A method for preparing a high 3-hydroxyvalerate content PHBV copolymer using a halophilic mixed bacterial population

Abstract

This invention provides a method for preparing high-3-hydroxyvalerate (PHBV) copolymers using a halophilic mixed bacterial community, relating to the field of biodegradable polymer materials technology. The invention uses halophilic sludge from the shallow waters of Qinghai Lake (May-August) as the bacterial source, and agricultural waste hydrolysate as the sole organic carbon source for the domestication and synthesis stages. Halophilic activated sludge is obtained through initial screening with low-concentration hydrolysate and increased load domestication in an SBR reactor. Then, a sequencing batch reactor is used for cyclical operation, allowing the halophilic mixed bacterial community to metabolize multi-component reduced low-carbon sugars in the hydrolysate and accumulate PHA intracellularly. This invention achieves a stable conversion of multi-component lignocellulose hydrolysate into high-3-hydroxyvalerate (PHBV) copolymers, showing promising prospects for reducing PHBV production costs, improving the resource utilization level of agricultural waste, and promoting the industrial application of biodegradable materials.

Description

Technical Field

[0001] This invention relates to the field of biodegradable polymer materials technology, and in particular to a method for preparing a PHBV copolymer with high 3-hydroxyvalerate content using a halophilic mixed bacterial community. Background Technology

[0002] With the large-scale use of petroleum-based plastics, the resulting problems of resource consumption, environmental pollution, and the difficulty in degrading waste have become increasingly prominent. Developing renewable, environmentally friendly, and biodegradable polymer materials has become an important development direction in the fields of polymer materials and biomanufacturing. Polyhydroxyalkanoates (PHBVs) are a class of intracellular polyesters synthesized by microorganisms, possessing processing properties, biocompatibility, and biodegradability similar to traditional plastics, making them highly valuable for applications in packaging materials, agricultural films, and other disposable biodegradable products. Among them, poly(3-hydroxybutyrate-co-3-hydroxyvalerate), or PHBV copolymer, has attracted widespread attention due to the improved toughness, crystallinity, and processing adaptability of the material after the introduction of 3-hydroxyvalerate units.

[0003] Existing biosynthetic processes for PHA or PHBV typically employ pure microbial fermentation systems. These methods usually require expensive and relatively simple organic substrates such as glucose, propionic acid, and acetic acid, and necessitate strict sterilization and aseptic control during fermentation to prevent contamination. While these processes can yield polymers to a certain extent, the high substrate costs, sterilization energy consumption, and operational management costs hinder the large-scale, low-cost production of PHA-based materials. In particular, the preparation of PHBV copolymers often requires the addition of 3-hydroxyvalerate precursors such as propionate to increase the PHA unit content, further increasing raw material costs and process complexity.

[0004] Agricultural waste is a widely available and inexpensive lignocellulosic biomass resource. After pretreatment and enzymatic hydrolysis, it can form hydrolysates containing multiple low-carbon sugars such as glucose, xylose, arabinose, galactose, and mannose, which can serve as a potential carbon source for microbial synthesis of PHA. Compared with easily degradable industrial wastewater such as molasses alcohol wastewater, hydrolysates from agricultural waste such as corn stalks are characterized by complex sugar compositions, large fluctuations in carbon source concentration, and the potential presence of certain fermentation-inhibiting byproducts. Ordinary microbial systems struggle to achieve long-term stable growth and efficient PHA accumulation in these hydrolysates. Therefore, adapting microbial systems to the polysaccharide components and potential inhibitors in agricultural waste hydrolysates and stably converting them into PHBV copolymers with high 3-hydroxyvalerate content remains a significant technical challenge limiting the resource-based production of biodegradable plastics from agricultural waste.

[0005] In recent years, open fermentation using salt-tolerant or halophilic microorganisms has been considered an effective way to reduce the production cost of PHA. Halophilic microorganisms can grow in high-salinity environments, which inhibit most contaminating microorganisms, thus reducing the need for strict sterilization and making them suitable for continuous, open, or low-sterilization-intensity fermentation operations. However, existing research and processes for PHA production using halophilic mixed microbial communities mostly use easily degradable industrial wastewater such as molasses alcohol wastewater as substrates. The microbial acclimatization conditions, salinity control, and sequencing batch reactor (SBR) parameters are mainly designed around the organic load of industrial wastewater. They cannot effectively solve the problem of synergistic adaptation of halophilic mixed microbial communities to the multi-component low-carbon sugars and potential inhibitors in agricultural waste lignocellulose hydrolysates, especially corn straw hydrolysates. It is also difficult to stably obtain PHBV copolymers with high 3-hydroxyvalerate content without the addition of propionate precursors.

[0006] Therefore, there is still a need for a method that can use agricultural waste hydrolysate as the main or sole organic carbon source, and utilize halophilic mixed microbial communities for targeted domestication and sequential batch synthesis culture, so that the microbial communities can adapt to the complex carbon source composition of corn straw hydrolysate under open or non-strict sterilization conditions, and achieve efficient conversion of hydrolysate into PHBV copolymer with high 3-hydroxyvalerate content, thereby reducing the production cost of PHA-type materials and improving the resource utilization level of agricultural waste. Summary of the Invention

[0007] In view of this, the present invention provides a method for preparing PHBV copolymers with high 3-hydroxyvalerate content using a halophilic mixed bacterial community. This invention utilizes agricultural waste hydrolysate as the sole organic carbon source for the entire process of halophilic mixed bacterial community domestication and PHBV synthesis. Combined with the directed domestication of the halophilic mixed bacterial community from Qinghai Lake, open-cell culture in saline conditions, and SBR cycle operation regulation, it achieves stable conversion of multi-component lignocellulose hydrolysate into PHBV copolymers with high 3-hydroxyvalerate content. This method shows promising prospects for reducing PHBV production costs, improving the resource utilization level of agricultural waste, and promoting the industrial application of biodegradable materials.

[0008] The present invention relates to a method for producing PHBV copolymers using a halophilic mixed bacterial community from Qinghai Lake, comprising the following steps: S1. Harmophilic sludge from the shallow water area of ​​Qinghai Lake from May to August is put into the SBR reactor. The first culture medium is prepared with corn straw hydrolysate and sodium chloride. The reactor is run in a fully aerobic aeration mode to screen and activate halophilic activated sludge. S2. Add the second culture medium to the SBR reactor containing the halophilic activated sludge obtained in step S1, and continue the aerobic sequencing batch acclimation culture until the MLSS of the halophilic activated sludge in the SBR reactor reaches more than 7000 mg / L. S3. The halophilic activated sludge obtained in step S2 is transferred to the synthesis reactor to start the synthesis culture. The initial sludge concentration MLSS is 1500 mg / L. Corn straw hydrolysate is used as the only organic carbon source in the synthesis stage. The sequencing batch operation is carried out in a 12-hour cycle, which includes 11 hours of aeration and 1 hour of sedimentation. S4. After 8 consecutive cycles of operation, PHA is extracted from the residual halophilic activated sludge rich in PHA discharged from the reactor to obtain PHBV copolymer. The domestication and synthesis cultures are not subjected to high-pressure steam sterilization or chemical sterilization, and the corn stalk hydrolysate is not supplemented with glucose, propionic acid, acetic acid, or molasses alcohol wastewater as an auxiliary carbon source.

[0009] Preferably, in step S1, the shallow water area is a nearshore lakeside area with a water depth of no more than 3 m.

[0010] Preferably, in step S1, the corn stalk hydrolysate contains multiple reducing low-carbon sugars with a total reducing sugar content of 18.14 g / L, specifically including: 0.36 g / L arabinose, 0.57 g / L galactose, 13.11 g / L glucose, 3.71 g / L xylose and 0.39 g / L mannose; when used with water, the corn stalk hydrolysate is diluted to 500~3000 mg / L based on the total reducing sugar content as the carbon source for the water inlet.

[0011] Preferably, in step S1, the preparation method of the corn stalk hydrolysate is as follows: 30 g of 40-mesh wood flour is placed in a 500 ml high-temperature and high-pressure reactor at a solid-liquid ratio of 1:4 for high-temperature and high-pressure hydrothermal pretreatment. The pretreatment temperature is 170℃, and the pretreatment time is 50 min. After the temperature reaches the predetermined reaction temperature, the holding time is calculated. After the reaction is completed, the mixture is naturally cooled to room temperature. The pretreated material is taken out, and ultrapure water is added to 1 L. The pH is adjusted to 5 with 1 mol / L NaOH solution. 2 mL of cellulase (enzyme activity ≥1000 U / g) is added. The enzymatic hydrolysis temperature is 37℃, and the enzymatic hydrolysis time is 48 h. After the enzymatic hydrolysis is completed, the hydrolysate is heated in a 90℃ constant temperature water bath for 20 min to remove cellulase activity. The treated hydrolysate is then separated by vacuum filtration to obtain the corn stalk hydrolysate.

[0012] Preferably, in step S1, the first culture medium comprises: corn straw hydrolysate (calculated as total reducing sugar) at 500 mg / L, ammonium chloride at 25 mg / L, potassium dihydrogen phosphate at 5 mg / L, sodium chloride at 10 g / L, magnesium sulfate at 100 mg / L, and trace element solution at 1 mL / L; the trace element solution comprises: CaCl2 at 40 mg / L, MnCl2·4H2O at 0.3 mg / L, FeCl3·3H2O at 3 mg / L, H3BO3 at 0.3 mg / L, ZnSO4·7H2O at 0.24 mg / L, CuSO4·5H2O at 0.06 mg / L, CoCl2·6H2O at 0.3 mg / L, and KI at 0.06 mg / L.

[0013] Preferably, in step S1, the dissolved oxygen concentration of the fully aerobic aeration operation is ≥5 mg / L, the aeration time is 11h, the sedimentation time is 1h, and then the supernatant is discharged; when the sludge color changes from dark brown to light yellow, the sludge concentration MLSS increases to 4000mg / L, and the SVI reaches 40 mL / g, the acclimatization and cultivation in step S2 begins.

[0014] Preferably, in step S2, the second culture medium comprises: corn straw hydrolysate with a total reducing sugar content of 1000 mg / L, ammonium chloride of 50 mg / L, potassium dihydrogen phosphate of 10 mg / L, sodium chloride of 10 g / L, magnesium sulfate of 100 mg / L, and trace element solution of 1 mL / L; in step S2, the reaction conditions are controlled as follows: room temperature, dissolved oxygen ≥ 5 mg / L, aeration time of 11 h, and sedimentation time of 1 h, then the supernatant is drained, and the mixture is run for 20-30 days.

[0015] Preferably, in step S3, the process parameters for synthesis cultivation are: corn straw hydrolysate concentration of 1000~3000 mg / L, ammonium chloride concentration of 40~120 mg / L, sodium chloride concentration of 0~20 g / L (and not 0), and pH value of 6~8; more preferably, in the synthesis stage, the corn straw hydrolysate concentration is 2000~2700 mg / L, ammonium chloride concentration is 60~100 mg / L, sodium chloride concentration is 5~10 g / L, and pH value is 7~8; even more preferably, the corn straw hydrolysate concentration is 2500 mg / L, ammonium chloride concentration is 75 mg / L, sodium chloride concentration is 5 g / L, and pH value is 8.

[0016] Preferably, step S4 includes: collecting the residual halophilic activated sludge rich in PHA discharged after continuous operation by centrifugation, washing it with deionized water to remove high-salt culture medium residue, and then freeze-drying it to obtain mixed bacterial cell freeze-dried powder; mixing the mixed bacterial cell freeze-dried powder with 0.1% SDS solution at a mass-volume ratio of 1 g: 10 mL, stirring in a water bath at 37°C for 2 h, centrifuging at 5000 r / min for 30 min or 10000 r / min for 15 min, collecting the precipitate and freeze-drying it for 24 h; mixing the freeze-dried precipitate with chloroform at a mass-volume ratio of 1 g: 10 mL, and treating it at 100°C for 4 h under sealed conditions; cooling to room temperature and adding an equal volume of deionized water, centrifuging at 5000 r / min for 5 min, taking the lower organic phase and filtering it; slowly pouring the obtained organic phase into pre-cooled ethanol at -20°C, stirring until a white precipitate precipitates, collecting the precipitate and drying it at room temperature in a fume hood to constant weight to obtain PHBV solid.

[0017] Preferably, the yield of PHA converted from the corn stalk hydrolysate is 456~3079 mg / L, the PHA is a PHBV copolymer, the maximum PHA content reaches 59.9% of the dry weight of the bacterial cells, and the 3-hydroxybutyrate unit and 3-hydroxyvalerate unit (i.e. 3HV unit) in the PHBV copolymer are all derived from the intracellular polymerization accumulation of corn stalk hydrolysate after metabolism by halophilic mixed bacterial groups, without the need to add propionate as a comonomer precursor.

[0018] The second aspect of the present invention provides a PHBV copolymer prepared by the above method, wherein the 3-hydroxybutyrate unit and the 3-hydroxyvalerate unit in the PHBV copolymer are both derived from the intracellular polymerization accumulation of corn straw hydrolysate after being metabolized by the halophilic mixed bacterial community of Qinghai Lake, and no propionate is added as a comonomer precursor during the preparation process.

[0019] Preferably, the 3HV unit content in the PHBV copolymer is 42.5 wt.% to 95.0 wt.%.

[0020] A third aspect of the present invention provides the application of the PHBV copolymer in the preparation of biodegradable materials.

[0021] Preferably, the biodegradable material is a green packaging material or an agricultural mulch film material.

[0022] The fourth aspect of this invention provides the application of a halophilic mixed microbial community from Qinghai Lake in the production of PHBV copolymers using agricultural waste hydrolysate, wherein the agricultural waste hydrolysate is corn stalk hydrolysate, and the halophilic mixed microbial community from Qinghai Lake is obtained through directional domestication via steps S1 and S2.

[0023] Compared with the prior art, the beneficial technical effects of the present invention are as follows: This invention uses corn stalk hydrolysate as the sole organic carbon source for the entire process of halophilic mixed microbial culture domestication and PHBV synthesis, without adding auxiliary carbon sources such as glucose, propionic acid, acetic acid, or molasses alcohol wastewater. It can directly convert the multi-component low-carbon sugars formed from the hydrolysis of agricultural waste such as corn stalks into PHBV copolymers. Therefore, this invention reduces the dependence on commercial sugars and exogenous comonomer precursors in traditional PHA or PHBV production processes, reduces raw material costs, and simultaneously increases the added value and resource utilization level of agricultural waste.

[0024] This invention utilizes a halophilic mixed microbial community from Qinghai Lake as the production microbial community, and conducts activation, domestication, and synthetic culture in a saline environment. Because the halophilic mixed microbial community can adapt to high-salinity environments, which inhibit common bacteria, this invention eliminates the need for autoclaving or chemical sterilization during domestication and synthetic culture. This reduces equipment requirements, energy consumption, and operational costs associated with strictly aseptic fermentation, facilitating open and low-cost production of PHBV copolymers.

[0025] This invention employs a two-stage targeted acclimatization method combining primary screening and activation with sequencing batch acclimatization (SBR), enabling the halophilic mixed microbial community of Qinghai Lake to gradually adapt to the multi-component reducing sugars (glucose, xylose, arabinose, galactose, and mannose) in corn stalk hydrolysate, as well as potential fermentation-inhibiting byproducts present in the hydrolysate. The acclimatized halophilic activated sludge can stably utilize corn stalk hydrolysate for growth and intracellular polymer accumulation under SBR cyclic operation conditions, solving the problem that existing halophilic mixed microbial community processes are mostly applicable to easily degradable industrial wastewater and difficult to directly adapt to lignocellulosic hydrolysate from agricultural waste.

[0026] This invention controls the concentration of corn stalk hydrolysate, nitrogen source concentration, salinity, pH value, and aeration and sedimentation cycles during the synthesis stage to enable a halophilic mixed microbial community to efficiently accumulate PHA during a sequencing batch reactor process. The resulting PHA is a PHBV copolymer. The 3-hydroxybutyrate and 3-hydroxyvalerate units in the PHBV copolymer are derived from the intracellular polymerization accumulation of corn stalk hydrolysate after metabolism by the halophilic mixed microbial community, eliminating the need for external propionate as a 3-hydroxyvalerate precursor. Therefore, it is possible to obtain a PHBV copolymer with a high 3-hydroxyvalerate content while reducing the cost of the comonomer precursor.

[0027] The PHBV copolymer prepared by this invention can be used in the preparation of green packaging materials, agricultural mulch films, and other biodegradable materials. This method combines the advantages of agricultural waste resource utilization, open culture of halophilic mixed microbial communities, low-cost fermentation operation, and the preparation of PHBV copolymers with high 3-hydroxyvalerate content, providing a new approach for the large-scale production of bio-based biodegradable plastics. Detailed Implementation

[0028] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0029] Unless otherwise stated, the corn stalk hydrolysate in the examples is a corn stalk hydrolysate containing multiple reducing low-carbon sugars. The total reducing sugar content in the hydrolysate is 18.14 g / L, specifically including 0.36 g / L arabinose, 0.57 g / L galactose, 13.11 g / L glucose, 3.71 g / L xylose and 0.39 g / L mannose.

[0030] The trace element solution (mg / L) contains CaCl2 40, MnCl2·4H2O 0.3, FeCl3·3H2O 3, H3BO3 0.3, ZnSO4·7H2O 0.24, CuSO4·5H2O 0.06, CoCl2·6H2O 0.3 and KI 0.06.

[0031] All experiments were repeated three times, and the results are expressed as averages.

[0032] Example 1: A method for preparing PHBV copolymers with high 3-hydroxyvalerate content using a halophilic mixed bacterial community, the steps of which are as follows: S1. In mid-May, halophilic sludge from the shallow waters of Qinghai Lake was introduced into an SBR reactor (SBR reactor volume: 5 L, liquid volume: 3 L, inoculated activated sludge MLSS: 2000 mg / L, sludge retention method: sedimentation for 1 h followed by discharge of 1.5 L of supernatant). A culture medium was prepared using corn straw hydrolysate and sodium chloride. The culture medium contained 500 mg / L corn straw hydrolysate, 25 mg / L ammonium chloride, 5 mg / L potassium dihydrogen phosphate, 10 g / L sodium chloride, 100 mg / L magnesium sulfate, and 1 mL / L trace element solution. Halophilic activated sludge was screened and activated. The system was operated under fully aerobic aeration, with a dissolved oxygen concentration ≥5 mg / L, aeration time of 11 h, sedimentation for 1 h, and then the supernatant was discharged. When the sludge color changed from dark brown to light yellow, the MLSS concentration increased to 4000 mg / L, and the SVI reached 40 mL / g, the next stage of acclimatization and cultivation began. This step uses corn stalk hydrolysate as the sole organic carbon source for initial screening, without adding molasses alcohol wastewater or single commercial sugar.

[0033] S2. Add culture medium to the SBR reactor containing the halophilic activated sludge from S1. The culture medium contains 1000 mg / L corn straw hydrolysate, 50 mg / L ammonium chloride, 10 mg / L potassium dihydrogen phosphate, 10 g / L sodium chloride, 100 mg / L magnesium sulfate, and 1 mL / L trace element solution. Control the reaction conditions as follows: room temperature, dissolved oxygen ≥5 mg / L, aeration time 11 h, sedimentation time 1 h, and then drain the supernatant; run for 20-30 days until the MLSS of the halophilic activated sludge in the SBR reaches above 7000 mg / L, and then proceed to the next stage of cultivation; after this stage of acclimatization, a stable mixed halophilic bacterial community from Qinghai Lake that metabolizes multi-component reducing sugars from corn straw hydrolysate is obtained.

[0034] S3. The halophilic activated sludge from S2 was transferred to the synthesis reactor to start the synthesis culture. The initial sludge concentration (MLSS) was 1500 mg / L. The concentration of corn straw hydrolysate was 2500 mg / L, ammonium chloride was 100 mg / L, sodium chloride was 10 g / L, and the pH was 7. The synthesis cycle was 12 h, with 11 h of aeration and 1 h of sedimentation. This was repeated for 8 cycles. During this synthesis stage, corn straw hydrolysate continued to be used as the sole carbon source to form the PHBV copolymer.

[0035] S4. After eight consecutive cycles of operation, the yield of PHA converted from corn stalk hydrolysate was 1544 mg / L. The produced PHA was a copolymer PHBV, with a 3HV unit content of 42.5 wt.%. The PHA content reached 23.9% of the cell dry weight. This example demonstrates that under the above parameter combination, the halophilic mixed bacterial community of Qinghai Lake can convert lignocellulose hydrolysate into PHA, and is not only applicable to easily degradable industrial wastewater substrates.

[0036] The extraction method for PHA / PHBV is as follows: After 8 consecutive cycles, the PHA-rich mixed bacterial cells discharged from the synthesis reactor are collected by centrifugation, washed with deionized water to remove high-salt culture medium residue, and then freeze-dried. The freeze-dried mixed bacterial cell powder is weighed and mixed with 0.1% SDS solution at a ratio of 1 g:10 mL, and stirred in a water bath at 37℃ for 2 h. Then, it is centrifuged at 10000 r / min for 15 min, the precipitate is collected and freeze-dried for 24 h. The obtained precipitate is mixed with chloroform at a ratio of 1 g:10 mL and dissolved at 100℃ for 4 h under sealed conditions. After cooling to room temperature, an equal volume of deionized water is added, and the mixture is centrifuged at 5000 r / min for 5 min. The lower organic phase is taken out and filtered. The obtained organic phase is slowly poured into pre-cooled ethanol at -20℃ and stirred until a white precipitate is formed. The precipitate is collected and dried at room temperature in a fume hood to constant weight to obtain white PHBV solid.

[0037] The detection method for the PHV (3HV unit) content is as follows: Take 10 mL of activated sludge mixture and centrifuge at 10000 rpm for 10 min. Remove the supernatant and freeze the lower sludge layer in a -80℃ freezer for 12 h. Freeze-dry the frozen sludge at -50℃ for 24 h. Weigh a certain amount of dried sludge into an esterification tube, add 2 mL of chloroform and 1 mL of esterification reaction solution, react at 103℃ for 4 h, cool to room temperature, add 1 mL of high-purity water to the esterification tube, shake vigorously, and let stand in a 4℃ freezer for 1 h. Use a 1 mL disposable syringe to draw about 1 mL of the lower organic phase and transfer it to a GC-MS sample vial for GC-MS analysis. Preparation of PHA esterification solution: Accurately weigh 1.0 g of benzoic acid (analytical grade), dissolve it in 100 mL of methanol (chromatographic grade), add 10 mL of concentrated sulfuric acid (analytical grade), and stir until completely dissolved. This is the esterification reaction solution for PHA detection. The GC-MS instrument used was a Thermo Fisher Scientific ISQ LT, with a TG-WAXMS quartz capillary column and a flame ionization detector (FID). The GC-MS conditions were as follows: initial temperature 70℃, hold for 2 min; ramp to 140℃ at a rate of 7℃ / min, hold for 1 min; ramp to 220℃ at a rate of 16℃ / min, hold for 1 min. The entire temperature program took 19 min. Since PHV standards were unavailable, PHBV standards were used to determine the PHB and PHV standard curves. PHBV standards of 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, and 6 mg were weighed for standard curve determination. The PHBV standards contained 92 wt.% PHB and 8 wt.% PHV. PHV content was calculated as HV / (HB+HV)×100%.

[0038] Example 2 The difference from Example 1 is that in S3, the concentration of corn straw hydrolysate is 2000 mg / L, the concentration of ammonium chloride is 80 mg / L, the concentration of sodium chloride is 5 g / L, and the pH value is 7. After 8 consecutive cycles, the yield of PHA converted from corn straw hydrolysate is 1488 mg / L. The produced PHA is a PHBV copolymer, in which the 3HV unit content is 67.9 wt.%, and the PHA content reaches 28.9% of the cell dry weight.

[0039] Example 3 The difference from Example 1 is that in S3, the concentration of corn straw hydrolysate is 2000 mg / L, the concentration of ammonium chloride is 60 mg / L, the concentration of sodium chloride is 10 g / L, and the pH value is 7. After 8 consecutive cycles, the yield of PHA converted from corn straw hydrolysate is 2073 mg / L. The produced PHA is a PHBV copolymer, in which the 3HV unit content is 93.4 wt.%, and the PHA content reaches 48.3% of the dry weight of the bacterial cells.

[0040] Example 4 The difference from Example 1 is that in S3, the concentration of corn straw hydrolysate is 2000 mg / L, the concentration of ammonium chloride is 80 mg / L, the concentration of sodium chloride is 10 g / L, and the pH value is 8. After 8 consecutive cycles, the yield of PHA converted from corn straw hydrolysate is 2219 mg / L. The produced PHA is a PHBV copolymer, in which the 3HV unit content is 89.5 wt.%, and the PHA content reaches 49.3% of the dry weight of the bacterial cells.

[0041] Example 5 The difference from Example 1 is that in S3, the concentration of corn straw hydrolysate is 2500 mg / L, the concentration of ammonium chloride is 75 mg / L, the concentration of sodium chloride is 5 g / L, and the pH value is 8. After 8 consecutive cycles, the yield of PHA converted from corn straw hydrolysate is 3079 mg / L. The produced PHA is a PHBV copolymer, in which the 3HV unit content is 94.3 wt.%, and the PHA content reaches 52.6% of the cell dry weight.

[0042] Example 6 The difference from Example 1 is that in S3, the concentration of corn straw hydrolysate is 2700 mg / L, the concentration of ammonium chloride is 75 mg / L, the concentration of sodium chloride is 5 g / L, and the pH value is 8. After 8 consecutive cycles, the yield of PHA converted from corn straw hydrolysate is 2396 mg / L. The produced PHA is a PHBV copolymer, in which the 3HV unit content is 95.0 wt.%, and the PHA content reaches 59.9% of the cell dry weight.

[0043] Comparative Example 1 The difference from Example 1 is that in S3, the concentration of corn straw hydrolysate is 2000 mg / L, the concentration of ammonium chloride is 80 mg / L, the concentration of sodium chloride is 10 g / L, and the pH value is 6.5. After 8 consecutive cycles, the yield of PHA converted from corn straw hydrolysate is 456 mg / L. The produced PHA is a PHBV copolymer, in which the 3HV unit content is 64.4 wt.%, and the PHA content reaches 6.83% of the cell dry weight.

[0044] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A method for producing PHBV copolymer using a mixed halophilic bacterial community from Qinghai Lake, characterized in that, Includes the following steps: S1. Harmophilic sludge from the shallow water area of ​​Qinghai Lake from May to August is put into the SBR reactor. The first culture medium is prepared with corn straw hydrolysate and sodium chloride. The reactor is run in a fully aerobic aeration mode to screen and activate halophilic activated sludge. S2. Add the second culture medium to the SBR reactor containing the halophilic activated sludge obtained in step S1, and continue the aerobic sequencing batch acclimation culture until the MLSS of the halophilic activated sludge in the SBR reactor reaches more than 7000 mg / L. S3. The halophilic activated sludge obtained in step S2 is transferred to the synthesis reactor to start the synthesis culture. The initial sludge concentration MLSS is 1500 mg / L. Corn straw hydrolysate is used as the only organic carbon source in the synthesis stage. The sequencing batch operation is carried out in a 12-hour cycle, which includes 11 hours of aeration and 1 hour of sedimentation. S4. After 8 consecutive cycles of operation, PHA is extracted from the residual halophilic activated sludge rich in PHA discharged from the reactor to obtain PHBV copolymer. In all steps S1 to S3, corn straw hydrolysate is used as the sole organic carbon source, and no propionate, acetate, glucose, or molasses alcohol wastewater is added as an auxiliary carbon source; the domestication culture and synthesis culture are not subjected to high-pressure steam sterilization or chemical sterilization treatment.

2. The method for producing PHBV copolymer using a halophilic mixed bacterial community from Qinghai Lake according to claim 1, characterized in that, The corn stalk hydrolysate contains multiple components of reducing low-carbon sugars, specifically including arabinose, galactose, glucose, xylose, and mannose. When used as an influent carbon source, the corn stalk hydrolysate is diluted to 500-3000 mg / L based on total reducing sugars.

3. The method for producing PHBV copolymer using a halophilic mixed bacterial community from Qinghai Lake according to claim 1, characterized in that, In step S1, the first culture medium comprises: corn straw hydrolysate with a total reducing sugar content of 500 mg / L, ammonium chloride of 25 mg / L, potassium dihydrogen phosphate of 5 mg / L, sodium chloride of 10 g / L, magnesium sulfate of 100 mg / L, and trace element solution of 1 mL / L.

4. The method for producing PHBV copolymer using a halophilic mixed bacterial community from Qinghai Lake according to claim 1, characterized in that, In step S1, the dissolved oxygen concentration of the fully aerobic aeration operation is ≥5 mg / L, the aeration time is 11 h, the sedimentation time is 1 h, and then the supernatant is discharged; when the sludge color changes from dark brown to light yellow, the sludge concentration MLSS increases to 4000 mg / L, and the SVI reaches 40 mL / g, the acclimatization and cultivation in step S2 begins.

5. The method for producing PHBV copolymer using a halophilic mixed bacterial community from Qinghai Lake according to claim 1, characterized in that, In step S2, the second culture medium comprises: corn straw hydrolysate (total reducing sugar 1000 mg / L), ammonium chloride 50 mg / L, potassium dihydrogen phosphate 10 mg / L, sodium chloride 10 g / L, magnesium sulfate 100 mg / L, and trace element solution 1 mL / L. In step S2, the reaction conditions are controlled as follows: room temperature, dissolved oxygen ≥5 mg / L, aeration time 11 h, and sedimentation time 1 h. Then, the supernatant is drained, and the mixture is run for 20-30 days.

6. The method for producing PHBV copolymer using a halophilic mixed bacterial community from Qinghai Lake according to claim 1, characterized in that, In step S3, the process parameters for synthesis and cultivation are as follows: the concentration of corn straw hydrolysate is 1000~3000 mg / L, the concentration of ammonium chloride is 40~120 mg / L, the concentration of sodium chloride is 0~20 g / L, and the pH value is 6~8.

7. The method for producing PHBV copolymer using a halophilic mixed bacterial community from Qinghai Lake according to claim 1, characterized in that, In step S4, the cell walls of halophilic microorganisms are disrupted by utilizing their tendency to lyse in low-salt or fresh water. The resulting product is then extracted with an organic solvent, centrifuged or filtered, precipitated, and dried to obtain the PHA product.

8. The PHBV copolymer prepared by the method according to any one of claims 1 to 7, characterized in that, The 3-hydroxybutyrate unit and 3-hydroxyvalerate unit in the PHBV copolymer are both derived from the intracellular polymerization accumulation of corn straw hydrolysate after being metabolized by the mixed halophilic bacteria of Qinghai Lake. Furthermore, no propionate is added as a comonomer precursor during the preparation process. The 3HV unit content in the PHBV copolymer is 42.5 wt.% to 95.0 wt.%.

9. The use of the PHBV copolymer of claim 8 in the preparation of biodegradable materials.

10. The application of a halophilic mixed microbial community from Qinghai Lake in the production of PHBV copolymers using agricultural waste hydrolysate, characterized in that, The agricultural waste hydrolysate is corn straw hydrolysate, and the Qinghai Lake halophilic mixed microbial community is obtained through directional domestication via steps S1 and S2 as described in any one of claims 1 to 7.