Preparation method for producing PHBV (polyhydroxybutyrate valerate) copolymer by using methane-oxidizing bacteria-heterotrophic bacteria mixed flora

Abstract

The invention belongs to the technical field of biosynthesis, and discloses a preparation method for producing a PHBV copolymer by using methane-oxidizing bacteria-heterotrophic bacteria mixed flora. The method comprises a step of synthesizing PHBV by using methane and a trace amount of co-substrate as a carbon source and a mixed flora through a specific microbial metabolic pathway, and a step of extracting the PHBV copolymer from thalli. According to the method, the maximum content of the synthesized PHBV accounts for 42.8% of the dry weight of the mixed bacteria by introducing a co-substrate, fermenting the mixed bacteria and optimizing the fermentation conditions of a mixed bacteria system, the content of 3-HV monomers in the synthesized PHBV is increased, the melting temperature and the crystallinity are good, the effect of improving the flexibility and the processing performance of the PHBV is achieved, and the method has a good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biosynthesis, and relates to a method for synthesizing a PHBV copolymer by using a mixed flora of methanotrophic bacteria and heterotrophic bacteria and adding a specific co-substrate, and application of the method in improving the quality of PHB products. Background Art

[0002] The widespread use of traditional plastics is accompanied by serious environmental problems, which has prompted people to actively seek sustainable and environmentally friendly alternatives. Polyhydroxyalkanoates (PHA), especially poly-β-hydroxybutyrate (PHB), are regarded as ideal substitutes for traditional plastics due to their excellent biocompatibility, biodegradability and a series of outstanding physical properties. However, the brittleness of PHB materials limits the further expansion of their application range. In order to overcome this limitation, researchers developed PHBV copolymers, which effectively improved the brittleness problem of PHB by introducing 3-hydroxyvalerate (3-HV) monomers, and improved the flexibility and processing performance of the material.

[0003] In the biosynthesis of PHB and PHBV, microbial fermentation technology has always been a hot topic of research. Early studies mainly focused on the use of pure microorganisms for fermentation. For example, some specific bacteria can use carbon sources such as glucose to synthesize PHB. However, these pure microorganisms have limitations in terms of substrate utilization range, fermentation efficiency and cost control. As a type of microorganism that can use methane as the only carbon source and energy source, methanotrophic bacteria have gradually attracted attention in the field of biosynthesis. As a greenhouse gas, the effective use of methane can not only reduce greenhouse gas emissions, but also promote the recycling of resources. Studies have shown that some methanotrophic bacteria have the ability to synthesize PHB, but they can usually only use methane as a carbon source to synthesize PHB, but cannot directly synthesize PHBV.

[0004] Based on the above background, in view of the limitations of methanotrophic bacteria in synthesizing PHBV, the present invention proposes an innovative PHBV copolymer production strategy: using a mixed community of methanotrophic bacteria and heterotrophic bacteria to synthesize PHBV through a combined fermentation process of methane and organic co-substrates. For the synthesis of PHBV, the key is to introduce 3-HV monomers to improve the brittleness of PHB. In the mixed fermentation system, methanotrophic bacteria have the ability to synthesize PHBV using organic matter and intermediate metabolites, and heterotrophic bacteria also have the ability to synthesize 3-HV using specific co-substrates. Therefore, by regulating the fermentation conditions and the types and proportions of co-substrates, the microorganisms in the mixed system can be guided to collaboratively synthesize PHBV copolymers.

[0005] In the present invention, methane and organic matter have significant advantages as co-substrates. First, methane, a greenhouse gas, as the main carbon source greatly reduces production costs, promotes the recycling of resources, and helps achieve the goal of carbon neutrality; secondly, this method broadens the range of raw material selection, reduces dependence on specific resources, and enhances the flexibility and stability of the production process. Through reasonable regulation of fermentation conditions, the production efficiency of PHBV copolymers can be significantly improved, and product performance can be further optimized. In summary, the present invention not only improves the performance of PHBV copolymers through a mixed bacteria fermentation system, but also achieves a double leap in economic and environmental benefits through the low-cost co-substrate application of methane and organic matter, laying a solid foundation for the wide application of PHBV copolymers and the sustainable development of the entire field of biodegradable materials. Summary of the invention

[0006] The present invention provides a method for synthesizing PHBV copolymers using a methanotrophic bacteria-heterotrophic bacteria mixed flora and a co-substrate. Through specific acclimation conditions, co-substrate selection and concentration optimization, the obtained PHBV copolymer has good thermal properties and improves the flexibility and processing performance of the product.

[0007] The technical solution of the present invention:

[0008] A method for preparing a PHBV copolymer produced by a mixed flora of methanotrophic bacteria and heterotrophic bacteria, comprising the following steps:

[0009] (1) Obtaining a mixed bacterial community of methanotrophs and heterotrophs

[0010] The mixed flora of methanotrophs and heterotrophs is derived from activated sludge rich in microorganisms, which is obtained after an enrichment and acclimatization process under fully aerobic conditions with methane as the only carbon source;

[0011] (2) Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymers using mixed bacterial communities

[0012] a) Inoculation and fermentation: The mixed bacterial flora is inoculated into a container containing a suitable culture medium for fermentation. The composition and content of the suitable culture medium are: Na 2 HPO 4 0.28g / L,KH 2 PO 4 0.27g / L,CaCl 2 0.2g / L,MgSO 4 7H 2 O 1g / L,CuSO 4 ·5H 2 O 1.25mg / L and trace element storage solution 1mL / L. Trace element storage solution components and content: FeSO 4500mg / L,NaMO 4 260mg / L,MnCl2·4H 2 O 20mg / L, 2Na-EDTA 250mg / L, H 3 BO 3 15mg / L,.NiCl 2 6H 2 O 10mg / L,Fe-EDTA 380mg / L,ZnSO 4 7H 2 O 400mg / L.

[0013] b) Fermentation condition control: During the fermentation period, the culture medium needs to be replaced regularly to ensure the freshness of the culture medium components. The pH value of the mixed bacterial flora culture process is monitored to 5.5-6.5. The fermentation culture temperature is 28-30°C, and a shaker, stirring system or ventilation system is set up to promote gas-liquid transfer and promote bacterial flora growth and metabolism.

[0014] c) Carbon source management: The carbon source in the fermentation process includes methane and at least one organic matter as a co-substrate. The volume ratio of methane gas to oxygen in the fermentation system is 1:1, and fresh gas needs to be added regularly to ensure sufficient carbon source and oxygen; the organic matter includes but is not limited to common carbon sources (glucose, lactose, etc.), VFAs carbon sources (acetic acid, propionic acid, etc.), and organic matter related to metabolism (malic acid, citric acid, etc.). The concentration of organic matter in the fermentation system is controlled at 0-0.8mmol / L to regulate the synthesis of PHBV copolymers;

[0015] (3) Extraction and performance analysis of PHBV copolymers from bacteria

[0016] The method for extracting PHBV from bacteria comprises the following steps: collecting the bacteria by centrifugation after fermentation, freeze-drying, dissolving the PHBV in the bacteria by using chloroform and sodium hypochlorite, filtering to remove insoluble matter, precipitating the PHBV by using cold ethanol, and finally drying by nitrogen blowing to obtain the PHBV copolymer. The method for characterizing the PHBV copolymer includes but is not limited to FT-IR spectroscopy, differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA).

[0017] The beneficial effects of the present invention are as follows: the maximum content of synthesized PHBV accounts for 42.8% of the dry weight of the mixed bacteria by introducing a co-substrate, mixed bacterial fermentation, and optimizing the fermentation conditions of the mixed bacterial system, the content of 3-HV monomer in the synthesized PHBV is increased, and the melting temperature and crystallinity are good, thereby achieving the effect of improving its flexibility and processing performance, and having good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1This is the FT-IR spectrum of PHA synthesized by mixed bacteria under methane + propionic acid conditions.

[0019] Figure 2 This is the FT-IR spectrum of PHA synthesized by the mixed bacterial community under methane + citric acid conditions. DETAILED DESCRIPTION

[0020] The specific implementation of the present invention is further described below in conjunction with the accompanying drawings and technical solutions.

[0021] Example 1

[0022] Enrichment and acclimatization of mixed bacterial communities of methanotrophs and heterotrophs and identification of community structure:

[0023] The activated sludge was collected from the secondary sedimentation tank of the Lingshui River Sewage Treatment Plant in Dalian City, Liaoning Province. The activated sludge was used as the domesticated seed sludge, and methane was used as the only carbon source. The sludge was enriched and domesticated under sufficient aerobic conditions.

[0024] After the acclimation, the methanotrophic-heterotrophic microbial community of the enriched bacterial community was analyzed. In the obtained mixed bacterial community, the methanotrophs were composed of Methylophilus, Methylosinus, Methylocella, etc., and the heterotrophic bacteria were composed of Mycobacterium, Thiomonas, etc.

[0025] Example 2

[0026] PHBV is synthesized by fermentation of methane and co-substrate using a mixed community of methanotrophs and heterotrophs:

[0027] The mixed flora of methane oxidizing bacteria and heterotrophic bacteria was inoculated into the liquid culture medium for fermentation, and a mineral salt culture medium without nitrogen was prepared, and its composition and content were as follows: Na 2 HPO 4 0.28g / L,KH 2 PO 4 0.27g / L,CaCl 2 0.2g / L,MgSO 4 7H 2 O 1g / L,CuSO 4 ·5H 2 O 1.25mg / L and trace element storage solution 1mL / L. Trace element storage solution components and content: FeSO 4 500mg / L,NaMO 4 260mg / L,MnCl2·4H 2 O 20mg / L, 2Na-EDTA 250mg / L, H 3 BO 315mg / L,.NiCl 2 6H 2 O 10mg / L,Fe-EDTA 380mg / L,ZnSO 4 7H 2 O 400mg / L.

[0028] The fermentation experiment was carried out in 125 mL serum bottles, each containing 10 mL of liquid. The fermentation time was set at 48 to 72 hours to ensure sufficient microbial growth and metabolism.

[0029] During the entire fermentation process, we strictly control the pH value of the fermentation broth to maintain it in the neutral or weakly acidic range (5.5-6.5) to optimize the activity of the microorganisms. At the same time, the serum bottle is placed in a shaker at a speed of 150r / min and a temperature of 30°C to promote full contact and reaction between the microorganisms and the substrate.

[0030] In order to explore the effect of co-substrate on the synthesis of PHBV copolymers, we controlled the methane-oxygen ratio to 1:1 during the fermentation process, filled it into the serum bottle, and added different concentrations of propionic acid and citric acid as co-substrates using a disposable injection needle. The changes in the monomer ratio and content of PHBV copolymers during the fermentation process were monitored by gas chromatography. The results showed that after the addition of propionic acid or citric acid, the PHB synthesis content increased significantly and the synthesis of PHV was successfully monitored, and the PHV content increased significantly with the increase in substrate concentration (Table 1).

[0031] Table 1 Synthesis of PHBV by mixed bacterial flora at different propionic acid and citric acid concentrations

[0032]

[0033]

[0034] Example 3

[0035] Characterization of the structure and thermal properties of synthesized PHBV:

[0036] After the fermentation, the cells were collected by centrifugation, and 1 g of freeze-dried cells containing PHBV were taken to synthesize and extract PHBV under appropriate conditions to obtain PHBV copolymers. The structure and thermal properties of PHA were analyzed by FT-IR spectroscopy, DSC and TGA.

[0037] The suitable extraction conditions are as follows: take 1g of freeze-dried PHBV-containing bacteria, add 50mL of chloroform and 50mL of 5% sodium hypochlorite aqueous solution, shake for 60-90min, stand for stratification, take the organic phase containing PHA and other fat-soluble substances, and filter it with a 0.22μm polytetrafluoroethylene membrane, then transfer the chloroform solution to a 1000mL separatory funnel, add five times the volume of cold ethanol while stirring, and precipitate PHA from the organic phase. Take the solution containing the precipitate, place it in a glass tube at 4°C, and after about 24h, suck out the upper organic solvent, dry it with nitrogen blowing, dissolve the dried powder again with 2mL of chloroform, filter it with a 0.22μm polytetrafluoroethylene membrane, and then dry it again with nitrogen blowing to obtain a PHBV copolymer.

[0038] The molecular structure of the prepared PHBV copolymer was tested and analyzed by Fourier transform infrared spectroscopy (FT-IR). The FT-IR spectrum showed that the PHA ( Figure 1 , Figure 2 ) is very similar to that of the PHBV standard, and the attribution of the sample's characteristic peaks is consistent with that of the standard, which indicates that the PHA film sample prepared in this experiment is indeed a PHBV copolymer, rather than a mixture of PHB and PHV.

[0039] The thermal stability of the prepared material was analyzed using a thermogravimetric analyzer. By measuring its thermal decomposition temperature, we obtained the TGA curve and DTG curve of PHA synthesized by the mixed bacterial community under two co-substrate conditions. Curve data analysis shows (as shown in Table 2) that the thermal decomposition temperature of the synthesized PHA is higher than the initial thermal decomposition temperature of the PHB standard (237°C). This result shows that the addition of HV components helps to improve the heat resistance and thermal stability of PHA and broaden its application scenarios.

[0040] Table 2

[0041]

[0042]

[0043] Note: Onset: The temperature at which the sample begins to have a significant thermal effect; Peak: The temperature at which the sample thermal effect reaches its maximum value; Endset: The temperature at which the sample thermal effect ends significantly.

[0044] in conclusion:

[0045] The present invention proposes a method for synthesizing PHBV copolymers by co-substrate fermentation using a mixed flora of methanotrophic bacteria and heterotrophic bacteria. This method not only significantly reduces production costs and increases yields, but is also easy to operate, bringing a new solution to the field of biodegradable plastics. In addition, the PHBV copolymer synthesized by this method has significantly improved performance compared to PHB synthesized by traditional methanotrophic bacteria using methane, providing strong support for performance optimization in practical applications. In general, the present invention has opened up a new path for the industrial production of PHBV copolymers and has made an important contribution to promoting the development of biodegradable plastics.

Claims

1. A method for preparing PHBV copolymers by using a mixed flora of methanotrophic bacteria and heterotrophic bacteria, characterized in that: Here are the steps: (1) Obtaining a mixed bacterial community of methanotrophs and heterotrophs The mixed flora of methanotrophs and heterotrophs is derived from activated sludge rich in microorganisms, which is obtained after an enrichment and acclimatization process under fully aerobic conditions with methane as the only carbon source; (2) Synthesis of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) copolymers using a mixed community of methanotrophs and heterotrophs a) Inoculation and fermentation: inoculating a mixed bacterial community of methanotrophic bacteria and heterotrophic bacteria into a container containing a suitable culture medium for fermentation; b) Fermentation condition control: During the fermentation period, the culture medium needs to be replaced regularly to ensure the freshness of the culture medium components; the pH value of the mixed bacterial flora of methanotrophs and heterotrophs is monitored to 5.5-6.5, the fermentation temperature is 28-30°C, and a shaker, stirring system or ventilation system is set to promote gas-liquid transfer and promote bacterial growth and metabolism; c) Carbon source management: The carbon source in the fermentation process includes methane and at least one organic matter as a co-substrate; the volume ratio of methane gas to oxygen in the fermentation system is 1:1, and fresh gas needs to be added regularly to ensure sufficient carbon source and oxygen; the organic matter concentration in the fermentation system is controlled to be 0-0.8 mmol / L to regulate the synthesis of PHBV copolymer; (3) Extraction and performance analysis of PHBV copolymers from bacteria The method of extracting PHBV from bacteria is as follows: after the fermentation is completed, the bacteria are collected by centrifugation, freeze-dried, and the PHBV in the bacteria is dissolved by chloroform and sodium hypochlorite. After filtering to remove the insoluble matter, the PHBV is precipitated by cold ethanol, and finally dried by nitrogen blowing to obtain the PHBV copolymer.

2. The method for preparing PHBV copolymer by using a mixed flora of methanotrophic bacteria and heterotrophic bacteria according to claim 1, characterized in that: The composition and content of the suitable culture medium are: Na2HPO4 0.28g / L, KH2PO4 0.27g / L, CaCl2 0.2g / L, MgSO4·7H2O 1g / L, CuSO4·5H2O 1.25mg / L and trace element storage solution 1mL / L; the components and contents of the trace element storage solution are: FeSO4 500mg / L, NaMO4 260mg / L, MnCl2·4H2O 20mg / L, 2Na-EDTA 250mg / L, H3BO3 15mg / L, .NiCl2·6H2O 10mg / L, Fe-EDTA 380mg / L, ZnSO4·7H2O 400mg / L.

3. The method for preparing PHBV copolymer by using a mixed flora of methanotrophic bacteria and heterotrophic bacteria according to claim 1, characterized in that: The organic matter includes carbon sources and organic matter related to metabolism.

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