Construction method and application of liquid metal-based biological hybrid system

Abstract

The invention discloses a construction method and application of a liquid metal-based biological hybrid system. The method comprises the following steps: S1, preparing liquid metal microparticles; s2, combining methanogens with the liquid metal microparticles to construct a liquid metal-based biological hybrid system; s3, performing illumination culture to promote methane generation. By constructing the liquid metal-based biological hybrid system, the defects of limited material proton and electron transfer, unstable combination, high cost and the like in the hybrid system are overcome, and the efficiency of photoelectrically promoting methane generation is effectively improved. The method is relatively simple, low in preparation cost, high in operability and high in applicability, provides an efficient and stable bioconversion way for solar-driven conversion of CO2 into methane, and has wide application prospects in the fields of environmental engineering, renewable energy sources and the like.

Description

Technical Field

[0001] The present invention relates to the field of renewable energy technology, and more specifically, to a method for constructing a liquid metal-based biohybrid system and its application. Background Art

[0002] Solar-driven conversion of CO2 into high-value fuels such as methane has become a key strategy for addressing the global energy crisis. Nanobiohybrids combine the superior photosensitivity of semiconductors with the efficient catalytic performance of whole-cell microorganisms, showing unique advantages in the field of CO2 photocatalytic conversion. However, the application of biohybrids constructed based on traditional nanosemiconductors faces challenges. For example, patent CN109666703B discloses a method for light-driven methanosarcina to reduce carbon dioxide to produce methane. This method uses traditional inorganic semiconductors such as CdS to construct nanobiohybrids, which have problems such as poor biocompatibility, low proton-electron transfer efficiency and low methane yield. In order to solve these problems, patent CN112237839 B proposes a transition metal-mediated nanobiohybrid construction method, which uses doped transition metals to improve the stability and photocatalytic efficiency of semiconductor nanoparticles. However, this method mainly enhances the transfer efficiency of photogenerated electrons, but fails to effectively solve the problem of poor methane selectivity. In contrast, patent CN114085826B discloses a method for constructing a sandwich-structured photoelectromethanogenic biohybrid, which constructs a bimetallic intermediate layer between microorganisms and inorganic semiconductors to act as an "electron bridge" and promote photoelectron transfer and surface catalytic hydrogen reaction, thereby improving methane selectivity. However, the construction process of this method is complicated, and it is difficult to effectively regulate the bio-inorganic interface, which limits the promotion and application of nano-biohybrids. Therefore, it is urgent to develop a method for constructing a new biohybrid system with strong compatibility, high stability, and excellent conversion efficiency. Summary of the invention

[0003] The purpose of the present invention is to provide a method for constructing a liquid metal-based biohybrid system and its application.

[0004] According to one aspect of the present invention, a method for constructing a liquid metal-based biohybrid system is provided, comprising the following steps:

[0005] S1. Ultrasonic treatment of liquid metal to prepare liquid metal microparticles with a "core-shell" structure;

[0006] S2. Cultivate methanogens until the logarithmic growth phase or OD 600 When the value is 0.1 to 0.3, the liquid metal microparticles are added to construct a liquid metal-based biohybrid system;

[0007] S3. The liquid metal-based biohybrid system is cultured under light to promote the growth and metabolism of methanogens and the production of methane.

[0008] In some embodiments, the liquid metal in step S1 is single-element gallium or gallium-indium alloy, and the mass ratio of gallium element to indium element in the gallium-indium alloy is 75.5:24.5 to 80:20.

[0009] In some embodiments, step S1 is: placing the liquid metal in a phosphate buffer solution, and performing ultrasonic treatment at 25-40° C. for 10-80 min, with a power of 220-360W.

[0010] In some embodiments, the particle size of the liquid metal microparticles in step S1 ranges from 8 micrometers to 10 nanometers, and the surface oxygen content thereof ranges from 1% to 20%.

[0011] In some embodiments, the methanogen in step S2 is at least one of Methanobacterium formicicum, Methanosarcina mazei, Methanosarcina barkeri, or Methanospirillum hungatei.

[0012] In some embodiments, the liquid metal-based biohybrid system in step S2 is constructed by self-deposition or physical adhesion-self-assembly.

[0013] In some embodiments, the loading amount of liquid metal microparticles in the liquid metal-based biohybrid system in step S2 is 0.25-1 g / L.

[0014] In some embodiments, the conditions for light culture in step S3 are: light wavelength: 395±5nm or full spectrum of sunlight, light intensity: 0.8-100mW·cm -2 , lighting duration: 12h / day, lighting time: 7~28 days, lighting temperature: 33~37℃.

[0015] According to another aspect of the present invention, a method for constructing a liquid metal-based biohybrid system is provided for use in promoting efficient growth and metabolism of methanogens and methane production.

[0016] Beneficial effects of the present invention:

[0017] 1. The present invention utilizes liquid metal materials to construct an adaptive photoelectric conversion system, thereby promoting the metabolic activity of methanogens under light conditions, thereby improving the efficiency of methane generation and solving the problems of low efficiency and poor stability of photoelectron transfer. Liquid metal can form a unique "core-shell" structure through oxidation by oxygen in the air, which includes a self-limiting oxidation layer as a photosensitive layer and a liquid metal core with high conductivity and proton transfer as an electron-proton transfer layer. The liquid metal microparticles prepared in the present invention provide a photosensitive layer and an electron-proton transfer layer, and the thickness of the two is adjusted. The core-shell structure formed during the oxidation process provides superior catalytic performance and biocompatibility for light-driven methane generation, thereby promoting the efficient conversion of CO2 into CH4.

[0018] 2. After the liquid metal comes into contact with oxygen, a layer of stable oxide is quickly formed on its surface, which essentially creates a local anoxic microenvironment. This is because the oxidation reaction consumes the surrounding oxygen and reduces the oxygen partial pressure in the environment, thus forming a low oxygen / anaerobic area. This characteristic is very beneficial to anaerobic microorganisms and can reduce the inhibitory or interfering effects that oxygen may bring. The method of the present invention is applicable to a variety of methanogens and liquid metal materials, has good versatility and scalability, and provides a new technical path for the field of bioenergy;

[0019] 3. The method of the present invention is low-cost, has the advantages of simple process, self-energy supply, self-adjustment, and high selectivity. The relative simplicity of the liquid metal-based biohybrid system makes it easier to assemble and improve in a modular manner, and can be promoted and applied on a large scale. The method and application provided by the present invention have significant advantages in improving the efficiency of photosynthetic methane production, enhancing system stability, and expanding the scope of application, and are of great significance for promoting the development of bioenergy technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a comparison chart of the methane production performance of the Mb-EGaIn biohybrid system of Example 9 of the present invention under different EGaIn loading conditions.

[0021] Figure 2 For the Mb-EGaIn biohybrid system of Example 10 of the present invention 13 Results of the C isotope labeling test of the carbon source of methane.

[0022] Figure 3 This is a graph showing the results of four 7-day consecutive cycles of methane production and methane selectivity tests of the Mb-EGaIn biohybrid system of Example 11 of the present invention under light / dark cycle conditions.

[0023] Figure 4This is a comparison chart of the methane production performance of the liquid metal-based biohybrid system of Comparative Example 2 of the present invention under different conditions. DETAILED DESCRIPTION

[0024] The present invention is further described in detail through specific implementation cases, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art are all limited by the claims attached to this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0025] Example 1

[0026] A method for constructing a liquid metal-based biohybrid system comprises the following steps:

[0027] S1. 50 mg of gallium-indium alloy (LM-GaIn) with a mass ratio of gallium to indium of 75.5:24.5 was added to 1 mL of 0.1 M phosphate buffer (PBS, pH 7.0), and then ultrasonic treatment was performed for 20 min at 25°C to 30°C using a bath ultrasonic generator with an ultrasonic power of 360 W and an ultrasonic frequency of 39 kHz to disperse larger LM-GaIn droplets into smaller droplets, thereby preparing liquid metal microparticles with a "core-shell" structure, namely EGaIn, with a particle size range of 600 nm to 800 nm and a surface oxygen content of 3.04%;

[0028] S2. Methanosarcina barkeri (Mb) was obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). According to standard aseptic operation procedures, the Mb strain was inoculated into a heterotrophic medium (SHM) containing sodium bicarbonate (NaHCO3) and sodium acetate (NaAc) as carbon sources at a volume ratio of 5%. The formula of SHM is shown in Table 1. The pH value of the medium is 7.2 to maintain suitable growth conditions for the Mb strain. The inoculated medium was placed in a constant temperature incubator at 37°C for initial culture until the number of Mb bacteria reached the logarithmic growth phase or OD 600 When the value was 0.2, 50 mg of liquid metal microparticles EGaIn were added and cultured in a shaker at 37°C and 120 rpm for 5 min to promote the self-sedimentation combination of Mb and EGaIn to construct a Mb-EGaIn biohybrid system. The loading amount of EGaIn in the Mb-EGaIn biohybrid system was 0.5 g / L.

[0029] S3. After the Mb-EGaIn biohybrid system constructed in step S2 was cultured in a shaker at 37°C and 120 rpm for 24 h, the Mb-EGaIn biohybrid system was centrifuged at 6500 rpm for 7 min using a high-speed centrifuge, the supernatant was removed, the precipitate was washed three times with 0.9% saline, and resuspended in 1 mL of 0.9% saline to obtain a resuspended Mb-EGaIn biohybrid system; the resuspended Mb-EGaIn biohybrid system was transferred to a 50 mL anaerobic bottle containing 24 mL of organic carbon source-free medium (SAM), the SAM formula is shown in Table 1. The medium contains 2.52 g·L -1 NaHCO3 was added to maintain the carbon source supply required for its growth, and cystine (Cysteine-HCl), a hole scavenger, was added to make its final concentration 0.24 g·L -1 , and the optimized Mb-EGaIn biohybrid system was obtained.

[0030] Table 1 Formulations of heterotrophic medium (SHM) and organic carbon-free medium (SAM)

[0031]

[0032] Note: *Each liter of culture medium contains:

[0033]

[0034] **Each liter of culture medium contains:

[0035]

[0036] ***Each liter of culture medium contains:

[0037]

[0038] S5. After the optimized Mb-EGaIn biohybrid system was statically cultured for 6 h, the ultraviolet light (LED) with a wavelength of 395 ± 5 nm and a light intensity of 0.8 ± 0.1 mW cm -2 The Mb-EGaIn biohybrid system was cultured for 8 days under 35°C with a 12h light / 12h dark cycle to facilitate the production of methane by the Mb-EGaIn biohybrid system. During the light culture period, the gas samples in the system were sampled every day and the methane concentration was measured by gas chromatograph. All culture and sampling operations were carried out in an anaerobic glove box with a nitrogen atmosphere (oxygen content <1ppm). After sampling and testing, the Mb-EGaIn biohybrid system produced 194.83±5.14μmol / g of methane after 8 days of continuous illumination.

[0039] Example 2

[0040] The preparation method in this embodiment is the same as that in Example 1, except that: in this embodiment, the gallium and the gallium-indium alloy with a mass ratio of 80:20 between the gallium element and the indium element (i.e., GaIn80:20) are used as the test objects, replacing the gallium-indium alloy with a mass ratio of 75.5:24.5 between the gallium element and the indium element in step S2 (i.e., GaIn75.5:25.5). Due to the different freezing points and melting points of different liquid metals, different temperature ranges need to be maintained during the ultrasonic process to maintain their liquid state, otherwise liquid metal microparticles cannot be prepared. Referring to the method in Example 1, ultrasonic treatment is performed under different temperature conditions to determine the ultrasonic treatment temperature range required for preparing microparticles suitable for single-element gallium, GaIn75.5:25.5, and GaIn80:20, as shown in Table 2 below.

[0041] Table 2 Ultrasonic temperature required for preparing microparticles with different liquid metals

[0042] Liquid metal type Temperature range Ga 35～40℃ GaIn (75.5:24.5) 25～30℃ GaIn (80:20) 30～35℃

[0043] Example 3

[0044] The preparation method in this embodiment is the same as that in Example 1, except that the ultrasonic duration of 20 min in step S2 is replaced by an ultrasonic duration of 10 min, 40 min or 80 min, and then the main particle size range and surface oxygen content of EGaIn obtained with different ultrasonic treatment times are respectively measured, as shown in Table 3.

[0045] Table 3 Different ultrasonic treatment time and the main particle size range and surface oxygen content of EGaIn

[0046] Ultrasound duration Main particle size range Surface oxygen content 10min 1μm~8μm 1.01％ 20min 600nm~800nm 3.04％ 40min 50nm~300nm 7.91％ 80min 10nm~200nm 10.86％

[0047] Example 4

[0048] The preparation method of this embodiment is the same as that of Embodiment 1, except that the ultrasonic power in step S2 is set to 220W.

[0049] Example 5

[0050] The preparation method of this embodiment is the same as that of embodiment 1, except that: the OD 600 The Mb culture with a value of 0.2 was replaced by OD 600 Mb cultures with values ​​of 0.1 or 0.3.

[0051] Example 6

[0052] The preparation method of this embodiment is the same as that of embodiment 1, except that the Methanosarcina barkeri (Mb) in step S2 is replaced by Methanobacterium formicicum (Mf), Methanosarcina mazei (Mm) or Methanospirillum hungatei (Mh).

[0053] Example 7

[0054] The preparation method of this embodiment is the same as that of embodiment 1, except that the self-deposition bonding in step S2 is replaced by a physical adhesion-self-assembly method. The specific construction method includes the following steps:

[0055] (1) preparing liquid metal microparticles, which is the same as step S1 in the embodiment;

[0056] (2) Take 50 mg of EGaIn prepared in step (1) and add it to 1 mL to prepare 1 mL of EGaIn suspension for later use;

[0057] (3) Isolation and purification of bacteria: OD of Mb culture 600 When the value reaches about 0.2, centrifuge Mb at 6500 rpm for 7 min in a high-speed centrifuge, remove the supernatant, wash the precipitate three times with 0.9% saline, and resuspend the washed Mb bacteria in 1 mL of 0.9% saline to obtain a resuspended Mb bacterial solution;

[0058] (4) Construction of biohybrid system: The above-mentioned resuspended Mb bacterial solution and 1 mL of EGaIn suspension were transferred to a 50 mL new culture system, and an appropriate amount of NaHCO3 was added to maintain the carbon source supply required for its growth. The hole scavenger cystine was added to a final concentration of 1.5 wt% to help eliminate harmful free radicals that may be generated. The culture was carried out at 37 ° C and 120 rpm in a shaking environment for 5 min to promote the construction of the Mb-EGaIn biohybrid system by physical adhesion-self-assembly between Mb and EGaIn.

[0059] Example 8

[0060] The preparation method of this embodiment is the same as that of embodiment 1, except that: the near-ultraviolet light (395±5nm ultraviolet light, LED, light intensity of 0.8±0.1mW·cm) in step S2 is -2 ) and replaced with full spectrum sunlight (full spectrum wavelength, xenon lamp, light intensity of 100±5mW·cm -2 ), and the other steps are the same as those in Example 1.

[0061] Example 9

[0062] The preparation method of this embodiment is the same as that of embodiment 1, except that the loading amount of EGaIn in the hybrid system in step S2 is 0.5 g / L, and the amount of EGaIn added is replaced by 0.125 g / L, 0.25 g / L, 0.75 g / L and 1 g / L respectively, and the methane production is compared with that of embodiment 1. The specific results are as follows Figure 1 shown.

[0063] Example 10

[0064] The preparation method of this embodiment is the same as that of embodiment 1, except that: NaH 13 CO3 replaced NaHCO3 as the sole carbon source, and a stable isotope labeling experiment was conducted to verify whether the generation of methane in the Mb-EGaIn biohybrid system originated from the reduction process of CO2. After 8 days of light culture, the gas produced by the Mb-EGaIn biohybrid system was collected and analyzed using a mass spectrometer. The specific results are shown in Figure 2 .

[0065] Embodiment 11

[0066] The preparation method of this example is the same as that of Example 1, except that: the optimized Mb-EGaIn biohybrid system is prepared and cultured under light; each cycle lasts for 7 days, after which the Mb-EGaIn biohybrid system is collected and then added to fresh culture medium. This process is repeated for four 7-day cycles, and the total illumination time is 28 days. The specific results are shown in FIG. Figure 3 .

[0067] Comparative Example 1

[0068] The preparation method of this comparative example is the same as that of Example 1, except that in step S1, cadmium sulfide (CdS) is used to replace the gallium-indium alloy (LM-GaIn) with a mass ratio of 75.5:24.5 between gallium and indium to prepare liquid metal microparticles. The preparation method of CdS is as follows:

[0069] Weigh 10mmol CdCl2 and 17mmol 3-mercaptopropionic acid (MPA) and disperse them in 200mL water. Adjust the pH to 10 by adding 5M sodium hydroxide. Transfer to a 500mL three-necked flask, pass argon for 10min to remove the top air, and slowly add 1mmol Na2S solution under stirring. Then heat the mixed system to 100℃ under condensation reflux, and keep the temperature for 30min to promote the dispersion of quantum dots. After the insulation is completed, add anhydrous ethanol to form a precipitate. Centrifuge, remove the supernatant, wash with ultrapure water 3 times, and freeze-dry for 48h to obtain a CdS semiconductor.

[0070] Comparative Example 2

[0071] The preparation method of this comparative example is the same as that of Example 1, except that: Example 1 is used as the treatment group, marked as the Mb-EGaIn (illumination) group, and three different control groups are set according to the method of Example 1, including: the Mb (illumination) group, that is, no EGaIn is added, and other operations are the same as those in Example 1; the Mb-EGaIn (no illumination) group, that is, the hybrid system is not subjected to illumination treatment, and other operations are the same as those in Example 1; the EGaIn (illumination) group, that is, the Mb added is inactivated bacteria, and other operations are the same as those in Example 1. The specific results are shown in Figure 4 .

[0072] The methane production of Examples 1 to 8 and Comparative Example 1 was compared, and the results are shown in Table 4.

[0073] Table 4 Comparison of methane production in the systems of Examples 1 to 8 and Comparative Example 1

[0074]

[0075] The results are shown in Table 4. From Examples 1 and 2 to 4, it can be seen that different types of liquid metals are used to prepare microparticles to construct biohybrids, and the methanogenic performance is different. 75.5:24.5 , that is, the gallium-indium alloy with a mass ratio of 75.5:24.5 between gallium and indium has the highest yield, indicating that GaIn 75.5:24.5 The high In content ratio is beneficial to the photoelectric reaction and thus promotes methane production; when the ultrasonic treatment time for preparing liquid metal microparticles is greater than 20 minutes, there is no significant difference in the methane production, indicating that 20 minutes of ultrasonic treatment is the shortest effective time, and the methane production increases with the increase of ultrasonic power.

[0076] Further analysis shows that from Examples 1 and 5-6, different OD 600 The mixture of Mb bacteria cultured under different conditions to construct biohybrids showed different methanogenic performances, among which OD 600When it reaches 0.2, the Mb bacteria are in the growth stage, which is conducive to the construction of an efficient liquid metal-based biohybrid system with the highest methane production, which is the optimal condition for constructing a biohybrid. Different types of methanogens can effectively produce methane, including Mh bacteria that only have a hydrogen metabolism pathway, indicating the effective transfer of electrons and protons in the liquid metal shell-core structure in the biohybrid.

[0077] Further analysis shows that from Example 1 and Examples 7-8, the methane production of the biohybrid system constructed by the self-deposition adsorption hybridization method is better than that of the biohybrid system constructed by the physical adhesion-self-assembly hybridization method; and different typical light sources and light intensities, near-ultraviolet light (0.8 mW·cm -2 ) and full spectrum of sunlight (100mW·cm -2 ) can drive the Mb-EGaIn biohybrid system to effectively produce methane.

[0078] Further analysis shows that from Examples 1 and 9, the methane production of the Mb-EGaIn biohybrid system after 8 days of continuous illumination culture is different under different EGaIn loading conditions, among which the biohybrid system constructed with an addition amount of 50 mg EGaIn, i.e., a loading amount of 0.5 g / L of the Mb-EGaIn biohybrid system, has the highest methane production.

[0079] Further analysis showed that the methane produced by the Mb-EGaIn biohybrid system in Example 10 contained characteristic 13 The CH4 peak proves that the generation of methane is indeed derived from the reduction process of CO2, and further confirms that the Mb-EGaIn biohybrid system uses CO2 as a carbon source to reduce CO2 to produce methane.

[0080] Further analysis shows that the Mb-EGaIn biohybrid system in Example 11 maintained high methane production and stability during long-term operation of four consecutive 7-day cycles, with a methane yield of up to 455.64±15.99μmol / g, and a methane selectivity of over 99%, that is, the proportion of the target product methane in the total product (methane and byproduct hydrogen). These data demonstrate the high efficiency and long-term stability of the Mb-EGaIn biohybrid system in the process of solar-driven CO2 conversion to methane.

[0081] Further analysis shows that the methane production performance of the liquid metal-based biohybrid system is much better than that of the CdS-Mb biohybrid constructed in Comparative Example 1. The methane production of the Mb-EGaIn (illumination) group after 8 days of continuous illumination treatment was 194.83±5.14 μmol / g, while the methane production of the other control groups, the Mb (illumination) group, the Mb-EGaIn (no illumination) group and the EGaIn (illumination) group, was always 0, indicating that the method of Example 1 of the present invention successfully constructed the Mb-EGaIn biohybrid system, further proving the effectiveness of the hybrid system.

[0082] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

1. A method for constructing a liquid metal-based biohybrid system, characterized in that: The following steps are involved: S1. Ultrasonic treatment of liquid metal to prepare liquid metal microparticles having a "core-shell" structure; S2. Cultivate methanogens until the logarithmic growth phase or OD 600 When the value is 0.1 to 0.3, the liquid metal microparticles are added to construct a liquid metal-based biohybrid system; S3. The liquid metal-based biohybrid system is cultured under light to promote the growth and metabolism of methanogens and the production of methane.

2. The method for constructing a liquid metal-based biohybrid system according to claim 1, characterized in that: The liquid metal in step S1 is single-element gallium or gallium-indium alloy, and the mass ratio of gallium element to indium element in the gallium-indium alloy is 75.5:24.5-80:

20.

3. The method for constructing a liquid metal-based biohybrid system according to claim 1, characterized in that: The step S1 is: placing the liquid metal in a phosphate buffer solution, and performing ultrasonic treatment at 25-40° C. for 10-80 minutes, with the power of the ultrasonic treatment being 220-360W.

4. The method for constructing a liquid metal-based biohybrid system according to claim 1, characterized in that: The liquid metal microparticles with a "core-shell" structure are composed of a surface self-limiting oxide layer and a liquid metal layer.

5. The method for constructing a liquid metal-based biohybrid system according to claim 1, characterized in that: The particle size of the liquid metal microparticles ranges from 8 micrometers to 10 nanometers, and the surface oxygen content thereof ranges from 1 to 20%.

6. The method for constructing a liquid metal-based biohybrid system according to claim 1, characterized in that: The methanogen is at least one of Methanobacterium formicicum, Methanosarcinamazei, Methanosarcina barkeri or Methanospirillumhungatei.

7. The method for constructing a liquid metal-based biohybrid system according to claim 1, characterized in that: In step S2, the liquid metal-based biohybrid system is constructed by self-deposition or physical adhesion-self-assembly.

8. The method for constructing a liquid metal-based biohybrid system according to claim 1, characterized in that: The loading amount of the liquid metal microparticles in the liquid metal-based biohybrid system in step S2 is 0.25-1 g / L.

9. The method for constructing a liquid metal-based biohybrid system according to claim 1, characterized in that: The conditions for light culture in step S3 are: light wavelength: 395±5nm or full spectrum of sunlight, light intensity: 0.8-100mW·cm -2 , lighting duration: 3~24h / day, lighting time: 7~28 days, lighting temperature: 33~37℃.

10. Application of the method for constructing a liquid metal-based biohybrid system according to any one of claims 1 to 9 in promoting efficient growth and metabolism of methanogens and methane production.

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