WO2023016058A1 - Halomonas lutescens strain and use thereof - Google Patents

Halomonas lutescens strain and use thereof Download PDF

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WO2023016058A1
WO2023016058A1 PCT/CN2022/095741 CN2022095741W WO2023016058A1 WO 2023016058 A1 WO2023016058 A1 WO 2023016058A1 CN 2022095741 W CN2022095741 W CN 2022095741W WO 2023016058 A1 WO2023016058 A1 WO 2023016058A1
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fermentation
content
medium
halomonas
lutescens
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French (fr)
Chinese (zh)
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沈宏伟
吕金艳
银会娟
司徒卫
余柳松
何世琪
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珠海麦得发生物科技股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/62—Carboxylic acid esters
    • C12P7/625—Polyesters of hydroxy carboxylic acids

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  • the invention relates to the field of microbial fermentation, in particular to a halomonas strain and its application.
  • the degradation rate of chemical-based plastics is slow and the cycle is long, especially after the formation of microplastics, it is more difficult to handle, and its large-scale use will cause serious pollution to the environment. Therefore, the development of bio-based degradable plastics is conducive to the healthy development of the environment.
  • Polyhydroxyalkanoates are biological materials that are completely synthesized and stored intracellularly by microorganisms under the conditions of excess carbon source and limited other nutrients. According to the carbon chain length of the monomer, it is divided into short-chain PHA (4-6C, scl-PHA) and medium-long chain PHA ( ⁇ 6C, mcl-PHA).
  • Poly- ⁇ -hydroxybutyrate poly- ⁇ -hydroxybutyrate, PHB
  • PHA has the characteristics of degradability, biocompatibility and texture diversity, and has broad application prospects in chemical products, medical implant materials, drug sustained-release carriers and other fields.
  • the present invention proposes a strain of Halomonas and its application.
  • the strain is Halomonas lutescens MDF-9, and its preservation number is GDMCC NO.61850.
  • the Halomonas of the present invention can efficiently accumulate polyhydroxyalkanoate (PHA) in the culture medium of the present invention, which provides a good guarantee for the biosynthesis of PHA.
  • PHA polyhydroxyalkanoate
  • the invention provides a strain of Halomonas lutescens MDF-9, and the preservation number of the Halomonas lutescens is GDMCC NO.61850.
  • the present invention also provides the application of the Halomonas in the preparation of polyhydroxyalkanoate.
  • polyhydroxyalkanoate is poly- ⁇ -hydroxybutyric acid.
  • the present invention also provides a method for preparing polyhydroxyalkanoate, comprising the steps of: fermenting Halomonas lutescens MDF-9 GDMCC NO.61850 to obtain polyhydroxyalkanoate, and the fermentation used in the fermentation Culture medium and fermentation vessels are not sterilized.
  • the purpose of non-sterilization is to save process and cost, and to better reflect the value of the present invention. Sterilization can also achieve the technical effect of the present invention.
  • the sodium chloride concentration of the fermentation medium is 10-20 g/L.
  • the salt concentration of the fermentation medium of the invention is low, which is more conducive to the treatment of waste water.
  • the pH value of the fermentation is 8-10
  • the stirring speed is 50-800 rpm
  • the ventilation volume is 0.1-2vvm
  • the fermentation temperature is 25-40°C.
  • the yield of PHA is higher under the above fermentation conditions.
  • the pH value of the fermentation is 9-10
  • the ventilation rate is 0.8-2vvm
  • the fermentation temperature is 37°C-40°C.
  • the fermentation method comprises the following steps:
  • Another aspect of the present invention is to provide a medium for Halomonas fermentation.
  • the fermentation medium comprises the following components:
  • Carbon source any one or more of glucose, fructose, and sodium gluconate; the bacterial strain of the present invention can not only use glucose as a carbon source, but also utilize fructose and sodium gluconate.
  • Nitrogen source corn steep liquor dry powder, yeast powder, tryptone, urea, ammonium sulfate, ammonium chloride;
  • potassium dihydrogen phosphate disodium hydrogen phosphate, magnesium sulfate
  • the fermentation medium does not contain cobalt chloride and nickel chloride.
  • Cobalt chloride and nickel chloride are toxic metal compounds, which may cause sensitization by inhalation and skin contact. Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment. Therefore, the medium without cobalt chloride and nickel chloride is safer and more environmentally friendly.
  • the total concentration of the carbon source is 90-150 g/L.
  • the content of the corn steep liquor dry powder is 1-8%
  • the content of the yeast powder is 0.5-5%
  • the content of the tryptone is 0.1-3%
  • the content of the urea is 0.1-3%
  • the content of the ammonium sulfate is 0.1-3%
  • the content of the ammonium chloride is 0.1-3%.
  • the content of potassium dihydrogen phosphate is 0.1-2%
  • the content of disodium hydrogen phosphate is 0.05-1%
  • the content of magnesium sulfate is 0.05-1%
  • the content is mass volume ratio .
  • the sodium chloride concentration of the fermentation medium is 10-20 g/L.
  • polyhydroxyalkanoate is poly- ⁇ -hydroxybutyric acid.
  • the concentration of sodium chloride is low during the fermentation process, which is 10-20g/L, and the wastewater in the later stage is easy to treat.
  • Fermentation nutrients are simple, and no toxic and harmful metal compounds are used.
  • the dry weight of the bacteria can reach 198.5g/L, and the content of PHA can reach 79.5%.
  • Figure 1 is a single colony diagram of the bacterial strain in Example 1 of the present invention after being coated on a plate.
  • Fig. 2 is an image of microscopic observation of the bacterial strain in Example 1 of the present invention before fermentation.
  • Fig. 3 is an image of microscopic observation of the bacterial strain in Example 1 of the present invention after fermentation.
  • Fig. 4 is the FTIR spectrum of the PHB standard product of sigma company in the embodiment 1 of the present invention.
  • Fig. 5 is the FTIR spectrum of the PHB sample prepared in Example 1 of the present invention.
  • Fig. 6 is the GC spectrogram of the standard sample in Example 1 of the present invention.
  • Fig. 7 is the GC spectrogram of the sample tested in Example 1 of the present invention.
  • Fig. 8 is the FTIR spectrum of the PHB sample prepared in Example 2 of the present invention.
  • Fig. 9 is a GC spectrum of a PHB sample prepared using medium 1 without sterilization in Example 2 of the present invention.
  • Fig. 10 is the GC spectrum of the PHB sample prepared without sterilization and using medium 2 in Example 2 of the present invention.
  • Fig. 11 is the GC chromatogram of the PHB sample prepared without sterilization and using medium 3 in Example 2 of the present invention.
  • Fig. 12 is a GC spectrum of a PHB sample prepared without sterilization and using medium 4 in Example 2 of the present invention.
  • Fig. 13 is the GPC chart of the PHB sample prepared without sterilization and using medium 1 in Example 2 of the present invention.
  • Fig. 14 is the GPC chart of the PHB sample prepared without sterilization and using medium 2 in Example 2 of the present invention.
  • Fig. 15 is the GPC chart of the PHB sample prepared without sterilization and using medium 3 in Example 2 of the present invention.
  • Fig. 16 is the GPC chart of the PHB sample prepared without sterilization and using medium 4 in Example 2 of the present invention.
  • Fig. 17 is the gas chromatographic result of the PHB sample prepared under the culture condition 1 without sterilization in Example 3 of the present invention.
  • Fig. 18 is the gas chromatographic result of the PHB sample prepared under the culture condition 2 without sterilization in Example 3 of the present invention.
  • Fig. 19 is the gas chromatography result of the PHB sample prepared under the non-sterilized and culture condition 3 in Example 3 of the present invention.
  • Fig. 20 is the gas chromatographic result of the PHB sample prepared under non-sterile and culture condition 4 in Example 3 of the present invention.
  • Figure 21 is the electrophoresis result of the 486bp product in Example 4.
  • Figure 22 is the electrophoresis result of the 432bp product in Example 4.
  • the invention provides a strain of Halomonas lutescens MDF-9, which can accumulate PHA in the cell.
  • Culture conditions sodium chloride concentration 10-20g/L, pH 8-10, ventilation volume 0.1-2vvm, temperature 25-40°C.
  • Carbon source any one or more of glucose, fructose, and sodium gluconate, with a total concentration of 90-150 g/L.
  • Nitrogen source corn steep liquor dry powder 1-8%, yeast powder 0.5-5%, tryptone 0.1-3%, urea 0.1-3%, ammonium sulfate 0.1-3%, ammonium chloride 0.1-3% (m/v) .
  • potassium dihydrogen phosphate 0.1-2% disodium hydrogen phosphate 0.05-1%
  • magnesium sulfate 0.05-1% m/v
  • the Halomonas of the present invention is inoculated into a liquid medium, and the medium components include one or a mixture of two or three of the above carbon sources, the sugar concentration is 90-150g/L, sodium chloride 10-20g/L, 1-8% corn steep liquor dry powder, 0.5-5% yeast powder, 0.1-3% tryptone, 0.1-3% urea, 0.1-3% ammonium sulfate, 0.1-3% ammonium chloride (m /v), potassium dihydrogen phosphate 0.1-2%, disodium hydrogen phosphate 0.05-1%, magnesium sulfate 0.05-1% (m/v).
  • the 3 kinds of culture medium used in the following examples are flat solid medium and 2 kinds of liquid medium, and the flat solid medium is used to coat bacterial strains.
  • the seed culture medium and the fermentation medium are used for shake flask culture and 5L fermenter culture Halomonas of the present invention respectively.
  • Their specific composition is as follows:
  • Plate solid medium yeast powder, 0.5-1%; tryptone, 0.5-1%; sodium chloride 1-2%, agar powder 18g/L, pH 9.0.
  • Seed medium glucose solution, 30-50g/L; sodium chloride 10-20g/L, corn steep liquor dry powder 1-8%, yeast powder 0.5-5%, tryptone 0.1-3%, urea 0.1-3%, Ammonium sulfate 0.1-3%, ammonium chloride 0.1-3% (m/v), potassium dihydrogen phosphate 0.1-2%, disodium hydrogen phosphate 0.05-1%, magnesium sulfate 0.05-1% (m/v).
  • Sodium hydroxide was used to adjust the pH of the medium to 8.0-10, and the volume of the medium was 50 mL (250 mL Erlenmeyer flask).
  • Fermentation medium glucose solution, 90-150g/L; sodium chloride 10-20g/L, corn steep liquor dry powder 1-8%, yeast powder 0.5-5%, tryptone 0.1-3%, urea 0.1-3%, Ammonium sulfate 0.1-3%, ammonium chloride 0.1-3% (m/v), potassium dihydrogen phosphate 0.1-2%, disodium hydrogen phosphate 0.05-1%, magnesium sulfate 0.05-1% (m/v).
  • Sodium hydroxide was used to adjust the pH of the medium to 8-10.
  • the invention is verified by a 5L fermentation tank experiment, and the culture is 30-40 hours, the dry weight of the bacteria in the fermentation liquid at the end of fermentation is 90-200g/L, and the PHA content in the dry bacteria is 50-80%.
  • Figure 1 is round, white, slightly raised in the middle, and the edges are complete and opaque.
  • Figure 2 is the morphology of the thalli was observed under a microscope.
  • Figure 2 The thalli morphology before fermentation was shown in Figure 2, and the thalli were short rods, non-spore-free, and Gram-negative.
  • Figure 3 The morphology of the thalline in the later stage of fermentation is shown in Figure 3. It can be seen that the middle of the thallus is white and transparent, with colored ends, flagella, and can move. Detect the 16S rDNA sequence of the bacterial strain, and the measured sequence is shown in SEQ ID NO.1.
  • the strain has been preserved in the Guangdong Provincial Microbial Culture Collection Center (GDMCC for short, address: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Institute of Microbiology, Zip code 510070) on August 2, 2021.
  • the deposit number is GDMCC NO.61850.
  • the strain name is MDF-9, and the classification is named Halomonas lutescens.
  • seed culture medium composition glucose 90g/L; Sodium chloride 35g/L, corn steep liquor dry powder 1%, yeast powder 0.5%, urea 0.1%, ammonium sulfate 0.1%, Ammonium chloride 0.1%, potassium dihydrogen phosphate 0.3%, disodium hydrogen phosphate 0.1%, magnesium sulfate 0.1%, (m/v).
  • Sodium hydroxide regulates the pH of the medium to 9.0, and the volume of the medium is 30mL (250mL Erlenmeyer flask ), using shake flask culture to verify whether intracellular PHA is synthesized.
  • Culture conditions 35°C, 200rpm, culture time 40h.
  • the detection steps of intracellular products in the strain are as follows:
  • Detector FID, column temperature 140°C, injection temperature 200°C, detector temperature 220°C, temperature programming, 10°C/min, temperature rise to 220°C, capillary column SP-2560, injection volume 0.5 ⁇ L.
  • the GC spectra of standard samples and test samples are shown in Figures 6 and 7. It can be seen from the standard sample that the peak at retention time about 3.5min is 3-hydroxybutyric acid (3HB), and the peak at about 5.2min is the benzoic acid standard substance. Therefore, in conjunction with the FTIR verification results, the intracellular metabolism of the bacterial strain of the present invention
  • the product is a polymer of 3-hydroxybutyric acid, ie poly(3-hydroxybutyrate), ie PHB.
  • Embodiment 2 utilizes the method for Halomonas of the present invention to produce PHA in fermentor tank
  • Bacteria were taken from a 4°C refrigerator in the laboratory, and a single colony was picked with an inoculation loop to streak and inoculate on a solid plate, using solid plate medium, and cultured for 24 hours. Repeat the above operation, inoculate the second stage of the plate, and cultivate for 24 hours.
  • Primary bacteria liquid Take the secondary plate, pick a single colony and inoculate it in the seed medium, place the culture solution on a shaker at 25-40°C, 150-220rpm and cultivate for 12 hours.
  • Secondary bacterial solution absorb 500 ⁇ L of primary bacterial solution (1% inoculum size), inoculate it into the seed medium, place the culture solution on a shaker at 37° C., and incubate at 220 rpm for 12 hours.
  • Table 1 Components of four different fermentation media and their corresponding bacterial dry weight, PHA content and weight average molecular weight
  • CDW is calculated after M1, M2 and M3 are basically unchanged.
  • the detection steps of PHB content in the strain are as follows:
  • 3HB standard sample preparation (PHB is polymerized from 3HB monomer): Weigh five PHB standard samples, and perform pre-injection treatment according to the PHA digestion procedure.
  • Detector FID, column temperature 140°C, injection temperature 200°C, detector temperature 220°C, temperature programming, 10°C/min, temperature rise to 220°C, capillary column SP-2560, injection volume 0.5 ⁇ L.
  • the dry weight of the bacteria fermented with medium 1 was 93.3g/L, and the PHA content was 69.5%.
  • the dry weight of the thalline fermented with medium 2 was 112.7g/L, and the PHA content was 62%.
  • the dry weight of the thalline fermented with medium 3 was 105.5g/L, and the PHA content was 55.1%.
  • the dry weight of the thalline fermented with medium 4 was 198.5g/L, and the PHA content reached 75.1%.
  • the results of the GC spectrum are shown in Figure 9 (medium 1), Figure 10 (medium 2), Figure 11 (medium 3), and Figure 12 (medium 4).
  • Sample pretreatment method 100°C, pressure-resistant seal, 15min
  • the molecular weight test results are shown in Figure 13 (medium 1), Figure 14 (medium 2), Figure 15 (medium 3), and Figure 16 (medium 4).
  • the weight average molecular weight of PHB obtained using medium 1 was 104003Da
  • the weight average molecular weight of PHB obtained using medium 2 was 90126Da
  • the weight average molecular weight of PHB obtained using medium 3 was 121083Da
  • the weight average molecular weight of PHB obtained using medium 4 was The weight average molecular weight is 83168Da.
  • Different applications require PHAs with different molecular weights, and the method of the present invention can prepare PHAs with different molecular weights when using different medium components, which is suitable for various purposes.
  • Example 2 the medium 4 in Example 2 was used, and the other operation steps were the same, only 4 sets of comparative experiments were set up on the culture conditions to verify whether different culture conditions had an impact on the yield of PHB products.
  • the 4 different culture conditions are as follows:
  • the gas chromatography results of products under culture conditions 1-4 are shown in Figure 17 (medium 1), Figure 18 (medium 2), Figure 19 (medium 3), and Figure 20 (medium 4).
  • the peak with a retention time of about 3.5 minutes in the figure is 3-hydroxybutyric acid (3HB), and PHB is formed by the polymerization of 3HB monomer.
  • 3HB 3-hydroxybutyric acid
  • PHB is formed by the polymerization of 3HB monomer.
  • the peak with a retention time of about 3.5 min in Figure 20 is higher and has a larger area. Therefore, under culture condition 4, a higher content of PHB can be obtained.
  • the two selected sequences are highly conserved sequences of Halomonas lutescens MDF-9. Among them, the two pairs of primers are:
  • the fragment length is 486bp
  • R5'-CGCTCCGTTTACGGTAGTGTTGT-3' (SEQ ID NO.5), the fragment length is 432bp.
  • the PCR detection results are shown in Figure 21 and Figure 22.
  • A is the sample taken after 12 hours of fermentation
  • B is the sample taken after 48 hours of fermentation.
  • a total of 60 samples were taken for PCR verification.
  • M is the marker
  • C is the positive control of Halomonas lutescens MDF-9 purebred as the template
  • 1-10 is the partial sampling sample.
  • the results of colony morphology and PCR verification showed that no other bands were amplified except for the band of the target fragment, indicating that there was no bacterial infection during the fermentation process, indicating that non-sterile fermentation is feasible, which greatly reduces the energy consumption of the production process , reduces the cost and simplifies the fermentation process.
  • Halomonas lutescens MDF-9 For halophilic bacteria, high salt concentration is necessary for growth. At the same time, the growth of Halomonas lutescens MDF-9 also depends on a high pH value, and high salt and high pH medium can inhibit the growth of other non-halophilic bacteria. Therefore, a sterile production process is possible.
  • the NaCl concentration of the disclosed medium components in the prior art is 20-200g/L, while the NaCl concentration of the present invention is only 10-20g/L, which is lower than the higher salt concentration at a lower salt concentration.
  • the culture method of the present invention can also realize non-sterile fermentation, reduce the energy consumption produced by the sterilization process, and minimize the damage to the microbial culture medium, thereby to a greater extent cut costs.
  • the low salt concentration will also lead to easier wastewater treatment in the later stage and save production costs.
  • the culture medium of the present invention has simple components and does not contain cobalt chloride and nickel chloride, which may be sensitized by inhalation or skin contact. Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment. Therefore, the medium without cobalt chloride and nickel chloride is safer and more environmentally friendly.

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Abstract

Disclosed in the present invention are a Halomonas lutescens strain and the use thereof. The Halomonas lutescens strain is Halomonas lutescens MDF-9 with the deposit number of GDMCC NO. 61850. The Halomonas lutescens strain of the present invention can efficiently accumulate polyhydroxyalkanoate (PHA) in a culture medium of the present invention. A fermentation process of the Halomonas lutescens strain of the present invention does not require a sterilization, can be performed continuously, and is easy to control. In the fermentation process, the concentration of sodium chloride is low and is 10-20 g/L, the late-stage wastewater is easily treated, the nutrient components of a culture medium used in the fermentation are simple, and a toxic and harmful metal compound is not used. A method in the present invention for preparing PHA by means of using Halomonas lutescens MDF-9 reduces the production cost and increases the yield of PHA.

Description

一株盐单胞菌及其应用A Strain of Halomonas and Its Application 技术领域technical field
本发明涉及微生物发酵领域，特别涉及一株盐单胞菌及其应用。The invention relates to the field of microbial fermentation, in particular to a halomonas strain and its application.
背景技术Background technique
化基塑料降解速度慢、周期长，特别是形成微塑料后更加难以处理，其大量使用会对环境造成严重污染。因此开发生物基可降解塑料有利于环境健康发展。The degradation rate of chemical-based plastics is slow and the cycle is long, especially after the formation of microplastics, it is more difficult to handle, and its large-scale use will cause serious pollution to the environment. Therefore, the development of bio-based degradable plastics is conducive to the healthy development of the environment.
聚羟基脂肪酸酯(polyhydroxyalkanoates，PHA)是一种完全由微生物在碳源过量而其他营养物质限制的条件下胞内合成并储存的生物材料。根据单体碳链长度不同分为短链PHA(4-6C，scl-PHA)和中长链PHA(≥6C，mcl-PHA)。聚-β-羟丁酸(poly-β-hydroxybutyrate，PHB)为短链PHA中的一种。PHA具有可降解性、生物相容性以及质地多样性等特点，在化工产品、医用植入材料、药物缓释载体等领域具有广泛的应用前景。Polyhydroxyalkanoates (PHA) are biological materials that are completely synthesized and stored intracellularly by microorganisms under the conditions of excess carbon source and limited other nutrients. According to the carbon chain length of the monomer, it is divided into short-chain PHA (4-6C, scl-PHA) and medium-long chain PHA (≥6C, mcl-PHA). Poly-β-hydroxybutyrate (poly-β-hydroxybutyrate, PHB) is a kind of short-chain PHA. PHA has the characteristics of degradability, biocompatibility and texture diversity, and has broad application prospects in chemical products, medical implant materials, drug sustained-release carriers and other fields.
虽然近十年来PHA产业链发展迅猛，但PHA制造过程的复杂性使其制造成本高昂，无法与以石油为基础的传统材料竞争。主要原因包括原材料成本、能耗、下游处理成本等。所以目前试图采取很多方法来降低PHA的生产成本，比如，通过基因工程改造或代谢途径改造来获得性状优良的高产菌、探索简单有效的下游提取纯化方法、利用价格低廉的原材料等。现有技术公开了一些盐单胞菌，能够实现在非灭菌条件下的PHA生产，可有效降低生产成本，消除无菌操作复杂性。但是也存在明显的缺陷：(1)NaCl浓度高达20～200g/L，导致后期废水难以处理；(2)培养过程中使用氯化钴和氯化镍，均为有毒有害物质；(3)培养基成分复杂。因此，目前亟需一种能够在低盐浓度和无有害物质的发酵条件下生产PHA的盐单胞菌和发酵方法。Although the PHA industry chain has developed rapidly in the past decade, the complexity of the PHA manufacturing process makes its manufacturing cost high and cannot compete with traditional petroleum-based materials. The main reasons include raw material cost, energy consumption, downstream processing cost, etc. Therefore, many methods are currently being tried to reduce the production cost of PHA, such as obtaining high-yielding bacteria with excellent properties through genetic engineering or metabolic pathway modification, exploring simple and effective downstream extraction and purification methods, and using low-cost raw materials. The prior art discloses some Halomonas, which can realize the production of PHA under non-sterile conditions, which can effectively reduce the production cost and eliminate the complexity of aseptic operation. But there are also obvious defects: (1) the NaCl concentration is as high as 20-200g/L, which makes it difficult to treat the wastewater in the later stage; (2) cobalt chloride and nickel chloride are used in the cultivation process, which are poisonous and harmful substances; (3) the cultivation The basic components are complex. Therefore, there is an urgent need for a Halomonas and a fermentation method capable of producing PHA under low-salt concentration and fermentation conditions without harmful substances.
发明内容Contents of the invention
针对现有技术中的缺陷，本发明提出了一株盐单胞菌及其应用，该菌株为盐单胞菌(Halomonas lutescens)MDF-9，其保藏编号为GDMCC NO.61850。本发明的盐单胞菌可在本发明的培养基中高效积累聚羟基脂肪酸酯(PHA)，为PHA的生物合成提供了良好的保证。Aiming at the defects in the prior art, the present invention proposes a strain of Halomonas and its application. The strain is Halomonas lutescens MDF-9, and its preservation number is GDMCC NO.61850. The Halomonas of the present invention can efficiently accumulate polyhydroxyalkanoate (PHA) in the culture medium of the present invention, which provides a good guarantee for the biosynthesis of PHA.
本发明提供一株盐单胞菌(Halomonas lutescens MDF-9)，所述盐单胞菌的保藏编号为GDMCC NO.61850。The invention provides a strain of Halomonas lutescens MDF-9, and the preservation number of the Halomonas lutescens is GDMCC NO.61850.
本发明还提供所述的盐单胞菌在制备聚羟基脂肪酸酯中的应用。The present invention also provides the application of the Halomonas in the preparation of polyhydroxyalkanoate.
进一步的，所述聚羟基脂肪酸酯为聚-β-羟丁酸。Further, the polyhydroxyalkanoate is poly-β-hydroxybutyric acid.
本发明还提供一种制备聚羟基脂肪酸酯的方法，包括如下步骤：发酵盐单胞菌(Halomonas lutescens MDF-9)GDMCC NO.61850，得到聚羟基脂肪酸酯，所述发酵中使用的发酵培养基及发酵容器不灭菌。不灭菌是为了节约流程和成本，更能体现本发明的价值，灭菌同样可以实现本发明的技术效果。The present invention also provides a method for preparing polyhydroxyalkanoate, comprising the steps of: fermenting Halomonas lutescens MDF-9 GDMCC NO.61850 to obtain polyhydroxyalkanoate, and the fermentation used in the fermentation Culture medium and fermentation vessels are not sterilized. The purpose of non-sterilization is to save process and cost, and to better reflect the value of the present invention. Sterilization can also achieve the technical effect of the present invention.
进一步的，所述发酵培养基的氯化钠浓度为10～20g/L。本发明的发酵培养基的盐浓度低，更有利于废水的处理。Further, the sodium chloride concentration of the fermentation medium is 10-20 g/L. The salt concentration of the fermentation medium of the invention is low, which is more conducive to the treatment of waste water.
进一步的，所述发酵的pH值为8～10，搅拌速度为50～800rpm，通气量为0.1～2vvm， 发酵温度为25～40℃。在上述发酵条件下PHA的产量更高。Further, the pH value of the fermentation is 8-10, the stirring speed is 50-800 rpm, the ventilation volume is 0.1-2vvm, and the fermentation temperature is 25-40°C. The yield of PHA is higher under the above fermentation conditions.
进一步优选为：所述发酵的pH值为9～10，通气量为0.8～2vvm，发酵温度为37℃～40℃。More preferably: the pH value of the fermentation is 9-10, the ventilation rate is 0.8-2vvm, and the fermentation temperature is 37°C-40°C.
进一步的，所述发酵的方法包括如下步骤：Further, the fermentation method comprises the following steps:
(1)将盐单胞菌(Halomonas lutescens MDF-9)GDMCC NO.61850接入发酵培养基，进行发酵培养；(1) Insert Halomonas lutescens MDF-9 GDMCC NO.61850 into the fermentation medium for fermentation;
(2)发酵过程中检测发酵液中糖的浓度低于10g/L时，补加糖使其维持在30g/L；(2) When the concentration of sugar in the fermentation broth is detected to be lower than 10g/L during the fermentation process, add sugar to maintain it at 30g/L;
(3)继续培养30～40h，待发酵液中糖浓度低于0.2g/L时，结束发酵。(3) Continue culturing for 30-40 hours, and stop the fermentation when the sugar concentration in the fermentation broth is lower than 0.2 g/L.
本发明的另一个方面是提供一种用于盐单胞菌发酵的培养基。Another aspect of the present invention is to provide a medium for Halomonas fermentation.
所述发酵培养基包括如下组分：The fermentation medium comprises the following components:
碳源：葡萄糖、果糖、葡萄糖酸钠中的任意一种或多种；本发明的菌株不仅能够以葡萄糖作为碳源，还可以利用果糖和葡萄糖酸钠。Carbon source: any one or more of glucose, fructose, and sodium gluconate; the bacterial strain of the present invention can not only use glucose as a carbon source, but also utilize fructose and sodium gluconate.
氮源：玉米浆干粉、酵母粉、胰蛋白胨、尿素、硫酸铵、氯化铵；Nitrogen source: corn steep liquor dry powder, yeast powder, tryptone, urea, ammonium sulfate, ammonium chloride;
其他营养成分：磷酸二氢钾、磷酸氢二钠、硫酸镁；Other nutrients: potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate;
氯化钠；Sodium chloride;
所述发酵培养基中不包含氯化钴和氯化镍。氯化钴和氯化镍均为有毒金属化合物，吸入及皮肤接触可能致敏。对水生生物有极高毒性，可能对水体环境产生长期不良影响。因此去除了氯化钴和氯化镍的培养基更加安全和环境友好。The fermentation medium does not contain cobalt chloride and nickel chloride. Cobalt chloride and nickel chloride are toxic metal compounds, which may cause sensitization by inhalation and skin contact. Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment. Therefore, the medium without cobalt chloride and nickel chloride is safer and more environmentally friendly.
进一步的，所述碳源的总浓度为90～150g/L。Further, the total concentration of the carbon source is 90-150 g/L.
优选地，按照质量体积比，所述玉米浆干粉的含量为1～8％，所述酵母粉的含量为0.5～5％，所述胰蛋白胨的含量为0.1～3％，所述尿素的含量为0.1～3％，所述硫酸铵的含量为0.1～3％，所述氯化铵的含量为0.1～3％。Preferably, according to the mass volume ratio, the content of the corn steep liquor dry powder is 1-8%, the content of the yeast powder is 0.5-5%, the content of the tryptone is 0.1-3%, and the content of the urea 0.1-3%, the content of the ammonium sulfate is 0.1-3%, and the content of the ammonium chloride is 0.1-3%.
优选地，所述磷酸二氢钾的含量为0.1～2％，所述磷酸氢二钠的含量为0.05～1％，所述硫酸镁的含量为0.05～1％，所述含量为质量体积比。Preferably, the content of potassium dihydrogen phosphate is 0.1-2%, the content of disodium hydrogen phosphate is 0.05-1%, the content of magnesium sulfate is 0.05-1%, and the content is mass volume ratio .
所述发酵培养基的氯化钠浓度为10～20g/L。The sodium chloride concentration of the fermentation medium is 10-20 g/L.
进一步的，所述聚羟基脂肪酸酯为聚-β-羟丁酸。Further, the polyhydroxyalkanoate is poly-β-hydroxybutyric acid.
综上，与现有技术相比，本发明达到了以下技术效果：In summary, compared with the prior art, the present invention achieves the following technical effects:
1、发酵过程中氯化钠浓度低，为10～20g/L，后期废水易于处理。1. The concentration of sodium chloride is low during the fermentation process, which is 10-20g/L, and the wastewater in the later stage is easy to treat.
2、发酵营养成分简单，不使用有毒有害的金属化合物。2. Fermentation nutrients are simple, and no toxic and harmful metal compounds are used.
3、本发明的菌株使用本发明的发酵培养基在发酵罐培养后，菌体干重可达到198.5g/L，PHA的含量可达到79.5％。3. After the bacterial strain of the present invention is cultivated in a fermenter using the fermentation medium of the present invention, the dry weight of the bacteria can reach 198.5g/L, and the content of PHA can reach 79.5%.
附图说明Description of drawings
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。In order to illustrate the technical solutions of the embodiments of the present invention more clearly, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention, and thus It should be regarded as a limitation on the scope, and those skilled in the art can also obtain other related drawings based on these drawings without creative work.
图1为本发明实施例1中菌株涂布平板后的单菌落形态图。Figure 1 is a single colony diagram of the bacterial strain in Example 1 of the present invention after being coated on a plate.
图2为本发明实施例1中菌株发酵前显微镜观察的图像。Fig. 2 is an image of microscopic observation of the bacterial strain in Example 1 of the present invention before fermentation.
图3为本发明实施例1中菌株发酵后显微镜观察的图像。Fig. 3 is an image of microscopic observation of the bacterial strain in Example 1 of the present invention after fermentation.
图4为本发明实施例1中sigma公司的PHB标准品FTIR图谱。Fig. 4 is the FTIR spectrum of the PHB standard product of sigma company in the embodiment 1 of the present invention.
图5为本发明实施例1中制备PHB样品的FTIR图谱。Fig. 5 is the FTIR spectrum of the PHB sample prepared in Example 1 of the present invention.
图6为本发明实施例1中标准样品的GC谱图。Fig. 6 is the GC spectrogram of the standard sample in Example 1 of the present invention.
图7为本发明实施例1中检测样品的GC谱图。Fig. 7 is the GC spectrogram of the sample tested in Example 1 of the present invention.
图8为本发明实施例2中制备PHB样品的FTIR图谱。Fig. 8 is the FTIR spectrum of the PHB sample prepared in Example 2 of the present invention.
图9为本发明实施例2中不灭菌、使用培养基1制备PHB样品的GC图谱。Fig. 9 is a GC spectrum of a PHB sample prepared using medium 1 without sterilization in Example 2 of the present invention.
图10为本发明实施例2中不灭菌、使用培养基2制备PHB样品的GC图谱。Fig. 10 is the GC spectrum of the PHB sample prepared without sterilization and using medium 2 in Example 2 of the present invention.
图11为本发明实施例2中不灭菌、使用培养基3制备PHB样品的GC图谱。Fig. 11 is the GC chromatogram of the PHB sample prepared without sterilization and using medium 3 in Example 2 of the present invention.
图12为本发明实施例2中不灭菌、使用培养基4制备PHB样品的GC图谱。Fig. 12 is a GC spectrum of a PHB sample prepared without sterilization and using medium 4 in Example 2 of the present invention.
图13为本发明实施例2中不灭菌、使用培养基1制备PHB样品的GPC图谱。Fig. 13 is the GPC chart of the PHB sample prepared without sterilization and using medium 1 in Example 2 of the present invention.
图14为本发明实施例2中不灭菌、使用培养基2制备PHB样品的GPC图谱。Fig. 14 is the GPC chart of the PHB sample prepared without sterilization and using medium 2 in Example 2 of the present invention.
图15为本发明实施例2中不灭菌、使用培养基3制备PHB样品的GPC图谱。Fig. 15 is the GPC chart of the PHB sample prepared without sterilization and using medium 3 in Example 2 of the present invention.
图16为本发明实施例2中不灭菌、使用培养基4制备PHB样品的GPC图谱。Fig. 16 is the GPC chart of the PHB sample prepared without sterilization and using medium 4 in Example 2 of the present invention.
图17为本发明实施例3中不灭菌、培养条件1下制备PHB样品的气相色谱结果。Fig. 17 is the gas chromatographic result of the PHB sample prepared under the culture condition 1 without sterilization in Example 3 of the present invention.
图18为本发明实施例3中不灭菌、培养条件2下制备PHB样品的气相色谱结果。Fig. 18 is the gas chromatographic result of the PHB sample prepared under the culture condition 2 without sterilization in Example 3 of the present invention.
图19为本发明实施例3中不灭菌、培养条件3下制备PHB样品的气相色谱结果。Fig. 19 is the gas chromatography result of the PHB sample prepared under the non-sterilized and culture condition 3 in Example 3 of the present invention.
图20为本发明实施例3中不灭菌、培养条件4下制备PHB样品的气相色谱结果。Fig. 20 is the gas chromatographic result of the PHB sample prepared under non-sterile and culture condition 4 in Example 3 of the present invention.
图21为实施例4中486bp产物的电泳结果。Figure 21 is the electrophoresis result of the 486bp product in Example 4.
图22为实施例4中432bp产物的电泳结果。Figure 22 is the electrophoresis result of the 432bp product in Example 4.
具体实施方式Detailed ways
为了使本技术领域的人员更好地理解本发明方案，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分的实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所获得的所有其他实施例，都应当属于本发明保护的范围。In order to enable those skilled in the art to better understand the solutions of the present invention, the following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only It is an embodiment of a part of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by persons of ordinary skill in the art without making creative efforts shall fall within the protection scope of the present invention.
本发明提供了一株盐单胞菌(Halomonas lutescens MDF-9)，其可在胞内积累PHA。The invention provides a strain of Halomonas lutescens MDF-9, which can accumulate PHA in the cell.
一、培养条件：氯化钠浓度10～20g/L，pH 8～10，通气量为0.1～2vvm，温度25～40℃。1. Culture conditions: sodium chloride concentration 10-20g/L, pH 8-10, ventilation volume 0.1-2vvm, temperature 25-40℃.
二、培养基组成：2. Culture medium composition:
碳源：葡萄糖、果糖、葡萄糖酸钠中的任意一种或多种，总浓度为90～150g/L。Carbon source: any one or more of glucose, fructose, and sodium gluconate, with a total concentration of 90-150 g/L.
氮源：玉米浆干粉1～8％、酵母粉0.5～5％、胰蛋白胨0.1～3％、尿素0.1～3％、硫酸铵0.1～3％、氯化铵0.1～3％(m/v)。Nitrogen source: corn steep liquor dry powder 1-8%, yeast powder 0.5-5%, tryptone 0.1-3%, urea 0.1-3%, ammonium sulfate 0.1-3%, ammonium chloride 0.1-3% (m/v) .
氯化钠10～20g/L。Sodium chloride 10-20g/L.
其他营养成分：磷酸二氢钾0.1～2％、磷酸氢二钠0.05～1％、硫酸镁0.05～1％(m/v)。Other nutrients: potassium dihydrogen phosphate 0.1-2%, disodium hydrogen phosphate 0.05-1%, magnesium sulfate 0.05-1% (m/v).
三、利用本发明的盐单胞菌生产PHA的方法如下：Three, the method utilizing Halomonas of the present invention to produce PHA is as follows:
(1)将盐单胞菌(Halomonas lutescens MDF-9)GDMCC NO.61850接入发酵培养基，进行发酵培养；(1) Insert Halomonas lutescens MDF-9 GDMCC NO.61850 into the fermentation medium for fermentation;
(2)发酵过程中检测发酵液中糖的浓度低于10g/L时，补加糖使其维持在30g/L；(2) When the concentration of sugar in the fermentation broth is detected to be lower than 10g/L during the fermentation process, add sugar to maintain it at 30g/L;
(3)继续培养30～40h，待发酵液中糖浓度低于0.2g/L时，结束发酵。(3) Continue culturing for 30-40 hours, and stop the fermentation when the sugar concentration in the fermentation broth is lower than 0.2 g/L.
具体的，将本发明的盐单胞菌接种到液体培养基中，培养基成分包括上述碳源中的一种或两种或三种的混合，糖浓度为90～150g/L，氯化钠10～20g/L，玉米浆干粉1～8％、酵母粉0.5～5％、胰蛋白胨0.1～3％、尿素0.1～3％、硫酸铵0.1～3％、氯化铵0.1～3％(m/v)，磷酸二氢钾0.1～2％、磷酸氢二钠0.05～1％、硫酸镁0.05～1％(m/v)。Specifically, the Halomonas of the present invention is inoculated into a liquid medium, and the medium components include one or a mixture of two or three of the above carbon sources, the sugar concentration is 90-150g/L, sodium chloride 10-20g/L, 1-8% corn steep liquor dry powder, 0.5-5% yeast powder, 0.1-3% tryptone, 0.1-3% urea, 0.1-3% ammonium sulfate, 0.1-3% ammonium chloride (m /v), potassium dihydrogen phosphate 0.1-2%, disodium hydrogen phosphate 0.05-1%, magnesium sulfate 0.05-1% (m/v).
在pH 8～10，通气量为0.1～2vvm，搅拌50～800rpm，温度25～40℃条件下培养30～40h，当发酵液中糖浓度低于10g/L时，通过补加1500～800g/L的葡萄糖和0.5～10g/L的氯化铵溶液，维持糖浓度在10～50g/L，结束时糖浓度为小于0.2g/L。Cultivate for 30-40 hours at a pH of 8-10, ventilation rate 0.1-2vvm, stirring 50-800rpm, and temperature 25-40°C. When the sugar concentration in the fermentation broth is lower than 10g/L, add 1500-800g/L L of glucose and 0.5-10g/L ammonium chloride solution to maintain the sugar concentration at 10-50g/L, and the sugar concentration at the end is less than 0.2g/L.
以下实施例中使用的3种培养基有平板固体培养基和2种液体培养基，平板固体培养基用来涂布菌株。种子培养基和发酵培养基分别用来摇瓶培养和5L发酵罐培养本发明的盐单胞菌。它们的具体组成如下：The 3 kinds of culture medium used in the following examples are flat solid medium and 2 kinds of liquid medium, and the flat solid medium is used to coat bacterial strains. The seed culture medium and the fermentation medium are used for shake flask culture and 5L fermenter culture Halomonas of the present invention respectively. Their specific composition is as follows:
平板固体培养基：酵母粉，0.5～1％；胰蛋白胨，0.5～1％；氯化钠1～2％，琼脂粉18g/L，pH 9.0。Plate solid medium: yeast powder, 0.5-1%; tryptone, 0.5-1%; sodium chloride 1-2%, agar powder 18g/L, pH 9.0.
种子培养基：葡萄糖溶液，30～50g/L；氯化钠10～20g/L，玉米浆干粉1～8％、酵母粉0.5～5％、胰蛋白胨0.1～3％、尿素0.1～3％、硫酸铵0.1～3％、氯化铵0.1～3％(m/v)，磷酸二氢钾0.1～2％、磷酸氢二钠0.05～1％、硫酸镁0.05～1％(m/v)。氢氧化钠调节培养基pH至8.0～10，培养基体积为50mL(250mL三角瓶)。Seed medium: glucose solution, 30-50g/L; sodium chloride 10-20g/L, corn steep liquor dry powder 1-8%, yeast powder 0.5-5%, tryptone 0.1-3%, urea 0.1-3%, Ammonium sulfate 0.1-3%, ammonium chloride 0.1-3% (m/v), potassium dihydrogen phosphate 0.1-2%, disodium hydrogen phosphate 0.05-1%, magnesium sulfate 0.05-1% (m/v). Sodium hydroxide was used to adjust the pH of the medium to 8.0-10, and the volume of the medium was 50 mL (250 mL Erlenmeyer flask).
发酵培养基：葡萄糖溶液，90～150g/L；氯化钠10～20g/L，玉米浆干粉1～8％、酵母粉0.5～5％、胰蛋白胨0.1～3％、尿素0.1～3％、硫酸铵0.1～3％、氯化铵0.1～3％(m/v)，磷酸二氢钾0.1～2％、磷酸氢二钠0.05～1％、硫酸镁0.05～1％(m/v)。氢氧化钠调节培养基pH至8～10。Fermentation medium: glucose solution, 90-150g/L; sodium chloride 10-20g/L, corn steep liquor dry powder 1-8%, yeast powder 0.5-5%, tryptone 0.1-3%, urea 0.1-3%, Ammonium sulfate 0.1-3%, ammonium chloride 0.1-3% (m/v), potassium dihydrogen phosphate 0.1-2%, disodium hydrogen phosphate 0.05-1%, magnesium sulfate 0.05-1% (m/v). Sodium hydroxide was used to adjust the pH of the medium to 8-10.
本发明经过5L发酵罐实验验证，培养30～40h，发酵终点发酵液中菌体干重为90～200g/L，干菌体中PHA含量为50～80％。The invention is verified by a 5L fermentation tank experiment, and the culture is 30-40 hours, the dry weight of the bacteria in the fermentation liquid at the end of fermentation is 90-200g/L, and the PHA content in the dry bacteria is 50-80%.
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.
实施例1菌株的分离、纯化的鉴定Identification of the isolation and purification of embodiment 1 bacterial strain
一、菌株分离1. Isolation of strains
取100μL的青海湖水涂布到分离纯化固体培养基中(培养基成分：酵母粉10g/L，胰蛋白胨5g/L，氯化钠35g/L，琼脂粉18g/L，pH＝9.0)，33℃静止培养24h。Take 100 μL of Qinghai Lake water and apply it to the separation and purification solid medium (medium components: yeast powder 10g/L, tryptone 5g/L, sodium chloride 35g/L, agar powder 18g/L, pH=9.0), Incubate statically at 33°C for 24h.
二、菌株纯化2. Strain purification
挑去固体培养基中长出的单菌落，用35g/L的盐水稀释100～10000倍，涂布于上述固体培养基上，33℃静止培养24h。反复重述上述过程，直至固体培养基上长出的单菌落形体一致，完成菌株纯化。Pick out the single colony grown in the solid medium, dilute it 100-10000 times with 35g/L saline, spread it on the above-mentioned solid medium, and culture it statically at 33°C for 24h. Repeat the above process repeatedly until the single colony grown on the solid medium has the same shape, and the strain purification is completed.
三、菌株的鉴定3. Identification of strains
肉眼可见，菌落形态如图1所示，呈圆形，白色，中间有些许凸起，边缘完整，不透明。革兰氏染色后利用显微镜观察菌体形态，发酵前的菌体形态如图2所示，菌体呈短杆、无芽孢、革兰氏阴性。发酵后期的菌体形态如图3所示，可见菌体中间呈白色透明状，两端着色，有鞭毛，能运动。检测该菌株的16S rDNA序列，测得的序列如SEQ ID NO.1所示。将该序列在NCBI数据库中比对，结果该菌株与Halomonas lutescens Q1U相似度达99％。综合以上鉴定结果，将该菌株鉴定为盐单胞菌(Halomonas lutescens)。该菌株已于2021年8月2日保藏于广东省微生物菌种保藏中心(简称GDMCC，地址：广州市先列中路100号大院59号楼5楼，广东省微生物研究所，邮编510070)。保藏号为GDMCC NO.61850。菌株名称为MDF-9，分类命名为盐单胞菌(Halomonas lutescens)。Visible to the naked eye, the shape of the colony is shown in Figure 1, which is round, white, slightly raised in the middle, and the edges are complete and opaque. After Gram staining, the morphology of the thalli was observed under a microscope. The thalli morphology before fermentation was shown in Figure 2, and the thalli were short rods, non-spore-free, and Gram-negative. The morphology of the thalline in the later stage of fermentation is shown in Figure 3. It can be seen that the middle of the thallus is white and transparent, with colored ends, flagella, and can move. Detect the 16S rDNA sequence of the bacterial strain, and the measured sequence is shown in SEQ ID NO.1. The sequence was compared in the NCBI database, and the similarity between the strain and Halomonas lutescens Q1U was 99%. Based on the above identification results, the strain was identified as Halomonas lutescens. The strain has been preserved in the Guangdong Provincial Microbial Culture Collection Center (GDMCC for short, address: 5th Floor, Building 59, Compound, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Institute of Microbiology, Zip code 510070) on August 2, 2021. The deposit number is GDMCC NO.61850. The strain name is MDF-9, and the classification is named Halomonas lutescens.
SEQ ID NO.1SEQ ID NO.1
Figure PCTCN2022095741-appb-000001
Figure PCTCN2022095741-appb-000001
四、菌株产PHA验证实验4. Verification experiment of strain producing PHA
将纯化后的本发明菌株接种到种子培养基(种子培养基成分：葡萄糖90g/L；氯化 钠35g/L，玉米浆干粉1％、酵母粉0.5％、尿素0.1％、硫酸铵0.1％、氯化铵0.1％，磷酸二氢钾0.3％、磷酸氢二钠0.1％、硫酸镁0.1％，(m/v)。氢氧化钠调节培养基pH至9.0，培养基体积为30mL(250mL三角瓶)中，利用摇瓶培养进行验证是否合成胞内PHA。培养条件：35℃，200rpm，培养时间40h。The bacterial strain of the present invention after purifying is inoculated to seed culture medium (seed culture medium composition: glucose 90g/L; Sodium chloride 35g/L, corn steep liquor dry powder 1%, yeast powder 0.5%, urea 0.1%, ammonium sulfate 0.1%, Ammonium chloride 0.1%, potassium dihydrogen phosphate 0.3%, disodium hydrogen phosphate 0.1%, magnesium sulfate 0.1%, (m/v).Sodium hydroxide regulates the pH of the medium to 9.0, and the volume of the medium is 30mL (250mL Erlenmeyer flask ), using shake flask culture to verify whether intracellular PHA is synthesized. Culture conditions: 35°C, 200rpm, culture time 40h.
(一)Halomonas lutescens胞内代谢产物FTIR验证(1) FTIR verification of intracellular metabolites of Halomonas lutescens
取发酵结束后的发酵液30mL离心8000rpm，5min，去掉上清液，然后加入去离子水25mL，震荡摇匀后8000rpm，5min，去掉上清液，然后加入去离子水25mL，震荡摇匀后8000rpm，5min，去掉上清液，然后加入去离子水5mL，再加入10mL三氯甲烷，充分混合震荡60min，8000rpm离心5min，取下层三氯甲烷相，旋转蒸发去掉三氯甲烷，收集所得白色粉末，80℃干燥24h。进行FTIR分析，结果如图5所示。After the fermentation, take 30mL of the fermentation broth and centrifuge it at 8000rpm for 5min, remove the supernatant, then add 25mL of deionized water, shake it up at 8000rpm, 5min, remove the supernatant, then add 25mL of deionized water, shake it up at 8000rpm , 5min, remove the supernatant, then add 5mL of deionized water, then add 10mL of chloroform, mix and shake for 60min, centrifuge at 8000rpm for 5min, remove the lower chloroform phase, remove the chloroform by rotary evaporation, collect the white powder obtained, Dry at 80°C for 24h. FTIR analysis was performed and the results are shown in Figure 5.
Sigma公司的PHB标准品FTIR图谱如图4所示，C＝O峰(1723-1740)为PHB特征峰。可见图5中在1723-1740有PHB特征峰，初步证明本发明的菌株的确可以合成PHA，且含量为46％(w/w)。The FTIR spectrum of the PHB standard product of Sigma Company is shown in Figure 4, and the C=O peak (1723-1740) is a characteristic peak of PHB. It can be seen that there are PHB characteristic peaks at 1723-1740 in Fig. 5, which preliminarily proves that the bacterial strain of the present invention can indeed synthesize PHA, and the content is 46% (w/w).
(二)Halomonas lutescens胞内代谢产物GC验证(2) GC verification of intracellular metabolites of Halomonas lutescens
菌株中胞内产物检测步骤如下：The detection steps of intracellular products in the strain are as follows:
(1)消解(1) digestion
(a)向消解杯中分别加入2mL液体A(由15mL浓硫酸+485mL甲醇+0.5g苯甲酸配制而成)和2mL三氯甲烷，混匀后加入发酵结束后的干菌体(约含100mg干菌体)。(a) Add 2mL of liquid A (prepared from 15mL of concentrated sulfuric acid + 485mL of methanol + 0.5g of benzoic acid) and 2mL of chloroform into the digestion cup, mix well and add the dried bacteria after fermentation (about 100mg dried bacteria).
(b)将消解杯放入消解仪器中，温度100℃，消解时间3～4小时。(b) Put the digestion cup into the digestion apparatus, the temperature is 100°C, and the digestion time is 3 to 4 hours.
(c)消解结束后，待消解杯冷却至室温，取出消解杯。往消解杯中加入1mL纯水，振荡1～2min后，静置0.5h。(c) After the digestion is finished, wait for the digestion cup to cool down to room temperature, and then take out the digestion cup. Add 1mL of pure water into the digestion cup, shake for 1-2min, and let stand for 0.5h.
(2)标准品制备：(2) Standard product preparation:
3HB标准样制备：称取50mg的PHB标准样品，按照PHA消解步骤进行进样前处理。3. Preparation of HB standard sample: Weigh 50 mg of PHB standard sample and perform pre-injection treatment according to the PHA digestion procedure.
(3)气相色谱检测：(3) Gas chromatography detection:
检测器：FID，柱温140℃，进样温度200℃，检测器温度220℃，采用程序升温，10℃/min，升温至220℃，毛细管柱SP-2560，进样量0.5μL。Detector: FID, column temperature 140°C, injection temperature 200°C, detector temperature 220°C, temperature programming, 10°C/min, temperature rise to 220°C, capillary column SP-2560, injection volume 0.5μL.
标准样品和检测样GC谱图如图6和7所示。由标准样品可知，在保留时间3.5min左右的峰为3-羟基丁酸(3HB)，5.2min左右的峰为苯甲酸标准品，因此，结合FTIR验证结果，本发明所述菌株的胞内代谢产物为3-羟基丁酸的聚合物，即聚(3-羟基丁酸)酯，即为PHB。The GC spectra of standard samples and test samples are shown in Figures 6 and 7. It can be seen from the standard sample that the peak at retention time about 3.5min is 3-hydroxybutyric acid (3HB), and the peak at about 5.2min is the benzoic acid standard substance. Therefore, in conjunction with the FTIR verification results, the intracellular metabolism of the bacterial strain of the present invention The product is a polymer of 3-hydroxybutyric acid, ie poly(3-hydroxybutyrate), ie PHB.
实施例2利用本发明的盐单胞菌在发酵罐中生产PHA的方法 Embodiment 2 utilizes the method for Halomonas of the present invention to produce PHA in fermentor tank
一、菌种活化1. Activation of bacteria
实验室4℃冰箱取菌种，用接种环挑取单菌落划线接种于固体平板上，使用固体平板培养基，培养24h。重复上述操作，接种平板二级，培养24h。Bacteria were taken from a 4°C refrigerator in the laboratory, and a single colony was picked with an inoculation loop to streak and inoculate on a solid plate, using solid plate medium, and cultured for 24 hours. Repeat the above operation, inoculate the second stage of the plate, and cultivate for 24 hours.
二、种子液制备2. Preparation of seed solution
一级菌液：取二级平板，挑取单菌落接种于种子培养基中，将培养液置于摇床 25～40℃、150～220rpm培养12h。Primary bacteria liquid: Take the secondary plate, pick a single colony and inoculate it in the seed medium, place the culture solution on a shaker at 25-40°C, 150-220rpm and cultivate for 12 hours.
二级菌液：吸取一级菌液500μL(1％接种量)，接种于种子培养基中将培养液置于摇床37℃、220rpm培养12h。Secondary bacterial solution: absorb 500 μL of primary bacterial solution (1% inoculum size), inoculate it into the seed medium, place the culture solution on a shaker at 37° C., and incubate at 220 rpm for 12 hours.
三、发酵罐培养3. Fermentation tank culture
接种前30分钟，配置发酵培养基，将种子液按1～20％的接种量接种到5L发酵罐中，使用4种不同配比的发酵培养基(培养基1、培养基2培养基3和培养基4)，见表1，调节并维持pH至9.0，通气量到0.5vvm，搅拌500rpm，温度32℃，发酵过程中检测发酵液中糖的浓度低于10g/L时，补加糖使其维持在30g/L。培养30～40h，待发酵液中糖浓度低于0.2g/L时，结束发酵。整个发酵过程中使用的发酵培养基及发酵容器均无需灭菌。30 minutes before the inoculation, configure the fermentation medium, inoculate the seed liquid into a 5L fermenter with an inoculum size of 1-20%, and use 4 kinds of fermentation medium with different ratios (medium 1, medium 2, medium 3 and Culture medium 4), see Table 1, adjust and maintain pH to 9.0, ventilation rate to 0.5vvm, stirring 500rpm, temperature 32°C, when the concentration of sugar in the fermentation liquid detected during fermentation is lower than 10g/L, add sugar to make it Maintained at 30g/L. Cultivate for 30-40 hours, and stop the fermentation when the sugar concentration in the fermentation liquid is lower than 0.2g/L. The fermentation medium and the fermentation container used in the whole fermentation process do not need to be sterilized.
表1 4种不同的发酵培养基的组分及其对应的菌体干重、PHA含量和重均分子量Table 1 Components of four different fermentation media and their corresponding bacterial dry weight, PHA content and weight average molecular weight
Figure PCTCN2022095741-appb-000002
Figure PCTCN2022095741-appb-000002
发酵结束后对菌体干重进行分析，方法如下：After the fermentation, the dry weight of the thalline was analyzed, and the method was as follows:
(1)取50ml离心管，80℃烘干两个小时，干燥器中至常温后称重，计M0；(1) Take a 50ml centrifuge tube, dry it at 80°C for two hours, put it in the desiccator to normal temperature, weigh it, and calculate M0;
(2)准确量取20mL发酵液，放入称重后50mL离心管中；(2) Accurately measure 20mL fermentation broth and put it into a weighed 50mL centrifuge tube;
(3)根据重量对称放置到离心机中，8000rpm离心5min；(3) symmetrically placed in the centrifuge according to the weight, and centrifuged at 8000rpm for 5min;
(4)去掉上清液，在菌体中加入20mL纯水，漩涡震荡至所有菌体全部呈悬浮状态；(4) Remove the supernatant, add 20 mL of pure water to the cells, and vortex until all the cells are suspended;
(5)再次进行离心洗涤，8000rpm离心5min，重复洗涤3次；(5) Carry out centrifugal washing again, 8000rpm centrifugal 5min, repeat washing 3 times;
(6)将离心后菌体放入80℃鼓风干燥箱中，干燥24h；(6) Put the centrifuged bacteria into an 80°C blast drying oven and dry for 24 hours;
(7)取出称重，计为M1；(7) Take it out and weigh it, and count it as M1;
(8)再次放入80℃鼓风干燥箱中干燥2h后，取出称重计为M2；(8) Put it into the blast drying oven at 80°C for 2 hours, take it out and weigh it as M2;
(9)第三次放入80℃鼓风干燥箱中干燥2h后，取出称重计为M3；(9) After drying for 2 hours in a blast drying oven at 80°C for the third time, take it out and weigh it as M3;
(10)M1、M2和M3基本没有变化后计算CDW。(10) CDW is calculated after M1, M2 and M3 are basically unchanged.
菌体内PHB提取方法：In vivo PHB extraction method:
取1～4号培养基发酵结束后的发酵液30mL离心8000rpm，5min，去掉上清液，然后加入去离子水25mL，震荡摇匀后8000rpm，5min，去掉上清液，然后加入去离子水25mL，震荡摇匀后8000rpm，5min，去掉上清液，然后加入去离子水5mL，再加入10mL三氯甲烷，充分混合震荡60min，8000rpm离心5min，取下层三氯甲烷相，旋转蒸发去掉三氯甲烷，收集所得白色粉末，即PHB。进行FTIR分析，图8所示。可见图8中有1723-1740的PHB特征峰，证明本发明的菌株的确可以合成PHB。Take 30mL of the fermentation liquid after the fermentation of No. 1-4 culture medium, centrifuge at 8000rpm for 5min, remove the supernatant, then add 25mL of deionized water, oscillate and shake well at 8000rpm for 5min, remove the supernatant, then add 25mL of deionized water , after oscillating and shaking at 8000rpm for 5min, remove the supernatant, then add 5mL of deionized water, then add 10mL of chloroform, mix and shake for 60min, centrifuge at 8000rpm for 5min, take the lower chloroform phase, and remove the chloroform by rotary evaporation , collect the resulting white powder, namely PHB. Perform FTIR analysis, as shown in Figure 8. It can be seen that there are PHB characteristic peaks at 1723-1740 in Fig. 8, which proves that the bacterial strain of the present invention can indeed synthesize PHB.
菌株中PHB含量检测步骤如下：The detection steps of PHB content in the strain are as follows:
(1)消解(1) digestion
(a)向消解杯中分别加入2mL液体A(由15mL浓硫酸+485mL甲醇+0.5g苯甲酸配制而成)和2mL三氯甲烷，混匀后加入发酵结束后的干菌体(约含100mg干菌体)。(a) Add 2mL of liquid A (prepared from 15mL of concentrated sulfuric acid + 485mL of methanol + 0.5g of benzoic acid) and 2mL of chloroform into the digestion cup, mix well and add the dried bacteria after fermentation (about 100mg dried bacteria).
(b)将消解杯放入消解仪器中，温度100℃，消解时间3～4h。(b) Put the digestion cup into the digestion apparatus, the temperature is 100°C, and the digestion time is 3-4 hours.
(c)消解结束后，待消解杯冷却至室温，取出消解杯。往消解杯中加入1mL纯水，振荡1～2min后，静置0.5h。(c) After the digestion is finished, wait for the digestion cup to cool down to room temperature, and then take out the digestion cup. Add 1mL of pure water into the digestion cup, shake for 1-2min, and let stand for 0.5h.
(2)标准品制备：(2) Standard product preparation:
3HB标准样制备(PHB由3HB单体聚合而成)：称量五个PHB标准样品，按照PHA消解步骤进行进样前处理。3HB standard sample preparation (PHB is polymerized from 3HB monomer): Weigh five PHB standard samples, and perform pre-injection treatment according to the PHA digestion procedure.
(3)气相色谱检测：(3) Gas chromatography detection:
检测器：FID，柱温140℃，进样温度200℃，检测器温度220℃，采用程序升温，10℃/min，升温至220℃，毛细管柱SP-2560，进样量0.5μL。Detector: FID, column temperature 140°C, injection temperature 200°C, detector temperature 220°C, temperature programming, 10°C/min, temperature rise to 220°C, capillary column SP-2560, injection volume 0.5μL.
经过以上分析和检测，使用培养基1发酵的菌体干重为93.3g/L，PHA含量为69.5％。使用培养基2发酵的菌体干重为112.7g/L，PHA含量为62％。使用培养基3发酵的菌体干重为105.5g/L，PHA含量为55.1％。使用培养基4发酵的菌体干重为198.5g/L，PHA含量达到75.1％。GC图谱结果如图9(培养基1)，图10(培养基2)，图11(培养基3)，图12(培养基4)所示。After the above analysis and detection, the dry weight of the bacteria fermented with medium 1 was 93.3g/L, and the PHA content was 69.5%. The dry weight of the thalline fermented with medium 2 was 112.7g/L, and the PHA content was 62%. The dry weight of the thalline fermented with medium 3 was 105.5g/L, and the PHA content was 55.1%. The dry weight of the thalline fermented with medium 4 was 198.5g/L, and the PHA content reached 75.1%. The results of the GC spectrum are shown in Figure 9 (medium 1), Figure 10 (medium 2), Figure 11 (medium 3), and Figure 12 (medium 4).
分子量检测分析方法Molecular weight detection and analysis method
仪器：Shimadzu LC 20ADInstrument: Shimadzu LC 20AD
检测器：RID-10ADetector: RID-10A
样品预处理方法：100℃，抗压密封，15minSample pretreatment method: 100°C, pressure-resistant seal, 15min
分析条件：Analysis conditions:
Figure PCTCN2022095741-appb-000003
Figure PCTCN2022095741-appb-000003
分子量测试结果如图13(培养基1)，图14(培养基2)，图15(培养基3)，图16(培养基4)所示。使用培养基1获得的PHB的重均分子量为104003Da，使用培养基2获得的PHB的重均分子量为90126Da，使用培养基3获得的PHB的重均分子量为121083Da，使用培养基4获得的PHB的重均分子量为83168Da。不同用途需要不同分子量的PHA，本发明的方法使用不同的培养基组分时能够制备出不同分子量的PHA，适应于多种用途。The molecular weight test results are shown in Figure 13 (medium 1), Figure 14 (medium 2), Figure 15 (medium 3), and Figure 16 (medium 4). The weight average molecular weight of PHB obtained using medium 1 was 104003Da, the weight average molecular weight of PHB obtained using medium 2 was 90126Da, the weight average molecular weight of PHB obtained using medium 3 was 121083Da, and the weight average molecular weight of PHB obtained using medium 4 was The weight average molecular weight is 83168Da. Different applications require PHAs with different molecular weights, and the method of the present invention can prepare PHAs with different molecular weights when using different medium components, which is suitable for various purposes.
实施例3不同培养条件生产PHA的探索The exploration of embodiment 3 different culture conditions produces PHA
本实施例采用实施例2中的培养基4，其他操作步骤相同，仅在培养条件上设置了4组对比实验，来验证不同的培养条件对PHB产物的产量是否有影响。4种不同的培养条件如下：In this example, the medium 4 in Example 2 was used, and the other operation steps were the same, only 4 sets of comparative experiments were set up on the culture conditions to verify whether different culture conditions had an impact on the yield of PHB products. The 4 different culture conditions are as follows:
培养条件1：pH＝8.0，通气量为0.1vvm，温度25℃Culture condition 1: pH = 8.0, ventilation volume 0.1vvm, temperature 25°C
培养条件2：pH＝10，通气量为2vvm，温度40℃Culture condition 2: pH = 10, ventilation volume 2vvm, temperature 40°C
培养条件3：pH＝8.8，通气量为1vvm，温度32℃Culture condition 3: pH = 8.8, ventilation volume 1vvm, temperature 32°C
培养条件4：pH＝9.2，通气量为0.8vvm，温度37℃Culture condition 4: pH = 9.2, ventilation rate 0.8vvm, temperature 37°C
培养条件1～4条件下产物的气相色谱结果如图17(培养基1)，图18(培养基2)，图19(培养基3)，图20(培养基4)所示。图中保留时间3.5min左右的峰为3-羟基丁酸(3HB)，PHB由3HB单体聚合而成。结果可见图20中保留时间3.5min左右的峰更高，面积更大。因此在培养条件4下，能够获得含量更高的PHB。The gas chromatography results of products under culture conditions 1-4 are shown in Figure 17 (medium 1), Figure 18 (medium 2), Figure 19 (medium 3), and Figure 20 (medium 4). The peak with a retention time of about 3.5 minutes in the figure is 3-hydroxybutyric acid (3HB), and PHB is formed by the polymerization of 3HB monomer. As a result, it can be seen that the peak with a retention time of about 3.5 min in Figure 20 is higher and has a larger area. Therefore, under culture condition 4, a higher content of PHB can be obtained.
实施例4杂菌的检测The detection of embodiment 4 miscellaneous bacteria
从发酵开始时计起，每隔4小时取发酵液稀释至10 -4，取100μL涂布于LB平板(配制同平板固体培养基)，37℃培养箱培养24小时，观察菌落形态，随机挑选60个菌落进行PCR验证，确定不灭菌条件下发酵是否染菌。 From the beginning of the fermentation, take the fermentation liquid every 4 hours and dilute it to 10 -4 , take 100 μL and spread it on the LB plate (prepared as the solid medium on the same plate), culture in the incubator at 37°C for 24 hours, observe the colony shape, and select randomly 60 colonies were verified by PCR to determine whether the fermentation was contaminated under non-sterile conditions.
选取的两段序列为Halomonas lutescens MDF-9的高度保守序列。其中，两对引物分别为：The two selected sequences are highly conserved sequences of Halomonas lutescens MDF-9. Among them, the two pairs of primers are:
F5’-GCACACCAAGTCACATCGTCCAG-3’(SEQ ID NO.2)F5'-GCACACCAAGTCACATCGTCCAG-3' (SEQ ID NO.2)
R5’-CGGCGTGTAGCTGGCGAGCTTGG-3’(SEQ ID NO.3)，R5'-CGGCGTGTAGCTGGCGAGCTTGG-3' (SEQ ID NO.3),
片段长度为486bpThe fragment length is 486bp
F5’-CAGACTTGACGCGGTCGGCAATT-3’(SEQ ID NO.4)F5'-CAGACTTGACGCGGTCGGCAATT-3' (SEQ ID NO.4)
R5’-CGCTCCGTTTACGGTAGTGTTGT-3’(SEQ ID NO.5)，片段长度为432bp。R5'-CGCTCCGTTTACGGTAGTGTTGT-3' (SEQ ID NO.5), the fragment length is 432bp.
PCR检测结果如图21和图22所示。图中，A为发酵12小时取样样品，B为发酵48小时取样样品，共取60个样品进行PCR验证。M为marker，C为Halomonas lutescens MDF-9纯种做模板的正对照，1～10为部分取样样品。菌落形态和PCR验证结果显示，除了目标片段的条带没有扩增出其他条带，说明发酵过程中并没有染菌，表明不灭菌发酵是可行的，这极大地降低了生产过程的能耗，降低了成本，简化了发酵过程。The PCR detection results are shown in Figure 21 and Figure 22. In the figure, A is the sample taken after 12 hours of fermentation, and B is the sample taken after 48 hours of fermentation. A total of 60 samples were taken for PCR verification. M is the marker, C is the positive control of Halomonas lutescens MDF-9 purebred as the template, and 1-10 is the partial sampling sample. The results of colony morphology and PCR verification showed that no other bands were amplified except for the band of the target fragment, indicating that there was no bacterial infection during the fermentation process, indicating that non-sterile fermentation is feasible, which greatly reduces the energy consumption of the production process , reduces the cost and simplifies the fermentation process.
对于嗜盐菌而言，高盐浓度是生长所必须的，同时Halomonas lutescens MDF-9生长还依赖较高的pH值，而高盐及高pH的培养基能抑制其他非嗜盐菌的生长，因此让无灭菌生产工艺成为可能。然而现有技术中有公开的培养基组分的NaCl浓度为20～200g/L，而本发明的NaCl浓度仅为10～20g/L，在更低的盐浓度下比更高的盐浓度下更利于其他非嗜盐菌的生长，但是本发明的培养方法也能够实现不灭菌发酵，降低灭菌过程产生的能耗，最大限度地减少对微生物培养基的损害，从而从更大程度上降低成本。低盐浓度也会导致后期废水处理更加简单，也能节约生产的成本。For halophilic bacteria, high salt concentration is necessary for growth. At the same time, the growth of Halomonas lutescens MDF-9 also depends on a high pH value, and high salt and high pH medium can inhibit the growth of other non-halophilic bacteria. Therefore, a sterile production process is possible. However, the NaCl concentration of the disclosed medium components in the prior art is 20-200g/L, while the NaCl concentration of the present invention is only 10-20g/L, which is lower than the higher salt concentration at a lower salt concentration. It is more conducive to the growth of other non-halophilic bacteria, but the culture method of the present invention can also realize non-sterile fermentation, reduce the energy consumption produced by the sterilization process, and minimize the damage to the microbial culture medium, thereby to a greater extent cut costs. The low salt concentration will also lead to easier wastewater treatment in the later stage and save production costs.
本发明的培养基成分简单，不含有氯化钴和氯化镍，氯化钴和氯化镍吸入及皮肤接触可能致敏。对水生生物有极高毒性，可能对水体环境产生长期不良影响。因此去除了氯化钴和氯化镍的培养基更加安全和环境友好。The culture medium of the present invention has simple components and does not contain cobalt chloride and nickel chloride, which may be sensitized by inhalation or skin contact. Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment. Therefore, the medium without cobalt chloride and nickel chloride is safer and more environmentally friendly.
以上所述仅为本发明的较佳实施例，并不用以限制本发明，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection of the present invention. within range.

Claims (14)

  1. 一株盐单胞菌(Halomonas lutescens MDF-9)，所述盐单胞菌的保藏编号为GDMCC NO.61850。A strain of Halomonas lutescens MDF-9, the preservation number of which is GDMCC NO.61850.
  2. 权利要求1所述的盐单胞菌在制备聚羟基脂肪酸酯中的应用。The application of Halomonas described in claim 1 in the preparation of polyhydroxyalkanoate.
  3. 根据权利要求2所述的应用，其特征在于，所述聚羟基脂肪酸酯为聚-β-羟丁酸。The application according to claim 2, characterized in that, the polyhydroxyalkanoate is poly-β-hydroxybutyric acid.
  4. 一种制备聚羟基脂肪酸酯的方法，其特征在于，包括如下步骤：A method for preparing polyhydroxyalkanoate, is characterized in that, comprises the steps:
    将盐单胞菌(Halomonas lutescens MDF-9)GDMCC NO.61850，接入发酵培养基，进行发酵培养，得到聚羟基脂肪酸酯。Halomonas lutescens MDF-9 (Halomonas lutescens MDF-9) GDMCC NO.61850 was inserted into the fermentation medium and fermented to obtain polyhydroxyalkanoate.
  5. 根据权利要求4所述的方法，其特征在于，所述发酵培养基的氯化钠浓度为10～20g/L。The method according to claim 4, characterized in that the sodium chloride concentration of the fermentation medium is 10-20 g/L.
  6. 根据权利要求4所述的方法，其特征在于，所述发酵的pH值为8～10，搅拌速度为50～800rpm，通气量为0.1～2vvm，发酵温度为25～40℃。The method according to claim 4, characterized in that the pH value of the fermentation is 8-10, the stirring speed is 50-800rpm, the ventilation volume is 0.1-2vvm, and the fermentation temperature is 25-40°C.
  7. 根据权利要求4～6任一项所述的方法，其特征在于，所述发酵的方法包括如下步骤：The method according to any one of claims 4 to 6, wherein the fermentation method comprises the steps of:
    (1)将盐单胞菌(Halomonas lutescens MDF-9)GDMCC NO.61850接入发酵培养基，进行发酵培养；(1) Insert Halomonas lutescens MDF-9 GDMCC NO.61850 into the fermentation medium for fermentation;
    (2)发酵过程中检测发酵液中糖的浓度低于10g/L时，补加糖使其维持在30g/L；(2) When the concentration of sugar in the fermentation broth is detected to be lower than 10g/L during the fermentation process, add sugar to maintain it at 30g/L;
    (3)继续培养30～40h，待发酵液中糖浓度低于0.2g/L时，结束发酵。(3) Continue culturing for 30-40 hours, and stop the fermentation when the sugar concentration in the fermentation broth is lower than 0.2 g/L.
  8. 根据权利要求7所述的方法，其特征在于，所述发酵培养基包括如下组分：The method according to claim 7, wherein the fermentation medium comprises the following components:
    碳源：葡萄糖、果糖、葡萄糖酸钠中的任意一种或多种；Carbon source: any one or more of glucose, fructose, and sodium gluconate;
    氮源：玉米浆干粉、酵母粉、胰蛋白胨、尿素、硫酸铵、氯化铵；Nitrogen source: corn steep liquor dry powder, yeast powder, tryptone, urea, ammonium sulfate, ammonium chloride;
    其他营养成分：磷酸二氢钾、磷酸氢二钠、硫酸镁；Other nutrients: potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate;
    氯化钠；Sodium chloride;
    所述发酵培养基中不包含氯化钴和氯化镍。The fermentation medium does not contain cobalt chloride and nickel chloride.
  9. 根据权利要求8所述的方法，其特征在于，所述碳源的总浓度为90～150g/L；The method according to claim 8, characterized in that the total concentration of the carbon source is 90-150g/L;
    所述玉米浆干粉的含量为1～8％，所述酵母粉的含量为0.5～5％，所述胰蛋白胨的含量为0.1～3％，所述尿素的含量为0.1～3％，所述硫酸铵的含量为0.1～3％，所述氯化铵的含量为0.1～3％；The content of the corn steep liquor dry powder is 1-8%, the content of the yeast powder is 0.5-5%, the content of the tryptone is 0.1-3%, and the content of the urea is 0.1-3%. The content of ammonium sulfate is 0.1-3%, and the content of the ammonium chloride is 0.1-3%;
    所述磷酸二氢钾的含量为0.1～2％，所述磷酸氢二钠的含量为0.05～1％，所述硫酸镁的含量为0.05～1％，所述含量为质量体积比。The content of the potassium dihydrogen phosphate is 0.1-2%, the content of the disodium hydrogen phosphate is 0.05-1%, and the content of the magnesium sulfate is 0.05-1%, and the content is mass volume ratio.
  10. 根据权利要求4～9任一项所述的方法，其特征在于，所述聚羟基脂肪酸酯为聚-β-羟丁酸。The method according to any one of claims 4-9, characterized in that the polyhydroxyalkanoate is poly-β-hydroxybutyric acid.
  11. 根据权利要求6所述的方法，其特征在于，所述发酵的pH值为9～10，通气量为0.8～2vvm，发酵温度为37℃～40℃。The method according to claim 6, characterized in that the pH value of the fermentation is 9-10, the ventilation rate is 0.8-2vvm, and the fermentation temperature is 37°C-40°C.
  12. 一种用于盐单胞菌发酵的培养基，其特征在于，所述发酵培养基包括如下组分：A kind of medium that is used for Halomonas fermentation, is characterized in that, described fermentation medium comprises following component:
    碳源：葡萄糖、果糖、葡萄糖酸钠中的任意一种或多种；Carbon source: any one or more of glucose, fructose, and sodium gluconate;
    氮源：玉米浆干粉、酵母粉、胰蛋白胨、尿素、硫酸铵、氯化铵；Nitrogen source: corn steep liquor dry powder, yeast powder, tryptone, urea, ammonium sulfate, ammonium chloride;
    其他营养成分：磷酸二氢钾、磷酸氢二钠、硫酸镁；Other nutrients: potassium dihydrogen phosphate, disodium hydrogen phosphate, magnesium sulfate;
    氯化钠，浓度为10～20g/L；Sodium chloride, the concentration is 10～20g/L;
    所述发酵培养基中不包含氯化钴和氯化镍；Cobalt chloride and nickel chloride are not included in the fermentation medium;
    所述碳源的总浓度为90～150g/L。The total concentration of the carbon source is 90-150 g/L.
  13. 根据权利要求12所述的培养基，其特征在于，所述玉米浆干粉的含量为1～8％，所述酵母粉的含量为0.5～5％，所述胰蛋白胨的含量为0.1～3％，所述尿素的含量为0.1～3％，所述硫酸铵的含量为0.1～3％，所述氯化铵的含量为0.1～3％。The medium according to claim 12, characterized in that the content of the corn steep liquor dry powder is 1-8%, the content of the yeast powder is 0.5-5%, and the content of the tryptone is 0.1-3% , the content of the urea is 0.1-3%, the content of the ammonium sulfate is 0.1-3%, and the content of the ammonium chloride is 0.1-3%.
  14. 根据权利要求12或13所述的培养基，其特征在于，所述磷酸二氢钾的含量为0.1～2％，所述磷酸氢二钠的含量为0.05～1％，所述硫酸镁的含量为0.05～1％，所述含量为质量体积比。The medium according to claim 12 or 13, characterized in that the content of potassium dihydrogen phosphate is 0.1-2%, the content of disodium hydrogen phosphate is 0.05-1%, and the content of magnesium sulfate It is 0.05～1%, and described content is mass volume ratio.
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