A method for using a biological agent under low temperature conditions and its application

Abstract

The present invention provides a method for using a biological agent under low temperature conditions and its application, comprising the following steps: (1) using Arthrobacter australis, Bacillus terracotta, aquatic psychrophilic bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria to prepare a composite agent; (2) adding the composite agent to a bucket containing 9-12L of water, the water temperature of which is controlled at 20-25°C, continuing to add 35-45L of a muddy water mixture to the bucket, aerating for 3-5 hours, and then adding 35-45L of a muddy water mixture, aerating for 3-5 hours, adding the material in the bucket to the water inlet end of an aerobic pool, and controlling the water temperature at 20-25°C. The present invention provides a method for using a biological agent under low temperature conditions and its application, and the method of the present invention has high efficiency and strong pertinence in treating domestic sewage at low temperature, and has very important social and economic benefits.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a method for using a biological bacterial agent under low temperature conditions and an application thereof. Background Art

[0002] With the rapid development of social economy and the continuous improvement of living standards, domestic sewage is mainly

[0003] It includes fecal water and washing water, which contain a large amount of organic matter, suspended solids, nutrients and pathogens, etc. Compared with other types of sewage, domestic sewage contains more feces, fiber, protein, grease, sugar, starch and other substances. If domestic sewage is discharged directly without treatment, it will have a great impact on the natural environment or human body.

[0004] Microbial sewage treatment technology has the advantages of simple operation, low operating cost, good treatment effect, no secondary pollution, etc., and it is the most promising method. Most sewage treatment microorganisms are mesophilic bacteria, and the optimal growth temperature is above 25°C to achieve good treatment effects. However, in a low temperature environment of 20-25°C, the biological activity and metabolic efficiency of microorganisms drop sharply, which seriously hinders the use of microbial sewage treatment technology, resulting in poor treatment effects on domestic sewage under low temperature environments. At present, there are many processes or methods for treating domestic sewage, but the methods currently used rarely treat the characteristics of domestic sewage components, and cannot treat feces, fiber, protein, oil, sugar, starch and other substances in domestic sewage in a targeted manner. Therefore, it is of great social and economic benefit to develop methods that grow well under low temperature conditions and can efficiently treat domestic sewage at low temperatures.

[0005] In the existing technology, the improvement of microbial wastewater treatment methods often focuses on the discovery of new strains, but the discovery process of new strains is time-consuming and costly, and the effect in actual production applications is not necessarily ideal. The purpose of this study is to study commercially available strains and improve the treatment efficiency of domestic sewage at low temperatures on the basis of reducing R&D costs. Summary of the invention

[0006] The purpose of the present invention is to provide a method for using a biological agent under low temperature conditions and its application. The method of the present invention is highly efficient and targeted in treating domestic sewage at low temperatures and has very important social and economic benefits.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A method for using a biological agent under low temperature conditions comprises the following steps:

[0009] (1) Antarctic Arthrobacter, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria are selected and inoculated into slant culture medium respectively, and the culture temperature is 20-25°C for 3-5 days; each bacterial species is inoculated into a triangular flask culture medium respectively, and shaken culture is carried out for 48 hours at a culture temperature of 20-25°C to obtain a primary culture solution of each bacterial species; the primary culture solution of each bacterial species is re-inoculated into the respective triangular flask culture medium, and shaken culture is carried out for 48 hours at a culture temperature of 20-25°C to obtain a secondary culture solution of each bacterial species; the secondary culture solution of each bacterial species is inoculated into an expansion culture medium according to the CFU ratio, and the dissolved oxygen in the fermentation tank is controlled at 5-6 mg / L, and aerobic fermentation is carried out for 1-2 hours; after aerobic fermentation, the fermentation is sealed for 20-25 hours to obtain a composite bacterial agent;

[0010] (2) Use 9-12L of water to dissolve 500g of the required compound bacterial agent in the barrel for 30-40 minutes, and control the water temperature at 20-25℃; add 35-45L of mud-water mixture to the barrel, aerate for 3-5 hours, then add 35-45L of mud-water mixture, and aerate for another 3-5 hours. Then, add the material in the barrel to the water inlet of the aerobic pool, and control the water temperature at 20-25℃.

[0011] Furthermore, in step (1), the volume ratio of the secondary culture medium to the expansion culture medium is 1:6-8.

[0012] The inventors found that the number of colonies per unit volume in the composite bacterial agent obtained by conventional culture methods was not ideal. The inventors speculated that it was due to the mixture of multiple bacterial species. The culture conditions had a great influence on the composition of various bacterial species in the composite bacterial agent, and also affected the number of colonies per unit volume of the composite bacterial agent. The present invention uses a culture temperature of 20-25°C and cooperates with corresponding aerobic fermentation and sealed fermentation conditions, and the number of colonies per unit volume of the composite bacterial agent is significantly improved. Under this condition, the composite bacterial agent obtained by compounding various bacterial species has a better sewage treatment effect. The present invention uses the optimal culture conditions of six bacterial species, namely, Antarctic Bacillus, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria, to obtain a composite bacterial agent that can achieve 1×10 9 -5×10 9 Colony count / ml is of great significance for saving costs and improving processing efficiency.

[0013] Furthermore, in step (1), the CFU ratio is: the CFU ratio of Antarctic Bacillus, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria is (1.5-2.5): (1-2): (0.5-1.5): (3-4): (1-2): (1-2).

[0014] Furthermore, the CFU ratio of the Antarctic Arthrobacter, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria is 2:1.5:1:3.5:1.5:1.5.

[0015] The single low-temperature strains used in most existing technologies are often difficult to achieve the purification effect of domestic sewage. The present invention selects commercially available specific low-temperature resistant strains, which are cultured into composite bacterial agents, which can be directly put into use without complex engineering processes and are easy to operate. The strains selected by the present invention are mixed with a specific CFU ratio to form a composite bacterial agent. The strains have a synergistic effect. The removal effect of ammonia nitrogen, nitrate nitrogen, total nitrogen, total phosphorus, COD, and SS in domestic sewage under low-temperature environment is significantly higher than that of a single strain, and the removal rate can reach more than 95%, which is better than the treatment effect of the composite bacterial agent on the market.

[0016] Furthermore, the sludge-water mixture in step (2) is activated sludge or dewatered sludge from a sewage treatment plant.

[0017] Furthermore, in step (2), the dissolved oxygen in the aerobic tank is controlled at 2-4 mg / L.

[0018] The present invention also provides the application of the method for using the biological agent under low temperature conditions in treating sewage.

[0019] Furthermore, the sewage is domestic sewage.

[0020] Furthermore, a total amount of 2×10 7 -4×10 7 CFU compound bacterial agent.

[0021] Furthermore, the removal rates of ammonia nitrogen, nitrate nitrogen, total nitrogen, total phosphorus, COD and SS in the domestic sewage are all greater than 95%.

[0022] Arthrobacter antarcticus was purchased from Wuhan Huizao Biotechnology Co., Ltd.

[0023] Bacillus bingmayongensis was purchased from Wuhan Huizao Biotechnology Co., Ltd.

[0024] Psychrobacter glacincola was purchased from Wuhan Huizao Biotechnology Co., Ltd.

[0025] Lactobacillus brevis was purchased from Shanghai AiYan Biotechnology Co., Ltd., ATCC8287.

[0026] Nitrifying bacteria were purchased from Guangzhou Gandew Biotechnology Co., Ltd., GANDEW-NI.

[0027] Denitrifying bacteria were purchased from Guangzhou GANDEW-DEN Biotechnology Co., Ltd.

[0028] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0029] 1. The present invention provides a method for using a biological agent under low temperature conditions and its application. The method of the present invention is highly efficient and targeted in treating domestic sewage at low temperatures and has very important social and economic benefits.

[0030] 2. The present invention uses a culture temperature of 20-25°C, as well as corresponding aerobic fermentation and sealed fermentation conditions, and the number of colonies per unit volume of the composite bacterial agent is significantly improved. The present invention uses the optimal culture conditions of six species of bacteria, namely, Antarctic Arthrobacter, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria, to obtain a composite bacterial agent that can achieve 1×10 9 -5×10 9 Colony count / ml is of great significance for saving costs and improving efficiency.

[0031] 3. The present invention selects commercially available specific low-temperature resistant bacteria, which are cultured into composite bacterial agents, which can be directly put into use without complex engineering processes and are easy to operate. The bacteria selected by the present invention are mixed with a specific CFU ratio to form a composite bacterial agent. The various bacteria have a synergistic effect. The removal effect of ammonia nitrogen, nitrate nitrogen, total nitrogen, total phosphorus, COD, and SS in domestic sewage under low temperature environment is significantly higher than that of a single strain, and the removal rate can reach more than 95%, which is better than the treatment effect of composite bacterial agents on the market. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Slant culture medium: ordinary agar slant culture medium purchased from Nantong Kaiheng Biotechnology Development Co., Ltd.

[0034] Erlenmeyer flask culture medium: including glucose 0.22 g / L, beef extract 0.15 g / L, hydrolyzed milk protein 0.11 g / L, soluble starch 0.14 g / L, CH3COONa 0.17 g / L, peptone 0.14 g / L, (NH4)2SO4 0.04 g / L, KH2PO4 0.08 g / L, Na2CO3 0.06 g / L, pH 7.1-7.5.

[0035] Expansion culture medium: comprising, by mass, 1.3 parts of rice vinegar, 1.3 parts of brown sugar, 1.5 parts of 36wt% edible alcohol and 60 parts of water.

[0036] Example 1

[0037] This embodiment provides a method for using a biological agent under low temperature conditions, comprising the following steps:

[0038] (1) Antarctic Arthrobacter, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria were selected and inoculated into slant culture medium respectively, and the culture temperature was 23°C for 4 days; each bacterial species was inoculated into a triangular flask culture medium respectively, and shaken cultured for 48 hours at a culture temperature of 23°C to obtain a primary culture solution of each bacterial species; the primary culture solution of each bacterial species was re-inoculated into the respective triangular flask culture medium, and shaken cultured for 48 hours at a culture temperature of 23°C to obtain a secondary culture solution of each bacterial species; the secondary culture solution of each bacterial species was inoculated into an expansion culture medium according to the CFU ratio, and the dissolved oxygen in the fermenter was controlled at 5.5 mg / L, and aerobic fermentation was performed for 1.5 hours; after aerobic fermentation, the fermentation was sealed for 22 hours to obtain a composite bacterial agent;

[0039] (2) Add the composite bacterial agent to a bucket containing 10L of water, and control the water temperature at 23°C. Continue to add 40L of mud-water mixture to the bucket. After aeration for 4 hours, add another 40L of mud-water mixture. After aeration for 4 hours, add the material in the bucket to the water inlet of the aerobic pool. The total amount of 3×10 7 CFU compound bacterial agent, water temperature is controlled at 23℃.

[0040] In step (1), the volume ratio of the secondary culture medium to the expansion culture medium is 1:7.

[0041] The CFU ratio of the Antarctic Arthrobacter, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria is 2:1.5:1:3.5:1.5:1.5.

[0042] The sludge-water mixture in step (2) is activated sludge from a sewage treatment plant.

[0043] In step (2), the dissolved oxygen in the aerobic tank is controlled at 3 mg / L.

[0044] Example 2

[0045] This embodiment provides a method for using a biological agent under low temperature conditions, comprising the following steps:

[0046] (1) Antarctic Arthrobacter, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria were selected and inoculated into slant culture medium respectively, and the culture temperature was 25°C for 5 days; each bacterial species was inoculated into triangular flask culture medium respectively, and shaken cultured for 48 hours at a culture temperature of 25°C to obtain a primary culture solution of each bacterial species; the primary culture solution of each bacterial species was re-inoculated into the respective triangular flask culture medium, and shaken cultured for 48 hours at a culture temperature of 25°C to obtain a secondary culture solution of each bacterial species; the secondary culture solution of each bacterial species was inoculated into the expansion culture medium according to the CFU ratio, and the dissolved oxygen in the fermentation tank was controlled at 6 mg / L, and aerobic fermentation was carried out for 2 hours; after aerobic fermentation, the fermentation was sealed for 25 hours to obtain a composite bacterial agent;

[0047] (2) Add the composite bacterial agent to a bucket containing 12L of water, control the water temperature at 25°C, and continue to add 45L of mud-water mixture to the bucket. After aeration for 5 hours, add another 45L of mud-water mixture. After aeration for 5 hours, add the material in the bucket to the water inlet of the aerobic pool. The total amount of 3×10 7 CFU compound bacterial agent, water temperature is controlled at 25℃.

[0048] In step (1), the volume ratio of the secondary culture medium to the expansion culture medium is 1:8.

[0049] The CFU ratio in step (1) is: the CFU ratio of Antarctic Bacillus, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria is 2.5: 2: 1.5: 4: 2: 2.

[0050] The sludge-water mixture in step (2) is activated sludge or dewatered sludge from a sewage treatment plant.

[0051] In step (2), the dissolved oxygen in the aerobic tank is controlled at 4 mg / L.

[0052] Example 3

[0053] This embodiment provides a method for using a biological agent under low temperature conditions, comprising the following steps:

[0054] (1) Antarctic Arthrobacter, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria were selected and inoculated into slant culture medium respectively, and the culture temperature was 20°C for 3 days; each bacterial species was inoculated into triangular flask culture medium respectively, and shaken cultured for 48 hours at a culture temperature of 20°C to obtain a primary culture solution of each bacterial species; the primary culture solution of each bacterial species was re-inoculated into the respective triangular flask culture medium, and shaken cultured for 48 hours at a culture temperature of 20°C to obtain a secondary culture solution of each bacterial species; the secondary culture solution of each bacterial species was inoculated into the expansion culture medium according to the CFU ratio, and the dissolved oxygen in the fermentation tank was controlled at 5 mg / L, and aerobic fermentation was carried out for 1 hour; after aerobic fermentation, the fermentation was sealed for 20 hours to obtain a composite bacterial agent;

[0055] (2) Add the composite bacterial agent to a bucket containing 9L of water, and control the water temperature at 20°C. Continue to add 35L of mud-water mixture to the bucket. After aeration for 3 hours, add another 35L of mud-water mixture. After aeration for 3 hours, add the material in the bucket to the water inlet of the aerobic pool. The total amount of 3×10 7 CFU compound bacterial agent, water temperature is controlled at 20℃.

[0056] In step (1), the volume ratio of the secondary culture medium to the expansion culture medium is 1:6.

[0057] The CFU ratio in step (1) is: the CFU ratio of Antarctic Bacillus, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria is 1.5: 1: 0.5: 3: 1: 1.

[0058] The sludge-water mixture in step (2) is dewatered sludge.

[0059] In step (2), the dissolved oxygen in the aerobic tank is controlled at 2 mg / L.

[0060] Comparative Example 1

[0061] The difference between this comparative example and Example 1 is that the composite bacterial agent is replaced by Antarctic Bacillus.

[0062] Comparative Example 2

[0063] The difference between this comparative example and Example 1 is that the composite bacterial agent is replaced by Bacillus terracotta.

[0064] Comparative Example 3

[0065] The difference between this comparative example and Example 1 is that the composite bacterial agent is replaced by aquatic psychrophilic bacillus.

[0066] Comparative Example 4

[0067] The difference between this comparative example and Example 1 is that the composite bacterial agent is replaced by Lactobacillus brevis.

[0068] Comparative Example 5

[0069] The difference between this comparative example and Example 1 is that the CFU ratio of the Antarctic Arthrobacter, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria is 1.5:1.5:3.5:1:2:1.5.

[0070] Comparative Example 6

[0071] The difference between this comparative example and Example 1 is that Arthrobacter antarctica is replaced by Bacillus coagulans (ATCC No. 15949), and Lactobacillus brevis is replaced by Lactobacillus plantarum (ATCC No. 14917).

[0072] Comparative Example 7

[0073] The difference between this comparative example and Example 1 is that the culture conditions of the composite bacterial agent are different.

[0074] Specifically, Antarctic Arthrobacter, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria are selected and inoculated into slant culture medium respectively, and the culture temperature is 30°C for 4 days; each bacterial species is inoculated into a triangular flask culture medium respectively, and shaken culture is carried out for 48 hours at a culture temperature of 30°C to obtain a primary culture solution of each bacterial species; the primary culture solution of each bacterial species is re-inoculated into the respective triangular flask culture medium, and shaken culture is carried out for 48 hours at a culture temperature of 30°C to obtain a secondary culture solution of each bacterial species; the secondary culture solution of each bacterial species is inoculated into an expansion culture medium according to the CFU ratio, and the dissolved oxygen in the fermenter is controlled at 4 mg / L, and aerobic fermentation is carried out for 3 hours; after aerobic fermentation, the fermentation is sealed for 30 hours to obtain a composite bacterial agent.

[0075] Performance Testing

[0076] Domestic sewage was collected from a domestic sewage treatment plant in Jiangsu Province as the domestic sewage to be treated. The methods of Examples 1-3 and Comparative Examples 1-7 were used to add a total amount of 3×10 7 CFU composite bacterial agent. Determine the removal rate of ammonia nitrogen, nitrate nitrogen, total nitrogen, total phosphorus, COD, and SS (suspended solids) in domestic sewage. The calculation formula is: (concentration before treatment - concentration after treatment) / concentration before treatment * 100%.

[0077] Table 1 Nitrogen and phosphorus removal rate in domestic sewage (%)

[0078]

[0079] The results show that the methods of Examples 1-3 of the present invention are effective in treating domestic sewage. Comparative Examples 1-7 show that the treatment effects decrease to varying degrees when the bacterial species, ratios and culture conditions are changed.

[0080] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for using a biological agent under low temperature conditions, the method is used to simultaneously remove ammonia nitrogen, nitrate nitrogen, total nitrogen, total phosphorus, COD, and SS in domestic sewage, characterized in that: The following steps are involved: (1) Antarctic Arthrobacter, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria are selected and inoculated into slant culture medium respectively, and the culture temperature is 20-25°C for 3-5 days; each bacterial species is inoculated into a triangular flask culture medium respectively, and shaken culture is carried out for 48 hours at a culture temperature of 20-25°C to obtain a primary culture solution of each bacterial species; the primary culture solution of each bacterial species is re-inoculated into the respective triangular flask culture medium, and shaken culture is carried out for 48 hours at a culture temperature of 20-25°C to obtain a secondary culture solution of each bacterial species; The secondary culture fluid of each bacterial species is inoculated into the expansion culture medium according to the CFU ratio, the dissolved oxygen in the fermentation tank is controlled at 5-6 mg / L, and aerobic fermentation is performed for 1-2 hours; after aerobic fermentation, the fermentation is sealed for 20-25 hours to obtain a composite bacterial agent; (2) Add the composite bacterial agent to a bucket containing 9-12L of water, and control the water temperature at 20-25°C; continue to add 35-45L of mud-water mixture to the bucket, aerate for 3-5 hours, and then add another 35-45L of mud-water mixture. After aeration for 3-5 hours, add the material in the bucket to the water inlet of the aerobic pool, and control the water temperature at 20-25°C; In step (1), the volume ratio of the secondary culture medium to the expansion culture medium is 1:6-8; In step (1), the CFU ratio is: the CFU ratio of antarctic bacterium, terracotta bacillus, aquatic psychrophilic bacillus, lactobacillus brevis, nitrifying bacteria and denitrifying bacteria is (1.5-2.5): (1-2): (0.5-1.5): (3-4): (1-2): (1-2); The sludge-water mixture in step (2) is activated sludge or dewatered sludge from a sewage treatment plant; In step (2), the dissolved oxygen in the aerobic tank is controlled at 2-4 mg / L; The Lactobacillus brevis is ATCC8287; the nitrifying bacteria is GANDEW-NI, and the denitrifying bacteria is GANDEW-DEN; The expansion culture medium comprises, by weight, 1.3 parts of rice vinegar, 1.3 parts of brown sugar, 1.5 parts of 36wt% edible alcohol and 60 parts of water.

2. The method for using biological agents under low temperature conditions according to claim 1, characterized in that: The CFU ratio of the Antarctic Arthrobacter, Terracotta Bacillus, Aquatic Psychrophilic Bacillus, Lactobacillus brevis, nitrifying bacteria and denitrifying bacteria is 2:1.5:1:3.5:1.5:1.

5.

3. Application of the method for using the biological agent under low temperature conditions as described in any one of claims 1-2 in treating sewage.

4. The use according to claim 3, characterized in that: The sewage is domestic sewage.

5. The use according to claim 3, characterized in that: The total amount of 2×10 7 -4×10 7 CFU compound bacterial agent.

6. The use according to claim 4, characterized in that: The removal rates of ammonia nitrogen, nitrate nitrogen, total nitrogen, total phosphorus, COD and SS in the domestic sewage are all greater than 95%.

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