Nitrosomonas mobilis and application thereof in removing ammonia nitrogen in sewage at low temperature

Abstract

The invention relates to the field of microbiological sewage treatment, in particular to nitrosomonas mobilis and application thereof in low-temperature sewage treatment. The strain is classified and named as Nitrosomonas mobilis JMS02, and the preservation number of the strain is CCTCC (China Center For Type Culture Collection) NO: M20241937. The nitrosomonas mobilis can grow under the aerobic condition of 10 DEG C, and can effectively treat ammonia nitrogen in municipal sewage and black and odorous water at the low temperature of 12 DEG C.

Description

Technical Field

[0001] The invention relates to the field of sewage treatment by microorganisms, and in particular to a strain of Nitrosomonas mobilis and application thereof in removing ammonia nitrogen in sewage under low temperature conditions. Background Art

[0002] Ammonia nitrogen (NH3-N) is one of the important components of water pollution, mainly from agricultural fertilization, industrial wastewater discharge and domestic sewage. With the development of industrialization and the acceleration of urbanization, the problem of ammonia nitrogen pollution has become increasingly serious and has become the main factor of global water pollution. Ammonia nitrogen pollution not only leads to eutrophication of water bodies, triggers algae outbreaks, affects aquatic ecosystems, but also poses a threat to drinking water sources. Therefore, the removal of ammonia nitrogen has become a key issue that needs to be solved in the field of sewage treatment.

[0003] Nitrosomonas spp. is a type of autotrophic bacteria that can convert ammonium salts into nitrites. This type of bacteria can effectively reduce the concentration of ammonia nitrogen in water bodies without adding additional carbon sources, which helps to achieve economical and sustainable sewage treatment. Although Nitrosomonas has broad application prospects in sewage treatment, the effect of existing technologies on removing ammonia nitrogen under low temperature conditions is still limited. In winter or cold areas, the drop in water temperature will significantly reduce the activity of microorganisms in activated sludge, resulting in reduced ammonia nitrogen removal efficiency and even stagnation. According to the Pollutant Discharge Standard for Urban Sewage Treatment Plants (GB 18918-2002), the discharge standard of ammonia nitrogen when the water temperature is ≤12°C is relaxed compared to when the water temperature is normal, which further illustrates the difficulty and importance of reducing ammonia nitrogen in sewage at low temperatures. Therefore, screening microorganisms that can efficiently remove ammonia nitrogen from sewage under low temperature conditions is an important research direction in the current field of sewage treatment. The mobile Nitrosomonas of the present invention aims to provide a microorganism that still has good ammonia nitrogen removal ability under low temperature conditions to address this technical bottleneck. Summary of the invention

[0004] The purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art, and to provide a low-temperature resistant nitrosomonas, Nitrosomonas mobilis, screened from industrial park sewage (including domestic sewage and part of industrial wastewater), and its application in removing ammonia nitrogen from sewage under low temperature conditions.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A cold-resistant nitrosomonas mobilis, named JMS02. The strain was deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on September 6, 2024, and its classification name is Nitrosomonas mobilis JMS02, and the deposit number is CCTCC NO: M20241937.

[0007] The above-mentioned mobile Nitrosomonas can grow under aerobic conditions at 10°C, and can effectively treat ammonia nitrogen in municipal sewage and black and smelly water bodies under low temperature conditions of 12°C.

[0008] Compared with the prior art, the present invention has the following advantages and beneficial effects: the growth temperature of pure cultured Nitrosomonas mobilis strains reported at home and abroad is not lower than 12°C (including 12°C), while the Nitrosomonas mobilis JMS02 involved in the present invention has a shortest generation time of 30h in a 10°C culture medium, and can treat ammonia nitrogen in municipal sewage and black and odorous water bodies at 12°C to reach the Class A emission standard (8mg / L). BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 This is the growth of Nitrosomonas mobilis JMS02 at 10℃.

[0010] Figure 2 Deammonification effect of Nitrosomonas mobilis JMS02 in municipal wastewater.

[0011] Figure 3 It is the deammonification effect of Nitrosomonas mobilis JMS02 in black and smelly water bodies. DETAILED DESCRIPTION

[0012] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0013] The culture medium used in the following examples was prepared as follows. All culture media were sterilized at high temperature and high pressure (121° C., 20 min) before use:

[0014] AOB culture medium: FeSO4·7H2O 0.4 g / L, MgSO4·7H2O 0.5 g / L, K2HPO4 0.1 g / L, NaHCO3 1.5 g / L, CaCl2·2H2O 1.5 g / L, NaCl 2.0 g / L, NH4Cl 0.39 g / L, trace element solution 1 mL / L, pH 7.5.

[0015] Trace element solution: EDTA 0.5g / L, CuSO4·5H2O 0.075g / L, ZnSO4·7H2O 0.3g / L, CoCl2·6H2O 0.375g / L, MnCl2·2H2O 0.3g / L, H3BO4 0.014g / L, NaMoO4·2H2O 0.22g / L.

[0016] The water quality indicators involved are tested using national standard methods, among which the ammonia nitrogen detection method adopts the "Water Quality-Determination of Ammonia Nitrogen-Nessler's Reagent Spectrophotometric Method" (HJ 535-2009); the nitrite nitrogen determination adopts the "Water Quality-Determination of Nitrite Nitrogen-Spectrophotometric Method" (GB / T 7493-1987).

[0017] The calculation of the shortest generation time is based on the accumulation of nitrite nitrogen. The specific calculation formula is as follows:

[0018] Nitrite accumulation rate

[0019] Where V2 is the nitrite accumulation rate on day t2, mg / (L / d); V1 is the nitrite accumulation rate on day t1, mg / (L·d), C t2 is the nitrite nitrogen concentration on day t2, C t1 is the nitrite nitrogen concentration on day t1.

[0020] Example 1: Screening and identification of bacterial strains

[0021] 5 mL of activated sludge from the biochemical treatment system of a sewage treatment plant in Jiamusi City, Heilongjiang Province was placed in 100 mL of AOB medium and cultured at 12°C and 120 r / min for about 15 days until ammonia nitrogen decreased significantly and nitrite nitrogen accumulated significantly; the enriched culture solution was transferred to AOB medium at a volume ratio of 1% for continuous culture, and a total of 3 rounds of continuous culture were carried out. The bacterial solution of the third round of enrichment culture was diluted to different concentrations by a 10-fold gradient dilution method, added to a 96-well plate, and cultured at 30°C. After 30 days, samples were taken to detect the accumulation of nitrite nitrogen, and the culture solution in the nitrite nitrogen-positive well was transferred to 1 mL of AOB medium and cultured at 30°C and 120 r / min. The purified culture was PCR amplified for 16s rDNA and amoA gene fragments, and molecular biological identification was performed by sequencing and comparison. The primer sequences used for amplification are as follows:

[0022] 16s rDNA upstream primer 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ (SEQ ID NO. 1).

[0023] 16s rDNA downstream primer 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO. 2).

[0024] amoA upstream primer 1F: 5′-GGGGTTTCTACTGGTGGT-3′ (SEQ ID NO. 3).

[0025] amoA upstream primer 2R: 5′-CCCCTCKGSAAAGCCTTCTTC-3′ (SEQ ID NO. 4).

[0026] The sequencing of the PCR product was completed by Shanghai Sangon Biotechnology Co., Ltd., and the sequencing results of the 16s rDNA sequence and amoA sequence are shown in SEQ ID NO.5 and SEQ ID NO.6, respectively. After comparison of the 16s rDNA sequence with the NCBI database, it was shown that the isolated strain was Nitrosomonas mobilis. In the amoA sequence comparison results, the only known species was Nitrosomonas mobilis strain Nc2, with a similarity of 97.8%, which confirmed the above 16s rDNA comparison results and showed that the bacteria had relatively unique genetic characteristics compared with the known Nitrosomonas mobilis. The strain was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on September 6, 2024, and its classification name was Nitrosomonas mobilis JMS02, and the collection number was CCTCC NO: M20241937.

[0027] Example 2: Growth of Nitrosomonas mobilis JMS02 at 10°C

[0028] Preparation of inoculum: inoculate 1 mL of bacterial solution into 100 mL of AOB medium and culture at 30°C and 120 r / min for 5 days.

[0029] Figure 1 The implementation method is: take 1mL of inoculum into 100mL of AOB culture medium, culture at 10℃ and 120r / min, and take samples every 24h to determine the concentrations of ammonia nitrogen and nitrite nitrogen.

[0030] according to Figure 1The deammonification rate of Nitrosomonas mobilis JMS02 in 10℃ culture medium reached the fastest 0.64mg / L / h; and while deammonifying, it accumulated a large amount of nitrite-nitrogen, with the ratio of nitrite-nitrogen accumulation of 57.68mg / L to ammonia nitrogen removal of 63.15mg / L exceeding 90%, indicating that the main mechanism of deammonification was ammonia oxidation; according to the nitrite-nitrogen accumulation rate, Nitrosomonas mobilis JMS02 could reach a generation time of 30h in 10℃ culture medium, indicating that Nitrosomonas mobilis JMS02 still had a faster growth rate and higher ammonia oxidation activity under these conditions.

[0031] Example 3: Deammoniation effect of Nitrosomonas mobilis JMS02 on municipal sewage at low temperature

[0032] Preparation of inoculation solution: 1 mL of Nitrosomonas mobilis JMS02 and 1 mL of Nitrosomonas eutropha CZ-4 bacterial suspension (strain collection number: CCTCC NO: M 2018813) were inoculated into 100 mL of AOB medium, and cultured at 30°C and 120 r / min for 7 days; the culture solution was concentrated 10 times by centrifugation at 5000 r / min, 5 min to obtain the inoculum solution.

[0033] CZ-4 is an excellent nitrosomonas in the industry, first disclosed in Chinese patent 201811529529.7, and its nitrosation ability is mainly reflected in the 27°C experiment. The CZ-4 strain in this application was obtained from Hubei University of Technology.

[0034] Figure 2 The implementation method is: take municipal sewage (including domestic sewage and a small amount of industrial wastewater) from Chongyang County, Xianning City, Hubei Province, detect ammonia nitrogen in the sewage, use NH4Cl to add ammonia nitrogen to about 80mg / L, add 5mL of Nitrosomonas mobilis JMS02 inoculum to 100mL of municipal sewage in the experimental group, add 5mL of Nitrosomonas mobilis CZ-4 inoculum to 100mL of township sewage in the CZ-4 control group, and do not inoculate bacteria in the blank control group. Each group is set up with 2 parallels. Shake the culture at 12℃ and 120r / min, and take samples every 24h to detect the concentration of ammonia nitrogen and nitrite nitrogen.

[0035] according to Figure 2 After Nitrosomonas mobilis JMS02 was inoculated into municipal sewage, its deammoniation rate increased by 2.47 mg / L / h compared with the blank control group and by 1.80 mg / L / h compared with the CZ-4 control group. The results showed that Nitrosomonas mobilis JMS02 can effectively treat ammonia nitrogen in municipal sewage at low temperatures.

[0036] Example 4: Deammoniation effect of Nitrosomonas mobilis JMS02 on black and odorous water at low temperature

[0037] The preparation method of the inoculum solution is the same as that in Example 3.

[0038] Figure 3 The implementation method is: take the black and smelly water from the river of Guanggu Qilu Road, Wuhan City, Hubei Province, detect the ammonia nitrogen in it, use NH4Cl to add the ammonia nitrogen to about 60mg / L, add 5mL of Nitrosomonas JMS02 inoculum to 100mL of black and smelly water in the experimental group, add 5mL of Nitrosomonas CZ-4 inoculum to 100mL of black and smelly water in the CZ-4 control group, and do not inoculate bacteria in the blank control group. Each group is set up with 2 parallels. Cultivate in a shaking table at 12℃ and 120r / min, and take samples every 24h to detect the concentrations of ammonia nitrogen and nitrite nitrogen.

[0039] according to Figure 3 After Nitrosomonas mobilis JMS02 was inoculated into municipal sewage, its deammoniation rate increased by 1.96 mg / L / h compared with the blank control group and by 0.86 mg / L / h compared with the CZ-4 control group. The results showed that Nitrosomonas mobilis JMS02 can effectively treat ammonia nitrogen in black and smelly water bodies at low temperatures.

[0040] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A cold-resistant nitrosomonas, whose classification name is Nitrosomonas mobilis JMS02 and the deposit number is CCTCC NO: M20241937.

2. Use of the low-temperature resistant and motile Nitrosomonas according to claim 1 in sewage treatment.

3. The use according to claim 2, characterized in that: The mobile Nitrosomonas is used to remove ammonia nitrogen from sewage under aerobic conditions.

4. The use according to claim 3, characterized in that: The sewage water temperature is 12°C.

5. The use according to claim 3 or 4, characterized in that: The sewage is municipal sewage or black and smelly water.

Images