Low-temperature-resistant aerobic denitrifying bacterium and application thereof

Abstract

The invention belongs to the technical field of microorganisms, and discloses a low-temperature-resistant aerobic denitrifying bacterium and application thereof. The low-temperature-resistant aerobic denitrifying bacterium is Enterobacter ludwigii CW-01, and the preservation number of the low-temperature-resistant aerobic denitrifying bacterium is CGMCC (China General Microbiological Culture Collection Center) No.27126. The low-temperature-resistant aerobic denitrifying bacterium can be used for preparing the The enterobacter lewis CW-01 strain disclosed by the invention can efficiently realize aerobic denitrification degradation of ammonia nitrogen, nitrate and nitrite within the temperature range of 4-35 DEG C, and is especially suitable for wastewater treatment in which the ammonia nitrogen, nitrate and nitrite are difficult to remove due to low temperature; the strain also has good salt resistance, and provides a good microbial material for biological nitrogen removal treatment of low-temperature, high-salt and high-ammonia nitrogen polluted water. The nitrogen is efficiently removed, meanwhile, the use amount of a carbon source is remarkably reduced, the enterprise cost is greatly reduced, and the method has a good application prospect in the field of sewage treatment.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a low-temperature-resistant aerobic denitrifying bacterium and application thereof. Background Art

[0002] Traditional sewage treatment process flow Sewage nitrification-denitrification denitrification treatment is a sewage microbial denitrification treatment method using nitrifying bacteria and denitrifying bacteria. This method is divided into two stages: nitrification and denitrification. Under aerobic conditions, nitrifying bacteria in sewage are used to convert nitrogen-containing substances (including organic nitrogen and inorganic nitrogen) into nitrates, and then under anoxic conditions (dissolved oxygen <0.5 mg / L), denitrifying bacteria in sewage are used to reduce nitrates to gaseous nitrogen. Nitrification reaction can be carried out by primary nitrification or two-stage nitrification. In primary nitrification, carbon oxidation process is also carried out; in secondary nitrification, carbonization and nitrification processes can be carried out in separate tanks. Nitrification tanks can be in the form of aeration tanks. Two-stage biological denitrification method is an effective method for sewage microbial denitrification. As a standard biological denitrification method, it has been widely used. However, the anaerobic and aerobic requirements in the process are strict, and an appropriate amount of organic matter is required as an electron acceptor. The aerobic denitrification process refers to the biochemical process in which some bacteria use aerobic denitrification enzymes to reduce nitrates to nitrogen gas under aerobic conditions. This process consists of a series of enzyme-catalyzed reaction steps, including the participation of nitrate reductase, nitrite reductase, nitric oxide reductase and nitrous oxide reductase. Therefore, aerobic denitrification can better remove ammonia nitrogen and nitrate nitrogen from sewage, thereby improving water quality and benefiting environmental protection.

[0003] Biological treatment of sewage denitrification is a green, efficient and low-cost treatment method, which has been widely studied and applied in recent years. The core issue of this method is the development of efficient denitrification strains. Through the physiological metabolic process of functional strains, ammonia nitrogen in sewage is efficiently converted and degraded, thereby achieving sewage denitrification. However, biological denitrification is sensitive to environmental conditions and is easily affected by temperature changes. The normal growth temperature of most microorganisms is 20~35℃. Low temperature will affect the activity of enzymes in microbial cells, thereby reducing the treatment effect of sewage. After the process is put into operation, due to the alternation of the four seasons and the influence of the geographical location, it is difficult to maintain a suitable temperature without artificial regulation. Temperature regulation consumes a lot of energy. Therefore, low temperature becomes a limiting factor for microbial denitrification in winter. At present, there is still little research on denitrification in sewage under low temperature restrictions. Summary of the invention

[0004] In view of the above-mentioned shortcomings of the prior art, the object of the present invention is to provide a low-temperature resistant aerobic denitrification strain to solve the problems in the prior art.

[0005] To achieve the above purpose, the present invention specifically adopts the following technical solutions.

[0006] One of the purposes of the present invention is to protect a strain of Enterobacter ludwigii CW-01, whose deposit number is CGMCC No.27126.

[0007] In certain embodiments, the 16S rDNA sequence of Enterobacter ludwigii CW-01 is shown as SEQ ID No.1.

[0008] In certain embodiments, the Enterobacter ludwigii CW-01 has a tolerance temperature of 4 to 35°C.

[0009] In certain embodiments, the Enterobacter ludwigii CW-01 tolerates salt at a concentration of 0 to 80 g / L.

[0010] In certain embodiments, the Enterobacter ludwigii CW-01 has the ability of nitrification and aerobic denitrification.

[0011] The second object of the present invention is to protect a culture obtained by culturing the aforementioned Enterobacter ludwigii CW-01.

[0012] In certain embodiments, the culture contains Enterobacter ludwigii CW-01.

[0013] In certain embodiments, the culture is a fermentation product obtained by culturing Enterobacter ludwigii CW-01 in a microbial culture medium.

[0014] In certain embodiments, the culture comprises the Enterobacter ludwigii CW-01 and / or a metabolite of Enterobacter ludwigii CW-01.

[0015] In certain embodiments, the microbial culture medium may be a solid culture medium or a liquid culture medium.

[0016] In certain embodiments, the culture temperature is 4-35°C.

[0017] The term "culture" refers to a general term for liquid or solid products (all substances in the culture container, i.e., fermentation products) that have a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying the microorganisms, which may be a biologically pure culture of the microorganisms, or may contain a certain amount of culture medium, metabolites, and / or other components produced during the culture process. The term "culture" also includes a subculture obtained by subculturing the microorganisms, which may be a culture of a certain generation, or a mixture of several generations.

[0018] Herein, the metabolite can be obtained from the fermentation broth of Enterobacter ludwigii CW-01. The metabolite of Enterobacter ludwigii CW-01 can be a sterile metabolite of Enterobacter ludwigii CW-01 or a bacterial metabolite of Enterobacter ludwigii CW-01. The sterile metabolite (sterile fermentation filtrate) of Enterobacter ludwigii CW-01 can be prepared specifically according to the following method: culturing Enterobacter ludwigii CW-01 in a liquid culture medium, filtering and removing Enterobacter ludwigii CW-01 in the liquid culture (fermentation broth), and obtaining the sterile metabolite of Enterobacter ludwigii CW-01. The bacterial metabolites of Enterobacterludwigii CW-01 can be prepared specifically according to the following method: Enterobacterludwigii CW-01 is cultured in a liquid fermentation medium, and the fermentation broth (containing Enterobacterludwigii CW-01 and substances secreted into the liquid medium) is collected. The fermentation broth is the bacterial metabolites of Enterobacterludwigii CW-01.

[0019] The third object of the present invention is to protect a microbial agent comprising Enterobacter ludwigii CW-01 as described above or the culture as described above.

[0020] In certain embodiments, the microbial agent further comprises a carrier.

[0021] In certain embodiments, the carrier comprises a solid carrier or a liquid carrier.

[0022] In certain embodiments, the solid carrier comprises a mineral material, a plant material and / or a polymer compound; the mineral material may be at least one of clay, talc, medical stone, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, bean flour, rice husk flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol.

[0023] In certain embodiments, the liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane.

[0024] In certain embodiments, the bacterial agent may be in a variety of dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0025] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0026] In certain embodiments, the effective viable count of Enterobacter ludwigii CW-01 in the microbial agent is at least 1×10 9 CFU / g. CFU / g is interpreted as the common meaning in the art, indicating the number of microbial colonies contained in gram of test sample, and CFU means colony forming unit.

[0027] In certain embodiments, in the bacterial agent, the Enterobacter ludwigii CW-01 or / and the culture of Enterobacter ludwigii CW-01 may be present in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture, or a mixture of cells and filtrate.

[0028] The present application also provides a method for preparing the bacterial agent as described above, comprising the following steps:

[0029] 1) inoculating Enterobacter ludwigii CW-01 into a culture medium and culturing the culture medium to obtain a fermentation liquid;

[0030] 2) Drying the fermentation liquid to obtain the microbial agent.

[0031] In certain embodiments, during culturing, the culture medium used includes, but is not limited to, MS medium, LB medium, PDA medium, Czapek medium, and beef extract peptone medium.

[0032] In certain embodiments, the culture medium used includes tryptone and yeast powder.

[0033] In some specific embodiments, the culture medium further comprises one or both of sodium chloride and agar.

[0034] In certain specific embodiments, the culture medium comprises 5-15 g / L tryptone, 1-10 g / L yeast powder, and 5-15 g / L sodium chloride, and the pH value of the culture medium is 7.0-7.2.

[0035] In certain embodiments, the culture temperature is 4-35°C.

[0036] In certain embodiments, the culturing time is 2 to 10 days.

[0037] In certain embodiments, the culture rotation speed is 100-300 r / min.

[0038] A fourth object of the present invention is to protect the use of Enterobacter ludwigii CW-01 as described above, the culture of Enterobacter ludwigii CW-01 as described above, or the microbial agent as described above in at least one of the following:

[0039] a1) Reduce inorganic nitrogen in the environment;

[0040] a2) preparing products for reducing inorganic nitrogen in the environment;

[0041] a3) Reduce COD in the environment;

[0042] a4) preparing products that reduce COD in the environment;

[0043] a5) Reduce total nitrogen in the environment;

[0044] a6) preparing products for reducing total nitrogen in the environment;

[0045] a7) Reduce total phosphorus in the environment;

[0046] a8) Preparation of products that reduce total phosphorus in the environment.

[0047] In certain embodiments, the environment may be one or more of water, soil, and water sediment.

[0048] In some embodiments, the body of water can be wastewater.

[0049] In certain embodiments, the working temperature of the Enterobacter ludwigii CW-01 or the culture or the microbial agent is 4-35°C.

[0050] In some specific embodiments, the inorganic nitrogen includes one or more of ammonia nitrogen, nitrate, and nitrite.

[0051] The fifth object of the present invention is to protect any one of b1) to b3) by a method comprising: treating an object to be treated with Enterobacter ludwigii CW-01 as described above, or a culture as described above, or a microbial agent as described above;

[0052] b1) Biological denitrification;

[0053] b2) Biological phosphorus removal;

[0054] b3) Biodegradation of COD.

[0055] In certain embodiments, the object to be processed may be one or more of water, soil, and water sediment.

[0056] In some embodiments, the body of water can be wastewater.

[0057] In some embodiments, based on the volume of the water body, the amount of the microbial agent added is 0.1 g / m 3 ~100g / m 3 water.

[0058] In certain embodiments, the treatment temperature is 4-35°C.

[0059] The present invention also protects a product; the product comprises the Enterobacterludwigii CW-01 as described above or the culture as described above or the microbial agent as described above; the function of the product is at least one of the following:

[0060] c1) Reduce inorganic nitrogen in the environment;

[0061] c2) Reduce COD in the environment;

[0062] c3) Reduce total nitrogen in the environment;

[0063] c4) Reduce total phosphorus in the environment.

[0064] In certain embodiments, the environment may be one or more of water, soil, and water sediment.

[0065] In some embodiments, the body of water can be wastewater.

[0066] In certain embodiments, the working temperature of the Enterobacter ludwigii CW-01 or the culture or the microbial agent is 4-35°C.

[0067] In certain embodiments, the inorganic nitrogen includes one or more of ammonia nitrogen, nitrate, and nitrite.

[0068] In certain embodiments, the product may be a nitrogen scavenger.

[0069] Compared with the prior art, the present invention has the following beneficial effects:

[0070] The present invention provides a strain of Enterobacter ludwigii CW-01 that is resistant to low-temperature aerobic denitrification. The Enterobacter ludwigii CW-01 strain can efficiently realize aerobic denitrification degradation of ammonia nitrogen, nitrate, nitrite, etc. within the range of 4 to 35°C, and is particularly suitable for wastewater treatment in which ammonia nitrogen, nitrate, and nitrite are difficult to remove due to low temperature; and the strain has good salt tolerance, and provides a good microbial material for biological denitrification treatment of low-temperature, high-salt, and high-ammonia nitrogen polluted water bodies. While efficiently removing total nitrogen, the use of carbon sources is also significantly reduced, which greatly reduces enterprise costs and has good application prospects in the field of sewage treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0071] Figure 1 This is a morphological diagram of the Enterobacter ludwigii CW-01 strain.

[0072] Figure 2 This is the growth graph of Enterobacter ludwigii CW-01 strain at 4℃.

[0073] Figure 3 This is the growth graph of Enterobacter ludwigii CW-01 strain at 8℃.

[0074] Figure 4 This is the growth graph of Enterobacter ludwigii CW-01 strain at 15℃.

[0075] Figure 5 This is the growth chart of Enterobacter ludwigii CW-01 strain at 28℃. DETAILED DESCRIPTION

[0076] The present invention separates and screens a low-temperature-resistant aerobic denitrification strain, Enterobacter ludwigii CW-01, from a sewage treatment pool, provides bacterial resources for nitrogen removal in a low-temperature environment, and contributes to the application of biological methods for nitrogen removal and water quality improvement.

[0077] One of the purposes of the present invention is to protect a strain of Enterobacter ludwigii CW-01, whose deposit number is CGMCC No.27126.

[0078] The strain is classified as Enterobacter ludwigii, with a deposit number of CGMCC No. 27126. It was deposited at the General Microbiology Center of China Microbiological Culture Collection Administration on April 17, 2023, at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. It was isolated from a sewage treatment pool.

[0079] In certain embodiments, the 16S rDNA sequence of Enterobacter ludwigii CW-01 is shown as SEQ ID No.1.

[0080] The morphological characteristics of the Enterobacter ludwigii CW-01 of the present application are: the colonies are milky white, large and convex, and the homology of its 16S rDNA with the model strain Enterobacter ludwigii in GenBank is 99.86%.

[0081] In certain embodiments, the Enterobacter ludwigii CW-01 has a tolerance temperature of 4 to 35°C.

[0082] In certain embodiments, the Enterobacter ludwigii CW-01 tolerates salts at a concentration of 0 to 80 g / L. The Enterobacter ludwigii CW-01 of the present application can grow in a culture medium with a salinity of 0 to 80 g / L.

[0083] In certain embodiments, the salt is sodium chloride.

[0084] In certain embodiments, the Enterobacter ludwigii CW-01 has the ability of nitrification and / or aerobic denitrification.

[0085] The second object of the present invention is to protect a culture obtained by culturing the aforementioned Enterobacter ludwigii CW-01.

[0086] In certain embodiments, the culture contains Enterobacter ludwigii CW-01.

[0087] In certain embodiments, the culture is a fermentation product obtained by culturing Enterobacter ludwigii CW-01 in a microbial culture medium.

[0088] In certain embodiments, the culture comprises the Enterobacter ludwigii CW-01 and / or a metabolite of Enterobacter ludwigii CW-01.

[0089] In certain embodiments, the microbial culture medium may be a solid culture medium or a liquid culture medium.

[0090] In certain embodiments, the culture temperature is 4-35°C, or 15-35°C, or 20-28°C, or 25°C, 28°C, or 30°C.

[0091] The term "culture" refers to a general term for liquid or solid products (all substances in the culture container, i.e., fermentation products) that have a microbial population after artificial inoculation and culture. That is, the product obtained by growing and / or amplifying the microorganisms, which may be a biologically pure culture of the microorganisms, or may contain a certain amount of culture medium, metabolites, and / or other components produced during the culture process. The term "culture" also includes a subculture obtained by subculturing the microorganisms, which may be a culture of a certain generation, or a mixture of several generations.

[0092] Herein, the metabolite can be obtained from the fermentation broth of Enterobacter ludwigii CW-01. The metabolite of Enterobacter ludwigii CW-01 can be a sterile metabolite of Enterobacter ludwigii CW-01 or a bacterial metabolite of Enterobacter ludwigii CW-01. The sterile metabolite (sterile fermentation filtrate) of Enterobacter ludwigii CW-01 can be prepared specifically according to the following method: culturing Enterobacter ludwigii CW-01 in a liquid culture medium, filtering and removing Enterobacter ludwigii CW-01 in the liquid culture (fermentation broth), and obtaining the sterile metabolite of Enterobacter ludwigii CW-01. The bacterial metabolites of Enterobacterludwigii CW-01 can be prepared specifically according to the following method: Enterobacterludwigii CW-01 is cultured in a liquid fermentation medium, and the fermentation broth (containing Enterobacterludwigii CW-01 and substances secreted into the liquid medium) is collected. The fermentation broth is the bacterial metabolites of Enterobacterludwigii CW-01.

[0093] In certain embodiments, during culturing, the culture medium used includes, but is not limited to, MS medium, LB medium, PDA medium, Czapek medium, and beef extract peptone medium.

[0094] In certain embodiments, the culture medium used includes tryptone and yeast powder.

[0095] In some specific embodiments, the culture medium further comprises one or both of sodium chloride and agar.

[0096] In some specific embodiments, the culture medium contains 5-15 g / L tryptone, 1-10 g / L yeast powder, and 5-15 g / L sodium chloride. In a specific embodiment, the formula of the culture medium is 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and 1 L water. The pH value of the culture medium is 7.0-7.2.

[0097] The third object of the present invention is to protect a microbial agent comprising Enterobacter ludwigii CW-01 as described above or the culture as described above.

[0098] In certain embodiments, the bacterial agent may be in a variety of dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0099] In one embodiment, the bacterial agent is in the form of a powder.

[0100] In certain embodiments, the effective viable count of Enterobacter ludwigii CW-01 in the microbial agent is at least 1×10 9 CFU / g; can also be 1×10 9 CFU / g ~1×10 12 CFU / g; can also be 6×10 9 CFU / g. CFU / g is interpreted as the common meaning in the art, indicating the number of microbial colonies contained in gram of test sample, and CFU means colony forming unit.

[0101] In certain embodiments, the microbial agent further comprises a carrier.

[0102] In certain embodiments, the carrier comprises a solid carrier or a liquid carrier.

[0103] In certain embodiments, the solid carrier comprises a mineral material, a plant material and / or a polymer compound; the mineral material may be at least one of clay, talc, medical stone, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the plant material may be at least one of corn flour, bean flour, rice husk flour and starch; the polymer compound may be polyvinyl alcohol and / or polyglycol.

[0104] In certain embodiments, the liquid carrier may be an organic solvent, vegetable oil, mineral oil or water; the organic solvent may be decane and / or dodecane.

[0105] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0106] In the bacterial agent, the Enterobacter ludwigii CW-01 or / and the culture of Enterobacter ludwigii CW-01 may be present in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or a mixture of cells and filtrate.

[0107] The present application also provides a method for preparing the bacterial agent as described above, comprising the following steps:

[0108] 1) inoculating Enterobacter ludwigii CW-01 into a culture medium and culturing the culture medium to obtain a fermentation liquid;

[0109] 2) Drying the fermentation liquid to obtain the microbial agent.

[0110] In certain embodiments, during culturing, the culture medium used includes, but is not limited to, MS medium, LB medium, PDA medium, Czapek medium, and beef extract peptone medium.

[0111] In certain embodiments, the culture medium used includes tryptone and yeast powder.

[0112] In some specific embodiments, the culture medium further comprises one or both of sodium chloride and agar.

[0113] In some specific embodiments, the culture medium comprises 5-15 g / L tryptone, 1-10 g / L yeast powder, and 5-15 g / L sodium chloride. In a specific embodiment, it is 10 g / L tryptone, 5 g / L yeast powder, 10 g / L sodium chloride, and 1 L of water. The pH value of the culture medium is 7.0-7.2.

[0114] In certain embodiments, the culture temperature is 4-35°C; it can also be 15-35°C, 20-28°C, or 25°C, 28°C, or 30°C.

[0115] In certain embodiments, the culture time is 2 to 10 days, or 2 to 5 days, or 4 to 8 days, or 7 to 10 days, or 3 days.

[0116] In certain embodiments, the culture rotation speed is 100-300 r / min, or 100-180 r / min, or 230-300 r / min, or 170-240 r / min, or 100, 150, or 200 r / min.

[0117] The fourth object of the present invention is to protect the use of Enterobacter ludwigii CW-01 as described above, or the culture as described above, or the microbial agent as described above in at least one of the following:

[0118] a1) Reduce inorganic nitrogen in the environment;

[0119] a2) preparing products for reducing inorganic nitrogen in the environment;

[0120] a3) Reduce COD in the environment;

[0121] a4) preparing products that reduce COD in the environment;

[0122] a5) Reduce total nitrogen in the environment;

[0123] a6) preparing products for reducing total nitrogen in the environment;

[0124] a7) Reduce total phosphorus in the environment;

[0125] a8) Preparation of products that reduce total phosphorus in the environment.

[0126] In certain embodiments, the environment may be one or more of water, soil, and water sediment.

[0127] In some embodiments, the body of water can be wastewater.

[0128] In certain specific embodiments, the operating temperature of the Enterobacter ludwigii CW-01 or the culture or the microbial agent is 4-35°C; it can also be 15-35°C, it can also be 20-28°C, or it can also be 25°C, 28°C, or 30°C.

[0129] In some specific embodiments, the inorganic nitrogen includes one or more of ammonia nitrogen, nitrate, and nitrite.

[0130] The fifth object of the present invention is to protect any one of b1) to b3) by a method comprising: treating an object to be treated with Enterobacter ludwigii CW-01 as described above, or a culture as described above, or a microbial agent as described above;

[0131] b1) Biological denitrification;

[0132] b2) Biological phosphorus removal;

[0133] b3) Biodegradation of COD.

[0134] In certain embodiments, the object to be processed may be one or more of water, soil, and water sediment.

[0135] In some embodiments, the body of water can be wastewater.

[0136] In certain embodiments, the treatment temperature is 4-35°C; it can also be 15-35°C, it can also be 20-28°C, it can also be 25°C, 28°C, or 30°C.

[0137] In certain embodiments, the nitrogen is total nitrogen or inorganic nitrogen; the inorganic nitrogen includes one or more of ammonia nitrogen, nitrate, and nitrite.

[0138] In some embodiments, based on the volume of the water body, the amount of the microbial agent added is 0.1 g / m 3 ~100g / m 3 Water, can also be 0.2g / m 3 ~30g / m 3 Water, can also be 50 / m 3 ~100g / m 3 Water, can also be 1g / m 3 ~60g / m 3 Water, can also be 1g / m 3 ~20g / m 3 Water, can also be 1g / m 3 , 10g / m 3 water.

[0139] The present invention also protects a product; the product comprises the Enterobacterludwigii CW-01 as described above or the culture as described above or the microbial agent as described above; the function of the product is at least one of the following:

[0140] c1) Reduce inorganic nitrogen in the environment;

[0141] c2) Reduce COD in the environment;

[0142] c3) Reduce total nitrogen in the environment;

[0143] c4) Reduce total phosphorus in the environment.

[0144] In certain embodiments, the environment may be one or more of water, soil, and water sediment.

[0145] In some embodiments, the body of water can be wastewater.

[0146] In certain embodiments, the operating temperature of the Enterobacter ludwigii CW-01 or the culture or the microbial agent is 4-35°C; it can also be 15-35°C, 20-28°C, or 25°C, 28°C, or 30°C.

[0147] In certain embodiments, the inorganic nitrogen includes one or more of ammonia nitrogen, nitrate, and nitrite.

[0148] In certain embodiments, the product may be a nitrogen scavenger.

[0149] The fermentation culture of Enterobacter ludwigii CW-01 of the present application was inoculated into nitrification medium, and after 24 hours of treatment, the concentration of ammonia nitrogen decreased by 99.62%; the fermentation culture of Enterobacter ludwigii CW-01 of the present application was inoculated into denitrification medium, and the concentration of nitrite decreased by 99.96%, and the concentration of nitrate decreased by 99.79%; the fermentation culture of Enterobacter ludwigii CW-01 of the present application was inoculated into denitrification medium, and the concentration of nitrite decreased by 99.96%, and the concentration of nitrate decreased by 99.79%; The fermentation culture of CW-01 was inoculated into the denitrification medium and treated under low temperature environment, and it could still carry out good aerobic denitrification. After being treated at a low temperature of 4°C for 6 days, the nitrate removal rate reached about 98.85%, after being treated at 8°C for 6 days, the nitrate removal rate reached about 99.69%, after being treated at 15°C for 2 days, the nitrate removal rate reached about 99.49%, and after being treated at 28°C for 1 day, the nitrate removal rate reached about 99.81%. When the microbial agent of the present application was applied to the water body, the ammonia nitrogen in the water body was reduced by 95.74% after 25 days, and the amount of carbon source added was greatly reduced.

[0150] The Enterobacter ludwigii CW-01, culture or microbial agent of the present application has the function of degrading inorganic nitrogen (such as ammonia nitrogen, nitrate, and nitrite). The above effect enables the strain of the present application to significantly improve the effect of nitrogen-containing wastewater, and provides a new strain or new method for biological denitrification of nitrogen-containing wastewater.

[0151] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0152] Before further describing the specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the examples of the present invention are intended to describe specific embodiments, rather than to limit the scope of protection of the present invention. The test methods in the following examples without specifying specific conditions are generally carried out under conventional conditions or under conditions recommended by the manufacturers.

[0153] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, both endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0154] The culture medium formula involved in the examples of the present application is as follows:

[0155] The formula of LB liquid medium is: tryptone 10g / L, yeast powder 5g / L, sodium chloride 10g / L, water 1L, pH 7.0-7.2, sterilized at 121℃ for 30min. Add 1.5-2% agar when solid.

[0156] Aerobic denitrification medium 1 includes: sodium nitrite 0.2 / L, potassium dihydrogen phosphate 0.2g / L, dipotassium hydrogen phosphate 0.1g / L, glucose 1.5g / L, magnesium chloride 0.1g / L, ferrous sulfate 0.02g / L, pH 7.0-7.4, sterilized at 121℃ for 30min. Add 1.5-2% agar when solid.

[0157] Aerobic denitrification medium II includes: sodium nitrate 0.6 g / L, potassium dihydrogen phosphate 0.2 g / L, potassium hydrogen phosphate 0.1 g / L, glucose 1.5 g / L, magnesium chloride 0.1 g / L, ferrous sulfate 0.02 g / L, pH 7.0 ± 0.2, sterilized at 121 ° C for 30 min. When solid, add 1.5-2% agar.

[0158] Nitration medium: ammonium chloride 0.2g / L, potassium dihydrogen phosphate 0.2g / L, glucose 1.5g / L, magnesium chloride 0.1g / L, ferrous sulfate 0.02g / L, pH 7.0±0.2, sterilized at 121℃ for 30min. Add 1.5-2% agar when solid.

[0159] Example 1 Screening and Isolation of Enterobacter reuteri

[0160] The separation process of Enterobacter reuteri isolated in this embodiment is as follows:

[0161] 1.1 Sample collection

[0162] Use a sterile water sample collection bag to collect sewage from the sewage treatment pool of Dalian Xihai Sewage Treatment Company, take 5 mL and add it to 100 mL of sterile liquid LB culture medium that has been sterilized in advance, place it in a constant temperature shaker at 28°C, and carry out enrichment culture at 150 r / min for 2 days. Take 40 mL of the enrichment liquid and add it to 400 mL of aerobic denitrification culture medium 2, place it in a constant temperature shaker at 28°C, and carry out enrichment culture at 150 r / min for 2 days. This is the first acclimation cycle; take 40 mL of the culture solution from the previous cycle and inoculate it into the acclimation culture medium of the second cycle, place it in a constant temperature shaker at 28°C, and culture it for 2 days. And so on, carry out 5 acclimation cycles.

[0163] The culture medium and bacterial solution of the fifth acclimation were diluted: 150 μL of the culture medium and bacterial solution after acclimation were spread and separated on the plate of solid aerobic denitrification culture medium II with sterilized glass beads, and cultured in an incubator at 28°C, and the pure colonies on the culture medium were picked and separated by streaking on a new culture medium, and cultured in a constant temperature incubator at 28°C to obtain several strains, which were tested for aerobic denitrification respectively, and finally a strain capable of efficient denitrification was obtained, named strain CW-01, whose colonies on the LB plate were moist, milky white, and large and convex; Figure 1 shown.

[0164] 1.2. Species determination by 16S rDNA gene sequencing

[0165] The purified strain CW-01 was sent to Shanghai Saiheng Biotechnology Co., Ltd. for 16S rDNA sequencing. The obtained sequence was compared with the National Center for Bioinformation (NCBI) database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome), and it was found that the 16S rDNA sequence was 99.86% homologous to the model strain Enterobacter ludwigii in GenBank.

[0166] The sequence of 16S rDNA of strain CW-01 is as follows:

[0167]

[0168] Based on the strain morphology and 16S rDNA, the strain CW-01 was identified as Enterobacter ludwigii. The classification name of the strain is Enterobacter ludwigii, and the deposit number is CGMCC No.27126. It was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on April 17, 2023, with the address of No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

[0169] Example 2 Study on the low temperature and salt tolerance of strain CW-01

[0170] 2.1 Study on low temperature resistance

[0171] The strain CW-01 was streaked onto LB plates and cultured in constant temperature incubators at 4°C, 8°C, 15°C, and 28°C for 3 days. After three days of culture, the colony growth rate was as follows: Figure 2 , Figure 3 , Figure 4 , Figure 5 shown.

[0172] Experiments have shown that strain CW-01 grows well at ultra-low temperature of 4°C, indicating that the strain has low-temperature resistance.

[0173] 2.2 Study on salt tolerance

[0174] Use an inoculation needle to dip a ring of strain CW-01 single colony into the pre-sterilized LB liquid medium, place it in a constant temperature shaker at 28℃, 150r / min, and culture for three days to obtain the fermentation liquid. The fermentation liquid was inoculated into different concentrations of sodium chloride LB liquid medium (experimental group A1 (10g / L), experimental group A2 (20g / L), experimental group A3 (40g / L), experimental group A4 (60g / L), experimental group A5 (80g / L), experimental group A6 (100g / L)) at an inoculation rate of 1v / v%, 28℃, shaker 150r / min, and after three days of culture, the absorbance of each fermentation liquid at a wavelength of 600nm was measured with an ultraviolet spectrophotometer. Three parallel experiments were set up for each group. At the same time, a blank LB medium with the same salinity and no strain was inoculated was used as a blank control group; the results are shown in Table 1.

[0175] Table 1

[0176]

[0177] From the above data, it can be seen that strain CW-01 has good salt tolerance and can still grow well at a salinity of 80g / L.

[0178] Example 3 Study on the nitrification and aerobic denitrification performance of strain CW-01

[0179] 3.1 The fermented CW-01 LB fermentation liquid was inoculated into 150mL nitrification medium at a rate of 5v / v%, placed in a 28℃ constant temperature shaker, 150r / min, cultured for one day, and the ammonia nitrogen value was measured every 6 hours. Three parallel groups were set up for each group (experimental group B1, experimental group B2, experimental group B3), and one blank control group. The measured data are shown in the following table.

[0180] The ammonia nitrogen measurement method uses Nessler's reagent photometry. Take 5 mL of the fermentation product, add it to a 50 mL colorimetric tube, dilute to the mark, add 1.0 mL of potassium tartrate solution, and measure the absorbance at 420 nm. For details, refer to the method for Nessler's reagent photometry (A) in the "Water and Wastewater Monitoring and Analysis Methods (Fourth Edition)".

[0181] The detection principle is: the alkaline solution of mercuric iodide and potassium iodide of Nessler's reagent reacts with ammonia to form a light reddish brown colloidal compound, which has strong absorption in a wider wavelength range.

[0182] Table 2

[0183]

[0184] The results showed that the ammonia nitrogen concentration decreased significantly. For example, the ammonia nitrogen concentration in the experimental group B1 was degraded from 52.61 mg / L to 0.2 mg / L, the ammonia nitrogen concentration in the experimental group B2 was degraded from 52.42 mg / L to 0.21 mg / L, and the ammonia nitrogen concentration in the experimental group B3 was degraded from 52.50 mg / L to 0.18 mg / L. Combining the three groups, the overall ammonia nitrogen concentration decreased by 99.62%.

[0185] 3.2 The fermented CW-01 LB fermentation broth was inoculated into 150 mL of aerobic denitrification medium 1 at an inoculation rate of 5 v / v%, and placed in a constant temperature shaker at 28°C, 150 r / min, and cultured for 1 day. The nitrite value was measured every 6 hours. Three parallel groups were set up in each group (experimental group C1, experimental group C2, and experimental group C3), and one blank control group. The measured data are shown in the following table.

[0186] The measurement method is to use N-(1-naphthyl)-ethylenediamine photometric method (A) for detection. Take 5mL of fermentation product, add it to a 50mL colorimetric tube, dilute to the mark, add 1.0mL of color developer, and measure the absorbance at 540nm. For details, refer to "Water and Wastewater Monitoring and Analysis Methods (Fourth Edition)".

[0187] The detection principle is: in a phosphoric acid medium, when the pH value is 1.8±0.3, nitrite reacts with p-aminobenzenesulfonamide to generate a diazonium salt, which is then coupled with N-(1-naphthyl)-ethylenediamine to generate a red dye with maximum absorption at a wavelength of 540nm.

[0188] Table 3

[0189]

[0190] The results showed that the nitrite concentration decreased significantly. For example, 24 hours after inoculation, the nitrite concentration in the experimental group C1 degraded from 21.6 mg / L to 0.01 mg / L, the nitrite concentration in the experimental group C2 degraded from 21.59 mg / L to 0.01 mg / L, and the nitrite concentration in the experimental group C3 degraded from 21.59 mg / L to 0. Combining the three groups, the overall nitrite concentration decreased by 99.96%.

[0191] 3.3 The fermented CW-01 LB fermentation broth was inoculated into 150 mL of aerobic denitrification medium 2 at an inoculum size of 5 v / v%, and placed in a constant temperature shaker at 28°C, 150 r / min, and cultured for 1 day. The nitrate value was measured every 6 hours. Three parallel groups were set up in each group (experimental group D1, experimental group D2, and experimental group D3), and one blank control group. The measured data are shown in the following table.

[0192] The measurement method uses ultraviolet spectrophotometry, and the principle of the method is to quantitatively determine nitrate nitrogen by using the absorption of nitrate ions at a wavelength of 220nm. Dissolved organic matter also absorbs at 220nm, while nitrate ions do not absorb at 275nm. Therefore, another measurement is made at 275nm to correct the nitrate nitrogen value. For details, please refer to the "Water and Wastewater Monitoring and Analysis Methods (Fourth Edition)".

[0193] Table 4

[0194]

[0195] The results showed that the nitrate concentration decreased significantly. For example, 24 hours after inoculation, the nitrate concentration in the experimental group D1 degraded from 103.1 mg / L to 0.21 mg / L, the nitrate concentration in the experimental group D2 degraded from 103.2 mg / L to 0.21 mg / L, and the nitrate concentration in the experimental group D3 degraded from 103.1 mg / L to 0.22 mg / L. Combining the three groups, the overall nitrate concentration decreased by 99.79%.

[0196] Example 4 Effect of low temperature on aerobic denitrification performance of strain CW-01

[0197] The fermentation broth of strain CW-01 was inoculated into aerobic denitrification medium II at an inoculation rate of 5 v / v%, and cultured in a constant temperature shaker at 4°C, 8°C, 15°C, and 28°C, respectively. Three groups of experiments were set up in each group (repeat 1, repeat 2, and repeat 3, respectively). At the same time, a group of blank experiments was set up to observe the turbidity of the aerobic denitrification medium after inoculation. It was found that on the first day, the culture medium at 15°C and 28°C became turbid, on the third day, the culture medium at 8°C became turbid, and on the fourth day, the culture medium at 4°C became turbid. The data measured by ultraviolet spectrophotometer are shown in the following table.

[0198] Table 5

[0199]

[0200] Table 6

[0201]

[0202] It can be seen from Tables 5 and 6 that temperature has a certain effect on the efficiency of aerobic denitrification of strain CW-01. However, as time goes on, aerobic denitrification can still be carried out well at a low temperature of 4°C on the sixth day, and the nitrate removal rate reaches about 98.85%. The nitrate removal rate reaches about 99.69% at 8°C on the sixth day, and the nitrate removal rate reaches about 99.49% at 15°C on the second day. The nitrate removal rate reaches about 99.81% at 28°C on the first day.

[0203] Example 5 Application of strain CW-01 in the preparation of bacterial agent in sewage treatment

[0204] The LB fermentation broth of CW-01 cultured at 28℃ and 150r / min for 3 days was prepared into bacterial powder by low-temperature drying process. The live bacteria content in the bacterial powder was 6×10 9 CFU / g; 1g of bacterial powder was added to each cubic meter of sewage and added to two sewage treatment pools (No. 1 sewage treatment pool and No. 2 sewage treatment pool) of a sewage treatment plant in Northeast China. The sewage pool data before using bacterial powder are as follows.

[0205] Table 7

[0206]

[0207] One week after adding bacterial powder, the sewage data is as follows:

[0208] Table 8

[0209]

[0210] After adding bacterial powder for 25 days, the sewage data is as follows:

[0211] Table 9

[0212]

[0213] By comparing the data in Tables 7 to 9, it is found that when the temperature in Northeast China is low in March and April, the various indicators in the sewage have been significantly improved after adding CW-01 bacterial agent. Moreover, in actual operation, the amount of carbon source added has been greatly reduced after adding CW-01 bacterial agent. At most, the sewage station adds 12 bags of glucose per day, and the total nitrogen in the nitrification and denitrification effluent still fluctuates around 600 mg / L. After adding the bacterial agent, after a 20-day acclimation process, the total nitrogen in the nitrification and denitrification effluent showed a significant downward trend. In about a month, the total nitrogen can be stably controlled within 300 mg / L, and it still shows a stable downward trend. The amount of carbon source added is greatly reduced. At present, only 3 bags of glucose are needed per day. Under the condition of the same carbon source addition, the total nitrogen index removal advantage is obvious. While efficiently removing total nitrogen, it also greatly reduces the cost of the enterprise.

[0214] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A strain of Enterobacter ludwigii CW-01, whose deposit number is CGMCC No.27126.

2. The Enterobacter ludwigii CW-01 according to claim 1, characterized in that The 16S rDNA sequence of Enterobacter ludwigii CW-01 is shown in SEQ ID No.

1.

3. A culture, characterized in that The culture is obtained by culturing the Enterobacter ludwigii CW-01 according to any one of claims 1 to 2.

4. A microbial agent, characterized in that: The microbial agent comprises the Enterobacter ludwigii CW-01 according to any one of claims 1 to 2 or the culture according to claim 3.

5. The microbial agent according to claim 4, characterized in that: The effective viable count of Enterobacter ludwigii CW-01 in the microbial agent is at least 1×10 9 CFU / g.

6. Use of the Enterobacter ludwigii CW-01 according to any one of claims 1 to 2, the culture according to claim 3, or the microbial agent according to any one of claims 4 to 5 in at least one of the following: a1) Reduce inorganic nitrogen in the environment; a2) preparing products for reducing inorganic nitrogen in the environment; a3) Reduce COD in the environment; a4) preparing products that reduce COD in the environment; a5) Reduce total nitrogen in the environment; a6) preparing products for reducing total nitrogen in the environment; a7) Reduce total phosphorus in the environment; a8) Preparation of products that reduce total phosphorus in the environment.

7. The use according to claim 6, characterized in that: The working temperature of the Enterobacter ludwigii CW-01 or the culture or the microbial agent is 4 to 35° C.; and / or the environment includes one or more of water, soil, and water sediment; and / or the inorganic nitrogen includes one or more of ammonia nitrogen, nitrate, and nitrite.

8. A method for biological denitrification, comprising: The object to be treated is treated with the Enterobacter ludwigii CW-01 according to any one of claims 1 to 2, the culture according to claim 3, or the microbial agent according to any one of claims 4 to 5.

9. The method according to claim 8, characterized in that The object to be treated includes one or more of water, soil, and water sediment; and / or the treatment temperature is 4 to 35°C.

10. The method according to claim 9, characterized in that Based on the volume of the water body, the amount of microbial agent added is 0.1 g / m 3 ~100g / m 3 water.

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