Culture method of autotrophic microorganism for producing poly (beta-hydroxybutyrate)

Abstract

The invention relates to the technical field of microorganism culture, in particular to a culture method of autotrophic microorganisms for producing poly (beta-hydroxybutyrate). According to the method, carbon dioxide which is wide in source and low in obtaining cost is used as a carbon source for producing poly-beta-hydroxybutyrate (PHB), through a two-stage culture method of nitrogen sufficiency and nitrogen deficiency, firstly, hydrogen oxidizing bacteria (especially Rhodoblastus sp. TH20) are subjected to biomass enrichment, then intracellular biopolyester PHB accumulation is carried out, the obtained biopolyester has good biodegradability, and the biopolyester can be used for producing the poly-beta-hydroxybutyrate (PHB). The greenhouse gas emission reduction can be realized, the bioplastic product with high additional value can be produced, and the method has both ecological and economic benefits.

Description

Technical Field

[0001] The present invention relates to the field of microbial culture technology, in particular to a method for producing a polymer β Method for cultivating autotrophic microorganisms producing β-hydroxybutyrate. Background Art

[0002] Gather β -Hydroxybutyrate (Poly- β Bioplastics based on poly(hexyl-hydroxybutyrate) (PHB) are promising alternatives to petroleum-based chemical plastics. PHB is an intracellular polyester that is used by microorganisms as a carbon source and energy storage. Compared to traditional petroleum-based chemical plastics, PHB offers excellent properties such as high crystallinity, biocompatibility, biodegradability, and low toxicity, and holds great potential for bioplastic applications.

[0003] Bacteria are important producers of biodegradable polyester (PHB). Under specific growth conditions, most bacteria can accumulate 40–50% of their cell volume in PHB. Currently, research on PHB biosynthesis focuses on the construction of genetically engineered fermentation bacteria, which present challenges such as complex genetic modification, demanding fermentation conditions, and high energy consumption. Therefore, developing cost-effective, autotrophic microbial methods that utilize readily available and inexpensive carbon dioxide as a carbon source for PHB production could achieve both greenhouse gas emissions reductions and the production of high-value-added bioplastic products, achieving both ecological and economic benefits. Summary of the Invention

[0004] In order to solve the above problems, the present invention provides a method for producing a β The present invention provides a method for cultivating autotrophic microorganisms that produce -hydroxybutyrate. The cultivation method provided by the present invention can not only reduce greenhouse gas emissions, but also produce high-value-added bioplastic products, achieving both ecological and economic benefits.

[0005] In order to achieve the above object, the present invention provides the following technical solutions: The present invention provides a method for producing a β A method for cultivating an autotrophic microorganism that produces hydroxybutyrate, comprising the following steps: The hydrogen oxidizing bacteria seed liquid is inoculated into a culture medium containing nitrogen to carry out enrichment culture to obtain enriched hydrogen oxidizing bacteria; the hydrogen oxidizing bacteria include Rhodoblastus sp. TH20; The hydrogen oxidizing bacteria obtained by the enrichment culture are inoculated into a culture medium without nitrogen element for synthetic culture to obtain a polymer containing β -hydroxybutyrate culture medium; the synthetic culture conditions include: temperature of 30 ° C, initial pH of 7.0, and rotation speed of 200 rpm.

[0006] Preferably, the nitrogen-containing culture medium comprises components with 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 trace element solution comprises components with the following concentrations: CoCl2·6H2O 0.119 g / L, NiCl2·6H2O 0.118 g / L and CuSO4·5H2O 0.156 g / L.

[0007] Preferably, a gas mixture is introduced during the enrichment culture process; the flow rate of the gas mixture is 200 mL / min, and the ventilation time is 2 min; the gas mixture consists of the following volume percentages of gases: H2 70%, O2 20% and CO2 10%.

[0008] Preferably, the nitrogen-free culture medium comprises the following components at the following concentrations: 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 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.

[0009] Preferably, a gas mixture is introduced during the synthetic culture process; the flow rate of the gas mixture is 200 mL / min, and the ventilation time is 2 min; the gas mixture consists of the following volume percentages of gases: H2 70%, O2 20% and CO2 10%.

[0010] Preferably, the volume ratio of the hydrogen oxidizing bacteria seed solution to the culture medium containing nitrogen is 5:95; and the OD600 of the hydrogen oxidizing bacteria seed solution is 1.20.

[0011] Preferably, the preparation method of the hydrogen oxidizing bacteria seed solution comprises: inoculating hydrogen oxidizing bacteria into a selective culture medium for activation culture to obtain the hydrogen oxidizing bacteria seed solution; the selective culture medium comprises components with the following concentrations: (NH4)2SO40.47 g, KH2PO40.5 g / L, MgSO4·7H2O 0.2 g / L, CaCl20.003 g / L, ammonium ferrous sulfate 0.02 g / L and trace element solution 0.5 mL / L; the trace element solution comprises components with the following concentrations: CoCl2·6H2O0.119 g / L, NiCl2·6H2O 0.118 g / L and CuSO4·5H2O 0.156 g / L.

[0012] Preferably, the activation culture conditions include: temperature of 25°C, rotation speed of 200 rpm, gas introduction every 72 h, gas flow rate of 200 mL / min, and ventilation time of 2 min; the introduced gas is composed of the following volume percentages: H2 85%, O2 5% and CO2 10%.

[0013] Preferably, the pH value of the selective culture medium is 7.0.

[0014] Preferably, the OD600 of the hydrogen oxidizing bacteria in the enrichment culture is 0.80-1.20.

[0015] Beneficial effects: The present invention utilizes carbon dioxide, which is widely available and has low acquisition cost, as the carbon source for producing PHB, and adopts a two-stage culture method of "nitrogen sufficient-nitrogen deficient" ( Figure 1 ), first hydrogen oxidizing bacteria (especially Rhodoblastus sp. TH20) in a nitrogen-sufficient culture medium for biomass enrichment, and then hydrogen-oxidizing bacteria accumulate intracellular biopolyester PHB in a nitrogen-deficient culture medium. The resulting biopolyester has good biodegradability, which can not only achieve greenhouse gas emission reductions but also produce high-value-added bioplastic products, with both ecological and economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments are briefly introduced below.

[0017] Figure 1 Flow chart of the "nitrogen sufficient ‒ nitrogen deficient" two-stage culture method used in Example 1; Figure 2 This is a mass spectrometry image of PHB produced in Example 1; Figure 3 This is a transmission electron micrograph of PHB produced by the TH20 strain in Example 1. DETAILED DESCRIPTION

[0018] The present invention provides a method for producing a β A method for cultivating an autotrophic microorganism that produces hydroxybutyrate, comprising the following steps: The hydrogen oxidizing bacteria seed liquid is inoculated into a culture medium containing nitrogen to carry out enrichment culture to obtain enriched hydrogen oxidizing bacteria; the hydrogen oxidizing bacteria include Rhodoblastus sp. TH20; The hydrogen oxidizing bacteria obtained by the enrichment culture are inoculated into a culture medium without nitrogen element for synthetic culture to obtain a polymer containing β -hydroxybutyrate culture medium; the synthetic culture conditions include: temperature of 30 ° C, initial pH of 7.0, and rotation speed of 200 rpm.

[0019] The present invention Rhodoblastus sp. TH20 (GenBank No. MK968713.1, abbreviated as TH20) is deposited in the School of Environment and Energy, Shenzhen Graduate School, Peking University, and is publicly available from the literature [Tao Huchun, Xie Yong, Zhang Lijuan, et al. Study on the growth conditions of a hydrogen oxidizing 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.].

[0020] In one embodiment, the method for preparing the hydrogen oxidizing bacteria seed solution includes: inoculating hydrogen oxidizing bacteria into a selective culture medium for activation culture to obtain the hydrogen oxidizing bacteria seed solution; the selective culture medium includes 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 includes 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 one embodiment, the pH value of the selective culture medium is 7.0. As an embodiment, the activation culture conditions include: temperature of 25°C, rotation speed of 200 rpm, gas introduction every 72 hours, gas flow rate of 200 mL / min, and ventilation time of 2 minutes; the introduced gas is composed of the following volume percentages: H285%, O25% and CO210%.

[0021] In one embodiment, the volume ratio of the hydrogen oxidizing bacteria seed solution to the nitrogen-containing culture medium is 5:95; the OD600 of the hydrogen oxidizing bacteria seed solution is 1.20. The present invention adopts a specific inoculation ratio to ensure the initial number of strains while preventing resource competition caused by excessive number.

[0022] In one embodiment, the nitrogen-containing culture medium includes 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 a trace element solution 0.5 mL / L; the trace element solution includes 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 one embodiment, a gas mixture is introduced during the enrichment culture; the gas mixture has a flow rate of 200 mL / min and a ventilation duration of 2 minutes; the gas mixture is composed of the following gas percentages by volume: H2 70%, O2 20%, and CO2 10%.

[0023] The enrichment culture described in the present invention is biomass enrichment, that is, hydrogen oxidizing bacteria grow in a "nitrogen-sufficient" culture medium, so that the cell number can increase rapidly.

[0024] 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, Mg / LSO4·7H2O 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. In one embodiment, a gas mixture is introduced during the synthetic culture process; the gas mixture has a flow rate of 200 mL / min and a ventilation duration of 2 minutes; the gas mixture is composed of the following gas percentages by volume: H2 70%, O2 20%, and CO2 10%. As an embodiment, the OD600 of the hydrogen oxidizing bacteria in the enrichment culture is 0.80-1.20.

[0025] The synthetic culture of the present invention is the biopolyester PHB accumulation stage, that is, the hydrogen oxidizing bacteria grow in a "nitrogen-deficient" culture medium that does not contain nitrogen, so that the PHB content in the cells accumulates rapidly.

[0026] The method provided by the present invention can enable the biopolyester PHB produced in TH20 cells to reach a maximum amount on the 6th to 8th day. The obtained biopolyester has good biodegradability and is suitable for the production of high-molecular biodegradable plastics. It can not only achieve greenhouse gas emission reduction, but also produce high-value-added bioplastic products, with both ecological and economic benefits.

[0027] In order to further illustrate the present invention, the following is a method for producing a polymer provided by the present invention in conjunction with the accompanying drawings and embodiments. β The cultivation method of the autotrophic microorganism of β-hydroxybutyrate is described in detail, but they should not be understood as limiting the scope of protection of the present invention.

[0028] Example 1 A method for culturing autotrophic microbial cells for producing biodegradable biopolyester (PHB) comprises the following steps: 1) First stage of culture: 66.5 mL of mineral culture medium solution was placed in a 250 mL serum bottle, sterilized at 121°C for 20 minutes, and cooled to room temperature. Then, the TH20 bacterial solution was inoculated into the mineral culture medium at a 5% (v / v) inoculum ratio and cultured in a closed manner. The entire operation was performed in a sterile laminar flow hood. After inoculation, a gas mixture consisting of the following volume percentages: H2 70%, O2 20%, and CO2 10% was introduced at a flow rate of 200 mL / min for 2 minutes. The serum bottle was shaken in an air bath. The TH20 bacterial solution was prepared as follows: the TH20 strain was inoculated into a selective culture medium and activated and cultured until OD600 = 1.20 to obtain the TH20 bacterial solution; the activation culture conditions were: temperature of 25°C, rotation speed of 200 rpm, aeration of H2:O2:CO2 gas mixture (volume ratio of 85:5:10) every 72 hours, gas flow rate of 200 mL / min, and ventilation time of 2 minutes; The selective culture 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 value of the selective culture medium is 7.0; 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 value of the mineral culture medium solution is 7.0; the ferrous ammonium sulfate is sterilized separately and then filtered through a 0.22 μm membrane at room temperature before being added to the culture medium; 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; 2) Second stage of culture: The TH20 bacterial suspension (OD600 = 0.80) obtained from the first stage of enrichment was centrifuged at 1000 × g, the supernatant discarded, and the entire pellet transferred to a nitrogen-deficient mineral medium for culture. After inoculation, a gas mixture consisting of 70% H₂, 20% O₂, and 10% CO₂ (volume percentages) was introduced at a flow rate of 200 mL / min for 2 minutes. The serum bottle was shaken in an air bath, and the entire process was performed in a sterile laminar flow hood. The nitrogen-deficient mineral medium differed from the mineral medium only in that it did not contain KNO₃. The culture conditions for this second stage of culture were: a temperature of 30°C, an initial pH of 7.0, and a rotation speed of 200 rpm.

[0029] Example 2 Comparison of TH20 cell dry weight, PHB production and PHB yield under different culture conditions (Table 1) in the second stage of culture. The flow chart is shown in Figure 1 .

[0030] Table 1 Different culture conditions for the second stage of culture

[0031] 50 mL of culture medium were sampled on days 1, 2, 3, 4, 5, 6, 7, and 8, and the dry weight and PHB content of TH20 cells were measured to determine the optimal growth conditions of TH20.

[0032] The extraction and determination methods of the PHB are as follows: 1. Centrifuge 50 mL of culture medium at 10,000 rpm / min for 15 min at room temperature to collect the bacteria, and then wash them three times with sterile enzyme-free water.

[0033] 2. Place the collected bacteria in a freeze dryer and freeze-dry at −80°C for 48 h. Then take them out, accurately weigh the dry weight of the bacteria, and place them in a pressure-resistant, high-pressure reaction tube with a screw cap for reaction.

[0034] 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. Cover tightly and place in a 105°C oven for 8 h.

[0035] 4. After the reaction is complete, remove the sample and fully cool it to room temperature or lower. Add 2 mL of deionized water to the reaction mixture and shake thoroughly. This process utilizes the different solubility of methanol in chloroform and water to achieve extraction. Methanol has a low solubility in chloroform and a high solubility in water. After shaking and extraction, the mixture will separate into two layers: the upper aqueous layer and the lower chloroform layer.

[0036] 5. Place the chloroform layer in a centrifuge tube and add an appropriate amount of anhydrous sodium sulfate desiccant. Shake thoroughly to allow the anhydrous sodium sulfate to absorb the water. Centrifuge at 4°C and 5000 rpm for 5 min. Remove 1 mL of the supernatant using a 1 mL syringe. Filter the PHB using a 0.25 μm organic filter membrane. Quantitative analysis is performed by gas chromatography-mass spectrometry (GC-MS) according to the method described in [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 by esterification to convert it into methyl ester compounds. Benzoic acid was used as the internal standard. The ion fragments with charge-to-mass ratios (m / z) of 43.1 and 74.0 were monomers produced by TH20 cells. β -Characteristic ion fragments of hydroxybutyric acid ( Figure 2 ).

[0037] The measurement results are shown in Tables 2~3.

[0038] Table 2 Cell dry weight, PHB production and PHB yield at different culture times under different culture conditions (Groups 1 to 6)

[0039] Table 3 Cell dry weight, PHB production and PHB yield at different culture times under different culture conditions (Groups 7 to 12)

[0040] Note: PHB yield = PHB yield / cell dry weight × 100%.

[0041] The results showed that the culture conditions in Example 1 were the optimal culture conditions. The maximum cell dry weight of the TH20 strain 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).

[0042] In the "nitrogen-sufficient-nitrogen-deficient" two-stage culture system provided by the present invention, biomass is enriched in the first stage, and bacterial cells do not accumulate PHB during this process; in the second stage, TH20 cells begin to produce PHB, and the accumulation of PHB reaches its maximum on the 6th to 8th day. Biopolyester PHB particles mainly exist in the form of aggregates within TH20 cells, and their distribution accounts for 70% to 80% of the slice area ( Figure 3 ).

[0043] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. A production method β A method for cultivating an autotrophic microorganism that produces -hydroxybutyrate, characterized in that The following steps are involved: The hydrogen oxidizing bacteria seed liquid is inoculated into a culture medium containing nitrogen to carry out enrichment culture to obtain enriched hydrogen oxidizing bacteria; the hydrogen oxidizing bacteria include Rhodoblastus sp. TH20; The hydrogen oxidizing bacteria obtained by the enrichment culture are inoculated into a culture medium without nitrogen element for synthetic culture to obtain a polymer containing β -hydroxybutyrate culture medium; The synthetic culture conditions include: temperature of 30° C., initial pH of 7.0, and rotation speed of 200 rpm.

2. The culture method according to claim 1, wherein The nitrogen-containing culture medium includes components with 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 trace element solution includes components with the following concentrations: CoCl2·6H2O 0.119 g / L, NiCl2·6H2O 0.118 g / L and CuSO4·5H2O 0.156 g / L.

3. The culture method according to claim 1, wherein A gas mixture was introduced during the enrichment culture process; the flow rate of the gas mixture was 200 mL / min, and the ventilation time was 2 min; the gas mixture consisted of the following gas volumes: 70% H2, 20% O2, and 10% CO2.

4. The culture method according to claim 1, wherein The nitrogen-free culture medium includes the following components at the following concentrations: 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 a trace element solution 0.5 mL / L; the trace element solution includes 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.

5. The culture method according to claim 1, wherein A gas mixture was introduced during the synthetic culture process; the flow rate of the gas mixture was 200 mL / min, and the ventilation time was 2 min; the gas mixture consisted of the following gas volumes: 70% H2, 20% O2, and 10% CO2.

6. The culture method according to claim 1, wherein The volume ratio of the hydrogen oxidizing bacteria seed solution to the culture medium containing nitrogen elements is 5:95; the OD600 of the hydrogen oxidizing bacteria seed solution is 1.

20.

7. The culture method according to claim 1 or 6, characterized in that The preparation method of the hydrogen oxidizing bacteria seed liquid comprises: inoculating hydrogen oxidizing bacteria into a selective culture medium for activation culture to obtain the hydrogen oxidizing bacteria seed liquid; the selective culture medium comprises components with the following concentrations: (NH4)2SO4 0.47 g, KH2PO4 0.5 g / L, MgSO4·7H2O0.2 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 components with the following concentrations: CoCl2·6H2O 0.119 g / L, NiCl2·6H2O 0.118 g / L and CuSO4·5H2O 0.156 g / L.

8. The culture method according to claim 7, characterized in that The activation culture conditions include: a temperature of 25°C, a rotation speed of 200 rpm, gas introduction every 72 hours, a gas flow rate of 200 mL / min, and a ventilation time of 2 minutes; the introduced gas is composed of the following volume percentages: H2 85%, O2 5% and CO2 10%.

9. The culture method according to claim 7, characterized in that The pH value of the selective culture medium is 7.

0.

10. The culture method according to claim 1, wherein The OD600 of the hydrogen oxidizing bacteria in the enriched culture is 0.80-1.20.

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