A composite microbial agent, a preparation method and application thereof
By mixing compound microbial agents in a specific ratio and optimizing culture conditions, the problems of low degradation efficiency and long composting cycle of Polygonum cuspidatum residue were solved, resulting in a high-efficiency and stable organic fertilizer and realizing the resource utilization of Polygonum cuspidatum residue.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUBEI UNIV OF CHINESE MEDICINE
- Filing Date
- 2026-03-24
- Publication Date
- 2026-05-29
Smart Images
Figure SMS_1
Abstract
Description
Technical Field
[0001] This invention relates to the technical field of microbial degradation of Polygonum cuspidatum residue, specifically to a composite microbial agent, its preparation method, and its application. Background Technology
[0002] Polygonum cuspidatum, rich in resveratrol, polydextrin, and other secondary metabolites with anti-inflammatory, antioxidant, and anti-aging effects, has been widely used in medicine, health products, and cosmetics. In recent years, its cultivation and processing have expanded rapidly, resulting in a large amount of solid waste from Polygonum cuspidatum residue. This residue is mainly high-lignocellulose biomass remaining after the extraction of bioactive components, with a total organic matter content as high as 46.98% and a crude fiber content of 35.39%. It also possesses characteristics such as uniform particle size, good aeration, and natural resistance to pests and diseases. However, due to its dense lignocellulose composite structure and long natural degradation cycle, current disposal methods mostly rely on landfill or stockpiling. This not only occupies land and breeds pollution but also causes a serious waste of biomass resources, becoming a key bottleneck restricting the green and sustainable development of the Polygonum cuspidatum industry.
[0003] In recent years, scholars both domestically and internationally have actively explored ways to utilize solid waste from medicinal herbs. Studies have shown that the residue can be used for cultivating edible fungi, producing biomass fuel, or as a soil conditioner. Zhu Jie confirmed that Polygonum cuspidatum residue can effectively replace some of the main ingredients in Pleurotus eryngii cultivation and shows potential in tailings remediation. In addition to the above methods, given the high lignocellulose content of Polygonum cuspidatum residue, converting it into organic fertilizer through composting is another important pathway that combines environmental benefits with resource value. Functional microorganisms play a crucial role in the biotransformation of medicinal residue. Lu Zuyu isolated a highly efficient compound bacterium, HQW, from Salvia miltiorrhiza residue and improved its degradation efficiency by optimizing conditions. In the field of composting, compound microbial agents are widely recognized as being able to accelerate the maturation of organic waste during the composting process. He Zhouyang's research on sheep manure composting found that, compared to not inoculating with exogenous bacteria or only inoculating with a single strain, compound microbial agents can significantly improve composting efficiency and quality through the synergistic effect of multiple functional strains. The enzyme systems secreted by these strains, such as cellulase, hemicellulase, ligninase, and protease, work together to drive substrate decomposition. However, existing microbial agents are mostly designed for general waste (kitchen waste, livestock manure, straw), lacking functional strains specifically targeting the high lignocellulose and recalcitrant characteristics of Polygonum cuspidatum residue. Furthermore, they generally neglect the ratio of microbial communities and the antagonistic effects between agents. In fact, the composition and ratio of microbial communities have a decisive impact on composting efficiency: the composition and ratio of the microbial community affect the degradation efficiency of the residue, the maturation period, and the stability of the fertilizer product quality.
[0004] Current research has three major shortcomings: First, there is a lack of functional microbial strains that can efficiently utilize the Polygonum cuspidatum substrate; second, the antagonistic effects among microbial communities in compound microbial agents are often overlooked, affecting synergistic degradation efficiency; and third, unreasonable formulation of compound microbial agents makes it difficult to achieve a dual improvement in degradation efficiency and composting quality. Therefore, there is an urgent need to develop a highly efficient, stable, and non-antagonistic compound microbial agent specifically for Polygonum cuspidatum residue to meet the needs of resource utilization of Polygonum cuspidatum solid waste. Summary of the Invention
[0005] The purpose of this invention is to overcome the problems of poor degradation effect, long composting period and unstable quality of fertilizer products after composting in the existing technology of Polygonum cuspidatum residue degradation microbial agents, and to provide a compound microbial agent that has a good degradation efficiency for Polygonum cuspidatum residue, and at the same time has a short composting period for degradation of Polygonum cuspidatum residue and stable quality of fertilizer products after composting.
[0006] To achieve the above objectives, the present invention provides a compound microbial agent containing Trichoderma reesei, Aspergillus niger, Trichoderma longibranchii, Bacillus amyloliquefaciens, Bacillus vesiclei, and Bacillus subtilis. The ratio of spores or sporozoites of Trichoderma reesei, Aspergillus niger, Trichoderma longibranchii, Bacillus amyloliquefaciens, Bacillus belyceae, and Bacillus subtilis in the compound microbial agent is 1-6:1-6:1-6:1-6:1-6:1-6:1-6:1-6.
[0007] Preferably, the viable bacteria concentration of the compound microbial agent is 3.50 × 10⁻⁶. 9 ~9.62×10 9 CFU / g.
[0008] A second aspect of the present invention provides a method for preparing the above-mentioned composite microbial agent, the method comprising: Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus belye, and Bacillus subtilis were inoculated into liquid culture medium to obtain their respective seed solutions. The obtained seed solutions were then inoculated into solid fermentation substrates for culture. Subsequently, the spores or buds of each strain were mixed in a ratio of 1–6:1–6:1–6:1–6:1–6:1–6:1–6.
[0009] Preferably, the solid-state fermentation culture conditions for the bacterial strain include: a time of 48–96 h and a temperature of 28–37 °C.
[0010] Preferably, the solid-state fermentation culture conditions for the fungal strain include: a time of 168–216 h and a temperature of 25–32 °C.
[0011] Preferably, the solid fermentation substrates are bacterial solid culture media, Trichoderma longifolia solid culture media, Aspergillus niger solid culture media, and Trichoderma reesei solid culture media; The bacterial solid culture medium contains wheat bran, corn flour, calcium hydroxide, and water; the Trichoderma longifolia solid culture medium contains wheat bran, corn flour, and water; the Aspergillus niger solid culture medium contains wheat bran, soybean meal, and water; and the Trichoderma reesei solid culture medium contains rice straw powder, wheat bran, corn flour, ammonium sulfate, brown sugar, and water.
[0012] Preferably, in the bacterial solid culture medium, the total weight of the bacterial solid culture medium is 100%, the content of wheat bran is 40-60 wt%, the content of corn flour is 2-3 wt%, the content of calcium hydroxide is 0.5-1 wt%, and the content of water is 35-55 wt%.
[0013] Preferably, in the Trichoderma longicornis solid culture medium, the total weight of the Trichoderma longicornis solid culture medium is 100%, the content of wheat bran is 40-60 wt%, the content of corn flour is 10-15 wt%, and the content of water is 30-50 wt%.
[0014] Preferably, in the Aspergillus niger solid culture medium, the total weight of the Aspergillus niger solid culture medium is 100%, the content of wheat bran is 50-60 wt%, the content of soybean meal is 5-10 wt%, and the content of water is 30-45 wt%.
[0015] Preferably, in the Trichoderma reesei solid culture medium, the total weight of the Trichoderma reesei solid culture medium is 100%, the content of wheat bran is 30-45 wt%, the content of rice straw powder is 15-20 wt%, the content of corn flour is 5-10 wt%, the content of brown sugar is 0.5-2 wt%, the content of ammonium sulfate is 0.3-0.6 wt%, and the content of water is 30-40 wt%.
[0016] A third aspect of the present invention provides a method for degrading Polygonum cuspidatum residue, the method comprising: inoculating the above-mentioned compound microbial agent into Polygonum cuspidatum residue compost for fermentation.
[0017] Preferably, the inoculation amount of the compound microbial agent is 1-3%.
[0018] Preferably, the moisture content of the Polygonum cuspidatum residue compost is 50-70%.
[0019] Preferably, the composting time is 30 to 45 days.
[0020] Compared with the prior art, the technical solution of the present invention has the following advantages: The compound microbial agent of this invention is a combination of six microbial agents: Trichoderma reesei, Aspergillus niger, Trichoderma longibranchii, Bacillus amyloliquefaciens, Bacillus belye, and Bacillus subtilis. The ratio of spores or buds of each component is 1-6:1-6:1-6:1-6:1-6:1-6:1-6. Based on this, it has a very high degradation efficiency for Polygonum cuspidatum residue, and can accelerate the composting process of Polygonum cuspidatum residue, shorten the composting cycle, and the resulting compost product has stable quality and is a high-quality organic fertilizer. Detailed Implementation
[0021] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.
[0022] The endpoints and any values of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of the various ranges, the endpoint values of the various ranges and individual point values, and individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.
[0023] In the description of this application, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating relative importance or implying the number of technical features indicated. Therefore, unless otherwise stated, a feature defined as "first" or "second" may explicitly or implicitly include one or more of that feature; "multiple" means two or more. The term "comprising" and any variations thereof mean non-exclusive inclusion, where one or more other features, integers, steps, operations, units, components, and / or combinations thereof may be present or added.
[0024] In one aspect, the present invention provides a compound microbial agent containing Trichoderma reesei, Aspergillus niger, Trichoderma longibranchii, Bacillus amyloliquefaciens, Bacillus bellis, and Bacillus subtilis; The ratio of spores or sporozoites of Trichoderma reesei, Aspergillus niger, Trichoderma longibranchii, Bacillus amyloliquefaciens, Bacillus belyceae, and Bacillus subtilis in the compound microbial agent is 1-6:1-6:1-6:1-6:1-6:1-6:1-6:1-6.
[0025] In this invention, the composite microbial agent is based on a combination of six microbial agents: Trichoderma reesei, Aspergillus niger, Trichoderma longibranchii, Bacillus amyloliquefaciens, Bacillus belye, and Bacillus subtilis. These agents do not exhibit antagonistic effects against each other. Furthermore, the ratio of spores or endospores in each component is limited to 1–6:1–6:1–6:1–6:1–6:1–6:1–6, thereby achieving efficient degradation of Polygonum cuspidatum residue, accelerating the composting process, shortening the composting cycle, and producing a high-quality organic fertilizer with stable compost quality. This not only provides a new and efficient treatment method for Polygonum cuspidatum residue but also allows the treated residue to be utilized as a high-quality organic fertilizer, achieving effective resource utilization.
[0026] In a preferred embodiment, to further improve the degradation efficiency of Polygonum cuspidatum by the composite microbial agent, while simultaneously improving the quality of the product after composting the Polygonum cuspidatum residue and shortening the composting cycle, the viable bacteria concentration of the composite microbial agent is 3.50 × 10⁻⁶. 9 ~9.62×10 9 CFU / g.
[0027] A second aspect of the present invention provides a method for preparing the above-mentioned composite microbial agent, the method comprising: Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus belye, and Bacillus subtilis were inoculated into liquid culture medium to obtain their respective seed solutions. The obtained seed solutions were then inoculated into solid fermentation substrates for culture. Subsequently, the spores or buds of each strain were mixed in a ratio of 1–6:1–6:1–6:1–6:1–6:1–6:1–6.
[0028] In the method described in this invention, the liquid culture medium is a liquid culture medium commonly used in the field of bacterial culture, for example, it can be one or more of beef extract peptone medium and nutrient broth medium.
[0029] In a preferred embodiment, in order to improve the survival rate, viable cell concentration and reproduction rate of the bacterial strain, the culture conditions for solid-state fermentation of the bacterial strain include: a time of 48 to 96 hours and a temperature of 28 to 37°C.
[0030] In a preferred embodiment, in order to improve the survival rate, viable cell concentration and reproduction rate of the fungal strain, the culture conditions for solid-state fermentation of the fungal strain include: a time of 168-216 h and a temperature of 25-32 °C.
[0031] In a preferred embodiment, to further increase the viable cell concentration of all strains, the solid-state fermentation substrates are respectively bacterial solid culture medium, Trichoderma longifolia solid culture medium, Aspergillus niger solid culture medium, and Trichoderma reesei solid culture medium; wherein, the bacterial solid culture medium contains wheat bran, corn flour, calcium hydroxide, and water; the Trichoderma longifolia solid culture medium contains wheat bran, corn flour, and water; the Aspergillus niger solid culture medium contains wheat bran, soybean meal, and water; and the Trichoderma reesei solid culture medium contains rice straw powder, wheat bran, corn flour, ammonium sulfate, brown sugar, and water; based on the above different solid-state fermentation substrates, the six bacterial agents can be fermented and cultured in a targeted manner to obtain spores or buds of the six bacterial agents.
[0032] In a preferred embodiment, in order to further increase the spore content produced by Bacillus amyloliquefaciens, Bacillus belye and Bacillus subtilis, the bacterial solid culture medium contains, with the total weight of the bacterial solid culture medium being 100%, the content of wheat bran being 40-60 wt%, the content of corn flour being 2-3 wt%, the content of calcium hydroxide being 0.5-1 wt%, and the content of water being 35-55 wt%.
[0033] In a preferred embodiment, in order to further increase the spore content of the Trichoderma longifolia strain, the Trichoderma longifolia solid culture medium contains, with the total weight of the Trichoderma longifolia solid culture medium being 100%, the content of wheat bran being 40-60 wt%, the content of corn flour being 10-15 wt%, and the content of water being 30-50 wt%.
[0034] In a preferred embodiment, in order to further increase the spore content of the Aspergillus niger strain, the Aspergillus niger solid culture medium contains, with the total weight of the Aspergillus niger solid culture medium being 100%, the content of wheat bran being 50-60 wt%, the content of soybean meal being 5-10 wt%, and the content of water being 30-45 wt%.
[0035] In a preferred embodiment, to further increase the spore content of the Trichoderma reesei strain, the Trichoderma reesei solid culture medium comprises, with the total weight of the Trichoderma reesei solid culture medium being 100%, the content of wheat bran being 30-45 wt%, the content of rice straw powder being 15-20 wt%, the content of corn flour being 5-10 wt%, the content of brown sugar being 0.5-2 wt%, the content of ammonium sulfate being 0.3-0.6 wt%, and the content of water being 30-40 wt%.
[0036] In the method described in this invention, there are no special requirements for the wheat bran, corn flour, soybean meal, and rice straw powder; any commonly used materials in the art are acceptable.
[0037] A third aspect of the present invention provides a method for degrading Polygonum cuspidatum residue, the method comprising: inoculating the above-mentioned compound microbial agent into Polygonum cuspidatum residue compost for fermentation; based on the above method, the compound microbial agent can effectively degrade Polygonum cuspidatum residue, making it decomposed and composted into high-quality organic fertilizer.
[0038] In this invention, the Polygonum cuspidatum residue is the solid waste remaining after extracting the chemical components from the Polygonum cuspidatum medicinal material.
[0039] In a preferred embodiment, in order to improve the degradation effect of the microbial agent and shorten the composting cycle, the inoculation amount of the compound microbial agent is 1-3%.
[0040] In the method described in this invention, the "inoculation amount" refers to the weight ratio of the compound microbial agent and the residue of Polygonum cuspidatum.
[0041] In a preferred embodiment, in order to improve the quality stability of the product after composting with Polygonum cuspidatum residue, the moisture content of the composted Polygonum cuspidatum residue is 50-70%; preferably 55-65%.
[0042] In a preferred embodiment, to further improve the quality stability of the product after composting Polygonum cuspidatum residue, the composting time is 30 to 45 days.
[0043] The following examples further illustrate the composite microbial agent, its preparation method, and its application according to the present invention. These examples are implemented based on the technical solution of the present invention, providing detailed implementation methods and specific operating procedures; however, the scope of protection of the present invention is not limited to the following examples.
[0044] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods in the art. Unless otherwise specified, the experimental materials used in the following embodiments are commercially available.
[0045] Example 1 Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus belye, and Bacillus subtilis were inoculated into liquid culture medium to obtain their respective seed cultures. These seed cultures were then inoculated into solid-state fermentation substrates for cultivation. Subsequently, a mixture of spores or endospores of Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus belye, and Bacillus subtilis in a ratio of 5:3:1:6:4:2 was prepared to obtain compound microbial agent F1 (viable cell concentration of 5.87 × 10⁻⁶). 9 CFU / g); In the bacterial solid culture medium, the total weight of the bacterial solid culture medium is 100%, and the contents of wheat bran, corn flour, calcium hydroxide, and water are 50%, 2.2%, 0.8%, and 47%, respectively. The solid-state fermentation time is 72 hours, and the temperature is 32°C. In the Trichoderma longicornis solid culture medium, the total weight of the Trichoderma longicornis solid culture medium is 100%, and the contents of wheat bran, corn flour, and water are 53%, 14%, and 33%, respectively. The solid-state fermentation time is 192 hours, and the temperature is 28°C. The Aspergillus niger solid culture medium... In the culture medium, the total weight of the Aspergillus niger solid culture medium was 100%, and the contents of wheat bran, soybean meal and water were 60%, 7% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃. In the Trichoderma reesei solid culture medium, the total weight of the Trichoderma reesei solid culture medium was 100%, and the contents of wheat bran, rice straw powder, corn flour, brown sugar, ammonium sulfate and water were 40%, 20%, 6%, 0.7%, 0.3% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃.
[0046] Example 2 Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus bellies, and Bacillus subtilis were inoculated separately into liquid culture medium to obtain their respective seed cultures. These seed cultures were then inoculated into solid-state fermentation substrates for cultivation. Subsequently, a mixture of spores or endospores of Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus bellies, and Bacillus subtilis in a ratio of 4:1:5:2:6:3 was prepared to obtain compound microbial agent F2 (viable cell concentration of 4.42 × 10⁻⁶). 9 CFU / g) In the bacterial solid culture medium, the total weight of the bacterial solid culture medium is 100%, and the contents of wheat bran, corn flour, calcium hydroxide, and water are 50%, 2.2%, 0.8%, and 47%, respectively. The solid-state fermentation time is 72 hours, and the temperature is 32°C. In the Trichoderma longicornis solid culture medium, the total weight of the Trichoderma longicornis solid culture medium is 100%, and the contents of wheat bran, corn flour, and water are 53%, 14%, and 33%, respectively. The solid-state fermentation time is 192 hours, and the temperature is 28°C. The Aspergillus niger solid culture medium... In the culture medium, the total weight of the Aspergillus niger solid culture medium was 100%, and the contents of wheat bran, soybean meal and water were 60%, 7% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃. In the Trichoderma reesei solid culture medium, the total weight of the Trichoderma reesei solid culture medium was 100%, and the contents of wheat bran, rice straw powder, corn flour, brown sugar, ammonium sulfate and water were 40%, 20%, 6%, 0.7%, 0.3% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃.
[0047] Example 3 Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus bellies, and Bacillus subtilis were inoculated separately into liquid culture medium to obtain their respective seed cultures. These seed cultures were then inoculated into solid-state fermentation substrates for cultivation. Subsequently, a mixture of spores or endospores of Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus bellies, and Bacillus subtilis in a ratio of 6:5:4:3:2:1 was prepared to obtain compound microbial agent F3 (viable cell concentration of 3.59 × 10⁻⁶). 9 CFU / g); In the bacterial solid culture medium, the total weight of the bacterial solid culture medium is 100%, and the contents of wheat bran, corn flour, calcium hydroxide, and water are 50%, 2.2%, 0.8%, and 47%, respectively. The solid-state fermentation time is 72 hours, and the temperature is 32°C. In the Trichoderma longicornis solid culture medium, the total weight of the Trichoderma longicornis solid culture medium is 100%, and the contents of wheat bran, corn flour, and water are 53%, 14%, and 33%, respectively. The solid-state fermentation time is 192 hours, and the temperature is 28°C. The Aspergillus niger solid culture medium... In the culture medium, the total weight of the Aspergillus niger solid culture medium was 100%, and the contents of wheat bran, soybean meal and water were 60%, 7% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃. In the Trichoderma reesei solid culture medium, the total weight of the Trichoderma reesei solid culture medium was 100%, and the contents of wheat bran, rice straw powder, corn flour, brown sugar, ammonium sulfate and water were 40%, 20%, 6%, 0.7%, 0.3% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃.
[0048] Example 4 Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus bellies, and Bacillus subtilis were inoculated separately into liquid culture medium to obtain their respective seed cultures. These seed cultures were then inoculated into solid-state fermentation substrates for cultivation. Subsequently, a mixture of spores or endospores of Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus bellies, and Bacillus subtilis in a ratio of 3:6:2:5:1:4 was prepared to obtain compound microbial agent F4 (viable cell concentration of 5.93 × 10⁻⁶). 9 CFU / g); In the bacterial solid culture medium, the total weight of the bacterial solid culture medium is 100%, and the contents of wheat bran, corn flour, calcium hydroxide, and water are 50%, 2.2%, 0.8%, and 47%, respectively. The solid-state fermentation time is 72 hours, and the temperature is 32°C. In the Trichoderma longicornis solid culture medium, the total weight of the Trichoderma longicornis solid culture medium is 100%, and the contents of wheat bran, corn flour, and water are 53%, 14%, and 33%, respectively. The solid-state fermentation time is 192 hours, and the temperature is 28°C. The Aspergillus niger solid culture medium... In the culture medium, the total weight of the Aspergillus niger solid culture medium was 100%, and the contents of wheat bran, soybean meal and water were 60%, 7% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃. In the Trichoderma reesei solid culture medium, the total weight of the Trichoderma reesei solid culture medium was 100%, and the contents of wheat bran, rice straw powder, corn flour, brown sugar, ammonium sulfate and water were 40%, 20%, 6%, 0.7%, 0.3% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃.
[0049] Example 5 Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus bellies, and Bacillus subtilis were inoculated separately into liquid culture medium to obtain their respective seed cultures. These seed cultures were then inoculated into solid-state fermentation substrates for cultivation. Subsequently, a mixture of spores or endospores of Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus bellies, and Bacillus subtilis in a ratio of 2:4:6:1:3:5 was prepared to obtain compound microbial agent F5 (viable cell concentration of 4.45 × 10⁻⁶). 9 CFU / g); In the bacterial solid culture medium, the total weight of the bacterial solid culture medium is 100%, and the contents of wheat bran, corn flour, calcium hydroxide, and water are 50%, 2.2%, 0.8%, and 47%, respectively. The solid-state fermentation time is 72 hours, and the temperature is 32°C. In the Trichoderma longicornis solid culture medium, the total weight of the Trichoderma longicornis solid culture medium is 100%, and the contents of wheat bran, corn flour, and water are 53%, 14%, and 33%, respectively. The solid-state fermentation time is 192 hours, and the temperature is 28°C. The Aspergillus niger solid culture medium... In the culture medium, the total weight of the Aspergillus niger solid culture medium was 100%, and the contents of wheat bran, soybean meal and water were 60%, 7% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃. In the Trichoderma reesei solid culture medium, the total weight of the Trichoderma reesei solid culture medium was 100%, and the contents of wheat bran, rice straw powder, corn flour, brown sugar, ammonium sulfate and water were 40%, 20%, 6%, 0.7%, 0.3% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃.
[0050] Example 6 Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus belye, and Bacillus subtilis were inoculated separately into liquid culture medium to obtain their respective seed cultures. These seed cultures were then inoculated into solid-state fermentation substrates for cultivation. Subsequently, a mixture of spores or endospores of Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus belye, and Bacillus subtilis in a ratio of 1:2:3:4:5:6 was prepared to obtain compound microbial agent F6 (viable cell concentration of 8.61 × 10⁻⁶). 9 CFU / g); In the bacterial solid culture medium, the total weight of the bacterial solid culture medium is 100%, and the contents of wheat bran, corn flour, calcium hydroxide, and water are 50%, 2.2%, 0.8%, and 47%, respectively. The solid-state fermentation time is 72 hours, and the temperature is 32°C. In the Trichoderma longicornis solid culture medium, the total weight of the Trichoderma longicornis solid culture medium is 100%, and the contents of wheat bran, corn flour, and water are 53%, 14%, and 33%, respectively. The solid-state fermentation time is 192 hours, and the temperature is 28°C. The Aspergillus niger solid culture medium... In the culture medium, the total weight of the Aspergillus niger solid culture medium was 100%, and the contents of wheat bran, soybean meal and water were 60%, 7% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃. In the Trichoderma reesei solid culture medium, the total weight of the Trichoderma reesei solid culture medium was 100%, and the contents of wheat bran, rice straw powder, corn flour, brown sugar, ammonium sulfate and water were 40%, 20%, 6%, 0.7%, 0.3% and 33%, respectively. The solid-state fermentation time was 192 h and the temperature was 28℃.
[0051] Comparative Example 1 The procedure was carried out in accordance with Example 1, except that Trichoderma reesei was not added.
[0052] Comparative Example 2 The procedure was carried out in accordance with Example 1, except that Bacillus belesiensis was not added.
[0053] Comparative Example 3 The implementation was carried out in accordance with Example 1, except that the ratio of spores or sporozoites of Trichoderma reesei, Aspergillus niger, Trichoderma longibranchii, Bacillus amyloliquefaciens, Bacillus vesicularis, and Bacillus subtilis was 8:0.5:0.5:7:6:0.5.
[0054] Comparative Example 4 A blank control group (without any microbial agents) was set up as a control. Except for not adding any microbial agents, all other steps were the same as in Example 1.
[0055] Test case The composite microbial agents prepared in the examples and comparative examples were inoculated at a 1% inoculation rate into a composting system containing Polygonum cuspidatum residue (the initial moisture contents of Examples 1-6 were 59.35%, 56.56%, 57.35%, 59.34%, 57.64%, and 57.75%, respectively; the initial moisture contents of Control Examples 1-4 were 58.18%, 57.17%, 57.19%, and 56.41%, respectively). Fermentation was carried out for 43 days, and the temperature was recorded daily. 200g samples were collected from the upper, middle, and lower layers of each pile on days 1, 8, 15, 22, 29, 36, and 43. After thorough mixing, the samples were stored at -80℃ for analysis. The piles were manually turned after each sampling to ensure the material decomposition was as uniform as possible. The physicochemical properties of the samples were measured after each sampling, according to the national standard for organic fertilizer NY. The determination method of 525-2021 is to determine moisture content, pH, electrical conductivity (EC), seed germination index (GI), organic matter content (OM), total carbon and total nitrogen. The composting effect of different proportions of compound microbial agents is evaluated through composting experiments. The optimal proportion of compound microbial agents is determined by composting physicochemical indicators such as T value, maximum temperature, thermophilic period time and seed germination index. T value = final carbon-nitrogen ratio / initial carbon-nitrogen ratio. Analysis of composting test results: Examples 1-6 and Comparative Examples 1-4 all showed an initial temperature increase followed by a gradual decrease. The thermophilic period began on day 2 and lasted until day 13, but the duration of the thermophilic period varied among the groups: the durations for Examples 1-6 were 12, 10, 10, 11, 9, and 11 days, respectively, while the durations for Comparative Examples 1-4 were 9, 9, 8, and 5 days, respectively. The peak temperatures for Examples 1-6 were 71.7℃, 70.6℃, 69.6℃, 67.6℃, 67.1℃, and 67.7℃, respectively, while the peak temperatures for Comparative Examples 1-4 were 69.4℃, 65.5℃, 67.5℃, and 57.3℃, respectively. All inoculated groups in Examples 1-6 maintained a thermophilic phase of ≥9 days, meeting the criteria for effective composting. At the start of the experiment, the initial moisture contents of Examples 1-6 were 59.35%, 56.56%, 57.35%, 59.34%, 57.64%, and 57.75%, respectively; the initial moisture contents of Comparative Examples 1-4 were 58.18%, 57.17%, 57.19%, and 56.41%, respectively. At the end of composting, the moisture contents of Examples 1-6 were 29.95%, 29.17%, 31.82%, 31.72%, 30.89%, and 31.72%, respectively; the moisture contents of Comparative Examples 1-4 were 30.07%, 31.71%, 30.73%, and 33.68%, respectively. These results indicate that, compared with the blank control group, microbial inoculants were more effective in reducing moisture content during composting. This may be related to microbial metabolic activity and the heat of the compost pile, thereby promoting the reduction of moisture content. The pH values of Examples 1-6 and Comparative Examples 1-4 all initially increased and then decreased. The overall trend showed a sharp increase from day 1 to day 22, indicating active ammoniation. Subsequently, the pH values decreased rapidly from day 23 to day 29, followed by a slow decrease from day 30 to day 43, reflecting the onset and continuation of nitrification. On day 1, the initial pH values of Examples 1-6 were 7.29, 7.25, 7.32, 7.50, 7.62, and 7.71, respectively, while the initial pH values of Comparative Examples 1-4 were 7.27, 7.26, 7.38, and 8.11, respectively. On day 22, the pH values of Examples 1-6 were 9.42, 9.31, 9.37, 9.36, 9.37, and 9.25, respectively, while the pH values of Comparative Examples 1-4 were 8.88 and 9. 07, 8.91, and 8.78; at the end of composting, the pH values of Examples 1-6 were 8.12, 8.18, 8.52, 8.10, 7.98, and 8.09, respectively, while the pH values of Comparative Examples 1-4 were 7.68, 7.76, 7.59, and 7.69, respectively. It can be seen that the final pH values of Examples 1-6 were significantly higher than those of Comparative Examples 1-4, and the pH values of Examples 1-6 all met the standard for mature compost (pH≤8.5). The EC values of Examples 1-6 and Comparative Examples 1-4 all showed a consistent trend: a steady increase in the early stages of composting, followed by a stabilization at the end of composting. In the initial stage of composting, the EC values of Examples 1-6 were 2.93 mS / cm, 2.97 mS / cm, 3.02 mS / cm, 2.98 mS / cm, 2.97 mS / cm, and 3.04 mS / cm, respectively, while the EC values of Comparative Examples 1-4 were 2.75 mS / cm, 2.83 mS / cm, 3.01 mS / cm, and 2.76 mS / cm, respectively. At the end of composting, the EC values of Examples 1-6 were 3.84 mS / cm, 2.97 mS / cm, 2.98 mS / cm, 2.97 mS / cm, and 3.04 mS / cm, respectively. The EC values of Examples 1-6 were 3.98 mS / cm, 4.00 mS / cm, 3.98 mS / cm, 3.89 mS / cm, and 4.00 mS / cm, respectively, while those of Comparative Examples 1-4 were 3.91 mS / cm, 3.70 mS / cm, 3.83 mS / cm, and 3.28 mS / cm, respectively. Analysis shows that the final EC values of Examples 1-6 were all significantly higher than those of Comparative Examples 1-4. This indicates that inoculation with the compound microbial agent described in this invention can accelerate the conversion of organic nitrogen into inorganic nitrogen that can be used by plants, thereby promoting compost maturation. Moreover, the EC values of Examples 1-6 were all ≤4 mS / cm, reaching the threshold for mature compost. The glycemic index (GI) of Examples 1-6 and Comparative Examples 1-4 increased continuously from the early to middle stages of composting and then stabilized in the later stage. At the beginning of composting, the GI values of Examples 1-6 were 20.63%, 19.10%, 19.82%, 20.67%, 21.33%, and 20.06%, respectively, while the GI values of Comparative Examples 1-4 were 21.07%, 22.14%, 20.95%, and 20.68%, respectively. At the end of composting, the GI values of Examples 1-6 rose to 93.35%, 89.11%, and 83%, respectively. The GI values of Examples 1-6 were 15%, 82.24%, 87.67%, and 81.47%, respectively, while those of Comparative Examples 1-4 were 76.07%, 77.14%, 77.95%, and 71.15%. Analysis showed that the final GI values of Examples 1-6 were significantly higher than those of Comparative Examples 1-4. This indicates that inoculation with the compound microbial agent described in this invention can reduce the content of phytotoxic substances in compost and accelerate the humification process. Furthermore, the final GI values of Examples 1-6 were all ≥80%, indicating that they had no toxic effect on plant growth and met the standards for mature compost. The organic matter content of Examples 1-6 and Comparative Examples 1-4 decreased continuously from the early to middle stages of the composting process, and tended to stabilize in the later stage. At the beginning of composting, the organic matter content of Examples 1-6 was 61.61%, 61.27%, 58.58%, 60.75%, 61.58%, and 57.97%, respectively, while the organic matter content of Comparative Examples 1-4 was 60.92%, 60.05%, 59.81%, and 59.24%. At the end of composting, the organic matter content of Examples 1-6 decreased to [a lower value]. The organic matter content of Examples 1-6, inoculated with the compound microbial agent of the present invention, was 41.38%, 42.68%, 42.60%, 40.71%, 39.51%, and 40.07%, respectively, while that of Comparative Examples 1-4 was reduced to 44.03%, 43.92%, 44.33%, and 45.74%. Analysis shows that the reduction in organic matter in Examples 1-6 inoculated with the compound microbial agent of the present invention was higher than that in Comparative Examples 1-4. This indicates that inoculation with the compound microbial agent of the present invention can promote the decomposition of organic matter and accelerate the humification process in composting. The T values (final carbon-nitrogen ratio / initial carbon-nitrogen ratio) of Examples 1-6 were 0.83, 0.88, 1.01, 1.00, 0.92, and 0.91, respectively, while the T values (final carbon-nitrogen ratio / initial carbon-nitrogen ratio) of Comparative Examples 1-4 were 1.06, 1.17, 1.03, and 1.05, respectively. This indicates that the T values of Examples 1-6 inoculated with the compound microbial agent described in this invention were significantly lower than those of Comparative Examples 1-4, and the T values of Examples 1-6 all met the standards for mature composting. The physicochemical indicators such as organic matter content, total nutrient content, moisture content, pH, and GI of the Polygonum cuspidatum residue after composting in Examples 1-6 and Comparative Examples 1-4 are shown in Table 1. Table 1
[0056] The results analysis above and the results in Table 1 show that the compound microbial agent described in this invention has a good degradation effect on Polygonum cuspidatum residue. At the same time, it can accelerate the composting process of Polygonum cuspidatum residue, shorten the composting cycle, and the compost product obtained by inoculating the compound microbial agent described in this invention has stable quality. It is a high-quality organic fertilizer and meets the national standard for organic fertilizer NY525-2021, thereby realizing the utilization of resources.
[0057] The preferred embodiments of the present invention have been described in detail above; however, the present invention is not limited thereto. Within the scope of the inventive concept, various simple modifications can be made to the technical solutions of the present invention, including combinations of various technical features in any other suitable manner. These simple modifications and combinations should also be considered as the content disclosed in the present invention and are all within the protection scope of the present invention.
Claims
1. A compound microbial agent, characterized in that, The compound microbial agent contains Trichoderma reesei, Aspergillus niger, Trichoderma longibranchii, Bacillus amyloliquefaciens, Bacillus vesalis, and Bacillus subtilis; The ratio of spores or sporozoites of Trichoderma reesei, Aspergillus niger, Trichoderma longibranchii, Bacillus amyloliquefaciens, Bacillus belyceae, and Bacillus subtilis in the compound microbial agent is 1-6:1-6:1-6:1-6:1-6:1-6:1-6:1-6.
2. The compound microbial agent according to claim 1, characterized in that, The viable cell concentration of the compound microbial agent is 3.50 × 10⁻⁶. 9 ~9.62×10 9 CFU / g.
3. A method for preparing the composite microbial agent according to claim 1 or 2, characterized in that, The method includes: Trichoderma reesei, Aspergillus niger, Trichoderma longifolia, Bacillus amyloliquefaciens, Bacillus belye, and Bacillus subtilis were inoculated into liquid culture medium to obtain their respective seed solutions. The obtained seed solutions were then inoculated into solid fermentation substrates for culture. Subsequently, the spores or buds of each strain were mixed in a ratio of 1–6:1–6:1–6:1–6:1–6:1–6:1–6.
4. The method according to claim 3, characterized in that, The solid-state fermentation culture conditions for the bacterial strains include: a time of 48–96 h and a temperature of 28–37 °C.
5. The method according to claim 3 or 4, characterized in that, The solid-state fermentation culture conditions for fungal strains include: time of 168–216 h and temperature of 25–32 °C.
6. The method according to any one of claims 3-5, characterized in that, The solid fermentation substrates are bacterial solid culture medium, Trichoderma longifolia solid culture medium, Aspergillus niger solid culture medium, and Trichoderma reesei solid culture medium, respectively. The bacterial solid culture medium contains wheat bran, corn flour, calcium hydroxide, and water; the Trichoderma longifolia solid culture medium contains wheat bran, corn flour, and water; the Aspergillus niger solid culture medium contains wheat bran, soybean meal, and water; and the Trichoderma reesei solid culture medium contains rice straw powder, wheat bran, corn flour, ammonium sulfate, brown sugar, and water.
7. The method according to claim 6, characterized in that, In the bacterial solid culture medium, with the total weight of the bacterial solid culture medium being 100%, the content of wheat bran is 40-60 wt%, the content of corn flour is 2-3 wt%, the content of calcium hydroxide is 0.5-1 wt%, and the content of water is 35-55 wt%; and / or In the *Trichoderma longicornis* solid culture medium, the total weight of the *Trichoderma longicornis* solid culture medium is 100%, the content of wheat bran is 40-60 wt%, the content of corn flour is 10-15 wt%, and the content of water is 30-50 wt%; and / or In the Aspergillus niger solid culture medium, with the total weight of the Aspergillus niger solid culture medium being 100%, the content of wheat bran is 50-60 wt%, the content of soybean meal is 5-10 wt%, and the content of water is 30-45 wt%; and / or In the Trichoderma reesei solid culture medium, with the total weight of the Trichoderma reesei solid culture medium being 100%, the content of wheat bran is 30-45 wt%, the content of rice straw powder is 15-20 wt%, the content of corn flour is 5-10 wt%, the content of brown sugar is 0.5-2 wt%, the content of ammonium sulfate is 0.3-0.6 wt%, and the content of water is 30-40 wt%.
8. A method for degrading Polygonum cuspidatum residue, characterized in that, The method includes: inoculating the compound microbial agent according to claim 1 or 2 into the compost of Polygonum cuspidatum residue for fermentation.
9. The method according to claim 8, characterized in that, The inoculation amount of the compound microbial agent is 1-3%; and / or The moisture content of the Polygonum cuspidatum residue compost is 50-70%.
10. The method according to claim 8 or 9, characterized in that, The composting time is 30 to 45 days.