Preparation method of fe3o4 nanomaterial and application thereof in synthesis of driving type phb high-efficiency synthesis method

Abstract

The application discloses a preparation method of Fe3O4 nanomaterial and application of the Fe3O4 nanomaterial in a synthesis driving type PHB efficient synthesis method. Cupriavidus necator The method utilizes excellent biocompatibility and metabolic regulation characteristics of the Fe3O4 nanomaterial to strengthen the H2 / CO2 carbon fixation and PHB synthesis process in a "synthesis driving" mode The application discloses a preparation method of Fe3O4 nanomaterial and application of the Fe3O4 nanomaterial in a synthesis driving type PHB efficient synthesis method. Through system characterization of material-microorganism interaction characteristics, optimization of fermentation parameters, and combination of transcriptome and metabolite analysis, it is determined that the Fe3O4 nanomaterial activates key genes of the Calvin cycle and the PHB synthesis path in a specific manner, synchronously improves the level of core metabolites, and significantly improves the PHB yield; the material has good recycling performance, and the strengthening effect is significant under light inhibition conditions. The application does not need genetic modification, has high strengthening efficiency and strong stability, and provides key technical support for industrialized efficient production of PHB and resource utilization of CO2.

Description

Technical Field

[0001] This invention relates to the fields of low-carbon biomanufacturing, microbial metabolic regulation, and biodegradable plastic synthesis technology, specifically to a method for preparing Fe3O4 nanomaterials and its application in a synthesis-driven, high-efficiency PHB synthesis method. Background Technology

[0002] As a representative material of biodegradable bio-based plastics, polyhydroxybutyrate (PHB) is prepared via a biosynthetic pathway using carbon dioxide by insecticidal copper-killing bacteria, providing core technological support for achieving carbon neutrality goals and addressing the problem of white pollution. However, the low carbon dioxide fixation efficiency and insufficient energy and reducing power supply of chemoautotrophic microorganisms are key bottlenecks restricting the yield of PHB. The Calvin cycle, as the core pathway for carbon dioxide fixation, has limited activity of its key enzymes, resulting in insufficient carbon skeleton supply. At the same time, the ATP and NADPH generated by hydrogen oxidation are insufficient to meet the high demand for PHB synthesis, limiting the targeted allocation of metabolic flow to PHB.

[0003] While existing enhancement strategies, such as genetic engineering, can target and regulate the Calvin cycle or polyhydroxybutyrate (PHB) synthesis pathway, they suffer from technical complexity, high cost, poor genetic stability, and environmental risks. Traditional nanomaterials are mostly used as nutritional supplements or carriers, making it difficult to fundamentally achieve synergistic enhancement of carbon fixation, energy supply, and PHB synthesis. Fe3O4 nanomaterials, due to their combination of Fe... 2+ with Fe 3+ Due to its redox properties, it possesses excellent biocompatibility, electron transport capacity, and gas adsorption performance, making it widely used in biomedicine, environmental remediation, and other fields. However, its application in microbial carbon fixation synthesis has not been fully explored. In particular, how to utilize its physicochemical properties to specifically activate the Calvin cycle, simultaneously enhance energy and reducing power supply, and construct a highly efficient "synthesis-driven" metabolic mode is a key focus of current technological research.

[0004] Furthermore, existing research lacks a systematic analysis of the interaction mechanism between Fe3O4 nanomaterials and microorganisms, its concentration-dependent effect, recycling performance, and multi-omics molecular mechanisms, which limits its industrial application. Therefore, developing a synthesis-driven, efficient method for the synthesis of polyhydroxybutyrate (PHB) is of great significance for overcoming existing technological bottlenecks and promoting the industrial production of PHB.

[0005] In conclusion, a new technical solution is urgently needed to address the problems existing in the current technology. Summary of the Invention

[0006] Based on this, the present invention provides a method for preparing Fe3O4 nanomaterials and its application in a synthesis-driven, high-efficiency synthesis method for PHB (polyhydroxybutyrate). The present invention aims to provide a method for preparing Fe3O4 nanomaterials and a synthesis-driven, high-efficiency synthesis method for PHB. Utilizing the unique physicochemical properties of Fe3O4 nanomaterials, it specifically activates the Calvin cycle and the PHB synthesis pathway, simultaneously enhancing energy and reducing power supply, thereby significantly improving PHB yield. Through systematic characterization of the material-microorganism interaction characteristics, optimization of fermentation parameters, and verification of the material's recycling performance and light adaptability, the invention provides technical support for the efficient industrial production of PHB.

[0007] One object of the present invention is to provide a method for preparing Fe3O4 nanomaterials, comprising the following steps: FeCl3·6H2O and FeSO4·7H2O were mixed in water, the pH of the solution was adjusted with alkali, and then the reaction was carried out by heating in the dark. After purification, Fe3O4 nanomaterials were obtained.

[0008] Furthermore, the mass ratio of FeCl3·6H2O to FeSO4·7H2O is 2:(1-5).

[0009] Furthermore, the heating temperature is 50-70°C.

[0010] Furthermore, the pH value is 8-10.

[0011] Furthermore, the Fe3O4 nanomaterial has a granular structure with a particle size of 1-200 nm.

[0012] Another objective of this invention is to provide a synthesis-driven, high-efficiency method for synthesizing PHB, comprising the aforementioned Fe3O4 nanomaterial, and including the following steps: S1, will Cupriavidus necator (C.necator ; Cn) The preserved strain was inoculated into broth medium, activated, and then amplified in fructose medium to the logarithmic phase. After that, it was transferred to nitrogen-limited basal medium and acclimated in an H2 atmosphere to obtain a highly active seed culture. S2. The highly active seed culture is inoculated into a nitrogen-limited basal medium, and a mixture of H2, CO2 and air is filled to form a fermentation system. S3. Add Fe3O4 nanomaterials to the fermentation system for fermentation to obtain PHB.

[0013] Furthermore, the synthesis-driven high-efficiency PHB synthesis method further includes the following steps: S4. After fermentation, Fe3O4 nanomaterials were recovered by magnetic separation technology, washed and dried, and reused. The bacterial cells were collected and PHB was extracted and purified by concentrated sulfuric acid digestion-ultra-high performance liquid chromatography. S5. Verify the effect of material concentration, adaptability to light conditions, and recycling performance to ensure the stability and practicality of the method.

[0014] Furthermore, in the H2, CO2, and air mixture, the volume ratio of H2, CO2, and air is (7-9):(1-3):(8-10).

[0015] Furthermore, in step S3, the concentration of Fe3O4 nanomaterials added to the fermentation system is 0.1-10 g / L.

[0016] Furthermore, in step S3, the fermentation conditions are: fermentation at 30℃, pH 7.0, and 200 rpm for 24-48 h.

[0017] The present invention has the following beneficial effects: This invention provides a method for preparing Fe3O4 nanomaterials and their application in a synthesis-driven, high-efficiency PHB synthesis method. This invention elucidates for the first time the molecular mechanism by which Fe3O4 nanomaterials enhance PHB synthesis in a "synthesis-driven" mode. Combined with transcriptomics and metabolite analysis, it confirms that the material specifically activates the Calvin cycle and the PHB synthesis pathway, simultaneously increasing the levels of core metabolites and achieving a significant increase in PHB yield, overcoming the limitations of traditional methods that can only regulate a single step.

[0018] The Fe3O4 nanomaterial of this invention has a particulate structure, is uniformly attached to the surface of bacteria, has excellent biocompatibility, and has no significant cytotoxicity. The material can be efficiently recycled and reused through magnetic separation technology, and still maintains a good strengthening effect after secondary use, thereby reducing production costs.

[0019] The synthesis method of this invention exhibits significant enhancement effects under light-inhibited conditions and excellent light adaptability; it requires no genetic modification, is simple to operate, highly reproducible, and suitable for long-term continuous fermentation production; at the same time, the regulatory logic of this method can be extended to the carbon fixation synthesis process of other chemoautotrophic microorganisms, providing a general technical framework for the resource utilization of CO2 to prepare high-value compounds, and has important academic and industrial value. Attached Figure Description

[0020] Figure 1 An experimental flowchart of the present invention is shown.

[0021] Figure 2 TEM images of bacteria and Fe3O4 are shown; in, Figure 2a shows a SEM image of Fe3O4 nanomaterials; Figure 2 b shows a SEM image of bacterial-Fe3O4 gas fermentation for 24 h; Figure 2 c shows a SEM image of the bacterial-Fe3O4 gaseous fermentation after 48 h.

[0022] Figure 3 EDS diagrams of bacteria and Fe3O4 are shown; in, Figure 3 a shows the EDS diagram of Fe3O4 nanomaterials; Figure 3 b shows the EDS diagram of bacterial-Fe3O4 gas fermentation for 24 h; Figure 3 c shows the EDS diagram of bacterial-Fe3O4 gaseous fermentation after 48 h.

[0023] Figure 4 TEM images of negatively stained sections of bacteria and Fe3O4 are shown. in, Figure 4 a shows a TEM image of bacteria with negative staining; Figure 4 b shows a TEM image of bacteria stained with Fe3O4; Figure 4 c shows a TEM image of a bacterial section; Figure 4 Image d shows a TEM image of a bacterial-Fe3O4 section.

[0024] Figure 5 The effect of 1 g / L Fe3O4 on PHB synthesis is shown in the figure. in, Figure 5 a shows OD 600 Test results; Figure 5 b shows the results of the PHB production test.

[0025] Figure 6 The time-curve of Fe3O4 on bacterial growth and PHB synthesis is shown. in, Figure 6 a shows OD 600 Test results; Figure 6 b shows the results of the PHB production test.

[0026] Figure 7 The diagram shows the effect of different concentrations of Fe3O4 on the synthesis of PHB. in, Figure 7 a shows OD 600 Test results; Figure 7 b shows the results of the PHB production test.

[0027] Figure 8 The diagram shows the effect of light irradiation on the Fe3O4-enhanced PHB synthesis. in, Figure 8 a shows OD 600 Test results; Figure 8 b shows the results of the PHB production test.

[0028] Figure 9 The recycling performance of Fe3O4 material was demonstrated; in, Figure 9 a shows OD 600 Test results; Figure 9 b shows the results of the PHB production test.

[0029] Figure 10 This diagram illustrates the mechanism by which Fe3O4 promotes PHB synthesis as revealed by the transcriptome. in, Figure 10 a shows the DEGs volcano plot (red dots represent upregulated DEGs, green dots represent downregulated DEGs, and blue dots represent differentially expressed genes; screening criteria: |log2FC|≥1, padj<0.05). Figure 10 b shows a heatmap of core differentially expressed genes; Figure 10 c shows a bubble chart of GO functional enrichment; Figure 10 d shows a bubble diagram of KEGG pathway enrichment.

[0030] Figure 11 The diagram shows the concentration of core metabolites under the influence of Fe3O4 nanomaterials; in, Figure 11 a shows a graph of NADPH concentration; Figure 11 b shows a graph of pyruvate concentration; Figure 11 c shows the ATP concentration graph; Figure 11 d shows a graph of acetyl-CoA concentration. Detailed Implementation

[0031] To more clearly illustrate the technical solution of the present invention, the following embodiments are provided. Unless otherwise stated, the raw materials, reactions, and post-processing methods appearing in the embodiments are all commercially available raw materials and technical methods well known to those skilled in the art.

[0032] The terms "preferred," "more preferably," and "more suitable" used in this invention refer to embodiments of the invention that provide certain beneficial effects under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the description of one or more preferred embodiments does not imply that other embodiments are unavailable, nor is it intended to exclude other embodiments from the scope of this invention.

[0033] It should be understood that, except in any operational instance or otherwise indicated, the amounts or all figures representing ingredients used, for example, in the specification and claims, should be understood to be modified by the term "about" in all cases. Therefore, unless otherwise stated, the numerical parameters set forth in the following specification and appended claims are approximate values ​​varying according to the desired performance to be obtained according to the invention.

[0034] The present invention uses the following raw materials: Cupriavidus necator Strains: Cupriavidus necator H16; sourced from ATCC Cupriavidus necator 17699 TM ;337 [ATCC 23440, H16, NCIB 10442, S-10-1].

[0035] Nitrogen-limiting basal medium, pH 7.0, consists of the following components: C6H5FeO7, concentration 0.5741 mg / L; NaHCO3, concentration 0.2 g / L; MgSO4·7H2O, concentration 80 mg / L; Na2HPO4, concentration 3.57 g / L; KH2PO4, concentration 1.5 g / L; (NH4)2SO4, concentration 0.167 g / L; CaSO4·2H2O, concentration 1 mg / L; NiSO4·7H2O, concentration 0.56 mg / L; water.

[0036] Fructose medium, pH 7.0, consists of the following components: C6H5FeO7, concentration 0.5741 mg / L; NaHCO3, concentration 0.2 g / L; MgSO4·7H2O, concentration 80 mg / L; fructose, concentration 10 g / L; (NH4)2SO4, concentration 0.167 g / L; Na2HPO4, concentration 3.57 g / L; KH2PO4, concentration 1.5 g / L; CaSO4·2H2O, concentration 1 mg / L; NiSO4·7H2O, concentration 0.56 mg / L; water.

[0037] The nutrient broth medium (LB broth medium), with a pH of 7.0, consists of the following components: The concentration of NaCl was 10 g / L; the concentration of tryptone was 10 g / L; the concentration of yeast extract was 5 g / L; and the concentration of water was 10 g / L.

[0038] The mixture of H2, CO2, and air is obtained by mixing H2, CO2, and air in a volume ratio of 8:2:9.

[0039] The present invention uses the following testing methods: Biomass OD 600 The absorbance at 600 nm was measured using a spectrophotometer.

[0040] PHB yield: The bacterial culture was centrifuged at 1000 rpm for 10 min, then dried overnight in an oven at 60℃, digested with 98wt% concentrated sulfuric acid for 90 min, and then diluted with water. The results were detected by ultra-high performance liquid chromatography under the following conditions: the mobile phase was an aqueous solution of acetic acid (0.1 wt%) and acetonitrile at a volume ratio of 80:20, the flow rate was 0.30 mL / min, and the detection wavelength was 210 nm.

[0041] All water used in this invention is deionized water.

[0042] In this invention, "parts" refers to parts by mass.

[0043] Example A method for preparing Fe3O4 nanomaterials includes the following steps: FeCl3·6H2O (2.43 g) and FeSO4·7H2O (1.25 g) were dissolved in 150 mL of deionized water, and the pH was measured. The solution was vigorously stirred, and 25 wt% ammonia solution was added dropwise using a syringe pump to gradually adjust the pH to 9.0. Then, the glassware containing the solution was wrapped with aluminum foil and stirred at a constant temperature of 60 °C for 2 h in a heating mantle. Subsequently, the solution was filtered through Whatman No. 1 filter paper, and the pH was washed with deionized water to 7. After centrifugation and dehydration, the sample was frozen at -80 °C and then freeze-dried in a nitrogen atmosphere for 6 h to remove water and other volatile substances from the solution, yielding Fe3O4 nanomaterials with a particulate structure and a particle size of approximately 50 nm.

[0044] Application examples A synthesis-driven, high-efficiency method for synthesizing PHB includes the following steps: S1, will Cupriavidus necator The preserved strain was inoculated into broth medium and activated at 30℃ and 200 rpm for 12 h; after being amplified and cultured to the logarithmic phase on fructose medium, it was transferred to nitrogen-limited basal medium and acclimatized in H2 atmosphere for 24 h to obtain a highly active seed culture. S2. Inoculate the highly active seed culture into a 100 mL serum bottle containing 10 mL of nitrogen-limiting basal medium, and control the initial OD. 600 The concentration is 0.2, and the space at the top of the bottle is filled with a mixture of H2, CO2, and air to form a fermentation system; S3. Add Fe3O4 nanomaterials to the fermentation system, control the final concentration to 1 g / L, and ferment for 48 h at 30℃, pH 7.0 and 200 rpm to obtain PHB.

[0045] Comparative application examples The difference between this comparative application example and the application example is that Fe3O4 nanomaterials are not added in step S3, and fermentation is carried out under the same conditions. Other steps and dosages are the same as in the application example.

[0046] Figure 1 An experimental flowchart of the present invention is shown.

[0047] Test Example 1 Fe3O4 nanomaterials and C.necator Observations on interactions.

[0048] Test method: (1) SEM and EDS were used to prepare and observe Fe3O4 nanomaterials.

[0049] (2) According to the application example method, C.necator Bacterial suspension was mixed with fresh nitrogen-limiting basal medium to adjust OD 600The solution was diluted to 0.2, and then 1 g / L of Fe3O4 nanomaterials was added. H2 and CO2 were injected, and gaseous fermentation was carried out in a closed environment. After 24 h and 48 h of fermentation, the cells were collected by centrifugation and observed by SEM, EDS, and TEM.

[0050] Test results are as follows Figure 2-4 As shown.

[0051] Figure 2 TEM images of bacteria and Fe3O4 are shown; in, Figure 2 a shows a SEM image of Fe3O4 nanomaterials; Figure 2 b shows a SEM image of bacterial-Fe3O4 gas fermentation for 24 h; Figure 2 c shows a SEM image of the bacterial-Fe3O4 gaseous fermentation after 48 h.

[0052] Figure 3 EDS diagrams of bacteria and Fe3O4 are shown; in, Figure 3 a shows the EDS diagram of Fe3O4 nanomaterials; Figure 3 b shows the EDS diagram of bacterial-Fe3O4 gas fermentation for 24 h; Figure 3 c shows the EDS diagram of bacterial-Fe3O4 gaseous fermentation after 48 h.

[0053] Figure 4 TEM images of negatively stained sections of bacteria and Fe3O4 are shown. in, Figure 4 a shows a TEM image of bacteria with negative staining; Figure 4 b shows a TEM image of bacteria stained with Fe3O4; Figure 4 c shows a TEM image of a bacterial section; Figure 4 Image d shows a TEM image of a bacterial-Fe3O4 section.

[0054] The test results above show that the SEM images show that the cell surface is smooth and intact, and the Fe3O4 nanomaterial is uniformly attached to the cell surface; the TEM ultrathin section shows that the cell structure is intact, the number and size of PHB particles are increased, and the intracellular PHB particles are more dense after Fe3O4 treatment. The section image confirms that the material does not penetrate into the bacterial interior, but only plays a regulatory role through surface interaction; the EDS surface scan confirms that the iron element is uniformly distributed, and the material can be uniformly attached to the bacterial surface after co-culturing with the bacterial cells for 24 h and 48 h.

[0055] Test Example 2 1 g / L Fe3O4 nanomaterials C.necator Effects on growth and PHB synthesis.

[0056] Test method: Set up the Fe3O4 group according to the application example method, and C.necator Inoculated into nitrogen-limiting basal medium containing a mixture of H2, CO2, and air, initial OD 600 =0.2, cultured at 30℃. 1 g / L Fe3O4 nanomaterials were added, and OD was measured after 24 h and 48 h of gas fermentation. 600 and PHB concentration.

[0057] A control group was set up according to the comparative application example method, and OD was measured under the same conditions. 600 and PHB concentration.

[0058] Test results are as follows Figure 5 As shown.

[0059] Figure 5 The effect of 1 g / L Fe3O4 on PHB synthesis is shown in the figure. in, Figure 5 a shows OD 600 Test results; Figure 5 b shows the results of the PHB production test. Labels in the figure: Fe3O4: Fe3O4 group; Blank: control group.

[0060] The test results above show that, at 24 hours, the OD of the control group was... 600 =1.428, PHB was 347.536 mg / L, OD of Fe3O4 group 600 =1.640, PHB was 428.955 mg / L; at 48 h, the OD of the control group was... 600 =2.099, PHB was 807.094 mg / L, OD of Fe3O4 group 600 =2.254, PHB is 945.063 mg / L.

[0061] Test Example 3 Fe3O4 nanomaterials C.necator The influence of growth and PHB synthesis curves.

[0062] Test method: Set up the Fe3O4 group according to the application example method, and C.necator Inoculated into nitrogen-limiting basal medium containing a mixture of H2, CO2, and air, initial OD 600 =0.2, incubated at 30℃. 1 g / L Fe3O4 nanomaterial was added, and OD was measured every 6 h. 600 Draw the growth curve.

[0063] A control group was set up according to the comparative application example method, and OD was measured under the same conditions. 600 and PHB concentration.

[0064] Test results are as follows Figure 6 As shown.

[0065] Figure 6 The time-curve of Fe3O4 on bacterial growth and PHB synthesis is shown. in, Figure 6 a shows OD 600 Test results; Figure 6 b shows the results of the PHB production test. Labels in the figure: Fe3O4: Fe3O4 group; Blank: control group.

[0066] The test results above show that the biomass OD of the Fe3O4 group... 600 Both the yield of Fe3O4 and the yield of PHB were significantly higher than those of the control group, indicating that Fe3O4 can accelerate the growth of microorganisms and the rapid synthesis of PHB.

[0067] Test Example 4 The effect of Fe3O4 nanomaterial concentration on C.necator Effects on growth and PHB synthesis.

[0068] Test method: According to the application examples and comparative application examples, C.necator Inoculated into nitrogen-limiting basal medium containing a mixture of H2, CO2, and air, initial OD 600 =0.2, cultured at 30℃. Different concentrations of Fe3O4 were set up as treatment groups (concentrations of 0, 0.5, 1, 2, 3, 4, 5, 8, and 10 g / L).

[0069] Test results are as follows Figure 7 As shown.

[0070] Figure 7The diagram shows the effect of different concentrations of Fe3O4 on the synthesis of PHB. in, Figure 7 a shows OD 600 Test results; Figure 7 b shows the results of the PHB production test.

[0071] The test results above show that OD 600 The yield increased with increasing material concentration, which is due to the absorbance of the material in the solution. The yield of PHB changed little with increasing concentration. At a concentration of 1 g / L, the yield of PHB after 48 h of gas fermentation was 939.662±40.197 mg / L, which was the optimal concentration. At this concentration, the yield of PHB was 15.24% higher than that of the control group (815.368±9.552 mg / L).

[0072] Test Example 5 Experiment on the effects of light.

[0073] Test method: Following the methods of the application examples and comparative application examples, light groups and dark groups were set up, cultured under the same conditions, and PHB yield was detected.

[0074] (1) Dark blank group: set up according to the method of the comparative application example, fermented under dark conditions; (2) Dark Fe3O4 group: set up according to the application example method, fermentation under dark conditions; (3) Light control group: Fermentation was carried out under light conditions (with an additional LED light source in the incubator, light intensity 3 mW) according to the method of the comparative application example; (4) Light-illuminated Fe3O4 group: Fermentation was carried out under light conditions (with an additional LED light source in the incubator, with a light intensity of 3mW) according to the application example.

[0075] Test results are as follows Figure 8 As shown.

[0076] Figure 8 The diagram shows the effect of light irradiation on the Fe3O4-enhanced PHB synthesis. in, Figure 8 a shows OD 600 Test results; Figure 8 b shows the results of the PHB production test.

[0077] The test results above show that light inhibited bacterial growth and PHB synthesis, but the material itself promoted both. Under light conditions, after 24 h of gas fermentation, the PHB yield in the Fe3O4 group was 183.90±9.97 mg / L, which was 2.83 times that of the light-controlled group (65.02±21.47 mg / L). After 48 h of gas fermentation, the PHB yield in the Fe3O4 group was 707.29±46.02 mg / L, which was 1.42 times that of the light-controlled group (496.27±11.71 mg / L), demonstrating excellent light adaptability.

[0078] Test Example 6 Material recycling experiment.

[0079] Test method: The Fe3O4 nanomaterials recovered by magnetic separation after the reaction in the application example were washed and reused (after the first use, they were recovered and washed using the same method before a second use). A control group was set up according to the method in the comparative application example, and the biomass OD was measured. 600 And PHB production.

[0080] Test results are as follows Figure 9 As shown.

[0081] Figure 9 The recycling performance of Fe3O4 material was demonstrated; in, Figure 9 a shows OD 600 Test results; Figure 9 b shows the PHB yield test results. The labels in the figure indicate: Blank: control group; Fe3O4: material used for the first time; Fe3O4 repeat: material used for the second time.

[0082] The test results above show that after 48 h of gas fermentation, the PHB yield in the control group was 629.057±65.90 mg / L, the PHB yield in the first use of the material was 831.35±25.54 mg / L, and the PHB yield in the second use of the material was 759.87±18.83 mg / L.

[0083] Test Example 7 Transcriptomics analysis.

[0084] Test method: The Fe3O4 group was set up according to the application example method, and the control group was set up according to the control application example method. Bacterial cells were collected after 24 h of culture, total RNA was extracted, and Illumina sequencing was performed. Differentially expressed gene screening criteria: |log2FC|≥1, p<0.05.

[0085] Test results are as follows Figure 10 As shown.

[0086] Figure 10 This diagram illustrates the mechanism by which Fe3O4 promotes PHB synthesis as revealed by the transcriptome. in, Figure 10 a shows the DEGs volcano plot (red dots represent upregulated DEGs, green dots represent downregulated DEGs, and blue dots represent differentially expressed genes; screening criteria: |log2FC|≥1, padj<0.05). Figure 10 b shows a heatmap of core differentially expressed genes; Figure 10 c shows a bubble chart of GO functional enrichment; Figure 10 d shows a bubble diagram of KEGG pathway enrichment.

[0087] The test results show that after treatment with Fe3O4 nanomaterials, a total of 315 differentially expressed genes (DEGs) were identified, of which 161 were significantly upregulated and 154 were significantly downregulated. The core differentially expressed genes included: highly significant upregulation of the Calvin cycle key genes cbbP2 (log2FC≈4.8) and cbbL2 (log2FC≈1.8), enhancing CO2 fixation efficiency; specific activation of the key PHB synthesis genes phaC2 (log2FC≥2.2) and phaB2 (log2FC≥2.4); and explosive upregulation of the copper resistance operon (copABCDRS), maintaining intracellular metal ion homeostasis. GO functional enrichment showed that DEGs focused on catalytic activity, transporter activity, and metabolic processes; KEGG pathway enrichment pointed to core pathways such as the Calvin cycle and oxidative phosphorylation.

[0088] Test Example 8 Key metabolite analysis.

[0089] Test method: The Fe3O4 group was set up according to the application example method, and the control group was set up according to the comparative application example method. Bacterial cells were collected after 24 h of culture. The intracellular metabolites NADPH, ATP, pyruvate and acetyl-CoA were measured using the corresponding kits (Beyotime, catalog numbers S0179, S0026, S0299S; mlbio, catalog number ml077327) and according to the instructions.

[0090] Test results are as follows Figure 11 As shown.

[0091] Figure 11 The diagram shows the concentration of core metabolites under the influence of Fe3O4 nanomaterials; in, Figure 11 a shows a graph of NADPH concentration; Figure 11 b shows a graph of pyruvate concentration; Figure 11 c shows the ATP concentration graph; Figure 11 Figure d shows the acetyl-CoA concentration graph. Labels in the graph: Fe3O4: Fe3O4 group; Blank: control group.

[0092] The test results show that the NADPH concentration in the Fe3O4 group was 394.22±98.97 nM, which was 7.3 times that of the control group (53.81±18.56 nM); the pyruvate concentration in the Fe3O4 group was 17.87±1.60 μM, which was similar to that of the control group (17.91±1.23 μM); the ATP concentration in the Fe3O4 group was 325.95±74.35 nM, which was 31.25% less than that of the control group (427.82±9.22 nM), indicating that the material group consumed more ATP for PHB synthesis; and the acetyl-CoA concentration in the Fe3O4 group was 2.78±1.63 nM, which was 1.4 times that of the control group (1.92±0.84 nM).

[0093] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

[0094] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A method for preparing Fe3O4 nanomaterials, characterized in that, Includes the following steps: FeCl3·6H2O and FeSO4·7H2O were mixed in water, the pH of the solution was adjusted with alkali, and then the reaction was carried out by heating in the dark. After purification, Fe3O4 nanomaterials were obtained.

2. The preparation method based on Fe3O4 nanomaterials according to claim 1, characterized in that, The mass ratio of FeCl3·6H2O to FeSO4·7H2O is 2:(1-5).

3. The preparation method based on Fe3O4 nanomaterials according to claim 1, characterized in that, The heating temperature is 50-70℃.

4. The preparation method based on Fe3O4 nanomaterials according to claim 1, characterized in that, The pH value is 8-10.

5. A Fe3O4 nanomaterial prepared by the preparation method based on Fe3O4 nanomaterials according to any one of claims 1-4, characterized in that, The Fe3O4 nanomaterial has a granular structure with a particle size of 1-200 nm.

6. A synthesis-driven, high-efficiency method for synthesizing PHB, comprising the Fe3O4 nanomaterial as described in claim 5, characterized in that, Includes the following steps: S1, will Cupriavidus necator The preserved strain was inoculated into broth medium, activated, and then amplified in fructose medium to the logarithmic phase. After that, it was transferred to nitrogen-limited basal medium and acclimated in an H2 atmosphere to obtain a highly active seed culture. S2. The highly active seed culture is inoculated into a nitrogen-limited basal medium, and a mixture of H2, CO2 and air is filled to form a fermentation system. S3. Add Fe3O4 nanomaterials to the fermentation system for fermentation to obtain PHB.

7. The synthesis-driven high-efficiency synthesis method for PHB according to claim 6, characterized in that, In the mixture of H2, CO2, and air, the volume ratio of H2, CO2, and air is (7-9):(1-3):(8-10).

8. The synthesis-driven high-efficiency synthesis method for PHB according to claim 6, characterized in that, In step S3, the concentration of Fe3O4 nanomaterials added to the fermentation system is 0.1-10 g / L.

9. The synthesis-driven high-efficiency synthesis method for PHB according to claim 6, characterized in that, In step S3, the fermentation conditions are: fermentation at 30℃, pH 7.0, and 200 rpm for 24-48 h.

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