Nitrosomonas with high ammonia oxidation activity and application thereof

Abstract

The invention discloses nitrosomonas with high ammonia oxidation activity and application thereof, and relates to the technical field of microorganisms and environmental protection. The bacterial strain is preserved in Guangdong Microbial Culture Collection Center on October 21, 2024, the name of the bacterial strain is Nitrosomonas sp. LY-1, and the preservation number is GDMCC No: 65307. The invention also discloses a preparation method of the bacterial strain. The strain LY-1 is a new strain, expands the variety and diversity of ammonia oxidizing bacteria nitrosomonas strains, can perform biological nitrogen removal under aerobic conditions, can quickly and efficiently convert ammonia nitrogen into nitrite nitrogen, and can be used for treatment of wastewater or soil containing high ammonia nitrogen, namely, the strain has a biological function of biological nitrogen removal, and has a good application prospect. Wide application prospects are realized.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms and environmental protection, and in particular to a strain of Nitrosomonas with high ammonia oxidation activity and application thereof. Background Art

[0002] Nitrogen is essential for life and food production. Nevertheless, nitrogen losses from unused nitrogen in wastewater, air, freshwater and oceans have caused nitrogen pollution, which affects the environment and leads to eutrophication, climate change, biodiversity loss and ozone depletion. It also causes cardiopulmonary problems in humans. Human activities such as food processing, fertilizer production, mining, etc. affect the global biogeochemical nitrogen cycle. The most important advantage of biological denitrification technology is that the biological treatment process can be carried out at ambient temperature and atmospheric pressure, the operation is simple, the capital and operating costs are low but the treatment efficiency is high, and it is cleaner in ecology than physical and chemical methods, which makes up for the disadvantages of chemical and physical methods such as high cost and generation of undesirable by-products.

[0003] Ammonia oxidation is the first and rate-limiting step of nitrification. Ammonia oxidizing bacteria (AOB) are one of the important microorganisms involved in the ammonia oxidation process. Since the first isolation of AOB in 1890, a large number of studies have been conducted to determine its phylogenetic diversity. Through these efforts, five AOB genera have been identified and classified into the class Proteobacteria. Four of them belong to the subclass β-Proteobacteria, including Nitrosomonas (including Nitrosococcus motileus), Nitrosospira, Nitrosomonas, and Nitrosovibrio, while a cluster of Nitrosococci belongs to the subclass α-Proteobacteria. However, the investigation of the count, diversity, and abundance of AOB in engineered systems or their natural environments remains a complicated process using traditional culture-dependent techniques due to their complexity.

[0004] In summary, due to the difficulty in isolating and purifying AOB strains, it is difficult to obtain AOB strains. The isolation and screening of ammonia oxidizing bacteria is beneficial to the types and diversity of ammonia oxidizing bacteria, increasing the application potential of chemoautotrophic ammonia oxidizing bacteria, and the research on this type of functional microorganisms will also provide microbial resource guarantees for ecological environment governance and resource geochemical cycles. Summary of the invention

[0005] In order to overcome the shortcomings and deficiencies of the prior art, the primary purpose of the present invention is to provide a Nitrosomonas with high ammonia oxidation activity, which solves the problem that the existing isolated ammonia oxidizing bacteria are of few species and small quantities.

[0006] Another object of the present invention is to provide the application of the above-mentioned Nitrosomonas.

[0007] The purpose of the present invention is to control the O2 concentration of the sludge sample in the liquid culture medium and the solid plate so that the AOB strain grows faster and better, thereby obtaining a strain of Nitrosomonas. The present invention verifies through experiments that the strain Nitrosomonas sp.LY-1 of the present invention has a strong ability to quickly convert ammonia nitrogen into nitrite nitrogen, so it can be used to remove ammonia nitrogen and has a wide range of application prospects.

[0008] The purpose of the present invention is achieved through the following technical solutions:

[0009] A strain of nitrosomonas with high ammonia oxidation activity, named Nitrosomonas sp. LY-1, with a preservation number of GDMCC No: 65307, was preserved on October 21, 2024 at the Guangdong Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, China.

[0010] The use of the above-mentioned Nitrosomonas with high ammonia oxidation activity in removing ammonia nitrogen, and further in converting ammonia nitrogen into nitrite nitrogen; preferably comprises the following steps: placing the above-mentioned Nitrosomonas with high ammonia oxidation activity in a medium containing ammonia nitrogen (NH4 + -N) environment.

[0011] The growth is performed under an oxygen concentration of 3 to 21%; further, the growth is performed under an oxygen concentration of 5 to 21%.

[0012] The ammonia nitrogen includes at least one of ammonia nitrogen compounds such as ammonium chloride, ammonium sulfate and urea.

[0013] The initial ammonia nitrogen concentration in the environment is 1-100 mM; preferably 1.5-100 mM; further 1.5-50 mM; further 5-50 mM; further 5-10 mM.

[0014] The salinity of the environment is 0-200 mM (excluding 200 mM); further 0-100 mM.

[0015] The environment preferably includes at least one of water and soil.

[0016] The growth temperature is 18-37°C, more preferably 30-37°C.

[0017] Compared with the prior art, the present invention has the following advantages and effects:

[0018] The chemoautotrophic nitrosomonas LY-1 provided by the present invention is a new species, which expands the types and diversity of ammonia-oxidizing bacteria Nitrosomonas strains. The strain LY-1 can perform biological denitrification under aerobic conditions and can quickly and efficiently convert ammonia nitrogen into nitrite nitrogen. It can be used for the treatment of wastewater or soil containing high ammonia nitrogen, that is, the strain has the biological function of biological denitrification. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 The colonies and transparent circles formed by the ammonia-oxidizing bacteria enriched liquid of the sludge sample in Longtan Park, Liuzhou City, in a double-layer serum bottle sealed solid plate under the headspace of CaCO3 and 5% O2; among them, the left: cultured for 10 days; the right: cultured for 30 days.

[0020] Figure 2 This is a phylogenetic tree diagram based on the 16S rDNA of Nitrosomonas LY-1.

[0021] Figure 3 The cell morphology and colony morphology of Nitrosomonas LY-1; A is a Gram staining photo (100×), scale: 200 pixels; B is a colony morphology photo.

[0022] Figure 4 This is a graph showing the ammonia oxidation metabolism and growth curve results of Nitrosomonas LY-1 under normoxia, 1.5mM NH4Cl, and 2.5mM NaHCO3 growth conditions.

[0023] Figure 5 This is the nitrite nitrogen production of Nitrosomonas LY-1 under normoxia, 1.5mM NH4Cl, 2.5mM NaHCO3, and 7 different NaCl concentrations (10, 50, 100, 200, 300, 400, 500mM).

[0024] Figure 6 The nitrite nitrogen production of Nitrosomonas LY-1 under normoxia, 2.5mM NaHCO3, and 6 different NH4Cl concentrations (1, 5, 10, 20, 50, 100mM).

[0025] Figure 7 The nitrite nitrogen production of Nitrosomonas LY-1 under normoxia, 1.5mM NH4Cl, 2.5mM NaHCO3, and four different culture temperatures (18, 20, 30, and 37°C). DETAILED DESCRIPTION

[0026] The present invention is further described in detail below in conjunction with the examples and drawings, but the embodiments of the present invention are not limited thereto. The experimental methods in the following examples where specific experimental conditions are not specified are usually carried out under conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used are reagents and materials obtained from commercial sources unless otherwise specified.

[0027] Example 1

[0028] Enrichment, Screening, Isolation and Identification of Chemoautotrophic Nitrosomonas sp.LY-1

[0029] (1) Enrichment culture of ammonia oxidizing bacteria: Sludge samples were collected from the Longtan Park site in Liuzhou City (E110°01′, N23°29′) using a sampler and placed in a 50mL centrifuge tube for later use. 5mL of sludge sample was inoculated into 40mL of inorganic culture medium containing CaCO3 for enrichment culture and cultured in a shaker at 30°C and 150rpm in the dark. Samples were taken every 2 days to measure the ammonia nitrogen consumption and nitrite nitrogen production. Under this condition, the transfer was repeated for 3-6 generations to obtain a stable ammonia oxidizing bacteria (AOB) flora. The components of the inorganic culture medium containing CaCO3 were: MgSO4·7H2O 0.05g / L, KH2PO4 0.1g / L, NaCl 0.5g / L (8.5mM), CaCO3 0.5g / L, NaHCO3 0.21g / L (2.5mM), NH4Cl 5-10mM, and trace element solution 1mL / L. The composition of the trace element solution: EDTA-Na2 5.2g / L, FeSO4·7H2O 2.1g / L, H3BO3 0.03g / L, CuCl2·2H2O0.002g / L, MnC12·4H2O 0.1g / L, Na2MoO4·2H2O 0.036g / L, ZnSO4 0.144g / L, CoCl2·6H2O 0.19g / L, NiCl2·6H2O 0.024g / L, pH adjusted to 6.0 with NaOH. Among them, NaHCO3, NH4Cl mother liquor, and trace element solution were sterilized by filtering with a 0.22μm filter membrane and then added to the sterilized liquid culture medium.

[0030] (2) Isolation and purification of ammonia oxidizing bacteria: After obtaining the stable ammonia oxidizing bacterial flora in step (1) above, take 100 μL of the enrichment solution, dilute it 10 times in a gradient, and then spread it on a plate (the upper layer is a solid inorganic culture medium plate containing CaCO3), seal it with a butyl silicone stopper and an aluminum cap, and then use a vacuum pump to evacuate the headspace gas, and replace the headspace oxygen with nitrogen, so that the oxygen concentration in the headspace of the sealed serum bottle solid plate is 5%. The serum bottle is then placed upright in a 30°C constant temperature incubator and cultured in the dark. After 10 days and 30 days of culture, AOB colonies grow on the culture medium, and transparent circles are formed around the colonies ( Figure 1 ). Pick the above transparent circle and streak culture on the sealed solid plate of serum bottle, and control the oxygen concentration of the headspace to 5% in the same way. After 10 days of culture, when a single colony grows on the plate, the colony can be picked.

[0031] The above-mentioned colonies were picked up in an inorganic culture medium containing CaCO3, cultured in a shaker at 30°C and 150 rpm in the dark, and the ammonia nitrogen consumption and nitrite nitrogen production were tested every day. If nitrite nitrogen was produced steadily, it could be preliminarily determined to be the target ammonia oxidizing bacteria.

[0032] Among them, solid inorganic culture medium containing CaCO3: add 15-20g / L agar on the basis of inorganic culture medium containing CaCO3. After high temperature sterilization, add NaHCO3, NH4Cl mother solution and trace element solution filtered and sterilized with 0.22μm filter membrane; pour 30-40mL solid inorganic culture medium containing CaCO3 into serum bottle, cover with sealing film, shake blue glass sand (25g / bottle) evenly, spread it horizontally on the side of the bottle, and place the bottle to wait for agar to solidify.

[0033] NH4 + Detection:

[0034] 1. Staining solution A: 50 mL water + 8.5 g sodium salicylate + 0.6 g NaOH + 63.9 mg sodium nitrosoferrocyanide dihydrate;

[0035] 2. Staining solution B: 0.2g sodium dichloroisocyanurate + 50mL water;

[0036] 3. 100μL sample + 100μL staining solution A + 100μL staining solution B + 1700μL water;

[0037] 4. Measure the absorbance at a wavelength of 660nm 30 minutes after color development.

[0038] NO2 - Detection:

[0039] 1. Staining solution: Sulfonamide solution: add 600mL of water to a 1000mL volumetric flask, then add 200mL of concentrated phosphoric acid, then add 80g of sulfonamide, dilute to 1000mL, and store at 4℃ for 1 year; N-(1-naphthyl)-ethylenediamine hydrochloride solution: dissolve 0.4g of N-(1-naphthyl)-ethylenediamine hydrochloride in 100mL of water, store at 4℃, and re-prepare the solution if it becomes concentrated; Color developer: 20mL of sulfonamide solution + 20mL of N-(1-naphthyl)-ethylenediamine hydrochloride solution + 20mL of concentrated phosphoric acid in a 100mL brown bottle, store at 4℃, and re-prepare the solution if the color becomes darker;

[0040] 2. 100 μL bacterial supernatant, 1850 μL water, 50 μL color developer;

[0041] 3. After 10 minutes, measure the absorbance at a wavelength of 543 nm.

[0042] (3) 16S rDNA identification of strain LY-1: Pick the single colony obtained above that can perform normal ammonia oxidation reaction in inorganic medium containing CaCO3, use the single colony as a template, and perform PCR amplification with 16S rDNA universal primer 27F / 1492R. The PCR reaction system is: single colony, 0.5μL of upstream primer (10μmol / L), 0.5μL of downstream primer (10μmol / L), 12.5μL of DreamTaq Green PCR Master Mix (2×), and add ddH2O to 25μL. The PCR reaction conditions are: 94℃ pre-denaturation for 5min; 94℃ denaturation for 30s, 54℃ annealing for 30s, 72℃ extension for 90s, 35 cycles; 72℃ extension for 10min. The amplified PCR product was stored in a refrigerator at 4℃. The PCR product was sent to Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. for 16S rDNA sequencing, and the results are as follows:

[0043] 27F: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0044] 1492R: 5′-GGTTACCTTGTTACGACTT-3′.

[0045] Sequencing results:

[0046]

[0047] The strain was identified as AOB by 16S rDNA Blast analysis. Phylogenetic tree analysis showed that AOB strain LY-1 was most similar to Nitrosomonas nitrosa. The similarities between strain LY-1 and 16S rDNA of N. nitrosa, N. communis, N. aestuarii, N. halophila, N. marina and Nitrosopira sp. were 97.91%, 97.14%, 93.75%, 93.67%, 93.68% and 93.43%, respectively. According to reference (Kook JK, et al. Genome-Based Reclassification of Fusobacterium nucleatum Subspecies at the Species Level. Curr Microbiol (2017) 74: 1137-1147.), the strain's 16S The rDNA sequence similarity of 98.65% can be used as the threshold for distinguishing the two strains. Therefore, strain LY-1 is a new species of the genus Nitrosomonas ( Figure 2 The strain was named Nitrosomonas sp.LY-1 and deposited in Guangdong Microbiological Culture Collection Center (GDMCC) with the deposit number GDMCC No: 65307. The deposit date is October 21, 2024. The deposit address is Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong, China.

[0048] In summary, a strain LY-1 was obtained. The colony morphology of the strain and the Gram staining photos under 100× oil microscope are shown in Figure 3 As shown, the bacteria are short rod-shaped, the colonies are colorless and transparent (cultured for 10 days), the edges are complete, the surface is shiny, and it does not produce spores. It is a Gram-negative bacterium, an obligate chemoautotrophic bacterium, with the optimal growth temperature of 30-37°C and the optimal growth pH of 7.0-7.5.

[0049] The carbon source and nitrogen source utilization of the strain LY-1 are shown in Table 1. The data in Table 1 show that the strain LY-1 can grow and metabolize using ammonium chloride, urea, ammonium sulfate and the like as substrates; it can grow and metabolize using lactic acid, glycerol, sodium pyruvate, sodium bicarbonate and the like as carbon sources, among which sodium bicarbonate has the best effect; the strain LY-1 cannot grow and metabolize using organic carbon sources such as sodium formate, sodium acetate, methanol and ethanol.

[0050] Table 1 Utilization of carbon and nitrogen sources of LY-1

[0051]

[0052] Note: "+" means positive, "-" means negative; under normal oxygen conditions.

[0053] Example 2

[0054] The results of the test on the nitrogen metabolism characteristics and growth characteristics of Nitrosomonas sp. LY-1 in the inorganic medium containing CaCO3 and 1.5mM NH4Cl and 2.5mM NaHCO3 under normal oxygen conditions are as follows:

[0055] A single colony of Nitrosomonas sp. LY-1 isolated, purified and identified above was picked up and inoculated into an inorganic culture medium containing CaCO3 (see Example 1 for ingredients) containing 1.5 mM NH4Cl and 2.5 mM NaHCO3 for activation. Under normal oxygen conditions, when the nitrite nitrogen generation concentration reached 0.6 mM, it was transferred into a fresh liquid culture medium containing 1.5 mM NH4Cl and 2.5 mM NaHCO3 at a 5% inoculum. Under normal oxygen conditions, the experimental group was placed in a shaker at 30°C and 150 rpm in a dark shaking culture. Samples were taken every 12 hours to measure the ammonia nitrogen consumption and nitrite nitrogen generation, and the number of cells was calculated by plating. Each parallel group had three groups, and the total reaction time was 108 h.

[0056] The results of the test on the nitrogen metabolism characteristics and growth characteristics of Nitrosomonas sp. LY-1 in the inorganic medium containing CaCO3 and 1.5mM NH4Cl and 2.5mM NaHCO3 under normal oxygen conditions are as follows Figure 4 The results showed that as the culture time increased, the ammonia nitrogen concentration continued to decrease, the nitrite nitrogen concentration continued to increase, and the cell growth rate also continued to increase, reaching 2.1×10 6 CFU / mL.

[0057] Example 3

[0058] The results of the ammonia oxidation metabolism characteristics of Nitrosomonas sp. LY-1 in an inorganic medium containing CaCO3 under normal oxygen conditions, 1.5mM NH4Cl, 2.5mM NaHCO3, and different NaCl concentrations (10, 50, 100, 200, 300, 400, 500mM) are as follows:

[0059] The single colony of Nitrosomonas sp. LY-1 isolated, purified and identified above was picked up, and inoculated into an inorganic culture medium containing CaCO3 containing 1.5 mM NH4Cl and 2.5 mM NaHCO3 for activation. Under normal oxygen conditions, when the nitrite nitrogen production concentration reached 0.6 mM, it was transferred into a fresh liquid culture medium containing 1.5 mM NH4Cl, 2.5 mM NaHCO3, and different NaCl concentrations (10, 50, 100, 200, 300, 400, 500 mM) at a 5% inoculum amount. Under normal oxygen conditions, the experimental group was placed in a shaker at 30°C and 150 rpm in a dark shaking culture. Samples were taken every 24 hours to measure the ammonia nitrogen consumption and nitrite nitrogen production. Each group was paralleled to three groups, and the total reaction time was 6 days.

[0060] The results of the ammonia oxidation metabolism characteristics of Nitrosomonas sp. LY-1 in an inorganic medium containing CaCO3 under normal oxygen conditions, 1.5mM NH4Cl, 2.5mM NaHCO3, and different NaCl concentrations (10, 50, 100, 200, 300, 400, 500mM) are shown in the figure. Figure 5 The results showed that when the NaCl concentration was ≥200mM, LY-1 stopped growing and metabolizing; when the NaCl concentration was <200mM, LY-1 grew and metabolized normally, and when the NaCl concentration was 50mM, the ammonia oxidation rate of LY-1 was the best.

[0061] Example 4

[0062] The results of the ammonia oxidation characteristics test of Nitrosomonas sp. LY-1 in inorganic medium containing CaCO3 and 2.5 mM NaHCO3 and different concentrations of NH4Cl (1, 5, 10, 20, 50, 100 mM) under normal oxygen conditions are as follows:

[0063] The single colony of Nitrosomonas sp. LY-1 isolated, purified and identified above was picked up and inoculated into an inorganic culture medium containing CaCO3 containing 1.5 mM NH4Cl and 2.5 mM NaHCO3 for activation. Under normal oxygen conditions, when the concentration of nitrite nitrogen generated reached 0.6 mM, it was transferred into fresh liquid culture medium containing different concentrations of NH4Cl (1, 5, 10, 20, 50, 100 mM) and 2.5 mM NaHCO3 at a 5% inoculum. Under normal oxygen conditions, the experimental group was placed in a shaker at 30°C and 150 rpm in a dark shaking culture. Samples were taken every 24 hours to measure the ammonia nitrogen consumption and nitrite nitrogen generation. Each group was paralleled to three groups, and the total reaction time was 8 days.

[0064] The results of the ammonia oxidation metabolism characteristics of Nitrosomonas sp. LY-1 in inorganic medium containing CaCO3 and 2.5 mM NaHCO3 and different concentrations of NH4Cl (1, 5, 10, 20, 50, 100 mM) under normal oxygen conditions are shown in the figure. Figure 6 The results showed that when the NH4Cl concentration was greater than 100 mM, the ammonia oxidation rate of LY-1 was inhibited. When the NH4Cl concentration was less than or equal to 100 mM, the ammonia oxidation rate of LY-1 increased with the increase of substrate concentration, but the final product (nitrite nitrogen concentration) was maintained at 5 mM. It is speculated that 2.5 mM NaHCO3 may only provide LY-1 with 5 mM NH4Cl consumption.

[0065] Example 5

[0066] The results of the ammonia oxidation characteristics test of Nitrosomonas sp. LY-1 in inorganic medium containing CaCO3 at different temperatures (18, 20, 30, 37°C) under normal oxygen conditions are as follows:

[0067] The single colony of Nitrosomonas sp. LY-1 isolated, purified and identified above was picked up, and inoculated into an inorganic culture medium containing CaCO3 containing 1.5 mM NH4Cl and 2.5 mM NaHCO3 for activation. Under normal oxygen conditions, when the nitrite nitrogen production concentration reached 0.6 mM, it was transferred into fresh liquid culture medium containing 1.5 mM NH4Cl and 2.5 mM NaHCO3 at different temperatures (18, 20, 30, 37°C) at a 5% inoculum. Under normal oxygen conditions, the experimental group was placed in a shaker at 30°C and 150 rpm in a dark shaking culture. Samples were taken every 24 hours to measure the ammonia nitrogen consumption and nitrite nitrogen production. Each group was paralleled to three groups, and the total reaction time was 6 days.

[0068] The results of the ammonia oxidation metabolism characteristics of Nitrosomonas sp. LY-1 in inorganic medium containing CaCO3 at normal oxygen conditions, 1.5mM NH4Cl, 2.5mM NaHCO3, and different temperatures (18, 20, 30, 37°C) are shown in Figure 7 The results showed that as the culture temperature increased, the ammonia nitrogen concentration continued to decrease, the nitrite nitrogen concentration continued to increase, and the ammonia oxidation rate was: 37℃>30℃>18℃>20℃.

[0069] 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 Nitrosomonas with high ammonia oxidation activity, characterized in that: The strain is named Nitrosomonas sp. LY-1, with a deposit number of GDMCC No: 65307. It was deposited on October 21, 2024 at the Guangdong Microbial Culture Collection Center, Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou City, Guangdong Province, China.

2. Use of the Nitrosomonas with high ammonia oxidation activity according to claim 1 in deammoniation.

3. The use according to claim 2, characterized in that: Application in converting ammonia nitrogen into nitrite nitrogen.

4. The use according to claim 2 or 3, characterized in that: The method comprises the following steps: placing the nitrosomonas with high ammonia oxidation activity as claimed in claim 1 in an environment containing ammonia nitrogen for growth.

5. The use according to claim 4, characterized in that: The ammonia nitrogen includes at least one of ammonium chloride, ammonium sulfate and urea.

6. The use according to claim 4, characterized in that: The growth is performed under an oxygen concentration of 3 to 21%; further, the growth is performed under an oxygen concentration of 5 to 21%.

7. The use according to claim 4, characterized in that: The initial ammonia nitrogen concentration in the environment is 1-100 mM; further 1.5-100 mM; further 1.5-50 mM; further 5-50 mM; further 5-10 mM.

8. The use according to claim 4, characterized in that: The salinity of the environment is 0-200 mM, excluding 200 mM; further, 0-100 mM.

9. The use according to claim 4, characterized in that: The environment includes at least one of water and soil.

10. The use according to claim 4, characterized in that: The growth temperature is 18-37°C; further 30-37°C.

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