Compound microbial agent for rapidly detecting BOD (Biochemical Oxygen Demand) and preparation method of compound microbial agent
By preparing composite microbial agents, using Clostridium butyrate and Bacillus subtilis as biological sensitive elements, the problems of poor biofilm stability and low efficiency of traditional five-day methods are solved, and rapid and accurate water BOD detection is achieved, which is suitable for a wide range of applications. water monitoring scenarios and reduce production costs.
Patent Information
- Application Number
- CN202310850033.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-07-12
- Publication Date
- 2025-01-14
AI Technical Summary
The biofilm in existing BOD rapid detection instruments has poor stability and is difficult to apply on a large scale. The traditional five-day method has a long test cycle and low efficiency, which cannot meet the daily monitoring needs.
Complex microbial agents are prepared by biofermentation, boiling, drying, crushing and mixing of Clostridium butyrate and Bacillus subtilis as biosensitive reaction elements, replacing biofilms, and used to rapidly detect the biochemical oxygen demand of water.
It realizes fast and accurate BOD detection of water bodies, shortens detection time, improves detection accuracy, is suitable for a wide range of water body monitoring scenarios, and reduces production costs.
Smart Images
Figure CN119307391A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of rapid detection of biochemical oxygen demand (BOD) of water bodies, and specifically relates to a method for preparing a composite microbial agent. Background Art
[0002] Among the water environment monitoring indicators, biochemical oxygen demand (BOD) is a comprehensive indicator that characterizes the degree of organic pollution. It is widely used in water pollution monitoring and operation control of sewage treatment plants, and is a must-check item for water quality monitoring. After the implementation of the policy of "one report per day" for the main indicators of relevant water quality, the market demand in the field of BOD rapid detection will rise sharply. The current BOD measurement method is mostly the traditional "five-day method". Its test cycle is long and the efficiency is low, which is not suitable for the development of daily monitoring work. Therefore, BOD rapid detection instruments and equipment have great scientific research and market prospects. At present, most of the BOD rapid testers on the market are based on biofilms, and their poor stability makes it difficult to apply and promote them on a large scale. Microbial agents are relatively stable and can replace biofilms as important biological sensitive elements of BOD rapid testers. Based on this, the development and promotion of microbial agents used in conjunction with BOD rapid detection instruments will fill the market gap and bring new breakthroughs to my country's water environment monitoring field. Summary of the invention
[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a composite microbial agent, that is, to use the microbial agent to replace the biofilm in the current microbial sensor for rapid detection of the biochemical oxygen demand (BOD) of water bodies. As a biological sensitive reaction element, the problems of small number of microorganisms in the biofilm and difficulty in maintaining a stable working state are solved. The microbial agent can better react with organic matter in water samples, accurately and quickly react to the content of organic matter in water samples, and achieve the purpose of rapid detection of the biochemical oxygen demand (BOD) of water bodies.
[0004] In order to solve the above technical problems, the technical solution adopted by the present invention is: A composite microbial agent for rapid BOD detection and a preparation method thereof, wherein the composite microbial agent is compounded according to the bacterial content ratio, wherein the effective viable count of Clostridium butyricum is ≥2.0×10 10 cfu / g, the effective viable count of Bacillus subtilis is ≥1.0×10 10 cfu / g.
[0005] The preparation method of the composite microbial agent comprises the following steps: (1) Biological fermentation: subjecting the Clostridium butyricum and the Bacillus subtilis to biological fermentation respectively to obtain a large number of microbial cells; (2) Boiling drying: The fermented products were dried at 70°C to obtain a microbial agent product with a moisture content of ≤5%; (3) Crushing: crushing the dried biological fermentation products respectively; (4) Mixing: The biological fermentation products of the pulverized Clostridium butyricum and the Bacillus subtilis are mixed and compounded according to the proportion of bacterial content, and auxiliary materials are added.
[0006] The method of the Clostridium butyricum biofermentation adopts the following steps: (1) Shake flask culture: inoculate the Clostridium butyricum strain from a slant into a shake flask medium and culture it statically for 12 hours; (2) Seed culture: Inoculate the shake flask into the seed tank for culture at a temperature of 33°C ± 1°C, an initial pH value of pH 7.0 ± 0.2, and a rotation speed of 30 r / min for continuous stirring for 12 h; (3) Biofermentation: The seed liquid is inoculated into a fermentation tank for culturing at a temperature of 33°C ± 1°C, an initial pH value of pH 7.0 ± 0.2, and a rotation speed of 30 r / min for continuous stirring for 48 hours to obtain the biofermentation product of Clostridium butyricum.
[0007] Furthermore, the culture medium of the shake flask, the seed tank and the fermentation tank is the same, and the formula is 0.5% peptone, 0.3% beef extract, trace element solution (0.5g / LCaCl 2 •2H 2 O, 0.2g / LK 2 HPO 4 、3.0g / L(NH 4 ) 2 HPO 4 、0.1g / LMgSO 4 •7H 2 O, 0.1g / LMnSO 4 •H 2 O), agar 1.5%-2%, pH 7.0±0.2, biological fermentation medium formula is brown sugar 10g / L, starch 2g / L, soybean meal 12g / L, bran 2g / L, yeast extract 1g / L, KH 2 PO 4 2g / L, K 2 HPO 4 1g / L, CaCO 3 0.5g / L, MgSO 4 0.5g / L, CaCl 2 0.5g / L, MnSO 4 0.2g / L, FeCl 3 0.01g / L, EDTA0.01g / L, pH7.0-7.5.
[0008] The method for the Bacillus subtilis biofermentation adopts the following steps: (1) Shake flask culture: inoculate the Bacillus subtilis strain from the preserved slant into the shake flask culture medium at a temperature of 36°C ± 1°C, an initial pH value of pH 6.5-7.0, and a rotation speed of 120 r / min for continuous stirring for 12-14 hours; (2) Seed culture: Inoculate the shake flask culture into the seed tank at an inoculum rate of 1.0%, the temperature is 36℃±1℃, the dissolved oxygen is greater than 8%, the initial pH value is pH6.5-7.0, and the speed is 180r / min and continuous stirring is performed for 10-12h; (3) Biological fermentation: The seed liquid is inoculated into the fermentation tank for fermentation at a temperature of 36°C ± 1°C, dissolved oxygen > 8%, an initial pH value of pH 6.5-7.0, and a speed of 200 r / min with continuous stirring for 48 h.
[0009] Further, the culture medium of the shake flask, the seed tank and the fermentation tank is the same, the formula is 25g of peptone, 15g of glucose, 1000mL of water, pH7.5-7.8, the seed culture medium formula is 10-15g of peptone, 10-15g of beef extract, 1000mL of water, pH7.5-7.8, and the biological fermentation culture medium formula is 2g / L of brown sugar, 10g / L of starch, 1g / L of soybean powder, 0.3g / L of yeast extract, (NH 4 ) 2 SO 4 2g / L, KH 2 PO 4 2g / L, MgSO 4 0.5g / L, CaCO 3 0.2g / L, FeCl 3 0.01g / L, pH7.0.
[0010] Compared with the prior art, the present invention can replace the current microbial sensor to quickly detect the biofilm in the biochemical oxygen demand (BOD) of water bodies. As a biological sensitive reaction element, it solves the problems of small number of microorganisms in the biofilm, difficulty in maintaining a stable working state and high production cost. The microbial agent can better react with the organic matter in the water sample, stably, accurately and quickly react to the content of organic matter in the water sample, and achieve the purpose of quickly detecting the biochemical oxygen demand (BOD) of water bodies.
[0011] Its main advantages are as follows: (1) The composite microorganism is suitable for a wide range of scenarios for rapid detection of BOD in water bodies; (2) The composite microorganism can realize rapid detection of BOD in water bodies; (3) The composite microbial agent has a higher microbial load, higher microbial activity, faster and more complete reaction with organic matter in water, and a wider range of upper and lower limits of detection; (4) The composite microbial agent has low-cost raw materials, is easy to store and transport, and has a simple process; (5) The composite microorganisms have great scientific research value and good market application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0012] Figure 1 The present invention is a flow chart of the preparation method of the composite microbial agent.
[0013] Figure 2 This is Experimental Chart 1 of the present invention.
[0014] Figure 3 This is Experimental Chart 2 of the present invention. Implementation
[0015] In order to make the purpose and advantages of the present invention more clearly understood, the present invention is further described below in conjunction with embodiments and experimental examples. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.
[0016] Please refer to the attached Figure 1 The preferred embodiment of the present invention is described below. Figure 1 The schematic diagram is simplified to illustrate the basic process of the present invention in a schematic manner, so it only shows the process related to the present invention. It should be understood by those skilled in the art that these embodiments are only used to explain the technical principles of the present invention and are not intended to limit the scope of protection of the present invention. Example
[0017] The following is combined with Figure 1 The process flow chart for implementing the first technical solution of the present invention is described in detail. In this embodiment, the preparation of the composite microbial agent is achieved as follows: A method for preparing a composite microbial agent for rapid BOD detection, wherein the composite microbial agent is compounded from biological fermentation products of Clostridium butyricum and Bacillus subtilis, wherein the effective viable count of Clostridium butyricum is ≥2.0×10 10 cfu / g, the effective viable count of Bacillus subtilis is ≥1.0×10 10 cfu / g.
[0018] The preparation method of the composite microbial agent comprises the following steps: (1) Biological fermentation: subjecting the Clostridium butyricum and the Bacillus subtilis to biological fermentation respectively to obtain a large number of microbial cells; (2) Boiling drying: The fermented products were dried at 70°C to obtain a microbial agent product with a moisture content of ≤5%; (3) Crushing: crushing the dried biological fermentation products respectively; (4) Mixing: The biological fermentation products of the pulverized Clostridium butyricum and the Bacillus subtilis are mixed and compounded according to the proportion of bacterial content, and auxiliary materials are added.
[0019] The method of the Clostridium butyricum biofermentation adopts the following steps: (1) Shake flask culture: inoculate the Clostridium butyricum strain from a slant into a shake flask medium and culture it statically for 12 hours; (2) Seed culture: Inoculate the shake flask into the seed tank for culture at a temperature of 33°C ± 1°C, an initial pH value of pH 7.0 ± 0.2, and a rotation speed of 30 r / min for continuous stirring for 12 h; (3) Biofermentation: The seed liquid is inoculated into a fermentation tank for culturing at a temperature of 33°C ± 1°C, an initial pH value of pH 7.0 ± 0.2, and a rotation speed of 30 r / min for continuous stirring for 48 hours to obtain the biofermentation product of Clostridium butyricum.
[0020] Furthermore, the culture medium of the shake flask, the seed tank and the fermentation tank is the same, and the formula is 0.5% peptone, 0.3% beef extract, trace element solution (0.5g / LCaCl 2 •2H 2 O, 0.2g / LK 2 HPO 4 、3.0g / L(NH 4 ) 2 HPO 4 、0.1g / LMgSO 4 •7H 2 O, 0.1g / LMnSO 4 •H 2 O), agar 1.5%-2%, pH 7.0±0.2, biological fermentation medium formula is brown sugar 10g / L, starch 2g / L, soybean meal 12g / L, bran 2g / L, yeast extract 1g / L, KH 2 PO 4 2g / L, K 2 HPO 4 1g / L, CaCO 3 0.5g / L, MgSO 4 0.5g / L, CaCl 2 0.5g / L, MnSO 4 0.2g / L, FeCl 3 0.01g / L, EDTA0.01g / L, pH7.0-7.5.
[0021] The method for the Bacillus subtilis biofermentation adopts the following steps: (1) Shake flask culture: inoculate the Bacillus subtilis strain from the preserved slant into the shake flask culture medium at a temperature of 36°C ± 1°C, an initial pH value of pH 6.5-7.0, and a rotation speed of 120 r / min for continuous stirring for 12-14 hours; (2) Seed culture: Inoculate the shake flask culture into the seed tank at an inoculum rate of 1.0%, the temperature is 36℃±1℃, the dissolved oxygen is greater than 8%, the initial pH value is pH6.5-7.0, and the speed is 180r / min and continuous stirring is performed for 10-12h; (3) Biological fermentation: The seed liquid is inoculated into the fermentation tank for fermentation at a temperature of 36°C ± 1°C, dissolved oxygen > 8%, an initial pH value of pH 6.5-7.0, and a rotation speed of 200 r / min with continuous stirring for 48 hours.
[0022] Further, the culture medium of the shake flask, the seed tank and the fermentation tank is the same, the formula is 25g of peptone, 15g of glucose, 1000mL of water, pH7.5-7.8, the seed culture medium formula is 10-15g of peptone, 10-15g of beef extract, 1000mL of water, pH7.5-7.8, and the biological fermentation culture medium formula is 2g / L of brown sugar, 10g / L of starch, 1g / L of soybean powder, 0.3g / L of yeast extract, (NH 4 ) 2 SO 4 2g / L, KH 2 PO 4 2g / L, MgSO 4 0.5g / L, CaCO 3 0.2g / L, FeCl 3 0.01g / L, pH7.0.
[0023] Further, experimental comparison.
[0024] Experimental Example 1 Application test of the composite microbial agent of the present invention on domestic sewage: Trial period: June 7, 2022 to June 12, 2022; Test location: Licunhe Wastewater Treatment Plant, Qingdao; Experimental treatments: Treatment (1) was the traditional five-day biochemical method as the standard control, treatment (2) was the biofilm method, and treatment (3) was the composite microbial agent method. Test process: On June 7, 2022, samples were collected from the water inlet of the Licunhe Wastewater Treatment Plant in Qingdao City to test the biochemical oxygen demand (BOD) in domestic sewage. Each treatment was tested three times, and the test data of each treatment was recorded; Test results: In terms of detection time, treatment (1) takes a full five days, treatment (2) takes 2 hours, and treatment (3) takes only 1 hour. The use of composite microbial agents can significantly shorten the detection time. In terms of accuracy, Figure 2 It can be seen that, taking the test results of treatment (1) as the control, the relative error of treatment (2) is 18.9%, and that of treatment (3) is 2.5%. The use of composite microbial agents for detection can greatly improve the detection accuracy.
[0025] Experimental Example 2 Application test of composite microbial agents on industrial sewage: Test period: June 5, 2023 to June 10, 2023; Test location: Qingdao Yongxing Chemical Plant; Experimental treatments: Treatment (1) was the traditional five-day biochemical method as the standard control, treatment (2) was the biofilm method, and treatment (3) was the composite microbial agent method. Test process: On June 5, 2022, samples were collected from the outlet of Qingdao Yongxing Chemical Plant to test the biochemical oxygen demand (BOD) in industrial wastewater. Each treatment was tested three times, and the test data for each treatment was recorded; Test results: In terms of detection time, treatment (1) takes a full five days, treatment (2) takes 2 hours, and treatment (3) takes only 1 hour. The use of composite microbial agents can significantly shorten the detection time. In terms of accuracy, Figure 3 It can be seen that, taking the test results of treatment (1) as the control, the relative error of treatment (2) is 9.8%, and that of treatment (3) is 2.6%. The use of composite microbial agents for detection can greatly improve the detection accuracy.
[0026] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.
Claims
1. A composite microbial agent for rapid detection of BOD and a preparation method thereof, characterized in that: The composite microbial agent is compounded according to the bacterial content ratio, wherein the effective live bacteria count of Clostridium butyricum is ≥2.0×10 10 cfu / g, the effective viable count of Bacillus subtilis is ≥1.0×10 10 cfu / g.
2. The method for preparing the composite microbial agent according to claim 1, characterized in that: The following steps are involved: (1) Biological fermentation: subjecting the Clostridium butyricum and the Bacillus subtilis to biological fermentation respectively to obtain a large number of microbial cells; (2) Boiling drying: Dry the fermented product at 70°C to obtain a microbial agent product with a moisture content of ≤5%; (3) Crushing: crushing the dried biological fermentation products respectively; (4) Mixing: The biological fermentation products of the pulverized Clostridium butyricum and the Bacillus subtilis are mixed and compounded according to the proportion of bacterial content, and auxiliary materials are added.
3. The method for preparing the composite microbial agent according to claim 2, wherein the Clostridium butyricum biofermentation is characterized by adopting the following steps: (1) Shake flask culture: inoculate the Clostridium butyricum strain from a slant into a shake flask medium and culture it statically for 12 hours; (2) Seed culture: Inoculate the shake flask into the seed tank for culture at a temperature of 33°C ± 1°C, an initial pH value of pH 7.0 ± 0.2, and a rotation speed of 30 r / min for continuous stirring for 12 h; (3) Biofermentation: The seed liquid is inoculated into a fermentation tank for culturing at a temperature of 33°C ± 1°C, an initial pH value of pH 7.0 ± 0.2, and a rotation speed of 30 r / min for continuous stirring for 48 hours to obtain the biofermentation product of Clostridium butyricum.
4. The preparation method of the composite microbial agent according to claim 3, wherein the Clostridium butyricum biofermentation is characterized in that the culture medium of the shake flask, the seed tank and the fermentation tank is the same, the formula is 0.5% peptone, 0.3% beef extract, trace element liquid (0.5g / L CaCl2•2H2O, 0.2g / L K2HPO4, 3.0g / L (NH4)2HPO4, 0.1g / LMgSO4•7H2O, 0.1g / LMnSO4•H2O), 1.5%-2% agar, pH is 7.0±0.2, and the formula of the biofermentation culture medium is brown sugar 10 g / L, starch 2g / L, soybean meal 12g / L, bran 2g / L, yeast extract 1g / L, KH2PO42g / L, K2HPO41g / L, CaCO30.5g / L, MgSO40.5g / L, CaCl20.5g / L, MnSO40.2g / L, FeCl30.01g / L, EDTA0.01g / L, pH7.0-7.
5.
5. The method for preparing the composite microbial agent according to claim 2, wherein the Bacillus subtilis biofermentation is characterized by adopting the following steps: (1) Shake flask culture: inoculate the Bacillus subtilis strain from the preserved slant into the shake flask culture medium at a temperature of 36°C ± 1°C, an initial pH value of pH 6.5-7.0, and a rotation speed of 120 r / min for continuous stirring for 12-14 hours; (2) Seed culture: Inoculate the shake flask culture into the seed tank at an inoculum rate of 1.0%, the temperature is 36℃±1℃, the dissolved oxygen is greater than 8%, the initial pH value is pH6.5-7.0, and the speed is 180r / min and continuous stirring is performed for 10-12h; (3) Biological fermentation: The seed liquid is inoculated into the fermentation tank for fermentation at a temperature of 36°C ± 1°C, dissolved oxygen > 8%, an initial pH value of pH 6.5-7.0, and a rotation speed of 200 r / min with continuous stirring for 48 h.
6. The preparation method of the composite microbial inoculant as claimed in claim 5, wherein the Bacillus subtilis biological fermentation is characterized in that the culture medium of the shake flask, the seed tank and the fermentation tank is the same, the formula is 25g peptone, 15g glucose, 1000mL water, pH7.5-7.8, the seed culture medium formula is 10-15g peptone, 10-15g beef extract, 1000mL water, pH7.5-7.8, and the biological fermentation culture medium formula is 2g / L brown sugar, 10g / L starch, 1g / L soybean powder, 0.3g / L yeast extract, (NH4)2SO42g / L, KH2PO42g / L, MgSO40.5g / L, CaCO30.2g / L, FeCl30.01g / L, pH7.0.