CN118006514A - Method for enriching polyhydroxyalkanoate synthetic flora and producing polyhydroxyalkanoate by utilizing lactic acid-rich agricultural waste fermentation liquor - Google Patents
Method for enriching polyhydroxyalkanoate synthetic flora and producing polyhydroxyalkanoate by utilizing lactic acid-rich agricultural waste fermentation liquor Download PDFInfo
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- C12N1/20—Bacteria; Culture media therefor
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Abstract
The invention belongs to the field of agricultural waste utilization, and provides a method for enriching polyhydroxyalkanoate synthetic flora by utilizing lactic acid-rich agricultural waste fermentation liquor and producing polyhydroxyalkanoate. The method comprises the following steps: firstly, utilizing agricultural waste to directionally ferment to produce lactic acid fermentation liquor, then utilizing the lactic acid fermentation liquor to synthesize polyhydroxyalkanoate flora for enrichment by aerobic sludge, and utilizing the enriched flora to synthesize polyhydroxyalkanoate.
Description
Technical Field
The invention belongs to the field of agricultural waste utilization, and particularly relates to a method for enriching polyhydroxyalkanoate synthetic flora by utilizing lactic acid-rich agricultural waste fermentation liquor and producing polyhydroxyalkanoate.
Background
The common waste treatment mainly realizes the purposes of harmless treatment, fertilizer treatment and energy treatment of the waste through composting or anaerobic fermentation and other processes. Agricultural waste can also be used for producing Volatile Fatty Acid (VFA) and Lactic Acid (LA) through anaerobic fermentation, and the organic acid can be used for producing a high value-added product, namely a biodegradable material Polyhydroxyalkanoate (PHA) besides being used in the fields of medicines, foods and the like.
PHA is one of important biodegradable plastics, can be used as a potential substitute for traditional petroleum-based plastics, and can be used in the fields of agriculture, green packaging, medicine, biofuel and the like. The PHA production raw materials in the current market mainly comprise grain crops (DUTT TRIPATHI ET al, 2021; TAN et al, 2021) such as glucose, sucrose, edible vegetable oil (such as rapeseed oil, palm oil and sunflower seed oil) and starch, and the PHA production cost is high, and the PHA production by using the raw materials has the potential problem of competing with people for grain, so that the popularization and the application of the PHA production raw materials are further limited. While the use of mixed flora to synthesize PHA is capable of converting a large number of available low cost carbon waste sources into value added biocompatible and biodegradable plastic products, achieving sustainable environmental and economic (Nguyenhuynh et al., 2021). The use of waste to produce PHA has therefore become a very promising strategy for the production of PHA in industry (Liu et al 2021). The prior researches mainly focus on the enrichment of mixed flora for synthesizing PHA by taking activated sludge and VFA of municipal sewage treatment plants as substrates. CN114410565A discloses a method for enriching PHA-synthesizing bacteria by utilizing sludge anaerobic fermentation liquid, which can enhance the enrichment of bacterial groups with strong PHA-synthesizing capability by pretreatment of fermentation liquid. The flora for synthesizing PHA by using waste fermentation liquor mainly comes from activated sludge of municipal sewage treatment plants, while the agricultural waste fermentation liquor is different from other wastes, which can lead to different dominant flora for producing PHA by using livestock and poultry manure, and no method for enriching PHA synthesizing flora by using homologous sludge is seen at present.
Disclosure of Invention
The invention aims to provide a method for enriching polyhydroxyalkanoate synthetic flora by utilizing lactic acid-rich agricultural waste fermentation liquor and producing polyhydroxyalkanoate.
The invention takes agricultural wastes such as livestock and poultry manure and the like as raw materials, and the PHA bacterial colony is efficiently synthesized by enriching the homologous aerobic sludge, so that the PHA synthesis effect is improved. Provides a new way and selection for the value-added utilization (such as PHA) of pig manure and apple waste.
Wherein the PHA mainly comprises PHB and PHV copolymer PHBV.
In order to achieve the aim of the invention, the invention provides a method for enriching polyhydroxyalkanoate synthetic flora by using lactic acid-rich agricultural waste fermentation liquor and producing polyhydroxyalkanoate, which comprises the following steps:
A. The first stage: taking livestock and poultry manure, crop straws, fruit and vegetable wastes or a mixture thereof as a fermentation substrate (wherein the livestock and poultry manure is an essential component, namely the fermentation substrate is a mixture of the livestock and poultry manure and at least one of agricultural wastes such as the crop straws, the fruit and vegetable wastes, and the like), and carrying out anaerobic fermentation by using lactobacillus as fermentation bacteria to obtain fermentation liquor rich in lactic acid and volatile fatty acid;
B. and a second stage: c, putting the aerobic sludge into an enrichment reactor for synthesizing PHA flora, and domesticating the aerobic sludge by utilizing the fermentation liquor obtained in the step a to enrich polyhydroxyalkanoate synthesis flora;
C. and a third stage: and (3) taking the fermentation broth obtained in the step (A) as a substrate, and synthesizing polyhydroxyalkanoate by utilizing the flora enriched in the step (B).
Further, the carbon to nitrogen ratio of the fermentation substrate in the step A is (35-40): 1.
The livestock manure is pig manure, cow manure, sheep manure, chicken manure and the like; the crop straw is one or more of corn straw, wheat straw and rice straw; the fruit and vegetable waste is decayed fruits, seedlings, vines and leaves generated in the planting process of fruits or vegetables and tails in the processing process; the total solid content of the crop straw is 40-70% TS, the volatile solid content is 80-90% TS (based on the total solid content), the crude fiber content is 30-50%, and the carbon-nitrogen ratio is (45-60): 1; the total solid content of the fruit and vegetable waste is 1-20% TS, the volatile solid content is 90-98% TS (based on the total solid content), the total sugar content is 10-20% (based on fresh weight), and the carbon-nitrogen ratio is (40-100): 1.
Further, the lactic acid bacteria in the step a include one or more of Bifidobacterium (Bifidobacterium), streptococcus thermophilus (Streptococcus thermophilus), lactobacillus plantarum (Lactobacillus plantarum), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus bulgaricus (Lactobacillus bulgaricus), and the like.
Further, step a includes: adding water into fermentation substrate, mixing, adding into anaerobic fermentation tank (preferably adding water into fermentation substrate according to feed liquid ratio of 1g (1-2)) and inoculating lactobacillus solution, fermenting at 35-55deg.C and pH of 3.0-7.0.
Further, the fermentation concentration in step A is 2% VS-15% VS (by mass); the inoculation amount of the lactobacillus liquid is 5-10% v/v, and the viable count in the lactobacillus liquid is 10 6-109 CFU/mL.
Further, step B includes:
B1, pretreatment of aerobic sludge
The aerobic sludge is added into an enrichment reactor for synthesizing PHA flora after inorganic particles are removed by sieving with a sieve with the aperture of 0.2-0.5 mm (preferably 0.25 mm), the fermentation temperature is room temperature, aeration is carried out for 5-10 days (preferably 7 days) to restore the activity of the sludge, the nutrient substances in the sludge are exhausted, and the aeration rate is 0.6-1.0L/L/min (preferably 1.0L/L/min); no material is added in the process;
b2, adaptation stage of polyhydroxyalkanoate-synthesizing flora
Putting pretreated aerobic sludge into an enrichment reactor for synthesizing PHA flora, and performing domestication according to 1 domestication period of water inlet-aerobic aeration-water outlet, wherein the period of continuous domestication is 8-20 periods;
wherein, the concentration of the sludge added into the enrichment reactor for synthesizing PHA flora is 0.5-5 g/L calculated by MLSS; the water inlet time is 1-10 min (preferably 10 min), the aerobic aeration time is 660-1500 min (preferably 1420 min), the water outlet time is 1-10 min (preferably 10 min), and the hydraulic retention time and the sludge retention time are 1-4 days (preferably 2 days);
b3, stage of formal enrichment of polyhydroxyalkanoate-synthesizing flora
In the enrichment reactor for synthesizing PHA flora, the domestication is carried out according to 1 domestication period of water inlet, aerobic aeration, precipitation and water outlet, and the continuous domestication is carried out for 60-200 periods;
The water inlet time is 1-10 min (preferably 10 min), the aerobic aeration time is 330-1500 min (preferably 660 min), the sedimentation time is 20-60 min (preferably 40 min), the water outlet time is 1-10 min (preferably 10 min), the hydraulic retention time is 1-4 days (preferably 2 days), and the sludge retention time is 2-10 days (preferably 8 days).
Further, in B2 and B3, the fermentation broth obtained in the step A is diluted with water to have a COD concentration of about 1000-3000 mg/L (preferably 1600 mg/L) as the water for the inflow.
Further, the aeration rate in B2 and B3 is 0.6-1.0L/L/min.
Further, the period of time for the B2 adaptation phase is 4-20 days (preferably 16 days).
Further, after enrichment for 30-100 days (preferably 96 days), the enriched polyhydroxyalkanoate synthetic flora is mainly at least one of Paracoccus (Paracoccus), agrimonia (Gemmobacter), monoblongus (Brevundimonas) and Cytophaga hydrocytophaga (Hydrogenophaga), and PHA content is 25-50% of bacterial cell dry weight.
Further, step C includes: and (C) regulating the pH value of the fermentation liquor obtained in the step (A) to 7.0-8.0 as a substrate, wherein the fermentation temperature is 20-30 ℃.
Further, the step A adopts a batch fermentation process, and the number of fermentation days in each batch is 6-10 days.
By means of the technical scheme, the invention has at least the following advantages and beneficial effects:
The method for enriching PHA bacterial colony synthesized by agricultural wastes provided by the invention is to utilize homologous activated sludge to enrich PHA bacterial colony, so that efficient PHA bacterial colony can be enriched in a short period of time.
The method for synthesizing PHA flora from the agricultural wastes can utilize the agricultural wastes such as livestock and poultry manure, fruits and vegetables to produce PHA products with high added value, and realize the dual purposes of harmless and high-value utilization of the agricultural wastes such as livestock and poultry manure. The method has the advantages of wide sources of raw materials, simple process operation, realization of enrichment of the PHA synthesizing flora in a short period and good application prospect.
Drawings
FIG. 1 is a graph showing the comparison of PHA content during enrichment of PHA flora synthesized from different aerobic sludge in examples 2-3 of the present invention.
FIG. 2 is a graph showing the comparison of PHA synthesis results from PHA-enriched colonies of different aerobic sludge in examples 2-3 of the present invention.
Detailed Description
Aiming at the problems of poor domestication and enrichment of PHA flora synthesized by agricultural wastes such as livestock and poultry manure, the invention provides a method for enriching PHA synthesis flora by utilizing agricultural waste fermentation liquor.
The invention adopts the following technical scheme:
the invention provides a method for enriching PHA (polyhydroxyalkanoate) synthetic flora by utilizing agricultural waste fermentation liquor, which aims at enriching the mixed flora of the polyhydroxyalkanoate synthesized by agricultural waste, and specifically comprises the following steps:
A. The first stage: agricultural wastes such as livestock and poultry manure, fruit and vegetable wastes and the like are used as substrates, and zymophytes are lactic acid bacteria, so that fermentation liquor rich in lactic acid and volatile fatty acid is obtained; namely, agricultural waste is taken as a substrate, and lactic acid bacteria are utilized for directional fermentation to produce lactic acid fermentation liquor;
B. And a second stage: using fermentation liquor rich in lactic acid as a substrate, and enriching and synthesizing PHA mixed flora by using aerobic sludge; namely, different sewage treatment aerobic sludge is enriched by utilizing agricultural waste acidizing fluid to synthesize PHA flora;
C. a third stage; taking fermentation liquor rich in lactic acid as a substrate, and utilizing the enriched mixed flora to synthesize PHA; namely, the enriched bacterial population utilizes lactic acid fermentation broth to efficiently synthesize PHA.
Wherein, in the step A, a batch process is adopted, the fermentation temperature is 33-55 ℃, the initial pH is 3.0-7.0, the competitive advantage of lactic acid production is improved by adding lactobacillus, meanwhile, the lactic acid produced by fermentation can reduce the pH in a fermentation system, the metabolic activity of other microorganisms is further inhibited, the oriented lactic acid and volatile fatty acid production of agricultural wastes such as livestock and poultry manure is favorably realized, and each batch fermentation time is 6-10 days.
Wherein, the PHA flora is enriched in the step B by adopting activated sludge of aerobic tanks of different sewage treatment plants, and the fermentation liquor in the step A is enriched to obtain the flora for efficiently synthesizing PHA, and the fermentation temperature is 20-30 ℃.
Wherein, the fermentation temperature in the step C is 20-30 ℃, the pH of the lactic acid fermentation liquor is 7.0-8.0, and the PHA synthesis effect of different mixed bacteria groups is compared.
The following examples are illustrative of the invention and are not intended to limit the scope of the invention. Unless otherwise indicated, the technical means used in the examples are conventional means well known to those skilled in the art, and all raw materials used are commercially available.
Example 1 oriented fermentation of livestock and poultry feces and apple waste to produce lactic acid
The embodiment mainly comprises the following steps:
1. Culture of lactic acid bacteria the lactic acid bacteria culture medium is selected MRS broth (Hirudo high tech Industrial science and technology Co., ltd.) and is inoculated with Baisheng's preferred lactic acid bacteria powder (4 g/L, commercially available from Shankang Biotechnology Co., ltd.) at 35-40deg.C to obtain lactic acid bacteria inoculation liquid (viable count about 10 7 CFU/mL) for use.
2. Pig manure (TS: 26.9% fresh weight, VS:20.4% fresh weight and C/N=11.3) and apple waste (TS: 14.9% fresh weight, VS13.2% fresh weight and C/N=62.4) are uniformly mixed according to a certain proportion, added into an anaerobic fermentation tank, and uniformly mixed with a proper amount of water, wherein the fermentation concentration of a substrate after mixing is 4% (based on volatile solids), the carbon nitrogen ratio of the material is 40:1, and the lactobacillus inoculation amount is 10% (volume ratio). The fermentation temperature is 35-40 ℃, and the initial fermentation pH is 6.5+/-0.1. After 10 days of continuous anaerobic fermentation, the concentrations of lactic acid, ethanol, acetic acid, propionic acid, butyric acid and isovaleric acid were 24.28, 097, 2.12, 0.31, 0.64 and 0.06 g/L, respectively.
EXAMPLE 2 PHA flora enrichment by pig farm activated sludge Using lactic acid fermentation broth
The example includes the following steps:
1. taking aerobic sludge of a pig farm sewage treatment plant, removing inorganic particles by a sieve with the aperture of 0.25 mm, adding the sludge into an enrichment reactor for synthesizing PHA flora, performing aeration for 7 days at the fermentation temperature of room temperature (24+/-2 ℃) to restore the activity of the sludge, exhausting nutrient substances in the sludge, and ensuring that the aeration rate is 1.0L/L/min, wherein no material is added in the process.
2. Enrichment of polyhydroxyalkanoate synthetic flora
(1) PHA flora adaptation phase (16 days)
The concentration of the sludge fed into the enrichment reactor for the PHA-synthesizing flora was 2.89 g/L in terms of MLSS. The diluted lactic acid fermentation broth of example 1 (COD concentration of about 1600 mg/L) was added with a cycle of 24: 24 h each, including feed (10: 10 min), continuous aeration (1420: 1420 min, aeration rate of 1: 1L/L/min) and discharge (10: 10 min), with a hydraulic retention time and a sludge retention time of 2 days.
(2) Formally enriching phase of synthesizing PHA flora
Each cycle was 12 h, including feed (10 min), aeration (660 min, aeration 1L/L/min), precipitation (40 min), and discharge (10 min). The hydraulic retention time of the enrichment reactor was 2 days and the sludge retention time was 8 days.
3. After 96 days of enrichment, the mixed flora was mainly Paracoccus (41.45%) and Agrimonia (Gemmobacter) (1.31%) and PHA synthesis was performed using the lactic acid fermentation broth of example 1 (pH adjusted to 7.0-8.0) as substrate, at 20-30deg.C. The volume ratio of mixed flora and diluted lactic acid fermentation liquid in the domestication reactor added into the reactor for synthesizing PHA is 3:1, the COD concentration of the diluted lactic acid fermentation liquid is about 1600 mg/L, the aeration rate is 1L/L/min, and the rotation speed of a stirrer is 125 rpm.
After continuous fermentation for 1 day, the comparative graph of PHA content in the PHA flora enrichment process of different aerobic sludge is shown in figure 1, and the comparative graph of PHA synthesis effect after PHA flora enrichment of different aerobic sludge is stabilized is shown in figure 2.
The PHA content reached about 35.97% of the dry weight of the cells. The PHA produced is PHBV copolymer, PHB is mainly PHB, and PHV is 1.55%.
Example 3 Synthesis of PHA Fu enrichment Using lactic acid fermentation broth from municipal aerobic sludge
The process is identical to example 2, except that the activated sludge enriched in PHA-synthesizing flora is municipal sewage treatment plant activated sludge. The embodiment comprises the following steps:
1. Taking aerobic sludge of a municipal sewage treatment plant, removing inorganic particles by a sieve with the aperture of 0.25 mm, adding the sludge into a fermentation bottle, and performing aeration for 7 days to recover the activity of the sludge, depleting the nutrient substances in the sludge, wherein the aeration rate is 1.0L/L/min, and no material is added in the process.
2. Enrichment of polyhydroxyalkanoate synthetic flora
(1) PHA flora adaptation phase (16 days)
The concentration of the sludge fed into the enrichment reactor for the PHA-synthesizing flora was 6.26 g/L in terms of MLSS. The diluted lactic acid fermentation broth of example 1 (COD concentration of about 1600 mg/L) was added with a cycle of 24: 24 h each, including feed (10: 10 min), continuous aeration (1420: 1420 min, aeration rate of 1: 1L/L/min) and discharge (10: 10 min), with a hydraulic retention time and a sludge retention time of 2 days.
(2) Formally enriching phase of synthesizing PHA flora
The PHA-synthesizing flora formal enrichment stage, each cycle being 12 h, comprises feeding (10 min), aeration (660 min, aeration rate 1L/L/min), precipitation (40 min) and draining supernatant (10 min). The hydraulic retention time of the enrichment reactor was 2 days and the sludge retention time was 8 days.
3. After 96 days of enrichment, the mixed flora was mainly Paracoccus (31.28%) and Agrimonia (Gemmobacter) (1.90%) and the mixed flora was subjected to PHA synthesis using the lactic acid fermentation broth of example 1 (pH adjusted to 7.0-8.0) at 20-30deg.C. The volume ratio of the mixed flora and the diluted lactic acid fermentation liquid in the domestication reactor is 3:1, the COD concentration of the diluted lactic acid fermentation liquid is about 1600 mg/L, the aeration rate is 1L/L/min, and the rotation speed of the stirrer is 125 rpm
The comparative graph of PHA content in the PHA flora enrichment process of different aerobic sludge after continuous fermentation for 1 day is shown in figure 1, and the comparative graph of PHA synthesis effect after PHA flora enrichment of different aerobic sludge is stabilized is shown in figure 2
The PHA content reaches about 29.01% of the dry cell weight. The PHA is PHBV copolymer, PHB is the main ingredient, and the PHV content is 2.13%.
While the invention has been described in detail in the foregoing general description and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that modifications and improvements can be made thereto. Accordingly, such modifications or improvements may be made without departing from the spirit of the invention and are intended to be within the scope of the invention as claimed.
Claims (10)
1. The method for enriching polyhydroxyalkanoate synthetic flora and producing polyhydroxyalkanoate by using lactic acid-rich agricultural waste fermentation liquor is characterized by comprising the following steps:
A. the first stage: livestock and poultry manure, crop straws, fruit and vegetable waste or a mixture thereof are used as fermentation substrates, fermentation bacteria are lactic acid bacteria, and anaerobic fermentation is carried out to obtain fermentation liquor rich in lactic acid and volatile fatty acid;
B. and a second stage: c, putting the aerobic sludge into an enrichment reactor for synthesizing PHA flora, and domesticating the aerobic sludge by utilizing the fermentation liquor obtained in the step a to enrich polyhydroxyalkanoate synthesis flora;
C. and a third stage: and (3) taking the fermentation broth obtained in the step (A) as a substrate, and synthesizing polyhydroxyalkanoate by utilizing the flora enriched in the step (B).
2. The method according to claim 1, wherein the carbon to nitrogen ratio of the fermentation substrate in step A is (35-40): 1.
3. The method of claim 1, wherein the lactic acid bacteria in step a comprise one or more of bifidobacteria (bifidobacteria), streptococcus thermophilus (Streptococcus thermophilus), lactobacillus plantarum (Lactobacillus plantarum), lactobacillus acidophilus (Lactobacillus acidophilus), lactobacillus rhamnosus (Lactobacillus rhamnosus), lactobacillus bulgaricus (Lactobacillus bulgaricus).
4. The method of claim 1, wherein step a comprises: adding water into fermentation substrate, mixing, adding into anaerobic fermentation tank, and inoculating lactobacillus solution, wherein the fermentation temperature is 35-55deg.C, and pH is 3.0-7.0.
5. The method according to claim 4, wherein the fermentation concentration in step A is 2% vs-15% vs; the inoculation amount of the lactobacillus liquid is 5-10% v/v, and the viable count in the lactobacillus liquid is 10 6-109 CFU/mL.
6. The method of claim 1, wherein step B comprises:
B1, pretreatment of aerobic sludge
The aerobic sludge is added into an enrichment reactor for synthesizing PHA flora after inorganic particles are removed by sieving with a sieve with the aperture of 0.2-0.5 mm, the fermentation temperature is room temperature, aeration is carried out for 5-10 days to restore the activity of the sludge, the nutrient substances in the sludge are exhausted, and the aeration rate is 0.6-1.0L/L/min;
b2, adaptation stage of polyhydroxyalkanoate-synthesizing flora
Putting pretreated aerobic sludge into an enrichment reactor for synthesizing PHA flora, and performing domestication according to 1 domestication period of water inlet-aerobic aeration-water outlet, wherein the period of continuous domestication is 8-20 periods;
Wherein, the concentration of the sludge added into the enrichment reactor for synthesizing PHA flora is 0.5-5 g/L calculated by MLSS; the water inlet time is 1-10 min, the aerobic aeration time is 660-1500 min, the water outlet time is 1-10 min, and the hydraulic retention time and the sludge retention time are 1-4 days;
b3, stage of formal enrichment of polyhydroxyalkanoate-synthesizing flora
In the enrichment reactor for synthesizing PHA flora, the domestication is carried out according to 1 domestication period of water inlet, aerobic aeration, precipitation and water outlet, and the continuous domestication is carried out for 60-200 periods;
The water inlet time is 1-10 min, the aerobic aeration time is 330-1500 min, the sedimentation time is 20-60 min, the water outlet time is 1-10 min, the hydraulic retention time is 1-4 days, and the sludge retention time is 2-10 days;
In B2 and B3, diluting the fermentation liquor obtained in the step A with water until the COD concentration is 1000-3000 mg/L, and taking the fermentation liquor as water for domestication of the PHA synthesizing flora;
the aeration rate in B2 and B3 is 0.6-1.0L/L/min.
7. The method of claim 6, wherein the B2 adaptation phase is for a period of 4-20 days.
8. The method according to claim 6, wherein the polyhydroxyalkanoate-synthesizing bacteria enriched in step B after 30-100 days of enrichment are mainly at least one of Paracoccus (Paracoccus), agrobacter (Gemmobacter), monobacter shortwave (Brevundimonas) and Cytophaga hydrocarpa (Hydrogenophaga), and the PHA content is 25-50% of the dry weight of the bacterial cells.
9. The method of claim 1, wherein step C comprises: and (C) regulating the pH value of the fermentation liquor obtained in the step (A) to 7.0-8.0 as a substrate, wherein the fermentation temperature is 20-30 ℃.
10. The method according to any one of claims 1 to 9, wherein step a employs a batch fermentation process with a number of days per batch of fermentation of 6 to 10 days.
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| CN104651420A (en) * | 2015-03-11 | 2015-05-27 | 北京工商大学 | Method for synthesizing PHBV by using VFAs from residual sludge fermentation solution |
| CN113337550A (en) * | 2021-08-02 | 2021-09-03 | 中国农业科学院农业环境与可持续发展研究所 | Method for producing polyhydroxyalkanoate by fermenting agricultural wastes |
| WO2023010787A1 (en) * | 2021-08-02 | 2023-02-09 | 中国农业科学院农业环境与可持续发展研究所 | Method for producing polyhydroxyalkanoate by fermentation of agricultural wastes |
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