A salt-reducing and mineralizing dual-effect bacterial strain and its cultivation method and application

Abstract

The invention discloses a salt-reducing and mineralizing dual-effect bacterial strain and a cultivation method and application thereof. The salt-reducing and mineralizing dual-effect bacterial strain is classified and named as sulfide mineralizing halomonas Halomonas sulfidearis. ZN2#, deposited in the General Microbiology Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.32033. Its optimal culture conditions are: culture temperature 30 °C, culture speed 200 r / min, and culture time 48 h. This strain has the dual-effect performance of mineralization and salt reduction, and can reduce the salt content in the soil while solidifying the soil. This strain combines microbial induced calcium carbonate precipitation technology to achieve the improvement of soil strength while reducing the salt content in saline soil, and is suitable for the improvement and modification of saline-alkali soils. This strain avoids the impact of exogenous strains on the soil environment while repairing and improving saline-alkali soil, and realizes the sustainability of saline soil improvement.

Description

Technical Field

[0001] The invention relates to the technical field of preparation of salt-reducing and mineralizing dual-effect bacterial strains, and in particular to a salt-reducing and mineralizing dual-effect bacterial strain and a cultivation method and application thereof. Background Art

[0002] Saline soil is a soil with a soluble salt content greater than 0.3% and engineering properties such as dissolution, salt swelling, and corrosion. Its special physical and mechanical properties have caused great damage to the construction and operation of shallow surface light structures such as industrial and civil buildings, water conservancy, railways, and highways. Different types of saline soils have different effects on structural facilities. Sulfate saline soils mainly destroy the structure of infrastructure through crystallization and expansion, while chloride saline soils cause uneven foundation settlement and other hazards through moisture absorption and softening, and chloride salts accelerate the corrosion of steel bars. In addition, soil salinization and sub-salinization cause waste of land resources, greatly restrict the development of agriculture, and cause huge economic losses. However, traditional saline soil improvement measures often have problems such as high material cost, large limitations, damage to the original soil structure, unsuitable for large-scale use, poor sustainability, large side effects, secondary soil pollution, and waste of water resources, making it difficult to achieve sustainable development of saline soil improvement. Biological improvement measures have been gradually promoted due to their environmental friendliness and sustainable development. Among them, the application of microbial solidification combined with microbial induced calcium carbonate precipitation (MICP) technology to the improvement of saline soil is an emerging, green, environmentally friendly and sustainable soil improvement technology. However, there are few reports on the application of microbial salt reduction combined with microbial solidification based on microbial induced calcium carbonate precipitation technology to the improvement and restoration of saline soil. Summary of the invention

[0003] The purpose of the present invention is to isolate and screen a salt-reducing and mineralizing dual-effect strain and its application method in saline soil. The strain can solidify the soil and improve the soil strength while reducing the salt content in the soil to solve the problems raised in the above background technology. The purpose of the present invention is achieved through the following technical solutions:

[0004] A salt-reducing and mineralizing dual-effect strain, the salt-reducing and mineralizing dual-effect strain is classified and named as sulfide mineralizing Halomonas ( Halomonassulfidaeris. ) ZN2#, deposited in the General Microbiological Center of China Microbiological Culture Collection Administration, with the deposit number CGMCC No.32033 and the deposit date September 20, 2024. Its 16S rRNA gene sequence is shown in SEQ ID NO.1. Halomonassulfidaeris Esulfide 1 (or the same genus) has a homology of 99.08%.

[0005] The salt-reducing and mineralizing dual-effect strain is isolated from a salt lake. The strain is a Gram-positive bacterium, a moderately halophilic bacterium and an alkali-resistant bacterium, and a urease-producing mineralizing bacterium. The salt-reducing and mineralizing dual-effect strain has an optimal growth range of chloride concentrations of 10% to 15%; a chloride concentration tolerance range of 3% to 20%; a sulfate concentration tolerance range of 0.4 to 1.5 mol / L; and an alkali tolerance range of pH 5.5 to 13. The strain appears as an irregular liquid droplet-like morphology on a solid culture medium, is white in color, and has a rod-shaped body with a length of 1 μm to 3 μm, such as Figure 1 shown.

[0006] The logarithmic growth period of the salt-reducing and mineralizing dual-effect strain is 48 h, and the optimal culture conditions are: culture temperature 30 °C, culture speed 200 r / min, and culture time 48 h.

[0007] The separation and screening method of the salt-reducing and mineralizing dual-effect bacterial strain comprises the following steps:

[0008] Step 1: Take a water sample within the range of 0-10 cm below the surface of the salt lake, put it into a half-volume beef extract peptone culture medium after high temperature sterilization, and shake it at a constant temperature of 28-32°C, a shaking frequency of 150-200 r / min, and a shaking time of 24 hours. After taking it out, let it stand for 12 hours; culture it to obtain a bacterial enrichment solution;

[0009] Step 2: Collect the bacterial enrichment solution and prepare 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 dilution of the dilution solution;

[0010] Step 3: The diluted solutions of different concentrations are respectively coated on a plate with a solid culture medium on the surface and cultured at a constant temperature of 28°C to 32°C for 2 days to 3 days. The plate is sealed and placed in a constant temperature incubator for culture;

[0011] Step 4: Pick a single white colony on the solid culture medium and continue to inoculate it into the liquid culture medium for shaking culture. The shaking temperature is 28-32°C, the shaking frequency is 150-200 r / min, the pH is the natural pH of the culture medium, and the culture time is 24-48 hours;

[0012] Step 5: Take the bacterial solution to verify the mineralization performance;

[0013] Step 6: Take the bacterial solution to verify the salt reduction performance;

[0014] Step 7: Select a single colony with both mineralization and salt reduction effects and continue to inoculate it into a solid culture medium. After repeated streaking and purification, the obtained single colony is the salt reduction and mineralization dual-effect strain;

[0015] The half-strength beef extract peptone culture medium comprises: 1-2 g beef extract, 5-10 g peptone, and 2-3 g NaCl per 1000 mL deionized water;

[0016] The solid culture medium comprises: 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, 5-15 mL of SL-6 trace element solution, and 10-15 g of agar per 1000 mL of deionized water;

[0017] The liquid culture medium comprises: 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, and 5-15 mL of SL-6 trace element solution per 1000 mL of deionized water.

[0018] Further, the mineralization performance verification: after different single colonies are placed in liquid culture medium and cultured for 24-48 hours, 5-10 ml of bacterial solution is inoculated into the calcium source solution, the mixed solution with white precipitation is allowed to stand for 24 hours, the supernatant is poured out, and the residual bacteria in the precipitation are washed with sterile distilled water, and the sediment sample is obtained at 40-50°C for 12-24 hours; the sediment is detected with 1 mol / L dilute hydrochloric acid. If a large number of bubbles are generated immediately after adding dilute hydrochloric acid to the sediment, the sediment is judged to be calcium carbonate precipitation, and the single colony is a colony with mineralization performance; the calcium source solution includes 0.5-1 mol / L urea and 0.5-1 mol / L dihydrate calcium chloride solution. The microscopic morphology of the sediment is observed and judged by scanning electron microscopy (SEM); the crystal structure of the sediment is analyzed by X-ray diffractometer (XRD); and the chemical composition of the sediment is analyzed by energy dispersive spectrometer (EDS).

[0019] The salt reduction performance verification is as follows: different single colonies are placed in liquid culture medium and cultured for 3-7 days, the cultured bacterial solution is centrifuged and the supernatant is collected; 0.5 ml of the supernatant is placed in a conical flask, 3-5 drops of potassium chromate indicator are added, and titration is performed using a standard silver nitrate solution. If the chloride ion content in the cultured microbial solution is reduced, it is judged that there is a salt reduction effect, and the single colony is a colony with salt reduction performance.

[0020] The method for culturing the salt-reducing and mineralizing dual-effect strain is as follows: the salt-reducing and mineralizing dual-effect strain is inoculated into a liquid culture medium and shaken for culture: the culture temperature is 30 ° C, the culture speed is 200 r / min, and the culture time is 48 h. The liquid culture medium composition is: every 1000 mL of deionized water includes 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, and 5-15 mL of SL-6 trace element solution.

[0021] The method for culturing the salt-reducing and mineralizing dual-effect strain is as follows: the salt-reducing and mineralizing dual-effect strain is inoculated into a solid culture medium for cultivation at a temperature of 30 °C for a culture time of 48 h; the solid culture medium components are: per 1000 mL of deionized water, 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, 5-15 mL of SL-6 trace element solution, and 10-15 g of agar.

[0022] The salt-reducing and mineralizing dual-effect bacterial strain is used to solidify saline soil and reduce soil salt content at the same time.

[0023] One application method includes the following steps:

[0024] Step 1: Prepare salt-reducing and mineralizing dual-effect bacterial solution and cementing solution for use in saline soil: The salt-reducing and mineralizing dual-effect bacterial solution is prepared by expanding the salt-reducing and mineralizing dual-effect bacterial strain, and the OD of the bacterial solution is 600 The value is 0.5~2.0, and the urease activity is 4~6 mMurea / min; the cementing fluid composition is: urea 30~60 g / L, CaCl 2 ·2H 2 O 73.5~147 g / L; the solvent of the cementing fluid is water;

[0025] Step 2: Perform in-situ microbial mixing to form a microbial mineralization layer, including the following steps:

[0026] 1) mixing the salt-reducing and mineralizing dual-effect bacterial solution and the cementing solution at a volume ratio of 1:1 to 3 and stirring evenly to obtain a bacterial solution and cementing solution mixture;

[0027] 2) mixing the bacterial liquid-cementing liquid mixture with the soil in the saline soil area at a mass ratio of 1:2-4 and stirring evenly;

[0028] 3) Compact the mixed saline soil and air-dry it.

[0029] In practical applications, it can be naturally air-dried until the strength reaches 1.5~5.0 MPa;

[0030] The salt-reducing and mineralizing dual-effect bacterial strain solidifies saline soil while reducing the salt content in the soil, thereby improving the soil strength and reducing the salt content in the soil, thereby achieving the restoration and improvement of the saline soil.

[0031] The salt-reducing and mineralizing dual-effect strain is suitable for the improvement and restoration of chlorine and subchlorine saline soil, sulfuric acid and sulfite saline soil, and chloride-sulfate mixed saline soil. The average salt content (in mass percentage) of chlorine and subchlorine saline soil is applicable to 3%~20%; the average salt content of sulfuric acid and sulfite saline soil is applicable to 1%~15%. The pH range of the salt-reducing and mineralizing dual-effect strain applicable to saline soil is: 5.5~13.

[0032] The advantages and beneficial effects of the present invention are:

[0033] 1. The salt-reducing and mineralizing dual-effect strain provided by the present invention is a sulfide mineralizing bacterium Halomonassulfidaeris. ZN2# is a urease-producing mineralizing bacterium and also a salt-reducing bacterium. This strain is an autologous strain of saline soil, has good environmental tolerance, high curing strength, and can avoid the impact of exogenous strains on the environment, and has great research and application value.

[0034] 2. Sulfide mineralizing bacteria Halomonassulfidaeris. ZN2# is a tolerant salt-reducing and mineralizing dual-effect bacterial strain that can not only tolerate high-salinity and alkaline environments, but also induce carbonate precipitation to solidify soil particles. It can also reduce the salinity of saline soils and can be used as a new type of microbial strain for soil solidification in special environments.

[0035] 3. The salt-reducing and mineralizing dual-effect strains are combined with microbial induced mineralization (MICP) technology to solidify the saline soil, improve the soil strength, and reduce the soil salt content, thereby achieving green restoration and improvement of the saline soil. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0037] Figure 1 The sulfide mineral salt monosaccharide provided in the embodiment of the present application Halomonassulfidaeris. ZN2# colony morphology and SEM scanning electron microscopy images;

[0038] Figure 2 The sulfide mineral salt monosaccharide provided in the embodiment of the present application Halomonassulfidaeris. Phylogenetic tree of ZN2#;

[0039] Figure 3 The sulfide mineral salt monosaccharide provided in the embodiment of the present application Halomonassulfidaeris. SEM, EDS and XRD spectra of ZN2# induced mineralization products; Figure 3 (a) is SEM, Figure 3 (b) for EDS, Figure 3 (c) is the XRD pattern;

[0040] Figure 4 The sulfide mineral salt monosaccharide provided in the embodiment of the present application Halomonassulfidaeris. Growth curve of ZN2#;

[0041] Figure 5 The sulfide mineral salt monosaccharide provided in the embodiment of the present application Halomonassulfidaeris. Chloride resistance characteristic bar graph of ZN2#;

[0042] Figure 6 The sulfide mineral salt monosaccharide provided in the embodiment of the present application Halomonassulfidaeris. Bar graph of sulfate resistance of ZN2#;

[0043] Figure 7 The sulfide mineral salt monosaccharide provided in the embodiment of the present application Halomonassulfidaeris. Bar graph of alkali resistance of ZN2#;

[0044] Figure 8 The sulfide mineral salt monosaccharide provided in the embodiment of the present application Halomonassulfidaeris. ZN2# salt reduction characteristic curve in chloride salt environment;

[0045] Fig. 9 The sulfide mineral salt monosaccharide provided in the embodiment of the present application Halomonassulfidaeris. Salt reduction characteristic curve of ZN2# in sulfur-chlorine mixed salt environment;

[0046] Fig.10 The UCS strength characteristic curve of the salt-reducing and mineralizing dual-effect bacterial strain solidifying saline soil provided in the embodiment of the present application;

[0047] Fig.11 This is a bar graph showing the changes in the soluble salt content in soil after the salt-reducing and mineralizing dual-effect bacterial strain provided in the examples of the present application solidified saline soil. DETAILED DESCRIPTION

[0048] Example 1

[0049] A salt-reducing and mineralizing dual-effect strain, the salt-reducing and mineralizing dual-effect strain is classified and named as sulfide mineralizing halomonas Halomonassulfidaeris. ZN2#, deposited in the General Microbiology Center of China Microbiological Culture Collection Committee, with the deposit number CGMCC No.32033 and the deposit date September 20, 2024. Address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Its 16S rRNA gene sequence is shown in SEQ ID NO.1. Halomonassulfidaeris Esulfide 1 (or the same genus) has a homology of 99.08%.

[0050] Halomonas sulfide Halomonassulfidaeris. ZN2# bacteria 16S rRNA gene sequence SEQ ID NO.1:

[0051]

[0052] The salt-reducing and mineralizing dual-effect strain is isolated from a salt lake. The strain is a Gram-positive bacterium, a moderately halophilic bacterium and a facultative alkaliphilic bacterium, a urease-producing mineralizing bacterium, and a salt-reducing bacterium. The salt-reducing and mineralizing dual-effect strain has an optimal growth range of chloride concentrations of 10% to 15%; a chloride concentration tolerance range of 3% to 20%; a sulfate concentration tolerance range of 0.4 to 1.5 mol / L; and an alkali tolerance range of pH 5.5 to 13. The strain appears as an irregular liquid water droplet-like morphology on a solid culture medium, is white in color, and has a rod-shaped body with a length of 1μm to 3μm, such as Figure 1 shown.

[0053] In this embodiment, the separation, screening and identification of the salt-reducing and mineralizing dual-effect strain includes the following steps:

[0054] Step 1: Isolation and screening of microorganisms from salt lakes, specifically including the following steps:

[0055] The samples used in this experiment mainly come from salt lakes. Salt lake samples were taken from Yuncheng Salt Lake in Shanxi.

[0056] Step 1: Take a water sample within the range of 0-10 cm below the surface of the salt lake, put it into a half-volume beef extract peptone culture medium after high temperature sterilization, and shake it at a constant temperature of 28-32°C, a shaking frequency of 150-200 r / min, and a shaking time of 24 hours. After taking it out, let it stand for 12 hours; culture it to obtain a bacterial enrichment solution;

[0057] The half-dosage beef extract peptone culture medium comprises: 1-2 g beef extract, 5-10 g peptone, and 2-3 g NaCl per 1000 mL deionized water;

[0058] Step 2: Collect the bacterial enrichment solution and prepare 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 dilution of the dilution solution;

[0059] Step 3: The diluted solutions of different concentrations are respectively coated on a plate with a solid culture medium on the surface and cultured at a constant temperature of 28°C to 32°C for 2 days to 3 days. The plate is sealed and placed in a constant temperature incubator for culture;

[0060] Wherein, the solid culture medium is LB solid culture medium: per 1000 mL of deionized water, 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, 5-15 mL of SL-6 trace element solution, and 10-15 g of agar;

[0061] Step 4: Pick a single white colony on the solid culture medium and continue to inoculate it into the liquid culture medium for shaking culture. The shaking temperature is 28-32°C, the shaking frequency is 150-200 r / min, the pH is the natural pH of the culture medium, and the culture time is 24-48 hours;

[0062] The liquid culture medium is LB liquid culture medium: every 1000 mL of deionized water is composed of 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, and 5-15 mL of SL-6 trace element solution;

[0063] Step 5: Take the bacterial solution to verify the mineralization performance;

[0064] The verification of the mineralization performance of the strain comprises the following steps:

[0065] a. Inoculate a single colony obtained during the isolation of any saline soil self-source mineralization strain into a liquid culture medium and culture for 24 to 48 hours, then take 5 to 10 ml of the bacterial solution and mix it with a calcium source solution, and let the mixed solution with a white precipitate stand for 24 hours, wherein the calcium source solution includes 0.5 to 1 mol / L urea and 0.5 to 1 mol / L calcium chloride dihydrate solution;

[0066] b. Pour off the supernatant, wash with sterile distilled water to remove the residual bacteria in the sediment, and dry at 40-50 °C for 12-24 h to obtain a sediment sample;

[0067] c. Use 1 mol / L dilute hydrochloric acid to detect the sediment. If a large number of bubbles are generated immediately after adding dilute hydrochloric acid to the sediment, it is judged that the sediment is calcium carbonate precipitation, and the single colony is a colony with mineralization performance;

[0068] d. Further, a scanning electron microscope (SEM) is used to observe and determine the microscopic morphology of the sediment; an X-ray diffractometer (XRD) is used to analyze the crystal structure of the sediment; and an energy dispersive spectrometer (EDS) is used to analyze the chemical composition of the sediment. The analysis results are as follows: Figure 3 . Figure 3 To characterize the mineralization products induced by the salt-reducing and mineralization-enhancing strains. Figure 3 (b) is the EDS spectrum, which shows that the elements that induce mineralization products are mainly C, O, and Ca. Figure 3 (a) is a SEM image showing that the induced mineralization product is square in shape. Combined with the XRD pattern (such as Figure 3 (c) It was determined that the crystal form of the mineralization product induced by the salt-reducing and mineralizing dual-effect strain was mainly calcite.

[0069] Step 6: Take the bacterial solution to conduct salt reduction performance verification experiment.

[0070] The verification of the salt reduction performance of the strain comprises the following steps:

[0071] a. Place different single colonies in liquid culture medium and culture for 3-7 days;

[0072] b. Centrifuge the cultured bacterial solution and collect the supernatant; take 0.5 ml of the supernatant and place it in a conical flask;

[0073] c. Add 3 to 5 drops of potassium chromate indicator and titrate with standard silver nitrate solution;

[0074] d. If the chloride ion content in the microbial solution after culture is reduced, it is judged that there is a salt-reducing effect.

[0075] Step 7: Select a single colony with both mineralization and salt reduction properties and continue to inoculate it into a solid culture medium. After repeated streaking and purification culture for 4 to 6 times, the obtained single colony is the salt reduction and mineralization dual-effect strain sulfide mineralizing bacteria. Halomonassulfidaeris. ZN2#;

[0076] The sulfide mineral salt mononas is in the form of irregular liquid water droplets on the solid culture medium, and the color is white. The SEM scanning electron microscope shows that the bacteria are rod-shaped, and the length is 1 μm to 3 μm. Figure 1 shown.

[0077] Step 2: Identification of bacterial species. The obtained strains were screened and the 16S rDNA sequence was determined, and compared with the known sequences in the gene bank (Gen Bank) established by the National Center for Biotechnology Information (NCBI, http: / / www.Ncbi.nlm.nih.gov) of the United States using the local alignment search tool (Blast). Based on the 16S rDNA sequencing results, a phylogenetic tree was constructed using MEGA4.1 software. This strain belongs to Gram-positive bacteria, and its 16S rDNA gene sequencing results are shown in SEQ ID NO.1. The phylogenetic tree constructed based on the gene sequencing results is shown in Figure 2 The bacteria screened were confirmed to be of the genus Halomonas and named Halomonas sulfide Halomonassulfidaeris. ZN2# was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 20, 2024, and the registration number of the collection center is CGMCC NO.32033.

[0078] Step 3: Sulfide mineralizing bacteria Halomonassulfidaeris. Growth characteristics of ZN2#. Figure 4 The logarithmic growth period of ZN2# bacteria is 2 to 48 h, and it enters the stable period after 48 h.

[0079] Step 4: Sulfide mineralizing bacteria Halomonassulfidaeris.ZN2# has anti-chloride salt properties. Figure 5 When salt was added to the LB medium, ZN2# bacteria could not grow in a salt-free or low-salt environment. The optimal growth chloride concentration range was 10% to 15%. According to the classification standards for salt-tolerant microorganisms, it was identified as a moderately halophilic bacterium.

[0080] Step 5: Sulfide mineralizing bacteria Halomonassulfidaeris. ZN2# has anti-sulfate properties. Figure 6 , salt was added to LB medium, and the sulfate concentration range for ZN2# bacteria was 0.4~1.5 mol / L.

[0081] Step 6: Sulfide mineralizing bacteria Halomonassulfidaeris. ZN2#'s anti-alkali properties. Figure 7 , the alkali resistance range of ZN2# bacteria is pH=5.5~13. According to the alkali resistance characteristics of microorganisms, ZN2# bacteria are facultative alkaliphiles.

[0082] Step 7: Sulfide mineralizing bacteria Halomonassulfidaeris. ZN2# has salt reduction characteristics in chloride salt environment. Figure 8 , adding salt to LB medium, the salt reduction range of ZN2# bacteria in chloride salt environment was 0~6.43%.

[0083] Step 8: Sulfide Halomonas Halomonassulfidaeris. ZN2# has salt reduction characteristics in sulfur-chlorine mixed salt environment. Fig. 9 , the salt reduction range of ZN2# bacteria in the sulfur-chlorine mixed salt environment is 1.25%~10.61%.

[0084] Mixed salt medium is LB medium with chloride and sulfate. Mix 1: 0.5mol / L sodium chloride + 0.2mol / L sodium sulfate; Mix 2: 1mol / L sodium chloride + 0.4mol / L sodium sulfate; Mix 3: 1.5mol / L sodium chloride + 0.8mol / L sodium sulfate; Mix 4: 2mol / L sodium chloride + 1mol / L sodium sulfate; Mix 5: 2.5mol / L sodium chloride + 1.5mol / L sodium sulfate; Mix 6: 3mol / L sodium chloride + 2mol / L sodium sulfate; Mix 7: 3.5mol / L sodium chloride + 2.5mol / L sodium sulfate.

[0085] Embodiment 2:

[0086] The method for applying the salt-reducing and mineralizing dual-effect strain to solidify saline soil and reduce the salt content in the soil comprises the following steps: This embodiment uses the separation and screening method of the salt-reducing and mineralizing dual-effect strain in Example 1 to separate the strain.

[0087] The saline soil from the Hetao region of Inner Mongolia is sieved and fully dried for later use;

[0088] Step 1: Prepare a salt-reducing mineralization dual-effect bacterial solution and a cementing solution for solidifying saline soil and reducing the salt content in the soil: the salt-reducing mineralization dual-effect bacterial solution is prepared by expanding the salt-reducing mineralization dual-effect bacterial strain Halomonas, and the OD of the bacterial solution is 600 The value is 1.7, and the urease activity is 4-6 mM urea / min; the cementing fluid composition is: urea 30.03 g / L, CaCl 2 ·2H 2 O 73.51 g / L; the solvent of the cementing fluid is water;

[0089] Step 2: preparing microbial mineralization specimens by mixing method, including the following steps:

[0090] Firstly, the salt-reducing and mineralizing dual-effect bacterial liquid and the cementing liquid are mixed in a ratio of 1:1 and stirred evenly;

[0091] Then, the bacterial liquid-cementing liquid mixture and the saline soil are mixed at a ratio of 1:3.17 and stirred evenly, and then loaded into a prefabricated mold and compacted;

[0092] Then let it air dry for 14 days.

[0093] The results show that the salt-reducing and mineralizing dual-effect strain can solidify saline soil while reducing the salt content in the saline soil, thereby improving the soil strength of the saline soil and reducing the salt content, thereby achieving the repair and improvement of the saline soil. Fig.10 The UCS strength characteristic curve of the salt-reducing and mineralizing dual-effect strain solidifying saline soil in the embodiment of the present application shows that the solidification strength can reach 3.498 MPa, which is 133.2% higher than the average solidification strength of the soil body of the traditional mineralizing strain (the traditional strain is shown in Comparative Example 1, and the parameters of the comparative example are the optimal data of the effect of Bacillus pasteurianus (ATCC 11859) within the test range); Fig.11 This is a bar chart of the change in the soluble salt content in the soil after the salt-reducing and mineralizing dual-effect strain solidified the saline soil in the embodiment of the present application. The total amount of soluble salt in the soil was reduced by 13.42%, among which the chloride ion content was reduced more significantly, by 24.68%.

[0094] Example 3

[0095] The method for applying the salt-reducing and mineralizing dual-effect strain to solidify saline soil and reduce the salt content in the soil comprises the following steps: This embodiment uses the separation and screening method of the salt-reducing and mineralizing dual-effect strain in Example 1 to separate the strain.

[0096] The saline soil from the Hetao region of Inner Mongolia is sieved and fully dried for later use;

[0097] Step 1: Prepare a salt-reducing mineralization dual-effect bacterial solution and a cementing solution for solidifying saline soil and reducing the salt content in the soil: the salt-reducing mineralization dual-effect bacterial solution is prepared by expanding the salt-reducing mineralization dual-effect bacterial strain Halomonas, and the OD of the bacterial solution is 600 The value is 1.0, and the urease activity is 4-6 mM urea / min; the cementing fluid composition is: urea 30.03 g / L, CaCl 2 ·2H 2 O 73.51 g / L; the solvent of the cementing fluid is water;

[0098] Step 2: preparing microbial mineralization specimens by mixing method, including the following steps:

[0099] Firstly, the salt-reducing and mineralizing dual-effect bacterial liquid and the cementing liquid are mixed in a ratio of 1:1 and stirred evenly;

[0100] Then, the bacterial liquid-cementing liquid mixture and the saline soil are mixed at a ratio of 1:3.17 and stirred evenly, and then loaded into a prefabricated mold and compacted;

[0101] Then let it air dry for 14 days.

[0102] The salt-reducing and mineralizing dual-effect strain solidifies saline soil and reduces the salt content in the saline soil, thereby improving the soil strength of the saline soil and reducing the salt content, thereby achieving the restoration and improvement of the saline soil. The test results of this example are not significantly different from those of Example 2.

[0103] Example 4

[0104] The method for applying the salt-reducing and mineralizing dual-effect strain to solidify saline soil and reduce the salt content in the soil comprises the following steps: This embodiment uses the separation and screening method of the salt-reducing and mineralizing dual-effect strain in Example 1 to separate the strain.

[0105] The saline soil from the Hetao region of Inner Mongolia is sieved and fully dried for later use;

[0106] Step 1: Prepare a salt-reducing mineralization dual-effect bacterial solution and a cementing solution for solidifying saline soil and reducing the salt content in the soil: the salt-reducing mineralization dual-effect bacterial solution is prepared by expanding the salt-reducing mineralization dual-effect bacterial strain Halomonas, and the OD of the bacterial solution is 600 The value is 0.5, and the urease activity is 4-6 mM urea / min; the cementing fluid composition is: urea 30.03 g / L, CaCl 2 ·2H 2 O 73.51 g / L; the solvent of the cementing fluid is water;

[0107] Step 2: preparing microbial mineralization specimens by mixing method, including the following steps:

[0108] Firstly, the salt-reducing and mineralizing dual-effect bacterial liquid and the cementing liquid are mixed in a ratio of 1:1 and stirred evenly;

[0109] Then, the bacterial liquid-cementing liquid mixture and the saline soil are mixed at a ratio of 1:3.17 and stirred evenly, and then loaded into a prefabricated mold and compacted;

[0110] Then let it air dry for 14 days.

[0111] The salt-reducing and mineralizing dual-effect strain solidifies saline soil and reduces the salt content in the saline soil, thereby improving the soil strength of the saline soil and reducing the salt content, thereby achieving the restoration and improvement of the saline soil. The test results of this example are slightly lower than those of Example 2, and the difference is not significant.

[0112] Example 5

[0113] The method for applying the salt-reducing and mineralizing dual-effect strain to solidify saline soil and reduce the salt content in the soil comprises the following steps: This embodiment uses the separation and screening method of the salt-reducing and mineralizing dual-effect strain in Example 1 to separate the strain.

[0114] The saline soil from the Hetao region of Inner Mongolia is sieved and fully dried for later use;

[0115] Step 1: Prepare a salt-reducing mineralization dual-effect bacterial solution and a cementing solution for solidifying saline soil and reducing the salt content in the soil: the salt-reducing mineralization dual-effect bacterial solution is prepared by expanding the salt-reducing mineralization dual-effect bacterial strain Halomonas, and the OD of the bacterial solution is 600 The value is 0.5, and the urease activity is 4-6 mM urea / min; the cementing fluid composition is: urea 60.06 g / L, CaCl 2 ·2H 2 O 147.02 g / L; the solvent of the cementing fluid is water;

[0116] Step 2: preparing microbial mineralization specimens by mixing method, including the following steps:

[0117] Firstly, the salt-reducing and mineralizing dual-effect bacterial liquid and the cementing liquid are mixed in a ratio of 1:2 and stirred evenly;

[0118] Then, the bacterial liquid-cementing liquid mixture is mixed with the saline soil in a ratio of 1:2 and stirred evenly, and then loaded into a prefabricated mold and compacted;

[0119] Then let it air dry for 14 days.

[0120] The salt-reducing and mineralizing dual-effect strain solidifies saline soil and reduces the salt content in the saline soil, thereby improving the soil strength of the saline soil and reducing the salt content, thereby achieving the restoration and improvement of the saline soil. The test results of this example are slightly lower than those of Example 2, and the difference is not significant.

[0121] Example 6

[0122] The method for applying the salt-reducing and mineralizing dual-effect strain to solidify saline soil and reduce the salt content in the soil comprises the following steps: This embodiment uses the separation and screening method of the salt-reducing and mineralizing dual-effect strain in Example 1 to separate the strain.

[0123] The saline soil from the Hetao region of Inner Mongolia is sieved and fully dried for later use;

[0124] Step 1: Prepare a salt-reducing mineralization dual-effect bacterial solution and a cementing solution for solidifying saline soil and reducing the salt content in the soil: the salt-reducing mineralization dual-effect bacterial solution is prepared by expanding the salt-reducing mineralization dual-effect bacterial strain Halomonas, and the OD of the bacterial solution is 600 The value is 0.5, and the urease activity is 4-6 mM urea / min; the cementing fluid composition is: urea 60.06 g / L, CaCl 2 ·2H 2 O 147.02 g / L; the solvent of the cementing fluid is water;

[0125] Step 2: preparing microbial mineralization specimens by mixing method, including the following steps:

[0126] Firstly, the salt-reducing and mineralizing dual-effect bacterial liquid and the cementing liquid are mixed in a ratio of 1:3 and stirred evenly;

[0127] Then, the bacterial liquid-cementing liquid mixture is mixed with the saline soil in a ratio of 1:4 and stirred evenly, and then loaded into a prefabricated mold and compacted;

[0128] Then let it air dry for 14 days.

[0129] The salt-reducing and mineralizing dual-effect strain solidifies saline soil and reduces the salt content in the saline soil, thereby improving the soil strength of the saline soil and reducing the salt content, thereby achieving the restoration and improvement of the saline soil. The test results of this example are slightly lower than those of Example 2, and the difference is not significant.

[0130] Comparative Example 1

[0131] Preparation of fine-grained soil for fine-grained soil biologically induced mineralization reinforcement: the fine-grained soil is saline soil with a particle size of less than 2 mm taken from a saline-alkali wasteland in the Hetao Irrigation Area of ​​Inner Mongolia, and the fine-grained saline soil is fully dried for later use.

[0132] Step 1: Preparation of mineralized bacterial solution and cementing solution for fine-grained soil biological induced mineralization reinforcement: The mineralized bacterial solution is prepared by the expansion of Bacillus pasteurianus (ATCC 11859), and the average OD of the mineralized bacterial solution is 600 The value is 0.7, and the average urease activity is 6.67 mM urea / min; the cementing fluid composition is: urea 30.03 g / L, CaCl 2·2H 2 O 73.51g / L; the solvent of the cementing fluid is water;

[0133] Step 2: Using a mixing method to induce microbial mineralization to consolidate fine-grained soil, including the following steps:

[0134] First, the mineralized bacterial solution and the cementing solution are mixed in a volume ratio of 1:1;

[0135] Then the mixed liquid and fine-grained soil were mixed in a mass ratio of 1:3.17 and stirred evenly;

[0136] Then put it into the prefabricated mold, compact it, and air dry it for 14 days;

[0137] Bacillus pasteurianus (ATCC 11859) biomineralizes fine-grained saline soil, with an average solidification strength of up to 1.5 MPa.

[0138] Finally, it should be noted that the above is only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred arrangement scheme, a person skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. A salt-reducing and mineralizing dual-effect bacterial strain, characterized in that: The salt-reducing and mineralizing dual-effect strain is classified and named as Sulfide Mineral Halomonas ( Halomonas sulfidaeris. ) ZN2#, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with the deposit number CGMCC No.32033 and the deposit date September 20, 2024.

2. A method for cultivating the salt-reducing and mineralizing dual-effect bacterial strain according to claim 1, characterized in that: The salt-reducing and mineralizing dual-effect strain was inoculated into a liquid culture medium and shaken for culture at a temperature of 30 °C, a speed of 200 r / min, and a culture time of 48 h. The composition of the liquid culture medium was as follows: 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, and 5-15 mL of SL-6 trace element solution were added to every 1000 mL of deionized water.

3. A method for cultivating the salt-reducing and mineralizing dual-effect bacterial strain according to claim 1, characterized in that: The salt-reducing and mineralizing dual-effect strain was inoculated into a solid culture medium for cultivation at a temperature of 30 °C for 48 h. The components of the solid culture medium were as follows: 10-20 g of tryptone, 5-10 g of yeast extract, 100-150 g of NaCl, 5-15 mL of SL-6 trace element solution and 10-15 g of agar were added to every 1000 mL of deionized water.

4. Use of the salt-reducing and mineralizing dual-effect strain described in claim 1 in solidifying saline soil and reducing soil chloride content at the same time.

5. The use according to claim 4, characterized in that: The following steps are involved: Step 1: Prepare salt-reducing and mineralizing dual-effect bacterial solution and cementing solution for use in saline soil: The salt-reducing and mineralizing dual-effect bacterial solution is prepared by expanding the salt-reducing and mineralizing dual-effect bacterial strain, and the OD of the bacterial solution is 600 The value is 0.5~2.0, and the urease activity is 4~6 mM urea / min; the composition of the cementing liquid is: urea 30~60 g / L, CaCl2·2H2O 73.5~147 g / L; the solvent of the cementing liquid is water; Step 2: Perform in-situ microbial mixing to form a microbial mineralization layer, including the following steps: 1) mixing the salt-reducing and mineralizing dual-effect bacterial solution and the cementing solution at a volume ratio of 1:1-3 and stirring evenly to obtain a bacterial solution-cementing solution mixture; 2) mixing the bacterial liquid-cementing liquid mixture with the soil in the saline soil area at a mass ratio of 1:2-4 and stirring evenly; 3) Compact the mixed saline soil and air-dry it.

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