JP7359309B2 - Encipher and its applications in biopower generation - Google Patents

Description

GDMCC GDMCC 60957

TECHNICAL FIELD The present invention relates to the technical field of environmental microorganisms and bioenergy, and specifically relates to an encipher that has the ability to generate electricity derived from the sediment of a black-smelling water body and its use in biopower generation.

Power-generating microorganisms (also called "extracellular power-generating bacteria, anodic respiration bacteria") are a type of microorganisms that have an extracellular electron transfer function that can transfer electrons generated through metabolism to extracellular electron acceptors. It is a sexual microorganism or a facultative anaerobic microorganism. Power-producing microorganisms are widely distributed in anaerobic or hypoxic environments such as soil, wastewater, lake, ocean and river sediments.

A microbial fuel cell is a bioelectrochemical reaction device that has been rapidly developing in recent years.It combines conventional biodegradation and electrochemical technology, and converts the chemical energy of organic matter that can be used by microorganisms into electrical energy through electron transfer by microorganisms. Convert. Microbial fuel cells have broad application prospects in areas such as pollution control, biopower generation, and more. Its working principle is that power-generating microorganisms oxidize available organic matter and at the same time generate electrons at the anode, and the electrons are transferred to the electron acceptors at the cathode through the corresponding electron transfer mechanism, and the electron acceptors at the cathode are generally oxygen and potassium ferricyanide.

Power-generating microorganisms are very important for the development and application of microbial fuel cells, but the types and numbers of power-generating microorganisms that have been reported in previous studies are still very limited, and at present, related research mainly focuses on Geobacter. They are concentrated in the genus Geobacter and Shewanella. In addition, the power generation efficiency of most power-generating microorganisms is low, and the number of power-generating microorganisms that can be applied to microbial fuel cells is very limited, and the development and utilization of power-generating microorganism resources has only just begun. 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 Encipher, there have been no research reports in the field of biopower generation.

It is an object of the present invention to provide an encipher and its use in biopower generation, taking into account the current situation where resources of power-generating microbial strains are relatively scarce.

The first objective of the present invention is to provide Ensifer sesbaniae Y5 with biopower generation function, and this strain was acquired on January 14, 2020 by the Microbial Species Storage Center of Guangdong Province, China (Address: China. Guangzhou, China (Guangdong Provincial Institute of Microbiology, Postal code: 510070), and the storage center registration number is GDMCC No: 60957.

The Ensifer sesbaniae Y5 originates from the deposits of the Black Smell River in Ronggui Wenta Park, Foshan, Guangdong, China. Microbial fuel cell enrichment and gradient dilution-separated by azo reduction screening method. This strain is a facultative anaerobe, rod-shaped, approximately 3 μm long, 0.8 to 0.9 μm wide, has flagella, and is Gram-negative. It can grow within a wide range of pH (5.5-9.5) and temperature (4-45°C). A distinctive feature of this bacterium is that it has power generating activity.

Encypha is a Gram-negative, facultative anaerobic bacterium. Currently, the Ensifer genus consists of 14 systematically named bacterial species: Ensifer adhaerens (typical strain: LMG 20216), Ensifer alkalisoli (typical strain: YIC4027), and Ensifer aridi (typical strain: LMG 20216). Strain: RMT3), Ensifer collicola (typical strain: Mol 12), Ensifergaramanticus (typical strain: ORS 1400), Ensiferglycinis (typical strain: CCBAU 23380), Ensifermaghr ebium (typical strain: ORS 1410), Ensifermexicanus (typical strain: ITTG to R7), Ensifer numidicus (typical strain: ORS 1407), Ensifer psoraleae (typical strain: CCBAU 65732), Ensifer sesbaniae (typical strain: CCBAU 65732) Strain: CCBAU 65729), Ensifer shofinae (Typical strain: CCBAU 251167), Ensifer sojae (Typical strain: CCBAU 05684), and Ensifer xericitae (Typical strain: STM 354). However, to date, there have been no reports related to power generation in 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 Ensifer sesbaniae Y5 into a culture medium in an anode chamber of a microbial fuel cell, and causing the Ensifer sesbaniae Y5 to grow in suspension in the culture medium or adhere to the surface of the anode;
After inoculation, the step (2) includes injecting sterile nitrogen to remove oxygen in the anode chamber, sealing the anode chamber, performing anaerobic culture, and generating electricity from the microbial fuel cell.

Preferably, the composition of the culture medium in the anode chamber is 17.1-18 g/L of disodium hydrogen phosphate, 3-3.6 g/L of potassium dihydrogen phosphate, and 0.5-0.6 g of sodium chloride. /L, ammonium chloride 1-1.2g/L, yeast extract 0.5-0.6g/L, sodium acetate 0.82-1g/L or sodium formate 1.04-1.2g/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 Ensifer sesbaniae Y5, it is left to culture on an anaerobic workstation, and colonies are selected and uniformly distributed in a sterile PBS buffer solution. and after uniform mixing, the bacterial solution is inoculated into the culture medium in the anode chamber of the microbial fuel cell, and the Ensifer sesbaniae Y5 grows in suspension in the culture medium or adheres to the surface of the anode. do.

Preferably, the temperature of the anaerobic workstation is between 30 and 37°C.

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

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

Preferably, the OD ₆₀₀ of the culture solution in the anode chamber after inoculation is 0.05 to 0.06.

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

Compared with the prior art, the present invention has the following advantages: Ensifer sesbaniae Y5 of the present invention is a power-generating microorganism derived from the sediments of black-smelling water bodies. Able to grow within a range of temperatures (4-45° C.), this bacterium can also generate electricity using a variety of carbon sources. These characteristics indicate that Ensifer sesbaniae Y5 has strong environmental adaptation ability and can oxidize and decompose organic wastes to generate electrical energy under low temperature, acidic or alkaline conditions. This application not only realizes the treatment of environmental pollution, but also can obtain green electrical energy, and is expected to have a wide range of applications.

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

Electron microscopy image of colony morphology of Ensifer sesbaniae Y5. It is a power generation verification diagram of Ensifer (Ensifer sesbaniae) Y5, and Y5 in the figure represents Ensifer (Ensifer sesbaniae) Y5. This is a power generation characteristic (cyclic voltammetry chart) of Ensifer (Ensifer sesbaniae) Y5, and Y5 in the figure represents Ensifer (Ensifer sesbaniae) Y5. Figure 2 shows power generation analysis of Ensifer sesbaniae Y5 using various carbon sources: a: sodium formate; b: sodium acetate; c: sodium lactate; d: glucose.

The present invention will be 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 it in any way. Especially specified process parameters can be set with reference to conventional techniques.

Example 1
Screening and isolation of Ensifer sesbaniae Y5:

(1) Preparation of inoculum: River sediment was collected from the black-smelling water area of Foshan Ronggui Wenta Park, Guangdong, China, and stored in a cold room (5-8°C). An appropriate amount of sediment sample is subjected to sieving (80 meshes), stirred uniformly, and then prepared (uniformly mixed sediment).

(2) Structure and current collection system of the sediment microbial fuel cell device: The volume of the sediment microbial fuel cell is 1L, and it is a single chamber microbial fuel cell without proton exchange membrane. The cathode and anode are both carbon felt (5 x 5 cm) and connected to the electrodes via titanium wire. After the anode is connected to the titanium wire, placed at the bottom of the cell, add 600 mL of uniformly mixed deposit, then slowly drip 300 mL of tap water as top water. After the cathode is connected to the titanium wire, it floats on the upper water, and the cathode and anode are connected to a 1000Ω resistor through the titanium wire. The resistive ends of the battery are connected to a Kethley 2700 multi-channel data collector to collect battery power generation data (voltage).

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

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

(5) Identification of bacterial species and verification of power generation activity: As seen from the results of morphological analysis, Ensifer sesbaniae Y5 is rod-shaped, approximately 3 μm long, 0.8 to 0.9 μm wide, and has flagella. . This bacterium is a facultative anaerobe and Gram-negative. A variety of carbon sources can be used for growth, 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 the homology analysis of the 16S rRNA gene (SEQ ID NO. 1). However, among all the bacterial species belonging to this genus that have been discovered so far, there have been no reports of any strains capable of generating electricity. Therefore, this bacterium was tentatively named Ensifer sesbaniae Y5.

The Ensifer sesbaniae Y5 of the present invention was stored at the Guangdong Provincial Microbial Species Conservation Center (GDMCC) on January 14, 2020, and the address is Building 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, mixed thoroughly and homogeneously to obtain a bacterial solution, and the bacterial solution was added to the anode chamber of a dual chamber microbial fuel cell. The LM culture medium (anolyte) was inoculated and a power generation test was performed, and the OD ₆₀₀ of the culture solution in the anode chamber after inoculation was 0.05 to 0.06, and Ensifer sesbaniae Y5 was suspended in the culture medium. The cells grow in the state or adhere to the surface of the anode, and after removing oxygen in the anode chamber by injecting sterile nitrogen, the anode chamber is sealed to perform anaerobic culture. Test method: For the dual chamber microbial fuel cell, the cathode and anode of the cell are both high purity graphite electrodes with the same size (2 cm x 3.3 cm x 0.2 cm). The electrolyte in the cathode chamber is a 50 mM potassium ferricyanide solution, and the solvent is PBS buffer. The 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 0.6g/L, sodium acetate 1g/L, and the solvent is water.Preparation: Each component of the culture medium is mixed and dissolved in water, dissolved with stirring, sterilized, and cooled. ). As can be seen from the test results, as shown in Figure 2, this strain can stably generate electricity for a long period of time using microbial fuel cells.

Example 2
This example discloses the extracellular electron transport characteristics of Ensifer sesbaniae Y5. As shown in Example 1, the anode chamber 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. A cyclic voltammetry scan analysis is then performed on the anode chamber of the cell, using a CHI 700E potentiostat, an Ag/AgCl electrode as a reference electrode, and a cathode as a 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. 3.

According to existing literature reports, electron transport in power-generating microorganisms is mainly carried out by 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 called routes. As can be seen from Figure 3, there is a small redox peak in the cyclic voltammetry curve, indicating that Ensifer sesbaniae Y5 may produce and secrete soluble redox mediators during power generation. . Therefore, Ensifer sesbaniae Y5 has the potential for electron transfer by self-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. As can be seen from the experimental results, 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 the most favorable in the case of power generation using sodium acetate (Figure 4). This example discloses the power generation characteristics in a dual chamber microbial fuel cell of Ensifer sesbaniae Y5 and is intended to disclose that this bacterium is capable of generating power using a variety of carbon sources. The dual-chamber microbial fuel cells and processes used are not optimal, and many publications currently suggest that methods such as electrode improvements, optimization of reactors and reaction conditions can significantly improve the power generation capacity of power-generating microorganisms. has been reported.

Although the preferred embodiments of the present invention have been described above, the preferred embodiments described above do not limit the present invention, and the protection scope of the present invention should be in accordance with the claims. It is clear to those skilled in the art that some changes and improvements made without departing from the spirit and scope of the invention will all fall within the protection scope of the invention.

(Additional note)
(Additional note 1)
Ensifer sesbaniae Y5 whose storage number is GDMCC No: 60957.

(Additional note 2)
Use of Ensifer sesbaniae Y5 according to appendix 1 in the preparation of biopower or microbial fuel cells.

(Additional note 3)
(1) inoculating the Encipher sesbaniae Y5 into a culture medium in the anode chamber of the microbial fuel cell, and the Encipher sesbaniae Y5 grows in suspension in the culture medium or adheres to the surface of the anode; ,
After inoculation, the step (2) includes injecting sterile nitrogen to remove oxygen in the anode chamber, sealing the anode chamber, performing anaerobic culture, and generating electricity from the microbial fuel cell. The use described in Appendix 2, characterized by:

(Additional note 4)
The composition of the culture medium in the anode chamber is 17.1 to 18 g/L of disodium hydrogen phosphate, 3 to 3.6 g/L of potassium dihydrogen phosphate, 0.5 to 0.6 g/L of sodium chloride, 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 to 2.0 g/L, and the solvent is water.

(Appendix 5)
In step (1), a solid culture medium is inoculated with the Ensifer sesbaniae Y5, which is left to culture on an anaerobic workstation, and colonies are selected and uniformly mixed in a sterile PBS buffer solution. , after uniformly mixing, the bacterial solution is inoculated into the culture medium in the anode chamber of the microbial fuel cell, and the Ensifer sesbaniae Y5 grows in suspension in the culture medium or adheres to the surface of the anode. The use according to appendix 3, which is characterized by:

(Appendix 6)
The use according to appendix 5, characterized in that the temperature of the anaerobic workstation is 30 to 37°C.

(Appendix 7)
The solid culture medium is prepared by adding 0.15 to 0.2 mM of amaranth or 0.15 to 0.2 mM of methyl orange dye, and 15 to 18 g/L of agar to the culture medium in the anode chamber described in Appendix 4. The use according to appendix 5, which is characterized in that:

(Appendix 8)
The composition of the PBS buffer solution is 3.6 g/L of disodium hydrogen phosphate, 0.27 g/L of potassium dihydrogen phosphate, 8 g/L of sodium chloride, and 0.2 g/L of potassium chloride, and the solvent is water. The use according to appendix 5, characterized in that the pH value is 7.3 to 7.5.

(Appendix 9)
The use according to appendix 3, wherein the culture solution in the anode chamber after inoculation has an OD ₆₀₀ of 0.05 to 0.06.

(Appendix 10)
The microbial fuel cell is a dual-chamber microbial fuel cell separated by a proton exchange membrane, and the cathode and anode are both graphite plates, or a single-chamber microbial fuel cell, and the anode is a graphite plate; The use according to appendix 3, wherein the cathode is a platinum-supported air cathode.

Claims (10)

Ensifer sesbaniae Y5 whose storage number is GDMCC No: 60957. Use of Ensifer sesbaniae Y5 according to claim 1 in the preparation of biopower or microbial fuel cells. (1) inoculating the Encipher sesbaniae Y5 into the culture medium of the negative electrode chamber of the microbial fuel cell, and the Encipher sesbaniae Y5 proliferates in suspension in the culture medium or adheres to the surface of the negative electrode; ,
After the inoculation, sterile nitrogen is injected to remove oxygen in the negative electrode chamber, the negative electrode chamber is sealed, anaerobic culture is performed, and electricity is generated from the microbial fuel cell (2). Use according to claim 2, characterized in that: The composition of the culture medium in the negative electrode chamber is 17.1 to 18 g/L of disodium hydrogen phosphate, 3 to 3.6 g/L of potassium dihydrogen phosphate, 0.5 to 0.6 g/L of sodium chloride, 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 Use according to claim 3, characterized in that the concentration is ~1.5 g/L or glucose 1.8-2.0 g/L and the solvent is water. In step (1), a solid culture medium is inoculated with the Ensifer sesbaniae Y5, which is left to culture on an anaerobic workstation, and colonies are selected and uniformly mixed in a sterile PBS buffer solution. After uniformly mixing, the bacterial solution is inoculated into the culture medium in the negative electrode chamber of the microbial fuel cell, and the Ensifer sesbaniae Y5 grows in suspension in the culture medium or adheres to the surface of the negative electrode. 4. Use according to claim 3, characterized in that: Use according to claim 5, characterized in that the temperature of the anaerobic workstation is between 30 and 37°C. The solid culture medium includes 0.15 to 0.2 mM of amaranth or 0.15 to 0.2 mM of methyl orange dye, and 15 to 18 g/L of agar to the culture medium in the negative electrode chamber according to claim 4. The use according to claim 5, characterized in that it is an additive. The composition of the PBS buffer solution is 3.6 g/L of disodium hydrogen phosphate, 0.27 g/L of potassium dihydrogen phosphate, 8 g/L of sodium chloride, and 0.2 g/L of potassium chloride, and the solvent is water. Use according to claim 5, characterized in that the pH value is between 7.3 and 7.5. The use according to claim 3, wherein the OD600 of the culture solution in the negative electrode chamber after inoculation is 0.05 to 0.06. The microbial fuel cell is a dual-chamber microbial fuel cell separated by a proton exchange membrane, and the positive and negative electrodes are both graphite plates, or a single-chamber microbial fuel cell, and the negative electrode is a graphite plate; Use according to claim 3, characterized in that the positive electrode is a platinum-supported air positive electrode.

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