A method for maintaining hydrogen production by photosynthesis of microalgae under ultra-high light intensity

Abstract

A method for maintaining continuous photosynthetic hydrogen production in microalgae under ultra-high light intensity belongs to the field of bioenergy technology. The method comprises the following steps: culturing microalgae: transferring the microalgae species to TAP culture medium and culturing them under light until the number of microalgae cells reaches the logarithmic growth phase; synthesizing poly(N-isopropylacrylamide-butyl acrylate) copolymer: dissolving N-isopropylacrylamide, butyl acrylate, and azobisisobutyronitrile solution in anhydrous tetrahydrofuran, deoxygenating for 30 minutes under magnetic stirring, and reacting at 50°C for 24 hours to obtain PNIPAM-BA; taking the microalgae solution, centrifuging and collecting the microalgae, dispersing the microalgae in HM culture medium, adding PNIPAM-BA and graphene oxide, and 2000 μmol photons·m ‑2 ·s ‑1 The light intensity is 25℃ and continuous. The present invention uses graphene oxide and PNIPAM‑BA to construct a sunlight-driven intelligent microalgae photoreactor, which can maintain the survival rate and chlorophyll content of microalgae under ultra-high light intensity, so that the microalgae can survive at 2000μmol photons·m ‑2 ·s ‑1 Under ultra-high light intensity, it can maintain high-rate photosynthetic hydrogen production.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of bio-energy, and particularly relates to a method for maintaining microalgae to continuously produce hydrogen under super-high light intensity. BACKGROUND

[0002] As a clean and sustainable energy carrier, hydrogen has great potential and can be used as a future fuel. When burned, hydrogen gas produces water as a product, which is not polluting to the environment. The combustion heat value (3042 cal / m 3 ) of hydrogen gas is high, and it is the highest in weight energy density among all known fuels so far. Today, hydrogen gas is mainly produced by steam reforming of hydrocarbons, gasification of coal, electrolysis of water, and refining of chemical by-products, etc. However, these methods of producing hydrogen do not fundamentally get rid of the dependence on fossil energy, and do not fundamentally eliminate pollution to the environment, and the production cost is generally high. Using solar energy to split water to produce hydrogen is an ideal way of producing hydrogen.

[0003] In nature, green algae can effectively use solar energy to produce hydrogen by using photosynthetic pigments and hydrogenase in their bodies. Green algae are simple photosynthetic autotrophic organisms that can use light energy to carry out photosynthesis in a culture medium containing only water and inorganic salts. Under anaerobic conditions, they can use light energy to split water to produce electrons and protons, and through a series of electron transfer processes and redox reactions, ultimately produce hydrogen gas through hydrogenase. This process does not require any organic matter as a substrate and energy source, and is therefore considered to be a truly clean and renewable energy production method.

[0004] Current research on hydrogen production by microalgae mostly focuses on weak light or medium light intensity (0-200 μmol photons·m -2 ·s -1)[XU Z, QI J, WANG S, et al. Algal cell bionics as a step towards photosynthesis-independent hydrogen production[J]. Nat. Commun., 2023, 14(1): 1872; ZHU X, XU Z, TANG H, et al. Photosynthesis-Mediated Intracellular Biomineralization of Gold Nanoparticles inside Chlorella Cells towards Hydrogen Boosting under Green Light[J]. Angew. Chem. Int. Ed., 2023, 62(33): e202308437.] and the light intensity in nature can reach 2000 μmol photons·m -2 ·s -1 However, few researchers have improved the photosynthetic hydrogen production of microalgae to the range of 500-2000 μmol photons·m -2 ·s -1 Two reasons limit microalgae from continuously producing hydrogen under high light intensity: 1. The evolution rate of oxygen in microalgae increases with increasing light intensity, and oxygen will cause hydrogenase to inactivate and terminate hydrogen evolution; 2. Excessive light intensity will damage the photosynthetic reaction center, cause photoinhibition, and ultimately lead to cell apoptosis. If the above two problems are not solved, it will greatly limit the large-scale hydrogen production of microalgae outdoors. SUMMARY

[0005] The purpose of the present application is to solve the problem that microalgae can only produce hydrogen under weak light or medium light (0-200 μmol photons·m -2 ·s -1 ) in the prior art, and to provide a method for maintaining microalgae to continuously produce hydrogen under super high light intensity (2000 μmol photons·m -2 ·s -1 ) for a long time. Chlorella can produce 12.53 L H2 / L in 25 days.

[0006] To achieve the above purpose, the technical solutions adopted by the present application are as follows:

[0007] A method for maintaining microalgae to continuously produce hydrogen under super high light intensity, the method comprising:

[0008] Step one, culture of microalgae: 100 mL of microalgae seed is transferred to 1 L of TAP medium, and is cultured in a light incubator at a temperature of 20-30℃, wherein the light intensity is 50-200 μmol photons·m -2 ·s -1 , and the microalgae cell number reaches the logarithmic growth phase;

[0009] Step two, synthesis of poly(N-isopropylacrylamide-butyl acrylate) (PNIPAM-BA) copolymer: 3-5 g of N-isopropylacrylamide (NIPAM) monomer, 0.1-0.5 g of butyl acrylate (BA), and 10-40 mg of azobisisobutyronitrile solution are dissolved in 30-60 mL of anhydrous tetrahydrofuran, and under the action of magnetic stirring, the solution is deoxygenated by blowing inert gas for 30 min. After deoxygenation, the solution is reacted at a temperature of 50℃ for 24 h, and then cooled; the solution is added dropwise to a mixed solution of diethyl ether and petroleum ether to obtain a precipitate PNIPAM-BA; the precipitate is filtered and vacuum dried for 12 h to obtain a dried PNIPAM-BA product;

[0010] Step three, hydrogen production by microalgae under ultrahigh light intensity: 50 mL of the microalgae solution obtained in step one is centrifuged for 5 min (5000 rpm) to collect the microalgae, and the collected microalgae is dispersed in 50 mL of HM medium and transferred to a 50 mL photoreactor; then 40-200 mg of PNIPAM-BA and 2.5 mg of graphene oxide (GO) are added to the photoreactor, and the mixture is shaken to obtain a smart photoreactor; the smart photoreactor containing the microalgae is transferred to a light incubator with a light intensity of 2000 μmol photons·m -2 ·s -1 and a temperature of 25℃, and is continuously illuminated.

[0011] Further, in step one, the microalgae is green algae.

[0012] Further, in step one, the green algae is one of Chlorella vulgaris, Streptophyta, Chlamydomonas reinhardtii, Scenedesmus or Lemna. Chlorella vulgaris has been commercialized and is easier to obtain in actual experiments.

[0013] Further, in step one, the light is (1) continuous light, or (2) light for 12 h and darkness for 12 h.

[0014] Further, in step three, the ingredients contained in each liter of HM medium are as follows: glucose 10 g, urea 0.914 g, KH2PO40.211 g, MgSO4·7H2O 0.157 g, FeSO4·7H2O 12.51 mg, H3BO32.85 mg, CuSO4·5H2O 0.8 mg, ZnSO4·7H2O 1.11 mg, CoSO4·7H2O 0.97 mg, MnCl2·4H2O 1.3 mg, (NH4)6Mo7O 24 ·4H2O 0.46 mg, CaCl26.94 mg, NaCl 1.16 mg, BTP (1,3-bis(tris(hydroxymethyl) aminomethyl) propane) 0.565 g, Mg(OH)21.17 g.

[0015] The beneficial effects of the present application over the prior art are: by constructing a sunlight-driven intelligent microalgae photoreactor based on the photo-thermal material graphene oxide and the thermal-sensitive material PNIPAM-BA, the survival rate and chlorophyll content of microalgae under ultra-high light intensity can be maintained, so that the microalgae can maintain a high rate of photosynthetic hydrogen production under an ultra-high light intensity of 2000 μmol photons·m -2 ·s -1 The method of the present application is simple and has good application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 is a graph of the light transmittance of PNIPAM-BA at different temperatures;

[0017] Figure 2 is a graph of the temperature change of the intelligent photoreactor containing Chlorella under strong light;

[0018] Figure 3 is a graph of the cumulative hydrogen production of Chlorella in the intelligent photoreactor (circular curve) and Chlorella alone (square curve) under strong light;

[0019] Figure 4 is a graph of the hydrogen production rate (square) and chlorophyll content (circle) of Chlorella in the intelligent photoreactor;

[0020] Figure 5 is a graph of the survival rate of Chlorella in the photoreactor with / without PNIPAM-BA and graphene oxide under light intensities of 100-2000 μmol photons·m -2 ·s -1 ;

[0021] Figure 6 is a graph of the survival rate of Chlorella in the photoreactor with / without PNIPAM-BA and graphene oxide under light intensities of 100-2000 μmol photons·m-2 ·s -1 Figure 2 shows the chlorophyll content graph under different light intensities. DETAILED DESCRIPTION

[0022] The technical solutions of the present application are further described below in conjunction with the accompanying drawings and examples, but are not limited thereto. Any modification or equivalent replacement of the technical solutions of the present application, without departing from the spirit and scope of the technical solutions of the present application, shall be covered within the protection scope of the present application.

[0023] Below the lower critical phase transition temperature, PNIPAM-BA exhibits intermolecular hydrogen bonds between polymer chains and surrounding water molecules, showing a transparent state. Above the lower critical phase transition temperature, the intermolecular hydrogen bonds are broken, and intramolecular hydrogen bonds are formed between CO and NH groups inside the polymer chains, leading to phase separation, scattering of incident light, and an opaque state. The entire phase transition process is completely reversible. Graphene oxide (GO) is a light-to-heat conversion material that can efficiently convert light energy into heat energy. The smart photoreactor composed of the thermosensitive polymer PNIPAM-BA and the light-heat material graphene oxide can dynamically perceive the intensity of light and regulate the entry of incident light. When the light is relatively strong (2000 μmol photons·m -2 ·s -1 ), graphene oxide absorbs light energy and converts it into heat energy to drive the phase transition of PNIPAM-BA, making the system opaque. At this time, most of the light is reflected, the light received by the microalgae is weakened, the photosynthetic oxygen production rate decreases, the activity of green algae hydrogenase is ensured, and thus the hydrogen production rate of microalgae is maintained at a high level.

[0024] Example 1

[0025] Step one: Chlorella cultivation: Chlorella spores were transferred to 1 L of TAP medium and cultured in a light incubator at a temperature of 25℃ with a 12h light and 12h dark cycle, with a light intensity of 100 μmol photons·m -2 ·s -1 When the number of Chlorella cells reached the logarithmic growth phase, it was taken;

[0026] Step two, synthesis of poly(N-isopropylacrylamide-butyl acrylate) (PNIPAM-BA) copolymer: 5 g of N-isopropylacrylamide (NIPAM) monomer, 0.25 g of butyl acrylate (BA) and 22 mg of azobisisobutyronitrile solution were dissolved in 50 mL of anhydrous tetrahydrofuran, under the action of magnetic stirring, the solution was deoxygenated by blowing inert gas for 30 min, after deoxygenation, the solution was reacted at 50°C for 24 h, and then cooled. The solution was added dropwise into a mixed solution of diethyl ether and petroleum ether to obtain a precipitate PNIPAM-BA. The precipitate was filtered and vacuum dried for 12 h to obtain a dried PNIPAM-BA product. The transmittance of 4 mg / mL PNIPAM-BA at 25-30°C was measured by a spectrophotometer. As shown in Figure 1 , the transmittance of photosynthetically active radiation of PNIPAM-BA at 25°C was 94%, and the transmittance of photosynthetically active radiation of PNIPAM-BA at 30°C was 5%. PNIPAM-BA can achieve 89% light regulation on photosynthetically active radiation.

[0027] Step three, OD 680 = 2-5 Chlorella vulgaris was centrifuged and re-dispersed in 50 mL of HM medium, and then transferred to a 50 mL photoreactor. Then 200 mg of PNIPAM-BA and 2.5 mg of graphene oxide were added to the reactor and shaken to obtain a smart photoreactor. The smart photoreactor containing Chlorella vulgaris was pre-dark treated for 24 h to form an anaerobic environment, and then transferred to a light intensity of 2000 μmol photons·m -2 ·s -1 under light to produce hydrogen. Due to the presence of graphene oxide photothermal material, the temperature of the system can be maintained at 31°C under a light intensity of 2000 μmol photons·m -2 ·s -1 , as shown in Figure 2 , most of the light is reflected, so as not to damage the Chlorella vulgaris cells. The composition of the HM medium per liter is as follows: glucose 10 g, urea 0.914 g, KH2PO40.211 g, MgSO4·7H2O 0.157 g, FeSO4·7H2O 12.51 mg, H3BO32.85 mg, CuSO4·5H2O 0.8 mg, ZnSO4·7H2O 1.11 mg, CoSO4·7H2O 0.97 mg, MnCl2·4H2O 1.3 mg, (NH4)6Mo7O 24 ·4H2O 0.46 mg, CaCl26.94 mg, NaCl 1.16 mg, BTP (1,3-bis(tris(hydroxymethyl)methylamino)propane) 0.565 g, Mg(OH)21.17 g.

[0028] Step four, the amount of hydrogen produced in the photobioreactor at different times was detected by gas chromatograph. The results are shown in Figure 3 Figure 2 (circular curve), Chlorella produced 12.53 L H2 / L in 25 days, and the average hydrogen production rate was 17.53 μmol H2(mg chlorophyll) -1 h -1 .

[0029] Comparative Example 1

[0030] Step one, Chlorella culture: Chlorella spores were transferred to 1 L of TAP medium, and were subjected to a 12 h light and 12 h dark cycle in a light incubator at 25°C, with a light intensity of 100 μmol photons·m -2 ·s -1 When the number of Chlorella cells reached the logarithmic growth phase, they were taken;

[0031] Step two, Chlorella with OD 680 = 2-5 was centrifuged and then dispersed in 50 mL of HM medium, and was transferred to a 50 mL photobioreactor. The photobioreactor containing Chlorella (without PNIPAM-BA and graphene oxide) was subjected to a 24 h dark treatment to form an anaerobic environment, and then was transferred to a hydrogen production under light with a light intensity of 2000 μmol photons·m -2 ·s -1 .

[0032] Step three, the amount of hydrogen produced in the photobioreactor at different times was detected by gas chromatograph. The results are shown in Figure 3 Figure 2 (square curve), Chlorella terminated hydrogen production in 4 days due to strong light damage, and the cumulative hydrogen production was 0.32 L H2 / L.

[0033] Comparative Example 2

[0034] Step one, Chlorella culture: Chlorella spores were transferred to 1 L of TAP medium, and were subjected to a 12 h light and 12 h dark cycle in a light incubator at 25°C, with a light intensity of 100 μmol photons·m -2 ·s -1 When the number of Chlorella cells reached the logarithmic growth phase, they were taken;

[0035] Step two, Chlorella with OD 680=2-5 were centrifuged and re-dispersed in 50 mL of TAP medium and transferred to 50 mL of photobioreactor containing PNIPAM-BA and graphene oxide. The survival rate and the change of chlorophyll content of Chlorella sp. under different light intensities were measured. Hydrogen production by Chlorella sp. is a biological metabolic process, so the survival rate of Chlorella sp. directly affects the amount of hydrogen production. Chlorophyll, as a light-harvesting molecule, plays an important role in maintaining the operation of PS II reaction centers. As shown in Figure 5 and Figure 6 , it can be seen that Chlorella sp. in the photobioreactor can maintain a very high survival rate (> 90%) and high chlorophyll content (15.5 (μg chlorophyll / mg dry weight)) under light intensity of 100-2000 μmol photons·m -2 ·s -1 ;

[0036] Step three, Chlorella sp. with OD 680 =2-5 were centrifuged and re-dispersed in 50 mL of TAP medium and transferred to 50 mL of photobioreactor (without PNIPAM-BA and graphene oxide). The survival rate and the change of chlorophyll content of Chlorella sp. under different light intensities were measured. As shown in Figure 5 and Figure 6 , we found that Chlorella sp. in the photobioreactor (without PNIPAM-BA and graphene oxide) can maintain a very high survival rate (> 90%) and high chlorophyll content (15.5 (μg chlorophyll / mg dry weight)) under light intensity of 100-2000 μmol photons·m -2 ·s -1 ; the survival rate of Chlorella sp. and the chlorophyll content decreased with the increase of light intensity. Under the light intensity of 2000 μmol photons·m -2 ·s -1 , Chlorella sp. almost died and the chlorophyll content was very low (0.74 (μg chlorophyll / mg dry weight)), which was the main reason why Chlorella sp. could not continuously produce hydrogen.

Claims

1. A method for maintaining continuous photosynthetic hydrogen production by microalgae under ultra-high light intensity, characterized by: The method is: Step 1: Cultivation of Chlorella: Transfer 100 mL of Chlorella seed into 1 L of TAP medium and irradiate in a light incubator at a temperature of 20-30°C with a light intensity of 50-200 μmol photons·m -2 ·s -1 , when the number of Chlorella cells reaches the logarithmic growth phase; Step 2, Synthesis of N-isopropylacrylamide-butyl acrylate (PNIPAM-BA) copolymer: 3-5 g of N-isopropylacrylamide (NIPAM) monomer, 0.1-0.5 g of butyl acrylate (BA), and 10-40 mg of azobisisobutyronitrile solution were dissolved in 30-60 mL of anhydrous tetrahydrofuran. Under magnetic stirring, inert gas was introduced into the solution to deoxygenate for 30 min. After deoxygenation, the mixture was reacted at 50°C for 24 h and cooled. The solution was added dropwise to a mixed solution of diethyl ether and petroleum ether to obtain a precipitate of PNIPAM-BA; the precipitate was filtered and vacuum dried for 12 h to obtain a dry PNIPAM-BA product; Step 3: Chlorella produces hydrogen under ultra-high light intensity: Take 50 mL of the Chlorella solution obtained in step 1, centrifuge for 5 minutes to collect the Chlorella, disperse the collected Chlorella in 50 mL of culture medium, and transfer it to a 50 mL photoreactor; Then, 40-200 mg of PNIPAM-BA and 2.5 mg of graphene oxide (GO) were added to the photoreactor and shaken to obtain a smart photoreactor. The smart photoreactor containing Chlorella vulgaris was pre-dark-treated for 24 hours to form an anaerobic environment and then transferred to a light intensity of 2000 μmol photons·m -2 ·s -1 , in a light incubator at 25°C with continuous light.

2. The method of claim 1, wherein: In step 1, the illumination is (1) continuous illumination, or (2) a cycle of 12 h light and 12 h dark.

3. The method of claim 1, wherein: In step 3, the components of each liter of culture medium are as follows: glucose 10 g, urea 0.914 g, KH2PO4 0.211 g, MgSO4·7H2O 0.157 g, FeSO4·7H2O 12.51 mg, H3BO3 2.85 mg, CuSO4·5H2O 0.8 mg, ZnSO4·7H2O 1.11 mg, CoSO4·7H2O 0.97 mg, MnCl2·4H2O 1.3 mg, (NH4)6Mo7O 24 4H2O 0.46 mg, CaCl2 6.94 mg, NaCl 1.16 mg, BTP (1,3-bis(tris(hydroxymethyl)methylamino)propane) 0.565 g, Mg(OH)2 1.17 g.

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