Heterotrophic nitrification and aerobic denitrification arthrobacter MD1 and application thereof

Abstract

The invention relates to the technical field of microbial sewage treatment, in particular to heterotrophic nitrification and aerobic denitrification arthrobacter MD1 and application thereof. The bacterial strain is Arthrobacter sp. MD1 which is preserved in the China General Microbiological Culture Collection Center (CGMCC) on September 2, 2024, and the preservation number is CGMCC No.31808. The invention further discloses a preparation method of the bacterial strain. The arthrobacter MD1 provided by the invention has the functions of heterotrophic nitrification and aerobic denitrification at the same time; various organic carbon sources such as sodium citrate, glucose, sodium acetate and sodium succinate can be utilized, the arthrobacter MD1 can utilize NH4 < + >-N, NO3 <->-N and NO2 <->-N as sole nitrogen sources to perform aerobic nitrification and denitrification nitrogen and phosphorus removal under the aerobic condition, the requirements of nitrification and denitrification in different oxygen environments are met, and the arthrobacter MD1 has a good application prospect.

Description

Technical Field

[0001] The present invention relates to the technical field of microbial sewage treatment, in particular to a heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 and applications thereof. Background Art

[0002] Traditional biological denitrification is the mainstream denitrification process in sewage treatment plants at home and abroad. In view of the difference in the demand for dissolved oxygen and organic matter between nitrification and denitrification, sewage treatment plants need to organically combine the two to achieve complete biological denitrification. Specifically, denitrification is carried out through autotrophic nitrification (NH4 + -N to NO 3- -N / NO 2- -N) and heterotrophic denitrifying microorganisms convert NO3 - -N / NO2 - -N is reduced to N2. Meanwhile, phosphorus removal relies on phosphorus-accumulating organisms (PAOs) and denitrifying phosphorus-accumulating organisms (DPAOs), requiring alternating anaerobic and aerobic / anoxic conditions. Simultaneous removal of nitrogen and phosphorus limits the performance of wastewater treatment plants due to the different physiological and biochemical requirements of the functional bacteria.

[0003] Recently, many new strains have been discovered that can achieve heterotrophic nitrification and aerobic denitrification and simultaneous denitrification and phosphorus removal under aerobic conditions, and can overcome the requirements of different oxygen environments. However, there are currently only a few reports on heterotrophic nitrification and aerobic denitrification and denitrification and phosphorus removal strains. Summary of the Invention

[0004] In view of the above-mentioned defects in the prior art, the technical problem to be solved by the present invention is to provide a heterotrophic nitrification aerobic denitrification denitrification and phosphorus removal strain. 2 A new strain of Arthrobacter was isolated from activated sludge under aerobic conditions in the O treatment process. It has a wide adaptability to factors such as carbon source type, carbon-nitrogen ratio (C / N), phosphorus-nitrogen ratio (P / N), pH and dissolved oxygen (DO). Its nitrogen and phosphorus removal performance was evaluated, and its addition to actual sewage effectively improved its nitrogen and phosphorus removal efficiency.

[0005] The present invention aims to provide a heterotrophic nitrifying and aerobic denitrifying strain Arthrobacter MD1 and its applications. The Arthrobacter MD1 strain provided by the present invention possesses both heterotrophic nitrification and aerobic denitrification functions; it can utilize a variety of organic carbon sources, including sodium citrate, glucose, sodium acetate, and sodium succinate. The strain is suitable for treating nitrogenous wastewater with a high carbon / nitrogen (C / N) ratio of 15 to 20 and exhibits high pH tolerance. Within a pH range of 6 to 9, it can achieve nitrate and phosphate removal rates exceeding 96% and 80%, respectively.

[0006] In order to achieve the above objectives, the present invention provides the following technical solutions:

[0007] The present invention provides a heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1, wherein the strain is Arthrobacter (Arthrobacter sp.) MD1, which was deposited in the General Microbiology Center of the China Culture Collection Administration on September 2, 2024, with a deposit number of CGMCC No. 31808.

[0008] The heterotrophic nitrifying aerobic denitrifying bacterium MD1 was obtained by separation and screening of water samples collected from sewage treatment plants and can efficiently degrade NH4 + -N, NO3 - -N, NO2 - -N and PO4 3- -P.

[0009] The colony morphology of the heterotrophic nitrifying and aerobic denitrifying Arthrobacter MD1 is characterized by being a Gram-positive bacterium with round, light yellow, slightly raised colonies, a moist and smooth surface, and regular edges.

[0010] Preferably, the nucleotide sequence of 16S rDNA of the heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 is shown as SEQ ID NO.1.

[0011] The present invention also provides the use of the heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 in the treatment of nitrogen and phosphorus-containing wastewater.

[0012] Preferably, the heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 is applied to the treatment of nitrogen and phosphorus-containing wastewater, comprising the following steps:

[0013] The seed liquid of heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 is inoculated into sewage, and cultured for 20 to 28 hours at a temperature of 25° C. to 40° C. and a rotation speed of 90 rpm to 180 rpm to complete the treatment of nitrogen and phosphorus-containing sewage.

[0014] Preferably, the preparation method of the heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 seed solution is:

[0015] (1) Heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 was inoculated into LB medium to prepare a bacterial suspension;

[0016] (2) Centrifuge the bacterial suspension at 5000 rpm for 2-7 minutes, discard the supernatant, add sterile water for washing, and centrifuge again. Repeat this process 2-3 times to prepare the seed solution.

[0017] Preferably, the culture conditions in step (1) are 25-35° C., 120-180 rpm, and 12-36 h.

[0018] Preferably, the inoculation amount of the seed liquid is 0.2-0.6 g / 150 mL of sewage.

[0019] Preferably, the nitrogen contained in the sewage is at least one of nitrite nitrogen, ammonia nitrogen and nitrate nitrogen; and the carbon source contained in the sewage is at least one of sodium citrate, glucose, sodium acetate and sodium succinate.

[0020] Preferably, the C / N (carbon / nitrogen) ratio of the sewage is 2-25; more preferably, the C / N (carbon / nitrogen) ratio is 15-20.

[0021] Preferably, the pH value of the sewage is 6-10; more preferably, the pH value of the sewage is 7-9.

[0022] Preferably, the P / N (phosphorus / nitrogen) ratio of the sewage is 0.1 to 0.6; more preferably, the P / N ratio of the sewage is 0.2 to 0.4.

[0023] Preferably, the DO (dissolved oxygen) of the sewage is 3.0 to 6.5 mg / L; more preferably, the dissolved oxygen concentration of the sewage is 4.6 to 5.2 mg / L.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] (1) The present invention provides a heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 and its application in denitrification and phosphorus removal in sewage. The heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1, with a deposit number of CGMCC No.31808, can be used in the field of nitrogen and phosphorus-containing sewage treatment. Under aerobic conditions, the strain can utilize NH4 + -N, NO3 - -N and NO2 - -N is used as the only inorganic nitrogen source for heterotrophic nitrification, aerobic denitrification and denitrification and phosphorus removal. When ammonia nitrogen is used as the nitrogen source, NH4 + -N、PO4 3- -P removal rates can reach up to 93.77% and 89.67% respectively, and the maximum OD 600 is 1.132; when nitrate nitrogen is used as nitrogen source, NO3 - -N、PO4 3- -P removal rates can reach up to 100% and 93.88% respectively, and the maximum OD 600 is 0.899; when nitrite nitrogen is used as nitrogen source, NO2 - -N、PO4 3- The maximum P removal rates can reach 100% and 86.05% respectively, overcoming the requirements of different oxygen environments for nitrification and denitrification, and having good application prospects.

[0026] (2) The Arthrobacter strain MD1 provided by the present invention has both heterotrophic nitrification and aerobic denitrification functions; it can utilize a variety of organic carbon sources such as sodium citrate, glucose, sodium acetate and sodium succinate. The strain is suitable for the treatment of nitrogen-containing wastewater with a high C / N ratio of 15 to 20 and has a high tolerance to pH. Within the pH range of 6 to 9, the removal rates of nitrate nitrogen and phosphate can reach 96% and over 80%, respectively.

[0027] (3) The Arthrobacter strain MD1 provided by the present invention has NO3 when the P / N ratio is 0.1, 0.2, 0.4, and 0.6, that is, when the phosphorus concentration range is 5 mg / L to 30 mg / L. - -N removal rates were 99.89%, 100%, 100%, 99.77% respectively, PO4 3- The P removal rates were 100%, 99.52%, 97.55% and 87.41% respectively. In previous studies, when the P / N ratio was 0.5, the nitrogen and phosphorus removal efficiency decreased significantly. The strain isolated in the present invention has significantly improved its phosphorus tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a phylogenetic tree diagram of Arthrobacter MD1 in Example 2;

[0029] Figure 2 This is a morphological characterization diagram of Arthrobacter MD1 in Example 2;

[0030] Figure 3 is a scanning electron microscope (SEM) image of Arthrobacter MD1 in Example 2;

[0031] Figure 4 The growth of Arthrobacter MD1 under different carbon source conditions and the comparison of nitrogen and phosphorus removal effects in Example 3; Figure A is OD 600 Value, Figure B is NO3 - -N concentration, Figure C is PO4 3- -P concentration;

[0032] Figure 5 The growth of Arthrobacter MD1 under different C / N conditions and the comparison of nitrogen and phosphorus removal effects in Example 3 are shown in Figure A. 600 Value, Figure B is NO3 - -N concentration, Figure C is PO4 3- -P concentration;

[0033] Figure 6 The growth of Arthrobacter MD1 under different pH conditions and the comparison of nitrogen and phosphorus removal effects in Example 3 are shown in Figure A. 600 Value, Figure B is NO3 - -N concentration, Figure C is PO43- -P concentration;

[0034] Figure 7 The growth of Arthrobacter MD1 under different P / N conditions and the comparison of nitrogen and phosphorus removal effects in Example 3 are shown in Figure A. 600 Value, Figure B is NO3 - -N concentration, Figure C is PO4 3- -P concentration;

[0035] Figure 8 The growth of Arthrobacter MD1 under different DO conditions and the comparison of nitrogen and phosphorus removal effects in Example 3 are shown in Figure A. 600 Value, Figure B is NO3 - -N concentration, Figure C is PO4 3- -P concentration;

[0036] Figure 9 Figure 3 shows the growth of Arthrobacter MD1 under different nitrogen source conditions and the comparison of nitrogen and phosphorus removal effects; Figure A uses ammonia nitrogen as the nitrogen source, Figure B uses nitrate nitrogen as the nitrogen source, and Figure C uses nitrite nitrogen as the nitrogen source; Figure D uses a mixed nitrogen source (ammonia nitrogen and nitrate nitrogen) as the nitrogen source;

[0037] Figure 10 The comparison of nitrogen and phosphorus removal efficiency of Arthrobacter MD1 in oxidation pond sewage in Example 4; Figure A is NH4 + -N concentration, Figure B is NO3 - -N concentration, Figure C is NO2 - -N concentration, Figure D is PO4 3- -Total amount of P;

[0038] Figure 11 The comparison of nitrogen and phosphorus removal efficiency of Arthrobacter MD1 in the sewage of the effluent pool in Example 4; Figure A is NH4 + -N concentration, Figure B is NO3 - -N concentration, Figure C is NO2 - -N concentration, Figure D is PO4 3- -The total amount of P. DETAILED DESCRIPTION

[0039] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0040] In the present invention, NH4 + -N, NO3 - -N, NO2 - -N The determination and analysis methods of the three nitrogen elements are based on the national standard, among which:

[0041] NH4 + -N determination and analysis were in accordance with “Water quality—Determination of ammonia nitrogen—Nessler’s reagent spectrophotometric method” (GB HJ535-2009);

[0042] NO3 - -N determination and analysis were in accordance with "Water quality - Determination of nitrate nitrogen - Ultraviolet spectrophotometry" (GB HJ / T346-2007);

[0043] NO 2- -N determination and analysis were in accordance with "Water quality - Determination of nitrite nitrogen - Spectrophotometric method" (GB 7493-87);

[0044] PO4 3- -P using ammonium molybdate spectrophotometry.

[0045] Biological deposit information

[0046] The Arthrobacter MD1 provided by the present invention is classified and named Arthrobacter (Arthrobacter sp.), the strain number is MD1, the preservation number is CGMCC No. 31808, the preservation time is September 2, 2024, and the preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0047] Example 1

[0048] This example provides a strain of Arthrobacter MD1 (CGMCC No. 31808) that has both heterotrophic nitrification and aerobic denitrification and phosphorus removal functions. The breeding process includes the following steps:

[0049] 1. Sample collection

[0050] The experimental samples were collected from a municipal sewage treatment plant in Shijiazhuang in winter. 2 Activated sludge from different stages of the O treatment process is used to screen for aerobic denitrifying microorganisms. Sampling is done at a water temperature of 17°C in each stage, using sterilized, dried glassware at 121°C. Each stage receives a 1L sample. After collection, the sample is immediately placed in a portable low-temperature insulated container and transported to the laboratory for processing.

[0051] 2. The types and compositions of each culture medium are as follows:

[0052] Enrichment medium (EM): composed of 0.5g / L (NH4)2SO4, 0.36g / L KNO3, 4.0g / L sodium citrate, and 2mL / L trace element solution; each liter of trace element solution includes 6.5g / LK2HPO4·3H2O, 2.5g / L MgSO4·7H2O, 2.5g / LNaCl, 0.05g / L FeSO4·7H2O, and 0.04g / L MnSO4·H2O, and the rest is made up with water.

[0053] Heterotrophic nitrification medium (HNM): 4.0 g / L sodium citrate, 0.285 g / L (NH4)2SO4, 0.0877 g / L KH2PO4, 0.20 g / L MgSO4·7H2O, 2 mL / L trace element solution; each liter of trace element solution includes: 10.0 g EDTA, 0.2 g ZnSO4, 1.2 g MnCl2·4H2O, 1.0 g FeSO4·7H2O, 0.5 g CuSO4·5H2O, 0.3 g CoCl2·6H2O, 0.2 g Na2MoO4·2H2O, 0.1 g CaCl2, and the rest is made up with water.

[0054] Aerobic denitrification medium (DM-1): 4.0 g / L sodium citrate, 0.36 g / LKNO3, 0.0877 g / LKH2PO4, 0.20 g / LMgSO4·7H2O, 2 mL / L trace element solution; each liter of trace element solution includes: 10.0 g EDTA, 0.2 g ZnSO4, 1.2 g MnCl2·4H2O, 1.0 g FeSO4·7H2O, 0.5 g CuSO4·5H2O, 0.3 g CoCl2·6H2O, 0.2 g Na2MoO4·2H2O, 0.1 g CaCl2, and the rest is made up with water.

[0055] Aerobic denitrification medium (DM-2): 4.0 g / L sodium citrate, 0.493 g / LNaNO2, 0.0877 g / LKH2PO4, 0.20 g / LMgSO4·7H2O, 2 mL / L trace element solution; each liter of trace element solution includes: 10.0 g EDTA, 0.2 gZnSO4, 1.2 g MnCl2·4H2O, 1.0 g FeSO4·7H2O, 0.5 g CuSO4·5H2O, 0.3 g CoCl2·6H2O, 0.2 gNa2MoO4·2H2O, 0.1 gCaCl2, and the rest is made up with water.

[0056] Heterotrophic nitrification-aerobic denitrification medium (HNADM): 4.0 g / L sodium citrate, 0.118 g / L (NH4)2SO4, 0.18 g / L KNO3, 0.0877 KH2PO4, 0.20 MgSO4·7H2O, 2 mL / L trace element solution; each liter of trace element solution includes: 10.0 g EDTA, 0.2 g ZnSO4, 1.2 g MnCl2·4H2O, 1.0 g FeSO4·7H2O, 0.5 g CuSO4·5H2O, 0.3 g CoCl2·6H2O, 0.2 g Na2MoO4·2H2O, 0.1 g CaCl2, and the rest is made up with water.

[0057] Agar plates were prepared by adding agar powder to the culture medium at a volume ratio of 1.5%. All culture media were adjusted to pH 7.0 and autoclaved at 121°C for 30 minutes before use.

[0058] 3. The method for breeding Arthrobacter MD1 having both heterotrophic nitrification and aerobic denitrification and phosphorus removal functions of the present invention comprises the enrichment, isolation and screening of the original strain of Arthrobacter MD1, and specifically comprises the following steps:

[0059] (1) Enrichment culture of strains:

[0060] Add the collected activated sludge to sterile enrichment medium at a 10% volume ratio. The Erlenmeyer flasks were then placed in a constant-temperature incubator at 30°C and 150 rpm for 30 days. During the enrichment phase, 25 mL of the enrichment solution was discarded and replaced with 25 mL of fresh sterile EM medium every two days. Three replicates were set up for each activated sludge sample.

[0061] (2) Primary Screening: After strain enrichment, 25 mL of a mixed solution of DM medium and microorganisms was added to 250 mL of fresh aerobic denitrification medium (DM-1). The conical flask was placed in a constant temperature incubator at 30°C and 150 rpm for 14 days. Fresh DM medium was replaced every 24 hours at a 10% volume ratio. Three replicates were set up for each sample.

[0062] (3) Isolation, purification and rescreening of strains: After the initial screening of the mixed bacterial solution, the plate dilution method and plate streak method were used to separate and purify the strains. -2 ~10 -6Evenly spread 0.1 mL of a mixed strain suspension at varying concentrations onto a solid aerobic denitrification medium. Cultivate in a 30°C incubator for 2–3 days until distinct, non-adherent colonies develop. Using a platinum inoculation loop, select colonies with distinct morphological characteristics (e.g., shape, size, color, and smoothness) in a sterile operating environment. Streak cultures on LB-activated medium are then cultured in a 30°C incubator to purify the individual strains. Purified strains are stored in a 4°C refrigerator using the culture slant method.

[0063] Example 2

[0064] The steps of identifying the biological and other related characteristics of the Arthrobacter MD1 strain capable of performing heterotrophic nitrification and aerobic denitrification functions screened out in Example 1 include:

[0065] (1) 16S rDNA identification: The strains screened from the sewage treatment plant were isolated and purified. The effective length of the 16S rDNA sequence of strain MD1 was approximately 1411 bp, and the nucleotide sequence was shown in GenBank: PQ044809.1 (SEQ ID NO. 1). The sequence was entered into Genbank and BLAST analysis showed that the 16S rDNA sequence of this strain was highly similar to that of Arthrobacter sp. MN14 (JN082743.1), with a similarity of 99.93%. The phylogenetic tree of the strain is shown in Figure 1. Figure 1 shown.

[0066] (2) The strain was characterized by morphology and cultured on LB plates at 30°C for 24 h. Figure 2 As shown, the strain is a Gram-positive bacterium, and the colonies are round, light yellow, slightly convex, with a moist and smooth surface and regular edges, as shown in FIG. Figure 3 The scanning electron microscope (SEM) image shows that the nanostructured particles are in the shape of thin rods.

[0067] (3) The 16s rDNA sequencing and physiological and biochemical experiments above confirmed that the MD1 strain was Arthrobacter sp., which was deposited in the General Microbiology Center of the China Culture Collection Administration of Microorganisms (CGMCC) on September 2, 2024, with the deposit number CGMCC No. 31808 and the classification name Arthrobacter sp. The deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0068] >PQ044809.1Arthrobacter sp.strain MD116S ribosomal RNAgene,partialsequence(SEQ ID NO.1):

[0069]

[0070] Example 3

[0071] The aforementioned Arthrobacter MD1, which has both heterotrophic nitrification and aerobic denitrification functions, was applied to the field of sewage treatment to test and verify its optimal growth and nitrogen and phosphorus removal conditions, including the following steps:

[0072] Effects of different organic carbon sources on the growth and denitrification performance of Arthrobacter MD1

[0073] Sodium citrate, glucose, sodium acetate and sodium succinate were selected as four carbon sources, and the C / N (carbon / nitrogen) was fixed at 15, 30℃, 150r / min, and pH = 7. Based on DM-1 medium, sodium citrate, glucose, sodium acetate and sodium succinate were used as four organic carbon sources. Candidate strains were inoculated into LB medium respectively, cultured at 30℃, 150r / min for 1 day, and then inoculated into the above denitrification medium with different organic carbon sources at an inoculum of 1%, and the culture fluid was taken at 0h, 12h, 24h, 36h, 48h, 60h, and 72h to measure its OD 600 , 5000r / min, 4℃ low speed centrifugation for 10min, and then the supernatant was collected to determine NO 3- -N、PO4 3- -P content. Three technical replicates and a blank control group were set up. The control group was inoculated with an equal amount of ultrapure water. The effects of four different organic carbon sources—sodium citrate, glucose, sodium acetate, and sodium succinate—on the growth and nitrogen and phosphorus removal performance of strain MD1 were analyzed.

[0074] Figure 4 The nitrogen and phosphorus removal of Arthrobacter MD1 under different carbon source conditions. When sodium citrate was used as the carbon source, NO3 - -N、PO4 3- -P removal rates were 62.03% and 33.89% respectively, and the maximum OD 600 is 0.374; when glucose is used as carbon source, NO 3- -N、PO4 3- -P removal rates were 100% and 94.22% respectively, and the maximum OD 600 is 1.234; when sodium acetate is used as the carbon source, NO3 - -N、PO4 3- -P removal rates were 83.01% and 89.63% respectively, and the maximum OD 600 is 0.616; when sodium succinate is used as carbon source, NO3 - -N、PO4 3- -P removal rates were 100% and 91.13% respectively, and the maximum OD 600 is 1.060.

[0075] Effects of different C / N conditions on the growth and nitrogen and phosphorus removal performance of strain MD1

[0076] Glucose was selected as the carbon source for the denitrification medium, and the medium was fixed at 30°C, 150 r / min, and pH = 7. The medium was based on DM-1. The C / N gradient was set to 2, 5, 10, 15, 20, and 25. The candidate strains were inoculated into LB medium and cultured at 30°C and 150 r / min for 1 day. Then, 1% of the inoculum was inoculated into the denitrification medium with different C / N gradients. The culture fluid was collected at 0h, 36h, and 72h to measure its OD. 600 , 5000r / min, 4℃ low speed centrifugation for 10min, and then the supernatant was taken to determine NO3 - -N、PO4 3- -P content. Three technical replicates and a blank control group were set up. The control group was inoculated with an equal amount of ultrapure water. The effects of six C / N gradients (2, 5, 10, 15, 20, and 25) on the growth and nitrogen and phosphorus removal efficiency of strain MD1 were analyzed.

[0077] Figure 5 The nitrogen and phosphorus removal of Arthrobacter MD1 under different C / N conditions. When the C / N gradient was 2, 5, 10, 15, 20, and 25, the maximum OD 600 They are 0.447, 0.767, 1.093, 1.512, 1.653, 1.681, NO3 - -N removal rates were 33.91%, 40.56%, 87.59%, 100%, 98.79%, 98.49%, respectively, and PO4 3- The -P removal rates were 67.14%, 90.65%, 100%, 100%, 98.79% and 98.53% respectively.

[0078] Effects of different pH conditions on the growth and nitrogen and phosphorus removal performance of strain MD1

[0079] The C / N ratio was fixed at 15, 30°C, 150r / min, glucose was used as the carbon source of the denitrification medium, DM-1 medium was used as the basis, and the pH gradient was set to 6, 7, 8, 9, and 10. The candidate strains were inoculated into LB medium and cultured at 30°C and 150r / min for 1 day. Then, 1% of the inoculum was inoculated into the denitrification medium with different pH gradients. The culture fluid was taken at 0h, 12h, 24h, 36h, 48h, 60h, and 72h to measure its OD 600 , 5000r / min, 4℃ low speed centrifugation for 10min, and then the supernatant was collected to determine NO 3- -N、PO4 3--P content. Three technically replicate experimental groups and a blank control group were set up. The control group was inoculated with an equal amount of ultrapure water. The effects of five pH gradients (6, 7, 8, 9, and 10) on the growth and nitrogen and phosphorus removal efficiency of strain MD1 were analyzed.

[0080] Figure 6 The results show that Arthrobacter MD1 can remove nitrogen and phosphorus under different pH conditions. When the pH gradient is 6, 7, 8, 9, and 10, the maximum OD 600 They are 1.467, 1.506, 1.513, 1.526, 1.482, NO3 - -N removal rates were 96.29%, 100%, 100%, 100%, and 100%, respectively, and PO4 3- The -P removal rates were 87.74%, 90.65%, 90.77%, 91.03% and 82.10% respectively.

[0081] Effects of different P / N (phosphorus / nitrogen) conditions on the growth and nitrogen and phosphorus removal performance of strain MD1

[0082] The C / N ratio was fixed at 15, 30°C, 150r / min, pH=7, DM-1 medium was used as the basis, glucose was used as the carbon source of the denitrification medium, the nitrogen concentration was fixed at 50mg / L, and the P / N gradient was adjusted to 0.1, 0.2, 0.4, and 0.6 by changing the content of KH2PO4. The candidate strains were inoculated into LB medium and cultured at 30°C and 150r / min for 1 day. Then, the strains were inoculated into the denitrification medium with different pH gradients at a 1% inoculum. The culture fluid was taken at 0h, 12h, 24h, 36h, 48h, and 60h to measure the OD. 600 , 5000r / min, 4℃ low speed centrifugation for 10min, and then the supernatant was taken to determine NO3 - -N、PO4 3- -P content. Three technically replicate experimental groups and a blank control group were set up. The control group was inoculated with an equal amount of ultrapure water. The effects of four P / N gradients of 0.1, 0.2, 0.4, and 0.6 on the growth and nitrogen and phosphorus removal efficiency of strain MD1 were analyzed.

[0083] Figure 7 The nitrogen and phosphorus removal of Arthrobacter MD1 under different P / N conditions. When the P / N gradient was 0.1, 0.2, 0.4, and 0.6, the maximum OD 600 They are 1.335, 1.311, 1.261, 1.254, NO3 - -N removal rates were 99.89%, 100%, 100%, 99.77% respectively, PO4 3--P removal rates were 100%, 99.52%, 97.55% and 87.41% respectively.

[0084] Effects of different DO (dissolved oxygen) conditions on the growth and nitrogen and phosphorus removal performance of strain MD1. Fixed C / N at 15, 30℃, pH=7, P / N=0.2, glucose as the carbon source of the denitrification medium, based on DM-1 medium, by adjusting the rotation speed to 90rpm, 120rpm, 150rpm, set the dissolved oxygen (DO) gradient to 3.0mg / L, 4.8mg / L and 6.5mg / L. The candidate strains were inoculated into LB medium respectively, cultured at 30℃, 150r / min for 1 day, and then inoculated into the denitrification medium with different DO gradients with an inoculum of 1%, and the culture fluid was taken at 0h, 12h, 24h, 36h, 48h, and 60h to measure its OD 600 , 5000r / min, 4℃ low speed centrifugation for 10min, and then the supernatant was collected to determine NO 3- -N、PO4 3- -P content. Three technical replicates and a blank control group were set up. The control group was inoculated with an equal amount of ultrapure water. The effects of three DO gradients (3.0 mg / L, 4.8 mg / L, and 6.5 mg / L) on the growth and nitrogen and phosphorus removal of strain MD1 were analyzed.

[0085] Figure 8 The results of nitrogen and phosphorus removal by Arthrobacter MD1 under different DO conditions are shown. When the dissolved oxygen (DO) gradient was 3.0 mg / L, 4.8 mg / L and 6.5 mg / L, the maximum OD 600 They are 1.030, 1.238, 1.238, NO3 - -N removal rates were 91.4%, 99.41%, and 100%, respectively, and PO4 3- -P removal rates were 91.03%, 94.57% and 92.84% respectively.

[0086] Effects of different nitrogen source conditions on the growth and nitrogen and phosphorus removal performance of strain MD1

[0087] The C / N ratio was fixed at 15, 30℃, 150r / min, pH=7, P / N=0.2, glucose was used as the carbon source of the culture medium, and three single nitrogen sources (NH4 + -N, NO 3- -N, NO 2- -N) and a mixed nitrogen source (NH4 + -N and NO3 --N) as a substrate for batch testing. Candidate strains were inoculated into LB medium and cultured at 30°C, 150 rpm for 1 day. Then, at a 1% inoculum, they were inoculated into the four nitrogen source media mentioned above, namely HNM, DM-1, DM-2, and HNADM medium. The culture fluid was collected at 0 h, 12 h, 24 h, 36 h, and 48 h to measure the OD value. 600 , 5000r / min, 4℃ low speed centrifugation for 10min, and then the supernatant was taken to determine NH4 + -N, NO3 - -N, NO2 - -N, TN and PO4 3- -P content. Three technical replicates and a blank control group were set up. The control group was inoculated with an equal amount of ultrapure water. The effects of nitrogen source type on the growth of strain MD1 and its nitrogen and phosphorus removal efficiency were analyzed.

[0088] Figure 9 The nitrogen and phosphorus removal of Arthrobacter MD1 under different nitrogen source conditions. When ammonia nitrogen was used as the nitrogen source, NH4 + -N、PO4 3- -P removal rates were 93.77% and 89.67% respectively, and the maximum OD 600 is 1.132; when nitrate nitrogen is used as nitrogen source, NO3 - -N、PO4 3- -P removal rates were 100% and 93.88% respectively, and the maximum OD 600 is 0.899; when nitrite nitrogen is used as nitrogen source, NO2 - -N、PO4 3- -P removal rates were 100% and 86.05% respectively, and the maximum OD 600 is 0.952; when mixed nitrogen source (ammonia nitrogen and nitrate nitrogen) is used as nitrogen source, NH4 + -N, NO3 - -N、PO4 3- -P removal rates were 100%, 100%, and 94.88%, respectively, with maximum OD 600 It is 0.974.

[0089] Example 4

[0090] Application of heterotrophic nitrification and aerobic denitrification Arthrobacter MD1 in actual sewage treatment

[0091] In order to explore the application effect of heterotrophic nitrification-aerobic denitrification and denitrification Arthrobacter MD1 in actual sewage, MD1 was inoculated into LB medium and cultured at 30°C and 150rpm for 48h to obtain a bacterial suspension. The suspension was centrifuged at 5000rpm and 4°C for 5min to collect the bacteria. The bacteria were washed 3 times with sterile ddH2O to purify the bacteria. The resuspended seed culture was then inoculated into a conical flask containing 150mL of actual sewage at an inoculum size of 0.2g, 0.4g, and 0.6g (wet weight). The actual sewage of the experimental group was pretreated by adding sodium succinate to adjust the C / N of the actual sewage to 15, adding HCl / NaOH to adjust the pH to 7.0-7.2, and cultured at 30°C and 150rpm for 24h. Determine the OD in the sample 600 NH4 + -N, NO3 - -N, NO2 - -N and PO4 3- -P content. The experiment set up 3 replicate samples and a blank (untreated, uninoculated) as the control group. This example uses two groups of actual sewage samples, which were taken from the contact oxidation pond and the outlet pond of a water station in Shijiazhuang City. The water quality of the oxidation pond water is: temperature 23.8 ° C, pH 7.31, TN 60.91 mg / L, NH4 + -N38.3mg / L, NO3 - -N 28.4mg / L, NO2 - -N 2.08mg / L, PO4 3- -P 3.8mg / L. The water quality of the outlet pool is: temperature 21.8℃, pH 7.17, TN 61.8mg / L, NH4 + -N12.06mg / L, NO3 - -N 52.9mg / L, NO2 - -N0.45mg / L, PO4 3- -P 3.8mg / L.

[0092] Figure 10 The nitrogen and phosphorus removal results of oxidation pond wastewater treated with different inoculation amounts of Arthrobacter MD1 are shown. When the inoculation amount is 0.2, 0.4, and 0.6, NH4 + -N removal rates were 98.51%, 98.07%, 96.02%, and NO3 - -N removal rates were 100%, 100%, and 100%, respectively, and 24hNO2 - -N concentrations were all reduced to below 0.2 mg / L, with removal rates of 95.13%, 94.98%, and 94.50%, respectively. 3--P removal rates were 79.75%, 85.67% and 88.41% respectively. + -N removal rate was 83.97%, NO3 - -N removal rate was 30.14%, NO2 - -N removal rate was 95.72%, PO4 3- -P removal rate was 23.33%.

[0093] Figure 11 The nitrogen and phosphorus removal of the sewage from the effluent treated by Arthrobacter MD1 at different inoculation rates. When the inoculation rate was 0.2, 0.4, and 0.6, NH4 + -N removal rates were 90.03%, 88.42%, 91.07% and NO3 - -N removal rates were 100%, 100%, and 100%, respectively, and 24hNO2 - -N concentrations were all reduced to below 0.2 mg / L, with removal rates of 79.93%, 80.46%, and 80.60%, respectively. 3- -P removal rates were 89.94%, 90.18% and 89.65% respectively. + -N removal rate was 76.31%, NO3 - -N removal rate was 7.56%, NO2 - -N removal rate was 79.11%, PO4 3- -P removal rate was 20.18%.

[0094] By Example 3 ( Figure 4-Figure 9 ) and Example 4 ( Figure 10-11 ) It can be seen that Arthrobacter MD1 can grow using a variety of organic carbon sources such as sodium citrate, glucose, sodium acetate and sodium succinate, and its growth and nitrogen and phosphorus removal effects are best when using glucose. MD1 can grow under C / N of 2-25, pH of 6-10, P / N of 0.1-0.6, and DO (dissolved oxygen) of 3.0-6.5. The optimal denitrification conditions are glucose as the carbon source, C / N of 15, pH of 7-10, DO of 4.8-6.5, P / N of 0.2-0.4, and a mixed nitrogen source; the optimal phosphorus removal conditions are glucose as the carbon source, C / N of 10-15, pH of 9, DO of 4.8, P / N of 0.1-0.4, and a mixed nitrogen source. Strain MD1 also has heterotrophic nitrification, aerobic denitrification, denitrification and phosphorus removal capabilities. Under aerobic conditions, strain MD1 can utilize NH4 + -N, NO3 - -N, NO2 --N is the only nitrogen source for aerobic nitrification and denitrification to remove nitrogen and phosphorus, which overcomes the requirements of different oxygen environments for nitrification and denitrification and has good application prospects.

[0095] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1, characterized in that: The strain is Arthrobacter sp. MD1, which was deposited in the General Microbiology Center of China Microbiological Culture Collection Administration on September 2, 2024, with the deposit number CGMCC No.31808.

2. The heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 according to claim 1, characterized in that The nucleotide sequence of 16S rDNA of the Arthrobacter MD1 is shown in SEQ ID NO.

1.

3. Use of the heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 according to claim 1 or 2 in the treatment of nitrogen and phosphorus-containing wastewater.

4. The use according to claim 3, characterized in that: The following steps are involved: The seed liquid of heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 is inoculated into the sewage, and cultured for 20 to 28 hours at a temperature of 25°C to 40°C and a rotation speed of 90 rpm to 180 rpm to complete the treatment of the nitrogen and phosphorus-containing sewage.

5. The use according to claim 4, characterized in that: The preparation method of the heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 seed solution is: (1) inoculating heterotrophic nitrifying aerobic denitrifying Arthrobacter MD1 into LB medium to prepare a bacterial suspension; (2) Centrifuge the bacterial suspension at 5000 rpm for 2 to 7 minutes, discard the supernatant, add sterile water for washing, and centrifuge again. Repeat 2 to 3 times to prepare the seed solution.

6. The use according to claim 5, characterized in that: The culture conditions in step (1) are 25-35° C. and 120-180 rpm for 12-36 hours.

7. The use according to claim 4, characterized in that: The inoculation amount of the seed liquid is 0.2-0.6 g / 150 mL of sewage.

8. The use according to claim 4, characterized in that: The nitrogen contained in the sewage is at least one of nitrite nitrogen, ammonia nitrogen and nitrate nitrogen; the carbon source contained in the sewage is at least one of sodium citrate, glucose, sodium acetate and sodium succinate.

9. The use according to claim 4, characterized in that: The C / N of the sewage is 6-10, the P / N is 0.1-0.6, and the DO is 3.0-6.5 mg / L.

10. The use according to claim 4, characterized in that: The pH of the sewage is 7-10.

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