Bacterial strain for producing polyhydroxybutyrate and application thereof

Abstract

The invention provides a strain for producing polyhydroxybutyrate and application of the strain. The sequence of 16s rDNA of the strain is shown as any one of the following sequences: 1) a nucleotide sequence shown as SEQ ID NO: 1; 2) a nucleotide sequence obtained by modifying, substituting, deleting or adding one or more basic groups to the nucleotide sequence shown in 1); 3) a nucleotide sequence having at least 70% identity with 1), preferably a nucleotide sequence having at least 80% identity, preferably a nucleotide sequence having at least 85% identity, preferably a nucleotide sequence having at least 90% identity, preferably a nucleotide sequence having at least 95% identity, preferably a nucleotide sequence having at least 90% identity, preferably a nucleotide sequence having at least 90% identity, preferably a nucleotide sequence having at least 90% identity, preferably a nucleotide sequence having at least 90% identity, preferably a nucleotide sequence having at least 90% identity, preferably a nucleotide sequence having at least 90% identity and preferably a nucleotide sequence having at least 90% identity. The present invention relates to a nucleotide sequence having at least 97% identity, and more preferably, a nucleotide sequence having at least 99% identity. The strain is a Ralzonia bacterium, and the preservation number of the Ralzonia bacterium is CCTCC (China Center for Type Culture Collection) NO: M 20231548. The screened new strain capable of efficiently synthesizing the PHB by using the glycerol has PHB production potential, and fills the blank in the field of producing the PHB by using the strain by using the crude glycerol as a substrate.

Description

Technical Field

[0001] The invention relates to the technical field of bacterial separation and cultivation, and in particular to a strain for producing polyhydroxybutyrate and application thereof. Background Art

[0002] Polyhydroxyalkanoates (PHAs) are a class of biodegradable and biocompatible polyesters synthesized by various microorganisms and used as carbon and energy storage materials in bacteria. PHAs have a variety of chemical structures, physical properties and thermoplastic characteristics, and are potential substitutes for petroleum-based plastics, so they have attracted much attention. PHAs can be used in many fields such as biomedicine, agriculture, packaging and environment. PHB (poly 3-hydroxybutyrate) is a branch of PHAs. It is a biodegradable polyester synthesized intracellularly by some bacteria and archaea, and is also a carbon and energy storage material. PHB has good biocompatibility, water insolubility, oxygen permeability and UV resistance, so it has broad application prospects in biomedicine, food and environment.

[0003] At present, crude glycerol as a chemical by-product has reached a market size of US$700 million per year. Due to its low price and large output, it has attracted much attention in many fields such as biology and chemical industry to study the efficient utilization of crude glycerol. In the current PHB production process, the high cost of raw materials is an important factor limiting its commercial application. Therefore, the selection of carbon source raw materials with a wide range of sources and low value is the key to reducing the production cost of PHB. Glycerol, as a by-product of the production of various chemical products, has a wide range of sources. At the same time, glycerol has a higher reducing power as a carbon source for microbial fermentation, which is conducive to the synthesis of PHB. Therefore, it is of great significance to screen and separate production strains that can efficiently utilize glycerol to synthesize PHB.

[0004] In recent years, there have been many reports on strains used for industrial production of PHB, which can be divided into two categories: one is to rely on wild-type PHB production strains for industrial transformation, such as Ralstonia eutropha, Aquabacterium sp.A7-Y, Halomonas campaniensis LS21, Rhodospirillum rubrum, Pseudodonghicolaxiamenensis, Rhodococcus sp.strain BSRT1-1, Bacillus megaterium, Streptomyces sp. The other is to construct an efficient PHB synthesis pathway in existing engineered strains such as Escherichia coli.

[0005] Professor Chen Guoqiang's team published an article in 2014, describing that they screened a halophilic bacterium Halomonas campaniensis LS21 from a saline-alkali lake in Xinjiang. The strain could grow under the conditions of 1% (w / v) NaCl and pH 10, and finally achieved a PHB production of 4.674 g / L in a basic salt medium with glucose as the carbon source.

[0006] The strains currently reported for synthesizing PHB from glycerol have the disadvantages of slow glycerol metabolism, low PHB production rate, and unclear genetic background. Moreover, at this stage, the effective disposal of crude glycerol is far from reaching the peak level, and the field of using crude glycerol as a substrate to produce PHB is still at a relatively blank level. In the chemical industry, the production of PHB under various conditions of high temperature, high pressure and high-value catalysts is obviously too costly, and the biological world has not yet effectively connected glycerol utilization with PHB production. Summary of the invention

[0007] In view of the shortcomings of the prior art, the present invention provides a strain for producing polyhydroxybutyrate and its application, which solves the problems of slow glycerol metabolism rate, low PHB production rate and unclear genetic background in the prior art PHB production strain.

[0008] In a first aspect, the present invention provides a strain for producing polyhydroxybutyrate, wherein the sequence of 16s rDNA of the strain is any one of the following:

[0009] 1) having the nucleotide sequence shown in SEQ ID NO: 1;

[0010] 2) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in 1);

[0011] 3) A nucleotide sequence having at least 70% identity with 1), preferably a nucleotide sequence having at least 80% identity, preferably a nucleotide sequence having at least 85% identity, preferably a nucleotide sequence having at least 90% identity, preferably a nucleotide sequence having at least 95% identity, preferably a nucleotide sequence having at least 97% identity, more preferably, a nucleotide sequence having at least 99% identity.

[0012] In a specific embodiment of the present invention, the identity can be more than 70%, more than 80%, more than 85%, more than 90%, preferably more than 95%, more preferably 96%, 97%, 98%, 99% identity.

[0013] Methods for determining sequence identity that are well known to those of ordinary skill in the art include, but are not limited to, Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987 and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991 and Carillo, H. and Lipman, D., SIAM J. Applied Math., 48:1073 (1988). The preferred method for determining identity is to obtain the largest match between the sequences tested. Methods for determining identity are compiled in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to, the GCG program package (Devereux, J. et al., 1984), BLASTP, BLASTN and FASTA (Altschul, S, F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith Waterman algorithm can also be used to determine identity.

[0014] In a specific embodiment of the present invention, the strain is of the genus Ralstonia.

[0015] In a specific embodiment of the present invention, its deposit number is CCTCC NO:M 20231548.

[0016] The strain was collected from the soil environment of Dongli District, Tianjin to obtain the microbial community, enriched in the basic salt medium under high concentration of glycerol, and then obtained by plate streaking method to obtain a single colony. The strain was cultured on the plate and showed light yellow round colonies with clear edges and no burrs. After being placed for a long time (>3 days), the color of the colony will turn brown.

[0017] The strain is deposited in China Center for Type Culture Collection (CCTCC). Address: Collection Center of Wuhan University, Wuhan, 430072. The deposit date is August 31, 2023. The deposit number is CCTCC M 20231548. Strain name: Ralstonia RamessesRRA.1.

[0018] In a second aspect, the present invention provides the use of the above strain in any of the following:

[0019] 1) Production of poly 3-hydroxybutyrate;

[0020] 2) Preparation of products for producing poly 3-hydroxybutyrate

[0021] 3) Used for glycerol recycling or glycerol bioconversion.

[0022] In a specific embodiment of the present invention, the carbon source used to produce poly 3-hydroxybutyrate includes small molecule alcohols, sugars, small molecule fatty acids or their salts.

[0023] Preferably, the sugar is selected from glucose and fructose; the small molecule alcohol is selected from glycerol; and the small molecule fatty acid or its salt is selected from acetic acid or its salt.

[0024] In a third aspect, the present invention provides a method for producing poly 3-hydroxybutyrate, comprising the following steps: fermenting the above strain in a culture medium to obtain a fermentation product; obtaining poly 3-hydroxybutyrate from the fermentation product;

[0025] The culture medium includes the above-mentioned carbon source.

[0026] In a specific embodiment of the present invention, the culture medium comprises Na2HPO4, KH2PO4, NH4Cl, MgSO4 and a nutrient solution;

[0027] The nutrient solution contains FeCl3, CaCl2, CoCl2·6H2O, CuSO4·5H2O, NiCl3·6H2O and CrCl3·6H2O.

[0028] In a specific embodiment of the present invention, the amount of each component in the culture medium can be increased or decreased according to actual conditions. Preferably, the culture medium includes 2.44-3.8 g / L Na2HPO4, 1.5-3 g / L KH2PO4, 0.36-7.2 g / L NH4Cl, 0.1-0.2 g / L MgSO4, and 1 mL / L nutrient solution; each liter of nutrient solution contains 9.7 g FeCl3, 7.8 g CaCl2, 0.218 g CoCl2·6H2O, 0.156 g CuSO4·5H2O, 0.118 g NiCl3·6H2O and 0.105 g CrCl3·6H2O.

[0029] Further preferably, the culture medium comprises 3.8 g Na2HPO4, 2.65 g KH2PO4, 1.5 g NH4Cl, 0.2 g MgSO4, and 1 mL nutrient solution; each liter of nutrient solution contains 9.7 g FeCl3, 7.8 g CaCl2, 0.218 g CoCl2·6H2O, 0.156 g CuSO4·5H2O, 0.118 g NiCl3·6H2O and 0.105 g CrCl3·6H2O.

[0030] In a specific embodiment of the present invention, the concentration of the carbon source is 1-40 g / L. In a further specific embodiment of the present invention, the concentration of the carbon source is 1-20 g / L. In a further specific embodiment of the present invention, the concentration of the carbon source is 1-10 g / L. More preferably, the concentration of the carbon source is 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 / L, 8 g / L, 9 g / L, 10 g / L.

[0031] In a specific embodiment of the present invention, the carbon source is glycerol, and the concentration of the glycerol is 1-10 g / L.

[0032] In a specific embodiment of the present invention, the fermentation time is 1-2 days and the fermentation temperature is 30°C.

[0033] In a fourth aspect, the present invention provides a bacterial agent comprising the above-mentioned strain or its fermentation broth.

[0034] In a specific embodiment of the present invention, the bacterial agent may further include a carrier. The carrier may be a solid carrier or a liquid carrier. The solid carrier is a mineral material or a biological material; the mineral material may be at least one of peat, clay, talc, kaolin, montmorillonite, white carbon, zeolite, silica and diatomaceous earth; the biological material may be at least one of various crop straws, pine husks, straw, peanut shells, corn flour, soybean flour, starch, peat and animal feces; the liquid carrier may be water; in the bacterial agent, Ralstonia Ramesses RRA.1 bacteria or / and the metabolites of Ralstonia Ramesses RRA.1 bacteria may exist in the form of cultured living cells, fermentation broth of living cells, filtrate of cell culture or a mixture of cells and filtrate. The dosage form of the bacterial agent may be a variety of dosage forms, such as liquid, emulsion, suspension, powder, granule, wettable powder or water-dispersible granule.

[0035] As required, a surfactant (such as Tween 20, Tween 80, etc.), a binder, a stabilizer (such as an antioxidant), a pH adjuster, etc. may also be added to the bacterial agent.

[0036] Compared with the prior art, the present invention has the following beneficial effects:

[0037] (1) The novel strain screened by the present invention can efficiently utilize glycerol to synthesize PHB and has the potential for PHB production, thus filling the gap in the field of strains utilizing crude glycerol as a substrate to produce PHB.

[0038] (2) The strain of the present invention can grow normally and accumulate PHB in conventional carbon sources such as glycerol, glucose, fructose, and acetic acid. When glycerol is used as a carbon source, the dry weight of PHB reaches half of the cell dry weight after 48 hours of growth. Therefore, it has great potential for industrial use of glycerol to produce PHB. The specific growth rate of the wild-type strain using 1 g / L glycerol as a carbon source reaches 0.1867 h -1 , and 2.32 g / L PHB can be accumulated using 10 g / L glycerol as the carbon source.

[0039] (3) The present invention biolinks glycerol utilization and PHB production, achieving a high-efficiency result of both processing chemical by-product glycerol and producing high-value product PHB. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is a transmission electron microscope (TEM) image of Ralstonia Ramesses after being cultured under 10 g / L glycerol for 48 hours.

[0041] Figure 2This is a transmission electron microscope (TEM) image of Ralstonia Ramesses after being cultured under 10 g / L glucose for 48 hours.

[0042] Figure 3 The phylogenetic tree of Ralstonia Ramesses and other strains of the genus Ralstonia (left side) and the average nucleotide similarity (Average Nucleotide Identity, ANI) comparison diagram (right side) are shown.

[0043] Figure 4 This is the growth curve of Ralstonia Ramesses with glycerol, glucose, fructose and sodium acetate as carbon sources.

[0044] Figure 5 This is a bar graph of dry weight and PHB content of Ralstonia Ramesses using glycerol, glucose, fructose, and sodium acetate as carbon sources. DETAILED DESCRIPTION

[0045] The technical scheme of the present invention will be further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only exemplary descriptions and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are included in the scope that the present invention is intended to protect.

[0046] Unless otherwise specified, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods.

[0047] Technical terms

[0048] "Small molecule alcohols" refer to alcohols with a molecular weight less than 1000 Daltons (especially small molecules less than 400 Daltons), including but not limited to methanol, ethanol, ethylene glycol, isopropanol, n-propanol, propylene glycol, glycerol, etc.

[0049] From the perspective of molecular structure, "sugars" can be divided into polyhydroxy aldehydes, polyhydroxy ketones and their dehydrated condensates. Most of their compositions are represented by the general formula Cm(H2O)n. Sugars include monosaccharides: any sugar that cannot be hydrolyzed is called a monosaccharide. Such as glucose, fructose, ribose and deoxyribose. Oligosaccharides: 1 mol of oligosaccharides can produce 2-10 mol of monosaccharides after hydrolysis. If hydrolysis produces 2 mol of monosaccharides, it is called a disaccharide. Important disaccharides include maltose, lactose and sucrose. Polysaccharides: 1 mol of polysaccharides can produce many moles of monosaccharides after hydrolysis, such as starch, cellulose, etc.

[0050] "Small molecule fatty acid or its salt" refers to a fatty acid or its salt with a molecular weight of less than 1000 Dalton (especially a small molecule of less than 400 Dalton); including but not limited to formic acid and its salts, acetic acid and its salts, propionic acid and its salts.

[0051] "Crude glycerol" refers to the product with a glycerol content of more than 95% obtained by evaporating and concentrating glycerol water after secondary purification treatment. It is the main by-product of biodiesel production.

[0052] "Dry cell weight" refers to the net weight of cells after removing water, which is generally 10% to 20% of the wet weight. In the study of microbial growth, fermentation and its conditions, dry cell weight is an important measurement parameter.

[0053] Example 1 Isolation and identification of Ralstonia Ramesses strains

[0054] 1. Isolation of Ralstonia Ramesses strains

[0055] Strain collection: Microbial communities were obtained from the soil environment of Dongli District, Tianjin, enriched in basic salt medium under high concentration of glycerol, and then single colonies were obtained by plate streaking method.

[0056] 2. Identification of Ralstonia Ramesses strains

[0057] (1) Observation of strain culture characteristics

[0058] Ralstonia Ramesses is cultured on plates and appears as pale yellow round colonies with clear edges and no burrs. After being placed for a long time (>3 days), the color of the colonies will turn brown.

[0059] (2) Analysis of physiological and biochemical characteristics

[0060] The culture steps are as follows: a. Pick a single colony from the streaked plate and inoculate it into a 5 mL 2YT medium tube containing 10 μg / mL gentamicin and culture overnight.

[0061] b. Inoculate the above bacterial solution into a 250 mL conical flask containing 50 mL of basic salt medium at a final concentration of OD600 of 0.02 for cultivation.

[0062] The culture medium used was as follows:

[0063] 2YT medium (per liter): 16.0 g tryptone, 10.0 g yeast extract, 5.0 g sodium chloride.

[0064] Basic salt medium (per liter): contains 3.8g Na2HPO4, 2.65g KH2PO4, 1.5g NH4Cl, 0.2g MgSO4, and 1mL nutrient solution. Each liter of nutrient solution contains 9.7g FeCl3, 7.8g CaCl2, 0.218g CoCl2·6H2O, 0.156g CuSO4·5H2O, 0.118g NiCl3·6H2O and 0.105g CrCl3·6H2O. The antibiotic used is 10μg / mL gentamicin. The final concentration of glycerol added is 10g / L. The culture conditions are 30℃, 220rpm, and the culture time is 48 hours.

[0065] Ralstonia Ramesses was cultured with 10 g / L glycerol as the carbon source. The TEM showed that the elliptical shape of the bacteria contained clear PHB spherical aggregates. Figure 1 .

[0066] Ralstonia Ramesses was cultured with 10 g / L glucose as the carbon source. The TEM showed that the bacteria were rod-shaped, with clear PHB spherical aggregates inside. Figure 2 .

[0067] (3) 16S rDNA sequence analysis

[0068] The primers used for 16S rDNA sequence were:

[0069] F:5'-AGAGTTTGATCCTGGCTCAG-3'

[0070] R:5'-AAGGAGGTGATCCAGCC-3'

[0071] The PCR system was 25 μL 2×PhantaMax Master Mix, 2 μL primer F, 2 μL primer R, and 21 μL ddH2O. The PCR reaction program was 95°C for 3 min, (95°C for 15 sec, 56°C for 15 sec, 72°C for 1 min and 30 sec)*31 cycles, and 72°C for 10 min. The DNA sequence obtained by sequencing is shown in SEQ ID NO:1.

[0072] (4) Genome sequencing analysis

[0073] Genome analysis was performed by BGI-Scientific, and the average nucleotide identity (ANI) obtained from the sequencing results showed that its similarity with the adjacent Ralstonia species was less than 95%, which met the requirements for naming as a new strain. Figure 3 .

[0074] (5) Specific sequence amplification

[0075] The identification method is colony PCR, and the primers designed according to the specific genome sequence are

[0076] F:5'-GAATCTGTCTTATGGAATAACCTCCTC-3'

[0077] R:5'-CGCCTTTTTCGTGCCGGATGGAGGGC-3'

[0078] The PCR system was 25 μL 2×PhantaMax Master Mix, 2 μL primer F, 2 μL primer R, and 21 μL ddH2O. The PCR reaction program was 95°C for 3 min, (95°C for 15 sec, 56°C for 15 sec, 72°C for 1 min)*31 cycles, and 72°C for 10 min. The DNA sequence obtained by sequencing is shown in SEQ ID NO:2.

[0079] Based on the above morphological, physiological and biochemical characteristics, as well as the results of genome sequence determination, it was finally determined that the strain of the present invention was a new species isolated from the genus Ralstonia. According to its genome analysis and various physiological and biochemical parameter analyses, it was named Ralstonia Ramesses as a new branch of the genus Ralstonia.

[0080] The strain Ralstonia Ramesses has been deposited in the China Center for Type Culture Collection (CCTCC). Address: Wuhan University Collection Center, Wuhan, 430072. The deposit date is August 31, 2023. The deposit number is CCTCC NO: M20231548. Strain name: Ralstonia Ramesses RRA.1.

[0081] Example 2 Production of polyhydroxybutyrate using glycerol as carbon source

[0082] 1. Pick a single colony from the streaked plate and inoculate it into a 5 mL 2YT medium tube containing 10 μg / mL gentamicin and culture overnight.

[0083] 2. Inoculate the above bacterial solution into a 250 mL conical flask containing 50 mL of basic salt medium at a final concentration of OD600 of 0.02 for culture.

[0084] The culture medium used was as follows:

[0085] 2YT medium (per liter): 16.0 g tryptone, 10.0 g yeast extract, 5.0 g sodium chloride.

[0086] Basic salt medium (per liter): contains 3.8g Na2HPO4, 2.65g KH2PO4, 1.5g NH4Cl, 0.2g MgSO4, and 1mL nutrient solution. Each liter of nutrient solution contains 9.7g FeCl3, 7.8g CaCl2, 0.218g CoCl2·6H2O, 0.156g CuSO4·5H2O, 0.118g NiCl3·6H2O and 0.105g CrCl3·6H2O. The antibiotic used is 10μg / mL gentamicin. The final concentration of glycerol added is 10g / L.

[0087] The culture conditions were 30°C, 220 rpm, and the culture time was 48 hours. The dry weight and PHB content of Ralstonia Ramesses were detected.

[0088] The dry weight of Ralstonia Ramesses was measured as follows: 40 mL of the fermented bacterial solution was taken into a 50 mL centrifuge tube, and the bacterial cells were collected under the conditions of 6000 rpm for 10 min. The collected bacterial cells were washed twice with double distilled water and finally dried in a -30°C freeze dryer until the weight was constant.

[0089] The measurement method of PHB is as follows: 5 mg of freeze-dried bacteria are weighed with an electronic analytical balance, and the 5 mg of freeze-dried bacteria are mixed with 1 mL of concentrated sulfuric acid in a 1.5 mL EP tube. The EP tube is then placed in a metal bath instrument and derivatized at 95°C for 30 minutes. The derivatized liquid is analyzed by high performance liquid chromatography. The chromatographic column is Aminex HPX-87H, the mobile phase is 5 mM sulfuric acid, the flow rate is 0.6 mL / min, and the PHB product peak is collected at about 28 minutes.

[0090] The dry weight of Ralstonia Ramesses was 4.8 g / L and the PHB content was 2.32 g / L. Figure 5 shown.

[0091] Example 3 Production of polyhydroxybutyrate using glycerol as carbon source

[0092] 1. Pick a single colony from the streaked plate and inoculate it into a 5 mL 2YT medium tube containing 10 μg / mL gentamicin and culture overnight.

[0093] 2. The above bacterial solution was measured at OD 600 The final concentration was 0.02 and inoculated into a 48-deep-well plate containing 1 mL of minimal salt medium.

[0094] The culture medium formula used is as follows:

[0095] 2YT medium (per liter): 16.0 g tryptone, 10.0 g yeast extract, 5.0 g sodium chloride.

[0096] Basic salt medium (per liter): contains 3.8g Na2HPO4, 2.65g KH2PO4, 1.5g NH4Cl, 0.2g MgSO4, and 1mL nutrient solution. Each liter of nutrient solution contains 9.7g FeCl3, 7.8g CaCl2, 0.218g CoCl2·6H2O, 0.156g CuSO4·5H2O, 0.118g NiCl3·6H2O and 0.105g CrCl3·6H2O. The antibiotic used is 10μg / mL gentamicin. The final concentration of glycerol added is 1g / L.

[0097] The growth rate detection method is as follows: the growth rate is measured by a fully automatic microbial growth curve analyzer, and then the growth rate is calculated according to the following steps:

[0098] (1) Calculate the growth rate μ for each time period i ;

[0099]

[0100] Among them, N i+1 is the amount of microbial cells at the end of the time period; N i is the amount of microbial cells at the beginning of the same time period; T i+1 is the end time of the time period; T i is the start time of the same time period.

[0101] (2) Calculate the maximum growth rate μ in each time period max ;

[0102] Comparing the growth rates of all time periods, we can get the maximum growth rate:

[0103] μ max =max(μ1,μ2,μ3,......μ i+1 )

[0104] (3) Calculate the growth rate μ

[0105] To reduce the error, we can calculate the average growth rate using the following formula:

[0106]

[0107] Here μ max-2 to μ max+2 Represents the five growth rate values ​​before and after the maximum growth rate.

[0108] It was found that the specific growth rate could reach 0.1867h on the medium with 1g / L glycerol as carbon source. -1 ,like Figure 4 shown.

[0109] Example 4 Production of polyhydroxybutyrate using glucose as carbon source

[0110] The preparation method is similar to that of Example 2, except that glycerol is replaced by glucose.

[0111] According to the detection method of Example 2, the dry weight of Ralstonia Ramesses is 3.7 g / L and the PHB content is 1.45 g / L. The results are as follows Figure 4 , 5 shown.

[0112] Example 5 Production of polyhydroxybutyrate using sodium acetate as carbon source

[0113] The preparation method is similar to that of Example 2, except that glycerol is replaced by sodium acetate.

[0114] According to the detection method of Example 2, the dry weight of Ralstonia Ramesses is 1.2 g / L and the PHB content is 0.5 g / L. The results are as follows Figure 4 , 5 shown.

[0115] Example 6 Production of polyhydroxybutyrate using fructose as carbon source

[0116] The preparation method is similar to that of Example 2, except that glycerol is replaced by fructose.

[0117] According to the detection method of Example 2, the dry weight of Ralstonia Ramesses is 2.5 g / L and the PHB content is 1.03 g / L. The results are as follows Figure 4 , 5 shown.

[0118] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. A strain for producing polyhydroxybutyrate, characterized in that: The sequence of the 16s rDNA of the strain is shown in any of the following: 1) having the nucleotide sequence shown in SEQ ID NO: 1; 2) A nucleotide sequence obtained by modifying, replacing, deleting or adding one or more bases to the nucleotide sequence shown in 1); 3) A nucleotide sequence having at least 70% identity with 1), preferably a nucleotide sequence having at least 80% identity, preferably a nucleotide sequence having at least 85% identity, preferably a nucleotide sequence having at least 90% identity, preferably a nucleotide sequence having at least 95% identity, preferably a nucleotide sequence having at least 97% identity, more preferably, a nucleotide sequence having at least 99% identity.

2. A strain for producing polyhydroxybutyrate as claimed in claim 1, characterized in that: The strain is of the genus Ralstonia.

3. A polyhydroxybutyrate-producing strain as claimed in claim 1, characterized in that: Its deposit number is CCTCCNO:M 20231548.

4. Use of the strain according to any one of claims 1 to 3 in any of the following: 1) Production of poly 3-hydroxybutyrate; 2) preparing products for producing poly 3-hydroxybutyrate; 3) Used for glycerol recycling or glycerol bioconversion.

5. The use according to claim 4, characterized in that: The carbon sources used for producing poly 3-hydroxybutyrate include small molecule alcohols, sugars, small molecule fatty acids or their salts.

6. The use according to claim 5, characterized in that: The sugar is selected from glucose and fructose; the small molecule alcohol is selected from glycerol; the small molecule fatty acid or its salt is selected from acetic acid or its salt.

7. A method for producing poly 3-hydroxybutyrate, comprising the following steps: Fermenting the strain according to any one of claims 1 to 3 in a culture medium to obtain a fermentation product; obtaining poly 3-hydroxybutyrate from the fermentation product, The culture medium comprises the carbon source according to claim 5 or claim 6.

8. The method according to claim 7, characterized in that: The culture medium comprises Na2HPO4, KH2PO4, NH4Cl, MgSO4 and a nutrient solution; The nutrient solution contains FeCl3, CaCl2, CoCl2·6H2O, CuSO4·5H2O, NiCl3·6H2O and CrCl3·6H2O.

9. The method according to claim 7, characterized in that: The concentration of the carbon source is 1-40 g / L; Preferably, the carbon source is glycerol, and the concentration of the glycerol is 1-10 g / L; Preferably, the fermentation time is 1-2 days and the fermentation temperature is 30°C.

10. A bacterial agent for producing polyhydroxybutyrate, characterized in that: The invention comprises the strain or fermentation broth thereof according to any one of claims 1 to 3.

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