A method for cultivating autotrophic microorganisms for producing poly-beta-hydroxybutyrate

Abstract

This invention relates to the field of microbial culture technology, and in particular to a method for producing polymerases. β A method for cultivating autotrophic microorganisms for hydroxybutyrate. This invention utilizes carbon dioxide, which is widely available and inexpensive to obtain, as a microbial agent for the production of hydroxybutyrate. β‑ Hydroxybutyrate (Poly- β The carbon source of hydroxybutyrate (PHB) is obtained through a two-stage culture method of "nitrogen-sufficient" followed by nitrogen-deficient culture. Hydroxybutyrate bacteria (especially...) are first introduced into the culture. Rhodoblastus Biomass enrichment is carried out using sp. TH20, followed by intracellular accumulation of biopolyester PHB. The resulting biopolyester has good biodegradability, which can not only reduce greenhouse gas emissions, but also produce high-value-added bioplastic products, thus achieving both ecological and economic benefits.

Description

Technical Field

[0001] This invention relates to the field of microbial culture technology, and in particular to a method for producing polymerases. β Methods for culturing autotrophic microorganisms containing hydroxybutyrate. Background Technology

[0002] Gather β 2-hydroxybutyrate (Poly- β 2-hydroxybutyrate (PHB)-based bioplastics are ideal alternatives to petroleum-based chemical plastics. PHB is an intracellular polyester used by microorganisms as a carbon source and energy storage. Compared to traditional petroleum-based chemical plastics, PHB possesses superior properties such as high crystallinity, biocompatibility, biodegradability, and low toxicity, demonstrating great potential in bioplastic applications.

[0003] Bacteria are important producers of biodegradable biopolyesters (PHB). Under specific growth conditions, most bacteria can accumulate 40-50% of their cell volume in PHB. Currently, major research on PHB biosynthesis focuses on the construction of genetically engineered fermentation bacteria, which suffers from problems such as complex genetic modification processes, demanding fermentation conditions, and high energy consumption. Therefore, developing an economical and efficient autotrophic microbial method that utilizes widely available and low-cost carbon dioxide as a carbon source for PHB production can both reduce greenhouse gas emissions and produce high-value-added bioplastic products, achieving both ecological and economic benefits. Summary of the Invention

[0004] To address the above problems, the present invention provides a method for producing polymers. β A method for cultivating autotrophic microorganisms of hydroxybutyrate. The cultivation method provided by this invention can both reduce greenhouse gas emissions and produce high-value-added bioplastic products, thus achieving both ecological and economic benefits.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides a method for producing poly β The method for culturing autotrophic microorganisms containing hydroxybutyrate includes the following steps:

[0007] Hydroxyhydric bacteria seed culture was inoculated into a nitrogen-containing culture medium for enrichment culture to obtain enriched hydroxide bacteria; the hydroxide bacteria include Rhodoblastus sp. TH20;

[0008] The enriched hydroxide bacteria were inoculated into a nitrogen-free culture medium for synthesis culture to obtain a product containing polymers. βThe culture medium for hydroxybutyrate; the conditions for the synthesis culture include: a temperature of 30°C, an initial pH of 7.0, and a rotation speed of 200 rpm.

[0009] Preferably, the nitrogen-containing culture medium comprises the following components at the following concentrations: KNO3 0.58 g / L, KH2PO4 0.5 g / L, NaHCO3 0.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.003 g / L, ferrous ammonium sulfate 0.02 g / L, and a trace element solution of 0.5 mL / L; wherein the trace element solution comprises the following components at the following concentrations: CoCl2·6H2O 0.119 g / L, NiCl2·6H2O 0.118 g / L, and CuSO4·5H2O 0.156 g / L.

[0010] Preferably, a gas mixture is introduced during the enrichment culture process; the flow rate of the gas mixture is 200 mL / min, and the aeration time is 2 min; the gas mixture is composed of the following gases by volume percentage: H2 70%, O2 20%, and CO2 10%.

[0011] Preferably, the nitrogen-free culture medium comprises the following components at the following concentrations: KH₂PO₄ 0.5 g / L, NaHCO₃ 0.5 g / L, MgSO₄·7H₂O 0.2 g / L, CaCl₂ 0.003 g / L, ferrous ammonium sulfate 0.02 g / L, and a trace element solution 0.5 mL / L; the trace element solution comprises the following components at the following concentrations: CoCl₂·6H₂O 0.119 g / L, NiCl₂·6H₂O 0.118 g / L, and CuSO₄·5H₂O 0.156 g / L.

[0012] Preferably, a gas mixture is introduced during the synthesis culture process; the flow rate of the gas mixture is 200 mL / min, and the aeration time is 2 min; the gas mixture is composed of the following gases by volume percentage: H2 70%, O2 20%, and CO2 10%.

[0013] Preferably, the volume ratio of the hydrogen hydroxide bacterial seed solution to the nitrogen-containing culture medium is 5:95; the OD600 of the hydrogen hydroxide bacterial seed solution is 1.20.

[0014] Preferably, the method for preparing the hydroxide bacteria seed solution includes: inoculating hydroxide bacteria into a selective culture medium for activation culture to obtain the hydroxide bacteria seed solution; the selective culture medium comprises the following components at the following concentrations: (NH4)2SO4 0.47 g, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.003 g / L, ferrous ammonium sulfate 0.02 g / L and trace element solution 0.5 mL / L; the trace element solution comprises the following components at the following concentrations: CoCl2·6H2O 0.119 g / L, NiCl2·6H2O 0.118 g / L and CuSO4·5H2O 0.156 g / L.

[0015] Preferably, the activation culture conditions include: a temperature of 25°C, a rotation speed of 200 rpm, gas introduction every 72 h, a gas flow rate of 200 mL / min, and an aeration time of 2 min; the introduced gas consists of the following gas components by volume percentage: H2 85%, O2 5%, and CO2 10%.

[0016] Preferably, the pH value of the selective culture medium is 7.0.

[0017] Preferably, the OD600 of the enriched hydroxide bacteria is 0.80~1.20.

[0018] Beneficial effects:

[0019] This invention utilizes carbon dioxide, which is widely available and inexpensive to obtain, as the carbon source for the production of PHB, through a two-stage cultivation method of "nitrogen-sufficient" and "nitrogen-deficient" conditions. Figure 1 First, add hydroxide bacteria (especially) Rhodoblastus Biomass enrichment was carried out in a nitrogen-rich culture medium by sp. TH20, and then intracellular biopolyester PHB was accumulated by hydroxyl bacteria in a nitrogen-deficient culture medium. The resulting biopolyester has good biodegradability, which can not only reduce greenhouse gas emissions, but also produce high-value-added bioplastic products, thus achieving both ecological and economic benefits. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0021] Figure 1 This is a flowchart of the two-stage culture method of "nitrogen-sufficient" culture used in Example 1;

[0022] Figure 2 This is a mass spectrometry result of the PHB produced in Example 1;

[0023] Figure 3 This is a transmission electron microscope image of PHB produced by strain TH20 in Example 1. Detailed Implementation

[0024] This invention provides a method for producing poly β The method for culturing autotrophic microorganisms containing hydroxybutyrate includes the following steps:

[0025] Hydroxyhydric bacteria seed culture was inoculated into a nitrogen-containing culture medium for enrichment culture to obtain enriched hydroxide bacteria; the hydroxide bacteria include Rhodoblastus sp. TH20;

[0026] The enriched hydroxide bacteria were inoculated into a nitrogen-free culture medium for synthesis culture to obtain a product containing polymers. β The culture medium for hydroxybutyrate; the conditions for the synthesis culture include: a temperature of 30°C, an initial pH of 7.0, and a rotation speed of 200 rpm.

[0027] The present invention Rhodoblastus sp. TH20 (GenBank No. MK968713.1, abbreviated as TH20) is deposited at the School of Environment and Energy, Peking University Shenzhen Graduate School, and is publicly available from the literature [Tao Huchun, Xie Yong, Zhang Lijuan, et al. Growth conditions of a hydrolytic bacterium and its utilization of different nitrogen sources [J]. Journal of Peking University: Natural Science Edition, 2021, 57(4):9. DOI:10.13209 / j.0479-8023.2021.059.].

[0028] In one embodiment, the method for preparing the hydroxide-oxidizing bacteria seed solution includes: inoculating hydroxide-oxidizing bacteria into a selective culture medium for activation culture to obtain the hydroxide-oxidizing bacteria seed solution; the selective culture medium comprises the following components at concentrations: (NH4)2SO4 0.47 g, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.003 g / L, ferrous ammonium sulfate 0.02 g / L, and trace element solution 0.5 mL / L; the trace element solution comprises the following components at concentrations: CoCl2·6H2O 0.119 g / L, NiCl2·6H2O 0.118 g / L, and CuSO4·5H2O 0.156 g / L. In one embodiment, the pH value of the selective culture medium is 7.0. As one implementation method, the activation culture conditions include: a temperature of 25°C, a rotation speed of 200 rpm, gas introduction every 72 h, a gas flow rate of 200 mL / min, and an aeration time of 2 min; the introduced gas consists of the following gas composition by volume percentage: H2 85%, O2 5%, and CO2 10%.

[0029] In one embodiment, the volume ratio of the hydroxide-containing bacterial seed solution to the nitrogen-containing culture medium is 5:95; the OD600 of the hydroxide-containing bacterial seed solution is 1.20. This invention employs a specific inoculation ratio that ensures an initial quantity of strains without causing resource competition due to excessive numbers.

[0030] In one embodiment, the nitrogen-containing culture medium comprises the following components at the following concentrations: KNO3 0.58 g / L, KH2PO4 0.5 g / L, NaHCO3 0.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.003 g / L, ferrous ammonium sulfate 0.02 g / L, and a trace element solution of 0.5 mL / L; the trace element solution comprises the following components at the following concentrations: CoCl2·6H2O 0.119 g / L, NiCl2·6H2O 0.118 g / L, and CuSO4·5H2O 0.156 g / L. In another embodiment, a gas mixture is introduced during the enrichment culture process; the flow rate of the gas mixture is 200 mL / min, and the aeration time is 2 min; the gas mixture consists of the following gas components by volume percentage: H2 70%, O2 20%, and CO2 10%.

[0031] The enrichment culture described in this invention is a biomass enrichment, that is, the growth of hydroxide bacteria in a "nitrogen-sufficient" culture medium, which enables the cell number to increase rapidly.

[0032] In one embodiment, the nitrogen-free culture medium comprises the following components at the following concentrations: KH₂PO₄ 0.5 g / L, NaHCO₃ 0.5 g / L, MgSO₄·7H₂O 0.2 g / L, CaCl₂ 0.003 g / L, ferrous ammonium sulfate 0.02 g / L, and a trace element solution of 0.5 mL / L; the trace element solution comprises the following components at the following concentrations: CoCl₂·6H₂O 0.119 g / L, NiCl₂·6H₂O 0.118 g / L, and CuSO₄·5H₂O 0.156 g / L. In another embodiment, a gas mixture is introduced during the synthesis culture process; the flow rate of the gas mixture is 200 mL / min, and the aeration time is 2 min; the gas mixture consists of the following gas components by volume percentage: H₂ 70%, O₂ 20%, and CO₂ 10%. In one embodiment, the OD600 of the enriched hydroxide bacteria is 0.80~1.20.

[0033] The synthetic culture described in this invention is the biopolyester PHB accumulation stage, in which hydroxide bacteria grow in a nitrogen-deficient culture medium, causing the intracellular PHB content to accumulate rapidly.

[0034] The method provided by this invention enables the biopolyester PHB produced in TH20 cells to reach its maximum quantity on days 6-8. The resulting biopolyester has good biodegradability and is suitable for the production of high-molecular-weight biodegradable plastics. This method can achieve both greenhouse gas emission reduction and the production of high-value-added bioplastic products, thus achieving both ecological and economic benefits.

[0035] To further illustrate the present invention, the following description, in conjunction with the accompanying drawings and embodiments, describes a method for producing polymers according to the present invention. β The methods for culturing autotrophic microorganisms of hydroxybutyrate are described in detail, but they should not be construed as limiting the scope of protection of this invention.

[0036] Example 1

[0037] A method for cultivating autotrophic microbial cells to produce biodegradable biopolyester (PHB) comprises the following steps:

[0038] 1) First stage of culture: Measure 66.5 mL of mineral culture medium solution into a 250 mL serum bottle, sterilize at 121 ℃ for 20 min, and cool to room temperature; then, inoculate TH20 bacterial suspension into the mineral culture medium at an inoculation ratio of 5% (v / v) and incubate in a closed system. The entire operation is carried out in a sterile laminar flow hood. After inoculation, a gas mixture is introduced at a flow rate of 200 mL / min for 2 min. The gas mixture consists of the following gas components by volume percentage: H2 70%, O2 20%, and CO2 10%. Place the serum bottle in an air bath shaker and shake.

[0039] The TH20 bacterial suspension is prepared as follows: the TH20 strain is inoculated into a selective medium and activated until OD600=1.20 to obtain the TH20 bacterial suspension; the activation conditions are: temperature 25℃, rotation speed 200 rpm, H2:O2:CO2 gas mixture (volume ratio 85:5:10) is introduced every 72 h, gas flow rate is 200 mL / min, and aeration time is 2 min;

[0040] The selective medium contains the following components at the following concentrations: (NH4)2SO4 0.47 g, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.003 g / L, ferrous ammonium sulfate 0.02 g / L, and trace element solution 0.5 mL / L; the pH of the selective medium is 7.0.

[0041] The mineral culture medium solution contains the following components at the following concentrations: KNO3 0.58 g / L, KH2PO4 0.5 g / L, NaHCO3 0.5 g / L, Mg / LSO4·7H2O 0.2 g / L, CaCl2 0.003 g / L, ferrous ammonium sulfate 0.02 g / L, and trace element solution 0.5 mL / L; the pH of the mineral culture medium solution is 7.0; the ferrous ammonium sulfate is sterilized separately and then added to the culture medium at room temperature through a 0.22 μm membrane filter.

[0042] The trace element solution contains the following components at the following concentrations: CoCl2·6H2O 0.119 g / L, NiCl2·6H2O 0.118 g / L and CuSO4·5H2O 0.156 g / L;

[0043] 2) Second stage culture: The TH20 bacterial suspension (OD600=0.80) obtained in the first stage was centrifuged at 1000×g, the supernatant was discarded, and the precipitate was transferred to a nitrogen-deficient mineral culture medium for further culture. After inoculation, a gas mixture was introduced at a flow rate of 200 mL / min for 2 min. The gas mixture consisted of the following gas components by volume percentage: H2 70%, O2 20%, and CO2 10%. The serum bottle was placed in an air bath shaker and shaken. The entire operation was performed in a sterile laminar flow hood. The only difference between the nitrogen-deficient mineral culture medium and the mineral culture medium was that the latter did not contain KNO3. The culture conditions for the second stage were: a culture temperature of 30 ℃, an initial pH of 7.0, and a rotation speed of 200 rpm.

[0044] Example 2

[0045] The flowchart compares the dry weight of TH20 cells, PHB yield, and PHB production rate under different culture conditions in the second stage of culture (Table 1). Figure 1 .

[0046] Table 1 Different culture conditions for the second stage of cultivation

[0047]

[0048] On days 1, 2, 3, 4, 5, 6, 7 and 8, 50 mL of culture medium was collected to determine the dry weight and PHB content of TH20 cells and to determine the optimal growth conditions for TH20.

[0049] The extraction and determination methods for PHB are as follows:

[0050] 1. Centrifuge 50 mL of culture medium at 10,000 rpm / min for 15 min at room temperature, collect the bacterial cells, and then wash the bacterial cells three times with sterile, enzyme-free water.

[0051] 2. Place the collected bacterial cells in a freeze dryer and freeze-dry at -80℃ for 48 h. After that, take them out, accurately weigh the dry weight of the bacterial cells, and place them in a pressure-resistant reaction tube with a screw cap for reaction.

[0052] 3. Add 2 mL of chloroform, 2 mL of 7% methanol-sulfuric acid solution, and 200 μL of 3% methanol-benzoic acid solution to the reaction tube in sequence. After sealing, place the tube in an oven at 105℃ and react for 8 h.

[0053] 4. After the reaction is complete, remove the sample and allow it to cool fully to room temperature or lower. Add 2 mL of deionized water to the reaction solution and shake thoroughly. This process utilizes the difference in solubility of methanol in chloroform and water to achieve extraction. Methanol has low solubility in chloroform but high solubility in water. After shaking and extraction, the mixture will separate into two layers: an upper aqueous phase and a lower chloroform layer.

[0054] 5. Place the chloroform layer turbidity in a centrifuge tube, add an appropriate amount of anhydrous sodium sulfate desiccant, and shake thoroughly to allow the anhydrous sodium sulfate to fully absorb water. After centrifugation at 4℃ and 5000 r / min for 5 min, take 1 mL of the supernatant using a 1 mL syringe, filter the PHB through a 0.25 μm organic filter membrane, and then perform quantitative analysis by gas chromatography-mass spectrometry (GC-MS). The method is described in the reference [Yves Comeau, Kenneth J. Hall, William K. Oldham. Determination of Poly-3-Hydroxybutyrate and Poly-3-Hydroxyvalerate in Activated Sludge by Gas-Liquid Chromatography. Applied and Environmental Microbiology, 1988, 2325-2327.]. Before injection, PHB was derivatized using an esterification reaction to convert it into methyl ester compounds. Benzoic acid was used as an internal standard, and ion fragments with mass-to-charge ratios (m / z) of 43.1 and 74.0 were used to produce monomers for TH20 cells. β Characteristic ion fragments of hydroxybutyric acid (Hydroxybutyric acid) Figure 2 ).

[0055] The measurement results are shown in Tables 2 and 3.

[0056] Table 2. Cell dry weight, PHB yield, and PHB production rate at different time points under different culture conditions (Groups 1-6)

[0057]

[0058] Table 3. Cell dry weight, PHB yield, and PHB production rate at different time points under different culture conditions (groups 7-12)

[0059]

[0060] Note: PHB yield = PHB production / cell dry weight × 100%.

[0061] The results showed that the culture conditions in Example 1 were the optimal culture conditions. The maximum cell dry weight of strain TH20 was 686 mg / L (group 11), the maximum intracellular PHB content was 230.8 mg / L (group 1), and the maximum PHB yield was 40.07 wt.% (group 1).

[0062] In the nitrogen-sufficient / nitrogen-deficient two-stage culture system provided by this invention, biomass enrichment is achieved in the first stage, during which bacterial cells do not accumulate PHB; in the second stage, TH20 cells begin to produce PHB, with the maximum accumulation of PHB reaching its peak on days 6-8. The biopolyester PHB particles mainly exist in aggregated form within TH20 cells, accounting for 70%-80% of the slice area. Figure 3 ).

[0063] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for producing polymer β A method for culturing autotrophic microorganisms containing hydroxybutyrate, characterized in that, Includes the following steps: Hydroxyhydric bacteria seed culture was inoculated into a nitrogen-containing culture medium for enrichment culture to obtain enriched hydroxide bacteria; the hydroxide bacteria are... Rhodoblastus sp. TH20; the volume ratio of the hydroxide-containing bacterial seed culture to the nitrogen-containing culture medium is 5:95; the OD of the hydroxide-containing bacterial seed culture... 600 =1.20; The nitrogen-containing culture medium comprises the following components at the following concentrations: KNO3 0.58 g / L, KH2PO4 0.5 g / L, NaHCO3 0.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.003 g / L, ferrous ammonium sulfate 0.02 g / L, and trace element solution 0.5 mL / L; The pH of the nitrogen-containing culture medium is 7.0; The trace element solution comprises the following components at the following concentrations: CoCl2·6H2O 0.119 g / L, NiCl2·6H2O 0.118 g / L, and CuSO4·5H2O 0.156 g / L; A gas mixture is introduced during the enrichment culture process; The flow rate of the gas mixture is 200 mL / min, and the aeration time is 2 min; The gas mixture consists of the following gases by volume percentage: H2 70%, O2 20%, and CO2 10%; the OD of the enriched cultured hydroxide bacteria 600 =0.80~1.20; The enriched hydroxide bacteria were inoculated into a nitrogen-free culture medium for synthesis culture to obtain a product containing polymers. β The culture medium for hydroxybutyrate (H2PO4) was prepared under the following conditions: a temperature of 30°C, an initial pH of 7.0, and a rotation speed of 200 rpm. The nitrogen-free culture medium contained the following components at the following concentrations: KH2PO4 0.5 g / L, NaHCO3 0.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.003 g / L, ferrous ammonium sulfate 0.02 g / L, and a trace element solution of 0.5 mL / L. The trace element solution contained the following components at the following concentrations: CoCl2·6H2O 0.119 g / L, NiCl2·6H2O 0.118 g / L, and CuSO4·5H2O 0.156 g / L. A gas mixture was introduced during the synthesis culture process at a flow rate of 200 mL / min for 2 min. The gas mixture consisted of the following gas components by volume percentage: H2 70%, O2... 20% and CO2 10%.

2. The cultivation method according to claim 1, characterized in that, The method for preparing the hydrogen hydroxide bacteria seed solution includes: inoculating hydrogen hydroxide bacteria into a selective culture medium for activation culture to obtain the hydrogen hydroxide bacteria seed solution; the selective culture medium comprises the following components at the following concentrations: (NH4)2SO4 0.47 g, KH2PO4 0.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl2 0.003 g / L, ferrous ammonium sulfate 0.02 g / L and trace element solution 0.5 mL / L, and the pH value of the selective culture medium is 7.0; the trace element solution comprises the following components at the following concentrations: CoCl2·6H2O 0.119 g / L, NiCl2·6H2O 0.118 g / L and CuSO4·5H2O 0.156 g / L.

3. The cultivation method according to claim 2, characterized in that, The activation culture conditions include: a temperature of 25°C, a rotation speed of 200 rpm, gas introduction every 72 h, a gas flow rate of 200 mL / min, and an aeration time of 2 min; the introduced gas consists of the following volume percentages: H2 85%, O2 5%, and CO2 10%.

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