Cold-resistant nitrosomonas mobilis and application of cold-resistant nitrosomonas mobilis to removal of ammonia nitrogen in sewage under low-temperature condition

Abstract

The invention relates to the field of microbiological treatment of sewage, in particular to cold-resistant nitrosomonas mobilis and application of the cold-resistant nitrosomonas mobilis to removal of ammonia nitrogen in sewage under a low-temperature condition. The strain is classified and named as Nitrosomonas mobilis XY01, and the preservation number of the strain is CCTCC (China Center for Type Culture Collection) NO: M 20242855. When the strain grows in a 7 DEG C culture medium, the deamination rate has no obvious difference in the interval of 10-16 DEG C; the activity of the nitrosomonas eutropha CZ-4 at a low temperature is superior to that of the nitrosomonas eutropha CZ-4; ammonia nitrogen in landfill leachate and black and odorous water can be treated under the condition of 13 DEG C, and the problem of removing ammonia nitrogen in sewage under the condition of low temperature is well solved.

Description

Technical Field

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

[0002] Ammonia nitrogen (NH3-N) is one of the key indicators in sewage treatment and has attracted much attention due to its potential threat to eutrophication of water bodies, algal blooms and drinking water safety. Biological nitrification is widely used as the main means of deammonification in most sewage treatment plants due to its advantages of low cost and few side effects. However, low temperature environment poses a severe challenge to the nitrification process, especially in winter, when lower water temperatures significantly reduce the activity of autotrophic ammonia-oxidizing bacteria such as Nitrosomonas, which are the core participants in the nitrification and deammonification process. Studies have shown that when the temperature drops to 17°C, the nitrification rate of Nitrosomonas is only half of that at 30°C; and below 15°C, the proportion of oligotrophic Nitrosomonas in sewage treatment plants decreases, and the efficiency of ammonia nitrogen removal decreases significantly. In addition, low temperature may also inhibit the activity of ammonia-oxidizing bacteria by damaging the functional genome. In my country's current urban sewage treatment discharge standard (GB18918-2002), considering the impact of low temperature on nitrification, ammonia nitrogen is allowed to be discharged at a higher concentration when the water temperature is below 12°C. In order to ensure the effect of nitrification and deammonification in winter, sewage treatment plants usually adopt insulation, heating or immobilization technology to maintain microbial activity, but this undoubtedly increases operating costs. Therefore, the development of microorganisms that can maintain high efficiency in ammonia nitrogen removal under low temperature conditions has become the key to solving this problem.

[0003] The research on low-temperature sewage microorganisms at home and abroad is mainly concentrated on heterotrophic bacteria such as Pseudomonas and acinetobacter, and the report on low-temperature autotrophic bacteria is relatively less, and the research on Nitrosomonas mobilis (mobile nitrosomonas) pure strains has only two examples, and its low-temperature resistance is not outstanding. Domestic research in this regard is more scarce, and there is almost no document record about low-temperature nitrosomonas. In view of this, the mobile nitrosomonas of the present invention is intended to provide a microorganism with good ammonia nitrogen removal ability under low temperature conditions for this technical bottleneck. The bacterium not only fills the gap in domestic related fields, but also provides new ideas and technical support for improving the nitrification and deamination efficiency of sewage treatment plants under low temperature conditions, which is of great significance for promoting the development of low-temperature nitrification and deamination technology. 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 cold-resistant nitrosomonas strain, Nitrosomonas mobilis, screened from municipal sewage, 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 and mobile Nitrosomonas, whose classification name is Nitrosomonas mobilis XY01, was deposited in the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China) on December 25, 2024. Its classification name is Nitrosomonas mobilis XY01, and the preservation number is CCTCCNO:M 20242855.

[0007] The above-mentioned cold-resistant and mobile Nitrosomonas is used to remove ammonia nitrogen from sewage under low temperature conditions.

[0008] The above-mentioned cold-resistant and mobile Nitrosomonas is used to remove ammonia nitrogen from sewage under low temperature conditions. The cold-resistant and mobile Nitrosomonas grows at 7°C under aerobic conditions and is used to remove ammonia nitrogen from sewage under low temperature conditions.

[0009] The use of the above-mentioned cold-resistant and mobile Nitrosomonas for removing ammonia nitrogen from sewage under low temperature conditions is characterized in that the water temperature of the low-temperature water body in which it grows is 7°C and the water temperature of the sewage is 13°C.

[0010] The use of the above-mentioned cold-resistant and mobile Nitrosomonas for removing ammonia nitrogen from sewage under low temperature conditions is characterized in that the sewage is landfill leachate or black and smelly water.

[0011] Beneficial effects:

[0012] Compared with the pure cultured Nitrosomonas mobilis strains reported at home and abroad, whose growth temperature is not lower than 12°C (including 12°C), the Nitrosomonas mobilis XY01 involved in the present invention grows in a culture medium at 7°C, and there is no obvious difference in the deammoniation rate in the range of 10°C to 16°C; its activity at low temperature is better than that of Nitrosomonas eutropha CZ-4; it can treat ammonia nitrogen in landfill leachate and black and smelly water bodies at 13°C, and better solves the problem of removing ammonia nitrogen in sewage under low temperature conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is the growth of Nitrosomonas mobilis XY01 at 7-16℃.

[0014] Figure 2Activity of Nitrosomonas mobilis XY01 and Nitrosomonas eutropha CZ-4 under low temperature conditions.

[0015] Figure 3 It is the deammonification effect of Nitrosomonas mobilis XY01 in black and smelly water bodies.

[0016] Figure 4 It is the deammoniation effect of Nitrosomonas mobilis XY01 in landfill leachate. DETAILED DESCRIPTION

[0017] 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.

[0018] 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:

[0019] 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.

[0020] 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, H3BO40.014 g / L, NaMoO4·2H2O0.22g / L.

[0021] 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).

[0022] The calculation of the shortest generation time is based on the accumulation of nitrite nitrogen. The specific calculation formula is as follows:

[0023] Nitrite accumulation rate

[0024] 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 t2is the nitrite nitrogen concentration on day t2, C t1 is the nitrite nitrogen concentration on day t1.

[0025] Example 1

[0026] The activated sludge from the aerobic pool of a township sewage treatment station in Nanzhang County, Xiangyang City, Hubei Province was inoculated into 100 mL AOB culture medium at a ratio of 1‰, and enriched and cultured at 13°C and 140 r / min for about 7 days until ammonia nitrogen decreased significantly and nitrite nitrogen accumulated significantly; the enriched culture solution was transferred to AOB culture medium at a volume ratio of 5% for continuous culture, and a total of 5 rounds of continuous culture were carried out. The bacterial solution of the fifth 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 13°C. After 30 days, samples were taken to detect the accumulation of nitrite nitrogen, and the culture solution in the nitrite nitrogen-positive wells was transferred to 1 mL AOB culture medium, and cultured at 30°C and 140 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:

[0027] 16S rDNA upstream primer 27F: 5′-AGAGTTTGATCMTGGCTCAG-3′ (SEQ ID NO. 1).

[0028] 16S rDNA downstream primer 1492R: 5′-GGTTACCTTGTTACGACTT-3′ (SEQ ID NO. 2).

[0029] amoA upstream primer 1F: 5′-GGGGTTTCTACTGGTGGT-3′ (SEQ ID NO. 3).

[0030] amoA upstream primer 2R: 5′-CCCCTCKGSAAAGCCTTCTTC-3′ (SEQ ID NO. 4).

[0031] According to the sequencing results of the PCR products completed by Shanghai Sangon Biotechnology Co., Ltd. (SEQ ID NO.5 and SEQ ID NO.6), the isolated strain was determined to be Nitrosomonas mobilis by comparing the 16S rDNA sequence in the NCBI database. Further amoA gene sequence comparison results showed that the strain had a high similarity of 99.79% with the known species Nitrosomonas mobilis strain Nc2, which further verified the conclusion of the 16S rDNA comparison. The strain was deposited in the China Center for Type Culture Collection (Address: Wuhan University, Wuhan, China) on December 25, 2024, and its classification name was Nitrosomonas mobilis XY01, and the collection number was CCTCCNO:M 20242855.

[0032] Example 2

[0033] Preparation of inoculum: inoculate 1 mL of bacterial solution into 100 mL of AOB culture medium, culture at 30°C and 140 r / min. When the accumulation of nitrite nitrogen of XY01 reaches 2.0 mg / L / h, use it as inoculum.

[0034] The culture medium was inoculated with an initial ammonia nitrogen concentration of 200 mg / L and a salinity of 0.50% at a volume ratio of 3%, and cultured at 7°C, 10°C, 13°C and 16°C with shaking at 140 r / min. Each group had three parallels, and samples were taken every day to determine the nitrite nitrogen concentration.

[0035] Reference Figure 1 : Nitrosomonas mobilis XY01 can grow in 7℃ culture medium, and its fastest nitrite nitrogen rate reaches 0.053mg / L / h, indicating that its ammonia nitrogen removal is carried out through ammonia oxidation reaction; according to the nitrite nitrogen accumulation rate, Nitrosomonas mobilis XY01 can reach a generation time of 30h in 7℃ culture medium; at the same time, the maximum nitrite nitrogen rate of Nitrosomonas mobilis XY01 at 10-16℃ is 0.155mg / L / h, and there is no significant difference among the three rates (P>0.05), indicating that Nitrosomonas mobilis XY01 still has a faster growth rate and higher ammonia oxidation activity under these conditions.

[0036] Example 3

[0037] Preparation of inoculum: 1 mL of the patented strain Nitrosomonas eutropha CZ-4 was inoculated into 100 mL of liquid culture medium, and cultured at 27°C and 200 r / min with shaking until the nitrite nitrogen accumulation rate reached about 2.0 mg / L / h as the inoculum. The preparation method of the inoculum of Nitrosomonas mobilis XY01 was the same as in Example 2.

[0038] The inoculum of CZ-4 and Nitrosomonas mobilis XY01 were inoculated into the basal culture medium at a volume ratio of 3%, respectively. The initial ammonia nitrogen was 200 mg / L, the salinity was 0.25%, and the shaking speed was 140 r / min. The culture was shaken at 10°C and 19°C, and 3 parallels were set for each group. The culture was shaken at 13°C and 140 r / min, and the concentrations of ammonia nitrogen and nitrite nitrogen were sampled every 24 hours.

[0039] Reference Figure 2 :The maximum nitrite accumulation rate of CZ-4 and Nitrosomonas mobilis XY01 at 19℃ (1.0±0.1mg / L / h and 0.6±0.2mg / L / h, respectively) was defined as 100%, and the relative activity of CZ-4 at 10℃ was only 3.60±0.60%, while that of XY01 was 16.60±5.80%. The maximum specific growth rate of CZ-4 and XY01 at 19℃ (0.5±0.1d -1 and 1.2±0.5d -1 ) is defined as 100%, and the relative activity of CZ-4 at 10°C is calculated to be 0% (indicating that its growth is completely inhibited), while that of XY01 is 50%. The above results indicate that XY01 has better low temperature adaptability than CZ-4.

[0040] Example 4

[0041] Preparation of inoculum: Referring to the method for preparing inoculum in Example 2, the bacterial solution of Nitrosomonas mobilis XY01 was taken when the nitrite nitrogen accumulation reached 2.0 mg / L / h, and then concentrated by centrifugation (speed:

[0042] 5000r / min, 5min) 10 times and then the inoculum solution was obtained.

[0043] The supernatant of a river water body (black and smelly water) and a certain landfill leachate were taken respectively. 1 mL of Nitrosomonas mobilis XY01 inoculum was added to 100 mL of black and smelly water and landfill leachate in the experimental group, and no bacteria were inoculated in the blank control group. Three parallels were set for each group. The culture was shaken at 13°C and 140 r / min, and samples were taken every 24 hours to detect the concentrations of ammonia nitrogen and nitrite nitrogen.

[0044] according to Figure 3 , Figure 4: After 5 days of treatment at 13℃, the ammonia nitrogen consumption and nitrite nitrogen accumulation of each test group were basically the same, indicating that ammonia nitrogen was mainly removed by ammonia oxidation. Under low temperature conditions, the nitrite nitrogen accumulation of XY01 in landfill leachate was (31.7±0.8mg / L) and the nitrite nitrogen accumulation in Xunsi River water (black and smelly river water) was (4.0±0.5mg / L), both indicating that it can treat landfill leachate and black and smelly water under low temperature conditions.

[0045] 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 strain of cold-tolerant Nitrosomonas, named Nitrosomonas mobilis XY01, characterized by: The deposit number is CCTCC NO: M 20242855.

2. Use of the cold-resistant and mobile Nitrosomonas according to claim 1 in removing ammonia nitrogen from sewage under low temperature conditions.

3. The use of a cold-resistant nitrosomonas strain according to claim 2 for removing ammonia nitrogen from sewage under low temperature conditions, characterized in that: The cold-resistant mobile nitrosomonas grows at 7°C under aerobic conditions and is used for removing ammonia nitrogen from sewage under low temperature conditions.

4. The use of a cold-resistant nitrosomonas strain according to claim 3 for removing ammonia nitrogen from sewage under low temperature conditions, characterized in that: The water temperature of the low-temperature growth water body is 7°C, and the water temperature of the sewage is 13°C.

5. Use of a cold-resistant and mobile Nitrosomonas according to claim 3 or 4 to remove ammonia nitrogen from sewage under low temperature conditions, characterized in that: The sewage is garbage leachate or black and smelly water.

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