Culture medium for screening mineralizable microorganisms and screening method

Abstract

The invention relates to the technical field of microorganism culture and screening, and discloses a culture medium for screening mineralizable microorganisms and a screening method, and the culture medium for screening mineralizable microorganisms contains L-arginine, yeast extract powder, seawater salt, beta-sodium glycerophosphate, sodium silicate, phenol red and ferric sodium ethylene diamine tetraacetate. The culture medium is high in mineralizable microorganism screening pertinence and extremely low in infectious microbe content. According to the culture medium disclosed by the invention, L-arginine is taken as a main nutrient substance, and mineralizable microorganisms often preferentially utilize L-arginine (agmatine is produced through a decarboxylation reaction, the pH is indirectly increased, and the combination of Ca < 2 + > and CO3 < 2-> is promoted); beta-sodium glycerophosphate is added into the culture medium, so that'carbonate mineralizing bacteria '(utilizing Ca < 2 + > + CO3 < 2->) and'phosphate mineralizing bacteria' (utilizing Ca < 2 + > + phosphate radical) can be screened at the same time; the added sodium silicate is only utilized by silicate mineralizing bacteria. The types of mineralization bacteria screened by the culture medium cover three mineralization types of carbonate, phosphate and silicate.

Description

Technical Field

[0001] This invention relates to the field of microbial culture and screening technology, and more specifically, to a culture medium and screening method for screening mineralizable microorganisms. Background Technology

[0002] During navigation, ships easily accumulate marine organisms on their surfaces, increasing drag and causing corrosion of the metal. Offshore platforms and oil and gas pipelines, constantly exposed to seawater, frequently experience metal corrosion, often resulting in material perforation and potentially causing serious accidents and losses. Microbial mineralization is a unique natural process driven by the metabolic activities of microorganisms, promoting the deposition of inorganic minerals both inside and outside cells. This induces extracellular minerals to deposit on the metal substrate surface, forming a biomineralized layer. This layer acts as a natural anti-corrosion and anti-fouling barrier, effectively blocking seawater and marine organisms from corroding the metal and significantly reducing corrosion. Moreover, this protective layer forms spontaneously during the growth and reproduction of microorganisms, requiring no artificial chemical additives, thus eliminating the potential pollution hazards of traditional chemical coatings to the marine environment. Furthermore, due to the spontaneous and adaptive nature of its formation process, it can be well applied to irregularly shaped marine structural components, achieving comprehensive and effective anti-corrosion and anti-fouling. Therefore, screening for mineralizable microorganisms capable of forming biomineralized layers on metal surfaces is of paramount importance.

[0003] Currently, existing technologies for screening bacteria capable of precipitating calcium carbonate on metal substrates typically utilize common basal nutrient media, such as Luria-Bertani (LB) medium, Reasoner's 2A medium (R2A), or nutrient agar (NA), to facilitate the enrichment and isolation of various heterotrophic bacteria from environmental samples such as soil, water, or marine sediments. These media have relatively simple compositions and can support the growth of most non-obligate bacteria, thus providing strain resources for subsequent screening of mineralization properties. However, the following technical problems exist:

[0004] 1. Existing culture media lack specificity for screening mineralizable microorganisms and have a high content of contaminating bacteria. Traditional LB, R2A, or NA media mostly use urea and calcium chloride as core components. Existing culture media use peptone and yeast extract as general nutrient sources. These two are also nutrient sources that non-mineralizable bacteria can utilize, resulting in insufficient specificity during screening and a high proportion of contaminating bacteria.

[0005] 2. Existing culture media have limited screening capabilities for mineralizable microorganisms. Traditional LB, R2A, or NA media can only screen for calcium carbonate precipitating bacteria, and cannot screen for silicate-decomposing bacteria or phosphate-mineralizing bacteria. Summary of the Invention

[0006] This invention provides a culture medium for screening mineralizable microorganisms.

[0007] Another object of the present invention is to provide a method for preparing the culture medium for screening mineralizable microorganisms.

[0008] Another object of the present invention is to provide the application of the culture medium for screening mineralizable microorganisms in the screening of mineralizable microorganisms.

[0009] To solve the above-mentioned technical problems, the technical solution provided by the present invention is as follows:

[0010] A culture medium for screening mineralizable microorganisms contains L-arginine, yeast extract, seawater salt, sodium β-glycerophosphate, sodium silicate, agar powder, phenol red, and sodium iron EDTA.

[0011] The culture medium described in this invention has the following technical effects:

[0012] 1. The culture medium of this invention has high specificity for screening mineralizable microorganisms and extremely low content of miscellaneous bacteria.

[0013] In this invention, L-arginine is used as the main nutrient in the culture medium. Mineralizing microorganisms often preferentially utilize L-arginine (through decarboxylation to produce guanidinobutylamine, indirectly increasing pH and promoting calcium absorption). 2+ With CO3 2- (Combined); Non-mineralized bacteria have low utilization of L-arginine, creating "nutrient selection pressure", thus reducing the growth of miscellaneous bacteria by more than 60%, and increasing the proportion of mineralized bacteria in the colony from 30% to 80%. In addition, L-arginine metabolism is directly related to changes in mineralization pH, which can avoid misjudgment caused by non-mineralized bacteria using a common nitrogen source to change the color of the culture medium.

[0014] 2. The culture medium of this invention can screen for a variety of mineralizable microorganisms, including calcium carbonate precipitating bacteria, silicate-decomposing bacteria, and phosphate-mineralizing bacteria, offering diverse screening options. β-glycerophosphate sodium is an exclusive phosphorus source for mineralizing bacteria (difficult for non-mineralizing bacteria to decompose), and its decomposition product (phosphate) is similar to Ca... 2+ A white precipitate forms, which can simultaneously screen for "carbonate mineralizing bacteria" (using Ca). 2+ +CO3 2- ) and "phosphate mineralizing bacteria" (utilizing Ca 2+ +phosphate); sodium silicate cannot be metabolized by non-mineralizing bacteria and is only utilized by silicate mineralizing bacteria, and the precipitate is a white flocculent, which is easy to observe and can be used to further screen for silicate mineralizing bacteria. The culture medium described in this invention covers the three mineralization types of "carbonate, phosphate, and silicate".

[0015] The chelation sites of sodium iron ethylenediaminetetraacetate for Fe 3+ More specific, not related to Ca 2+ Mg2+ Combined, the formation efficiency of mineralized precipitates (such as CaCO3) is increased by 40%, and the precipitate morphology is more regular (without chelate interference), making it easier to observe the mineralization structure under a microscope.

[0016] Preferably, the culture medium also contains calcium chloride, sodium sulfate, sodium bicarbonate, potassium bromide, and boric acid.

[0017] Preferably, the culture medium comprises the following parts by weight:

[0018] L-arginine 0.5–2 parts,

[0019] 5-10 parts yeast extract,

[0020] 20-35 parts seawater salt

[0021] 1-3 parts of sodium β-glycerophosphate

[0022] 1 to 1.5 parts agar powder

[0023] Sodium silicate 0.1-1 part,

[0024] 0.01–0.1 parts of ferric sodium ethylenediaminetetraacetate,

[0025] Phenol red 0.1 to 1 part.

[0026] Preferably, the culture medium further comprises the following parts by weight:

[0027] 0.5–2 parts calcium chloride,

[0028] Sodium sulfate 2-5 parts,

[0029] Sodium bicarbonate 0.1-2 parts,

[0030] Potassium bromide 0.01–0.2 parts,

[0031] Boric acid 0.01 to 0.2 parts.

[0032] This invention also protects a method for preparing the culture medium described in any of the preceding claims, comprising the following steps:

[0033] First, L-arginine, yeast extract, seawater salt, sodium β-glycerophosphate, sodium silicate, and sodium iron ethylenediaminetetraacetate are added in sequence according to the specified ratio. The pH is adjusted to 7.2-7.7, and then the mixture is autoclaved at 120-130°C to obtain the culture medium. This culture medium is a solid culture medium.

[0034] Preferably, the method for preparing the culture medium includes the following steps:

[0035] First, add L-arginine, yeast extract, seawater salt, sodium β-glycerophosphate, sodium silicate, agar powder, sodium ferric EDTA, sodium chloride, calcium chloride, calcium chloride, sodium sulfate, sodium bicarbonate, potassium bromide, and boric acid in the specified proportions to adjust the pH to 7.2–7.7. Then, autoclave at 120–130°C. Finally, add phenol red after the temperature has dropped to 40–50°C to obtain the culture medium.

[0036] The preparation method of the liquid culture medium of the present invention is as follows: before sterilization, remove the agar powder, and when the temperature drops to room temperature, dispense the liquid culture medium into the enzyme-labeled plate in a sterile ultra-clean workbench.

[0037] This invention also protects the application of the culture medium in screening mineralizable microorganisms, wherein the method for screening mineralizable microorganisms is to select mineralizable microorganisms based on the color change of the culture medium.

[0038] Preferably, in the method for screening mineralizing microorganisms, the color change of the culture medium during the 24-hour culture of the mineralizing microorganisms is in the following order: red-orange-dark red-purple or red-orange-dark red.

[0039] Preferably, the mineralizable microorganisms are bacteria, fungi, or archaea.

[0040] Preferably, the mineralizable microorganisms are selected from calcium carbonate precipitating bacteria, silicate decomposing bacteria, or phosphate mineralizing bacteria.

[0041] Compared with the prior art, the present invention has the following technical effects:

[0042] Sodium ferric ethylenediaminetetraacetate not only accelerates cellular respiration and electron transport reactions, but its chelation sites also enhance the effects of Fe. 3+ More specific, not related to Ca 2+ Mg 2+ Combined, the formation efficiency of mineralized precipitates (such as CaCO3) is increased by 40%, and the precipitate morphology is more regular (without chelate interference), making it easier to observe the mineralization structure.

[0043] Specifically, the phenol red is a sterile phenol red solution.

[0044] Phenol red solution is yellow under acidic conditions, red under neutral conditions, and purple under alkaline conditions. It can clearly indicate the change in pH value of the culture medium caused by bacterial growth, making it easy to monitor the growth and metabolism of bacteria in real time. It eliminates the need to wait for the bacteria to react with the metal surface before using SEM and XRD for time-consuming and cumbersome component characterization. The culture medium described in this invention can simply and quickly screen mineralizable bacteria on a large scale from the natural environment and bacterial strain banks.

[0045] Seawater salt enhances the specificity of culture medium screening and inhibits the growth of contaminating bacteria. Naturally mineralized environments (such as oceans and salt lakes) contain complex seawater components (containing Sr...). 2+Ba 2+ Mineralizing bacteria are adapted to trace ions in natural sea salt, while non-mineralizing bacteria are not. The growth of miscellaneous bacteria (which are only adapted to pure sodium chloride) is inhibited.

[0046] Yeast extract provides organic nitrogen, vitamins (such as B vitamins), and growth factors to support the basic metabolism and proliferation of microorganisms.

[0047] Calcium chloride provides Ca 2+ It promotes the precipitation of calcium carbonate (CaCO3) and is a key ion in microbial mineralization (such as Pasteurella multocida).

[0048] Sodium sulfate provides SO4 2- It participates in sulfur metabolism and regulates osmotic pressure to maintain the ionic strength of the culture medium.

[0049] Sodium bicarbonate provides CO3 2- (pH 7.2–7.5), promotes calcium carbonate formation and neutralizes acid-producing metabolites.

[0050] Potassium bromide mimics the composition of seawater, providing Br - It may affect the membrane potential and enzyme activity of certain microorganisms.

[0051] Boric acid provides boron, which participates in cell wall synthesis and the regulation of the activity of certain enzymes, and is especially important for siliceous microorganisms such as diatoms.

[0052] In addition, there are interactions between the components of the culture medium:

[0053] Synergistic effects of carbon, nitrogen, phosphorus, and silicon sources. Yeast extract + L-arginine: This complex nitrogen source supports rapid growth; L-arginine may promote urease activity and accelerate the decomposition of urea into CO2 and NH3, the latter increasing pH and promoting calcium carbonate precipitation; Sodium β-glycerophosphate + sodium silicate: These provide P and Si respectively, and are precursors to hydroxyapatite (Ca). 10 (PO4)6(OH)2) and key components of siliceous minerals.

[0054] Ion balance and mineralization regulation. Ca 2+ +CO3 2- Direct formation of calcium carbonate is the main mechanism of microbial mineralization (such as by *Pasteurella multocida*); Mg 2+ +SO4 2- It stabilizes the mineral crystal structure and inhibits non-specific precipitation.

[0055] pH monitoring and adjustment. Phenol red indicator: reflects the acid / alkali production process in real time through color changes (red-orange-dark red-purple or red-orange-dark red); Sodium bicarbonate + sea salt: maintains an alkaline environment and promotes the dissolution of CO2 into CO3. 2- It also buffers pH fluctuations caused by metabolism.

[0056] Trace elements and cofactors. Sodium iron EDTA: Ensures iron availability; iron is a key component of cytochrome oxidase and nitrogenase. Boric acid + potassium bromide: Trace elements influence the metabolic pathways of specific microorganisms, such as meeting the boron requirements of diatoms.

[0057] Osmotic pressure and environmental simulation. Seawater salt + sodium chloride: simulates marine or high-salt environments, screens for salt-tolerant and mineralizing bacteria (such as certain Bacillus species), and provides a variety of trace elements; sodium sulfate + calcium chloride: adjusts the ionic strength of the culture medium to prevent cell dehydration or rupture. Attached Figure Description

[0058] Figure 1 The color change of the solid culture medium obtained in Example 1 within 24 hours.

[0059] Figure 2 The mineralization and preservation effect of the solid culture medium obtained in Example 1 was used to screen bacteria. Detailed Implementation

[0060] The present invention will be further described in detail below with reference to specific embodiments.

[0061] Example 1

[0062] A culture medium consists of the following components: 1.3 parts L-arginine, 7 parts yeast extract, 15 parts seawater salt, 2 parts sodium β-glycerophosphate, 1.5 parts agar powder, 0.5 parts sodium silicate, 0.05 parts sodium iron ethylenediaminetetraacetate, 0.05 parts phenol red solution, 15 parts sodium chloride, 1.2 parts calcium chloride, 1.3 parts calcium chloride, 3.5 parts sodium sulfate, 1.3 parts sodium bicarbonate, 0.15 parts potassium bromide, 0.12 parts boric acid, and a pH of 7.5.

[0063] The method for preparing the culture medium includes the following steps:

[0064] First, L-arginine, yeast extract, seawater salt, sodium β-glycerophosphate, sodium silicate, sodium iron ethylenediaminetetraacetate, sodium chloride, calcium chloride, calcium chloride, sodium sulfate, sodium bicarbonate, potassium bromide, and boric acid are added in sequence according to the specified proportions. The pH is adjusted to 7.2–7.7. Then, the mixture is autoclaved at 121°C. Finally, after the temperature drops to 40–50°C, sterile phenol red solution is added to obtain the culture medium.

[0065] Example 2

[0066] This embodiment is the second embodiment of the present invention. Unlike embodiment 1, the culture medium is composed of the following components: 0.5 parts L-arginine, 5 parts yeast extract, 18 parts seawater salt, 3 parts sodium β-glycerophosphate, 1 part agar powder, 0.1 parts sodium silicate, 0.01 parts ferric sodium ethylenediaminetetraacetate, 0.1 parts phenol red solution, 10 parts sodium chloride, 0.5 parts calcium chloride, 2 parts sodium sulfate, 0.1 parts sodium bicarbonate, 0.01 parts potassium bromide, 0.2 parts boric acid, and a pH value of 7.2.

[0067] Example 3

[0068] This embodiment is the third embodiment of the present invention. Unlike embodiment 1, the culture medium is composed of the following components: 2 parts L-arginine, 10 parts yeast extract, 10 parts seawater salt, 1 part sodium β-glycerophosphate, 1 part sodium silicate, 0.5 parts agar powder, 0.1 parts ferric sodium ethylenediaminetetraacetate, 1 part phenol red solution, 20 parts sodium chloride, 2 parts calcium chloride, 5 parts sodium sulfate, 2 parts sodium bicarbonate, 0.2 parts potassium bromide, 0.01 parts boric acid, and a pH value of 7.7.

[0069] Example 4

[0070] This embodiment is the fourth embodiment of the present invention. Unlike embodiment 1, the culture medium components do not contain sodium chloride, calcium chloride, sodium sulfate, sodium bicarbonate, potassium bromide, and boric acid.

[0071] Comparative Example 1

[0072] 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.

[0073] Comparative Example 2

[0074] 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).

[0075] Comparative Example 3

[0076] 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).

[0077] Comparative Example 4

[0078] This comparative example is the fourth comparative example of the present invention. Unlike Example 1, the culture medium component does not contain sodium β-glycerophosphate.

[0079] Comparative Example 5

[0080] This comparative example is the fifth comparative example of the present invention. Unlike Example 1, the culture medium component does not contain sodium silicate.

[0081] Comparative Example 6

[0082] This comparative example is the sixth comparative example of the present invention. The difference from Example 1 is that L-arginine in the culture medium component is replaced with peptone.

[0083] Comparative Example 7

[0084] This comparative example is the 7th comparative example of the present invention. Unlike Example 1, the culture medium components do not contain seawater salt.

[0085] Performance testing:

[0086] Screening of mineralizable bacterial strains: *Pseudoalteromonas spongiae*, *P. pseudoalcaligenes*, *Vibrio pomeroyi*, and *V. chagasii* strains were transferred to the culture medium obtained in Example 1 and incubated at 120 rpm and 20–28°C. 100 μL of bacterial culture was spread onto a solid selective culture medium, and the color change of the medium was observed over 32 hours. Figure 1 The culture methods for the culture medium strains obtained in Examples 2-4 and Comparative Examples 1-7 were the same as those in Example 1. The color changes and antiseptic effects of the culture medium strains obtained in each example and comparative example are shown in Table 1. The strains Pseudoalteromonas spongiae, P. pseudoalcaligenes, Vibrio pomeroyi, and V. chagasii are abbreviated as J2B10F, M2B2F, J2B6F, and M1B10F, respectively.

[0087] The criteria for judging the anti-corrosion effect are as follows:

[0088] (1) If the culture medium color changes from red to orange to dark red to purple after 24 hours of culture, it can be judged that the strain has a good antiseptic effect.

[0089] (2) If the culture medium color changes from red to orange to dark red after 24 hours of cultivation, it can be judged that the strain has a good antiseptic effect.

[0090] (3) If the culture medium changes from red to yellow after 24 hours of cultivation, it can be judged that the strain has a general antiseptic effect.

[0091] (4) If the culture medium changes from red to light red after 24 hours of incubation, it is determined that the strain has a poor preservative effect.

[0092] Table 1. Results of color changes and preservative effects of culture medium strains obtained in each example and comparative example.

[0093]

[0094] Contaminating microorganism percentage detection: Based on colony morphology and functional characteristics, the plate counting method utilizes the specific phenotypes of target mineralizing microorganisms on the screening medium (such as mineralization products, indicator reactions) to distinguish them from the non-specific phenotypes of contaminating microorganisms. Contaminating microbial colonies are directly counted, and the contaminating microorganism percentage is calculated. Specific operating steps are as follows:

[0095] Take the liquid culture and serially dilute it with sterile physiological saline, such as 10-1. -1 ~10 -6 Take 0.1–0.2 mL of bacterial suspension at each dilution, spread it onto the original screening medium plate, and incubate at 30–37°C for 24–72 h; observe the colonies:

[0096] Characteristics of the target bacteria: Precipitation may occur on the screening medium due to mineralization, including: calcium carbonate precipitation as a white turbidity ring, silicate decomposition as a clear ring, or the phenol red indicator may change color due to changes in pH due to metabolism (such as alkali / acid production) (color change is red-orange-dark red-purple or red-orange-dark red).

[0097] Characteristics of contaminating microorganisms: lacking the above-mentioned mineralization-related phenotypes (no precipitation, no clear zone, no color change of indicator), or growing on general culture media but not exhibiting the target characteristics on screening media.

[0098] The "total bacteria count" and "target bacteria count" are obtained by plate counting, and the proportion of miscellaneous bacteria is calculated after indirectly deducing the miscellaneous bacteria count.

[0099] Obtaining the total bacterial count: Spread the culture medium on a general nutrient medium (such as NA or LB agar), count all colonies (including target bacteria and other bacteria), and record the result as N total (unit: CFU / mL or CFU / g).

[0100] Obtaining the target bacterial count: Spread the culture medium on a screening medium and count the colonies with mineralization-specific phenotypes (such as colonies that produce precipitates or change the color of indicators). The result is recorded as N target (the unit is the same as N total).

[0101] Derivation of the number of miscellaneous bacteria: Number of miscellaneous bacteria N_miscellaneous = N_total - N_target (assuming that all colonies on the general culture medium can grow on the selection medium, or ignore miscellaneous bacteria that do not grow).

[0102] Percentage calculation: Percentage of miscellaneous bacteria content = (N_miscellaneous bacteria ÷ N_total) × 100% = [(N_total - N_target) ÷ N_total] × 100%. The percentage of miscellaneous bacteria in the culture media obtained from each example and comparative example is shown in Table 2.

[0103] Table 2 shows the percentage of contaminants in the culture media obtained from each example and comparative example.

[0104] Percentage of miscellaneous bacteria Example 1 0.2％ Example 2 0.5％ Example 3 0.4％ Example 4 0.7％ Comparative Example 1 5.1％ Comparative Example 2 4.8％ Comparative Example 3 4.3％ Comparative Example 4 1.2％ Comparative Example 5 1.5％ Comparative Example 6 2.8％ Comparative Example 7 2.1％

[0105] Steel corrosion testing:

[0106] 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.

[0107] The number of bacteria inoculated in the culture medium should be kept as consistent as possible; therefore, during the logarithmic growth phase, the O2 content should be measured. D600 nm 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 culture medium inoculated with bacteria 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, place it under a white light interferometer to observe the local corrosion morphology of the steel surface.

[0108] After soaking for 7 days, the steel samples were rinsed once with deionized water and then fixed in a 2.5% glutaraldehyde solution for 20–40 min. They were then dehydrated sequentially in 30%, 50%, 70%, 80%, 90%, and anhydrous ethanol for 10–20 min. After drying, the samples were sputtered with gold for 60–90 s using a vacuum sputtering instrument. The corrosion morphology and biomineralization layer structure of the steel sample surface were observed using a scanning electron microscope. The control group was compared with the blank control group, showing localized corrosion and surface morphology. Numerous corrosion products were observed on the sample surface, indicating severe damage to the matrix, including defects and pitting. *Pseudoalteromonas spongiae* and *P. pseudoalcaligenes* formed relatively complete biomineralization layers on the steel surface. Magnified images show that the biomineralization layers exhibit a nested structure of small triangles. *Vibrio pomeroyi* also formed a certain biomineralization layer, but with numerous pores and cracks, causing localized corrosion to some extent. Vibrio chagasii forms distinct yellow corrosion products on the steel surface, without the formation of a significant mineralization layer.

[0109] Combining Table 1 and Figure 1 It can be seen that the culture media obtained in Examples 1-4 showed the following color changes in P. spongiae and P. pseudoalcaligenes strains: medium red-orange-dark red-purple, or red-orange-dark red, indicating that these strains have good preservative effects. The V. chagasii strain showed a color change of red-light red, indicating that these strains have poor preservative effects. Figure 1The judgment results of Examples 1-4 in Table 1 are correct. However, the color changes of the strains cultured in the culture medium of Comparative Examples 1-7 do not fully meet the criteria for judging the preservative effect of this invention. Figure 1 This indicates that the judgment results of comparison examples 1 to 7 are incorrect.

[0110] 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 for screening mineralizable microorganisms, characterized in that, It contains L-arginine, yeast extract, seawater salt, sodium β-glycerophosphate, sodium silicate, agar powder, phenol red, and sodium iron EDTA.

2. The culture medium according to claim 1, characterized in that, It also contains sodium chloride, calcium chloride, sodium sulfate, sodium bicarbonate, potassium bromide, and boric acid.

3. The culture medium according to claim 1, characterized in that, Contains the following quantities: L-arginine 0.5–2 parts, 5-10 parts yeast extract, Sea salt 10-18 parts, 1-3 parts of sodium β-glycerophosphate 1 to 1.5 parts agar powder Sodium silicate 0.1-1 part, 0.01–0.1 parts of ferric sodium ethylenediaminetetraacetate, Phenol red 0.1 to 1 part.

4. The culture medium according to claim 3, characterized in that, It also contains the following components by weight: 10-20 parts sodium chloride, 0.5–2 parts calcium chloride, Sodium sulfate 2-5 parts, Sodium bicarbonate 0.1-2 parts, Potassium bromide 0.01–0.2 parts, Boric acid 0.01 to 0.2 parts.

5. A method for preparing a culture medium as described in any one of claims 1, characterized in that, Includes the following steps: First, L-arginine, yeast extract, seawater salt, sodium β-glycerophosphate, sodium silicate, agar powder, and sodium iron EDTA are added in sequence according to the specified ratio. The pH is adjusted to 7.2–7.

7. Then, the mixture is autoclaved at 120–130°C and cooled to 40–50°C before adding phenol red to obtain the culture medium. This culture medium is a solid culture medium.

6. The method for preparing the culture medium according to claims 2 to 6, characterized in that, Includes the following steps: First, add L-arginine, yeast extract, seawater salt, sodium β-glycerophosphate, sodium silicate, agar powder, sodium iron EDTA, sodium chloride, calcium chloride, calcium chloride, sodium sulfate, sodium bicarbonate, potassium bromide, and boric acid in the specified proportions to adjust the pH to 7.2–7.

7. Then, autoclave at 120–130°C. Finally, add phenol red after the temperature has decreased to obtain the culture medium.

7. The use of the culture medium according to any one of claims 1 to 4 in screening mineralizable microorganisms, characterized in that, The method for screening mineralizing microorganisms is to select them based on the color change of the culture medium.

8. The application of the culture medium according to claim 7 in screenable mineralizable microorganisms, characterized in that, In the method for screening mineralizing microorganisms, the color change of the culture medium during the 24-hour culture of mineralizing microorganisms is as follows: red-orange-dark red-purple or red-orange-dark red.

9. The application of the culture medium according to claim 7 or 8 in screening mineralizable microorganisms, characterized in that, The mineralizable microorganisms were screened as bacteria, fungi, or archaea.

10. The application of the culture medium according to claim 9 in screening mineralizable microorganisms, characterized in that, Mineralizing microorganisms are selected from calcium carbonate precipitating bacteria, silicate decomposing bacteria, or phosphate mineralizing bacteria.

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