Humic substance reinforced hydroxyapatite-bacillus subtilis semi-artificial photosynthetic material as well as preparation method and application thereof

Abstract

The invention discloses a humic substance reinforced hydroxyapatite-bacillus subtilis semi-artificial photosynthetic material and a preparation method and application thereof.The preparation method comprises the steps that firstly, hydroxyapatite microspheres are subjected to Fe2O3 doping, the Fe2O3 doped hydroxyapatite microspheres are combined with bacillus subtilis, and the hydroxyapatite-bacillus subtilis semi-artificial photosynthetic material is obtained; and the semi-artificial photosynthetic material of the bacillus subtilis-Fe2O3 doped hydroxyapatite microspheres is obtained. And then, adding the humic substance into the hydroxyapatite-bacillus subtilis semi-artificial photosynthetic system, so as to obtain the humic substance reinforced hydroxyapatite-bacillus subtilis semi-artificial photosynthetic material. The raw materials of the prepared material are easy to obtain, the method is simple, and when the material is applied to enhancing the activity of the antioxidant enzyme of the bacillus subtilis, the cost of producing the antioxidant enzyme by the bacillus subtilis is expected to be reduced; meanwhile, the bacillus subtilis can be used for fixing CO2 through photocatalytic reduction, carbon emission and the like in the aerobic fermentation process of the bacillus subtilis are expected to be reduced, and economic benefits and environmental benefits are achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic biology, and in particular relates to a humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material, a preparation method and an application thereof. Background Art

[0002] At present, the use of synthetic biology, that is, the use of microorganisms to produce enzyme preparations with various catalytic activities, has attracted more and more attention in the fields of agriculture, medicine, food, etc. Bacillus subtilis is a widely studied beneficial bacterium. Based on in-depth research on Bacillus subtilis and its related enzymes, researchers believe that Bacillus subtilis is expected to be used in the future to produce single-cell proteins and even enzyme preparations. For example, Bacillus subtilis is used to produce catalase under optimized conditions. However, Bacillus subtilis is an aerobic facultative, chemoheterotrophic bacterium. When aerobic fermentation is used in the production and application process, a large amount of biomass is consumed and a large amount of CO2 is emitted, which has a negative impact on the environment.

[0003] The semi-artificial photosynthesis system that appeared at the beginning of this century is a hybrid system composed of microorganisms and semiconductors. It can capture light and fix CO2. Due to the combination of the efficient capture of light energy by materials and the high selectivity of biocatalysis, it can achieve efficient and highly specific conversion of light energy to chemical energy. Mature technology for carbon fixation Plant photosynthesis is limited by space and has a slow rate, especially the light energy conversion rate of photosynthesis is only 1%; while the artificial photosynthesis technology of semiconductor materials capturing light and reducing CO2 is low in maturity and poor in stability. It usually only produces small molecules and it is difficult to achieve specific synthesis of high energy density and high value-added multi-carbon compounds. However, at present, the biological-semiconductor semi-artificial photosynthesis system is only used in autotrophic microorganisms because these microorganisms have enzyme systems for fixing CO2. Bacillus subtilis is a chemoheterotrophic organism and is generally considered difficult to be constructed as a biological semiconductor semi-artificial photosynthesis system. However, from an evolutionary perspective, organic matter evolved from inorganic matter chemically; unlike inorganic matter, organic matter is always scarce for the needs of organisms. In this way, early microorganisms may mainly adopt autotrophic systems, or autotrophic and heterotrophic parallel systems. At present, Bacillus subtilis actually still retains an enzyme system for fixing CO2, such as pyruvate carboxylase, which may be its degenerate carbon fixation system. This system may still be used, or may be used efficiently under certain conditions.

[0004] It is clear that semiconductor materials with wide applications usually have biocompatibility issues, and it is unlikely that microorganisms choose these semiconductors to transfer electrons during biological evolution. However, there are abundant inorganic mineral materials in the ancient earth environment, which can capture light, generate photogenerated electrons and transfer them to some degraded enzyme systems, thereby inducing and stimulating their activity.

[0005] First, most semiconductor materials are xenobiotics, such as nano-TiO2, nano-C3N4, and nano-CdS. These materials either release heavy metals when dissolved, or their own toxicity is still unclear. Usually, these semiconductor materials produce highly oxidizing photogenerated holes or free radicals when illuminated, which have strong biological toxicity.

[0006] Secondly, in most cases, it is impossible to simultaneously improve the photosensitivity of semiconductor materials and reduce their toxicity. For example, if you want to enhance the generation of photogenerated electrons in CdS and effectively use it for biological carbon fixation, you need to increase material defects and reduce its crystallinity; but to reduce the toxicity of semiconductor materials, materials such as CdS must have high crystallinity and few material defects, so that they are not easily dissolved and release heavy metal ions. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to provide a humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material, which effectively achieves carbon fixation and antioxidant enzyme activity enhancement of heterotrophic bacteria Bacillus subtilis. The present invention realizes a semi-artificial photosynthetic system of heterotrophic organisms, enriches the species of organisms in the semi-artificial photosynthetic system; enables the Bacillus subtilis aerobic fermentation system that may be used to produce enzyme preparations but has a large amount of CO2 emissions to fix CO2 and enhance the activity of antioxidant enzymes; at the same time, it solves the problems of poor biocompatibility of semiconductor materials in existing semi-artificial photosynthetic systems, and the inability to simultaneously improve the photosensitivity of semiconductor materials and reduce their toxicity.

[0008] Technical solution: To achieve the above-mentioned purpose, the present invention provides a humus-enhanced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material, which includes humus, Fe2O3-doped hydroxyapatite microspheres, and Bacillus subtilis; the Fe2O3-doped hydroxyapatite microspheres adsorb Bacillus subtilis, and the humus is distributed on the solid phase surface of the Fe2O3-doped hydroxyapatite microspheres or in the liquid phase of the culture medium.

[0009] Specifically, the material is formed by mixing humus with Bacillus subtilis-Fe2O3-doped hydroxyapatite microsphere hybrid semi-artificial photosynthetic material, and the Bacillus subtilis-Fe2O3-doped hydroxyapatite microsphere hybrid semi-artificial photosynthetic material is obtained by adding Fe2O3-doped hydroxyapatite to a culture medium containing Bacillus subtilis wet cells for adsorption culture.

[0010] Wherein, the humic substances include humic acid and fulvic acid.

[0011] Wherein, the Bacillus subtilis is a wild type Bacillus subtilis, and any commercially available conventional Bacillus subtilis can be used.

[0012] Preferably, the hydroxyapatite microspheres are conventional commercially available materials provided by Nanjing Eppre Nanomaterials Co., Ltd., and have a particle size of 30-38 μm.

[0013] The method for preparing the humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material of the present invention comprises the following steps:

[0014] (1) adding an iron salt solution to hydroxyapatite microspheres, stirring evenly, and then adding water to prepare a sol, aging the sol, and then washing and drying to obtain Fe2O3-doped hydroxyapatite microspheres;

[0015] (2) inoculating Bacillus subtilis into a culture medium, culturing at a constant temperature with shaking, and obtaining wet cells of Bacillus subtilis after centrifugation;

[0016] (3) adding Fe2O3-doped hydroxyapatite microspheres to a culture medium containing wet cells of Bacillus subtilis for adsorption culture, and obtaining Bacillus subtilis-Fe2O3-doped hydroxyapatite microsphere hybrid semi-artificial photosynthetic material by centrifugation after adsorption culture;

[0017] (4) adding the hybrid semi-artificial photosynthetic material to a culture medium, adding humus, and mixing to obtain a hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material enhanced by humus.

[0018] In which, an iron salt solution is added to the hydroxyapatite micron sphere powder in step (1), stirred evenly, and then water is added to prepare a sol, the sol is aged at a constant temperature of 70-80°C for 2-3 hours, and then washed and dried to obtain Fe2O3-doped hydroxyapatite with a mass ratio of Fe of 1.5-26%.

[0019] Preferably, the drying temperature is 105-120° C. for 2-4 hours.

[0020] Preferably, the iron salt is Fe(NO3)3.

[0021] Wherein, the pH value of the culture medium for the expanded culture of Bacillus subtilis in step (2) is 6-8, the culture time is 24-48h, the constant temperature culture temperature is 26-30°C, the oscillation frequency is 100-200rpm, the centrifugal speed is 5000-10000rpm, and the centrifugal time is 5-20min.

[0022] Wherein, in step (3), the Fe2O3-doped hydroxyapatite microspheres are added to the wet cells of Bacillus subtilis, and the concentration of the wet cells in the two-phase mixed solution is 1-10 g / L, the pH value of the adsorption culture medium is 6-8, the culture time is 24-48 h, the constant temperature culture temperature is 26-30 ° C, the oscillation frequency is 100-200 rpm, the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 5-20 min.

[0023] Wherein, in step (4), the hybrid semi-artificial photosynthetic material is added to a new culture medium, humus is added, and the mixture is mixed to obtain hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material reinforced with humus; the concentrations of Fe2O3-doped hydroxyapatite microspheres, Bacillus subtilis wet cells, humic acid, and fulvic acid in the culture medium are 0.1-1, 1-10, 0.8-1.2, and 0.8-1.2 g / L, respectively.

[0024] The humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material of the present invention is used in the photocatalytic reduction and fixation of CO2.

[0025] The humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material of the present invention is used to enhance the antioxidant enzyme activity of Bacillus subtilis and reduce the cost of producing antioxidant enzymes by Bacillus subtilis; at the same time, Bacillus subtilis can use photocatalytic reduction to fix CO2, thereby reducing carbon emissions during aerobic fermentation of Bacillus subtilis.

[0026] The illumination condition of the applied system is 0-3000 lux, the CO2 concentration of the system is 400-1200 μL / L, the temperature condition of the system is 25-40° C., and the culture time is 24-72 hours.

[0027] Furthermore, in the application, illumination, CO2 introduction, and constant temperature 25-40°C are used for culturing for 24-72 hours. The illumination conditions of the system are 0-3000 lux, and the CO2 concentration of the system is 400-1200 μL / L; the concentration of Bacillus subtilis in the culture system is 1-10 g / L; the concentration of the sacrificial agent humic acid in the culture system is 0.8-1.2 g / L; and the concentration of the electron shuttle fulvic acid in the culture system is 0.8-1.2 g / L, respectively.

[0028] The present invention provides a hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material strengthened by humus, a preparation method of the semi-artificial photosynthetic material, and a method for converting CO2 and enhancing the antioxidant enzyme activity of bacteria using the material. The present invention realizes carbon fixation and enhancement of antioxidant enzyme activity of Bacillus subtilis by using the hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material strengthened by humus.

[0029] In the present invention, hydroxyapatite microspheres are firstly doped with Fe2O3 to convert the insulating hydroxyapatite material into a semiconductor material with photocatalytic activity; then, the Fe2O3-doped hydroxyapatite microspheres are combined with Bacillus subtilis to obtain Bacillus subtilis-Fe2O3-doped hydroxyapatite microsphere semi-artificial photosynthetic material; then, humic substances, namely humic acid and fulvic acid, are added to the hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic system to obtain the hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material reinforced by humic substances.

[0030] The present invention utilizes the humic material-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material to convert CO2 and enhance the antioxidant enzyme activity of the bacteria. Specifically: Fe2O3-doped hydroxyapatite microsphere semiconductor is combined with Bacillus subtilis to achieve biological photocatalysis, which is simple, low-cost and environmentally friendly; Fe2O3-doped hydroxyapatite microsphere semiconductor has uniform size distribution, good biocompatibility, photoelectric conversion ability, and excellent stability, etc., and generates photogenerated electrons under visible light irradiation; in addition, since iron oxide can also be a photoanode material, a large number of electrons are also released under illumination conditions; electrons enter the interior of Bacillus subtilis through the assistance of electron shuttles to participate in the carbon fixation cycle, effectively improving the CO2 fixation efficiency. The Fe2O3-doped hydroxyapatite microsphere in the material is a semiconductor material with good biocompatibility and photocatalytic activity, and its photogenerated electrons can enter Bacillus subtilis, thereby proliferating Bacillus subtilis and enhancing the antioxidant enzyme activity. The humic substances in the material are humic acid and fulvic acid, among which humic acid is an excellent photogenerated hole scavenger and fulvic acid is an electron shuttle. Through the scavenging of photogenerated holes and the electron shuttling effect, the humic acid and fulvic acid in the humic substances can enhance the conversion of CO2 by hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material and enhance the activity of bacterial antioxidant enzymes.

[0031] The hydroxyapatite used in the present invention has excellent biocompatibility, especially spherical morphology and large particle size hydroxyapatite has better biocompatibility; the hydroxyapatite microspheres used are artificial bone materials for biomedical materials, and even if dissolved, only non-toxic Ca 2+ With PO4 3- Ions. Bacillus subtilis is an aerobic, facultative, chemoheterotrophic microorganism that is widely distributed and easy to obtain. It will enter a spore dormancy period in unsuitable environments such as harsh environments and nutrient deficiencies, forming spores that have extremely strong stress resistance and can survive in extreme environments such as high temperature, acid and alkali. It grows fast, has relatively low nutritional requirements, can efficiently secrete many proteins and metabolites, and does not produce toxins. It is a non-pathogenic and safe microorganism.

[0032] The humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material of the present invention utilizes the principle of converting CO2 and enhancing the antioxidant enzyme activity of the bacteria, based on:

[0033] First of all, although Bacillus subtilis is a heterotrophic bacterium, it usually does not have the ability to fix carbon. It only has the potential to utilize inorganic carbon sources under special circumstances. For example, if it contains a carbon-fixing enzyme system, its activity may be activated under specific conditions.

[0034] Secondly, doping with biocompatible semiconductor materials gives hydroxyapatite semiconductor properties. Hydroxyapatite has a wide bandgap and is an insulator; but Fe2O3 doping can narrow the bandgap of hydroxyapatite, transforming it from an insulator to a semiconductor; this doping may also enhance the absorption of hydroxyapatite in the visible light band.

[0035] Thirdly, it is known that Ti-doped hydroxyapatite is a p-type semiconductor, and it is speculated that high-valent trivalent Fe-doped hydroxyapatite also has p-type semiconductor properties; and Fe2O3 is a common n-type semiconductor photoanode, so theoretically, the contact interface between Fe-doped hydroxyapatite and Fe2O3 can form a PN heterojunction, thereby retaining the Fe2O3 conduction band electrons with stronger redox properties and the Fe-doped hydroxyapatite valence band holes, reducing the recombination of photogenerated electron-hole pairs and improving the utilization rate of light energy.

[0036] In addition, in the humic substance-enhanced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material system, Fe-doped hydroxyapatite-Fe2O3 captures visible light to generate photogenerated electrons and photogenerated holes. Subsequently, the photogenerated holes are scavenged by the sacrificial agent humic acid. This is because the solubility of humic acid is relatively small and it can exist relatively more at the liquid-solid interface; at the same time, humic acid has a relatively strong ability to scavenge photogenerated holes. The photogenerated electrons are transferred to the Bacillus subtilis cell by the electron shuttle fulvic acid. The solubility of fulvic acid is relatively small and it can exist relatively more in the liquid phase; at the same time, fulvic acid has a strong electron transfer ability. Subsequently, the photogenerated electrons enter the Bacillus subtilis, and the cell uses carboxylase to fix CO2, realizing efficient and highly specific conversion of light energy to chemical energy.

[0037] At the same time, since the present invention adopts a hybrid system of organisms and semiconductors, the photogenerated electron holes of the semiconductor have strong redox properties, resulting in enhanced activity of bacterial antioxidant enzymes.

[0038] The raw materials involved in the humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material of the present invention are easy to obtain, the method is simple, and it is suitable for Bacillus subtilis photocatalytic reduction and fixation of CO2, production of various antioxidant enzymes, etc., and has economic and environmental benefits.

[0039] The present invention proposes for the first time that a P-type semiconductor Fe-doped hydroxyapatite forms a PN junction with impurity iron oxide, and utilizes the composite structure of the interface between the P-type semiconductor Fe-doped hydroxyapatite and the N-type semiconductor iron oxide to reduce the recombination of photogenerated electron-hole pairs and improve the utilization rate of light energy. In the present invention, iron oxide is an N-type semiconductor well known in the art. Ti (a high-valent element, Fe and Ti are both higher in valence than Ca) doped hydroxyapatite is a P-type semiconductor. In the present invention, electrons are provided to microorganisms by semiconductors, the carboxylase of the microorganisms is activated, and carbon is fixed by photogenerated electrons. At the same time, the enhanced activity of antioxidant enzymes may come from the hybridization of biological semiconductors. The present invention achieves carbon fixation and enhanced antioxidant enzyme activity through a hybrid system.

[0040] At present, only autotrophic organisms can be used to optimize the carbon fixation of these organisms, while Bacillus subtilis is a heterotrophic organism. The present invention realizes carbon fixation by heterotrophic organisms. At the same time, the present invention adopts a non-toxic and harmless biocompatible system, and elements such as Fe, Ca, and P are non-toxic nutrient elements. Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0041] (1) The hybrid system selected by the present invention is non-toxic and harmless. The Bacillus subtilis used is a common probiotic, which is widely distributed and easy to obtain. It will enter a spore dormancy period in unsuitable environments such as harsh environments and nutrient deficiencies, forming spores with extremely strong stress resistance and the ability to survive in extreme environments such as high temperature, acid and alkali. It has a fast growth rate and relatively low nutritional requirements. It can efficiently secrete many proteins and metabolites, and it does not produce toxins. It is a non-pathogenic and safe microorganism. The hydroxyapatite used in the present invention has excellent biocompatibility, especially spherical morphology and large particle size hydroxyapatite has better biocompatibility. The hydroxyapatite microspheres used are artificial bone materials for biomedical materials, and even if dissolved, they only release non-toxic Ca 2+ With PO4 3- Ions are environmentally safe materials.

[0042] (2) The present invention reduces the recombination of photogenerated electron-hole pairs and improves the utilization rate of light energy through material hybridization. First, Fe2O3 doping can narrow the band gap of hydroxyapatite, transforming it from an insulator to a semiconductor; this doping may also enhance the absorption of hydroxyapatite in the visible light band. Secondly, Fe-doped hydroxyapatite also has p-type semiconductor properties; and Fe2O3 is a common n-type semiconductor photoanode. In theory, the contact interface between Fe-doped hydroxyapatite and Fe2O3 can form a PN heterojunction, so that the Fe2O3 conduction band electrons with stronger redox properties and the Fe-doped hydroxyapatite valence band holes can be retained, reducing the recombination of photogenerated electron-hole pairs and improving the utilization rate of light energy.

[0043] (3) The humic substances used in the present invention to strengthen the semi-artificial photosynthesis of hydroxyapatite-Bacillus subtilis are non-toxic, easy to obtain and effective. Humic acid acts as a sacrificial agent for removing photogenerated holes. Humic acid is relatively present in the liquid-solid interface and has a strong ability to remove photogenerated holes. Fulvic acid is relatively present in the liquid phase and has a strong electron transfer ability. The photogenerated electrons are transferred to the Bacillus subtilis cells by the electron shuttle fulvic acid. The cells use carboxylase to fix CO2, realizing efficient and highly specific conversion of light energy to chemical energy. In addition, due to the use of a hybrid system of biological and semiconductors, the photogenerated electron holes of the semiconductor have a strong redox property, resulting in enhanced activity of the antioxidant enzymes of the cells.

[0044] (4) The present invention utilizes solar energy through a semi-artificial photosynthetic system, and microorganisms accept photogenerated electrons and use carboxylase to fix CO2; due to the use of a biological and semiconductor hybrid system, the photogenerated electron holes of the semiconductor have strong redox properties, resulting in enhanced antioxidant enzyme activity in the bacteria. The relevant technology is expected to be applied to the production of related peroxidases, reduce production costs, improve the quality of enzyme preparations, and produce high-value-added enzyme preparations, which also have environmental protection value and economic value. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 Microscope image of hydroxyapatite microspheres used in the present invention;

[0046] Figure 2 X-ray diffraction (XRD) patterns of hydroxyapatite microspheres and hydroxyapatite microspheres doped with different concentrations of Fe2O3;

[0047] Figure 3 The activity of SOD enzyme of Bacillus subtilis in hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material system enhanced by humus;

[0048] Figure 4 The POD enzyme activity of Bacillus subtilis in the hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material system enhanced by humus;

[0049] Figure 5 CAT enzyme activity of Bacillus subtilis in hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material system enhanced by humus.

[0050] The data without the same lowercase letters in the figures indicate that p<0.05 is statistically significant. DETAILED DESCRIPTION

[0051] To further illustrate the present invention, a humus-enhanced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material and its preparation, CO2 conversion and enhancement of bacterial antioxidant enzyme activity provided by the present invention are described in detail below in conjunction with the examples, but this should not be construed as limiting the scope of protection of the present invention.

[0052] Unless otherwise specified, the materials and reagents in the present invention are conventional materials and reagents available on the market.

[0053] In the embodiment, the hydroxyapatite microspheres are provided by Nanjing Apre Nanomaterials Co., Ltd., and have a particle size of 30-38 μm. The hydroxyapatite of the present invention is conventional hydroxyapatite microspheres available on the market, and can also be synthesized by conventional methods.

[0054] The Bacillus subtilis is Bacillus subtilis ATCC 6051.

[0055] Example 1

[0056] Preparation of hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic materials enhanced by humus:

[0057] 10 mL of 0.027 M Fe(NO3)3 solution was added dropwise to 1.00 g of hydroxyapatite microspheres, and 10 mL of water was added after stirring to prepare a sol. The sol was aged at 80°C for 2 hours, then washed with deionized water and dried at 105°C for 3 hours to obtain a Fe2O3-doped hydroxyapatite semiconductor material with photocatalytic activity containing 1.5% Fe (Fe mass: Fe2O3-doped hydroxyapatite mass is 1.5%). Microscope images of hydroxyapatite microsphere raw materials are shown in Figure 2. Figure 1 The X-ray diffraction (XRD) patterns of the prepared hydroxyapatite microspheres and the Fe2O3-doped hydroxyapatite microspheres containing 1.5% Fe are shown in FIG. Figure 2 shown.

[0058] Add 100 mL of culture medium to a 250 mL conical flask, the 100 mL culture medium contains 0.5 g beef extract, 1 g peptone, 0.5 g glucose, 0.5 g NaCl, and pH 7.0. Inoculate the logarithmic phase Bacillus subtilis bacterial solution at a volume ratio of 6% into the sterilized 100 mL culture medium under sterile conditions, expand and culture at 30°C and 150 rpm for 48 hours, centrifuge at 10,000 rpm for 10 minutes, collect the wet cells after expansion, usually larger than 0.3 g, and transfer 0.3 g to the new culture medium.

[0059] 0.1 g of Fe2O3-doped hydroxyapatite was added to 100 mL of culture medium in the above-mentioned 250 mL conical flask containing 0.3 g of wet bacteria. After adsorption culture at 30°C and an oscillation frequency of 150 rpm for 48 h, the mixture was centrifuged at 10,000 rpm for 10 min to obtain the Bacillus subtilis-Fe2O3-doped hydroxyapatite microsphere hybrid semi-artificial photosynthetic material.

[0060] Add all the above hybrid semi-artificial photosynthetic materials into the new 100 mL culture medium, add 0.1 g humic acid and 0.1 g fulvic acid, and mix quickly (this process is a rapid adsorption process) to obtain hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material enhanced by humus.

[0061] Example 2

[0062] According to the method in Example 1, 10 mL of 0.027 M, 50 mL of 0.018 M, 100 mL of 0.018 M, 100 mL of 0.024 M, or 100 mL of 0.047 M Fe(NO3)3 solution was added dropwise to each evaporating dish containing 1.00 g of hydroxyapatite microspheres, and water was added after stirring to prepare a sol. The sol was aged at a constant temperature of 80°C for 2 hours, then washed with deionized water and dried at 105°C for 3 hours to obtain Fe2O3-doped hydroxyapatite semiconductor materials with photocatalytic activity containing 1.5, 5, 10, 13, and 26% Fe (Fe mass: Fe2O3-doped hydroxyapatite mass). The X-ray diffraction (XRD) patterns of hydroxyapatite and Fe2O3-doped hydroxyapatite microspheres containing 1.5, 10, and 26% Fe are shown in Figure 2. Figure 2 As shown, it indicates that both Fe2O3-doped hydroxyapatite and Fe2O3 exist in the Fe2O3-doped hydroxyapatite material system.

[0063] Example 3

[0064] Humic substance-enhanced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material for CO2 conversion:

[0065] The composition of the culture medium described below is consistent with that of the culture medium in Example 1 above.

[0066] The Fe2O3-doped hydroxyapatite-Bacillus subtilis hybrid semi-artificial photosynthetic material containing 1.5% Fe obtained by centrifugation in the above Example 1 was all added to 100 mL of culture medium in a new 250 mL conical flask, 0.1 g of humic acid and 0.1 g of fulvic acid were added, and the mixture was quickly mixed to obtain hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material enhanced with humus.

[0067] CO2 gas was introduced into the conical flask containing the above-mentioned mixed system until the CO2 concentration of the gas in the conical flask reached 1200 μL / L; the conical flask was placed in a light incubator, the light condition was controlled to be 3000 lux, and the conical flask was cultured at a constant temperature of 30°C for 48 hours. 3 mL of gas from the conical flask was transferred using a gas chromatography injection needle, and the CO2 concentration of the gas in the conical flask was determined by gas chromatography.

[0068] The mixture was cultured for 48 hours and centrifuged at 1000 rpm for 5 minutes to obtain all the precipitates to obtain hydroxyapatite microspheres, the supernatant was retained, and the Bacillus subtilis adsorbed on the surface of the hydroxyapatite microspheres was washed away with new culture medium in small amounts for multiple times; the Bacillus subtilis eluted from the hydroxyapatite and in the supernatant of the culture system were combined and centrifuged at 10000 rpm for 10 minutes to obtain the wet cells of Bacillus subtilis cultured under light and carbon fixation.

[0069] The results are shown in Table 1. Under 1200μL / L CO2, the fresh weight of wet bacteria increased from 0.31 to 0.92g / 100mL with Fe2O3 doped with 1.5% Fe-hydroxyapatite-Bacillus subtilis hybrid semi-artificial photosynthetic material, and the CO2 concentration in the conical flask increased from 1200 to 2000μL / L. The experimental results show that under illumination conditions, the CO2 concentration in the high CO2 level system increases less, and the carbon fixation effect is better; under high CO2 level system conditions, the CO2 concentration in the illumination system increases less, and the carbon fixation effect is better.

[0070] Table 1 48h culture results of Fe2O3 doped hydroxyapatite (containing 1.5% Fe)-Bacillus subtilis material system enhanced by humic substances

[0071]

[0072] Example 4

[0073] On the basis of Example 3, the gas containing a certain concentration of CO2 was not introduced into the conical flask, and the CO2 concentration of the gas in the conical flask was about 400μL / L; when other conditions were the same, the hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material strengthened by humus was used for the CO2 conversion experiment. The experimental results are shown in Table 1. Under illumination, low CO2 level, and illumination culture for 48h, the fresh weight of wet bacteria increased from 0.34 to 0.84g / 100mL, and the CO2 concentration in the conical flask increased from 400 to 1400μL / L. It shows that high CO2 concentration level corresponds to higher strain growth and lower CO2 release, which is more conducive to CO2 fixation.

[0074] Example 5

[0075] On the basis of Example 3, the CO2 concentration of the gas in the conical flask was set to 1200 μL / L, and the culture was carried out in the dark; when other conditions were the same, the hydroxyapatite-Bacillus subtilis material reinforced with humus was used for the CO2 conversion experiment. The experimental results in Table 1 show that the fresh weight of the wet bacteria increased from 0.31 to 0.89 g / 100 mL after 48 hours of dark culture, and the CO2 concentration in the conical flask increased from 1200 to 2200 μL / L. Under light conditions, the fresh weight of the wet bacteria increased from 0.31 to 0.92 g / 100 mL after 48 hours of culture, and the CO2 concentration in the conical flask increased from 1200 to 2000 μL / L. Bacillus subtilis is a heterotrophic organism, and the increase of its biomass is proportional to the amount of CO2 produced by its decomposition of organic matter; the dark condition culture system does not have semi-artificial photosynthetic carbon fixation, therefore: compared with the light condition culture, the dark condition culture of Bacillus subtilis has a smaller biomass increment (biomass increment is dark 0.58g / 100mL: light 0.61g / 100mL), but the amount of CO2 produced is larger (CO2 production 1000μL / L: 800μL / L). Compared with the dark culture, the light culture has a higher strain growth and a lower CO2 release, which is more conducive to CO2 fixation, indicating that the material prepared by the present invention is conducive to the carbon fixation of Bacillus subtilis.

[0076] Example 6

[0077] Humic substance-enhanced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material is used to convert CO2 and enhance the antioxidant enzyme activity of bacteria:

[0078] The composition of the culture medium described below is consistent with that of the culture medium in Example 1 above.

[0079] The Fe2O3-doped hydroxyapatite-Bacillus subtilis hybrid semi-artificial photosynthetic material containing 1.5% Fe obtained by centrifugation in the above Example 1 was added to 100 mL of culture medium in a new 250 mL conical flask, 0.1 g of humic acid and 0.1 g of fulvic acid were added, and the mixture was quickly mixed to obtain the hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material reinforced with humic substances. The conical flask was placed in a light incubator under indoor air conditions, the light conditions were controlled to be 3000 lux, and the culture was kept at 30° C. for 48 h.

[0080] The mixture was cultured for 48 hours and centrifuged at 1000 rpm for 5 minutes to obtain all the precipitates to obtain hydroxyapatite microspheres, the supernatant was retained, and the Bacillus subtilis adsorbed on the surface of the hydroxyapatite microspheres was washed off with new culture medium in small amounts for multiple times; the Bacillus subtilis eluted from the hydroxyapatite and in the supernatant of the culture system were combined, centrifuged at 10000 rpm for 10 minutes, and 0.82 g / 100 mL of wet cells of Bacillus subtilis cultured under light were obtained.

[0081] Treat the wet cells of Bacillus subtilis and detect its superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). The SOD result is as follows Figure 3 、POD results are as follows Figure 4 ,CAT results are as follows Figure 5 The activity of superoxide dismutase (SOD) was 231.1U / gFW / min, that of peroxidase (POD) was 976.4U / gFW / min, and that of catalase (CAT) was 281.5U / gFW / min.

[0082] However, the experiment of the present invention found that hydroxyapatite without Fe2O3 is not a semiconductor material and cannot generate photogenerated electrons and photogenerated holes under light. Therefore, the biomass growth and antioxidant enzyme activity of Bacillus subtilis in the non-hybrid system and in the hybrid system formed with undoped hydroxyapatite, regardless of light or dark conditions, are not significantly different from those of the Bacillus subtilis system; for example, peroxidase (POD) is about 837.3±24.1U / gFW / min, and catalase (CAT) is about 248.4±4.1U / gFW / min. This is basically equivalent to the literature value, such as the catalase (CAT) here is converted to 3.03×10 4 U / mL. As mentioned in the literature (Yao Dandan, Liu Liming, Li Jianghua, Hua Zhaozhe, Du Guocheng, Chen Jian, Reactive oxygen stress promotes excessive synthesis of catalase in Bacillus subtilis WSHDZ-01, Chinese Journal of Biotechnology, 2009, 25(5): 786-792.), the CAT enzyme activity range is 0.3-1.4×10 4 U / mL. The literature (Cao Wenlong, Guo Yaqiong, Kang Zhen, Du Guocheng, Optimization and industrialization of catalase production by Bacillus subtilis, Journal of Food and Biotechnology, 2015, 34(5): 482-486.) shows that the catalase activity of Bacillus subtilis can reach 3.5×10 4 The above results show that the hybrid system of iron-doped hydroxyapatite and Bacillus subtilis of the present invention corresponds to higher peroxidase and catalase, which is caused by various reasons, but is helpful for the application in the production of related enzyme preparations.

[0083] Example 7

[0084] On the basis of Example 6, in the same manner, the Fe2O3-doped hydroxyapatite-Bacillus subtilis hybrid semi-artificial photosynthetic material containing 1.5% Fe obtained by centrifugation in the above Example 1 was added to 100 mL of culture medium in a new 250 mL conical flask, and 0.1 g of humic acid and 0.1 g of fulvic acid were added, and the mixture was quickly mixed to obtain humic substance-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material. When other conditions were the same, the conical flask was placed in an incubator under indoor air conditions for dark culture, and cultured at a constant temperature of 30°C for 48 hours to obtain 0.79 g of cultured Bacillus subtilis wet cells. The antioxidant enzyme activity of Bacillus subtilis was detected, and its superoxide dismutase (SOD) activity was 123.9 U / gFW / min ( Figure 3 ), peroxidase (POD) was 793.5U / gFW / min( Figure 4 ), catalase (CAT) was 313.1U / gFW / min( Figure 5 ). Compared with dark culture, light culture can enhance the activity of SOD and POD enzymes of Bacillus subtilis, but the activity of CAT enzyme is lower under light conditions. The mechanism of the increase of related enzyme activities under light conditions is speculated to be that photogenerated electrons promote carbon fixation and promote the expression of related enzymes.

[0085] Example 8

[0086] On the basis of Example 1 and Example 6, undoped hydroxyapatite and Fe2O3-doped hydroxyapatite with a mass ratio of 5, 10, 13, and 26% of Fe were used as inorganic (semiconductor) materials, 0.1 g of the above materials were added to 100 mL of culture medium in the above 250 mL conical flask containing 0.3 g of wet bacteria, and adsorbed and cultured for 48 h at 30 ° C and an oscillation frequency of 150 rpm, and then centrifuged at 10000 rpm for 10 min to obtain a precipitate of Bacillus subtilis-Fe2O3-doped hydroxyapatite microsphere hybrid semi-artificial photosynthetic material. The above hybrid semi-artificial photosynthetic material was added to the new 100 mL culture medium, 0.1 g of humic acid and 0.1 g of fulvic acid were added, and the humic substance-enhanced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material was obtained. When other conditions are the same, place the conical flask in a 3000 lux light incubator for light culture or in a dark incubator for culture at 30°C for 48 hours to obtain the cultured Bacillus subtilis wet cells. Detect the SOD, POD, and CAT enzyme activities of Bacillus subtilis. The results are shown in Figure 3 , Figure 4 , Figure 5. When Fe2O3-doped hydroxyapatite exists in the system, the enzyme activities of SOD and POD are basically enhanced under light conditions. When Fe2O3-doped hydroxyapatite and the Fe mass ratio content is greater than 10%, the CAT enzyme activity is also enhanced under light conditions. It shows that the hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material enhanced by humus can effectively convert CO2 and enhance the antioxidant enzyme activity of bacteria. At the same time, there is no effect under dark conditions without doping. In general, the Fe2O3-doped hydroxyapatite-Bacillus subtilis hybrid semi-artificial photosynthetic material with 1.5% Fe has the best carbon fixation and enhancement of antioxidant enzyme activity.

Claims

1. A humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material, characterized in that: The material comprises humic substances, Fe2O3-doped hydroxyapatite microspheres and Bacillus subtilis; the Fe2O3-doped hydroxyapatite microspheres adsorb Bacillus subtilis, and the humic substances are distributed on the solid phase surface of the Fe2O3-doped hydroxyapatite microspheres or in the liquid phase of the culture medium.

2. The humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material according to claim 1, characterized in that: The humic substances preferably include humic acid and fulvic acid.

3. The humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material according to claim 1, characterized in that: The Bacillus subtilis is a conventional wild-type Bacillus subtilis.

4. A method for preparing the humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material according to claim 1, characterized in that: The steps of the treasure house are as follows: (1) adding an iron salt solution to hydroxyapatite microspheres, stirring evenly, and then adding water to prepare a sol, aging the sol, and then washing and drying to obtain Fe2O3-doped hydroxyapatite microspheres; (2) inoculating Bacillus subtilis into a culture medium, culturing at a constant temperature with shaking, and obtaining wet cells of Bacillus subtilis after centrifugation; (3) adding Fe2O3-doped hydroxyapatite microspheres to a culture medium containing wet cells of Bacillus subtilis for adsorption culture, and obtaining Bacillus subtilis-Fe2O3-doped hydroxyapatite microsphere hybrid semi-artificial photosynthetic material by centrifugation after adsorption culture; (4) adding the hybrid semi-artificial photosynthetic material into a culture medium, adding humus, and mixing to obtain a hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material enhanced by humus.

5. The preparation method according to claim 4, characterized in that: In step (1), an iron salt solution is added to the hydroxyapatite micron sphere powder, stirred evenly, and then water is added to prepare a sol, the sol is aged at a constant temperature of 70-80° C. for 2-3 hours, and then washed and dried to obtain Fe2O3-doped hydroxyapatite with a mass ratio of Fe of 1.5-26%.

6. The preparation method according to claim 4, characterized in that: The pH value of the culture medium for the expanded culture of Bacillus subtilis in step (2) is 6-8, the culture time is 24-48 hours, the constant temperature culture temperature is 26-30° C., the oscillation frequency is 100-200 rpm, the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 5-20 minutes.

7. The preparation method according to claim 4, characterized in that In step (3), the Fe2O3-doped hydroxyapatite microspheres are added to the wet cells of Bacillus subtilis, the concentration of the wet cells in the two-phase mixed solution is 1-10 g / L, the pH value of the adsorption culture medium is 6-8, the culture time is 24-48 h, the constant temperature culture temperature is 26-30 ° C, the oscillation frequency is 100-200 rpm, the centrifugal speed is 5000-10000 rpm, and the centrifugal time is 5-20 min.

8. The preparation method according to claim 4, characterized in that, In step (4), the hybrid semi-artificial photosynthetic material is added to a new culture medium, humus is added, and the mixture is mixed to obtain hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material reinforced by humus; the concentrations of Fe2O3-doped hydroxyapatite microspheres, Bacillus subtilis wet cells, humic acid, and fulvic acid in the culture medium are 0.1-1, 1-10, 0.8-1.2, and 0.8-1.2 g / L, respectively.

9. Use of the humus-reinforced hydroxyapatite-Bacillus subtilis semi-artificial photosynthetic material according to claim 1 in fixing CO2 by photocatalytic reduction and enhancing the antioxidant enzyme activity of Bacillus subtilis.

10. The use according to claim 9, characterized in that: The lighting conditions in the applied system are 0-3000 lux, CO2 concentration is 400-1200 μL / L, temperature conditions are 25-40° C., and the culture time is 24-72 hours.

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