Bacillus licheniformis SDB04 and application thereof

Abstract

The invention discloses bacillus licheniformis SDB04, and the preservation number of the bacillus licheniformis SDB04 is CCTCC (China Center for Type Culture Collection) NO: M20241169. The invention further provides application of the bacillus licheniformis SDB04 in preparation of the PHA (polyhydroxyalkanoate) in a high-salt environment. The invention further provides the application of the bacillus licheniformis SDB04 in the preparation of the PHA in the high-salt environment. According to the invention, the production efficiency and economical efficiency of PHA are improved, and the market price of bioplastics is reduced. Pollution to the environment is reduced, and sustainable development and circular economy are promoted. And a new way is provided for comprehensive utilization of saline-alkali land resources. The application expansion of the biological plastic in the fields of packaging, medical treatment and the like is promoted.

Description

Technical Field

[0001] The present invention relates to the field of microbial applications and PHA production. Specifically, it relates to a Bacillus licheniformis and its isolation method and application. Background Art

[0002] Polyhydroxyalkanoates (PHA), as a renewable, biodegradable and biocompatible bioplastic, are regarded as environmentally friendly green materials to replace traditional petrochemical plastics. PHA shows great potential in alleviating the current serious environmental pollution problems. With the progress of science and technology and the enhancement of environmental awareness, the research on the production of PHA by microbial fermentation method has been continuously deepened. However, the production cost of PHA is still relatively high, about 2.2 - 5.0 euros per kilogram, which severely limits its wide application as an environmental protection material.

[0003] Currently, the production of PHA mainly relies on traditional strains such as Escherichia coli and Bacillus yeast. These strains cannot achieve continuous and open fermentation processes and must be cultured under strict aseptic conditions. To reduce the industrial production cost of PHA, developing open and continuous fermentation technology has become one of the key ways. Microorganisms that can tolerate extreme environmental conditions (such as high salt, high pH, etc.) show important value in realizing open and continuous fermentation processes. Halotolerant bacteria show potential in PHA production due to their advantages such as rapid reproduction in saline-alkali environments, wide substrate adaptability, and simple culture processes. Bacillus licheniformis, as a bacterium widely distributed in soil, has been widely used in the field of probiotics in recent years and also shows excellent PHA-producing ability.

[0004] The defects and deficiencies of the prior art mainly lie in that traditional PHA-producing strains require strict aseptic conditions, increasing the production cost and limiting industrial applications. In addition, many studies mainly focus on conventional strains, and the research on the application of halotolerant microorganisms in PHA production is limited. Summary of the Invention

[0005] The purpose of the present invention is to screen out strains with high halotolerance and high PHA yield to achieve open and continuous fermentation and reduce production costs. And optimize the PHA fermentation conditions of halotolerant Bacillus licheniformis to increase the PHA yield. Evaluate the actual application potential of halotolerant Bacillus licheniformis in PHA production and provide a reference for the application of halotolerant bacteria in PHA industrial production.

[0006] To achieve the above object, the present invention provides a strain of Bacillus licheniformis SDB04 with a preservation number of CCTCC NO: M20241169. The Bacillus licheniformis SDB04 has the ability to synthesize PHA and can grow under saline conditions.

[0007] Bacillus licheniformis SDB04 was deposited at the China Center for Type Culture Collection on June 7, 2024, with the deposit number CCTCC NO: M20241169, address: Wuhan University, Wuhan, China, 430072, phone: 02768754833.

[0008] Preferably, the amplification medium and / or fermentation medium of the strain is: carbon source 20 g / L to 25 g / L, yeast extract 1 g / L, NaCl 60 g / L, MgCl2·6H2O 10 g / L, MgSO4·7H2O 15 g / L, peptone 5 g / L, KCl 4 g / L, CaCl2·2H2O 1 g / L, NaHCO3 0.5 g / L.

[0009] Preferably, any of the above, the carbon source includes at least one of glucose, sucrose, starch, and glycerol.

[0010] Preferably, any of the above, the amplification medium and / or fermentation medium of the strain is: glucose 25 g / L, yeast extract 1 g / L, NaCl 10 - 120 g / L, MgCl2·6H2O 10 g / L, MgSO4·7H2O 15 g / L, peptone 5 g / L, KCl 4 g / L, CaCl2·2H2O 1 g / L, NaHCO3 0.5 g / L.

[0011] Preferably, any of the above, the amplification medium and / or fermentation medium of the strain is: glucose 25 g / L, yeast extract 1 g / L, NaCl 60 g / L, MgCl2·6H2O 10 g / L, MgSO4·7H2O 15 g / L, peptone 5 g / L, KCl 4 g / L, CaCl2·2H2O 1 g / L, NaHCO3 0.5 g / L.

[0012] Preferably, any of the above, the PHA content in Bacillus licheniformis SDB04 reaches 55.2% of its cell weight after fermentation culture.

[0013] Preferably, any of the above, the PHA yield of Bacillus licheniformis SDB04 in each liter of fermentation medium reaches 6.01 g / L after fermentation culture.

[0014] A strain of Bacillus licheniformis SDB04 with the ability to synthesize PHA and grow salt-tolerantly was screened from the soil beside the coastal salt pans in Shandong Province. Through the optimization of fermentation conditions, the PHA yield can reach 6.01 g / L under the optimal conditions. This discovery not only provides new ideas for the practical application of salt-tolerant bacteria in PHA production, but also provides important references for reducing the production cost of PHA and promoting its industrial application. The optimal conditions are preferably a fermentation medium with a NaCl concentration of 60 g / L to 120 g / L, preferably fermented and cultured at 37 °C, preferably cultured with shaking at 150 rpm; preferably fermented and cultured for 72 h.

[0015] The preferred medium for Bacillus licheniformis SDB04 is as follows:

[0016] Screening medium: 25 g / L of glucose, 1 g / L of yeast extract, 60 g / L of NaCl, 10 g / L of MgCl2·6H2O, 15 g / L of MgSO4·7H2O, 5 g / L of peptone, 4 g / L of KCl, 1 g / L of CaCl2·2H2O, 0.5 g / L of NaHCO3, 16 g / L of agar, 500 ml of water. Sterilize at 120 °C for 20 min, and add 50 μL of Nile red stock solution after cooling to 50 - 55 °C.

[0017] Amplification and fermentation medium: 25 g / L of glucose, 1 g / L of yeast extract, 60 g / L of NaCl, 10 g / L of MgCl2·6H2O, 15 g / L of MgSO4·7H2O, 5 g / L of peptone, 4 g / L of KCl, 1 g / L of CaCl2·2H2O, 0.5 g / L of NaHCO3.

[0018] Preferably, in the fermentation medium: 25 g / L of glucose, 1 g / L of yeast extract, 10 - 120 g / L of NaCl, 10 g / L of MgCl2·6H2O, 15 g / L of MgSO4·7H2O, 5 g / L of peptone, 4 g / L of KCl, 1 g / L of CaCl2·2H2O, 0.5 g / L of NaHCO3. The preferred NaCl concentrations are 10, 30, 60, 80, 100, 120 g / L. In a preferred embodiment of the present invention, the NaCl concentration can still grow within the range of 10 - 120 g / L, and has a high salt tolerance.

[0019] The present invention provides a method for screening salt-tolerant PHA-producing strains from high-salt soil, comprising the following steps:

[0020] Step 1: Add the soil sample to sterile physiological saline and mix. After shaking, add the suspension to the liquid medium and culture with shaking.

[0021] Step 2: Take the culture medium after culturing in Step 1, dilute it, spread it on the screening medium plate, and culture it in the dark for 2 days.

[0022] Step 3: Observe under 312 nm ultraviolet light, select the colonies showing orange-red fluorescence, and obtain pure strains.

[0023] Step 4: Pick a single colony to prepare a smear, fix it with Nile red stock solution, stain it in the dark, and observe the fluorescence under the excitation of 420 - 490 nm blue light. Select the strains with strong fluorescence for preservation for future use.

[0024] Preferably, in Step 1, the preferred oscillation conditions are 37 °C, oscillating at 150 rpm for 1 h.

[0025] Preferably, in Step 1, the preferred culture conditions are 37 °C, oscillating at 150 rpm for 20 h.

[0026] Preferably, in Step 1, add 5 g of soil sample to 95 mL of sterile physiological saline and mix. Oscillate at 37 °C and 150 rpm for 1 h. Take 5 mL of the suspension and add it to 20 mL of liquid medium, and oscillate at 37 °C and 150 rpm for 20 h.

[0027] Preferably, in Step 1, the liquid medium is: glucose 25 g / L, yeast extract 1 g / L, NaCl 60 g / L, MgCl2·6H2O 10 g / L, MgSO4·7H2O 15 g / L, peptone 5 g / L, KCl 4 g / L, CaCl2·2H2O 1 g / L, NaHCO3 0.5 g / L.

[0028] Preferably, in Step 2, the preferred dilution factor is 10 -3 dilution.

[0029] Preferably, in Step 2, the preferred culture conditions are culturing at 37 °C in the dark for 2 d.

[0030] Preferably, in Step 2, take 100 μL of the culture medium for gradient dilution, select 10 -3 dilution and spread 200 μL on the screening medium plate, and culture it at 37 °C in the dark for 2 d.

[0031] Preferably, in step 2, the screening culture medium plate is: 25 g / L of glucose, 1 g / L of yeast extract, 60 g / L of NaCl, 10 g / L of MgCl2·6H2O, 15 g / L of MgSO4·7H2O, 5 g / L of peptone, 4 g / L of KCl, 1 g / L of CaCl2·2H2O, 0.5 g / L of NaHCO3, 16 g / L of agar, and 500 ml of water. Sterilize at 120 °C for 20 min, and add 50 μL of nile red stock solution when cooled to 50 - 55 °C.

[0032] Preferably, in step 3, observe under 312 nm ultraviolet light, select the colonies showing orange - red fluorescence for streak isolation and purification to obtain pure strains. Number the pure strains and store them at 4 °C for later use.

[0033] Preferably, in step 4, perform rescreening of PHA - producing bacteria. Pick single colonies to prepare smears, after drying and fixing, add 1 - 2 drops of 0.5 μg / mL nile red stock solution, and stain in the dark for 15 min. Observe the fluorescence under 420 - 490 nm blue light excitation, and select the strains with strong fluorescence for preservation for later use.

[0034] The present invention also provides the application of Bacillus licheniformis SDB04 described in any one of the above in the preparation of polyhydroxyalkanoates.

[0035] Preferably, Bacillus licheniformis SDB04 prepares polyhydroxyalkanoates under high - salt conditions, and the high - salt conditions are that the concentration of NaCl in the fermentation culture system is 60 - 120 g / L.

[0036] The beneficial technical effects of the present invention are as follows:

[0037] 1. Strong salt tolerance: Bacillus licheniformis SDB04 can grow rapidly in a high - salt environment, significantly reducing the demand for fresh water resources and expanding the raw material sources for PHA production.

[0038] 2. High - efficiency PHA production: Under optimized conditions, the PHA content of this strain reaches 55.2%, that is, 6.01 g / L, with a high conversion efficiency.

[0039] 3. Simplified production process: Due to the salt - tolerant characteristics, open - type continuous fermentation can be achieved, simplifying the production process that traditionally requires strict aseptic conditions.

[0040] 4. Reduced production cost: The simplified process and the utilization of cheap saline - alkali resources effectively reduce the production cost of PHA.

[0041] 5. Environmentally friendly: The cultivation process of strain SDB04 is pollution-free, and the produced PHA can be completely biodegradable, reducing the negative impact on the environment.

[0042] 6. Wide substrate adaptability: This strain can utilize a variety of carbon sources, increasing the flexibility of raw material selection.

[0043] 7. Fast growth: The production cycle of PHA is shortened to 72 h with high yields, improving production efficiency.

[0044] 8. Easy to scale up: The simple cultivation process is conducive to large-scale industrial production.

[0045] The present invention improves the production efficiency and economy of PHA, helps to reduce the market price of bioplastics, reduces environmental pollution, promotes sustainable development and circular economy, provides a new way for the comprehensive utilization of saline-alkali land resources, and promotes the application expansion of bioplastics in the fields of packaging, medical treatment, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 It is the fluorescence microscope inspection image of Bacillus licheniformis SDB04 strain in the first preferred embodiment of the present invention.

[0047] Figure 2 It is the macroscopic observation of the single colony morphology of Bacillus licheniformis SDB04 strain in the second preferred embodiment of the present invention.

[0048] Figure 3 It is the observation result of Bacillus licheniformis SDB04 strain by optical microscope after Gram staining in the second preferred embodiment of the present invention.

[0049] Figure 4 It is the carbon source utilization, gelatin liquefaction and oxidase situation of Bacillus licheniformis SDB04 in the third preferred embodiment of the present invention.

[0050] Figure 5 It is the growth curve of Bacillus licheniformis SDB04 in the fourth preferred embodiment of the present invention.

[0051] Figure 6 It is the phylogenetic tree in the fifth preferred embodiment of the present invention.

[0052] Figure 7Growth of Bacillus licheniformis SDB04 in Example 6, a preferred embodiment of the present invention, at different NaCl concentrations.

[0053] Figure 8 Observation results of TEM after 72 hours of fermentation culture of Bacillus licheniformis SDB04 in Example 7, a preferred embodiment of the present invention.

[0054] Figure 9 PHA production in different carbon source fermentation media in Example 8, a preferred embodiment of the present invention.

[0055] Figure 10 PHA production at different salt concentrations in Example 7, a preferred embodiment of the present invention. Detailed implementation manners

[0056] The present invention will be further described in detail below with reference to the accompanying drawings and specific implementation manners.

[0057] Media used in the present invention:

[0058] 1. Screening medium: A screening medium improved on the basis of the TYS medium, containing 25 g / L glucose, 1 g / L yeast extract, 60 g / L NaCl, 10 g / L MgCl2·6H2O, 15 g / L MgSO4·7H2O, 5 g / L peptone, 4 g / L KCl, 1 g / L CaCl2·2H2O, 0.5 g / L NaHCO3, 16 g / L agar, and 500 ml of water. Sterilize at 120 °C for 20 min, and add 50 μL of nile red stock solution when cooled to 50 - 55 °C.

[0059] 2. Liquid medium: An amplification medium and a fermentation medium improved on the basis of the TYS medium, containing 25 g / L glucose, 1 g / L yeast extract, 60 g / L NaCl, 10 g / L MgCl2·6H2O, 15 g / L MgSO4·7H2O, 5 g / L peptone, 4 g / L KCl, 1 g / L CaCl2·2H2O, and 0.5 g / L NaHCO3.

[0060] 3. Preparation of nile red stock solution: Dissolve 50 mg of nile red dry powder in 100 mL of dimethyl sulfoxide (DMSO) solvent, with a final concentration of 0.5 μg / mL, and store at 4 °C in the dark.

[0061] Example 1

[0062] Example 1 provides a method for screening the strain Bacillus licheniformis SDB04.

[0063] 1. Sample collection: Soil samples were collected from a salt field in Huanghua City, Shandong Province, and the sampling depth was 2 cm.

[0064] 2. Primary screening of PHA-producing bacteria:

[0065] Mix 5 g of soil sample with 95 mL of sterile physiological saline. Shake at 37 °C and 150 rpm for 1 h. Take 5 mL of the suspension and add it to 20 mL of liquid medium. Shake at 37 °C and 150 rpm for 20 h. Take 100 μL of the culture solution for gradient dilution, and select the 10 -3 dilution to coat 200 μL on the screening medium plate, and culture it in the dark at 37 °C for 2 d. Observe under ultraviolet light at 312 nm, select the colonies showing orange-red fluorescence for streak isolation and purification to obtain pure strains. Number the pure strains and store them at 4 °C for later use.

[0066] 3. Rescreening of PHA-producing bacteria:

[0067] Pick a single colony to prepare a smear. After drying and fixing, add 1 - 2 drops of 0.5 μg / mL nile red stock solution and stain it in the dark for 15 min. Observe the fluorescence under blue light excitation at 420 - 490 nm, and select the strains with strong fluorescence for preservation, as Figure 1 shown.

[0068] Example 2

[0069] In Example 2, the colony morphology of Bacillus licheniformis SDB04 was observed and identified. Observe the single colony morphology with the naked eye (as Figure 2 shown), and observe it under an optical microscope after Gram staining (as Figure 3 shown).

[0070] Example 3

[0071] In Example 3, the physiological and biochemical identification of Bacillus licheniformis SDB04 was carried out. Use a reagent strip for the physiological and biochemical identification of Bacillus to detect the carbon source utilization, gelatin liquefaction and oxidase situation of Bacillus licheniformis SDB04 (the results are as Figure 4 shown, Figure 4 in which "+" indicates positive or utilizable; "-" indicates negative or non-utilizable). The results showed that Bacillus licheniformis SDB04 was Gram-positive, could utilize o-nitrophenyl-β-D-galactopyranoside (ONPG), glucose, mannitol, sucrose, arabinose, and could not utilize citric acid, lactose, inositol, sorbitol, rhamnose, melibiose. It could liquefy gelatin and had no oxidase activity.

[0072] Example 4

[0073] Example 4 provided the growth curve determination of Bacillus licheniformis SDB04.

[0074] 2.5 mL of the activated bacterial solution was inoculated into 60 mL of the fermentation medium and cultured at 37 °C and 150 rpm. Samples were taken every 2 h to measure the OD600 value and the growth curve was plotted ( Figure 5 ). The results showed that the lag phase was from 0 to 8 h, the logarithmic phase was from 8 to 26 h, and the stationary phase began after 26 h.

[0075] The components of the fermentation medium in Example 4 were: glucose 25 g / L, yeast extract 1 g / L, NaCl 60 g / L, MgCl2·6H2O 10 g / L, MgSO4·7H2O 15 g / L, peptone 5 g / L, KCl 4 g / L, CaCl2·2H2O 1 g / L, NaHCO3 0.5 g / L.

[0076] Example 5

[0077] Example 5 provided the results of 16S rRNA gene sequence analysis. Universal primers were used - 27F (5'-AGAGTTTGATCCTGGCTCAG-3') with the nucleotide sequence as shown in SEQ ID NO: 1 and 1492R (5'-TACGGCTACCTTGTTACGACTT-3') with the nucleotide sequence as shown in SEQ ID NO: 2 to amplify the 16S rRNA gene sequence. The PCR reaction system (50 μL): template DNA 5 μL, premixed enzyme 25 μL, each primer 2 μL, ddH2O 16 μL. PCR reaction conditions: 94 °C for 5 min; 94 °C for 30 s, 54 °C for 45 s, 72 °C for 2 min, 30 cycles; 72 °C for 7 min. The amplified product was sent for sequencing, and the sequence result was as shown in SEQ ID NO: 3. The sequencing result was compared with the NCBI database, and a phylogenetic tree was constructed using MEGA11 (as Figure 6 shown). The identification result showed that Bacillus licheniformis SDB04 was Bacillus licheniformis.

[0078] Example 6

[0079] Example 6 carried out the salt tolerance determination of Bacillus licheniformis SDB04.

[0080] The NaCl concentration of the screening medium was increased in a gradient of 20 g / L, and the bacterial suspension was diluted to 10 -3 and spread on the screening medium. It was cultured in the dark at 37 °C for 2 d, and the salt tolerance of the bacteria was tested by observing the growth of the bacteria. The results are as Figure 7 shown, Figure 7 wherein "+" indicates that it can grow; "-" indicates that it cannot grow. It can be seen that SDB04 can still grow at a NaCl concentration of 120 g / L, showing a high salt tolerance ability.

[0081] Example 7

[0082] In Example 7, the cell dry weight and PHA content were measured.

[0083] The bacterial liquid cultured at 37 °C for 72 h in the fermentation medium was taken, centrifuged at 12000 r / min for 10 min, the supernatant was discarded, and washed twice with ddH2O. After freezing at -80 °C for 60 min, it was freeze-dried in vacuo for 18 h, and the cell dry weight was calculated by weighing. The bacterial liquid cultured at 37 °C for 72 h in the fermentation medium was taken and centrifuged at 1000 r / min for 5 min, and washed twice with PBS. 1 mL of 2.5% glutaraldehyde was added and fixed overnight at 4 °C. The sample was observed by TEM (the results are as Figure 8 shown).

[0084] The PHA content was determined by ultraviolet spectrophotometry. 0.01 g of dry bacteria was added to 10 mL of chloroform-ethanol (V:V = 2:1) solution, reacted at 60 °C for 1 h and then filtered. 0.5 mL of the filtrate was evaporated to dryness at 70 °C, 5 mL of concentrated sulfuric acid was added and heated at 100 °C for 10 min, and after cooling, it was fixed to 25 mL. The OD value at 235 nm was measured, and compared with the PHA standard curve to detect the PHA content (%) contained in the bacteria. This result was multiplied by the cell dry weight to obtain the PHA yield (g / L).

[0085] The composition of the fermentation medium in Example 7 was: glucose 25 g / L, yeast extract 1 g / L, NaCl 60 g / L, MgCl2·6H2O 10 g / L, MgSO4·7H2O 15 g / L, peptone 5 g / L, KCl 4 g / L, CaCl2·2H2O 1 g / L, NaHCO3 0.5 g / L.

[0086] Under the optimized conditions of Example 7, the PHA content obtained by fermenting Bacillus licheniformis SDB04 reached 55.2%, that is, the PHA yield per liter of fermentation broth was 6.01 g / L, showing a high conversion efficiency.

[0087] In Example 7, the PHA yields at different salt concentrations were also studied, as Figure 10 shown for the PHA yields at different salt concentrations,Figure 10 The fermentation medium with different salt concentrations consists of: glucose 25 g / L, yeast extract 1 g / L, NaCl 40 - 120 g / L, MgCl2·6H2O 10 g / L, MgSO4·7H2O 15 g / L, peptone 5 g / L, KCl 4 g / L, CaCl2·2H2O 1 g / L, NaHCO3 0.5 g / L.

[0088] Example 8

[0089] Example 8 studied the carbon sources utilized by Bacillus licheniformis SDB04 for the fermentation production of PHA. The results showed that Bacillus licheniformis SDB04 could utilize various carbon sources for the fermentation production of PHA. The carbon sources include glucose, sucrose, starch, and glycerol. Figure 9 Shown are the yields of PHA in the fermentation media with different carbon sources. The fermentation media with different carbon sources consist of: carbon source 20 - 25 g / L, yeast extract 1 g / L, NaCl 60 g / L, MgCl2·6H2O 10 g / L, MgSO4·7H2O 15 g / L, peptone 5 g / L, KCl 4 g / L, CaCl2·2H2O 1 g / L, NaHCO3 0.5 g / L.

[0090] The above examples are only for clear illustration and not limitations on the implementation. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation manners here. The obvious changes or modifications derived therefrom are still within the protection scope of this invention.

Claims

1. A strain of Bacillus licheniformis SDB04, with a deposit number of CCTCC NO:M20241169.

2. The Bacillus licheniformis SDB04 according to claim 1, characterized in that The amplification medium and / or fermentation medium of the strain is: carbon source 20-25 g / L, yeast extract 1 g / L, NaCl 10-120 g / L, MgCl2·6H2O 10 g / L, MgSO4·7H2O 15 g / L, peptone 5 g / L, KCl 4 g / L, CaCl2·2H2O 1g / L, NaHCO3 0.5g / L.

3. The Bacillus licheniformis SDB04 according to claim 2, characterized in that The amplification medium and / or fermentation medium of the strain is: 25 g / L glucose, 1 g / L yeast extract, 60 g / L NaCl, 10 g / L MgCl2·6H2O, 15 g / L MgSO4·7H2O, 5 g / L peptone, 4 g / L KCl, 1 g / L CaCl2·2H2O, and 0.5 g / L NaHCO3.

4. The Bacillus licheniformis SDB04 according to any one of claims 1 to 3, characterized in that After fermentation, the PHA content of Bacillus licheniformis SDB04 reached 55.2% of its cell weight.

5. The Bacillus licheniformis SDB04 according to any one of claims 1 to 3, characterized in that After fermentation, the PHA production of Bacillus licheniformis SDB04 reached 6.01 g / L per liter of fermentation medium.

6. A method for preparing Bacillus licheniformis SDB04 having PHA synthesis ability and salt-tolerant growth, characterized in that: The following steps are involved: Step 1: Add the soil sample to sterile saline and mix, shake, then add the suspension to the liquid culture medium and shake and culture; Step 2: Take the culture solution after culturing in step 1, dilute it, spread it on the screening culture medium plate, and culture it in the dark for 2 days; Step 3: Observe under 312nm ultraviolet light and select colonies with orange-red fluorescence to obtain pure strains; Step 4: Pick a single colony to prepare a smear, fix it with Nile red storage solution, stain it in the dark, observe the fluorescence under 420-490nm blue light excitation, and select the strain with strong fluorescence for future use.

7. The preparation method according to claim 6, characterized in that: In step 1, the liquid culture medium is: glucose 25 g / L, yeast extract 1 g / L, NaCl 60 g / L, MgCl2·6H2O 10 g / L, MgSO4·7H2O 15 g / L, peptone 5 g / L, KCl4 g / L, CaCl2·2H2O 1 g / L, NaHCO3 0.5 g / L.

8. The preparation method according to claim 6, characterized in that: In step 2, the screening culture medium plate is: glucose 25 g / L, yeast extract 1 g / L, NaCl 60 g / L, MgCl2·6H2O 10 g / L, MgSO4·7H2O 15 g / L, peptone 5 g / L, KCl 4 g / L, CaCl2·2H2O 1 g / L, NaHCO3 0.5 g / L, agar 16 g / L, and water 500 ml. Sterilize at 120℃ for 20 min, cool to 50-55℃ and add 50μL of Nile red stock solution.

9. Use of the Bacillus licheniformis SDB04 according to any one of claims 1 to 5 in the preparation of synthetic polyhydroxyalkanoates.

10. The use according to claim 9, characterized in that The Bacillus licheniformis SDB04 prepares and synthesizes polyhydroxyalkanoate under high-salt conditions, wherein the high-salt conditions are that the concentration of NaCl in the fermentation culture system is 60-120 g / L.

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