Fermentation method for halomonas sp. and use thereof in preparation of pha

Abstract

The present invention relates to the technical field of biological fermentation and particularly to a fermentation method for Halomonas sp. and use thereof in the preparation of PHA. Using Halomonas sp. as a fermentation strain, glucose and a derivative thereof as carbon sources, and an acetate (such as sodium acetate) as a pH neutralizer, the method provided by the present invention not only reduces the production cost, but also improves the overall rate of conversion of glucose to PHA or other products during production, achieving the effect of improving dry cell weight and product quality.

Description

Halomonas sp. fermentation method and its application in preparing PHA

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese Patent Application No. 2024106643145 entitled "Halomonas sp. fermentation method and its application in preparing PHA" filed on May 27, 2024, the entire disclosure of which is incorporated herein by reference in its entirety. TECHNICAL FIELD

[0003] The present application relates to the technical field of biological fermentation, and in particular to a Halomonas sp. fermentation method and its application in preparing PHA. BACKGROUND

[0004] Currently, the industrial large-scale production of biopolymers usually uses glucose or sodium gluconate as the carbon source. Glucose or sodium gluconate is first converted into pyruvate through the glycolysis pathway, and then acetyl-CoA is generated from pyruvate, and then various polymer monomers are synthesized from acetyl-CoA. However, during the fermentation process, various organic acids are produced by cell metabolism and released into the environment, affecting the pH of the cell growth environment, and usually causing the pH of the environment to decrease until the cell cannot tolerate the conditions (environmental pH < cell limit tolerance acidic environment pH), affecting cell growth.

[0005] Halomonas sp. is a type of moderately halophilic bacteria isolated from the Aiding Salt Lake in Xinjiang, China, with an optimal growth salt concentration of 6% and an optimal growth pH of 9.0. Using Halomonas sp. to ferment and produce biopolymers such as polyhydroxyalkanoate (PHA) has the advantages of no need for sterilization, high yield, and easy extraction. During the process of using Halomonas sp. to ferment and produce biopolymers, if the environmental pH is not maintained, the pH will quickly drop to a weakly acidic environment of about 6, which will seriously affect the growth of the bacterial cells and the synthesis of related products.

[0006] The currently commonly used solution is to add sodium hydroxide / potassium hydroxide as a pH neutralizer in the fermentation process to maintain the optimum pH environment required for cell growth. However, the use of sodium hydroxide / potassium hydroxide brings a series of production problems: due to the high price of sodium hydroxide / potassium hydroxide, it will lead to the increase of production cost; a special alkali tank needs to be prepared, and the depreciation of the alkali tank is very serious; in addition, such substances are hazardous goods, which will increase the risk in the production process. Other alkaline substances such as ammonia, potassium hydrogen phosphate, sodium hydrogen phosphate, ammonium sulfate, etc. are used as pH neutralizers, which have the disadvantages of weak alkalinity and are also used as nitrogen source, phosphorus source and other nutrients by cells, thereby affecting the product yield. Especially in the process of halomonas fermentation for producing PHA products, it will have a very serious negative impact on the yield of PHA products.

[0007] Therefore, the present application is proposed. SUMMARY

[0008] The present application provides a halomonas fermentation method and its application in preparing PHA, which uses acetate (such as sodium acetate) as a pH neutralizer, which has obvious technical effects superior to conventional solutions in the art.

[0009] Specifically, the technical solutions of the present application are as follows:

[0010] In a first aspect, the present application provides a fermentation method, which uses halomonas as a fermentation strain and glucose and its derivatives as a carbon source; a pH neutralizer is added when the pH value of the fermentation system is lower than the set value during the fermentation process; the pH neutralizer is selected from acetate; and the set value is 8±1.

[0011] The present application uses halomonas as a fermentation strain, which can be subjected to open fermentation without additional sterilization. At the same time, various air filters and complex sterile operations are not required during the fermentation process to reduce the possibility of contamination, thereby greatly saving production costs and manpower and resources.

[0012] In the fermentation process of halomonas, the present application uniquely uses acetate as a pH neutralizing agent. When acetate is dissolved in water, it will release acetate ions and positive metal ions. The acetate ions will be accompanied by hydrogen ions in the metabolic process of microorganisms, thereby consuming a large amount of hydrogen ions and increasing the pH of the solution.

[0013] The present application adds a pH neutralizer when the pH value of the fermentation system is lower than a set value. If sodium acetate is added from the beginning, the pH value of the fermentation system will quickly rise, which may on the one hand lead to a limitation on the cell growth rate and thus a reduction in yield, and on the other hand lead to difficulty in controlling the pH value in the overall fermentation process, which is not worth the cost. Therefore, the present application uses a method of regularly feeding sodium acetate to adjust the pH value of the fermentation system during fermentation.

[0014] Compared with the conventional scheme of using sodium hydroxide as a pH neutralization regulator, acetate, as a weak base, is safer, and reduces the depreciation of the lye tank and the production cost. At the same time, the amount of acetate is less than that of sodium hydroxide, which intuitively reduces the use cost of the neutralizer in terms of raw material prices. In addition, the present application shows that, compared with the use of sodium hydroxide, the use of sodium acetate as a pH neutralization regulator can more significantly improve the overall conversion rate from glucose to PHA or other products in the production process, thereby improving the cell dry weight and product yield.

[0015] In the present application:

[0016] Preferably, the fermentation product is PHA. In addition to the advantages mentioned above, the use of acetate as a pH neutralizer not only plays a role in pH neutralization, but also can be utilized by Halomonas to convert PHA, thereby improving the conversion rate.

[0017] Preferably, the PHA is a homopolymer and / or a copolymer; the homopolymer is preferably at least one selected from the group consisting of PHP, PHB, PHO and PHV, more preferably at least one selected from the group consisting of P3HB, P4HB, P3HP and P3HV; the copolymer is preferably at least one selected from the group consisting of P34HB, PHBHHx, PHBV, P3HO3HHx, PHBVHHx and P3HB4HB3HV.

[0018] Preferably, the pH neutralizer is added when the pH value of the fermentation broth is lower than a set value; the set value is 8±0.8.

[0019] The metabolic pathway of acetate to acetyl-CoA is shorter than that of glucose, and due to its high oxidation state, it cannot provide sufficient reducing power and energy for Halomonas. The fermentation method of the present application does not add acetate to the initial culture medium, which not only helps to avoid the waste of raw materials caused by the metabolism of acetate by Halomonas in the early fermentation stage, avoids the reduction in yield caused by the limitation on the cell growth rate, but also helps to maintain the pH stability of the overall fermentation process, avoids the loss of control of the high pH in the fermentation process, and is not worth the cost.

[0020] In the present application:

[0021] Preferably, the fermentation time is 48±12h; and / or, the fermentation temperature is 37±3℃.

[0022] Preferably, based on a total fermentation broth volume of 3L, the total amount of pH neutralizing agent added during the entire fermentation process is 24-25.5g. If a larger amount of sodium acetate is added, the residual sodium acetate can still play a role in subsequent wastewater treatment processes, reducing the overall amount of sodium acetate used and lowering process costs.

[0023] The pH neutralizing agent (acetate) described in this invention can be added in solid form or prepared as an aqueous acetate solution. The preferred mass percentage concentration of the aqueous acetate solution is 20%-35%. In the embodiments provided by this invention, the acetate is sodium acetate, with a concentration of 200 g / L to 300 g / L, preferably a saturation concentration of 300 g / L (the solubility of sodium acetate is 300 g / L).

[0024] Preferably, the *Haloxymonas* strain is selected from at least one of the following strains:

[0025] Halomonas bluephagenesis TD01;

[0026] Halomonas bluephagenesis LS21;

[0027] Halomonas bluephagenesis WZY254;

[0028] Halomonas bluephagenesis TD68-194;

[0029] Halomonas bluephagenesis TDH4.

[0030] Among them, Halomonas bluephagenesis TD01, also known as Halomonas sp. TD01, was deposited on November 19, 2010, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 4353 and classified as Halomonas. It is described in patent application publication number CN102120973A. The public can obtain this bacterium from Tsinghua University.

[0031] Halomonas campaniensis LS21 is a Gram-negative halophilic bacterium screened in our laboratory. It has a very good prospect for industrial production and application. Its preservation number is CGMCC No.6593. The public can obtain this bacterium from Tsinghua University. See the literature: Ling C, Qiao GQ, Shuai BW, Song KN, Yao WX, Jiang XR, Chen GQ. Engineering self-flocculating Halomonas campaniensis for wastewaterless open and continuous fermentation. Biotechnol Bioeng. 2019 Apr; 116(4):805-815. doi:10.1002 / bit.26897. Epub 2019 Feb 8. PMID:30537067.

[0032] Halomonas bluephagenesis WZY254 is an outer membrane-defective strain that can produce PHB and P3HB4HB in a 7L fermenter; see reference: Ziyu Wang et al., 2022. Hyperproduction of PHAcopolymers containing high fractions of 4-hydroxybutyrate (4HB) by outer membrane-defected Halomonas bluephagenesis grown in bioreactors[J]. Microbial Biotechnology.

[0033] Halomonas bluephagenesis TD68-194 is a recombinant strain of TD01 that has been metabolically engineered to synthesize P(3HB-co-4HB) from glucose; see reference: Stimulus response-based fine-tuning of polyhydroxyalkanoate pathway in Halomonas (Ye et al, Metabolic Engineering, 2020).

[0034] Halomonas bluephagenesis TDH4, reference: Shen R, Yin J, Jian-Wen Ye, et al. Promoter Engineering for Enhanced P(3HB-co-4HB) Production by Halomonas bluephagenesis[J]. ACS Synthetic Biology, 7(8): 1897-1906.; can be obtained from Tsinghua University.

[0035] The fermentation method described in this invention can be achieved using conventional open fermentation, which relies on a high-salt, high-pH environment to inhibit the growth of other contaminating microorganisms. This invention adds glucose as a carbon source to the basal culture medium and adds sodium chloride to provide the high osmotic pressure environment required for the growth of *Halomonas*.

[0036] Specifically, the present invention preferably uses MM medium as the base medium, and adds 30±10g / L of glucose as a carbon source and 5-100g / L of sodium chloride to provide the osmotic pressure environment required for the growth of halophilic bacteria; and adjusts the pH to about 9.0.

[0037] In a more specific embodiment provided by the present invention, the specific formulation of MM medium for culturing Halomonas is as follows:

[0038] Urea 0.5 g / L; MgSO4 0.2 g / L; KH2PO4 1.5 g / L; and a total of <0.1 g / L of Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, and NaMoO4·2H2O.

[0039] Secondly, this invention provides the application of the fermentation method in the production of PHA. This application helps to reduce the production cost of PHA, improve the overall conversion rate from glucose to PHA in the production process, and increase cell dry weight and product yield.

[0040] Thirdly, the present invention provides a method for producing PHA, wherein the fermentation method is used to produce PHA. After fermentation, the sugar conversion rate is ≥32%; the mass content of PHA in the dry bacteria is ≥70%. Beneficial effects:

[0041] This invention provides a method for fermenting Halomonas bacteria and its application in the preparation of PHA. The method uses Halomonas bacteria as the fermentation strain, glucose and its derivatives as the carbon source, and acetate (such as sodium acetate) as the pH neutralizer. This method not only reduces production costs but also improves the overall conversion rate from glucose to PHA or other products in the production process, thereby improving cell dry weight and product quality. Detailed Implementation

[0042] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the embodiments are all conventional methods; the materials and reagents used are all commercially available.

[0043] In the following examples, the basal culture medium used to culture Halomonas is MM medium, and its formula is as follows:

[0044] Urea 0.5 g / L; MgSO4 0.2 g / L; KH2PO4 1.5 g / L; and a total of <0.1 g / L of Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, and NaMoO4·2H2O.

[0045] The aforementioned MM medium was used as the basal medium, with the addition of glucose 30±10 g / L and sodium chloride 5-100 g / L, for fermentation experiments. A 7L fermenter was used, with a fermentation broth volume of 3L, an initial pH of 9.0, and a temperature maintained at 37℃.

[0046] In the following embodiments, gas chromatography was used to detect the content of polyhydroxyalkanoates (PHA). The specific methods are as follows:

[0047] The furnace temperature was set to 80℃, the injector temperature to 200℃, the detector temperature to 220℃, and the column head pressure to 0.25 MPa. The programmed temperature rise conditions were: 80℃ for 1.5 minutes, then ramped up to 140℃ at a rate of 30℃ / min, followed by ramping up to 220℃ at a rate of 40℃ / min and holding at this temperature for 0.5 minutes. The sample injection volume was 1 μl, using a microsyringe manufactured by Agilent Technologies.

[0048] The preparation method for gas chromatograph samples is as follows: Take 40-60 mg of stem cells from the sample to be tested (centrifuge the bacterial culture at 10000 rpm at room temperature for 10 minutes, wash the resulting cell pellet once with water, and then freeze-dry to obtain stem cells; the homopolymer is produced in the cells), add 2 ml of chloroform and 2 ml of esterification solution (pure methanol containing 3% (v / v) concentrated sulfuric acid and 1 g / L benzoic acid as an internal standard) to an esterification tube, seal the tube, and heat at 100℃ for 4 hours. After cooling, add 1 ml of distilled water, shake thoroughly, and let stand until the chloroform phase and aqueous phase completely separate. Then, inject 1 μl of the lower chloroform phase into the gas chromatograph (HP Hewlett Packard 6890) for chromatographic analysis. Operate the gas chromatograph according to the instruction manual of the HP Hewlett Packard 6890 gas chromatograph.

[0049] The preparation method for standard samples is as follows: Take 10-20 mg of standard sample into an esterification tube, add 2 ml of chloroform and 2 ml of esterification solution, seal the tube, and esterify at 100℃. The result analysis method is as follows: Using the standard sample as a control, if the esterified sample of the cells to be tested (the test sample) has a clear peak at the standard sample, the mass of each monomer can be calculated based on the peak area, and then the molar ratio can be calculated based on the mass fraction of each monomer; the proportion of polymer contained in the dry weight of the cells (wt%) can be calculated based on the amount of sample added.

[0050] In the following examples, the cell dry weight (CDW, g / L) mentioned is the ratio of the mass of the dried cells to the volume of the fermentation product. PHA specifically refers to P3HB, and its content (wt%) is the proportion of P3HB to the mass of the dried cells.

[0051] Example 1

[0052] This embodiment provides a method for producing P3HB using Halomonas campaniensis fermentation of glucose. Halomonas campaniensis TD01 was selected as the fermentation strain, and MM medium was used as the basal culture medium. Its formula is: urea 0.5 g / L; MgSO4 0.2 g / L; KH2PO4 1.5 g / L; and a total of <0.1 g / L of Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, and NaMoO4·2H2O. Glucose 30 g / L and sodium chloride 73 g / L were added to the basal culture medium. The fermentation experiment was conducted in a 7L fermenter: the fermentation broth volume was 3 L, the initial pH was set at 9.0, and the fermentation temperature was maintained at 37℃. During fermentation, microorganisms produce organic acids, which lowers the overall pH. When the pH of the fermentation broth is lower than the set value of 8.0, a 300g / L sodium acetate solution is added dropwise using a peristaltic pump to neutralize the pH.

[0053] This embodiment also includes a control group experiment, which differs from the previous method only in that the neutralizing agent (300g / L sodium acetate) is replaced with 300g / L sodium hydroxide.

[0054] After 48 hours of fermentation, the cell dry weight, sugar conversion rate, PHA content, and neutralizing agent cost were calculated, and the results are shown in Table 1.

[0055] Table 1

[0056] It is evident that using sodium acetate instead of sodium hydroxide in the fermentation process of Halomonas bluephagenesis TD01 can slightly increase cell dry weight and PHA content, significantly reduce the cost of neutralizing agents, and significantly improve sugar conversion rate, demonstrating great application value.

[0057] Example 2

[0058] This embodiment provides a method for producing P3HB by fermenting glucose using Halomonas. The difference from Embodiment 1 is that Halomonas campaniensis LS21 is selected as the fermentation strain in this embodiment.

[0059] In addition, the culture medium used in this embodiment has the following components:

[0060] The formula was as follows: urea 0.5 g / L; MgSO4 0.2 g / L; KH2PO4 1.5 g / L; and a total of <0.1 g / L of Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, and NaMoO4·2H2O. Glucose 30 g / L and sodium chloride 100 g / L were added to the basal medium. Fermentation experiments were conducted in a 7 L fermenter: the fermentation broth volume was 3 L, the initial pH was set at 9.0, and the fermentation temperature was maintained at 37℃. During fermentation, microorganisms produced organic acids, leading to a decrease in the overall pH. When the pH of the fermentation broth fell below the set value of 8.0, 270 g / L sodium acetate solution was added dropwise using a peristaltic pump to neutralize the pH.

[0061] The results are shown in Table 2.

[0062] Table 2

[0063] It is evident that using sodium acetate instead of sodium hydroxide in the fermentation of Halomonas campaniensis LS21 can slightly increase cell dry weight and PHA content, significantly reduce the cost of neutralizing agents, and significantly improve sugar conversion rate, demonstrating great application value.

[0064] Example 3

[0065] This embodiment provides a method for producing P3HB by fermenting glucose using Halomonas. The difference from Embodiment 1 is that Halomonas campaniensis WZY254 is selected as the fermentation strain in this embodiment.

[0066] In addition, the culture medium used in this embodiment has the following components:

[0067] The formula was as follows: urea 0.5 g / L; MgSO4 0.2 g / L; KH2PO4 1.5 g / L; and a total of <0.1 g / L of Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, and NaMoO4·2H2O. Glucose 30 g / L and sodium chloride 53 g / L were added to the basal medium. Fermentation experiments were conducted in a 7 L fermenter: the fermentation broth volume was 3 L, the initial pH was set at 9.0, and the fermentation temperature was maintained at 37 °C. During fermentation, microorganisms produced organic acids, leading to a decrease in the overall pH. When the pH of the fermentation broth fell below the set value of 8.0, a 300 g / L sodium acetate solution was added dropwise using a peristaltic pump to neutralize the pH.

[0068] The results are shown in Table 3.

[0069] Table 3

[0070] It is evident that using sodium acetate instead of sodium hydroxide in the fermentation process of Halomonas bluephagenesis WZY254 can slightly increase cell dry weight and PHA content, significantly reduce the cost of neutralizing agents, and significantly improve sugar conversion rate, demonstrating great application value.

[0071] Example 4

[0072] This embodiment provides a method for producing P3HB by fermenting glucose with Halomonas. The difference from Embodiment 1 is that Halomonas campaniensis TD68-194 is selected as the fermentation strain in this embodiment.

[0073] The formulation was as follows: urea 0.5 g / L; MgSO4 0.2 g / L; KH2PO4 1.5 g / L; and a total of <0.1 g / L of Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, and NaMoO4·2H2O. Glucose 30 g / L and sodium chloride 5 g / L were added to the basal medium. Fermentation experiments were conducted in a 7 L fermenter: the fermentation broth volume was 3 L, the initial pH was set at 9.0, and the fermentation temperature was maintained at 37 °C. During fermentation, microorganisms produced organic acids, leading to a decrease in the overall pH. When the pH of the fermentation broth fell below the set value of 8.0, 200 g / L sodium acetate solution was added dropwise using a peristaltic pump to neutralize the pH.

[0074] The results are shown in Table 4.

[0075] Table 4

[0076] It is evident that using sodium acetate instead of sodium hydroxide in the fermentation process of Halomonas bluephagenesis TD68-194 can slightly increase cell dry weight and PHA content, significantly reduce the cost of neutralizing agents, and significantly improve sugar conversion rate, demonstrating great application value.

[0077] Example 5

[0078] This embodiment provides a method for producing P3HB by fermenting glucose with Halomonas. The only difference from Embodiment 1 is that Halomonas campaniensis TDH4 is selected as the fermentation strain in this embodiment.

[0079] The formula was as follows: urea 0.5 g / L; MgSO4 0.2 g / L; KH2PO4 1.5 g / L; and a total of <0.1 g / L of Fe(III)-NH4-Citrate, CaCl2·2H2O, ZnSO4·7H2O, MnCl2·4H2O, H3BO3, CoCl2·6H2O, CuSO4·5H2O, NiCl2·6H2O, and NaMoO4·2H2O. Glucose 30 g / L and sodium chloride 24 g / L were added to the basal medium. Fermentation experiments were conducted in a 7 L fermenter: the fermentation broth volume was 3 L, the initial pH was set at 9.0, and the fermentation temperature was maintained at 37 °C. During fermentation, microorganisms produced organic acids, leading to a decrease in the overall pH. When the pH of the fermentation broth fell below the set value of 8.0, 300 g / L sodium acetate solution was added dropwise using a peristaltic pump to neutralize the pH.

[0080] The results are shown in Table 5.

[0081] Table 5

[0082] It is evident that using sodium acetate instead of sodium hydroxide in the fermentation process of Halomonas bluephagenesis TDH4 can slightly increase cell dry weight and PHA content, significantly reduce the cost of neutralizing agents, and significantly improve sugar conversion rate, demonstrating great application value.

[0083] The embodiments described above are merely illustrative of several implementations of the present invention, designed to facilitate a detailed understanding of the technical solutions of the present invention, but should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Industrial applicability

[0084] This invention provides a method for fermenting *Haloxylon ammodendron* and its application in the preparation of pharmacohydrate (PHA). The method uses *Haloxylon ammodendron* as the fermentation strain, glucose and its derivatives as the carbon source, and acetate (such as sodium acetate) as the pH neutralizer. This not only reduces production costs but also improves the overall conversion rate from glucose to PHA or other products, thereby increasing cell dry weight and product quality. It has significant economic value and promising application prospects.

Claims

1. A fermentation process, characterized in that, Halomonas as the fermentation strain, glucose and its derivatives as the carbon source; adding a pH neutralizer when the pH value of the fermentation system is lower than the set value during the fermentation process; the pH neutralizer is selected from acetate; and the set value is 8±1.

2. The fermentation process of claim 1, wherein, The fermentation product is PHA.

3. The fermentation process of claim 2, wherein, The PHA is a homopolymer and / or a copolymer; the homopolymer is preferably at least one selected from PHP, PHB, PHO and PHV, and more preferably at least one selected from P3HB, P4HB, P3HP and P3HV; and the copolymer is preferably at least one selected from P34HB, PHBHHx, PHBV, P3HO3HHx, PHBVHHx and P3HB4HB3HV.

4. The fermentation process according to any one of claims 1 to 3, characterized in that, The pH neutralizer is added when the pH value of the fermentation broth is lower than the set value; and the set value is 8±0.

8.

5. The fermentation process according to any one of claims 1 to 4, characterized in that, The fermentation time is 48±12 h; and / or the fermentation temperature is 37±3℃.

6. The fermentation method according to any one of claims 1-5, characterized in that, The total amount of the pH neutralizer added during the whole fermentation process is 24-25.5 g, based on the total volume of the fermentation broth of 3 L.

7. The fermentation process according to any one of claims 1 to 6, characterized in that, The Halomonas is selected from at least one of the following strains: Halomonas bluephagenesis TD01; Halomonas bluephagenesis LS21; Halomonas bluephagenesis WZY254; Halomonas bluephagenesis TD68-194; Halomonas bluephagenesis TDH4.

8. The fermentation process according to any one of claims 1 to 7, characterized in that, The MM medium is used as the basic medium, and 30±10 g / L of glucose and 5-100 g / L of sodium chloride are added.

9. Use of the fermentation method according to any one of claims 1-8 in the production of PHA.

10. A method for producing PHA, characterized by, The fermentation method according to any one of claims 1-8 is used to produce PHA; the sugar conversion rate after the fermentation is completed is ≥32%; and the mass content of PHA in the dry bacteria is ≥70%.

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