A composite microbial agent, a preparation method thereof and application thereof in degradation of organic waste

Abstract

The application belongs to the technical field of microorganisms, and particularly relates to a compound microbial agent, a preparation method thereof and application of the compound microbial agent in degradation of organic waste. The compound microbial agent comprises an active component composed of 11 specific strains and a porous carrier modified by beta-cyclodextrin, and the carrier has a hydrophilic-hydrophobic gradient. In the active component, Saccharomyces cerevisiae is responsible for low-temperature start of fermentation, six Bacillus spp. are used for synergistic degradation of protein and starch, Lactobacillus plantarum is used for acid production and bacterium inhibition, Aspergillus niger is used for decomposition of grease, and two Trichoderma spp. are used for degradation of fiber and whole-process bacterium inhibition, so as to form a complete degradation chain of segmented relay. The carrier forms a grafting density gradient by immersing one end in a beta-cyclodextrin solution, so that bacteria and fungi are planted on the hydrophilic end and the hydrophobic end, respectively. The compound microbial agent can be applied to the treatment of garbage transfer stations, septic tanks, oil separation tanks and toilet fecal pollution, and effectively solves problems such as low-temperature start difficulty, long degradation period, incomplete harmless treatment and difficult elimination of odor.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a composite microbial agent, its preparation method, and its application in the degradation of organic waste. Background Technology

[0002] With rapid urbanization and continuous population growth, the amount of organic waste generated by garbage transfer stations, septic tanks, grease traps, and other similar facilities is increasing daily. This type of waste is high in organic matter and complex in composition. Improper treatment not only occupies a large amount of space but also produces malodorous gases such as ammonia and hydrogen sulfide, seriously affecting the surrounding environmental quality and residents' lives. Microbial degradation technology, due to its environmental friendliness, ease of operation, and relatively low treatment costs, is gradually becoming an important approach for organic waste treatment.

[0003] Currently, numerous studies have applied composite microbial agents to the degradation and treatment of organic waste. For example, Chinese patent CN104193430A discloses a straw composting agent composed of Bacillus subtilis, Saccharomyces cerevisiae, Aspergillus niger, and Trichoderma longifolia, used for the composting of agricultural waste such as straw. Chinese patent CN105733975A discloses a multifunctional and efficient organic material composting agent containing Bacillus amyloliquefaciens, Aspergillus oryzae, Trichoderma viride, and Saccharomyces cerevisiae, which can be used for the fermentation and transformation of various organic materials such as livestock and poultry manure, crop straw, and kitchen waste. However, these existing agents generally suffer from the problem of a limited number of strains and incomplete functional coverage. Taking CN104193430A as an example, it only uses four strains, and its degradation function is mainly concentrated in limited areas such as cellulose decomposition and starch decomposition, with insufficient ability to degrade other types of organic matter such as proteins, oils, and pectins. In practical applications, organic waste is often a mixture of multiple components, and it is difficult to achieve comprehensive and thorough degradation with only a few strains.

[0004] The difficulty of starting up under low-temperature conditions is another long-standing technical challenge in this field. In northern my country and most other regions, the autumn and winter seasons are characterized by prolonged periods of low temperatures. When the ambient temperature drops below 20°C, composting or degradation processes require a start-up phase from low temperature to warm-up, which is crucial for the mass reproduction of microorganisms and the initiation of the degradation process. Most existing microbial agents exhibit significantly reduced activity under low-temperature conditions, leading to longer processing cycles, reduced efficiency, and even material stagnation and failure to decompose. Although some studies have attempted to address this problem by screening for low-temperature resistant strains, these studies are often limited to single-function low-temperature degrading bacteria, lacking consideration for the simultaneous degradation of multiple organic substances.

[0005] Furthermore, the functions of existing microbial carriers are relatively limited. Most carriers only serve to adsorb and carry microbial strains, lacking functional designs to maintain strain activity and assist in the degradation process. The carrier itself does not participate in the degradation reaction, and the microbial strains require a long adaptation period before they can take effect, thus affecting treatment efficiency.

[0006] Therefore, it is evident that developing a composite microbial agent with a more comprehensive strain configuration, rapid activation under low-temperature conditions, and carrier with auxiliary degradation function is of great significance for solving the problems of slow activation, long cycle, and incomplete degradation in the existing organic waste treatment. Summary of the Invention

[0007] The purpose of this invention is to provide a composite microbial agent, its preparation method, and its application in the degradation of organic waste. By screening a variety of functional strains in specific combinations and using a porous carrier with auxiliary degradation function, the agent can quickly start fermentation under low temperature conditions, achieve comprehensive degradation of various organic matter, effectively shorten the treatment cycle, and reduce the generation of malodor.

[0008] To achieve the above objectives, the present invention provides the following technical solution: The first aspect of this invention provides a composite microbial agent, comprising an active component and a carrier, wherein the active component is composed of the following bacterial species: Bacillus licheniformis (… Bacillus licheniformis Lactobacillus plantarum ( Lactobacillus plantarum ), gelatinous spore-forming bacteria ( Paenibacillus mucilaginosus ), Polymyxin Bacillus ( Paenibacillus polymyxa ), Bacillus retroflexus ( Brevibacillus laterite Bacillus subtilis ( Bacillus subtilis ), Bacillus pumilus ( Bacillus pumilus Trichoderma harzianum ( Trichoderma harzianum ), Trichoderma acicularis ( Trichoderma asperellum Aspergillus niger ( ) Aspergillus niger ), brewer's yeast ( Saccharomyces cerevisiae ); The carrier is a β-cyclodextrin-modified porous carrier with a hydrophilic-hydrophobic gradient.

[0009] Furthermore, the β-cyclodextrin-modified porous support is prepared by a method comprising the following steps: (a) Chitosan and polyvinyl alcohol are dissolved in an acetic acid solution with a mass concentration of 1-3% and then freeze-dried to prepare a porous carrier; (b) Dissolve β-cyclodextrin in water to prepare a β-cyclodextrin solution; (c) Immerse one end of the porous carrier in a β-cyclodextrin solution and use capillary action to allow the solution to permeate along the carrier channels from the immersed end to the unimmersed end, forming a concentration gradient of β-cyclodextrin inside the carrier. Then add a crosslinking agent to the solution. (d) Heat to 50-70℃ to carry out the grafting reaction. After the reaction is complete, remove the support, wash it, and dry it to obtain the final product.

[0010] Further, the mass ratio of chitosan to polyvinyl alcohol in step (a) is (2-5):1.

[0011] Further, the mass concentration of the β-cyclodextrin solution in step (b) is 8-15%.

[0012] Further, the crosslinking agent in step (c) is glutaraldehyde, and the amount of glutaraldehyde added is 5-20% of the mass of β-cyclodextrin.

[0013] Furthermore, the immersion length in step (c) is 1 / 3 to 1 / 2 of the total length of the carrier, and the immersion time is 4-8 hours.

[0014] Furthermore, based on the total mass of the active components, the effective viable count of *Bacillus licheniformis* is 11-30 billion / g, the effective viable count of *Lactobacillus plantarum* is above 9.2 billion / g, the effective viable count of *Bacillus jellyii* is above 2.6 billion / g, the effective viable count of *Bacillus polymyxa* is above 12 billion / g, the effective viable count of *Bacillus laterosporus* is above 5 billion / g, the effective viable count of *Bacillus subtilis* is above 5 billion / g, the effective viable count of *Bacillus pumilus* is above 11 billion / g, the effective viable count of *Trichoderma harzianum* is above 5.6 billion / g, the effective viable count of *Trichoderma echinospora* is 5.6-11 billion / g, the effective viable count of *Aspergillus niger* is above 10 billion / g, and the effective viable count of *Saccharomyces cerevisiae* is above 12 billion / g.

[0015] Furthermore, the weight ratio of the carrier to the active component is (5-15):1.

[0016] Saccharomyces cerevisiae initially decomposes sugars and generates heat at low temperatures, causing the material to heat up initially. As the temperature rises, hydrolytic enzymes such as proteases and amylases secreted by six Bacillus species hydrolyze proteins and starches into smaller organic molecules, releasing a large amount of metabolic heat and driving up the pile temperature. The high-temperature environment can kill pathogens and insect eggs. During fermentation, Lactobacillus plantarum lowers the pH value of the material by producing lactic acid, inhibiting the reproduction of putrefactive and gas-producing bacteria and reducing the release of ammonia and hydrogen sulfide. Aspergillus niger continuously secretes lipases and pectins during the temperature maintenance and settling stages, decomposing oils and pectin-like substances. In the later stages of fermentation, Trichoderma echinococcus and Trichoderma harzianum secrete cellulase systems to finally degrade the residual fibrous substances in the material, while simultaneously secreting antibacterial substances to inhibit the growth of putrefactive bacteria. The four types of microbial communities have clear functional divisions and are sequential in time, corresponding to different temperature stages and different organic matter components, forming a complete degradation chain. The hydrophilic-hydrophobic gradient of the β-cyclodextrin-modified carrier enables bacteria to spontaneously colonize the hydrophilic end and fungi to colonize the hydrophobic end, reducing interspecies competition. The same carrier particle becomes a seed source pool for the continuous release of multiple bacterial species. The β-cyclodextrin cavity also enriches hydrophobic organic matter such as lipids, forming a local high-concentration microenvironment around the bacterial species and shortening the adaptation period of the species.

[0017] A second aspect of this invention provides a method for preparing the above-mentioned composite microbial agent, comprising the following steps: (1) Bacillus licheniformis, Lactobacillus plantarum, Bacillus jellyoidis, Bacillus polymyxa, Bacillus brevicaulis, Bacillus subtilis, Bacillus brevicaulis, Trichoderma harzianum, Trichoderma echinosporum, Aspergillus niger, and Saccharomyces cerevisiae were inoculated into a culture medium for fermentation culture to obtain fermentation broth of each strain, and after drying, they were made into single strain powders. (2) Mix the bacterial powders obtained in step (1) in proportion to obtain the active components; (3) Add the active component to sterile water at 2-5 times its mass and stir to prepare a bacterial suspension; add the β-cyclodextrin modified porous carrier to the bacterial suspension and stir at 4-25℃ for 2-6 hours to allow the bacterial strain to be loaded onto the carrier. After drying, the composite microbial agent is obtained.

[0018] The third aspect of this invention provides the application of the above-mentioned composite microbial agent in the degradation of organic waste.

[0019] Furthermore, the organic waste includes at least one of the following: garbage from a garbage transfer station, septic tank waste, grease trap grease, and toilet waste.

[0020] Furthermore, the application includes directly adding or diluting the compound microbial agent and spraying it onto organic waste.

[0021] Furthermore, when used in garbage transfer stations, the compound microbial agent is diluted with water at a ratio of 1:(10-50) and sprayed evenly on the surface of the garbage, with a dosage of 0.05-0.2 kg of agent per cubic meter of garbage; when used in septic tanks, the compound microbial agent is directly added to the septic tank, with a dosage of 0.05-0.2 kg of agent per cubic meter of fecal sludge, added 1-4 times per month; when used in grease traps, the compound microbial agent is diluted with water at a ratio of 1:(10-50) and injected into the grease trap, with a dosage of 0.1-0.5 kg of agent per cubic meter of oil sludge, added 1-2 times per week; when used in toilet sludge, the compound microbial agent is directly added or diluted and added to the toilet sewage system, with a dosage of 0.1-0.5 kg of agent per ton of fecal sludge.

[0022] Compared with the prior art, the advantages and beneficial effects of the present invention are as follows: This invention employs a composite system composed of 11 functional bacterial strains, covering the entire degradation chain from low to high temperatures, from aerobic to anaerobic processes, and from easily degradable substances to stubborn macromolecules, thus solving the problem of narrow functional coverage in traditional single-strain or limited-strain bacterial agents. The hydrophilic-hydrophobic gradient structure within the carrier allows bacteria with different surface properties to automatically migrate and colonize to the most suitable area during adsorption, avoiding spatial competition between strains and improving the synergistic efficiency of the bacterial community per unit carrier. The inclusion and enrichment effect of β-cyclodextrin molecules on hydrophobic substrates such as oils effectively creates a substrate reserve layer around the bacteria, enabling the bacterial agent to skip the lengthy substrate adaptation period and directly enter a highly efficient degradation state after addition. Practical engineering applications have verified the effectiveness of this invention in the treatment of septic tanks and toilets in large transportation hubs. After treatment, the concentrations of ammonia, hydrogen sulfide, and methane all decreased significantly, indicating that this bacterial agent has practical application value in degrading organic matter, eliminating odors, and reducing greenhouse gas emissions. Detailed Implementation

[0023] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] Unless otherwise specified, all raw materials used in the embodiments are commercially available products. The following sources are illustrative examples.

[0025] Bacillus licheniformis was purchased from the China General Microbiological Culture Collection Center (CGMCC), catalog number: CGMCC 1.3649; Bacillus subtilis was purchased from the China General Microbiological Culture Collection Center (CGMCC), catalog number: CGMCC 1.2166; Bacillus pumilus was purchased from the China General Microbiological Culture Collection Center (CGMCC), catalog number: CGMCC 1.1847; Bacillus laterosporus was purchased from the China Industrial Microbiological Culture Collection Center (CICC), catalog number: CICC 24775; Bacillus polymyxa was purchased from the China Industrial Microbiological Culture Collection Center (CICC), catalog number: CICC 21781; Bacillus gelatinosa was purchased from the China General Microbiological Culture Collection Center (CGMCC), catalog number: CGMCC 1.232; Lactobacillus plantarum was purchased from the China General Microbiological Culture Collection Center (CGMCC), catalog number: CGMCC 1.1856; *Trichoderma harzianum* was purchased from the China General Microbiological Culture Collection Center (CGMCC), number: CGMCC3.1541; *Trichoderma echinococcus* was purchased from the China Industrial Microbiological Culture Collection Center (CICC), number: CICC 40993; *Aspergillus niger* was purchased from the China General Microbiological Culture Collection Center (CGMCC), number: CGMCC 3.11598; *Saccharomyces cerevisiae* was purchased from the China Industrial Microbiological Culture Collection Center (CICC), number: CICC 30015; *Candida utilis* was purchased from the China Industrial Microbiological Culture Collection Center (CICC), number: CICC 1769; *Trichoderma reesei* was purchased from the China General Microbiological Culture Collection Center (CGMCC), number: CGMCC 3.3711.

[0026] Example 1 This embodiment provides a composite microbial agent, the preparation method of which includes the following steps: (1) Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, Bacillus pumilus laterosporus, Bacillus polymyxa, and Bacillus pumilus were inoculated into nutrient broth medium (10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, pH 7.0) and cultured at 37℃ and 180 r / min for 36 hours with shaking. Lactobacillus plantarum was inoculated into MRS medium (10 g / L casein peptone, 10 g / L beef extract, 5 g / L yeast extract, 5 g / L glucose, 5 g / L sodium acetate, 2 g / L diammonium citrate, 1 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, pH 6.8) and incubated statically at 37°C for 36 hours. Trichoderma harzianum, Trichoderma echinococcus, and Aspergillus niger were inoculated into potato glucose medium (1 L potato extract, 20 g glucose, pH natural) and cultured at 28 °C and 160 r / min for 72 hours with shaking. Saccharomyces cerevisiae was inoculated into YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) and cultured at 30℃ and 180 r / min for 36 hours with shaking.

[0027] After the above cultivation was completed, the fermentation broth of each strain was collected, and after centrifugation and washing, the bacterial sludge of each strain was obtained. After low-temperature drying, it was made into single strain bacterial powder.

[0028] (2) Mix the bacterial powders of each strain evenly according to the mass ratio to obtain the active component. The effective viable count of Bacillus licheniformis in the active component was 28 billion / g, the effective viable count of Lactobacillus plantarum was 9.8 billion / g, the effective viable count of Bacillus jellyoidus was 3.2 billion / g, the effective viable count of Bacillus polymyxa was 13.5 billion / g, the effective viable count of Bacillus brevis was 7.2 billion / g, the effective viable count of Bacillus subtilis was 5.5 billion / g, the effective viable count of Bacillus brevis was 11.8 billion / g, the effective viable count of Trichoderma harzianum was 6.8 billion / g, the effective viable count of Trichoderma echinospora was 8.5 billion / g, the effective viable count of Aspergillus niger was 11.5 billion / g, and the effective viable count of Saccharomyces cerevisiae was 14.2 billion / g.

[0029] (3) Add the active component to sterile water in a volume of 3 times its weight and stir to prepare a bacterial suspension; add the β-cyclodextrin-modified porous carrier to the bacterial suspension and stir at 40 r / min at 15℃ for 4 hours to adsorb the bacteria onto the carrier. The weight ratio of the carrier to the active component is 10:1. After adsorption, vacuum dry the carrier loaded with bacteria at 35℃ until the water content is less than 8%, then pulverize and sieve to obtain the composite microbial agent.

[0030] The β-cyclodextrin-modified porous support is prepared according to the following method: (a) Chitosan and polyvinyl alcohol were mixed at a mass ratio of 3:1 and dissolved in a 2% acetic acid solution to prepare a mixed solution with a total solids mass concentration of 2%. After stirring until completely dissolved, the mixture was allowed to stand to remove bubbles, poured into a mold, pre-frozen at -40°C for 12 hours, and then transferred to a vacuum freeze dryer and freeze-dried at a vacuum degree of 10 Pa for 48 hours to obtain a porous carrier. (b) Dissolve β-cyclodextrin in water to prepare a β-cyclodextrin solution with a mass concentration of 12%; (c) Cut the porous carrier obtained in step (a) into rectangular strips, immerse half of the strip lengthwise into the β-cyclodextrin solution obtained in step (b), and let it stand at 25°C for 6 hours. Use capillary action to allow the solution to permeate along the carrier pores from the immersed end to the unimmersed end, forming a concentration gradient of β-cyclodextrin inside the carrier. Then add glutaraldehyde to the solution, with the amount of glutaraldehyde added being 10% of the mass of β-cyclodextrin. (d) Heat to 60℃ and carry out the grafting reaction for 4 hours. After the reaction is completed, take out the support, wash it three times with deionized water, and dry it under vacuum at 40℃ to constant weight to obtain the β-cyclodextrin modified porous support.

[0031] Example 2 This embodiment provides a composite microbial agent, the preparation method of which includes the following steps: (1) Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, Bacillus pumilus, Bacillus polymyxa, and Bacillus pumilus were inoculated into nutrient broth medium (10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, pH 7.0) and cultured with shaking at 35℃ and 160 r / min for 40 hours. Lactobacillus plantarum was inoculated into MRS medium (10 g / L casein peptone, 10 g / L beef extract, 5 g / L yeast extract, 5 g / L glucose, 5 g / L sodium acetate, 2 g / L diammonium citrate, 1 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, pH 6.8) and incubated statically at 35°C for 40 hours. Trichoderma harzianum, Trichoderma echinococcus, and Aspergillus niger were inoculated into potato glucose medium (1 L potato extract, 20 g glucose, pH natural) and cultured at 26 °C and 150 r / min for 80 hours with shaking. Saccharomyces cerevisiae was inoculated into YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) and cultured at 29℃ and 170 r / min for 40 hours with shaking.

[0032] After the above cultivation was completed, the fermentation broth of each strain was collected, and after centrifugation and washing, the bacterial sludge of each strain was obtained. After low-temperature drying, it was made into single strain bacterial powder.

[0033] (2) Mix the bacterial powders of each strain evenly according to the mass ratio to obtain the active component. The effective viable count of Bacillus licheniformis in the active component was 21.8 billion / g, the effective viable count of Lactobacillus plantarum was 10.2 billion / g, the effective viable count of Bacillus jellyoidus was 4.3 billion / g, the effective viable count of Bacillus polymyxa was 14.7 billion / g, the effective viable count of Bacillus lateralis was 6.2 billion / g, the effective viable count of Bacillus subtilis was 5.8 billion / g, the effective viable count of Bacillus pumilus was 13.3 billion / g, the effective viable count of Trichoderma harzianum was 7.8 billion / g, the effective viable count of Trichoderma echinospora was 6.8 billion / g, the effective viable count of Aspergillus niger was 12.8 billion / g, and the effective viable count of Saccharomyces cerevisiae was 15.2 billion / g.

[0034] (3) Add the active component to sterile water at 4 times its weight and stir to prepare a bacterial suspension; add the β-cyclodextrin-modified porous carrier to the bacterial suspension and stir at 35 r / min at 10℃ for 5 hours to adsorb the bacteria onto the carrier. The weight ratio of the carrier to the active component is 12:1. After adsorption, vacuum dry the carrier loaded with bacteria at 32℃ until the water content is less than 8%, then pulverize and sieve to obtain the composite microbial agent.

[0035] The β-cyclodextrin-modified porous support is prepared according to the following method: (a) Chitosan and polyvinyl alcohol were mixed at a mass ratio of 4:1 and dissolved in a 1.5% acetic acid solution to prepare a mixed solution with a total solids mass concentration of 2.5%. After stirring until completely dissolved, the mixture was allowed to stand to remove bubbles, poured into a mold, pre-frozen at -45°C for 14 hours, and then transferred to a vacuum freeze dryer and freeze-dried at a vacuum degree of 8 Pa for 44 hours to obtain a porous carrier. (b) Dissolve β-cyclodextrin in water to prepare a 10% (w / w) β-cyclodextrin solution; (c) Cut the porous carrier obtained in step (a) into rectangular strips, immerse 2 / 5 of the length of the strips into the β-cyclodextrin solution obtained in step (b), and let it stand at 28°C for 5 hours. Use capillary action to allow the solution to permeate along the carrier channels from the immersed end to the unimmersed end, forming a concentration gradient of β-cyclodextrin inside the carrier. Then add glutaraldehyde to the solution, with the amount of glutaraldehyde added being 15% of the mass of β-cyclodextrin. (d) Heat to 55℃ and carry out the grafting reaction for 5 hours. After the reaction is completed, take out the support, wash it three times with deionized water, and dry it under vacuum at 38℃ to constant weight to obtain the β-cyclodextrin modified porous support.

[0036] Example 3 This embodiment provides a composite microbial agent, the preparation method of which includes the following steps: (1) Bacillus licheniformis, Bacillus subtilis, Bacillus pumilus, Bacillus pumilus, Bacillus polymyxa, and Bacillus pumilus were inoculated into nutrient broth medium (10 g / L peptone, 3 g / L beef extract, 5 g / L sodium chloride, pH 7.0) and cultured at 30℃ and 200 r / min for 30 hours with shaking. Lactobacillus plantarum was inoculated into MRS medium (10 g / L casein peptone, 10 g / L beef extract, 5 g / L yeast extract, 5 g / L glucose, 5 g / L sodium acetate, 2 g / L diammonium citrate, 1 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, pH 6.8) and incubated statically at 30°C for 48 hours. Trichoderma harzianum, Trichoderma echinococcus, and Aspergillus niger were inoculated into potato glucose medium (1 L potato extract, 20 g glucose, pH natural) and cultured at 30 °C and 180 r / min for 64 hours with shaking. Saccharomyces cerevisiae was inoculated into YPD medium (10 g / L yeast extract, 20 g / L peptone, 20 g / L glucose) and cultured at 28℃ and 190 r / min for 48 hours with shaking.

[0037] After the above cultivation was completed, the fermentation broth of each strain was collected, and after centrifugation and washing, the bacterial sludge of each strain was obtained. After low-temperature drying, it was made into single strain bacterial powder.

[0038] (2) Mix the bacterial powders of each strain evenly according to the mass ratio to obtain the active component. The effective viable count of Bacillus licheniformis in the active component was 16.3 billion / g, the effective viable count of Lactobacillus plantarum was 11.8 billion / g, the effective viable count of Bacillus jellyoidus was 4.8 billion / g, the effective viable count of Bacillus polymyxa was 14.2 billion / g, the effective viable count of Bacillus brevis was 7.7 billion / g, the effective viable count of Bacillus subtilis was 7.2 billion / g, the effective viable count of Bacillus brevis was 13.8 billion / g, the effective viable count of Trichoderma harzianum was 8.7 billion / g, the effective viable count of Trichoderma echinospora was 9.3 billion / g, the effective viable count of Aspergillus niger was 11.3 billion / g, and the effective viable count of Saccharomyces cerevisiae was 13.6 billion / g.

[0039] (3) Add the active component to 2.5 times its weight of sterile water and stir to prepare a bacterial suspension; add the β-cyclodextrin-modified porous carrier to the bacterial suspension and stir at 50 r / min at 20℃ for 3 hours to adsorb the bacteria onto the carrier. The weight ratio of the carrier to the active component is 7:1. After adsorption, vacuum dry the carrier loaded with bacteria at 38℃ until the water content is less than 8%, then pulverize and sieve to obtain the composite microbial agent.

[0040] The β-cyclodextrin-modified porous support is prepared according to the following method: (a) Chitosan and polyvinyl alcohol were mixed at a mass ratio of 2.5:1 and dissolved in a 2.5% acetic acid solution to prepare a mixed solution with a total solids mass concentration of 1.8%. After stirring until completely dissolved, the mixture was allowed to stand to remove bubbles, poured into a mold, pre-frozen at -38°C for 10 hours, and then transferred to a vacuum freeze dryer and freeze-dried at a vacuum degree of 12 Pa for 52 hours to obtain a porous carrier. (b) Dissolve β-cyclodextrin in water to prepare a β-cyclodextrin solution with a mass concentration of 14%; (c) Cut the porous carrier obtained in step (a) into rectangular strips, immerse 3 / 7 of the length of the strips into the β-cyclodextrin solution obtained in step (b), and let it stand at 22°C for 7 hours. Use capillary action to allow the solution to permeate along the carrier channels from the immersed end to the unimmersed end, forming a concentration gradient of β-cyclodextrin inside the carrier. Then add glutaraldehyde to the solution, with the amount of glutaraldehyde added being 8% of the mass of β-cyclodextrin. (d) Heat to 65℃ and carry out the grafting reaction for 3.5 hours. After the reaction is completed, take out the support, wash it three times with deionized water, and dry it under vacuum at 35℃ to constant weight to obtain the β-cyclodextrin modified porous support.

[0041] Comparative Example 1 The difference between this comparative example and Example 1 is that the β-cyclodextrin-modified porous carrier was prepared according to the following method: (a) Chitosan and polyvinyl alcohol were mixed at a mass ratio of 3:1 and dissolved in a 2% acetic acid solution to prepare a mixed solution with a total solids mass concentration of 2%. After stirring until completely dissolved, the mixture was allowed to stand to remove bubbles, poured into a mold, pre-frozen at -40°C for 12 hours, and then transferred to a vacuum freeze dryer and freeze-dried at a vacuum degree of 10 Pa for 48 hours to obtain a porous carrier. (b) Dissolve β-cyclodextrin in water to prepare a 12% (w / w) β-cyclodextrin solution, and add glutaraldehyde in an amount equal to 10% of the mass of β-cyclodextrin. (c) Immerse the porous support obtained in step (a) completely in the solution obtained in step (b) and soak it at 25°C for 6 hours to allow β-cyclodextrin and glutaraldehyde to fully penetrate into the pores of the support. (d) Heat to 60℃ and carry out the grafting reaction for 4 hours. After the reaction is completed, take out the support, wash it three times with deionized water, and dry it under vacuum at 40℃ to constant weight to obtain the β-cyclodextrin modified porous support.

[0042] The remaining steps are the same as in Example 1.

[0043] Comparative Example 2 The difference between this comparative example and Example 1 is that the β-cyclodextrin-modified porous carrier is replaced with a chitosan-polyvinyl alcohol porous carrier. The preparation method of the chitosan-polyvinyl alcohol porous carrier is as follows: Chitosan and polyvinyl alcohol were mixed at a mass ratio of 3:1 and dissolved in a 2% acetic acid solution to prepare a mixed solution with a total solids concentration of 2%. After stirring until completely dissolved, the solution was allowed to stand to remove bubbles, poured into a mold, pre-frozen at -40℃ for 12 hours, and then transferred to a vacuum freeze dryer and freeze-dried at a vacuum degree of 10 Pa for 48 hours to obtain a chitosan-polyvinyl alcohol porous carrier.

[0044] The remaining steps are the same as in Example 1.

[0045] Comparative Example 3 The difference between this comparative example and Example 1 is that the β-cyclodextrin-modified porous support was replaced with diatomaceous earth. Commercially available diatomaceous earth was dried to constant weight at 105°C and used directly as a support after cooling.

[0046] The remaining steps are the same as in Example 1.

[0047] Comparative Example 4 The difference between this comparative example and Example 1 is that the brewer's yeast was replaced with Candida utilis (…). Useful Candida The fermentation culture of *Candida utilis* was carried out on YPD medium at 28°C and 180 r / min for 36 hours with shaking. The effective viable count of *Candida utilis* in the active component was determined to be 19.5 billion CFU / g.

[0048] The remaining steps are the same as in Example 1.

[0049] Comparative Example 5 The difference between this comparative example and Example 1 is that Bacillus jelly-like and Bacillus lateralis are omitted.

[0050] Comparative Example 6 The difference between this comparative example and Example 1 is that *Trichoderma harzianum* and *Trichoderma echinococcus* were replaced with *Trichoderma reesei*. Trichoderma reesei The fermentation culture of *Trichoderma reesei* was carried out on potato dextrose medium at 28°C and 160 r / min for 72 hours with shaking. The effective viable count of *Trichoderma reesei* in the active component was determined to be 15 billion CFU / g.

[0051] The remaining steps and conditions are the same as in Example 1.

[0052] Performance testing The simulated organic waste used in the performance tests was a mixture of kitchen waste and straw at a mass ratio of 7:3, with the moisture content adjusted to 60%. The simulated manure was a mixture of well-rotted cow manure and deionized water at a mass ratio of 1:3. After stirring thoroughly, the mixture was allowed to stand for 24 hours, filtered to remove large particles, and the supernatant was collected for later use. The same simulated organic waste and simulated manure were used in all examples and comparative tests, with a microbial agent dosage of 0.1 kg / m³. 3 .

[0053] 1. Time required to heat to 40℃ Simulated organic waste was placed into a composting reactor, and the test microbial agent was added. After thorough mixing, the reactor was placed in a 10°C constant temperature chamber. The temperature at the center of the compost pile was monitored in real time using a temperature probe, and the time (in hours) required for the pile to heat up from 10°C to 40°C was recorded. Three parallel samples were set up for each group, and the average value was taken.

[0054] 2. Organic matter degradation rate Simulated organic waste was placed in a composting reactor, and the test inoculant was added. After mixing thoroughly, the mixture was fermented at room temperature for 15 days. After fermentation, the organic matter content of the fermented material was determined. The organic matter content was determined using the ignition method: the sample was dried to constant weight at 105℃, and a dried sample was placed in a muffle furnace and ignited at 550℃ for 4 hours. The organic matter content was calculated based on the mass difference before and after ignition. The organic matter degradation rate was calculated using the following formula: Organic matter degradation rate (%) = (Initial organic matter content - Organic matter content after fermentation) / Initial organic matter content × 100%. Three parallel samples were set up for each group, and the average value was taken.

[0055] 3. Ammonia removal rate Simulated fecal waste was placed in a sealed container, and the test bacterial agent was added. The container was treated at 25°C for 7 days. The ammonia concentration in the container was measured before and after treatment. The ammonia removal rate was calculated using the following formula: Ammonia removal rate (%) = (Initial concentration - Post-treatment concentration) / Initial concentration × 100%. Three parallel samples were set up for each group, and the average value was taken.

[0056] 4. Hydrogen sulfide removal rate Simulated fecal waste was placed in a sealed container, and the test bacterial agent was added. The container was treated at 25°C for 7 days. The hydrogen sulfide concentration in the container was measured before and after treatment. The hydrogen sulfide removal rate was calculated using the following formula: Hydrogen sulfide removal rate (%) = (Initial concentration - Post-treatment concentration) / Initial concentration × 100%. Three parallel samples were set up for each group, and the average value was taken.

[0057] 5. Seed Germination Index (GI) The material fermented for 15 days in the organic matter degradation rate test was used as a sample. The sample was mixed with deionized water at a ratio of 1:10 (w / v), and extracted by shaking for 2 hours. The extract was then filtered. 10 mL of the extract was added to a petri dish lined with filter paper, and 20 cabbage seeds were evenly placed on each dish. The dishes were incubated in the dark at 25℃ for 72 hours, with deionized water as a blank control. After incubation, the germination rate of the seeds in each petri dish was recorded, and the root length was measured. The seed germination index was calculated using the following formula: Seed germination index (%) = (germination rate of treatment group × root length of treatment group) / (germination rate of control group × root length of control group) × 100%. Three parallel samples were set up for each group, and the average value was taken.

[0058] The test results are shown in Table 1.

[0059] Table 1 Performance Test Results The performance test results above show that Examples 1-3 performed well in all four dimensions: heating rate, organic matter degradation, odor removal, and maturity. In Comparative Example 1, the carrier lacked a hydrophilic-hydrophobic gradient, leading to random distribution and competition between bacteria and fungi on the carrier surface, resulting in a significant decrease in both degradation and deodorization effects. In Comparative Example 2, the carrier was not modified with β-cyclodextrin, and its function was limited to physical adsorption, resulting in a significant reduction in all indicators. In Comparative Example 3, a conventional diatomaceous earth carrier was used, which lacked sufficient protection and enrichment capabilities for microorganisms, leading to a significant decrease in all performance indicators. In Comparative Example 4, replacing *Saccharomyces cerevisiae* with *Candida utilis* significantly slowed the heating rate and reduced low-temperature start-up capability. In Comparative Example 5, the reduction in the number of *Bacillus* species resulted in an incomplete enzyme system, leading to a decrease in both degradation rate and deodorization effect. In Comparative Example 6, replacing two *Trichoderma* species with a single *Trichoderma reesei*, while maintaining acceptable cellulose degradation capability, lacked overall antibacterial function, increasing the risk of contaminant growth in the later stages of fermentation and significantly reducing maturity.

[0060] Application examples The composite microbial agent prepared in Example 1 of this invention was used for the treatment of septic tanks and toilet sewage in a large transportation hub in Shenzhen.

[0061] Septic tank treatment method: A static treatment method is used, where the bacterial agent is directly added to the septic tank. The initial application rate is 0.15 kg / m³. 3 Add the initial dose, then supplement with 1-2 doses per month, each time at half the initial dosage. A test report from a third-party testing agency showed that the ammonia concentration in the septic tank before treatment was 1.02 mg / m³. 3 After treatment, the concentration dropped to 0.08 mg / m³. 3 The removal rate was 92.2%; hydrogen sulfide was reduced from 0.011 mg / m³. 3 The odor concentration decreased to undetectable levels, achieving a removal rate of 100%; the odor concentration decreased from 55 dimensionless to below 10 dimensionless, achieving a removal rate of 81.8%; and the methane concentration decreased from 1620 mg / m³. 3 Decreased to 1.68 mg / m³ 3 The removal rate was 99.9%.

[0062] Bathroom treatment method: A dynamic treatment approach is adopted. The disinfectant is diluted with water at a ratio of 1:30, and then sprayed onto the bathroom floor, walls, and toilet surface using a sprayer. Treatment is conducted twice a week, with approximately 500 mL of diluted solution sprayed per bathroom each time. After treatment, the bathroom odor is fundamentally eliminated, and the concentrations of ammonia and hydrogen sulfide both reach or fall below the relevant national standards.

[0063] The above description represents the preferred embodiments of the present invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A compound microbial agent, characterized in that, It includes an active component and a carrier, wherein the active component is composed of the following microbial species: Bacillus licheniformis, Lactobacillus plantarum, Bacillus jellyoidis, Bacillus polymyxa, Bacillus brevis lateralis, Bacillus subtilis, Bacillus brevis, Trichoderma harzianum, Trichoderma echinospora, Aspergillus niger, and Saccharomyces cerevisiae; The carrier is a β-cyclodextrin-modified porous carrier with a hydrophilic-hydrophobic gradient.

2. The compound microbial agent according to claim 1, characterized in that, The β-cyclodextrin-modified porous support is prepared by a method comprising the following steps: (a) Chitosan and polyvinyl alcohol are dissolved in an acetic acid solution with a mass concentration of 1-3% and then freeze-dried to prepare a porous carrier; (b) Dissolve β-cyclodextrin in water to prepare a β-cyclodextrin solution; (c) Immerse one end of the porous carrier in a β-cyclodextrin solution and use capillary action to allow the solution to permeate along the carrier channels from the immersed end to the unimmersed end, forming a concentration gradient of β-cyclodextrin inside the carrier. Then add a crosslinking agent to the solution. (d) Heat to 50-70℃ to carry out the grafting reaction. After the reaction is complete, remove the support, wash it, and dry it to obtain the final product.

3. The compound microbial agent according to claim 2, characterized in that, The mass ratio of chitosan to polyvinyl alcohol in step (a) is (2-5):

1.

4. The compound microbial agent according to claim 2, characterized in that, The mass concentration of the β-cyclodextrin solution in step (b) is 8-15%.

5. The compound microbial agent according to claim 2, characterized in that, The crosslinking agent mentioned in step (c) is glutaraldehyde, and the amount of glutaraldehyde added is 5-20% of the mass of β-cyclodextrin.

6. The compound microbial agent according to claim 2, characterized in that, The immersion length in step (c) is 1 / 3 to 1 / 2 of the total length of the carrier, and the immersion time is 4-8 hours.

7. The compound microbial agent according to claim 1, characterized in that, Of the active components, based on the total mass of the active components, the effective viable count of Bacillus licheniformis is 11-30 billion / g, the effective viable count of Lactobacillus plantarum is above 9.2 billion / g, the effective viable count of Bacillus jellyii is above 2.6 billion / g, the effective viable count of Bacillus polymyxa is above 12 billion / g, the effective viable count of Bacillus brevis is above 5 billion / g, the effective viable count of Bacillus subtilis is above 5 billion / g, the effective viable count of Bacillus brevis is above 11 billion / g, the effective viable count of Trichoderma harzianum is above 5.6 billion / g, the effective viable count of Trichoderma echinospora is 5.6-11 billion / g, the effective viable count of Aspergillus niger is above 10 billion / g, and the effective viable count of Saccharomyces cerevisiae is above 12 billion / g.

8. The compound microbial agent according to claim 1, characterized in that, The weight ratio of the carrier to the active component is (5-15):

1.

9. The method for preparing the composite microbial agent according to any one of claims 1-8, characterized in that, Includes the following steps: (1) Bacillus licheniformis, Lactobacillus plantarum, Bacillus jellyoidis, Bacillus polymyxa, Bacillus brevicaulis, Bacillus subtilis, Bacillus brevicaulis, Trichoderma harzianum, Trichoderma echinosporum, Aspergillus niger, and Saccharomyces cerevisiae were inoculated into a culture medium for fermentation culture to obtain fermentation broth of each strain, and after drying, they were made into single strain powders. (2) Mix the bacterial powders obtained in step (1) in proportion to obtain the active components; (3) Add the active component to sterile water at 2-5 times its mass and stir to prepare a bacterial suspension; add the β-cyclodextrin modified porous carrier to the bacterial suspension and stir at 4-25℃ for 2-6 hours to allow the bacterial strain to be loaded onto the carrier. After drying, the composite microbial agent is obtained.

10. The application of the composite microbial agent according to any one of claims 1-8 in the degradation of organic waste.

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