Compound microbial agent for degrading chemical oxygen demand and preparation method thereof

Abstract

The invention provides a compound microbial agent for degrading chemical oxygen demand and a preparation method thereof. The compound microbial agent comprises an effective microbial flora, a carrier and a protective agent, wherein the effective microbial flora is a mixture of at least two of bacillus subtilis, saccharomyces cerevisiae and pseudomonas putida; the carrier is a mixture of bentonite and bran; the protective agent is a mixture of trehalose and skim milk powder. The composite microbial agent can adapt to different application scenes such as water and soil, secondary pollution is avoided in the degradation process, the composite microbial agent has practicability and environmental friendliness, and the production cost and the COD removal cost can be effectively reduced by integrally adopting the raw materials which are low in cost and easy to obtain.

Description

Technical Field

[0001] This invention belongs to the field of wastewater treatment technology, specifically to a composite microbial agent for degrading chemical oxygen demand and its preparation method. Background Technology

[0002] Chemical oxygen demand (COD) is a key indicator for measuring the degree of organic pollution in water bodies. Reducing COD is one of the core objectives in the treatment of industrial wastewater (such as food processing, papermaking, printing and dyeing, and pharmaceutical wastewater) and domestic sewage. Currently, commonly used COD removal methods include physical, chemical, and biological methods.

[0003] Biological methods are widely used due to their advantages such as low cost, no secondary pollution, and thorough treatment. Traditional activated sludge processes suffer from problems such as limited microbial species, slow start-up, poor tolerance to shock loads and toxic substances, and large amounts of residual sludge. Although some commercially available microbial agents exist, they often have limited functionality, lack versatility for specific wastewater types, and are prone to inactivation in complex wastewater environments, leading to unstable COD removal efficiency.

[0004] Therefore, there is currently a lack of a COD removal agent that is highly efficient, broad-spectrum, adaptable, and can reduce the production of residual sludge.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The primary objective of this invention is to provide a composite microbial agent for degrading chemical oxygen demand (COD). This composite microbial agent, through the synergistic mixing of specific raw materials, can be adapted to different application scenarios such as water bodies and soil. The degradation process is free from secondary pollution, combining practicality and environmental friendliness. Furthermore, the overall use of low-cost and readily available raw materials can effectively reduce production costs and COD removal costs.

[0007] The second objective of this invention is to provide the above-mentioned preparation method, which is simple to operate and produces a microbial agent with the advantages of high efficiency, broad spectrum, strong adaptability, and the ability to reduce COD in the production of residual sludge.

[0008] To achieve the above-mentioned objectives of this invention, the following technical solution is adopted: This invention provides a composite microbial agent for degrading chemical oxygen demand, comprising: an effective microbial community, a carrier, and a protectant; The effective microbial community is a mixture of at least two of Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putidae. The carrier is a mixture of bentonite and wheat bran; The protective agent is a mixture of trehalose and skim milk powder.

[0009] The composite microbial agent of the present invention consists of an effective microbial community, a carrier, and a protectant. The effective microbial community is a mixture of at least two of Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida. The carrier is a mixture of bentonite and wheat bran. The protectant is a mixture of trehalose and skim milk powder. This agent can efficiently degrade chemical oxygen demand (COD) and has strong stability and wide applicability.

[0010] In the composite microbial agent of this invention, the synergistic effect of at least two of the three bacterial strains—Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida—effectively solves the problem of traditional microbial agents easily failing under conditions of high organic matter content. Bacillus subtilis secretes abundant extracellular enzymes such as proteases, amylases, and lipases, decomposing large organic molecules such as proteins, polysaccharides, and fats in wastewater into smaller organic molecules and organic acids, providing usable substrates for other microorganisms. Simultaneously, its spores exhibit strong resistance, helping the agent survive in harsh environments. Saccharomyces cerevisiae readily utilizes small-molecule sugars and organic acids for rapid growth, directly consuming dissolved COD in the water. Its metabolic products also promote the growth of other bacterial communities, forming a stable micro-ecosystem. Pseudomonas putida possesses extremely strong degradation capabilities for aromatic compounds, hydrocarbons, and other recalcitrant organic matter, compensating for the degradation blind spots of the first two strains and broadening the agent's degradation spectrum. The combination of two or more strains can functionally promote and synergistically enhance each other, forming a highly efficient "decomposition-utilization-purification" chain, achieving efficient degradation of complex organic matter in industrial water treatment.

[0011] In this invention, the carrier is specifically a mixture of bentonite and wheat bran. Bentonite, as an inorganic carrier, leverages its unique porous structure to effectively adsorb functional strains such as Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida, forming a stable microbial immobilization system. Simultaneously, it resists shear force damage generated during aeration in industrial water treatment, preventing microbial loss. Wheat bran, as an organic carrier, slowly releases carbohydrates and other nutrients, providing Bacillus subtilis and Pseudomonas putida with the energy and nutrients needed for initial growth, promoting rapid proliferation and the formation of a dominant microbial community. The carrier composed of bentonite and wheat bran immobilizes microorganisms, prevents loss, and provides nutritional support. The synergistic application of bentonite and wheat bran helps ensure the survival stability of microorganisms and lays a nutritional foundation for the synergistic degradation of organic matter by the microbial community, achieving a dual synergistic function of "immobilizing and protecting microorganisms" and "providing energy and promoting growth."

[0012] In this invention, the protective agents specifically employ trehalose and skim milk powder. The protective agent formed by these two ingredients reduces damage to microorganisms from the external environment, further ensuring the shelf life and effectiveness of the microbial agent. Specifically, trehalose focuses on basic protection at the microbial cellular level. Its unique molecular structure allows it to penetrate the cell membrane, replacing water in a dry environment to form a hydrogen bond network, maintaining cell membrane integrity, and resisting the impact of high osmotic pressure on the cells. This fundamentally reduces the risk of cell rupture and death, building a solid "first line of defense" for microbial survival. Skim milk powder, on the other hand, combines external protection with growth enhancement. The protein protective film it forms can construct a physical barrier outside the cells, specifically resisting shear forces and oxidative damage during aeration, compensating for the shortcomings of trehalose in protecting against external mechanical damage. Simultaneously, the released amino acids provide microorganisms with readily available nutrients, overcoming the limitation of trehalose only providing protection and not energy, thus promoting rapid colonization and proliferation of microorganisms in complex water treatment environments. The synergy between the two is not a simple superposition, but rather a dual protection formed by the "internal protective membrane" of trehalose and the "external protective barrier" of skim milk powder. The cell activity maintained by trehalose provides the basis for the nutritional supply of skim milk powder, while the nutritional support of skim milk powder further enhances the microorganisms' tolerance to environmental stress, ultimately greatly improving the survival rate of live bacteria. This ensures that the three functional strains can stably exert the synergistic degradation effect of "decomposition-utilization-purification", avoiding the decline in the degradation efficiency of the bacterial agent due to bacterial inactivation, and achieving continuous and efficient degradation of complex organic matter.

[0013] In summary, this invention, by using specific raw materials for synergistic mixing, can be adapted to different application scenarios such as water bodies and soil. The degradation process is free of secondary pollution, combining practicality and environmental protection. Furthermore, the use of low-cost and readily available raw materials can effectively reduce production costs and COD removal costs.

[0014] Preferably, the effective microbial flora includes Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida.

[0015] The above-mentioned scheme preferentially combines three bacterial groups: Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida. The core advantage lies in constructing a complete degradation system covering "macromolecule decomposition - small molecule utilization - recalcitrant purification" through functional complementarity and synergistic effects. This completely solves the problems of incomplete degradation and limited efficiency of traditional single or dual bacterial groups: Bacillus subtilis secretes a variety of extracellular enzymes that can decompose macromolecular organic matter such as proteins, polysaccharides, and fats in wastewater into small molecule organic matter and organic acids, providing usable substrates for the entire bacterial group. Moreover, its highly resilient spore structure can ensure the survival of the bacterial group in harsh environments. Saccharomyces cerevisiae rapidly utilizes these small molecule sugars, organic acids, and other carbon sources for rapid growth, directly consuming soluble COD. At the same time, its metabolites can promote the proliferation of other bacterial groups and stabilize the micro-ecosystem. Pseudomonas putida specializes in aromatic compounds, hydrocarbons, and other recalcitrant organic matter, precisely filling the degradation blind spots of the first two strains and broadening the degradation spectrum of the bacterial agent. The combination of these three elements is not simply a functional addition, but rather forms a synergistic closed loop of "decomposition-energy supply-specialized treatment". This not only improves the degradation efficiency of complex organic matter, but also enhances the adaptability and stability of the microbial agent under high organic matter content and harsh working conditions, ensuring comprehensive and efficient degradation of COD in complex water bodies such as industrial wastewater.

[0016] Preferably, the mass ratio of Bacillus subtilis, Saccharomyces cerevisiae and Pseudomonas putida is (3~6):(1~3):(2~4).

[0017] The above scheme optimizes the mass ratio of Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida to (3-6):(1-3):(2-4). The core advantage is that the precise ratio maximizes the synergistic function of the microbial community, ensuring both degradation efficiency and environmental adaptability. Bacillus subtilis, as the "core of macromolecular decomposition," has the highest proportion (3-6 parts) and can fully secrete extracellular enzymes to efficiently break down large organic molecules, providing sufficient substrates for the entire microbial community. Its strong resistance also lays a solid foundation for the system's environmental tolerance. Saccharomyces cerevisiae has a moderate proportion (1-3 parts) and can efficiently utilize the small molecule substrates produced by Bacillus subtilis decomposition to quickly consume soluble COD, while avoiding competition for degradation resources of difficult-to-degrade substances due to an excessively high proportion. Pseudomonas putida has a reasonable proportion (2-4 parts) and can specifically degrade aromatic compounds, hydrocarbons, and other pollutants that are difficult for the first two types of bacteria to treat, precisely filling the degradation blind spots. This ratio allows the three bacteria to each play to their strengths while supporting each other. The substrate supply of Bacillus subtilis meets the growth needs of Saccharomyces cerevisiae and Pseudomonas putida, the metabolites of Saccharomyces cerevisiae promote the stability of the microbial community, and the specialized ability of Pseudomonas putida broadens the degradation range. Ultimately, the optimal balance between degradation rate, degradation range and microbial community stability is achieved, ensuring that the microbial agent can continuously and efficiently play a role in COD degradation under high organic matter content and complex working conditions.

[0018] Preferably, the mass ratio of Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida is 5:2:3.

[0019] The above scheme specifically selects a mass ratio of 5:2:3 for the three bacterial groups. This helps to optimize the synergistic effect of the three bacterial groups and achieve the best balance between degradation efficiency, microecological stability, and environmental adaptability. Bacillus subtilis accounts for 5 parts, serving as the "core of macromolecular decomposition." The amount of extracellular enzymes it secretes is sufficient to efficiently break down various macromolecular organic matter, providing sufficient and balanced small molecule substrates for subsequent bacterial groups. At the same time, its strong resistance can fully guarantee the survival of the bacterial groups under complex working conditions. Saccharomyces cerevisiae accounts for 2 parts, which is just right to match the substrate supply produced by Bacillus subtilis. It can quickly consume soluble COD and improve the degradation rate, without competing with the recalcitrant degradation resources needed by Pseudomonas malodorosa due to an excessively high proportion. Pseudomonas malodorosa accounts for 3 parts, which can specifically tackle recalcitrant pollutants such as aromatic compounds and hydrocarbons, accurately making up for the degradation blind spots of the first two bacteria. Moreover, this proportion allows it to obtain sufficient nutrients while forming a stable competitive and cooperative relationship with the other two bacteria. This specific ratio allows the three bacteria to function in a closed loop without redundancy or shortcomings: the substrate decomposition efficiency of Bacillus subtilis is highly matched with the small molecule utilization rate of Saccharomyces cerevisiae, and the degradation capacity of recalcitrant bacteria is fully released. The three promote each other without interfering with each other, ultimately achieving a better COD degradation rate and thoroughness than other ratios. It is especially suitable for complex high-organic-pollution scenarios such as industrial wastewater, and can stably maintain a high-efficiency degradation effect.

[0020] Preferably, the mass ratio of bentonite to wheat bran in the carrier is 1:(1~3); preferably, the mass ratio of bentonite to wheat bran in the carrier is 1:2.

[0021] In the above scheme, the preferred mass ratio of bentonite to wheat bran in the carrier is 1:(1~3). This specific ratio of compounding can balance adsorption and nutrient supply, avoiding the defects of a single inorganic carrier lacking nutrition and a single organic carrier being prone to mold growth. This improves the survival rate of microorganisms and the colonization rate of the inoculant in the aeration tank, ensuring the efficient degradation of complex organic matter by the compound inoculant of this invention, and overcoming the problem of low survival rate of traditional microbial inoculants in complex industrial water environments. Moreover, this specific ratio of compounding can achieve precise matching of the functions of inorganic and organic carriers, allowing the synergistic effect of "solidifying and protecting bacteria" and "supplying energy and promoting growth" to reach the optimal level. Specifically, within the range of 1:(1~3), the porous structure of bentonite can fully adsorb and fix various functional microorganisms, resisting aeration shear force damage, while wheat bran can release sufficient carbohydrates to meet the initial nutritional needs of Bacillus subtilis and Pseudomonas putida, avoiding the problems of insufficient nutrition due to excessive bentonite content or loose carrier and easy loss of microorganisms due to excessive wheat bran content. In a further proposed solution, a 1:2 ratio was specifically chosen. At this ratio, an optimal balance is achieved, where the adsorption and retention capacity of bentonite is highly compatible with the nutrient supply efficiency of wheat bran. This not only forms a stable microbial immobilization system, ensuring the survival stability of the microorganisms under complex working conditions, but also slowly and continuously provides energy for the growth of the microbial community, promoting the rapid proliferation of functional strains and the formation of a dominant microbial community. This lays a solid foundation for the synergistic degradation of complex organic matter by various microorganisms. Compared to other ratios, this approach better balances the structural stability of the carrier and the sustainability of nutrient supply, further improving the degradation efficiency and environmental adaptability of the microbial agent.

[0022] Preferably, the amount of carrier used is 30% to 50% of the mass of the effective microbial community; preferably, the amount of carrier used is 40% of the mass of the effective microbial community.

[0023] In the above scheme, the preferred carrier dosage is 30% to 50% of the effective microbial community mass. This is to achieve a precise balance between carrier function and microbial activity, maximize the degradation efficiency of the microbial agent, and overcome the problem of low survival rate of traditional microbial agents in complex industrial water environments. Specifically, within the range of 30% to 50%, the mixed carrier of sufficient bentonite and bran can fully adsorb and fix functional microorganisms and resist damage from the external environment, while also releasing sufficient nutrients to promote microbial community proliferation. This avoids the problems of insufficient fixation capacity and nutrient supply due to too low a carrier dosage, or too high a dosage crowding out the proportion of effective microbial communities and reducing the core degradation efficiency. In a further proposed solution, a dosage of 40% was selected. At this dosage, the carrier's functions of "solidifying and protecting bacteria" and "providing energy and promoting growth" are perfectly matched with the degradation activity of the effective microbial community. This not only provides stable survival and growth support for the three strains but also ensures that the community occupies a reasonable proportion in the agent, ensuring that it can quickly exert a synergistic degradation effect in the application scenario. Compared with other dosages, this solution can better balance the storage stability, transportation convenience, and actual degradation efficiency of the agent, allowing the composite microbial agent to maintain a high COD degradation capacity in complex water treatment scenarios.

[0024] Preferably, the mass ratio of trehalose to skim milk powder in the preservative is (1~2):(1~2); preferably, the mass ratio of trehalose to skim milk powder in the preservative is 1:1.

[0025] The above scheme optimizes the mass ratio of trehalose to skim milk powder in the protectant to be (1~2):(1~2). The core advantage is that it achieves a precise balance between physical protection and nutritional supply, maximizing the synergistic effect of the protectant. Specifically, within the range of (1~2):(1~2), trehalose can fully protect the cell membrane integrity of the three microorganisms, resisting dryness and osmotic pressure shocks, while skim milk powder can form a protein protective film, isolating shear forces and oxidants, and simultaneously providing amino acid nutrition. This avoids the problem of insufficient protection and nutritional deficiencies caused by either an excessively high proportion of a certain component leading to a single function or an excessively low proportion. In a further formulation, a specific 1:1 ratio is selected. Under this specific ratio, the "internal protective film" effect of trehalose and the "external protective barrier + nutritional supply" function of skim milk powder complement each other perfectly. The physical protection under the synergistic effect of the two is more comprehensive and the nutritional supply is more balanced, which can maximize the survival rate of live bacteria and ensure that the functional flora maintains high activity during storage, transportation and application. Compared with other ratios, it can better balance the protective effect of the protectant and the nutritional support capacity, and provide a stable guarantee for the efficient degradation of complex organic matter by various microorganisms.

[0026] Preferably, the amount of the protectant is 5% to 10% of the mass of the effective microbial community; preferably, the amount of the protectant is 8% of the mass of the effective microbial community.

[0027] The above-mentioned scheme optimizes the dosage of the protectant to 5%–10% of the effective microbial community's mass. The core advantage is achieving a precise balance between the protective effect and the core efficacy of the microbial agent. Specifically, within the 5%–10% range, sufficient trehalose and skim milk powder can work synergistically to ensure the cell membrane integrity of each functional microorganism and resist damage from the external environment. Simultaneously, it provides necessary nutritional support, preventing insufficient protection and decreased viable bacterial survival rates due to excessively low protectant dosage. Conversely, it prevents excessive dosage from crowding out the effective microbial community and carrier, avoiding resource waste or affecting the synergistic degradation efficiency of the microbial community. In a further refinement, 8% was determined to be the optimal dosage. At this dosage, the "physical protection + nutrient supply" function of the protectant is perfectly matched with the role of the microbial community and the carrier. This not only minimizes the risk of microbial inactivation during storage, transportation and application, but also ensures that the effective microbial community occupies a reasonable proportion, enabling it to quickly colonize in water treatment scenarios and play a synergistic degradation role. Compared with other dosages, this method better balances the storage stability, application compatibility and COD degradation efficiency of the microbial agent, allowing the compound microbial agent to maintain its high-efficiency degradation performance.

[0028] The present invention also provides a method for preparing the above-mentioned composite microbial agent, comprising: Each bacterial community in the effective microbial community was activated and cultured in a large-scale manner to obtain a high-concentration bacterial solution; High-concentration bacterial solutions of each bacterial group in the effective microbial community are mixed to obtain a composite bacterial solution; Bentonite and wheat bran are mixed to obtain a carrier; Trehalose and skim milk powder are mixed to obtain a preservative; The carrier and the protective agent are mixed evenly and then added to the composite bacterial solution to obtain a mixture; The mixture is dried at low temperature, pulverized, sieved, and packaged to obtain the compound microbial agent.

[0029] The above preparation method is simple and can fully guarantee the activity and synergistic degradation efficiency of the bacterial agent. Specifically, the above scheme first activates and expands the culture of each bacterial community in the effective microbial community, allowing each strain to reach a high concentration and high activity state independently, avoiding competitive inhibition during mixed culture and laying the foundation for subsequent synergistic effects of the bacterial community. Then, the high-concentration bacterial solutions are mixed to obtain a composite bacterial solution, while the carrier component and the protective agent component are premixed separately to ensure that the carrier and protective agent are evenly dispersed. When they are fused with the composite bacterial solution, the bacterial community, carrier, and protective agent can be fully contacted, avoiding functional failure caused by uneven local concentrations. Finally, a low-temperature drying process is used, combined with the synergistic protective effect of trehalose and skim milk powder, to minimize the mortality rate of microorganisms during the drying process and extend the shelf life of the bacterial agent. The subsequent pulverization and sieving processes ensure that the bacterial agent has a uniform morphology, which is convenient for storage, transportation, and quantitative dosing in actual application. The entire process does not require complex equipment, is suitable for industrial production, and can completely preserve the "decomposition-utilization-purification" synergistic chain of the effective bacterial community and the core functions of the carrier and protective agent, ensuring the efficient degradation effect of the finished bacterial agent on COD.

[0030] Preferably, the drying temperature is 35~40℃. This specific selection of a drying temperature of 35~40℃ ensures the bacterial agent is dried and formed while maximizing the preservation of the activity of the complex microbial community, achieving an optimal balance between drying efficiency and viable cell survival rate. Specifically, drying at this temperature meets the dryness requirements for storage and transportation while avoiding damage to the cell membranes of the bacterial agent from high temperatures, reducing bacterial protein denaturation and enzyme activity loss. Combined with the synergistic protective effect of trehalose and skim milk powder, this further reduces the bacterial mortality rate during the drying process. Simultaneously, this temperature does not damage the porous structure and nutrient release performance of the carrier (bentonite + bran), nor does it affect the physical protection and nutrient supply functions of the preservative. This ensures that the dried bacterial agent maintains the complete effectiveness of "synergistic degradation of the microbial community + carrier-based bacterial fixation and energy supply + preservative-based bacterial protection." Compared to lower temperatures, this improves drying efficiency and shortens the production cycle; compared to higher temperatures, it significantly increases the viable cell survival rate, allowing the finished bacterial agent to maintain a consistently high COD degradation capacity during application.

[0031] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. High efficiency: This microbial agent uses a variety of complementary bacterial groups to rapidly degrade a variety of organic matter. Compared with single microbial agents or traditional activated sludge methods, the COD removal efficiency can be increased by 10% to 15% or more. 2. Good stability: This bacterial agent uses a solid carrier and protectant, resulting in a long shelf life, high survival rate of live bacteria, and easy transportation and storage; 3. Environmentally friendly: This microbial agent does not contain chemical agents, has no secondary pollution, and can effectively reduce the production of residual sludge, thus having environmentally friendly characteristics; 4. Rapid start-up: Adding the bacterial agent of this invention can significantly shorten the start-up or recovery time of the biochemical system and improve the emergency response capability of the treatment facility; 5. Broad spectrum: Applicable to a variety of industrial wastewater (such as food, brewing, and chemical) and domestic sewage, it has a good removal effect on COD of different water qualities. Detailed Implementation

[0032] The technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. However, those skilled in the art will understand that the embodiments described below are some embodiments of the present invention, but not all embodiments, and are only used to illustrate the present invention, and should not be regarded as limiting the scope of the present invention. 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. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall be followed. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0033] Example 1 First, activate and expand the bacterial strains: Take Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida, respectively, and inoculate them into their respective liquid culture media. Incubate at 30-37℃ with shaking for 18-36 hours until the bacterial concentration reaches 3×10⁻⁶. 9 CFU / g.

[0034] Then, take 18g of Bacillus subtilis, 3g of Saccharomyces cerevisiae, and 9g of Pseudomonas putida, mix and stir evenly to obtain a compound bacterial solution.

[0035] Meanwhile, take 4g of bentonite and 8g of wheat bran, mix them evenly to obtain a carrier. Take 1.2g of trehalose and 1.2g of skim milk powder, mix them evenly to obtain a preservative.

[0036] Next, the carrier and protectant are mixed evenly and then slowly added to the composite bacterial solution while stirring, so that the bacterial solution is fully adsorbed onto the carrier, thus obtaining the mixture.

[0037] Finally, the mixture is dried under low temperature vacuum at 35-40℃ to reduce its moisture content to below 10%, then crushed, sieved, and packaged to obtain the finished solid powder bacterial agent.

[0038] Tests showed that the total number of live bacteria in the finished product of this microbial agent was ≥2 billion / gram.

[0039] Example 2 The only difference between this embodiment and Embodiment 1 is that the compound bacterial solution contains 18g of Bacillus subtilis, 6g of Saccharomyces cerevisiae, and 6g of Pseudomonas putida.

[0040] Example 3 The only difference between this embodiment and Embodiment 1 is that the compound bacterial solution contains 15g of Bacillus subtilis, 3g of Saccharomyces cerevisiae, and 12g of Pseudomonas putida.

[0041] Example 4 The only difference between this embodiment and Embodiment 1 is that the compound bacterial solution contains 15g of Bacillus subtilis, 6g of Saccharomyces cerevisiae, and 9g of Pseudomonas putida.

[0042] Example 5 The only difference between this embodiment and Embodiment 1 is that the compound bacterial solution contains 12g of Bacillus subtilis, 9g of Saccharomyces cerevisiae, and 9g of Pseudomonas putida.

[0043] Example 6 The only difference between this embodiment and Embodiment 4 is that *Pseudomonas putida* is not used. The mass ratio of *Bacillus subtilis* to *Saccharomyces cerevisiae* in the compound bacterial solution remains unchanged, that is, the mass ratio of *Bacillus subtilis* to *Saccharomyces cerevisiae* is 5:2.

[0044] Example 7 The only difference between this embodiment and Embodiment 4 is that brewer's yeast is not used. The mass ratio of Bacillus subtilis to Pseudomonas putida in the compound bacterial solution remains unchanged, that is, the mass ratio of Bacillus subtilis to Pseudomonas putida is 5:3.

[0045] Example 8 The only difference between this embodiment and Embodiment 4 is that Bacillus subtilis is not used. The mass ratio of Saccharomyces cerevisiae to Pseudomonas putida in the compound bacterial solution remains unchanged, that is, the mass ratio of Saccharomyces cerevisiae to Pseudomonas putida is 2:3.

[0046] Example 9 The only difference between this embodiment and embodiment 4 is that the total weight of the carrier is adjusted to 10% of the effective microbial community (i.e., the compound bacterial solution).

[0047] Example 10 The only difference between this embodiment and embodiment 4 is that the total weight of the carrier is adjusted to 30% of the effective microbial community (i.e., the compound bacterial solution).

[0048] Example 11 The only difference between this embodiment and embodiment 4 is that the total weight of the carrier is adjusted to 50% of the effective microbial community (i.e., the compound bacterial solution).

[0049] Example 12 The only difference between this embodiment and embodiment 4 is that the total weight of the carrier is adjusted to 80% of the effective microbial community (i.e., the compound bacterial solution).

[0050] Example 13 The only difference between this embodiment and embodiment 4 is that the mass ratio of the carrier bentonite to the bran in the carrier is adjusted to 1:1.

[0051] Example 14 The only difference between this embodiment and embodiment 4 is that the mass ratio of the carrier bentonite to the bran in the carrier is adjusted to 1:3.

[0052] Example 15 The only difference between this embodiment and Embodiment 4 is that the total weight of the protective agent is adjusted to 2% of the effective microbial community (i.e., the compound bacterial solution).

[0053] Example 16 The only difference between this embodiment and embodiment 4 is that the total weight of the protective agent is adjusted to 5% of the effective microbial community (i.e., the compound bacterial solution).

[0054] Example 17 The only difference between this embodiment and Embodiment 4 is that the total weight of the protective agent is adjusted to 10% of the effective microbial community (i.e., the compound bacterial solution).

[0055] Example 18 The only difference between this embodiment and Embodiment 4 is that the total weight of the protective agent is adjusted to 15% of the effective microbial community (i.e., the compound bacterial solution).

[0056] Example 19 The only difference between this embodiment and embodiment 4 is that the mass ratio of trehalose and skim milk powder in the preservative is adjusted to 2:1.

[0057] Example 20 The only difference between this embodiment and embodiment 4 is that the mass ratio of trehalose and skim milk powder in the preservative is adjusted to 1:2.

[0058] Experimental Example 1 The microbial agents prepared in Examples 1-20, as well as commercially available microbial agents A, B, and C (the groups corresponding to the three commercially available microbial agents are referred to as Comparative Example 1, Comparative Example 2, and Comparative Example 3, respectively), were used to treat wastewater from the equalization tank of a food factory. The initial COD of the wastewater was 1500 mg / L.

[0059] After processing, the supernatant after centrifugation was taken and the COD of each group was tested. The results are shown in Table 1 below.

[0060] Table 1 Experimental Results

[0061] Based on the above data, the composite microbial agent of the present invention can achieve good COD removal effect. Under the premise of using environmentally friendly formulation, its COD removal efficiency is no less than that of commercially available products. Among them, Example 4 has the best removal efficiency, and its COD removal efficiency can be increased by more than 10% to 15% compared with Comparative Examples 1-3.

[0062] Comparing the experimental results of Examples 1-5, it can be found that Example 4 has the best effect, with a COD removal rate of 88.0%, which is significantly better than the other four examples. This is because the advantage of Example 4 lies in its optimized component ratio, and the dosage and ratio of the carrier and protective agent are within the optimal range. The performance data of Example 4 shows that the three functionally complementary microorganisms work synergistically to achieve efficient degradation of complex organic matter, effectively overcoming the shortcomings of traditional biological agents in high organic matter environments. These five sets of data confirm that the ratio of viable bacteria of Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida is 5:2:3 to exert the efficient degradation of complex organic matter by this composite microbial agent.

[0063] Examples 6-8 show that the lack of synergistic effects among the three complementary microorganisms affects the degradation performance in high-organic-matter environments. Data from Example 6 indicates that the absence of *Pseudomonas putida* easily leads to degradation blind spots, significantly reducing the degradation efficiency of the prepared bio-agent. While Example 7 is slightly better than Example 6, it is still far inferior to Example 4, indicating that *Pseudomonas putida* has a higher priority than *Saccharomyces cerevisiae*. Example 8 demonstrates that *Bacillus subtilis* plays a crucial role in degradation within this bio-agent. *Bacillus subtilis* secretes abundant extracellular enzymes such as proteases, amylases, and lipases, breaking down large organic molecules (such as proteins, polysaccharides, and fats) in wastewater into smaller organic molecules and organic acids, providing usable substrates for other microorganisms. Simultaneously, it can form spores, exhibiting strong resistance and contributing to the survival of the bio-agent in harsh environments. *Saccharomyces cerevisiae*, on the other hand, readily utilizes small-molecule sugars and organic acids as carbon sources for rapid growth, directly consuming dissolved COD in the water. Its metabolites can also promote the growth of other bacterial communities, forming a stable micro-ecosystem. *Pseudomonas putida* has an extremely strong ability to degrade aromatic compounds, hydrocarbons, and other recalcitrant organic matter, filling the degradation blind spots of the first two bacteria and broadening the degradation spectrum of the microbial agent. These three sets of data confirm that the synergistic effect of the three bacteria is necessary for the prepared composite microbial agent to possess the property of efficiently degrading complex organic matter.

[0064] Data from Examples 9-12 show that the amount of carrier also affects the COD treatment performance of the microbial agent. Example 9 indicates that insufficient carrier leads to fewer microbial attachment points and insufficient nutrient supply, thus affecting the organic matter degradation performance of the microbial agent. Example 10 shows that excessive carrier dilutes the effective bacterial concentration, weakening the degradation of complex organic matter. Only when the carrier amount is maintained within the range of 30%–50% of the effective microbial community can a relatively good synergistic effect be achieved. The optimal synergistic effect is achieved when the carrier amount is 40% of the effective microbial community.

[0065] By comparing Example 4 with Examples 13-14, it can be seen that the COD removal effect of Example 4 is far superior to that of Examples 13 and 14. This indicates that the ratio and amount of bentonite and wheat bran, the carriers of the composite microbial agent of the present invention, affect the COD degradation performance of the microbial agent. Example 13 shows that when the proportion of inorganic carrier bentonite is too high, insufficient nutrients lead to slow microbial proliferation and a decrease in COD removal efficiency. Example 14 shows that when the proportion of organic carrier wheat bran is too high, the carrier is easily washed away by aeration, resulting in insufficient colonization. Therefore, maintaining the ratio of the two at 1:2 is necessary to achieve the optimal synergistic effect.

[0066] Comparing Examples 15-18, it can be seen that the dosage of the protectant also affects the COD treatment performance of the microbial agent. The experimental results of Example 15 show that insufficient protectant dosage leads to inadequate protection of microorganisms, thus affecting the survival rate and cycle of the microbial agent. Example 18 shows that excessive protectant dosage easily leads to moisture absorption and clumping of the agent or osmotic pressure imbalance, affecting the normal proliferation of the agent and weakening or even abnormally degrading complex organic matter. Therefore, only by maintaining the protectant dosage within the range of 5% to 10% can a relatively good protective effect be achieved. The protective effect is optimal at a dosage of 8%.

[0067] By comparing Example 4 with Examples 19-20, it can be seen that the COD removal effect of Example 4 is significantly better than that of Examples 19 and 20. It is evident that the ratio of trehalose to skim milk powder in the preservative affects the COD degradation performance of the microbial agent. As shown in Example 19, when the trehalose content as a sugar preservative is too high, there is a lack of a protective film formed by proteins. Microorganisms are easily damaged by mechanical shear forces during transportation and application, affecting cell survival. Simultaneously, high sugar concentrations easily absorb moisture and clump, leading to mold growth and reduced storage time. As shown in Example 20, when the skim milk powder content as a protein preservative is too high, the resistance to drying and freezing is weak. Proteins themselves are easily denatured due to dehydration or low temperatures, causing microbial cell membrane rupture and increasing the loss rate of live bacteria in the dried agent. Excessive protein also easily breeds other bacteria, especially increasing the risk of mold growth in high humidity environments. Therefore, the optimal protective effect is achieved when the ratio of the two preservatives is 1:1.

[0068] In summary, the embodiments of the present invention provide a COD removal agent that is highly efficient, broad-spectrum, adaptable, and can reduce the production of residual sludge. This agent is both practical and environmentally friendly, and can effectively reduce production costs and COD removal costs.

[0069] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A compound microbial agent for degrading chemical oxygen demand, comprising: Effective microbial communities, carriers, and protectants; The effective microbial community is a mixture of at least two of Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putidae. The carrier is a mixture of bentonite and wheat bran; The protective agent is a mixture of trehalose and skim milk powder.

2. The compound microbial agent according to claim 1, characterized in that, The effective microbial community includes Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida.

3. The compound microbial agent according to claim 2, characterized in that, The mass ratio of Bacillus subtilis, Saccharomyces cerevisiae and Pseudomonas putida is (3~6):(1~3):(2~4).

4. The compound microbial agent according to claim 3, characterized in that, The mass ratio of Bacillus subtilis, Saccharomyces cerevisiae, and Pseudomonas putida is 5:2:

3.

5. The compound microbial agent according to claim 1, characterized in that, The mass ratio of bentonite to wheat bran in the carrier is 1:(1~3); Preferably, the mass ratio of bentonite to wheat bran in the carrier is 1:

2.

6. The compound microbial agent according to claim 1, characterized in that, The amount of carrier used is 30% to 50% of the mass of the effective microbial community; Preferably, the amount of carrier used is 40% of the mass of the effective microbial community.

7. The compound microbial agent according to claim 1, characterized in that, The mass ratio of trehalose to skim milk powder in the protective agent is (1~2):(1~2); Preferably, the mass ratio of trehalose to skim milk powder in the protective agent is 1:

1.

8. The compound microbial agent according to claim 1, characterized in that, The amount of the protective agent used is 5% to 10% of the mass of the effective microbial community; Preferably, the amount of the protective agent is 8% of the mass of the effective microbial community.

9. A method for preparing the composite microbial agent as described in any one of claims 1-8, characterized in that, include: Each bacterial community in the effective microbial community was activated and cultured in a large-scale manner to obtain a high-concentration bacterial solution; High-concentration bacterial solutions of each bacterial group in the effective microbial community are mixed to obtain a composite bacterial solution; Bentonite and wheat bran are mixed to obtain a carrier; Trehalose and skim milk powder are mixed to obtain a preservative; The carrier and the protective agent are mixed evenly and then added to the composite bacterial solution to obtain a mixture; The mixture is dried at low temperature, pulverized, sieved, and packaged to obtain the compound microbial agent.

10. The preparation method according to claim 9, characterized in that, The drying temperature is 35~40℃.

Images