CN110511882B - Salt-tolerant bacterial strain with electrogenesis characteristic and application thereof in microbial fuel cell - Google Patents

Abstract

The invention relates to a salt-tolerant bacterial strain with electrogenesis property, which is named as E-1 and classified as: shewanella alga Shewanella algae with a collection number of: CGMCC No.17857, preservation date: 27 days 5 months in 2019, west road No.1 hospital No. 3, tokyo, yoyo, north, and depository: china general microbiological culture Collection center. The strain is a salt-tolerant electrogenesis microorganism. The method is the first report that the internal microorganism has the electrogenesis characteristic, enriches the diversity of the salt-tolerant electrogenesis microorganism, and provides a new experimental material for the application of the microbial fuel cell in the aspect of seawater resource treatment.

Description

Salt-tolerant bacterial strain with electrogenesis characteristic and application thereof in microbial fuel cell

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a salt-tolerant Shewanella algae strain with electrogenesis characteristic and application thereof in a microbial fuel cell.

Background

Microbial Fuel Cells (MFCs) are a novel device for directly converting chemical energy into electrical energy by oxidizing organic substances (including pollutants in wastewater) through the catalytic action of microorganisms. Because of the dual functions of wastewater treatment and electric energy generation, the method has great application prospect in the fields of clean energy production, wastewater resource treatment, biosensor development, environmental bioremediation and the like.

The electricity-generating microorganism is the core component of a microbial fuel cell system, and the electrochemical activity of the electricity-generating microorganism shows obvious difference due to different mechanisms of generating electrons and transferring electrons by strains. At present, most of the electricity-generating microorganisms are from activated sludge, sediments, soil, biological garbage and the like of sewage treatment plants. The electrogenic microorganisms found are mainly concentrated in Proteobacteria (Proteobacteria) and Firmicutes (Firmicutes). Proteobacteria mainly include Pseudomonas (Pseudomonas), Shewanella (Shewanella), Klebsiella (Klebsiella), Geobactrum (Geobactor), Ochrobactrum (Ochrobactrum), Rhodococcus (Rhodoferax), Acidophilum (Acidiphilium), Aeromonas (Aeromonas), Citrobacter (Citrobacter), Arcobacter (Arcobacter), Desulfovibrio (Desulfovibrio), and the like, and Thelephora mainly includes Bacillus (Bacillus) and Clostridium (Clostridium), and the like. Although researchers have begun in recent years to isolate and screen marine-derived electricity-producing microorganisms from offshore sludge samples and intertidal surface sediments in the sea, these strains lack further analysis of their electricity-producing characteristics under salt conditions.

Shewanella alga was first isolated from the surface of red algae in 1985 by Yuichi Kotati et al, and was then named OK-1. In 1990, Simidu analyzed the G + C content of OK-1 and the base sequence characteristics of 16S rRNA, and found that OK-1 has a close relationship with Shewanella, but OK-1 was defined as a new Shewanella alga, namely Shewanella alga, because OK-1 and Shewanella putreferae (Shewanella putrefacesiens) have a significant difference in base sequence. Currently, Shewanella alga is mostly applied to the aspects of fermentation production of tetrodotoxin and inhibition of corrosion performance on metal materials. The salt resistance and the electricity generation characteristics of the endophyte and the application of the endophyte in microbial fuel cells are not found in relevant reports at home and abroad.

Through searching, no patent publication related to the present patent application has been found.

Disclosure of Invention

The invention aims to overcome the defect that few salt-tolerant electrogenesis microorganisms are reported in the prior art, and provides a salt-tolerant Shewanella algae strain with electrogenesis characteristics and application thereof in a microbial fuel cell. The method is the first report that the internal microorganism has the electrogenesis characteristic, enriches the diversity of the salt-tolerant electrogenesis microorganism, and provides a new experimental material for the application of the microbial fuel cell in the aspect of seawater resource treatment.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a Shewanella algae strain with salt tolerance and electrogenic property, which is named as E-1 and classified as: shewanella alga Shewanella algae with a collection number of: CGMCC No.17857, preservation date: 27 days 5 months in 2019, west road No.1 hospital No. 3, tokyo, yoyo, north, and depository: china general microbiological culture Collection center.

Moreover, the 16S rDNA gene of the strain has a nucleotide sequence shown as a sequence SEQ ID No.1, the sequence length is 1450bp, and the accession number on Genbank is MK 787267.1.

Furthermore, the strain is selected from seawater of the south sea; the seawater desalination device is obtained by carrying out enrichment, separation, purification and screening on the seawater in the south China sea after simple precipitation treatment.

Moreover, the strain is facultative anaerobe, short rod-shaped, 2.5um long, 0.5um wide and gram negative; the pH range suitable for growth is 5-10, the temperature range is 25-40 ℃, and the growth can be carried out in the range of NaCl mass concentration of 0-7%.

Moreover, the strain has growth properties under salt stress; the strain has electrogenesis performance.

Moreover, the strain has electrogenesis performance under the condition of containing salt.

The strain can utilize lactic acid, acetic acid, succinamic acid, L-alanine, uridine, and cannot utilize L-arabinose, maltose, sucrose, or glycerol.

The Shewanella algae strain with salt tolerance and electrogenesis property is applied to the electrogenesis of the microbial fuel cell.

The application of the Shewanella algae strain with salt resistance and electrogenesis property in the aspect of manufacturing the microbial fuel cell.

Moreover, the application method comprises the following steps:

firstly, activating a strain: selecting a ring of Shewanella algae strains from an LB slant culture medium, inoculating the Shewanella algae strains into 50mL of LB liquid culture medium, carrying out shake culture at 30-37 ℃ and 200r/min at 160-48 h, taking 6000 Xg of bacterial liquid, centrifuging for 10min, collecting the bacterial cells, and washing the bacterial cells for 3 times by using physiological saline with the mass concentration of 0.9%;

preparing an inoculation liquid: resuspending the cells in anolyte containing different NaCl concentrations, and using 0.8-2.0g/L glucose as substrate to make initial OD₆₀₀0.8-1.5, and then inoculating into a reactor for MFCs;

and thirdly, detecting the electricity generation performance: the reactor is connected with an external resistor and a data acquisition device respectively, and runs at the constant temperature of 37 +/-1 ℃, and inoculation liquid is replaced periodically in the running process; when two continuous stable voltages appear, the starting is considered to be successful; when the monitored output voltage is below 50mV, the anolyte change is started until a stable and repeatable output voltage is obtained.

The invention has the advantages and positive effects that:

1. the temperature range of the strain suitable for growth is 25-40 ℃, the pH range is 5-10, the strain can grow in the NaCl concentration range of 0-7%, and the salt tolerance is good. The strain is inoculated in a microbial fuel cell which is not added with NaCl and is added with NaCl to generate electric energy, which shows that the strain is a salt-tolerant electrogenesis microorganism. The method is the first report that the internal microorganism has the electrogenesis characteristic, enriches the diversity of the salt-tolerant electrogenesis microorganism, and provides a new experimental material for the application of the microbial fuel cell in the aspect of seawater resource treatment.

2. The salt-tolerant electrogenesis bacteria from the sea are respectively inoculated into microbial fuel cells which are not added with NaCl and are added with 6.6 percent of NaCl to show electrogenesis capability, and the power density respectively reaches 51.69 mW.m^-2And 26.56 mW.m^-2. The temperature range suitable for growth of the strain is 25-40 ℃, the pH range is 5-10, and the strain shows various substrate utilization capacities, which are reports of the electricity generation performance of microorganisms in S.algae species and the application of the microorganisms in microbial fuel cells for the first timeAnd provides a new experimental material for the application of the microbial fuel cell in the aspect of seawater recycling treatment.

Drawings

FIG. 1 is a graph showing the voltage change curve (a), polarization curve and power density curve (b) of the strain Shewanella algae E-1 of the present invention when MFCs were operated without exogenous salt-adding pressure;

FIG. 2 is a graph showing the growth curves of the strain Shewanella algae E-1 of the present invention at different salt concentrations;

FIG. 3 is a graph showing the voltage change curve (a), polarization curve and power density curve (b) of the strain Shewanella algae E-1 of the present invention when MFCs were operated under the condition of exogenous salt addition pressure of 6.6%;

FIG. 4 is a colony morphology map of Shewanella algae E-1 of the strain of the present invention (a, b); gram stain results (c); scanning electron microscope image (d) of thallus;

FIG. 5 is a phylogenetic tree constructed by homology alignment of the Shewanella algae E-1 strain of the present invention with 16S rDNA sequences of closely related strains recorded in GenBank.

Detailed Description

The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.

The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.

A Shewanella algae strain with salt tolerance and electrogenic property, which is named as E-1 and classified as: shewanella alga Shewanella algae with a collection number of: CGMCC No.17857, preservation date: 27 days 5 months in 2019, west road No.1 hospital No. 3, tokyo, yoyo, north, and depository: china general microbiological culture Collection center.

Preferably, the 16S rDNA gene of the strain has a nucleotide sequence shown as a sequence SEQ ID No.1, the sequence length is 1450bp, and the accession number on Genbank is MK 787267.1.

Preferably, the strain is selected from seawater of the south sea; the seawater desalination device is obtained by carrying out enrichment, separation, purification and screening on the seawater in the south China sea after simple precipitation treatment.

Preferably, the strain is facultative anaerobe, short rod-shaped, 2.5um long, 0.5um wide, gram negative; the pH range suitable for growth is 5-10, the temperature range is 25-40 ℃, and the growth can be carried out in the range of NaCl mass concentration of 0-7%. The strain can be used as an anode catalyst of a microbial fuel cell to catalyze a substrate to generate electricity, thereby achieving the aim of the invention.

Preferably, the strain has good growth performance under salt stress; the strain has good electricity generating performance.

Preferably, the strain has good electrogenesis performance under the condition of containing salt.

Preferably, the strain is capable of utilizing lactic acid, acetic acid, succinamic acid, L-alanine, uridine, and not L-arabinose, maltose, sucrose, glycerol.

The Shewanella algae strain which is salt-tolerant and has electrogenic properties as described above can be applied in the aspect of electricity generation of microbial fuel cells.

The Shewanella algae strain which is salt-tolerant and has electrogenic properties as described above can be applied in the fabrication of microbial fuel cells.

Preferably, the application method comprises the following steps:

firstly, activating a strain: selecting a ring of Shewanella algae strains from an LB slant culture medium, inoculating the Shewanella algae strains into an LB liquid culture medium filled with 50mL, carrying out shake culture at 30-37 ℃ and 200r/min at 160-48 h, taking 6000 Xg of bacterial liquid, centrifuging for 10min, collecting the bacterial cells, and washing the bacterial cells for 3 times by using physiological saline with the mass concentration of 0.9%;

preparing an inoculation liquid: resuspending the cells in anolyte containing different NaCl concentrations, and using 0.8-2.0g/L glucose as substrate to make initial OD₆₀₀0.8-1.5, and then inoculating into a reactor for MFCs;

and thirdly, detecting the electricity generation performance: the reactor is connected with an external resistor and a data acquisition device respectively, and runs at the constant temperature of 37 +/-1 ℃, and inoculation liquid is replaced periodically in the running process; when two continuous stable voltages appear, the starting is considered to be successful; when the monitored output voltage is below 50mV, the anolyte change is started until a stable and repeatable output voltage is obtained.

The Shewanella alga (Shewanella algae) E-1 has 100% homology with the known strain Shewanella algae Hiro-1 by 16SrDNA sequence alignment analysis. The Genbank accession number of the 16S rRNA of the strain is MK 787267.1.

Shewanella alga (Shewanella algae) E-1 is a gram-negative, facultative anaerobic bacterium. To date, there has been no report of Shewanella alga capable of generating electricity. The Shewanella alga (Shewanella algae) E-1 can generate electricity under the concentration of 0-7% NaCl.

More specifically, the application of Shewanella alga (Shewanella algae) E-1 in the preparation of the microbial fuel cell specifically comprises the following steps:

1) activating strains: a ring of bacteria is selected from an LB slant culture medium and inoculated into a 250mL triangular flask filled with 50mL of LB liquid culture medium, the shaking culture is carried out for 20-48h at 30-37 ℃ and at 160-200r/min, the bacteria is collected after 6000 Xg of bacteria liquid is centrifuged for 10min and washed for 3 times by 0.9 percent physiological saline.

Preparing an inoculation solution: the cells were resuspended in anolyte containing different NaCl concentrations (0.8-2.0 g/L glucose as substrate) to give initial OD₆₀₀0.8-1.5, and then inoculated into a reactor for MFCs.

And (3) detecting the electricity generation performance: the reactor is connected with an external resistor and a data acquisition device respectively, and runs at the constant temperature of (37 +/-1) DEG C, and the inoculation liquid is replaced periodically in the running process. And when two continuous stable voltages appear, the starting is considered to be successful. When the monitored output voltage was below 50mV, the anolyte was started to be replaced. And when stable and repeatable output voltage is obtained, measuring power density, calculating internal resistance of a system and the like.

Specifically, the steps related to the invention are as follows:

separation and purification of microorganisms on MFCs anode biomembrane

Passing seawater from south sea through simple sedimentationAnd then inoculating the mixed solution into an anode chamber of MFCs, and continuously operating under two conditions that anolyte contains NaCl with the mass concentration of 0% and 6.6% respectively. The reactor main body is an organic glass cylinder with the length of 2.0cm and the diameter of the cross section of 3.0cm, the effective volume is 14mL, and the effective areas of the cathode and the anode are all 7cm². The anode and the cathode are respectively arranged on two sides of the MFCs main body and are sealed and fixed by a rubber ring. One end of the anode is covered by an organic glass cover, the cover at one end of the cathode is open, the cathode carbon cloth carries platinum, the cathode and the anode are connected with a titanium wire to lead electrons out or in, and finally the whole device is screwed up and fixed by screws and lead screws. After the system is continuously operated for 125h and 60h at 37 ℃, the voltage at two ends of the load resistor is gradually stabilized, and a macroscopic thick biological film is formed on the anode. Scraping all thalli on the biomembrane by using an inoculating needle, suspending the thalli in physiological saline with the mass concentration of 0.9% to prepare uniform bacterial suspension, coating the bacterial suspension on a separated LB solid culture medium after gradient dilution, culturing for 2d under the constant temperature condition of 37 ℃, selecting bacterial colonies with obvious characteristic difference according to the characteristics of the morphology, the color, the size, the surface and the edge of the bacterial colonies, respectively inoculating the bacterial colonies in the LB liquid culture medium for enrichment culture again, repeatedly carrying out passage for 5-6 times in the way to obtain a pure cultured bacterial strain, and numbering and naming the bacterial strain.

Secondly, analyzing the electrogenesis performance of pure microorganisms on the MFCs anode biomembrane

And (3) taking each isolate separated and screened in the step one as an object, respectively inoculating the isolate into an LB liquid culture medium, carrying out shaking culture at 37 ℃ at 200r/min for 24h, taking 6000 Xg of bacterial liquid, centrifuging for 10min, collecting the bacterial body, and washing for 3 times by using physiological saline with the mass concentration of 0.9%.

Separately, bacterial cells of the same mass from different isolates were resuspended in an anolyte (1 g/L glucose as substrate) to obtain a stock solution (anolyte inoculated with bacterial cells of the corresponding isolate) and the initial OD of the stock solution was adjusted₆₀₀1.0, and then inoculated into a MFCs reactor. The reactor is connected with an external resistor (the external resistor is fixed at 1000 omega when no special description exists) and a data acquisition device respectively, the reactor operates under the constant temperature condition of (37 +/-1) DEG C, and the inoculation liquid is replaced periodically in the operation process. When two continuous stable voltages appearThe start-up is considered successful. And entering a formal operation period after the successful start, and only replacing the anolyte when the monitored output voltage is lower than 50 mV. The electrochemical parameters were measured after the output voltage again reached the highest and a stable and reproducible output voltage was obtained, after which the MFCs operation was stopped. In the whole process, a PISO-813 type data acquisition system is used for carrying out real-time online data monitoring and recording on the voltage, the average value within 30min is taken when the data is analyzed, and the sampling precision is 0.001V. Finally screening the isolate E-1 with better electrogenesis performance according to the voltage curve of each isolate.

Thirdly, the electrogenesis performance of the strain E-1 under the pressure without exogenous salt addition

As can be seen from FIG. 1, under the condition of no exogenous salt-adding pressure, the isolate E-1 was stable after running for 415h after being inoculated on MFCs, the stable voltage was about 554mV, and the power density was 192.14mA/m²The time is maximum and reaches 51.69 mW.m^-2。

Fourth, the growth performance of the strain E-1 under the salt pressure

The overnight-cultured bacterial suspension of isolate E-1 was transferred to 50mL LB liquid medium containing 0%, 2%, 4%, 6.6% NaCl by mass, and the initial OD values were adjusted to be the same. Shaking culture at 37 deg.C and 200r/min, sampling at different times to determine OD₆₀₀And a growth curve is plotted.

As can be seen from FIG. 2, under the condition that the NaCl concentration is 0%, the lag phase of the strain is 0-2.5 h, the log phase is 2.5-16 h, and the OD is obtained after 21h of culture₆₀₀The value reached a maximum of 1.66. With the increase of NaCl concentration, the lag phase of the strain is obviously prolonged, and the stable OD is reached₆₀₀The value decreases accordingly. When the NaCl concentration is 6.6%, the lag phase of the strain is 0-6 h, the logarithmic phase is 6-23 h, and the OD is obtained after 25h of culture₆₀₀The value reached a maximum of 1.53. Therefore, the above data indicate that strain E-1 has strong tolerance to salt and good growth conditions under salt pressure.

Fifthly, the electrogenesis performance of the strain E-1 under the salt pressure

As can be seen from FIG. 3, isolate E-1 was inoculated into exogenous MFCs having a salt mass concentration of 6.6% and allowed to stand for 98 hoursThe stable voltage is about 384mV, and the power density is 79.54mA/m²The time is the maximum and reaches 26.56 mW.m^-2. Compared with the non-pressure condition, the time for the system to reach the stability after the NaCl is added is shortened by 76.4 percent. The strain is shown to be a salt-tolerant electrogenesis microorganism and shows great application potential in the aspect of seawater resource treatment.

Sixthly, biological identification of the strain E-1

1. Morphological characteristics of the Strain

(1) And (3) colony morphology characteristics: after the strain E-1 is cultured on an LB solid culture medium at the constant temperature of 37 ℃ for 24-48h, the colony is round, light tan, neat in edge, transparent and smooth in surface, greasy in shape, 2mm in diameter and capable of secreting a certain brown substance to make the culture medium light tan (see a and b in figure 4)

(2) Morphological characteristics of the thallus: the results of gram staining indicated that the strain E-1 was a gram-negative bacterium. The strain was observed by a scanning electron microscope, and the bacterial cells were in the form of short rods having a length of about 2.5 μm and a width of about 0.5. mu.m (see FIG. 4(c, d)).

Biolog analysis

Suitable culture conditions were selected according to the Biolog identification parameters of gram-negative bacteria. The reference performs Biolog analysis. The specific method comprises the following steps: inoculating a single colony on a BUA + B culture plate, culturing at 37 ℃ for 24h, picking a small amount of fresh colonies with a sterile toothpick to prepare a bacterial suspension, comparing the bacterial suspension with a standard bacterial suspension, controlling the error range to be less than +/-2, inoculating a Biolog GNII plate by using eight pipette guns, inoculating 150 mu L of bacterial suspension in each hole, covering the bacterial suspension with a cover at 30 ℃ for 24h, opening a microporous plate cover, putting into an automatic result reading instrument, and comparing and analyzing the color development result with a Biolog database. Biolog identification results there are three important parameters to consider: probability (probability), (prob), similarity (sim) and distance (dist). When DIST <5.0, SIM >0.75 is a good match; the closer the SIM value is to 1, the higher the reliability of the verification result.

As shown in table 1, the SIM value of isolate E-1 was 0.94> 0.75; DIS value 0.89< 5.0; PROB is 100%. The three important parameters obtained by the system are ideal and are well matched with the database, and the closer the SIM value is to 1.00, the higher the reliability of the identification result is. The species column shows a best match name: shewanella algae. The strains identified positive reaction 33, negative reaction 50, boundary reaction 12. The positive reaction indicates that the matching degree of the target strain and the database is more than 80 percent, the negative reaction indicates that the matching degree of the sample strain and the database is less than 20 percent, and the boundary reaction is more than 15, so that the identification result is not good, and the result is 12 in the experiment. Various data indexes show that the identification result is accurate, the database is well matched, and the bacterial strain E-1 is preliminarily identified to be Shewanella alga.

TABLE 1 Biolog identification System identification results

3. 16S rDNA sequencing analysis of strains

A bacterial genome DNA extraction kit is adopted, the genome DNA of the extracted strain E-1 is used as a template, a universal primer (27F, 1492R) of the bacterial 16Sr DNA is used as a primer, and a 16S rDNA fragment of the strain E-1 is amplified. The sequence length of the 16S rDNA gene of the strain is 1450bp, the obtained 16S rDNA gene sequence is input into GenBank, and all sequences in a database are compared and analyzed by a Blast program. A phylogenetic tree is constructed by using MEGA7.0 software and a Neighbor-joining method (Neighbor-joining method) based on a Kimura 2-parameter model, and the branching stability of the phylogenetic tree is analyzed by Bootstrap and repeated for 1000 times. As a result, the 16S rDNA gene sequence of the strain is found to have higher similarity with the Shewanella algae Hiro-1 strain of Shewanella (Shewanella), and the homology reaches 100%. As can be seen in FIG. 5, the isolate E-1 is in the same branch of the phylogenetic tree as Shewanella algae Hiro-1, and the relationship is closest.

The strain E-1 was identified as Shewanella algae by morphological observation, biologics and 16S rDNA sequence alignment of the strain.

Seventhly, carbon source utilization condition and growth condition characteristics of the strain E-1

According to the metabolic microplate by contrasting corresponding carbon sources, the strain E-1 is respectively coated on basic inorganic salt solid culture media containing different carbon sources, and the utilization degree of the carbon sources is analyzed by observing whether the strain grows or not. As is clear from Table 2, the strains can utilize carbon sources such as lactic acid, acetic acid, succinamic acid, L-alanine, uridine, etc., but cannot utilize carbon sources such as L-arabinose, maltose, sucrose, glycerol, etc. (Table 3). In addition, the growth conditions of the strain under different temperature and pH conditions show that the temperature range suitable for growth is 25-40 ℃, and the pH range is 5-10.

TABLE 2 carbon sources that the strain E-1 can utilize

TABLE 3 carbon sources not utilizable by Strain E-1

The 16S rDNA gene of the strain has a nucleotide sequence shown as a sequence SEQ ID No.1, the sequence length is 1450bp, and the accession number on Genbank is MK 787267.1.

SEQ ID No.1：

CGCTTGCGCAGCTACACATGCAGTCGAGCGGTAACATTTCAAAAGCTTGCTTTTGAAGATGACGAGCGGCGGACGGGTGAGTAATGCCTGGGAATTTGCCCATTTGTGGGGGATAACAGTTGGAAACGACTGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGGACCTTCGGGCCTTGCGCTGATGGATAAGCCCAGGTGGGATTAGCTAGTAGGTGAGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAgGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGKTGTAAAGCACTTTCAGCGAGGAGGAAAGGGTGTAAGTTAATACCTTACATCTGTGACGtTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACCGCATTTCGAACTGGCAAACTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTCGGAGTTTGGTGTCTTGAACACTGGGCTCTCAAGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAAcCCTTACCTACTCTTGACATCCASAGAACTTKSCtAGAGATGSATYGGTGCCTTCGGGAACTSTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTACTTGCCAGCGGGTAATGcCCGGGAACTTTAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGTCGGTACAGAGGGTTGCGAAGCCGCGAGGTGGAGCTAATCCCATAAAGCCGGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTACCACGGTTGGTCTGCAT。

Although the embodiments of the present invention have been disclosed for illustrative purposes, those skilled in the art will appreciate that: various substitutions, changes and modifications are possible without departing from the spirit and scope of the invention and the appended claims, and therefore the scope of the invention is not limited to the disclosure of the embodiments and the accompanying drawings.

Sequence listing

<110> Tianjin science and technology university

<120> a salt-tolerant bacterial strain with electrogenesis characteristic and application thereof in microbial fuel cell

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<170> SIPOSequenceListing 1.0

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<211> 1450

<212> DNA/RNA

<213> 16S rDNA Gene of Strain (Unknown)

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cgcttgcgca gctacacatg cagtcgagcg gtaacatttc aaaagcttgc ttttgaagat 60

gacgagcggc ggacgggtga gtaatgcctg ggaatttgcc catttgtggg ggataacagt 120

tggaaacgac tgctaatacc gcatacgccc tacgggggaa agcaggggac cttcgggcct 180

tgcgctgatg gataagccca ggtgggatta gctagtaggt gaggtaaagg ctcacctagg 240

cgacgatccc tagctggtct gagaggatga tcagccacac tgggactgag acacggccca 300

gactcctacg ggaggcagca gtggggaata ttgcacaatg ggggaaaccc tgatgcaggc 360

catgccgcgt gtgtgaagaa ggccttcggg ktgtaaagca ctttcagcga ggaggaaagg 420

gtgtaagtta ataccttaca tctgtgacgt tactcgcaga agaagcaccg gctaactccg 480

tgccagcagc cgcggtaata cggagggtgc gagcgttaat cggaattact gggcgtaaag 540

cgtgcgcagg cggtttgtta agcgagatgt gaaagccccg ggctcaacct gggaaccgca 600

tttcgaactg gcaaactaga gtcttgtaga ggggggtaga attccaggtg tagcggtgaa 660

atgcgtagag atctggagga ataccggtgg cgaaggcggc cccctggaca aagactgacg 720

ctcaggcacg aaagcgtggg gagcaaacag gattagatac cctggtagtc cacgccgtaa 780

acgatgtcta ctcggagttt ggtgtcttga acactgggct ctcaagctaa cgcattaagt 840

agaccgcctg gggagtacgg ccgcaaggtt aaaactcaaa tgaattgacg ggggcccgca 900

caagcggtgg agcatgtggt ttaattcgat gcaacgcgaa gaacccttac ctactcttga 960

catccasaga acttksctag agatgsatyg gtgccttcgg gaactstgag acaggtgctg 1020

catggctgtc gtcagctcgt gttgtgaaat gttgggttaa gtcccgcaac gagcgcaacc 1080

cctatcctta cttgccagcg ggtaatgccc gggaacttta gggagactgc cggtgataaa 1140

ccggaggaag gtggggacga cgtcaagtca tcatggccct tacgagtagg gctacacacg 1200

tgctacaatg gtcggtacag agggttgcga agccgcgagg tggagctaat cccataaagc 1260

cggtcgtagt ccggattgga gtctgcaact cgactccatg aagtcggaat cgctagtaat 1320

cgtggatcag aatgccacgg tgaatacgtt cccgggcctt gtacacaccg cccgtcacac 1380

catgggagtg ggctgcacca gaagtagata gcttaacctt cgggagggcg ttaccacggt 1440

tggtctgcat 1450

Claims (4)

1. A salt-tolerant Shewanella alga (Shewanella algae) strain E-1 with electrogenic properties, characterized in that: the preservation number is: CGMCC No.17857, preservation date: 27 days 5 months in 2019, west road No.1 hospital No. 3, tokyo, yoyo, north, and depository: china general microbiological culture Collection center.

2. Use of the salt-tolerant Shewanella alga (Shewanella algae) strain E-1 with electrogenic properties according to claim 1 for the electrogenesis of microbial fuel cells.

3. Use of the salt-tolerant and electrogenic Shewanella alga (Shewanella algae) strain E-1 according to claim 1 for the manufacture of microbial fuel cells.

4. The use of Shewanella alga (Shewanella algae) strain E-1, which is salt-tolerant and has electrogenic properties, according to claim 3, for the manufacture of microbial fuel cells, characterized in that: the application method comprises the following steps:

firstly, activating a strain: selecting a ring of Shewanella algae strains from an LB slant culture medium, inoculating the Shewanella algae strains into 50mL of LB liquid culture medium, carrying out shake culture at 30-37 ℃ and 200r/min at 160-48 h, taking 6000 Xg of bacterial liquid, centrifuging for 10min, collecting the bacterial cells, and washing the bacterial cells for 3 times by using physiological saline with the mass concentration of 0.9%;

preparing an inoculation liquid: resuspending the cells in anolyte containing different NaCl concentrations, and using 0.8-2.0g/L glucose as substrate to make initial OD₆₀₀0.8-1.5, and then inoculating into a reactor for MFCs; wherein, the mass concentration of NaCl is 0-7%;

and thirdly, detecting the electricity generation performance: the reactor is connected with an external resistor and a data acquisition device respectively, and runs at the constant temperature of 37 +/-1 ℃, and inoculation liquid is replaced periodically in the running process; when two continuous stable voltages appear, the starting is considered to be successful; when the monitored output voltage is below 50mV, the anolyte change is started until a stable and repeatable output voltage is obtained.

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