A low-temperature-tolerant aerobic denitrifying bacterium and its application

Abstract

The present invention belongs to the field of microbial technology, and discloses a low-temperature-tolerant aerobic denitrifying bacterium and its application; the low-temperature-tolerant aerobic denitrifying bacterium is Enterobacter ludwigii CW-01, and its preservation number is CGMCC No. 27126. The Enterobacter ludwigii CW-01 strain of the present invention can efficiently achieve aerobic denitrification to degrade ammonia nitrogen, nitrate, and nitrite within the range of 4-35 °C, and is particularly suitable for wastewater treatment where it is difficult to remove ammonia nitrogen, nitrate, and nitrite due to low temperature; this strain also has good salt tolerance, providing a good microbial material for the biological denitrification treatment of low-temperature, high-salt, and high-ammonia-nitrogen polluted water bodies; moreover, while efficiently removing nitrogen, it also significantly reduces the usage amount of carbon sources, greatly reducing the costs of enterprises, and having good application prospects in the field of sewage treatment.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganisms, and particularly to a low-temperature-tolerant aerobic denitrifying bacterium and its application. Background Art

[0002] The traditional sewage treatment process for sewage nitrification - denitrification nitrogen removal is a method for sewage microbial nitrogen removal using nitrifying bacteria and denitrifying bacteria. This method is divided into two stages: nitrification and denitrification. Under aerobic conditions, nitrifying bacteria in the 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 the sewage are used to reduce nitrates to gaseous nitrogen. The nitrification reaction can be carried out by single-stage nitrification or two-stage nitrification. In single-stage nitrification, the carbon oxidation process also occurs simultaneously; in two-stage nitrification, the carbonization and nitrification processes can be carried out in separate tanks. The nitrification tank can be in the form of an aeration tank. The two-stage biological nitrogen removal method is an effective method for sewage microbial nitrogen removal and has been widely used as a standard biological nitrogen removal method. However, its process has strict anaerobic and aerobic requirements and requires an appropriate amount of organic matter as an electron acceptor. The aerobic denitrification process refers to the biochemical process in which certain bacteria reduce nitrates to nitrogen gas under aerobic conditions using aerobic denitrifying enzymes. This process consists of a series of reaction steps catalyzed by enzymes, 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 being beneficial to environmental protection.

[0003] The biological treatment of sewage denitrification is a microbial treatment method that is green, efficient, and low in input cost, and has been widely studied and applied in recent years. The core problem of this method is the research and development of highly efficient denitrifying strains, which can efficiently convert and degrade ammonia nitrogen in sewage through the physiological metabolism process of functional strains, thereby achieving sewage denitrification. However, biological nitrogen removal is sensitive to environmental conditions and is easily affected by temperature changes. The normal growth temperature of the vast majority of microorganisms is 20 - 35 °C. Low temperature will affect the activity of enzymes in microbial cells, thereby reducing the treatment effect on sewage. After the process is put into operation, due to the alternation of seasons and the influence of the geographical location, it is difficult to maintain a suitable temperature without artificial regulation. And temperature regulation will consume a large amount of energy. Therefore, low temperature has become a limiting factor for microbial denitrification nitrogen removal in winter, and currently, there is still relatively little research on the denitrification in sewage under low-temperature constraints. Summary of the Invention

[0004] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a low-temperature-tolerant aerobic denitrifying 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 objectives of the present invention is to protect a strain of Enterobacter ludwigii CW-01, with a preservation number of CGMCC No. 27126.

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

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

[0009] In certain embodiments, the Enterobacter ludwigii CW-01 tolerates salts 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 for nitrogen removal.

[0011] Another objective 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 includes the Enterobacter ludwigii CW-01 and / or metabolites of the Enterobacter ludwigii CW-01.

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

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

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

[0018] In this article, 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 bacterium-containing metabolite of Enterobacter ludwigii CW-01. The sterile metabolite (sterile fermentation filtrate) of Enterobacter ludwigii CW-01 can be specifically prepared as follows: Cultivate Enterobacter ludwigii CW-01 in a liquid medium, and filter out Enterobacter ludwigii CW-01 in the liquid culture (fermentation broth), then the sterile metabolite of Enterobacter ludwigii CW-01 is obtained. The bacterium-containing metabolite of Enterobacter ludwigii CW-01 can be specifically prepared as follows: Cultivate Enterobacter ludwigii CW-01 in a liquid fermentation medium, and collect the fermentation broth (containing Enterobacter ludwigii CW-01 and substances secreted into the liquid medium), and this fermentation broth is the bacterium-containing metabolite of Enterobacter ludwigii CW-01.

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

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

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

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

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

[0024] In some embodiments, the dosage form of the microbial agent may be various dosage forms, such as liquid agent, emulsion, suspending agent, powder, granule, wettable powder, or water dispersible granule.

[0025] According to needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. may also be added to the microbial agent.

[0026] In some 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 in the usual sense in this field, indicating the number of microbial colonies contained in g of the test sample, and CFU represents colony forming unit.

[0027] In some embodiments, in the microbial agent, Enterobacter ludwigii CW-01 or / and the culture of Enterobacter ludwigii CW-01 may exist in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture, or a mixture of cells and filtrate.

[0028] This application also provides a preparation method of the microbial agent as described above, including the following steps:

[0029] 1) Inoculate Enterobacter ludwigii CW-01 into a culture medium for cultivation to obtain a fermentation broth;

[0030] 2) Dry the fermentation broth to obtain the microbial agent.

[0031] In some embodiments, when culturing, the culture medium used includes but is not limited to MS medium, LB medium, PDA medium, Czapek medium, and nutrient broth medium.

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

[0033] In certain 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 of tryptone, 1-10 g / L of yeast extract, and 5-15 g / L of sodium chloride. The pH value of the culture medium is 7.0-7.2.

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

[0036] In certain embodiments, the time of the culture is 2-10 d.

[0037] In certain embodiments, the rotation speed of the culture 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 inoculant as described above in at least one of the following;

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

[0040] a2) Preparing a product for reducing inorganic nitrogen in the environment;

[0041] a3) Reducing COD in the environment;

[0042] a4) Preparing a product for reducing COD in the environment;

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

[0044] a6) Preparing a product for reducing total nitrogen in the environment;

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

[0046] a8) Preparing a product for reducing total phosphorus in the environment.

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

[0048] In certain specific embodiments, the water body may be wastewater.

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

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

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

[0052] b1) Biological denitrification;

[0053] b2) Biological phosphorus removal;

[0054] b3) Biological degradation of COD.

[0055] In some embodiments, the object to be treated may be one or more of water bodies, soil, and water body sediments.

[0056] In some specific embodiments, the water body may be wastewater.

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

[0058] In some embodiments, the temperature of the treatment is 4 to 35 °C.

[0059] The present invention also protects a product; the product includes Enterobacter ludwigii CW-01 as described above, or the culture as described above, or the microbial inoculum as described above; the function of the product is at least one of the following;

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

[0061] c2) Reducing COD in the environment;

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

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

[0064] In some embodiments, the environment may be one or more of water bodies, soil, and water body sediments.

[0065] In some specific embodiments, the water body may be wastewater.

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

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

[0068] In some embodiments, the product can be a nitrogen remover.

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

[0070] The present invention provides Enterobacter ludwigii CW-01, a strain of psychrophilic aerobic denitrifying bacteria. Enterobacter ludwigii CW-01 can efficiently achieve aerobic denitrification to degrade ammonia nitrogen, nitrate, nitrite, etc. within the range of 4 to 35 °C, and is particularly suitable for wastewater treatment where it is difficult to remove ammonia nitrogen, nitrate, and nitrite due to low temperature. Moreover, this strain has good salt tolerance, providing a good microbial material for the biological denitrification treatment of water bodies contaminated by low temperature, high salt, and high ammonia nitrogen. While efficiently removing total nitrogen, it also significantly reduces the usage amount of carbon source, greatly reducing the cost of enterprises and having good application prospects in the field of sewage treatment. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0072] Figure 2 It is a growth diagram of Enterobacter ludwigii CW-01 strain at 4 °C.

[0073] Figure 3 It is a growth diagram of Enterobacter ludwigii CW-01 strain at 8 °C.

[0074] Figure 4 It is a growth diagram of Enterobacter ludwigii CW-01 strain at 15 °C.

[0075] Figure 5 It is a growth diagram of Enterobacter ludwigii CW-01 strain at 28 °C. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0076] The present invention isolates and screens a strain of cold-tolerant aerobic denitrifying bacterium - Enterobacter ludwigii CW-01 from a sewage treatment pool, providing a strain resource for nitrogen removal in low-temperature environments and contributing to the application of biological methods for nitrogen removal and water quality improvement.

[0077] One of the objectives of the present invention is to protect a strain of Enterobacter ludwigii CW-01 with a preservation number of CGMCC No. 27126.

[0078] The taxonomic name of this strain is Enterobacter ludwigii, with a preservation number of CGMCC No. 27126. It was preserved at the General Microbiological Center of the China General Microbiological Culture Collection Center on April 17, 2023, at the address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It was isolated from a sewage treatment pool.

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

[0080] The morphological characteristics of Enterobacter ludwigii CW-01 in this application are as follows: the colonies are milky white, large in colony morphology, and convex. Its 16S rDNA has a homology of 99.86% with the type strain Enterobacter ludwigii in GenBank.

[0081] In some embodiments, the temperature tolerance of Enterobacter ludwigii CW-01 is 4 - 35°C.

[0082] In some embodiments, Enterobacter ludwigii CW-01 tolerates salts with a concentration of 0 - 80 g / L. Enterobacter ludwigii CW-01 in this application can grow in a medium with a salinity of 0 - 80 g / L.

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

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

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

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

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

[0088] In some embodiments, the culture includes Enterobacter ludwigii CW-01 and / or metabolites of Enterobacter ludwigii CW-01.

[0089] In some embodiments, the microbial medium can be a solid medium or a liquid medium.

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

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

[0092] In this article, the metabolite can be obtained from the fermentation broth of Enterobacter ludwigii CW-01. The metabolite of Enterobacter ludwigii CW-01 can be the sterile metabolite of Enterobacter ludwigii CW-01 or the bacterium-containing metabolite of Enterobacter ludwigii CW-01. The sterile metabolite (sterile fermentation filtrate) of Enterobacter ludwigii CW-01 can be specifically prepared by the following method: culturing Enterobacter ludwigii CW-01 in a liquid medium and filtering out Enterobacter ludwigii CW-01 in the liquid culture (fermentation broth), thus obtaining the sterile metabolite of Enterobacter ludwigii CW-01. The bacterium-containing metabolite of Enterobacter ludwigii CW-01 can be specifically prepared by the following method: culturing Enterobacter ludwigii CW-01 in a liquid fermentation medium and collecting the fermentation broth (containing Enterobacter ludwigii CW-01 and substances secreted into the liquid medium), and this fermentation broth is the bacterium-containing metabolite of Enterobacter ludwigii CW-01.

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

[0094] In some embodiments, the medium used includes tryptone and yeast extract.

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

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

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

[0098] In some embodiments, the dosage form of the microbial agent can be various dosage forms, such as liquid agent, emulsion, suspension, powder, granule, wettable powder or water dispersible granule.

[0099] In a specific embodiment, the dosage form of the microbial agent is powder.

[0100] In some embodiments, the effective viable count of Enterobacter ludwigii CW-01 in the microbial agent is at least 1×10 9 CFU / g; it can also be 1×10 9 CFU / g ~ 1×10 12 CFU / g; it can also be 6×10 9 CFU / g. CFU / g is interpreted in the usual sense in this field, indicating the number of microbial colonies contained in g of the test sample, and CFU represents colony forming unit.

[0101] In some embodiments, the microbial agent further includes a carrier.

[0102] In some embodiments, the carrier includes a solid carrier or a liquid carrier.

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

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

[0105] According to needs, surfactants (such as Tween 20, Tween 80, etc.), binders, stabilizers (such as antioxidants), pH regulators, etc. can also be added to the microbial agent.

[0106] In the microbial agent, Enterobacter ludwigii CW-01 or / and the culture of Enterobacter ludwigii CW-01 can exist in the form of cultured live cells, fermentation broth of live cells, filtrate of cell culture or a mixture of cells and filtrate.

[0107] This application also provides a preparation method of the microbial agent as described above, including the following steps:

[0108] 1) Inoculate Enterobacter ludwigii CW-01 into a culture medium for cultivation to obtain a fermentation broth;

[0109] 2) Dry the fermentation broth to obtain the microbial inoculant described above.

[0110] In some embodiments, during cultivation, the culture medium used includes but is not limited to MS medium, LB medium, PDA medium, Czapek medium, and nutrient broth medium.

[0111] In some embodiments, the culture medium used includes tryptone and yeast extract.

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

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

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

[0115] In some embodiments, the cultivation time is 2 - 10 d, it can also be 2 - 5 d, or 4 - 8 d, or 7 - 10 d, or 3 d.

[0116] In some embodiments, the cultivation rotation speed is 100 - 300 r / min, it can also be 100 - 180 r / min, or 230 - 300 r / min, or 170 - 240 r / min, or 100, 150, 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 inoculant as described above in at least one of the following;

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

[0119] a2) Preparing a product for reducing inorganic nitrogen in the environment;

[0120] a3) Reducing COD in the environment;

[0121] a4) To prepare a product for reducing COD in the environment;

[0122] a5) To reduce total nitrogen in the environment;

[0123] a6) To prepare a product for reducing total nitrogen in the environment;

[0124] a7) To reduce total phosphorus in the environment;

[0125] a8) To prepare a product for reducing total phosphorus in the environment.

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

[0127] In some specific embodiments, the water body may be wastewater.

[0128] In some specific embodiments, the working temperature of Enterobacter ludwigii CW-01 or the culture or the microbial inoculum as described above is 4-35 °C; it can also be 15-35 °C, or 20-28 °C, or 25 °C, 28 °C, 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 a method of any one of b1)-b3), including: treating the object to be treated with Enterobacter ludwigii CW-01 as described above or the culture as described above or the microbial inoculum as described above;

[0131] b1) Biological denitrification;

[0132] b2) Biological phosphorus removal;

[0133] b3) Biological degradation of COD.

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

[0135] In some specific embodiments, the water body may be wastewater.

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

[0137] In some 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 addition amount of the microbial inoculum is 0.1 g / m 3 ~100 g / m 3 water, or it can be 0.2 g / m 3 ~30 g / m 3 water, or it can be 50 / m 3 ~100 g / m 3 water, or it can be 1 g / m 3 ~60 g / m 3 water, or it can be 1 g / m 3 ~20 g / m 3 water, or it can be 1 g / m 3 、10 g / m 3 water.

[0139] The present invention also protects a product; the product includes Enterobacter ludwigii CW-01 as described above or the culture as described above or the microbial inoculum as described above; the function of the product is at least one of the following;

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

[0141] c2) Reducing COD in the environment;

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

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

[0144] In some embodiments, the environment can be one or more of a water body, soil, and water sediment.

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

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

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

[0148] In some embodiments, the product can be a nitrogen removal agent.

[0149] The fermentation culture of Enterobacter ludwigii CW-01 of the present application was inoculated into a nitrification medium. 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 a 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 a denitrification medium and treated under low-temperature conditions, and good aerobic denitrification could still be carried out. When treated at a low temperature of 4°C for 6 days, the nitrate removal rate reached about 98.85%. When treated at 8°C for 6 days, the nitrate removal rate reached about 99.69%. When treated at 15°C for 2 days, the nitrate removal rate reached about 99.49%. When treated at 28°C for 1 day, the nitrate removal rate reached about 99.81%. The microbial inoculant of the present application was applied to water. After 25 days, the ammonia nitrogen in the water decreased by 95.74%, and the carbon source dosage was greatly reduced.

[0150] Enterobacter ludwigii CW-01, the culture or the microbial inoculant of the present application has the effect of degrading inorganic nitrogen (such as ammonia nitrogen, nitrate, nitrite). The above effects make the strain of the present application have a significant effect on improving nitrogen-containing wastewater, providing a new strain or a new method for biological nitrogen removal of nitrogen-containing wastewater.

[0151] The following specific embodiments illustrate the implementation manners of the present invention. Those skilled in the art can easily understand the other advantages and effects of the present invention from the content disclosed in this specification.

[0152] Before further describing the specific implementation manners of the present invention, it should be understood that the protection scope of the present invention is not limited to the specific implementation manners described below; it should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific implementation manners, rather than limiting the protection scope of the present invention. The test methods without specific conditions noted in the following embodiments are usually carried out under conventional conditions or according to the conditions recommended by each manufacturer.

[0153] When numerical ranges are given in the embodiments, it should be understood that unless otherwise specified in the present invention, both endpoints of each numerical range and any 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 commonly understood by those skilled in the technical field. In addition to the specific methods, equipment, and materials used in the embodiments, according to the knowledge of those skilled in the technical field of the prior art and the description of the present invention, any methods, equipment, and materials of the prior art similar to or equivalent to those described in the embodiments of the present invention can also be used to implement the present invention.

[0154] The culture medium formulations involved in the embodiments of the present application are as follows:

[0155] The LB liquid culture medium formulation is: tryptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, water 1 L, pH 7.0 - 7.2, sterilized at 121 °C for 30 min. When solid, 1.5 - 2% agar is added.

[0156] The aerobic denitrification culture medium I includes: sodium nitrite 0.2 / L, potassium dihydrogen phosphate 0.2 g / L, dipotassium 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 - 7.4, sterilized at 121 °C for 30 min. When solid, 1.5 - 2% agar is added.

[0157] The aerobic denitrification culture medium II includes: sodium nitrate 0.6 g / L, potassium dihydrogen phosphate 0.2 g / L, dipotassium 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, 1.5 - 2% agar is added.

[0158] The nitrification culture medium: ammonium chloride 0.2 g / L, potassium dihydrogen phosphate 0.2 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, 1.5 - 2% agar is added.

[0159] Example 1 Screening and isolation of Enterobacter ludwigii

[0160] The isolation process of Enterobacter ludwigii isolated in this example is as follows:

[0161] 1.1 Sample collection

[0162] Collect the sewage in the sewage treatment pool of Dalian Xihai Sewage Treatment Company with a sterile water sample collection bag. Take 5 mL and transfer it into a pre-sterilized 100 mL sterile liquid LB medium. Place it in a constant temperature shaker at 28 °C and enrich for 2 days at 150 r / min. Take 40 mL of the enriched solution and add it to 400 mL of aerobic denitrification medium II. Place it in a constant temperature shaker at 28 °C and enrich for 2 days at 150 r / min. This is the first domestication cycle. Take 40 mL of the culture solution from the previous cycle and inoculate it into the domestication medium of the second cycle. Place it in a constant temperature shaker at 28 °C and culture for 2 days. And so on, carry out domestication 5 times.

[0163] Dilute the culture solution of the medium after the fifth domestication: Spread and separate 150 μL of the domesticated culture solution of the medium with sterilized glass beads on the plate of solid aerobic denitrification medium II. Culture it in an incubator at 28 °C. Pick the pure colonies on the medium and streak-separate them on a new medium. Place it in a constant temperature incubator at 28 °C to culture, and obtain several strains. Conduct aerobic denitrification tests on them respectively. Finally, obtain a strain that can efficiently perform aerobic denitrification, named strain CW-01. Its colonies are moist, milky white, large in colony morphology, and convex on the LB plate; as Figure 1 shown.

[0164] 1.2. Determine the species by 16S rDNA gene sequencing

[0165] Send the purified strain CW-01 to Shanghai Saiheng Biotechnology Co., Ltd. for 16S rDNA sequencing. Compare the obtained sequence in the database of the National Center for Biotechnology Information (NCBI) in the United States (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastn&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome). It is found that the homology of the 16S rDNA sequence with the type strain Enterobacter ludwigii in GenBank is 99.86%.

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

[0167]

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

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

[0170] 2.1 Study on low-temperature tolerance

[0171] The strain CW-01 was streaked onto an LB plate and incubated in a constant temperature incubator at 4°C, 8°C, 15°C, and 28°C for 3 days. After three days of incubation, the colony growth was as Figure 2 , Figure 3 , Figure 4 , Figure 5 shown.

[0172] The experiment proved that the strain CW-01 grew well at the ultra-low temperature of 4°C, indicating that this strain has the function of low-temperature tolerance.

[0173] 2.2 Study on salt tolerance

[0174] A loopful of the single colony of strain CW-01 was picked with an inoculation needle and inoculated into a pre-sterilized LB liquid medium, placed in a constant temperature shaker at 28°C, 150 r / min, and cultured for three days to obtain a fermentation broth. The fermentation broth was inoculated into LB liquid media with different concentrations of sodium chloride (experimental groups A1 (10 g / L), A2 (20 g / L), A3 (40 g / L), A4 (60 g / L), A5 (80 g / L), A6 (100 g / L)) at an inoculation amount of 1 v / v%. After culturing at 28°C, 150 r / min for three days, the absorbance of each fermentation broth at a wavelength of 600 nm was measured using an ultraviolet spectrophotometer. Three parallel experiments were set for each group. At the same time, a blank LB medium with the same salinity without inoculating any strain 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 the strain CW-01 has good salt tolerance and can still grow well at a salinity of 80 g / L.

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

[0179] 3.1 Inoculate the fermented CW-01 LB fermentation broth into 150 mL of nitrification medium at an inoculation amount of 5 v / v%, place it in a constant temperature shaker at 28 °C, 150 r / min, culture for one day, measure the ammonia nitrogen value every 6 hours, set three parallel groups in each group (experimental groups B1, B2, and B3 respectively), and one blank control group. The measured data are shown in the following table.

[0180] Among them, the ammonia nitrogen measurement method is detected by the Nessler's reagent spectrophotometry. 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 specific details, refer to the method of Nessler's reagent spectrophotometry (A) in "Water and Wastewater Monitoring and Analysis Methods (Fourth Edition)".

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

[0182] Table 2

[0183]

[0184] It was found that the ammonia nitrogen concentration decreased significantly. For example, the ammonia nitrogen concentration in experimental group B1 decreased from 52.61 mg / L to 0.2 mg / L, the ammonia nitrogen concentration in experimental group B2 decreased from 52.42 mg / L to 0.21 mg / L, and the ammonia nitrogen concentration in experimental group B3 decreased from 52.50 mg / L to 0.18 mg / L; considering the three groups together, the overall ammonia nitrogen concentration decreased by 99.62%.

[0185] 3.2 Inoculate the fermented CW-01 LB fermentation broth into 150 mL of aerobic denitrification medium I at an inoculation amount of 5 v / v%, place it in a constant temperature shaker at 28 °C, 150 r / min, culture for 1 day, measure the nitrite value every 6 hours, set three parallel groups in each group (experimental groups C1, C2, and C3 respectively), and one blank control group; the measured data are shown in the following table.

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

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

[0188] Table 3

[0189]

[0190] It was found that the nitrite concentration decreased significantly. For example, after 24 h of inoculation: the nitrite concentration in experimental group C1 decreased from 21.6 mg / L to 0.01 mg / L, the nitrite concentration in experimental group C2 decreased from 21.59 mg / L to 0.01 mg / L, and the nitrite concentration in experimental group C3 decreased from 21.59 mg / L to 0; Considering the three groups together, the overall nitrite concentration decreased by 99.96%.

[0191] 3.3 Inoculate the fermented LB fermentation broth of CW-01 into aerobic denitrification medium II at an inoculation amount of 5 v / v%, place it in a constant temperature shaker at 28 °C, 150 r / min, culture for 1 day, measure the nitrate value every 6 hours, set three parallel groups for each group (experimental groups D1, D2, and D3 respectively), and one blank control group; The measured data are shown in the following table.

[0192] The measurement method uses ultraviolet spectrophotometry. The method principle is to quantitatively determine nitrate nitrogen by using the absorption of nitrate ions at a wavelength of 220 nm. Dissolved organic matter also has absorption at 220 nm, while nitrate ions have no absorption at 275 nm. Therefore, another measurement is made at 275 nm to correct the nitrate nitrogen value. For details, refer to "Water and Wastewater Monitoring and Analysis Methods (Fourth Edition)".

[0193] Table 4

[0194]

[0195] It was found that the nitrate concentration decreased significantly. For example, after 24 h of inoculation: the nitrate concentration in experimental group D1 decreased from 103.1 mg / L to 0.21 mg / L, the nitrate concentration in experimental group D2 decreased from 103.2 mg / L to 0.21 mg / L, and the nitrate concentration in experimental group D3 decreased from 103.1 mg / L to 0.22 mg / L; Considering the three groups together, the overall nitrate concentration decreased by 99.79%.

[0196] Example 4 Influence 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 amount of 5 v / v%, and cultured in a constant temperature shaker at 4°C, 8°C, 15°C, and 28°C respectively. Three experiments were set for each group (replicates 1, 2, and 3 respectively), and a blank test was set at the same time. The turbidity of the aerobic denitrification medium after inoculation was observed. It was found that on the first day, the media at 15°C and 28°C became turbid, on the third day the medium at 8°C became turbid, and on the fourth day the medium at 4°C became turbid. The data measured by the ultraviolet spectrophotometer method are statistically shown in the following table.

[0198] Table 5

[0199]

[0200] Table 6

[0201]

[0202] It can be seen from Table 5 and Table 6 that temperature has a certain influence on the aerobic denitrification efficiency of strain CW-01. However, with the extension of time, good aerobic denitrification can still be carried out 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, about 99.49% at 15°C on the second day, and about 99.81% at 28°C on the first day.

[0203] Example 5 Application of strain CW-01 prepared into a microbial agent in sewage treatment

[0204] The LB fermentation broth of CW-01 cultured at 28°C and 150 r / min for 3 days was prepared into a microbial powder through a low-temperature drying process. The viable bacteria content in the microbial powder was 6×10 9 CFU / g; 1 g of the microbial powder was added per cubic meter of sewage and added to two sewage treatment ponds (No. 1 sewage treatment pond and No. 2 sewage treatment pond respectively) of a sewage treatment plant in Northeast China. The data of the sewage ponds before using the microbial powder are shown in the following table.

[0205] Table 7

[0206]

[0207] One week after adding the microbial powder, the sewage data are as follows:

[0208] Table 8

[0209]

[0210] Twenty-five days after adding the microbial powder, the sewage data are as follows:

[0211] Table 9

[0212]

[0213] By comparing the data in Table 7 - Table 9, it is found that when the temperature is relatively low in March and April in Northeast China, after adding the CW - 01 bacterial agent, various indicators in the sewage have been significantly improved. Moreover, in actual operation, after only adding the CW - 01 bacterial agent, the carbon source dosage has been greatly reduced. At most, the sewage treatment station used to add 12 packs of glucose per day, and the total nitrogen in the nitrification and denitrification effluent still fluctuated around 600 mg / L. After adding the bacterial agent, through a 20 - day acclimation process, the total nitrogen in the nitrification and denitrification effluent showed an obvious downward trend. In about one month, the total nitrogen could be stably controlled within 300 mg / L, and it still showed a stable downward trend. The carbon source dosage was greatly reduced, and currently only 3 packs of glucose need to be added per day. Under the condition of the same carbon source dosage, the total nitrogen index removal has obvious advantages. While efficiently removing the total nitrogen, it also greatly reduces the enterprise cost.

[0214] The above embodiments are only illustrative of the principles and effects of the present invention, and are not used to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes completed by those with ordinary knowledge in the technical field without departing from the spirit and technical ideas disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A strain of Enterobacter reuteri ( Enterobacter ludwigii ) CW-01, its deposit number is CGMCC No.27126.

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

1.

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

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

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

6. The Enterobacter reuteri according to any one of claims 1 to 2 Enterobacter ludwigii Use of CW-01 or 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: Enterobacter reuteri Enterobacter ludwigii The working temperature of 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: Using the Enterobacter reuteri described in any one of claims 1 to 2 Enterobacter ludwigii CW-01 or the culture according to claim 3 or the microbial agent according to any one of claims 4 to 5 treats the object to be treated.

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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