A process for the preparation of polyhydroxyalkanoates

Abstract

The present application relates to a kind of polyhydroxyaliphatic ester preparation method, comprising the following steps: (1) with organic waste as substrate preparation containing medium chain fatty acid acid-producing liquid;(2) with the acid-producing liquid containing medium chain fatty acid as substrate, inoculate mixed flora, carry out reaction in continuous flow reactor to prepare the polyhydroxyaliphatic ester;Wherein, the sum of the relative abundance of pseudomonas, alkaligenes, halomonas, burkholderia and bacillus in the mixed flora is not less than 80%.The substrate cost of the present application is low, the PHA yield is high, the proportion of medium and long chain fatty acid monomer in the product is high, and the material performance of PHA is effectively improved.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and biosynthesis technology, and specifically relates to a method for preparing polyhydroxy fatty acid esters. Background Technology

[0002] This section is intended to provide background or context for embodiments of the present invention. The description herein is not intended to imply that it is prior art simply because it is included in this section.

[0003] Polyhydroxyalkanoates (PHAs), as a fully biodegradable and environmentally friendly material, have physicochemical properties similar to traditional petroleum-based plastics and can be degraded into carbon dioxide and water by microorganisms in the natural environment, making them one of the core alternative materials for solving "white pollution." However, the commercial production of PHA currently faces the bottleneck of high costs: industrially, refined carbon sources such as glucose, fructose, and propionic acid are mostly used as fermentation raw materials, and the cost of these substrates accounts for 40%-60% of the total cost of PHA; at the same time, pure microbial fermentation processes require strict sterilization to avoid contamination by other microorganisms, further increasing energy consumption and process complexity, resulting in high prices for PHA products and making large-scale promotion and application difficult.

[0004] To overcome these challenges, the industry has gradually focused on the conversion pathway of "waste biomass - low-cost PHA". However, this pathway faces core technological bottlenecks: a lack of functional strains that can efficiently degrade complex components of organic waste (especially difficult-to-degrade kitchen waste oil) and stably produce high levels of PHA. Pure bacteria have weak degradation capabilities for oils in kitchen waste, requiring the addition of emulsifiers or complex pretreatment. Ordinary mixed bacteria suffer from low PHA synthesis efficiency, poor product purity, and low content of medium- and long-chain fatty acid monomers in PHA products, which cannot meet industrial needs.

[0005] Therefore, developing a method for preparing PHA using organic waste as a substrate to achieve targeted high-yield PHA and increase the content of medium- and long-chain fatty acids is not only key to solving the problem of organic waste pollution, but also a core breakthrough for promoting the industrialization of PHA and reducing its production costs, which has important environmental, economic and social significance. Summary of the Invention

[0006] The purpose of this invention is to provide a method for preparing polyhydroxy fatty acid esters that has low substrate cost, high PHA yield, and high content of medium and long chain fatty acid monomers.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides a method for preparing a polyhydroxy fatty acid ester, comprising the following steps: (1) Preparation of acid-producing liquid containing medium-chain fatty acids using organic waste as substrate; (2) Using the acid-producing liquid containing medium-chain fatty acids as a substrate, a mixed bacterial community is inoculated and the reaction is carried out in a continuous flow reactor to prepare the polyhydroxy fatty acid ester; wherein, the sum of the relative abundances of Pseudomonas, Alcaligenes, Halomonas, Burkholderia and Bacillus in the mixed bacterial community is not less than 80%.

[0008] In some embodiments, the sum of the relative abundances of Pseudomonas, Alcaligenes, Halomonas, Burkholderia, and Bacillus in the mixed microbial community is 80%-85%.

[0009] In some embodiments, the Shannon diversity index of the mixed microbial community is not less than 4.

[0010] Furthermore, the Shannon diversity index of the mixed microbial community is 4-5.

[0011] In some embodiments, the mixed microbial community is obtained by mixing and enriching inoculum from activated sludge, liquor cellar mud, mangrove soil, saline-alkali soil, park woodland humus, park soil, farmland soil, and lakeside silt.

[0012] In some embodiments, the mixed microbial community is obtained by separately treating inoculum from activated sludge, liquor cellar mud, mangrove soil, saline-alkali soil, park woodland humus, park soil, farmland soil, and lakeside silt, allowing them to stand, separate solids and liquids, and then mixing them to obtain the initial inoculum. Then, in a sequencing batch reactor, using the acid-producing liquid containing medium-chain fatty acids as the substrate, the initial inoculum is enriched and cultured by a stepwise increase in organic load. Each organic load is operated for 30-50 cycles, and each cycle includes a feeding stage, a reaction stage, and a sludge discharge stage.

[0013] Furthermore, inoculum from different environments is subjected to settling, solid-liquid separation, and filtration, and then mixed in equal mass ratios to obtain the initial inoculum.

[0014] Furthermore, the specific methods for the settling, solid-liquid separation and filtration treatment are as follows: inoculum from different environments is settling in a constant temperature environment of 20-30℃ for 20-30 hours. After the solid and liquid naturally separate into layers, the upper clear liquid is removed, and the lower solid material is repeatedly filtered 2-4 times through a 0.18-0.3mm nylon screen.

[0015] Furthermore, the specific methods for the settling, solid-liquid separation and filtration treatment are as follows: inoculum from different environments is settling at a constant temperature of 25°C for 24 hours. After the solid and liquid naturally separate into layers, the upper clear liquid is removed, and the lower solid material is repeatedly filtered through a 0.3mm nylon screen 2-4 times.

[0016] Furthermore, the organic loads increased in stages were 200-400 mg COD / L, 500-700 mg COD / L, 1100-1300 mg COD / L, 2200-2600 mg COD / L, 3400-3800 mg COD / L, and 5200-5600 mg COD / L.

[0017] Furthermore, the organic loads were increased in stages to 300 mg COD / L, 600 mg COD / L, 1200 mg COD / L, 2400 mg COD / L, 3600 mg COD / L and 5400 mg COD / L.

[0018] Furthermore, during the feeding stage, NH4Cl and KH2PO4 are added as nitrogen and phosphorus sources, respectively, and the C:N:P mass ratio is controlled at 90-110:9-11:1.

[0019] Furthermore, the running time for each cycle is 10-14 hours.

[0020] Furthermore, each cycle takes 12 hours to run.

[0021] Furthermore, the time ratio of the feeding stage, the reaction stage, and the sludge discharge stage is 4-6:350-360:1, and even more specifically, 5:354:1.

[0022] Furthermore, during the enrichment culture, the reaction temperature was controlled at 25±3℃ and the dissolved oxygen concentration was above 4 mg / L.

[0023] Further, the initial inoculum is adjusted to a suspended solids concentration of 1-3 g / L using distilled water and then inoculated into the sequencing batch reactor.

[0024] Furthermore, the amount of sludge discharged during the sludge discharge stage is 0.08-0.12 of the effective solvent in the sequencing batch reactor.

[0025] In some embodiments, in step (2), the reaction temperature is controlled at 25±3℃ and the dissolved oxygen concentration is 1.5-2.5mg / L.

[0026] In some embodiments, in step (2), the inoculation amount of the mixed microbial community is 4-6 g / L, and more specifically 5 g / L.

[0027] In some embodiments, in step (1), the mass of hexanoic acid in the acid-producing liquid containing medium-chain fatty acids accounts for 45% or more of the total mass of the medium-chain fatty acids.

[0028] In some embodiments, the specific method of step (1) is as follows: using organic waste as substrate, a mixed inoculum of activated sludge and liquor cellar mud is inoculated, and anaerobic fermentation is carried out under the conditions of pH 6.2-7.2, temperature 32-38℃, organic load 10-30g COD / L / d, and sludge retention time 5-7 days to obtain an acid-producing liquid containing short-chain fatty acids; then, using the acid-producing liquid containing short-chain fatty acids as substrate, ethanol is added, and anaerobic fermentation is carried out under the conditions of pH 6.5±0.1, temperature 35℃±3℃, organic load 2-6 g COD / L / d, and sludge retention time 18-22 days to synthesize the acid-producing liquid containing medium-chain fatty acids.

[0029] Further, the specific method of step (1) is as follows: using organic waste as substrate, a mixed inoculum of activated sludge and liquor cellar mud is inoculated, and anaerobic fermentation is carried out under the conditions of pH 6.2-7.2, temperature 32-38℃, organic load 10-30g COD / L / d, and sludge retention time of 6 days to obtain acid-producing liquid containing short-chain fatty acids; then, using the acid-producing liquid containing short-chain fatty acids as substrate, ethanol is added, and anaerobic fermentation is carried out under the conditions of pH 6.5±0.1, temperature 35℃±0.5℃, organic load 2-6 g COD / L / d, and sludge retention time of 20 days to synthesize the acid-producing liquid containing medium-chain fatty acids.

[0030] Furthermore, the mass ratio of the activated sludge and the liquor cellar mud in the mixed inoculation species is 1:0.8-1.2, and even more specifically 1:1.

[0031] Furthermore, the organic waste is inoculated with the mixed inoculum at a volume ratio of 2-4:1, and even further at a volume ratio of 3:1.

[0032] Furthermore, the concentration of ethanol added is 50-100 mmol / L.

[0033] In some embodiments, the organic waste is one or more of the following: kitchen waste, beer wastewater, molasses wastewater, food processing wastewater, paper mill wastewater, or municipal sludge.

[0034] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art: This invention uses organic waste as a substrate, obtaining an acid-producing liquid through anaerobic fermentation, which is then efficiently synthesized into PHA using a mixed microbial community. The substrate cost is low, the PHA yield is high, and the product contains a high proportion of medium- and long-chain fatty acid monomers, effectively improving the material properties of PHA. Furthermore, this method can simultaneously degrade organic matter such as fatty acids, carbohydrates, and proteins in the organic waste during PHA synthesis. This invention provides a new approach for the resource utilization of organic waste and low-cost PHA production, and is applicable to large-scale industrial production of PHA and resource-based treatment of organic waste. Attached Figure Description

[0035] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0036] Figure 1 The figure shows the experimental results of PHA accumulation rate and 3HHx monomer ratio in Example 1. Detailed Implementation

[0037] To address the shortcomings of existing PHA production technologies, such as high substrate costs, weak microbial degradation capabilities, poor product performance, and complex processes, this invention provides a method for the efficient synthesis of PHA using a mixed microbial community. In this mixed community, the sum of the relative abundances of *Pseudomonas*, *Alcaligenes*, *Haloxylon*, *Burkholderia*, and *Bacillus* is not less than 80%, achieving metabolic synergy and complementarity among the microbial communities. This solves the problems of low yield, weak stress resistance, and poor product performance associated with single or dual microbial communities. Simultaneously, the invention provides an enrichment method for this mixed microbial community and a PHA synthesis process, offering a new solution for low-cost, large-scale PHA production and the resource utilization of organic waste.

[0038] Furthermore, this invention forms a complete and synergistic mixed microbial community system by combining eight functional microbial communities from different environmental sources with specific functions. This system can effectively obtain a mixed microbial community with high relative abundance of core microbial communities and high Shannon diversity index, which is more conducive to efficient synthesis of PHA, further increasing the yield of PHA and the proportion of medium and long chain fatty acid monomers, thereby improving the material properties of PHA.

[0039] Furthermore, this method employs a sequencing batch reactor (SBR) as an acclimatization platform to selectively enrich microbial communities capable of PHA synthesis through a "dynamic substrate supply strategy." The core of this strategy lies in utilizing the instantaneous and gradient nature of carbon source supply to effectively screen dominant strains with efficient PHA storage capabilities.

[0040] Furthermore, in the preparation of acid-producing liquid containing medium-chain fatty acids (MCFA), the activity of functional enzymes and the abundance of functional microorganisms are enhanced by mixing inoculum and a two-stage fermentation process, which alleviates product inhibition and achieves efficient synthesis of MCFA, thereby helping to increase the content of medium-chain fatty acid monomers in PHA products.

[0041] In some embodiments, the PHA yield of the present invention can account for more than 40% of the dry weight of cells, the 3-hydroxyhexanoic acid (3HHx) monomer content in the product is more than 2.0 wt%, and the carbon source conversion rate is not less than 65%.

[0042] Specifically, the method of the present invention has the following beneficial effects: (1) Environmental benefits: Realize the resource utilization of organic waste such as kitchen waste and liquor wastewater, reduce secondary pollution caused by landfill / incineration, and at the same time produce biodegradable PHA to replace petroleum-based plastics to alleviate "white pollution"; (2) Economic benefits: Using inexpensive organic waste as substrate, there is no need for refined carbon source and strict sterilization, thus reducing the production cost of PHA; the mixed microbial community is enriched through sequencing batch reactor (SBR) to achieve high-density cultivation; (3) Technical advantages: The mixed microbial community has complementary functions and strong resistance (tolerance to high fatty acid concentrations); (4) Improved product performance: Increase the content of medium-chain monomer 3HHx to improve the tensile strength and elongation at break of PHA, thus meeting the needs of downstream plastic processing.

[0043] In the following description, only certain exemplary embodiments are briefly described. As those skilled in the art will recognize, the described embodiments can be modified in various ways without departing from the spirit or scope of the embodiments of the invention. Therefore, the drawings and description are considered to be exemplary in nature and not restrictive.

[0044] All features disclosed in this invention, or steps in all methods or processes disclosed, may be combined in any way, except for mutually exclusive features or steps.

[0045] The technical solutions of the present invention will be further described below with reference to specific embodiments. However, the present invention should not be limited to these embodiments. Unless specifically stated otherwise, all features can be replaced by other equivalent or similar alternative features. Unless specifically stated otherwise, each feature is only one example of a series of equivalent or similar features. The terminology used in the present invention, unless otherwise stated, generally has the meaning commonly understood by those skilled in the art. The implementation conditions adopted in the embodiments can be further adjusted according to different requirements of specific use. Implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0046] In this invention, operations without specific instructions are performed at room temperature. The raw materials used in this invention are commercially available or can be prepared using conventional methods in the prior art. Unless otherwise specified, the solutions in this invention are methods in the prior art.

[0047] Example 1: Preparation of acid-producing solution rich in medium-chain fatty acids (MCFA) Inoculum: Activated sludge (taken from the secondary sedimentation tank of an urban wastewater treatment plant) and baijiu cellar sludge (taken from the bottom mud of baijiu fermentation pits) were mixed in equal mass ratio after being allowed to stand to remove supernatant and filtered through a 0.25mm sieve to remove impurities. The physicochemical properties were determined to be TCOD 76500±2300mg COD / L, SCOD 23800±2100mg COD / L, and VSS 32500±1500mg / L.

[0048] Substrate pretreatment: The kitchen waste is taken from the city's organic waste treatment center. After removing hard objects such as bones, it is shredded to about 2mm using a food processor.

[0049] First stage fermentation: Inoculum and kitchen waste were inoculated at a volume ratio of 1:3. The culture conditions were: 35℃, pH 6.4, organic load of 18g COD / L / d, and sludge retention time (SRT) of 6 days, to obtain an acid-producing liquid rich in SCFA with a concentration of 23.6g COD / L.

[0050] Second-stage fermentation: The acid-producing liquid rich in SCFA obtained in the first stage was centrifuged, and the supernatant was used as substrate. 90 mmol / L ethanol was added, and the pH was controlled at 6.5±0.1, the temperature at 35℃, the organic loading at 4.5 g COD / L / d, and the SRT at 20 days to synthesize an acid-producing liquid rich in MCFA. The liquid was collected, filtered through a membrane module, and stored at 4℃ for later use. The yield of MCFA was 23.2 g COD / L, of which hexanoic acid accounted for 45% of the total mass of medium-chain fatty acids.

[0051] Example 2: Enrichment of PHA Mixed Microbial Community This embodiment provides a method for enriching mixed bacterial communities for the efficient synthesis of polyhydroxy fatty acid esters, which includes the following steps: 1. Sampling and Pretreatment: Eight types of inoculum were collected: activated sludge (from the secondary sedimentation tank of urban wastewater treatment at Gaobeidian Water Plant in Beijing, collected at a depth of 1.2m), baijiu cellar mud (from the bottom mud of fermentation pits for strong-aroma baijiu at Yanghe Distillery in Jiangsu, collected at a depth of 1.0m), mangrove soil (from the intertidal zone of mangrove wetlands in Zhanjiang Mangrove Nature Reserve in Guangdong, collected from the 0-20cm topsoil / mud-water mixture at the tidal line), saline-alkali soil (from the topsoil (0-20cm) or mud-water mixture at the groundwater level in moderately saline-alkali land in the Hetao Plain of Inner Mongolia, with a pH of 8.5-9.5 and a salt content of 0.3%-0.8%), and park humus soil (from the humus layer of coniferous / broadleaf forests in Shanghai Century Park, collected at a depth of 5-15cm, selecting those with a humus content ≥30%). The samples were collected from various locations, including: park soil (from urban park green space in Suzhou Tiger Hill Wetland Park, topsoil 0-15cm), farmland soil (topsoil from farmland in Suzhou where vegetables have been grown for a long time, collected at a depth of 0-20cm, selected from plots where no highly toxic pesticides have been used), and lake silt (bottom mud (0-10cm) in shallow water area near the shore of Taihu Lake, selected from clean areas without cyanobacterial blooms).

[0052] Each of the collected inoculum was placed in a constant temperature environment of 25℃ and allowed to stand for 24 hours. After solid-liquid separation, the upper clear liquid was removed (to remove free impurities and small molecule organic matter). The lower solid was then filtered twice through a 0.28mm nylon screen to remove solid impurities such as stones and plant residues. The pretreated inoculum was mixed in equal mass ratios to obtain the initial inoculum for the mixed microbial community.

[0053] 2. Microbial community enrichment: A 5 L effective volume acrylic SBR reactor was used as the acclimation device, equipped with an aeration system, a stirring device, and a constant temperature water bath system. During inoculation, the initial inoculum of the mixed bacterial population was introduced into the reactor, and distilled water was used to adjust the suspended solids concentration of the mixture to 2 g / L. During operation, the dissolved oxygen concentration was maintained above 4 mg / L by adjusting the aeration rate to ensure optimal PHA synthase activity and inhibit the growth of anaerobic competitive bacteria; the temperature was controlled at 25℃.

[0054] A stepwise approach was adopted to increase the organic loading rate. This involved gradually increasing the carbon source concentration by increasing the flow rate of MCFA-rich acid-producing solution to screen for PHA-synthesizing bacteria adapted to high loading rates. The initial organic loading rate was set at 300 mg COD / L, subsequently increased to 600, 1200, 2400, 3600, and 5400 mg COD / L. Each loading gradient was run for 30 SBR cycles. The PHA accumulation rate (≥25%) and the relative abundance of core bacterial genera (≥50%) were monitored at the end of each cycle. If these conditions were met, the cycle proceeded to the next gradient; otherwise, the cycle was extended to 50 cycles.

[0055] Each SBR operating cycle is set to 12 h, specifically including three stages: the feeding stage (10 min) involves rapidly adding acid-producing liquid as a carbon source, while simultaneously adding NH4Cl and KH2PO4 as nitrogen and phosphorus sources, controlling the C:N:P mass ratio to be 100:10:1, so that the carbon source concentration in the reactor quickly reaches its peak, forming a bountiful period; then the reaction stage (11 h 48 min) begins, where the microbial community rapidly synthesizes PHA when the carbon source is sufficient, and enters a scarcity period after the carbon source is depleted, causing non-PHA-synthesizing bacteria to be eliminated due to insufficient energy reserves; finally, the sludge discharge stage (2 min) involves simultaneously discharging sludge and supernatant, with the discharge volume being 1 / 10 of the effective reactor volume.

[0056] Detection using 16S high-throughput sequencing revealed that the core functional bacteria (Pseudomonas spp.) Pseudomonas Alcaligenes Alcaligenes spp. Halomonas Burkholderia Burkholderia Bacillus Bacillus The sum of relative abundance reached 81.6%, the PHA accumulation rate remained stable at over 35% (fluctuation ≤5%), and the carbon source removal rate remained above 80%, thus ceasing enrichment.

[0057] Example 3: Preparation of Synthetic PHA Using MCFA-rich acid-producing liquid as a substrate, the mixed bacterial population enriched in Example 2 (inoculation biomass 5 g / L) was inoculated into a continuous flow bioreactor, cultured at 25°C, DO=2 mg / L, and reacted for 12 h.

[0058] Synthesis results: such as Figure 1 As shown, the PHA accumulation rate was 41.8 wt%, the 3HHx monomer content was 2.1 wt%, and the carbon source conversion rate was 65.4%. The PHA accumulation rate refers to the percentage of PHA mass in the dry weight of the cells.

[0059] Comparative Example 1: This example is basically the same as Example 3, except that only a single activated sludge is inoculated, instead of the mixed microbial community enriched in Example 2.

[0060] Microbial community characteristics: The biomass was slightly lower than that of the mixed microbial community, and the total abundance of core genera was only 45.2%.

[0061] Synthesis results: PHA accumulation rate was 28.5 wt%, 3HHx monomer content was 0.8 wt%, and carbon source conversion rate was 42.3%, all of which were significantly lower than those in Example 3.

[0062] It is evident that mixed microbial communities are significantly superior to single microbial communities in terms of microbial abundance, stress resistance, and PHA synthesis performance.

[0063] Comparative Example 2: This example is basically the same as Example 3, except that: the inoculated material is a mixed microbial community obtained by enriching 6 types of inoculants according to the method of Example 2. The 6 types of inoculants are 6 types other than mangrove soil and saline-alkali soil.

[0064] Microbial abundance and diversity: 16S high-throughput sequencing was used to analyze the community structure of the mixed microbial community at the end of domestication. Results showed that the core functional genera (Pseudomonas) were abundant and diverse. Pseudomonas Alcaligenes Alcaligenes spp. Halomonas Burkholderia Burkholderia Bacillus Bacillus The total relative abundance of the microbial community decreased to 63.4%, significantly lower than 81.6% in Example 2. Simultaneously, the Shannon diversity index decreased from 4.52 in Example 2 to 3.38, indicating a significant reduction in microbial diversity.

[0065] Synthesis results: The PHA accumulation rate was only 32.3 wt%, the 3HHx monomer ratio decreased to 1.2 wt%, and the carbon source conversion rate decreased to 51.6%, all of which were significantly lower than the carbon source conversion rates of 41.8 wt%, 2.1 wt%, and 65.4% in Example 3.

[0066] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A method for preparing a polyhydroxyalkanoate, characterized in that: Includes the following steps: (1) Preparation of acid-producing liquid containing medium-chain fatty acids using organic waste as substrate; (2) Using the acid-producing liquid containing medium-chain fatty acids as a substrate, a mixed bacterial community is inoculated and the reaction is carried out in a continuous flow reactor to prepare the polyhydroxy fatty acid ester; wherein, the sum of the relative abundances of Pseudomonas, Alcaligenes, Halomonas, Burkholderia and Bacillus in the mixed bacterial community is not less than 80%.

2. The method for preparing polyhydroxyalkanoate according to claim 1, characterized in that: The Shannon diversity index of the mixed microbial community is not less than 4.

3. The method for preparing polyhydroxyalkanoate according to claim 1, characterized in that: The mixed microbial community is obtained by separately treating inoculants from activated sludge, liquor cellar mud, mangrove soil, saline-alkali soil, park woodland humus, park soil, farmland soil, and lakeside silt, allowing them to stand, separate solids and liquids, and then mixing them to obtain the initial inoculum. Then, in a sequencing batch reactor, using the acid-producing liquid containing medium-chain fatty acids as the substrate, the initial inoculum is enriched and cultured by stepwise increasing the organic load. Each organic load is operated for 30-50 cycles, and each cycle includes a feeding stage, a reaction stage, and a sludge discharge stage.

4. The method for preparing polyhydroxyalkanoate according to claim 3, characterized in that: Inoculum from different environments, after being allowed to stand, undergoing solid-liquid separation and filtration, is mixed in equal mass ratios to obtain the initial inoculum; and / or, The specific methods for settling, solid-liquid separation and filtration are as follows: inoculum from different environments is settling at a constant temperature of 20-30℃ for 20-30 hours. After the solid and liquid naturally separate into layers, the upper clear liquid is removed, and the lower solid is filtered repeatedly 2-4 times through a 0.18-0.3mm nylon screen.

5. The method for preparing polyhydroxyalkanoate according to claim 3, characterized in that: The organic loading rates were increased in stages: 200-400 mg COD / L, 500-700 mg COD / L, 1100-1300 mg COD / L, 2200-2600 mg COD / L, 3400-3800 mg COD / L, and 5200-5600 mg COD / L; and / or, During the feeding stage, NH4Cl and KH2PO4 are added as nitrogen and phosphorus sources, respectively, and the C:N:P mass ratio is controlled at 90-110:9-11:1; and / or, The runtime for each cycle is 10-14 hours; and / or, The time ratio of the feeding stage, the reaction stage, and the sludge removal stage is 4-6:350-360:1; and / or, During enrichment culture, the reaction temperature should be controlled at 25±3℃, and the dissolved oxygen concentration should be above 4 mg / L; and / or, The initial inoculum is adjusted to a suspended solids concentration of 1-3 g / L using distilled water and then inoculated into the sequencing batch reactor; and / or... The amount of sludge discharged during the sludge discharge stage is 0.08-0.12 of the effective solvent in the sequencing batch reactor.

6. The method for preparing polyhydroxyalkanoate according to claim 1, characterized in that: In step (2), the reaction temperature is controlled at 25±3℃, and the dissolved oxygen concentration is 1.5-2.5 mg / L; and / or, In step (2), the inoculation amount of the mixed microbial community is 4-6 g / L.

7. The method for preparing polyhydroxyalkanoate according to claim 1, characterized in that: In step (1), the mass of hexanoic acid in the acid-producing liquid containing medium-chain fatty acids accounts for 45% or more of the total mass of the medium-chain fatty acids.

8. The method for preparing polyhydroxyalkanoate according to claim 1, characterized in that: The specific method of step (1) is as follows: using organic waste as substrate, a mixed inoculum of activated sludge and liquor cellar mud is inoculated and anaerobic fermentation is carried out under the conditions of pH 6.2-7.2, temperature 32-38℃, organic load 10-30g COD / L / d, and sludge retention time 5-7 days to obtain acid-producing liquid containing short-chain fatty acids; then, using the acid-producing liquid containing short-chain fatty acids as substrate, ethanol is added and anaerobic fermentation is carried out under the conditions of pH 6.5±0.1, temperature 35℃±3℃, organic load 2-6 g COD / L / d, and sludge retention time 18-22 days to synthesize the acid-producing liquid containing medium-chain fatty acids.

9. The method for preparing polyhydroxyalkanoate according to claim 8, characterized in that: The mass ratio of the mixed inoculated species, namely the activated sludge and the liquor cellar mud, is 1:0.8-1.2; and / or, The organic waste and the mixed inoculum are inoculated at a volume ratio of 2-4:1; and / or, The concentration of ethanol added is 50-100 mmol / L.

10. The method for preparing polyhydroxyalkanoate according to claim 1 or 8, characterized in that: The organic waste is one or more of the following: kitchen waste, beer wastewater, molasses wastewater, food processing wastewater, paper mill wastewater, or municipal sludge.

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