Efficient nitrogen and phosphorus removal flora as well as construction method and application thereof

Abstract

The invention discloses a high-efficiency nitrogen and phosphorus removal flora as well as a construction method and application thereof, and belongs to the technical field of environmental microorganisms. On one hand, the invention provides an efficient nitrogen and phosphorus removal flora which comprises stenotrophomonas maltophilia X03 and / or halomonas W06, and on the other hand, the invention provides a construction method of the efficient nitrogen and phosphorus removal flora and application of the efficient nitrogen and phosphorus removal flora in sewage treatment. The efficient mixed bacteria screened and cultured through the construction method integrate nitrogen and phosphorus removal performance, the optimal nitrogen and phosphorus removal effect is 96.67% and 93.18% when aerobic culture is conducted for 30 h under the conditions that sodium succinate serves as the optimal carbon source, the C / N is 14, the pH is 8, the temperature is 35 DEG C, the rotating speed is 220 rpm, and the phosphorus content is 20 mg, meanwhile, the bacterial strain has the good removal effect on ammonia nitrogen and nitrite nitrogen, and the nitrogen and phosphorus removal efficiency is greatly improved. The method has important practical significance for developing an efficient denitrification biological agent and reducing the treatment cost of microbial denitrification.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental microorganisms, and specifically relates to a highly efficient nitrogen removal and phosphorus removal bacterial community and a construction method and application thereof. Background Art

[0002] With the continuous growth of human demand for aquatic products, aquaculture has developed rapidly and has become an important economic industry around the world. However, the wastewater generated in the aquaculture process contains a large amount of organic and inorganic pollutants, such as bait residues, drug residues, animal excrement, etc., which seriously affect the ecological balance of water bodies and the quality and safety of aquatic products. Nitrogen and phosphorus are the main pollutants in aquaculture wastewater, accounting for more than 75% and 98% of the total respectively. After about 9% and 17% of them are consumed by the metabolism of aquatic products, most of the nutrients are precipitated in the form of nitrate nitrogen, nitrite nitrogen, phosphoric acid, etc. Excessive release of these pollutants will lead to eutrophication of water bodies, promote the proliferation of algae, cause algal blooms and other problems, and thus affect the health of the water ecosystem.

[0003] At present, the main aquaculture wastewater purification technologies include physical methods, chemical methods and biological methods. Physical treatment technology is mainly used to remove suspended particles, chemical oxygen demand and biochemical oxygen demand in aquaculture wastewater. It has limited removal effects on soluble nitrogen, phosphorus and other substances, and has certain limitations in practical applications; chemical methods mainly remove soluble substances or colloids in sewage by adding agents to coagulate or cause redox reactions in sewage. The treatment effect is good, but the cost is high and there is a hidden danger of secondary pollution; biological methods mainly use microorganisms and autotrophic plants such as algae and aquatic plants to improve water quality, solve residual bait and aquatic animal metabolites, and thus achieve a decontamination effect. Biological methods have the characteristics of low cost, wide adaptability of process technology, no secondary pollution, strong renewability and ecology, and have been widely used in practice.

[0004] In recent years, the research focus of new wastewater biological treatment technologies has gradually focused on biological denitrification and phosphorus removal. Experimental research on nitrogen and phosphorus removal by denitrifying polyphosphate bacteria has provided a research direction for biotechnology water treatment. However, traditional denitrification and phosphorus removal technologies have problems such as the contradiction between carbon source and sludge age and carbon source competition, making it difficult to meet the increasingly stringent discharge standards for nitrogen and phosphorus in sewage. Denitrifying polyphosphate bacteria are a type of polyphosphate bacteria with both denitrification and phosphorus removal characteristics. They can use nitrate as an electron acceptor to carry out simultaneous denitrification (denitrification) and excessive phosphorus absorption (phosphorus removal), successfully solving the problem of competition for carbon sources between denitrification bacteria and polyphosphate bacteria in traditional processes, and at the same time providing a possibility to solve the contradiction of sludge age differences, so that nitrogen and phosphorus removal can be carried out simultaneously. Therefore, screening and isolating more DPAOs and exploring their species and characteristics, and clarifying their growth influencing factors are enriching and supplementing the theory of biological denitrification and phosphorus removal, which will help the research, development and application of wastewater biological denitrification and phosphorus removal processes.

[0005] Traditional microbial nitrogen and phosphorus removal methods mainly use single microorganisms or natural mixed bacteria systems, which face problems such as low efficiency, long cycle, poor stability of bacterial community structure and low ability to resist environmental load. Compared with single bacteria, artificial multicellular systems composed of multiple bacterial species are based on intercellular communication, division of labor, energy and material transfer. Their composition is clearer and their functions are more stable. They can accurately control and monitor the structure and function of the bacterial community, which has significant advantages over natural microbial systems. Therefore, constructing artificial multicellular systems with clearer composition and more stable functions is a new development direction and strategy for dealing with complex environmental problems. Summary of the invention

[0006] In view of the problems existing in the prior art, the purpose of the present invention is to design and provide a technical solution for a highly efficient denitrification and phosphorus removal bacterial community and its construction method and application. By the method of the present invention, a highly efficient denitrification and phosphorus removal bacterial community can be constructed, which can efficiently denitrify and remove phosphorus from nitrogen-containing wastewater.

[0007] The present invention is specifically implemented by the following technical solutions:

[0008] The first aspect of the present invention provides a high-efficiency denitrification and phosphorus removal bacterial community, which includes Stenotrophomonas maltophilia X03 and / or Halomonas sp. W06, the preservation number of Stenotrophomonas maltophilia X03 is CCTCC NO: M20242132, and the preservation number of Halomonas sp. W06 is CCTCC NO: M20242131.

[0009] The second aspect of the present invention provides the use of the above-mentioned high-efficiency denitrification and dephosphorization bacterial community in the denitrification and dephosphorization treatment of nitrogen-phosphorus-containing wastewater.

[0010] Furthermore, the application is to inoculate the bacterial community into wastewater containing nitrogen and phosphorus, add a carbon source to a C / N ratio of 4 to 16, and culture at pH 5 to 9, 20°C to 40°C, 80 to 240rpm, and an initial phosphorus content of 5 to 25 mg to remove nitrogen and phosphorus from the wastewater.

[0011] Furthermore, the carbon source is sodium succinate, and the carbon source is added to a C / N ratio of 14, and the culture is carried out at pH 8, 35° C., 220 rpm, and a phosphorus content of 20 mg.

[0012] The third aspect of the present invention provides a method for constructing the above-mentioned efficient nitrogen removal and phosphorus removal bacterial community, which comprises the following steps:

[0013] 1) Enrichment and acclimatization: Take the bacterial sample sludge and add it to a conical flask containing 100 mL of sterile water. After shaking on a shaker for 3 hours, use a pipette to draw 2 mL of liquid and inoculate it into the sterilized LB medium. Culture it at 30°C and 180 rpm for 2 to 3 days.

[0014] 2) Isolation and initial screening: Take the bacterial solution and -1 Serial dilutions up to 10 -6 , take 0.1 mL and spread it on bromothymol blue medium and nitrogen-rich and phosphorus-rich medium for culture, pick the colonies with blue light rings and streak the strains several times until a single colony is obtained;

[0015] 3) Rescreening: The single bacteria obtained in step 2) were further tested for nitrogen and phosphorus removal. They were inoculated into the corresponding rescreening culture medium of the pure bacteria, and a non-inoculated control was set up to measure the NO3 in the culture medium. - -N and TP contents, and determined that the strains capable of efficient nitrogen and phosphorus removal were Stenotrophomonas maltophilia X03 and Halomonas W06.

[0016] Furthermore, the initial screening conditions in step 2) are: culturing in a biochemical incubator at 30° C. for 72 hours.

[0017] Furthermore, the bromothymol blue culture medium in step 2) includes: 1.01 g potassium nitrate, 8.5 g sodium succinate, 1.0 g magnesium sulfate heptahydrate, 1.0 g potassium dihydrogen phosphate, 0.59 g ferrous sulfate heptahydrate, 0.09 g calcium chloride, 1 mL 1% bromothymol blue ethanol solution, distilled water to 1000 mL, 20 g agar, and adjusted to pH 7.0-7.3 with 1 mol / L NaOH.

[0018] Further, the nitrogen-rich and phosphorus-rich culture medium in step 2) includes 5 g of sodium acetate, 0.0659 g of potassium dihydrogen phosphate, 0.607 g of sodium nitrate, 0.2 g of magnesium sulfate heptahydrate, 2 ml of trace elements, 0.1 g of EDTA-Na, distilled water to 1000 mL, 20 g of agar, and natural pH;

[0019] The above trace elements include: zinc sulfate 4.4 mg, manganese chloride tetrahydrate 10.2 mg, calcium chloride 11 mg, ferrous sulfate heptahydrate 10 mg, copper sulfate pentahydrate 3.2 mg, ammonium molybdate tetrahydrate 2.2 mg, cobalt chloride hexahydrate 3.2 mg, EDTA-Na 144 mg, distilled water 1000 mL, natural pH.

[0020] Furthermore, the rescreening conditions in step 3) are: shaking culture in a constant temperature shaking incubator at 30° C. and 160 r / min for 72 hours.

[0021] Further, in step 3), the rescreening medium includes 3.583 g of sodium citrate, 0.075 g of dipotassium hydrogen phosphate trihydrate, 0.607 g of sodium nitrate, 0.05 g of magnesium sulfate heptahydrate, 0.01 g of ferrous sulfate heptahydrate, 0.12 g of sodium chloride, 2 mL of trace elements, 0.1 g of EDTA-Na, and the volume is adjusted to 1000 mL with distilled water, and the pH is adjusted to 8 with 1 mol / L NaOH;

[0022] The above trace elements include: zinc sulfate 4.4 mg, manganese chloride tetrahydrate 10.2 mg, calcium chloride 11 mg, ferrous sulfate heptahydrate 10 mg, copper sulfate pentahydrate 3.2 mg, ammonium molybdate tetrahydrate 2.2 mg, cobalt chloride hexahydrate 3.2 mg, EDTA-Na 144 mg, distilled water 1000 mL, natural pH.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The high-efficiency mixed bacteria screened and cultured by the construction method of the present invention have the performance of nitrogen removal and phosphorus removal as one, and have the best nitrogen removal and phosphorus removal effect of 96.67% and 93.18% when aerobic culture is carried out for 30 hours under the conditions of sodium succinate as the optimal carbon source, C / N of 14, pH of 8, temperature of 35°C, rotation speed of 220rpm, and phosphorus content of 20mg. At the same time, the strain has a good removal effect on ammonia nitrogen and nitrite nitrogen, which is of great practical significance for developing high-efficiency denitrification biological bacteria and reducing the treatment cost of microbial denitrification. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The degradation rate of different strains prepared in Example 1 of the present invention;

[0026] Figure 2 is the degradation rate of the two pure strains mixed cultured in Example 2 of the present invention;

[0027] Figure 3 This is a colony characteristic diagram of the denitrifying bacteria X03 in Example 3 of the present invention;

[0028] Figure 4 This is a colony characteristic diagram of the phosphorus removal bacteria W06 in Example 3 of the present invention;

[0029] Figure 5 Schematic diagram of nitrogen and phosphorus removal efficiency of the bacterial community of the present invention under different carbon sources;

[0030] Figure 6 Schematic diagram of nitrogen and phosphorus removal efficiency of the bacterial community of the present invention at different carbon-nitrogen ratios;

[0031] Figure 7 Schematic diagram of nitrogen and phosphorus removal efficiency of the bacterial community of the present invention at different initial phosphorus contents;

[0032] Figure 8 Schematic diagram of nitrogen and phosphorus removal efficiency of the bacterial community of the present invention at different pH values;

[0033] Fig. 9 This is a schematic diagram of the nitrogen and phosphorus removal efficiency of the bacterial community of the present invention at different temperatures;

[0034] Fig.10 It is a schematic diagram of the nitrogen and phosphorus removal efficiency of the bacterial community of the present invention at different rotation speeds. DETAILED DESCRIPTION

[0035] The present invention is further described below in conjunction with specific preferred embodiments, but the protection scope of the present invention is not limited thereby. In the embodiments, all are conventional methods unless otherwise specified, and the reagents used are conventional reagents or reagents prepared according to conventional methods unless otherwise specified.

[0036] Example 1: Screening and cultivation of efficient nitrogen and phosphorus removal strains

[0037] Screening method for denitrifying bacteria: Take 2-3g of biological turntable mud sample from a sewage treatment company in Hangzhou and add it to a conical flask containing 100mL of sterile water. After shaking on a shaker for 3h, use a pipette to draw 2mL of liquid and inoculate it into sterilized LB medium. Culture it at 30℃ and 180rpm for 2-3d. Take the culture solution of the strain and inoculate it into a test tube containing sterile water. Dilute it according to the proportion to obtain strain concentrations of 10 -1 -10 -8 Gradient bacterial suspension; then, 100 μL of each gradient bacterial suspension was taken and spread on bromothymol blue (BTB) solid culture medium by plate spreading method, and cultured at 30℃ for 4 days. The blue colonies were picked for streaking separation, and separated and purified for more than 3 times until the characteristics of the colonies were basically consistent. Then, they were inoculated into 100 mL of rescreening culture medium and placed in a constant temperature shaking incubator at 30℃ and 160r / min for shaking culture for 72h. At the same time, an uninoculated control was set up to determine the NO3 in the culture medium. - -N and TP contents, strains with high degradation rates were screened out, namely X02, X03, X04 and XL, as shown in Table 1.

[0038] Screening method for phosphorus removal bacteria: Take 2-3g of biological turntable mud sample from a sewage treatment company in Hangzhou and add it to a conical flask containing 100mL of sterile water. After shaking on a shaker for 3h, use a pipette to draw 2mL of liquid and inoculate it into sterilized LB medium. Culture it at 30℃ and 180rpm for 2-3d. Take the culture solution of the bacteria and inoculate it into a test tube containing sterile water. Dilute it according to the proportion to obtain a bacterial concentration of 10 -1 ~10 -8Gradient bacterial suspension; then, the plate coating method was used to take 100 μL from each gradient bacterial suspension and spread it on a nitrogen-rich and phosphorus-rich solid culture medium, and cultured at 30°C for 3 days. The blue colonies were picked for streaking separation, and the separation and purification were repeated for more than 3 times until the colony characteristics were basically consistent. Then, they were inoculated into 100 mL of rescreened culture medium and placed in a constant temperature shaking incubator at 30°C and 160 r / min for shaking culture for 72 hours. At the same time, an uninoculated control was set up to determine the NO3 in the culture medium. - -N and TP contents, strains with high degradation rates were screened out, namely Y01, Y02, Y03 and W06, as shown in Table 1.

[0039] Table 1 Bacterial strain screening results

[0040]

[0041] The culture medium involved in Example 1 is specifically:

[0042] LB medium includes: tryptone 10 g / L, yeast powder 5 g / L, and sodium chloride 10 g / L.

[0043] Bromothymol blue (BTB) culture medium includes: potassium nitrate (KNO3) 1.01g, sodium succinate (C4H4Na2O4) 8.5g, magnesium sulfate heptahydrate (MgSO4·7H2O) 1.0g, potassium dihydrogen phosphate (KH2PO4) 1.0g, ferrous sulfate heptahydrate (FeSO4·7H2O) 0.59g, calcium chloride (CaCl2) 0.09g, 1% bromothymol blue ethanol solution 1mL, distilled water to 1000mL, agar 20g, and adjusted to pH 7.0-7.3 with 1mol / L NaOH.

[0044] The nitrogen-rich and phosphorus-rich culture medium includes: 5 g sodium acetate (CH3COONa), 0.0659 g potassium dihydrogen phosphate (KH2PO4), 0.607 g sodium nitrate (NaNO3), 0.2 g magnesium sulfate heptahydrate (MgSO4·7H2O), 2 ml trace elements, 0.1 g EDTA-Na, distilled water to 1000 mL, 20 g agar, and natural pH.

[0045] The above-mentioned trace elements include: zinc sulfate (ZnSO4) 4.4 mg, manganese chloride tetrahydrate (MnCl2·4H2O) 10.2 mg, calcium chloride (CaCl2) 11 mg, ferrous sulfate heptahydrate (FeSO4·7H2O) 10 mg, cupric sulfate pentahydrate (CuSO4·5H2O) 3.2 mg, ammonium molybdate tetrahydrate ((NH4)6Mo7O2·4H2O) 2.2 mg, cobalt chloride hexahydrate (CoCl2·6H2O) 3.2 mg, EDTA-Na 144 mg, distilled water 1000 mL, natural pH.

[0046] The rescreening medium includes: sodium citrate (C6H5Na3O7) 3.583g, potassium dihydrogen phosphate trihydrate (K2HPO4·3H2O) 0.075g, sodium nitrate (NaNO3) 0.607g, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.05g, ferrous sulfate heptahydrate (FeSO4·7H2O) 0.01g, sodium chloride (NaCl) 0.12g, trace elements 2mL, EDTA-Na 0.1g, distilled water to 1000mL, and adjusted to pH 8 with 1mol / L NaOH;

[0047] The above-mentioned trace elements include: zinc sulfate (ZnSO4) 4.4 mg, manganese chloride tetrahydrate (MnCl2·4H2O) 10.2 mg, calcium chloride (CaCl2) 11 mg, ferrous sulfate heptahydrate (FeSO4·7H2O) 10 mg, cupric sulfate pentahydrate (CuSO4·5H2O) 3.2 mg, ammonium molybdate tetrahydrate ((NH4)6Mo7O2·4H2O) 2.2 mg, cobalt chloride hexahydrate (CoCl2·6H2O) 3.2 mg, EDTA-Na 144 mg, distilled water 1000 mL, natural pH.

[0048] Example 2: Construction of efficient nitrogen and phosphorus removal bacterial community

[0049] The bacterial suspensions of two strains with different functions were cultured to the logarithmic growth phase, and then compounded in pairs. They were inoculated into the mixed culture medium at a ratio of 1:1 with an inoculum of 2%. The culture was carried out in a constant temperature shaking incubator at 30°C and 160 rpmin. Samples were taken regularly, and the NO3--N, TP and OD600 values ​​in the supernatant were determined and the removal rate was calculated. The results are shown in Figure 1 .

[0050] The mixed culture medium includes: sodium succinate (C4H4Na2O4) 8g, sodium nitrate (NaNO3) 1.21g, potassium dihydrogen phosphate (KH2PO4) 0.1g, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.2g, sodium chloride (NaCl) 0.12g, trace elements 1ml, EDTA-Na 0.1g, distilled water to 1000mL, natural pH.

[0051] Conclusion: It can be seen that the combinations with better nitrogen and phosphorus degradation effects are X03+W06, X04+W06, and XL+W06. Among them, the degradation rate of X03+W06 is significantly better than that of other combinations. Figure 2The degradation rate of the pure mixed culture of X03 and W06 strains shows that the bacterial community is in the logarithmic growth period from 0 to 24 hours, and the degradation rates of nitrate nitrogen and total phosphorus are 95.54% and 89.83% respectively, and no accumulation of nitrite nitrogen was found in the end, indicating that the bacterial community has a good denitrification and phosphorus removal effect. Therefore, X03 and W06 were selected as the combination of efficient denitrification and phosphorus removal strains.

[0052] Example 3: Identification of strain X03 and strain W06

[0053] (1) Identification of strain X03

[0054] The colonies of strain X03 are light yellow, round, convex in the middle, with neat, smooth, moist edges, and easy to pick. Figure 3 shown.

[0055] The 16S rDNA sequence of strain X03 was uploaded to the Ezbiocloud.net website, compared with the standard strains on the website, and the bacterial developmental evolution tree was constructed using the Neighbor-Joining method using MEGA-X software, and evaluated using the Bootstrap method (repeated 1000 times). The constructed phylogenetic tree determined that the strain X03 belonged to the genus Stenotrophomonas, named Stenotrophomonas maltophilia X03, and was deposited in the China Center for Type Culture Collection, with a preservation date of September 30, 2024, and a preservation number of CCTCC No: M 20242132. The address is: Wuhan University, Wuhan, China, Postal Code 430072.

[0056] (2) Identification of strain W06

[0057] The colonies of strain W06 are light yellow, smooth, translucent, round and convex, and the surface of the colonies is wet and not conducive to picking. Figure 4 shown.

[0058] The 16S rDNA sequence of strain W06 was uploaded to the Ezbiocloud.net website, compared with the standard strains on the website, and the bacterial developmental evolution tree was constructed using the Neighbor-Joining method using MEGA-X software, and evaluated using the Bootstrap method (repeated 1000 times). The constructed phylogenetic tree determined that the strain W06 belonged to the genus Halomonas and was named Halomonas sp. W06, which was deposited in the China Center for Type Culture Collection on September 30, 2024, with a deposit number of CCTCC No: M 20242131. The address is: Wuhan University, Wuhan, China, Postal Code 430072.

[0059] Example 4: Effects of different environmental factors on the efficiency of bacterial flora in nitrogen and phosphorus removal

[0060] Basic culture medium: sodium nitrate (NaNO3) 1.21g, magnesium sulfate heptahydrate (MgSO4·7H2O) 0.2g, sodium chloride (NaCl) 0.12g, trace elements 1ml, EDTA-Na 0.1g, distilled water to 1000mL, natural pH. The seed liquid is the bacterial suspension after culturing in LB medium at 30℃, 160rpm for 12h.

[0061] Trace elements include: zinc sulfate 4.4 mg, manganese chloride tetrahydrate 10.2 mg, calcium chloride 11 mg, ferrous sulfate heptahydrate 10 mg, copper sulfate pentahydrate 3.2 mg, ammonium molybdate tetrahydrate 2.2 mg, cobalt chloride hexahydrate 3.2 mg, EDTA-Na 144 mg, distilled water 1000 mL, natural pH.

[0062] (1) Effects of different carbon sources on the efficiency of nitrogen and phosphorus removal by bacterial communities

[0063] The fixed carbon source content was 2.4 g / L (in terms of C). The carbon source types were changed to glucose, sucrose, sodium succinate, sodium citrate, and sodium acetate as carbon sources. 2% seed solution was inoculated, and 0.1 g / L of potassium dihydrogen phosphate (KH2PO4) was added. The culture was carried out at 30°C and 160 rpm for 48 hours, and the nitrogen and phosphorus removal efficiency was determined. The results are as follows Figure 5 It shows that when sodium succinate is used as the carbon source, the degradation rates of nitrate nitrogen and total phosphorus are 97.85% and 90.33% respectively, which are much higher than the nitrogen and phosphorus degradation rates when glucose, sodium citrate and sodium acetate with the same content (in terms of C) are used as carbon sources.

[0064] (2) Effects of different C / N conditions on the efficiency of bacterial community in nitrogen and phosphorus removal

[0065] Based on the above basic culture medium, 0.1g / L of potassium dihydrogen phosphate was added, and the designed amount of sodium succinate added was 2.7g / L, 4.1g / L, 5.4g / L, 6.75g / L, 8.1g / L, 9.45g / L, and 10.8g / L, respectively, so that the C / N ratio was 4, 6, 8, 10, 12, 14, and 16, respectively. Then, 2% seed liquid was inoculated and cultured at 30°C and 160rpm for 36h, and then the nitrogen and phosphorus removal efficiency was determined. The results are as follows Figure 6 As shown, when the C / N mass ratio is 14, the nitrogen and phosphorus removal rates are the highest, reaching 96.21% and 89.67% respectively. In addition, when the C / N ratio is 8-12, there is also a good nitrogen and phosphorus removal capacity, the nitrate nitrogen removal rate is more than 90%, and the total phosphorus removal rate is more than 60%.

[0066] (3) Effects of different initial phosphorus contents on the efficiency of bacterial community in nitrogen and phosphorus removal

[0067] Based on the above carbon-free basal medium, when 0.1 g / L potassium dihydrogen phosphate and 9.45 g / L sodium succinate were added, the C / N ratio was 14, and the designed amounts of potassium dihydrogen phosphate added were 0.022 g / L, 0.044 g / L, 0.0659 g / L, 0.088 g / L, and 0.11 g / L, respectively, so that the phosphorus contents were 5, 10, 15, 20, and 25 mg / L, respectively. After inoculation with 2% seed solution, the medium was cultured at 30°C and 160 rpm for 36 h, and its nitrogen and phosphorus removal abilities were determined. Figure 7 It can be seen that the phosphorus content has little effect on the denitrification effect of the bacterial community, all reaching more than 90%, but when the phosphorus content is 20 mg, the phosphorus removal effect is the best, which is 82.89%.

[0068] (4) Effects of different pH values ​​on the efficiency of bacterial denitrification and phosphorus removal

[0069] When 9.45 g / L sodium succinate and 0.088 g / L potassium dihydrogen phosphate were added to the above basic culture medium, the C / N ratio was 14, the pH values ​​were adjusted to 5.0, 6.0, 7.0, 8.0, and 9.0, respectively, and 2% seed solution was inoculated. The culture was carried out at 30°C and 160 rpm for 36 h, and the nitrogen and phosphorus removal capacity was determined. Figure 8 When the pH of the culture medium is greater than 7, the denitrification and phosphorus removal capabilities of the bacterial community reach a higher level, indicating that the bacterial community is more adapted to the alkaline environment.

[0070] (5) Effects of different temperatures on the efficiency of bacterial flora in nitrogen and phosphorus removal

[0071] When 9.45 g / L sodium succinate and 0.088 g / L potassium dihydrogen phosphate were added to the above basic culture medium, the C / N ratio was 14, the pH value was adjusted to 8, 2% seed solution was inoculated, and the culture temperature was changed to 20°C, 25°C, 30°C, 35°C, and 40°C at 160 rpm. After 36 hours, the residual nitrogen and phosphorus removal rates were detected as follows: Fig. 9 As shown, when the temperature is 25℃, the denitrification and phosphorus removal efficiency is the highest, reaching 95.47% and 92.92% respectively.

[0072] (6) Effects of different rotation speeds on the efficiency of bacterial flora in nitrogen and phosphorus removal

[0073] When the above basic culture medium was added with 9.45 g / L sodium succinate and 0.088 g / L potassium dihydrogen phosphate, the C / N ratio was 14, the pH value was adjusted to 8, 2% seed solution was inoculated, and the shaking speed was changed to 80, 120, 160, 200, and 240 rpm at 25°C. After 30 hours, the residual nitrogen and phosphorus removal rate was detected. Fig.10As shown, when the rotation speed is 200rpm, the denitrification and phosphorus removal efficiency is the highest, reaching 96.67% and 93.18% respectively.

[0074] In summary, the high-efficiency denitrification and phosphorus removal bacteria have the strongest denitrification and phosphorus removal efficiency, reaching 96.67% and 93.18% when sodium succinate is used as the optimal carbon source and the C / N ratio is 14, the pH is 8, the culture temperature is adjusted to 25°C, and the culture is aerobic for 30 hours at 200rpm.

[0075] Example 5: Application of high-efficiency nitrogen and phosphorus removal bacteria in sewage

[0076] The sewage samples were taken from a sewage treatment plant in Hangzhou. Take 1L of the above sewage sample, package it according to 100mL of sewage per bottle, inoculate 2% seed liquid (seed liquid is the bacterial suspension after culturing LB medium at 30℃, 160rpm for 12h), add 30.37g of sodium succinate (calculated by C / N=14); culture in a shaking table at 35℃, 220rpm, and regularly sample and determine the concentrations of total nitrogen, ammonia nitrogen, nitrate nitrogen and total phosphorus.

[0077] Table 2 Sewage water quality test data before and after the test

[0078]

[0079] The results show that the present invention can effectively reduce the TN and NH4 + The removal efficiencies of -N, NO3--N and TP were 48%, 98.16%, 48.51% and 92.94%, respectively.

Claims

1. A highly efficient nitrogen and phosphorus removal bacterial community, characterized in that: The bacterial community includes Stenotrophomonas maltophilia X03 and / or Halomonas sp. W06, wherein the deposit number of Stenotrophomonas maltophilia X03 is CCTCCNO: M20242132, and the deposit number of Halomonas sp. W06 is CCTCCNO: M20242131.

2. Use of a high-efficiency denitrification and dephosphorization bacterial community as described in claim 1 in the denitrification and dephosphorization treatment of nitrogen-phosphorus-containing wastewater.

3. The use according to claim 2, characterized in that The application is to inoculate the bacterial community into sewage containing nitrogen and phosphorus, add a carbon source to a C / N ratio of 4 to 16, and culture at a pH of 5 to 9, 20°C to 40°C, 80 to 240 rpm, and an initial phosphorus content of 5 to 25 mg to remove nitrogen and phosphorus from the wastewater.

4. The use according to claim 3, characterized in that The carbon source is sodium succinate, and the carbon source is added until the C / N ratio is 14. The culture is carried out at pH 8, 35° C., 220 rpm, and 20 mg phosphorus content.

5. The method for constructing a highly efficient nitrogen removal and phosphorus removal bacterial community according to claim 1, characterized in that: The following steps are involved: 1) Enrichment and acclimatization: Take the bacterial sample sludge and add it to a conical flask containing 100 mL of sterile water. After shaking on a shaker for 3 hours, use a pipette to draw 2 mL of liquid and inoculate it into the sterilized LB medium. Culture it at 30°C and 180 rpm for 2 to 3 days. 2) Isolation and initial screening: Take the bacterial solution and -1 Serial dilutions up to 10 -6 , take 0.1 mL and spread it on bromothymol blue medium and nitrogen-rich and phosphorus-rich medium for culture, pick the colonies with blue light rings and streak the strains several times until a single colony is obtained; 3) Rescreening: The single bacteria obtained in step 2) were further tested for nitrogen and phosphorus removal. They were inoculated into the corresponding rescreening culture medium of the pure bacteria, and a non-inoculated control was set up to measure the NO3 in the culture medium. - -N and TP contents, and determined that the strains capable of efficient nitrogen and phosphorus removal were Stenotrophomonas maltophilia X03 and Halomonas W06.

6. The method for constructing a highly efficient nitrogen removal and phosphorus removal bacterial community according to claim 5, characterized in that: The initial screening condition in step 2) is: culturing in a biochemical incubator at 30° C. for 72 hours.

7. The method for constructing a highly efficient nitrogen removal and phosphorus removal bacterial community according to claim 5, characterized in that: The bromothymol blue culture medium in step 2) includes: 1.01g potassium nitrate, 8.5g sodium succinate, 1.0g magnesium sulfate heptahydrate, 1.0g potassium dihydrogen phosphate, 0.59g ferrous sulfate heptahydrate, 0.09g calcium chloride, 1mL 1% bromothymol blue ethanol solution, distilled water to 1000mL, 20g agar, and adjusted to pH 7.0-7.3 with 1mol / L NaOH.

8. The method for constructing a highly efficient nitrogen removal and phosphorus removal bacterial community according to claim 5, characterized in that: The nitrogen-rich and phosphorus-rich culture medium in step 2) includes 5 g of sodium acetate, 0.0659 g of potassium dihydrogen phosphate, 0.607 g of sodium nitrate, 0.2 g of magnesium sulfate heptahydrate, 2 ml of trace elements, 0.1 g of EDTA-Na, distilled water to 1000 mL, 20 g of agar, and natural pH; The above trace elements include: zinc sulfate 4.4 mg, manganese chloride tetrahydrate 10.2 mg, calcium chloride 11 mg, ferrous sulfate heptahydrate 10 mg, copper sulfate pentahydrate 3.2 mg, ammonium molybdate tetrahydrate 2.2 mg, cobalt chloride hexahydrate 3.2 mg, EDTA-Na 144 mg, distilled water 1000 mL, natural pH.

9. The method for constructing a highly efficient nitrogen removal and phosphorus removal bacterial community according to claim 5, characterized in that: The rescreening conditions in step 3) are: shaking culture in a constant temperature shaking incubator at 30° C. and 160 r / min for 72 hours.

10. The method for constructing a highly efficient nitrogen removal and phosphorus removal bacterial community according to claim 5, characterized in that: The rescreening medium in step 3) includes 3.583 g of sodium citrate, 0.075 g of dipotassium hydrogen phosphate trihydrate, 0.607 g of sodium nitrate, 0.05 g of magnesium sulfate heptahydrate, 0.01 g of ferrous sulfate heptahydrate, 0.12 g of sodium chloride, 2 mL of trace elements, 0.1 g of EDTA-Na, and distilled water to 1000 mL, and adjusted to pH 8 with 1 mol / L NaOH; The above trace elements include: zinc sulfate 4.4 mg, manganese chloride tetrahydrate 10.2 mg, calcium chloride 11 mg, ferrous sulfate heptahydrate 10 mg, copper sulfate pentahydrate 3.2 mg, ammonium molybdate tetrahydrate 2.2 mg, cobalt chloride hexahydrate 3.2 mg, EDTA-Na 144 mg, distilled water 1000 mL, natural pH.

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