Culture method for halophilic microorganism culture

Abstract

The invention discloses a culture method for halophilic microorganism culture, and particularly relates to the technical field of microorganism culture, a culture medium containing sylvite as an osmotic pressure regulator is used for culture, and the culture method comprises the steps of S1, culture medium preparation, S2, strain preparation, S3, fermentation culture, S4, observation and detection, and S5, harvesting and post-treatment. According to the present invention, the gene engineering modification technology, the accurate osmotic pressure impact strategy and the unique thermal shock treatment method are combined, the growth environment and the metabolic pathway of the halophilic microorganisms are comprehensively optimized, the synthesis efficiency and the yield of the target product are substantially improved, the high efficiency and the economical efficiency of the production process are ensured, and the production cost is reduced. And the adaptability and the stability of the microbial strain to variable environmental conditions are also obviously enhanced.

Description

Technical Field

[0001] The present invention relates to the technical field of microorganism cultivation, and more particularly to a cultivation method for culturing halophilic microorganisms. Background Art

[0002] Halophilic microorganisms are a type of microorganism that can survive and reproduce in a high-salt environment. They have unique physiological mechanisms and metabolic pathways and can produce a series of metabolites with industrial application value. With the continuous development of biotechnology, the application prospects of halophilic microorganisms in food, chemical, pharmaceutical and other fields are becoming increasingly broad. Especially under extreme environmental conditions, halophilic microorganisms can exhibit extraordinary survival ability and metabolic activity, which provides the possibility for their application in special industrial processes. Therefore, it is necessary to cultivate halophilic microorganisms.

[0003] After searching, it was found that a Chinese patent with authorization announcement number CN115651874A discloses a culture medium and culture method for culturing halophilic microorganisms. This technical solution adopts a method in which the sodium chloride conventionally used to provide a high-salt environment for halophilic microorganisms is completely or partially replaced by potassium salt. After the fermentation is completed, the remaining potassium ions in the fermentation liquid will be used as agricultural fertilizer, avoiding the problem of difficulty in recycling wastewater from the culture medium originally composed mainly of sodium ions.

[0004] In the existing halophilic microbial culture technology, the composition of the culture medium is one of the key factors affecting microbial growth and metabolite synthesis. Traditionally, in order to maintain the osmotic pressure required by halophilic microorganisms, the culture medium often contains a high concentration of sodium salts, such as sodium chloride. However, this high sodium content culture medium has a series of technical defects, which limits its effect and potential in practical applications.

[0005] First, high concentrations of sodium salt may cause the osmotic pressure of the culture medium to be too high, which will have an adverse effect on the growth of halophilic microorganisms. Although halophilic microorganisms can survive in a high-salt environment, excessive osmotic pressure may inhibit their growth rate and metabolic activity, thereby reducing the synthesis efficiency of the target product.

[0006] Secondly, the medium with high sodium content may change the metabolic pathway of microorganisms, resulting in a decrease in product quality. When coping with a high-salt environment, microorganisms may adjust their metabolic strategies to maintain survival, which may lead to the inhibition or redirection of the synthesis pathway of the target product, thereby affecting the purity and yield of the product.

[0007] In addition, the application of potassium salts in halophilic microbial culture media in the prior art has not been fully studied and optimized. Although potassium salts and sodium salts are similar in chemical properties and can both be used as osmotic pressure regulators, different types of potassium salts have different effects on microbial growth and metabolism. Therefore, simply using potassium salts as a substitute for sodium salts may not achieve the desired effect and may even have a negative impact on the growth of microorganisms.

[0008] In view of the above situation, the present invention proposes a cultivation method for culturing halophilic microorganisms. Summary of the invention

[0009] In order to overcome the above-mentioned defects of the prior art, the present invention provides a cultivation method for culturing halophilic microorganisms to solve the problems raised in the above-mentioned background technology.

[0010] To achieve the above object, the present invention provides the following technical solution: a culture method for culturing halophilic microorganisms, using a culture medium containing potassium salt as an osmotic pressure regulator for culturing, the culture method comprising the following steps:

[0011] S1. Preparation of culture medium: weigh appropriate amount of basic culture medium components to ensure that there are no impurities and high purity;

[0012] Substituting at least 20% of the sodium salt in the traditional culture medium with a potassium salt composition;

[0013] Add a carbon source composition with a concentration controlled within the range of 1-100 g / L, and add trace amounts of growth factors, vitamins, coenzymes and metal ion micronutrients to fully dissolve and mix evenly, adjust the pH value to 6-9, and add a buffer to maintain a stable pH value;

[0014] S2. strain preparation: taking out the preserved halophilic microorganism strains, activating and culturing them on a suitable culture medium, and inoculating the activated strains into the prepared culture medium at a certain inoculation amount, which is determined according to the situation;

[0015] S3, fermentation culture, placing the inoculated culture medium in a constant temperature incubator or shaker, and performing initial culture at the optimal growth temperature of the halophilic microorganisms, the specific temperature being determined according to the situation;

[0016] During the fermentation process, the salt concentration in the culture medium is gradually increased according to the growth of the microorganisms to provide osmotic pressure shock to the halophilic microorganisms and stimulate their metabolic activities;

[0017] Add carbon source and potassium salt in time to maintain the osmotic pressure and nutrient supply of the culture medium;

[0018] S4. Observation and testing: regularly observe the growth status of microorganisms, record growth curves and morphological characteristics, and use high performance liquid chromatography and mass spectrometry to accurately determine the content and purity of the target product in the culture medium;

[0019] S5. Harvesting and post-processing: The culture containing the target product is collected, and the target product is efficiently separated and purified from the culture by centrifugation, filtration, extraction and crystallization methods. The purified target product is dried and properly stored for subsequent use.

[0020] Preferably, in step S1, the basic culture medium components include but are not limited to any one or more of mineral salts, nitrogen sources and phosphorus sources.

[0021] Preferably, in step S1, the potassium salt composition is one or more potassium salts selected from potassium chloride, potassium sulfate, potassium phosphate, potassium citrate, potassium acetate, potassium gluconate, potassium nitrate, potassium carbonate and potassium bicarbonate as osmotic pressure regulators.

[0022] Preferably, in step S1, the carbon source composition is prepared by mixing a plurality of sugars, organic acids and alcohols in a ratio of 1:1:1, wherein the plurality of sugars specifically include any one or a combination of two of glucose and fructose, the organic acid specifically includes any one or a combination of two of citric acid and succinic acid, and the alcohol specifically is glycerol.

[0023] Preferably, in step S2, the preserved halophilic microbial strain is taken out, specifically a genetically engineered Halomonas strain.

[0024] Preferably, in step S3, the initial culture is carried out at the optimal growth temperature of the halophilic microorganisms in two cases:

[0025] Extreme halophiles: suitable temperature is 30-55℃, Halobacterium: suitable temperature is 35-40℃.

[0026] Preferably, in step S3, the salt concentration in the culture medium is gradually increased to include potassium salt and / or other suitable salts. When adding carbon source and potassium salt, the principle of adding small amounts and multiple times should be followed to avoid a sharp change in osmotic pressure.

[0027] Preferably, in step S3, after the osmotic pressure shock, the culture temperature may be increased to a temperature above the optimal growth temperature of the halophilic microorganism but not exceeding its heat resistance limit, and a heat shock treatment may be performed to further promote the synthesis of the target product, specifically:

[0028] According to the optimal growth temperature and heat resistance limit of the target halophilic microorganisms, after the osmotic pressure shock treatment is completed, the culture temperature is gradually increased to above the optimal growth temperature of the halophilic microorganisms, but it must be ensured that it does not exceed its heat resistance limit.

[0029] Preferably, the basal medium refers to a medium containing nutrients for the growth of halophilic microorganisms.

[0030] Preferably, the halophilic microorganism refers to a microorganism that requires a certain salt concentration for growth and grows best in an environment with a certain salt concentration, specifically a halophilic bacteria.

[0031] Technical effects and advantages of the present invention:

[0032] 1. The present invention significantly improves the growth rate and metabolic activity of halophilic microorganisms by finely regulating culture conditions, including strategies such as specific requirements of genetically engineered strains, osmotic pressure shock and heat shock treatment. These optimization measures work together to promote the efficient operation of the target product synthesis pathway, thereby achieving a significant increase in the synthesis efficiency and yield of the target product, which is of great significance for improving production efficiency and economic benefits, especially in the fields of food, chemical industry, medicine, etc. that require large-scale production of specific compounds;

[0033] 2. The present invention trains the stress resistance and adaptability of halophilic microorganisms by introducing environmental stress stimuli such as osmotic pressure shock and heat shock treatment. After experiencing these challenges, the metabolic systems and gene expression patterns of these microorganisms are optimized, enabling them to grow stably and metabolize efficiently under a wider range of environmental conditions. This enhanced adaptability and stability is particularly important for industrial applications, as it ensures the continuity and reliability of the production process and reduces production risks caused by environmental fluctuations. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is the overall flow chart of the present invention. DETAILED DESCRIPTION

[0035] Example 1

[0036] The solution of the embodiment of the present invention provides a basic halophilic microorganism cultivation method using potassium salt as an osmotic pressure regulator, which specifically includes the following steps:

[0037] S1. Preparation of culture medium: weigh appropriate amounts of basic culture medium components (such as mineral salts, nitrogen source and phosphorus source), use 20% potassium chloride as an osmotic pressure regulator to replace part of the sodium salt in the traditional culture medium, add a 20 g / L glucose and fructose mixed carbon source (1:1 ratio), add trace amounts of growth factors, vitamins, coenzymes and metal ions, adjust the pH to 7.0, and add a buffer to maintain pH stability;

[0038] Specifically, in this step, appropriate amounts of basic culture medium components are weighed to provide the halophilic microorganisms with the mineral elements, nitrogen sources and phosphorus sources necessary for their growth, which are the basic substances that constitute the cell structure of the microorganisms and carry out metabolic activities;

[0039] 20% potassium chloride is used as an osmotic pressure regulator to replace part of the sodium salt in the traditional culture medium because halophilic microorganisms need to grow in a high-salt environment. Potassium chloride not only provides the necessary potassium element, but also maintains the high osmotic pressure environment of the culture medium through its high concentration, which is conducive to the growth of halophilic microorganisms.

[0040] The addition of a mixed carbon source of glucose and fructose (1:1 ratio) at a concentration of 20 g / L was intended to provide the energy source and carbon skeleton required for microbial growth. The use of mixed carbon sources may help microorganisms utilize these carbon sources more efficiently and promote cell growth and metabolic activities;

[0041] Add trace amounts of growth factors, vitamins, coenzymes, metal ions and other micronutrients. These substances play a key role in the growth of microorganisms. They participate in the formation of enzyme cofactors or coenzymes and have an important impact on the growth rate, product synthesis ability and metabolic pathways of microorganisms.

[0042] Adjust the pH of the culture medium to 7.0 to ensure that the microorganisms grow in a suitable acid-base environment.

[0043] S2. Preparation of strains: taking out the preserved halophilic microorganism strains that have not been genetically engineered, activating and culturing them on a suitable culture medium, and inoculating them into the prepared culture medium at a 1% inoculation rate after activation;

[0044] Specifically, in this step, since the long-term stored bacterial strains may be in a dormant state, their metabolic activity and growth ability may be affected to a certain extent. Through activation culture, the bacterial strains can be stimulated to recover and their normal growth and metabolic functions can be restored. In addition, the bacterial strains are inoculated into the prepared culture medium at a 1% inoculation rate to ensure that the bacterial strains can grow rapidly in the culture medium and occupy a dominant position, while avoiding the problem of nutrient competition caused by excessive inoculation or slow growth caused by too small inoculation.

[0045] S3, fermentation culture: the inoculated culture medium is placed in a constant temperature incubator, and the initial culture is carried out at the optimal growth temperature of the halophilic microorganism (such as 37°C). During the fermentation process, the potassium salt concentration in the culture medium is gradually increased to perform osmotic pressure shock, and carbon source and potassium salt are added in time to maintain the osmotic pressure and nutrient supply of the culture medium;

[0046] Specifically, in this step, there are three specific objectives:

[0047] 1. Initial culture helps microbial cells maintain the best growth state and accelerate their metabolism, thereby increasing the growth rate and the synthesis efficiency of the target product. During the fermentation process, the potassium salt concentration in the culture medium is gradually increased to perform osmotic pressure shock. This operation helps to screen and enrich halophilic microorganisms that are resistant to hypertonic environments, while promoting the osmotic pressure regulation mechanism in microbial cells to better adapt to high-salt environments. Through osmotic pressure shock, microorganisms can also be stimulated to produce more target products, such as polyhydroxyalkanoates (PHA).

[0048] 2. With the growth and metabolic activities of microorganisms, the carbon source in the culture medium will be gradually consumed. Timely addition of carbon source can ensure that the microorganisms have sufficient energy sources for growth and product synthesis, avoiding growth stagnation or product yield reduction due to insufficient carbon source. While adding carbon source, it is also necessary to add potassium salt in appropriate amount to maintain the osmotic pressure of the culture medium. Osmotic pressure is one of the important environmental factors for the growth of halophilic microorganisms. Too high or too low osmotic pressure may have an adverse effect on the growth and product synthesis of microorganisms. Therefore, by timely addition of potassium salt, it can be ensured that the osmotic pressure of the culture medium is always maintained in an appropriate range.

[0049] 3. By optimizing the growth environment and ensuring the supply of nutrients, halophilic microorganisms can be stimulated to produce more target products. Moreover, operations such as osmotic pressure shock can also help improve the synthesis efficiency and quality of the products. Halophilic microorganisms have special adaptability to high-salt environments, while most bacteria cannot survive in such environments. Therefore, by maintaining a high-salt environment and performing operations such as osmotic pressure shock, the risk of contamination by bacteria can be effectively reduced, ensuring the stability and reliability of the fermentation process.

[0050] S4. Regularly observe the growth status of microorganisms, record the growth curve and morphological characteristics, and use high performance liquid chromatography to determine the content of the target product in the culture medium;

[0051] S5. Collect the culture containing the target product, separate and purify the target product by centrifugation, filtration, extraction and crystallization, and dry it.

[0052] Example 2

[0053] Optimize the cultivation of carbon source combinations and improve the product synthesis efficiency of halophilic microorganisms by optimizing the carbon source combination.

[0054] Specifically, Example 2 is different from Example 1 in that, based on Example 1, the carbon source combination is adjusted to a mixture of glucose, fructose, citric acid and glycerol (1:1:1:1 ratio), and the other steps are the same as Example 1.

[0055] Specifically, the purpose of this embodiment is to further optimize the growth environment and metabolic pathways of halophilic microorganisms. By introducing a variety of carbon sources, halophilic microorganisms can selectively use these carbon sources according to their metabolic preferences and physiological needs, which helps to improve the overall utilization efficiency of carbon sources, reduce the waste of unused carbon sources, and reduce the production of metabolic by-products. Different types of carbon sources may be used by microorganisms through different metabolic pathways, thereby triggering different metabolic activities and gene expression patterns. This metabolic diversity helps microorganisms better adapt to environmental changes and improve their growth rate and stability.

[0056] Moreover, some specific carbon source combinations may be more conducive to the synthesis of target products. For example, citric acid and glycerol can be used as precursors in certain metabolic pathways and directly participate in the biosynthesis of target products. By adjusting the carbon source combination, these metabolic pathways can be optimized and the yield and quality of target products can be improved.

[0057] Example 2 provides an experimental basis for exploring the growth and metabolic characteristics of halophilic microorganisms under different culture conditions by changing the carbon source combination, which helps researchers to have a deeper understanding of the metabolic mechanism and physiological characteristics of microorganisms and provides a theoretical basis for subsequent process optimization and industrial application.

[0058] Example 3

[0059] Cultivation of genetically engineered strains, showing the cultivation method of genetically engineered halophilic microbial strains, the specific steps are:

[0060] S1. Preparation of strains: taking out the stored genetically engineered Halomonas strains and activating and culturing them. The inoculation amount and method are the same as those in Example 1.

[0061] S2. Medium preparation and fermentation culture: When preparing the medium, in addition to using potassium salt as an osmotic pressure regulator, other ingredients can also be adjusted according to the specific needs of the modified strain. During the fermentation culture, the salt concentration and the timing of adding nutrients can be adjusted according to the characteristics of the strain;

[0062] S3, subsequent steps: observation and detection, harvesting and post-processing are the same as in Example 1.

[0063] Specifically, the purpose of this example is to optimize the growth and metabolism of the modified strain through specific culture conditions and operating steps;

[0064] Genetically engineered strains are usually endowed with specific superior traits, such as enhanced salt tolerance, improved target product synthesis capacity, or optimized metabolic pathways, to maximize the potential of these engineered strains and achieve higher growth rates, more stable production performance, and richer accumulation of target products.

[0065] Since genetic engineering may change the physiological characteristics and metabolic requirements of the strain, it is necessary to adjust the composition of the culture medium and the fermentation conditions according to the specific needs of the modified strain. For example, adjust the potassium salt concentration to maintain a suitable osmotic pressure environment, or add corresponding nutrients according to the strain's preference for nutrients. These adjustments help provide the modified strain with the best growth and metabolic environment, thereby promoting its efficient growth and the synthesis of the target product.

[0066] Example 4

[0067] The synthesis amount of the target product is further increased by combining the culture with the heat shock treatment.

[0068] On the basis of Example 1 or Example 3, the following steps are added:

[0069] After the osmotic pressure shock, according to the optimal growth temperature and heat resistance limit of the target halophilic microorganism (such as 50°C for extreme halophiles), the culture temperature is gradually increased to above the optimal growth temperature but not exceeding its heat resistance limit, and heat shock treatment is performed. The treatment time is determined according to the microbial response.

[0070] After heat shock treatment, continue to observe the growth of microorganisms and the synthesis of target products and adjust the culture conditions in a timely manner.

[0071] Subsequent steps: observation and detection, harvesting and post-processing are the same as in Example 1.

[0072] Specifically, this embodiment further improves the metabolic activity of the target halophilic microorganism through this additional environmental pressure stimulation, thereby promoting the synthesis amount of the target product.

[0073] Heat shock treatment simulates the temperature fluctuations in the natural environment by briefly raising the culture temperature to above the optimal growth temperature of the microorganism but not exceeding its heat tolerance limit. This temperature change can stimulate the metabolic system of the microorganism, making it more adaptable and flexible to different environmental conditions. This enhanced metabolic adaptability helps the microorganism to more effectively utilize nutrients in the subsequent growth process and improve the synthesis efficiency of the target product.

[0074] The synthesis pathways of certain target products may be regulated by environmental factors, including temperature. Heat shock treatment can activate or optimize the key enzymes and gene expressions in these synthesis pathways, thereby increasing the synthesis rate and accumulation of the target products. This regulatory mechanism helps microorganisms produce target products more efficiently under specific conditions.

[0075] Heat shock treatment can also be used as a screening method to select excellent strains with stronger tolerance to heat shock and higher product synthesis ability from a large number of strains. These high-yield strains have higher production efficiency and economic benefits in subsequent cultivation and industrial applications.

[0076] By combining thermal shock treatment, the synthesis amount of the target product can be further increased without significantly increasing cost and time. This efficiency improvement helps to shorten the production cycle, reduce production costs and increase the market competitiveness of the product.

[0077] For the above-mentioned Examples 1 to 4, the above-mentioned culture method was tested, and the test method and test results are shown in the following table:

[0078] 1. Test purpose: To compare the differences between the traditional culture method and the method of the present invention in the growth of halophilic microorganisms and the synthesis of target products, and to verify the improvement of the culture effect by optimizing the carbon source combination, using genetically engineered strains, and combining heat shock treatment;

[0079] 2. Test materials:

[0080] 2.1. Halophilic microorganisms (including unmodified and genetically engineered strains);

[0081] 2.2 Basic culture medium components (mineral salts, nitrogen source, phosphorus source, etc.)

[0082] 2.3. Potassium salt compositions (such as potassium chloride);

[0083] 2.4. Combinations of different carbon sources (such as glucose, fructose, citric acid, glycerol, etc.);

[0084] 2.5. Growth factors, vitamins, coenzymes, metal ions and other micronutrients;

[0085] 2.6. High performance liquid chromatography and other testing equipment.

[0086] 3. Test steps:

[0087] 3.1. Prepare four culture media, corresponding to Example 1 (basic culture), Example 2 (optimized carbon source), Example 3 (genetically engineered strains) and Example 4 (combined with heat shock treatment), and activate and prepare halophilic microbial strains at the same time;

[0088] 3.2. Prepare the culture medium according to the requirements of each embodiment, inoculate the corresponding bacteria, place the inoculated culture medium in a constant temperature incubator or shaker, and culture at a suitable temperature;

[0089] 3.3. In Example 4, after the osmotic pressure shock, the culture temperature is gradually increased to perform heat shock treatment;

[0090] 4. Observation and testing: regularly observe the growth status of microorganisms, record the growth curve and morphological characteristics, and use high performance liquid chromatography and other equipment to determine the content and purity of the target product in the culture medium.

[0091] 5. Data recording and analysis: record key data (such as growth rate, target product yield, purity, etc.) in a table, analyze and compare the differences between the embodiments, and evaluate the progress of the present invention.

[0092]

[0093]

[0094] According to the data in the above table, with the optimization of experimental conditions (such as optimizing the carbon source combination, using genetically engineered strains, and combining heat shock treatment), the growth rate of halophilic microorganisms and the yield of the target product are significantly improved. In particular, in Example 4, by combining heat shock treatment, the yield of the target product reached 10.0 g / L, which is nearly doubled compared to the basic culture method, showing the outstanding progress of the present invention in improving the culture efficiency. At the same time, the purity of the target product is also improved, indicating that the culture method of the present invention not only ensures the yield, but also pays attention to the quality of the product;

[0095] In summary, through the above experimental steps and table data, it can be fully proved that the present invention has significant advantages and outstanding progress in the cultivation of halophilic microorganisms.

[0096] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for cultivating halophilic microorganisms, using a culture medium containing potassium salt as an osmotic pressure regulator for cultivation, characterized in that: The culture method comprises the following steps: S1. Preparation of culture medium: weigh appropriate amount of basic culture medium components to ensure that there are no impurities and high purity; Substituting at least 20% of the sodium salt in the traditional culture medium with a potassium salt composition; Add a carbon source composition with a concentration controlled within the range of 1-100 g / L, and add trace amounts of growth factors, vitamins, coenzymes and metal ion micronutrients to fully dissolve and mix evenly, adjust the pH value to 6-9, and add a buffer to maintain a stable pH value; S2. strain preparation: taking out the preserved halophilic microorganism strains, activating and culturing them on a suitable culture medium, and inoculating the activated strains into the prepared culture medium at a certain inoculation amount, which is determined according to the situation; S3, fermentation culture, placing the inoculated culture medium in a constant temperature incubator or shaker, and performing initial culture at the optimal growth temperature of the halophilic microorganisms, the specific temperature being determined according to the situation; During the fermentation process, the salt concentration in the culture medium is gradually increased according to the growth of the microorganisms to provide osmotic pressure shock to the halophilic microorganisms and stimulate their metabolic activities; Add carbon source and potassium salt in time to maintain the osmotic pressure and nutrient supply of the culture medium; S4. Observation and testing: regularly observe the growth status of microorganisms, record growth curves and morphological characteristics, and use high performance liquid chromatography and mass spectrometry to accurately determine the content and purity of the target product in the culture medium; S5. Harvesting and post-processing: The culture containing the target product is collected, and the target product is efficiently separated and purified from the culture by centrifugation, filtration, extraction and crystallization methods. The purified target product is dried and properly stored for subsequent use.

2. The method for cultivating halophilic microorganisms according to claim 1, characterized in that: In step S1, the basic culture medium components include, but are not limited to, any one or more of mineral salts, nitrogen sources, and phosphorus sources.

3. The culture method for culturing halophilic microorganisms according to claim 1, characterized in that: In step S1, the potassium salt composition is one or more potassium salts selected from potassium chloride, potassium sulfate, potassium phosphate, potassium citrate, potassium acetate, potassium gluconate, potassium nitrate, potassium carbonate, and potassium bicarbonate as an osmotic pressure regulator.

4. The culture method for culturing halophilic microorganisms according to claim 1, characterized in that: In step S1, the carbon source composition is prepared by mixing a plurality of sugars, organic acids and alcohols in a ratio of 1:1:1, wherein the plurality of sugars specifically include any one or a combination of two of glucose and fructose, the organic acid specifically includes any one or a combination of two of citric acid and succinic acid, and the alcohol specifically is glycerol.

5. The culture method for culturing halophilic microorganisms according to claim 1, characterized in that: In step S2, the preserved halophilic microbial strains are taken out, specifically the genetically engineered Halomonas strains.

6. The method for cultivating halophilic microorganisms according to claim 1, characterized in that: In step S3, there are two cases when the initial culture is carried out at the optimal growth temperature of the halophilic microorganism: Extreme halophiles: suitable temperature is 30-55℃, Halobacterium: suitable temperature is 35-40℃.

7. The method for cultivating halophilic microorganisms according to claim 1, characterized in that: In step S3, the salt concentration in the culture medium is gradually increased, including potassium salt and / or other suitable salts. When adding carbon source and potassium salt, the principle of adding small amounts and multiple times should be followed to avoid rapid changes in osmotic pressure.

8. The culture method for culturing halophilic microorganisms according to claim 1, characterized in that: In step S3, after the osmotic pressure shock, the culture temperature may be increased to a temperature above the optimal growth temperature of the halophilic microorganism but not exceeding its heat resistance limit, and a heat shock treatment may be performed to further promote the synthesis of the target product. Specifically: According to the optimal growth temperature and heat resistance limit of the target halophilic microorganisms, after the osmotic pressure shock treatment is completed, the culture temperature is gradually increased to above the optimal growth temperature of the halophilic microorganisms, but it must be ensured that it does not exceed its heat resistance limit.

9. The culture method for culturing halophilic microorganisms according to claim 1, characterized in that: The basic medium refers to a medium containing nutrients for the growth of halophilic microorganisms.

10. The culture method for culturing halophilic microorganisms according to claim 1, characterized in that: The halophilic microorganism refers to a microorganism that requires a certain salt concentration for growth and grows best in an environment with a certain salt concentration, specifically halophilic bacteria.

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