Prosteria megatherium and method for producing PHA (polyhydroxyalkanoate) by semi-solid fermentation of Prosteria megatherium

Abstract

The invention provides a Pichia megaterium BA1251 and a method for producing polyhydroxyalkanoate (PHA) by utilizing the Pichia megaterium BA1251 through semi-solid shallow tray fermentation. The Pichia megaterium BA1251 has the advantages that the Pichia megaterium BA1251 can be used for producing the polyhydroxyalkanoate (PHA) by the aid of the Pichia megaterium BA1251; the dry weight of zymophyte produced by the semi-solid fermentation method can reach 100g / kg, and the semi-solid fermentation method has the advantages of high yield, short period, low cost, less waste and environmental friendliness.

Description

Technical Field

[0001] The present invention relates to a Priestia megaterium and a method for semi-solid fermentation of polyhydroxyalkanoate (PHA) using the bacterium, belonging to the field of microbial fermentation. Background Art

[0002] Polyhydroxyalkanoate (PHA) is a completely bio-based and biodegradable bioplastic with great potential to replace traditional plastics. PHA is synthesized by microorganisms through fermentation of sugars, vegetable oils or other carbon sources. PHA can be degraded by microorganisms in natural environments such as soil, seawater and compost, and finally decomposed into water and carbon dioxide without causing pollution. PHA has good biocompatibility, processability and mechanical properties, and has a wide range of uses, and can be used in fields such as packaging, agriculture, medicine, 3D printing, etc.

[0003] There are many problems in the industrialization of polyhydroxyalkanoate, such as low fermentation level. The accumulation of PHA in bacteria is affected by the metabolism of the strains themselves, and is also affected by the environment and nutrients, and the energy consumption is too high. To solve these problems, people have tried to solve them from many aspects, improve the fermentation level, use synthetic biology means to transform the metabolic pathways of existing strains, knock out the secondary metabolic pathways unrelated to PHA synthesis, and improve the metabolic flux of PHA; in addition, try to change the cell morphology and size to accumulate more PHA in the cells, or screen relatively extreme environments, such as PHA-producing bacteria under high-salt environments.

[0004] The PHA production processes disclosed in the prior art are as follows: Chinese invention patents with application publication numbers CN115807043A, CN117535356A, CN111593078A, and CN111394398A disclose the production of PHA using continuous fermentation or batch fermentation in a fermenter; Chinese invention patent with application publication number CN118813725A discloses a semi-continuous fermentation process for producing PHA using Halomonas as the base strain; Chinese invention patent with application publication number CN118813726A discloses a continuous fermentation process for producing PHA using Halomonas as the base strain to eliminate the influence of toxic metabolites in each stage on fermentation; Chinese invention patent with application publication number CN118726499A discloses continuous fermentation by continuously adding a carbon source and a nutrient solution in a cell proliferation tank, and realizing the continuous production of PHA by controlling the carbon-nitrogen ratio in two series-connected fermenters; Chinese invention patent with application publication number CN118685466A discloses the use of a mixed bacterial population at high density to increase the PHA yield; Chinese invention patent with application publication number CN119351482A discloses a two-step fermentation method for producing PHA using organic waste and Cupriavidus necator. Application publication number CN116042447A uses a liquid medium to ferment Priestia megaterium MIBE00004 to produce PHA. The cell dry weight in the fermentation broth is 13.8 g / L, and the proportion of accumulated PHA in the cell dry weight can reach 65.8%.

[0005] The PHA production processes disclosed in the prior art generally involve fermentation in a large fermenter and then separating PHA from the fermentation broth. The separation usually requires expensive and energy-consuming continuous centrifugation, microfiltration, or time-consuming gravity sedimentation, etc. Although halophilic bacteria have been engineered into PHA-producing bacteria, there are still some problems to be solved, such as corrosion of steel equipment caused by high salt, high cost of the induction system, high-salt wastewater treatment, and inconvenient gene manipulation, etc. Summary of the Invention

[0006] To solve the problems existing in the prior art, the present invention provides a Priestia megaterium BA1251 and a method for producing polyhydroxyalkanoates (PHA) by semi-solid fermentation using this bacterium.

[0007] The present invention is achieved through the following technical solutions:

[0008] As the first aspect of the present invention, the present invention provides a Priestia megaterium BA1251, which is taxonomically named Priestia megaterium. This strain has been deposited as a patented strain at the General Microbiology Center of the China Center for Type Culture Collection (CGMCC) located in Beijing, China. The deposit number is CGMCC NO. 33762, and the deposit date is March 10, 2025.

[0009] After accumulating PHA, this bacterium can form a thick layer of sand-like cells, and the cells can be collected by scraping the cells on the surface of the culture medium. Therefore, PHA can be produced through a semi-solid fermentation mode. In one embodiment of the present application, the dry weight of the cells obtained by semi-solid fermentation of this strain to produce PHA can reach about 100 grams per kilogram, and the proportion of the accumulated PHA in the dry weight of the cells can reach 70.0%.

[0010] As the second aspect of the present invention, the present invention provides the application of the Priestia megaterium BA1251 in the production of PHA.

[0011] As the third aspect of the present invention, the present invention provides a method for producing polyhydroxyalkanoates (PHA) by semi-solid fermentation. The method is to use Priestia megaterium BA1251 to produce PHA through semi-solid fermentation culture. The Priestia megaterium has been deposited as a patented strain at the General Microbiology Center of the China Center for Type Culture Collection (CGMCC) located in Beijing, China. The deposit number is CGMCC NO. 33762, and the deposit date is March 10, 2025.

[0012] Furthermore, the method includes the following steps:

[0013] Step 1, inoculation and culture: inoculate the liquid strain of Priestia megaterium BA1251 into a semi-solid medium for culture;

[0014] Step 2, harvesting and extraction: collect the cells on the surface of the semi-solid medium, add lysozyme and perform mechanical crushing to extract PHA.

[0015] Preferably, the semi-solid medium in Step 1 contains a carbon source, a nitrogen source, and inorganic salts; wherein, the carbon source is selected from any one or more of corn straw, wheat straw, rice straw, weeds enzymatically hydrolyzed by cellulase and xylanase, or corn flour, sweet potato flour, cassava flour, fruit residue, molasses, and grease; the nitrogen source is selected from any one or more of ammonium sulfate, ammonium chloride, ammonium acetate, and ammonium citrate; the inorganic salts are selected from any one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, sodium chloride, calcium chloride, and potassium chloride;

[0016] More preferably, the carbon source is a combination of corn flour and enzymatically hydrolyzed straw, or a combination of corn flour, molasses and fruit residue. Even more preferably, the carbon source is a combination of 2% - 3% corn flour and 20% - 40% enzymatically hydrolyzed straw, or a combination of 2% - 3% corn flour, 2% - 5% molasses and 2% - 5% fruit residue.

[0017] More preferably, the content of the nitrogen source is 0.2% - 0.4%.

[0018] More preferably, the sodium chloride content is 1% - 10%; even more preferably, the sodium chloride content is 2% - 8%.

[0019] More preferably, the culture medium further comprises any one of agar, glutinous rice flour or kudzu root powder; the content is 0.5% - 1.0%.

[0020] More preferably, the culture medium further comprises trace elements, and the trace elements are selected from any one or several of KI, H3BO3, MnSO4, ZnSO4, Na2MoO4, CuSO4, CoCl.

[0021] More preferably, after the culture medium solidifies, 0.5% - 2.0% of starch, rice bran, wheat bran or sodium alginate is evenly sprinkled on the surface.

[0022] Most preferably, the semi-solid culture medium formula is: 1 liter of culture medium contains 20 - 30 grams of corn flour, 200 - 400 ml of enzymatically hydrolyzed straw, 10 - 80 grams of sodium chloride, 3 - 10 grams of potassium dihydrogen phosphate, 2 - 5 grams of magnesium sulfate, 1 - 2 grams of ammonium sulfate, 1 - 2 grams of ammonium citrate, 1 - 2 grams of potassium chloride, trace elements (0.8 mg of KI, 6.0 mg of H3BO3, 22.0 mg of MnSO4, 10.0 mg of ZnSO4, 0.3 mg of Na2MoO4, 0.3 mg of CuSO4, 0.025 mg of CoCl), 5 - 10 grams of agar, glutinous rice flour or kudzu root powder, the balance being water, and the pH is adjusted to 7.0 - 7.5;

[0023] Or, 1 liter of culture medium contains 20 - 30 grams of corn flour, 20 - 50 grams of molasses, 20 - 50 grams of fruit residue, 10 - 100 grams of sodium chloride, 5 - 10 grams of potassium dihydrogen phosphate, 2 - 5 grams of magnesium sulfate, 1 - 2 grams of ammonium sulfate, 1 - 2 grams of ammonium citrate, 1 - 2 grams of potassium chloride, trace elements (0.8 mg of KI, 6.0 mg of H3BO3, 22.0 mg of MnSO4, 10.0 mg of ZnSO4, 0.3 mg of Na2MoO4, 0.3 mg of CuSO4, 0.025 mg of CoCl), 5 - 10 grams of agar or kudzu root powder, the balance being water, and the pH is adjusted to 7.0 - 7.5.

[0024] Preferably, the inoculation amount in Step 1 is 10% - 20% of the volume of the semi-solid medium. More preferably, the inoculation amount is 15% of the volume of the semi-solid medium.

[0025] Preferably, the culture temperature in Step 1 is 22°C - 45°C; more preferably, for energy-saving considerations, the temperature can be controlled separately in winter and summer. Specifically: the culture temperature in winter is 22°C - 33°C, the relative humidity is controlled at 90 - 95%, and the culture lasts for 50 - 55 hours; the culture temperature in summer is 33°C - 45°C, the relative humidity is controlled at 90 - 95%, and the culture lasts for 40 - 48 hours.

[0026] Preferably, after harvesting the bacterial cells in Step 2, nutrient solution is added to the medium, and fermentation to produce polyhydroxyalkanoates is continued for 2 consecutive cultures. The nutrient solution is a semi-solid fermentation medium formula without agar / rice flour / kudzu root powder.

[0027] Advantages of the present invention:

[0028] 1. The semi-solid fermentation culture method of the present application is one of the innovations of the present application. During the research process, the inventors found that only the bacterium Priestia megaterium BA1251 of the present invention can form a thick layer of sand-like bacterial cells on the surface of the medium after accumulating PHA, and the bacterial cells can be collected by scraping the bacterial cells on the surface of the medium (the bacterial cells fermented by other strains are all colloidal and adhere to the solid medium and cannot be scraped). Therefore, the semi-solid fermentation mode was proposed, which is the first time in the field of fermentative cultivation of Priestia megaterium for PHA.

[0029] 2. Based on the fermentation characteristics of Priestia megaterium BA1251, the present application adopts semi-solid shallow pan fermentation, and the dry weight of the fermented bacterial cells can reach 100 g / kg, which is much higher than the traditional liquid fermentation method (the concentration of bacterial cells in traditional liquid fermentation is only 13 - 15 g / L). There is no need for operations such as centrifugation, filtration, and gravity sedimentation, and it can be directly dried. Compared with the traditional PHA liquid fermentation process, the method of the present invention produces less wastewater and reduces the pressure of wastewater treatment.

[0030] 3. The semi-solid medium of the present application does not require autoclaving: First, the strain BA1251 of the present invention grows rapidly, has a wide adaptability to the temperature range, and has strong anti-pollution ability; second, the strain has the characteristic of salt tolerance and can grow on a medium containing 10% sodium chloride, while most microorganisms are not salt-tolerant and cannot grow on a medium containing sodium chloride. Therefore, the medium of the present application does not require autoclaving; third, the semi-solid medium needs to be boiled during the preparation process, and most of the miscellaneous bacteria have been killed, so there is no need for separate sterilization; fourth, a specific inoculation ratio (10 - 20% of the solid medium) can make the bacterial liquid completely cover the surface of the medium, greatly eliminating the risk of contamination by miscellaneous bacteria. Therefore, the medium of the present application does not require autoclaving, which greatly reduces the cost.

[0031] 4. The semi-solid culture medium of this application can be reused (twice), which not only reduces costs, but also avoids waste of nutrients and excessive accumulation of waste. After the solid culture medium residue is dehydrated by a screw press, the residual PHA is removed through composting fermentation. The residue after composting is still rich in Priestia megaterium, which is an environmentally friendly strain that can dissolve phosphate rock and is an excellent soil conditioner. Brief Description of the Drawings

[0032] Figure 1 Fermentation bacteria of Priestia megaterium BA1251 producing PHA (close-up shot);

[0033] Figure 2 Fermentation bacteria of Priestia megaterium BA1187 producing PHA (close-up shot);

[0034] Figure 3 Normal microscopic structure of Priestia megaterium BA1251;

[0035] Figure 4 Microscopic structure of Priestia megaterium BA1251 producing PHA;

[0036] Figure 5 Culture diagram of Priestia megaterium BA1251 on insoluble inorganic phosphorus plate medium;

[0037] Figure 6 Agarose gel electrophoresis diagram of phaC gene of Priestia megaterium BA1251;

[0038] Figure 7 Bacteria of Priestia megaterium BA1251 producing PHA in shallow pan culture;

[0039] Figure 8 Harvested bacteria of Priestia megaterium BA1251 producing PHA in shallow pan culture. Detailed Description of the Invention

[0040] The present invention will be further described below through specific examples. The present invention is not limited to the following examples. Changes, combinations or substitutions made within the scope of the present invention or without departing from the content, spirit and scope of the present invention are obvious to those skilled in the art and are included within the scope of the present invention.

[0041] Example 1 Strain Collection, Isolation and Screening of Semi-solid Fermentation Culture

[0042] 1. Collection and Isolation of Strains

[0043] The strain was collected from the saline-alkali land by the Yellow Sea.

[0044] Isolation medium: 1 liter of medium contains 10 g of glucose, 5 g of peptone, 2.5 g of yeast powder, 100 g of sodium chloride, 2 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate, 1.5 g of potassium chloride, 0.1 g of calcium chloride, trace elements (0.8 mg of KI, 6.0 mg of H3BO3, 22.0 mg of MnSO4, 10.0 mg of ZnSO4, 0.3 mg of Na2MoO4, 0.3 mg of CuSO4, 0.025 mg of CoCl), and the pH is adjusted to 7.0. The medium is filled into shake flasks and sterilized for later use.

[0045] Isolation method: Weigh 1 g of soil samples collected from different locations, add 10 ml of sterile water and shake well. Take 1 ml and inoculate it into a shake flask for culture. After culturing and enriching at 30 °C and 150 rpm for 2 days, use an inoculation loop to pick samples from the enrichment shake flask and streak-culture them on the same plate medium for 2 - 3 days for purification to obtain strains BA1251 and BA1187.

[0046] 2. Screening of strains by semi-solid fermentation culture

[0047] Preparation of medium: 1 liter of medium contains 20 g of corn flour, 300 ml of enzymatically hydrolyzed straw juice of cellulase and xylanase, 10 g of sodium chloride, 5 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate, 2 g of ammonium sulfate, 1 g of ammonium citrate, 1 g of potassium chloride, 10 g of agar, and the balance is water. The pH is adjusted to 7.0; Pump the ingredients into a boiling pot, add half the amount of tap water, boil and stir continuously. After boiling, simmer for 15 minutes, then supplement tap water to the required concentration for culture, cool to 60 - 70 °C, pump it into a culture tray through a pipeline, turn on the air conditioner for precooling to promote the medium to quickly cool and solidify to 25 - 30 °C, and evenly sprinkle 1% corn flour on the surface for later use.

[0048] Spray the liquid seeds of strains BA1251 and BA1187 evenly on the surface of the semi-solid medium for fermentation culture to accumulate PHA. In the later stage of fermentation, the fermented bacteria of strain BA1251 are dense granular substances in the culture tray (see Figure 1 ); while the fermented bacteria of strain BA1187 are densely coagulated into a colloidal state and do not form sand-like particles (see Figure 2 ). Based on the characteristic that strain BA1251 can form "sand-like bacterial cells" after accumulating PHA, the bacterial cells can be collected by scraping the bacterial cells on the surface of the medium. Therefore, the semi-solid fermentation culture mode can be used to produce PHA.

[0049] Example 2 Identification of Megasphaera elsdenii BA1251

[0050] 1. Morphological characteristics

[0051] The obtained BA1251 strain bacterial suspension was dropped onto a slide and observed under a fiber optic microscope. It was found that the bacterial cells were rod-shaped, single or arranged in short chains. The cells were large, 1.2 - 1.5 × 2.0 - 4.0 micrometers (see Figure 3 ).

[0052] The bacterial suspension of BA1251 strain producing PHA was dropped onto a slide and observed under a microscope. It was found that the bacterial cells were swollen or distorted. The cells were 1.5 - 2.5 × 4.0 - 5.0 micrometers (see Figure 4 ).

[0053] 2. Physicochemical properties

[0054] Identification of phosphorus solubilization effect: The BA1251 strain was spot-inoculated on an insoluble inorganic phosphorus plate medium. The medium formula was: 1 liter of medium contained 10 grams of glucose, 0.3 grams of magnesium sulfate, 5 grams of tricalcium phosphate, 0.5 grams of ammonium sulfate, 0.2 grams of sodium chloride, 0.2 grams of potassium chloride, 0.03 grams of manganese sulfate, 0.01 grams of ferrous sulfate, 0.5 grams of yeast powder, pH 7.0, and 20 grams of agar. After culturing at 30°C for 4 days, observing from the back of the plate, a transparent zone was visible along the outer edge of the colony, and it was determined to have the phosphorus solubilization effect (see Figure 5 ).

[0055] Identification of high salt tolerance characteristics: This strain could grow on a medium containing 10% sodium chloride and had high salt tolerance characteristics.

[0056] 3. Sequence analysis

[0057] 16S rDNA sequence analysis: Bacterial 16S rDNA gene primers were used, F27:

[0058] 5′-AGAGTTTGATCMTGGCTCAG-3′, R1492: 5′-TACGYTACCTTGTTACGACT-3′ for PCR amplification. PCR reaction system: LA Taq enzyme (5U / μL) 0.5μL, 10×LA Taq buffer 5μL, dNTP Mix (2.5mmol / L) 8μL, forward and reverse primers (10μmol / L) 2.5μL each, template (1ng / μL) 1μL, supplemented with ddH2O to 50μL. PCR reaction conditions: 94°C for 5 min; 94°C for 60 s, 55°C for 60 s, 72°C for 90 s, 35 cycles; 72°C for 5 min; stored at 4°C. The PCR products were detected by 1% agarose gel electrophoresis, purified using a product recovery kit, and then sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The differences between the sequencing results and the strain sequences publicly available on NCBI were compared. The comparison showed that the 16S rDNA gene sequence of the BA1251 strain was highly homologous to the sequences of multiple Priestia megaterium strains publicly available in GeneBank. Primer F27:

[0059] The sequences amplified by 5′-AGAGTTTGATCMTGGCTCAG-3′ and R1492: 5-TACGYTACCTTGTTACGACT-3′ have 100% homology with multiple publicly available Priestia megaterium. Through the comparison of 16S rDNA gene homology, strain BA1251 was identified as Priestia megaterium.

[0060] Example 3 Identification of PHA Production by Priestia megaterium BA1251

[0061] 1. Primary Screening

[0062] PHA primary screening medium: 1 liter of medium contains 20 grams of glucose, 20 grams of sodium chloride, 10 grams of potassium dihydrogen phosphate, 2 grams of magnesium sulfate, 2 grams of ammonium sulfate, 1 gram of ammonium citrate, 1 gram of potassium chloride, trace elements (0.8 milligram of KI, 6.0 milligrams of H3BO3, 22.0 milligrams of MnSO4, 10.0 milligrams of ZnSO4, 0.3 milligram of Na2MoO4, 0.3 milligram of CuSO4, 0.025 milligram of CoCl), 15 grams of agar, and the balance is water, with the pH adjusted to 7.0.

[0063] After preparing and sterilizing the primary screening medium, cool it to about 60°C, add Nile blue dye solution, and control the final concentration of Nile blue at 0.5 micrograms per milliliter to prepare a plate medium. Streak-culture the isolated strain BA1251 on the primary screening medium at a culture temperature of 30°C. After 3 days of culture, place the plate under an ultraviolet lamp for observation. The strain shows yellow-green, and it is preliminarily determined that strain BA1251 produces PHA.

[0064] 2. Secondary Screening

[0065] PHA secondary screening medium: 1 liter of medium contains 20 grams of glucose, 20 grams of sodium chloride, 10 grams of potassium dihydrogen phosphate, 2 grams of magnesium sulfate, 2 grams of ammonium sulfate, 1 gram of ammonium citrate, 1 gram of potassium chloride, trace elements (0.8 milligram of KI, 6.0 milligrams of H3BO3, 22.0 milligrams of MnSO4, 10.0 milligrams of ZnSO4, 0.3 milligram of Na2MoO4, 0.3 milligram of CuSO4, 0.025 milligram of CoCl). Fill it into a triangular flask and sterilize it for standby.

[0066] For the BA1251 strain obtained from the primary screening, pick the bacterial cells with an inoculation loop and inoculate them into the secondary screening medium. Culture at a temperature of 30°C and a rotation speed of 150 rpm. After 2 days of culture, take 2 mL of the bacterial liquid cultured for 2 days, centrifuge at 12000 rpm / min for 5 minutes to collect the bacterial cells, add 1 ml of double-distilled water to suspend, add 50 μL of Nile red dye solution with a concentration of 80 μg / mL for staining for 30 minutes. If the bacterial liquid and the bacterial cells turn orange-red, it is determined that the bacterium produces PHA.

[0067] 3. Detection of PHA polymerase gene

[0068] Primers (F: 5′-CGTGCAAGAGTGGGAAAAAT-3′;

[0069] R: 5′-TCGCAATATGATCACGGCTA-3′), genomic DNA was extracted as a template for PCR amplification. The PCR reaction system: LA Taq enzyme (5 U / μL) 0.5 μL, 10×LA Taq buffer 5 μL, dNTP Mix (2.5 mmol / L) 8 μL, forward and reverse primers (10 μmol / L) 2.5 μL each, template (1 ng / μL) 1 μL, supplemented with ddH2O to 50 μL. PCR reaction conditions: 94°C for 5 min; 94°C for 60 s, 57°C for 60 s, 72°C for 90 s, 35 cycles; 72°C for 50 min; stored at 4°C. The PCR products were detected by 1% agarose gel electrophoresis. The bands of the PCR products were detected by agarose gel electrophoresis, and obvious bands were produced at 750 - 1000 bp in agarose electrophoresis (see Figure 6 ). After purifying the products with a product recovery kit, they were sent to Suzhou Genewiz Biotechnology Co., Ltd. for sequencing. The sequencing results were uploaded to GeneBank for comparison. The phaC gene of type IV of strain BA1251 of Priestia megaterium was highly homologous to those of multiple Priestia megaterium and Priestia aryabhattai publicly available in GeneBank, indicating that strain BA1251 of Priestia megaterium has the ability to produce PHA.

[0070] Example 4 Production of PHA using Priestia megaterium BA1251

[0071] 1. Bacterial strain

[0072] The bacterial strain used in the present invention is Priestia megaterium BA1251, which is preserved in the China General Microbiological Culture Collection Center with the preservation number of CGMCC.NO.33762.

[0073] 2. Liquid seed medium and its preparation

[0074] 1 liter of liquid seed medium contains 20 g of glucose, 10 g of peptone, 15 g of yeast powder, 10 g of sodium chloride, 5 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate, 1.5 g of potassium chloride, 0.1 g of calcium chloride, trace elements (0.8 mg of KI, 6.0 mg of H3BO3, 22.0 mg of MnSO4, 10.0 mg of ZnSO4, 0.3 mg of Na2MoO4, 0.3 mg of CuSO4, 0.025 mg of CoCl), and the pH is adjusted to 7.0.

[0075] 3. Solid Medium and Its Preparation

[0076] The medium for PHA production is as follows: 1 liter of the medium contains 20 g of corn flour, 300 ml of enzymatically hydrolyzed straw juice of cellulase and xylanase, 20 g of sodium chloride, 5 g of potassium dihydrogen phosphate, 2 g of magnesium sulfate, 2 g of ammonium sulfate, 1 g of ammonium citrate, 1 g of potassium chloride, 10 g of agar, with the balance being water, and the pH is adjusted to 7.0;

[0077] Pump the ingredients required for PHA cultivation into the cooking pot, add half the amount of tap water, boil while stirring continuously, after boiling, simmer for 15 minutes on a low heat, then supplement tap water to the required concentration for cultivation, cool to 60 - 70 °C, pump it into the culture tray through a pipeline, turn on the air conditioner for precooling to promote the rapid cooling and solidification of the medium to 25 - 30 °C, and evenly sprinkle 1% rice bran on the surface for standby.

[0078] 4. Inoculation and Cultivation

[0079] Open the pipeline of the liquid seed fermenter. In a relatively clean environment, evenly spray the liquid strain onto the surface of the solid medium at a ratio of 15% of the volume of the solid medium, and transfer it to the fermentation culture room for fermentation culture to accumulate polyhydroxyalkanoates.

[0080] Solid culture conditions: In winter, the culture temperature is controlled at 22 - 33 °C, the relative humidity is controlled at 90 - 95%, the carbon dioxide is controlled below 2000 ppm, and the culture lasts for 50 - 55 hours. In the later stage of fermentation, a large amount of PHA accumulates in the cells and appears as dense granular substances in the culture tray (see Figure 1 7 ).

[0081] 5. Harvesting of Bacteria

[0082] After solid fermentation culture for 50 - 55 hours, move the culture tray full of bacteria to the harvesting workshop, scrape the bacteria on the surface of the medium with an automated blade and collect the bacteria (wet bacteria, see Figure 8 ). Spray the PHA - producing medium without agar on the surface of the culture tray after scraping off the bacteria, transfer it back to the culture room for continuous cultivation to accumulate polyhydroxyalkanoates, and conduct continuous cultivation 2 times.

[0083] Transfer the collected bacteria to the extraction tank, add lysozyme and combine with mechanical crushing to extract polyhydroxyalkanoates.

[0084] 6. PHA Detection

[0085] ​Accurately weigh 10 g of wet bacterial cells, dry them in an oven at 105 °C until constant weight (repeat 3 times) to obtain dry bacterial cells, weigh them, and calculate that the dry weight of the fermented bacterial cells reaches 96 g / kg. Dissolve Nile red solid in dimethyl sulfoxide to a concentration of 80 μg / mL. Take 0.1 g of dried bacterial cells, add 2 mL of double-distilled water, centrifuge at 12000 r / min for 5 min to collect the bacterial cell precipitate, and vortex with double-distilled water; add 50 μL of Nile red staining solution, stain at room temperature for 30 min; centrifuge at 12000 r / min for 5 min, discard the supernatant; resuspend with double-distilled water and repeat once; add 2 mL of double-distilled water, and measure the fluorescence intensity at 535 nm with an enzyme-labeled instrument. Using the PHA standard product as a control, calculate the PHA content. The result shows that the PHA concentration is 67.2 g / kg, and the PHA content is 70.0%.

[0086] Dry weight of fermented bacterial cells = dry bacterial cells (g) / wet bacterial cells (kg);

[0087] PHA content % = PHA concentration (g / kg) / dry weight of fermented bacterial cells (g / kg) × 100%.

[0088] 7. Treatment of culture waste liquid and residue

[0089] The solid medium with fully utilized nutrients can be transferred through a conveyor system to a screw press dehydrator for dehydration treatment. The dehydrated culture residue is subjected to composting fermentation to degrade the residual PHA for 20 - 30 days. The residual culture medium after composting treatment can be used as organic fertilizer. The wastewater is aerobically oxidized by the sewage treatment system and recycled after precipitation.

[0090] Example 5 Production of PHA using Priestia megaterium BA1251

[0091] 1. Strain

[0092] The strain used in this invention is Priestia megaterium, and this strain is preserved in the China General Microbiological Culture Collection Center with the preservation number CGMCC NO. 33762.

[0093] 2. Liquid seed medium and its preparation

[0094] 1 L of the medium contains 25 g of glucose, 15 g of peptone, 10 g of yeast extract, 10 g of sodium chloride, 2 g of potassium dihydrogen phosphate, 1 g of magnesium sulfate, 1.5 g of potassium chloride, 0.2 g of calcium chloride, trace elements (0.8 mg of KI, 6.0 mg of H3BO3, 22.0 mg of MnSO4, 10.0 mg of ZnSO4, 0.3 mg of Na2MoO4, 0.3 mg of CuSO4, 0.025 mg of CoCl), and the pH is adjusted to 7.0.

[0095] 3. Solid medium and its preparation

[0096] The PHA production medium consists of: in 1 liter of medium, there are 20 grams of corn flour, 20 grams of molasses, 30 grams of fruit residue, 80 grams of sodium chloride, 8 grams of potassium dihydrogen phosphate, 3 grams of magnesium sulfate, 2 grams of ammonium sulfate, 2 grams of ammonium citrate, 1 gram of potassium chloride, trace elements (0.8 mg of KI, 6.0 mg of H3BO3, 22.0 mg of MnSO4, 10.0 mg of ZnSO4, 0.3 mg of Na2MoO4, 0.3 mg of CuSO4, 0.025 mg of CoCl), 10 grams of glutinous rice flour, with the balance being water, and the pH is adjusted to 7.0.

[0097] Pump the required ingredients into the boiling pan, add half the amount of tap water, boil while constantly stirring, and simmer for 20 minutes after boiling. Then add tap water to the concentration required for cultivation, cool to 60 - 70 °C, pump it into the cultivation tray through a pipeline, turn on the air conditioner for pre-cooling to promote the rapid cooling and solidification of the medium to 25 - 30 °C, and evenly sprinkle 1.0% starch on the surface for standby.

[0098] 4. Inoculation and Cultivation

[0099] Open the pipeline of the liquid seed fermenter. In a relatively clean environment, evenly spray the liquid strain onto the surface of the solid medium at a ratio of 20% of the volume of the solid medium, and transfer it to the fermentation cultivation room for fermentation cultivation to accumulate polyhydroxyalkanoates.

[0100] Solid cultivation conditions: In summer, the cultivation temperature is controlled at 33 - 45 °C, the relative humidity is controlled at 90 - 95%, the carbon dioxide is controlled below 2500 ppm, and the cultivation lasts for 40 - 48 hours. After the fermentation ends, a large amount of PHA accumulates in the cells and appears as dense granular substances in the cultivation tray.

[0101] 5. Harvesting of Bacteria

[0102] After 40 - 48 hours of solid fermentation cultivation, move the tray covered with bacteria to the harvesting workshop, scrape the bacteria on the surface of the medium with an automated scraper and collect the bacteria (wet bacteria). After spraying the PHA production medium without glutinous rice flour on the surface of the tray from which the bacteria have been scraped, transfer it back to the cultivation room for continued cultivation to accumulate polyhydroxyalkanoates, and conduct continuous cultivation 2 times.

[0103] Transfer the collected bacteria to an extraction tank, add lysozyme and combine with mechanical crushing to extract polyhydroxyalkanoates.

[0104] 6. PHA Detection

[0105] Accurately weigh 10 g of wet bacterial cells, dry them in an oven at 105 °C until constant weight (repeat 3 times) to obtain dry bacterial cells, weigh them, and calculate that the dry weight of the fermented bacterial cells reaches 101.3 g / kg. Dissolve Nile red solid in dimethyl sulfoxide to a concentration of 80 μg / mL. Weigh 0.1 g of dried bacterial cells, add 2 mL of double-distilled water, centrifuge at 12000 r / min for 5 min to collect the bacterial cell precipitate, and vortex with double-distilled water; add 50 μL of Nile red staining solution and stain at room temperature for 30 min; centrifuge at 12000 r / min for 5 min and discard the supernatant; resuspend with double-distilled water and repeat once; add 2 mL of double-distilled water, and measure the fluorescence intensity at 535 nm with a microplate reader. Using the PHA standard product as a control, calculate the PHA content. The result shows that the PHA concentration can reach 70.6 g / kg, and the PHA content can reach 69.7%.

[0106] 7. Treatment of culture waste residue

[0107] The solid medium with fully utilized nutrients can be transferred through a conveyor system to a screw press dehydrator for dehydration treatment. The dehydrated culture waste residue is subjected to composting fermentation for 20 - 30 days to degrade the residual PHA. The residual culture medium after composting treatment can be used as organic fertilizer. The wastewater is aerobically oxidized in the sewage treatment system and recycled after precipitation.

Claims

1. A new strain of Priestia megaterium, BA1251, is classified and named Priestia megaterium. The strain has been deposited as a patented strain in the General Microbiology Center of China Microbiological Culture Collection Administration in Beijing, my country, with a deposit number of CGMCC NO.33762 and a deposit date of March 10, 2025.

2. Use of Priesterol megaterium BA1251 as claimed in claim 1 in producing PHA.

3. A method for producing polyhydroxyalkanoates by semi-solid tray fermentation, characterized in that: The method is to produce PHA by semi-solid fermentation culture using Priococcus giganteus BA1251. The Priococcus giganteus has been deposited as a patented strain in the General Microbiological Collection Center of the China Microbiological Culture Collection Administration in Beijing, my country, with a deposit number of CGMCC NO.33762 and a deposit date of March 10, 2025.

4. The method according to claim 3, characterized in that The method comprises the following steps: Step 1, inoculation and culturing: inoculating the liquid strain of Priesterol BA1251 into a semi-solid culture medium for culturing; Step 2: Harvesting and extraction: Collect the bacteria on the surface of the semi-solid culture medium, add lysozyme and mechanically break them to extract PHA.

5. The method according to claim 4, characterized in that In step 1, the semi-solid culture medium contains a carbon source, a nitrogen source, and an inorganic salt; wherein the carbon source is selected from any one or more of corn stalks, wheat stalks, rice straw, weeds hydrolyzed by cellulase or xylanase, or corn flour, sweet potato flour, cassava flour, pomace, molasses, and oil; the nitrogen source is selected from any one or more of ammonium sulfate, ammonium chloride, ammonium acetate, and ammonium citrate; and the inorganic salt is selected from any one or more of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, magnesium chloride, sodium chloride, calcium chloride, and potassium chloride.

6. The method according to claim 5, characterized in that 0.5% to 1.0% agar, glutinous rice flour or kudzu root powder is added to the culture medium; after the culture medium solidifies, 0.5% to 2.0% starch, rice bran, bran or sodium alginate is evenly sprinkled on the surface.

7. The method according to claim 5, characterized in that The culture medium also includes trace elements, which are selected from any one or more of KI, H3BO3, MnSO4, ZnSO4, Na2MoO4, CuSO4, and CoCl.

8. The method according to claim 4, characterized in that In step 1, the inoculation amount is 10% to 20% of the volume of the semi-solid culture medium; preferably, the inoculation amount is 15% of the volume of the semi-solid culture medium.

9. The method according to claim 4, characterized in that Step 1: The culture temperature is 22°C to 45°C.

10. The method according to claim 9, characterized in that The cultivation temperature in winter is controlled at 22°C-33°C, the relative humidity is controlled at 90-95%, and the cultivation time is 50-55 hours; the cultivation temperature in summer is controlled at 33°C-45°C, the relative humidity is controlled at 90-95%, and the cultivation time is 40-48 hours.

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