Microalgae semi-artificial photosynthesis hydrogen production system as well as preparation method and application thereof

Abstract

The invention relates to the field of hydrogen production through semi-artificial photosynthesis, and discloses a microalgae semi-artificial photosynthesis hydrogen production system as well as a preparation method and application thereof. The microalgae semi-artificial photosynthesis hydrogen production system comprises a microalgae-cadmium-based chalcogenide semiconductor heterogeneous aggregate; the microalgae-cadmium-based chalcogenide semiconductor heterogeneous aggregate is composed of a three-dimensional multicellular structure and a cadmium-based chalcogenide semiconductor layered structure, wherein the three-dimensional multicellular structure is formed by mutual adhesion and agglomeration of internal microalgae cells, the cadmium-based chalcogenide semiconductor layered structure is formed on the outer surface of microalgae in situ, and a cadmium-based chalcogenide semiconductor is obtained by in-situ generation of cadmium ions and chalcogenide anions on the surface of the microalgae. According to the microalgae-cadmium-based chalcogenide semiconductor heterogeneous aggregate semi-artificial photosynthesis hydrogen production system provided by the invention, the problem that the hydrogen production time and the hydrogen production rate of a conventional microalgae aggregate photosynthesis hydrogen production system are difficult to consider at the same time is solved through endogenous oxygen inhibition and exogenous photo-induced electron synergy; and the development of semi-artificial photosynthesis hydrogen production applied to large-scale commercial hydrogen preparation is expected to be promoted.

Description

Technical Field

[0001] The present invention relates to the technical field of semi-artificial photosynthesis hydrogen production, and in particular to a microalgae semi-artificial photosynthesis hydrogen production system, a preparation method thereof, and applications thereof. Background Art

[0002] As global energy demand and environmental crises intensify, the search for clean, renewable energy sources to replace traditional fossil fuels has become a pressing priority. Hydrogen, with its high energy density and the fact that its only byproduct during combustion is water, is considered an ideal clean energy source and is expected to play a vital role in the future global energy system. However, current commercial hydrogen production is almost exclusively derived from traditional fossil fuels like natural gas, failing to fully realize its clean, renewable properties.

[0003] In nature, microalgae can produce hydrogen through photosynthesis. The principle is that hydrogenase or nitrogenase in the microalgae directly or indirectly obtains some electrons from the photosynthetic electron transport chain to catalyze the reaction and produce hydrogen. The energy source of microalgae photosynthesis hydrogen production is solar energy, and the substrate is water or organic matter. The carbon dioxide produced can be reabsorbed and utilized by the microalgae, so it has excellent clean renewable properties and environmental sustainability. However, due to the oxygen sensitivity of hydrogenase and nitrogenase and the limitations of the light energy utilization of the photosystem, the efficiency of microalgae photosynthesis hydrogen production is low, which hinders its application and development.

[0004] To address the issue of oxygen sensitivity of hydrogen-producing enzymes, CN104962585B provides a method for sustainable photosynthetic hydrogen production by microalgae under natural aerobic conditions. It uses biosilicification to form free-floating Chlorella into aggregate structures, resulting in spatial functional differentiation. The Chlorella outside the aggregate receives light and performs conventional oxygen-releasing photosynthesis; the Chlorella inside lacks light, thus ceasing photosynthesis and releasing oxygen. However, the Chlorella's respiration is unaffected by light and can proceed normally, thus continuously consuming oxygen, ultimately forming an anaerobic environment inside the aggregate to achieve hydrogen production. This idea of ​​forming microalgae into aggregates effectively addresses the limitations of the inherent oxygen sensitivity of hydrogen-producing enzymes on photosynthetic hydrogen production by microalgae.

[0005] However, although microalgae aggregates can produce hydrogen through photosynthesis under aerobic conditions, the energy source of the microalgae with hydrogen production activity within the aggregates is severely limited, resulting in low hydrogen production efficiency. Therefore, although the addition of dimethyl sulfoxide to enhance microalgae respiration to increase the anaerobic zone in the aggregates (CN107267395B) and the addition of glucose to enable microalgae aggregates to enter a mixotrophic culture mode to obtain additional energy supplementation (CN114410694B) have both effectively improved the hydrogen production efficiency of microalgae aggregates, neither of these strategies addresses how to improve the microalgae's efficiency in converting light energy into hydrogen energy, resulting in a still-low efficiency in the system's conversion of light energy to hydrogen energy.

[0006] Semi-artificial photosynthesis is an innovative strategy for the conversion and utilization of light energy. By integrating the high efficiency of light energy utilization of artificial photosensitizers with the high selectivity and sustainability of biocatalytically active ingredients, it provides a new paradigm for overcoming the inherent limitations of natural photosynthetic systems. Among them, inorganic semiconductors such as cadmium-based chalcogenides significantly surpass the biological photosynthetic systems of microalgae in terms of light energy capture and photogenerated carrier yield due to their broad-spectrum absorption characteristics and high quantum efficiency limits. In addition, the synthesis process of cadmium-based chalcogenides is mature and simple, and the preparation cost is relatively low. Therefore, they are regarded as a promising artificial photosensitizer that can be used to construct semi-artificial photosynthesis systems with biocatalytically active ingredients.

[0007] Positively charged cadmium ions can induce aggregation of negatively charged microalgae cells through electrostatic interactions. Furthermore, cadmium ions can disrupt the function of photosystem II (PSII) in microalgae, inhibiting oxygen release. Consequently, the microalgae aggregates formed by cadmium ions can maintain an anaerobic environment for extended periods, thereby sustaining the activity of hydrogenases or nitrogenases. Subsequently, cadmium-based chalcogenides are formed in situ outside the microalgae aggregates through the reduction of sulfide anions (including sulfide, selenium, and tellurium ions), forming microalgae-cadmium-based chalcogenides heteroaggregates and constructing a microalgae-cadmium-based chalcogenide heteroaggregate system for semi-artificial photosynthetic hydrogen production. This designed and constructed semi-artificial photosynthetic hydrogen production system features long-lasting catalytic hydrogen production activity in microalgae and efficient light-to-hydrogen conversion of cadmium-based chalcogenides, providing sufficient photogenerated electrons for hydrogen production. This system combines a long hydrogen production time with a high hydrogen production rate and light-to-hydrogen conversion efficiency. Summary of the Invention

[0008] In order to solve the technical problem that it is difficult to balance the hydrogen production time and hydrogen production rate in the existing microalgae aggregate photosynthesis hydrogen production system, the present invention provides a microalgae semi-artificial photosynthesis hydrogen production system and its preparation method and application.

[0009] The first object of the present invention is to provide a microalgae semi-artificial photosynthesis hydrogen production system, comprising a microalgae-cadmium-based chalcogenide semiconductor heteroaggregate; the microalgae-cadmium-based chalcogenide semiconductor heteroaggregate is composed of a three-dimensional multicellular structure formed by the mutual adhesion and aggregation of internal microalgae cells, and a cadmium-based chalcogenide semiconductor layered structure formed in situ on the external surface of the microalgae, wherein the cadmium-based chalcogenide semiconductor is generated in situ by cadmium ions and chalcogen anions on the surface of the microalgae.

[0010] The microalgae-cadmium-based chalcogenide heteroaggregate herein refers to a composite system consisting of a three-dimensional multicellular structure formed by the adhesion and aggregation of internal microalgae cells, and a cadmium-based chalcogenide layered structure formed in situ on the external surface of the microalgae. Once the microalgae-cadmium-based chalcogenide heteroaggregate is formed, the function of the microalgae's Photosystem II within the aggregate is inhibited, ceasing oxygen release. The microalgae consume oxygen through their own respiration, thereby maintaining the anaerobic environment necessary for hydrogen production over the long term. The cadmium-based chalcogenide on the aggregate's exterior efficiently absorbs light energy to generate high-energy electrons that supply hydrogen production to the internal microalgae, thereby ensuring a high light energy utilization efficiency. The cadmium-based chalcogenide is generated in situ within the microalgae, and the microalgae-cadmium-based chalcogenide heteroaggregate is composed of the microalgae and its in situ generated cadmium-based chalcogenide.

[0011] In some embodiments of the present invention, the size of the microalgae-cadmium-based chalcogenide semiconductor heteroaggregates is 50 to 3000 μm; The microalgae include one or two of Chlamydomonas reinhardtii, Chlorella vulgaris or Chlorella pyrenoidosa. However, it should be understood that other microorganisms that can produce hydrogen and adsorb cadmium ions under anaerobic conditions are also suitable for the present invention.

[0012] In some embodiments of the present invention, the cadmium ions are selected from one or more of cadmium chloride, cadmium sulfate and cadmium nitrate.

[0013] In the present invention, the function of the microalgae photosystem II in the microalgae-cadmium-based chalcogenide heteroaggregate is inhibited, the photolysis of water stops, and oxygen and electrons cannot be released. The electron source of the microalgae hydrogen production is mainly the high-energy electrons generated by the cadmium-based chalcogenide semiconductor absorbing light energy.

[0014] In some embodiments of the present invention, the sulfide anion is a sulfide ion, which is generated from one or more compounds selected from sodium sulfide, cysteine, and thiourea. However, it should be understood that other sulfide anions capable of in situ generation of cadmium-based chalcogenides on the surface of microalgae through chemical reactions can also be used in the present invention.

[0015] In some embodiments of the present invention, the molar ratio of the cadmium ion to the sulfide anion is (1:10) to (1:50). For example, it can be 1:10, 1:15, 1:20, 1:25, 1:30, 1:35, 1:40, 1:45, 1:50, etc., or any interval value between any two values.

[0016] The second object of the present invention is to provide a method for preparing a microalgae semi-artificial photosynthesis hydrogen production system, comprising the following steps: Cadmium ions and sulfide anions are added to the liquid culture of microalgae growing in the logarithmic phase. The positively charged cadmium ions induce the aggregation of negatively charged microalgae through electrostatic interaction. The sulfide anions generate cadmium-based sulfide semiconductors in situ on the surface of the microalgae through chemical reactions, thereby forming microalgae-cadmium-based sulfide semiconductor heteroaggregates, and ultimately forming a microalgae semi-artificial photosynthesis hydrogen production system.

[0017] In some embodiments of the present invention, in step S1, the OD750 of the microalgae liquid culture is 0.1-1.0; The culture conditions of the microalgae liquid culture are as follows: the culture temperature is 15-30° C. and the illumination is 1000-10000 Lux; The pH of the microalgae liquid culture of the present invention is 6 to 8. For example, it can be 6, 6.5, 7, 7.5, 8, etc., or an interval between any two values. The present invention utilizes the fact that the photolysis water function of the microalgae photosystem II in the microalgae-cadmium-based chalcogenide semiconductor heteroaggregate is inhibited, stopping the release of oxygen. The microalgae consume oxygen through respiration, so that the culture system can maintain an anaerobic environment for a long time under light culture conditions. The pH is stably between 6 and 8, ensuring that the microalgae have the activity of efficient hydrogen production. The cadmium-based chalcogenide semiconductor efficiently absorbs light energy, generating a large amount of high-energy electrons to supply the microalgae with hydrogen production, thereby obtaining a long-term, efficient and stable semi-artificial photosynthesis hydrogen production system. The catalytic activity of the microalgae hydrogenase or nitrogenase is ensured within this pH range. This is because the microalgae hydrogenase or nitrogenase first requires an anaerobic environment to be activated, and then requires a suitable pH to catalyze hydrogen production.

[0018] In some embodiments of the present invention, the microalgae liquid culture is cultured in any one of TAP medium, SE medium and BG11 medium.

[0019] In some embodiments of the present invention, the concentration of cadmium ions is 0.25~50 mM, illustratively 0.25, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15, 20, 25, 30, 35, 40, 45, 50 mM, etc., or any interval value directly between any two values, more preferably 1~10 mM. The concentration of the sulfide anion is 1.25-250 mM, and can be illustratively 1.25, 2, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 mM, or any range between any two values. In practice, the amounts of cadmium ions and sulfide anions can be adjusted according to the specific microalgae cell concentration, as long as the system can ensure that clear microalgae-cadmium-based chalcogenide semiconductor heteroaggregates are produced.

[0020] The third object of the present invention is to provide the application of the microalgae semi-artificial photosynthesis hydrogen production system in hydrogen energy development.

[0021] The above technical solution of the present invention has the following advantages over the prior art: 1. The present invention uses cadmium ions and sulfide anions to directly form microalgae-cadmium-based sulfide semiconductor heteroaggregates in microalgae liquid culture, thereby realizing the construction of a low-cost and long-term semi-artificial photosynthesis hydrogen production system. On the one hand, cadmium ions block the oxygen source of the culture system from the source by inhibiting the oxygen-releasing activity of microalgae photosystem II; on the other hand, they consolidate the anaerobic environment of the culture system by inducing microalgae to form aggregate structures. On the one hand, sulfide anions react with cadmium ions to generate cadmium-based sulfide semiconductors through chemical reactions to alleviate the toxicity of cadmium ions to microalgae cells; on the other hand, the cadmium-based sulfide semiconductors generated in situ on the surface of microalgae aggregates can efficiently capture light energy and generate a large amount of photogenerated electrons to supply microalgae hydrogenase or nitrogenase, thereby improving the efficiency of microalgae hydrogen production.

[0022] 2. The microalgae-cadmium-based chalcogenide semiconductor heteroaggregate semi-artificial photosynthetic hydrogen production system provided by this invention solves the problem of balancing hydrogen production time and rate in conventional microalgae aggregate photosynthetic hydrogen production systems by synergizing endogenous oxygen suppression with exogenous photogenerated electrons. Experimental results show that this semi-artificial photosynthetic hydrogen production system can produce hydrogen continuously for more than 4 days, with an average hydrogen production rate of 17.82 µmol·H2·h -1 (mg chlorophyll) -1This invention is expected to promote the development of semi-artificial photosynthesis of microalgae for hydrogen production in large-scale commercial hydrogen production. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings, wherein: Figure 1 A schematic diagram of the construction process of microalgae-cadmium-based chalcogenide semiconductor heteroaggregates and their semi-artificial photosynthesis hydrogen production in the method provided by the present invention; Figure 2 This is a microscopic observation result of Chlamydomonas reinhardtii-cadmium sulfide heteroaggregates in Example 1 of the present invention, where the scale bar is 300 μm; Figure 3 This is a microscopic observation result of normally cultured Chlamydomonas reinhardtii in Comparative Example 1 of the present invention, with the scale bar in the figure being 300 μm; Figure 4 This is a microscopic observation result of the homogeneous aggregates of Chlamydomonas reinhardtii after adding cadmium chloride in Comparative Example 2 of the present invention, and the scale bar in the figure is 300 μm; Figure 5 This is a microscopic observation result of Chlamydomonas reinhardtii after adding sodium sulfide in Comparative Example 3 of the present invention, and the scale bar in the figure is 300 μm; Figure 6 This is the UV-visible absorption spectrum of the Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate in Example 2 of the present invention, the circle-marked curve is Example 1, and the square-marked curve is Comparative Example 1; Figure 7 3 is a graph showing changes in oxygen content in Example 3 of the present invention. The circular curve is for Example 1, the regular triangle curve is for Comparative Example 2, the inverted triangle curve is for Comparative Example 3, and the square curve is for Comparative Example 1. Figure 8 3 is a graph showing the change in hydrogen content in Example 3 of the present invention. The circular curve is for Example 1, the regular triangle curve is for Comparative Example 2, the inverted triangle curve is for Comparative Example 3, and the square curve is for Comparative Example 1. Figure 9 The pH change curve in Example 4 of the present invention is shown in FIG. 4. The circle-marked curve is the experimental group, the regular triangle-marked curve is the comparative example 2, the inverted triangle-marked curve is the comparative example 3, and the square-marked curve is the comparative example 1. Figure 10 This is a graph showing the change in chlorophyll content in Chlamydomonas reinhardtii in Example 5 of the present invention, wherein the circle-marked curve is Example 1, the regular triangle-marked curve is Comparative Example 2, the inverted triangle-marked curve is Comparative Example 3, and the square-marked curve is Comparative Example 1; Figure 11Graph showing the change in cell activity of Chlamydomonas reinhardtii in Example 6 of the present invention, wherein the dotted line represents Comparative Example 1, which is set to 100% cell activity, the circular curve represents Example 1, the equilateral triangle curve represents Comparative Example 2, and the inverted triangle curve represents Comparative Example 3; Figure 12 The graph of the change in photosynthetic activity of Chlamydomonas reinhardtii in Example 7 of the present invention is shown in FIG. 7 , wherein the circle-marked curve is Example 1, the regular triangle-marked curve is Comparative Document 2, the inverted triangle-marked curve is Comparative Example 3, and the square-marked curve is Comparative Example 1. Figure 13 Schematic diagram of the photosynthetic electron transport chain and electron inhibitor action sites of Chlamydomonas reinhardtii in Example 8 of the present invention, wherein the direction of the arrow represents the direction of electron flow; Figure 14 This is a statistical graph of the cumulative hydrogen production results after the addition of an electron inhibitor in Example 8 of the present invention. The horizontal line represents the control group, that is, the cumulative hydrogen production of the semi-artificial photosynthesis hydrogen production system (Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate) without the addition of a photosynthetic electron inhibitor; the slash represents the cumulative hydrogen production of the semi-artificial photosynthesis hydrogen production system (Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate + DCMU) with the addition of DCMU; and the backslash represents the cumulative hydrogen production of the semi-artificial photosynthesis hydrogen production system (Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate + DBMIB) with the addition of DBMIB. DETAILED DESCRIPTION

[0024] The present invention will be further described below with reference to the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it. However, the embodiments are not intended to limit the present invention.

[0025] In the present invention, a simple, inexpensive and long-lasting method for preparing a semi-artificial photosynthesis hydrogen production system is provided. It should be understood that this is only for example and not limitation. In this embodiment, cadmium chloride and sodium sulfide are selected to be combined with Chlamydomonas reinhardtii cultured in TAP medium to generate Chlamydomonas reinhardtii-cadmium sulfide heteroaggregates. The scale of the semi-artificial photosynthesis hydrogen production system is 1 to 15 mL, the amount of cadmium chloride used is preferably 5 to 75 μmol, and the amount of sodium sulfide used is preferably 20 to 300 μmol. The content of oxygen and hydrogen in the system is monitored using a gas chromatograph, and the following examples specifically illustrate the implementation effect of the present invention. However, it should be understood that the semi-artificial photosynthesis hydrogen production system provided by the present invention has the potential for large-scale expansion and is suitable for larger scales far exceeding 15 mL. The scale is not limited under the condition of sufficient raw material supply.

[0026] Cadmium chloride, sodium sulfide, and 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) were purchased from Sinopharm Chemical Reagent Co., Ltd.; 2,5-dibromo-6-isopropyl-3-methyl-1,4-benzoquinone (DBMIB) was purchased from Shanghai Titan Technology Co., Ltd.; 5-[3-(carboxymethoxy)phenyl]-3-(4,5-dimethyl-2-thiazolyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt (MTS) and a chlorophyll content analysis kit were purchased from Beijing Biolab Technology Co., Ltd.; the remaining reagents were purchased from Sigma-Aldrich China; and Chlamydomonas reinhardtii (FACHB-479) was purchased from the Freshwater Algae Seed Bank of the Chinese Academy of Sciences.

[0027] The gas chromatograph used was an Agilent 8860 gas chromatograph (Agilent Technologies, USA), equipped with a TCD detector capable of simultaneously analyzing the oxygen and hydrogen contents. The carrier gas was high-purity nitrogen with a flow rate of 3-30 mL / min.

[0028] The working principle of the present invention is to introduce cadmium ions to selectively inhibit the activity of microalgae photosystem II to completely shut down the endogenous photolysis water oxygen production pathway, and at the same time use the positively charged cadmium ions to promote the aggregation of microalgae cells through electrostatic interactions with negatively charged groups such as carboxyl and phosphate groups in the microalgae cell wall, and then introduce sulfide anions to react with cadmium ions to in situ generate cadmium-based sulfide semiconductor layered structures with a wide spectrum response on the surface of microalgae cells. The photogenerated electron flow serves as the main electron source for hydrogen production by microalgae. The constructed microalgae-cadmium-based sulfide semiconductor heterogeneous aggregates have both long-term anaerobic environment self-sustaining ability and high light energy utilization efficiency, such as Figure 1 As shown; then, a gas chromatograph was used to monitor the content of oxygen and hydrogen in the microalgae-cadmium-based chalcogenide heteroaggregate semi-artificial photosynthesis hydrogen production system.

[0029] Example 1 This embodiment provides a microalgae semi-artificial photosynthesis hydrogen production system and a preparation method thereof, as shown below: 13.75 mg of cadmium chloride was added to a centrifuge tube containing 15 mL of Chlamydomonas reinhardtii culture (OD750 = 0.5). The mixture was shaken at 300 rpm for 2 hours at room temperature, during which time the cadmium ions were adsorbed on the cell walls of the Chlamydomonas reinhardtii cells. The mixture was then centrifuged at 3000 rpm for 3 minutes to remove free cadmium ions. The precipitate was transferred to a 65 mL clear glass tube and 15 mL of TAP medium was added to obtain the cadmium-adsorbed Chlamydomonas reinhardtii cells. 23.41 mg of sodium sulfide was then added to form the Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate, a semi-artificial photosynthetic hydrogen production system. Using a gas-tight needle, 10 μL of the Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate liquid culture was dropped onto a glass slide. After covering with a coverslip, the Chlamydomonas reinhardtii biomorphology in the system was observed under an Olympus inverted fluorescence microscope.

[0030] The results are as follows Figure 2 As shown in the figure (the scale bar in the figure is 300 μm), it can be seen from the figure that a large number of large-sized and compactly structured Chlamydomonas reinhardtii-cadmium sulfide heteroaggregates are produced in the semi-artificial photosynthesis hydrogen production system obtained according to the preparation method of the present invention.

[0031] Comparative Example 1 This comparative example is similar to Example 1, except that no other reagents were added to the liquid culture system of Chlamydomonas reinhardtii after culture, i.e., the microscopic observation results of Chlamydomonas reinhardtii cultured normally are as follows. Figure 3 As shown, no aggregates of Chlamydomonas reinhardtii were observed in the culture system.

[0032] Comparative Example 2 This comparative example is similar to Example 1, except that only cadmium chloride was added to the liquid culture system of Chlamydomonas reinhardtii without sodium sulfide. The microscopic observation results are as follows: Figure 4 As shown, a certain number of Chlamydomonas reinhardtii homogeneous aggregates with relatively small size and relatively loose structure were formed in the system.

[0033] Comparative Example 3 This comparative example is similar to Example 1, except that only sodium sulfide was added to the liquid culture system of Chlamydomonas reinhardtii without adding cadmium chloride. The microscopic observation results are as follows: Figure 5 As shown, no obvious aggregates were observed in the system.

[0034] Example 2 The semi-artificial photosynthesis hydrogen production system prepared in Example 1 was subjected to ultraviolet-visible absorption spectral analysis of the Chlamydomonas reinhardtii-cadmium sulfide heteroaggregates. 200 μL of the Chlamydomonas reinhardtii-cadmium sulfide heteroaggregates were placed in a cuvette, and the ultraviolet-visible absorption spectrum of the culture was measured using a double-beam spectrophotometer (Hitachi, Japan).

[0035] The results are as follows Figure 6 As shown, the Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate obtained according to the preparation method of the present invention has good light absorption ability.

[0036] Example 3 The transparent glass test tube of Example 1 was sealed and cultured at 25°C and 6300 lux illumination. 100 μL of the headspace gas in the test tube was extracted using a gas-tight needle and injected into the inlet of a gas chromatograph for analysis of the oxygen and hydrogen contents in the culture system.

[0037] The results are as follows Figure 7 , the semi-artificial photosynthesis hydrogen production system obtained by the preparation method of the present invention is in an anaerobic environment for 4 days; Figure 8 As shown, the semi-artificial photosynthesis hydrogen production system obtained by the preparation method of the present invention continuously and efficiently produces hydrogen for 4 days, accumulating 58.26 μmol of hydrogen. In contrast, in Comparative Example 2, because there is no cadmium sulfide semiconductor to supply photoelectrons, the microalgae hydrogenase has no electron source when the photolysis water function of photosystem II is inhibited, so no hydrogen is produced within 4 days. In Comparative Examples 1 and 3, since the microalgae photosystem II in the system functions normally, but no obvious microalgae aggregates are formed in the system, only a small amount of microalgae in the local anaerobic environment can perform short-term photosynthesis hydrogen production, so only 2.30 μmol of hydrogen and 2.72 μmol of hydrogen are accumulated in 4 days, respectively. It can be seen from this that only by forming a microalgae-cadmium-based chalcogenide semiconductor heteroaggregate and constructing a robust semi-artificial photosynthesis system can the system achieve efficient conversion of light energy into hydrogen.

[0038] Example 4 The pH of the semi-artificial photosynthesis hydrogen production system prepared in Example 1 was measured using a pH meter to obtain the corresponding pH value of the system.

[0039] The results are as follows Figure 9 As shown, the pH of the experimental group, i.e., the semi-artificial photosynthetic hydrogen production system forming Chlamydomonas reinhardtii-cadmium sulfide heteroaggregates, is relatively stable, maintained at 7.4~7.7, which is similar to the pH change in Comparative Example 1. The pH of Comparative Example 2 is lowered due to the hydrolysis of cadmium chloride, and the pH of Comparative Example 3 is increased due to the hydrolysis of sodium sulfide, indicating that the pH of the semi-artificial photosynthetic hydrogen production system prepared according to the present invention is in the pH range for optimal hydrogen production catalyzed by hydrogenase.

[0040] Example 5 Place 200 μL of a Chlamydomonas reinhardtii culture (C. reinhardtii-cadmium sulfide heteroaggregate) from a semi-artificial photosynthetic hydrogen production system, a C. reinhardtii culture with only cadmium chloride added (C. reinhardtii + cadmium chloride), and a C. reinhardtii culture (C. reinhardtii) in an EP tube. Add 200 μL of chlorophyll extraction buffer and shake at room temperature in the dark for 4 hours. Then, centrifuge at 10,000 g for 15 minutes in a high-speed centrifuge. The supernatant is used as the chlorophyll extract. Transfer 200 μL of the chlorophyll extract to a transparent 96-well plate and measure the absorbance of the sample at 665 nm and 649 nm using a microplate reader. According to the following empirical formula: Total chlorophyll content (mg / L) = CT × N; CT = 6.63 × A 665 + 18.08 × A 649 ; N = dilution factor; A 665 and A 649 Represent the absorbance values ​​at 665 nm and 649 nm, respectively.

[0041] Get the total chlorophyll content results of Chlamydomonas reinhardtii.

[0042] The results are as follows Figure 10 In Comparative Example 2 where only cadmium chloride was added, the total chlorophyll of Chlamydomonas reinhardtii decreased continuously over time due to the toxicity of cadmium ions, and the chlorophyll content on the 4th day was 0.70 mg / L. In Comparative Example 3, the total chlorophyll decreased continuously over time due to the toxicity of sulfur ions, and the chlorophyll content on the 4th day was 1.73 mg / L. In the experimental group, that is, the total chlorophyll content of Chlamydomonas reinhardtii in the semi-artificial photosynthetic hydrogen production of Chlamydomonas reinhardtii was also affected by the toxicity of cadmium ions and decreased continuously over time, and was 1.89 mg / L on the 4th day. This shows that in the semi-artificial photosynthetic hydrogen production system prepared according to the present invention, the formation of cadmium sulfide reduces the effect of cadmium ion toxicity on Chlamydomonas reinhardtii, so that the chlorophyll content of Chlamydomonas reinhardtii increases relative to Comparative Examples 2 and 3.

[0043] Example 6 Take 200 μL of the Chlamydomonas reinhardtii culture of the semi-artificial photosynthesis hydrogen production system obtained in Example 1 (Example 1, Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate), the Chlamydomonas reinhardtii culture obtained in Comparative Example 2 (Chlamydomonas reinhardtii + cadmium chloride), the Chlamydomonas reinhardtii culture obtained in Comparative Example 3 (Comparative Example 3, Chlamydomonas reinhardtii + sodium sulfide), and the normally cultured Chlamydomonas reinhardtii culture obtained in Comparative Example 1 in an EP tube, add 20 μL of MTS solution, shake in the dark at room temperature for 4 h, and then use a high-speed centrifuge to centrifuge at a centrifugal force of 10,000 g for 15 min, leaving the supernatant for cell activity test. Take 150 μL of the supernatant and transfer it to a transparent 96-well plate, and measure the light absorbance of the sample at 490 nm using a microplate reader. According to the following empirical formula: Cell viability (%) = (OD490 s -OD490 b ) / (OD490 c -OD490 b )×100; OD490 s is the light absorbance at a wavelength of 490 nm of the supernatant of the Chlamydomonas reinhardtii culture obtained in Examples and Comparative Examples after being treated with MTS; OD490 c The absorbance of the supernatant of the normally cultured Chlamydomonas reinhardtii culture obtained in Comparative Example 1 at a wavelength of 490 nm after being treated with MTS; OD490 b The absorbance of blank TAP culture medium after treatment with MTS at 490 nm.

[0044] The results are as follows Figure 11 As shown, by comparing with the results of Comparative Example 1, the relative cell activity of Chlamydomonas reinhardtii in the semi-artificial photosynthetic hydrogen production of Chlamydomonas reinhardtii-cadmium sulfide heteroaggregates obtained in Example 1 is close to that of Comparative Example 1, and is higher than that of Comparative Example 2 and Comparative Example 3, indicating that in the semi-artificial photosynthetic hydrogen production system prepared according to the present invention, the effect of cadmium ion toxicity on cell activity is reduced, and Chlamydomonas reinhardtii has relatively good cell activity.

[0045] Example 7 Take 1 mL of the Chlamydomonas reinhardtii culture of the semi-artificial photosynthesis hydrogen production system obtained in Example 1 (Example 1, Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate), the Chlamydomonas reinhardtii culture obtained in Comparative Example 2 (Chlamydomonas reinhardtii + cadmium chloride), the Chlamydomonas reinhardtii culture obtained in Comparative Example 3 (Comparative Example 3, Chlamydomonas reinhardtii + sodium sulfide), and the normally cultured Chlamydomonas reinhardtii culture obtained in Comparative Example 1 in a cuvette and dark-adapt for 15 minutes to ensure that the reaction center of photosystem II is fully open and the photosynthetic electron transfer chain is completely oxidized. The photosynthetic activity of the dark-adapted Chlamydomonas reinhardtii culture is tested using a portable modulated chlorophyll fluorescence instrument, that is, the F v / F m ; F v is the variable fluorescence amount; F m is the maximum fluorescence yield; The results are as follows Figure 12 , Chlamydomonas reinhardtii obtained in comparative example 2 with only cadmium chloride added (Chlamydomonas reinhardtii + cadmium chloride) F v / F m Because cadmium ions destroy the proteins of photosystem II and are close to 0, the Fv / Fm of Chlamydomonas reinhardtii in the semi-artificial photosynthetic hydrogen production system of Chlamydomonas reinhardtii-cadmium sulfide heteroaggregates obtained in Example 1 is also close to 0, indicating that in the semi-artificial photosynthetic hydrogen production system prepared according to the present invention, cadmium ions selectively inhibit the activity of green algae photosystem II, thereby completely shutting down its endogenous photolysis water oxygen evolution pathway.

[0046] Example 8 After monitoring the oxygen and hydrogen contents on the first day of cultivation in Example 3, photosynthetic electron inhibitors DCMU (10 μL) and DBMIB (10 μL) were added to the semi-artificial photosynthesis hydrogen production system. DCMU acts on the QB site of the terminal plastoquinone of photosystem II, and DBMIB acts on the cytochrome b6f complex site. Figure 13 Subsequently, the contents of oxygen and hydrogen in the system after adding the above-mentioned photosynthetic electron inhibitor were monitored by gas chromatography.

[0047] The results are as follows Figure 14As shown, by comparing the cumulative hydrogen production of the above-mentioned semi-artificial photosynthetic hydrogen production system with the addition of DCMU (Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate + DCMU) with the addition of photosynthetic electron inhibitors, the cumulative hydrogen production of the above-mentioned semi-artificial photosynthetic hydrogen production system (Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate + DBMIB) was reduced by 58.7%, and the cumulative hydrogen production of the above-mentioned semi-artificial photosynthetic hydrogen production system (Chlamydomonas reinhardtii-cadmium sulfide heteroaggregate + DBMIB) was reduced by 87.9%, indicating that the photogenerated electrons of cadmium sulfide efficiently entered the photosynthetic electron transport chain of green algae, and most of the electrons entered the photosynthetic electron transport chain from photosystem II and the plastoquinone pool or other pathways before photosystem I, and only a small amount of electrons could be directly transferred to hydrogenase.

[0048] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A microalgae semi-artificial photosynthesis hydrogen production system, characterized in that: It includes a microalgae-cadmium-based chalcogenide semiconductor heteroaggregate; the microalgae-cadmium-based chalcogenide semiconductor heteroaggregate is composed of a three-dimensional multicellular structure formed by the mutual adhesion and aggregation of internal microalgae cells, and a cadmium-based chalcogenide semiconductor layered structure formed in situ on the external surface of the microalgae, and the cadmium-based chalcogenide semiconductor is obtained by in situ generation of cadmium ions and chalcogen anions on the surface of the microalgae.

2. The microalgae semi-artificial photosynthesis hydrogen production system according to claim 1, characterized in that: The size of the microalgae-cadmium-based chalcogenide semiconductor heterogeneous aggregates is 50 to 3000 μm; The microalgae include one or two of Chlamydomonas reinhardtii, Chlorella vulgaris or Chlorella pyrenoidosa.

3. The microalgae semi-artificial photosynthesis hydrogen production system according to claim 1, characterized in that: The cadmium ions are selected from one or more of cadmium chloride, cadmium sulfate and cadmium nitrate.

4. The microalgae semi-artificial photosynthesis hydrogen production system according to claim 1, characterized in that: The sulfide anion is a sulfide ion, and the sulfide ion is generated from one or more compounds selected from sodium sulfide, cysteine ​​and thiourea.

5. The microalgae semi-artificial photosynthesis hydrogen production system according to claim 1, characterized in that: The molar ratio of the cadmium ions to the sulfide anions is (1:10) to (1:50).

6. A method for preparing a microalgae semi-artificial photosynthesis hydrogen production system according to any one of claims 1 to 5, characterized in that: The following steps are involved: Cadmium ions and sulfide anions are added to the liquid culture of microalgae growing in the logarithmic phase. The positively charged cadmium ions induce the aggregation of negatively charged microalgae through electrostatic interaction. The sulfide anions generate cadmium-based sulfide semiconductors in situ on the surface of the microalgae through chemical reactions, thereby forming microalgae-cadmium-based sulfide semiconductor heteroaggregates, and ultimately forming a microalgae semi-artificial photosynthesis hydrogen production system.

7. The preparation method according to claim 6, characterized in that In step S1, the OD750 of the microalgae liquid culture is 0.1-1.0; The culture conditions of the microalgae liquid culture are as follows: the culture temperature is 15-30° C., and the illumination is 1000-10000 Lux; The pH of the microalgae liquid culture is 6-8.

8. The preparation method according to claim 6, characterized in that The microalgae liquid culture is cultured in any one of TAP medium, SE medium and BG11 medium.

9. The preparation method according to claim 6, characterized in that The concentration of cadmium ions is 0.25~50 mM, and the concentration of sulfide anions is 1.25~250 mM.

10. Use of the microalgae semi-artificial photosynthesis hydrogen production system according to any one of claims 1 to 5 in hydrogen energy development.

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