxygen content was 2-5 mg / L, and the reaction time was 12-24 h.

[0025] In another preferred embodiment, the dosage of the mushroom residue immobilized Bacillus mucilaginosus encapsulation agent is 5-15 g / L.

[0026] In another preferred embodiment, a sequencing batch reaction or a continuous flow reaction is used to treat pharmaceutical wastewater. After the reaction is completed, the mushroom residue immobilized Bacillus mucilaginosus encapsulation agent is recovered by filtration or sedimentation and reused 3-5 times.

[0027] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0028] Figure 1 Schematic diagram of the preparation process of mushroom residue modified biochar.

[0029] Figure 2 The removal rate of different pollutants in norfloxacin production wastewater after the use of bacterial agents. Detailed Implementation

[0030] Studies have shown that *Bacillus mucilaginosus* can still produce flocculants after being encapsulated. Encapsulation, by trapping microorganisms within a polymeric carrier grid, prevents cell loss while allowing their metabolic activity to continue. Microbial flocculants (such as polysaccharides and proteins), as metabolic products, can freely diffuse outside the carrier to exert their effects. For example, extracellular polymers produced by immobilized bacteria can flocculate particles through bridging, and the porous structure of the encapsulation material does not affect mass transfer between the substrate and the product. Therefore, encapsulation technology effectively maintains bacterial activity, ensuring continuous flocculant production.

[0031] This invention discloses an immobilized Bacillus mucilaginosus strain from mushroom residue and its application in pharmaceutical wastewater, belonging to the field of solid waste resource utilization and wastewater treatment technology. The preparation method of this immobilized microbial strain includes three steps: pretreatment and modification of the mushroom residue carrier, activation and cultivation of Bacillus mucilaginosus, and immobilization reaction. This invention uses widely available and inexpensive mushroom residue as the raw material for nano-biochar. The selected solid waste mushroom residue is pretreated with an inorganic salt culture solution before drying. The carrier performance is further optimized through material modification, and then it is adsorbed and immobilized with a high-concentration Bacillus mucilaginosus bacterial solution. Specific excipients and processes are selected to improve the survival rate and performance stability of the immobilized bacterial agent. The *Paenibacillus mucilaginosus* K1-1 selected in this invention was deposited on April 25, 2021, at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, with accession number CCTCC NO.M2021439 and name: *Paenibacillus mucilaginosus* K1-1. The inventors have discovered that *Paenibacillus mucilaginosus* K1-1 is a flocculant-producing bacterium that can effectively remove pollutants from pharmaceutical wastewater, especially residual mycelium, COD, ammonia nitrogen, and heavy metal ions. The mushroom residue immobilized Bacillus mucilaginosus agent prepared by this invention exhibits strong stability, good tolerance, and easy recycling and reuse. When applied to pharmaceutical wastewater treatment, it achieves COD removal rates exceeding 85%, ammonia nitrogen removal rates exceeding 80%, and heavy metal ion removal rates exceeding 75%, maintaining high treatment efficiency even after 3-5 reuses. This invention realizes the resource utilization of mushroom residue waste, reduces wastewater treatment costs, eliminates secondary pollution, and has broad industrial application prospects.

[0032] Before describing this invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can be varied. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0033] 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 invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0034] While any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention, preferred methods and materials are exemplified herein.

[0035] The purpose of this invention is to develop a method for preparing immobilized Bacillus mucilaginosus using mushroom residue as a carrier, thereby improving the survival rate of Bacillus mucilaginosus in the immobilized agent and increasing the density of Bacillus mucilaginosus per unit space. This method can be applied to pharmaceutical wastewater treatment to achieve efficient removal of pollutants and solve the problem of resource utilization of mushroom residue waste, which has important practical significance and application value.

[0036] The objective of this invention is achieved through the following technical solution: Mushroom residue was used to make biochar as an immobilization carrier to fix flocculants and produce Bacillus spp.

[0037] This invention defines the preparation method of mushroom residue composite carrier, which uses agricultural waste mushroom residue (one or more of oyster mushroom residue, shiitake mushroom residue, and enoki mushroom residue, preferably oyster mushroom residue) as raw material, and prepares a high-performance carrier through a three-step process of pretreatment, carbonization, and magnesium sulfate modification.

[0038] In a preferred embodiment of the present invention, the preparation of the immobilized carrier includes the following steps: a. Pretreatment: Take fresh mushroom residue, remove impurities such as stones and plastics, dry it in an oven at 60-80℃ until constant weight, pulverize it and pass it through a 40-80 mesh sieve to obtain uniform mushroom residue powder, which improves the uniformity of subsequent carbonization. b. Carbonization: The mushroom residue powder is placed in a tube furnace and heated to 400-600 ℃ at a heating rate of 5-10℃ / min under a nitrogen inert atmosphere. It is then kept at a constant temperature for 2-4 h and cooled to obtain the original mushroom residue biochar. The carbonization process constructs a porous structure, providing basic sites for microbial attachment. c. Magnesium sulfate modification: The original mushroom residue biochar was added to a 0.5-2 mol / L magnesium sulfate solution, with the solid-liquid ratio controlled at 1:10-1:20 (g / mL). Modification was carried out at 30-50℃ with constant temperature shaking at 120-180 r / min for 4-8 hours. After modification, the mixture was repeatedly washed with distilled water until the filtrate was neutral, and then dried in a 60-80℃ oven to constant weight to obtain the magnesium sulfate-modified mushroom residue biochar carrier. Magnesium sulfate modification significantly optimizes the carrier's pore structure, increases its specific surface area, and introduces active functional groups, improving the carrier's adsorption performance and biocompatibility, providing a more stable growth microenvironment for Bacillus mucilaginosus.

[0039] Preferably, the immobilized Bacillus mucilaginosus agent of the present invention is prepared into a bacterial suspension before immobilization, and the ratio of the bacterial suspension to the modified mushroom residue biochar carrier is 0.1-0.3 ml: 1 g.

[0040] Preferably, the *Bacillus mucilaginosus* immobilized from mushroom residue mentioned in this invention should be activated and cultured before encapsulation. For example, the *Bacillus mucilaginosus* strain is inoculated into an activation medium and cultured with shaking at 30-37°C and 150-200 r / min for 18-24 hours to obtain an activated bacterial solution. The activated bacterial solution is then inoculated into an expansion medium at an inoculation rate of 5-10% and cultured with shaking under the same conditions for 24-36 hours to obtain a high-concentration bacterial solution. After centrifugation and filtration, the lower bacterial precipitate is washed three times with sterile water to remove cell growth metabolites and residual culture medium. The solution is then diluted to volume with sterile physiological saline to prepare a bacterial suspension with a concentration of 10... 8 -10 9 CFU / mL.

[0041] Furthermore, the specific composition of the liquid culture medium is as follows: The composition of each 1000 mL culture medium is as follows: 15-20 g / L sucrose, 1000-1200 mg Na2SO4, 1000-1200 mg NH4Cl, 80-100 mg CaCl2·2H2O, 1800-2000 mg MgSO4·7H2O, 500-600 mg KH2PO4, 800-1000 mg yeast extract, 800-1000 mg CH3COONa, 400-500 mg FeSO4·7H2O, 5 mL trace element stock solution, and the remainder is water. The pH is 7.0-7.5. The medium is sterilized at 121 °C for 30 min before use.

[0042] Preferably, the trace element stock solution includes KI, FeCl3·6H2O, H3BO3, CuSO4, MnSO4·H2O, and ZnSO4·7H2O.

[0043] Furthermore, the trace element supplementation culture medium consists of the following per 1000 mL: 0.25-0.30 mg KI, 5.5-6.0 mg FeCl3·6H2O, 0.5-0.6 mg H3BO3, 0.5-0.6 mg CuSO4, 0.75-0.80 mg MnSO4·H2O, 0.375-0.400 mg ZnSO4·7H2O, with the remainder being water. The medium is filtered and sterilized before use.

[0044] Preferably, the immobilized Bacillus mucilaginosus agent of the present invention is prepared into a bacterial suspension before immobilization, and the ratio of the bacterial suspension to the modified mushroom residue biochar carrier is 0.5-0.8 ml: 1 g.

[0045] Preferably, the selected microbial agent also includes other excipients acceptable in the field of microbial agents, such as liquid culture medium, filler and carrier.

[0046] Compared with the prior art, the beneficial effects of the present invention are: This invention enables the resource-based recycling of solid waste, especially mushroom residue, with significant cost advantages: using agricultural waste mushroom residue as raw material to prepare a carrier can effectively achieve the "high-value transformation of waste" and effectively solve the environmental pollution problem caused by the random disposal of mushroom residue; compared with traditional adsorption materials such as activated carbon and ion exchange resin, the raw material cost is reduced by more than 50%, and the price of magnesium sulfate modifier is low, which greatly reduces the cost of immobilization preparation and wastewater treatment, making it suitable for large-scale industrial applications.

[0047] The immobilized bacterial agent used in this invention exhibits excellent performance, with strong survival ability of Bacillus mucilaginosus in the obtained immobilized bacterial agent and strong product stability. The mushroom residue biochar modified with magnesium sulfate has a richer pore structure and surface active groups, a larger specific surface area, and better biocompatibility, which can provide sufficient attachment sites and a stable growth microenvironment for Bacillus mucilaginosus. Combined with sodium alginate cross-linking enhancement, the immobilized strain has high mechanical strength and can effectively resist the toxic stress of heavy metal wastewater. Its stability is significantly better than that of free strains and original mushroom residue immobilized strains, and it still maintains a high heavy metal removal efficiency after being reused 3-5 times.

[0048] The immobilized bacterial agent obtained by this invention has high removal efficiency for pollutants, including residual mycelium and heavy metals released from it, with outstanding synergistic effects: the immobilized strains remove heavy metal ions through the synergistic effect of "carrier adsorption + microbial bioadsorption"; the modified mushroom residue biochar can quickly adsorb heavy metal ions through pore interception and functional group complexation; the functional groups on the surface of Bacillus mucilaginosus can further adsorb the remaining heavy metal ions through electrostatic attraction and complexation, achieving "dual adsorption" synergistic effect.

[0049] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions as described in *Molecular Cloning: A Laboratory Manual* by Sambrook J. et al. (translated by Huang Peitang et al., Beijing: Science Press, 2002), or as recommended by the manufacturer. Percentages and parts are by weight unless otherwise stated. Unless otherwise specified, all experimental materials and reagents used in the following embodiments are commercially available.

[0050] Example 1: Preparation of mushroom residue modified biochar and method of magnesium sulfate modification Includes the following steps: Pretreatment of mushroom residue powder: Take fresh oyster mushroom secondary strain residue, remove impurities, dry it in a 70℃ oven until constant weight, pulverize it and pass it through a 60-mesh sieve to obtain mushroom residue powder. Preparation of biochar from raw mushroom residue: The mushroom residue powder was placed in a tube furnace and heated to 500°C at a heating rate of 8°C / min under a nitrogen atmosphere. After pyrolysis for 3 hours, the raw mushroom residue biochar was obtained after cooling. Magnesium sulfate modification of mushroom residue biochar: The original mushroom residue biochar was added to a 1 mol / L magnesium sulfate solution at a solid-liquid ratio of 1:15 (g / mL), and modified by constant temperature shaking at 40℃ for 6 h at a shaking rate of 150 r / min. After modification, it was washed with distilled water until neutral and dried in a 70℃ oven to constant weight to obtain magnesium sulfate modified mushroom residue biochar carrier.

[0051] A schematic diagram of the preparation process of mushroom residue modified biochar is attached. Figure 1 As shown.

[0052] Example 2: Preparation of Bacillus inoculum immobilized on mushroom residue modified biochar Before embedding, the immobilized *Paenibacillus mucilaginosus* strain in mushroom residue must first be activated and cultured. *Paenibacillus mucilaginosus* Kl-1 strain is inoculated into activation medium and cultured with shaking at 37℃ and 180 rpm for 24 hours to obtain an activated bacterial solution. The activated bacterial solution is then inoculated into the medium at a 10% inoculation rate and cultured with shaking for 24 hours under the same conditions to obtain a high-concentration bacterial solution. After centrifugation and filtration, the lower bacterial precipitate is washed three times with sterile water to remove cell growth metabolites and residual culture medium. The solution is then diluted to volume with sterile physiological saline to prepare a bacterial suspension with a concentration of 10% *Paenibacillus mucilaginosus*. 8 CFU / mL or higher.

[0053] The formulation of the culture medium used for activation and expansion culture is as follows: The composition of each 1000 mL culture medium is as follows: 20 g / L sucrose, 1000 mg Na2SO4, 1000 mg NH4Cl, 100 mg CaCl2·2H2O, 2000 mg MgSO4·7H2O, 500 mg KH2PO4, 1000 mg yeast extract, 1000 mg CH3COONa, 500 mg FeSO4·7H2O, 5 mL trace element stock solution, and the remainder is water. The pH is 7.2, and the medium is sterilized at 121 °C for 15 minutes.

[0054] The trace element supplementation culture medium mentioned above is: The composition per 1000 mL is: 0.25 mg KI, 5.5 mg FeCl3·6H2O, 0.6 mg H3BO3, 0.5 mg CuSO4, 0.75 mg MnSO4·H2O, 0.375 mg ZnSO4·7H2O, with the remainder being water. It is used after filtration through a 22 μm filter membrane.

[0055] Dissolve 3.0 g of sodium alginate, 7.0 g of modified mushroom biochar, and 9.0 g of polyvinyl alcohol separately in 40 mL of pure water and mix thoroughly. Then add 5 mL of bacterial suspension. Add pure water to make up to 100 mL and mix the solutions thoroughly to obtain a mixed suspension.

[0056] Dissolve 4.5g of calcium chloride in 100mL of pure water.

[0057] Place the calcium chloride solution on a magnetic stirrer, and add the mixed suspension dropwise into the calcium chloride solution using a disposable syringe to form small spherical immobilized bacteria. Let it stand at room temperature for 24 hours to complete the cross-linking process.

[0058] After cross-linking, discard the calcium chloride solution and rinse the immobilized microspheres with physiological saline to remove impurities and free bacteria from the surface of the microspheres. Immerse the washed microspheres in physiological saline and store them in a refrigerator at 4°C for later use.

[0059] Case Study 3: Application of Mushroom Residue Modified Biochar Immobilized Bacillus subtilis Agent in Pharmaceutical Wastewater Wastewater from a pharmaceutical factory producing norfloxacin was collected, and its water quality indicators were tested. The mushroom residue immobilized Bacillus mucilaginosus agent prepared in Case 2 was added to the wastewater at a dosage of 10 g / L. The pH of the wastewater was adjusted to 7.0, the reaction temperature was controlled at 30 ℃, and the dissolved oxygen content was continuously maintained at 3 mg / L through aeration. A sequencing batch reaction was adopted, and the reaction time was 18 h.

[0060] Subsequently, samples were taken to test the main pollutants in the water, including COD, ammonia nitrogen, and Cu. 2+ Indicators of pollutants such as suspended solids.

[0061] The data before and after treatment with the microbial agent are as follows: After the reaction, the COD of the antibiotic production wastewater decreased from 3200 mg / L before the reaction to 420 mg / L. The ammonia nitrogen content in the water decreased from 120 mg / L before treatment to 22 mg / L; The total suspended solids decreased from 489 mg / L before the reaction to 46 mg / L; Norfloxacin decreased from 3.2 mg / L before the reaction to 0.1 mg / L; Cu 2+The concentration decreased from 30 mg / L before the reaction to 6.2 mg / L.

[0062] After treatment with Bacillus mucilaginosus immobilized bacterial agent, the levels of COD, ammonia nitrogen, total suspended solids, norfloxacin, and Cu in the norfloxacin production wastewater were reduced. 2+ The degradation rates were 86.8%, 81.7%, 90.6%, 96.8%, and 79.3%, respectively. For example... Figure 2 As shown.

[0063] After being reused up to the 5th time, the COD, ammonia nitrogen, total suspended solids, norfloxacin, and Cu in the norfloxacin production wastewater were reduced. 2+ The degradation rates were 79.7%, 76.3%, 86.2%, 90.1%, and 78.6%, respectively.

[0064] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A mushroom residue immobilized Bacillus mucilaginosus embedding inoculant, characterized by, The mushroom residue immobilized gelatinous Bacillus agent includes an immobilization carrier made from mushroom residue and gelatinous Bacillus embedded in the immobilization carrier.

2. The bacteria-embedded agent of claim 1, wherein The method for preparing the immobilized carrier includes the following steps: (1) Take mushroom residue, dry it to constant weight, crush it to obtain mushroom residue powder, place the mushroom residue powder in a tube furnace, heat it to 400-600℃ at a heating rate of 5-10℃ / min under nitrogen atmosphere, pyrolyze for 2-4 h, cool it to room temperature and take it out to obtain mushroom residue biochar. (2) Add mushroom residue biochar to a 0.5-2 mol / L magnesium sulfate solution with a solid-liquid ratio of 1:10-1:20 (g / mL) and modify it by constant temperature shaking at 30-50℃ for 4-8 h at a shaking rate of 120-180 r / min. After modification, wash with distilled water until neutral and dry to constant weight to obtain the immobilized carrier.

3. The encapsulating agent as described in claim 2, characterized in that, The preparation method of the encapsulated bacterial agent includes the following steps: a. Disperse sodium alginate, polyvinyl alcohol and immobilization carrier separately in pure water, then add bacterial suspension and mix thoroughly to obtain mixed suspension; b. Prepare a calcium chloride solution by dissolving calcium chloride in pure water; c. Place the calcium chloride solution on a magnetic stirrer, and add the mixed suspension dropwise into the calcium chloride solution. Let it stand at room temperature for 24-36 h to allow cross-linking. d. After cross-linking is completed, the immobilized microspheres are rinsed with physiological saline to remove impurities and free bacteria from the surface of the microspheres, thereby obtaining the encapsulated bacterial agent.

4. The encapsulating agent as described in claim 3, characterized in that, In step a, the weight-to-volume ratio of sodium alginate, polyvinyl alcohol, immobilization carrier and bacterial suspension is 2.0-5.0 (w): 8.0-10.0 (w): 6.0-10.0 (w): 5 (v).

5. The encapsulating agent as described in claim 3, characterized in that, In step b, the mass concentration of the calcium chloride solution is 4%-5%.

6. The encapsulating agent as described in claim 3, characterized in that, The concentration of Bacillus mucilaginosus in the bacterial suspension is 10 8 -10 9 CFU / mL.

7. The encapsulating agent as described in claim 1, characterized in that, The Bacillus colloidis is Bacillus colloidis Kl-1, with accession number CCTCC No. M 2021439.

8. The application of the mushroom residue immobilized Bacillus mucilaginosus encapsulation agent as described in claim 1 in pharmaceutical wastewater treatment.

9. The application as described in claim 8, characterized in that, The pharmaceutical wastewater is antibiotic pharmaceutical wastewater, chemically synthesized pharmaceutical wastewater, or traditional Chinese medicine pharmaceutical wastewater.

10. A method for treating pharmaceutical wastewater using the mushroom residue immobilized Bacillus mucilaginosus encapsulation agent as described in claim 1, characterized in that, The method includes the following steps: The mushroom residue immobilized Bacillus mucilaginosus encapsulation agent was added to pharmaceutical wastewater, the pH of the wastewater was adjusted to 6.5-8.0, the reaction temperature was controlled at 25-35℃, the dissolved oxygen content was 2-5 mg / L, and the reaction time was 12-24 h.

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