Methanobacteria / nano-material hybrid biological membrane system construction and reinforcement method

Abstract

The invention discloses a method for constructing and reinforcing a hybrid biological membrane system composed of methane bacteria / carbon nitride. According to the method, a hybrid system composed of methanogens and an extracellular polymeric substance (EPS) semiconductor material (modified graphite-phase carbon nitride) is constructed, and the hybrid system is used for preparing the carbon nitride-modified graphite-phase carbon nitride hybrid system. The carbon nitride-modified graphite-phase carbon nitride hybrid system has the advantages that the carbon nitride-modified graphite-phase carbon nitride hybrid system is used for preparing the carbon nitride-modified graphite-phase carbon nitride hybrid system, and the carbon nitride-modified graphite-phase carbon nitride hybrid system; the extracellular polymeric substance in the biological membrane obviously improves the light energy utilization efficiency and the operation stability of the system; nano conductive particles are introduced to the surfaces of methane bacteria cells, so that the utilization rate of extracellular electrons by the cells is improved. The method effectively solves the problems of low efficiency, high energy consumption, low product selectivity and the like in the process of reducing carbon dioxide into methane in a traditional biological hybrid system, and has the advantages of simple and convenient process, low energy consumption and high product selectivity.

Description

Technical Field

[0001] This invention relates to a method for constructing and enhancing a hybrid biofilm system composed of methanogens and nano-carbon nitride. Background Technology

[0002] To address the energy shortage challenge brought about by the depletion of fossil fuels, it is essential to fully utilize renewable energy sources in nature, with obtaining energy from the sun being one of the best options. Compared to chemical catalysts, microbial systems can convert energy from sunlight in a more complex and efficient manner; however, microorganisms have relatively low light-harvesting efficiency. Recent research has shown that non-photosynthetic microorganisms can metabolize by acquiring photoelectron energy generated by solar photocatalytic semiconductor materials. This microbial hybrid photosynthetic system (semi-artificial photosynthetic system), which effectively combines the highly selective and efficient metabolism of microorganisms with the superior light-harvesting properties of inorganic semiconductors, has enormous potential for CO2 reduction.

[0003] Although many systems discovered so far can achieve selective CO2 conversion, they exhibit some significant drawbacks in experiments. For example, microbial hybrid suspension systems suffer from severe light attenuation in their turbid liquid phase, leading to low efficiency in the utilization of light energy by the catalyst and microbial cells; the low-wavelength ultraviolet light typically used to excite photoelectrons in the catalyst can easily damage microorganisms, preventing the system from operating stably for extended periods; the quantum efficiency of microbial hybrid systems is currently low; and they are difficult to design into intensive reactors with practical value. Summary of the Invention

[0004] The purpose of this invention is to address the numerous shortcomings of current microbial / photocatalytic hybrid systems, such as low light utilization efficiency, low quantum yield, poor microbial activity, and unsuitability for constructing intensive reactors. This invention proposes a method for constructing and enhancing a hybrid biofilm system composed of methanogens and nano-carbon nitride. This method offers advantages such as high light energy utilization efficiency, high quantum yield, stable microbial activity, and methanogenic performance, demonstrating significant potential for developing practical carbon dioxide reduction and methanogenesis devices.

[0005] The purpose of this invention is to provide a method for constructing and enhancing a hybrid biofilm system composed of methanogens and nano-carbon nitride.

[0006] The technical solution adopted in this invention is as follows:

[0007] A method for constructing a hybrid biofilm system composed of methanogens / nano-carbon nitride includes the following steps:

[0008] Surface-modified carbon nitride materials exhibit good affinity for methanogenic bacteria cells and extracellular polymers.

[0009] Methanogens, when cultured in a medium rich in organic nitrogen and carbon sources, develop abundant extracellular polymers. Adding exogenous polymers after cell culture achieves the same effect.

[0010] Nanoparticles are added during the methanogenic bacteria culture process, so that the surface of the methanogenic bacteria cells is covered with nanoparticles after the culture is completed.

[0011] Methanogens, extracellular polymers, and carbon nitride materials are combined under anaerobic conditions to form a stable biofilm with a three-dimensional structure.

[0012] Light is applied to the side of the biofilm.

[0013] A method for constructing a hybrid biofilm reactor includes the following steps:

[0014] The hybrid biomembrane was attached to a porous gel to construct a hybrid biomembrane / porous transparent substrate reactor unit.

[0015] The hybrid biomembrane / porous transparent substrate reactor units are combined in series / parallel and carbon dioxide is introduced to achieve large-scale reactions;

[0016] Preferably, the graphitic carbon nitride is modified and then grafted with cyanamide groups or carboxyl groups on the material surface, or the modified carbon nitride surface has a positive Zeta potential.

[0017] Preferably, the culture medium for culturing methanogens contains both organic carbon and organic nitrogen sources. The culture medium formulation is as follows:

[0018]

[0019]

[0020] Each liter of FeSO4·7H2O solution (0.1% w / v) contains 1 g FeSO4·7H2O and 1000 ml of 0.05 mol / L H2SO4.

[0021] Each liter of trace element solution SL-10 contains 10 ml of 25% HCl, 1.35 g of FeCl2·4H2O, 63 mg of ZnCl2, 90 mg of MnCl2·4H2O, 5.4 mg of H3BO3, 171 mg of CoCl2·6H2O, 1.8 mg of CuCl2·2H2O, 21.6 mg of NiCl2·6H2O, and 32.4 mg of Na2MoO4·2H2O;

[0022] Each liter of Wolin vitamin solution (10x concentration) contains 20.00 mg biotin, 20.00 mg folic acid, 100.00 mg pyridoxine hydrochloride, 50.00 mg thiamine hydrochloride, 50.00 mg riboflavin, 50.00 mg niacin, 50.00 mg D-calcium pantothenate, 1.00 mg vitamin B12, 50.00 mg para-aminobenzoic acid, and 50.00 mg (DL)-α-lipoic acid;

[0023] Preferably, the particle size of the nano-conductive particles added to the system is 2-10 nm, and the characteristic absorbance value of the nano-conductive particles corresponding to an added concentration of 10 mL of bacterial solution is 0.25-0.85 Units / ml.

[0024] Preferably, the nano-conductive particles added to the system shown are at least one of nano-gold, nano-silver, nano-copper, nano-copper oxide, and carbon quantum dots.

[0025] Preferably, the conductive nanoparticles are uniformly distributed on the surface of the cell as sites for electron transport.

[0026] Preferably, the thickness of the stable hybrid biofilm is between 10 μm and 300 μm.

[0027] Preferably, the light intensity range is 0.01 mW / cm². 2 -0.2mW / cm 2 .

[0028] Preferably, the concentration of extracellular proteins in the hybrid biological membrane is 1-15 μg / mL, and the concentration of extracellular polysaccharides is 10-150 μg / mL;

[0029] Preferably, the key component material of the porous gel is at least one of cellulose nanofibers, cellulose nanocrystals, and bacterial cellulose.

[0030] Preferably, according to the method of claim 2, the porous gel material has a pore size of 10 μm-500 μm and a porosity of 80%-100%.

[0031] Preferably, the presence of the gel simultaneously enhances the efficiency of light transmission and solute transport, thereby strengthening the carbon dioxide conversion efficiency of the hybrid biomembrane attached thereto.

[0032] Preferably, the reactor units are connected in series or in parallel to achieve large-scale application.

[0033] The beneficial effects of this invention are as follows:

[0034] A method for constructing and enhancing a hybrid biofilm system composed of methanogens and nano-carbon nitride has been developed, solving the problems of low light energy utilization efficiency and susceptibility to oxidative damage under ultraviolet light in microbial / semiconductor hybrid systems, while simultaneously improving quantum yield and product selectivity. The constructed hybrid biofilm / transparent gel reactor with efficient light and solute transport provides a solid foundation for large-scale applications. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the construction and enhancement process of the present invention.

[0036] Figure 2 This is a schematic diagram of the microstructure of the methanogenic bacteria / carbon nitride hybrid biofilm of the present invention.

[0037] Figure 3 This is a schematic diagram showing the methane production of the methanogenic bacteria / carbon nitride hybrid biofilm of the present invention under different ultraviolet light irradiation.

[0038] Figure 4 This is a schematic diagram of the methane quantum yield of the methanogenic bacteria / carbon nitride hybrid biomembrane of the present invention under different ultraviolet light irradiation.

[0039] Figure 5 This is a schematic diagram of the methane selectivity of the methanogenic bacteria / carbon nitride hybrid biofilm of the present invention under different ultraviolet light irradiation.

[0040] Figure 6 This is a schematic diagram illustrating the stability of the methane production performance of the methanogenic bacteria / carbon nitride hybrid biofilm of the present invention under high / low ultraviolet irradiation.

[0041] Figure 7 This is a schematic diagram of the methane generation performance of the methanogenic bacteria / carbon nitride hybrid biofilm of the present invention after the addition of nano-conductive particles.

[0042] Figure 8 This is a schematic diagram showing the distribution of various materials on the surface of methanogenic bacteria cells after the addition of nano-conductive particles to the methanogenic bacteria / carbon nitride hybrid biofilm of the present invention.

[0043] Figure 9 This is a schematic diagram illustrating the reaction between extracellular polymers and reactive oxygen species in the methanogenic / carbon nitride hybrid biofilm of the present invention.

[0044] Figure 10 This is a schematic diagram illustrating the level of oxidative stress experienced by cells in the methanogenic / carbon nitride hybrid biomembrane of the present invention under the protection of extracellular polymers.

[0045] Figure 11 This is a schematic diagram illustrating the mechanical stability of the methanogen / carbon nitride hybrid biofilm of the present invention in maintaining its morphology under hydraulic shear.

[0046] Figure 12This is a schematic diagram comparing the methanogenic performance of the methanogenic bacteria / carbon nitride hybrid biofilm of the present invention under light irradiation after the exogenous addition of extracellular polymers with that of the control group without the addition of extracellular polymers.

[0047] Figure 13 This is a schematic diagram comparing the conductivity of the methanogenic bacteria / carbon nitride hybrid biofilm of the present invention with and without the addition of nano-conductive particles.

[0048] Figure 14 This invention provides a comparative study on the selectivity of methane production by methanogenic bacteria / carbon nitride hybrid biofilms under extracellular polymer protection. Detailed Implementation

[0049] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0050] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0051] The formulation of the culture medium containing organic nitrogen and organic carbon described in the embodiments of the present invention is shown in Table 1.

[0052] Table 2 Culture medium formulation

[0053]

[0054]

[0055] Each liter of FeSO4·7H2O solution (0.1% w / v) contains 1 g FeSO4·7H2O and 1000 mL of 0.05 mol / L H2SO4.

[0056] Each liter of trace element solution SL-10 contains 10 mL of 25% HCl, 1.35 g of FeCl2·4H2O, 63 mg of ZnCl2, 90 mg of MnCl2·4H2O, 5.4 mg of H3BO3, 171 mg of CoCl2·6H2O, 1.8 mg of CuCl2·2H2O, 21.6 mg of NiCl2·6H2O, and 32.4 mg of Na2MoO4·2H2O;

[0057] Each liter of Wolin vitamin solution (10x concentration) contains 20.00 mg biotin, 20.00 mg folic acid, 100.00 mg pyridoxine hydrochloride, 50.00 mg thiamine hydrochloride, 50.00 mg riboflavin, 50.00 mg niacin, 50.00 mg D-calcium pantothenate, 1.00 mg vitamin B12, 50.00 mg para-aminobenzoic acid, and 50.00 mg (DL)-α-lipoic acid;

[0058] Example 1

[0059] (1) Inoculate methanogens at a volume ratio of 5% into a culture medium with a pH of 6.9 and incubate at a constant temperature of 35°C. (2) Wait until the above culture medium reaches the OD value. 600 When the value is >0.5, centrifuge at 7500 rpm / min, wash three times with PBS buffer, and resuspend in 20 mL of PBS buffer solution.

[0060] (3) Add 0.06g of modified graphitic carbon nitride to the cell suspension above after ultrasonic treatment for 10min, shake well and let stand for 24h.

[0061] (4) Add the above hybrid solution to the bottle of new culture medium and let it stand for 24 hours. After the biofilm formed is stably attached to the bottom of the bottle, wrap the bottle with light-proof tape around the body, and only use the outer side of the biofilm at the bottom as the surface to receive light.

[0062] (5) The bottle containing the hybrid biomembrane was placed at 395 nm and 0.1 mW / cm at 35 °C. 2 -1.2mW / cm 2 The reaction begins under light.

[0063] A comparative analysis was conducted on hybrid biomembrane systems that received light and those that did not, as well as on hybrid biomembranes exposed to different light intensities. The results showed that at 0.1 mW / cm², the optimal light intensity was [not specified]. 2 The hybrid biomembrane under illumination exhibited the best quantum yield, reaching approximately 1.5%. Figure 4 As light intensity increases, the amount of methane produced gradually increases (…). Figure 3 However, the quantum yield gradually decreases. Figure 4 This demonstrates that hybrid biomembranes exhibit good light transmission and biotransformation efficiency at low light intensities. Figure 2 ).

[0064] Example 2

[0065] (2) Inoculate methanogens at a volume ratio of 5% into a culture medium with a pH of 6.9 and incubate at a constant temperature of 35°C. (3) Add nano-conductive particles to the cell culture medium.

[0066] (2) Wait until the above culture medium reaches OD 600 When the value is >0.5, centrifuge at 7500 rpm / min, wash three times with PBS buffer, and resuspend in 20 mL of PBS buffer solution.

[0067] (3) Add 0.06g of modified graphitic carbon nitride to the cell suspension above after ultrasonic treatment for 10min, shake well and let stand for 24h.

[0068] (4) Add the above hybrid solution to the bottle of new culture medium and let it stand for 24 hours. After the biofilm formed is stably attached to the bottom of the bottle, wrap the bottle with light-proof tape around the body, and only use the outer side of the biofilm at the bottom as the surface to receive light.

[0069] (5) The bottle containing the hybrid biomembrane was placed at 395 nm and 0.1 mW / cm at 35 °C. 2 The reaction begins under light.

[0070] The results showed that the added conductive nanoparticles during the culture process did not significantly affect cell viability, and the added conductive nanoparticles adhered to the cell surface. Figure 8 In terms of methanogenesis performance, the hybrid biomembrane with added conductive nanoparticles showed an approximately 30% improvement in methanogenesis performance compared to the one without. Figure 7 The corresponding quantum conversion efficiency also increased by about 30%. The conductivity of the hybrid biomembrane increased by about 20%. Figure 13 )

[0071] Example 3

[0072] (4) Inoculate the methanogens at a volume ratio of 5% into a culture medium with a pH of 6.9 and incubate at a constant temperature of 35°C. (2) Wait until the above culture medium reaches the OD value. 600 When the value is >0.5, centrifuge at 7500 rpm / min, wash three times with PBS buffer, and resuspend in 20 mL of PBS buffer solution.

[0073] (3) Add 0.06g of modified graphitic carbon nitride to the cell suspension above after ultrasonic treatment for 10min, shake well and let stand for 24h.

[0074] (4) Add the above hybrid solution to the bottle of new culture medium and let it stand for 24 hours. After the biofilm formed is stably attached to the bottom of the bottle, wrap the bottle with light-proof tape around the body, and only use the outer side of the biofilm at the bottom as the surface to receive light.

[0075] (5) The bottle containing the hybrid biomembrane was placed at 395 nm and 0.1 mW / cm at 35 °C. 2and 1.2mW / cm 2 The reaction begins under light.

[0076] (6) On the seventh day of the reaction, change the culture medium and start light exposure again.

[0077] The results showed that the hybrid biomembrane exhibited higher methane production performance under higher light intensities during the first 7-day cycle compared to lower light intensities. However, in the second 7-day cycle, the methane production performance under high light intensities showed a significant decline, due to cell damage caused by high-intensity ultraviolet radiation. Conversely, at 0.1 mW / cm², the performance under higher light intensities was significantly reduced. 2 Under illumination, the methanogenesis performance in the second cycle remained above 90% of that in the first cycle, demonstrating the stability of the hybrid biomembrane's methanogenesis performance under light intensity. This is attributed to the protective effect of abundant EPS in the hybrid biomembrane against cellular oxidative stress. Figure 10 )

[0078] Example 4

[0079] (5) Inoculate the methanogens at a volume ratio of 5% into a culture medium with a pH of 6.9 and incubate at a constant temperature of 35°C. (2) Wait until the above culture medium reaches the OD value. 600 When the value is >0.5, centrifuge at 7500 rpm / min, wash three times with PBS buffer, and resuspend in 20 mL of PBS buffer solution.

[0080] (3) Add 0.06g of modified graphitic carbon nitride to the cell suspension above after ultrasonic treatment for 10min, shake well and let stand for 24h.

[0081] (4) Add the above hybrid solution to the bottle of new culture medium and let it stand for 24 hours. After the biofilm formed is stably attached to the bottom of the bottle, wrap the bottle with light-proof tape around the body, and only use the outer side of the biofilm at the bottom as the surface to receive light.

[0082] (5) Add exogenous extracellular polymers to the experimental group and add deionized water to the control group to replenish the volume.

[0083] (6) The bottle containing the hybrid biomembrane was placed at 395 nm and 2.7 mW / cm at 35 °C. 2 The reaction begins under light.

[0084] The results showed that the experimental group with added exogenous extracellular polymeric substances exhibited better methanogenesis performance. This is because exogenous extracellular polymeric substances reduced the consumption of reactive oxygen species under ultraviolet light, thus alleviating the degree of oxidative stress experienced by the cells. Figure 10 This maintained better biological activity. Furthermore, cells supplemented with exogenous extracellular polymeric substances exhibited higher methane selectivity on their surface under light irradiation. Figure 14 This indicates that protected cells convert more photogenerated electrons into methane.

[0085] Example 5

[0086] (6) Inoculate the methanogens at a volume ratio of 5% into a culture medium with a pH of 6.9 and incubate at a constant temperature of 35°C. (2) Wait until the above culture medium reaches the OD value. 600 When the value is >0.5, centrifuge at 7500 rpm / min, wash three times with PBS buffer, and resuspend in 20 mL of PBS buffer solution.

[0087] (3) Add 0.06g of modified graphitic carbon nitride to the cell suspension above after ultrasonic treatment for 10min, shake well and let stand for 24h.

[0088] (4) Inject the hybrid liquid into the gel skeleton and wait for the hybrid biomembrane to attach to the gel pores.

[0089] (5) Place the gel with the hybrid biomembrane attached in the culture medium, connect multiple identical gel systems, introduce carbon dioxide, and start irradiation.

[0090] The results showed that the hybrid biomembrane exhibited good conformability, allowing it to adhere well to the complex pore structure of the gel. Furthermore, the hybrid biomembrane possessed sufficient structural strength to withstand certain hydraulic shear in the liquid phase without breaking. Figure 11 ).

[0091] In summary, this invention comprises five core elements: methanogens, graphitic carbon nitride, conductive nanoparticles, a biofilm, and a gel. This method effectively overcomes the limitations of semi-artificial photosynthesis systems, improving quantum conversion efficiency and long-term operational stability. The system's simplicity makes it easy to improve and scale up production.

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for constructing and enhancing a methanogen / carbon nitride hybrid biofilm system, characterized in that... Includes the following steps: Step 1. The carbon nitride material underwent surface modification to improve its interaction energy with methanogenic bacteria cells and extracellular polymers; Step 2. Methanogens are cultured in a medium rich in organic nitrogen and carbon sources, resulting in abundant extracellular polymers. Adding exogenous polymers after cell culture achieves the same effect. Step 3. During the methanogenic bacteria culture process, nano-conductive particles are added so that the surface of the methanogenic bacteria cells is covered with nano-conductive particles after the culture is completed. Step 4. Methanogens, extracellular polymers, and carbon nitride materials are combined under anaerobic conditions to form a stable biofilm with a three-dimensional structure; Step 5. Apply light to the side of the biofilm.

2. A method for constructing a hybrid biofilm reactor, characterized in that... Includes the following steps: Step 1. Attach the above hybrid biomembrane to a porous gel to construct a hybrid biomembrane / porous transparent substrate reactor unit; Step 2. Combine the hybrid biomembrane / porous transparent substrate reactor units in series / parallel and introduce carbon dioxide to achieve continuous large-scale reaction.

3. The method according to claim 1, characterized in that, After surface modification, carbon nitride can be grafted with cyano or carboxyl groups, or the modified carbon nitride surface can have a local zeta potential that is positive.

4. The method according to claim 1, characterized in that, The culture medium formula containing organic carbon and organic nitrogen is as follows: Table 1 Culture medium formulation Each liter of FeSO4·7H2O solution (0.1% w / v) contains 1 g of FeSO4·7H2O, 1000 ml 0.05 mol / L H2SO4; Each liter of trace element solution SL-10 contains 10 ml of 25% HCl, 1.35 g of FeCl2·4H2O, 63 mg of ZnCl2, 90 mg of MnCl2·4H2O, 5.4 mg of H3BO3, 171 mg of CoCl2·6H2O, 1.8 mg of CuCl2·2H2O, 21.6 mg of NiCl2·6H2O, and 32.4 mg of Na2MoO4·2H2O; Each liter of Wolin vitamin solution (10x concentration) contains 20.00 mg biotin, 20.00 mg folic acid, 100.00 mg pyridoxine hydrochloride, 50.00 mg thiamine hydrochloride, 50.00 mg riboflavin, 50.00 mg niacin, 50.00 mg D-calcium pantothenate, 1.00 mg vitamin B12, 50.00 mg para-aminobenzoic acid, and 50.00 mg (DL)-α-lipoic acid; According to the method of claim 1, the concentration of extracellular protein in the hybrid system containing extracellular polymer is 1-15 μg / mL, and the concentration of extracellular polysaccharide is 10-150 μg / mL.

5. The method according to claim 1, characterized in that, The nano-conductive particles have a particle size of 2-10 nm. When added at a concentration of 10 mL of bacterial solution in the logarithmic growth phase, the characteristic absorbance value of the nano-conductive particles is 0.25-0.85 Units / ml.

6. The method according to claim 1, characterized in that, The conductive nanoparticles added to the system shown are at least one of nano-gold, nano-silver, nano-copper, nano-copper oxide, and carbon quantum dots; the conductive nanoparticles are uniformly distributed on the surface of the cells as sites for electron transport.

7. The method according to claim 1, characterized in that, The thickness of the stable biofilm is between 10 μm and 300 μm.

8. The method according to claim 1, characterized in that, The lateral illumination intensity ranges from 0.01 mW / cm². 2 -0.2mW / cm 2 .

9. The method according to claim 2, characterized in that, The key component material of porous gel is at least one of cellulose nanofibers, cellulose nanocrystals, and bacterial cellulose.

10. The method according to claim 2, characterized in that, The porous gel material has a pore size of 10μm-500μm; the porous gel material has a porosity of 80%-100%; the presence of the gel simultaneously enhances the efficiency of light transmission and solute transport, thereby enhancing the carbon dioxide conversion efficiency of the hybrid biofilm attached to it; the reactor unit can be connected in series or in parallel to achieve continuous large-scale application.

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