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WO2023016058A1 - Souche d'halomonas lutescens et son utilisation - Google Patents

Souche d'halomonas lutescens et son utilisation Download PDF

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Publication number
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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Prior art keywords
fermentation
content
medium
halomonas
lutescens
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Chinese (zh)
Inventor
沈宏伟
吕金艳
银会娟
司徒卫
余柳松
何世琪
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Zhuhai Maidefa Biotechnology Co Ltd
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Zhuhai Maidefa Biotechnology Co Ltd
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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

Definitions

  • 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

La présente invention concerne une souche d'Halomonas lutescens et son utilisation. La souche Halomonas lutescens est Halomonas lutescens MDF-9 et son numéro de dépôt est GDMCC NO. 61850. La souche Halomonas lutescens de la présente invention peut accumuler efficacement le polyhydroxyalcanoate (PHA) dans un milieu de culture de la présente invention. Un processus de fermentation de la souche Halomonas lutescens de la présente invention ne nécessite pas de stérilisation, peut être réalisé en continu et est facile à maîtriser. Dans le processus de fermentation, la concentration de chlorure de sodium est faible et se situe entre 10 et 20 g/L, les eaux usées de stade avancé sont facilement traitées, les composants nutritifs d'un milieu de culture utilisé dans la fermentation sont simples, et un composé métallique toxique et nocif n'est pas utilisé. Le procédé de la présente invention pour préparer du PHA en utilisant le MDF-9 de Halomonas lutescens réduit le coût de production et augmente le rendement du PHA.
PCT/CN2022/095741 2021-08-13 2022-05-27 Souche d'halomonas lutescens et son utilisation Ceased WO2023016058A1 (fr)

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CN118497246A (zh) * 2024-06-05 2024-08-16 北京微构工场生物技术有限公司 一种盐单胞重组菌及其构建方法和应用

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CN117384933B (zh) * 2023-12-12 2024-04-02 珠海麦得发生物科技股份有限公司 利用木糖生产3-羟基丙酸的菌株及其构建方法和应用

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