Pseudomonas stutzeri and application thereof in treatment of nitrogen-containing sewage

Abstract

The invention relates to the field of sewage treatment, in particular to pseudomonas stutzeri and application of the pseudomonas stutzeri in nitrogen-containing sewage treatment. The invention provides Pseudomonas stutzeri, and the preservation number of the Pseudomonas stutzeri is CGMCC (China General Microbiological Culture Collection Center) No.25807. The invention also provides a preparation method of the Pseudomonas stutzeri. The invention provides a method for screening heterotrophic nitrification-aerobic denitrification bacteria for treating aquaculture tail water and evaluating the denitrification performance of the heterotrophic nitrification-aerobic denitrification bacteria, which is suitable for treating the heterotrophic nitrification-aerobic denitrification bacteria of the aquaculture tail water, and eliminates adverse factors of traditional biological denitrification bacteria in the prior art. The strain is separated from activated sludge in Qiu Lake of Hainan University, and has remarkable denitrification capacity.

Description

Technical Field

[0001] The present invention relates to the field of sewage treatment, and particularly to Pseudomonas stutzeri and its application in treating nitrogen-containing sewage. Background Art

[0002] With the continuous development of society, people's living standards are also constantly improving. To meet people's needs, the aquaculture industry in China has developed rapidly in recent years, and the scale of high-density aquaculture has been expanding day by day, resulting in excessive accumulation of bait residues and metabolites of cultured animals in the aquaculture system, generating a large amount of aquaculture nitrogen-polluted wastewater, increasing the nitrogen content in the aquaculture water body, continuously deteriorating the water quality, seriously damaging the environment in the aquaculture water body, having a toxic effect on cultured animals, and significantly increasing the occurrence frequency of diseases of cultured animals; the direct discharge of aquaculture tail water rich in feces and residual bait will cause serious ecological and environmental problems such as eutrophication of the coastal waters and outbreaks of harmful bacteria and algae.

[0003] Currently, the common methods for removing ammonia nitrogen, nitrate, and nitrite in aquaculture tail water are physical methods, chemical methods, and biological methods.

[0004] Physical methods mainly include water replacement, filtration, adsorption, aeration, stripping, air stripping method, etc., but nitrogen is not really removed, and the nitrogen pollution problem cannot be fundamentally solved.

[0005] Chemical methods mainly include ion exchange, breakpoint chlorination method, etc. The water body treated by chemical methods is easy to detect chemical residues, and these residues are transmitted through the food chain, which may cause carcinogenic effects on the human body and may also cause secondary pollution to the aquaculture water body.

[0006] Biological nitrogen removal is to utilize the ammonification, nitrification, and denitrification of microorganisms themselves to transform the form of nitrogen in the water body, transform it into the protein of the bacteria themselves, or transform ammonia nitrogen and nitrite nitrogen harmful to aquaculture animals into nitrate nitrogen harmless to aquaculture animals, and even transform it into nitrogen-containing gas. Compared with the above physical methods and chemical methods, biological nitrogen removal is more economical and effective, easy to operate, and does not cause secondary pollution.

[0007] Currently, the nitrifying bacteria and denitrifying bacteria used in biological methods have the disadvantages of slow growth rate, long reaction time, and low treatment efficiency. Summary of the Invention

[0008] In view of this, the present invention provides Pseudomonas stutzeri and its application in treating nitrogen-containing sewage. The present invention provides a screening method for heterotrophic nitrification-aerobic denitrifying bacteria applied to treat aquaculture tail water and an evaluation method for their nitrogen removal performance, which is applicable to heterotrophic nitrification-aerobic denitrifying strains for treating aquaculture tail water, eliminating the adverse factors of traditional biological nitrogen-removing bacteria in the prior art. This bacterium is isolated from the activated sludge of Qiu Haihu in Hainan University and has remarkable nitrogen removal ability.

[0009] To achieve the above-mentioned invention object, the present invention provides the following technical solutions:

[0010] The present invention provides Pseudomonas stutzeri with a preservation number of: CGMCC No. 25807.

[0011] The present invention also provides a microbial inoculum, comprising: the above-mentioned Pseudomonas stutzeri and an acceptable auxiliary agent.

[0012] The present invention also provides the application of the above-mentioned Pseudomonas stutzeri and / or the above-mentioned microbial inoculum in treating nitrogen-containing sewage.

[0013] In some embodiments of the present invention, in the above application, the nitrogen-containing sewage includes: aquaculture tail water and / or domestic sewage.

[0014] The present invention also provides a method for treating nitrogen-containing sewage, inoculating the above-mentioned Pseudomonas stutzeri and / or the above-mentioned microbial inoculum into the nitrogen-containing sewage and culturing.

[0015] In some embodiments of the present invention, in the above treatment method, the carbon source for culturing includes: one or more of sodium acetate, sucrose, glucose, sodium succinate, sodium citrate, sodium pyruvate, and potassium sodium tartrate.

[0016] In some embodiments of the present invention, in the above treatment method, the carbon source for culturing includes: one or more of sodium acetate, sodium citrate, and sodium pyruvate.

[0017] In some embodiments of the present invention, in the above treatment method, the carbon source for culturing is: sodium acetate.

[0018] In some embodiments of the present invention, in the above treatment method, the concentration of the carbon source is 3 - 20 g / L.

[0019] In some embodiments of the present invention, in the above treatment method, the concentration of the carbon source is 6 - 15 g / L.

[0020] In some embodiments of the present invention, in the above treatment method, the carbon source is sodium acetate with a concentration of 6 - 15 g / L.

[0021] In some embodiments of the present invention, in the above treatment method, the pH value of the culture is 4 - 9.

[0022] In some embodiments of the present invention, in the above-mentioned treatment method, the pH value of the culture is 7 to 9.

[0023] In some embodiments of the present invention, in the above-mentioned treatment method, the pH value of the culture is 8.5.

[0024] In some embodiments of the present invention, in the above-mentioned treatment method, the temperature of the culture is 15 to 40 °C.

[0025] In some embodiments of the present invention, in the above-mentioned treatment method, the temperature of the culture is 30 to 40 °C.

[0026] In some embodiments of the present invention, in the above-mentioned treatment method, the temperature of the culture is 30 °C.

[0027] In some embodiments of the present invention, in the above-mentioned treatment method, the rotation speed of the culture is 120 to 200 r / min.

[0028] In some embodiments of the present invention, in the above-mentioned treatment method, the rotation speed of the culture is 160 r / min.

[0029] In some embodiments of the present invention, in the above-mentioned treatment method, the salinity of the culture is 0 to 50 g / L.

[0030] In some embodiments of the present invention, in the above-mentioned treatment method, the salinity of the culture is 0 to 10 g / L.

[0031] In some embodiments of the present invention, in the above-mentioned treatment method, the inorganic salt ions in the culture include: Zn 2+ , Ca 2+ , Cu 2+ , Mn 2+ , Mg 2+ and Fe 2+ or more than one of them.

[0032] In some embodiments of the present invention, in the above-mentioned treatment method, the inorganic salt ion in the culture is: Mg 2+ .

[0033] In some embodiments of the present invention, in the above-mentioned treatment method, the inorganic salt ions in the culture are added in the form of inorganic salt compounds, and the inorganic salt compounds include: one or more of CaCl2, MnCl2, CuSO4, FeSO4, ZnSO4 and MgSO4.

[0034] In some embodiments of the present invention, in the above-mentioned treatment method, the inorganic salt ions in the culture are added in the form of inorganic salt compounds, and the inorganic salt compound is: MgSO4.

[0035] In some embodiments of the present invention, in the above treatment method, the viable cell count at the time of inoculation is 5.9×10 3 ~11.8×10 8 CFU / mL.

[0036] In some embodiments of the present invention, in the above treatment method, the viable cell count at the time of inoculation is 5.9×10 8 CFU / mL.

[0037] In some embodiments of the present invention, in the above treatment method, the C / N in the nitrogen-containing sewage is 3 - 30.

[0038] In some embodiments of the present invention, in the above treatment method, the C / N in the nitrogen-containing sewage is 6.

[0039] In some embodiments of the present invention, in the above treatment method, the cultivation uses a culture medium; the culture medium includes: 2.0 g of (NH4)2SO4, 3.0 g of KNO3, 15 g of CH3COONa, 0.2 g of MgSO4·7H2O, 1.0 g of KH2PO4, and 2.0 mL of trace element solution.

[0040] The beneficial effects of the present invention include:

[0041] (1) The strain L27 provided by the present invention has a high denitrification rate. When the conditions are optimal and the initial ammonia nitrogen concentration is 346.78 mg / L and the nitrate concentration is 513.88 mg / L, the denitrification rate reaches over 90.00%, and there is no obvious accumulation of nitrite nitrogen, indicating that the strain L27 has the potential to treat nitrogen-containing wastewater.

[0042] (2) The strain L27 provided by the present invention adapts to low-concentration C / N and has a wide range. When the C / N is 15 - 80, the denitrification rates all reach over 90.00%, which can save the cost of carbon source. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art.

[0044] Figure 1 Shows the phylogenetic tree diagram of the 16S rDNA of strain L27;

[0045] Figure 2 Shows the growth and denitrification effect diagrams of Pseudomonas stutzeri L27 of the present invention under different carbon source conditions; where: A shows the effect of different carbon sources on the nitrogen removal rate of strain L27; B shows the effect of different carbon sources on the growth of strain L27;

[0046] Figure 3 Show the growth and denitrification effect diagrams of Pseudomonas stutzeri L27 of the present invention under different rotation speed conditions; wherein: A is the effect of different rotation speeds on the nitrogen removal rate of strain L27; B is the effect of different rotation speeds on the growth of strain L27;

[0047] Figure 4 Show the growth and denitrification effect diagrams of Pseudomonas stutzeri L27 of the present invention under different pH conditions; wherein: A is the effect of different pH values on the nitrogen removal rate of strain L27; B is the effect of different pH values on the growth of strain L27;

[0048] Figure 5 Show the growth and denitrification effect diagrams of Pseudomonas stutzeri L27 of the present invention under different temperature conditions; wherein: A is the effect of different temperatures on the nitrogen removal rate of strain L27; B is the effect of different temperatures on the growth of strain L27;

[0049] Figure 6 Show the growth and denitrification effect diagrams of Pseudomonas stutzeri L27 of the present invention under different carbon source concentration conditions; wherein: A is the effect of different carbon source concentrations on the nitrogen removal rate of strain L27; B is the effect of different carbon source concentrations on the growth of strain L27;

[0050] Figure 7 Show the growth and denitrification effect diagrams of Pseudomonas stutzeri L27 of the present invention under different salinity conditions; wherein: A is the effect of different salinities on the nitrogen removal rate of strain L27; B is the effect of different salinities on the growth of strain L27;

[0051] Figure 8 Show the growth and denitrification effect diagrams of Pseudomonas stutzeri L27 of the present invention under different inoculum size conditions; wherein: A is the effect of different inoculum sizes on the nitrogen removal rate of strain L27; B is the effect of different inoculum sizes on the growth of strain L27;

[0052] Figure 9 Show the growth and denitrification effect diagrams of Pseudomonas stutzeri L27 of the present invention under different inorganic salt ion conditions; wherein: A is the effect of different inorganic salt ions on the nitrogen removal rate of strain L27; B is the effect of different inorganic salt ions on the growth of strain L27;

[0053] Figure 10 Show the denitrification effect result diagram of Pseudomonas stutzeri L27 of the present invention in open aquaculture tail water; wherein: the upper part is the NH4 + -N removal effect of strain L27 on open aquaculture tail water; the middle part is the NO3 - -N removal effect of strain L27 on open aquaculture tail water; the lower part is the NO2 - -N removal effect of strain L27 on open aquaculture tail water.

[0054] Biological Deposit Description

[0055] Biological material: L27; Taxonomic name: Pseudomonas stutzeri; Deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on September 26, 2022; Address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; Deposit number: CGMCC No. 25807. Detailed Implementation Modes

[0056] The present invention discloses Pseudomonas stutzeri and its application in treating nitrogen-containing sewage.

[0057] It should be understood that the expression "one or more of..." individually includes each of the objects recited after the said expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0058] The terms "comprising", "having" or "containing", including the use of their grammatical synonyms, should generally be understood as open and non-restrictive, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0059] It should be understood that as long as the present invention is still operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be carried out simultaneously.

[0060] The use of any and all examples or exemplary language such as "for example" or "including" herein is merely intended to better illustrate the present invention and does not limit the scope of the present invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential for the practice of the present invention.

[0061] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant values in the specific embodiments have been presented as precisely as possible herein. However, any value inherently and inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, values and percentages used in this disclosure are modified by "about". Herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range.

[0062] The strain provided by the present invention is a Gram-negative bacterium. The colony surface is light yellow, opaque, round or nearly round, with a neat edge, a diameter of 1-2 mm, the colony surface is prominent and smooth, smooth and moist, and sticky when picked up. This bacterium was deposited in the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on September 26, 2022, with the deposit number: CGMCC NO. 25807. The address of the deposit unit is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. Contact phone number: 010-64807355.

[0063] The phylogenetic tree of Pseudomonas stutzeri L27 described in the present invention is as Figure 1 shown.

[0064] The above-mentioned Pseudomonas stutzeri with heterotrophic nitrification-aerobic denitrification function can efficiently degrade ammonia nitrogen, nitrate nitrogen and nitrite nitrogen, and has heterotrophic nitrification function and aerobic denitrification function. This strain has good environmental adaptability and high safety. Therefore, it has a wide application prospect in the field of biological denitrification treatment of aquaculture tail water or other nitrogen-containing sewage.

[0065] The application of the above-mentioned Pseudomonas stutzeri with heterotrophic nitrification-aerobic denitrification function in the denitrification treatment of nitrogen-containing sewage preferably includes the following steps: inoculating the above-mentioned Pseudomonas stutzeri with heterotrophic nitrification-aerobic denitrification function in the HN-AD medium and culturing to obtain the denitrified wastewater.

[0066] The nitrogen-containing sewage is preferably aquaculture tail water and domestic sewage.

[0067] The carbon source in the culture is preferably sodium acetate.

[0068] The C / N of the sewage in the culture is 3-30, and the better condition is C / N = 6.

[0069] The pH value of the sewage in the culture is 4-9, and preferably pH = 8.5.

[0070] The temperature in the culture is 20°C-40°C, and the better condition is T = 30°C.

[0071] The salinity of the medium is 0-50, and the better condition is salinity = 0.

[0072] The rotation speed of the medium is 120 r / min-200 r / min, and the better condition is 160 r / min.

[0073] The inoculation amount in the medium is 5.9×10 3 cfu / mL-11.8×10 8 cfu / mL, and the better condition is 5.9×108 CFU / mL or 11.8×10 8 CFU / mL.

[0074] At least one of the inorganic salt ions CaCl2, MnCl2, CuSO4, FeSO4, ZnSO4, MgSO4 in the medium, and the preferred condition is MgSO4.

[0075] The determination and analysis methods of the three nitrogen elements in the experiments involved in the present invention all refer to national standards. Among them, the determination and analysis of ammonia nitrogen are based on "Water Quality - Determination of Ammonia Nitrogen - Ammonia Nitrogen Indophenol Blue Method" (GB17378.4 - 2007); the determination and analysis of nitrate nitrogen are based on "Water Quality - Determination of Nitrate Nitrogen - Cadmium Column Reduction Method" (GB / T5750.6 - 2006); the determination and analysis of nitrite nitrogen are based on "Water Quality - Determination of Nitrite Nitrogen - Spectrophotometry" (GB 7493 - 1987).

[0076] The medium used in the experiments involved in the present invention is sterilized at 121 °C for 20 min using a high-pressure steam sterilizer, and its formula is as follows:

[0077] Trace elements (g / L): Na2EDTA 50; ZnSO4·7H2O 2.2; CaCl2 5.5; MnCl2·4H2O 5.06; FeSO4·7H2O 5.0; CuSO4 1.01; CoCl2·6H2O 1.61;

[0078] Nitrifying medium HM: 2.0 g (NH4)2SO4, 16.4 g CH3COONa, 0.2 g MgSO4·7H2O, 1.0 g KH2PO4, 2.0 mL trace element solution.

[0079] Denitrifying medium DM: 3.0 g KNO3, 16.4 g CH3COONa, 0.2 g MgSO4·7H2O, 1.0 g KH2PO4, 2.0 mL trace element solution.

[0080] Heterotrophic nitrification-aerobic denitrification medium HN-AD: 2.0 g (NH4)2SO4, 3.0 g KNO3, 15 g CH3COONa, 0.2 g MgSO4·7H2O, 1.0 g KH2PO4, 2.0 mL trace element solution.

[0081] LB medium: 10.0 g peptone, 5.0 g yeast extract, 10.0 g NaCl.

[0082] Among them, the C / N of the sewage in Example 2 and Example 3 is 3 to 30, and the preferred condition is C / N = 6.

[0083] In Examples 1 to 3 and the comparative example of the present invention, the raw materials and reagents used can be purchased from the market.

[0084] The present invention will be further described below in conjunction with examples:

[0085] Example 1

[0086] (1) Sample collection

[0087] In this experiment, the samples for screening heterotrophic nitrification-aerobic denitrification strains were collected from the activated sludge in Qiu Lake of Hainan University. The activated sludge was put into a sterilized bag and taken back to the laboratory for storage in a 4°C refrigerator for subsequent use.

[0088] (2) Enrichment, isolation and screening of heterotrophic nitrification-aerobic denitrification strains

[0089] 1) Enrichment culture: The obtained activated sludge was put into an Erlenmeyer flask, 250 mL of sterile water was added, and it was cultured overnight in a shaker at 30°C and 180 r / min, and then left to stand to absorb the supernatant, which was added to the LB medium and cultured for 24 hours.

[0090] 2) Sample plate coating: The bacterial solution cultured in LB was inoculated into the heterotrophic nitrification liquid medium (HM) and cultured in a constant temperature shaker at 30°C and 180 r / min for 24 h. The cultured bacterial solution was coated on the heterotrophic nitrification solid culture plate.

[0091] 3) Single colonies were picked from the heterotrophic nitrification solid culture plate and inoculated into the aerobic denitrification medium (DM), cultured in a shaker at 30°C and 180 r / min for 24 h, and then coated on the aerobic denitrification medium plate.

[0092] 4) Isolation and purification: Single colonies from the aerobic denitrification plate in step 3) were picked and inoculated into the HN-AD liquid medium, cultured in a shaker at 30°C and 180 r / min for 24 h. The cultured strains were coated on the HN-AD solid medium plate. The plate was sealed with a sealing film and inverted and placed in a 30°C incubator. The single colonies grown on the HNAD solid plate were picked into the HNAD liquid medium, and then the cultured bacteria were glycerol-preserved and placed in an -80°C refrigerator.

[0093] (5) Identification of nitrification and denitrification performance: The selected strains were first inoculated in LB medium for activation. The bacterial liquid in the logarithmic growth phase was aspirated and inoculated into 100 mL of medium with ammonium sulfate and potassium nitrate as the common nitrogen source and sodium acetate as the carbon source at an inoculation amount of 3%. The culture was carried out in a shaker at 30 °C and 180 r / min for 48 h, and the contents of ammonia nitrogen, nitrate, and nitrite in the medium before and after inoculation were detected respectively.

[0094] (3) Identification

[0095] (1) Identification of morphological characteristics

[0096] On the HN-AD medium plate, the colonies of the strain were protruding and smooth on the surface, round or approximately round, light yellow, opaque, smooth and moist on the surface, with neat edges, and were sticky when picked up.

[0097] (2) Extraction of bacterial genomic DNA

[0098] The TIANamp Bacteria DNA kit (TIANGEN) was used to extract bacterial DNA, and the integrity of the extracted DNA was verified by agarose gel electrophoresis.

[0099] The DNA of the strain was extracted as a PCR template for amplification. The primers used were universal primers: upstream 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (as shown in SEQ ID NO:1), downstream 1492R: 5'-GGTTACCTTGTTACGACTT-3' (as shown in SEQ ID NO:2), and the reaction was carried out in a PCR amplifier.

[0100] PCR amplification system (Total 50 μL): Green Tap Mix 25 μL, 27F 1 μL, 1492R 1 μL, template DNA 2 μL, sterile water 23.5 μL.

[0101] The PCR reaction program was set as shown in Table 1:

[0102] Table 1

[0103]

[0104] After the PCR products were detected to be qualified by agarose gel electrophoresis, they were sent to BGI Tech Solutions Co., Ltd. (Beijing) for 16S rDNA sequencing, and the phylogenetic tree of the strain was obtained by homology analysis of the sequencing results.

[0105]

[0106] Based on various identification items such as its 16S rDNA, bacterial morphology, colony morphology, and physiological and biochemical characteristics, it was determined that strain L27 is Pseudomonas stutzeri. This strain was deposited in the China General Microbiological Culture Collection Center on September 26, 2022, with the deposit number: CGMCC NO.25807, and the address of the depository unit is Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0107] Example 2 Effects of Different Environmental Factors on the Growth and Denitrification Performance of Strain L27

[0108] Carbon source: Under the condition of keeping other conditions the same, sodium acetate, sucrose, glucose, sodium succinate, sodium citrate, sodium pyruvate, and potassium sodium tartrate were used as the carbon sources of the HN-AD basal medium respectively. After culturing in a constant temperature shaking incubator at 30 °C and 180 r / min for 48 h, the supernatant was taken to detect ammonia nitrogen, nitrate, nitrite, and OD600nm. As Figure 2 shown; the strain had different removal rates of nitrate and ammonia nitrogen under different carbon source conditions. When sodium acetate, sodium citrate, and sodium pyruvate were used as carbon sources, the strain had the best removal rates of ammonia nitrogen and nitrate nitrogen. The removal rate of nitrate nitrogen reached more than 99%, and the removal rate of ammonia nitrogen was more than 89%, which was higher than the other three groups.

[0109] Rotation speed: Under the condition of keeping other conditions the same, by adjusting the rotation speed of the constant temperature shaking incubator (120, 140, 160, 180, 200 r / min), the above optimized medium and culture conditions were used. After culturing in a constant temperature shaking incubator at 30 °C for 48 h, the supernatant was taken to detect ammonia nitrogen, nitrate, nitrite, and OD600nm. The results were as Figure 3 shown. Between 120 r / min and 200 r / min, the difference in the denitrification effect of the strain was small. When the rotation speed was 160 r / min, the maximum removal rate was achieved. The removal rates of ammonia nitrogen and nitrate nitrogen were 73.34% and 100% respectively, and OD600nm was 1.36.

[0110] pH: Under the condition of keeping other conditions the same, the initial pH of the HN-AD medium was adjusted to 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0 respectively, and the other components remained the same. It was placed in a constant temperature shaking incubator at 30 °C and 180 r / min for 48 h, and then the supernatant was taken to detect ammonia nitrogen, nitrate, nitrite, and OD600. The results were as Figure 4As shown in the figure, the initial pH value of the culture medium has a great influence on the removal rates of ammonia nitrogen and nitrate nitrogen. When the initial pH value of the culture medium is 8.5, the denitrification effect of the strain is the best, and the removal rates of ammonia nitrogen and nitrate nitrogen are 97.04% and 99.31% respectively, and the OD600nm is 1.85. When the pH value is between 7 and 9, the strain can remove nitrogen well, indicating that the strain is suitable for growing under neutral and alkaline conditions. When the pH value is lower than 7.0, the growth of the strain is inhibited, and ammonia nitrogen and nitrate nitrogen cannot be removed, indicating that too low pH value will inhibit the growth of the strain and the removal of nitrogen, probably because the activities of nitrate reductase and nitrite reductase decrease under acidic conditions, affecting the removal of ammonia nitrogen and nitrate nitrogen.

[0111] Temperature: Under the condition of keeping other conditions the same, by adjusting the temperature of the constant temperature shaking incubator (15°C, 20°C, 25°C, 30°C, 35°C, 40°C), using the above optimized culture medium, culture at the condition of 180 r / min. After 48 h, take the supernatant to detect ammonia nitrogen, nitrate, nitrite, and OD600nm. From Figure 5 The results show that the strain has high adaptability to temperature and is suitable for culturing between 30°C and 40°C, and is suitable for use in tropical regions such as Hainan. When the culture temperature is 30°C, the removal rates of ammonia nitrogen and nitrate nitrogen reach the highest, which are 91.18% and 99.80% respectively; when the culture temperature is lower than 30°C, the removal rates of ammonia nitrogen and nitrate nitrogen decrease rapidly. Therefore, select the temperature of 30°C for subsequent experiments.

[0112] Different carbon source concentrations: Under the condition of keeping other conditions the same, by changing the dosage of the best carbon source to adjust the carbon source concentration of the HN-AD culture medium (3, 6, 9, 12, 15, 20) g / L, place it in a constant temperature shaking incubator at 30°C and 180 r / min for culture. After 48 h, take the supernatant to detect ammonia nitrogen, nitrate, nitrite, and OD600nm. The results Figure 6 show that when the sodium acetate concentration is between 6 and 15 g / L, the removal rates of ammonia nitrogen and nitrate nitrogen are both relatively high, and reach the highest at 15 g / L, which are 98.92% and 88.40% respectively. When the sodium acetate concentration is greater than 15 g / L or less than 6 g / L, the nitrogen removal rate decreases. The denitrification characteristics of the strain are different at different concentrations, probably because when the carbon source concentration is too low, the carbon in the culture medium is not enough for the growth of the strain, and the growth ability of the strain decreases, while too high carbon source concentration will hinder the growth and reproduction of the strain.

[0113] Salinity: Under the condition of keeping other conditions the same, by adjusting the initial salinity of the HN-AD culture medium (0, 10, 20, 30, 40, 50) g / L, using the above optimized culture medium and culture conditions. After culturing in a constant temperature shaking incubator at 30°C for 48 h, take the supernatant to detect ammonia nitrogen, nitrate, nitrite, and OD600nm. FromFigure 7 It can be seen that under the condition of 0 - 50 g / L NaCl, as the NaCl concentration increases, the denitrification performance of the strain is gradually affected and becomes worse, the growth of the bacteria is restricted, and almost no ammonia nitrogen and nitrate nitrogen are removed when the NaCl concentration is 50 g / L. It can be seen that the strain growth and denitrification are suitable for an environment with a NaCl concentration of 0 - 10 g / L.

[0114] Inoculum size: Under the condition of keeping other conditions the same, by adjusting the inoculum size of the HN - AD medium (11.8×10 8 CFU / mL, 5.9×10 8 CFU / mL, 5.9×10 7 CFU / mL, 5.9×10 6 CFU / mL, 5.9×10 5 CFU / mL, 5.9×10 4 CFU / mL, 5.9×10 3 CFU / mL), using the above - optimized medium and culture conditions. After culturing in a 30℃ constant - temperature shaking incubator for 48 h, take the supernatant to detect ammonia nitrogen, nitrate, nitrite, and OD600nm. From Figure 8 the results, it can be seen that as the inoculum size increases, the denitrification ability of the strain gradually becomes stronger. When the viable bacteria count in the inoculum before inoculation is 11.8×10 8 CFU / mL, the strain has the best effect on ammonia nitrogen and denitrification, and the removal rates of ammonia nitrogen and nitrate nitrogen are 87.28% and 99.30% respectively. When the viable bacteria count in the inoculum is lower than 5.9×10 8 CFU / mL, the removal rates of ammonia nitrogen and nitrate nitrogen by the strain are significantly reduced. It may be that the low concentration after the strain is inoculated into the medium leads to an extended growth and reproduction time, and the lag phase affects the removal of ammonia nitrogen and nitrate nitrogen. However, when the inoculum size exceeds 5.9×10 8 CFU / mL, the changes in the removal rates of ammonia nitrogen and nitrate nitrogen are not significant. Therefore, the appropriate inoculum size of the strain is about 5.9×10 8 CFU / mL.

[0115] Inorganic salt ions: Under the condition of keeping other conditions the same, by adjusting the inorganic salt ions (CaCl2, MnCl2, CuSO4, FeSO4, ZnSO4, MgSO4) of the HN - AD medium, using the above - optimized medium and culture conditions. After culturing in a 30℃ constant - temperature shaking incubator for 48 h, take the supernatant to detect ammonia nitrogen, nitrate, nitrite, and OD600nm. The results are as Figure 9 shown. It can be seen from the figure that Mg 2+ and Fe 2+ can promote the removal of ammonia nitrogen and nitrate nitrogen by the strain, while Zn2+ , Ca 2+ , Cu 2+ , Mn 2+ All inhibited the growth of the strain and the removal of ammonia nitrogen and nitrate nitrogen to varying degrees. Cu 2+ showed a more significant inhibitory effect on the growth of the strain. When Mg 2+ was used as an inorganic salt ion, the removal rates of ammonia nitrogen and nitrate nitrogen were the highest, reaching 88.46% and 99.67% respectively. Mg 2+ was selected for subsequent research.

[0116] Example 3 Practical application research of heterotrophic nitrification-aerobic denitrification bacterium L27 in open aquaculture tail water

[0117] Shrimp culture tail water collected from the Coral Museum of the College of Ocean Sciences, Hainan University, 500 L per portion (three parallels and a blank control were set). The screened strain P. stutzeri L27, with a viable bacteria content of 1×10 10 CFU / mL detected, was centrifuged and then put into the aquaculture tail water and cultured for 144 h. The changes in ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen in the aquaculture tail water were detected every 6 h (the detection method was the same as described above). The results were as Figure 10 shown. After 144 h, the content of NH4 + -N in the experimental group decreased from 2.04 mg / L to 0.05 mg / L, with a removal rate of 97.44%; the content of NH4 + -N in the blank control decreased from 2.28 mg / L to 0.51 mg / L, with a removal rate of 77.49%; the content of NO3 - -N in the experimental group decreased from 4.52 mg / L to 0.19 mg / L, with a removal rate of 96.12%; the content of NO3 - -N in the blank control was 4.61 mg / L, with a removal rate close to 0%; the content of NO2 - -N in the experimental group decreased from 1.45 mg / L to 0.16 mg / L, with a removal rate of 88.98%. The content of NO2 - -N in the blank control decreased from 1.42 mg / L to 1.33 mg / L, with a removal rate of 6.79%. It shows that the strain P. stutzeri L27 can significantly reduce the nitrogen content in the aquaculture tail water during the treatment of aquaculture tail water, has the effect of enhancing nitrogen removal, and has a certain application prospect in the treatment of nitrogen-containing sewage.

[0118] Comparative example

[0119] The differences between the present invention and the prior art are shown in Table 2:

[0120] Table 2

[0121]

[0122] The above data comparison results show that the strain L27 of the present invention is particularly suitable for the treatment of aquaculture tail water with low-concentration pollutants, and also maintains a high treatment capacity for nitrite, while ZH-14 is suitable for the livestock and poultry polluted environment with high-concentration pollutants. At the same time, ZH-14 has an accumulation effect on nitrite, and nitrite is an important pollutant index for aquaculture. High-concentration nitrite (greater than 1 mg / L) will cause the poisoning and death of aquatic animals. Since aquaculture is generally carried out in areas close to lakes, bays, etc., the discharge of nitrite is a key index. Therefore, compared with ZH-14, the strain L27 is more suitable for the treatment of aquaculture wastewater with the above characteristics.

[0123] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Pseudomonas stutzeri, characterized in that, Its preservation number is: CGMCC No. 25807.

2. Microbial inoculant, characterized in that, It includes: Pseudomonas stutzeri as described in claim 1 and acceptable auxiliaries.

3. Use of the Pseudomonas stutzeri as described in claim 1 and / or the microbial inoculum as described in claim 2 in treating nitrogen-containing sewage.

4. The application according to claim 3, characterized in that, The nitrogen-containing sewage includes: aquaculture tail water and / or domestic sewage.

5. A method for treating nitrogen-containing sewage, characterized in that, Inoculate the Pseudomonas stutzeri as described in claim 1 and / or the microbial inoculum as described in claim 2 into the nitrogen-containing sewage and culture.

6. The processing method according to claim 5, characterized in that, The carbon source for the culture includes: one or more of sodium acetate, sucrose, glucose, sodium succinate, sodium citrate, sodium pyruvate, and potassium sodium tartrate; the concentration of the carbon source is 3 - 20 g / L.

7. The processing method according to claim 5 or 6, characterized in that, The pH value of the culture is 4 - 9; the temperature of the culture is 15 - 40 °C; the rotation speed of the culture is 120 - 200 r / min; the salinity of the culture is 0 - 50 g / L.

8. The processing method according to any one of claims 5 to 7, characterized in that The cultivated inorganic salt ions include: Zn 2+ , Ca 2+ , Cu 2+ , Mn 2+ , Mg 2+ and Fe 2+ or one or more of them.

9. The processing method according to any one of claims 5 to 8, characterized in that, The viable count at the time of inoculation is 5.9×10 3 ~11.8×10 8 CFU / mL.

10. The processing method according to any one of claims 5 to 9, characterized in that, The C / N in the nitrogen-containing sewage is 3 - 30.

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