JP2022545580A - Enciphers and their uses in biopower - Google Patents

Abstract

Enciphers and their use in biopower generation. The Ensifer sesbaniae Y5 is stored in Guangdong Microbial Species Conservation Center (GDMCC) of China, the collection number is GDMCC No: 60957, and the collection date is January 14, 2020. Ensifer (Ensifer sesbaniae) Y5 was screened and isolated from black-smelling water body sediment, this strain has a high power generating capacity and can be tolerated in a wide range of pH (5.5-9.5), temperature (4- 45° C.) range, at the same time, it can also generate electricity using various carbon sources, and has good application prospects in the fields of environmental treatment and energy production. [Selection drawing] Fig. 2

Description

TECHNICAL FIELD The present invention relates to the technical field of environmental microorganisms and bioenergy, and specifically relates to an encipher with power generation capability derived from sediments of dark water bodies and its application in biopower generation.

Power-generating microorganisms (also called "extracellular power-generating bacteria, anode-breathing bacteria") refer to a type of microorganism with extracellular electron transfer function that can transfer electrons produced by metabolism to extracellular electron acceptors, mainly anaerobic are facultative or facultative anaerobes. Power-generating microorganisms are widely distributed in anaerobic or hypoxic environments such as soils, wastewaters, lakes, marine and river sediments.

Microbial fuel cells are bioelectrochemical reactors that have been rapidly developing in recent years. By combining conventional biodegradation and electrochemical technology, the electron transfer of microorganisms converts the chemical energy of organic matter that can be used by microorganisms into electrical energy. Convert. Microbial fuel cells have broad application prospects in areas such as pollution control, biopower generation and the like. Its working principle is that the electricity-generating microorganisms oxidize available organic matter while simultaneously producing electrons at the anode, and the electrons are transferred to the cathode electron acceptor through the corresponding electron transfer mechanism, and the cathode electron acceptor is generally oxygenated. and potassium ferricyanide.

Power-generating microorganisms are very important for the development and application of microbial fuel cells. (Geobacter) and Shewanella genera. In addition, most power-generating microorganisms have low power-generating efficiency, and the number of power-generating microorganisms that can be applied to microbial fuel cells is very limited. Therefore, there is an urgent need to select and isolate more and more efficient power-generating bacterial species.

The genus Ensifer is a Gram-negative bacterium that can be widely found in natural environments such as soil and river sediments. However, although there have been many studies on the decomposition of environmental pollutants by enciphers so far, there have been no research reports in the field of biopower generation.

SUMMARY OF THE INVENTION It is an object of the present invention to provide an encipher and its use in biopower generation in view of the current relatively scarce resource of power-generating microbial species.

The first object of the present invention is to provide Ensifer sesbaniae Y5 with bioelectric power generation function, which strain was approved by Guangdong Microbial Species Storage Center of China on January 14, 2020 (Address: China. Guangzhou, Guangdong Institute of Microbiology, China, Zip code: 510070), and the registration number of the storage center is GDMCC No: 60957.

Said Ensifer (Ensifer sesbaniae) Y5 originates from the black stink river sediments of Yonggui Wenta Park, Foshan, Guangdong, China. Microbial fuel cells are separated by enrichment and gradient dilution-azo reduction screening methods. This strain is a facultative anaerobe, rod-shaped, approximately 3 μm long, 0.8-0.9 μm wide, flagellated, and Gram-negative. It can grow within a wide range of pH (5.5-9.5) and temperature (4-45°C). A distinguishing feature of this bacterium is its electrical activity.

Enciphers are Gram-negative, facultative anaerobic bacteria. At present, the genus Ensifer has 14 systematically named fungal species, respectively Ensifer adhaerens (typical strain: LMG 20216), Ensifer alkalisoli (typical strain: YIC4027), Ensifer aridi (typical strain: YIC4027). Strain: RMT3), Ensifer collicola (typical strain: Mol 12), Ensifergaramanticus (typical strain: ORS 1400), Ensiferglycinis (typical strain: CCBAU 23380), Ensifermaghrebium (typical strain: ORS 1410), Ensifermexicanus (typical strain: ITTG-R7), Ensifer numidicus (typical strain: ORS 1407), Ensifer psoraleae (typical strain: CCBAU 65732), Ensifer sesbaniae (typical strain: CCBAU 65729), Ensifer ash (typical strain: CCBAU 251167), Ensifer sojae (typical strain: CCBAU 05684), Ensifer xericitae (typical strain: STM 354). However, to date there have been no reports related to the power generation of the genus Encipher.

A second object of the present invention is to provide the use of Ensifer sesbaniae Y5 in the preparation of biopower or microbial fuel cells.

In particular,
(1) inoculating a culture medium in the anode compartment of a microbial fuel cell with Ensifer Ensifer sesbaniae Y5, said Ensifer Ensifer sesbaniae Y5 growing in suspension in said culture medium or adhering to the surface of the anode;
(2) after inoculation, injecting sterile nitrogen to remove oxygen in the anode chamber and sealing the anode chamber for anaerobic cultivation and generating electricity from the microbial fuel cell.

Preferably, the composition of said culture medium in said anode chamber is disodium hydrogen phosphate 17.1-18 g/L, potassium dihydrogen phosphate 3-3.6 g/L, sodium chloride 0.5-0.6 g /L, ammonium chloride 1-1.2 g/L, yeast extract 0.5-0.6 g/L, sodium acetate 0.82-1 g/L or sodium formate 1.04-1.2 g/L or sodium lactate 1.12-1.5 g/L or glucose 1.8-2.0 g/L, and the solvent is water.

Preferably, in said step (1), a solid culture medium is inoculated with said encifer Ensifer sesbaniae Y5, which is left to culture in an anaerobic workstation, colonies are selected and homogenized in a sterile PBS buffer solution. and inoculating the culture medium in the anode chamber of the microbial fuel cell with the bacterial solution after mixing to homogeneity, the Encifer Ensifer sesbaniae Y5 growing in suspension in the culture medium or adhering to the surface of the anode do.

Preferably, the temperature of said anaerobic workstation is 30-37°C.

Preferably, the solid culture medium is the culture medium in the anode compartment supplemented with 0.15-0.2 mM amaranth or 0.15-0.2 mM methyl orange dye and 15-18 g/L agar. is.

The composition of the PBS buffer solution is disodium hydrogen phosphate 3.6 g/L, potassium dihydrogen phosphate 0.27 g/L, sodium chloride 8 g/L, potassium chloride 0.2 g/L, and the solvent is water. and the pH value is 7.3-7.5.

Preferably, the OD ₆₀₀ of said anode chamber culture after inoculation is between 0.05 and 0.06.

Preferably, said microbial fuel cell is a dual chamber microbial fuel cell separated by a proton exchange membrane, the cathode and anode both being graphite plates, or a single chamber microbial fuel cell, the anode being a graphite plate. and the cathode is a platinum-supported air cathode.

Compared with the prior art, the present invention has the following advantages. Ensifer (Ensifer sesbaniae) Y5 of the present invention is an electricity-generating microorganism derived from the sediment of a black-smelling water area. It can grow within a range of temperatures (4-45° C.), and in addition the bacterium can use various carbon sources to generate electricity. These characteristics indicate that Ensifer sesbaniae Y5 has strong environmental adaptability, oxidizing and decomposing organic waste under low temperature, acidic or alkaline conditions to generate electrical energy. This application can not only realize the treatment of environmental pollution, but also obtain green electric energy, and has a wide range of potential applications.

The Ensifer (Ensifer sesbaniae) Y5 of the present invention is stored in Guangdong Microbial Species Conservation Center (GDMCC), China on January 14, 2020, which is located at No. 59 Building, No. 100 Yard, Xianlie Middle Road, Guangzhou City; 5th floor, storage number is GDMCC No: 60957.

Electron microscope image of Ensifer sesbaniae Y5 colony morphology. It is a power generation verification diagram of Ensifer (Ensifer sesbaniae) Y5, where Y5 in the diagram represents Ensifer (Ensifer sesbaniae) Y5. Fig. 2 is a power generation characteristic (cyclic voltammetry chart) of Ensifer sesbaniae Y5, where Y5 in the figure represents Ensifer sesbaniae Y5; Power generation analysis of Ensifer sesbaniae Y5 using different carbon sources is shown: a: sodium formate; b: sodium acetate; c: sodium lactate; d: glucose.

The invention is further described below with reference to examples. The examples are intended to illustrate the invention by way of example and are not intended to limit the invention in any way. The process parameters specifically specified can be set with reference to the prior art.

Example 1
Screening and isolation of Ensifer sesbaniae Y5:

(1) Preparation of inoculum: River sediment was collected from the black-smelling water area of Yonggui Wenta Park, Foshan, Guangdong, China, and stored in a cold room (5-8°C). An appropriate amount of sediment sample is sieved (80 meshes) and prepared after homogenous agitation (homogeneously mixed sediment).

(2) Sedimentary microbial fuel cell device structure and current collection system: The volume of the sedimentary microbial fuel cell is 1 L, which is a single-chamber microbial fuel cell without a proton exchange membrane. Both the cathode and anode are carbon felt (5 x 5 cm) and are connected to the electrodes via titanium wires. After the anode is connected to the titanium wire, it is placed on the bottom of the battery, and 600 mL of evenly mixed sediment is added, and then 300 mL of tap water is slowly dripped as top water. Floating in the upper water after the cathode is connected to the titanium wire, the cathode and anode are connected to a 1000Ω resistor through the titanium wire. Across the resistance of the battery is connected to a Kethley 2700 multi-channel data collector to collect the power generation data (voltage) of the battery.

(3) Concentration of power-generating microorganisms in sediment microbial fuel cells: After a certain lag period, the cell voltage continues to rise with time and eventually stabilizes. When the voltage of the battery stabilizes, the battery completes concentration of power-generating microorganisms.

(4) Screening and isolation of power-generating microorganisms: Transfer the concentrated battery to an ultra-clean workbench and take out the anode carbon felt. The carbon felt was placed in sterile PBS buffer (the composition of the PBS solution was disodium hydrogen phosphate 3.6 g/L, potassium dihydrogen phosphate 0.27 g/L, sodium chloride 8 g/L, potassium chloride 0.2 g/L). , the solvent is water, the pH value is 7.3-7.5, and the preparation method is to uniformly mix each component according to the content, and adjust the pH value to 7.3-7.5 and sterilized), and the vortex vibration drives the microbes on the electrode surface into the solution. After gradient dilution with this solution as inoculum, different dilutions of the inoculum are applied to sterile solid culture medium (composition of culture medium: disodium hydrogen phosphate 17.1 g/L, potassium dihydrogen phosphate 3 g/L, sodium chloride 0.5 g/L, ammonium chloride 1 g/L, yeast extract 0.5 g/L, sodium acetate 0.82 g/L, amaranth 0.15 mM, agar 15 g/L, and the solvent is water. , and preparation: mixing each component of the culture medium, dissolving in water, dissolving with stirring, sterilizing, and cooling after cooling). The coated solid culture medium (plate) is placed in an anaerobic workstation and cultured at a culture temperature of 30-37°C. After culturing for a period of time, remove the culture medium, select a single colony on the plate culture medium and streak it into a new culture medium for purification, then place the new culture medium in an anaerobic workstation for cultivation. The temperature is 30-37°C. The streaking and purification process is repeated to perform multiple rounds of isolation and purification until all colonies are consistent in morphology and color. Finally, a pure culture was obtained, this pure culture is Ensifer sesbaniae Y5 with high electrical activity and its morphology is shown in FIG.

(5) Identification of fungal species and verification of power generation activity: As can be seen from the results of morphological analysis, this Ensifer (Ensifer sesbaniae) Y5 is rod-shaped, about 3 μm long and 0.8-0.9 μm wide, and has flagella. . This bacterium is a facultative anaerobe and Gram-negative. It can be grown using a variety of carbon sources such as sodium formate, sodium acetate, sodium lactate and glucose. It can grow within a wide range of pH (5.5-9.5) and temperature (4-45°C), reflecting the high environmental adaptability of this bacterium.

This bacterium reached 99.97% similarity with Ensifer sesbaniae CCBAU 65729, as shown by homology analysis of the 16S rRNA gene (SEQ ID NO.1). However, among all the species belonging to this genus that have been discovered so far, there have been no related reports of strains capable of generating electricity. Therefore, this bacterium was temporarily named Ensifer sesbaniae Y5.

The Ensifer sesbaniae Y5 of the present invention is stored in Guangdong Microbial Species Conservation Center (GDMCC), China on January 14, 2020, at No. 59, Yard 100, Xianlie Middle Road, Guangzhou, China. It is on the 5th floor and the storage number is GDMCC No: 60957.

A pure culture of Ensifer sesbaniae Y5 was scraped into sterile PBS buffer with a sterile spatula and mixed thoroughly and evenly to obtain a bacterial solution, which was applied to the anode compartment of a dual-chamber microbial fuel cell. Inoculated into the LM culture medium (anolyte), a power generation test was performed, the OD ₆₀₀ of the culture solution in the anode chamber after inoculation was 0.05-0.06, and Ensifer (Ensifer sesbaniae) Y5 was suspended in the culture medium. It grows in a state or adheres to the surface of the anode, and after injecting sterile nitrogen to remove oxygen in the anode chamber, the anode chamber is sealed and anaerobic culture is performed. Test Method: For a dual-chamber microbial fuel cell, both the cathode and anode of the cell are high purity graphite electrodes of the same size (2 cm x 3.3 cm x 0.2 cm). The electrolyte in the cathode compartment is 50 mM potassium ferricyanide solution and the solvent is PBS buffer. Anolyte is LM culture medium (composition of culture medium: disodium hydrogen phosphate 18 g/L, potassium dihydrogen phosphate 3.6 g/L, sodium chloride 0.6 g/L, ammonium chloride 1.2 g/L, Yeast extract 0.6 g/L, sodium acetate 1 g/L, solvent is water.Preparation: Mix each component of culture medium and dissolve in water, dissolve with stirring, sterilize, cool. ). As can be seen from the test results, this strain can stably generate electricity in a microbial fuel cell for a long period of time, as shown in FIG.

Example 2
This example discloses the extracellular electron transfer characteristics of Ensifer sesbaniae Y5. As shown in Example 1, the anode compartment of a dual-chamber microbial fuel cell is inoculated with Ensifer sesbaniae Y5 and the cell is operated until the voltage reaches a peak value and remains stable. Cyclic voltammetry scan analysis was then performed on the anode compartment of the cell, the potentiostat was CHI 700E, Ag/AgCl electrode was used as the reference electrode, and the cathode was the counter electrode. The scan voltage was −0.6 to −0.2 V, the scan speed was 10 mV/s and the results are shown in FIG.

According to existing literature reports, the electron transport of power-generating microorganisms mainly consists of the extracellular transport pathway of outer membrane cytochromes, the extracellular electron transport pathway of nanowires on the extracellular membrane, and the electron transport secreting soluble redox electron mediators. There are three methods of routing. As can be seen from Figure 3, the cyclic voltammetry curve has a small redox peak, indicating that Ensifer sesbaniae Y5 may produce and secrete soluble redox mediators during power generation. . Therefore, Ensifer sesbaniae Y5 is an electron transfer potential that transfers electrons through its own secretion of soluble redox electron mediators.

Example 3
This example discloses the power generation characteristics of Ensifer sesbaniae Y5 using various carbon sources. The test method follows step 5 of Example 1 above. It can be seen from the experimental results that Ensifer sesbaniae Y5 can generate electricity using carbon sources such as sodium formate, sodium acetate, sodium lactate and glucose, but the electricity generation effect is not the same. Among them, the power generation effect is most favorable in the case of power generation by sodium acetate (Fig. 4). In this example, we intend to disclose the power generation characteristics in a dual-chamber microbial fuel cell of Ensifer sesbaniae Y5, and that this bacterium is capable of generating power using a variety of carbon sources. The dual-chamber microbial fuel cell and process used are not optimal, and currently many literatures show that the power generating capacity of power generating microorganisms can be greatly improved by methods such as electrode improvement, reactor and reaction condition optimization. have been reported.

Although the preferred embodiments of the present invention have been described above, the above preferred embodiments should not limit the present invention, and the protection scope of the present invention should be subject to the claims. It is obvious for those skilled in the art that any modifications and improvements made without departing from the spirit and scope of the present invention are all included in the protection scope of the present invention.

(Appendix)
(Appendix 1)
Encifer Ensifer sesbaniae Y5 with holding number GDMCC No: 60957.

(Appendix 2)
Use of the encifer Ensifer sesbaniae Y5 according to Supplementary Note 1 in the preparation of a biopower or microbial fuel cell.

(Appendix 3)
(1) inoculating the culture medium of the anode compartment of the microbial fuel cell with the Ensifer Ensifer sesbaniae Y5, wherein the Encifer Ensifer Ensifer sesbaniae Y5 grows in suspension in the culture medium or adheres to the surface of the anode; ,
after inoculation, injecting sterile nitrogen to remove oxygen in the anode chamber and sealing the anode chamber for anaerobic culture and generating electricity from the microbial fuel cell (2); The use according to appendix 2, characterized in that

(Appendix 4)
The composition of the culture medium in the anode chamber is disodium hydrogen phosphate 17.1-18 g/L, potassium dihydrogen phosphate 3-3.6 g/L, sodium chloride 0.5-0.6 g/L, Ammonium chloride 1-1.2 g/L, yeast extract 0.5-0.6 g/L, sodium acetate 0.82-1 g/L or sodium formate 1.04-1.2 g/L or sodium lactate 1.12 1.5 g/L or 1.8-2.0 g/L glucose and the solvent is water.

(Appendix 5)
In the step (1), a solid culture medium is inoculated with the Ensifer Ensifer sesbaniae Y5, which is left to culture in an anaerobic workstation, colonies are selected and mixed evenly in a sterile PBS buffer solution. and inoculating the culture medium in the anode compartment of the microbial fuel cell with the bacterial solution after being evenly mixed, the Encifer Ensifer sesbaniae Y5 growing in suspension in the culture medium or adhering to the surface of the anode. The use according to appendix 3, characterized in that

(Appendix 6)
Use according to clause 5, characterized in that the temperature of the anaerobic workstation is between 30 and 37°C.

(Appendix 7)
The solid culture medium is the culture medium in the anode chamber of Appendix 4 supplemented with 0.15-0.2 mM amaranth or 0.15-0.2 mM methyl orange dye and 15-18 g/L agar. The use according to appendix 5, characterized in that it is a

(Appendix 8)
The composition of the PBS buffer solution is disodium hydrogen phosphate 3.6 g/L, potassium dihydrogen phosphate 0.27 g/L, sodium chloride 8 g/L, potassium chloride 0.2 g/L, and the solvent is water. and the pH value is 7.3 to 7.5.

(Appendix 9)
Use according to claim 3, characterized in that the OD ₆₀₀ of the culture in the anode chamber after inoculation is 0.05-0.06.

(Appendix 10)
said microbial fuel cell is a dual-chamber microbial fuel cell separated by a proton exchange membrane, the cathode and anode are both graphite plates, or a single-chamber microbial fuel cell, the anode is a graphite plate, 3. Use according to claim 3, characterized in that the cathode is a platinum-supported air cathode.

Claims (10)

Encifer Ensifer sesbaniae Y5 with holding number GDMCC No: 60957. Use of the encifer Ensifer sesbaniae Y5 according to claim 1 in the preparation of biopower or microbial fuel cells. (1) inoculating the culture medium of the anode compartment of the microbial fuel cell with the Ensifer Ensifer sesbaniae Y5, wherein the Encifer Ensifer Ensifer sesbaniae Y5 grows in suspension in the culture medium or adheres to the surface of the anode; ,
after inoculation, injecting sterile nitrogen to remove oxygen in the anode chamber and sealing the anode chamber for anaerobic culture and generating electricity from the microbial fuel cell (2); 3. Use according to claim 2, characterized in that The composition of the culture medium in the anode chamber is disodium hydrogen phosphate 17.1-18 g/L, potassium dihydrogen phosphate 3-3.6 g/L, sodium chloride 0.5-0.6 g/L, Ammonium chloride 1-1.2 g/L, yeast extract 0.5-0.6 g/L, sodium acetate 0.82-1 g/L or sodium formate 1.04-1.2 g/L or sodium lactate 1.12 1.5 g/L or 1.8-2.0 g/L glucose and the solvent is water. In the step (1), a solid culture medium is inoculated with the Ensifer Ensifer sesbaniae Y5, which is left to culture in an anaerobic workstation, colonies are selected and mixed evenly in a sterile PBS buffer solution. and inoculating the culture medium in the anode compartment of the microbial fuel cell with the bacterial solution after being evenly mixed, the Encifer Ensifer sesbaniae Y5 growing in suspension in the culture medium or adhering to the surface of the anode. 4. Use according to claim 3, characterized in that Use according to claim 5, characterized in that the temperature of the anaerobic workstation is 30-37°C. The solid culture medium comprises 0.15-0.2 mM amaranth or 0.15-0.2 mM methyl orange dye and 15-18 g/L agar in the culture medium in the anode chamber of claim 4. 6. Use according to claim 5, characterized in that it is added. The composition of the PBS buffer solution is disodium hydrogen phosphate 3.6 g/L, potassium dihydrogen phosphate 0.27 g/L, sodium chloride 8 g/L, potassium chloride 0.2 g/L, and the solvent is water. The use according to claim 5, characterized in that the pH value is between 7.3 and 7.5. The use according to claim 3, characterized in that the OD ₆₀₀ of the culture in the anode chamber after inoculation is 0.05-0.06. said microbial fuel cell is a dual-chamber microbial fuel cell separated by a proton exchange membrane, the cathode and anode are both graphite plates, or a single-chamber microbial fuel cell, the anode is a graphite plate, 4. Use according to claim 3, characterized in that the cathode is a platinum-supported air cathode.

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