Method for maintaining continuous photosynthetic hydrogen production of microalgae under ultrahigh light intensity

Abstract

The invention discloses a method for maintaining continuous photosynthetic hydrogen production of microalgae under ultrahigh light intensity, and belongs to the technical field of biological energy sources. The method comprises the following steps: culturing microalgae: transferring microalgae species into a TAP culture medium, and carrying out illumination culture until the number of microalgae cells reaches a logarithmic phase; the preparation method comprises the following steps: synthesizing a poly (N-isopropylacrylamide-butyl acrylate) copolymer, namely dissolving N-isopropylacrylamide, butyl acrylate and an azodiisobutyronitrile solution in anhydrous tetrahydrofuran, removing oxygen for 30 minutes under the action of magnetic stirring, and reacting for 24 hours at the temperature of 50 DEG C after the oxygen removal is finished, so as to obtain PNIPAM-BA (poly (N-isopropylacrylamide-butyl acrylate)-BA (poly (N-isopropylacrylamide-butyl acrylate))-BA (poly (N-isopropylacrylamide)-BA); the method comprises the following steps: taking a microalgae solution, centrifugally collecting microalgae, dispersing the microalgae in an HM culture medium, adding PNIPAM-BA and graphene oxide, and continuously illuminating at 25 DEG C under the light intensity of 2000 [mu] mol photons.m <-2 >. S <-1 >; the sunlight-driven intelligent microalgae photoreactor is constructed through graphene oxide and PNIPAM-BA, the survival rate and chlorophyll content of microalgae under the ultrahigh light intensity can be maintained, and the microalgae can maintain high-rate photosynthetic hydrogen production under the ultrahigh light intensity of 2000 micromoles photons * m <-2 > * s <-1 >.

Description

Technical Field

[0001] The present invention belongs to the technical field of bioenergy, and in particular relates to a method for maintaining continuous photosynthetic hydrogen production by microalgae under ultra-high light intensity. Background Art

[0002] As a clean and sustainable energy carrier, hydrogen has great potential as a future fuel. When burned, hydrogen produces water and is environmentally friendly. The calorific value of hydrogen (3042 cal / m 3 ) is high, and has the highest weight energy density of all fuels known so far. Today, hydrogen is mainly produced by steam reforming of hydrocarbons, gasification of coal, electrolysis of water, and refining of chemical by-products. However, these hydrogen production methods have not fundamentally gotten rid of the dependence on fossil energy, nor have they fundamentally eliminated environmental pollution, and the production costs are generally high. Using solar energy to photolyze water to produce hydrogen is an ideal way to produce hydrogen.

[0003] In nature, green algae can effectively use solar energy to produce hydrogen by using photosynthetic pigments and hydrogenases in their bodies. Green algae are simple photosynthetic autotrophic organisms that can use light energy to photosynthesize 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, they ultimately produce hydrogen through hydrogenase. This process does not require any organic matter as a substrate or energy source, so it is considered a truly clean and renewable energy production method.

[0004] At present, most of the research on hydrogen production by microalgae is focused on weak light or medium-intensity light range (0–200 μmolphotons·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.], while the light intensity in nature can reach 2000 μmol photons·m -2 ·s -1 However, few researchers have increased the light intensity of photosynthetic hydrogen production in microalgae to 500-2000 μmol photons·m -2 ·s -1 There are two main reasons that limit the continuous production of hydrogen by microalgae under high light intensity: 1. The rate of oxygen evolution in microalgae increases with the increase of light intensity, while oxygen will inactivate hydrogenase and terminate hydrogen evolution; 2. Excessive light intensity will destroy the photoreaction center, leading to photoinhibition and ultimately cell apoptosis. If the above two problems are not solved, the large-scale outdoor hydrogen production application of microalgae will be greatly limited. Summary of the invention

[0005] The purpose of the present invention is to solve the problem that the microalgae in the prior art can only be grown under weak light or medium strong light (0-200 μmol photons·m -2 ·s -1 ) to produce hydrogen, providing a method to maintain microalgae under ultra-high light intensity (2000 μmolphotons·m -2 ·s -1 ) Using the continuous photosynthetic hydrogen production method, Chlorella produced a total of 12.53LH2 / L of hydrogen within 25 days.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

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

[0008] Step 1: Cultivation of microalgae: 100 mL of microalgae seed was transferred into 1 L of TAP medium and exposed to light in a light incubator at a temperature of 20-30 °C, where the light intensity was 50-200 μmol photons·m -2 ·s -1 , when the number of microalgae cells reaches the logarithmic growth phase;

[0009] Step 2, 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 inert gas is introduced into the solution for deoxygenation for 30 min under magnetic stirring. After deoxygenation, the solution is reacted at 50° C. for 24 h and cooled; the solution is added dropwise to a mixed solution of ether and petroleum ether to obtain a precipitate PNIPAM-BA; the precipitate is filtered and vacuum dried for 12 h to obtain a dry PNIPAM-BA product;

[0010] Step 3: Microalgae produce hydrogen under ultra-high light intensity: Take 50 mL of the microalgae solution obtained in step 1, centrifuge for 5 minutes (5000 rpm) to collect the microalgae, disperse the collected microalgae in 50 mL of HM medium, and transfer them to a 50 mL photoreactor; then add 40-200 mg PNIPAM-BA and 2.5 mg of graphene oxide (GO) to the photoreactor, shake well to obtain a smart photoreactor; transfer the smart photoreactor containing the microalgae to a light intensity of 2000 μmol photons·m -2 ·s -1 , in a light incubator at 25°C with continuous light.

[0011] Furthermore, in step 1, the microalgae is green algae.

[0012] Furthermore, in step 1, the green algae is one of Chlorella vulgaris, Cellulose algae, Chlamydomonas reinhardtii, Scenedesmus or Lunar algae. Chlorella vulgaris has been commercialized and is easier to obtain in actual experiments.

[0013] Furthermore, in step 1, the illumination is (1) continuous illumination, or (2) a cycle of 12 hours of light and 12 hours of darkness.

[0014] Further, in step 3, the components of each liter of HM 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.

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

[0016] Figure 1 The transmittance variation diagram of PNIPAM-BA at different temperatures;

[0017] Figure 2 This is a temperature change diagram of the smart photoreactor containing Chlorella under strong light;

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

[0019] Figure 4 The graphs are the changes in hydrogen production rate (square) and chlorophyll content (circle) of Chlorella in the smart photoreactor;

[0020] Figure 5 The photoreactors for Chlorella vulgaris with and without PNIPAM-BA and graphene oxide were used at 100-2000 μmol photons·m -2 ·s -1 Survival rate diagram under light intensity of ;

[0021] Figure 6 The results show that the growth of Chlorella vulgaris in a photoreactor with or without PNIPAM-BA and graphene oxide was 100-2000 μmol photons·m-2 ·s -1 Chlorophyll content at different light intensities. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and embodiments, but is not limited thereto. Any modification or equivalent replacement of the technical solution of the present invention without departing from the spirit and scope of the technical solution of the present invention should be included in the protection scope of the present invention.

[0023] When PNIPAM-BA is below the lower critical phase transition temperature, there are intermolecular hydrogen bonds between the polymer chain and the surrounding water molecules, showing a transparent state; when PNIPAM-BA is above the lower critical phase transition temperature, the intermolecular hydrogen bonds are broken, and intramolecular hydrogen bonds are formed between the CO and NH groups inside the polymer chain, resulting in phase separation, scattering the incident light, and being in an opaque state. And the entire phase transition process is completely reversible. Graphene oxide (GO) is a photothermal conversion material that can efficiently convert light energy into thermal energy. The smart photoreactor composed of the thermosensitive polymer PNIPAM-BA and the photothermal material graphene oxide can dynamically sense the intensity of light and regulate the entry of incident light. When the light is relatively strong (2000μmol photons·m -2 ·s -1 ), graphene oxide absorbs light energy and converts it into heat energy to drive the phase change of PNIPAM-BA, making the system opaque. At this time, most of the light is reflected, the light received by the microalgae is weakened, and the photosynthetic oxygen production rate decreases, ensuring the activity of green algae hydrogenase, thereby maintaining a high hydrogen production rate of the microalgae.

[0024] Example 1

[0025] Step 1: Cultivation of Chlorella: Transfer the Chlorella species to 1 L of TAP medium and place in a light incubator at 25 °C for 12 h of light and 12 h of darkness with a light intensity of 100 μmol photons·m -2 ·s -1 , when the number of Chlorella cells reaches the logarithmic growth phase;

[0026] Step 2. Synthesis of poly(N-isopropylacrylamide-butyl acrylate) (PNIPAM-BA) copolymer: Dissolve 5 g of N-isopropylacrylamide (NIPAM) monomer, 0.25 g of butyl acrylate (BA) and 22 mg of azobisisobutyronitrile solution in 50 mL of anhydrous tetrahydrofuran. Under magnetic stirring, introduce inert gas into the solution for deoxygenation for 30 minutes. After deoxygenation, react at 50°C for 24 hours and cool. Add the solution dropwise to a mixed solution of ether and petroleum ether to obtain a precipitate PNIPAM-BA. Filter the precipitate and vacuum dry it for 12 hours to obtain a dry PNIPAM-BA product. Use a spectrophotometer to measure the transmittance of 4 mg / mL PNIPAM-BA at 25-30°C. Figure 1 As shown in the figure, the transmittance of photosynthetically active radiation of PNIPAM-BA is 94% at 25°C, and the transmittance of photosynthetically active radiation of PNIPAM-BA is 5% at 30°C. PNIPAM-BA can achieve 89% light regulation of photosynthetically active radiation.

[0027] Step 3: OD 680 =2-5 of Chlorella were centrifuged and dispersed in 50 mL of HM medium and 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 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 Hydrogen production under light. Due to the presence of graphene oxide photothermal material, at 2000 μmol photons·m -2 ·s -1 Under light intensity, such as Figure 2 As shown, the system temperature can be maintained at 31 ° C, so that PNIPAM-BA undergoes a phase transition and most of the light is reflected, thereby not damaging the Chlorella cells. The components of each liter of HM medium are as follows: glucose 10g, urea 0.914g, KH2PO40.211 g, MgSO4·7H2O0.157g, FeSO4·7H2O12.51mg, H3BO32.85 mg, CuSO4·5H2O 0.8mg, ZnSO4·7H2O 1.11mg, CoSO4·7H2O 0.97mg, MnCl2·4H2O 1.3mg, (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.

[0028] Step 4: Use gas chromatograph to detect the amount of hydrogen produced in the photoreactor at different times. Figure 3 (circular curve) shows that Chlorella produced a total of 12.53 LH2 / L of hydrogen in 25 days, and the average hydrogen production rate was 17.53 μmol H2 (mgchlorophyll) -1 h -1 .

[0029] Comparative Example 1

[0030] Step 1: Cultivation of Chlorella: Transfer the Chlorella species to 1 L of TAP medium and place in a light incubator at 25 °C for 12 h of light and 12 h of darkness with a light intensity of 100 μmol photons·m -2 ·s -1 , when the number of Chlorella cells reaches the logarithmic growth phase;

[0031] Step 2: OD 680 =2-5 of Chlorella were centrifuged and redispersed in 50 mL of HM medium and transferred to a 50 mL photoreactor. The photoreactor containing Chlorella (without PNIPAM-BA and graphene oxide) 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 Produces hydrogen under light.

[0032] Step 3: Use gas chromatograph to detect the amount of hydrogen produced in the photoreactor at different times. Figure 3 As shown in the square curve, due to the damage caused by strong light, Chlorella stopped producing hydrogen within 4 days, and the cumulative hydrogen production was 0.32LH2 / L.

[0033] Comparative Example 2

[0034] Step 1: Cultivation of Chlorella: Transfer the Chlorella species to 1 L of TAP medium and place in a light incubator at 25 °C for 12 h of light and 12 h of darkness with a light intensity of 100 μmol photons·m -2 ·s -1 , when the number of Chlorella cells reaches the logarithmic growth phase;

[0035] Step 2: OD 680= 2-5 of Chlorella were centrifuged and dispersed in 50 mL of TAP medium, and then transferred to a 50 mL photoreactor containing PNIPAM-BA and graphene oxide. The survival rate of Chlorella under different light intensities and the changes in chlorophyll content were measured. Chlorella hydrogen production is a biological metabolic process, so the survival rate of Chlorella directly affects the amount of hydrogen produced. Chlorophyll, as a light-harvesting molecule, plays an important role in maintaining the operation of the PS II reaction center. Figure 5 and Figure 6 It can be seen that the Chlorella in the photoreactor has a high -2 ·s -1 Under light intensity of 1.5 ℃, it can maintain a very high survival rate (>90%) and a high chlorophyll content (15.5 (μg chlorophyll / mg dry weight));

[0036] Step 3: OD 680 = 2-5 of Chlorella were centrifuged and redispersed in 50 mL of TAP medium and transferred to a 50 mL photoreactor (without PNIPAM-BA and graphene oxide). The survival rate and chlorophyll content of Chlorella under different light intensities were measured. Figure 5 and Figure 6 We found that Chlorella in the photoreactor (without PNIPAM-BA and graphene oxide) had a high activity at 100-2000 μmol photons·m -2 ·s -1 At 2000 μmol photons·m -2 ·s -1 Under the light intensity of , almost all Chlorella died and the chlorophyll content was extremely low (0.74 (μg chlorophyll / mg dry weight)), which was the main reason why Chlorella could not continuously produce hydrogen.

Claims

1. A method for maintaining continuous photosynthetic hydrogen production by microalgae under ultra-high light intensity, characterized in that: The method is: Step 1: Cultivation of microalgae: 100 mL of microalgae seed was transferred into 1 L of TAP medium and exposed to light in a light incubator at a temperature of 20-30 °C, where the light intensity was 50-200 μmolphotons·m -2 ·s -1 , when the number of microalgae cells reaches the logarithmic growth phase; Step 2, 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 inert gas is introduced into the solution for deoxygenation for 30 min under magnetic stirring. After deoxygenation, the mixture is reacted at 50° C. for 24 h and 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 hours to obtain a dry PNIPAM-BA product; Step 3: Microalgae produce hydrogen under ultra-high light intensity: Take 50 mL of the microalgae solution obtained in step 1, centrifuge for 5 minutes (5000 rpm) to collect the microalgae, disperse the collected microalgae in 50 mL of HM medium, and transfer to a 50 mL photoreactor; Then, 40-200 mg PNIPAM-BA and 2.5 mg graphene oxide (GO) were added to the photoreactor and shaken to obtain a smart photoreactor. The smart photoreactor containing microalgae was transferred to a light intensity of 2000 μmol photons·m -2 ·s -1 , in a light incubator at 25°C with continuous light.

2. A method for maintaining continuous photosynthetic hydrogen production by microalgae under ultra-high light intensity according to claim 1, characterized in that: In step 1, the microalgae is green algae.

3. The method for maintaining continuous photosynthetic hydrogen production by microalgae under ultra-high light intensity according to claim 1, characterized in that: In step 1, the green algae is one of Chlorella vulgaris, Cellulose algae, Chlamydomonas reinhardtii, Scenedesmus or Lunar algae.

4. A method for maintaining continuous photosynthetic hydrogen production by microalgae under ultra-high light intensity according to any one of claims 1 to 3, characterized in that: In step 1, the illumination is (1) continuous illumination, or (2) a cycle of 12 h of light and 12 h of darkness.

5. The method for maintaining continuous photosynthetic hydrogen production of microalgae under ultra-high light intensity according to claim 1, characterized in that: In step 3, the components of each liter of HM 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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