Alkali-resistant, facultatively aerobic Pseudomonas XYH-2 and its denitrification performance application

Abstract

The invention discloses an alkali-resistant, facultative aerobic Pseudomonas XYH‑2 and a denitrification performance application thereof. The Pseudomonas (Pseudomonas guariconensis) XYH‑2 is deposited in China Center for Type Culture Collection, address: Wuhan University, Wuhan, China, postal code: 430072, deposit number: CCTCC NO: M 2022459, deposit date: April 22, 2022. The invention provides an alkali-resistant, facultative aerobic Pseudomonas (Pseudomonas guariconensis) XYH‑2, which can achieve a nitrate nitrogen reduction rate of 100% with an initial concentration of 100 mg / L within 48 hours. The discovery of the strain is of great significance for further research on biological denitrification.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to an alkali-resistant, facultatively aerobic Pseudomonas XYH-2 and its denitrification performance application. Background Art

[0002] Nitrate has become the main nitrogen-containing pollutant in water due to its high water solubility. High concentrations of nitrate can cause a series of environmental problems, such as eutrophication of rivers, and ultimately threaten human health. Therefore, nitrate remediation has become a major topic that has received widespread attention in recent years.

[0003] At present, the methods for removing nitrate from wastewater mainly include biological denitrification, ion exchange, electrodialysis, reverse osmosis, zero-valent iron and zero-valent magnesium physical and chemical reduction, etc. Among them, biological denitrification is currently the most effective way to reduce nitrogen pollution. Many reports have shown that biological denitrification has higher removal efficiency, lower operating costs, and environmental friendliness compared with physical and chemical methods. The biological denitrification process is mainly completed by anaerobic / aerobic denitrifying bacteria, which use oxygen and nitrate as terminal electron acceptors for aerobic respiration and denitrification.

[0004] The present invention is a new facultative aerobic denitrifying bacteria, named Pseudomonas XYH-2. The present invention uses nitrate as the sole nitrogen source and studies the impact resistance of strain XYH-2 to medium and high temperatures, extremely high / low dissolved oxygen, high C / N ratio and pH. The test results show that strain XYH-2 has good tolerance to medium and high alkalinity, with pH = 9 being the best, and 100 mg / L of nitrate nitrogen is completely removed within 20 hours. In addition, the growth rate of the strain is sensitive to the dissolved oxygen concentration. The higher the dissolved oxygen content, the higher the OD of the bacteria. 600 The larger the C / N ratio, the higher the removal rate of nitrate nitrogen. And under the condition of high C / N ratio, the cell density and denitrification performance of strain XYH-2 have been greatly improved. Therefore, strain XYH-2 can be used for biological denitrification of wastewater with high C / N ratio and high alkalinity. Summary of the invention

[0005] The purpose of the present invention is to provide an alkali-resistant, facultative aerobic Pseudomonas XYH-2 and its denitrification performance application in view of the deficiencies of the prior art.

[0006] The objective of the present invention is achieved through the following technical solutions:

[0007] In the first aspect, the present invention provides an alkali-resistant, facultative aerobic Pseudomonas XYH-2, which is classified as Pseudomonas guariconensis XYH-2 and has been deposited in the China Center for Type Culture Collection on April 22, 2022, with a deposit number of CCTCC NO: M 2022459; the 16S rRNA sequence of XYH-2 is shown in SEQ ID NO.1.

[0008] In a second aspect, the present invention provides an application of an alkali-resistant, facultatively aerobic Pseudomonas XYH-2 in biological denitrification.

[0009] Furthermore, the application is specifically as follows: inoculating the alkali-resistant, facultative aerobic Pseudomonas XYH-2 into a first DM liquid selective culture medium with nitrate nitrogen as the only nitrogen source, and culturing at 25-50°C and 120-200rpm to achieve degradation of nitrate nitrogen; the final concentration composition of the first DM liquid selective culture medium is: nitrogen source 100-1000 mg / L, carbon source 400-15000 mg / L, KH2PO4 1500 mg / L, MgSO4·7H2O 100 mg / L, Na2HPO4·12H2O 7715 mg / L, trace elements, the solvent is water, and the pH value is 7.0-9.0; the final concentration composition of the trace elements is: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O240mg / L, MnCl2·4H2O 990mg / L, H3BO4 14mg / L, the solvent is water.

[0010] Furthermore, the alkali-resistant, facultative aerobic Pseudomonas XYH-2 is measured by OD 600 The inoculation is carried out in the form of a bacterial suspension with a value of 0.9 to 0.95, and the volume inoculation amount is 1%.

[0011] Furthermore, the carbon source is sodium acetate, glucose, sodium succinate, sodium oxalate or ethanol.

[0012] Furthermore, the carbon-nitrogen mass ratio is 7:1 to 15:1.

[0013] Furthermore, the carbon-nitrogen mass ratio is 15:1, and the carbon source is 1500 mg / L sodium succinate.

[0014] Furthermore, the bacterial suspension is prepared according to the following steps:

[0015] (1) DM medium culture: the alkali-resistant, facultative aerobic Pseudomonas XYH-2 was inoculated into the second DM liquid selective medium for culture at 30°C for 1 day to obtain the culture medium bacteria; the final concentration composition of the second DM liquid selective medium was: NaNO3 100 mg / L, sodium succinate 700 mg / L, KH2PO4 1500 mg / L, MgSO4·7H2O 100 mg / L, Na2HPO4·12H2O7715 mg / L, trace elements, the solvent was water, and the pH value was 7.0; the final concentration composition of the trace elements was: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430 mg / L, CoCl2·6H2O240 mg / L, MnCl2·4H2O 990mg / L, H3BO4 14mg / L, solvent is water;

[0016] (2) Seed culture: A colony was picked from the culture medium and inoculated into LB seed culture medium, and cultured at 30° C. for 12 h to obtain a bacterial suspension; the final concentration of the LB seed culture medium was as follows: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, the solvent was water, and the pH value was 7.0.

[0017] The beneficial effects of the present invention are as follows: the present invention provides an alkali-resistant, facultative aerobic Pseudomonas guariconensis XYH-2 which can achieve a 100% reduction rate of nitrate nitrogen with an initial concentration of 100 mg / L within 48 hours, and the discovery of the strain is of great significance for further research on biological denitrification. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the transmission electron micrograph of strain XYH-2;

[0019] Figure 2 This is the phylogenetic tree diagram of strain XYH-2;

[0020] Figure 3 NO3 of strain XYH-2 under different carbon source conditions — -N reduction performance comparison chart, where Figure 3 a) is the NO3 of strain XYH-2 under the condition that the carbon source is sodium succinate — -N reduction performance comparison chart, Figure 3 b) is the NO3 of strain XYH-2 when the carbon source is sodium acetate — -N reduction performance comparison chart, Figure 3 c) is the NO3 of strain XYH-2 when the carbon source is ethanol — -N reduction performance comparison chart, Figure 3 d) is the NO3 of strain XYH-2 when the carbon source is glucose — -N reduction performance comparison chart, Figure 3 e) is the NO3 of strain XYH-2 under the condition that the carbon source is sodium oxalate — -N reduction performance comparison chart;

[0021] Figure 4 is the NO3 of strain XYH-2 under different C / N mass ratios — -N reduction performance comparison chart, where Figure 4 a) is the NO3 of strain XYH-2 under the condition of C / N mass ratio of 2:1 — -N reduction performance comparison chart, Figure 4 b) NO3 of strain XYH-2 under the condition of C / N mass ratio of 4:1 — -N reduction performance comparison chart, Figure 4 c) is the NO3 of strain XYH-2 under the condition of C / N mass ratio of 10:1 — -N reduction performance comparison chart, Figure 4 d) is the NO3 of strain XYH-2 under the condition of C / N mass ratio of 15:1 — -N reduction performance comparison chart;

[0022] Figure 5 is the NO3 of strain XYH-2 under different initial pH conditions — -N reduction performance comparison chart, Figure 5 a) is the NO3 of strain XYH-2 under the condition of initial pH value of 4.0 — -N reduction performance comparison chart, Figure 5 b) is the NO3 of strain XYH-2 under the condition of initial pH value of 5.0 — -N reduction performance comparison chart, Figure 5 c) is the NO3 of strain XYH-2 under the condition of initial pH value of 6.0 — -N reduction performance comparison chart, Figure 5 d) is the NO3 of strain XYH-2 under the condition of initial pH value of 7.0 — -N reduction performance comparison chart, Figure 5 e) is the NO3 of strain XYH-2 under the condition of initial pH value of 8.0 — -N reduction performance comparison chart, Figure 5 f) is the NO3 of strain XYH-2 under the condition of initial pH value of 9.0 — -N reduction performance comparison chart, Figure 5 g) is the NO3 of strain XYH-2 under the condition of initial pH value of 10.0 — -N reduction performance comparison chart;

[0023] Figure 6 is the NO3 of strain XYH-2 under different initial nitrogen source concentrations — -N reduction performance comparison chart, Figure 6 a) is the NO3 of strain XYH-2 under the condition of initial nitrogen source concentration of 100 mg / L — -N reduction performance comparison chart, Figure 6 b) is the NO3 of strain XYH-2 under the condition of initial nitrogen source concentration of 200 mg / L — -N reduction performance comparison chart, Figure 6 c) is the NO3 of strain XYH-2 under the condition of initial nitrogen source concentration of 400 mg / L — -N reduction performance comparison chart, Figure 6 d) is the NO3 of strain XYH-2 under the condition of initial nitrogen source concentration of 600 mg / L — -N reduction performance comparison chart, Figure 6 e) is the NO3 of strain XYH-2 under the condition of initial nitrogen source concentration of 800 mg / L — -N reduction performance comparison chart, Figure 6 f) is the NO3 of strain XYH-2 under the condition of initial nitrogen source concentration of 1000 mg / L — -N reduction performance comparison chart;

[0024] Figure 7 NO3 of strain XYH-2 under different initial temperature conditions — -N reduction performance comparison chart, Figure 7 a) is the NO3 of strain XYH-2 at an initial temperature of 25℃ — -N reduction performance comparison chart, Figure 7 b) is the NO3 of strain XYH-2 at an initial temperature of 30°C — -N reduction performance comparison chart, Figure 7 c) is the NO3 of strain XYH-2 at an initial temperature of 37°C — -N reduction performance comparison chart, Figure 7 d) is the NO3 of strain XYH-2 at an initial temperature of 40°C — -N reduction performance comparison chart, Figure 7 e) is the NO3 of strain XYH-2 at an initial temperature of 45°C — -N reduction performance comparison chart, Figure 7 f) is the NO3 of strain XYH-2 at an initial temperature of 50°C — -N reduction performance comparison chart;

[0025] Figure 8NO3 of strain XYH-2 under different shaking speed conditions — -N reduction performance comparison chart, Figure 8 a) is the NO3 of strain XYH-2 under the condition of shaking speed of 0 rpm — -N reduction performance comparison chart, Figure 8 b) NO3 of strain XYH-2 at a shaking speed of 40 rpm — -N reduction performance comparison chart, Figure 8 c) is the NO3 of strain XYH-2 under the condition of shaking speed of 80 rpm — -N reduction performance comparison chart, Figure 8 d) is the NO3 of strain XYH-2 under the condition of shaking speed of 120 rpm — -N reduction performance comparison chart, Figure 8 e) is the NO3 of strain XYH-2 under the condition of shaking speed of 160 rpm — -N reduction performance comparison chart, Figure 8 f) is the NO3 of strain XYH-2 under the condition of shaking speed of 200 rpm — -N reduction performance comparison chart. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0027] The experimental methods below that do not specify specific experimental conditions are usually carried out under conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials and reagents used, unless otherwise specified, are those obtained from commercial sources.

[0028] The present invention provides an alkali-resistant, facultative aerobic Pseudomonas XYH-2, which is named Pseudomonas guariconensis XYH-2 by microbial classification, and has been deposited in the China Center for Type Culture Collection on April 22, 2022, with the address being: Wuhan University, Wuhan, China, with a postal code of 430072; the deposit number is CCTCC NO: M 2022459; the 16S rRNA sequence of the XYH-2 is shown in SEQ ID NO.1.

[0029] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0030] Example 1 Isolation, purification and identification of Pseudomonas guariconensis XYH-2

[0031] 1. Isolation and purification of Pseudomonas guariconensis XYH-2

[0032] Pseudomonas guariconensis XYH-2 was screened from the aerobic pool sludge of Hangzhou Qige Sewage Treatment Plant. The specific steps are as follows:

[0033] (1) Sampling: Sampling was performed at multiple points from the sludge in the aerobic pool of the Qige Wastewater Treatment Plant in Hangzhou as the raw material for screening the alkali-resistant, facultatively aerobic Pseudomonas XYH-2;

[0034] (2) Strain isolation: Take an appropriate amount of activated sludge from the aerobic pool sludge, let it stand for 2 hours, take 10 mL of the supernatant and inoculate it into a 250 mL culture bottle containing 100 mL of the second DM liquid selection medium, shake and culture it on a constant temperature shaker at 30°C and 160 rpm for 24 hours, repeat 3 times; dilute the above bacterial solution by 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 The obtained bacterial liquid was separated and purified by multiple plate streaking using DM solid selection medium to obtain a single colony, which was recorded as strain XYH-2.

[0035] The final concentration composition of the second DM liquid selection culture medium is: NaNO3 100mg / L, sodium succinate 700mg / L, KH2PO4 1500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, the solvent is water, and the pH value is 7.0; the final concentration composition of the trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O990mg / L, H3BO4 14mg / L, and the solvent is water.

[0036] The final concentration composition of the DM solid selection medium is: NaNO3 100mg / L, sodium succinate 700mg / L, KH2PO4 1500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, agar 20g / L, trace elements, the solvent is water, and the pH value is 7.0; the final concentration composition of the trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O 250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO4 14mg / L, and the solvent is water.

[0037] 2. Identification of strain XYH-2

[0038] a. Physiological and biochemical characteristics of strain XYH-2

[0039] The obtained strain XYH-2 was subjected to morphological observation and physiological and biochemical identification. The colony color was white, the colony was small single colony, without spores, transparent, with smooth surface and neat edges. The morphology of the bacteria was observed under transmission electron microscope, such as bacillus. Figure 1 The optimum pH value for its growth is 9.0 and the optimum temperature is 30℃.

[0040] b. 16S rRNA sequence analysis of strain XYH-2

[0041] The DNA of strain XYH-2 was extracted and purified using a 3S column centrifugal environmental sample DNA recovery kit (V2.2, Zhejiang Tianke Biotechnology Co., Ltd.) and stored at 4°C. The 16S rRNA was amplified by PCR using bacterial universal primers 27F (forward primer 27F, 5AGAGTTTGA TCCTGG CTC AG-3') and 1492R (reverse primer 1492R5-GGT TAC CTT GTT ACG ACT T-3').

[0042] The PCR reaction system (50uL) included 1.75uL of template DNA, 1uL of primers 27f and 1492r, 3uL of MgC12 (25mmol / L), 0.25uL of Tag enzyme (5U / uL), 5uL of PCR buffer (10x), 4uL of dNTP (2.5mmol / L), and 34mL of redistilled water.

[0043] PCR amplification program: 94°C pre-denaturation for 4 min: 4°C denaturation for 1 min, 59°C annealing for 1 min, 72°C extension for 1.5 min, 35 cycles; 72°C extension for 10 min: 4°C hold for 10 min.

[0044] The PCR product was subjected to 16S RNA sequence testing (Zhejiang Tianke), and the 16S RNA sequence of strain XYH-2 is shown in SEQ ID NO.1.

[0045] The 16S rRNA sequence of strain XYH-2 was uploaded to the gene sequence in Genbank for homology comparison, and it was found that it belonged to the genus Pseudomonas, and had the highest homology with Pseudomonas guariconensis, reaching 99.6%. Figure 2 The phylogenetic tree of strain XYH-2. To further determine the reliability of the identification results, after physiological and biochemical experiments, it was finally determined that strain XYH-2 belonged to Pseudomonas guariconensis. Therefore, strain XYH-2 was named Pseudomonas guariconensis XYH-2 and deposited in the China Center for Type Culture Collection. Its deposit number is CCTCC NO: M2022459, and the deposit date is April 22, 2022. The address is: Wuhan University, Wuhan, China, and the postal code address is: 430072.

[0046] Example 2: Preparation of bacterial suspension with alkali-resistant, facultative aerobic Pseudomonas XYH-2 as active ingredient

[0047] The preparation process of the bacterial suspension is as follows:

[0048] (1) DM medium culture: the alkali-resistant, facultative aerobic Pseudomonas XYH-2 was inoculated into the second DM liquid selective medium for culture at 30°C for 1 day to obtain the culture medium bacteria;

[0049] (2) Seed culture: A colony was picked from the culture medium and inoculated into LB seed culture medium, and cultured at 30° C. for 12 h to obtain a bacterial suspension; the final concentration of the LB seed culture medium was as follows: NaCl 10 g / L, tryptone 10 g / L, yeast extract 5 g / L, the solvent was water, and the pH value was 7.0.

[0050] Example 3: Denitrification performance test of Pseudomonas guariconensis XYH-2

[0051] 1. Denitrification performance test of Pseudomonas guariconensis XYH-2 under different carbon source conditions

[0052] Pseudomonas guariconensis XYH-2 reduces NO3 — -N experiment, the experimental results are as follows Figure 3shown; from Figure 3 It can be seen that the best carbon source is sodium succinate.

[0053] The specific experimental plan is as follows:

[0054] Using NaNO3 as the sole nitrogen source, the OD 600 The bacterial suspension with a concentration of 0.93 was inoculated into DM liquid selective medium A (carbon source is sodium succinate, C / N mass ratio is 7:1), DM liquid selective medium B (carbon source is sodium acetate, C / N mass ratio is 7:1), DM liquid selective medium C (carbon source is ethanol, C / N mass ratio is 7:1), DM liquid selective medium D (carbon source is glucose, C / N mass ratio is 7:1), DM liquid selective medium E (carbon source is sodium oxalate, C / N mass ratio is 7:1) at a volume concentration of 1% inoculation amount, and cultured at 30°C and 160rpm under shaking conditions, and culture solutions were obtained at culture times of 0h, 8h, 16h, 24h, 32h, 40h and 48h, respectively, and 3mL of the culture solution was taken as a test sample, and the bacterial density (OD 600 value) and nitrate (NO3 - ), nitrite (NO2 - ) and ammonia nitrogen (NH4 + ) concentration, the experimental results are as follows Figure 3 shown.

[0055] Carbon sources provide important energy for the growth of microorganisms and constitute the cell material of microorganisms. Different carbon sources have different effects on microbial reduction due to their different structures and molecular weights. Figure 3 It can be seen that Pseudomonas guariconensis XYH-2 produces NO3 when sodium succinate is used as the carbon source. — -N degradation rate is relatively high, and the degradation rate is the lowest when sodium oxalate is used as the carbon source. The conclusion drawn from the study is that the chemical structure and molecular weight of the carbon source used have a great influence on the reduction efficiency. Generally, the simpler the structure and the smaller the molecular weight of the carbon source, the more conducive it is to the utilization of microorganisms.

[0056] Final concentration composition of DM liquid selection medium A: NaNO3 100mg / L, sodium succinate 700mg / L, KH2PO4 1500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements 2mg / L, solvent is water, pH value is 7.0;

[0057] The final concentration of trace elements is: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430 mg / L, CoCl2·6H2O 240 mg / L, MnCl2·4H2O 990 mg / L, H3BO4 14 mg / L, and the solvent is water.

[0058] Final concentration composition of DM liquid selection medium B: NaNO3 100 mg / L, sodium acetate 700 mg / L, KH2PO4 1500 mg / L, MgSO4·7H2O 100 mg / L, Na2HPO4·12H2O 7715 mg / L, trace elements 2 mg / L, solvent is water, pH value is 7.0;

[0059] The final concentration of trace elements is: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430 mg / L, CoCl2·6H2O 240 mg / L, MnCl2·4H2O 990 mg / L, H3BO4 14 mg / L, and the solvent is water.

[0060] The final concentration of DM liquid selection medium C is as follows: NaNO3 100mg / L, ethanol 700mg / L, KH2PO4 1500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements 2mg / L, solvent is water, pH value is 7.0;

[0061] The final concentration of trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O 250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO414mg / L, and the solvent is water.

[0062] The final concentration of DM liquid selection medium D is as follows: NaNO3 100mg / L, glucose 700mg / L, KH2PO4 1500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements 2mg / L, solvent is water, pH value is 7.0;

[0063] The final concentration of trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O 250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO414mg / L, and the solvent is water.

[0064] Final concentration composition of DM liquid selection medium E: NaNO3 100 mg / L, sodium oxalate 700 mg / L, KH2PO4 1500 mg / L, MgSO4·7H2O 100 mg / L, Na2HPO4·12H2O 7715 mg / L, trace elements 2 mg / L, solvent is water, pH value is 7.0;

[0065] The final concentration of trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O 250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO414mg / L, and the solvent is water.

[0066] 2. Denitrification performance test of Pseudomonas guariconensis XYH-2 under different C / N mass ratios

[0067] Reduction of NO3 by Pseudomonas guariconensis XYH-2 under different C / N mass ratios - -N experiment, the experimental results are as follows Figure 4 shown; from Figure 4 It can be seen that the optimal C / N mass ratio is 15:1.

[0068] The specific experimental plan is as follows:

[0069] Using NaNO3 as the sole nitrogen source and sodium succinate as the carbon source, OD prepared by the method of Example 2 was 600The bacterial suspension with a concentration of 0.93 was inoculated into 5 culture media, namely, DM liquid selective medium A1 (C / N ratio of 2:1), DM liquid selective medium B1 (C / N ratio of 4:1), DM liquid selective medium C1 (C / N ratio of 7:1), DM liquid selective medium D1 (C / N ratio of 10:1), and DM liquid selective medium E1 (C / N ratio of 15:1), at a volume concentration of 1%, and cultured at 30°C and 160 rpm with shaking, and culture solutions were obtained at culture times of 0 h, 8 h, 16 h, 24 h, 32 h, 40 h, and 48 h, respectively, and 3 mL of the culture solution was taken as a test sample, and the bacterial density (OD 600 value) and nitrate (NO3 - ), nitrite (NO2 - ) and ammonia nitrogen (NH4 + ) concentration, the experimental results are as follows Figure 4 shown.

[0070] from Figure 4 It can be seen that Pseudomonas guariconensis XYH-2 is under the condition of C / N mass ratio of 15:1. When sodium succinate is less than 2360 mg / L, since the carbon source plays an important role in the growth and reduction efficiency of the bacteria, if the carbon source is insufficient, there will not be enough electron donors for the growth of the bacteria. When C / N>7, the reduction efficiency reaches the maximum and is stable.

[0071] The final concentration composition of DM liquid selection medium A1 is: NaNO3 100mg / L, sodium succinate 200mg / L, KH2PO41500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0072] The final concentration of trace elements is: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430 mg / L, CoCl2·6H2O 240 mg / L, MnCl2·4H2O 990 mg / L, H3BO4 14 mg / L, and the solvent is water.

[0073] The final concentration composition of DM liquid selection medium B1 is: NaNO3 100mg / L, sodium succinate 400mg / L, KH2PO41500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0074] The final concentration of trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O 250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO4 14mg / L, and the solvent is water.

[0075] The final concentration composition of DM liquid selection medium C1 is: NaNO3 100mg / L, sodium succinate 700mg / L, KH2PO41500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0076] The final concentration of trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O 250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO4 14mg / L, and the solvent is water.

[0077] The final concentration composition of DM liquid selection medium D1 is: NaNO3 100mg / L, sodium succinate 1000mg / L, KH2PO41500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0078] The final concentration of trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O 250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO4 14mg / L, and the solvent is water.

[0079] The final concentration composition of DM liquid selection medium E1 is: NaNO3 100mg / L, sodium succinate 1500mg / L, KH2PO41500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0080] The final concentration of trace elements is: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430 mg / L, CoCl2·6H2O 240 mg / L, MnCl2·4H2O 990 mg / L, H3BO4 14 mg / L, and the solvent is water.

[0081] 3. Denitrification performance of Pseudomonas guariconensis XYH-2 under different initial pH conditions

[0082] Reduction of NO3 by Pseudomonas guariconensis XYH-2 at different initial pH values - -N experiment, the experimental results are as follows Figure 5 shown; from Figure 5 It can be seen that the optimal initial pH is 9.0.

[0083] The specific experimental plan is as follows:

[0084] Using NaNO3 as the sole nitrogen source and sodium succinate as the carbon source, OD prepared by the method of Example 2 was 600 The bacterial suspension with a concentration of 0.93 was inoculated into 7 DM liquid selective culture media E2 with different pH values ​​(pH values ​​were 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 or 10.0, respectively) at a volume concentration of 1%, and cultured at 30°C and 160 rpm with shaking. Culture fluids were obtained at 0 h, 8 h, 16 h, 24 h, 32 h, 40 h and 48 h of culture time, and 3 mL of culture fluid was taken as test samples. The bacterial density (OD 600 value) and nitrate (NO3 - ), nitrite (NO2 - ) and ammonia nitrogen (NH4 + ) concentration, the experimental results are as follows Figure 5 shown.

[0085] from Figure 5 It can be seen that Pseudomonas guariconensis XYH-2 has relatively poor adaptability to strong acid and alkali environments, but when the pH value is 7.0, 8.0 or 9.0, Pseudomonas XYH-2 has a strong adaptability to NO3 —-N degradation rate can reach 72.2%, 68.8% or 100%. Therefore, the optimal initial pH of Pseudomonas XYH-2 is 9.0. It can be seen that Pseudomonas guariconensis XYH-2 is an alkali-resistant strain.

[0086] The final concentration composition of DM liquid selection medium E2 is: NaNO3 100mg / L, sodium succinate 1500mg / L, KH2PO41500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, and the solvent is water;

[0087] The final concentration of trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O 250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO414mg / L, and the solvent is water.

[0088] 4. Denitrification performance test of Pseudomonas guariconensis XYH-2 under different initial nitrogen source concentrations

[0089] Reduction of NO3 by Pseudomonas guariconensis XYH-2 under different initial nitrogen source concentrations - -N experiment, the experimental results are as follows Figure 6 shown; from Figure 6 It can be seen that the optimal initial concentration of nitrogen source is 100 mg / L.

[0090] The specific experimental plan is as follows:

[0091] Using NaNO3 as the sole nitrogen source and sodium succinate as the carbon source, OD prepared by the method of Example 2 was 600The bacterial suspension with a volume concentration of 0.93 was inoculated into 5 culture media, including DM liquid selective medium A3 (NaNO3 1220 mg / L, C / N mass ratio of 15:1), DM liquid selective medium B3 (NaNO3 2440 mg / L, C / N mass ratio of 15:1), DM liquid selective medium C3 (NaNO3 3660 mg / L, C / N mass ratio of 15:1), DM liquid selective medium D3 (NaNO3 4880 mg / L, C / N mass ratio of 15:1), and DM liquid selective medium E3 (NaNO3 6100 mg / L, C / N mass ratio of 15:1), at a volume concentration of 1%, and cultured at 30°C and 160 rpm with shaking. Culture fluids were obtained at culture times of 0 h, 8 h, 16 h, 24 h, 32 h, 40 h, and 48 h, respectively. 3 mL of the culture fluid was taken as a test sample, and the bacterial density (OD 600 value) and nitrate (NO3 - ), nitrite (NO2 - ) and ammonia nitrogen (NH4 + ) concentration, the experimental results are as follows Figure 6 shown.

[0092] from Figure 6 It can be seen that Pseudomonas guariconensis XYH-2 has a strong affinity for NO3 — The degradation rate of -N decreased with the increase of the initial concentration of nitrogen source.

[0093] The final concentration composition of DM liquid selection medium A3 is: NaNO3 100mg / L, sodium succinate 1500mg / L, KH2PO41500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0094] The final concentration of trace elements is: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430 mg / L, CoCl2·6H2O 240 mg / L, MnCl2·4H2O 990 mg / L, H3BO4 14 mg / L, and the solvent is water.

[0095] The final concentration composition of DM liquid selection medium B3 is: NaNO3 200mg / L, sodium succinate 3000mg / L, KH2PO41500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0096] The final concentration of trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O 250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO414mg / L, and the solvent is water.

[0097] The final concentration composition of DM liquid selection medium C3 is: NaNO3 400mg / L, sodium succinate 6000mg / L, KH2PO41500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0098] The final concentration of trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O 250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO414mg / L, and the solvent is water.

[0099] The final concentration composition of DM liquid selection medium D3 is: NaNO3 600mg / L, sodium succinate 9000mg / L, KH2PO41500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0100] The final concentration of trace elements is: EDTA 5000mg / L, CaCl2 5500mg / L, CuSO4·5H2O 250mg / L, FeSO4·7H2O 500mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO414mg / L, and the solvent is water.

[0101] The final concentration composition of DM liquid selection medium E3 is: NaNO3 800mg / L, sodium succinate 12000mg / L, KH2PO4 1500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0102] The final concentration of trace elements is: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430 mg / L, CoCl2·6H2O 240 mg / L, MnCl2·4H2O 990 mg / L, H3BO4 14 mg / L, and the solvent is water.

[0103] The final concentration composition of DM liquid selection medium F3 is: NaNO3 1000mg / L, sodium succinate 15000mg / L, KH2PO4 1500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0104] The final concentration of trace elements is: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430 mg / L, CoCl2·6H2O 240 mg / L, MnCl2·4H2O 990 mg / L, H3BO4 14 mg / L, and the solvent is water.

[0105] 5. Denitrification performance test of Pseudomonas guariconensis XYH-2 under different initial temperature conditions

[0106] Reduction of NO3 by Pseudomonas guariconensis XYH-2 at different initial temperatures - -N experiment, the experimental results are as follows Figure 7 shown; from Figure 7 It can be seen that the optimal initial temperature is 37°C.

[0107] The specific experimental plan is as follows:

[0108] Using NaNO3 as the sole nitrogen source and sodium succinate as the carbon source, OD prepared by the method of Example 2 was 600The bacterial suspension with a concentration of 0.93 was inoculated into 6 DM liquid selective culture media A4 with different initial temperatures (the initial temperatures were 25°C, 30°C, 37°C, 40°C, or 45°C) at a volume concentration of 1%, and cultured at 160 rpm. Culture fluids were obtained at 0 h, 8 h, 16 h, 24 h, 32 h, 40 h, and 48 h of culture time, and 3 mL of culture fluid was taken as test samples. The bacterial density (OD 600 value) and nitrate (NO3 - ), nitrite (NO2 - ) and ammonia nitrogen (NH4 + ) concentration, the experimental results are as follows Figure 7 shown.

[0109] from Figure 7 It can be seen that Pseudomonas guariconensis XYH-2 does not have good tolerance to medium and high temperatures, with 30°C being the best. The removal rate of nitrate nitrogen decreases significantly above 45°C.

[0110] The final concentration composition of DM liquid selection medium A4 is: NaNO3 100mg / L, sodium succinate 1500mg / L, KH2PO41500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0111] The final concentration of trace elements is: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430 mg / L, CoCl2·6H2O 240 mg / L, MnCl2·4H2O 990 mg / L, H3BO4 14 mg / L, and the solvent is water.

[0112] 6. Denitrification performance test of Pseudomonas guariconensis XYH-2 at different shaking speeds

[0113] Reduction of NO3 by Pseudomonas guariconensis XYH-2 under different shaking speed conditions — -N experiment, the experimental results are as follows Figure 8 shown; from Figure 8 It can be seen that the optimal shaking speed is 200 rpm.

[0114] The specific experimental plan is as follows:

[0115] Using NaNO3 as the sole nitrogen source and sodium succinate as the carbon source, the OD prepared in Example 2 was 600 The bacterial suspension of 0.93 was inoculated into 5 identical DM liquid selection medium A5 at a volume concentration of 1%. The 5 DM liquid selection medium A5 were shaken and cultured at different shaking speeds (0 rpm, 40 rpm, 80 rpm, 120 rpm, 160 rpm or 200 rpm) at 37°C. The culture fluids were obtained at 0 h, 8 h, 16 h, 24 h, 32 h, 40 h and 48 h of culture time, and 3 mL of the culture fluid was taken as the test sample. The bacterial density (OD 600 value) and nitrate (NO3 - ), nitrite (NO2 - ) and ammonia nitrogen (NH4 + ) concentration, the experimental results are as follows Figure 8 shown.

[0116] The culture medium is cultured at different shaking speeds, that is, different oxygen is introduced into the culture medium, that is, the dissolved oxygen concentration in the culture medium cultured at different shaking speeds is different; the higher the shaking speed, the greater the dissolved oxygen concentration. Figure 8 As can be seen from the figure, with the increase of the shaking speed, the OD of Pseudomonas guariconensis XYH-2 600 The larger the value, the more sensitive Pseudomonas guariconensis XYH-2 is to dissolved oxygen concentration. 600 The larger the value, the better for NO3 - -N degradation rate is greater. It can be seen that Pseudomonas guariconensis XYH-2 is an aerobic strain.

[0117] DM liquid selection medium A5 contains NaNO3 100mg / L, sodium succinate 1500mg / L, KH2PO4 1500mg / L, MgSO4·7H2O 100mg / L, Na2HPO4·12H2O 7715mg / L, trace elements, solvent is water, pH value is 7.0;

[0118] The final concentration of trace elements is: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430 mg / L, CoCl2·6H2O 240 mg / L, MnCl2·4H2O 990 mg / L, H3BO4 14 mg / L, and the solvent is water.

[0119] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An alkali-resistant, facultatively aerobic Pseudomonas strain XYH-2, characterized in that: The microorganism is classified and named Pseudomonas guariconensis XYH-2, which has been deposited in the China Center for Type Culture Collection on April 22, 2022, and its deposit number is CCTCC NO: M 2022459; the 16S rRNA sequence of XYH-2 is shown in SEQ ID NO.

1.

2. Use of the alkali-resistant, facultative aerobic Pseudomonas XYH-2 according to claim 1 in biological denitrification.

3. The use according to claim 2, characterized in that: The application is specifically as follows: inoculating the alkali-resistant, facultatively aerobic Pseudomonas XYH-2 into a first DM liquid selective culture medium with nitrate nitrogen as the only nitrogen source, and culturing at 25-50° C. and 120-200 rpm to achieve degradation of nitrate nitrogen; the final concentration composition of the first DM liquid selective culture medium is: nitrogen source 100-1000 mg / L, carbon source 400-15000 mg / L, KH2PO4 1500 mg / L, MgSO4·7H2O 100 mg / L, Na2HPO4·12H2O 7715 mg / L, trace elements, the solvent is water, and the pH value is 7.0-9.0; the final concentration composition of the trace elements is: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430mg / L, CoCl2·6H2O 240mg / L, MnCl2·4H2O 990mg / L, H3BO4 14mg / L, the solvent is water.

4. The use according to claim 3, characterized in that: The alkali-resistant, facultative aerobic Pseudomonas XYH-2 was measured by OD 600 The inoculation is carried out in the form of a bacterial suspension with a value of 0.9 to 0.95, and the volume inoculation amount is 1%.

5. The use according to claim 3, characterized in that: The carbon source is sodium acetate, glucose, sodium succinate, sodium oxalate or ethanol.

6. The use according to claim 3, characterized in that: The mass ratio of the carbon source to the nitrogen source is 7 to 15:

1.

7. The use according to claim 3, characterized in that: The mass ratio of the carbon source to the nitrogen source is 15:1, and the carbon source is 1500 mg / L sodium succinate.

8. The use according to claim 4, characterized in that: The bacterial suspension was prepared according to the following steps: (1) DM medium culture: the alkali-resistant, facultative aerobic Pseudomonas XYH-2 was inoculated into the second DM liquid selective medium for culture at 30°C for 1 day to obtain the culture medium bacteria; the final concentration composition of the second DM liquid selective medium was: NaNO3 100 mg / L, sodium succinate 700 mg / L, KH2PO4 1500 mg / L, MgSO4·7H2O 100 mg / L, Na2HPO4·12H2O7715 mg / L, trace elements, the solvent was water, and the pH value was 7.0; the final concentration composition of the trace elements was: EDTA 5000 mg / L, CaCl2 5500 mg / L, CuSO4·5H2O 250 mg / L, FeSO4·7H2O 500 mg / L, ZnSO4 430 mg / L, CoCl2·6H2O240 mg / L, MnCl2·4H2O 990mg / L, H3BO4 14mg / L, solvent is water; (2) Seed culture: Pick colonies from the culture medium and inoculate them into LB seed medium. Culture at 30°C for 12 h to obtain the bacteria. suspension; the final concentration of the LB seed medium is: NaCl 10g / L, tryptone 10g / L, yeast extract 5g / L, The solvent is water, pH 7.0.

Images