Heterotrophic nitrification-aerobic denitrification mucus lactobacillus reuteri D1 and application thereof

Abstract

The invention discloses a lactobacillus reuteri D1 for heterotrophic nitrification-aerobic denitrification and application of the lactobacillus reuteri D1, the strain is separated from river water, can grow by taking ammonia nitrogen, nitrate nitrogen and nitrite nitrogen as a unique nitrogen source or a mixed nitrogen source, and has the functions of heterotrophic nitrification and aerobic denitrification at the same time. The strain has very high treatment efficiency on sewage with the nitrogen concentration of 500 mg / L or below, can tolerate high-salinity sewage with the concentration of 30 g / L, has wide environmental adaptability, is non-toxic and harmless in the treatment process and eco-friendly, and has great application potential.

Description

Technical Field

[0001] The invention relates to the field of environmental microorganisms, and in particular to a heterotrophic nitrification-aerobic denitrification Lactobacillus reuteri D1 and an application thereof. Background Art

[0002] With the rapid development of population and social economy, sewage discharge has become a key factor in water eutrophication and water ecological degradation. Large amounts of sewage discharge have caused the nitrogen content in rivers and lakes to exceed the standard. Nitrogen pollution in my country's water bodies mainly comes from domestic pollution, agricultural pollution and industrial pollution sources. Nitrogen pollution is one of the most important pollution problems facing mankind. When the content of nitrogen compounds in water bodies is too high, it will not only damage the ecological environment, but also have adverse effects on human health. The hazards caused by nitrogen pollution are mainly in the following aspects: (1) Eutrophication of water bodies. Excessive nitrogen in water bodies causes the number of photosynthetic microorganisms (mainly algae) to increase, that is, eutrophication of water bodies occurs, reducing water transparency, consuming dissolved oxygen in water bodies, releasing toxic substances into water bodies, deteriorating water quality, and affecting the growth and reproduction of aquatic organisms. (2) It poses a threat to human health. Increased nitrate content in water increases the risk of methemoglobinemia in infants and the risk of cancer in adults. (3) Ecosystem imbalance. Nitrogen pollution in water bodies causes changes in the structure and function of water ecosystems, which in turn leads to problems such as reduced biodiversity, decline in ecological functions, expansion of invasive species and secondary pollution. The excessive reproduction of algae has destroyed the original ecological balance and weakened the self-purification ability of the water body, resulting in the exclusion of native species and the rapid expansion of invasive species. The secondary pollution caused by the death of algae has further deteriorated the water environment, seriously threatening the health and stability of the water ecosystem.

[0003] Denitrification is an indispensable part of most sewage treatment systems. At present, denitrification technologies mainly include: (1) Physical denitrification: ammonia stripping and ion exchange. (2) Chemical denitrification: chemical precipitation and breakpoint chlorination. (3) Biological denitrification: traditional biological denitrification and new biological denitrification. Physical and chemical denitrification has high operating costs, high energy consumption, and is prone to secondary pollution. The biological denitrification process has attracted much attention in recent decades due to its significant advantages such as economic efficiency, simple process and no secondary pollution. Traditional biological denitrification nitrification and denitrification must be carried out separately, and have disadvantages such as long process flow, large land area, external carbon source, high energy consumption and high cost.

[0004] The discovery of heterotrophic nitrification-aerobic denitrification bacteria has broken through the traditional denitrification theory. Nitrification and denitrification can occur simultaneously under heterotrophic and aerobic conditions. There are reports on heterotrophic nitrification-aerobic denitrification microorganisms in the prior art. Different microbial strains have different nitrogen removal effects and tolerance to different environments. Chinese patent document CN116062904A discloses a salt-tolerant heterotrophic nitrification aerobic denitrification tobacco glutamic acid bacteria (Glutamicibacternicotianae) XH-220 in wastewater treatment and plant growth promotion. The strain has acid resistance, salt resistance and heavy metal resistance, and also has a plant growth promotion effect, and has important application prospects in the fields of sewage treatment and plant growth promotion. Patent CN117987320A discloses a strain of Pseudomonas mendocina PM65 and its application in sewage denitrification, the ammonia nitrogen degradation rate of which can reach 82.6% in 24 hours, and the ammonia nitrogen degradation rate of the strain at a salt concentration of 30 g / L is 78.45%.

[0005] Heterotrophic nitrification-aerobic denitrification bacteria have the advantages of short generation time, fast denitrification rate, and strong environmental tolerance, which makes them have broad application prospects in sewage treatment. Therefore, the development of efficient heterotrophic nitrification-aerobic denitrification bacteria has important practical significance for the denitrification of sewage. Summary of the invention

[0006] In view of the above problems and shortcomings, the purpose of the present invention is to provide a heterotrophic nitrification-aerobic denitrification Lactobacillus reuteri and its application, which can grow under the condition of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen as the only nitrogen source, and can effectively remove them. It not only has efficient nitrogen removal ability, but also has strong high salt tolerance, and is suitable for sewage treatment in extreme environments.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] The present invention provides a strain of Limosilactobacillus reuteri D1, which has been deposited in the China Center for Type Culture Collection on October 25, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M 20242336.

[0009] The Lactobacillus reuteri is a Gram-negative bacterium obtained by sampling from river water and undergoing enrichment culture, separation and purification.

[0010] The effective sequence length of the 16S rDNA of Lactobacillus reuteri is 1464 bp, and its nucleic acid sequence is shown in SEQ ID NO.1.

[0011] Another object of the present invention is to prepare a denitrifying bacterial agent using the Lactobacillus reuteri D1 strain, and the preparation method comprises the following steps: (1) inoculating Lactobacillus reuteri D1 in a solid culture medium for activation and culture to obtain an activated strain; (2) inoculating the activated strain obtained in step (1) in a liquid culture medium for expansion and culture, and adjusting the OD value of the bacterial solution; 600 is 1.0, and the denitrification bacterial agent of Lactobacillus reuteri D1 is obtained.

[0012] Preferably according to the present invention, the solid culture medium is LB solid culture medium, and the liquid culture medium is LB liquid culture medium.

[0013] Preferably, according to the present invention, the culture conditions in step (1) are: culture in a constant temperature incubator at 28-32°C for 2-3 days.

[0014] Preferably, according to the present invention, the culture conditions in step (2) are: 28-32° C., 160-200 rpm shaking culture for 8-9 hours.

[0015] Another object of the present invention is to provide the use of the Lactobacillus reuteri D1 denitrifying bacterial agent obtained by the above preparation method in treating nitrogen-containing wastewater.

[0016] Preferably according to the present invention, in the application, the concentration of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen or mixed nitrogen sources in the nitrogen-containing wastewater is below 500 mg / L.

[0017] Preferably according to the present invention, in the application, the salinity of the nitrogen-containing wastewater is below 30 g / L.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The invention provides a strain of Lactobacillus reuteri D1 and application thereof in sewage treatment. The strain has both heterotrophic nitrification and aerobic denitrification functions, and realizes the simultaneous completion of nitrification and denitrification in the same reactor, which greatly saves floor space and investment costs. The strain has a highly efficient removal effect on nitrogen in water bodies, and can tolerate extreme environments such as high salt, and has low cost, high efficiency, and is environmentally friendly, has wide environmental adaptability, and has great application potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a bar chart showing the removal rates of ammonia nitrogen and nitric nitrogen by different strains;

[0021] Figure 2 Figure 1 is the colony morphology and Gram staining result of Lactobacillus reuteri D1;

[0022] Figure 3is the phylogenetic tree of Lactobacillus reuteri D1;

[0023] Figure 4 The growth and denitrification line graph of Lactobacillus reuteri D1 over time when ammonia nitrogen is the only nitrogen source;

[0024] Figure 5 The growth and nitrogen removal rate of Lactobacillus reuteri D1 when different concentrations of ammonia nitrogen were used as substrates;

[0025] Figure 6 The growth and denitrification line graph of Lactobacillus reuteri D1 over time when nitrate nitrogen is the only nitrogen source;

[0026] Figure 7 The growth and nitrogen removal rate of Lactobacillus reuteri D1 when different concentrations of nitrate nitrogen were used as substrates;

[0027] Figure 8 The growth and denitrification line graph of Lactobacillus reuteri D1 over time when nitrite nitrogen is the only nitrogen source;

[0028] Fig. 9 The growth and nitrogen removal rate of Lactobacillus reuteri D1 when different concentrations of nitrite nitrogen were used as substrates;

[0029] Fig.10 It is a line graph of the growth and denitrification of Lactobacillus reuteri D1 over time when ammonia nitrogen, nitrate nitrogen and nitrite nitrogen are used as mixed nitrogen sources;

[0030] Fig.11 The growth and nitrogen removal rate of Lactobacillus reuteri D1 when different concentrations of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen were used as mixed nitrogen source substrates;

[0031] Fig.12 This is a bar chart showing the growth of strains and nitrogen removal rates under different salinities during nitrification.

[0032] Fig.13 This is a bar chart showing the growth of strains and nitrogen removal rates under different salinities during denitrification (nitrogen source is nitric nitrogen);

[0033] Fig.14 This is a bar chart showing the growth of strains and nitrogen removal rates under different salinities during the denitrification process (nitrogen source is nitrite nitrogen);

[0034] Fig.15 This is a bar chart showing the strain growth and nitrogen removal rate under different salinities during the simultaneous nitrification and denitrification process. DETAILED DESCRIPTION

[0035] The present invention is described in detail below in conjunction with the embodiments and test examples. Obviously, the described embodiments are only embodiments of a part of the present invention, rather than all embodiments, and the protection scope of the present invention is not limited thereto. The test methods used in the following embodiments are conventional methods unless otherwise specified; the materials, reagents, etc. used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0036] Example 1

[0037] Screening, enrichment and isolation of heterotrophic nitrifying and aerobic denitrifying strains:

[0038] 5 mL of sample was taken from Shuxi River, Panhe Campus, Shandong Agricultural University, Tai'an City, Shandong Province, and added into 100 mL of enrichment medium. The bacteria in the sample were enriched under shaking culture conditions of 30 °C and 180 rpm;

[0039] The components of the enrichment medium are as follows: 0.5 g ammonium chloride, 3.5 g sodium acetate, 0.05 g magnesium sulfate heptahydrate, 0.2 g dipotassium hydrogen phosphate, 0.12 g sodium chloride, 0.01 g manganese sulfate monohydrate, 0.01 g ferrous sulfate heptahydrate, and deionized water is added to make the volume to 1 L; the pH value is 7.0-7.5.

[0040] After 48 hours of shaking culture, 100 μL of the enriched culture solution was taken and separated and purified on LB agar plates using the gradient dilution plate method and incubated overnight at 30°C.

[0041] Single colonies were selected according to their morphology, and the selected single colonies were streaked on nitrification medium plates for purification. Through multiple separation and purification, a total of 12 strains with heterotrophic nitrification-aerobic denitrification capabilities were obtained.

[0042] The nitrification medium components are: 0.47 g ammonium sulfate, 5.62 g sodium succinate, 50 mL Weiss salt solution, 15 g agar powder, and deionized water is added to make the volume 1 L; the pH value is 7.0-7.5;

[0043] The formula of Weiss salt solution is: 5.0g of dipotassium hydrogen phosphate, 2.5g of magnesium chloride hexahydrate, 2.5g of sodium chloride, 0.05g of ferrous sulfate heptahydrate, 0.05g of manganese sulfate, add deionized water to make up to 1L; the pH value is 7.0.

[0044] Example 2

[0045] Identification of ammonia nitrogen and nitrate nitrogen removal performance of strains:

[0046] The absorbance of ammonia nitrogen was determined by Nessler spectrophotometer, and the absorbance of nitric nitrogen was determined by ultraviolet spectrophotometer. The corresponding standard curve was made, and the contents of ammonia nitrogen and nitric nitrogen were calculated according to the standard curve. The removal rates of ammonia nitrogen and nitric nitrogen were calculated as follows:

[0047]

[0048] Where D0 represents the ammonia nitrogen / nitrate nitrogen content in the blank nitrification / denitrification medium; D1 represents the ammonia nitrogen / nitrate nitrogen content in the nitrification / denitrification medium after 48 hours of cultivation.

[0049] Preparation of denitrifying bacteria:

[0050] The strains obtained from the above screening were activated and cultured in LB solid medium at 30°C for 3 days, and single colonies were picked into 50mL Erlenmeyer flasks filled with 30mL LB liquid medium, and placed in a constant temperature shaker at 30°C and 180rpm for 8-9h. The OD value of the bacterial solution was adjusted. 600 It is 1.0 denitrifying agent of Lactobacillus mucilaginosus.

[0051] The denitrification bacteria agent obtained above was inoculated into 250mL conical flasks containing 100mL nitrification medium and denitrification medium DM1 at an inoculation rate of 1% (volume ratio), and three parallels were used for each treatment. After culturing at 30°C and 180rpm for 48h, samples were taken, and the ammonia nitrogen absorbance was measured at 625nm using a Nessler spectrophotometer, and the nitrate nitrogen absorbance was measured at 543nm using an ultraviolet spectrophotometer, and the ammonia nitrogen and nitrate nitrogen removal rates were calculated. The results are shown in Figure 2. Figure 1 As shown, all tested strains have heterotrophic nitrification and aerobic denitrification capabilities, among which D1 has the highest removal rates of ammonia nitrogen and nitric nitrogen.

[0052] The above culture medium components are as follows:

[0053] LB liquid culture medium: 10 g tryptone, 5 g yeast extract, 10 g sodium chloride, add deionized water to make up to 1 L; pH value is 7.0.

[0054] Nitration medium: 0.47 g ammonium sulfate, 5.62 g sodium succinate, 50 mL Wei's salt solution, add deionized water to make up to 1 L; pH value is 7.0-7.5.

[0055] Denitrification medium DM1: 0.72 g potassium nitrate, 5.62 g sodium succinate, 50 mL of Wei's salt solution, add deionized water to make up to 1 L; pH value is 7.0-7.5.

[0056] Example 3

[0057] Identification of Limosilactobacillus reuteri D1:

[0058] The morphology and Gram staining results of the strain D1 obtained by the above screening on LB solid medium are as follows: Figure 2 As shown in the figure, the colonies are milky white, smooth and moist, with irregular and opaque edges. Gram staining is red, and the strain is a Gram-negative bacterium.

[0059] The 16S rDNA gene sequence of the strain D1 obtained by the above screening was sequenced, and the specific steps were: extracting the DNA of the strain D1 with a kit, and performing PCR amplification with universal primers for bacterial 16S rDNA gene;

[0060] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'

[0061] 1492R:5'-GGTTACCTTGTTACGACTT-3'

[0062] The PCR amplification system (50 μL) was as follows: template DNA 2 μL, Mix (2×) 25 μL, primer 27F 2 μL, primer 1492R 2 μL, ddH O 19 μL;

[0063] The PCR amplification procedure was as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 1 min, annealing at 58°C for 1 min, extension at 72°C for 2 min, 30 cycles; final extension at 72°C for 10 min, and storage at 4°C;

[0064] The 16S rDNA gene sequence amplification product of strain D1 was commissioned to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The 16S rDNA sequencing result is shown in SEQ ID NO.1. The obtained 16S rDNA sequence was subjected to BLAST analysis with the existing sequences in the NCBI database, and strains with similar homology were selected. The phylogenetic tree was constructed using MEGA software using the Neighbor-joining method. The results are shown in Figure 3 As shown; the strain has a homology of 99.79% with Limosilactobacillus reuteri ATCC 53608 in the database, therefore, the strain D1 was confirmed to be Limosilactobacillus reuteri and named Limosilactobacillus reuteri D1.

[0065] It was deposited in the China Center for Type Culture Collection on October 24, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 20242336.

[0066] Example 4

[0067] Preparation of Limosilactobacillus reuteri D1 denitrifying bacteria agent:

[0068] The preserved strains were activated in LB solid medium, and single colony D1 was picked into a 50 mL Erlenmeyer flask containing 30 mL LB liquid medium. The culture was placed in a constant temperature shaker at 30 °C and 180 r / min for 8 to 9 h, and the OD of the bacterial solution was adjusted. 600 It is 1.0 denitrifying bacteria agent of Lactobacillus reuteri D1.

[0069] Test Example 1

[0070] Denitrification characteristics and nitrogen concentration tolerance of Lactobacillus reuteri D1 (when ammonia nitrogen is the only nitrogen source):

[0071] The denitrifying bacteria agent of Lactobacillus mucilaginosus D1 in Example 4 was inoculated into a 250 mL conical flask containing 100 mL of nitrification medium at a volume ratio of 1%, and cultured at 30°C and 180 rpm with shaking. Three parallel samples were collected for each treatment group. Samples were taken every six hours and the OD 600 The values ​​of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen were measured for 48 hours to analyze the removal of nitrogen sources (ammonia nitrogen, nitrate nitrogen and nitrite nitrogen) and the generation of intermediate products. The composition of the nitrification medium was the same as that in Example 2.

[0072] The Lactobacillus reuteri D1 denitrifying bacteria agent in Example 4 was inoculated into a 250 mL conical flask containing 100 mL of nitrification medium at an inoculum amount of 1%, 2%, 3%, 4%, and 5% (volume ratio). The ammonia nitrogen concentration of the nitrification medium was set to 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L, respectively. The culture was shaken at 30° C. and 180 rpm. After 48 h, samples were taken to determine the OD 600 The values ​​and ammonia nitrogen removal rates were measured to study the tolerance of the strains to different ammonia nitrogen concentrations.

[0073] according to Figure 4 It can be seen that when ammonia nitrogen is the only nitrogen source, the strain D1 is in the logarithmic growth phase from 0 to 18 hours. 600 The value increased sharply, reaching 1.514 at 18h. The amount of ammonia nitrogen also decreased rapidly at this stage. The removal rate of ammonia nitrogen was 96.28% at this time, and there was almost no accumulation of nitrate nitrogen and nitrite nitrogen intermediates by strain D1 during the removal of ammonia nitrogen.

[0074] The tolerance of strain D1 to different ammonia nitrogen concentrations is as follows Figure 5 As shown in the figure, when the initial ammonia nitrogen concentration was between 100 and 500 mg / L, the OD of the bacterial solution after 48 hours of culture was 600The values ​​showed no downward trend and were all above 1.3, indicating that strain D1 could tolerate ammonia nitrogen concentrations of 500 mg / L and below. At the same time, the removal rate of ammonia nitrogen by the strain was above 30%, and when the initial ammonia nitrogen concentrations were 100 and 200 mg / L, the removal rates reached 92.52% and 52.71%, respectively, indicating that strain D1 had a strong tolerance to high ammonia nitrogen and had a good removal effect on ammonia nitrogen concentrations of 500 mg / L and below.

[0075] Test Example 2

[0076] Denitrification characteristics and nitrogen concentration tolerance of Lactobacillus reuteri D1 (when nitrate nitrogen is the only nitrogen source):

[0077] The denitrifying bacterial agent Lactobacillus reuteri D1 in Example 4 was inoculated into a 250 mL conical flask containing 100 mL of denitrification medium DM1 at an inoculum amount of 1% (volume ratio), and the experimental conditions were the same as those in Experimental Example 1. The composition of the denitrification medium DM1 was as shown in Example 2.

[0078] The Lactobacillus reuteri D1 denitrifying bacteria agent in Example 4 was inoculated into a 250 mL conical flask containing 100 mL of denitrification medium DM1 at an inoculum amount of 1%, 2%, 3%, 4%, and 5% (volume ratio). The nitrate nitrogen concentration of the denitrification medium DM1 was set to 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L, respectively. The culture was shaken at 30° C. and 180 rpm. After 48 h, samples were taken to measure the OD 600 The values ​​and nitrate-nitrogen removal rates were used to analyze the tolerance and removal ability of strain D1 to different nitrate-nitrogen concentrations.

[0079] according to Figure 6 It can be seen that when nitrate nitrogen is the only nitrogen source, the strain D1 is in the logarithmic growth phase from 0 to 24 hours, and the OD 600 The value reached 1.329. During this stage, the nitrate nitrogen content decreased rapidly. The nitrate nitrogen removal rate reached 96.29% at 42 hours. In the process of nitrate nitrogen removal, strain D1 had almost no accumulation of nitrite nitrogen intermediates, but accumulated a small amount of ammonia nitrogen intermediates.

[0080] The tolerance of strain D1 to different nitrate concentrations is shown in Figure 7 As shown in the figure, when the initial nitrate concentration was between 100 and 500 mg / L, the OD of the bacterial solution after 48 hours of culture was 600The values ​​were all between 1.23 and 1.6, indicating that strain D1 can tolerate nitrate nitrogen concentrations of 500 mg / L and below. At the same time, the removal rate of nitrate nitrogen by the strain was above 37%, and when the initial nitrate nitrogen concentrations were 100 and 200 mg / L, the removal rates reached 90.61% and 78.80%, respectively, indicating that strain D1 can tolerate a high nitrate nitrogen concentration of 500 mg / L and has a good removal effect on nitrate nitrogen concentrations of 500 mg / L and below.

[0081] Test Example 3

[0082] Denitrification characteristics and nitrogen concentration tolerance of Lactobacillus reuteri D1 (when nitrite nitrogen is the only nitrogen source):

[0083] The Lactobacillus mucilaginosus D1 denitrifying bacteria agent in Example 4 was inoculated into a 250 mL conical flask containing 100 mL of denitrification medium DM2 at an inoculum amount of 1% (volume ratio), and the experimental conditions were the same as those in Experimental Example 1. The denitrification medium DM2 consisted of: 0.49 g of sodium nitrite, 5.62 g of sodium succinate, 50 mL of Weiss salt solution, and deionized water was added to make the volume 1 L, and the pH value was 7.0-7.5.

[0084] The Lactobacillus reuteri D1 denitrifying bacteria agent in Example 4 was inoculated into a 250 mL conical flask containing 100 mL of denitrification medium DM2 at an inoculum amount of 1%, 2%, 3%, 4%, and 5% (volume ratio). The nitrite nitrogen concentration of the denitrification medium DM2 was set to 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L, respectively. The culture was carried out at 30° C. and 180 rpm. After 48 h, samples were taken to determine the OD 600 Values ​​and nitrite-nitrogen removal rates.

[0085] according to Figure 8 It can be seen that when nitrite nitrogen is the only nitrogen source, the strain D1 is in the logarithmic growth phase from 0 to 18 hours, and reaches the maximum growth at 30 hours. 600 The value was 1.344, at which time the maximum removal rate of nitrite nitrogen reached 84.95%, and strain D1 accumulated a small amount of ammonia nitrogen and nitrate nitrogen intermediates during the removal process of nitrite nitrogen.

[0086] The growth and removal capacity of strain D1 under different nitrite nitrogen concentrations are shown in Fig. 9 As shown in Figure 2, when the initial nitrite nitrogen concentration was between 100 and 500 mg / L, the OD of strain D1 600 The value was maintained at 0.95-1.3, and the growth was good. At the same time, the removal rate of nitrite nitrogen by the strain was above 43%. Strain D1 could tolerate a wide range of nitrite nitrogen concentrations and had a good removal effect on nitrite nitrogen at a concentration of 500 mg / L and below.

[0087] Test Example 4

[0088] Denitrification characteristics and nitrogen concentration tolerance of Lactobacillus reuteri D1 (when ammonia nitrogen, nitrate nitrogen and nitrite nitrogen are used as mixed nitrogen sources):

[0089] The Lactobacillus mucilaginosus D1 denitrifying bacteria agent in Example 4 was inoculated into a 250 mL conical flask containing 100 mL of mixed nitrogen source culture medium at an inoculum amount of 1% (volume ratio), and the experimental conditions were the same as those in Experimental Example 1. The mixed nitrogen source composition was: 0.157 g of ammonium sulfate, 0.24 g of potassium nitrate, 0.163 g of sodium nitrite, 5.62 g of sodium succinate, 50 mL of Weiss salt solution, and deionized water was added to make the volume 1 L, and the pH value was 7.0-7.5.

[0090] The Lactobacillus reuteri D1 denitrifying bacteria agent in Example 4 was inoculated into a 250 mL conical flask containing 100 mL of mixed nitrogen source culture medium at an inoculum amount of 1%, 2%, 3%, 4%, and 5% (volume ratio), and the mixed nitrogen source concentration of the culture medium (ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen are evenly distributed) was set to 100 mg / L, 200 mg / L, 300 mg / L, 400 mg / L, and 500 mg / L, respectively. The culture was cultured at 30°C and 180 rpm, and samples were taken after 48 h to determine the OD 600 Values ​​and removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen.

[0091] like Fig.10 As shown in the figure, strain D1 showed the characteristic of preferential utilization of ammonia nitrogen in an environment with ammonia nitrogen, nitrate nitrogen and nitrite nitrogen as mixed nitrogen sources. In the first six hours, the content of ammonia nitrogen decreased rapidly, while nitrate nitrogen and nitrite nitrogen also decreased but to a lesser extent, indicating that the strain first consumed ammonia nitrogen as the main nitrogen source. The utilization of nitrate nitrogen and nitrite nitrogen was relatively slow, and they may be gradually utilized only after the ammonia nitrogen was exhausted; strain D1 was in the logarithmic growth period from 0 to 18 hours, and reached the maximum growth at 18 hours, and its OD 600 The value was 1.355. During the conversion process, the ammonia nitrogen content showed a trend of first decreasing and then increasing. The maximum removal rate was reached at 24 hours. The removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen were 85.57%, 96.79% and 97.44%, respectively. The strain D1 also had strong heterotrophic nitrification and aerobic denitrification capabilities in the mixed nitrogen source.

[0092] like Fig.11 As shown in Figure 2, when the mixed nitrogen concentration increased from 100 mg / L to 500 mg / L, the growth of the strain was not affected by the high nitrogen environment. 600The values ​​were all maintained between 1.2 and 1.4. The removal effect of strain D1 on ammonia nitrogen always remained at a high level, with removal rates of 90.37%, 92.67%, 94.81%, 83.10%, and 55.38%, respectively; the nitrate nitrogen removal rate of strain D1 in mixed nitrogen sources with nitrogen concentrations of 400 mg / L and below was above 48%; at mixed nitrogen concentrations of 100 to 500 mg / L, the removal rate of nitrite nitrogen remained above 50%. In summary, the strain can efficiently remove three forms of nitrogen sources, ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen, in a high-concentration mixed nitrogen source environment, which is in line with the actual situation and has good application prospects for the denitrification treatment of actual sewage.

[0093] Test Example 5

[0094] Removal effect of Lactobacillus reuteri D1 on ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and three mixed nitrogen sources at different salinities:

[0095] The denitrifying bacteria agent of Lactobacillus mucilaginosus D1 in Example 4 was inoculated at a volume ratio of 1% into a 250 mL conical flask containing 100 mL of nitrification and denitrification medium DM1, denitrification medium DM2 and three mixed nitrogen source mediums (ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen were evenly distributed), and cultured at 30°C and 180 rpm, with three parallel samples in each treatment group. The NaCl addition amount was controlled so that the concentration of NaCl in the denitrification medium was 0 g / L, 10 g / L, 20 g / L, and 30 g / L, respectively, to simulate saline wastewater, and cultured continuously for 4 days, and the OD of strain D1 was measured every day. 600 , ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and three mixed nitrogen sources, and the removal ability and tolerance of the strain to different nitrogen sources under different salinities were analyzed.

[0096] like Figures 12 to 15 As shown in the figure, during the nitrification process, due to the inhibition of high salinity, strain D1 grew slowly on the first day, and the ammonia nitrogen removal rate was low at 30 g / L high salinity, only 34.36%. As the culture time increased, strain D1 gradually adapted to the high salinity environment, reaching the highest removal rate (82.55%) and maximum growth (OD) at 30 g / L salinity on the third day. 600=1.16); In the denitrification process, the nitrate removal capacity of strain D1 at 30g / L high salinity decreased significantly. At 20g / L and 30g / L salinity, the nitrate removal rates of strain D1 were 96.30% and 55.89%, respectively. Under different salinities, the nitrite removal rate of strain D1 was relatively stable, maintaining at about 60%; In the simultaneous nitrification and denitrification process, strain D1 had a high removal rate for the three nitrogen sources. With the extension of the culture time, the ammonia nitrogen removal capacity decreased, while the nitrate nitrogen and nitrite nitrogen removal capacities increased. Under lower salinity, the strain had a stronger nitrogen removal capacity. The high salinity of 30g / L inhibited the metabolic activity of the strain. At this time, the removal rates of ammonia nitrogen, nitrate nitrogen and nitrite nitrogen were 91.82%, 41.96% and 48.33%, respectively.

[0097] In summary, the denitrification ability of Limosilactobacillus reuteri D1 of the present invention is strong, and ammonia nitrogen, nitrate nitrogen, and nitrite nitrogen can be effectively removed, and the nitrogen content of water body can be reduced. Ammonia nitrogen and nitrite nitrogen can be effectively removed under 30g / L salinity conditions, and nitrate nitrogen can be effectively removed under 20g / L salinity conditions. In the case of mixed nitrogen sources, ammonia nitrogen can be effectively removed below 30g / L salinity, and nitrate nitrogen and nitrite nitrogen can be effectively removed below 20g / L salinity. In a word, D1 can tolerate 30g / L high salinity environment, can efficiently process ammonia nitrogen, nitrate nitrogen, nitrite nitrogen and mixed nitrogen under 20g / L salinity, and is environmentally friendly, has low operating cost, and has good application prospects.

Claims

1. A strain of Limosilactobacillus reuteri D1 was deposited in the China Center for Type Culture Collection on October 25, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M20242336.

2. A method for preparing a denitrifying bacterial agent of Lactobacillus reuteri D1, characterized in that: The steps are as follows: (1) inoculating the Lactobacillus reuteri D1 described in claim 1 into a solid culture medium for activation and culture to obtain an activated strain; (2) inoculating the activated strain obtained in step (1) into a liquid culture medium for expansion and culture, and adjusting the OD of the bacterial solution 600 is 1.0, and the denitrification bacterial agent of Lactobacillus reuteri D1 is obtained.

3. The method for preparing the denitrifying bacterial agent of Lactobacillus reuteri D1 according to claim 2, characterized in that: The solid culture medium is LB solid culture medium, and the liquid culture medium is LB liquid culture medium.

4. The method for preparing the denitrifying bacterial agent of Lactobacillus reuteri D1 according to claim 2, characterized in that: The culture conditions in step (1) are: culture in a constant temperature incubator at 28-32°C for 2-3 days.

5. The method for preparing the denitrifying bacterial agent of Lactobacillus reuteri D1 according to claim 2, characterized in that: The culture conditions in step (2) are: 28-32° C., 160-200 rpm shaking culture for 8-9 hours.

6. Use of the Lactobacillus reuteri D1 denitrifying bacterial agent obtained by the preparation method according to any one of claims 2 to 5 in treating nitrogen-containing wastewater.

7. The use according to claim 6, characterized in that: The concentration of ammonia nitrogen, nitrate nitrogen, nitrite nitrogen or mixed nitrogen sources in the nitrogen-containing wastewater is below 500 mg / L.

8. The use according to claim 6, characterized in that: The salinity of the nitrogen-containing wastewater is below 30 g / L.

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