A strain of Utah halomonas having both aerobic denitrification and ectoine production functions and its screening method and application

Abstract

The invention discloses a Utah halomonas having both aerobic denitrification and tetrahydropyrimidine production functions and a screening method and application thereof, relating 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 a deposit number of CGMCC No. 30540; the strain EH-01 was isolated from a salt pond bottom mud sample of a salt field in Dalian City, and is a strain that uses small molecular organic matter to reduce nitrate to nitrate under aerobic conditions. The invention is a strain capable of reducing nitrite to nitrogen gas. When the carbon source is sodium acetate, the nitrogen source is sodium nitrate, the nitrogen source concentration is 200 mgN / L, C / N=2, the salinity is 60 g / L and the rotation speed is 90 rpm, the denitrification efficiency is as high as 97%. The strain can produce compatible solute ectoine while carrying out heterotrophic denitrification, and the maximum accumulation of ectoine is 118 mg / g. The strain can not only be applied to environmental protection fields such as treatment of high-salt nitrogen-containing wastewater from ships, but also has broad application prospects in biological preparation of ectoine.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a strain of Utah halomonas having both aerobic denitrification and ectoine production functions, and a screening method 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 pollutants emitted by ships during navigation has become increasingly prominent. X ) is one of the main pollutants in ship exhaust emissions. After washing, nitrate (NO3 - ) will lead to eutrophication of water bodies, causing massive reproduction of plankton and algae, and death of aquatic animals due to lack of oxygen, thus affecting the biodiversity of the area. - When it enters the human body, it is reduced to nitrite (NO2 - ), which can form carcinogens with secondary amines in an acidic environment, thus endangering human health. Ship sewage also contains NO3 - , and its discharge will also affect the marine water environment. The above ship wastewater also has the characteristics of high salinity, which greatly increases the difficulty of combined in-situ treatment of wastewater.

[0003] Common methods for treating high-salinity and high-nitrogen wastewater from ships include physical, chemical and biological methods. Physical and chemical methods can effectively remove nitrogen from wastewater, but their disadvantages are that they are expensive and prone to secondary pollution. - It is to use the coordination of heterotrophic denitrifying bacteria to convert NO3 in wastewater - The biological method is widely used to treat nitrogen-containing wastewater due to its low cost, no side effects and thorough denitrification. Traditional theory holds that denitrification can only be carried out under anaerobic conditions, but in recent decades, aerobic denitrification processes have been continuously reported, providing a new idea for biological denitrification. Aerobic denitrification refers to the process in which aerobic denitrifying bacteria convert NO3 - The process of converting nitrogen into N2. Compared with the traditional anaerobic denitrification technology, the aerobic denitrification process has the advantages of high reaction rate, simple operation and high sewage treatment efficiency.

[0004] In the process of ship wastewater treatment, there is often the problem of high salinity inhibition. The hypertonic environment will inhibit microbial metabolism, making it difficult for ordinary denitrifying microorganisms to effectively denitrify in a high-salt environment, affecting their denitrification effect. Therefore, separating salt-tolerant denitrifying bacteria is the best way to solve this problem. In order to balance the osmotic pressure inside and outside the cell, salt-tolerant denitrifying bacteria will produce a certain concentration of compatible solutes inside the cell. Tetrahydropyrimidine, also known as Ectoine, has a chemical name (S)-2-methyl-3,4,5,6-tetrahydropyrimidine-4-carboxylic acid, which is an osmotic pressure compensation solute. The biological functions of tetrahydropyrimidine discovered so far include: maintaining the osmotic pressure balance inside and outside the microbial cell under hypertonic conditions, and stabilizing the structure of proteins in the cell; acting as a stress protector for the cell under heat or cold shock conditions; and maintaining the vitality of biological macromolecules in the cell under extreme environments. At the same time, in a high osmotic pressure environment, non-salt-tolerant bacteria can absorb exogenous tetrahydropyrimidine to improve their own stability.

[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 strain of Utah Halomonas having both aerobic denitrification and tetrahydropyrimidine production functions and a screening method and application thereof to solve the above problems.

[0007] The first aspect of the present invention provides a strain of Utah Halomonas having both aerobic denitrification and tetrahydropyrimidine production functions. The strain belongs to Utah Halomonas, named (Vreelandella utahensis) EH-01, with a preservation number of CGMCC No. 30540, a preservation date of May 9, 2024, and a preservation address of No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0008] The Utah Halomonas (Vreelandella utahensis) EH-01 grows on a liquid culture medium with sodium acetate as a carbon source and nitrate nitrogen as a nitrogen source. The colonies are round, smooth and sticky on the surface, and opaque light yellow in color. The Utah Halomonas (Vreelandella utahensis) EH-01 can use small molecular organic compounds as electron donors under aerobic conditions to convert NO3 - Through the denitrification process, it is first reduced to NO2 - , and then reduced to N2, thereby achieving NO3 - The removal of is a complete denitrification process;

[0009] The 16S rDNA gene sequence of Vreelandella utahensis EH-01 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 Vreelandella utahensis EH-01 and the gene sequence of Vreelandella utahensis is 98.48%.

[0010] The second aspect of the present invention provides a screening method for the Vreelandella utahensis EH-01, the screening method comprising the following steps:

[0011] 10 ml of mud sample was extracted from the salt pond bottom mud sample and placed in 90 ml of liquid enrichment culture medium, and then cultured in a constant temperature incubator at 120 rpm and 30°C for 3-4 days; then the supernatant was taken for gradient dilution and spread on the surface of solid screening culture medium, and after constant temperature culture at 30°C for 3-4 days, colonies with clear plaques and larger diameters were selected for streaking separation, and purer single colonies were obtained after 4-6 generations of separation and purification. Denitrification experiments were carried out to screen out strains that can denitrify by aerobic denitrification, and then the ectoine production of the screened strains was detected, and then the strains that can produce ectoine were selected, and finally Utah Halomonas EH-01 with aerobic denitrification function and ectoine production was obtained.

[0012] Furthermore, the culture medium for screening the Vreelandella utahensis EH-01 is prepared according to the following ratio:

[0013] The liquid enrichment medium: 50 g / L sodium chloride, 9.7 g / L crystalline magnesium sulfate, 2 g / L potassium chloride, 3 g / L sodium citrate, 0.2 g / L anhydrous calcium chloride, 1.5 g / L yeast extract powder, 0.6 g / L sodium nitrate, 10 g / L peptone, deionized water;

[0014] The solid screening medium includes: 50 g / L sodium chloride, 9.7 g / L crystalline magnesium sulfate, 2 g / L potassium chloride, 3 g / L sodium citrate, 0.2 g / L anhydrous calcium chloride, 1.5 g / L yeast extract powder, 0.6 g / L sodium nitrate, 10 g / L peptone, 12 g / L agar, and deionized water.

[0015] The third aspect of the present invention provides a method for extracting ectoine using the Utah Halomonas having both aerobic denitrification and ectoine production functions in the present application, comprising the following steps:

[0016] S1: Strain activation: Inoculate the strain EH-01 stored in glycerol at -20°C into 50 mL of sterilized liquid enrichment 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 preparation is complete;

[0017] S2: Aerobic culture: Inoculate the bacterial suspension into 50 mL of natural seawater denitrification medium at a bacterial inoculum volume of 2% (v / v), and culture at 30°C and 120 rpm for 48 h to obtain a bacterial solution containing intracellular ectoine;

[0018] S3: Crushing and extraction: Pour the intracellular ectoine-containing bacterial solution obtained in S2 into a 50 mL centrifuge tube, centrifuge at 4°C, 8000 rpm for 10 min, discard the supernatant, collect the precipitated bacteria, wash the bacteria with dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer with a pH of 7.0, centrifuge again under the same conditions, discard the supernatant, and collect the precipitated bacteria; finally, use 30 mL of deionized water to disperse the precipitated bacteria and crush them to release intracellular ectoine; after crushing, centrifuge at 4°C, 8000 rpm for 10 min, collect the supernatant containing ectoine and the bacterial precipitate, and dry and weigh the bacterial precipitate to obtain the ectoine yield per unit mass of bacteria in mg / g.

[0019] Furthermore, the disruption conditions are: using an ultrasonic cell disruptor, with the parameters set to a power of 500 W and a disruption time of 10 min.

[0020] The fourth aspect of the present invention provides the use of the Utah Halomonas having both aerobic denitrification and ectoine production functions in the field of ship high-salinity nitrogen-containing wastewater treatment and biological preparation of ectoine.

[0021] 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 200 mgN / L, C / N=2-3, the salinity is 35-60 g / L, and the rotation speed is 90-120 rpm.

[0022] The beneficial effects of the present invention are:

[0023] (1) The Utah Halomonas (Vreelandella utahensis) EH-01 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 Halomonas. Its denitrification efficiency is as high as 97%;

[0024] (2) The Utah Halomonas (Vreelandella utahensis) EH-01 provided by the present invention is good at using small molecular organic matter such as sodium acetate as an electron donor for denitrification reaction, and can be used in combination with strains that are good at using large molecular organic matter to achieve efficient removal of organic matter and nitrate in ship wastewater;

[0025] (3) The Vreelandella utahensis EH-01 provided by the present invention can achieve NO3 - The large-scale removal of nitrogen-containing wastewater breaks the previous situation that denitrification efficiency is high only under high carbon-nitrogen ratio conditions, which can greatly reduce the cost of strains in treating nitrogen-containing wastewater.

[0026] (4) The Vreelandella utahensis EH-01 provided by the present invention also has a high biological denitrification capacity 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;

[0027] (5) The Vreelandella utahensis EH-01 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 118 mg / g, which has a very broad development and application prospect in the field of biosynthesis of ectoine. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] Figure 1 This is the colony morphology of strain EH-01 on the culture medium;

[0030] Figure 2 This is the phylogenetic tree of strain EH-01 based on 16srDNA;

[0031] Figure 3 Characteristic peak diagram of ectoine standard detected by HPLC and standard curve diagram drawn according to peak area;

[0032] Figure 4 This is the characteristic peak diagram of ectoine produced by strain EH-01 detected by HPLC;

[0033] Figure 5The nitrogen removal effect and ectoine production of strain EH-01 in natural seawater denitrification medium under different carbon sources (a is NO3 - The removal rate of NO2 - concentration; c is the comparison of ectoine production under different carbon sources);

[0034] Figure 6 The nitrogen removal effect and ectoine production of strain EH-01 in natural seawater denitrification medium under different nitrogen sources (a is NO3 - and NO2 - b is the comparison of tetrahydropyrimidine production under different nitrogen sources);

[0035] Figure 7 The nitrogen removal effect and ectoine production of strain EH-01 in natural seawater denitrification medium under different nitrogen concentrations (a is NO3 - The removal rate of NO2 - concentration; c is the comparison of tetrahydropyrimidine production under different nitrogen concentrations);

[0036] Figure 8 The nitrogen removal effect and ectoine production of strain EH-01 in natural seawater denitrification medium under different carbon-nitrogen ratios (a is NO3 - The removal rate of NO2 - concentration; c is the comparison of tetrahydropyrimidine yield under different carbon-nitrogen ratios);

[0037] Fig. 9 The nitrogen removal effect and ectoine production of strain EH-01 in artificial seawater denitrification medium at different salinities (a is NO3 - The removal rate of NO2 - concentration; c is the comparison of tetrahydropyrimidine production under different salinities);

[0038] Fig.10 The nitrogen removal effect and ectoine production of strain EH-01 in artificial seawater denitrification medium under different dissolved oxygen (rotation speed) (a is NO3 - The removal rate of NO2 - concentration; c is the comparison of ectoine production under different dissolved oxygen (rotation speed). DETAILED DESCRIPTION

[0039] 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.

[0040] The culture medium used in the examples of this application is as follows:

[0041] 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;

[0042] Liquid activation medium: The composition is the same as that of liquid enrichment medium;

[0043] 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;

[0044] Natural seawater denitrification medium: sodium nitrate 0.6-1.5g / L, sodium acetate 1-10.8g / 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;

[0045] Artificial seawater denitrification culture medium: sodium nitrate 1.2g / L, sodium acetate 2.4g / 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.

[0046] Example 1: Isolation and identification of strain EH-01

[0047] 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 taken 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 Utah Halomonas EH-01 with aerobic denitrification function and ectoine production was obtained.

[0048] Morphological identification of strain EH-01: based on colony morphology Figure 1 It can be seen that the colonies of strain EH-01 on the solid screening medium are round, smooth and sticky on the surface, and opaque light yellow in color.

[0049] The strain EH-01 was subjected to physiological and biochemical identification, and the results are shown in the following table:

[0050] 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 +

[0051] Molecular biological identification of strain EH-01: DNA of the selected strain was extracted using a bacterial genome extraction kit, and PCR amplification of strain EH-01 was performed using universal primers 27F and 1492R of the 16S rDNA gene. The PCR reaction system consisted of 1.0 μL of genomic DNA (20 ng / μL), 10× Buffer (containing 2.5 mM Mg 2+) 5.0μL, Taq polymerase (5u / μL) 1.0μL, dNTP (10mM) 1.0μL, 27F primer (SEQ ID NO.2: 5'-AGAGTTTGATCCTGGCTCAG-3') (10μM) 1.5μL, 1492R primer (SEQ ID NO.3: 5'-GGCTACCTTGTTACGACTT-3') (10μM) 1.5μL, ddH2O 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 of PCR product was taken for 1% agarose gel electrophoresis detection to confirm the PCR amplification fragment, and the PCR product sequencing was completed by Qingdao Weilai Biotechnology Co., Ltd.

[0052] The 16s rDNA sequence of strain EH-01 is 1442 bp in length, and the nucleotide sequence is SEQ ID NO.1, SEQ ID NO.1:

[0053]

[0054] 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-01 is shown in Figure 2 As shown, the results showed that the strain EH-01 had a similarity of 98.48% with Vreelandella utahensis DSM 3051 (GenBank accession number: NR 042068.1); based on the above identification results, the strain was identified as Vreelandella utahensis.

[0055] Example 2: Extraction of ectoine from bacterial culture of strain EH-01

[0056] Extraction of ectoine from the bacterial solution of strain EH-01 comprises the following steps:

[0057] S1: Strain activation: Inoculate the strain EH-01 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.

[0058] S2: Aerobic culture: 2% (v / v) bacterial solution was inoculated into 50 mL 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)), and cultured at 30°C, 120 rpm for 48 h to obtain a bacterial solution containing intracellular ectoine;

[0059] 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 (pH7.0), 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 the supernatant is used 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.

[0060] Example 3: Detection of ectoine synthesis by strain EH-01

[0061] The detection steps for the synthesis of ectoine by strain EH-01 are as follows:

[0062] (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;

[0063] (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 ectoine with a concentration of 0.04 g / L. Figure 3 c is the liquid chromatogram of the standard 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;

[0064] (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 its ectoine production; HPLC detection of ectoine produced by strain EH-01 characteristic peak diagram as shown in Figure 4 As shown, in order to determine the characteristic that the strain can produce ectoine during growth;

[0065] (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-01 was calculated, and the result was 89.08 mg / g.

[0066] Example 4: Evaluation of nitrogen removal effect and ectoine production of strain EH-01 in natural seawater denitrification medium under different carbon sources

[0067] The specific method includes the following steps:

[0068] (1) Activation: The strain EH-01 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.

[0069] (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 the NO2 - 、NO3 - 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;

[0070] Depend on Figure 5 The results show that when the carbon source is small molecular organic acids such as sodium acetate, sodium propionate and sodium butyrate, NO3 - The removal rate increased to more than 80% in 48 hours, and the removal rate of NO2 - It also achieved efficient removal, indicating that under aerobic conditions, the strain is more suitable for aerobic denitrification using small molecular organic matter, while the utilization of large molecular organic matter is low and the denitrification efficiency is poor. Comparing the denitrification reaction efficiency with three small molecular organic acids as carbon sources, it can be seen that sodium acetate is the best carbon source for the strain to react. At 48h, NO3 - The removal rate can reach 87.53%, while making NO2 - The concentration dropped to 25 mgN / L at 48 h, indicating that the strain was able to achieve a higher NO3 - and NO2 -removal; by comparison, it was found that when sodium acetate was used as the carbon source, the strain could produce a higher concentration of ectoine while achieving efficient denitrification; when sodium propionate, sodium butyrate and glucose were used as carbon sources, the output of ectoine was relatively small; due to its complex molecular structure, peptone was poorly utilized as a carbon source, and the least ectoine was produced in the same time.

[0071] Example 5: Evaluation of nitrogen removal effect and ectoine production of strain EH-01 in natural seawater denitrification medium under different nitrogen sources

[0072] The specific method includes the following steps:

[0073] (1) Activation: the method is the same as in Example 4;

[0074] (2) Grouping: Use 4 conical flasks and divide them into 2 groups, with 2 conical flasks in each group. Add 50mL of natural seawater denitrification culture medium into each conical flask. Among them, the nitrogen sources in each group of conical flasks are 0.6g / L sodium nitrate and 0.6g / L sodium nitrite, and the other ingredients are consistent in each group (1g / L sodium acetate, 1g / L potassium dihydrogen phosphate, 1L natural seawater (salinity 35g / L)); after sterilization and cooling to room temperature, take 1mL of the bacterial solution at a 2% (v / v) inoculation amount and inoculate it into 4 50mL conical flasks. Then culture at a constant temperature of 30℃ and 120rpm for 48h, and take water samples every 12 hours to measure NO2 - 、NO3 - 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;

[0075] The results showed that the denitrification efficiency was higher when sodium nitrate was used as nitrogen source. - The removal rate can reach 87.62% in 48 hours, with NO2 - When NO2 was used as nitrogen source, its removal rate could only reach 66.65% in 48 hours, indicating that sodium nitrate was the best nitrogen source for aerobic denitrification of the strain. - Too high a content will have a certain inhibitory effect on the strain, resulting in a decrease in denitrification efficiency; the production of tetrahydropyrimidine when sodium nitrate is used as a nitrogen source is much higher than that of nitrite, further indicating that sodium nitrate is the best nitrogen source for this strain.

[0076] Example 6: Evaluation of nitrogen removal effect and ectoine production of strain EH-01 in natural seawater denitrification medium at different nitrogen concentrations

[0077] The specific method is:

[0078] (1) Activation: the method is the same as in Example 4;

[0079] (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, and then cultured at a constant temperature of 30°C and 120 rpm for 72 h. Water samples were taken every 12 hours to determine NO2 - 、NO3 - 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;

[0080] The results showed that when the initial nitrogen concentration was 200 mgN / L, the denitrification effect was the best, and NO3 - The removal rate reached 95.32% after 48 hours. At the same time, NO2 - The residual amount is 37.66 mgN / L; in comparison, when the initial nitrogen concentration is 250 mgN / L, the NO3 - The removal rate decreased slightly, while NO2 - The remaining amount was as high as 69.66 mgN / L. The two groups with initial nitrogen concentrations below 200 mgN / L had 48hNO3 - The removal rates were all lower than 90%, indicating that 200 mg N / L was the optimal initial nitrogen concentration for the strain to carry out denitrification reaction. Too high or too low nitrogen concentration would reduce the denitrification effect. With the increase of initial nitrogen concentration, the ectoine production of the strain gradually increased, and the production reached the maximum when the nitrogen concentration was 200 mg N / L; when the nitrogen concentration rose to 250 mg N / L, the ectoine production decreased significantly, indicating that the initial nitrogen concentration was too high and did not promote the growth of the strain.

[0081] Example 7: Evaluation of nitrogen removal effect and ectoine production of strain EH-01 in natural seawater denitrification medium at different carbon-nitrogen ratios

[0082] The specific method is:

[0083] (1) Activation: the method is the same as in Example 4;

[0084] (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 culture 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 concentration of sodium acetate in each group of conical flasks was 1.2 g / L, 2.4 g / L, 3.6 g / L, 6 g / L, 8.4 g / L, and 10.8 g / L, respectively. The other components of each group remained the same (1.2 g / L of sodium nitrate, 1 g / L of potassium dihydrogen phosphate, and 1 L of 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 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 measure NO2 - 、NO3 - 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;

[0085] The results showed that when C / N=2, the strain had the best denitrification effect, NO3 - The removal rate reached 95.87% after 48 hours. At the same time, NO2 - The residual amount dropped to 20.19 mgN / L. In comparison, the NO3 - The removal rates did not exceed 95%, but when C / N>2, the removal rates of NO2 - The residual amount after 48 hours was less than 20 mgN / L, indicating that the presence of a large amount of organic matter would promote NO2 - After comprehensive comparison, it was found that the optimal C / N value for the aerobic denitrification reaction of the strain was 2; the ectoine production was the largest when C / N=2, and it decreased slightly after rising to 3. When the carbon-nitrogen ratio was less than 2 or greater than 3, the ectoine production of the strain decreased significantly; by comparing the denitrification efficiency under different carbon-nitrogen ratios, it was found that the strain's ability to produce ectoine was highly correlated with its denitrification ability.

[0086] Example 8: Evaluation of nitrogen removal effect and ectoine production of strain EH-01 in artificial seawater denitrification medium at different salinities

[0087] The specific method is:

[0088] (1) Activation: the method is the same as in Example 4;

[0089] (2) Grouping: Use 10 conical flasks and divide them into 5 groups, with 2 conical flasks in each group. Add 50 mL of artificial seawater denitrification culture medium into each conical flask, where the sodium chloride concentration in each group of conical flasks is 35 g / L, 60 g / L, 80 g / L, 100 g / L, and 120 g / L respectively, and the other components are kept consistent in each group (sodium nitrate 1.2 g / L, sodium acetate 2.4 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, take 1 mL of the bacterial solution at a 2% (v / v) inoculation amount and inoculate it into 10 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 NO2 - 、NO3 - 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;

[0090] The results showed that the strain had the best denitrification effect when the salinity was 60g / L, and NO3 - The removal rate reached 96.48% after 48 hours. At the same time, NO2 - The concentration dropped to 23.47 mgN / L. In contrast, when the salinity was as low as 35 g / L or increased to 80 g / L, the denitrification of the strain would drop to 90% or below; when the environmental salinity increased to 100 g / L or above, the denitrification ability of the strain was affected, and NO3 - The removal rate will drop below 85%, and the NO2 - The concentration will also increase to about 40mgN / L. As a halophilic bacterium, the strain needs a certain salinity in its living environment. Low salinity will affect the growth, reproduction and denitrification ability of the strain to a certain extent. When the environmental salinity is too high, the concentration of the solution inside the bacteria is lower than that of the outside, resulting in a large loss of internal water, which affects the growth of the strain and inhibits its denitrification ability. In comprehensive comparison, the strain has the most suitable salinity of 60g / L, at which its tetrahydropyrimidine production is the highest, and the production decreases slightly when the salinity is 35g / L; when the salinity is higher than 60g / L, the strain's tetrahydropyrimidine production decreases significantly, indicating that higher salinity affects the survival of the strain while also affecting its ability to produce tetrahydropyrimidine.

[0091] Example 9: Evaluation of nitrogen removal effect and ectoine production of strain EH-01 in artificial seawater denitrification medium under different dissolved oxygen (rotation speed)

[0092] The specific method is:

[0093] (1) Activation: the method is the same as in Example 4;

[0094] (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 5.18-5.2 2mg / 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 1.2g / L, sodium acetate 2.4g / L, potassium dihydrogen 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 NO2 - 、NO3 - 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;

[0095] The results show that when the rotation speed is 90rpm and 120rpm, the denitrification capacity is high and close, 48hNO3 - The removal rate can reach 96-97%, NO2 - The concentration can be reduced to 20mgN / L. However, when the speed is below 90rpm, NO3 - The removal rate decreased slightly, NO2 - The concentration will increase to 28-38mgN / L; when the speed is increased to 150rpm, NO3 - The removal rate dropped to 90%, NO2 - The residual amount will increase to 40mgN / L; when the speed drops to 0rpm and 30rpm, the NO3 - The removal rate dropped to about 91%, and the NO2 -The residual concentrations were 38 mg N / L and 32 mg N / L respectively; the decrease in rotation speed would reduce the dissolved oxygen concentration in the environment, thus affecting the growth and function of the strain; the good denitrification performance at 0 rpm and 30 rpm showed that the strain could still ensure good denitrification efficiency in an environment with low dissolved oxygen; the denitrification efficiency dropped significantly at 150 rpm because the excessively high rotation speed brought about a strong centrifugal force, which caused the bacteria in the denitrification medium to agglomerate, resulting in a reduction in the contact area between the strain and the medium, causing the denitrification efficiency to decrease under higher dissolved oxygen concentrations; as the rotation speed increased from 0 rpm to 90 rpm, the strain's ectoine production continued to increase, reaching a maximum value at 90 rpm; when the rotation speed was further increased, the ectoine production began to decrease, indicating that while the increase in rotation speed increased the dissolved oxygen content, the high centrifugal force affected the strain's denitrification ability and the production of ectoine.

[0096] 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 Utah Halomonas that has both aerobic denitrification and ectoine production functions ( Vreelandella utahensis ) EH-01, 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.30540. The base length of 16S rDNA is 1442bp.

2. A method of using the Utah Halomonas bacteria having both aerobic denitrification and tetrahydropyrimidine production functions as claimed in claim 1 ( Vreelandella utahensis ) The method for extracting ectoine of EH-01 is characterized in that, The following steps are involved: S1: Strain activation: Inoculate the strain EH-01 stored in glycerol at -20°C into 50 mL of sterilized liquid enrichment 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. After centrifugation, harvest the bacteria and dilute with sterile water to OD 600 The value is 0.1, and the bacterial suspension preparation is complete; S2: Aerobic culture: Inoculate the bacterial suspension into 50 mL of natural seawater denitrification medium at a bacterial inoculum volume of 2% (v / v), and culture at 30°C and 120 rpm for 48 h to obtain a bacterial solution containing intracellular ectoine; S3: Crushing and extraction: Pour the intracellular ectoine-containing bacterial solution obtained in S2 into a 50 mL centrifuge tube, centrifuge at 4°C, 8000 rpm for 10 min, discard the supernatant, collect the precipitated bacteria, wash the bacteria with dipotassium hydrogen phosphate-potassium dihydrogen phosphate buffer with a pH of 7.0, centrifuge again under the same conditions, discard the supernatant, and collect the precipitated bacteria; finally, use 30 mL of deionized water to disperse the precipitated bacteria, crush them, and release the intracellular ectoine; after crushing, centrifuge at 4°C, 8000 rpm for 10 min, collect the supernatant containing ectoine and the bacterial precipitate, and dry and weigh the bacterial precipitate to obtain the ectoine yield per unit mass of bacteria.

3. The Utah halomonas bacteria having both aerobic denitrification and tetrahydropyrimidine production functions according to claim 2 ( Vreelandella utahensis ) The method for extracting ectoine of EH-01 is characterized in that, The crushing conditions are: using an ultrasonic cell crusher, with the parameters set to a power of 500 W and a crushing time of 10 min.

4. The Utah halomonas bacteria having both aerobic denitrification and tetrahydropyrimidine production functions according to claim 1 ( Vreelandella utahensis )The application of EH-01 in the treatment of high-salinity nitrogen-containing wastewater from ships and the biological preparation of tetrahydropyrimidine is characterized by: In the ship high-salinity nitrogen-containing wastewater, sodium acetate is the carbon source, sodium nitrate is the nitrogen source, the concentration of the nitrogen source is 200 mgN / L, C / N=2-3, the salinity is 35-60 g / L, and the shaking table speed is 90-120 rpm.

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