Special compound microbial inoculant for processing waste water of trichosanthes fruit and preparation method thereof

Abstract

This invention discloses a compound microbial agent for processing wastewater from Trichosanthes kirilowii seeds and its preparation method, relating to the field of environmental microbiology. The compound microbial agent is composed of saponin-degrading strains, polysaccharide-degrading strains, and decolorizing strains in a volume ratio of 3:2:1. The saponin-degrading strains are Bacillus strains, the polysaccharide-degrading strains are Pseudomonas strains, and the decolorizing strains are Rhodococcus strains. The preparation method includes: isolating and screening functional strains from Trichosanthes kirilowii planting soil and wastewater from the processing workshop; expanding the culture to a bacterial concentration of CFU / mL or higher; mixing them in a volume ratio of 3:2:1; adding a protective agent; and drying to prepare a powder with a viable count greater than CFU / g. This compound microbial agent is added to the biochemical treatment unit at 0.01%-0.05% of the wastewater volume. Microbial community acclimatization can be completed within 7-14 days, effectively improving COD removal rate, saponin degradation rate, and color removal rate. It is suitable for the efficient biological treatment of Trichosanthes kirilowii seed processing wastewater.

Description

Technical Field

[0001] This invention relates to the field of environmental microbiology, specifically to a compound microbial agent for processing wastewater from Trichosanthes kirilowii seeds and its preparation method. Background Technology

[0002] The existing compound microbial agents and their preparation methods specifically for processing wastewater from Trichosanthes kirilowii seeds still have the following drawbacks in practical use:

[0003] Trichosanthes kirilowii, a traditional Chinese medicinal herb, has significant medicinal value in its fruit, seeds, pericarp, and rhizome. With the large-scale development of the Trichosanthes kirilowii industry, the wastewater treatment problem generated during seed processing has become increasingly prominent. The wastewater from Trichosanthes kirilowii seed processing mainly originates from washing, soaking, shelling, and pressing processes. This wastewater contains a large amount of organic substances such as saponins, polysaccharides, proteins, and natural pigments, and is characterized by high COD concentration, deep color, and poor biodegradability.

[0004] Saponins are a class of complex glycoside compounds with surface-active properties. They exhibit hemolytic and toxic effects on microbial cell membranes, inhibiting the activity of microorganisms in conventional activated sludge and leading to difficulties in sludge acclimation. Studies have shown that the biodegradation of saponins requires the participation of specific microbial glycosidase systems, which are lacking in conventional activated sludge. Furthermore, the high viscosity and low mass transfer efficiency of polysaccharides in the wastewater from Trichosanthes kirilowii seed processing further complicate biological treatment. In addition, the natural pigments in the wastewater make color removal difficult, and conventional biological treatment is insufficient to meet emission standards.

[0005] Currently, the main technologies for treating wastewater from Trichosanthes kirilowii seed processing fall into two categories: physicochemical methods and biological methods. Physicochemical methods, such as coagulation sedimentation, adsorption, and membrane separation, can reduce COD and color to some extent, but they suffer from problems such as high reagent consumption, high operating costs, and the potential for secondary pollution. The conventional activated sludge process, a mainstream technology in biological methods, is the treatment of organic wastewater, utilizing the metabolic activity of microbial communities in an aeration tank to degrade organic matter. However, due to the inhibitory effect of saponins and polysaccharides in Trichosanthes kirilowii seed processing wastewater on microorganisms, conventional activated sludge processes suffer from drawbacks such as a long microbial acclimatization period (typically 30-60 days), unstable degradation efficiency, and poor resistance to shock loads.

[0006] For the biological treatment of saponin-containing wastewater, existing technologies mainly employ a combination of electrochemical pretreatment and biological treatment. Electrochemical methods are used to reduce saponin toxicity before biochemical degradation. However, this technology involves high equipment investment and energy consumption, making it unsuitable for large-scale application in Trichosanthes kirilowii seed processing enterprises. In addition, some studies have used functional strains screened from specific environments to construct compound microbial agents for treating special wastewaters; however, no research reports have been found on dedicated compound microbial agents specifically for Trichosanthes kirilowii seed processing wastewater.

[0007] Therefore, developing a specialized compound microbial agent tailored to the characteristics of wastewater from Trichosanthes kirilowii seed processing, enabling rapid microbial acclimatization and efficient degradation of specific organic pollutants such as saponins and polysaccharides, is of great significance for improving the treatment efficiency of wastewater from Trichosanthes kirilowii seed processing, reducing treatment costs, and promoting the green and sustainable development of the Trichosanthes kirilowii industry. Summary of the Invention

[0008] The purpose of this invention is to provide a special compound microbial agent for wastewater from Trichosanthes kirilowii seed processing and its preparation method, so as to solve the above-mentioned problems.

[0009] To achieve the above objectives, the present invention provides the following technical solution: a compound microbial agent specifically for processing wastewater from Trichosanthes kirilowii seeds, comprising a mixture of saponin-degrading strains, polysaccharide-degrading strains, and decolorizing strains in a volume ratio of 3:2:1; wherein the saponin-degrading strains are Bacillus strains, the polysaccharide-degrading strains are Pseudomonas strains, and the decolorizing strains are Rhodococcus strains; the bacterial concentration of each strain after expanded culture reaches... The compound bacterial agent is in powder form with a CFU / mL or higher and a viable count greater than [value missing]. CFU / g.

[0010] Furthermore, the Bacillus, Pseudomonas, and Rhodococcus strains were all isolated from the soil in which Trichosanthes kirilowii was planted or from the wastewater in the Trichosanthes kirilowii processing workshop.

[0011] Furthermore, the powder is prepared by mixing bacterial liquid with a protective agent and then spray drying or freeze drying. The protective agent is maltodextrin and skim milk powder, and the amount added is 5-15% of the volume of the mixed bacterial liquid, wherein the mass ratio of maltodextrin to skim milk powder is 2:1.

[0012] A method for preparing the compound microbial agent as described above, characterized by comprising the following steps:

[0013] (1) Bacillus strains with saponin degradation ability, Pseudomonas strains with polysaccharide degradation ability and Rhodococcus strains with pigment degradation ability were isolated and screened from the soil of Trichosanthes kirilowii planting and sewage from the processing workshop.

[0014] (2) Each strain was cultured separately in an optimized culture medium until the logarithmic growth phase was reached, so that the bacterial concentration reached the target value. CFU / mL or higher;

[0015] (3) Mix Bacillus, Pseudomonas and Rhodococcus at a volume ratio of 3:2:1 to obtain a mixed bacterial solution;

[0016] (4) Add a protectant to the mixed bacterial solution, and then spray-dry or freeze-dry to make a powder, so that the viable count is greater than 100%. CFU / g.

[0017] Further, the optimized culture medium in step (2) is: peptone 10-15 g / L, yeast extract 5-8 g / L, sodium chloride 5-10 g / L, pH 7.0-7.5, culture temperature 28-35℃, and shaker speed 150-200 r / min.

[0018] Application of a compound microbial agent as described above in the treatment of wastewater from Trichosanthes kirilowii seed processing.

[0019] Furthermore, the compound bacterial agent is added to the biochemical treatment unit at 0.01% to 0.05% of the wastewater volume, and in conjunction with nutrient supplementation and aeration conditions, the bacterial community is established within 7 to 14 days.

[0020] Furthermore, the nutrient supplementation includes supplementing nitrogen and phosphorus sources, and controlling the influent C:N:P mass ratio to be 100:5:1; the aeration conditions are to control the dissolved oxygen concentration at 2-4 mg / L and the hydraulic retention time at 12-24 h.

[0021] Furthermore, the treated wastewater exhibits a COD removal rate greater than 90%, a saponin degradation rate greater than 95%, a color removal rate greater than 85%, and the effluent meets the rural domestic sewage treatment and discharge standards.

[0022] Furthermore, the application also includes the combined use of the compound microbial agent with other Trichosanthes kirilowii processing wastewater treatment processes, wherein the other processes include coagulation sedimentation, anaerobic treatment or membrane separation.

[0023] Compared with the prior art, the compound microbial agent for processing wastewater of Trichosanthes kirilowii seeds and its preparation method provided by the present invention have the following beneficial effects:

[0024] (1) The microbial community domestication cycle is significantly shortened: This invention targets specific pollutants such as saponins and polysaccharides in the wastewater from Trichosanthes kirilowii seed processing. Functional strains are screened from the soil of Trichosanthes kirilowii planting and wastewater from the processing workshop and compounded in a specific ratio of 3:2:1. This overcomes the technical defect of long domestication cycle of conventional activated sludge and can complete the establishment of microbial community domestication in 7-14 days, thereby improving domestication efficiency.

[0025] (2) High pollutant degradation efficiency: The Bacillus, Pseudomonas and Rhodococcus strains in the compound bacterial agent of this invention work together to specifically degrade saponins, polysaccharides and pigments. The COD removal rate, saponin degradation rate and color removal rate of the treated wastewater are effectively improved, and the effluent can stably meet the rural domestic sewage treatment discharge standards.

[0026] (3) Strong resistance to shock load: The present invention uses native functional strains isolated from the processing environment of Trichosanthes kirilowii, which have natural adaptability and tolerance to the wastewater of Trichosanthes kirilowii seed processing. With the use of maltodextrin and skim milk powder protectant, the bacterial agent has good stability and can still maintain high efficiency degradation performance under conditions of salinity fluctuation and organic load change.

[0027] (4) Low cost and easy operation: The dosage of the compound microbial agent of this invention is only 0.01%-0.05% of the wastewater volume, which is far lower than the dosage ratio of 2%-6% of conventional microbial agents. It can be directly added to the existing biochemical treatment unit without the need to add special equipment, thus reducing the wastewater treatment cost of Trichosanthes kirilowii seed processing enterprises.

[0028] (5) Long shelf life and easy transportation: The present invention uses spray drying or freeze drying processes to prepare powder, with a viable count greater than [missing information]. With a CFU / g content, it can be stored at 4℃ for more than 12 months, which is convenient for industrial production and long-distance transportation, overcoming the shortcomings of short shelf life and easy decline in activity of liquid bacterial agents. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0030] Figure 1 This is a flowchart illustrating the overall process flow of the present invention. Detailed Implementation

[0031] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0032] Please see Figure 1 A compound microbial agent for processing wastewater from Trichosanthes kirilowii seeds and its preparation method are disclosed. The agent comprises a mixture of saponin-degrading strains, polysaccharide-degrading strains, and decolorizing strains in a volume ratio of 3:2:1. The saponin-degrading strains are Bacillus strains, the polysaccharide-degrading strains are Pseudomonas strains, and the decolorizing strains are Rhodococcus strains. The concentration of each strain in the culture solution after expansion reaches [a certain level]. CFU / mL or higher, compound bacterial agent in powder form, viable count greater than CFU / g.

[0033] First, it should be noted that "combination" refers to mixing microbial strains with different functions in a specific ratio to form a synergistic microbial community. The volume ratio of 3:2:1 here refers to the volume ratio of Bacillus spp., Pseudomonas spp., and Rhodococcus spp. culture media. This ratio was obtained through extensive experimental optimization, enabling the three types of strains to form stable ecological niches in the wastewater treatment system and avoiding competitive inhibition.

[0034] Regarding "CFU / mL," this is an abbreviation for "colony forming units per milliliter" in microbiology, a standard unit for measuring the number of viable bacteria. The determination method is as follows: After serially diluting the bacterial suspension, take an appropriate amount of the diluted solution and spread it on a solid culture medium plate. After incubating at a suitable temperature for a certain period of time, count the number of colonies formed on the plate, and then multiply it by the dilution factor to calculate the viable bacteria concentration in the original bacterial suspension. CFU / mL indicates that each milliliter of bacterial solution contains 1 billion live bacteria.

[0035] This invention selects three strains of bacteria—Bacillus, Pseudomonas, and Rhodococcus—based on the unique water quality characteristics of wastewater from the processing of Trichosanthes kirilowii seeds. This wastewater contains a large amount of saponins, which are surface-active glycosides that have a toxic inhibitory effect on microorganisms in conventional activated sludge. Bacillus strains can secrete glycosidases to hydrolyze the sugar chains of saponin molecules, thereby reducing their toxicity and achieving degradation. Pseudomonas strains have a strong polysaccharide degradation capacity, secreting extracellular polysaccharide enzymes to break down polysaccharides in the wastewater into smaller sugar molecules, reducing wastewater viscosity and improving mass transfer efficiency. Rhodococcus strains have excellent pigment degradation capabilities, decomposing natural pigments in the wastewater and achieving decolorization.

[0036] First, specific functional bacterial strains were screened for specific pollutants in the wastewater from the processing of Trichosanthes kirilowii seeds, rather than using general activated sludge strains. Second, a specific ratio of 3:2:1 was used to achieve synergistic effects among the three types of strains: Bacillus preferentially degrades saponins to detoxify them, creating a suitable living environment for Pseudomonas and Rhodococcus; Pseudomonas degrades polysaccharides to reduce viscosity and promote mass transfer; and Rhodococcus focuses on decolorization. Third, the compound bacterial agent was prepared in powder form, achieving a viable count of [missing information]. With a concentration of CFU / g or higher, it is easy to store, transport, and use.

[0037] First, the specialized functional strains have a high efficiency in degrading target pollutants, overcoming the problems of poor tolerance to saponins and low degradation efficiency of conventional activated sludge. Second, the specific ratio ensures the stable coexistence and synergistic effect of the three types of strains in the wastewater treatment system, avoiding the problems of insufficient function of a single strain or mutual inhibition of multiple strains. Third, the high viable count powder ensures that the dominant bacterial community can be established quickly after addition, shortening the acclimatization time.

[0038] Bacillus, Pseudomonas, and Rhodococcus strains were all isolated from the soil in which Trichosanthes kirilowii was planted or from wastewater in the Trichosanthes kirilowii processing workshop.

[0039] This further clarifies the source of the functional strains. The emphasis on strains originating from the soil used for Trichosanthes kirilowii cultivation or wastewater from Trichosanthes kirilowii processing plants is based on the principle of "biological adaptation": microorganisms that have long existed in the Trichosanthes kirilowii ecological environment have adapted to the unique secondary metabolites of Trichosanthes kirilowii through natural selection, possessing the genetic basis and metabolic capacity to degrade these substances. Compared to strains isolated from other environments, these "native strains" exhibit natural tolerance and highly efficient degradation performance against Trichosanthes kirilowii seed processing wastewater.

[0040] The principle behind isolating bacterial strains from the soil in which Trichosanthes kirilowii is grown is that the roots of Trichosanthes kirilowii secrete various organic substances, including saponins and polysaccharides. These substances accumulate in the rhizosphere soil, forming specific microbial niches. Therefore, the rhizosphere soil of Trichosanthes kirilowii is an ideal source for screening functional bacterial strains capable of degrading saponins and polysaccharides. The principle behind isolating bacterial strains from wastewater in Trichosanthes kirilowii processing workshops is that the wastewater contains high concentrations of Trichosanthes kirilowii processing residues over a long period. Microorganisms that can survive and reproduce in this extreme environment must possess the ability to degrade these pollutants.

[0041] Among them, the compound microbial agent constructed using native strains exhibits strong adaptability to wastewater from Trichosanthes kirilowii seed processing. After addition, it can exert its degradation effect without a long period of acclimatization, further shortening the system start-up time. Simultaneously, the native strains possess ecological safety in the Trichosanthes kirilowii processing environment, and will not pose any potential risks to the local ecosystem due to the introduction of exogenous microorganisms.

[0042] The powder is prepared by mixing bacterial liquids, adding a protectant, and then spray drying or freeze drying. The protectant is maltodextrin and skim milk powder, and the amount added is 5-15% of the volume of the mixed bacterial liquid, wherein the mass ratio of maltodextrin to skim milk powder is 2:1.

[0043] The specific preparation process and protectant formulation for the compound microbial agent powder are defined. A key technical challenge in converting liquid microbial agents into powder is maintaining microbial activity during the drying process. Drying causes cell dehydration; direct drying will cause cell membranes to rupture due to water loss, leading to bacterial death. Therefore, a protectant must be added.

[0044] The protective agent works by using maltodextrin, a polysaccharide that forms a glassy protective layer on the surface of bacterial cells, reducing mechanical damage to the cells from ice crystals during drying. It also dissolves rapidly upon rehydration, releasing the bacterial cells. Skim milk powder is rich in protein and lactose. Protein forms a protective film on the bacterial cell surface, while lactose acts as a filler and protectant, maintaining cell membrane integrity. A 2:1 mass ratio of maltodextrin and skim milk powder provides synergistic protection: maltodextrin offers structural support and rapid resolubility, while skim milk powder provides nutritional protection and cell membrane stabilization.

[0045] Spray drying is a drying method that disperses liquid materials into fine droplets through an atomizer and rapidly evaporates the moisture in hot air to obtain powder. It has the advantages of fast drying speed and continuous production. Freeze drying, on the other hand, first freezes the material into a solid state, and then removes the moisture by directly sublimating the ice under vacuum conditions. This method can maximize the preservation of microbial activity, but it is more expensive and has a longer cycle time.

[0046] The amount of protective agent added is limited to 5-15% of the volume of the mixed bacterial solution, based on the following considerations: when the amount added is less than 5%, the protective agent is insufficient to form a complete protective layer on the surface of the bacteria, resulting in a low survival rate after drying; when the amount added is more than 15%, the concentration of the protective agent is too high, which will increase the viscosity of the solution, affect the atomization effect, or form an excessively thick glassy layer, affecting the rehydration performance, while also increasing the cost.

[0047] By using specific protective agent formulations and dosages, the compound microbial agent can maintain a high number of viable bacteria even after drying. After the powder is stored at 4°C for 12 months, the number of viable bacteria can still be maintained at more than 80% of the initial value. This solves the problems of short shelf life and easy decline in activity of liquid microbial agents, making it convenient for industrial production and long-distance transportation.

[0048] A method for preparing the above-mentioned compound microbial agent includes the following steps:

[0049] (1) Bacillus strains with saponin degradation ability, Pseudomonas strains with polysaccharide degradation ability and Rhodococcus strains with pigment degradation ability were isolated and screened from the soil of Trichosanthes kirilowii planting and sewage from the processing workshop.

[0050] (2) Each strain was cultured separately in an optimized culture medium until the logarithmic growth phase was reached, so that the bacterial concentration reached the target value. CFU / mL or higher;

[0051] (3) Mix Bacillus, Pseudomonas and Rhodococcus at a volume ratio of 3:2:1 to obtain a mixed bacterial solution;

[0052] (4) Add a protectant to the mixed bacterial solution, and then spray-dry or freeze-dry to make a powder, so that the viable count is greater than 100%. CFU / g.

[0053] Step (1) is the strain isolation and screening step, which is the foundation of the entire preparation method. The purpose of this step is to obtain target strains with specific degradation functions from the natural environment. "Isolation and screening" refers to the process of isolating the target strains from the mixed microbial community using microbiological techniques and screening out strains with excellent functions through performance testing.

[0054] The specific operating procedure is as follows: First, samples of soil from Trichosanthes kirilowii cultivation or wastewater from processing workshops are collected, serially diluted, and then spread onto selective culture medium plates. For screening Bacillus strains, a medium with saponins as the sole carbon source is used; only strains capable of using saponins as a carbon source can form colonies. For screening Pseudomonas strains, a medium with polysaccharides as the sole carbon source is used. For screening Rhodococcus strains, an induction medium containing natural pigments from Trichosanthes kirilowii processing wastewater is used. After obtaining pure cultures using the streak plate method, colony morphology is observed, Gram staining is performed, physiological and biochemical identification is conducted, and 16S rRNA gene sequence analysis is performed to determine the genus and species of the strains.

[0055] This step yielded specialized strains with clear genetic backgrounds and well-defined functions, providing functional assurance for subsequent compounding. Compared to model strains or general strains, strains screened from the Trichosanthes kirilowii ecological environment exhibit better adaptability and degradation efficiency to Trichosanthes kirilowii seed processing wastewater.

[0056] Step (2) is the scaling-up step, the purpose of which is to cultivate the target strain obtained in step (1) on a large scale to obtain a sufficient number of cells. The "logarithmic growth phase" is a stage in the microbial growth curve. During this stage, microorganisms divide and reproduce at the maximum rate, their cellular metabolic activity is the strongest, and the number of cells increases exponentially. Cells in the logarithmic growth phase are chosen for the preparation of the compound microbial agent because the cells are most vigorous at this time, have better resistance to the external environment, and have the highest survival rate after drying.

[0057] "Optimized culture medium" refers to a culture medium formula determined through experiments based on the nutritional requirements of each strain, providing the optimal carbon source, nitrogen source, inorganic salts, vitamins, and other nutrients needed for the growth and reproduction of the strains. Culture conditions have also been optimized to ensure that the strains rapidly grow to the target concentration.

[0058] The connection between this step and step (1) is that step (1) obtains a small amount of pure culture at the laboratory scale, while step (2) scales it up to the production scale, with the bacterial concentration reaching [a certain level]. The concentration of CFU / mL or higher provides sufficient bacterial volume for mixing in step (3). The technical effect of this step is that a high concentration and high activity bacterial solution is obtained, which lays the foundation for the subsequent preparation of high viable bacterial powder.

[0059] Step (3) is the mixing step, which involves mixing the culture media of the three functional strains in a specific ratio to form a composite bacterial solution. The volume ratio of 3:2:1 was determined through extensive experimental optimization.

[0060] The formulation was determined based on the following: In the wastewater from the processing of Trichosanthes kirilowii seeds, saponins are the most significant inhibitory substances and must be preferentially degraded to eliminate toxicity; therefore, saponin-degrading strains constitute the largest proportion. Polysaccharides are the next most abundant, and their degradation helps reduce viscosity and promote mass transfer; therefore, polysaccharide-degrading strains constitute the second largest proportion. While pigments affect color, their concentration is relatively low, and Rhodococcus bacteria are highly tolerant of the environment; therefore, decolorizing strains constitute the smallest proportion. This formulation ensures that the three types of strains can form a stable microecological relationship in the wastewater treatment system: Bacillus rapidly degrades saponins, relieving the toxic inhibitory effects on Pseudomonas and Rhodococcus; Pseudomonas degrades polysaccharides, reducing system viscosity and providing better mass transfer conditions for Bacillus and Rhodococcus; and Rhodococcus efficiently degrades pigments in an environment with reduced toxicity and suitable viscosity.

[0061] The connection between this step and step (2) is that step (2) provides a high concentration of single-strain bacterial solution, and step (3) mixes them according to functional requirements to form a functionally complementary compound bacterial community. The technical effect of this step is that the synergistic effect of the three types of strains is achieved through a specific ratio, and the degradation effect of the compound bacterial agent is significantly better than that of a single strain or a bacterial agent mixed in equal proportions.

[0062] Step (4) is the drying and shaping step, which is the key step in preparing the mixed bacterial solution obtained in step (3) into a powder product. In this step, a protective agent is first added to the mixed bacterial solution. The protective agent forms a glassy or film-like protective layer during the drying process to reduce damage to the bacteria. Then, the moisture is removed by spray drying or freeze drying to obtain the powder product.

[0063] The connection between this step and step (3) is that step (3) provides a mixed bacterial solution with a reasonable functional ratio, while step (4) converts it into a solid dosage form that is easy to store and use, while ensuring bacterial activity through a protectant. The technical effect of this step is that it yields a viable bacterial count greater than [a certain number]. This highly active powder, containing CFU / g, has a low moisture content, which inhibits microbial metabolic activity and extends its shelf life. The powder form facilitates precise measurement and dosing; it simply needs to be dissolved in water according to the specified ratio, making it easy to use.

[0064] The optimized culture medium in step (2) is: 10-15 g / L peptone, 5-8 g / L yeast extract, 5-10 g / L sodium chloride, pH 7.0-7.5, culture temperature 28-35℃, and shaker speed 150-200 r / min.

[0065] The specific process parameters for the scale-up culture in step (2) were further defined. These parameters were determined through single-factor experiments and orthogonal experiments to provide optimal growth and reproduction conditions for the three strains.

[0066] Peptone and yeast extract are commonly used organic nitrogen sources in microbial culture media, providing nutrients such as amino acids, peptides, and vitamins. Sodium chloride maintains the osmotic pressure of the culture medium and simulates the salinity of the strain's natural growth environment. pH 7.0-7.5 is the optimal pH range for most bacteria, within which bacterial enzyme activity is highest and growth rate is fastest. A culture temperature of 28-35℃ is the optimal growth temperature for mesophilic bacteria, compatible with the temperature of the Trichosanthes kirilowii processing environment and consistent with the operating temperature of most biochemical processing systems. A shaking speed of 150-200 r / min controls the dissolved oxygen level in the culture system. This speed range provides sufficient oxygen to meet the growth requirements of aerobic bacteria while avoiding shear force damage to the bacterial cells caused by excessively high speeds.

[0067] By optimizing the culture medium composition and culture conditions, all three strains were able to reach the logarithmic growth phase in a relatively short time, and the bacterial concentration was stabilized at a certain level. A CFU / mL or higher ensures the supply of bacteria and the stability of quality in subsequent steps.

[0068] Application of a compound microbial agent as described above in the treatment of wastewater from Trichosanthes kirilowii seed processing.

[0069] Add the compound microbial agent to the biological treatment unit at a rate of 0.01% to 0.05% of the wastewater volume, and with the addition of nutrients and aeration, complete the establishment of microbial community acclimatization within 7 to 14 days.

[0070] The specific application method and process parameters of the compound microbial agent are further defined. "Dosage of 0.01% to 0.05%" refers to adding 0.1-0.5 liters of compound microbial agent per cubic meter of wastewater. This dosage is much lower than the dosage ratio of conventional microbial agents because the compound microbial agent of this invention is a special high-efficiency agent with a high number of viable bacteria and strong adaptability to the target wastewater. A small amount can quickly establish a dominant bacterial community.

[0071] "Nutrient supplementation" refers to adding nitrogen and phosphorus sources to wastewater to meet the nutritional needs of microbial growth. Therefore, it is necessary to maintain an appropriate carbon-nitrogen-phosphorus ratio. "Aeration conditions" refer to supplying oxygen to the biochemical treatment unit through aeration equipment to maintain the dissolved oxygen concentration and meet the metabolic needs of aerobic microorganisms.

[0072] "Completion of microbial community acclimatization within 7 to 14 days" refers to the time required from the addition of the compound microbial agent to the system reaching a stable operating state. This time is significantly shorter than the acclimatization cycle of conventional activated sludge processes because the functional strains in the compound microbial agent of this invention have natural adaptability and efficient degradation capabilities to Trichosanthes kirilowii seed processing wastewater, eliminating the need for a long period of acclimatization.

[0073] By optimizing the dosage and operating conditions, the system achieved rapid start-up and efficient treatment of wastewater from Trichosanthes kirilowii seed processing, reducing operating costs and improving the stability and reliability of the treatment system.

[0074] Nutrient supplementation includes supplementing nitrogen and phosphorus sources, and controlling the influent C:N:P mass ratio to be 100:5:1; aeration conditions include controlling the dissolved oxygen concentration at 2-4 mg / L and the hydraulic retention time at 12-24 h.

[0075] The specific parameters for nutrient supplementation and aeration conditions were further defined. "C:N:P mass ratio 100:5:1" refers to a carbon, nitrogen, and phosphorus mass ratio of 100:5:1 in the wastewater, which is the optimal nutrient ratio for microbial growth in aerobic biological treatment. Wastewater from Trichosanthes kirilowii seed processing is typically rich in carbon but relatively deficient in nitrogen and phosphorus; therefore, nitrogen and phosphorus sources need to be supplemented to maintain this ratio and ensure normal microbial growth and metabolic activity.

[0076] The optimal dissolved oxygen concentration for aerobic biological treatment using activated sludge is 2-4 mg / L. Below 2 mg / L, microbial metabolic activity is limited, leading to decreased degradation efficiency; above 4 mg / L, it not only increases energy consumption but may also cause excessive oxidation and disintegration of sludge flocs. The hydraulic retention time of 12-24 h refers to the average residence time of wastewater in the biological treatment unit. This time range ensures sufficient contact and reaction time between pollutants and microorganisms, while balancing treatment efficiency and tank volume economy.

[0077] By precisely controlling the nutrient ratio and aeration conditions, the compound microbial agent is provided with an optimal growth environment, which effectively improves the COD removal rate, saponin degradation rate and color removal rate, and ensures that the effluent consistently meets the discharge standards.

[0078] The treated wastewater exhibits a COD removal rate of over 90%, a saponin degradation rate of over 95%, a color removal rate of over 85%, and meets the discharge standards for rural domestic sewage treatment.

[0079] The technical performance indicators for treating Trichosanthes kirilowii seed processing wastewater using the compound microbial agent of this invention are defined. "COD removal rate" refers to the percentage of chemical oxygen demand removed from the wastewater; "saponin degradation rate" refers to the percentage of saponin degradation in the wastewater; and "color removal rate" refers to the percentage of color removal from the wastewater.

[0080] The "Rural Domestic Sewage Treatment Discharge Standard" refers to the water quality limits specified in the "Rural Domestic Sewage Treatment Discharge Standard" (GB5749 or local standards), which typically require COD to be less than 100 mg / L and color to be less than 50 times. The raw wastewater from the processing of Trichosanthes kirilowii seeds usually has a COD of 2000-5000 mg / L and a color of 200-500 times. After treatment with the compound microbial agent of this invention, the COD is reduced to below 100 mg / L and the color is reduced to below 50 times, meeting the discharge standards.

[0081] The treatment effect of the compound microbial agent of this invention on wastewater from Trichosanthes kirilowii seed processing has been clarified, proving its industrial application value. The high removal rate indicates that this invention can effectively solve the problem of wastewater treatment in Trichosanthes kirilowii seed processing, achieving compliant discharge and resource utilization of the wastewater.

[0082] Applications also include the combined use of compound microbial agents with other wastewater treatment processes for Trichosanthes kirilowii processing, such as coagulation sedimentation, anaerobic treatment, or membrane separation.

[0083] The application of compound microbial agents has been expanded to include combined use with other wastewater treatment processes. "Coagulation and sedimentation" is a physicochemical method that uses coagulants to aggregate colloids and suspended solids in wastewater into larger particles for sedimentation and removal. It can be used for pretreatment to remove suspended solids and some colloidal substances from wastewater, reducing the load on subsequent biological treatment. "Anaerobic treatment" is a biological treatment method that utilizes anaerobic microorganisms to degrade organic matter under anaerobic conditions. It is suitable for high-concentration organic wastewater and can convert large organic molecules into smaller organic acids and methane. "Membrane separation" is a method that uses the selective permeability of membranes to separate pollutants in wastewater, such as ultrafiltration, nanofiltration, and reverse osmosis. It can be used for advanced treatment to achieve wastewater reuse.

[0084] The combined use of the compound microbial agent of the present invention with these processes includes: coagulation and sedimentation + aerobic treatment with compound microbial agent; aerobic treatment with compound microbial agent + membrane separation; anaerobic treatment + aerobic treatment with compound microbial agent, etc.

[0085] This invention improves the applicability and flexibility of the compound microbial agent, allowing for the selection of the optimal combination process based on the specific water quality characteristics and discharge requirements of the Trichosanthes kirilowii seed processing wastewater, thereby achieving the best economic and environmental benefits.

[0086] Example: Treatment of Trichosanthes kirilowii seed processing wastewater with compound microbial agents

[0087] 1. Wastewater quality

[0088] The wastewater sample was taken from a Trichosanthes kirilowii seed processing enterprise in Anhui Province, mainly originating from the washing, soaking, and shell-breaking processes. The water quality characteristics were: COD 2850 mg / L, saponin concentration 450 mg / L, color 320 times, and pH 6.8.

[0089] 2. Processing device

[0090] A sequencing batch reactor (SBR) with an effective volume of 10L was used. The reactor was equipped with a stirring mechanism.

[0091] Agitator, aeration head, dissolved oxygen meter, and online pH monitor.

[0092] 3. Test methods

[0093] The prepared compound microbial agent was added to the reactor at a dosage of 0.03% (v / v), i.e., 3g of powder. Urea and dipotassium hydrogen phosphate were added simultaneously to adjust the influent C:N:P ratio to 100:5:1. The operating cycle was as follows: 0.5h influent, 8h aeration reaction, 1h sedimentation, 0.5h effluent discharge, 0h idle time, total cycle 10h, hydraulic retention time 20h.

[0094] 4. Test Results

[0095] Acclimation period: From the start of adding the compound microbial agent, effluent COD was continuously monitored. On day 3, the effluent COD decreased to 450 mg / L; on day 7, it decreased to 280 mg / L; and from day 10 onwards, it stabilized below 220 mg / L, indicating the system had reached stable operation. The acclimation period was 10 days, significantly shorter than the 30-60 days required for conventional activated sludge processes.

[0096] Treatment results: After the system stabilized, the average effluent COD was 245 mg / L, with a COD removal rate of 91.4%; the effluent saponin concentration was 18 mg / L, with a saponin degradation rate of 96.0%; and the effluent color was 38 times higher, with a color removal rate of 88.1%. All indicators met the rural domestic sewage treatment and discharge standards.

[0097] Shock load resistance: On day 25, the influent COD was increased to 4500 mg / L. After 2 days of adaptation, the effluent COD recovered to below 280 mg / L, indicating that the compound bacterial agent has good shock load resistance.

[0098] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. A compound microbial agent specifically for wastewater from Trichosanthes kirilowii seed processing, characterized in that, The culture medium is composed of saponin-degrading strains, polysaccharide-degrading strains, and decolorizing strains in a volume ratio of 3:2:1; the saponin-degrading strains are Bacillus strains, the polysaccharide-degrading strains are Pseudomonas strains, and the decolorizing strains are Rhodococcus strains; the bacterial concentration of each strain after scale-up culture reaches... The compound bacterial agent is in powder form with a CFU / mL or higher and a viable count greater than [value missing]. CFU / g.

2. The compound microbial agent for processing wastewater of Trichosanthes kirilowii seeds according to claim 1, characterized in that, The Bacillus, Pseudomonas, and Rhodococcus strains were all isolated from the soil in which Trichosanthes kirilowii was planted or from the wastewater in the Trichosanthes kirilowii processing workshop.

3. The compound microbial agent for processing wastewater of Trichosanthes kirilowii seeds according to claim 1, characterized in that, The powder is prepared by mixing bacterial liquid, adding a protective agent, and then spray drying or freeze drying. The protective agent is maltodextrin and skim milk powder, and the amount added is 5-15% of the volume of the mixed bacterial liquid, wherein the mass ratio of maltodextrin to skim milk powder is 2:

1.

4. A method for preparing the compound microbial agent as described in any one of claims 1-3, characterized in that, Includes the following steps: (1) Bacillus strains with saponin degradation ability, Pseudomonas strains with polysaccharide degradation ability and Rhodococcus strains with pigment degradation ability were isolated and screened from the soil of Trichosanthes kirilowii planting and sewage from the processing workshop. (2) Each strain was cultured separately in an optimized culture medium until the logarithmic growth phase was reached, so that the bacterial concentration reached the target value. CFU / mL or higher; (3) Mix Bacillus, Pseudomonas and Rhodococcus at a volume ratio of 3:2:1 to obtain a mixed bacterial solution; (4) Add a protectant to the mixed bacterial solution, and then spray-dry or freeze-dry to make a powder, so that the viable count is greater than 100%. CFU / g.

5. The preparation method according to claim 4, characterized in that, The optimized culture medium in step (2) is: 10-15 g / L peptone, 5-8 g / L yeast extract, 5-10 g / L sodium chloride, pH 7.0-7.5, culture temperature 28-35℃, and shaker speed 150-200 r / min.

6. The application of the compound microbial agent as described in any one of claims 1-3 in the treatment of wastewater from the processing of Trichosanthes kirilowii seeds.

7. The application according to claim 6, characterized in that, The compound microbial agent is added to the biochemical treatment unit at a rate of 0.01% to 0.05% of the wastewater volume. With the addition of nutrients and aeration, the microbial community is established within 7 to 14 days.

8. The application according to claim 7, characterized in that, The nutrient supplementation includes supplementing nitrogen and phosphorus sources, and controlling the influent C:N:P mass ratio to be 100:5:1; the aeration conditions are to control the dissolved oxygen concentration at 2-4 mg / L and the hydraulic retention time at 12-24 h.

9. The application according to claim 7, characterized in that, The treated wastewater exhibits a COD removal rate of over 90%, a saponin degradation rate of over 95%, a color removal rate of over 85%, and meets the discharge standards for rural domestic sewage treatment.

10. The application according to claim 6, characterized in that, The application also includes the combined use of the compound microbial agent with other Trichosanthes kirilowii processing wastewater treatment processes, including coagulation sedimentation, anaerobic treatment, or membrane separation.

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