TW202043162A - Microbial fuel cell device and method for immobilizing algae strain - Google Patents

Abstract

A microbial fuel cell device and a method for immobilizing algae strain are provided. The microbial fuel cell device comprises: an anode tank, a cathode tank, a proton exchange membrane, an anode electrode, and a cathode electrode. The anode tank is filled with a first nutrient source comprising bacteria. The cathode tank is filled with a second nutrient source comprising Wu-G23 algae strain (SEQ ID NO：2). The proton exchange membrane is disposed between the anode tank and the cathode tank. The anode electrode is disposed in the anode tank. The cathode electrode is disposed in the cathode tank, and the anode electrode and the cathode electrode are respectively connected to the two ends of a conductive wire with a load. The bacteria undergo a respiration to produce electrons and protons, and the electrons pass through the anode electrode into the cathode tank to be electrochemically coupled with electron acceptors, and the protons pass through the proton exchange membrane into the cathode tank to generate current.

Description

Microbial fuel cell device and method for fixing algae strains

The present invention relates to a fuel cell device and a method for fixing algae strains, in particular to a microbial fuel cell device that uses bacteria and algae strains to generate electricity and a method for fixing algae strains to form particles.

Due to global warming and the exhaustion of fossil fuels, humans are actively and urgently seeking new alternative energy sources, such as microbial fuel cells (MFC). Moreover, the microbial fuel cell device is a device that converts chemical energy into electrical energy. In detail, in the anode tank, the microbial fuel cell uses microorganisms to decompose organic matter to produce protons, electrons and metabolites. The generated electrons reach the cathode via the external wire, and the generated protons reach the cathode tank through the proton exchange membrane to generate a reduction reaction in the cathode tank to provide electrical energy. The conventional microbial fuel cell device has achieved considerable results in generating electricity. However, the conventional microbial fuel cell device still has a huge technical bottleneck for its ability to supply power, produce high biodiesel and purify water at the same time.

In order to improve the above-mentioned problems, according to one objective of the present invention, a microbial fuel cell device is provided, which includes an anode tank, a cathode tank, a proton exchange membrane, an anode and a cathode. Wherein, the anode tank is filled with a first nutrient source containing bacteria. The cathode tank is filled with the second nutrient source of Wu-G23 algae strain (SEQ ID No: 2). The proton exchange membrane is arranged between the anode tank and the cathode tank. The anode is arranged in the anode tank. The cathode is arranged in the cathode tank, and the anode and the cathode are respectively connected to the two ends of the conductive wire with the load. Bacteria perform respiration to generate electrons and protons. The electrons pass through the anode to the cathode tank and are electrochemically coupled with the electron acceptor; and the protons pass through the proton exchange membrane to the cathode tank to generate electric current.

Preferably, it further includes a light source for illuminating the Wu-G23 algae strain.

Preferably, it further comprises a gas output device in which air is introduced into the cathode tank at a predetermined aeration rate, and the aeration rate ranges from 0.0 to 1.5 L/min.

Preferably, the first nutrient source contains sludge.

Preferably, the second nutrient source includes a nitrogen source, and the nitrogen source includes Peptone, ammonium sulfate ((NH4)SO4), potassium nitrate (KNO3), Yeast extract, Urea, or any combination thereof .

Preferably, the second nutrient source includes wastewater, sea water or kitchen waste.

Preferably, the NH ₄ ⁺ -N removal rate of the microbial fuel cell device ranges from 40 to 70%.

Preferably, the COD removal rate of the microbial fuel cell device ranges from 30% to 80%.

According to another object of the present invention, there is provided a method for immobilizing algae strains, which is applied to the Wu-G23 algae strains in the cathode tank of the aforementioned microbial fuel cell device, and includes the following steps: sterilizing a predetermined concentration of sodium alginate The aqueous solution is stirred and mixed with the Wu-G23 algae strain to form a mixed solution; and the mixed solution is injected into the forming solution through an injection needle to form immobilized particles of the Wu-G23 algae strain with a predetermined diameter. Wherein, the predetermined concentration ranges from 0.0 to 6.0 w.t%.

Preferably, the predetermined diameter ranges from 4.0 mm to 5.0 mm.

The microbial fuel cell device and the method for fixing algae strains of the present invention have one or more of the following advantages:

(1) In the microbial fuel cell device and the method of fixing algae strains of the present invention, the Wu-G23 algae strain has the characteristics of high lipid content, so it can produce high biological quality while providing electricity, thereby increasing production Quality diesel.

(2) In the microbial fuel cell device and method of fixing algae strains of the present invention, because Wu-G23 algae strain has the characteristics of high pH resistance, high CO ₂ concentration, high temperature resistance, and the use of organic matter and organic acid as a carbon source , So it can provide electricity and at the same time achieve a high chemical oxygen demand (COD) removal rate to purify water.

The following describes each example in detail.

Refer to Figure 1, which is a schematic diagram of the microbial fuel cell device of the present invention.

As shown in the figure, the microbial fuel cell device 1 of the present invention may include an anode tank 10, a cathode tank 20, a proton exchange membrane 30, an anode 11, a cathode 21, and a conductive wire 40 with a load 41. In addition, the microbial fuel cell device 1 may further include a light source 50. Among them, the proton exchange membrane 30 is an exchange membrane that only allows the passage of protons and does not allow the passage of water molecules. In terms of electrodes, the anode 11 and the cathode 21 may be carbonaceous or graphite. Specifically, the anode 11 and the cathode 21 may be carbon cloth, carbon fiber, or carbon rod. In terms of external circuits, the conductive wires 40 may be copper wires or silver wires. Moreover, the load 41 may be a resistor or any element that can consume power; for example, the load 41 may be a light bulb. Furthermore, preferably, the microbial fuel cell device 1 of the present invention can be provided with a reference electrode or an auxiliary electrode in the anode tank 10 and the cathode tank 20 respectively. Specifically, the reference electrode may be a silver/silver chloride reference electrode; the auxiliary electrode may be a platinum electrode. In some embodiments of the microbial fuel cell device 1 of the present invention, the microbial fuel cell device 1 may further include a light source 50, and the light source 50 may illuminate the Wu-G23 algae strain 23. Specifically, the light condition of the cathode tank 20 may be 2670 Lux for 24 hours a day. Among them, the light source 50 can affect the growth rate of the Wu-G23 algae strain 23 and the lipid composition formed, such as fatty acid methyl esters.

In addition, the cathode tank 20 of the microbial fuel cell device 1 of the present invention may further include a gas device 60, and the input air must consider the impact on the bacteria 13 in the anode tank 10. For example, if the bacteria 13 is an anaerobic bacteria, it may be When a large amount of air is input, oxygen enters the anode tank 10, which is not conducive to the growth of bacteria 13 on the contrary.

In the microbial fuel cell device 1 of the present invention, the bacteria 13 can perform respiration to generate electrons and protons. Wherein, the electrons pass through the anode 11 to the cathode cell 20 to be electrochemically coupled with the electron acceptor, and the protons pass through the proton exchange membrane 30 to the cathode cell 20, thereby generating a reduction reaction, so that electric current is generated to provide electric energy.

In addition, the anode tank 10 can be filled with bacteria 13 and the first nutrient source 12; the cathode tank 20 can be filled with the Wu-G23 algae strain 23 of Chlorella vulgaris and the second nutrient source 22. Among them, the first nutrient source 12 may be sludge; the second nutrient source 22 may include wastewater, seawater or kitchen waste. In some embodiments, the aforementioned bacteria 13 may be a strain domesticated in an anaerobic flask from the waste sludge of the Tairong Food Fructose Wastewater Treatment Plant, and its strain is named Sulfate-reducing bacteria. In addition, the algae strain (Wu-G23) of Chlorella vulgaris is a strain that has been screened from water quality samples collected from the coast of Taiwan, and its screening and sequencing will be detailed later. In addition, the aforementioned algae strain (Wu-G23) can use the oil in the second nutrient source 22 to produce fatty acid methyl esters (FAMEs), and thereby accumulate oil to generate biofuel. Therefore, the algae strain (Wu-G23) can simultaneously treat wastewater, reduce CO ₂ emissions and produce biomass fuel.

On the other hand, a nitrogen source can be added to the cathode tank 20 of the microbial fuel cell device 1 of the present invention. Wherein, the nitrogen source may include eggplant, ammonium sulfate, potassium nitrate, yeast extract, urea or any combination thereof to provide conditions favorable for the growth of the algae strain (Wu-G23).

Materials and Methods

1.1 Location selection and sample collection

The sources of microalgae samples screened by the present invention are seawater, deciduous mangroves, sandbars, intertidal zones and other different marine habitats including rocky coasts and coral reefs, estuaries, swamps, wetlands, soils and salt pan sediments around Taiwan. The microalgae samples were stored in sterile seawater, and most of the zooplankton were filtered out with a 60μm filter. And put it in the appropriate liquid medium at a temperature below 30°C.

1.2 Screening of microalgae water samples and identification of algae species

1.2.1 Screening of microalgae water samples

Place the microalgae sample on the sieve algae rotating disk and incubate it at 30°C and 50 rpm for one to several weeks, then take 0.1 mL of the liquid culture suspension and apply it to different solid mediums, and incubate them at 30°C. One week to two weeks, and reuse the solid medium to separate a single algae colony. Then, select a single algae to settle on the same medium for purification, the intensity of the fluorescent lamp is about 6017 lux, and the light cycle is 24 hours of light. The present invention selects several commonly used nutrient components of the medium for algae as examples of the medium for screening microalgae. For example, BG-11 growth medium, Zarrouck medium, etc.

1.2.2 Microalgae concentration analysis

Use UV/Visible spectrophotometer (UV/Visible spectrophotometer) to set the wavelength at 680nm to determine the algae concentration. If the optical density (optical density) is greater than 1.0, it is necessary to dilute each sample to be tested and control its absorbance within the range of 0.1-1.0. When the optical density exceeds the calibration curve range, it means that its biomass is too high and needs to be further diluted. Next, the culture solution of the sample was removed by centrifugation and washed twice with sterile water. Finally, the sterile water was removed by centrifugation, and the microalgae bodies were collected. After the treated microalgae was dried at 105°C for 16 hours, the dry cell weight (DCW, g/L) of the algae was measured.

1.2.3 Identification and analysis of algae species

The extraction of microalgae DNA (genomic DNA extraction) is performed using the Plant Gnomic DNA Purification Kit (Genmac). Next, polymerase chain reaction (PCR) is performed. Place the reaction tube in a DNA amplification instrument (Major Science Cycler-25, Saratoga, CA.95070, USA), and the operating conditions are: 94℃ dissociation for 3 minutes; 94℃ dissociation for 40 seconds; 55℃ primer bonding for 1 minute; 72 Enzyme synthesis at ℃ for 2.5 minutes; then repeat 33 cycles of dissociation, primer bonding, and synthesis steps, and finally fill up at 72 ℃ for 7 minutes, then take some DNA for electrophoresis. The primer conditions are shown in Table 1.

Table 1 Primer name 5'Chlo-rbcL Sequence 5' 5'Cgg gCA gAK Tgc AAg ATC gTA A 3' Primer name 3'Chlo-rbcL Sequence 3' 5'TTA AAg AgT ATC gAT WgT TTC gAA TTC 3'

Next, the purified DNA is mixed with the yT&A vector (Yeastern Biotech Ca, Ltd), T4 DNA ligase and related buffer solutions to perform a ligation reaction. Then, E-coli (E-coli XL-1) is used as a Competent cell for transformation. Furthermore, quick screening is used to screen out competent cells containing plastids. Then, after culturing the competent cells for 14-16 hours, the plasmid of the competent cells is extracted. And use the nucleic acid restriction enzyme (HindⅢ) of the yT&A vector to cut the extracted plastid DNA out of the vector and the selected algae strain fragment DNA, and then subject the aforementioned vector and the algae strain fragment DNA to 0.8% agaric gel Electrophoresis (agarose gel electrophoresis).

Use the 18S rRNA gene sequence of the unknown algae strain in NCBI GenBank database, and use BLAST software to compare the gene sequence of the known algae strain. Then draw the correlation of the phylogenetic tree based on the aligned sequence.

1.3 Lipid extraction

The total fat content of the selected microalgae is calculated by the following method: Take 0.05g sample and soak it in a 20mL glass culture tube containing 15mL chloroform: methanol (1:2, v/v) , After homogenization, use ultrasonic vibration for extraction. After filtering by suction, rinse with chloroform. After adding 9 mL of sterile water, make the solvent ratio 9:10:10. The culture tube was shaken and centrifuged to remove the methanol aqueous solution layer on the top, and then the chloroform/fat layer was dried with anhydrous sodium sulfate. The solvent is evaporated in nitrogen, the fat is placed in a pre-weighed bottle, and the weight of the fat is measured. 10 µL of tripentadecanoin contains chloroform: methanol (1:1) as an internal standard for the fat extracted from microalgae.

1.4 Lipid saponification (saponification) and esterification (esterification)

Saponification and fatty acid methylation are carried out by the following methods: fatty acid methyl esters (FAME) derived fatty acids, 14% boron trifluoride (BF ₃ ) added to methanol, and then added to the sample, Heat at 100°C. After cooling, a saturated NaCl solution was added to prevent emulsification of FAME, and then FAME was extracted with hexane. After the FAME in hexane was dried with nitrogen, chloroform was added to prepare fatty acids, and then gas chromatography (GC) (column: DB-WAX (30 m×0.25 mm); flame ion detector: Flame) ionization detector, FID) for analysis.

1.5 Analysis of fatty acids

Use GC to analyze FAME. Nitrogen was used as the carrier, the flow rate was 1 mL/min, and the sample volume was 1 μL. Maintain the oven temperature at 150°C for 5 min, and then increase the temperature to 200°C at a rate of 5°C min ^-1 . The temperature of the injection needle and the detector are 200°C and 220°C, respectively. Comparing the residence time of standard and experimental samples can be used to judge the cumulative content of FAME. Use n-heptadecanic acid as the internal standard. The peak can be compared with the internal standard to get the fatty acid content in the sample. Calculate the cumulative content of each fatty acid by comparing the residence time of the experimental sample and the C16-C24 methyl ester compound.

1.6 Algae morphology and the formation of algae oil droplets

1.6.1 Observation using optical microscope

Observation of general algae patterns

Before observation, the algae solution was washed three times with water and centrifuged for sampling. The algae solution was dropped on a glass slide and observed with an inverted microscope. The appropriate field of view was found at a low magnification (400 x), and then a higher magnification (1,000 x ) Observe and understand the morphology of algae cells under an inverted microscope.

General observation of algal lipids (Sudan Black Staining)

Centrifuge the algae to remove the supernatant, suspend and wash three times with a phosphate buffer solution, take the suspended algae solution and smear it on the slide. When the algae is slightly dry, soak the slide in 30, 50, 70% alcohol for 2 minutes each, dissolve The chlorophyll is fixed, and then immersed in a freshly prepared 0.3% Sudan Black B dye in a 40℃ water bath. After dyeing for 30-60 minutes, the alcohol is decolorized in the order of 70, 50, 30%, and finally Slowly buffer washed with distilled water, add a cover glass to observe the oil droplets of microalgae cells under an optical microscope at high magnification, and their lipids appear blue-black.

Fluorescence microscope observation (Nile red Staining)

Centrifuge 10-15 mL of algae solution at 9000 rpm to remove the supernatant, add 10 mL of sterile water to cover the solution and wash three times, then add 40 μL of Nile red (concentration: 250 mg/L acetone) to avoid After mixing with light shaking for 1 minute, take out the mixed algae solution and smear it on a glass slide for observation under a fluorescent microscope. After dyeing, the oil droplets and the dye will combine to give a fluorescent golden yellow, so it can be compared with the absence of oil. Light red to distinguish.

Scanning electron microscope observation

The sample undergoes pre- and post-fixation and a series of dehydration processes, the same as the penetrating sample processing step, and amyl acetate is used as the intermediate replacement fluid to replace 100% alcohol, because the replacement effect of amyl acetate and liquid carbon dioxide is better than that of alcohol and acetone. The sample will not shrink or deform, that is, the sample is dried. A critical point dryer is used to replace the amyl ester with liquid carbon dioxide. The temperature is raised to 31°C and the pressure is 1,070 psi for 8 minutes. The carbon dioxide is slowly discharged completely. When the pressure drops to normal pressure, take out the dried sample, and stick it on the aluminum table with double-sided tape. At this time, the sample must be evenly distributed on the double-sided tape. Do not overlap to avoid affecting the observation of the sample. The most important thing is to avoid electrical charges. produce. Move to the coating machine for gold plating under vacuum to make the sample easy to conduct electricity and facilitate observation. Finally, use a scanning electron microscope to observe the surface and internal morphological structure of the sample particles (immobilized microalgae particles).

1.7 Preparation of immobilized algal strain particles

1.7.1 Algae collection

Place the microalgae to be prepared into particles at appropriate temperature, light intensity, and medium for the first activation, and then transfer the cultured algae strain to a medium containing a suitable substrate, and cultivate the algae liquid before the insertion. The amount of algae inoculated is 10% (v/v), and the second activation culture is carried out for 7 days under appropriate temperature and light intensity. Finally, when the algae strain is stable, the algae liquid is concentrated by centrifugation, and the algae bodies are collected and prepared for immobilized algae strain particles.

1.7.2 Preparation of algae granules and determination of their basic properties

A certain concentration of sodium alginate (sodium alginate) is dissolved in distilled water and sterilized. Then add the algae liquid after centrifugal concentration, and stir evenly. This mixed solution is injected into the forming solution through an injection needle to form particles about 4 mm in diameter. After the granules are stirred in the forming solution for 2 hours, use sterile water to wash the granules several times and then stir the formed granules with sterile water at room temperature for one day.

1.8 Strength analysis of immobilized particles

The prepared immobilized particles were subjected to strength analysis, and this analysis was performed using a physical analyzer (Brookfield). Collect data by means of data acquisition with a strength tester connected to a computer. Place the particles in the groove of the platform, adjust the platform and use the probe type (TA44*) to align the particles to be tested, and then directly perform the compression process (Compression), the initial depression depth is set to 2 g, the initial depression The pressure speed is 0.5 mm s ^-1 ; as the probe rod moves down, the fixed needle will exert force on the particles, and the colloidal particles will be concave and deformed. The data of the force and the deformation distance are collected by the computer, and the parameters are converted A graph of the relationship between stress and strain is formed, and the result will be drawn as a line graph by taking the maximum stress point of the object under test. The mechanical strength of the particles is defined as: when the colloid produces 50% strain force, it is expressed in Kg/cm ² .

1.9MFC device and pre-processing

Using bipolar MFC (microbial fuel cell), a 64cm ² proton permeable membrane separates the anode and the cathode. The anode tank (capacity is 100 mL) and the cathode tank (capacity 100 mL) are composed of the proton permeable membrane. Before use, soak in 1% NaCl solution for 24 hours for pretreatment. The anode and cathode are carbon fiber cloth, the size is 5x3cm ² , and a fixed external resistor of 100Ω is connected between the electrodes.

The anode tank is inoculated with 20% of the bacteria cultured in the anaerobic bottle from the waste sludge of the Tairong Food Fructose Wastewater Treatment Plant, and is exposed to nitrogen for 5 minutes before incubation to ensure that the tank body reaches anaerobic conditions. The amount of inoculation in the cathode tank is 10%.

1.10 Discussion on different aeration rates of cathode

The cathode air aeration rate is (1) no aeration; (2) 0.3 L/min; (3) 0.5 L/min; and (4) 1 L/min.

1.11 Discussion on different nitrogen sources at cathode

Respectively use different nitrogen sources (1) no addition (Blank); (2) Peptone; (3) (NH ₄ ) SO ₄ ; (4) KNO ₃ ; (5) Urea; and (6) Yeast extract (YE) was discussed.

1.12 Voltage measurement

The anode chamber is connected to the cathode chamber through a 100Ω resistor and an electrical signal data collector is used and the potential difference between the anode and the cathode is recorded every 1 minute.

1.13 Cyclic Voltammetry Measurement

The 5640 multi-channel electrochemical test system was used after 7 days of culture under different aeration conditions at the cathode, the scanning range was -1.0V to 1.0V, the reference electrode was an Ag/AgC electrode, the auxiliary electrode was a platinum electrode, and the CV scan rate was 5 mV/s.

1.14 Measurement of polarization curve

Using a two-pole system, measure the open circuit voltage (OCV) before measuring the battery, that is, the voltage value under the condition of no resistance, and use the open circuit voltage as the extreme value of the potential sweep (for example: the open circuit voltage is 0.05V, set the polarization curve sweep The aiming range is 0.05-0 V), and the scan rate is the open circuit voltage divided by 100.

Results and discussion

2.1 Screen algae strains that can remove wastewater pollutants and synthetic oils

Refer to Figure 2, which is an analysis diagram of the algal strain lipid content of the present invention.

After picking out the same algae and purifying them in the culture medium, different algae species are screened and purified from various media, and then a culture experiment of high-efficiency microalgae removal of waste water and simultaneous production of algae oil screening is carried out, and the purified ones are different. The algae species were first activated and cultivated, and then 50 strains of algae strains selected from it were cultivated for 7 days using actual wastewater. They are: (1) Chlorella vulgaris sp. G2-3-2, (2) Chlorella vulgaris sp. W24-1-1, (3) Chlorella vulgaris sp. ) W20-2-1, (4) Chlorella vulgaris sp. W31-1-1, (5) Chlorella vulgaris sp. Pu24-1, (6) Chlorella vulgaris sp.) Pu8-2, (7) Chlorella vulgaris sp. Pu29-1-1, (8) Chlorella vulgaris sp. W27-2-1, (9) Chlorella ( Chlorella vulgaris sp.) Pu10-1, (10) Chlorella vulgaris sp. W26-1-3, (11) Chlorella vulgaris sp.) W24-1-2, (12) Globules Algae ( Chlorella vulgaris sp.) W20-2-1-1, (13) Chlorella vulgaris sp. W26-1-1, (14) Chlorella vulgaris sp. W25-2-1 , (15) Chlorella vulgaris sp. W29-4-1, (16) Chlorella vulgaris sp. Y8-2, (17) Chlorella vulgaris sp. P12-1 , (18) Chlorella vulgaris sp. G11, (19) Chlorella vulgaris sp. G22, (20) Chlorella vulgaris sp. P15-2, (21) Globules Chlorella vulgaris sp. P25-1, (22) Chlorella vulgaris sp. P12-1-2, (23) Chlorella vulgaris sp. G2-1, (24) pellets Chlorella vulgaris sp. P24-2, (25) Chlorella vulgaris sp. R15-1, (26 ) Chlorella vulgaris sp. R23-1-2, (27) Chlorella vulgaris sp. R26-1, (28) Chlorella vulgaris sp. R26-2, (29) ) Chlorella vulgaris sp. O8-1-2, (30) Chlorella vulgaris sp. O7-1-1, (31) Chlorella vulgaris sp. O10-1- 1. (32) Chlorella vulgaris sp. O23-1-1, (33) Chlorella vulgaris sp. O18-1-1, (34) Chlorella vulgaris sp. O2-1-2, (35) Chlorella (Chlorella vulgaris sp.) Y8-1, (36) Chlorella (Chlorella vulgaris sp.) P2-1, (37) Chlorella (Chlorella vulgaris sp.) O8-1-1, (38) Chlorella vulgaris sp. P29-1, (39) Chlorella vulgaris sp. W31-1-1 white, (40) Tetraselmis sp. ), (41) Chlorella vulgaris sp. G3H3-1-2, (42) Chlorella vulgaris sp. Green 2 iso 2-2, (43) Chlorella vulgaris sp. ) Green 2, (44) Chlorella vulgaris sp. Green 14, (45) Chlorella vulgaris sp. Green 10, (46) Chaetoceros sp., (47) Pseudomonas Nannochloropsis sp. DYU1, (48) Chlorella vulgaris sp. Wu-G23, (49) Chlorella vulgaris sp., different green 2-1, (50) Nannochloropsis sp. ( Nannochloropsis sp.) DYU2. Among them, 37 strains of algae can withstand the tolerance of wastewater and grow. The preliminary differences are: 29 strains of Chlorella sp. series, 6 strains of Oscillatoria series, Spirulina sp. series, and hot springs. For the algae and red algae, in order to select the algae strains with higher oil content from the 37 algae strains, they were preliminarily screened by Sudan black staining. Because Sudan black stain is a fat-soluble dye-Sudan B can specifically stain the neutral triglycerides, lipids and lipoproteins in the tissues dark blue-black, so you can determine whether the algae cells are There is accumulation of grease, and the result is shown in Figure 3.

Refer to Figure 3, which is the staining analysis diagram of the present invention. As shown in the figure, the four algae strains with high oil content selected by Sudan black staining and Nile red staining are: sample No. 18 G11, sample No. 19 G22, sample No. 48 G23, and sample No. 23 G2-1.

2.2 Screen the algae strains with good removal effect and oil accumulation for sequence identification

Refer to Figure 4, which is the analysis diagram of the DNA identification electrophoresis film of the present invention. Among them, (a) is the Pro-Taq PCR product; (b) is the Super-Taq PCR product; (c) is the elution of Super-Taq PCR DNA fragments (Elution); (d) is the rapid screening micro Algae DNA; (e) is a plastid derived from a variant of microalgae; (f) DNA inspection by Hind III. M stands for DNA molecular marker (Mark).

The four strains of microalgae obtained G22 and G23 have better FAME content and removal effect in the process of experimental research. Therefore, G22 and G23 were subjected to nucleotide sequencing and algae provenance identification. The rbcL (ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit) gene is one of the important genes related to photosynthesis in plant/algae chloroplasts, and is a commonly used gene marker in the classification of plants/algae. Therefore, polymerase chain reaction was used to amplify the rbcL gene sequences of the two microalgae Chlorella sp. In the present invention, a special rbcL primer for algae is designed, and then PCR reaction (polymerase chain reaction) is used for amplification to obtain fragmented DNA products corresponding to G22 and G23 rbcL. When performing PCR, first use Pro-Taq-DNA polymerase with no proofreading function to determine whether the size of the DNA fragment amplified by PCR is as expected. The result is shown in Figure 4(a). After PCR amplification The length is about 1.3k. After confirming that the G22 and G23 fragments are correct in size, the Super-Taq polymerase with correction function is used to perform PCR amplification again. The result is shown in Figure 4(b). DNA length is also about 1.3k. The result is consistent with the expected size. Therefore, the product of DNA amplified by Super-Taq PCR is further processed for subsequent processing.

2.3 Screening of rbcL for algae species and identification results

Use the clusterw system to list the rbcL gene sequence in the GenBank database and compare the gene sequences of known algae strains with BLAST software. The result shows that the algae strain is the same genus as Chlorella revealed by the system. According to the results of the phylogenetic tree, the two algae strains were classified into a monophylogenetic group based on the results of the rbcL gene comparison, and they are all closely related to Chlorella sp. Finally, according to the results of the parental analysis, the present invention named the G22 and G23 algae strains as Chlorella vulgaris Wu-G22 (SEQ ID NO: 1) and Chlorellasp. Wu-G23 (SEQ ID NO: 2), respectively. Chlorella sp. is a typical spherical cell with a diameter of about 3-5μm. It has a wide range of pH tolerance and heat resistance. It also has the presence of chlorophyll a and b and can accumulate oil in microalgae cells. In addition, Chlorella sp . It is a microalgae strain commonly used to produce oil, therefore, these characteristics are consistent with the results of the algae strain used in the present invention.

2.4 Choose the best immobilization conditions to prepare microalgae cell particles

2.4.1 Explore the influence of different alginate concentrations on immobilized particles

Alginate, a hydrophilic polysaccharide, is one of the most abundant natural biosynthetic materials, mainly from marine plants and bacteria. In commercial applications, brown algae are the main source of alginate, which can form a solid gel under mild conditions. In addition, studies have shown that immobilized algae can effectively and quickly remove nitrogen and phosphorus from wastewater.

In order to explore the influence of different alginate concentrations on particle formation, the alginate concentrations of 0.5%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, 4.0%, 4.5%, and 5.0% were configured. The solution was dropped into the CaCl ₂ forming solution and the particles were formed to be observed. The result is shown in Figure 5.

Refer to Figure 5, which is an analysis graph of alginate concentration of the present invention.

When the concentration is 0.5%, when the concentration is too low, the particles cannot be formed, resulting in irregular and extremely soft particle shapes, and low mechanical strength. When the concentration is 1.0%, although the particles can be formed, the particles are also relatively soft and the mechanical strength is not high. When the concentration is higher than 1.5%, the viscosity of the solution also increases, but it will cause the needle to block and make the production of particles difficult. In addition, in terms of particle diameter, the diameter of the immobilized particles at other concentrations is about 4.5mm except that when the concentration is 0.5%, the dropped particles cannot be spherical. In terms of particle mechanical strength, as the concentration is higher, the particle strength is higher, but when the concentration is 3.5-5.0%, the prepared particles are too viscous and the particle forming time must be longer. Therefore, the concentration of alginate for immobilization into algal cell particles is set at 3.0%.

2.4.2 Explore the influence of different oyster shell powder concentrations on immobilized particles

After the alginate is cross-linked with calcium chloride to form calcium alginate, the calcium ions in it will gradually lose over time, causing the particles to become soft, and even unable to shape the particles, leading to possible disintegration. In order to prevent this phenomenon, this experiment tried to add the discarded and ashed oyster shell powder and mix it with the best alginate solution (3%) discussed in the above experiment, so that the additional oyster shell powder was added to supplement the alginate Calcium that particles may lose. The results are shown in Table 2. When additional ashed oyster shells with a concentration of 1%, 2%, 3%, 4% and 5% were added, it was found that the additional addition of oyster shell powder could not increase the strength of the particles, but caused the original 3% of the formed alginate could not be smoothly formed into a spherical shape, and only the control group could be formed smoothly. Therefore, when the concentrated algae solution is mixed with alginate to prepare immobilized microalgae cell particles, oyster shell powder will not be added as a material to enhance the physical properties of the particles.

Table 2. Characteristics and feasibility of immobilized Chlorella Wu-G22 and Chlorella Wu-G23

2.4.3 Explore the impact of immobilization on the feasibility of microalgae

Since the alginate concentration discussed above is 3.0%, it is observed whether the alginate concentration is 3.0%, whether it has any effect on the prepared microalgae cell particles. The results are shown in Table 3.

table 3

As shown in the table, Chlorella vulgaris Wu-G22 and Chlorella sp. Wu-G23 (hereinafter referred to as G22 and G23) and 3% alginate were selected to make immobilized particles, and their cells were embedded in them. The shapes of G22 and G23 freshly made particles are regular round particles with a diameter of about 4.3mm. The cell density in the particles is 2.84 and 3.58 g/cm ³ , and the particle strength is 602.3 g and 447.3 g, respectively. In addition, the coating degree of immobilized particles was about 0.0104 and 0.0087 (biomass g/beads g) respectively. G22 and G23 were cultured for 10 days under appropriate medium, light intensity and temperature. The appearance of the particles can be clearly seen from the photos of the particles in Table 3. The particles after 10 days of cultivation are greener than the initial particles, and the diameter is not significantly larger than the initial particles. The changes are about 4.3mm, and the cell density in the particles has a significant increase to 3.94 and 4.89 g/cm ³ , and the particle strength also has a significant increase in strength to 805.9 and 781.3g, respectively. In addition, the coating degree of the immobilized particles is obvious. The increase increased to 0.109 and 0.0933 (g/g) respectively. After culturing the prepared pellets for 10 days and discovering these changes, it is speculated that the algal cells grow well in the immobilized pellets and have a high mass transfer efficiency for nutrients. Therefore, the cells in the immobilized pellets can be fully The absorption and utilization of nutrients in the cultivated chicken to achieve the best growth and metabolism.

Refer to Figure 6, which is the SEM analysis image of the present invention. Among them, (a) is the result graph of 25 times magnification on day 0 of G22; (b) is the result graph of 25 times magnification on day 10 of G22; (c) is the result graph of 1000 times magnification on day 0 of G22; (d) It is the result of 1000 times magnification on the 10th day of G22; (e) is the result of 25 times magnification on the 0th day of G23; (f) is the result of 25 times on the 10th day of G23; (g) is the 0th day of G23 Figure of 1000 times magnification result of G23; and (h) is the figure of 1000 times magnification result of G23 on the 10th day.

As shown in the figure, in terms of the observation of the microstructure of the immobilized particles, the G22 and G23 algae cell particles were observed by scanning electron microscope (SEM) before and after the culture and the conditions of the cross-linking space and the growth of the algae cells and The influence of the internal structure of the particles and the number of algae cells. In the case of G22 and G23 algae cells, on the 0th day of culture, the internal structure of the particles has more pores and larger pore sizes. On the 10th day of culture, the internal pore structure of the particles is tighter and the pore size is also larger than that of no day 0 particles. , On the contrary, it is denser, and the algae cells inside the granules are relatively more on the 10th day of culture than on the 0th day, and the growth of the algal cells in the granules is not very obvious. In addition, inside the immobilized particles, the denser the pores and the smaller the pore size, the greater the density of the immobilized cell particles. Compared with the photographs of culture from day 0 to day 10, it is found that this corresponds to this speculation, so when fixing After 10 days of culturing the chemical cell particles, the pore structure will be denser, the pore size will be smaller, the number of algae cells will increase, and the particle density will increase. In addition, the three-dimensional space formed by the internal cross-linking of the particles can fully provide for the growth and operation of algae cells inside.

2.5 The influence of different cathode aeration rates on the voltage and electric power of the bipolar microbial fuel cell (BA-MFC)

Refer to Figure 7, which is the BA-MFC voltage-time diagram (Figure 7(a)) and electric power density-time diagram (Figure 7(b)) for different cathode aeration rates. The first three days are due to BA-MFC. MFC is unstable, so its data is not used. According to the data on the third day, the voltage of BA-MFC with a cathode aeration rate of 0.3 L/min is the best. It may be that the biofilm has been attached, but its voltage will continue to decrease. This may be due to accelerating the attachment of the cathode biofilm to the electrode during aeration, but after the biofilm is attached, the shear stress caused by the aeration may cause irreversible biofilm separation and power drop, so the later voltage is maintained at 5 To 8 mV and there is no rising trend. In addition, this trend is also observed in BA-MFC with a cathode aeration rate of 0.5 L/min and a cathode aeration rate of 1 L/min. Although the initial voltage of the BA-MFC without aeration of the cathode is not high, the voltage rises significantly after the seventh day and is higher than other conditions. It may be that the biofilm slowly adheres to the electrode, thereby increasing the electrical activity without other external forces. Affect biofilm. Therefore, for the judgment of overall voltage output and electric power density, BA-MFC without aeration is the best condition.

Although not intended to be bound by any theory, according to reports in the literature, in an algae suspension, the medium can transport electrons received by the cathode to the algae. These reducing media will penetrate the algae cells to provide electrons, thereby being oxidized, and finally released from the algae cells back into the catholyte. In addition, because the power output of the microbial fuel cell depends on the low resistance of the external circuit, the high permeability ion exchange membrane, the reducing conditions and the high concentration of oxygen in the cathode tank, when the biofilm on the cathode can be used as a medium for electron transfer, it can Reduce the ohmic value in the MFC, and reduce the resistance loss when the charge is transferred.

2.6 The cyclic voltammogram of BA-MFC with different cathode aeration rates

Refer to Figure 8, which is the anode cyclic voltammetry (CV) diagram (Figure 8(a)) and the cathode cyclic voltammetry (CV) diagram of the BA-MFC fuel cell system cultured for 14 days under different aeration conditions at the cathode (Figure 8(b)). As shown in the result of Figure 8(a), the anode of the BA-MFC without aeration of the cathode shows a higher oxidation current than the anodes of the other three fuel cells. There is oxidation at 190 mV (58 mA) in the forward scan. The peak (such as the peak a in Figure 8(a)), the peak exhibited by the anode biofilm may be due to the medium produced by the bacterial culture, but there is no obvious reduction peak, which indicates that this may be an irreversible reaction. The cathode aeration rate of 0.3 L/min BA-MFC and the cathode aeration rate of 1 L/min BA-MFC have no obvious redox peak at the anode, which may be due to the biological activity of anaerobic sludge caused by cathode aeration through the permeable membrane It is not high, which results in the formation of biofilms. Cathode aeration rate 0.5L/min BA-MFC anode oxidation peak (such as the b peak in Figure 8 (a)) in the forward scan appears at 370 mV (27 mA). In the reverse scan, a reduction peak (such as the c wave peak in Figure 8 (a)) appears at −390 mV (-29 mA).

As shown in the result of Figure 8(b), the cathode of BA-MFC without aeration of the cathode has an oxidation peak at 230mV (59m A) (such as the peak a in Figure 8(b)), which is reduced in the reverse scan The peak (such as the e-wave peak in Figure 8(b)) appears at -750mV (-83 mA), which may be due to the fact that there is no flow in the tank and the probability of the algal biofilm attached to the electrode is small, and most of the algae precipitates At the bottom of the tank. In addition, the oxidation peak of the cathode of BA-MFC with a cathode aeration rate of 0.3 L/min in the forward scan (such as the d wave peak in Figure 8 (b)) appears at 500 mV (108 mA), and the reduction peak in the reverse scan (As shown in Figure 8(b), the h wave peak) appears at -510 mV (-135mA), and the cathode aeration rate of 0.5 L/min BA-MFC’s cathode has an oxidation peak in the forward scan (as shown in Figure 8 ( The b peak in b) appears at 490 mV (67 mA), and the reduction peak (such as the f peak in Figure 8 (b)) appears at -730 mV (-100 mA) in the reverse scan, but both Not obvious. Furthermore, the cathode aeration rate of 1 L/min BA-MFC has an oxidation peak at 380mV (82 mA) in the forward scan (such as the c wave peak in Figure 8(b)), which is reduced in the reverse scan The peak (such as the g-wave peak in Figure 8(b)) appears at -740 mV (-126 mA). As mentioned above, because the biofilm of algae may fall off when the aeration rate is too high, the result is consistent with the above. The aeration rate is 0.5 L/min and 1 L/min for algae. The adhesion of the biofilm is more unfavorable, and for the cathode Chorella sp.Wu-G23, the aeration rate of 0.3 L/min is the best.

2.7 Ammonia nitrogen analysis and pH of BA-MFC with different cathode aeration rates

Refer to Figure 9, which shows the anode ammonium ion concentration (Figure 9(a)) and cathode ammonium ion concentration of the BA-MFC fuel cell system under different aeration conditions at the cathode on the 7th day (Figure 9(b)) ), anode pH (Figure 9(c)) and cathode pH (Figure 9(d)). As shown in Figure 9 (a) and (c), on the anode side, except that the ammonium concentration of the BA-MFC without aeration at the cathode increased by 0.13 g/L, the ammonium concentration under other aeration conditions decreased. It may be due to the degradation of anaerobic sludge from urea, which is the main source of ammonia nitrogen in synthetic wastewater. Therefore, according to the above results, the activity of anaerobic sludge is higher under the condition of no aeration at the cathode, but there is no significant change in pH value.

As shown in Figure 9 (b) and (d), in terms of the cathode, the ammonium concentration of BA-MFC without aeration at the cathode increased by 0.04 g/L, and the ammonium concentration of other aeration conditions decreased. The decrease in concentration may be due to the direct assimilation of Chlorella sp.Wu-G23. In addition, the ammonium concentration decreased by 0.13g/L under the condition of cathode aeration rate of 0.3 L/min, which is the largest decrease in ammonium concentration under all conditions. Therefore, according to the above results, Chlorella sp.Wu-G23 has higher activity under the condition of cathode aeration rate of 0.3 L/min. At the same time, it can be seen from the pH trend that the higher the activity of Chlorella sp.Wu-G23, the higher the pH The value also rises higher.

Although not intended to be bound by any theory, according to reports in the literature, urease is an enzyme that catalyzes the conversion of urea into ammonia, ammonium, and bicarbonate. The catholyte lacking C. vulgaris produces less ammonium than the anode tank because the anode tank has already absorbed part of the urea. The presence of C. vulgaris in the cathode tank improves the ammonium removal rate, and the ammonium concentration is reduced by 24 ± 8 mg/L. Considering the degradation of urea, it is presumed that this may produce about 20 mg/L of ammonium. C.vulgaris can consume more than 40 mg/L of ammonium, which is equivalent to a removal rate of 5 mg/L/ during an 8-day residence period. d. For C. vulgaris grown in wastewater, the highest ammonium removal rate is 200 mg/L/d, and the lowest is 0.65 mg/L/d, which may depend on the growth rate of C. vulgaris in different wastewater types.

2.8 Analysis of BA-MFC ion chromatography with different cathode aeration rates

Referring to FIG. 10, day 7 and cultured as BA-MFC NO anode of the fuel cell system under different aeration conditions cathode ₃ ^- concentration (FIG. 10 (A)), a cathode NO ₃ ^- concentration (FIG. 10 ( b)), anode NH ₄ ⁺ concentration (Figure 10 (c)) and cathode NH ₄ ⁺ concentration (Figure 10 (d)). After the BA-MFC fuel cell is cultured with different aeration rates of the cathode, the culture solution of the anode and the cathode is taken out to detect the NO ₃ ^- concentration by IC ion chromatography and the NH ₄ ⁺ concentration by ammonia nitrogen analysis.

As shown in Figure 10 (a) and (b), according to the anode NO ₃ ^- concentration results, it can be seen that the anode NO ₃ ^- concentration of all aeration rates has a downward trend. In the aeration rate of 0 L/min, 0.3 L/min and 1.0 L/min, it decreased by 11.76 mg/L, 13.81 mg/L and 7.02 mg/L, respectively, and at the aeration rate of 0.5 L/min. Check out. In terms of the cathode NO ₃ ^- probably because the concentration of ammonium nitrogen is oxidized to NO ₃ ^- is not high when the concentration of alginate, and ammonium alginate preferred initial inoculation, leading to ₃ NO ^- upward trend to the concentration. According to literature reports, NO ₃ ^- can not only be assimilated by algae, but can also be removed as an electron acceptor on the cathode, so it is deduced that if the experiment time is extended, NO ₃ ^-may still be removed.

As shown in Figure 10 (c) and (d), according to the NH ₄ ⁺ concentration results, the NH ₄ ⁺ concentration can be seen to decrease in the anode, but except for the cathode aeration rate of 0 L/min, BA-MFC has increased Therefore, it is judged that the increase of cathode aeration may affect the growth and metabolism of anode anaerobic sludge. In terms of the cathode, the NH ₄ ⁺ concentration showed a downward trend. The largest decrease was the cathode aeration rate of 0.3 L/min (a decrease of 13.61 mg/L), which means that within 7 days, the cathode aeration rate was 0.3 L/min. The growth is best at L/min. The decrease in ammonium concentration may be due to the direct assimilation of C. vulgaris .

2.9 Chemical oxygen demand (COD) and biomass quality of BA-MFC with different cathode aeration rates

Refer to Figure 11, which shows the anode COD value of the BA-MFC fuel cell system under different aeration conditions of the cathode (Figure 11(a)), cathode COD value (Figure 11(b)), anode Biomass (Figure 11(c)) and cathode biomass (Figure 11(d)). On the anode side, the COD of BA-MFC without aeration at the cathode decreased from 7.1 g/L to 1.04 g/L, and the COD removal rate was 85%. The COD of BA-MFC with a cathode aeration rate of 0.3 L/min decreased from 7.1 g/L dropped to 2.47 g/L, the COD removal rate was 65%, the COD of BA-MFC at the cathode aeration rate of 0.5 L/min dropped from 7.1 g/L to 0.99 g/L, and the COD removal rate was 86%. At the cathode aeration rate of 1 L/min, the COD of BA-MFC decreased from 7.1 g/L to 6.88 g/L, and the COD removal rate was 5.9%, which corresponds to the biomass. At the cathode aeration rate of 1 L/min BA-MFC MFC may be due to too high aeration rate, a large amount of gas passing through the permeable membrane leads to a decrease in the activity of anaerobic sludge, and BA-MFC under other conditions has obvious COD removal.

As for the cathode, because the voltage started to be relatively stable on the third day, there were points and medium supplementation, and the growth rate of algae was slow, resulting in an increase in COD. In the biomass, it was found that the aeration rate of 0.3 L/min was Chlorella sp.Wu-G23 The best growth conditions, COD is also the least.

2.10 The influence of different cathode nitrogen sources on BA-MFC voltage and electric power

Refer to Figure 12, which shows the voltage-time diagram (Figure 12(a)) and the electric power density-time diagram (Figure 12(b)) of the BA-MFC with different nitrogen sources at the cathode. Since BA-MFC is not stable on the first day of the experiment, its data is not used. In addition, the BA-MFC whose nitrogen source is urea can reach 44 mV on the second day, which is the best condition. This reason may be due to the fact that urea is the best nitrogen source for Chlorella sp. Wu-G23, so that Wu-G23 can grow rapidly, leading to faster biofilm growth and adhesion, and thus the effect of increasing the voltage relatively. Followed by BA-MFC was not added and nitrogen as a nitrogen source of the KNO BA-MFC _3, though it is not Chlorella sp. Wu-G23 of the best nitrogen source, but the growth rate of Chlorella sp.Wu-G23 not promote BA- The only reason for the MFC voltage rise. In addition, BA-MFC under all conditions has a low voltage in the later stage, which may be because the suspension system is not stable enough or because the suspension system has poor voltage endurance.

2.11 Cyclic voltammogram of BA-MFC with different nitrogen sources at the cathode

Refer to Figure 13, which is the cyclic voltammogram of the anode anaerobic sludge (Figure 13(a)) and the cyclic voltammogram of the cathode anaerobic sludge of the BA-MFC fuel cell system under different nitrogen sources at the cathode for seven days. Figure 13(b)). As shown in Figure 13(a), in terms of anode, the anode without the addition of nitrogen source BA-MFC has no obvious redox peak, indicating that the electrochemical activity of anaerobic sludge is not good. The cathode nitrogen source is a BA-MFC anode. The oxidation peak in the forward scan (such as the peak a in Figure 13 (a)) appears at 435 mV (35 mA), and in the reverse scan, at −480 mV ( -22 mA), there is a reduction peak (such as the b peak in Figure 13 (a)), but the redox peak is still not obvious, and the adhesion of the anaerobic sludge biofilm to the electrode may be incomplete. The cathode nitrogen source is (NH ₄ ) ₂ SO ₄ BA-MFC anode in the forward scan, the oxidation peak (such as the c wave peak in Figure 13 (a)) appears at 670 mV (16 mA) in the reverse scan , There is a reduction peak at −520mV (-17 mA) (such as the d wave peak in Figure 13(a)). The reason for this phenomenon is the same as that of BA-MFC where the cathode nitrogen source is a protein. The oxidation peak of the BA-MFC anode whose cathode nitrogen source is KNO ₃ (such as the e wave peak in Figure 13 (a)) appears at 165 mV (31 mA) in the forward scan, and at −460 mV ( -31mA) a reduction peak (such as the f wave peak in Figure 13(a)) appears, which may be due to the formation of stable anaerobic sludge biofilm and adhere to the electrode without falling off. The oxidation peak of the anode of BA-MFC with urea as the cathode nitrogen source appears at 115mV (8 mA) in the forward scan (such as the g peak in Figure 13(a)), and at −430 mV (- 10mA) A reduction peak (such as the h wave peak in Figure 13 (a)) appears. The cathode nitrogen source is a BA-MFC anode of yeast extract. The oxidation peak (such as the i-wave peak in Figure 13 (a)) appears at 720 mV (14 mA) in the forward scan, and in the reverse scan, at − A reduction peak appears at 470 mV (-13 mA) (such as the j wave peak in Figure 13 (a)). According to the cyclic voltammetry diagram, it is judged that the MFC whose nitrogen source is KNO ₃ is the best.

As shown in Figure 13(b), as for the cathode, the cathode without adding a nitrogen source BA-MFC has no obvious redox peak, which may be caused by the poor growth of Chlorella sp. Wu-G23. The cathodic nitrogen source is a BA-MFC cathode with protein. In the forward scan, the oxidation peak (such as the peak a in Figure 13(b)) appears at 615 mV (22mA), and in the reverse scan, at −810 mV(- 51 mA) A reduction peak (such as the b peak in Figure 13 (b)) appears. Although there is a redox peak, it can be found from the reverse scan that the reaction is not completely stable. The BA-MFC cathode whose cathode nitrogen source is (NH ₄ ) ₂ SO ₄ has no obvious redox peak. The cathode nitrogen source is KNO ₃ BA-MFC cathode without obvious redox peak. The oxidation peak of the BA-MFC cathode whose cathode nitrogen source is urea (such as the c wave peak in Figure 13(b)) appears at -250 mV (34 mA) in the forward scan, and at −690 in the reverse scan The mV (-81 mA) has a reduction peak (such as the d wave peak in Figure 13(b)), so it is judged that Chlorella sp. Wu-G23 grows best, which may be due to the stable biofilm adhesion. The cathode nitrogen source is a BA-MFC cathode of yeast extract. In the forward scan, the oxidation peak (such as the e wave peak in Figure 13 (b)) appears at 515 mV (53mA), and in the reverse scan, at −599 The mV (-66 mA) has a reduction peak (such as the f peak in Figure 13(b)). Yeast extract is also a good nitrogen source for Chlorella sp. Wu-G23, and its growth is relatively fast. On the whole, BA-MFC with urea as the cathode nitrogen source is the best and has obvious redox peaks.

2.12 Polarization curves of BA-MFC with different nitrogen sources at the cathode

Refer to Figure 14, which is the polarization curve of BA-MFC fuel cell system cultured under different nitrogen sources at the cathode for seven days. As shown in FIG. 14, the cathode without the addition of a nitrogen source in the BA-MFC 2.5 mV, 0.002mA / cm ^2, the maximum power of 0.059 mW / m ^2. The cathode of the nitrogen source is peptone BA-MFC in 24.86 mV, 0.014mA / cm ² when the maximum power of 3.776 mW / m ^2. Nitrogen source _{(NH 4) 2 SO BA-} MFC 4 of the 26.06 mV, 0.015mA / cm ^2, the maximum power of 4.13 mW / m ^2. When the nitrogen source is KNO ₃ , BA-MFC has a maximum power of 8.074 mW/m ² at 11 mV and 0.072 mA/cm ² , and when the nitrogen source is urea BA-MFC has a maximum power of 19.72 mV and 0.005 mA/cm ² power 1.049 mW / m ^2, BA-MFC nitrogen source is yeast extract of 4.4 mV, 0.006 mA / cm ^2, the maximum power of 0.286 mW / m ^2.

According to the above polarization curve, the difference in power generation under different conditions is mainly caused by the anode potential, which corresponds to the cyclic voltammogram of the anode. The nitrogen source is protein, (NH ₄ ) ₂ SO ₄ , and urea BA-MFC. Higher open circuit voltage, but the current density is reduced due to excessive resistance. The possible cause of the resistance is that the anode anaerobic sludge biofilm is not completely attached to the electrode, and the solution (waste water) cannot be efficiently performed due to the space between the electrode and the electrode. For electron transfer, BA-MFC with KNO ₃ nitrogen source may be completely attached to the anode anaerobic sludge biofilm. Although the open circuit voltage is low, the current density is the highest, and the electrical power density is also the best among all conditions.

2.13 Screened microalgae Chlorella sp. Wu-G23 wastewater treatment and oil content

Using G23 culture in wastewater, the result is shown in Figure 15. When the dilution rate is 0-80% under wastewater aeration, the pH value of the final culture is close to pH 9. In terms of NH ₄ ⁺ -N removal rate, under aeration conditions, the NH ₄ ⁺ -N removal rate is the best (up to 84%) when the dilution rate is 10%, followed by 78% under undiluted conditions, and in static conditions. Setting conditions, when the dilution rate is 0%, the NH ₄ ⁺ -N removal rate is 74%. In addition, in the part of COD removal rate, under aeration and standing conditions, the COD removal rate is the best when the dilution rate is 0%, and both are as high as 60% or more. Especially under the standing condition, the COD removal rate is in the dilution When the rate is 0%, it can even reach 77%. Regarding the chroma removal rate, the chroma removal efficiency is higher than 70% regardless of the aeration or standing conditions. In addition, as shown in Figure 16, when the wastewater dilution rate is 0%, the total FAME content has the highest FAME content of 12.5%.

in conclusion

In order to screen out the nutrients (pollutants) that can be used in wastewater to grow, achieve the effect of removing wastewater pollutants and accumulate grease, therefore, from the 50 algae strains in our laboratory, 4 strains of their algae strains were selected. They are: G2-1, G11, G22 and G23. In addition, four microalgae in the process of experimental exploration, because G22 and G23 have better total FAME content and removal effect, therefore, G22 and G23 were subjected to a series of 18S rDNA sequence reaction procedures and the two strains were obtained by NCBI GenBank. Microalgae 18 S rRNA gene comparison, it is classified into a monophylogenetic group, and they are all closely related to Chlorella sp. Finally, according to the results of the parental analysis, the present invention named the G22 and G23 algae strains as Chlorella vulgaris Wu-G22 and Chlorella sp. Wu-G23, respectively, and Chlorella sp. Wu-G23 was used as the cathode in the experiment.

Explore the material for the immobilization of the preparation matrix (the particles prepared are not coated with microalgae cells), and the results found that the alginic acid concentration is 3% as the best condition. Next, the immobilized particles with the alginic acid concentration of 3% are discussed. The effect of the viability of microalgae cells, the test results show that the algae cells grow very well in the carrier granular material with alginic acid concentration of 3%, and the nutrients can be fully absorbed and utilized by the algae cells to achieve optimal growth and metabolism. The process.

In the experiment of exploring different cathode aeration rates, although cyclic voltammetry analysis, ammonia nitrogen analysis and COD removal are used to judge, the aeration rate 0.3 L/min BA-MFC is the best, but because aeration may cause air to pass through the membrane It also affects the electrochemical activity of the anode bacteria, and the algae biofilm falls off due to the shearing force caused by aeration, which causes the overall voltage to be low. In contrast, BA-MFC does not aerate although the algae biofilm adheres slowly However, the results of CV analysis show that the electrochemical activity of the anode bacteria is better, and the COD removal is as high as 85%. Aeration can bring a better growth environment for the cathode algae, but it affects the electrochemical activity of the anode bacteria. The overall judgment is that the BA-MFC without aeration is the best condition for this experiment.

In the discussion of different cathode nitrogen sources, urea is the best nitrogen source for the cathode Chlorella sp. Wu-G23. Due to the incomplete attachment of the anode biofilm, the cyclic voltammetry and polarization curve are not ideal, but according to the voltage-time According to the figure, there are still good results. The overall judgment is that the nitrogen source is urea. This is the best condition for the experiment. If the adhesion of the anode anaerobic sludge biofilm can be more stable, it is believed that the cyclic voltammetry and polarization curve can have the best As a result, there will be a higher breakthrough in voltage.

In addition, referring to Fig. 17, in the microbial fuel cell device 1 of the present invention, the method of fixing the algae strain (Wu-G23) used may include: Step S1: Combining a sterilized sodium alginate aqueous solution of a predetermined concentration with algae The strain (Wu-G23) is stirred and mixed to form a mixed solution; and Step S2: the mixed solution is injected into the forming solution through an injection needle to form immobilized particles of the algae strain (Wu-G23) with a predetermined diameter. Wherein, the predetermined concentration can be in the range of 0.0~6.0 w.t%; it is preferably 1.5~4.5 w.t%; and it is most preferably 2.5~3.5 w.t%. Also, the predetermined diameter may range from 4.0 to 5.0 mm; it is preferably 4.5 mm. In addition, the forming solution may be 1 to 5 w.t% calcium chloride aqueous solution; it is preferably 4 w.t%.

In some embodiments of the microbial fuel cell device 1 of the present invention, the anode tank 10 is filled with the bacteria 13 of the sulfate reducing bacteria and the first nutrient source 12, and the cathode tank 20 is filled with the algae strain (Wu-G23) 23 and the second Nutrition source 22. In addition, both the anode 11 and the cathode 21 are carbon fiber cloth, and in the method of fixing the algae strain (Wu-G23), a 3.0 w.t% sodium alginate solution is used. After the aforementioned microbial fuel cell device 1 is assembled, the voltage is measured. First connect the anode 11 and the cathode 21 to both ends of a 100 Ω resistor respectively, and use an electrical signal data collector to record the potential difference between the anode 11 and the cathode 21, and continue recording for 21 days. The result is displayed on the 18th Figure. This figure shows that the microbial fuel cell device 1 can supply a larger voltage after the 9th day.

Further, the cyclic voltammetry measurement is also performed on the microbial fuel cell device 1. Among them, add in the cathode tank 20 The algae strain (Wu-G23) and the algae strain (Wu-G23) were cultured for seven days. In addition, the measurement conditions and instruments are shown later, using a multi-channel electrochemical test system, the scanning range is -1.0V to 1.0V, and the reference electrode is Ag/AgCl. The auxiliary electrode is a platinum electrode, and the CV scan rate is 5 mV/s. Refer to Figure 19(a), which is the cyclic voltammogram after 21 days of battery operation. On the anode side, in the forward scan, the oxidation peak appears at 114 mV (32 mA), and in the reverse scan, the reduction peak Appears at 505 mV (-0.16 mA). Refer to Figure 19(b). On the cathode side, in the forward scan, the oxidation peak appears at 10 mV (37 mA), and in the reverse scan, the reduction peak appears at -390 mV (-0.62 mA).

Refer to Figure 20, which is a polarization curve diagram of the microbial fuel cell device 1 after 21 days of operation. In the polarization curve measurement method, a two-pole system is used, and before measuring the polarization curve, the battery first measures the open circuit voltage (OCV), and uses the open circuit voltage as the extreme value of the potential scan, and the scan rate is the open circuit voltage 0.01 times. The result shows that at 49.9 mV and 0.035 mA/cm ² , there is a maximum power of 17.4 mW/m ² .

Furthermore, the above-mentioned microbial fuel cell device 1 is subjected to the measurement of the NH4+-N removal rate and the COD removal rate. Wherein, wastewater is added to the cathode tank 20, the dilution rate of the wastewater is 10 times, and the algae strain (Wu-G23) is cultivated in the wastewater for seven days. Then, the ammonia nitrogen analysis method was used to detect the NH4+-N removal rate, and the potassium dichromate reflux method was used to detect the COD removal rate. Refer to Figure 21 (a) and (b), which are the water quality analysis results of the microbial fuel cell device 1 after 21 days of operation. In terms of anode, NH4+-N removal rate is 5%, and COD removal rate is 15%. In the cathode, the removal rate of NH4+-N is 1.5%, and the removal rate of COD is 90%. From the above results, it can be shown that the microbial fuel cell device 1 of the present invention can purify water quality.

1: battery device 10: Anode tank 11: anode 12: The first source of nutrition 13: Bacteria 20: Cathode tank 21: Cathode 22: The second source of nutrition 23: Algae strain 30: Proton exchange membrane 40: conductive thread 41: Load 50: light source 60: Gas device S1, S2: steps

Figure 1 is a schematic diagram of the microbial fuel cell device of the present invention.

Figure 2 is an analysis diagram of the lipid content of the algae strain of the present invention.

Figure 3 is the staining analysis diagram of the present invention.

Figure 4 is an analysis diagram of the DNA identification electrophoresis film of the present invention.

Figure 5 is a graph showing the concentration of alginate in the present invention.

Figure 6 is the SEM analysis image of the present invention.

Figure 7 is a graph of BA-MFC voltage versus time for different cathode aeration rates.

Figure 8 is the analysis diagram of the BA-MFC fuel cell system with different aeration rates at the cathode.

Figure 9 is the analysis diagram of the BA-MFC fuel cell system with different aeration rates at the cathode.

Figure 10 is an analysis diagram of the BA-MFC fuel cell system with different aeration rates at the cathode.

Figure 11 is an analysis diagram of the BA-MFC fuel cell system with different aeration rates at the cathode.

Figure 12 is the voltage-time diagram of BA-MFC with different nitrogen sources at the cathode.

Figure 13 is an analysis diagram of different nitrogen sources at the cathode of the BA-MFC fuel cell system.

Figure 14 shows the polarization curve of the BA-MFC fuel cell system under different nitrogen sources at the cathode for seven days.

Figure 15 is the analysis diagram of BA-MFC fuel cell system under wastewater aeration conditions.

Figure 16 is the analysis diagram of the BA-MFC fuel cell system under wastewater aeration conditions.

Figure 17 is a flow chart of the method of fixing the algae strain (Wu-G23).

Figure 18 is the voltage-time relationship diagram of the BA-MFC fuel cell device.

Figure 19 is the cyclic voltammogram of the BA-MFC fuel cell device.

Figure 20 is the polarization curve of the BA-MFC fuel cell device.

Figure 21 is a bar graph of the NH ₄ ⁺ -N removal rate and COD removal rate of the BA-MFC fuel cell device.

1: battery device

10: Anode tank

11: anode

12: The first source of nutrition

13: Bacteria

20: Cathode tank

21: Cathode

22: The second source of nutrition

Wu-G23: Algae strain

30: Proton exchange membrane

40: conductive thread

41: Load

50: light source

60: Gas device

Claims (10)

A microbial fuel cell device, which comprises: An anode tank filled with a first nutrient source containing a bacteria; A cathode tank filled with a second nutrient source containing Wu-G23 algae strain (SEQ ID No: 2); A proton exchange membrane arranged between the anode tank and the cathode tank; An anode arranged in the anode tank; and A cathode, which is arranged in the cathode tank, and the anode and the cathode are respectively connected to two ends of a conductive wire with a load; Wherein, the bacterium undergoes a respiration to produce an electron and a proton, the electron passes through the anode to the cathode tank and is electrochemically coupled with an electron acceptor, and the proton passes through the proton exchange membrane to the cathode tank to Generate current. The microbial fuel cell device described in the first item of the scope of patent application further comprises a light source for illuminating the Wu-G23 algae strain. The microbial fuel cell device described in the first item of the scope of the patent application further includes a gas device, which blows air into the cathode tank at a predetermined aeration rate, and the predetermined aeration rate ranges from 0.0 to 1.5 L/min. The microbial fuel cell device according to the first item of the patent application, wherein the first nutrient source includes sludge. The microbial fuel cell device described in item 1 of the scope of patent application, wherein the second nutrient source includes a nitrogen source, and the nitrogen source includes Peptone, ammonium sulfate ((NH4)SO ₄ ), potassium nitrate (KNO ₃ ), Yeast extract, Urea or any combination thereof. The microbial fuel cell device according to the first item of the scope of patent application, wherein the second nutrient source includes wastewater, seawater or kitchen waste. According to the microbial fuel cell device described in item 1 of the scope of patent application, the NH ₄ ⁺ -N removal rate of the microbial fuel cell device ranges from 40 to 70%. According to the microbial fuel cell device described in the first item of the scope of patent application, the COD removal rate of the microbial fuel cell device ranges from 30 to 80%. A method for immobilizing Wu-G23 algae strains used in the microbial fuel cell device described in item 1 of the scope of patent application, which comprises: Stirring and mixing a sterilized aqueous sodium alginate solution of a predetermined concentration with the Wu-G23 algae strain to form a mixed solution; and Inject the mixed solution into the forming solution through an injection needle to form an immobilized particle of Wu-G23 algae strain with a predetermined diameter; Wherein, the predetermined concentration ranges from 0.0 to 6.0 w.t%. The method described in item 9 of the scope of patent application, wherein the predetermined diameter ranges from 4.0 to 5.0 mm.

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