A strain with low temperature resistance and aerobic denitrification function and its application

Abstract

The invention relates to the field of microbial technology, and discloses a strain with low-temperature resistance and aerobic denitrification function and an application thereof. The strain is named Pseudomonas sp. BWFA71, and the preservation unit is Guangdong Microbial Culture Collection Center (GDMCC), the preservation date is February 22, 2022, and the preservation number is GDMCCNO:62257. The strain of the invention has strong applicability and a wide range of temperature tolerance. The maximum and minimum tolerance temperatures are both wider than those of the same genus strains in the prior art. The strain can have good denitrification ability under conditions of 6 to 30°C, and the dissolved oxygen in the water has no obvious effect on its denitrification process. The strain is particularly suitable for sewage treatment and denitrification systems in areas with relatively large temperature changes, and for total nitrogen control in ecological restoration projects such as aquaculture water bodies and landscape water bodies where anaerobic denitrification cannot be performed.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain with low temperature resistance and aerobic denitrification function and application thereof. Background Art

[0002] As an economical and effective sewage treatment method, biological denitrification has been widely used in the treatment of nitrogen-containing wastewater. However, in actual application, traditional denitrifying bacteria will inhibit the growth activity of microorganisms due to low temperature, resulting in poor treatment effect. However, low-temperature denitrifying bacteria are at a disadvantage in the system community structure because their growth and reproduction temperature is usually below 20°C and it is difficult for them to compete with normal-temperature denitrifying bacteria in summer. Therefore, both artificial and natural purification systems need to supplement low-temperature denitrifying bacteria in winter to allow the bacterial community to quickly adapt to the low temperature state, thereby solving the problem of treating nitrate nitrogen.

[0003] Low-temperature denitrifying bacteria are also disturbed by many external factors during actual application, resulting in instability, among which dissolved oxygen is the most common influencing factor. The altitude difference between the east and west of my country is as high as thousands of meters, resulting in different dissolved oxygen in the western plateau and the northern plains under the same low-temperature environment due to atmospheric pressure. The dissolved oxygen in the plains is high and the dissolved oxygen in the plateau is low. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes a strain with low temperature resistance and aerobic denitrification function and its application, which has stronger tolerance to oxygen and temperature and can improve the applicability and stability of the biological nitrogen removal process.

[0005] The first aspect of the present invention provides a strain, named Pseudomonas sp. BWFA71, deposited with Guangdong Microbiological Culture Collection Center (GDMCC), date of deposit: February 22, 2022, deposit number: GDMCCNO:62257.

[0006] The inventors collected water samples and sludge from sewage treatment facilities in many places in autumn and winter, and finally isolated a low-temperature resistant aerobic denitrifying bacteria through enrichment, primary screening and secondary screening. The strain belongs to the genus Pseudomonas sp., and its biological characteristics are: Gram-negative, rod-shaped or short rod-shaped (such as Figure 1 A); on the nitrate screening plate, the colonies are smooth, moist, and slightly raised round colonies in the middle. The colonies are light yellow in the middle and white around them (as shown in Figure 1B and C). The 16S rDNA of the strain was amplified by PCR using universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'), and the amplified product was sequenced by Guangzhou Aiki Biotechnology Co., Ltd. The sequence was compared with NCBI by BLAST, and the maximum likelihood method was used to construct a phylogenetic tree analysis (as shown in Figure 2 The results showed that the strain had the highest similarity with Pseudomonas sp. JCM 5482, but was not completely the same. Combined with the morphological characteristics of the strain, it was confirmed that the strain belonged to a new strain of Pseudomonas sp. The strain was named Pseudomonas sp. BWFA71 and was deposited in the Guangdong Microbial Strain Collection Center (GDMCC for short, address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Institute of Microbiology, Guangdong Academy of Sciences, Postal Code 510075) on February 22, 2022, with the deposit number GDMCC NO: 62257. This strain has strong applicability and a wide range of temperature tolerance. Both the highest and lowest tolerance temperatures are wider than those of the same genus strains in the prior art. It has good denitrification ability under conditions of 6 to 30°C, and the dissolved oxygen in the water has no obvious effect on its denitrification process. It is particularly suitable for sewage treatment and denitrification systems in areas with large temperature changes, and for total nitrogen control in ecological restoration projects such as aquaculture water bodies and landscape water bodies where anaerobic denitrification cannot be carried out.

[0007] Preferably, the 16S rRNA sequence of the strain is as shown in SEQ ID No: 1.

[0008] SEQ ID No:1

[0009]

[0010]

[0011] Preferably, the strain has a temperature tolerance of 6 to 30°C.

[0012] Preferably, the strain has aerobic denitrification capability.

[0013] The second aspect of the present invention provides a microbial agent, wherein the microbial agent contains the strain described in the first aspect of the present invention.

[0014] Preferably, the microbial agent further contains other auxiliary materials, and the auxiliary materials include at least one of a lyophilization protective agent, a solvent, a carrier, and a culture matrix.

[0015] A third aspect of the present invention provides the use of a reagent comprising at least one of the following in denitrification treatment of a water body;

[0016] (1) The strain described in the first aspect of the present invention;

[0017] (2) a bacterial agent of the strain described in the first aspect of the present invention;

[0018] (3) a live bacterial liquid of the strain described in the first aspect of the present invention;

[0019] (4) a dead liquid of the strain described in the first aspect of the present invention;

[0020] (5) metabolites of the strain described in the first aspect of the present invention;

[0021] (6) An extract of the strain described in the first aspect of the present invention.

[0022] Preferably, the water denitrification includes removing nitrate nitrogen, ammonium nitrogen or nitrite nitrogen from the water.

[0023] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0024] The strain of the present invention has strong applicability and a wide range of temperature tolerance. The highest and lowest tolerance temperatures are both wider than those of the same genus strains in the prior art, and the strain has good denitrification performance under the condition of 6 to 30°C. Compared with normal-temperature denitrifying bacteria, the strain of the present invention does not affect the denitrification effect under low-temperature conditions of 9 to 15°C, and still has obvious denitrification effect at 6 to 9°C; compared with cold-loving denitrifying bacteria, the strain of the present invention will not be inactivated by temperature increase, and the denitrification effect does not change much under normal temperature conditions of 15 to 30°C; and the dissolved oxygen in the water has no obvious effect on the denitrification process of the strain of the present invention, and is particularly suitable for sewage treatment and denitrification systems in areas with relatively large temperature changes, as well as total nitrogen control in ecological restoration projects such as aquaculture water bodies and landscape water bodies where anaerobic denitrification cannot be performed. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Gram staining microscopy and colony diagram of the strain of the present invention;

[0026] Figure 2 is a phylogenetic tree diagram of the strain of the present invention;

[0027] Figure 3 This is a comparison chart of the denitrification performance of the strain of the present invention at different temperatures;

[0028] Figure 4 It is a comparison chart of the growth and denitrification capabilities of the strain of the present invention under aerobic and anaerobic conditions. DETAILED DESCRIPTION

[0029] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be pointed out that the following embodiments do not limit the protection scope of the present invention.

[0030] Unless otherwise specified, the raw materials, reagents or instruments used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0031] The following is the culture medium formula used in the embodiments of the present invention:

[0032] Denitrification culture medium: potassium nitrate 1.5 g / L, sodium citrate 5.1 g / L, potassium dihydrogen phosphate 1.36 g / L, magnesium sulfate heptahydrate 0.4 g / L, trace elements (200×) 0.5% (v / v); pH was adjusted to 7.0-7.5.

[0033] The formula of trace elements (200×) is: EDTA·2Na2 2.8 g / L, ferrous sulfate heptahydrate 2 g / L, zinc sulfate heptahydrate 1.56 g / L, anhydrous calcium chloride 2.2 g / L, manganese sulfate monohydrate 1.76 g / L, ammonium paramolybdate tetrahydrate 0.44 g / L, copper sulfate pentahydrate 0.64 g / L, and cobalt chloride hexahydrate 0.64 g / L.

[0034] Denitrification culture medium plate: 1.5% to 2% agar is added to the above denitrification culture medium to prepare a solid plate.

[0035] Example 1: Screening and performance verification of strains with low temperature resistance and aerobic denitrification function

[0036] Sample collection and enrichment: In autumn and winter, water samples and sludge were collected from sewage treatment facilities in many places, supplemented with appropriate amounts of carbon source, nitrate, phosphate buffer, and pH was adjusted, and placed in a 6°C incubator for enrichment culture for 3 to 7 days. Regularly test the changes in nitrate, nitrite and total nitrogen in the enrichment culture solution, and select samples with good denitrification performance in a low temperature environment for strain screening.

[0037] Initial screening of strains: The enriched culture fluid was diluted with physiological saline in a gradient manner and spread on denitrification culture medium plates, placed in a 4°C incubator and cultured for 3 days. 12 low-temperature-resistant strains were selected for re-screening to verify the denitrification performance.

[0038] Strain rescreening: The primary screened strains were inoculated into denitrification culture medium, placed in a 6°C shaker at 200r / min for 3 days, the mixed culture solution was taken to determine the total nitrogen, and the centrifuged supernatant of the culture solution was taken to determine the nitrate and nitrite, and the nitrate degradation rate, nitrite accumulation rate, and total nitrogen degradation rate within 3 days were calculated. A strain with a nitrate degradation rate of 100.0%, a nitrite accumulation rate of 7.1%, and a total nitrogen degradation rate of 36.2% was obtained by rescreening.

[0039] Strain purification: Pick the colonies of the rescreened strains, separate them by streaking on the screening medium plate using the partition streaking method, and culture them in a 6°C incubator for 72 hours. Observe the morphology of the isolated colonies, and select 3 to 4 colonies with the same morphology for rescreening test. Select the strain with the highest total nitrogen degradation rate in the rescreening test for the next round of purification until the colony morphology on the streaking separation plate is completely consistent. Select several colonies for rescreening test and the test results are basically the same, and similar to the results of the previous round of rescreening test. After 3 rounds of purification, a purified strain numbered BWFA71 was obtained, with a nitrate degradation rate of 100.0%, a nitrite accumulation rate of 1.0%, and a total nitrogen degradation rate of 66.2%.

[0040] Molecular identification of strains: PCR amplification of 16S rDNA of the strain was performed using universal primers (27F: 5'-AGAGTTTGATCCTGGCTCAG-3' and 1492R: 5'-GGTTACCTTGTTACGACTT-3'), and the amplified product was sequenced by Guangzhou Aiki Biotechnology Co., Ltd. The sequencing result is shown in SEQ ID NO: 1. The sequence was compared with NCBI by BLAST, and the maximum likelihood method was used to construct a phylogenetic tree analysis (such as Figure 2 The results showed that the strain had the highest similarity with Pseudomonas sp. JCM5482, but was not completely the same. Combined with the morphological characteristics of the strain, it was confirmed that the strain belonged to a new strain of the genus Pseudomonas sp. The strain was named Pseudomonas sp. BWFA71.

[0041] Example 2: Effect of temperature on strains

[0042] Preparation of logarithmic phase bacterial suspension: Pseudomonas sp. BWFA71 of the present invention was picked from the slant and inoculated into 5 250 mL conical flasks containing 100 mL of denitrification medium, and cultured under shaking conditions at different temperatures (6-30°C) for several days, and samples were taken regularly to measure OD 600 , until the growth reaches the mid-logarithmic phase (OD 600 =0.4-0.8). The cells in the culture medium were collected by centrifugation and prepared into OD 600 =1.0 bacterial suspension.

[0043] Take 5 250mL conical flasks containing 200mL of denitrification culture medium, pass nitrogen to reduce the dissolved oxygen to below 0.5mg / L, add the above bacterial suspension at a 1% inoculum amount, take the initial sample, seal it and place it in a shaker at the corresponding temperature to continue shaking culture, and take samples regularly to test the changes in biomass and nitrogen. The test results are as follows Figure 3 shown.

[0044] from Figure 3 It can be seen that when the temperature is between 12 and 30 °C, the denitrification performance and total nitrogen removal rate of the strain of the present invention do not change much, and the nitrate degradation rate is between 0.06 and 0.08 h -1 , the total nitrogen removal efficiency is maintained at more than 75%; when the temperature is between 9 and 12 °C, the denitrification capacity does not change much, and the nitrate degradation rate can be maintained at 0.06h -1 However, under low temperature conditions, more nitrogen was used for the synthesis of bacterial substances, and the total nitrogen removal rate showed a downward trend. Under low temperature conditions of 6°C, the denitrification performance decreased, and the nitrate degradation rate was 0.042h -1 The total nitrogen removal rate was 39.7%, and there was still obvious denitrification.

[0045] The denitrification performance of general activated sludge decreases 2 to 3 times every 10°C, while the strain of the present invention can maintain a relatively stable denitrification effect between 12 and 30°C. Although the denitrification performance decreases below 12°C, it can still maintain about half of the denitrification performance at 6°C. Therefore, the strain of the present invention is particularly suitable for low-temperature or temperature-fluctuating sewage treatment denitrification and denitrification processes.

[0046] Example 3: Effect of oxygen on bacterial strains

[0047] Pseudomonas sp. BWFA71 of the present invention was picked from the slant and inoculated into a conical flask containing 100 mL of denitrification medium, and cultured at 15°C and 150 r / min for 3 days. The bacterial cells in the culture solution were collected by centrifugation and prepared into OD 600 =1.0 bacterial suspension.

[0048] Take three 500mL conical flasks filled with 200ml of denitrification culture medium, add the above bacterial suspension at a 1% inoculation rate, and seal with a breathable sealing film as an aerobic group; take another three 250mL conical flasks filled with 200ml of denitrification culture medium, add the above bacterial suspension at a 1% inoculation rate, pass nitrogen to reduce the dissolved oxygen to below 0.5mg / L, and then seal with a rubber stopper as an anaerobic group. Place the above 6 bottles in a shaker at 6, 15 and 30℃ respectively, and put one conical flask of the aerobic group and one conical flask of the anaerobic group in each shaker at each temperature. Oscillating and incubating at 150r / min, take samples regularly to test the changes in nitrogen and biomass. For each sampling of the anaerobic group, use nitrogen to maintain the dissolved oxygen below 0.5mg / L, and then seal after sampling. The measurement results are as follows Figure 4 shown.

[0049] from Figure 4It can be seen that after a period of cultivation, the nitrate in both the aerobic and anaerobic groups was completely degraded, and no nitrite accumulated. According to the data of nitrogen and biomass in each test group, the total nitrogen degradation rate at the end point and the average specific growth rate in the logarithmic period were calculated (as shown in Table 1). As can be seen from Table 1, at 6-30°C, the total nitrogen in each group decreased significantly under aerobic conditions. Since the strain grew faster under aerobic conditions than the anaerobic group at the same temperature, the nitrogen assimilated during growth was relatively large, and its total nitrogen degradation rate was lower than that of the anaerobic group. Therefore, it is judged that the strain has a significant denitrification effect under this condition; the aerobic and anaerobic denitrification effects are similar under low temperature conditions of 6-15°C; under normal temperature conditions of 15-30°C, the growth under aerobic conditions is faster, more nitrogen is used for assimilation, and the denitrification effect is reduced.

[0050] Table 1 Denitrification effect of aerobic and anaerobic conditions at different temperatures for 72 h

[0051]

[0052] Conventional denitrifying bacteria can only denitrify under anaerobic conditions. However, the strain of the present invention still exhibits obvious denitrification under aerobic conditions. Compared with anaerobic conditions, the nitrate degradation performance is improved, the bacterial count is increased, and the total nitrogen removal rate is reduced to a certain extent. In actual application, the bacteria can be removed by precipitation and other means, and the total nitrogen in the effluent can still be guaranteed to meet the standard. Therefore, the application conditions of the strain have no requirements for dissolved oxygen, and are particularly suitable for aquaculture water bodies, landscape water bodies, or denitrification and denitrification systems that are easily impacted by dissolved oxygen.

[0053] The preferred embodiments of the present invention are specifically described above, but the invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A strain, characterized in that The strain was named Pseudomonas Pseudomonas sp.)BWFA71, depository unit: Guangdong Microbiological Culture Collection Center (GDMCC), deposit date: February 22, 2022, deposit number: GDMCCNO:62257.

2. A microbial agent, characterized in that: The microbial agent contains the strain according to claim 1.

3. The microbial agent according to claim 2, characterized in that: The microbial agent also contains other auxiliary materials, which include at least one of a freeze-drying protective agent, a solvent, a carrier, and a culture matrix.

4. Use of a reagent containing at least one of the following in denitrification of water bodies; (1) The strain according to claim 1; (2) A bacterial agent containing the strain described in claim 1; (3) A live bacterial liquid containing the strain described in claim 1; The water denitrification includes removing nitrate nitrogen or nitrite nitrogen from the water.

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