Solid culture medium for separating and purifying thermoacidophilic bacteria as well as preparation method and application of solid culture medium

Abstract

The invention provides a thermoacidophilic bacterium separation and purification solid culture medium and a preparation method and application thereof, the solid culture medium is prepared by mixing a nutritional composition, a coagulator and deionized water, and the mass ratio of the coagulator in the solid culture medium is 9%-50%; wherein the coagulator is high-acyl gellan gum, and the number of acyl groups in the high-acyl gellan gum is 3-4; the concentration of the high-acyl gellan gum is 5g / L-30g / L. The solid culture medium is prepared from high-acyl gellan gum, and by adjusting the mass ratio of the high-acyl gellan gum, especially by limiting the concentration of the high-acyl gellan gum, the solid culture medium can still be stably formed for a long time in a high-temperature and high-acid environment, and better gel performance is kept; a stable high-temperature and high-acid growth environment can be provided for the thermoacidophilic bacteria, so that the thermoacidophilic bacteria are smoothly separated and purified. And the high-acyl gellan gum is wide in source and low in price, so that the culture cost is more favorably reduced.

Description

Technical Field

[0001] The present application relates to the technical field of microbial culture, and in particular to a solid culture medium for separating and purifying thermophilic and acidophilic bacteria, a preparation method and application thereof. Background Art

[0002] Thermophilic acidophiles are currently the focus of research in the biological field. Since most of them have iron and / or sulfur oxidation activity, they are widely used in the field of biometallurgy. In addition, some thermophilic acidophiles are used in technical fields such as AMD environmental governance and MCs degradation. Thermophilic acidophiles generally exist in sulfur-containing acidic hot springs and can survive above 60°C. Compared with mesophilic bacteria and medium thermophilic bacteria in leaching, their excellent iron and sulfur oxidation ability can greatly shorten the leaching cycle and improve the leaching efficiency. They are regarded as the most valuable microorganisms for the comprehensive utilization of mineral resources and environmental protection. However, due to the harsh living conditions of thermophilic acidophiles, it is difficult to keep the culture conditions consistent with their natural growth environment. Therefore, liquid mixed bacterial samples are generally cultured, and it is difficult to obtain pure cultures, resulting in very limited germplasm resources.

[0003] At present, it is still difficult to obtain pure cultures of thermoacidophiles, which is one of the "bottlenecks" that restricts their related research and application. With the application of molecular biology technology and high-throughput sequencing technology, more and more thermoacidophiles have been detected in leaching environments, and with the deepening of research and technological advances, many microorganisms have changed from uncultivable to cultivable states, and corresponding progress has been made in the study of the properties of newly isolated and purified thermoacidophiles.

[0004] For example, solid culture medium is used to separate and purify microorganisms, that is, to separate pure bacteria by using a single bacterium to form a single colony on a plate. Solid culture medium is mainly made of nutrients and gel materials. The gel materials include agar, low-acyl gellan gum, and Professor Xia Jinlan of Central South University once proposed to use jelly powder as a coagulant to prepare solid culture medium to separate and purify thermoacidophilic archaea. However, these raw materials have poor stability under long-term high temperature and high acid conditions, especially under lower pH conditions, their gel properties drop sharply and cannot be stably formed, resulting in the inability to purify and separate thermoacidophilic bacteria smoothly. Summary of the invention

[0005] To solve the above problems, the present application provides a solid culture medium for separating and purifying thermoacidophiles, a preparation method and its application. High acyl gellan gum is used as a gel material to prepare a solid culture medium for separating and purifying thermoacidophiles, so that the solid culture medium can be stably formed for a long time in a high temperature and high acid environment, and the gel effect is better, so that the purification and separation of thermoacidophiles can be carried out smoothly, thereby obtaining pure thermoacidophilic bacteria, which provides strong technical support for the protection of germplasm resources and in-depth research of thermoacidophiles.

[0006] In a first aspect, the present application provides a solid culture medium for separating and purifying thermophilic acidophilic bacteria, wherein the solid culture medium is prepared by mixing a nutrient composition, a coagulant, and deionized water, wherein the mass proportion of the coagulant in the solid culture medium is 9% to 50%; Wherein, the coagulant comprises high acyl gellan gum, and 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; Wherein, the concentration of the agar is 5 g / L~20 g / L; The mass ratio of the agar to the high acyl gellan gum is 0.5-0.6.

[0009] Further, the nutrient composition consists of CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, KSCN, FeSO4∙7H2O, HNO3 and H2SO4; The mass volume ratio of the 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] In a second aspect, the present application provides a method for preparing a solid culture medium for separating and purifying thermoacidophilic bacteria, the preparation method comprising: CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, HNO3, KSCN and H2SO4 in the nutrient composition are dissolved in deionized water, and the pH of the obtained mixed system is adjusted to 1.8-4 to prepare a first nutrient solution; Dissolve FeSO4∙7H2O in deionized water, adjust the pH of the resulting solution to 1.5-3, and filter and sterilize with a microporous filter membrane to obtain a second nutrient solution; Adding a coagulant to the first nutrient solution, stirring at 85°C to 200°C, filtering the obtained fluid, sterilizing it at high temperature, cooling it for 5 min to 10 min, and then mixing it with the second nutrient solution to obtain a culture medium colloid with a pH of 1.8 to 3.5, and obtaining a solid culture medium after gelation; Wherein, the coagulant includes high acyl gellan gum, and the concentration of the high acyl gellan gum is 5 g / L~30 g / L.

[0011] Further, 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 0.5-0.6.

[0012] Furthermore, the mass proportion of the coagulant in the solid culture medium is 16% to 27%.

[0013] Furthermore, the preparation method further comprises: The coagulant is added to the first nutrient solution, and ultrasonic stirring is performed at a frequency of 10 kHz to 30 kHz for 5 min to 30 min. The obtained fluid is filtered and sterilized at high temperature, then 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, and a solid culture medium is obtained after gelation.

[0014] Furthermore, the temperature of the high temperature sterilization is 110°C to 130°C, and the sterilization time is 20 min to 30 min.

[0015] In a third aspect, the present application provides an application of a solid culture medium for separating and purifying thermophilic acidophilic bacteria, wherein the solid culture medium is used to separate and purify thermophilic archaea, thermophilic / acidophilic bacteria, or moderate thermophilic / acidophilic bacteria.

[0016] In summary, the present application includes at least one of the following beneficial technical effects: 1. The present application provides a solid culture medium for separating and purifying thermophilic acidophilic bacteria. The solid culture medium is made of high acyl gellan gum as a coagulant, and by adjusting the mass proportion of the high acyl gellan gum, especially by limiting the concentration of the high acyl gellan gum, the solid culture medium maintains good gel properties in a high temperature and high acid environment, can be stably formed for a long time, and provides a stable growth environment for thermophilic acidophilic bacteria, thereby successfully separating and purifying thermophilic acidophilic bacteria; high acyl gellan gum is widely available and inexpensive (180 yuan / kg), which can effectively reduce the cost of culture; 2. The present application provides an application of a solid culture medium for separating and purifying thermophilic acidophilic bacteria. The solid culture medium prepared 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 continuously high temperature environment, and can withstand high temperature (65°C≤T≤100°C) and high acid (pH≥1.5) environments. The gel is stable and is a good medium for the growth of thermoacidophilic archaea, thermophilic / acidophilic bacteria or moderate thermophilic / acidophilic bacteria. It can stably and efficiently separate and purify thermoacidophilic archaea, thermophilic / acidophilic bacteria or moderate thermophilic / acidophilic bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the description of the embodiments of the present application will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative labor.

[0018] Figure 1 A flow chart of the method for preparing a solid culture medium for separating and purifying thermophilic and acidophilic bacteria proposed in an embodiment of the present application is shown; Figure 2 A schematic diagram showing the solid culture medium and bacterial strain growth at 65°C proposed in the embodiments of the present application; Figure 3 A schematic diagram of culturing thermoacidophilic bacteria on a solid culture medium proposed in Comparative Example 1 of the present application is shown. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, not all of the embodiments. Based on the embodiments in the present application, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of this application.

[0020] In the drawings, the size of the constituent elements, the thickness of the layer or the area may be exaggerated for the sake of clarity. Therefore, any implementation of the present disclosure is not necessarily limited to the size shown in the drawings, and the shapes and sizes of the components in the drawings do not reflect the true proportions. In addition, the drawings schematically show ideal examples, and any implementation of the present disclosure is not limited to the shapes or values ​​shown in the drawings.

[0021] Among the related technologies, thermoacidophiles are the focus of current research in the biological field. Since most of them have iron or (and) sulfur oxidation activity, they are widely used in the field of biometallurgy. Usually, the metabolic activity of thermoacidophiles is enhanced and the reproduction rate is accelerated in a high temperature (>65 ℃) and high acid (pH 1.5~3.5) environment, which is conducive to rapid enrichment and growth. Therefore, for the separation and purification of thermoacidophiles, the high acid and high temperature environment is unfavorable to most other microorganisms, and may even be fatal. Such an environment can inhibit the growth of other non-thermoacidophiles, thereby reducing competition and interference with thermoacidophiles, allowing thermoacidophiles to quickly enrich and grow, and become the dominant strain in the separation and purification process.

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

[0023] These technologies have promoted the relevant research on microorganisms, but the traditional sorting methods have frequent operations and poor separation and purification effects, resulting in less thermophilic and acidophilic bacteria. The emerging sorting technologies are difficult to promote in laboratories due to their demanding requirements, complex techniques or low output, and immature technology.

[0024] Therefore, most researchers still hope to use solid culture media to separate and purify microorganisms, that is, to use single bacteria to form single colonies on the plate to separate pure bacteria. Among them, agar has the advantages of high transparency, good water retention, non-toxicity, and not being liquefied by microorganisms, and is used as a coagulant for conventional solid culture media. However, at low pH values ​​(pH < 4), agar will be hydrolyzed into oligosaccharides and reducing sugars during sterilization, and lose its coagulability. Even if the acid solution and agar are sterilized separately and then mixed to prepare a solid culture medium, under high temperature (> 65 °C) culture conditions, the agar medium will also hydrolyze and lose its coagulability, and therefore cannot be used to stably purify and separate thermoacidophiles; Professor Xia Jinlan of Central South University once proposed using jelly powder as a coagulant to prepare solid culture medium to separate and purify thermoacidophilic archaea. However, the main raw materials of jelly powder are generally agar, gelatin powder (not heat-resistant and acid-resistant) or gelatin. These raw materials cannot form stably under high temperature and high acid conditions for a long time. As a new coagulant, low-acyl gellan gum 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 the amount of agar), high temperature resistance, and pH range of 3.4-7.5. However, under lower pH conditions (pH 1.5~3.5), a large amount of H + It will react with the functional groups in the low-acyl gellan gum molecules, destroy the original hydrogen bond network, and cause the stability of the gel network structure to decrease. It cannot be stably formed for a long time, and therefore cannot be used to separate thermophilic and acidophilic bacteria.

[0025] Based on the problems existing in the prior art, this application proposes a new solid culture medium for separating and purifying thermophilic acidophilic bacteria, which uses high acyl gellan gum as a coagulant, and overcomes the problems of low gel strength and poor hardness by adjusting its concentration and mass in the solid culture medium, so that the prepared solid culture medium can remain stable for a long time under high temperature (65 ℃ ≤ T ≤ 100 ℃) and high acid (3.5 ≥ pH ≥ 1.5) conditions, so that the separation and purification of thermophilic acidophilic bacteria can proceed smoothly, and pure thermophilic acidophilic bacteria can be obtained. In addition, the price of high acyl gellan gum is low (180 yuan / kg), which is much lower than that of low acyl gellan gum (about 1150 yuan / kg), thereby reducing the preparation cost and improving the economic benefits of strain cultivation.

[0026] In a first aspect, the present application provides a solid culture medium for separating and purifying thermophilic acidophilic bacteria, wherein the solid culture medium is prepared by mixing a nutrient composition, a coagulant, and deionized water, wherein the mass proportion of the coagulant in the solid culture medium is 9% to 50%; Wherein, the coagulant comprises high acyl gellan gum, and 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 the specific implementation, high acyl gellan gum was used as the gel material, and its mass proportion and concentration were precisely controlled to successfully overcome 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) environments. This new solid culture medium can maintain stable molding and sufficient hardness for a long time under extreme conditions, meeting the strict requirements for the separation and purification of thermoacidophilic bacteria, greatly improving the efficiency and success rate of separation and purification, and providing strong technical support for the protection and in-depth research of thermoacidophilic 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 high acyl gellan gum has one acyl group connected to the glucose molecule. The number of acyl groups of the high acyl gellan gum used in the embodiments of the present application is 3 to 4, and these acyl groups are divided into two categories, one is the acetyl group connected to the 6th carbon atom of the glucose molecule with a β-1,3 bond, and the other is the glyceryl group connected to the 2nd carbon atom of the glucose molecule with a β-1,3 bond, wherein the average ratio of the glyceryl group is 1 to 2 times that of the acetyl group.

[0029] In specific implementation, the main difference between high acyl gellan gum and low acyl gellan gum is that the number and nature of the acyl groups are different, and the number and arrangement of the acyl groups directly affect the strength and stability of the gel. The number of acyl groups in high acyl gellan gum is relatively large, which makes its gel structure more compact, the gel strength is higher, and the stability is better. + In an environment with high gel performance, it can still maintain high gel performance. In contrast, low acyl gellan gum has fewer acyl groups and a relatively loose gel structure, especially under high temperature and high acid conditions, the gel strength and stability are poor. Therefore, high acyl gellan gum can achieve better gel effect than low acyl gellan gum and is more suitable for growing and separating thermophilic acidophilic bacteria.

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

[0031] In specific implementation, the concentration and mass ratio of high acyl gellan gum will affect the looseness of its gel structure. The embodiment of the present application ensures that under extreme conditions of high temperature (65 ℃ ≤ T ≤ 100 ℃) and high acid (3.5 ≥ pH ≥ 1.5), it can maintain a tight structure and sufficient strength, and is not easy to hydrolyze or deform, thereby overcoming the defect that traditional thickeners are prone to failure under these conditions. Not only that, by precisely controlling its dosage, while ensuring the gel performance, it also achieves effective cost control. Compared with traditional coagulants, it reduces the overall cost and improves the efficiency of resource utilization.

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

[0033] In specific implementation, the present application uses agar and high acyl gellan gum in combination, and the two can 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 improves the elasticity and toughness of the solid culture medium, making it more durable and less prone to breakage during operation. The combined use can also optimize the water retention and air permeability of the solid culture medium, provide a more suitable environment, and reduce the mutual influence and interference between bacterial 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 nutritional composition consists of CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, KSCN, FeSO4∙7H2O, HNO3, and H2SO4; The mass volume ratio of the 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 specific implementation, high acyl gellan gum has a strong affinity for divalent ions (such as Ca 2+ Mg 2+ ) is very sensitive. Divalent ions will interact with functional groups such as carboxyl groups in high acyl gellan gum molecules to form more physical crosslinking points, making the gel network tighter and more stable, thus significantly enhancing the gel strength of high acyl gellan gum. By controlling the mass of divalent ion-containing components, the gel strength of high acyl gellan gum can be stabilized during the preparation of solid culture medium, reducing the loss of water molecules, improving the water retention of high acyl gellan gum gel, and reasonably controlling the amount of each component to avoid waste of resources. The presence of divalent ions can also slow down the stress relaxation rate of 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 necessary ions and trace elements for microorganisms; Yeast extract is mainly a substance obtained by crushing yeast cells after yeast growth and reproduction. It is refined after the proteins and nucleic acids in the yeast cells are degraded. It contains substances such as polypeptides, amino acids, vitamins and trace elements. 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 to help maintain the stability of the pH value of solid culture media. HNO3, as a strong acid, can further reduce the pH value of solid culture media, thereby creating a high acid environment, which is more conducive to the rapid growth and enrichment of thermophilic acidophilic bacteria.

[0038] In summary, the nutrient composition in the solid culture medium provides comprehensive nutritional support for microorganisms, which helps them grow and reproduce rapidly. By adjusting the physical and chemical properties (pH) of the solid culture medium and adding high acyl gellan gum, it is possible to create an environment most suitable for the growth of thermophilic acidophilic bacteria, thereby successfully separating and purifying thermophilic acidophilic bacteria.

[0039] Second, see Figure 1 The present application provides a method for preparing a solid culture medium for separating and purifying thermophilic and acidophilic bacteria as described in the first aspect above, the preparation method comprising: Step S1: taking CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, HNO3, KSCN and H2SO4 in the nutrient composition and dissolving them in deionized water, and adjusting the pH of the obtained mixed system to 1.8-4 to prepare a first nutrient solution; Step S2: dissolving FeSO4∙7H2O in deionized water, adjusting the pH of the resulting solution to 1.5-3, and filtering and sterilizing the solution with a microporous filter membrane to obtain a second nutrient solution; Step S3: adding a coagulant to the first nutrient solution, stirring at 85°C to 200°C, filtering the obtained fluid, sterilizing it at high temperature, cooling it for 5 min to 10 min, and then mixing it with the second nutrient solution to obtain a culture medium colloid with a pH of 1.8 to 3.5, and obtaining a solid culture medium after gelation; Wherein, the coagulant includes high acyl gellan gum, and the concentration of the high acyl gellan gum is 5 g / L~30 g / L.

[0040] In the specific implementation, by accurately proportioning the inorganic salts, H2SO4, yeast extract and other nutrients in the nutrient composition and adjusting the pH to the acidic range of 1.8-4, the solid culture medium has the nutritional basis required by thermoacidophiles and excludes the growth conditions of other non-thermoacidophiles, which is conducive to the rapid growth of thermoacidophiles; then FeSO4∙7H2O is dissolved in deionized water, and the pH is adjusted to 1.5-3.5, and the second nutrient solution is obtained after filtration and sterilization. This step provides the iron element required for the growth of thermoacidophiles, and ensures aseptic conditions by filtration and sterilization, avoiding contamination by other microorganisms; high acyl gellan gum can maintain the stability of the gel structure under lower pH conditions. High acyl gellan gum is added to the first nutrient solution, stirred at high temperature, so that the high acyl gellan gum is fully dissolved and forms a uniform gel system, and then filtered and sterilized at high temperature, cooled for 5 min-10 min 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, through the gelling effect of high-acyl gellan gum, prepares a solid culture medium that can remain stable for a long time in a high-temperature and high-acid environment, thereby achieving stable separation and purification of thermophilic and acidophilic bacteria.

[0041] The preparation method provided by the present application is simple to operate, avoids the cumbersome operation process in the traditional method, reduces the operation difficulty and cost. The prepared solid culture medium can be widely used in the fields of separation and purification of thermophilic acidophilic bacteria, biometallurgy, environmental governance, etc.

[0042] In specific implementation, FeSO4∙7H2O is easily oxidized to Fe2(SO4)3 during high temperature sterilization, and thus cannot provide energy substances for bacteria. Therefore, FeSO4∙7H2O is mixed with deionized water to form a second nutrient solution, and bacteria are removed through a microporous filtration membrane to maintain the stability of divalent iron ions and provide sufficient nutrients for the growth of thermophilic and acidophilic bacteria.

[0043] In specific implementation, the obtained culture medium colloid is a relatively soft and sticky dough, which is then divided into flat plates and quickly flattened to obtain a solid culture medium after gelation.

[0044] In some embodiments, the obtained fluid can be filtered using medical gauze or a filter screen with 180-220 meshes, as long as a homogenous effect can be achieved after filtration.

[0045] In some embodiments, since high acyl gellan gum is insoluble in water at room temperature, it is necessary to add 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 and avoid the presence of dry powder or lumps in the system.

[0046] In some embodiments, the pore size of the microporous filtration 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 solidifying agent is a mixture of the high acyl gellan gum and agar; Wherein, the concentration of the agar is 3 g / L~10 g / L; The mass ratio of the agar to the high acyl gellan gum is 0.5-0.6.

[0049] In specific implementation, agar and high acyl gellan gum are used in combination, and the two can produce a synergistic effect, which can not only overcome the problem of poor gelation of agar under high temperature and high acid, but also overcome the problem of low gel strength and insufficient hardness under low concentration of high acyl gellan gum, and is suitable for preparing a solid culture medium for separating and purifying thermophilic acidophilic bacteria. The use of agar and high acyl gellan gum in combination can also reduce the amount of high acyl gellan gum used, further reducing the preparation cost.

[0050] In some embodiments, the mass percentage of the coagulant in the solid culture medium is 16% to 27%.

[0051] By limiting the mass proportion of the coagulant in the solid culture medium, the problem of low gel strength and poor hardness at low concentrations of high acyl gellan gum can be overcome, so that the solid culture medium can still maintain a stable shape under high temperature and high acid conditions and is not easy to deform or dissolve. Using high acyl gellan gum within this mass proportion range can not only meet the stability and performance requirements of the solid culture medium, but also significantly reduce the preparation cost, which is of great significance for improving the economic benefits of strain culture and promoting industrial production.

[0052] In some embodiments, the preparation method further comprises: The coagulant is added to the first nutrient solution, and ultrasonic stirring is performed for 20 min to 30 min at a frequency of 10 kHz to 30 kHz. The obtained fluid is filtered and sterilized at high temperature, then 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, and a solid culture medium is obtained after gelation.

[0053] In specific implementation, ultrasonic stirring is used to homogenize the fluid, and sufficient stirring can prevent the uneven colloid of the culture medium after high-temperature heat sterilization treatment, and avoid the phenomenon that a part of the colloid is too thin and dries and hardens under high-temperature culture conditions.

[0054] In some embodiments, the high temperature sterilization temperature is 110°C to 130°C, and the sterilization time is 20 min to 30 min.

[0055] In a third aspect, the present application provides an application of a solid culture medium for separating and purifying thermophilic acidophilic bacteria as described in the first aspect or prepared in the second aspect, wherein the solid culture medium is used for separating and purifying thermophilic archaea, thermophilic / acidophilic bacteria or moderate thermophilic / acidophilic bacteria.

[0056] When the solid culture medium prepared in the present application is used, the water tray culture method (which means placing a plate of water in the incubator to increase the humidity in the air by evaporation of water to meet the growth needs of microorganisms) can be used to successfully separate thermophilic acidophilic bacteria that grow under conditions of 65 ℃~85 ℃ and pH value above 1.5. Thermophilic archaea, thermophilic / acidophilic bacteria or moderate thermophilic / acidophilic bacteria have the same thermophilic characteristics as thermophilic acidophilic bacteria, and their optimal growth temperature is also higher; thermophilic acidophilic bacteria, thermophilic / acidophilic bacteria and moderate thermophilic / acidophilic bacteria can survive and reproduce under high acid conditions. Although thermophilic archaea are mainly known for their thermophilic characteristics, they can also grow in acidic environments, especially those archaea that live in hot springs or boiling springs rich in sulfur, iron and other elements. Therefore, the solid culture medium provided in the present application can provide a good growth and reproduction environment for thermophilic archaea, thermophilic / acidophilic bacteria or moderate thermophilic / acidophilic bacteria, so as to smoothly separate and purify them.

[0057] In order to enable those skilled in the art to more clearly understand the present invention, the solid culture medium for separating and purifying thermophilic acidophilic bacteria, the preparation method and the application thereof described in the present invention are now described in detail through the following examples.

[0058] Example 1 Step 1, dissolving 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, stirring evenly, and then adjusting the pH of the obtained mixed system to 1.8 to prepare a first nutrient solution; Step 2, dissolve 44.7 g FeSO4∙7H2O in 300 mL deionized water, stir evenly, adjust the pH of the resulting solution to 1.5, and then filter and sterilize with a 0.22 μm microporous filter membrane to obtain a second nutrient solution; Step 3, adding 15 g of high acyl gellan gum (the number of acyl groups is 4, and the final concentration is 15 g / L) to the first nutrient solution, stirring at 95°C until a homogeneous fluid is obtained, and then filtering the obtained homogeneous fluid with gauze, placing it in a high pressure sterilizer after filtration, and sterilizing it at a temperature of 121°C for 25 min, and then cooling it for 10 min. The cooled fluid is then mixed with the second nutrient solution, and stirred evenly to obtain a culture medium colloid with a pH of 1.8; Step 4: quickly spread the culture medium colloid on a flat plate to obtain a solid culture medium after gelation.

[0059] Based on the solid culture medium prepared in Example 1, thermophilic acidophilic bacteria were cultured using a water tray culture method, specifically comprising: Step 1: Place an open large beaker filled with water in the incubator to keep the air in the incubator moist by evaporation of water; Step 2, pour the sterilized solid culture medium into a sterile culture dish and wait for it to solidify; Step 3, inoculating the thermophilic acidophilic bacteria sample to be cultured onto a solid culture medium, which can be done by a coating method, a streak method or a spot method; Step 4: Place the inoculated culture dish in a constant temperature incubator at 65°C and culture continuously for one week.

[0060] See also Figure 2 , Figure 2 The growth of the solid culture medium and the strains on the second day of culture at 65°C and pH 1.8 is shown. From the figure, it can be seen that the solid culture medium is stable in morphology, and thermoacidophilic bacteria are enriched in large quantities on the surface of the culture medium, proving that the solid culture medium prepared in Example 1 can successfully separate and purify thermoacidophilic bacteria.

[0061] Example 2 The difference between Example 2 and Example 1 is that step 3 is different, specifically: Add 6.5 g of high acyl gellan gum (the number of acyl groups is 4, the final concentration is 6.5 g / L) and 12.5 g of agar to the first nutrient solution, stir at 85°C until a homogeneous fluid is obtained, and then filter the obtained fluid with gauze, place it in an autoclave after filtration, sterilize it at 121°C for 25 min, and then cool it for 10 min. The cooled fluid is then 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 is that step 3 is different, specifically: Step 3, add 15 g of high acyl gellan gum (the number of acyl groups is 4, the final concentration is 15 g / L) to the first nutrient solution, and stir ultrasonically at a frequency of 20 kHz for 20 min until a homogeneous fluid is obtained, and then filter the obtained fluid with gauze, place it in an autoclave after filtration, and sterilize it at 121°C for 25 min, then cool it for 10 min, and mix the cooled fluid with the second nutrient solution, and stir it evenly to obtain a culture medium colloid with a pH of 1.8.

[0063] Example 4 Step 1, dissolving 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, stirring evenly, and then adjusting the pH of the obtained mixed system to 3 to prepare a first nutrient solution; Step 2, dissolve 50 g of FeSO4∙7H2O in 200 mL of deionized water, stir evenly, adjust the pH of the resulting solution to 2.8, and then filter and sterilize with a 0.22 μm microporous filter membrane to obtain a second nutrient solution; Step 3, adding 20 g of high acyl gellan gum (the number of acyl groups is 3, and the final concentration is 20 g / L) to the first nutrient solution, stirring at 100°C until a homogeneous fluid is obtained, and then filtering the obtained fluid with gauze, placing it in an autoclave after filtration, and sterilizing it at 130°C for 30 min, and then cooling it for 10 min. The cooled fluid is then mixed with the second nutrient solution, and stirred evenly to obtain a culture medium colloid with a pH of 3.1; Step 4: quickly spread the culture medium colloid on a flat plate to obtain a solid culture medium after gelation.

[0064] Example 5 Step 1, dissolving 0.05 g CaCl2, 0.05 g KCl, 0.2 g KH2PO4, 2.5 g (NH4)2SO4, 0.05 gKHSO4, 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, stirring evenly, and then adjusting the pH of the obtained mixed system to 2.1 to prepare a first nutrient solution; Step 2, dissolve 40 g of FeSO4∙7H2O in 400 mL of deionized water, stir evenly, adjust the pH of the resulting solution to 2.2, and then filter and sterilize with a 0.22 μm microporous filter membrane to obtain a second nutrient solution; Step 3, adding 10 g of high acyl gellan gum (the number of acyl groups is 4, and the final concentration is 10 g / L) to the first nutrient solution, stirring at 100°C until a homogeneous fluid is obtained, and then filtering the obtained fluid with gauze, placing it in an autoclave after filtration, and sterilizing it at a temperature of 115°C for 25 min, and then cooling it for 10 min. The cooled fluid is then mixed with the second nutrient solution, and stirred evenly to obtain a culture medium colloid with a pH of 2; Step 4: quickly spread the culture medium colloid on a flat plate to obtain a solid culture medium after gelation.

[0065] Comparative Example 1 The only difference between Comparative Example 1 and Example 1 is that the mass of 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 of the same concentration and quality as the coagulant.

[0067] Comparative Example 3 The difference between Comparative Example 3 and Example 1 is that Comparative Example 1 adopts agar of the same concentration and quality as a coagulant.

[0068] Experimental example Experiment 1 Based on Example 1 and Comparative Example 1, the morphology of the solid culture medium and the effect of separating and purifying thermophilic acidophilic bacteria were detected under the conditions of 65° C. to 85° C. and pH 1.5 to 3.5.

[0069] See also 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 1.5 ~ 3.5, and the whole will be in the form of rice paste, resulting in the solid culture medium being not hard enough and having a soft texture, which does not meet the hardness requirements for culturing thermophilic acidophilic bacteria and is not conducive to the stable cultivation of thermophilic acidophilic bacteria.

[0070] The solid culture medium prepared in Example 1 is shown in Figure 2 The solid culture medium was stable in morphology, and thermophilic and acidophilic bacteria were enriched on the surface of the culture medium. This indicates that the concentration of high acyl gellan gum affects its gel properties. Controlling the concentration of high acyl gellan gum between 5 g / L and 30 g / L can maintain good gel properties and thus maintain the stability of the solid culture medium.

[0071] Experiment 2 Based on Examples 1 to 5 and Comparative Example 2, the morphology of the solid culture medium and the effect of separating and purifying thermophilic acidophilic bacteria were detected under the conditions of 65° C. to 85° C. and pH 1.5 to 3.5.

[0072] According to the test, the solid culture medium prepared in Examples 1 to 5 can be stably formed at 65°C to 85°C and pH 1.5 to 3, and thermophilic acidophilic bacteria can be successfully separated. The morphology of the solid culture medium is similar to that of Figure 2 ; The solid culture medium prepared in Comparative Example 2 can only be stably formed under the conditions of pH greater than 3.4 and temperature less than 60°C. Under the above test conditions, its gel performance rapidly decreases, becoming a more fluid fluid and unable to be stably formed. In addition, due to the excessive concentration and high content of low-acyl gellan gum, part of the colloid gradually hardened, making the solid culture medium unusable for culturing thermophilic and acidophilic bacteria.

[0073] Experiment 3 1. Detect the morphology of solid culture media made of different concentrations of agar at 65 ℃ ~ 85 ℃ after use at different pH values, as follows: (1) The culture medium includes: 6% agar + 10 ml concentrated sulfuric acid, pH 1-1.5. After testing, the solid culture medium formed after the culture medium gelled dissolved after 2 hours of use and could not be recondensed; (2) The culture medium includes: 2% agar + 6 ml concentrated sulfuric acid, pH 1.5 to 2. After testing, the solid culture medium formed by the gelation of the culture medium was 1 / 2 dissolved after 2 hours of use and could not be recondensed.

[0074] It is proved that the gelling property of agar is greatly reduced in a high temperature and high acid environment with pH < 2, and agar is not suitable for preparing solid culture medium for culturing thermophilic and acidophilic bacteria.

[0075] 2. Based on Example 1 and Comparative Example 3, the morphology of the solid culture medium and the effect of separating and purifying thermophilic acidophilic bacteria were detected under the conditions of 65°C to 85°C and pH 1.5 to 3.5.

[0076] After testing, the solid culture medium prepared in Comparative Example 3 can only be stably formed under the conditions of pH greater than 3.4 and temperature below 60°C. Under the above test conditions, its gel performance rapidly decreases, becoming a more fluid fluid and unable to stably form. Therefore, it is proved that even by increasing the concentration of agar, the problem of agar dissolving under high temperature and high acid cannot be solved.

[0077] According to Example 2 and Comparative Example 3, it is explained that the compound use of high acyl gellan gum and agar can effectively solve the problem of agar dissolution under high temperature and high acid, and the prepared solid culture medium can provide a stable environment that satisfies the growth of thermophilic and acidophilic bacteria.

[0078] In summary, the present application uses high acyl gellan gum as a gel material and adds it to the solid culture medium, and then adjusts the concentration of high acyl gellan gum to significantly enhance the gel effect in a high acid and high temperature environment. The prepared solid culture medium can provide an environment that satisfies the rapid growth and enrichment of thermophilic acidophilic bacteria, and inhibit the growth of other bacterial species, and successfully separate thermophilic acidophilic bacteria, thermophilic archaea, thermophilic / acidophilic bacteria, or moderate thermophilic / acidophilic bacteria that grow under the conditions of 65 ℃ ~ 85 ℃ and pH 1.5 ~ 3.

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

[0080] Although the preferred embodiments of the present application have been described, those skilled in the art may make additional changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the embodiments of the present application.

[0081] Finally, it should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or terminal device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or terminal device. In the absence of further restrictions, the elements defined by the sentence "including one..." do not exclude the existence of other identical elements in the process, method, article or terminal device including the elements.

[0082] The above is a detailed introduction to a solid culture medium for separating and purifying thermophilic acidophilic bacteria, a preparation method and an application thereof provided in the present application. Specific examples are used herein to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method of the present application and its core idea. At the same time, for a person skilled in the art, according to the idea of ​​the present application, there will be changes in the specific implementation method and application scope. In summary, the content of this specification should not be understood as a limitation on the present application.

Claims

1. A solid culture medium for separating and purifying thermophilic acidophilic bacteria, characterized in that: The solid culture medium is prepared by mixing a nutrient composition, a coagulant and deionized water, wherein the mass proportion of the coagulant in the solid culture medium is 9% to 50%; Wherein, the coagulant comprises high acyl gellan gum, and 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.

2. The solid culture medium for separating and purifying thermophilic acidophilic bacteria according to claim 1, characterized in that: The concentration of the high acyl gellan gum is 10 g / L to 20 g / L.

3. The solid culture medium for separating and purifying thermophilic acidophilic bacteria according to claim 1, characterized in that: The solidifying agent also includes agar; Wherein, the concentration of the agar is 5 g / L~20 g / L; The mass ratio of the agar to the high acyl gellan gum is 0.5-0.

6.

4. The solid culture medium for separating and purifying thermophilic acidophilic bacteria according to claim 1, characterized in that: The nutrient composition consists of CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, KSCN, FeSO4∙7H2O, HNO3 and H2SO4; The mass volume ratio of the 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.

5. A method for preparing a solid culture medium for separating and purifying thermophilic and acidophilic bacteria according to any one of claims 1 to 4, characterized in that: The preparation method comprises: CaCl2, KCl, KH2PO4, (NH4)2SO4, KHSO4, MgSO4∙7H2O, yeast extract, HNO3, KSCN and H2SO4 in the nutrient composition are dissolved in deionized water, and the pH of the obtained mixed system is adjusted to 1.8-4 to prepare a first nutrient solution; Dissolve FeSO4∙7H2O in deionized water, adjust the pH of the resulting solution to 1.5-3, and filter and sterilize with a microporous filter membrane to obtain a second nutrient solution; Adding a coagulant to the first nutrient solution, stirring at 85°C to 200°C, filtering the obtained fluid, sterilizing it at high temperature, cooling it for 5 min to 10 min, and then mixing it with the second nutrient solution to obtain a culture medium colloid with a pH of 1.8 to 3.5, and obtaining a solid culture medium after gelation; Wherein, the coagulant includes high acyl gellan gum, and the concentration of the high acyl gellan gum is 5 g / L~30 g / L.

6. The method for preparing a solid culture medium for separating and purifying thermophilic acidophilic bacteria according to claim 5, characterized in that: 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 0.5-0.

6.

7. The method for preparing a solid culture medium for separating and purifying thermophilic and acidophilic bacteria according to claim 5, characterized in that: The mass proportion of the coagulant in the solid culture medium is 16% to 27%.

8. The method for preparing a solid culture medium for separating and purifying thermophilic and acidophilic bacteria according to claim 5, characterized in that: The preparation method further comprises: The coagulant is added to the first nutrient solution, and ultrasonic stirring is performed for 20 min to 30 min at a frequency of 10 kHz to 30 kHz. The obtained fluid is filtered and sterilized at high temperature, then 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, and a solid culture medium is obtained after gelation.

9. The method for preparing a solid culture medium for separating and purifying thermophilic and acidophilic bacteria according to claim 5, characterized in that: The temperature of the high temperature sterilization is 110°C to 130°C, and the sterilization time is 20 min to 30 min.

10. An application of a solid culture medium for separating and purifying thermophilic and acidophilic bacteria, characterized in that: The solid culture medium is used for separating and purifying thermophilic archaea, thermophilic / acidophilic bacteria or moderate thermophilic / acidophilic bacteria.

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