A human Staphylococcus aureus strain having both aerobic denitrification and ectoine production functions and its application

Abstract

The present invention discloses a human Staphylococcus with both aerobic denitrification and ectoine production functions and its application, which relates to the field of environmental protection microbial technology. The strain was deposited in the General Microbiological Center (CGMCC) of the China Microbiological Culture Collection Administration on May 9, 2024, with the deposit number of CGMCC No. 30539; the strain EH-02 was isolated from a salt pond bottom mud sample of a salt field in Dalian City. It is a strain that uses small molecular organic matter to convert NO3 into nitrification under aerobic conditions. ‑ The strain which first reduces to nitrite and then to nitrogen has a denitrification efficiency of up to 95.87% when the carbon source is sodium acetate, the nitrogen source is sodium nitrate, the nitrogen source concentration is 100 mgN / L, C / N=5, the salinity is 60 g / L, and the rotation speed is 120 rpm; the strain can also produce the compatible solute ectoine during heterotrophic denitrification, and the maximum accumulation of ectoine is 188.96 mg / g; the bacteria can not only be used in environmental protection fields such as ship high-salt nitrogen-containing wastewater treatment, but also has broad application prospects in the biological preparation of ectoine.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, in particular to a human staphylococcus with both aerobic denitrification and ectoine production functions and application thereof. Background Art

[0002] With the rapid development of the global shipping industry and the continuous increase in the number of ships, the problem of pollutant emissions from ships during navigation has become increasingly prominent. X ) is one of the main pollutants in ship exhaust emissions. After washing, nitrate (NO 3 - ) will cause eutrophication and black and smelly water, which has a bad impact on water functions. Traditional biological denitrification processes are relatively mature, but there are also problems such as large footprint, long treatment cycle and poor system stability, which have a lot of room for improvement. Aerobic denitrification is a new denitrification pathway developed in the past 30 years. It can denitrify under aerobic conditions. This technology can reduce the space required for biological denitrification treatment and reduce the addition of alkalinity, thereby reducing treatment costs.

[0003] In recent years, aerobic denitrifying bacteria have been found in various environments, and most of them are heterotrophic bacteria that can simultaneously remove organic matter and NO in sewage. 3 - . At present, the research on aerobic denitrifying bacteria is mostly focused on conventional environments, while less attention is paid to the denitrification characteristics of strains in extreme environments. In fact, salt mass concentration is an important factor affecting bacterial activity. Excessive salt content can cause cell dehydration or even lysis. In addition, the process of ship wastewater treatment is also accompanied by the problem of high salinity inhibition. Ordinary aerobic denitrifying bacteria are difficult to effectively denitrify in a high-salt environment, affecting their denitrification effect. In order to balance the osmotic pressure inside and outside the cell, salt-tolerant aerobic denitrifying bacteria will produce a certain concentration of compatible solutes in the cell.

[0004] Compatible solutes are solutes that participate in the intracellular osmotic pressure compensation system without affecting the normal life activities of cells. These solutes can accumulate to high concentrations in cells, regulate the osmotic pressure of cells, and thus prevent cell dehydration. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is a secondary metabolite of salt-tolerant microorganisms. It is a cyclic amino acid derivative with a molecular weight of 42.16 Da and a molecular formula of C 6 H 10 N 2 O 2Tetrahydropyrimidine is a highly effective biomacromolecule and cell protectant that can protect biomacromolecules such as proteins, nucleic acids, biomembranes, and entire cells from adverse environmental conditions such as high salt, high temperature, and freezing, and has no adverse effects on the structure of biomacromolecules and the normal life activities of cells.

[0005] Therefore, providing a strain that has both aerobic denitrification and ectoine production functions has important practical significance for the in-situ purification of high-salinity and nitrogen-containing wastewater from ships, and has broad application prospects. Summary of the invention

[0006] The present invention provides a human staphylococcus with both aerobic denitrification and ectoine production functions and application thereof to solve the above problems.

[0007] The first aspect of the present invention provides a human Staphylococcus with both aerobic denitrification and ectoine production functions. The bacterium belongs to Staphylococcus, named (Staphylococcus hominis) EH-02, and is deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration CGMCC, with a deposit number of CGMCC No.30539, a deposit date of May 9, 2024, and a deposit address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The Staphylococcus hominis EH-02 grows on a liquid culture medium with sodium acetate as a carbon source and nitrate nitrogen as a nitrogen source, and the colonies are round, smooth, sticky, and opaque light yellow. The Staphylococcus hominis EH-02 can use small molecular organic compounds as electron donors under aerobic conditions to convert NO 3 - Through the denitrification process, it is first reduced to NO 2 - , and then restored to N 2 , thus achieving NO 3 - The removal of is a complete denitrification process;

[0009] The 16S rDNA gene sequence of Staphylococcus hominis EH-02 was amplified by PCR, sequenced and compared with BLAST. The 16S rDNA sequence is shown in SEQ ID NO.1. The similarity between the 16S rDNA gene sequence of Staphylococcus hominis EH-02 and the gene sequence of Staphylococcus hominis is 99.73%.

[0010] Another aspect of the present invention provides the use of the human Staphylococcus aureus having both aerobic denitrification and ectoine production functions in the field of ship high-salt nitrogen-containing wastewater treatment and biological preparation of ectoine.

[0011] Furthermore, in the high-salt nitrogen-containing wastewater from the ship, sodium acetate is the carbon source, sodium nitrate is the nitrogen source, the concentration of the nitrogen source is 100-200 mg N / L, C / N=5, the salinity is 60 g / L, and the shaking table speed is 120 rpm.

[0012] The beneficial effects of the present invention are:

[0013] (1) The Staphylococcus hominis EH-02 provided by the present invention is isolated from a salt pond sludge sample of a salt field in Dalian City. It has a highly efficient salt-tolerant denitrification biological denitrification ability and is a high-salt-tolerant biological denitrification strain of the genus Staphylococcus. Its denitrification efficiency is as high as 95.87%;

[0014] (2) The Staphylococcus hominis EH-02 provided by the present invention is good at using small molecular organic matter such as sodium acetate as electron donors to carry out denitrification reaction, and can be used in combination with strains that are good at using large molecular organic matter to achieve the denitrification of organic matter and NO in ship wastewater. 3 - Efficient removal of

[0015] (3) The Staphylococcus hominis EH-02 provided by the present invention also has a high biological denitrification ability in ship sewage with low dissolved oxygen content, and the low oxygen demand during the denitrification process can greatly reduce the operating cost of ship wastewater treatment;

[0016] (4) The Staphylococcus hominis EH-02 provided by the present invention has a culture period of only 3-4 days and is capable of biosynthesizing ectoine with an ectoine yield of up to 188.96 mg / g, which has a very broad development and application prospect in the field of biosynthesis of ectoine. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0018] Figure 1 This is the colony morphology of strain EH-02 on the culture medium;

[0019] Figure 2 This is the phylogenetic tree of strain EH-02 based on 16srDNA;

[0020] Figure 3 Characteristic peak diagram of ectoine standard detected by HPLC and standard curve diagram drawn according to peak area;

[0021] Figure 4 This is the characteristic peak diagram of ectoine produced by strain EH-02 detected by HPLC;

[0022] Figure 5 The nitrogen removal effect and tetrahydropyrimidine production of strain EH-02 in natural seawater denitrification medium under different carbon sources ( Figure 5 (a) is NO 3 - Removal rate of Figure 5 (b) is NO 2 - concentration; Figure 5 (c) is a graph showing the comparison of ectoine production under different carbon sources);

[0023] Figure 6 The nitrogen removal effect and tetrahydropyrimidine production of strain EH-02 in natural seawater denitrification medium under different nitrogen sources ( Figure 6 (a) is NO 3 - and NO 2 - Removal rate of Figure 6 (b) is a comparison of the yield of tetrahydropyrimidine under different nitrogen sources);

[0024] Figure 7 The nitrogen removal effect and ectoine production of strain EH-02 in natural seawater denitrification medium under different nitrogen concentrations ( Figure 7 (a) is NO 3 - Removal rate of Figure 7 (b) is NO 2 - concentration; Figure 7 (c) is a graph showing the comparison of tetrahydropyrimidine production under different nitrogen concentrations);

[0025] Figure 8 The nitrogen removal effect and ectoine production of strain EH-02 in natural seawater denitrification medium under different carbon-nitrogen ratios ( Figure 8 (a) is NO 3 - Removal rate of Figure 8 (b) is NO 2 - concentration; Figure 8 (c) is a graph showing the comparison of tetrahydropyrimidine yields at different carbon-nitrogen ratios);

[0026] Fig. 9 The nitrogen removal effect and ectoine production of strain EH-02 in artificial seawater denitrification medium at different salinities ( Fig. 9 (a) is NO 3 - Removal rate of Fig. 9 (b) is NO 2 - concentration; Fig. 9 (c) is a comparison of the production of tetrahydropyrimidine under different salinities);

[0027] Fig.10 The nitrogen removal effect and tetrahydropyrimidine production of strain EH-02 in artificial seawater denitrification medium under different dissolved oxygen (rotation speed) ( Fig.10 (a) is NO 3 - Removal rate of Fig.10 (b) is NO 2 - concentration; Fig.10 (c) is a comparison of the production of ectoine under different dissolved oxygen (rotation speed). DETAILED DESCRIPTION

[0028] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] The culture medium used in the examples of this application is as follows:

[0030] Liquid enrichment medium: sodium chloride 50g / L, crystalline magnesium sulfate 9.7g / L, potassium chloride 2g / L, sodium citrate 3g / L, anhydrous calcium chloride 0.2g / L, yeast extract powder 1.5g / L, sodium nitrate 0.6g / L, peptone 10g / L, deionized water;

[0031] Liquid activation medium: The composition is the same as that of liquid enrichment medium;

[0032] Solid screening medium: sodium chloride 50g / L, crystalline magnesium sulfate 9.7g / L, potassium chloride 2g / L, sodium citrate 3g / L, anhydrous calcium chloride 0.2g / L, yeast extract powder 1.5g / L, sodium nitrate 0.6g / L, peptone 10g / L, agar 12g / L, deionized water;

[0033] Natural seawater denitrification medium: sodium nitrate 0.6-1.5g / L, sodium acetate 0.6-5.4g / L, dipotassium hydrogen phosphate 1g / L, natural seawater (salinity 35g / L); sodium nitrate can be replaced by sodium nitrite as a nitrogen source; sodium acetate can be replaced by one of peptone, sodium propionate, sodium butyrate or glucose as a carbon source;

[0034] Artificial seawater denitrification culture medium: sodium nitrate 0.6g / L, sodium acetate 3g / L, dipotassium hydrogen phosphate 1g / L, sodium chloride 35-120g / L, magnesium chloride 2.3g / L, crystalline magnesium sulfate 3.24g / L, anhydrous calcium chloride 1.14g / L, potassium chloride 0.72g / L, sodium bicarbonate 0.2g / L, deionized water.

[0035] Example 1: Isolation and identification of strain EH-02

[0036] From the salt pond bottom mud sample of a salt field in Dalian, 10 ml of mud sample was extracted and placed in 90 ml of liquid enrichment culture medium, and cultured in a constant temperature incubator at 120 rpm and 30 °C for 3-4 days. 1 ml of enriched bacterial solution was taken and 9 ml of sterile water was added to dilute the bacterial solution to a concentration of 10 -1 , and then dilute to 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 and 10 -8 Then, a diluted bacterial solution was obtained, and then 0.1 ml of the enriched bacterial solution and the diluted bacterial solution were respectively aspirated and spread on the solid screening culture medium by the plate spreading method, and then the culture medium was placed in a constant temperature incubator at 30°C for culture; after culturing for 3-4 days, single colonies with good growth, clear bacterial plaques and large diameters in each dilution gradient were selected for further purification; 4-6 generations of separation and purification were performed until the single colonies grown on the solid screening culture medium had the same shape, the strain purification was completed, and a pure culture was obtained, and the single strain was frozen in glycerol at -20°C for storage; different pure bacterial colonies were selected for denitrification experiments, and strains that can denitrify by aerobic denitrification were screened out, and then the screened strains were tested for ectoine production, and then strains that can produce ectoine were selected from them, and finally human Staphylococcus aureus EH-02 with aerobic denitrification function and ectoine production was obtained.

[0037] Morphological identification of strain EH-02: based on colony morphology Figure 1 It can be seen that the colonies of strain EH-02 on the solid screening medium are round, smooth, sticky, and opaque light yellow.

[0038] The strain EH-02 was subjected to physiological and biochemical identification, and the results are shown in the following table:

[0039] Physiology and Biochemistry Project result Physiology and Biochemistry Project result Nitrate reduction + glucose + Hydrogen sulfide - sucrose + Starch hydrolysis - maltose + Gelatin liquefaction - Cellobiose + Indole production - Mannose + Methyl red - lactose - Arabinose + fructose +

[0040] Molecular biological identification of strain EH-02: DNA of the selected strain was extracted using a bacterial genome extraction kit, and PCR amplification of strain EH-02 was performed using universal primers 27F and 1492R of the 16S rDNA gene. The PCR reaction system consisted of 1.0 μL genomic DNA (20 ng / μL), 10× Buffer (containing 2.5 mM Mg 2+ ) 5.0 μL, Taq polymerase (5u / μL) 1.0 μL, dNTP (10 mM) 1.0 μL, 27F primer (SEQ ID NO.2: 5'-AGAGTTTGATCCTGGCTCAG-3') (10 μM) 1.5 μL, 1492R primer (SEQ ID NO.3: 5'-GGCTACCTTGTTACGACTT-3') (10 μM) 1.5 μL, ddH 2 O 39.0μL, total volume 50.0μL; PCR amplification conditions: 95℃ pre-denaturation 5min; 95℃ denaturation 30s; 58℃ annealing 30s; 72℃ extension 1min30s; 72℃ final extension 7min; 35 cycles; after the reaction was completed, 3ul PCR product was taken for 1% agarose gel electrophoresis to confirm the PCR amplification fragment, and PCR product sequencing was completed by Qingdao Weilai Biotechnology Co., Ltd.

[0041] The 16s rDNA sequence of strain EH-02 is 1462 bp in length, and the nucleotide sequence is SEQ ID NO.1, SEQ ID NO.1:

[0042]

[0043] The sequence was submitted to the NCBI database for blast comparison and the phylogenetic tree was drawn using MEGA7.0. The phylogenetic tree of strain EH-02 is shown in Figure 2 As shown, the results showed that the strain EH-02 had a similarity of 99.73% with Staphylococcus hominis DM 122 (GenBank accession number: NR 036956.1); based on the above identification results, the strain was identified as Staphylococcus hominis.

[0044] Example 2: Extraction of ectoine from bacterial culture of strain EH-02

[0045] Extraction of ectoine from the bacterial solution of strain EH-02 comprises the following steps:

[0046] S1: Strain activation: Inoculate the strain EH-02 stored in glycerol at -20°C into 50 mL of sterilized liquid activation medium, place it in a shaker at 30°C and 120 rpm for 24 h to allow the bacteria to grow to a certain abundance, harvest the bacteria by centrifugation and dilute with sterile water to OD 600 The value is about 0.1, and the bacterial suspension is used for inoculation.

[0047] S2: Aerobic culture: The bacterial suspension was inoculated into 50 mL of natural seawater denitrification medium (sodium nitrate 0.6 g / L, sodium acetate 1 g / L, potassium dihydrogen phosphate 1 g / L, natural seawater (salinity 35 g / L)) at a bacterial suspension inoculation volume of 2% (v / v), and cultured at 30°C, 120 rpm for 48 h to obtain a bacterial suspension containing intracellular ectoine;

[0048] S3: Crushing and extraction: Pour the intracellular ectoine-containing bacterial solution obtained in S2 into a 50mL centrifuge tube, centrifuge at 4°C, 8000rpm for 10min, discard the supernatant, and collect the bacterial precipitate; wash the bacterial precipitate with dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer (pH=7.0), centrifuge again under the same conditions, discard the supernatant, collect the precipitate, and finally use 30mL deionized water to disperse the precipitated bacteria and crush them to release the intracellular ectoine. The crushing conditions are: use an ultrasonic cell crusher, set the parameters to 500W power, crushing time 10min, centrifuge at 4°C, 8000rpm for 10min after crushing, collect the supernatant containing ectoine, and use the supernatant as the sample for the next step of high performance liquid chromatography (HPLC) determination. At the same time, the crushed bacteria are dried and weighed to calculate the ectoine yield (mg / g) per unit mass of bacteria.

[0049] Example 3: Detection of ectoine synthesis by strain EH-02

[0050] The detection steps for the synthesis of ectoine by strain EH-02 are as follows:

[0051] (1) Selection of high performance liquid chromatography (HPLC) conditions: Agilent high performance liquid chromatograph was used to detect the content of ectoine in the sample: the injection volume of the supernatant sample obtained in Example 2 was 10 μL, the chromatographic column was a Diamonsil 5 μm C18 reverse phase chromatographic column (250×4.6 mm), the column temperature was 30° C., the mobile phase was an acetonitrile / water mixture (80:20, v / v), the flow rate was 1.0 mL / min, and the ultraviolet detection wavelength was 210 nm;

[0052] (2) Preparation of standard curve of ectoine: Weigh 0.1 g of ectoine standard and dissolve it in a 100 mL volumetric flask, dilute to volume with deionized water to prepare a 1 g / L ectoine standard solution, and then use gradient dilution to prepare 0.02 g / L, 0.04 g / L, 0.06 g / L, 0.08 g / L, and 0.1 g / L standard solutions, respectively. Perform HPLC detection under the conditions in step (1), record the corresponding peak areas, and draw a standard curve based on the peak areas. The results are shown in the figure. Figure 3 As shown, Figure 3 (a) is the standard curve of ectoine with peak area as the horizontal axis and concentration as the vertical axis. Figure 3 (b) is the liquid chromatogram of the standard substance of ectoine with a concentration of 0.04 g / L. Figure 3 (c) is the liquid chromatogram of the standard substance of ectoine with a concentration of 0.08 g / L. Figure 3 (b) Figure 3 (c) It can be seen that the detection peak time of ectoine is around 11.3-11.4 min;

[0053] (3) Detection of ectoine in bacteria: The supernatant obtained in Example 2 was subjected to HPLC determination using the conditions in step (1), and the retention time of the ectoine standard detected in step (2) was compared, and the corresponding peak area was recorded and substituted into the peak area in step (2). Figure 3 (a) Standard curve, calculate the ectoine production; HPLC detection of the characteristic peaks of ectoine produced by strain EH-02 is shown in Figure 4 As shown, in order to determine the characteristic that the strain can produce ectoine during growth;

[0054] (4) Calculation of the yield of ectoine synthesized by the bacteria: Based on the standard curve obtained in step (2) and the peak area in step (3), the ectoine yield of the bacteria EH-02 was calculated, and the result was 119.3 mg / g.

[0055] Example 4: Evaluation of nitrogen removal effect and ectoine production of strain EH-02 in natural seawater denitrification medium under different carbon sources

[0056] The specific method includes the following steps:

[0057] (1) Activation: The strain EH-02 stored in glycerol at -20°C was inoculated into 50 mL of sterilized liquid activation medium and cultured in a shaker at 30°C and 120 rpm for 24 h to allow the bacteria to grow to a certain abundance. The bacteria were harvested by centrifugation and diluted with sterile water to OD 600 The value is about 0.1, and the bacterial suspension is used for inoculation.

[0058] (2) Grouping: Ten conical flasks were divided into five groups, with two conical flasks in each group. 50 mL of natural seawater denitrification medium was added to each conical flask. The carbon sources in each group of conical flasks were 1 g / L sodium acetate, 1 g / L sodium propionate, 1 g / L sodium butyrate, 1 g / L peptone, and 1 g / L glucose, respectively. The other ingredients were kept consistent in each group (sodium nitrate 0.6 g / L, potassium dihydrogen phosphate 1 g / L, and natural seawater (salinity 35 g / L)). After sterilization and cooling to room temperature, 1 mL of the bacterial suspension was inoculated into ten 50 mL conical flasks at a 2% (v / v) inoculation volume. The flasks were then cultured at a constant temperature of 30°C and 120 rpm for 48 h. Water samples were taken every 12 hours to determine NO. 2 - 、NO 3 - After 48 hours, the cells of each group were collected by centrifugation, and the ectoine content was detected by the same detection method as in Example 3 after the cells were broken. The results are as follows Figure 5 As shown;

[0059] Figure 5 (a) shows that when the carbon source is small molecular organic acids such as sodium acetate, sodium propionate and sodium butyrate, NO 3 - The removal rate reached more than 80% after 42 hours. The denitrification effect was best when the carbon source was sodium acetate. 3 - The removal rate is 88.42%. Figure 5 (b) It can be seen that from NO 2 - The residual amount of NO in the small molecule organic acid group was also lower. 2 - The residual amount of each strain was less than 30 mgN / L, and the lowest was 21.19 mgN / L in the sodium acetate group, indicating that the strain was more suitable for aerobic denitrification using sodium acetate as a carbon source. Figure 5(c) It can be seen that when sodium acetate is used as the carbon source, the strain can achieve efficient denitrification and denitrification while producing a higher concentration of ectoine; when sodium propionate, sodium butyrate and glucose are used as carbon sources, the output of ectoine is relatively small; due to its complex molecular structure, peptone is less effective as a carbon source, and the least ectoine is produced in the same period of time.

[0060] Example 5: Evaluation of nitrogen removal effect and ectoine production of strain EH-02 in natural seawater denitrification medium under different nitrogen sources

[0061] The specific method includes the following steps:

[0062] (1) Activation: the method is the same as in Example 4;

[0063] (2) Grouping: Use 4 conical flasks and divide them into 2 groups, with 2 conical flasks in each group. Add 50 mL of natural seawater denitrification culture medium into each conical flask. The nitrogen sources in each group of conical flasks are 0.6 g / L sodium nitrate and 0.6 g / L sodium nitrite, and the other ingredients are kept consistent in each group (1 g / L sodium acetate, 1 g / L potassium dihydrogen phosphate, 1L natural seawater (salinity 35 g / L)); after sterilization and cooling to room temperature, take 1 mL of the bacterial solution at a 2% (v / v) inoculation amount and inoculate it into 4 50 mL conical flasks. Then, culture at a constant temperature of 30°C and 120 rpm for 48 hours, and take water samples every 12 hours to measure NO 2 - 、NO 3 - After 48 hours, the cells of each group were collected by centrifugation, and the ectoine content was detected by the same detection method as in Example 3 after the cells were broken. The results were as follows Figure 6 As shown;

[0064] Depend on Figure 6 (a) It can be seen that the denitrification efficiency is higher when the nitrogen source is sodium nitrate, and NO 3 - The removal rate can reach 87.99% in 48h. When the nitrogen source is sodium nitrite, the removal rate of NO 2 - The removal rate can only reach 64.15%, indicating that sodium nitrate is the best nitrogen source for aerobic denitrification reaction of this strain. Figure 6 (b) It can be seen that the ectoine production when sodium nitrate is used as the nitrogen source is 112 mg / g, which is almost twice that of nitrite, further indicating that sodium nitrate is the best nitrogen source for this strain.

[0065] Example 6: Evaluation of nitrogen removal effect and ectoine production of strain EH-02 in natural seawater denitrification medium at different nitrogen concentrations

[0066] The specific method is:

[0067] (1) Activation: the method is the same as in Example 4;

[0068] (2) Grouping: Eight conical flasks were divided into four groups, with two conical flasks in each group. 50 mL of natural seawater denitrification medium was added to each conical flask. The nitrogen concentrations in each group of conical flasks were 100 mg N / L, 150 mg N / L, 200 mg N / L, and 250 mg N / L, respectively, that is, the sodium nitrate concentrations were 0.6 g / L, 0.9 g / L, 1.2 g / L, and 1.5 g / L, respectively. The other components were kept consistent in each group (sodium acetate 1 g / L, potassium dihydrogen phosphate 1 g / L, and natural seawater 1L (salinity 35 g / L)). After sterilization and cooling to room temperature, 1 mL of the bacterial solution was inoculated into eight 50 mL conical flasks at a 2% (v / v) inoculation volume. The culture was then kept at a constant temperature of 30°C and 120 rpm for 72 h. Water samples were taken every 12 hours to determine NO. 2 - 、NO 3 - After 72 hours, the cells of each group were collected by centrifugation, and the ectoine content was detected by the same detection method as in Example 3 after the cells were broken. The results were as follows Figure 7 As shown;

[0069] Depend on Figure 7 (a) It can be seen that when the initial nitrogen concentration is 100-200 mgN / L, the denitrification effect is better, and the NO 3 - The removal rate was 87.53%-92.42%, tending to be stable after 48 hours, and the removal rate of NO was 72 hours. 3 - The removal rate was as high as 95.32%. When the initial nitrogen concentration was 250 mgN / L, the NO 3 - The removal rate is only 69.92%. Figure 7 (b) It can be seen that after 48 hours, NO 2 - In terms of the remaining amount, the remaining situation is 250mgN / L>200mgN / L>150mgN / L>100mgN / L, 100mgN / L of NO 2 - The minimum residual amount was 28.19 mgN / L, and the comprehensive NO 3 - Removal rate and NO 2 - In terms of residual amount, the denitrification effect is best when the nitrogen concentration is 100mgN / L. Figure 7(c) It can be seen that with the increase of initial nitrogen concentration, the ectoine production of the strain gradually decreased, and the highest ectoine production was 118.8 mg / g when the nitrogen concentration was 100 mgN / L.

[0070] Example 7: Evaluation of nitrogen removal effect and ectoine production of strain EH-02 in natural seawater denitrification medium at different carbon-nitrogen ratios

[0071] The specific method is:

[0072] (1) Activation: the method is the same as in Example 4;

[0073] (2) Grouping: 12 conical flasks were used and divided into 6 groups, with 2 conical flasks in each group. 50 mL of natural seawater denitrification medium was added to each conical flask, and the C / N value was controlled by adjusting the content of sodium acetate. The C / N value was set to 1, 2, 3, 5, 7, and 9, that is, the sodium acetate concentration in each group of conical flasks was 0.6 g / L, 1.2 g / L, 1.8 g / L, 3 g / L, 4.2 g / L, and 5.4 g / L, respectively. The other components of each group remained the same (sodium nitrate 0.6 g / L, potassium dihydrogen phosphate 1 g / L, 1L natural seawater (salinity 35 g / L)); after sterilization and cooling to room temperature, 1 mL of the bacterial solution was inoculated into 12 50 mL conical flasks at a 2% (v / v) inoculation amount. Then, the culture was maintained at a constant temperature of 30°C and 120 rpm for 48 h, and water samples were taken every 12 hours to determine NO 2 - 、NO 3 - After 48 hours, the cells of each group were collected by centrifugation, and the ectoine content was detected by the same detection method as in Example 3 after the cells were broken. The results are as follows Figure 8 As shown;

[0074] from Figure 8 (a) It can be seen that when C / N=5, the strain has the best denitrification effect. From 24h, NO 3 - The removal rate has been at the leading level and reached 94.74% at 48h. 3 - The removal rate did not exceed this value, indicating that too high or too low organic matter content is not conducive to the denitrification reaction of the bacteria. Figure 8 (b) It can be seen that from NO 2 - From the residual amount of each group, when C / N<5, 2 - The residual amount at 48h was higher, and when C / N>5, the NO 2 -The residual amount after 48 hours was less than 20 mgN / L. Figure 8 (c) The production of ectoine is roughly the same as the denitrification law summarized above. When C / N=5, the maximum ectoine production is 188.96 mg / g. Under other carbon-nitrogen ratios, the ectoine production of the strain decreases. When C / N=9, the ectoine production is less than 60 mg / g. Too high a carbon-nitrogen ratio is not conducive to the denitrification reaction of the strain, nor is it conducive to the production of ectoine by the strain.

[0075] Example 8: Evaluation of nitrogen removal effect and ectoine production of strain EH-02 in artificial seawater denitrification medium at different salinities

[0076] The specific method is:

[0077] (1) Activation: the method is the same as in Example 4;

[0078] (2) Grouping: 10 conical flasks were divided into 5 groups, with 2 conical flasks in each group. 50 mL of artificial seawater denitrification medium was added to each conical flask, where the sodium chloride concentration in each group of conical flasks was 35 g / L, 60 g / L, 80 g / L, 100 g / L, and 120 g / L, respectively. The other components were kept consistent in each group (sodium nitrate 0.6 g / L, sodium acetate 3 g / L, dipotassium hydrogen phosphate 1 g / L, magnesium chloride 2.3 g / L, crystalline magnesium sulfate 3.24 g / L, anhydrous calcium chloride 1.14 g / L, potassium chloride 0.72 g / L, sodium bicarbonate 0.2 g / L); after sterilization and cooling to room temperature, 1 mL of the bacterial solution was inoculated into 10 50 mL conical flasks at a 2% (v / v) inoculation volume. Then, the culture was maintained at a constant temperature of 30°C and 120 rpm for 48 h, and water samples were taken every 12 hours to determine NO 2 - 、NO 3 - After 48 hours, the cells of each group were collected by centrifugation, and the ectoine content was detected by the same detection method as in Example 3 after the cells were broken. The results are as follows Fig. 9 As shown;

[0079] like Fig. 9 As shown in (a), when the salinity is 60 g / L, the strain has the best denitrification effect, NO 3 - The removal rate reached 92.38% at 48h. 2 - The concentration dropped to 21 mgN / L. In contrast, the denitrification efficiency of the strain decreased to a certain extent when the salinity increased or decreased; when the environmental salinity increased to 120 g / L, the denitrification ability of the strain was greatly affected, and NO 3 -The removal rate dropped to 74%, and the NO 2 - The concentration will also increase to about 59mgN / L ( Fig. 9 (b)). Fig. 9 (c) In terms of the ectoine production, the ectoine production reached a maximum of 172 mg / g when the salinity was 60 g / L, and the ectoine production decreased under other salinity conditions, indicating that the higher the salinity, the higher the ectoine production, and the salinity and ectoine production were proportional within a certain limit. Salinity affects the denitrification ability of the strain as well as its ability to produce ectoine.

[0080] Example 9: Evaluation of nitrogen removal effect and ectoine production of strain EH-02 in artificial seawater denitrification medium under different dissolved oxygen (rotation speed)

[0081] The specific method is:

[0082] (1) Activation: the method is the same as in Example 4;

[0083] (2) Grouping: 12 conical flasks were divided into 6 groups, with 2 conical flasks in each group. 50 mL of artificial seawater denitrification medium was added to each conical flask, and the conical flasks were placed in a shaker. The dissolved oxygen content was controlled by adjusting the shaker speed, which was set to 0 rpm (dissolved oxygen concentration was 2.87-2.91 mg / L), 30 rpm (dissolved oxygen concentration was 3.25-3.29 mg / L), 60 rpm (dissolved oxygen concentration was 3.68-3.72 mg / L), 90 rpm (dissolved oxygen concentration was 4.27-4.31 mg / L), 120 rpm (dissolved oxygen concentration was 5.18-5.29 mg / L), and 120 rpm (dissolved oxygen concentration was 5.18-5.29 mg / L). 22mg / L), 150rpm (dissolved oxygen concentration is 6.05-6.09mg / L), and the other components of each group are consistent (sodium chloride 60g / L, sodium nitrate 0.6g / L, sodium acetate 3g / L, dipotassium hydrogen phosphate 1g / L, magnesium chloride 2.3g / L, crystalline magnesium sulfate 3.24g / L, anhydrous calcium chloride 1.14g / L, potassium chloride 0.72g / L, sodium bicarbonate 0.2g / L); after sterilization and cooling to room temperature, 1mL of the bacterial solution was inoculated into 12 50mL conical flasks at a 2% (v / v) inoculation amount, and then cultured at a constant temperature of 30℃ and 120rpm for 48h, and water samples were taken every 12 hours to determine NO 2 - 、NO 3 - After 48 hours, the cells of each group were collected by centrifugation, and the ectoine content was detected by the same detection method as in Example 3 after the cells were broken. The results were as follows Fig.10 As shown;

[0084] like Fig.10(a) and Fig.10 As shown in (b), when the rotation speed is 120 rpm, the denitrification effect is the best, 48hNO 3 - The removal rate can reach 95.87%, NO 2 - The concentration can be reduced to below 20 mgN / L. However, when the speed is lower or higher than 120 rpm, NO 3 - The removal rate will be reduced to a certain extent, NO 2 - The concentration will also increase by 5-25mgN / L; when the speed drops to 30rpm, 48hNO 3 - The removal rate dropped to 90.24%; when the rotation speed dropped to 0rpm, 48hNO 3 - The removal rate dropped to 82.25%, and the denitrification performance was still good. The decrease in rotation speed will reduce the dissolved oxygen concentration in the environment. The good denitrification performance at 0rpm and 30rpm shows that the strain can still ensure good denitrification efficiency in an environment with low dissolved oxygen. Fig.10 (c) It can be seen that the ectoine production of the strain fluctuates with the increase of the rotation speed. The ectoine production reaches a maximum of 185.3 mg / g at 120 rpm. When the rotation speed reaches 150 rpm, the ectoine production drops sharply to 96 mg / g. The high centrifugal force caused by the increase in rotation speed may affect the denitrification performance and ectoine production ability of the strain at the same time.

[0085] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A strain of Staphylococcus hominis EH-02 having both aerobic denitrification and ectoine production functions, characterized in that: The bacterium is deposited in the General Microbiology Center (CGMCC) of the China Microbiological Culture Collection Administration. The deposit date is May 9, 2024. The deposit number is CGMCC No.30539. The base length of 16S rDNA is 1462bp.

2. The human Staphylococcus aureus having both aerobic denitrification and ectoine production functions as claimed in claim 1 is used in the treatment of high-salinity nitrogen-containing wastewater from ships, characterized in that: In the ship's high-salt nitrogen-containing wastewater, the nitrogen source is sodium nitrate, the concentration of the nitrogen source is 100-200 mgN / L, and the salinity is 60 g / L; the carbon source is any one of sodium acetate, sodium propionate, sodium butyrate, peptone and glucose, and C / N=5.

3. The use of Staphylococcus aureus having both aerobic denitrification and ectoine production functions in the treatment of ship high-salinity nitrogen-containing wastewater according to claim 2, characterized in that: The shaking speed was 120 rpm.

4. Use of the human Staphylococcus aureus having both aerobic denitrification and ectoine production functions as claimed in claim 1 in the field of biological preparation of ectoine, characterized in that: The nitrogen source for preparing ectoine is sodium nitrate, the concentration of the nitrogen source is 100-200 mgN / L, and the salinity is 60 g / L; the carbon source is any one of sodium acetate, sodium propionate, sodium butyrate, peptone and glucose, and C / N=5.

5. The use of the human Staphylococcus aureus having both aerobic denitrification and ectoine production functions in the field of biological preparation of ectoine according to claim 4, characterized in that: The shaking speed was 120 rpm.

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