A pristinamycete and its application in producing pha from various cheap substrates

Abstract

The application discloses a pristinamycin and application of the pristinamycin in production of PHA by using various cheap substrates. The pristinamycin in the application is pristinamycin MIBE00003 with the preservation number CCTCC M 2022792. The pristinamycin MIBE00003 in the application can use various cheap carbon sources as single carbon source to produce polyhydroxyalkanoate efficiently, and the raw material cost of the polyhydroxyalkanoate production is reduced. In one implementation manner of the application, the proportion of the polyhydroxyalkanoate in the cell dry weight is as high as 68.5%, which provides a new strain resource for production of natural macromolecular biological materials to replace petroleum-based plastics, and provides a new scheme and way for efficient and low raw material cost production of polyhydroxyalkanoate.

Description

Technical Field

[0001] This application relates to the field of polyhydroxyalkanoate production technology, and in particular to a Priestella bacterium and its applications. Background Technology

[0002] Traditional petroleum-based plastics are inexpensive, simple to produce, and have excellent performance, making them widely used in daily life. However, because petroleum-based plastics are extremely difficult to degrade in the natural environment, and residual plastic fragments can adsorb other toxic and harmful pollutants, they pose a serious threat to the ecological environment and human health. Developing new biodegradable materials that can replace petroleum-based plastics is one of the most effective ways to solve petroleum-based plastic pollution.

[0003] Polyhydroxyalkanoates (PHA) are natural high-molecular-weight bio-based polymers synthesized by microorganisms under nutrient-unbalanced conditions and stored within cells as carbon and energy sources. In addition to the thermoplasticity and mechanical properties of traditional petroleum-based plastics, PHAs also possess excellent biocompatibility, biodegradability, and functional scalability, making them promising candidates for applications in daily packaging, chemical products, and medical implant materials.

[0004] Although PHA materials can effectively avoid the environmental harm caused by traditional petroleum-based plastics, their production cost is much higher than that of traditional petroleum-based plastics, which greatly limits their commercial application. Therefore, there is an urgent need to develop methods for the efficient production of PHA using inexpensive substrates. Summary of the Invention

[0005] The purpose of this application is to provide a novel Priestella bacterium and its application in producing PHA using a variety of inexpensive substrates.

[0006] The following technical solution is adopted in this application:

[0007] The first aspect of this application discloses a Priestella MIBE00003 with accession number CCTCC M 2022792.

[0008] It should be noted that this application isolated several strains with high cumulative PHA production from the surface soil of the mangrove reserve in Futian District, Shenzhen, and preserved and protected these strains under patent. One of these strains is *Priestia sp.* MIBE00003, which is the strain described in this application. In one implementation of this application, the biomass of the fermentation broth of *Priestia sp.* MIBE00003 reaches 15.0 g / L, and the PHA content reaches 68.5%. Furthermore, *Priestia sp.* MIBE00003 of this application has the ability to efficiently synthesize PHA using various inexpensive carbon sources, such as inexpensive sucrose, molasses, cassava starch, crude glycerol, or sodium acetate as a single carbon source. Using *Priestia sp.* MIBE00003 of this application, the inexpensive and efficient production of PHA is truly realized, providing important strain resources and technical means for the production of natural polymer biomaterials that can replace petroleum-based plastics.

[0009] It should also be noted that one of the advantages of the *Priscilla MIBE00003* strain of this application is that it can efficiently synthesize PHA using a variety of inexpensive substrates, such as sucrose, molasses, tapioca starch, and crude glycerol; while existing technologies generally can only use expensive glucose, and some can use a single inexpensive substrate, but cannot utilize multiple inexpensive substrates. Therefore, the *Priscilla MIBE00003* strain of this application has stronger and broader applicability, and has a significant advantage in reducing raw material costs.

[0010] The second aspect of this application discloses a microbial agent containing the Priestella MIBE00003 or its fermentation broth.

[0011] It should be noted that the microbial agent in this application refers to a live microbial preparation containing live Priestella MIBE00003. Since the Priestella MIBE00003 of this application can produce PHA efficiently and at low cost, Priestella MIBE00003 or its fermentation broth can be made into a microbial agent for sale or use.

[0012] In one implementation of this application, the bacterial agent contains polyhydroxyalkanoate, and the polyhydroxyalkanoate accounts for more than 68% of the dry weight of Priestella MIBE00003.

[0013] It should be noted that the *Primaterella* MIBE00003 of this application is characterized by its ability to efficiently produce PHA using a variety of inexpensive single carbon sources. For example, after fermentation and culture for 2 days in a liquid medium with molasses as the single carbon source, the accumulated PHA proportion reaches 68.5%, and after fermentation and culture for 2 days in a liquid medium with sucrose as the single carbon source, the accumulated PHA proportion reaches 83.3%. Therefore, preferably, the bacterial agent of this application contains polyhydroxyalkanoates with a cell dry weight proportion greater than 68%.

[0014] The third aspect of this application discloses the use of Priestella MIBE00003 or the microbial agent of this application in the production of polyhydroxyalkanoates.

[0015] The polyhydroxy fatty acid esters of this application particularly include poly(3-hydroxybutyrate).

[0016] The fourth aspect of this application discloses a method for producing polyhydroxy fatty acid esters, comprising inoculating the *Primaterella MIBE00003* of this application or the bacterial agent of this application into a liquid culture medium, culturing to obtain a fermentation broth, and separating the polyhydroxy fatty acid esters from the cultured fermentation broth.

[0017] It should be noted that the method of this application, by using the Priestella MIBE00003 of this application, or an inoculum containing this strain, can efficiently produce PHA using a variety of inexpensive carbon sources. For example, in one implementation of this application, the cell dry weight of the fermentation broth is 15.0 g / L, and the cumulative PHA accounts for 68.5% of the cell dry weight.

[0018] In one implementation of this application, the culture conditions for obtaining the fermentation broth are 25-35℃ and 150-220r / min for 1-3 days.

[0019] In one implementation of this application, the liquid culture medium is an inorganic salt culture medium that uses any one of glucose, sucrose, molasses, cassava starch, crude glycerol, and sodium acetate as a single carbon source.

[0020] It should be noted that one of the characteristics of the Priestella MIBE00003 in this application is that it can efficiently produce PHA using a variety of inexpensive single carbon sources, such as inexpensive sucrose, molasses, tapioca starch, crude glycerol or sodium acetate, and of course glucose can also be used as a single carbon source.

[0021] It should also be noted that, considering the cost of carbon sources, it is preferable to use any one of sucrose, molasses, tapioca starch, crude glycerol, and sodium acetate as the single carbon source for the liquid culture medium. Taking into account both the cost of carbon sources and the efficiency of PHA synthesis, it is preferable to use any one of molasses, sucrose, tapioca starch, and crude glycerol as the single carbon source for the liquid culture medium; more preferably, molasses is used as the single carbon source.

[0022] In one implementation of this application, the liquid culture medium also includes a nitrogen source.

[0023] In one implementation of this application, the nitrogen source is yeast extract and / or NH4Cl.

[0024] In one implementation of this application, the inorganic salt culture medium further includes Na2HPO4, KH2PO4, NaCl, MgSO4, CaCl2, ferric ammonium citrate, and trace element solution; wherein the trace element solution includes ZnSO4, MnCl2, H3BO3, CoCl2, CuCl2, NiCl2, and NaMoO4.

[0025] In one implementation of this application, the liquid culture medium comprises the following components.

[0026] (1) Carbon source: any one of glucose, sucrose, molasses, tapioca starch, crude glycerol and sodium acetate;

[0027] (2) Nitrogen source: yeast extract and NH4Cl;

[0028] (3) Other nutrients: Na2HPO4, KH2PO4, NaCl, MgSO4, CaCl2, ferric ammonium citrate and trace element solution, the trace element solution is composed of ZnSO4, MnCl2, H3BO3, CoCl2, CuCl2, NiCl2 and NaMoO4.

[0029] In one implementation of this application, the concentration of carbon source in the liquid culture medium is 30 g / L, the concentration of yeast extract in the nitrogen source is 1 g / L, the concentration of NH4Cl is 1 g / L, and other nutrients consist of 9 g / L Na2HPO4, 1.5 g / L KH2PO4, 30 g / L NaCl, 0.2 g / L MgSO4, 0.02 g / L CaCl2, 0.0012 g / L ferric ammonium citrate, and 100 μL of trace element solution. The trace element solution consists of 1 g / L ZnSO4, 0.3 g / L MnCl2, 3 g / L H3BO3, 2 g / L CoCl2, 0.1 g / L CuCl2, 0.2 g / L NiCl2, and 0.3 g / L NaMoO4.

[0030] It should be noted that the specific liquid culture medium formulations described above are merely one implementation method of this application, demonstrating the ability to efficiently produce PHA using an inexpensive single carbon source. For example, using the above formulation, after fermentation and culture with molasses as the single carbon source for 2 days, the cell dry weight was 15.0 g / L, with a cumulative PHA content of 68.5%. Molasses is low in cost and yields a high PHA production. It is understood that the key to the *Primaterella* MIBE00003 of this application lies in its ability to efficiently produce PHA using multiple inexpensive single carbon sources. Other components in the liquid culture medium can be adjusted experimentally according to needs or actual conditions, and are not limited to the specific formulations described above. Especially when carbon and nitrogen sources are ensured, other nutrients can be reduced or replaced according to actual conditions, and the amount of each component in other nutrients can also be adjusted accordingly.

[0031] The fifth aspect of this application discloses the application of the *Primaterella multocida* MIBE00003 of this application, or the bacterial agent of this application, or the method of producing polyhydroxyalkanoates of this application, in the preparation of packaging materials, adhesive materials, spraying materials or medical materials based on polyhydroxyalkanoates.

[0032] The beneficial effects of this application are as follows:

[0033] The *Primate* MIBE00003 strain described in this application can efficiently produce polyhydroxyalkanoates using a variety of inexpensive single carbon sources. In one implementation of this application, the cell dry weight in the fermentation broth reaches 15.0 g / L, and the proportion of polyhydroxyalkanoates in the cell dry weight is as high as 68.5%. This provides a new strain resource for the production of natural polymer biomaterials that can replace petroleum-based plastics, and offers a new scheme and approach for the efficient and low-cost production of polyhydroxyalkanoates. Attached Figure Description

[0034] Figure 1 Example 1 of this application shows the amplification of the phaC synthase gene by polymerase chain reaction (PCR) of Priscilla MIBE00003, and the gel image after separation of the PCR product by agarose gel electrophoresis.

[0035] Figure 2 This is a colony diagram of *Pristemonella MIBE00003* from Example 2 of this application, cultured at 30°C for 1 day.

[0036] Figure 3 This is a Gram staining image of Priestella MIBE00003 from Example 2 of this application;

[0037] Figure 4This is a phylogenetic tree constructed from the 16S sequence of Priestella MIBE00003 in Example 2 of this application;

[0038] Figure 5 This is a graph showing the PHA production results of *Primatelia MIBE00003* in Example 3 of this application.

[0039] Preservation information of the strain: The Priestia sp. strain MIBE00003 of this application was deposited on June 2, 2022 at the China Center for Type Culture Collection (CCTCC, address: No. 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, China, postal code: 430072), classified as Priestia sp., with accession number CCTCC M 2022792. Detailed Implementation

[0040] The present application will be further described in detail below through specific embodiments. The following embodiments are only for further illustration of the present application and should not be construed as limiting the present application.

[0041] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0042] The culture medium used in the examples includes:

[0043] (1) Oil and salt enrichment medium: Add 1-10% (v / v) of mixed vegetable oil (Golden Dragon Fish: Peanut oil: Rapeseed oil = 1:1:1) and 35-125‰ NaCl to the aged seawater for the isolation of strains.

[0044] According to GB / T 1534-2017, peanut oil contains more than 5% of the following components: palmitic acid (C16:0, 8.0-14.0), oleic acid (C18:1, 35.0-69.0), and linoleic acid (C18:2, 13.0-43.0). According to GB / T 1536-2004, rapeseed oil contains more than 5% of the following components: palmitic acid (C16:0, 1.5-6.0), oleic acid (C18:1, 8.0-60.0), linoleic acid (C18:2, 11.0-23.0), linolenic acid (C18:2, 5.0-13.0), erucic acid (C22:1, 3.0-60.0), and arachidonic acid (C20:1, 3.0-15.0).

[0045] (2) Ordinary seawater culture medium (2216E): 5.0 g / L peptone, 1.0 g / L yeast extract, 0.1 g / L ferric citrate, 19.45 g / L NaCl, 5.98 g / L MgCl2, 3.24 g / L Na2SO4, 1.8 g / L CaCl2, 0.55 g / L KCl, 0.16 g / L Na2CO3, 0.08 g / L KBr, 0.034 g / L SrCl2, 0.022 g / L H3BO3, 0.004 g / L Na2O·nSiO2, 0.0016 g / L NaNO3, 0.0024 g / L NaF and 0.008 g / L Na2HPO4, used for the purification culture of the strain and the preparation of seed culture.

[0046] (3) PHA-producing liquid culture media include inorganic salt media with glucose as the single carbon source or inorganic salt media with multiple inexpensive carbon sources such as sucrose, molasses, cassava starch, crude glycerol and sodium acetate as the single carbon source. The components are as follows:

[0047] Carbon source: any one of glucose, sucrose, molasses, tapioca starch, crude glycerol, and sodium acetate, at a concentration of 30 g / L.

[0048] Nitrogen source: 1 g / L yeast extract, 1 g / L NH4Cl.

[0049] Other nutrients: 9 g / L Na2HPO4, 1.5 g / L KH2PO4, 30 g / L NaCl, 0.2 g / L MgSO4, 0.02 g / L CaCl2, 0.0012 g / L ferric ammonium citrate, and 100 μL of trace element solution (1 g / L ZnSO4, 0.3 g / L MnCl2, 3 g / L H3BO3, 2 g / L CoCl2, 0.1 g / L CuCl2, 0.2 g / L NiCl2, 0.3 g / L NaMoO4).

[0050] The initial pH of the above culture medium was 7.0, and it was sterilized at 121°C for 25 min.

[0051] Example 1: Isolation and Screening of Priestella MIBE00003

[0052] This embodiment provides a method for isolating and screening Priscilla MIBE00003, and the specific steps are as follows:

[0053] 1. Isolation of culturable strains

[0054] Soil samples were collected from the top 10cm layer of the tidal flats in the mangrove reserve of Futian District, Shenzhen. The collected soil samples were added to Erlenmeyer flasks containing 100mL of sterilized oil- and salt-containing enrichment medium and incubated at 30℃ and 160r / min with constant temperature shaking for 77 days. Every 7 days, the samples were transferred to enrichment medium containing fresh medium at an inoculum rate of 1%, while simultaneously increasing the oil content by 1% and the salinity by 10‰. 1mL of soil suspension was collected on days 7, 42, and 77. The soil suspension was then serially diluted to prepare 10... -3 Up to 10 -5 Diluted suspensions with concentration gradients were prepared; the diluted suspensions were added to 2216E medium plates for plating and incubated at 30°C for 48 hours to obtain colonies; single colonies with different morphologies were picked for streak purification culture, numbered, and stored at low temperature.

[0055] 2. Screening of PHA-producing strains

[0056] Colony PCR was used to identify the phaC gene in the isolated strains. Single colonies were picked and added to a sterile PCR tube containing 50 μL of sterile water, and incubated at 95°C for 10 min to obtain the colony PCR template. The forward primer for the phaC gene was PHACGNF (SEQ ID NO. 2: 5'-CCYRGATCAACAAGTTCTAC-3'), and the reverse primer was PHACGNR (SEQ ID NO. 3: 5'-TTCCAGAACAGMAGGTCGAAGG-3').

[0057] PCR reaction system: 25 μL 2×PCR Master Mix; 1 μL each of forward and reverse primers; 1 μL template; ddH2O to make up to 50 μL.

[0058] PCR reaction conditions: 94℃ pre-denaturation for 6 min; 94℃ denaturation for 45 s, 54℃ annealing for 30 s; 72℃ extension for 90 s, 31 cycles; 72℃ extension for 10 min, storage at 4℃. The obtained PCR products were subjected to 120V, 30 min, 1% agarose gel electrophoresis. The gel was observed using a blue light transilluminator. Samples with bands were identified as phaC gene-positive strains, named MIBE00003. The electrophoresis pattern is shown below. Figure 1 As shown.

[0059] Example 2: Identification of Priestella MIBE00003

[0060] This embodiment identifies the *Priscilla MIBE00003* obtained in Example 1, including morphological observation, physiological and biochemical identification, and 16S rDNA sequence analysis. The specific steps are as follows:

[0061] 1. Morphological identification

[0062] The strain MIBE00003 isolated in Example 1 was streaked onto a 2216E medium plate. The plate was then inverted and incubated at 30°C for 24 hours. The growth of colonies on the plate was observed and recorded. The colony morphology of strain MIBE00003 is as follows: Figure 2 As shown. From Figure 2 The colonies of the strain are light yellow, round, with irregular edges and a moist, smooth surface.

[0063] Gram staining of strain MIBE00003 was performed using a kit, and the staining was observed under a 100x oil immersion microscope. The Gram staining image of strain MIBE00003 is shown below. Figure 3 As shown. From Figure 3 The strain appears purple, indicating it is a Gram-positive bacterium.

[0064] 2. Physiological and biochemical identification

[0065] Physiological and biochemical identification indicators were used to identify strain MIBE00003 isolated in Example 1 according to the "Manual of Systematic Identification of Common Bacteria" and "Bergey's Manual of Bacterial Identification" (9th edition).

[0066] The physiological and biochemical identification indicators provided in this embodiment include catalase activity, oxidase activity, methyl red MR test, VP test, starch hydrolysis activity, gelatin liquefaction activity, nitrate reduction activity, hydrogen sulfide production activity, citrate utilization activity, lysozyme activity, and mannitol acid production activity. The physiological and biochemical identification results of strain MIBE00003 are shown in Table 1.

[0067] Table 1. Physiological and biochemical identification results of the strains

[0068] catalase + Nitrate reduction - Oxidase + hydrogen sulfide + MR experiment + citrate utilization - VP Experiment - Lysozyme + Starch hydrolysis + Mannitol produces acid - Gelatin liquefaction -

[0069] In the table: + indicates that the strain is reactive or usable; - indicates that the strain is unreactive or unusable.

[0070] 3.16S rDNA sequence analysis

[0071] In this embodiment, DNA was extracted from strain MIBE00003 using the Ezup bacterial genomic DNA extraction kit. The forward primer for PCR amplification was 27F (SEQ ID NO.4: 5'-AGAGTTTGATCCTGGCTCAG-3'), and the reverse primer was 1492R (SEQ ID NO.5: 5'-GGTTACCTTGTTACGACTT-3').

[0072] PCR reaction system: 25 μL 2×PCR Master Mix; 1 μL each of forward and reverse primers; 1 μL template; ddH2O to make up to 50 μL.

[0073] PCR reaction conditions: 94℃ pre-denaturation for 4 min, 1 cycle; 94℃ denaturation for 45 s, 55℃ annealing for 45 s; 72℃ extension for 90 s, 30 cycles; store at 4℃.

[0074] The PCR products were sequenced by Shanghai Sangon Biotech Co., Ltd., and the sequencing results are shown in SEQ ID NO. 1. The obtained sequences were compared with GenBank using BLAST, yielding sequences with high similarity. A phylogenetic tree of strain MIBE00003 was constructed using MEGA 7.0 software. The 16S rDNA sequence of this strain showed 99.9% homology with *Priestiasp.*. The phylogenetic tree of strain MIBE00003 is shown below. Figure 4 As shown.

[0075] Based on the above morphological observations, physiological and biochemical identifications, and 16S rDNA sequence analysis, strain MIBE00003 was identified as *Priestia sp.*, and named *Priestia sp.* MIBE00003. This strain was deposited on June 2, 2022, at the China Center for Type Culture Collection (CCTCC, address: 299 Bayi Road, Wuchang District, Wuhan, Hubei Province, 430072, China), classified as *Priestia sp.*, with accession number CCTCC M 2022792.

[0076] Example 3: Determination of the ability of Priscilla MIBE00003 to produce PHA using inexpensive carbon sources

[0077] This embodiment measures the ability of Priscilla MIBE00003 to produce PHA using various inexpensive carbon sources. The measurement includes the following:

[0078] 1. Production of PHA

[0079] The *Priestella MIBE00003* strain provided in Example 1 was re-streaked onto a 2216E solid medium plate and cultured in a 30°C incubator for 1 day to obtain a reactivated strain. A single clone of the newly activated strain was picked and inoculated into a 250mL Erlenmeyer flask containing 50mL of sterilized 2216E liquid medium and cultured at 30°C and 180rpm for 1 day to obtain a seed culture. At an inoculation rate of 2%, the seed culture was inoculated into a 250mL Erlenmeyer flask containing 50mL of sterilized PHA-producing liquid medium and cultured at 30°C and 180rpm for 2 days to obtain a fermentation broth.

[0080] In this example, six liquid culture media for PHA production were prepared with glucose, sucrose, molasses, cassava starch, crude glycerol, or sodium acetate as carbon sources, and were used in experiments to test the efficiency of PHA production with different carbon sources. In all six PHA-producing liquid culture media, the carbon source concentration was 30 g / L; the other components were the same, namely, the nitrogen source consisted of 1 g / L yeast extract and 1 g / L NH4Cl; other nutrients included: 9 g / L Na2HPO4, 1.5 g / L KH2PO4, 30 g / L NaCl, 0.2 g / L MgSO4, 0.02 g / L CaCl2, 0.0012 g / L ferric ammonium citrate, and 100 μL of trace element solution (1 g / L ZnSO4, 0.3 g / L MnCl2, 3 g / L H3BO3, 2 g / L CoCl2, 0.1 g / L CuCl2, 0.2 g / L NiCl2, 0.3 g / L NaMoO4).

[0081] The initial pH of the above six PHA-producing liquid culture media was 7.0, and they were used after being sterilized at 121°C for 25 minutes.

[0082] 2. Extraction of PHA

[0083] The fermentation broth obtained after 2 days of culture was transferred to a 50 mL centrifuge tube and centrifuged at 6000 rpm for 10 min. The supernatant was removed, and then 30 mL of deionized water was added. After thorough mixing, the mixture was centrifuged again at 6000 rpm for 10 min, and the supernatant was removed to obtain the cell pellet of the strain. The cell pellet was pre-frozen at -80℃ for 12 h, and then freeze-dried to obtain the lyophilized sample of the strain. 20 mg of the lyophilized sample of the strain was weighed and placed in a 10 mL lipidization tube. Under fume hood conditions, 2 mL of chloroform (containing 0.5 mg / mL methyl benzoate) and 2 mL of methanol solution containing 15% (v / v) concentrated sulfuric acid were slowly added to the esterification tube, mixed thoroughly, and the cap was tightened. The esterification tube with the sample added was placed in a water bath at 100°C and sealed for 4 hours to carry out the methyl esterification reaction. After the reaction was completed, the tube was cooled in an ice bath for 10 minutes, and then 1 mL of deionized water was added to the esterification tube. The mixture was mixed thoroughly for 30 seconds and allowed to stand for layering to obtain the lower layer of methyl esterification product sample.

[0084] 3. Determination of PHA

[0085] The methyl esterification product samples were used to determine the PHA content. A DB-WAX column was selected as the stationary phase, and helium was used as the mobile phase. The injection volume was set to 1 μL, the injection temperature to 250℃, and the flow rate to 0.7 mL / min. Analytical grade poly(3-hydroxybutyrate) (PHB) was used as a standard for qualitative analysis of the PHA synthesized by the strain, and methyl benzoate was used as an internal standard for quantitative analysis. PHA products of a certain gradient mass were weighed for methyl esterification pretreatment. After gas chromatography analysis, the ratio of PHA monomer peak area to internal standard peak area and the ratio of monomer mass to internal standard mass were used to construct a standard curve. This standard curve was used for quantitative analysis of PHA content in stem cells.

[0086] The above standard curve is calculated using the following formula:

[0087] PHA content (%) = PHA concentration (g / L) / CDW (g / L) × 100%

[0088] CDW represents the dry weight of the strain's cells.

[0089] 4. PHA content

[0090] The results of the PHA production capacity determination of Priscilla MIBE00003 in this embodiment are as follows: Figure 5 As shown, from Figure 5 It can be seen that strain MIBE00003 can synthesize PHA when fermented in an inorganic salt medium with glucose as the single carbon source or an inorganic salt medium with any of the inexpensive carbon sources such as sucrose, molasses, cassava starch, crude glycerol and sodium acetate as the single carbon source.

[0091] After culturing for 2 days in an inorganic salt medium with glucose as the sole carbon source, the cell dry weight of the strain was 4.7 g / L, and the cumulative amount of PHA accounted for 57.3% of the cell dry weight.

[0092] After culturing for 2 days in an inorganic salt medium with sucrose as the sole carbon source, the cell dry weight of the strain was 6.5 g / L, and the cumulative amount of PHA accounted for 83.3% of the cell dry weight. Compared with glucose, sucrose has the advantage of being inexpensive and also has a higher PHA synthesis efficiency.

[0093] After culturing for 2 days in an inorganic salt medium with molasses as the sole carbon source, the cell dry weight of the strain was 15.0 g / L, and the cumulative amount of PHA accounted for 68.5% of the cell dry weight. Compared with glucose, molasses is not only cheaper, but also has a higher PHA synthesis efficiency.

[0094] After culturing the strain in an inorganic salt medium with cassava starch as the sole carbon source for 2 days, the cell dry weight was 6.4 g / L, and the cumulative amount of PHA accounted for 63.0% of the cell dry weight. Compared with glucose, cassava starch is not only cheaper, but also has a higher PHA synthesis efficiency.

[0095] After culturing for 2 days in an inorganic salt medium with crude glycerol as the sole carbon source, the cell dry weight of the strain was 6.8 g / L, and the cumulative amount of PHA accounted for 65.3% of the cell dry weight. Compared with glucose, crude glycerol is not only cheaper, but also has a higher PHA synthesis efficiency.

[0096] After culturing for 2 days in an inorganic salt medium with sodium acetate as the sole carbon source, the cell dry weight of the strain was 2.3 g / L, and the cumulative amount of PHA accounted for 22.5% of the cell dry weight.

[0097] The above results indicate that, in addition to utilizing glucose as a traditional fermentation substrate, *Priscilla MIBE00003* can also efficiently synthesize PHA by metabolizing various inexpensive carbon sources such as sucrose, molasses, cassava starch, and crude glycerol, thereby greatly reducing the raw material cost of PHA production and showing good prospects for industrial application.

[0098] The above description, in conjunction with specific embodiments, provides a further detailed explanation of this application and should not be construed as limiting the specific implementation of this application to these descriptions. Those skilled in the art to which this application pertains can make several simple deductions or substitutions without departing from the concept of this application.

Claims

1. A species of Priestella with accession number CCTCC M 2022792 ( Priestia The strain MIBE00003 was deposited on June 9, 2022, at the China Center for Type Culture Collection, No. 299, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

2. A microbial agent, characterized in that: Contains the Priestella as described in claim 1 ( Priestia sp.) MIBE00003 or its fermentation broth.

3. The microbial agent according to claim 2, characterized in that: In the bacterial agent, polyhydroxy fatty acid esters account for a certain percentage of the *Priscilla* ( Priestia The dry weight percentage of MIBE00003 (sp.) is greater than 68%.

4. The Priestella as described in claim 1 ( Priestia sp.) MIBE00003, or the application of the microbial agent as described in claim 2 or 3 in the production of polyhydroxy fatty acid esters.

5. A method for producing polyhydroxyalkanoates, characterized in that: Including the Priestella as described in claim 1 ( Priestia (spe.)MIBE00003, or the bacterial agent according to claim 2 or 3, is inoculated into a liquid culture medium, cultured to obtain a fermentation broth, and polyhydroxy fatty acid esters are isolated from the cultured fermentation broth.

6. The method according to claim 5, characterized in that: The culture conditions for obtaining the fermentation broth are 25-35℃ and 150-220r / min for 1-3 days.

7. The method according to claim 5, characterized in that: The liquid culture medium is an inorganic salt culture medium that uses any one of glucose, sucrose, molasses, cassava starch, or crude glycerol as a single carbon source.

8. The method according to claim 7, characterized in that: The liquid culture medium also includes a nitrogen source.

9. The method according to claim 8, characterized in that: The nitrogen source is yeast extract and / or NH4Cl.

10. The method according to claim 9, characterized in that: The inorganic salt culture medium also includes Na2HPO4, KH2PO4, NaCl, MgSO4, CaCl2, ferric ammonium citrate, and trace element solution; the trace element solution includes ZnSO4, MnCl2, H3BO3, CoCl2, CuCl2, NiCl2, and NaMoO4.

11. The method according to claim 10, characterized in that: The liquid culture medium consists of the following components. (1) Carbon source: any one of glucose, sucrose, molasses, tapioca starch, and crude glycerol; (2) Nitrogen source: yeast extract and NH4Cl; (3) Other nutrients: Na2HPO4, KH2PO4, NaCl, MgSO4, CaCl2, ferric ammonium citrate and trace element solution, wherein the trace element solution is composed of ZnSO4, MnCl2, H3BO3, CoCl2, CuCl2, NiCl2 and NaMoO4.

12. The method according to claim 11, characterized in that: The liquid culture medium contains a carbon source concentration of 30 g / L, a nitrogen source concentration of 1 g / L for yeast extract and 1 g / L for NH4Cl, and other nutrients consisting of 9 g / L Na2HPO4, 1.5 g / L KH2PO4, 30 g / L NaCl, 0.2 g / L MgSO4, 0.02 g / L CaCl2, 0.0012 g / L ferric ammonium citrate, and 100 μL of trace element solution. The trace element solution consists of 1 g / L ZnSO4, 0.3 g / L MnCl2, 3 g / L H3BO3, 2 g / L CoCl2, 0.1 g / L CuCl2, 0.2 g / L NiCl2, and 0.3 g / L NaMoO4.

13. The Priestella as described in claim 1 ( Priestia The use of (sp.)MIBE00003, or the microbial agent according to claim 2 or 3, or the method according to any one of claims 5-12, in the preparation of packaging materials, adhesive materials, spraying materials or medical materials based on polyhydroxyalkanoates.

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