Culture medium as well as preparation method and application thereof in screening mineralized microorganisms

Abstract

The invention relates to the technical field of microorganism culture and screening, and discloses a culture medium, a preparation method thereof and application of the culture medium in screening mineralized microorganisms, and the culture medium contains peptone, yeast extract powder, sodium chloride, magnesium chloride and calcium chloride. The culture medium takes peptone and yeast extract powder as core components, is not only suitable for screening urealysis type mineralized bacteria, but also provides nutrient substances required by growth of amino acid deamination, carbonic anhydrase catalysis or heterotrophic metabolism driven type mineralized microorganisms, comprehensively screens and prevents potential mineralized bacteria from being omitted. The culture medium can be used for accurately and efficiently screening metal surface mineralizable microorganisms. According to the culture medium, the ecological adaptability of marine strains is optimized, peptone and yeast extract powder supply nutrient substances needed by growth of marine bacteria, sodium chloride, magnesium chloride and calcium chloride are mainly used for simulating the marine salinity environment and adjusting the osmotic pressure and enzyme activity of the bacteria, the matching degree with the environment is high, and the enrichment efficiency is high.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microbial culture screening, and more particularly to a culture medium, a preparation method thereof and application thereof in screening mineralized microorganisms. BACKGROUND

[0002] The surface of a ship causes a large amount of energy loss due to marine organism attachment, and the corrosion of a marine platform and an oil and gas pipeline causes material perforation, resulting in serious loss. Microbial mineralization is a natural process driven by microbial metabolic activity, which deposits inorganic minerals in the cell or extracellularly, and plays a key role in the biodeposition of minerals such as calcium carbonate, phosphate and silicate. The deposition of extracellular minerals induced by microorganisms on the surface of a metal substrate can form a biomineralization layer as a natural corrosion and fouling barrier, which significantly reduces the corrosion behavior of the metal. The protective layer is spontaneously formed by microorganisms during growth and reproduction, which eliminates the harmfulness of chemical coatings to the marine environment from the source, and can be applied to irregular marine material structures for corrosion and fouling prevention.

[0003] At present, the existing technology for screening bacteria capable of depositing calcium carbonate on the surface of a metal substrate usually uses common basic nutrient culture media, such as Luria-Bertani (LB) medium, Reasoner's 2A medium (R2A) or nutrient agar (NA), etc., so as to enrich and isolate a variety of heterotrophic bacteria from environmental samples such as soil, water or marine sediments. These culture media have relatively simple compositions and can support the growth of most non-specific bacteria, thereby providing bacterial resources for subsequent screening of mineralization performance. However, the following technical problems exist:

[0004] (1) The existing culture medium cannot comprehensively screen metal surface mineralizable microorganisms. Traditional LB, R2A or NA medium mainly uses urea and calcium chloride as the core component, which is mainly suitable for the screening of urea-degrading mineralization bacteria, and lacks responsiveness design for other mineralization mechanisms such as amino acid deamination, carbonic anhydrase catalysis or heterotrophic metabolism driven mineralization, resulting in potential mineralization strains being missed.

[0005] (2) The existing culture medium cannot accurately and efficiently screen metal surface mineralizable microorganisms. The high salt, low temperature, alkaline or reducing conditions in the marine environment have an important influence on the physiology of microorganisms, and the existing culture medium is usually not optimized for the ecological adaptability of marine strains, resulting in low environmental matching degree and low enrichment efficiency. SUMMARY

[0006] The present application provides a culture medium.

[0007] Another object of the present application is to provide a preparation method of the culture medium.

[0008] Another object of the present application is to provide the use of the culture medium in screening mineralized microorganisms.

[0009] To solve the above technical problems, the technical solution provided by the present application is:

[0010] A culture medium contains peptone, yeast extract, sodium chloride, magnesium chloride and calcium chloride.

[0011] The culture medium has the following technical effects:

[0012] (1) The culture medium can comprehensively screen metal surface mineralizable microorganisms. The culture medium takes peptone and yeast extract as core components, which is suitable for screening urea-degrading mineralization bacteria and provides nutrients for the growth of microorganisms driven by amino acid deamination, carbonic anhydrase catalysis or heterotrophic metabolism, preventing potential mineralization bacteria from being missed.

[0013] (2) The culture medium accurately and efficiently screens metal surface mineralizable microorganisms. The culture medium is optimized for the ecological adaptability of marine strains, in which: peptone and yeast extract provide nutrients for the growth of marine bacteria, and the main role of sodium chloride, magnesium chloride and calcium chloride is to simulate the marine salinity environment, adjust the osmotic pressure and enzyme activity of bacteria, and match the environment with high efficiency.

[0014] Preferably, the culture medium further contains phenol red.

[0015] Specifically, it is a sterile phenol red solution.

[0016] Phenol red solution is yellow under acidic conditions, red under neutral conditions, and purple under alkaline conditions, which can clearly indicate the change of pH value of the culture medium caused by bacterial growth, and facilitate real-time monitoring of bacterial growth and metabolism. Without waiting for the reaction between the bacteria and the metal surface to be completed, the culture medium can be simply and quickly screened from the natural environment and the strain library.

[0017] Preferably, the culture medium further contains ferric citrate, sodium sulfate, sodium carbonate, potassium bromide and boric acid.

[0018] Ferric citrate accelerates cell respiration and electron transfer reactions.

[0019] Preferably, the culture medium contains the following mass fractions:

[0020] Peptone 1-10 parts,

[0021] Yeast extract 1-2 parts,

[0022] Sodium chloride 15-25 parts,

[0023] magnesium chloride 3-7 parts,

[0024] calcium chloride 2-5 parts.

[0025] More preferably, the medium further comprises 0.01-0.1 parts by mass of phenol red.

[0026] More preferably, the medium further comprises:

[0027] ferric citrate 0.1-1 part,

[0028] sodium sulfate 0.1-5 parts,

[0029] sodium bicarbonate 0.1-0.5 parts,

[0030] potassium bromide 0.01-0.1 parts,

[0031] boric acid 0.01-0.05 parts.

[0032] The present application also protects the preparation method of the medium described in any one of the above, comprising the following steps:

[0033] First, add the components in proportion except phenol red, and then autoclave at 120-130°C to obtain the medium; the medium is a solid or liquid medium.

[0034] The solid medium described in the present application can additionally add 10-20 parts of agar powder before sterilization, and add sterile phenol red when the temperature drops to about 45-55°C, and then divide the liquid medium into disposable sterile culture dishes in a sterile clean bench, and use after solidification.

[0035] Preferably, the preparation method of the medium comprises the following steps:

[0036] First, add the components in proportion except phenol red, and then autoclave at 120-130°C, and finally add sterile phenol red when the temperature drops to room temperature to obtain the medium.

[0037] The present application also protects the application of the medium in screening mineralized microorganisms.

[0038] Preferably, the mineralized microorganism screened is bacteria.

[0039] The medium described in the present application has the following beneficial effects:

[0040] 1. Basic nutrition supply: support microbial growth and reproduction

[0041] The metabolism and mineralization activities of mineralized microorganisms need to ensure their own growth first, so the medium needs to provide basic nutrients such as carbon, nitrogen, energy and growth factors, and the related components are:

[0042] (1) Peptone: As a core nitrogen and carbon source, it is made by hydrolyzing animal and plant proteins. It contains a variety of amino acids, short peptides and a small amount of carbohydrates. It can provide microorganisms with nitrogen (for the synthesis of proteins and nucleic acids) and carbon (as energy or metabolic substrates). At the same time, the small molecule peptides it contains are easily absorbed by microorganisms to meet the needs of rapid growth.

[0043] (2) Yeast extract: It supplements nutrition and growth factors. It is obtained by hydrolysis of yeast cells. In addition to amino acids and carbohydrates, it is also rich in vitamins (such as B vitamins), nucleotides and other growth factors. It can promote the synthesis of microbial metabolic enzymes (mineralization-related enzymes such as carbonic anhydrase depend on vitamin activation). It is crucial for the growth of mineralizing microorganisms with complex nutritional needs (such as certain bacteria and fungi).

[0044] 2. Mineral substrate supply: Provides the core raw materials for mineral synthesis.

[0045] The core function of mineralizing microorganisms is to form minerals (such as calcium carbonate, magnesium phosphate, etc.), requiring specific metal ions and anions as "mineralization substrates." The relevant components are:

[0046] (1) Calcium chloride (CaCl2) provides the core substrate for mineralization—calcium ions (Ca... 2+ Calcium is a key element in carbonate mineralization (the most common type of microbial mineralization) (e.g., the core component of calcium carbonate precipitation is Ca). 2+ Mineralizing microorganisms produce alkaline substances (such as OH-) through metabolism or convert CO2 into CO32-, which then react with Ca. 2+ Combined to form calcium carbonate precipitate, therefore Ca 2+ It is the "raw material" for mineralization reactions and also an essential substrate for screening mineralizing microorganisms (those that cannot utilize Ca). 2+ The microorganisms that perform mineralization are indirectly excluded.

[0047] (2) Magnesium chloride (MgCl2) and sodium sulfate (Na2SO4) provide auxiliary mineralizing ions:

[0048] Magnesium ions (Mg 2+ It can participate in the formation of various minerals (such as magnesium phosphate, dolomite, etc.), and some mineralizing microorganisms can synthesize magnesium-containing minerals; sulfate (SO42-) can serve as a sulfur source for some mineralizing microorganisms (used to synthesize sulfur-containing amino acids), and at the same time, it reacts with metal ions (such as Mg). 2+ They combine to maintain the ion balance in the culture medium and avoid excessive concentration of a single ion that inhibits microorganisms.

[0049] (3) Sodium bicarbonate (NaHCO3) provides key anions for mineralization (CO32-): as a "carbon source substrate" for carbonate mineralization, it provides CO32- for precipitation such as calcium carbonate; at the same time, it acts as a buffer to maintain the stability of the system.

[0050] 3. Environmental condition regulation: Maintaining the microenvironment required for mineralization

[0051] The metabolism and mineralization function of mineralizing microorganisms depend on a stable environment with osmotic pressure, pH, etc., and the relevant components are:

[0052] (1) Sodium chloride (NaCl) maintains osmotic pressure balance: The osmotic pressure inside microbial cells needs to be consistent with the external environment (to avoid cell dehydration or rupture due to water absorption). Sodium chloride provides Na+ by supplying Na+. + Cl- regulates the osmotic pressure of the culture medium, especially for certain mineralizing microorganisms adapted to low or medium salinity environments (such as mineralizing bacteria in soil), where a suitable salt concentration is the basis for their growth and mineralization.

[0053] (2) Phenol red indicates pH changes related to mineralization: Phenol red is a pH indicator (yellow in acidic conditions and red in alkaline conditions). Most mineralizing microorganisms produce alkaline substances during metabolism (e.g., OH- may be produced during mineralization through urea decomposition or respiration, raising the environmental pH), causing the culture medium pH to rise, and phenol red will change from yellow to red. By observing the color change, microorganisms that "can change pH through metabolism (indirectly indicating mineralization potential)" can be quickly screened, serving as a "visual marker" for screening.

[0054] 4. Trace elements and metabolic aids: ensuring enzyme activity and metabolic efficiency

[0055] The metabolic enzymes of mineralizing microorganisms (such as carbonic anhydrase and urease, which directly participate in the mineralization reaction) require activation by trace elements, and some components can also promote ion absorption. The relevant components are:

[0056] (1) Ferric citrate provides an iron source and promotes ion dissolution: Iron is an essential trace element for microorganisms (participating in the synthesis of cytochromes, catalase, etc., which are the core of respiratory metabolism, and the energy produced by metabolism is the basis of mineralization); citrate ions are chelating agents that can react with metal ions in the culture medium (such as Ca2+, Ca2+, etc.). 2+ Mg 2+ This combination prevents the ions from precipitating into insoluble substances, ensuring that microorganisms can absorb and utilize them.

[0057] (2) Potassium bromide (KBr) and boric acid (H3BO3) provide trace elements (bromine and boron): Bromine (Br -Boron (B) is a special nutritional requirement for some microorganisms (such as some actinomycetes) and participates in the redox reactions of enzymes. Boron (B) is an essential element for maintaining the cell wall structure of microorganisms (such as peptidoglycan synthesis) and the activity of metabolic enzymes, especially for microorganisms that rely on "extracellular mineral precipitation" during mineralization (which depends on the stability of the cell wall structure).

[0058] The culture medium described in this invention precisely screens mineralizing microorganisms through a synergistic process of "nutrient supply (peptone, yeast extract), mineralizing substrates (calcium chloride, sodium carbonate, etc.), environmental regulation (sodium chloride, phenol red), and metabolic assistance (ferric citrate, potassium bromide, etc.)." First, it ensures microbial survival through nutrition; then, it screens for microorganisms that can utilize these ions for mineralization using substrates such as calcium, magnesium, and carbonates; finally, it rapidly identifies target strains exhibiting "metabolic changes accompanied by alkaline conditions (mineralization characteristics)" through the pH indicator function of phenol red. Non-mineralizing microorganisms are excluded because they lack mineralization-related enzymes (cannot utilize specific ions), their metabolism does not change pH (phenol red does not change color), or they cannot adapt to an alkaline environment. This achieves efficient, convenient, and accurate screening of mineralizing microorganisms. Attached Figure Description

[0059] Figure 1 The wavelength is the maximum wavelength absorbed by the culture medium obtained in Example 1.

[0060] Figure 2 The mineralization and preservation effects of different bacteria. Detailed Implementation

[0061] To enable those skilled in the art to better understand the present invention, the present invention will now be further described in conjunction with specific embodiments.

[0062] In the following description, the embodiments of this application are for illustrative purposes and not for limiting purposes, so as to provide a thorough understanding of the embodiments. However, those skilled in the art will understand that the embodiments of this application can also be implemented in other embodiments without these specific details. In other instances, detailed descriptions of well-known preparation methods have been omitted so as not to obscure the description of the embodiments of this application with unnecessary details. Unless otherwise specified, the raw materials used in the following embodiments and comparative examples are all commercially available.

[0063] It should also be understood that the term "and / or" as used in the specification of embodiments of this application and the appended claims refers to any combination and all possible combinations of one or more of the associated listed items, and includes such combinations. The terms "comprising," "including," "containing," "having," and variations thereof all mean "including but not limited to," unless otherwise specifically emphasized. "A plurality" means two or more.

[0064] This section only introduces content related to the inventive points; other details can be obtained from relevant technologies and will not be described in detail here. The following embodiments only illustrate several implementation methods of this application, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of protection of this application. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of this application, and these all fall within the scope of protection of this application. Therefore, the scope of protection of this patent application should be determined by the appended claims. The invention will be further described in detail below with reference to specific embodiments.

[0065] Example 1

[0066] A culture medium consists of the following components: 5 parts peptone, 1.5 parts yeast extract, 0.5 parts ferric citrate, 20 parts sodium chloride, 3 parts sodium sulfate, 5 parts magnesium chloride, 3 parts calcium chloride, 0.3 parts sodium bicarbonate, 0.05 parts potassium bromide, 0.03 parts boric acid, 0.05 parts phenol red solution, and a pH of 7.0 ± 0.2.

[0067] The method for preparing the culture medium includes the following steps:

[0068] First, add all components except phenol red in proportion, then autoclave at 125°C, and finally add sterile phenol red after the temperature drops to room temperature to obtain the liquid culture medium.

[0069] Example 2

[0070] This embodiment is the second embodiment of the present invention. Unlike embodiment 1, the culture medium is composed of the following components: 1 part peptone, 1 part yeast extract, 0.1 part ferric citrate, 15 parts sodium chloride, 0.1 part sodium sulfate, 3 parts magnesium chloride, 2 parts calcium chloride, 0.1 part sodium bicarbonate, 0.01 part potassium bromide, 0.01 part boric acid, and 0.01 part phenol red solution.

[0071] Example 3

[0072] This embodiment is the third embodiment of the present invention. Unlike embodiment 1, the culture medium is composed of the following components: 10 parts peptone, 2 parts yeast extract, 1 part ferric citrate, 25 parts sodium chloride, 5 parts sodium sulfate, 7 parts magnesium chloride, 5 parts calcium chloride, 0.5 parts sodium bicarbonate, 0.1 parts potassium bromide, 0.05 parts boric acid, and 0.01 parts phenol red solution.

[0073] Example 4

[0074] This embodiment is the fourth embodiment of the present invention. Unlike embodiment 1, the culture medium components do not contain ferric citrate, sodium sulfate, sodium bicarbonate, potassium bromide, and boric acid.

[0075] Example 5

[0076] This embodiment is the fifth embodiment of the present invention. Unlike embodiment 1, the culture medium components do not contain phenol red.

[0077] Example 6

[0078] This embodiment is the sixth embodiment of the present invention. Unlike embodiment 1, the culture medium components do not contain ferric citrate, sodium sulfate, sodium bicarbonate, potassium bromide, boric acid, or phenol red.

[0079] Example 7

[0080] This embodiment is the 7th embodiment of the present invention. Unlike embodiment 1, the culture medium is composed of the following components: 5 parts peptone, 1.5 parts yeast extract, 0.5 parts ferric citrate, 20 parts sodium chloride, 3 parts sodium sulfate, 5 parts magnesium chloride, 3 parts calcium chloride, 0.3 parts sodium bicarbonate, 0.05 parts potassium bromide, 0.03 parts boric acid, 0.05 parts phenol red solution, and the pH value is 7.0±0.2.

[0081] The method for preparing the culture medium includes the following steps:

[0082] First, add all components except phenol red according to the proportion, and add 15 parts of agar powder. Then, autoclave at 125°C. Finally, when the temperature drops to about 50°C, dispense the liquid culture medium into disposable sterile culture dishes in a sterile laminar flow hood. After solidification, the solid culture medium is obtained.

[0083] Comparative Example 1

[0084] This comparative example is the first comparative example of the present invention. The difference between this example and Example 1 is that the culture medium is Luria-Bertani (LB) medium.

[0085] Comparative Example 2

[0086] This comparative example is the second comparative example of the present invention. The difference between this example and Example 1 is that the culture medium is Reasoner's 2A medium (R2A).

[0087] Comparative Example 3

[0088] This comparative example is the third comparative example of the present invention. The difference between this example and Example 1 is that the culture medium is nutrient agar (NA).

[0089] Performance testing:

[0090] The bacterial culture was diluted using the aforementioned selective culture medium to ensure a consistent final concentration. The bacteria then created an alkaline environment conducive to the deposition of calcium and magnesium carbonates.

[0091] This invention utilizes a 96-well microplate to achieve bacterial growth and high-throughput measurement of bacterial pH using optical density values. The maximum absorption wavelength in Example 1 is 560 nm. (See attached image.) Figure 1 The maximum absorption wavelengths of the culture media obtained in the remaining Examples 2-6 are similar to those in Example 1.

[0092] Uniform OD 600nm Add 200 μL of the bacteria to a sterile 96-well ELISA plate and incubate with shaking at 120 rpm in an incubator at 20–25°C. In the first stage, after culturing the bacterial culture for 16 hours, [the culture is then measured at OD]. 560nm The absorbance was measured at a specific wavelength; at this stage, the absorbance of the bacterial culture medium must be ≤1.0 OD. 560nm The OD value of the bacterial culture medium was ≤1.8 in the second phase (24h) as the incubation time increased. 560nm >3.0. In the final stage, the pH of the bacterial culture medium needs to be significantly increased, specifically, the OD value of the selective medium inoculated with bacteria should be >3.0 after 36 hours. 560nm The OD value of bacteria cultured in the culture media obtained in each example and comparative example needs to be above 3.7. 560nm The changes are shown in Table 1.

[0093] Table 1. OD values ​​of different bacteria cultured in the culture media obtained from each example and comparative example for 32 hours. 560nm change

[0094]

[0095]

[0096] Steel corrosion testing:

[0097] The steel sample uses Q235B carbon steel, which is economical but easily corroded. The steel is cut into 10*10*5mm square steel samples using a wire cutting machine. The surface is polished to 1000# after being sanded with silicon carbide sandpaper to remove rust. It is then placed in anhydrous ethanol and sonicated for 5 minutes to remove oil and impurities. It is then soldered to copper wire and sterilized for later use.

[0098] The number of bacteria inoculated in the culture medium should be kept as consistent as possible; therefore, OD is measured during the logarithmic growth phase. 600nm Dilute the bacterial culture to OD value using the supernatant of blank culture medium. 600nm ≈0.1, take 1 mL of diluted bacterial culture and add it to blank culture medium. Immerse the sterilized steel sample in the bacterial culture medium and hang it for 7 days. After 7 days, take out the sample and immerse it in concentrated hydrochloric acid solution, saturated sodium bicarbonate solution and deionized water solution in sequence to remove surface corrosion products / biomineralization layer. After drying with cold air, observe the local corrosion morphology of the steel surface under a white light interferometer. Example 1 obtained

[0099] After soaking for 7 days, the steel sample was rinsed once with deionized water and fixed in 2.5% glutaraldehyde solution for 20-40 min. Then, it was dehydrated in 30%, 50%, 70%, 80%, 90% and anhydrous ethanol for 10-20 min in sequence. After the sample was dried, gold was sputtered onto the sample surface for 60-90 s using a vacuum sputtering instrument. The corrosion morphology / biomineralization layer structure of the steel sample surface was observed using a scanning electron microscope.

[0100] Figure 2 The formation of biomineralization layers on steel surfaces and the anti-corrosion effect of different bacterial strains were investigated. The control group (blank control) showed localized corrosion and surface morphology. Numerous corrosion products were observed on the surface of the sterile group samples, indicating severe matrix damage, defects, and pitting. J2B10F and M2B2F formed relatively complete biomineralization layers on the steel surface. Magnified images showed that the biomineralization layers exhibited nested small triangular structures. J2B6F showed some calcium and magnesium carbonate deposits on its surface, but surface cracks were obvious, the mineralization layer was thin, and it showed a tendency to disintegrate. M2B4F and M1B1F formed obvious yellow corrosion products on the steel surface, without forming a significant mineralization layer.

[0101] Table 1 and the strain numbers mentioned above correspond to the following strains: J2B10F, M2B2F, J2B6F, and M1B1F, respectively: Pseudoalteromonas spongiae, P. pseudoalcaligenes, Vibrio pomeroyi, and V. chagasii.

[0102] contrast Figure 2 Based on the data in Table 1, it can be seen that the strains screened from the culture medium obtained in Example 1 meet the following conditions (16h): 1.0 ≤ OD 560nm OD in the range of ≤1.8 (24h) 560nm >3.0, (36h)OD 560nm The bacterial culture medium must be above 3.7 for the strains to form a biomineralized layer on the steel surface, thus exhibiting an anti-corrosion effect. Strains that do not meet this condition cannot form a biomineralized layer on the steel surface and therefore do not have an anti-corrosion effect. This verifies the rationality of using the culture medium obtained in Example 1 to screen for mineralizable strains. However, the conclusions drawn from the comparative example's culture medium regarding strains meeting the above conditions differ from those of the examples. This indicates that the comparative example's culture medium cannot accurately screen for strains that can form a biomineralized layer on the steel surface and exhibit an anti-corrosion effect.

[0103] The above description is merely an embodiment of the present invention and does not limit the scope of patent protection. Any non-substantial changes or substitutions made by those skilled in the art based on the present invention will still fall within the scope of patent protection.

Claims

1. A culture medium, characterized in that, It contains peptone, yeast extract, sodium chloride, magnesium chloride, and calcium chloride.

2. The culture medium according to claim 1, characterized in that, It also contains phenol red.

3. The culture medium according to claim 1 or 2, characterized in that, It also contains ferric citrate, sodium sulfate, sodium carbonate, potassium bromide, and boric acid.

4. The culture medium according to claim 1, characterized in that, Contains the following quantities: 1-10 parts peptone 1-2 parts yeast extract, 15-25 parts sodium chloride, 3-7 parts magnesium chloride Calcium chloride 2-5 parts.

5. The culture medium according to claim 2 or 4, characterized in that, It also contains 0.01 to 0.1 parts by weight of phenol red.

6. The culture medium according to claim 3, characterized in that, Also includes: Ferric citrate 0.1-1 part, Sodium sulfate 0.1–5 parts, Sodium bicarbonate 0.1–0.5 parts, Potassium bromide 0.01–0.1 parts, Boric acid 0.01 to 0.05 parts.

7. A method for preparing a culture medium as described in any one of claims 1 to 6, characterized in that, Includes the following steps: First, add all components except phenol red in proportion, then autoclave at 120-130°C. After the culture medium is cooled to room temperature, add sterile phenol red to obtain the culture medium; this culture medium is a liquid culture medium.

8. The method for preparing the culture medium according to claim 7, characterized in that, Includes the following steps: First, add all components except phenol red in proportion, then autoclave at 120-130°C, and finally add sterile phenol red after the temperature drops to room temperature to obtain the culture medium.

9. The use of the culture medium according to any one of claims 1 to 6 in screening mineralizing microorganisms.

10. The application of the culture medium according to claim 9 in screening mineralizing microorganisms, characterized in that, The mineralizing microorganisms were screened as bacteria.

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