Vibrio gigas for removing calcium ions from hard water and application thereof

Abstract

The present application relates to the field of microorganisms, and in particular to a strain of Vibrio gigas for removing calcium ions in high-salt hard water and its application. The strain is named Vibrio gigas DZWSW, and was preserved in the China General Microbiological Culture Collection Center on December 20, 2021, with the preservation number of CGMCC No. 24153. The Vibrio gigas DZWSW provided in the present application plays an important role in precipitating calcium ions in high-salt hard water, and has a broad application prospect in hard water softening. After the Vibrio gigas DZWSW provided in the present application precipitates calcium ions in high-salt hard water, carbonate mineral precipitates are formed, including calcite, single-water calcite and dolomite minerals; the formed mineral precipitates can be used in the field of building materials, and have a broad application prospect.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and in particular to a giant Vibrio strain that removes calcium ions from high-salt hard water and its applications. Background Technology

[0002] The discharge of domestic sewage, industrial manufacturing, and oil and gas extraction processes generate a large amount of high-salt hard water. The calcium ions contained in this water can form stable scale, clog pipes, cause equipment corrosion, and affect safe production. How to effectively remove calcium ions from high-salt hard water has become an important problem that urgently needs to be solved.

[0003] Commonly used methods for softening hard water include chemical softening, ion exchange softening, membrane separation, electromagnetic softening, and lime softening. Among these, chemical softening has high operating costs for large volumes of water, and the treated water quality is affected by the chemicals, limiting its application. Ion exchange softening requires specific cation exchange resins for ion replacement, resulting in high resin costs, poor stability, the need for regenerants and wastewater treatment, and difficulties in reuse. Membrane separation requires high influent pressure, leading to high operating costs. Electromagnetic softening is not very stable and has limited application, primarily used for commercial circulating cooling water treatment. Lime softening can treat large volumes of hard water, but its settling range is limited, resulting in unstable treated water quality.

[0004] Utilizing the metabolic activities of microorganisms to precipitate calcium ions can soften high-salinity hard water, which is more economical and environmentally friendly than traditional methods such as chemical and physical methods. However, because there are relatively few types of microorganisms that can survive in high-salinity environments, there is also limited research on the use of microbial mineralization to precipitate calcium ions in high-salinity environments. Summary of the Invention

[0005] The purpose of this invention is to provide a giant Vibrio bacterium that removes calcium ions from high-salt hard water. This giant Vibrio bacterium can settle calcium ions to achieve the purpose of softening high-salt hard water, and can also obtain calcite, monohydrate calcite, and dolomite minerals.

[0006] This invention also provides the application of a giant Vibrio strain that removes calcium ions from high-salt hard water in the precipitation of calcium ions in high-salt hard water.

[0007] This invention provides a Vibrio giantis strain that removes calcium ions from high-salt hard water. The strain is named Vibrio giantis DZWSW and was deposited on December 20, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24153.

[0008] Preferably, the sodium chloride concentration in the Vibrio megaterium growth environment is 5-30%.

[0009] Preferably, the sodium chloride concentration in the Vibrio megaterium growth environment is 20%.

[0010] The present invention also provides a microbial agent, wherein the active ingredient of the microbial agent is Vibrio giantis DZWSW CGMCC No.24153 as described in any one of claims 1-3.

[0011] The present invention also provides the application of the Vibrio giantis or the bacterial agent in the sedimentation and / or removal of calcium ions in high-salt hard water.

[0012] The present invention also provides the application of the product generated by the precipitation of calcium ions in high-salt hard water by the Vibrio giantis or the bacterial agent in the fields of building materials or oil and gas storage.

[0013] The present invention also provides a method for obtaining calcite, comprising the following steps: adding calcite to a solution containing 0.01 mol / L Ca... 2+ The Vibrio giantis DZWSW CGMCC No.24153 or the bacterial agent is added to the first liquid phase system.

[0014] The present invention also provides a method for obtaining monohydrate calcite, comprising the following steps: [The method involves adding 0.01 mol / L Ca...] 2+ and 0.03-0.06 mol / L Mg 2+ The Vibrio giantis DZWSW CGMCC No.24153 or the bacterial agent is added to the second liquid phase system.

[0015] This invention also provides a method for obtaining dolomite, comprising the following steps: adding a solution containing 0.01 mol / L Ca... 2+ and 0.1-0.12 mol / L Mg 2+ The Vibrio giantis DZWSW CGMCC No.24153 or the bacterial agent is added to the third liquid phase system.

[0016] Preferably, the first liquid phase system, the second liquid phase system, and the third liquid phase system all contain carbonate ions and / or bicarbonate ions.

[0017] Beneficial effects:

[0018] The *Vibrio giantis* DZWSW provided by this invention plays an important role in the precipitation of calcium ions in high-salinity hard water and has broad application prospects in water softening. After precipitating calcium ions in high-salinity hard water, *Vibrio giantis* DZWSW forms carbonate mineral precipitates, including calcite, monohydrate calcite, and dolomite. These mineral precipitates can be used in the building materials industry, showing broad application prospects. Utilizing the metabolic activity of microorganisms to precipitate calcium ions to reduce the hardness of high-salinity hard water is more environmentally friendly than other physicochemical methods. Attached Figure Description

[0019] Figure 1Phylogenetic tree of Vibrio giantis DZWSW constructed based on 16S rDNA sequence.

[0020] Figure 2 The optimal salt concentration curve for Vibrio megaterium DZWSW.

[0021] Figure 3 The growth curve and pH change curve of Vibrio megaterium DZWSW in 20% NaCl.

[0022] Figure 4 X-ray diffraction analysis of mineral precipitates in culture media A, B, C, D, and E.

[0023] Figure 5 Thermogravimetric-differential thermogravimetric analysis of minerals in culture medium E.

[0024] Figure 6 Fourier transform infrared analysis of mineral precipitates A, B, C, D, and E in the culture medium.

[0025] Figure 7 X-ray photoelectron spectroscopy analysis of mineral precipitates in culture medium B.

[0026] Figure 8 High-resolution transmission electron microscopy (TEM) and selected area electron diffraction (SED) images of dolomite mineral precipitated in culture medium E: (a) SED pattern; (b) TEM image.

[0027] Figure 9 Scanning electron microscopy and energy dispersive spectroscopy (EDS) images of mineral precipitates A, B, C, D, and E in the culture medium.

[0028] Figure 10 Analysis of extracellular polymers of Vibrio megaterium DZWSW: (a) percentage of amino acids; (b) protein and polysaccharide content.

[0029] Figure 11 Under the action of Vibrio megaterium DZWSW, the Ca in culture media A, B, C, D, and E... 2+ Curves showing changes in concentration, sedimentation rate, and sedimentation velocity. Detailed Implementation

[0030] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0031] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application. As used herein, the singular form includes the plural form unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this description, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, all reagents and instruments used are commercially available conventional products.

[0034] X-ray diffraction analysis used a copper target X-ray diffractometer, Rigaku Electric Co., Ltd., Japan, model D / Max-RC; scanning electron microscopy analysis used a Hitachi S-4800, Hitachi, Ltd., Japan; energy dispersive spectroscopy analysis used a GENESIS spectrometer, IDAX Inc., USA; high-resolution transmission electron microscopy analysis used a JEM-2100, JEOL Ltd., Japan; thermogravimetric analysis used a Mettler TGA 2, Mettler Toledo, Switzerland; atomic absorption spectrophotometer was a TAS-986, Beijing Purkinje General Instrument Co., Ltd.; Fourier transform infrared (FTIR) analysis used a Nicolet iS50 FT-IR, Thermo Fisher Scientific, USA; X-ray photoelectron spectroscopy analysis used an ESCALAB, Thermo Fisher Scientific, USA. 250XI; Amino acid analysis was performed using an amino acid analyzer, Hitachi L-8900 (Japan); 772 spectrophotometer, Shanghai Precision Instruments Co., Ltd.; Vacuum freeze dryer, Shanghai Binlong Instruments Co., Ltd., FD-1A-50; pH meter, Jiangsu Jiangfen Electrochemical Analytical Instruments Co., Ltd., PHS-3E.

[0035] Example 1

[0036] Isolation, purification, identification and preservation of Vibrio giantiflora DZWSW CGMCC No. 24153

[0037] I. Isolation and purification of Vibrio megaterium DZWSW

[0038] This study isolated and purified Vibrio giantis DZWSW from sediments collected from the Yinjiashan Salt Factory in Qingdao, China. The liquid culture medium required for isolation and purification consisted of the following components: 5 g / L beef extract, 10 g / L tryptone, 2 g / L potassium chloride, and 200 g / L sodium chloride, dissolved in 1 L of distilled water, and the pH was adjusted to 6.8. A solid culture medium was prepared by adding 20 g / L agar powder to the liquid medium.

[0039] Under aseptic conditions, after sterilizing and cooling the above liquid culture medium, 10g of sediment (from Qingdao Yinjiashan Salt Factory, China) was added to 150mL of liquid culture medium. The medium was placed in a shaking incubator (Harbin Donglian Electronic Technology Development Co., Ltd., model HZQ-F160) and incubated at 37℃ and 130r / min for 5 days. The liquid culture medium became turbid, indicating successful enrichment of halophilic bacteria. 20μL of the bacterial suspension was inoculated onto a solid plate using the spread plate method and incubated at 32℃ for 7 days. Single colonies were picked from the plates and purified repeatedly at least three times to screen for a single strain, which was named *Vibrio giantis* DZWSW.

[0040] II. Molecular Identification of Vibrio megaterium DZWSW

[0041] The purified Vibrio giantis DZWSW was evenly spread on a solid plate. After 7 days of incubation, orange-red single colonies grew on the plate. The plate was sent to Shanghai Sangon Biotech Co., Ltd. for 16S ribosomal deoxyribonucleotide (16S rDNA) sequencing. Neighbor-joining (NJ) was used, and a phylogenetic tree was constructed from the 16S rDNA sequences using MEGA 6 software. The phylogenetic tree results are as follows: Figure 1 As shown, this confirms that the strain belongs to the genus Vibrio. The 16S rDNA sequence of Vibrio megaterium DZWSW is shown in SEQ ID NO: 1.

[0042] III. Preservation of Vibrio giantiflora DZWSW

[0043] Strain name: Vibrio megaterium

[0044] Latin name: (Vibrio gigantis)

[0045] Strain number: DZWSW

[0046] Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee

[0047] Collection institution abbreviation: CGMCC

[0048] Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing

[0049] Deposit date: December 20, 2021

[0050] CGMCC Registration Number: CGMCC No. 24153

[0051] Example 2

[0052] Preparation of Vibrio giantiflora DZWSW inoculum

[0053] Vibrio giantis DZWSW was inoculated into a liquid culture medium containing 20% ​​NaCl and cultured with shaking at 37°C and 130 r / min. The absorbance of the obtained bacterial solution at 600 nm was measured using a visible spectrophotometer. The culture was stopped when the absorbance was around 1.0. The obtained bacterial solution is the above-mentioned bacterial agent.

[0054] Vibrio giantis DZWSW and its inoculants have the following uses: a) removal of calcium ions; b) sedimentation of calcium ions in water; c) use of the product after calcium ion sedimentation in the field of building materials; d) use of the product after calcium ion sedimentation in oil and gas storage.

[0055] Example 3

[0056] I. Optimal salt concentration of Vibrio megaterium DZWSW

[0057] Six liquid culture media were prepared with sodium chloride concentrations of 5%, 10%, 15%, 20%, 25%, and 30%, respectively. Three replicates were set up for each. *Vibrio megaterium* DZWSW inoculum was inoculated into the liquid culture media at a volume of 5%, and cultured with shaking at 37℃ and 130 rpm. After 150 hours, the bacterial concentration was measured using a visible spectrophotometer at a wavelength of 600 nm, and the average value was taken.

[0058] The experimental results are shown in Figure 2 OD of Vibrio giantiflora DZWSW at 20% NaCl concentration 600nm The highest value indicates that Vibrio giantis DZWSW has an optimal salt concentration of 20%, classifying it as an extreme halophile.

[0059] II. Growth curve and pH change curve of Vibrio giantiflora DZWSW at 20% NaCl concentration

[0060] Six 500mL Erlenmeyer flasks were used, each set to the optimal salt concentration condition, i.e., 300mL of liquid culture medium with a NaCl concentration of 20% was prepared and autoclaved at 121℃ for 30 minutes. Three replicates were prepared for both the control and experimental groups. In a clean bench, the experimental group was inoculated with 5% (v / v) DZWSW bacterial agent (OD). 600=1.0), and the control group was inoculated with the same volume of sterile ultrapure water. After inoculation, all conical flasks were placed in a constant temperature shaking incubator and cultured with shaking at 37℃ and 130 r / min. At regular intervals, 3 mL samples were taken to determine the bacterial concentration and pH value, and the average value was taken. Based on the experimental results, the growth curve and pH curve of Vibrio giantiflora DZWSW at a 20% NaCl concentration were plotted.

[0061] like Figure 3 As shown, the growth curve of Vibrio giantiflora DZWSW can be divided into four stages: lag phase (0–106 h), logarithmic phase (106–274 h), stationary phase (274–350 h), and decline phase (350–384 h).

[0062] During the lag phase, bacterial concentration is low, indicating that bacteria are adapting to the growth environment of the culture medium. In the logarithmic phase, bacterial concentration increases logarithmically, and bacteria are in an active state of division. In the stationary phase, bacterial growth is slow, as substances in the culture medium are consumed and toxic metabolites gradually accumulate. In the decline phase, bacterial catabolism exceeds anabolism, leading to mass bacterial death. Since no bacterial agent was added to the control group, no growth curve was observed. There were significant differences in the pH curves between the experimental and control groups. The pH curve of the control group remained almost unchanged, while the pH curve of the experimental group decreased to 6.88 after 20 hours and then gradually increased to 8.62.

[0063] Example 4

[0064] At a 20% NaCl concentration, Vibrio giantiflora DZWSW induced Ca2+. 2+ Sedimentation produces various carbonate minerals

[0065] 0.01 mol / L calcium chloride was added to a 20% salt concentration liquid culture medium. The medium was then dispensed and 0, 0.03, 0.06, 0.10, and 0.12 mol / L magnesium chloride were added respectively to obtain culture medium A, culture medium B, culture medium C, culture medium D, and culture medium E. The Mg / Ca ratios of these media were 0, 3, 6, 10, and 12, respectively. The culture media were sterilized at 121℃ for 30 minutes and then cooled to room temperature. 3 mL of 1 mol / L Na₂CO₃ and 3 mL of 1 mol / L NaHCO₃ aqueous solution were added to each conical flask (both Na₂CO₃ and NaHCO₃ aqueous solutions were sterilized by filtration through a 0.22 μm microporous membrane). The pH was then adjusted to 6.8 using hydrochloric acid and sodium hydroxide.

[0066] In a clean bench, Vibrio megaterium DZWSW inoculum was inoculated into liquid culture medium at a ratio of 5% as the experimental group; an equal volume of sterile 20% sodium chloride solution was inoculated into the liquid culture medium as the control group. Each gradient Mg / Ca ratio had both control and experimental groups, with three replicates in each group. After inoculation, all conical flasks were placed in a constant temperature shaking incubator and cultured with shaking at 37℃ and 130 rpm.

[0067] Example 5

[0068] Analysis of the mineral precipitate obtained in Example 4

[0069] I. X-ray Diffraction Analysis (XRD)

[0070] The precipitate from the bottom of the mineralized culture medium cultured for 7 days in Example 4 was washed three times each with distilled water and anhydrous ethanol to remove residual culture medium components and organic matter. The obtained minerals were then naturally dried and subjected to XRD analysis at a scanning angle (2θ) of 10–60°, a step size of 0.02°, and a scanning speed of 8° / min. No visible mineral precipitate was found in the control group; therefore, only the minerals in the experimental group were analyzed.

[0071] The experimental results are shown in Figure 4 The values ​​of Mg / Ca = 0, 3, 6, 10, and 12 represent culture media A, B, C, D, and E, respectively. The mineral precipitate produced in culture medium A shows diffraction peaks corresponding to calcite, indicating that the mineral is calcite. Culture media B and C contain monohydrate calcite. The mineral diffraction peaks in culture media D and E are close to those of dolomite, indicating that the product is dolomite.

[0072] Dolomite possesses highly interconnected intercrystalline pores, thus exhibiting excellent reservoir and drainage capabilities. This makes it significant not only for oil and gas storage but also as a crucial component of freshwater aquifers. Dolomite is the primary mineral composing dolomite. Under high salinity conditions, microbial metabolic activity leads to relatively high ion concentrations in the local microenvironment, which helps inhibit magnesium ion hydration. Furthermore, extremophiles secrete more extremophilic polysaccharides (EPS) to protect themselves from the effects of extreme environments. EPS can provide nucleation sites for carbonate minerals, and the carboxyl and hydroxyl groups they contain also play important roles.

[0073] II. Thermogravimetric Analysis (TG-DTG)

[0074] TG-DTG analysis was performed on dolomite minerals in culture medium E (Mg / Ca = 12) under the following conditions: nitrogen flow rate 50 cm⁻¹. 3 The study investigated the relationship between mineral content and temperature, as well as the changes in mineral content across different temperature ranges, using a heating rate of 15° / min, with a temperature range from room temperature to 1100°C.

[0075] The experimental results are shown in Figure 5 The maximum decomposition temperature of dolomite mineral is 700℃, with a mass loss of 52.19%. The thermal decomposition process can be divided into three stages: the first stage is from the initial temperature to T1, accompanied by the evaporation of adsorbed water and water of crystallization, and the loss of organic matter; the second stage is from T1 to T2, where the main cause of thermal weight loss is the thermal decomposition of calcium carbonate and magnesium carbonate, with T2 being the maximum thermal decomposition temperature of the sample; the third stage is from T3 to 1100℃, during which the mineral content remains essentially unchanged. The final results show that the mineral's thermal weight loss from the initial temperature to the final temperature is 52.19%.

[0076] III. Fourier Transform Infrared Spectroscopy (FTIR)

[0077] FTIR analysis was performed on the minerals induced by Vibrio megaterium DZWSW in media A, B, C, D, and E. The results are shown in [Figure 1]. Figure 6 .

[0078] In culture medium A ( Figure 6 a) 712cm -1 875cm -1 1397cm -1 1419cm -1 1489cm -1 and 2512cm -1 The characteristic bands confirmed the presence of calcite; culture media B and C ( Figure 6 In (b) and (c), 699cm -1 761cm -1 871cm -1 1067cm -1 1398cm -1 and 1508cm -1 The characteristic bands confirmed the presence of monohydrated calcite; culture medium D ( Figure 6 In d), 727cm -1 878cm -1 1445cm -1 2523cm -1 and 3423cm -1 The characteristic bands confirmed the presence of dolomite; culture medium E ( Figure 6 In e), 879cm -1 1443cm -1 1816cm -1 2523cm -1 and 3420cm -1 The characteristic banding confirms the presence of dolomite. Besides the mineral's characteristic banding, CH(1381cm) -1 C = O (1618cm) -1 1636cm-1 1701cm -1 1793cm -1 ), CO (1012cm) -1 This indicates the presence of organic functional groups, confirming the biogenic origin of the minerals.

[0079] IV. X-ray photoelectron spectroscopy (XPS) analysis

[0080] XPS analysis was performed on the minerals induced in culture medium B, and the results are shown below. Figure 7 The elements present on the surface of biominerals include C, O, Ca, Mg, N, P, and S. Figure 7 a). MgOH is present in Mg1s at 1305 eV ( Figure 7 b); O1s has C=O at 531.8 eV ( Figure 7 c) N1s contains NH4 at 402 eV. + exist( Figure 7 d) Two peaks appear in Ca2p, one at 346.75 eV and the other at Ca2p. 3 / 2 and Ca2p at 350.5 1 / 2 ( Figure 7 e); C1s has three peaks: CC at 285.2 eV, COC at 286.8 eV, and OC=O at 289 eV. Figure 7 f); S2p has SH at 162.56 eV. Figure 7 g); P2p has PO at 133.6 eV ( Figure 7 h). Based on XPS analysis of C, P, N, and S elements, it can be confirmed that organic matter does indeed participate in the crystallization and growth process of biominerals, further demonstrating the microbial origin of carbonate minerals.

[0081] V. High-resolution transmission electron microscopy and selected area electron diffraction images (HRTEM-SAED)

[0082] The precipitate from the bottom of culture medium E was placed in a 1.5 mL centrifuge tube and allowed to stand for 20 min. The supernatant was discarded, and sterile water was added to the original volume for washing. This process was repeated at least three times to remove various salt ions. After natural drying, the resulting mineral precipitate was thoroughly ground in an agate mortar and pestle, and then anhydrous ethanol was added. The precipitate was then resuspended by ultrasonic cleaning. The induced minerals were placed on a copper grid for HRTEM-SAED analysis. The results are shown in the figure. Figure 8 .

[0083] Figure 8 (a) Selected area electron diffraction (SAED) is used to calculate the interplanar spacing. This experimental result is consistent with the interplanar spacing of the dolomite standard card PDF#74-1687. They are close to each other and correspond to the crystal planes hkl(11-9), hkl(024), hkl(110), hkl(015), and hkl(006), respectively. Figure 8 (b, d, e) are high-resolution transmission electron microscopy images, corresponding to... Figure 8 The positions of b, d, and e in (c). Interplanar spacing. Crystal facets of standard cards They are similar and correspond to the crystal planes hkl(110), hkl(11-9), hkl(12-1), hkl(11-3), hkl(12-10), and hkl(300) in the standard dolomite card PDF#74-1687, respectively.

[0084] The above results further confirm that the mineral induced by Vibrio giantis DZWSW in culture medium E is dolomite.

[0085] VI. Scanning Electron Microscopy and Energy Dispersive Spectroscopy (SEM-EDS)

[0086] Mineral morphology and elemental composition at different Mg / Ca ratios in 20% NaCl were analyzed using SEM and EDS. Figure 9 The images show scanning electron microscopy and energy dispersive spectroscopy (EDS) spectra of the mineral precipitates.

[0087] Figure 9 a1-a3 represent the microstructure of mineral precipitates in culture medium A; the minerals are spherical and dumbbell-shaped. Figure 9 a3), composed of numerous tiny calcite particles. Figure 9 (a2), with a rough surface. The monohydrate calcite in culture medium B is a twisted aggregate composed of numerous platy minerals. Figure 9 b1-b3). The monohydrate calcite in culture medium C mostly formed twisted aggregates, which were more complete than those in culture medium B. Figure 9 c1) is composed of numerous tiny, flaky minerals, and the pores on its surface may be traces of bacterial activity. Figure 9 c2). The mineral surface of culture medium D is rough, composed of many nano-sized particulate minerals, and is dumbbell-shaped. Figure 9 d1) spherical ( Figure 9 d2-d4) contains a large number of pores, and good porosity is one of the important factors for a reservoir. Numerous small spheres adhere to the mineral surface (d2-d4). Figure 9 d3), the spheres may be in the early growth stage of dolomite. The culture medium minerals mainly appear as cauliflower-like or petal-like shapes. Figure 9 e2), spherical ( Figure 9e3-4), with a rough surface and numerous pores, is composed of many nano-sized granular minerals. The mineral surface also has a large number of small spheres ( Figure 9 e1), the cauliflower-shaped mineral is about 2 μm long and forms an aggregate with the petal-shaped mineral. Therefore, the petal-shaped mineral and the cauliflower-shaped mineral may be different stages of the same mineral. Figure 9 e2). The mineral surface contains pores, which may be formed by the shedding of small spheres attached to the mineral surface. Figure 9 e3-4). According to Figure 9 The e3 data shows that bacterial morphology can be observed on the mineral surface, further confirming the microbial origin of the mineral.

[0088] EDS results showed trace amounts of P and S elements in the minerals, possibly originating from bacterial metabolic activity. Ca, Mg, C, and O mainly came from the minerals. Figure 9 a4, 9b4, 9c3, 9d5, 9e5).

[0089] Example 6

[0090] Extraction of bacterial extracellular polymers and testing of amino acids, proteins, and polysaccharides in the extracellular polymers

[0091] I. Extraction of bacterial extracellular polymers

[0092] Extracellular polymers of Vibrio giantiflora DZWSW were extracted using a heating method. The specific steps are as follows:

[0093] 1. Take the bacterial agent that has grown to the stable phase and centrifuge it at 3000 r / min for 10 minutes;

[0094] 2. Discard the supernatant, add sterile water of the same salt concentration to the original volume, centrifuge at 2000 r / min for 3 minutes, repeat three times to remove impurities from the liquid culture medium and obtain the bacterial suspension;

[0095] 3. Place the bacterial suspension obtained in step 2 in a water bath and heat at 60°C for 30 minutes, then centrifuge at 10000 r / min for 10 minutes to obtain the sample;

[0096] 4. The supernatant of the sample obtained in step 3 is filtered through a microporous membrane with a pore size of 0.22 μm to obtain a colorless and transparent EPS solution.

[0097] 5. Transfer the EPS solution obtained in step 4 to a dialysis bag (DM=25mm, MwCO=500) and dialyze for 48 hours to remove sodium chloride from the solution;

[0098] 6. The sample obtained in step 5 is vacuum dried in a freezer for 48 hours to obtain extracellular polymer powder.

[0099] Extracellular polymeric substances (EPS) secreted by bacteria are polymers composed of high molecular weight substances such as polysaccharides, proteins, and nucleic acids. The carboxyl and hydroxyl functional groups in these EPS can effectively bind calcium and magnesium ions, reducing magnesium ion hydration and providing nucleation sites for carbonate minerals. Extreme halophiles can secrete even more EPS, thus protecting themselves from the effects of extreme environments.

[0100] II. Analysis of extracellular polymers (amino acids, polysaccharides, and proteins)

[0101] The obtained EPS powder was sent to Jiangsu Coastal Chemical Testing Technology Service Co., Ltd., where an amino acid analyzer was used to detect the amino acid composition in the EPS. Figure 10 a) Among the 15 amino acids detected, acidic amino acids (glutamic acid and aspartic acid) had the highest molar percentage content. Both aspartic acid and glutamic acid contain two carboxyl groups, which is conducive to the nucleation of dolomite minerals in culture medium E.

[0102] The protein content of EPS powder extracted from different culture media (A, B, C, D, and E) was analyzed using Coomassie Brilliant Blue staining, and the polysaccharide content was analyzed using the phenol-sulfuric acid method. The results are shown in [Figure 1]. Figure 10 b. As the magnesium-to-calcium ratio increases, the contents of polysaccharides and proteins gradually increase, especially the polysaccharide content, which increases most significantly. This indicates that the increased magnesium ion content can provide more carboxyl and hydroxyl groups, which is conducive to the nucleation of dolomite minerals.

[0103] Example 7

[0104] Analysis of calcium ions in high-salt, hard water by *Vibrio gigantis* DZWSW sedimentation

[0105] In Example 3, the pH of the culture medium increased under the action of the bacterial agent, providing a favorable environment for carbonate mineral precipitation. In Example 5, mineral analysis showed that carbonate minerals precipitated in media A, B, C, D, and E. In Example 6, the EPS of Vibrio giantis DZWSW provided nucleation sites for carbonate mineral precipitation, containing a large amount of acidic amino acids and polysaccharides. The extreme halophilic bacterium Vibrio giantis DZWSW can not only induce the formation of dolomite minerals but also precipitate Ca in hard water. 2+ This softens high-salinity hard water.

[0106] The changes in calcium ion concentration in hard water under the influence of Vibrio megaterium DZWSW were analyzed using atomic absorption spectrophotometry. Samples were taken at regular intervals, and the steps were as follows:

[0107] 1. Take 1 mL of supernatant from each of culture media A, B, C, D, and E;

[0108] 2. Dilute with sterile ultrapure water 10 times;

[0109] 3. Filter using a microporous membrane with a pore size of 0.22 μm;

[0110] 4. Measure the absorbance of the diluted sample using an atomic absorption spectrophotometer.

[0111] By analyzing samples collected over 16 days, curves showing changes in calcium ion concentration, calcium ion sedimentation rate, and calcium ion sedimentation velocity were plotted. The results are shown below. Figure 11 The calcium ion concentration decreased slowly over the first 5 days, possibly because the bacteria were in the lag phase at this time, followed by a rapid decrease. Figure 11 a). Among them, the calcium ion concentration in culture medium A decreased at a much higher rate than in other culture media from day 5 to day 8, and the sedimentation rate gradually decreased from day 8 to day 10, with the sedimentation rate tending to level off. This may be related to the presence or absence of magnesium ions. Figure 11 (b) and (c). On day 16, the calcium ion concentrations in media A, B, C, D, and E were 111.51 mg / L, 159.62 mg / L, 172.41 mg / L, 191.48 mg / L, and 200.39 mg / L, respectively, with sedimentation rates of 70.753%, 58.036%, 54.618%, 48.683%, and 47.594%, respectively. This confirms that Vibrio megaterium DZWSW can effectively soften high-salt hard water.

[0112] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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. sequence list <110> Shandong University of Science and Technology <120> A giant Vibrio strain that removes calcium ions from high-salt hard water and its application <160> 1 <170> SIPOSequenceListing 1.0 <210> 1 <211> 1459 <212> DNA <213> Vibrio gigantis ccggtcctaa gcatgctagt ggagcaggta actacacata acgagtcctt actgaggtgc 60 tgttaatggg cgtcgagcgg cggacgggtg agtaatgcct aggaaattgc cttgatgtgg 120 gggataacca ttggaaacga tggctaatac cgcataatgc ctacgggcca aagagggggga 180 ccttcgggcc tctcgcgtca agatatgcct aggtgggatt agctagttgg tgaggtaatg 240 gctcaccaag gcgacgatcc ctagctggtc tgagaggatg atcagccaca ctggaactga 300 gacacggtcc agactcctac gggaggcagc agtggggaat attgcacaat gggcgaaagc 360 ctgatgcagc catgccgcgt gtatgaagaa ggccttcggg ttgtaaagta ctttcagttg 420 tgaggaaggg ggtagcgtta atagcgctat ctcttgacgt tagcaacaga agaagcaccg 480 gctaactccg tgccagcagc cgcggtaata cggagggtgc gagcgttaat cggaattact 540 gggcgtaaag cgcatgcagg tggttcatta agtcagatgt gaaagcccgg ggctcaacct 600 cggaactgca tttgaaactg gtgaactaga gtactgtaga ggggggtaga atttcaggtg 660 tagcggtgaa atgcgtagag atctgaagga ataccagtgg cgaaggcggc cccctggaca 720 gatactgaca ctcagatgcg aaagcgtggg gagcaaacag gattagatac cctggtagtc 780 cacgccgtaa acgatgtcta cttggaggtt gtggccttga gccgtggctt tcggagctaa 840 cgcgttaagt agaccgcctg gggagtacgg tcgcaagatt aaaactcaaa tgaattgacg 900 ggggcccgca caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa gaaccttacc 960 tactcttgac atccagagaa gccagcggag acgcaggtgt gccttcgggga gctctgagac1020 aggtgctgca tggctgtcgt cagctcgtgt tgtgaaatgt tgggttaagt cccgcaacga1080 gcgcaaccct tatccttgtt tgccagcgag taatgtcggg aactccaggg agactgccgg1140 tgataaaccg gaggaaggtg gggacgacgt caagtcatca tggcccttac gagtagggct1200 acacacgtgc tacaatggcg catacagagg gcagcaagct agcgatagtg agcgaatccc1260 aaaaagtgcg tcgtagtcg gattggagtc tgcaactcga ctccatgaag tcggaatcgc1320 tagtaatcgt gaatcagaat gtcacggtga atacgttccc gggccttgta cacaccgccc1380 gtcacaccat gggagtggta tccataggaa gtgatgctag gctccagtaa gtcgtaagca1440 caggtaaccc catacggat 1459

Claims

1. A giant Vibrio strain that removes calcium ions from high-salt hard water, characterized in that, The strain was named Vibrio megaterium (Vibrio giantis) Vibrio gigantis DZWSW was deposited on December 20, 2021, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 24153.

2. The giant Vibrio according to claim 1, characterized in that, The sodium chloride concentration in the growth environment of Vibrio megaterium is 5-30%.

3. The giant Vibrio according to claim 1, characterized in that, The sodium chloride concentration in the growth environment of Vibrio megaterium is 20%.

4. A microbial agent, characterized in that, The active ingredient of the bacterial agent is Vibrio giantis DZWSW CGMCC No.24153 as described in any one of claims 1-3.

5. The application of Vibrio giantis according to any one of claims 1-3 or the bacterial agent according to claim 4 in sedimentation and / or removal of calcium ions in high-salt hard water.

6. The application according to claim 5, characterized in that, Applications of products obtained after calcium ions have settled in high-salt hard water in building materials or oil and gas storage.

7. A method for obtaining calcite, characterized in that, The steps include: adding a solution containing 0.01 mol / L Ca 2+ The Vibrio giantis DZWSW CGMCC No.24153 of any one of claims 1-3 or the bacterial agent of claim 4 is added to the first liquid phase system.

8. A method for obtaining monohydrated calcite, characterized in that, The steps include: adding a solution containing 0.01 mol / L Ca 2+ and 0.03-0.06 mol / L Mg 2+ The Vibrio giantis DZWSWCGMCC No.24153 of any one of claims 1-3 or the bacterial agent of claim 4 is added to the second liquid phase system.

9. A method for obtaining dolomite, characterized in that, The steps include: adding a solution containing 0.01 mol / L Ca 2+ and 0.1-0.12 mol / L Mg 2+ The Vibrio giantis DZWSWCGMCC No.24153 of any one of claims 1-3 or the bacterial agent of claim 4 is added to the third liquid phase system.

10. The method according to any one of claims 7-9, characterized in that, The liquid phase systems all contain carbonate ions and / or bicarbonate ions.

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