A solid culture medium for isolating and purifying thermophilic and acidophilic bacteria, a preparation method thereof, and application thereof

Abstract

This application provides a solid culture medium for the isolation and purification of thermophilic acidophilic bacteria, its preparation method, and its application. The solid culture medium is prepared by mixing a nutrient composition, a coagulant, and deionized water. The coagulant accounts for 9% to 50% of the solid culture medium by mass. The coagulant is a high-acyl gellan gum, containing 3 to 4 acyl groups. The concentration of the high-acyl gellan gum is 5 g / L to 30 g / L. This solid culture medium is made using high-acyl gellan gum. By adjusting the mass percentage of the high-acyl gellan gum, especially by limiting its concentration, the solid culture medium can remain stable for a long time under high temperature and high acid conditions, maintaining good gelation properties. This provides a stable high-temperature and high-acid growth environment for thermophilic acidophilic bacteria, thus facilitating the successful isolation and purification of these bacteria. Furthermore, high-acyl gellan gum is widely available and inexpensive, further reducing culture costs.

Description

Technical Field

[0001] This application relates to the field of microbial culture technology, and more specifically, to a solid culture medium for the isolation and purification of thermophilic and acidophilic bacteria, its preparation method, and its application. Background Technology

[0002] Thermophilic acidophilic bacteria are a key focus of current biological research. Due to their predominantly iron and / or sulfur oxidation activity, they are widely used in biometallurgy. In addition, some thermophilic acidophilic bacteria are used in technologies such as AMD environmental remediation and MCs degradation. These bacteria typically reside in sulfur-containing acidic hot springs and can survive above 60°C. Compared to mesophilic and moderately thermophilic bacteria in mineral leaching, their superior iron and sulfur oxidation capabilities significantly shorten the leaching cycle and improve leaching efficiency, making them highly valuable microorganisms for comprehensive mineral resource utilization and environmental protection. However, because thermophilic acidophilic bacteria have demanding survival conditions, it is difficult to replicate their natural growth environment in culture. Therefore, they are generally cultured as liquid mixed samples, making it difficult to obtain pure cultures, resulting in very limited germplasm resources.

[0003] Currently, pure cultures of thermophilic acidophilic bacteria remain difficult to obtain, which is one of the "bottlenecks" limiting their related research and applications. With the application of molecular biology techniques and high-throughput sequencing technology, more and more thermophilic acidophilic bacteria are being detected in leaching environments. Furthermore, with in-depth research and technological advancements, many microorganisms have transitioned from an unculturable state to a culturable state, and corresponding progress has been made in the study of the properties of newly isolated and purified thermophilic acidophilic bacteria.

[0004] For example, solid culture media are used to isolate and purify microorganisms, which involves using single bacteria to form single colonies on a plate to isolate pure bacteria. Solid culture media are mainly made of nutrients and gelling materials. Gelling materials include agar, low-acyl gellan gum, and, as proposed by Professor Xia Jinlan of Central South University, using gel powder as a solidifying agent to prepare solid culture media for the isolation and purification of acidophilic archaea. However, these raw materials have poor stability under prolonged high temperature and high acid conditions, especially at lower pH conditions, where their gelling properties decrease sharply and they cannot form stable gels, making it impossible to successfully purify and isolate thermophilic acidophilic bacteria. Summary of the Invention

[0005] To address the aforementioned issues, this application provides a solid culture medium for the isolation and purification of thermophilic acidophilic bacteria, its preparation method, and its application. Using high-acyl gellan gum as the gelling material, a solid culture medium for the isolation and purification of thermophilic acidophilic bacteria is prepared. This medium allows the bacteria to remain stable in a high-temperature, high-acid environment for an extended period, resulting in better gelation and facilitating the successful purification and isolation of the bacteria. This leads to the acquisition of pure thermophilic acidophilic strains, providing strong technical support for the protection and in-depth research of thermophilic acidophilic germplasm resources.

[0006] In a first aspect, this application provides a solid culture medium for the isolation and purification of thermophilic and acidophilic bacteria, wherein the solid culture medium is prepared by mixing a nutrient composition, a coagulant, and deionized water, and the mass percentage of the coagulant in the solid culture medium is 9% to 50%; The coagulant includes high acyl gellan gum, wherein the number of acyl groups in the high acyl gellan gum is 3 to 4. The concentration of the high-acyl gellan gum is 5 g / L to 30 g / L.

[0007] Furthermore, the concentration of the high-acyl gellan gum is 10 g / L to 20 g / L.

[0008] Furthermore, the coagulant also includes agar; The concentration of the agar is 5 g / L to 20 g / L; The mass ratio of the agar to the high-acyl gellan gum is 0.5 to 0.6.

[0009] Furthermore, the nutritional composition comprises CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, KSCN, FeSO4∙7H2O, HNO3 and H2SO4; The mass-to-volume ratio of CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, KSCN, FeSO4∙7H2O, HNO3, and H2SO4 is (0.05~0.1) g : (0.05~0.2) g : (0.2~1) g : (2.5~3.5) g : (0.05~0.1) g : (0.2~1) g : (0.01~0.05) g : (5~11) g : (42~46) g : (0.075~0.078) ml : (0.1~0.3) ml.

[0010] Secondly, this application provides a method for preparing a solid culture medium for the isolation and purification of thermophilic acidophilus, the preparation method comprising: The CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, HNO3, KSCN and H2SO4 in the nutrient composition were dissolved in deionized water, and the pH of the resulting mixture was adjusted to 1.8~4 to obtain the first nutrient solution. FeSO4∙7H2O was dissolved in deionized water, the pH of the resulting solution was adjusted to 1.5-3, and the solution was filtered through a microporous membrane to remove bacteria to obtain the second nutrient solution. Add the coagulant to the first nutrient solution and stir at 85 ℃~200 ℃. After the resulting fluid is filtered, sterilized at high temperature, and cooled for 5 min~10 min, it is mixed with the second nutrient solution to obtain a culture medium colloid with a pH of 1.8~3.5. After gelation, a solid culture medium is obtained. The coagulant includes high-acyl gellan gum, and the concentration of the high-acyl gellan gum is 5 g / L to 30 g / L.

[0011] Furthermore, the coagulant is a mixture of the high-acyl gellan gum and agar; The concentration of the agar is 3 g / L to 10 g / L; The mass ratio of the agar to the high-acyl gellan gum is 0.5 to 0.6.

[0012] Furthermore, the coagulant accounts for 16% to 27% of the mass of the solid culture medium.

[0013] Furthermore, the preparation method further includes: The coagulant is added to the first nutrient solution and ultrasonically stirred at a frequency of 10 kHz to 30 kHz for 5 min to 30 min. The resulting fluid is filtered, sterilized at high temperature, cooled for 5 min to 10 min, and then mixed with the second nutrient solution to obtain a culture medium colloid with a pH of 1.8 to 3.5. After gelation, a solid culture medium is obtained.

[0014] Furthermore, the high-temperature sterilization temperature is 110 ℃~130 ℃, and the sterilization time is 20 min~30 min.

[0015] Thirdly, this application provides an application of a solid culture medium for the isolation and purification of thermophilic and acidophilic bacteria, wherein the solid culture medium is used for the isolation and purification of thermophilic archaea, thermophilic / acidophilic bacteria, or moderately thermophilic / acidophilic bacteria.

[0016] In summary, this application includes at least one of the following beneficial technical effects: 1. This application provides a solid culture medium for the isolation and purification of thermophilic acidophilic bacteria. The solid culture medium is made using high-acyl gellan gum as a coagulant. By adjusting the mass ratio of high-acyl gellan gum, especially by limiting the concentration of high-acyl gellan gum, the solid culture medium maintains good gelation properties under high temperature and high acidity conditions, and can be stably formed for a long time, providing a stable growth environment for thermophilic acidophilic bacteria, thereby successfully isolating and purifying thermophilic acidophilic bacteria. High-acyl gellan gum is widely available and inexpensive (180 yuan / kg), which can effectively reduce the culture cost. 2. This application provides an application of a solid culture medium for the isolation and purification of thermophilic and acidophilic bacteria. The solid culture medium made with high-acyl gellan gum as a coagulant has no toxic effect on cells, has strong water retention, is not easy to dry out in a continuous high-temperature environment, and is resistant to high temperature (65 ℃≤T≤100 ℃) and high acid (pH≥1.5) environments. The gel is stable and is a good medium for the growth of acidophilic archaea, thermophilic / acidophilic bacteria or moderate thermophilic / acidophilic bacteria. It can stably and efficiently isolate and purify acidophilic archaea, thermophilic / acidophilic bacteria or moderate thermophilic / acidophilic bacteria. Attached Figure Description

[0017] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the description of the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 A flowchart of the method for preparing solid culture medium for isolating and purifying thermophilic and acidophilic bacteria according to an embodiment of this application is shown. Figure 2 A schematic diagram of the solid culture medium and bacterial growth at 65 °C as proposed in the embodiments of this application is shown; Figure 3 A schematic diagram of the solid culture medium for culturing thermophilic acidophilic bacteria proposed in Comparative Example 1 of this application is shown. Detailed Implementation

[0019] The technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of this application, not all embodiments. Based on the embodiments of this application, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0020] In the accompanying drawings, the size of constituent elements, the thickness of layers, or areas may sometimes be exaggerated for clarity. Therefore, any implementation of this disclosure is not necessarily limited to the dimensions shown in the drawings, and the shapes and sizes of the components in the drawings do not reflect true proportions. Furthermore, the drawings schematically illustrate ideal examples, and any implementation of this disclosure is not limited to the shapes or values ​​shown in the drawings.

[0021] In related technologies, thermophilic acidophilic bacteria are currently a key focus of research in the biological field. Due to their predominant iron and / or sulfur oxidation activity, they are widely applied in biometallurgy. Typically, thermophilic acidophilic bacteria exhibit enhanced metabolic activity and accelerated reproduction rates under high temperature (>65 °C) and high acid (pH 1.5–3.5) conditions, which facilitates rapid enrichment and growth. Therefore, for the isolation and purification of thermophilic acidophilic bacteria, a high-acid, high-heat environment is unfavorable, even potentially fatal, to most other microorganisms. Such an environment inhibits the growth of other non-thermophilic acidophilic bacteria, reducing competition and interference with the thermophilic acidophilic bacteria, allowing them to rapidly accumulate and grow, becoming the dominant species during the isolation and purification process.

[0022] However, traditional microbial culture methods are insufficient to meet the requirements for pure culture, and the efficiency of pure culture of microorganisms that have been achieved in the laboratory is not high. In general, existing methods for isolating and purifying thermophilic and acidophilic bacteria can be summarized as follows: 1) Liquid dilution method (MPN method): This method simulates the natural environment, prepares liquid culture medium, and obtains pure cultures through highly gradient dilution. 2) Based on the physiological and functional characteristics of microorganisms, isolation and purification are achieved by restricting their nutrients and growth conditions in shake flask culture; 3) Single cells are obtained and cultured into pure cultures by cell sorting methods such as labeling and tracking with fluorescent probes, separating single cells under a microscopic environment using a micromanipulator, or laser spraying single cells.

[0023] These technologies have driven related research on microorganisms, but traditional sorting methods are often too frequent and have poor separation and purification effects, resulting in fewer thermophilic and acidophilic bacteria. Meanwhile, emerging sorting technologies are difficult to promote in laboratories due to their demanding requirements, technical complexity, low output, or immaturity.

[0024] Therefore, most researchers still prefer to isolate and purify microorganisms using solid culture media, that is, to isolate pure bacteria by forming single colonies on a plate. Agar, with its advantages of high transparency, good water retention, non-toxicity, and resistance to microbial liquefaction, is commonly used as a solidifying agent in solid culture media. However, at low pH values ​​(pH < 4), agar is hydrolyzed into oligosaccharides and reducing sugars during sterilization, losing its solidifying properties. Even when solid culture media are prepared by separately sterilizing acid and agar and then mixing them, agar culture media will still hydrolyze and lose its solidifying form under high-temperature (> 65 °C) culture conditions, thus it cannot be used for the stable purification and isolation of thermophilic acidophilic bacteria. Professor Xia Jinlan of Central South University once proposed using gelatin powder as a coagulant to prepare solid culture medium for the isolation and purification of acidophilic thermoarchaea. However, the main raw materials of general gelatin powder are agar, gelatin powder (which is not heat-resistant and acid-resistant) or gelatin. These raw materials cannot be stably formed under long-term high temperature and high acid conditions. Low-acyl gellan gum, as an emerging gelling agent, has widely replaced agar in solid culture media due to its strong gelling ability, high transparency, and good stability. However, it is expensive (1 kg / 1150 ¥); the usage is 2 g / L~8 g / L (1 / 4-1 / 3 of agar), it is heat-resistant, and has a pH range of 3.4-7.5, but under lower pH conditions (pH 1.5~3.5), a large amount of H+ will be released. + It interacts with the functional groups in low-acyl gellan gum molecules, disrupting the original hydrogen bond network and causing a decrease in the stability of the gel network structure. It cannot be stably molded for a long time, and therefore cannot be used to isolate thermophilic acidophilic bacteria.

[0025] To address the problems existing in current technologies, this application proposes a novel solid culture medium for the isolation and purification of thermophilic acidophilic bacteria. High-acyl gellan gum is used as a gelling agent. By adjusting its concentration and mass in the solid culture medium, the problems of low gel strength and poor hardness are overcome. This allows the prepared solid culture medium to remain stable for extended periods under high temperature (65 ℃ ≤ T ≤ 100 ℃) and high acidity (3.5 ≥ pH ≥ 1.5) conditions, enabling the successful isolation and purification of thermophilic acidophilic bacteria and yielding pure thermophilic acidophilic strains. Furthermore, high-acyl gellan gum is inexpensive (180 RMB / kg), significantly lower than low-acyl gellan gum (approximately 1150 RMB / kg), thereby reducing preparation costs and improving the economic efficiency of bacterial culture.

[0026] In a first aspect, this application provides a solid culture medium for the isolation and purification of thermophilic and acidophilic bacteria, wherein the solid culture medium is prepared by mixing a nutrient composition, a coagulant, and deionized water, and the mass percentage of the coagulant in the solid culture medium is 9% to 50%; The coagulant includes high acyl gellan gum, wherein the number of acyl groups in the high acyl gellan gum is 3 to 4. The concentration of the high-acyl gellan gum is 5 g / L to 30 g / L.

[0027] In practice, high-acyl gellan gum was used as the gelling material. By precisely controlling its mass ratio and concentration, the problems of low gel strength, poor hardness, and easy hydrolysis and loss of coagulation of traditional coagulants (such as agar and low-acyl gellan gum) under high temperature (65 ℃≤T≤100 ℃) and high acid (3.5≥pH≥1.5) conditions were successfully overcome. This novel solid culture medium can maintain stable shape and sufficient hardness for a long time under extreme conditions, meeting the stringent requirements for the isolation and purification of thermophilic acidophilic bacteria. It greatly improves the efficiency and success rate of isolation and purification, and provides strong technical support for the protection and in-depth research of thermophilic acidophilic bacteria germplasm resources.

[0028] In some embodiments, the main chain structure of the high-acyl gellan gum molecule is composed of four sugars, namely (1,3)β-D-glucose, β(1,4)-D-glucuronic acid, (1,4)-D-glucose, and (1,4)α-L-rhamnose. Each repeating unit of the high-acyl gellan gum has one acyl group attached to a glucose molecule. In the embodiments of this application, the high-acyl gellan gum uses 3 to 4 acyl groups, which are divided into two categories: one is an acetyl group attached to the 6th carbon atom of the glucose molecule by a β-1,3 bond, and the other is a glycerol group attached to the 2nd carbon atom of the glucose molecule by a β-1,3 bond. The average proportion of glycerol groups is 1 to 2 times that of acetyl groups.

[0029] In practical application, the main difference between high-acyl gellan gum and low-acyl gellan gum lies in the number and properties of acyl groups. The number and arrangement of acyl groups directly affect the strength and stability of the gel. High-acyl gellan gum has a larger number of acyl groups, resulting in a more compact gel structure, higher gel strength, and better stability, even when containing a large amount of H... + In high-acyl gellan gum, high-acyl gellan gum maintains high gel performance even in harsh environments. In contrast, low-acyl gellan gum has fewer acyl groups and a relatively loose gel structure, resulting in poor gel strength and stability, especially under high-temperature and high-acid conditions. Therefore, high-acyl gellan gum achieves better gelation results than low-acyl gellan gum and is more suitable for the growth and isolation of thermophilic and acidophilic bacteria.

[0030] In some embodiments, the concentration of the high-acyl gellan gum is 10 g / L to 20 g / L.

[0031] In practical implementation, the concentration and mass ratio of high-acyl gellan gum affect the looseness of its gel structure. The embodiments of this application, through precise control, ensure that even under extreme conditions of high temperature (65 ℃ ≤ T ≤ 100 ℃) and high acidity (3.5 ≥ pH ≥ 1.5), it maintains a compact structure and sufficient strength, and is not easily hydrolyzed or deformed, thus overcoming the shortcomings of traditional thickeners that are prone to failure under these conditions. Furthermore, by precisely controlling its dosage, effective cost control is achieved while ensuring gel performance, reducing overall costs and improving resource utilization efficiency compared to traditional coagulants.

[0032] In some embodiments, the coagulant further includes agar; The concentration of the agar is 5 g / L to 20 g / L; The mass ratio of the agar to the high-acyl gellan gum is 0.5 to 0.6.

[0033] In practice, this application uses agar and high-acyl gellan gel in combination, which produce a synergistic effect to form a more complex and stable gel network. This not only improves the strength and stability of the gel but also enhances the elasticity and toughness of the solid culture medium, making it more durable and less prone to breakage during handling. The combined use also optimizes the water retention and aeration of the solid culture medium, providing a more suitable environment and reducing interactions and interference between colonies.

[0034] In some embodiments, preferably, the concentration of agar is 10 g / L to 15 g / L. When the coagulant consists of agar and high-acyl gellan gum, the concentration of high-acyl gellan gum can be 5 g / L to 20 g / L.

[0035] In some embodiments, the nutrient composition comprises CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, KSCN, FeSO4∙7H2O, HNO3, and H2SO4; The mass-to-volume ratio of CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, KSCN, FeSO4∙7H2O, HNO3, and H2SO4 is (0.05~0.1) g : (0.05~0.2) g : (0.2~1) g : (2.5~3.5) g : (0.05~0.1) g : (0.2~1) g : (0.01~0.05) g : (5~11) g : (42~46) g : (0.075~0.078) ml : (0.1~0.3) ml.

[0036] In practical implementation, high-acyl gellan gum resists divalent ions (such as Ca). 2+ Mg 2+ Highly sensitive to divalent ions, these ions interact with functional groups such as carboxyl groups in high-acyl gellan gum molecules, forming more physical cross-linking points. This results in a denser and more stable gel network, significantly enhancing the gel strength of the high-acyl gellan gum. By controlling the mass of components containing divalent ions during the preparation of solid culture media, the gel strength of the high-acyl gellan gum can be stabilized, reducing water molecule loss and improving the water retention of the gel. This also allows for reasonable control of the amount of each component used, avoiding resource waste. The presence of divalent ions can also slow down the stress relaxation rate of the high-acyl gellan gum, making the gel more stable.

[0037] In some embodiments, inorganic salts such as CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, and MgSO4∙7H2O provide microorganisms with the necessary ions and trace elements. Yeast extract is mainly the substance obtained by breaking yeast cells after yeast grows and multiplies. It is refined by degrading the proteins and nucleic acids in yeast cells. It contains peptides, amino acids, vitamins and trace elements, and as an organic nitrogen source, it helps the growth and reproduction of microorganisms. Sulfates such as KH₂PO₄ and KHSO₄ can also act as pH buffers, helping to maintain the stability of the pH value of solid culture media. HNO₃, as a strong acid, can further lower the pH value of solid culture media, thereby creating a highly acidic environment that is more conducive to the rapid growth and accumulation of thermophilic acidophilic bacteria.

[0038] In summary, the nutrient composition in solid culture media provides comprehensive nutritional support for microorganisms, which helps them grow and reproduce rapidly. By adjusting the physicochemical properties (pH) of solid culture media and adding high-acyl gellan gum, an environment most suitable for the growth of thermophilic acidophilic bacteria can be created, thus enabling the successful isolation and purification of thermophilic acidophilic bacteria.

[0039] Secondly, see Figure 1 This application provides a method for preparing a solid culture medium for isolating and purifying thermophilic and acidophilic bacteria as described in the first aspect above, the preparation method comprising: Step S1: Dissolve CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, HNO3, KSCN and H2SO4 in deionized water, and adjust the pH of the resulting mixture to 1.8~4 to obtain the first nutrient solution. Step S2: Dissolve FeSO4∙7H2O in deionized water, adjust the pH of the resulting solution to 1.5~3, and filter it with a microporous membrane to remove bacteria to obtain the second nutrient solution; Step S3: Add the coagulant to the first nutrient solution and stir at 85 ℃~200 ℃. After the resulting fluid is filtered, sterilized at high temperature, and cooled for 5 min~10 min, it is mixed with the second nutrient solution to obtain a culture medium colloid with a pH of 1.8~3.5. After gelation, a solid culture medium is obtained. The coagulant includes high-acyl gellan gum, and the concentration of the high-acyl gellan gum is 5 g / L to 30 g / L.

[0040] In practice, by precisely proportioning the inorganic salts, H2SO4, yeast extract, and other nutrients in the nutrient composition and adjusting the pH to an acidic range of 1.8–4, the solid culture medium provides the nutritional basis required by thermophilic acidophilic bacteria and eliminates the growth conditions of other non-thermophilic acidophilic bacteria, thus promoting the rapid growth of thermophilic acidophilic bacteria. Then, FeSO4∙7H2O is dissolved in deionized water, and the pH is adjusted to 1.5–3.5. After filtration and sterilization, a second nutrient solution is prepared. This step provides the iron element required for the growth of thermophilic acidophilic bacteria, and the sterilization process ensures aseptic conditions, avoiding contamination by other microorganisms. High-acyl gellan gum can maintain the stability of its gel structure under even lower pH conditions. The high-acyl gellan gum is added to the first nutrient solution and stirred at high temperature to fully dissolve the high-acyl gellan gum and form a homogeneous gel system. After filtration and high-temperature sterilization, it is cooled for 5–10 minutes and mixed with the second nutrient solution to obtain a culture medium colloid with a pH of 1.8–3.5. This step not only keeps the culture medium sterile, but also prepares a solid culture medium that can remain stable for a long time in a high-temperature and high-acid environment through the gelation effect of high-acyl gellan gum, thus achieving stable isolation and purification of thermophilic acidophilic bacteria.

[0041] The preparation method provided in this application is simple to operate, avoiding the cumbersome procedures of traditional methods, and reducing the difficulty and cost of operation. Furthermore, the resulting solid culture medium can be widely used in the isolation and purification of thermophilic and acidophilic bacteria, biometallurgy, environmental remediation, and other fields.

[0042] In practice, FeSO4∙7H2O is easily oxidized to Fe2(SO4)3 during high-temperature sterilization, thus failing to provide energy for bacteria. Therefore, FeSO4∙7H2O is mixed with deionized water to form a second nutrient solution, which is then filtered through a microporous membrane to remove unwanted bacteria, thereby maintaining the stability of ferrous ions and providing sufficient nutrients for the growth of thermophilic and acidophilic bacteria.

[0043] In practice, the obtained culture medium colloid is a soft, sticky dough, which is then dispensed into plates, quickly spread out, and gelled to obtain a solid culture medium.

[0044] In some embodiments, the obtained fluid can be filtered using medical gauze or a 180-220 mesh filter sieve, as long as the filtration achieves a homogeneous effect.

[0045] In some embodiments, since high-acyl gellan gum is insoluble in water at room temperature, it is necessary to add the high-acyl gellan gum to the first nutrient solution and stir it at a temperature above 85 °C to fully dissolve the high-acyl gellan gum by raising the temperature, so as to avoid the presence of dry powder or lumps in the system.

[0046] In some embodiments, the pore size of the microporous filter membrane is 0.2 μm to 0.5 μm, preferably 0.22 μm.

[0047] In some embodiments, a coagulant is added to the first nutrient solution and stirred at 95°C to 105°C.

[0048] In some embodiments, the coagulant is a mixture of the high-acyl gellan gum and agar; The concentration of the agar is 3 g / L to 10 g / L; The mass ratio of the agar to the high-acyl gellan gum is 0.5 to 0.6.

[0049] In practice, agar and high-acyl gellan gel are used in combination. The two work synergistically, overcoming both the poor gelation properties of agar under high temperature and high acidity conditions and the low gel strength and insufficient hardness of high-acyl gellan gel at low concentrations. This combination is suitable for preparing solid culture media for isolating and purifying thermophilic and acidophilic bacteria. Furthermore, using agar and high-acyl gellan gel in combination reduces the amount of high-acyl gellan gel required, further lowering preparation costs.

[0050] In some embodiments, the coagulant accounts for 16% to 27% of the mass of the solid culture medium.

[0051] By limiting the mass percentage of the coagulant in the solid culture medium, the problems of low gel strength and poor hardness at low concentrations of high-acyl gellan gum can be overcome. This allows the solid culture medium to maintain a stable form under high temperature and high acid conditions, without easily deforming or dissolving. Using high-acyl gellan gum within this mass percentage range not only meets the stability and performance requirements of the solid culture medium but also significantly reduces preparation costs. This is of great significance for improving the economic efficiency of microbial culture and promoting industrial production.

[0052] In some embodiments, the preparation method further includes: The coagulant is added to the first nutrient solution and ultrasonically stirred at a frequency of 10 kHz to 30 kHz for 20 min to 30 min. The resulting fluid is filtered, sterilized at high temperature, cooled for 5 min to 10 min, and then mixed with the second nutrient solution to obtain a culture medium colloid with a pH of 1.8 to 3.5. After gelation, a solid culture medium is obtained.

[0053] In practice, ultrasonic stirring is used to homogenize the fluid. Thorough stirring can prevent the culture medium colloid from becoming uneven after high-temperature heat sterilization, and avoid the phenomenon that some parts of the gel are too thin and dry and clump under high-temperature culture conditions.

[0054] In some embodiments, the high-temperature sterilization temperature is 110 ℃~130 ℃, and the sterilization time is 20 min~30 min.

[0055] Thirdly, this application provides an application of a solid culture medium for the isolation and purification of thermophilic and acidophilic bacteria prepared according to the first or second aspect above, wherein the solid culture medium is used for the isolation and purification of thermophilic archaea, thermophilic / acidophilic bacteria or moderately thermophilic / acidophilic bacteria.

[0056] The solid culture medium prepared in this application can be used in a water tray culture method (where a water-filled tray is placed in an incubator, and the humidity in the air is increased through water evaporation to meet the growth requirements of microorganisms). This method can successfully isolate thermophilic acidophilic bacteria that grow at 65℃~85℃ and a pH above 1.5. Thermophilic archaea, thermophilic / acidophilic bacteria, or moderately thermophilic / acidophilic bacteria, like thermophilic acidophilic bacteria, exhibit thermophilic characteristics and have relatively high optimal growth temperatures. All thermophilic acidophilic bacteria, thermophilic / acidophilic bacteria, and moderately thermophilic / acidophilic bacteria can survive and reproduce under highly acidic conditions. Although thermophilic archaea are primarily known for their thermophilic characteristics, they can also grow in acidic environments, especially those archaea living in hot springs or boiling springs rich in sulfur and iron. Therefore, the solid culture medium provided in this application can provide a favorable growth and reproduction environment for thermophilic archaea, thermophilic / acidophilic bacteria, or moderately thermophilic / acidophilic bacteria, facilitating their successful isolation and purification.

[0057] To enable those skilled in the art to more clearly understand the present invention, the following embodiments are provided to illustrate in detail the solid culture medium for the isolation and purification of thermophilic and acidophilic bacteria, its preparation method, and its application.

[0058] Example 1 Step 1: Dissolve 0.07 g CaCl2, 0.1 g KCl, 0.5 g KH2PO4, 3.09 g (NH4)2SO4, 0.08 g KHSO4, 0.5 g MgSO4∙7H2O, 0.02 g yeast extract, 0.078 mL HNO3, 8 g KSCN and 0.1 mL H2SO4 in 700 mL deionized water, stir well, and then adjust the pH of the resulting mixture to 1.8 to obtain the first nutrient solution. Step 2: Dissolve 44.7 g FeSO4∙7H2O in 300 mL of deionized water, stir well, adjust the pH of the resulting solution to 1.5, and then filter it through a 0.22 μm microporous membrane to remove bacteria to obtain the second nutrient solution. Step 3: Add 15 g of high-acyl gellan gum (acyl groups number 4, final concentration 15 g / L) to the first nutrient solution and stir at 95 ℃ until a homogeneous fluid is obtained. Filter the obtained homogeneous fluid with gauze and place it in an autoclave. Sterilize at 121 ℃ for 25 min and then cool for 10 min. Mix the cooled fluid with the second nutrient solution and stir evenly to obtain a culture medium colloid with a pH of 1.8. Step 4: Quickly spread the culture medium colloid onto a plate, and after gelation, obtain a solid culture medium.

[0059] Based on the solid culture medium prepared in Example 1, thermophilic acidophilic bacteria were cultured using the water tray culture method, specifically including: Step 1: Place a large, open beaker filled with water in the incubator to keep the air in the incubator humid through water evaporation; Step 2: Pour the sterilized solid culture medium into a sterile petri dish and allow it to solidify; Step 3: Inoculate the thermophilic acidophilic bacteria sample to be cultured onto a solid culture medium. This can be done by spreading, streak plating, or spot inoculation. Step 4: Place the inoculated culture dish in a constant temperature incubator and set the temperature to 65 ℃ for one week.

[0060] See Figure 2 , Figure 2 The image shows the growth of the solid culture medium and the bacterial strain on the second day of incubation at 65 °C and pH 1.8. The image shows that the solid culture medium was morphologically stable, and the thermophilic acidophilic bacteria were abundant on the surface, demonstrating that the solid culture medium prepared in Example 1 could successfully isolate and purify the thermophilic acidophilic bacteria.

[0061] Example 2 The difference between Example 2 and Example 1 lies in step 3, specifically: 6.5 g of high-acyl gellan gum (acyl groups numbered 4, final concentration 6.5 g / L) and 12.5 g of agar were added to the first nutrient solution and stirred at 85 °C until a homogeneous fluid was obtained. The resulting fluid was then filtered through gauze and placed in an autoclave for high-temperature sterilization at 121 °C for 25 min. After cooling for 10 min, the cooled fluid was mixed with the second nutrient solution and stirred evenly to obtain a culture medium colloid with a pH of 1.8.

[0062] Example 3 The difference between Example 3 and Example 1 lies in step 3, specifically: Step 3: Add 15 g of high-acyl gellan gum (acyl groups number 4, final concentration 15 g / L) to the first nutrient solution and sonicate at 20 kHz for 20 min until a homogeneous fluid is obtained. Filter the obtained fluid with gauze and place it in an autoclave for high-temperature sterilization at 121 ℃ for 25 min. Then cool for 10 min. Mix the cooled fluid with the second nutrient solution and stir evenly to obtain a culture medium colloid with a pH of 1.8.

[0063] Example 4 Step 1: Dissolve 0.1 g CaCl2, 0.2 g KCl, 1 g KH2PO4, 3.5 g (NH4)2SO4, 0.1 g KHSO4, 1 g MgSO4∙7H2O, 0.05 g yeast extract, 0.075 mL HNO3, 11 g KSCN and 0.3 mL H2SO4 in 800 mL deionized water, stir well, and then adjust the pH of the resulting mixture to 3 to obtain the first nutrient solution. Step 2: Dissolve 50 g FeSO4∙7H2O in 200 mL of deionized water, stir well, adjust the pH of the resulting solution to 2.8, and then filter it through a 0.22 μm microporous membrane to remove bacteria to obtain the second nutrient solution. Step 3: Add 20 g of high-acyl gellan gum (3 acyl groups, final concentration 20 g / L) to the first nutrient solution and stir at 100 °C until a homogeneous fluid is obtained. Filter the obtained fluid with gauze and place it in an autoclave for high-temperature sterilization at 130 °C for 30 min. Then cool it for 10 min. Mix the cooled fluid with the second nutrient solution and stir evenly to obtain a culture medium colloid with a pH of 3.1. Step 4: Quickly spread the culture medium colloid onto a plate, and after gelation, obtain a solid culture medium.

[0064] Example 5 Step 1: Dissolve 0.05 g CaCl2, 0.05 g KCl, 0.2 g KH2PO4, 2.5 g (NH4)2SO4, 0.05 g KHSO4, 0.2 g MgSO4∙7H2O, 0.01 g yeast extract, 0.078 mL HNO3, 5 g KSCN and 0.1 mL H2SO4 in 600 mL deionized water, stir well, and then adjust the pH of the resulting mixture to 2.1 to obtain the first nutrient solution. Step 2: Dissolve 40 g FeSO4∙7H2O in 400 mL of deionized water, stir well, adjust the pH of the resulting solution to 2.2, and then filter it through a 0.22 μm microporous membrane to remove bacteria to obtain the second nutrient solution. Step 3: Add 10 g of high-acyl gellan gum (acyl groups number 4, final concentration 10 g / L) to the first nutrient solution and stir at 100 ℃ until a homogeneous fluid is obtained. Filter the obtained fluid with gauze and place it in an autoclave for high-temperature sterilization at 115 ℃ for 25 min. Then cool it for 10 min. Mix the cooled fluid with the second nutrient solution and stir evenly to obtain a culture medium colloid with a pH of 2. Step 4: Quickly spread the culture medium colloid onto a plate, and after gelation, obtain a solid culture medium.

[0065] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that the mass of the high-acyl gellan gum in Comparative Example 1 is 2 g, and the final concentration is 2 g / L.

[0066] Comparative Example 2 The only difference between Comparative Example 2 and Example 1 is that Comparative Example 1 uses low-acyl gellan gum with the same concentration and mass as a coagulant.

[0067] Comparative Example 3 The only difference between Comparative Example 3 and Example 1 is that Comparative Example 1 uses agar with the same concentration and mass as a coagulant.

[0068] Experimental Example Experiment 1 Based on Example 1 and Comparative Example 1, the morphology of solid culture medium and its effectiveness in isolating and purifying thermophilic acidophilic bacteria were tested under the conditions of 65 ℃~85 ℃ and pH value 1.5~3.5.

[0069] See Figure 3 According to the test, due to the low concentration of high acyl gellan gum in Comparative Example 1, the solid culture medium will dissolve under the conditions of 65 ℃~85 ℃ and pH value 1.5~3.5, and the whole will be like rice paste. This results in the solid culture medium not being hard enough and having a soft texture, which does not meet the hardness conditions for culturing thermophilic acidophilic bacteria, and is not conducive to the stable culture of thermophilic acidophilic bacteria.

[0070] The solid culture medium prepared in Example 1 can be found here. Figure 2 The solid culture medium showed stable morphology, and thermophilic and acidophilic bacteria accumulated in large quantities on the surface of the medium. This indicates that the concentration of high-acyl gellan gum affects its gelation properties. Controlling the concentration of high-acyl gellan gum between 5 g / L and 30 g / L can maintain its good gelation properties, thereby maintaining the stability of the solid culture medium.

[0071] Experiment 2 Based on Examples 1-5 and Comparative Example 2, the morphology of solid culture media and the effect of isolating and purifying thermophilic acidophilic bacteria were tested under the conditions of 65 ℃~85 ℃ and pH value 1.5~3.5.

[0072] According to testing, the solid culture media prepared in Examples 1-5 were stable at 65℃-85℃ and pH 1.5-3, and thermophilic acidophilic bacteria were successfully isolated. The morphology of the solid culture media is shown in the reference. Figure 2 ; The solid culture medium prepared in Comparative Example 2 could only be stably formed under conditions of pH greater than 3.4 and temperature below 60 °C. Under these testing conditions, its gelation properties rapidly decreased, becoming a more fluid substance that could not be stably formed. Furthermore, due to the excessive concentration and content of low-acyl gelling gel, some of the colloids gradually hardened, making the solid culture medium unsuitable for culturing thermophilic and acidophilic bacteria.

[0073] Experiment 3 I. The morphological state of solid culture media prepared with agar of different concentrations at temperatures ranging from 65 ℃ to 85 ℃ and at different pH values ​​was examined, as detailed below: (1) The culture medium solution includes: 6% agar + 10 ml concentrated sulfuric acid, pH 1~1.5. Tests showed that the solid culture medium formed after gelation dissolved after 2 hours of use and did not re-gel. (2) The culture medium solution includes: 2% agar + 6 ml concentrated sulfuric acid, pH 1.5~2. Testing showed that after gelation, half of the solid culture medium formed by this solution dissolved within 2 hours of use and could not be re-gelled.

[0074] This demonstrates that the gelling properties of agar decrease significantly under high temperature and high acidity conditions with pH < 2, making it unsuitable for preparing solid culture media for thermophilic and acidophilic bacteria.

[0075] II. Based on Example 1 and Comparative Example 3, the morphology of the solid culture medium and its effect on the isolation and purification of thermophilic acidophilic bacteria were tested under the conditions of 65 ℃~85 ℃ and pH value 1.5~3.5.

[0076] Testing revealed that the solid culture medium prepared in Comparative Example 3 could only be stably formed under conditions of pH greater than 3.4 and temperature below 60 °C. Under these conditions, its gel properties rapidly decreased, becoming a more fluid substance that could not be stably formed. Therefore, this demonstrates that even increasing the agar concentration cannot solve the problem of agar dissolution under high temperature and high acidity conditions.

[0077] Furthermore, according to Example 2 and Comparative Example 3, it is shown that the combined use of high-acyl gellan gel and agar can effectively solve the problem of agar dissolution under high temperature and high acidity, and the resulting solid culture medium can provide a stable environment that meets the growth requirements of thermophilic acidophilic bacteria.

[0078] In summary, this application utilizes high-acyl gellan gum as a gelling material added to a solid culture medium. By adjusting the concentration of the high-acyl gellan gum, the gelation effect under high acid and high temperature conditions is significantly enhanced. The resulting solid culture medium provides an environment conducive to the rapid growth and enrichment of thermophilic acidophilic bacteria while inhibiting the growth of other bacterial species. Thermophilic acidophilic bacteria, thermophilic archaea, thermophilic / acidophilic bacteria, or moderately thermophilic / acidophilic bacteria that can grow at 65℃~85℃ and pH 1.5~3 have been successfully isolated.

[0079] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0080] Although preferred embodiments of the present application have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments as well as all changes and modifications falling within the scope of the embodiments of the present application.

[0081] Finally, it should be noted that in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or terminal device that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or terminal device. Without further limitations, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or terminal device that includes said element.

[0082] The above provides a detailed description of the solid culture medium for isolating and purifying thermophilic and acidophilic bacteria, its preparation method, and its applications. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the above embodiments are only for the purpose of helping to understand the method and core ideas of this application. At the same time, for those skilled in the art, there will be changes in the specific implementation methods and application scope based on the ideas of this application. Therefore, the content of this specification should not be construed as a limitation of this application.

Claims

1. A method for preparing a solid culture medium for the isolation and purification of thermophilic and acidophilic bacteria, characterized in that, The solid culture medium is prepared by mixing a nutrient composition, a coagulant, and deionized water; the nutrient composition consists of CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4•7H2O, yeast extract, KSCN, FeSO4•7H2O, HNO3, and H2SO4; the preparation method includes: Step 1: Dissolve 0.07 g CaCl2, 0.1 g KCl, 0.5 g KH2PO4, 3.09 g (NH4)2SO4, 0.08 g KHSO4, 0.5 g MgSO4•7H2O, 0.02 g yeast extract, 0.078 mL HNO3, 8 g KSCN and 0.1 mL H2SO4 in 700 mL deionized water, stir well, and then adjust the pH of the resulting mixture to 1.8 to obtain the first nutrient solution. Step 2: Dissolve 44.7 g FeSO4•7H2O in 300 mL of deionized water, stir well, adjust the pH of the resulting solution to 1.5, filter it through a microporous membrane to remove bacteria, and then obtain the second nutrient solution. Step 3: Add 6.5 g of high-acyl gellan gum and 12.5 g of agar to the first nutrient solution and stir at 85 °C until a homogeneous fluid is obtained. Filter the resulting fluid through gauze and place it in an autoclave for high-temperature sterilization at 121 °C for 25 min. Then cool for 10 min. Mix the cooled fluid with the second nutrient solution and stir evenly to obtain a culture medium colloid with a pH of 1.

8. Each repeating unit of the high-acyl gellan gum contains 4 acyl groups, and the final concentration is 6.5 g / L. Step 4: Quickly spread the culture medium colloid onto a plate, and after gelation, obtain a solid culture medium.

2. The application of a solid culture medium for isolating and purifying thermophilic and acidophilic bacteria obtained by the preparation method of claim 1, characterized in that, The solid culture medium is used to isolate and purify thermophilic archaea, thermophilic / acidophilic bacteria, or moderately thermophilic / acidophilic bacteria.

Images