Nanometer fe3o4 mineralized dunaliella salina, preparation method thereof and application of nanometer fe3o4 mineralized dunaliella salina in construction of microbial fuel cell

Abstract

The application discloses nano Fe3O4 mineralized Dunaliella salina, a preparation method thereof and application of the nano Fe3O4 mineralized Dunaliella salina in construction of a microbial fuel cell. The application adopts Dunaliella salina and nano Fe3O4 mineralized Dunaliella salina as anode electricity-producing microorganisms to construct a two-chamber microbial fuel cell (MFC), and the wild-type Dunaliella salina MFC is stable in electricity production and is an ideal electricity-producing microorganism; the nano Fe3O4 mineralized Dunaliella salina MFC helps the cells to be rapidly enriched on the electrode surface and improves the electricity production performance of the MFC, and the service life of the cell is increased. The Dunaliella salina has strong environmental tolerance and can survive under high salt concentration; in the photosynthesis process, the Dunaliella salina can grow and reproduce by synthesizing organic matters from simple inorganic matters. When the Dunaliella salina is used as an electricity-producing microorganism, the MFC is stable in electricity production and is an ideal electricity-producing microorganism. The Dunaliella salina culture medium can promote the growth of the electricity-producing microorganism and can also be used as an electrolyte of the MFC cell, and has a good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microalgae biotechnology, specifically relating to a nano-Fe3O4 mineralized Dunaliella salina, its preparation method, and its application in constructing microbial fuel cells. Background Technology

[0002] Microbial fuel cells (MFCs) are devices that directly convert the chemical energy of fuels (such as organic acids and sugars) into electrical energy through the catalytic action of microorganisms. MFCs generate bioelectricity by decomposing organic matter with electroactive bacteria on the anode. Electroactive bacteria attached to the electrode surface consume nutrients or organic pollutants to produce electrons and protons, which then enter the cathode cavity through the circuit and intermediate membrane to participate in the electrochemical reaction.

[0003] Microalgae, as a biomass energy fuel, has attracted attention for constructing micro-fuel cells (MFCs) for power generation due to its advantages such as high safety and low processing requirements. The microalgae-based MFC, constructed by combining microalgae with MFCs, can generate electricity using light energy and carbon dioxide. Electrons produced by algae's respiration and photosynthesis can be transferred to electrodes to generate electricity.

[0004] Dunaliella salina, a highly salt-tolerant eukaryotic green alga, lacks a cell wall. It is a photoautotrophic organism that uses water as an electron donor. During photosynthesis, it synthesizes organic matter from simple inorganic substances for growth and reproduction. It exhibits strong environmental tolerance, surviving in salinity ranges from 0.5 to 5 mol / L NaCl. Using Dunaliella salina to construct microcrystals (MFCs) may have two different applications: ① storing energy through photosynthesis under illumination; ② directly transferring electrons to the anode via the photosynthetic electron chain under illumination.

[0005] To meet different needs, it is necessary to modify microalgal cells or change the culture environment of microalgae. Summary of the Invention

[0006] To address the related technical problems, the primary objective of this invention is to provide a method for preparing nano-Fe3O4 mineralized Dunaliella salina.

[0007] Another object of the present invention is to provide nano-Fe3O4 mineralized Dunaliella salina obtained by the above preparation method.

[0008] Another object of the present invention is to provide the application of the above-mentioned nano-Fe3O4 mineralized Dunaliella salina in the construction of microbial fuel cells.

[0009] To achieve the above-mentioned objectives, the present invention adopts the following technical solution:

[0010] A method for preparing Dunaliella salina mineralized with nano-Fe3O4 involves co-culturing Dunaliella salina cells and nano-Fe3O4 in a Dunaliella salina culture medium to obtain Dunaliella salina mineralized with nano-Fe3O4.

[0011] Furthermore, the Dunaliella salina mentioned is Dunaliella salina CCAP19 / 18.

[0012] Furthermore, the Dunaliella salina cells are Dunaliella salina cells grown to the logarithmic growth phase in a Dunaliella salina culture medium.

[0013] Further, the Dunaliella salina culture medium is prepared as follows: NaNO3 0.4±0.1g / L, NaH2PO4·2H2O 0.1±0.1g / L, NaHCO3 0.8±0.1g / L, KCl 0.07±0.05g / L, MgSO4·7H2O 1.2±0.1g / L, CaCl2·2H2O 0.04±0.05g / L, 0.1% Fe-EDTA solution 0.5mL / L, and 1mL / L of A5 trace element solution is added to the culture medium to adjust the pH to 7.5; the Fe-EDTA solution composition is as follows: Na2EDTA 0.1±0.05g / L, FeCl3·6H2O 0.2±0.1g / L; the A5 trace element solution composition is as follows: H3BO3 2±0.5g / L, MnCl2·4H2O 1.8±0.1g / L, ZnSO4·7H2O0.2±0.1g / L, CuSO4·5H2O 0.08±0.005g / L, (NH4)6Mo7O 24 • 4H₂O 0.04±0.005g / L; Further details: NaNO₃ 0.420g / L, NaH₂PO₄·2H₂O 0.156g / L, NaHCO₃ 0.840g / L, KCl 0.074g / L, MgSO₄·7H₂O 1.230g / L, CaCl₂·2H₂O 0.044g / L, 0.1% Fe-EDTA solution 0.5mL / L, add A5 trace element solution 1mL / L to the culture medium, adjust pH to 7.5; Fe-EDTA solution composition: Na₂EDTA 0.189g / L, FeCl₃·6H₂O 0.244g / L; A5 trace element solution composition: H₃BO₃ 2.86g / L, MnCl₂·4H₂O 1.81g / L, ZnSO4·7H2O0.22g / L, CuSO4·5H2O0.08g / L, (NH4)6Mo7O 24 ·4H2O 0.04g / L.

[0014] The cultivation conditions were a temperature of 23±1℃, a light intensity of 3000±500lx, and a light-to-dark ratio of 16h:8h.

[0015] Furthermore, the nano-Fe3O4 is prepared as follows: in Fe 2+ and Fe 3+ An alkaline solution is added to a mixed solution of ions, and the mixture is stirred at high speed continuously to coprecipitate nano-Fe3O4. Preferably, 0.99±0.2g FeCl2(4H2O) and 2.7±0.5g FeCl3(6H2O) are added to 100mL of water, stirred until fully dissolved, and then 10±1mL of 25% NH4OH is added. The mixture is stirred at 20000±5000rpm for 30±5min to coprecipitate nano-Fe3O4.

[0016] Furthermore, the cell concentration of the Dunaliella salina cells is (0.1–2) × 10⁻⁶. 7 cells / mL; further, (1±0.2)×10 7 cells / mL.

[0017] Furthermore, the dispersion concentration of the nano-Fe3O4 is 0.5–8 mg / mL; preferably 2 ± 1 mg / mL.

[0018] Furthermore, the co-culture conditions are oscillation at 120±20 rpm for 30±5 min.

[0019] A nano-Fe3O4 mineralized Dunaliella salina was obtained by the above preparation method.

[0020] The above-mentioned application of nano-Fe3O4 mineralized Dunaliella salina in the construction of microbial fuel cells.

[0021] Furthermore, the application involves constructing dual-chamber MFCs made of plexiglass, with Dunaliella salina as the anolyte and inoculating it with nano-Fe3O4-mineralized Dunaliella salina, and a potassium persulfate solution as the cathode; even further, the anolyte is Dunaliella salina culture medium and inoculated with OD... 630 The electrode was mineralized from Dunaliella salina using nano-Fe3O4 with a thickness of 1.0 ± 0.2 μm. The cathode was a 50 ± 5 mmol / L potassium persulfate solution. Furthermore, the two electrode chambers were separated by a Nafion 117 proton exchange membrane, with a vacuum pad sandwiched between the membrane and the chambers to maintain a seal. Both the cathode and anode used a 24.63 cm² anode. 2 Circular carbon felt was used as an electrode, and it was led out with a titanium wire with a diameter of 1 mm and connected to an external resistor of 1 kΩ. All devices were placed in a light incubator with the temperature controlled at 23 ± 1 ℃ and the light intensity set at 3000 ± 500 lx.

[0022] The technical principle of this invention: Biomimetic mineralization of cell surfaces is a feasible method for artificially constructing a shell for cells. Biomimetic mineralization of cell surfaces refers to depositing a layer of inorganic minerals on the cell surface using the principle of biomineralization. The key is to introduce mineralizing functional groups or molecules onto the cell surface under mild conditions; its core is cell surface modification. Metal oxides have good stability and corrosion resistance. Among them, nano-ferric oxide has a large roughness and good biocompatibility, which is beneficial for its composite with algal cells. Compared with traditional carbon materials, metal materials have better electrical conductivity and mechanical strength. Therefore, this invention utilizes nano-ferric oxide to mineralize Dunaliella salina, thereby constructing a microbial fuel cell.

[0023] This invention uses Dunaliella salina as the research object and mimics the strategy of biomimetic mineralization to achieve direct and rapid composite of Dunaliella salina with iron tetroxide nanoparticles. The Dunaliella salina composite with nanoparticles can rapidly aggregate.

[0024] This invention screened different concentrations of iron oxide in mineralized Dunaliella salina and found that the best mineralization effect could be achieved at a concentration of 2 mg / mL of iron oxide nanoparticles.

[0025] The present invention found in experiments on encapsulating Dunaliella salina with iron oxide nanoparticles that the mineralized iron oxide nanoparticles do not damage the cell structure and have good biocompatibility.

[0026] This invention uses Dunaliella salina and Dunaliella salina mineralized with nano-ferric oxide as anode electrogenic microorganisms to construct a dual-chamber microbial fuel cell (MFC). Wild-type Dunaliella salina MFC has stable power generation and is an ideal electrogenic microorganism. The MFC constructed by Dunaliella salina mineralized with nano-ferric oxide helps cells to accumulate rapidly on the electrode surface and improves the power generation performance of the MFC, while also increasing battery life.

[0027] The MFC using Dunaliella salina DS-Wild as the anode electrogenic microorganism exhibits good electrogenic performance, with a maximum output voltage of 54.76mV, a stable voltage of 47.71±10mV, and a battery life of 274h.

[0028] The MFC using nano-ferric oxide mineralized Dunaliella salina DS-Wild as the anode electrogenic microorganism exhibits good electrogenic performance, with a maximum output voltage of 87.22mV, a stable voltage of 73.61±10mV, and a battery life of 311h.

[0029] The present invention has the following advantages and effects compared with the prior art:

[0030] (1) Dunaliella salina has strong environmental tolerance and can survive in high salt concentrations. During photosynthesis, it can synthesize organic matter from simple inorganic substances to grow and reproduce. When used as an electrogenic microorganism, its MFC (Metal-Cellular Filtration) produces stable electricity, making it an ideal electrogenic microorganism.

[0031] (2) The Dunaliella salina culture medium can promote the growth of electrogenic microorganisms and also serve as an electrolyte for MFC batteries.

[0032] (3) Fe3O4 mineralization of Dunaliella salina can significantly improve the power generation performance and battery life of MFC bio-batteries. Attached Figure Description

[0033] Figure 1 These are culture images of wild-type Dunaliella salina and nano-ferric oxide mineralized Dunaliella salina; (a) is a culture image of wild-type Dunaliella salina; (b) is a culture image of nano-ferric oxide mineralized Dunaliella salina.

[0034] Figure 2 These are optical microscope images of wild-type Dunaliella salina and mineralized Dunaliella salina; (a) is a cell image of wild-type Dunaliella salina; and (b) is a cell image of nano-ferric oxide mineralized Dunaliella salina.

[0035] Figure 3 These are cell diagrams of mineralization concentration gradients of Dunaliella salina nano-ferric oxide; where (a) is a cell diagram of wild-type Dunaliella salina; (b) 0.5 mg / mL, (c) 1 mg / mL, (d) 2 mg / mL, (e) 4 mg / mL, and (f) 8 mg / mL nano-ferric oxide mineralized Dunaliella salina cell diagrams.

[0036] Figure 4 The time-output voltage curves of the MFC constructed from wild-type Dunaliella salina (DS-Wild) and nano-ferric oxide mineralized Dunaliella salina (DS@Fe3O4) are shown. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0038] Unless otherwise specified, the test methods in the following examples are generally performed under standard experimental conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used are commercially available.

[0039] Fe3O4 was prepared by chemical coprecipitation; the electrolyte K2S2O3 was purchased from Tianjin Damao Chemical Reagent Factory (CAS: 7727-21-1); Dunaliella salina was purchased from the Culture Collection of Algae and Protozoa, UK, number CCAP19 / 18.

[0040] Example 1

[0041] 1. Preparation and cultivation of Dunaliella salina culture medium

[0042] Preparation of Dunaliella salina culture medium: NaNO3 0.420 g / L, NaH2PO4·2H2O 0.156 g / L, NaHCO3 0.840 g / L, KCl 0.074 g / L, MgSO4·7H2O 1.230 g / L, CaCl2·2H2O 0.044 g / L, 0.1% Fe-EDTA solution 0.5 mL / L. Add 1 mL / L of A5 trace element solution to the culture medium and adjust the pH to 7.5.

[0043] The composition of the Fe-EDTA solution is as follows: Na2EDTA 0.189 g / L, FeCl3·6H2O 0.244 g / L.

[0044] The composition of the A5 trace element solution is as follows: H3BO3 2.86 g / L, MnCl2·4H2O 1.81 g / L, ZnSO4·7H2O 0.22 g / L, CuSO4·5H2O 0.08 g / L, (NH4)6Mo7O 24 ·4H2O 0.04g / L.

[0045] Dunaliella salina culture: temperature 23±1℃, light intensity 3000 lx, light-dark ratio 16h:8h, cultured until the logarithmic growth phase (OD200). 630 =1.0).

[0046] 2. Preparation of nano-Fe3O4:

[0047] 100 mL of deionized water was heated to 80 °C in an electric furnace. 0.99 g of FeCl2(4H2O) and 2.7 g of FeCl3(6H2O) were added sequentially, and the mixture was continuously heated and stirred at high speed until fully dissolved. 10 mL of 25% NH4OH was quickly added, and the mixture was stirred continuously at 20,000 rpm for 30 min using a high-speed stirrer. Fe3O4 nanoparticles were precipitated using a hand-held magnet, washed three times with deionized water to achieve pH 7, and then dried in an oven. Before use, deionized water was added and the mixture was ultrasonically redispersed.

[0048] 3. Preparation of nano-Fe3O4 mineralized Dunaliella salina

[0049] After centrifuging the Dunaliella salina culture medium at 1000 rpm to remove the culture medium, the algae grown to the logarithmic growth phase were dispersed in fresh Dunaliella salina culture medium and the cell density was adjusted to 1×10⁻⁶ cells / day. 7 Cells / mL. Nano-Fe3O4 prepared by chemical co-precipitation was added to the dispersion of Dunaliella salina cells to achieve concentrations of 0, 0.5, 1, 2, 4, and 8 mg / mL. After shaking at 120 rpm for 30 min in a shaker, subsequent culturing was performed. Cell morphology was photographed under an optical microscope at 40X.

[0050] 4. Building an MFCs device

[0051] Construct two-chamber MFCs made of plexiglass, with Dunaliella salina as the anolyte and inoculated with OD. 630 Algal cells with a density of 1.0 μm were used. The cathode was a potassium persulfate solution (K₂S₂O₈, 50 mmol / L). Both the anode and cathode chambers had a volume of 100 mL, separated by a proton exchange membrane (Nafion 117). A vacuum gasket was placed between the proton exchange membrane and the two chambers to maintain a seal. Both the cathode and anode used a 24.63 cm² area algae. 2 A circular carbon felt (r = 2.8 cm) was used as the electrode, led out with titanium wire (1 mm in diameter), and connected to an external resistor of 1 kΩ. All apparatus were placed in a light incubator for the experiment, with the temperature controlled at 23 ± 1 °C and the light intensity set to 3000 lx.

[0052] Control group: The anolyte was a Dunaliella salina culture medium inoculated with OD. 630 Wild-type Dunaliella salina cells (DS-Wild) with a cathode of 1.0, and potassium persulfate solution (K2S2O8, 50 mmol / L).

[0053] Experimental group: The anolyte was Dunaliella salina culture medium and inoculated with OD. 630 =1.0 nanometer Fe3O4 mineralized Dunaliella salina cells (DS@Fe3O4), with potassium persulfate solution (K2S2O8, 50 mmol / L) as the cathode.

[0054] Experimental material pretreatment:

[0055] (1) Pretreatment of proton exchange membrane: Nafion 117 membrane needs to be pretreated before use to achieve better transfer effect. First, boil the membrane in 5% hydrogen peroxide at 80°C for 1 hour, then soak it in deionized water for 0.5 hours, then boil it in 5% dilute sulfuric acid at 100°C for 1 hour, and finally soak it in deionized water for 0.5 hours.

[0056] (2) Pretreatment of carbon felt: First soak it in 1 mol / L HCl and acetone for 2 hours, then soak it in deionized water for 1 hour to remove surface impurities, and finally dry it for use.

[0057] 5. Methods for analyzing the power generation performance of MFC batteries

[0058] The voltage signal generated by MFC is automatically acquired using a digital multimeter (VICTOR) at a frequency of once every 60 seconds and an acquisition accuracy of 0.01mV.

[0059] Figure 1 These are culture images of wild-type Dunaliella salina and nano-ferric oxide mineralized Dunaliella salina; (a) is a culture image of wild-type Dunaliella salina; (b) is a culture image of nano-ferric oxide mineralized Dunaliella salina.

[0060] Figure 2 These are optical microscope images of wild-type Dunaliella salina and mineralized Dunaliella salina; (a) is a cell image of wild-type Dunaliella salina; and (b) is a cell image of nano-ferric oxide mineralized Dunaliella salina.

[0061] Figure 3 These are cell diagrams of mineralization concentration gradients of Dunaliella salina nano-ferric oxide; where (a) is a cell diagram of wild-type Dunaliella salina; (b) 0.5 mg / mL, (c) 1 mg / mL, (d) 2 mg / mL, (e) 4 mg / mL, and (f) 8 mg / mL nano-ferric oxide mineralized Dunaliella salina cell diagrams.

[0062] Figure 4 This is a time-output voltage curve of the MFC constructed from wild-type Dunaliella salina (DS-Wild) and nano-ferric oxide mineralized Dunaliella salina (DS@Fe3O4). It can be seen that the MFC using wild-type Dunaliella salina cells as the anode electrogenic microorganism exhibits good electrogenic performance, with a maximum output voltage of 54.76 mV, a stable voltage of 47.71 ± 10 mV, and a battery life of 274 hours. The MFC using nano-Fe3O4 mineralized Dunaliella salina cells as the anode electrogenic microorganism exhibits even better electrogenic performance, with a maximum output voltage of 87.22 mV, a stable voltage of 73.61 ± 10 mV, and a battery life of 311 hours.

[0063] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the embodiments described above. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. An application of nano-Fe3O4 mineralized Dunaliella salina in the construction of microbial fuel cells, characterized in that: A two-chamber MFC made of plexiglass was constructed, with Dunaliella salina culture medium as the anolyte and Dunaliella salina mineralized with nano-Fe3O4 as the inoculum, and potassium persulfate solution as the cathode. The nano-Fe3O4 mineralized Dunaliella salina was prepared by the following method: Dunaliella salina cells and nano-Fe3O4 were co-cultured in Dunaliella salina culture medium to obtain nano-Fe3O4 mineralized Dunaliella salina. The cell concentration of Dunaliella salina cells is (0.1–2) × 10⁻⁶. 7 cells / mL; The dispersion concentration of the nano-Fe3O4 is 0.5–8 mg / mL; The co-culture conditions were oscillation at 120±20 rpm for 30±5 min.

2. The application according to claim 1, characterized in that: The Dunaliella salina cells mentioned above are Dunaliella salina cells grown to the logarithmic phase in Dunaliella salina culture medium; The Dunaliella salina culture medium was prepared as follows: NaNO3 0.4±0.1 g / L, NaH2PO4·2H2O 0.1±0.1 g / L, NaHCO3 0.8±0.1 g / L, KCl 0.07±0.05 g / L, MgSO4·7H2O 1.2±0.1 g / L, CaCl2·2H2O 0.04±0.05 g / L, 0.1% Fe-EDTA solution 0.5 mL / L. 1 mL / L of A5 trace element solution was added to the culture medium, and the pH was adjusted to 7.

5. The Fe-EDTA solution composition was as follows: Na2EDTA 0.1±0.05 g / L, FeCl3·6H2O 0.2±0.1 g / L. The A5 trace element solution composition was as follows: H3BO3 2±0.5 g / L, MnCl2·4H2O 1.8±0.1 g / L. g / L, ZnSO4·7H2O 0.2±0.1 g / L, CuSO4·5H2O 0.08±0.005 g / L, (NH4)6Mo7O 24 ·4H₂O 0.04±0.005 g / L; The preparation of the nano-Fe3O4 is as follows: in Fe 2+ and Fe 3+ An alkaline solution was added to a mixed solution of ions, and the mixture was stirred at high speed continuously to co-precipitate nano-Fe3O4.

3. The application according to claim 2, characterized in that: The Dunaliella salina culture medium was prepared as follows: NaNO3 0.420 g / L, NaH2PO4·2H2O 0.156 g / L, NaHCO3 0.840 g / L, KCl 0.074 g / L, MgSO4·7H2O 1.230 g / L, CaCl2·2H2O 0.044 g / L, 0.1% Fe-EDTA solution 0.5 mL / L. Add 1 mL / L of A5 trace element solution to the culture medium and adjust the pH to 7.

5. The Fe-EDTA solution composition is as follows: Na2EDTA 0.189 g / L, FeCl3·6H2O 0.244 g / L; the A5 trace element solution composition is as follows: H3BO3 2.86 g / L, MnCl2·4H2O 1.81 g / L, ZnSO4·7H2O 0.22 g / L. g / L, CuSO4·5H2O 0.08 g / L, (NH4)6Mo7O 24 ·4H₂O 0.04g / L; The cultivation conditions were a temperature of 23±1℃, a light intensity of 3000±500 lx, and a light-to-dark ratio of 16h:8h. The nano-Fe3O4 was prepared as follows: 0.99±0.2 g FeCl2(4H2O) and 2.7±0.5 g FeCl3(6H2O) were added to 100 mL of water and stirred until fully dissolved. Then, 10±1 mL of 25% NH4OH was added and stirred at 20000±5000 rpm for 30±5 min to co-precipitate nano-Fe3O4.

4. The application according to claim 1, characterized in that: The cell concentration of Dunaliella salina cells is (0.1–2) × 10⁻⁶. 7 cells / mL; The dispersion concentration of the nano-Fe3O4 is 0.5–8 mg / mL.

5. The application according to any one of claims 1-4, characterized in that: The Dunaliella salina mentioned is Dunaliella salina CCAP19 / 18.

6. The application according to claim 5, characterized in that: The application is as follows: constructing two-chamber MFCs made of plexiglass, using Dunaliella salina as the anolyte culture medium, and inoculating OD... 630 =1.0±0.2 nano-Fe3O4 mineralized Dunaliella salina; cathode: 50±5 mmol / L potassium persulfate solution; the two electrode chambers are separated by a Nafion 117 proton exchange membrane, with a vacuum pad sandwiched between the proton exchange membrane and the two electrode chambers to maintain a seal; both cathode and anode are 24.63 cm². 2 Circular carbon felt was used as an electrode and led out with titanium wire of 1 mm diameter, connected to an external resistor of 1 kΩ; all devices were placed in a light incubator with the temperature controlled at 23±1℃ and the light intensity set at 3000±500 lx.

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