High-activity autotrophic carbon-fixing bacteria freeze-dried powder based on microelectric field and flue gas stress and preparation method thereof

Abstract

The application discloses a high-activity chemotrophic autotrophic carbon-fixing bacteria freeze-dried powder based on microelectric field and flue gas stress and a preparation method, and belongs to the technical field of carbon capture and storage and microecological preparation. The method enriches and cultures seed liquid of the chemotrophic autotrophic carbon-fixing bacteria under a constant voltage by inputting simulated industrial flue gas. Inorganic nitrogen sources are flowed into the fermentation liquor to maintain the C / N ratio of the culture medium, and a redox mediator is added to the fermentation liquor, and the fermentation is carried out at a constant temperature. The fermentation liquor is cold-shocked before the fermentation is completed, and a permeation pressure regulator and metal ion cofactors are added in a pulse mode. After the cold-shock treatment is completed, centrifugation is carried out, and bacterial slurry is obtained. The bacterial slurry is mixed with skimmed milk powder and modified nano-silicon dioxide powder to obtain a resuspension. The resuspension is pre-frozen and then sublimation dried to obtain the high-activity chemotrophic autotrophic carbon-fixing bacteria freeze-dried powder. The method breaks the mass transfer and thermodynamic deadlock, realizes high-density propagation of the carbon-fixing bacteria, and achieves the purpose that the enzyme activity does not attenuate after freeze-drying.

Description

Technical Field

[0001] This invention belongs to the field of carbon capture and storage and microecological preparation technology, specifically relating to a highly active chemoautotrophic carbon-fixing bacteria freeze-dried powder based on micro-electric field and flue gas stress and its preparation method. Background Technology

[0002] With the intensification of global climate change, the efficient capture of carbon dioxide from industrial flue gas has become crucial for achieving deep decarbonization. However, traditional amine absorption or membrane separation methods generally suffer from high energy consumption, equipment corrosion, chemical consumption, and potential leakage risks, making it difficult to create value while achieving CO2 emission reduction. Therefore, conversion technologies that can directly and efficiently convert CO2 into high-value-added chemicals are of great significance for building a sustainable carbon cycle system.

[0003] Chemoautotrophic carbon-fixing bacteria (typically represented by Cupriavidus necator) can directly reduce and fix carbon dioxide into intracellular organic macromolecules (such as polyhydroxy fatty acid ester PHB) through chemoautotrophic metabolic pathways, using inorganic electron donors to provide energy and reducing power. They have shown great application potential in industrial flue gas emission reduction and the biosynthesis of high-value-added chemicals.

[0004] Chemoautotrophic carbon-fixing bacteria are typically industrially propagated using a two-stage fermentation process involving heterotrophic expansion and autotrophic / carbon-limited induction. The bacterial agent can be prepared as a liquid concentrate, freeze-dried powder, or solid adsorbent. The key to its preparation lies in selecting appropriate preservatives and a gentle drying process to maintain the catalytic activity of the core carbon-fixing enzymes while ensuring a high viable cell count, thus guaranteeing stable CO2 fixation after rehydration. However, current industrial propagation and powder preparation processes for chemoautotrophic carbon-fixing bacteria still face three major challenges: First, industrial flue gas often contains highly acidic gases (such as SO2 and NOx), which readily dissolve in the aqueous phase, leading to a large amount of H₂. + The chemoautotrophic carbon-fixing bacteria, flooding into the cell, are forced to consume large amounts of ATP to convert H into hydrogen. + First, the pumping out of the cell causes the Calvin cycle to stop due to energy depletion. Second, traditional gas-phase hydrogen power supply poses an extremely high risk of explosion and has very low solubility, while conventional direct electrochemical culture is limited by the physical contact area between the cell and the electrode, resulting in an electron transfer rate far lower than the theoretical requirements for bacterial division. Third, the catalytic activity of carbon-fixing core enzymes (represented by ribulose-1,5-bisphosphate carboxylase) is highly dependent on their metal cofactors and hydration network. In the freeze-drying process of preparing solid bacterial agents, the forced sublimation of free water in existing technologies directly strips the hydration layer on the protein surface, causing irreversible denaturation and collapse of the enzyme's secondary structure, resulting in poor carbon-fixing activity after rehydration. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a freeze-dried powder of highly active chemoautotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress, and its preparation method. It solves the problems of Calvin cycle cessation caused by strong acidic gases in industrial flue gas, electron transfer rate being far lower than the theoretical requirement for bacterial division, and poor carbon fixation activity after rehydration caused by forced sublimation of free water. It breaks the mass transfer and thermodynamic deadlock, and achieves the goal of high-density propagation of carbon-fixing bacteria without enzyme activity decay after freeze-drying.

[0006] This invention is achieved through the following technical solution: A method for preparing lyophilized powder of highly active chemoautotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress, specifically including the following steps: S1, the seed culture of chemoautotrophic carbon-fixing bacteria is enriched by passing simulated industrial flue gas through it under a constant voltage of 0.5-1.2V. The concentrations of CO2 and SO2 in the simulated industrial flue gas increase stepwise with the culture time, and the remainder is nitrogen and oxygen. S2, an inorganic nitrogen source is added to the fermentation broth enriched in S1 to maintain the C / N ratio of the culture medium. At the same time, a redox mediator with a final concentration of 5-20 μmol / L is added to the fermentation broth. The fermentation is carried out at a constant temperature for 8-12 days. Before the end of the fermentation, the fermentation broth is subjected to cold shock treatment. At the same time, an osmotic pressure regulator and a metal ion cofactor are added in a pulse. After the cold shock treatment, the mixture is centrifuged to obtain the bacterial sludge. S3. Skim milk powder and modified nano-silica powder are added to the bacterial sludge and mixed evenly to obtain a resuspended liquid. The resuspended liquid is pre-frozen and then sublimated and dried to obtain a highly active autotrophic carbon-fixing bacteria freeze-dried powder.

[0007] A further improvement of the present invention is that: S1 was cultured with chemoautotrophic carbon-fixing bacteria seed culture to the logarithmic growth phase, OD 600 The value is 0.8~1.5, and the seed liquid is then inoculated into an electrochemical fermenter for enrichment culture at an inoculation rate of 5%~15% of the fermentation liquid volume.

[0008] The ventilation ratio for simulating industrial flue gas in S1 is 0.1-0.5 vvm.

[0009] The enrichment culture in S1 lasted for 8–12 days. On days 1–3, the concentrations of CO2 and SO2 were 5%–10% and 10–20 ppm, respectively. On days 4–7, the concentrations of CO2 and SO2 were 10%–20% and 20–50 ppm, respectively. On days 8–12, the concentrations of CO2 and SO2 were 20%–30% and 50–100 ppm, respectively.

[0010] The redox mediator mentioned in S2 is anthraquinone-2,6-disulfonate, riboflavin, or neutral red.

[0011] S2 undergoes a cold shock treatment by rapidly lowering the temperature of the fermentation broth and maintaining it at 12-18°C 1-3 hours before the end of fermentation.

[0012] The osmotic pressure regulator described in S2 is one or more of trehalose, betaine, tetrahydropyrimidine, and fructooligosaccharides, with a final concentration of 0.1-0.3 M in the fermentation broth, and the metal ion cofactor is Mg²⁺. + or Mn² + The final concentration is 5-15 mM.

[0013] In S3, the mass of skim milk powder and modified nano silica powder are 5%~15% and 0.5%~2.0% of the bacterial sludge, respectively.

[0014] The pre-freezing treatment described in S3 is carried out at -60℃ to -40℃ for 2 to 4 hours, and the sublimation drying is carried out at a vacuum of 10 to 50 Pa and a temperature of 15 to 25℃ for 18 to 24 hours.

[0015] A highly active autotrophic carbon-fixing bacteria freeze-dried powder obtained by the preparation method of highly active autotrophic carbon-fixing bacteria freeze-dried powder based on micro-electric field and flue gas stress as described in any one of the above.

[0016] Compared with the prior art, the present invention has the following beneficial technical effects: This invention discloses a method for preparing highly active chemoautotrophic carbon-fixing bacteria freeze-dried powder based on micro-electric field and flue gas stress. During the enrichment culture of chemoautotrophic carbon-fixing bacteria, the micro-voltage is strictly limited to 0.5V-1.2V, below the water decomposition voltage (theoretical value 1.23V), preventing the production of hydrogen and oxygen (avoiding cell stripping and oxygen toxicity from bubbles), and precisely reducing only redox mediators at a concentration of 5-20 μmol / L. If the voltage is below 0.5V or the mediator is insufficient, the thermodynamic energy barrier of mediator reduction conversion cannot be overcome; if the voltage is above 1.2V, it will trigger anodic oxygen evolution and a burst of reactive oxygen species (ROS) free radicals, leading to cell membrane lipid peroxidation and death. The introduction of the mediator transforms mass transfer at the solid-liquid interface of the electrode surface into homogeneous bulk mass transfer. Through the shuttle movement of the redox mediator throughout the liquid within the fermenter, electron transfer, which previously only occurred at the electrode surface (2D), is extended to the entire liquid phase space of the fermenter (3D). This enables 3D spatial mass transfer in electrochemical fermenters, allowing for direct intracellular NADH supplementation across membranes, completely breaking the limits of gas-liquid mass transfer kinetics. In traditional microbial electrosynthesis reactors, bacteria must form a biofilm attached to the cathode surface (2D planar mass transfer) to acquire electrons. This leads to the limitation that the larger the fermenter, the smaller the electrode area per unit volume, making industrial-scale high-density propagation impossible. This solution addresses the long-standing technical challenge of scale-up electrofermentation. Cold shock inhibits intracellular protease activity, allowing osmotic pressure regulators to enter the cell and replace bound water on the enzyme protein surface, forming an amorphous glassy glycoprotein complex network. Simultaneously, the added metal ion cofactors precisely insert into the catalytic site of the RuBisCO enzyme, maintaining its three-dimensional conformation during dehydration and sublimation, preventing inactivation. This invention eliminates the dangerous hydrogen gas source and utilizes a micro-electric field and an electron shuttle (mediator) to construct a three-dimensional electron transport network, increasing the fermentation biomass per unit volume by 3 to 4 times. It solves the problems of Calvin cycle cessation caused by strong acidic gases in industrial flue gas, electron transfer rate far below the theoretical requirement for bacterial division, and poor carbon fixation activity after rehydration due to forced sublimation of free water. As a result, the survival rate of the final prepared bacterial powder after rehydration reaches more than 85%, and the RuBisCO enzyme activity retention rate reaches 85% to 90%, overcoming the problem of low live bacterial activity in traditional freeze-dried preparations. Attached Figure Description

[0017] Figure 1 The present invention provides a complete process flow diagram for the preparation of highly active carbon-fixing bacterial powder based on micro-electric field and flue gas stress.

[0018] Figure 2a This is the structure of the electrochemical fermenter for the high-density propagation stage enhanced by micro-electric field in step 2 of the present invention.

[0019] Figure 2b for Figure 2aA magnified schematic diagram of the microscopic mass transfer (homogeneous electron shuttle) mechanism in the dashed area.

[0020] Figure 3 This is a schematic diagram comparing the microscopic three-dimensional conformation of RuBisCO enzyme in the dehydrated state between the composite induction-locking freeze-drying stage of this invention and existing conventional freeze-drying technology.

[0021] Figure 4 The images show a comparison of the microstructures of the carbon-fixing bacteria after freeze-drying and rehydration in Example 1 at 20.00 kV-20000x.

[0022] Figure 5 The image shows a comparison of the microstructures of the carbon-fixing bacteria in Comparative Example 3 after freeze-drying and rehydration at 20.00 kV-20000x.

[0023] Figure 6 Comparison of the microstructures of carbon-fixing bacteria after freeze-drying and rehydration in Comparative Example 4 at 20.00 kV-20000x.

[0024] In the diagram: 1-Electrochemical fermenter with jacket; 2-Anode; 3-Cathode; 4-Annular gas distributor; 5-Bacteria. Detailed Implementation

[0025] The technical solutions and effects of the present invention will be further described in detail below with reference to specific embodiments and comparative examples. These descriptions are for explanation purposes only and are not intended to limit the scope of the invention.

[0026] A method for directed evolution, high-density propagation, and high-activity preservation of chemoautotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress, such as... Figure 1 As shown, it includes the following steps: S1, the seed culture of chemoautotrophic carbon-fixing bacteria was cultured to the logarithmic growth phase, OD 600 The value is 0.8~1.5. The seed liquid is inoculated into an electrochemical fermenter equipped with anode and cathode at an inoculation rate of 5%~15% (v / v) of the fermentation liquid volume. A constant DC micro voltage of 0.5V-1.2V is applied, and simulated industrial flue gas is introduced into the fermenter at an aeration ratio of 0.1-0.5 vvm for stepwise stress enrichment culture, thereby promoting directional evolution. The component concentrations of the simulated industrial flue gas increased in a stepwise manner with the cultivation time, specifically including the following three stress stages: (1) Acclimatization period (days 1-3): a mixed gas containing 5%-10% (v / v) CO2 and 10-20 ppm SO2 was introduced; (2) Adaptation period (days 4-7): a mixed gas containing 10%-20% (v / v) CO2 and 20-50 ppm SO2 was introduced; (3) High-pressure stress period (days 8-12): a mixed gas containing 20%-30% (v / v) CO2 and 50-100 ppm SO2 was introduced. The remainder of the mixed gas in each stage was nitrogen (N2) and oxygen (O2), with the volume concentration of O2 constantly controlled at 5%-10% to maintain the microaerobic metabolism requirements of the bacteria, and the remainder being N2.

[0027] S2, Inorganic nitrogen source is added to the fermentation broth enriched in step S1 to maintain the C / N ratio of the culture medium at 15:1-20:1. At the same time, a redox mediator with a final concentration of 5-20 μmol / L is added to the fermentation broth. The redox mediator is anthraquinone-2,6-disulfonate, riboflavin or neutral red. High-density propagation fermentation is carried out at a constant temperature of 25-35℃. The total fermentation cycle is 8-12 days. S3, 1-3 hours before the end of the high-pressure fermentation period in step S2, during the optimal time window for inducing cold shock protein (CSP) expression, the fermentation broth temperature is rapidly reduced (cooling rate ≥5℃ / min) and maintained at 12-18℃ for 1-3 hours to induce cold shock. Simultaneously, a complex induction and locking agent is pulsedly added to the fermenter. This agent contains an osmotic pressure regulator and a metal ion cofactor. The osmotic pressure regulator is selected from one or more of trehalose, betaine, tetrahydropyrimidine, and fructooligosaccharides, with a final concentration of 0.1-0.3 M in the fermentation broth. The metal ion cofactor is Mg²⁺. + or Mn² +The final concentration was 5-15 mM. After the cold shock treatment, the bacterial sludge was collected by centrifugation at 4°C. Based on the wet weight of the bacterial sludge, 5%-15% (w / w) of skim milk powder and 0.5%-2.0% (w / w) of modified nano-silica powder were added sequentially and mixed evenly to form a resuspension. The skim milk powder and modified nano-silica powder intertwined around the bacterial membrane to form an extracellular composite protective framework. The resuspension was then placed in a freeze dryer and pre-frozen at a cooling rate of 1-2°C / min at -40°C to -60°C for 2-4 hours (to allow free water to form tiny ice crystals without damaging the cell structure). Subsequently, the solution was frozen under a vacuum of 10-50°C. Sublimation drying at Pa and 15-25℃ for 18-24 hours allows the osmotic pressure regulator and metal cofactor to form a glassy locking network inside the cell, which, together with the extracellular composite protective framework, constructs a spatial dual-protection structure of intracellular chemical locking and extracellular physical isolation, ultimately producing a highly active carbon-fixing bacteria freeze-dried powder. The modified nano-silica prevents large ice crystals from piercing the cells through physical occupancy.

[0028] Example 1: Directed Evolution and High-Density Propagation of Chemoautotrophic Carbon-Fixing Bacteria (Optimal Parameters) Step (1), Vaccination and Initial Preparation: Hookworm copper-loving bacteria were activated in enrichment medium and cultured to the logarithmic growth phase. Seed culture of hookworm copper-loving bacteria in the middle of the logarithmic growth phase was selected, and the cell concentration was increased to OD0.05. 600 = 1.0 (Viable bacteria count approximately 2 × 10⁻⁶) 8 The inoculum (CFU / mL) was introduced into an electrochemical fermenter equipped with a carbon felt cathode and a titanium-ruthenium anode at an inoculum volume of 10% (v / v) of the effective volume of the fermenter. The DC regulated power supply was turned on to apply a constant DC micro-voltage of 0.8V, and the aeration ratio was set to 0.2 vvm.

[0029] Step (2), stepped simulated flue gas stress culture: Simulated industrial flue gas was introduced into the fermenter. The gas contained 8% oxygen (O2) by volume, with the remainder being nitrogen (N2). The concentrations of CO2 and SO2 were controlled in the following three stages: (1) Days 1-3 (acclimatization period): Maintain the CO2 concentration in the introduced gas at 5% and the SO2 concentration at 10 ppm; (2) Days 4-7 (acclimatization period): Increase and maintain the CO2 concentration in the introduced gas at 15%, and increase and maintain the SO2 concentration at 30 ppm; (3) Days 8-12 (high pressure stress period): Increase the CO2 concentration in the gas and apply maximum pressure to 25%, and increase the SO2 concentration and maintain it at 80 ppm.

[0030] Through the aforementioned stepwise pressure application, weak cells sensitive to toxic gases are eliminated, and mutant bacterial groups with extremely strong tolerance are screened and selectively enriched.

[0031] Step (3), cold shock activation and double-layer skeleton confined freeze-drying preservation: Ammonium sulfate was added to the fermentation broth enriched in step (2) to maintain a C / N ratio of 18:1. At the same time, sodium anthraquinone-2,6-disulfonic acid (AQDS) with a final concentration of 15 μmol / L was added as a redox mediator. The broth was cultured at a constant temperature of 30°C for a total fermentation period of 12 days.

[0032] Two hours before the end of fermentation, the fermenter jacket cooling system was activated to rapidly lower the fermentation broth temperature from 30°C to 15°C, stimulating the bacteria to express cold shock proteins. Simultaneously, a complex induction-locking agent (betaine and MgSO4) was added in a pulsed manner to achieve the following final concentrations in the fermentation broth: trehalose 0.2 M, Mg²⁺... + 10 mM.

[0033] After a 2-hour cold shock, the fermentation broth was centrifuged at 4°C and 6000 rpm for 10 minutes. The concentrated bacterial sludge at the bottom was collected by centrifugation. Based on the mass ratio of the bacterial sludge's wet weight, 10% skim milk powder and 1% modified nano-silica powder were added as a framework material. The mixture was gently homogenized using a homogenizer, allowing the nano-silica and skim milk powder to interweave around the bacteria to form an extracellular organic-inorganic composite framework. This extracellular composite framework, together with the amorphous glassy network formed earlier inside the cells, constitutes a complete spatial double-protection structure. The resuspended mixture was then spread into a freeze-drying tray and placed in a freeze dryer. The pre-freezing program was set as follows: the temperature was lowered to -50°C at a cooling rate of 1°C / min and held at this temperature for 3 hours to allow free water to form tiny ice crystals without damaging the cell structure. Subsequently, the vacuum pump was turned on, and the mixture was sublimated and dried for 20 hours under a vacuum of 20 Pa and a slow increase in the partition temperature to 20°C, ultimately yielding a highly active carbon-fixing bacterial freeze-dried powder.

[0034] Example 2 (lower limit of parameters and alternative materials) Step (1): Select the seed culture of Alcaligenes in the late logarithmic growth phase, and wait until the cell concentration reaches OD200. 600 = 1.2 (Viable bacteria count approximately 3 × 10⁻⁶) 8 CFU / mL), inoculated at 8% (v / v) of the effective volume of the fermenter into an electrochemical fermenter equipped with a carbon felt cathode and a titanium ruthenium anode, applied with a constant DC voltage of 1.2V, and aerated at a gas flow rate of 0.25 vvm (containing 5% oxygen by volume, with the remainder being nitrogen) for stress enrichment culture and evolution: (1) Days 1-3 (acclimatization period): Maintain the CO2 concentration in the introduced gas at 5% and the SO2 concentration at 10 ppm; (2) Days 4-7 (acclimatization period): Increase and maintain the CO2 concentration in the introduced gas at 15%, and increase and maintain the SO2 concentration at 30 ppm; (3) Days 8-12 (high pressure stress period): Increase the CO2 concentration in the gas and apply maximum pressure to 25%, and increase the SO2 concentration and maintain it at 80 ppm.

[0035] Step (2): Ammonium sulfate is added to the fermentation broth enriched in step (1) to maintain the C / N ratio at 18:1. At the same time, riboflavin with a final concentration of 5 μmol / L is added as a redox mediator. The broth is cultured at a constant temperature of 30℃ for a total fermentation period of 12 days.

[0036] Step (3): 3 hours before the end of fermentation, the temperature is suddenly dropped and maintained at 18°C, and a composite induction and locking agent with a final concentration of 0.1 M tetrahydropyrimidine and 5 mM MnSO4 is added in a single pulse.

[0037] Step (4): After a 3-hour cold shock, the fermentation broth was centrifuged at 4°C and 6000 rpm to collect the bacterial sludge. Based on the mass ratio of the bacterial sludge's wet weight, 10% skim milk powder and 1% modified nano-silica skeleton material were added. The mixture was gently homogenized using a homogenizer, allowing the nano-silica and skim milk powder to intertwine around the bacteria, forming an extracellular organic-inorganic composite skeleton. This extracellular composite skeleton, together with the amorphous glassy network formed earlier inside the cells, constitutes a complete spatial dual-protection structure. The resuspended mixture was then spread into a freeze-drying tray and placed in a freeze dryer. The pre-freezing program was set as follows: the temperature was lowered to -50°C at a rate of 1°C / min and held for 3 hours to allow free water to form tiny ice crystals without damaging the cell structure. Subsequently, the vacuum pump was turned on, and the mixture was sublimated and dried for 20 hours under a vacuum of 20 Pa and a slow increase in the partition temperature to 20°C, ultimately yielding highly active carbon-fixing bacterial freeze-dried powder.

[0038] Example 3 (Parameter Upper Limits and Alternative Materials) Step (1): Select Rhodococcus seed culture in the late logarithmic growth phase, and wait until the cell concentration reaches OD200. 600 = 1.5, inoculated into an electrochemical fermenter equipped with a carbon felt cathode and a titanium ruthenium anode at an inoculation rate of 15% (v / v) of the effective volume of the fermenter, applied a constant DC voltage of 1.2V, and aerated the fermenter at an aeration ratio of 0.2 vvm (containing 10% oxygen by volume and the remainder nitrogen) for stress enrichment culture.

[0039] (1) Days 1-3 (acclimatization period): Maintain the CO2 concentration in the introduced gas at 5% and the SO2 concentration at 10 ppm; (2) Days 4-7 (acclimatization period): Increase and maintain the CO2 concentration in the introduced gas at 15%, and increase and maintain the SO2 concentration at 30 ppm; (3) Days 8-12 (high pressure stress period): Increase the CO2 concentration in the gas and apply maximum pressure to 25%, and increase the SO2 concentration and maintain it at 80 ppm.

[0040] Step (2): Ammonium sulfate is added to the fermentation broth enriched in step (1) to maintain the C / N ratio at 18:1. At the same time, neutral red with a final concentration of 20 μmol / L is added as a redox mediator. The broth is cultured at a constant temperature of 30℃ for a total fermentation period of 8 days.

[0041] Step (3): One hour before the end of fermentation, the temperature is suddenly dropped and maintained at 12°C. A composite inducing and locking agent with a final concentration of 0.3 M fructooligosaccharide and 15 mM MgCl2 is added in a single pulse.

[0042] Step (4): After a 1-hour cold shock, the fermentation broth was centrifuged at 4°C and 6000 rpm to collect the bacterial sludge. Based on the mass ratio of the bacterial sludge's wet weight, 10% skim milk powder and 1% modified nano-silica skeleton material were added. The mixture was gently homogenized using a homogenizer, allowing the nano-silica and skim milk powder to intertwine around the bacteria, forming an extracellular organic-inorganic composite skeleton. This extracellular composite skeleton, together with the amorphous glassy network formed intracellularly previously, constitutes a complete spatial dual-protection structure. The resuspended mixture was then spread into a freeze-drying tray and placed in a freeze dryer. The pre-freezing program was set as follows: the temperature was lowered to -50°C at a rate of 1°C / min and held for 3 hours to allow free water to form tiny ice crystals without damaging the cell structure. Subsequently, the vacuum pump was turned on, and the mixture was sublimated and dried for 20 hours under a vacuum of 20 Pa and a slow increase in the partition temperature to 20°C, ultimately yielding highly active carbon-fixing bacterial freeze-dried powder.

[0043] Comparative Example 1 (Traditional Process Benchmark) Fermentation was carried out using the same hookworm copper-loving bacteria seed liquid as in Example 1, but in steps (1) and (2), electrodes were not installed, a micro electric field was not applied, and the redox mediator AQDS was not added. The culture was carried out under conventional conditions of 30°C and 0.2 vvm aeration until the end. In step (3), no cooling operation is performed (maintain 30°C), and no composite induction locking agent trehalose and MgSO4 are added. The bacterial sludge is collected directly by centrifugation, and only 10% skim milk powder and 1% modified nano silica are added before vacuum freeze drying.

[0044] Comparative Example 2 (Missing mediator features, verifying mass transfer deadlock) This comparative example aims to verify the necessity of redox mediators.

[0045] Steps (1) and (3) are exactly the same as in Example 1; however, in step (2), although a micro electric field of 0.8V is maintained, no redox mediator is added (mediator concentration is 0), which causes electrons to be transferred only on the physical surface of the electrode. The other conditions are the same as in Example 1.

[0046] Comparative Example 3 (conventional freeze-dried group, lacking locking features, verifying phase transition collapse) This comparative example aims to verify the necessity of enzyme conformation locking in step (3).

[0047] Steps (1) and (2) are exactly the same as in Example 1; however, before the end of fermentation in step (3), no sudden cooling operation is performed (maintaining 30°C), and no trehalose or MgSO4 is added. High-density bacterial sludge is collected directly by centrifugation, and after adding 10% skim milk powder and 1% modified nano silica skeleton material, it is vacuum freeze-dried.

[0048] Comparative Example 4 (Proof by contradiction regarding out-of-bounds parameters) This comparative example aims to verify the critical significance of the upper limit of 1.2V for the micro-voltage. Except for applying a DC voltage of 1.5V in step (1) (exceeding the upper limit of 1.2V), the subsequent steps are exactly the same as in Example 1.

[0049] Key performance indicator measurement basis and method description To ensure the accuracy and repeatability of the experimental data of this invention, the specific measurement methods for each indicator in Table 1 are as follows: 1. Fermentation endpoint biomass (dry weight, g / L): constant weight method was used. Take 10 mL of fermentation broth, centrifuge at 8000 rpm for 10 minutes, discard the supernatant, wash twice with sterile physiological saline, and dry in a 105℃ oven until constant weight. Weigh and calculate the total weight of stem cells per unit volume of fermentation broth.

[0050] 2. Rehydration survival rate (%) after freeze-drying: The plate coating count method (CFU) was used. Rehydration survival rate = (number of viable bacteria after rehydration of freeze-dried bacterial powder (CFU / g)) / (number of viable bacteria in the bacterial sludge before freeze-drying (CFU / g)) × 100%.

[0051] 3. RuBisCO enzyme activity retention rate (%) after rehydration: Determined using the spectrophotometric method (NADH oxidative coupling method) described by Geiger et al., or a commercially available RuBisCO enzyme activity assay kit. The rate of decrease in NADH absorbance was monitored at 340 nm to characterize the enzyme reaction rate. Activity retention rate = (specific enzyme activity after rehydration of lyophilized bacterial powder) / (specific enzyme activity extracted from the fermentation broth before lyophilization) × 100%.

[0052] 4. Flue gas CO2 fixation rate (mg / L / h): Gas chromatography (GC) was used. A fixed amount of rehydrated activated bacterial solution was placed in a sealed reaction bottle, and a CO2 / SO2 mixed gas of a set concentration was introduced. Headspace gas was extracted at set time intervals, and the decrease in CO2 concentration was measured by a gas chromatograph equipped with a TCD detector. The CO2 fixation per unit time and per unit volume of fermentation broth was calculated.

[0053] The data for each of the above groups were statistically analyzed, and the results are shown in Table 1: Table 1 Comparison of Technical Effects of Various Embodiments and Comparative Examples

[0054] Data Analysis: Comparing Example 1 with Comparative Examples 1 and 2, it can be seen that relying solely on electrode surface mass transfer (Comparative Example 2), the biomass only slightly increases to 6.2 g / L; while the micro-electric field of the present invention combined with the redox mediator (Example 1) completely breaks the gas-liquid / solid-liquid mass transfer polarization, and the biomass jumps to 18.5 g / L, proving the extremely strong synergistic effect of the front-end process of the present invention.

[0055] Comparing Example 1 and Comparative Example 3, it can be seen that although Comparative Example 3 showed excellent growth at the front end (18.2 g / L), due to the lack of cooling and locking agent intervention, the RuBisCO enzyme underwent a thermodynamic phase transition during dehydration, resulting in an activity of only 28.5% and a carbon fixation rate as low as 95.2 mg / L / h. The present invention significantly increases the enzyme activity retention rate from 28.5% to 89.5%, producing a significant improvement effect.

[0056] Comparing Example 1 and Comparative Example 4, it can be seen that when the voltage exceeds the upper limit to 1.5V, strong ROS lipid peroxidation causes large-scale cell death (biomass of only 2.1g / L), proving that the 1.2V critical value set in this invention is irreplaceable and necessary.

[0057] like Figure 2a As shown, the electrochemical fermenter of the present invention includes a jacketed electrochemical fermenter 1, an anode 2, a cathode 3, and an annular gas distributor 4. Under the action of a micro-electric field of 0.5-1.2V, as... Figure 2b As shown, the added redox mediator (taking AQDS as an example) captures electrons on the surface of cathode 3, becoming a reduced-state mediator. Subsequently, it actively migrates to the cell membrane of bacteria 5 through liquid-phase mass transfer, releasing electrons and reverting to the oxidized state. This homogeneous electron shuttle mechanism completely breaks the mass transfer area deadlock in traditional electrochemical fermentation, where bacteria must physically attach to the electrode surface to acquire electrons, achieving high-density liquid propagation throughout the entire tank volume.

[0058] Figure 3The left image is a schematic diagram of conformational collapse caused by the stripping of the hydration layer in the prior art. It shows that during the conventional freeze-drying process, due to the large-scale removal of bound water molecules, RuBisCO enzyme 10 loses the support of the hydration layer, resulting in the closure of its catalytic active center 11 and irreversible collapse and denaturation of its secondary structure. The right image is a schematic diagram of the conformation maintained by the mechanical locking of the cofactor and the glassy network in this invention. It shows that this invention uses pulsed injection of osmotic pressure regulator 12 (trehalose / betaine) and metal cofactor 13 (Mg²⁺). + / Mn² + In this process, osmolar regulator 12 forms a dense amorphous glassy network around the enzyme, replacing the hydration layer, while metal cofactor 13 precisely embeds and mechanically holds the catalytic center 11 in place. This dual protection mechanism creates a synergistic anchoring effect of extracellular encapsulation and intracellular cross-linking. The extracellular organic-inorganic composite framework forms a dense physical barrier, resisting the mechanical shearing and osmotic pressure shocks of the external ice crystal microenvironment; while the intracellular glassy network and cofactor support components construct a supporting structure within the cell / periplasmic space, maintaining the native active conformation of the core enzyme system, thus achieving extremely high activity retention after rehydration.

[0059] To further reveal the protective mechanism of the process of this invention at the cellular microstructure level, combined with Figure 4 , Figure 5 and Figure 6 As shown, the bacterial cells of Example 1 and the key comparative examples 3 and 4 after freeze-drying and rehydration were observed using scanning electron microscopy (SEM, magnification 20,000x). The specific microscopic morphology analysis is as follows: like Figure 4 As shown, the carbon-fixing bacteria treated in step 3 of this invention (pulse injection of the composite induction-locking agent and freeze-drying) exhibit extremely plump and regular short rod-shaped outlines after rehydration. The cell surface is smooth, without obvious mechanical damage or shrinkage marks, and the cell membrane has high integrity. This indicates that the osmotic pressure regulator in this invention not only regulates osmotic pressure in the liquid state, but also successfully constructs a robust amorphous glassy network on the cell membrane surface and around intracellular proteins during the freeze-drying process. This water-retaining skeletal structure effectively resists the enormous physical stress generated by ice crystal piercing and vacuum suction.

[0060] and Figure 4 In stark contrast, such as Figure 5As shown, the bacterial cells produced by the traditional freeze-drying process (without implementing the locking step 3 of this invention) exhibit severe deep wrinkles on their cell surface, with an overall shriveled and shrunken appearance, resembling typical raisins. They also show extensive tearing and folding of the cell membrane. This is because, during the vacuum sublimation process, due to the lack of combined protection from the osmotic pressure regulator and metal cofactor described in this invention, the bound water is forcibly extracted, and the hydration layer is completely stripped away, causing the cell wall and cell membrane to lose their mechanical support. Consequently, irreversible physical damage occurs under rapid changes in osmotic pressure.

[0061] like Figure 6 As shown, when the micro-electric field voltage in step 1 exceeds the upper limit threshold of this invention by 1.5V, the microstructure of the bacteria exhibits typical electrolethal characteristics. Irreversible micron-sized electroporation pores are densely distributed on the cell surface, and even intracellular flocculent material (cytoplasm, nucleic acid, and protein) can be seen leaking out of the cell in large quantities through the pores, proving that the excessively high voltage directly breaks down the insulating structure of the phospholipid bilayer, and the excessive ROS (reactive oxygen species) generated by hydrolysis causes severe peroxidative cleavage damage to the cell membrane.

Claims

1. A method for preparing freeze-dried powder of highly active autotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress, characterized in that, Includes the following steps: S1, the seed culture of chemoautotrophic carbon-fixing bacteria is enriched by passing simulated industrial flue gas through it under a constant voltage of 0.5-1.2V. The concentrations of CO2 and SO2 in the simulated industrial flue gas increase stepwise with the culture time, and the remainder is nitrogen and oxygen. S2, an inorganic nitrogen source is added to the fermentation broth enriched in S1 to maintain the C / N ratio of the culture medium. At the same time, a redox mediator with a final concentration of 5-20 μmol / L is added to the fermentation broth. The fermentation is carried out at a constant temperature for 8-12 days. Before the end of the fermentation, the fermentation broth is subjected to cold shock treatment. At the same time, an osmotic pressure regulator and a metal ion cofactor are added in a pulse. After the cold shock treatment, the mixture is centrifuged to obtain the bacterial sludge. S3. Skim milk powder and modified nano-silica powder are added to the bacterial sludge and mixed evenly to obtain a resuspended liquid. The resuspended liquid is pre-frozen and then sublimated and dried to obtain a highly active autotrophic carbon-fixing bacteria freeze-dried powder.

2. The method for preparing freeze-dried powder of highly active autotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress according to claim 1, characterized in that, S1 was cultured with chemoautotrophic carbon-fixing bacteria seed culture to the logarithmic growth phase, OD 600 The value is 0.8~1.5, and the seed liquid is then inoculated into an electrochemical fermenter for enrichment culture at an inoculation rate of 5%~15% of the fermentation liquid volume.

3. The method for preparing freeze-dried powder of highly active autotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress according to claim 1, characterized in that, In S1, the ventilation ratio of the simulated industrial flue gas is 0.1-0.5 vvm.

4. The method for preparing freeze-dried powder of highly active autotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress according to claim 1, characterized in that, The enrichment culture in S1 lasted for 8–12 days. On days 1–3, the concentrations of CO2 and SO2 were 5%–10% and 10–20 ppm, respectively. On days 4–7, the concentrations of CO2 and SO2 were 10%–20% and 20–50 ppm, respectively. On days 8–12, the concentrations of CO2 and SO2 were 20%–30% and 50–100 ppm, respectively.

5. The method for preparing freeze-dried powder of highly active autotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress according to claim 1, characterized in that, The redox mediator mentioned in S2 is anthraquinone-2,6-disulfonate, riboflavin, or neutral red.

6. The method for preparing freeze-dried powder of highly active autotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress according to claim 1, characterized in that, S2 involves a sudden drop in the temperature of the fermentation broth 1-3 hours before the end of fermentation, followed by a cold shock treatment at 12-18°C for 1-3 hours.

7. The method for preparing freeze-dried powder of highly active autotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress according to claim 1, characterized in that, The osmotic pressure regulator described in S2 is one or more of trehalose, betaine, tetrahydropyrimidine, and fructooligosaccharides, with a final concentration of 0.1-0.3 M in the fermentation broth, and the metal ion cofactor is Mg²⁺. + or Mn² + The final concentration is 5-15 mM.

8. The method for preparing freeze-dried powder of highly active autotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress according to claim 1, characterized in that, In S3, the mass of skim milk powder and modified nano silica powder are 5%~15% and 0.5%~2.0% of the bacterial sludge, respectively.

9. The method for preparing freeze-dried powder of highly active autotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress according to claim 1, characterized in that, The pre-freezing treatment described in S3 is carried out at -60℃ to -40℃ for 2 to 4 hours, and the sublimation drying is carried out at a vacuum of 10 to 50 Pa and a temperature of 15 to 25℃ for 18 to 24 hours.

10. A freeze-dried powder of highly active autotrophic carbon-fixing bacteria obtained by the preparation method of the freeze-dried powder of highly active autotrophic carbon-fixing bacteria based on micro-electric field and flue gas stress as described in any one of claims 1 to 9.

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