CN106754457A - Mixed bacterial and application thereof, the microorganism electricity generation system containing the mixed bacterial and microbiological fuel cell - Google Patents
Mixed bacterial and application thereof, the microorganism electricity generation system containing the mixed bacterial and microbiological fuel cell Download PDFInfo
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- CN106754457A CN106754457A CN201510812764.5A CN201510812764A CN106754457A CN 106754457 A CN106754457 A CN 106754457A CN 201510812764 A CN201510812764 A CN 201510812764A CN 106754457 A CN106754457 A CN 106754457A
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- 241000490596 Shewanella sp. Species 0.000 description 2
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- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 1
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- VVGWBARCAMGKOR-UHFFFAOYSA-M potassium dihydrogen phosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.P(=O)(O)(O)[O-].[K+] VVGWBARCAMGKOR-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01M—PROCESSES OR MEANS, e.g. BATTERIES, FOR THE DIRECT CONVERSION OF CHEMICAL ENERGY INTO ELECTRICAL ENERGY
- H01M8/00—Fuel cells; Manufacture thereof
- H01M8/16—Biochemical fuel cells, i.e. cells in which microorganisms function as catalysts
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
- Y02E60/30—Hydrogen technology
- Y02E60/50—Fuel cells
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- Chemical Kinetics & Catalysis (AREA)
- Electrochemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to microorganism electricity generation field, more particularly to mixed bacterial and application thereof, the microorganism electricity generation system containing the mixed bacterial and microbiological fuel cell.The present invention is by building the mixed bacterial being engineered, make it have the characteristics of dividing the work clear and definite, zymophyte 1 provides the carrier riboflavin of electron transmission for system, and the most frequently used, cheap glucose is converted into small molecule acid and is supplied to electricity production bacterium to use by zymophyte 2, expands the carbon source spectrum of electricity production bacterium.Present invention selection Escherichia coli or bacillus subtilis --- the type strain of prokaryotes Gram-negative bacteria and gram-positive bacteria, by means such as genetic engineering transformations, construct 2 kinds of zymophytes of different purposes, the carbon source spectrum of microbiological fuel cell has been expanded, the electron transmission efficiency of electricity production bacterium has been enhanced.Meanwhile, from material stream, energy stream, three important angles of information flow, build the mixed bacterium symbiosis electricity production system of stable, reasonable, efficient feature.
Description
Technical Field
The invention relates to the field of microbial electrogenesis, in particular to a mixed flora and application thereof, a microbial electrogenesis system containing the mixed flora and a microbial fuel cell.
Background
A Microbial Fuel Cell (MFC) is a device that directly converts chemical energy in organic matter into electrical energy using microorganisms. The basic working principle is as follows: under the anaerobic environment of the anode chamber, organic matters are decomposed under the action of microorganisms to release electrons and protons, the electrons are effectively transferred between biological components and an anode electrode by virtue of a proper electron transfer mediator and an electron transfer mechanism on a special cell membrane of an electrogenic bacterium and are transferred to a cathode through an external circuit to form current, the protons are transferred to the cathode through a proton exchange membrane, and an oxidant obtains the electrons at the cathode and is reduced to combine with the protons to form water.
Compared with other existing technologies utilizing organic energy, the microbial fuel cell has the advantages of operation and function: firstly, the substrate is directly converted into electric energy, so that high energy conversion efficiency is ensured; secondly, unlike all existing bioenergy treatments, the microbial fuel cell can effectively operate under the condition of normal temperature environment; third, the microbial fuel cell does not require waste gas treatment because the main component of the waste gas generated by the microbial fuel cell is carbon dioxide, and the microbial fuel cell does not have reusable energy under normal conditions; fourth, the microbial fuel cell does not require a large input of energy, since the cathode gas can be passively replenished by merely ventilating the single-cell microbial fuel cell; fifth, in local areas where there is a lack of electrical infrastructure, microbial fuel cells have the potential for widespread use, while also expanding the diversity of fuels that can be used to meet our energy needs.
In summary, the microbial fuel cell is a device for converting chemical energy into electric energy by using electricity-generating microbes which can transport electrons generated by metabolic reaction in cells through specific cell membrane proteins and output the cells, can overcome the limitation of renewable energy sources such as wind energy, solar energy and the like by the environment, plays an important role in the fields of environmental treatment and novel energy sources, and has a good development prospect. However, the growth environment of the electrogenic bacteria is harsh, the electrogenic efficiency is low, the electron transfer process is slow, and the industrial application cannot be realized at present.
The electricity-producing bacteria mainly comprising Shewanella bacteria have been widely researched by scientists, and people focus on the transformation of the electricity-producing bacteria and the optimization of electrode materials, but the effect is not obvious. For example: (1) the method cannot utilize a wide and cheap substrate as a carbon source (the carbon source spectrum is narrow); (2) in order to maintain the electricity generation quantity, a large amount of riboflavin needs to be added, so that the cost is high; (3) the genetically modified electrogenic bacteria grow more slowly, and the electrogenic activity is not as good as that of wild bacteria; (4) the duration is short, and the electricity generation is unstable; (5) the electrogenic bacteria are fragile, so that the metabolic burden of riboflavin production of the electrogenic bacteria is increased, and the electrogenesis of the electrogenic bacteria is influenced; (6) the fermentation-electricity generation multiple tasks are completed by single electricity generation bacteria; (7) complex mineral solution and complex vitamin solution are required to be added into the culture medium, so that the cost is high and the preparation is complex. The existing microbial fuel cell needs to continuously sample in the power generation process, detect the content of key substances in a system, and if the content is insufficient, the content needs to be added in time to maintain the activity of strains and the stability of the system. The system has poor stability and repeatability, short electricity generation duration, high cost and complex system, and dozens of mineral substances and vitamin solutions need to be added externally.
Therefore, it is important to provide a high-efficiency and stable microbial fuel cell.
Disclosure of Invention
In view of the above, the present invention provides a mixed bacterial population, a use thereof, a microbial power generation system and a microbial fuel cell containing the mixed bacterial population. The invention constructs the mixed flora to ensure the clear division of the work, the zymocyte 1 provides the carrier riboflavin for electron transfer for the system, and the zymocyte 2 converts the most common and cheap glucose into micromolecular acid to be provided for the electrogenesis bacteria. The invention selects the Escherichia coli-prokaryotic mode strain, constructs 2 kinds of zymogens with different purposes by means of genetic engineering transformation and the like, expands the carbon source spectrum of the microbial fuel cell and enhances the electron transfer efficiency of the electrogenic bacteria. Meanwhile, a stable, reasonable and efficient functional mixed bacteria symbiotic system is constructed from three important angles of material flow, energy flow and information flow.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a mixed flora, which is characterized by comprising zymocyte and electrogenesis bacteria.
In some embodiments of the invention, the fermentation tubes in the mixed population include high riboflavin-producing tubes.
In some embodiments of the present invention, the fermentation bacteria in the mixed bacterial population include bacteria that produce small molecule acids with pentose, hexose, cellobiose as carbon sources.
In the present invention, the small molecule acid is selected from lactic acid, formic acid, amino acid, and the like. Any small molecule acid known to those skilled in the art is within the scope of the present invention, and the present invention is not limited thereto.
In the present invention, the treatment fluid, in which no additional substance is required in the electricity generation process, glucose is used as a carbon source, and the substrate can be extended to xylose, cellobiose, or even cellulose, is within the protection scope of the present invention, and the present invention is not limited herein.
In some embodiments of the invention, the fermentation bacteria in the mixed population are escherichia coli;
the electrogenic bacteria are Shewanella.
In some embodiments of the present invention, the inoculation ratio of the riboflavin-highly producing bacteria to the electrogenic bacteria in the mixed population is not more than 1: 1.
The ratio of the inoculation of the bacteria for producing the micromolecular acid by taking the pentose and the hexose as carbon sources to the electricity-producing bacteria is not more than 1: 20.
in other embodiments of the present invention, the ratio of the zymocyte to the electrogenic bacteria in the mixed flora is 1: 20.
In some embodiments of the present invention, the inoculation ratio of the riboflavin-highly producing bacteria to the electrogenic bacteria in the mixed population is not less than 1: 10000;
the ratio of the inoculation of the bacteria for producing the micromolecular acid by taking the pentose and the hexose as carbon sources to the electricity-producing bacteria is 1: 20.
The invention also provides application of the mixed flora in conversion of chemical energy into electric energy.
The invention also provides a microbial electrogenesis system comprising the mixed flora.
In some embodiments of the invention, the mixed population of the microbial electrogenesis system has a inoculum size of electrogenesis bacteria of no more than 4OD in the microbial electrogenesis system.
In some embodiments of the invention, the microbial power generation system further comprises nitrate.
In some embodiments of the invention, the final concentration of the nitrate in the microbial electrogenesis system is no greater than 10 mM.
In some embodiments of the invention, the microbial power generation system has a pH of 6.2 to 7.2.
In some embodiments of the invention, the microbial electrogenesis system further comprises a buffer, the buffer being HEPES. Preferably 1 × HEPES.
In some embodiments of the invention, the concentration of the carbon source in the microbial power generation system is no more than 10 g/L.
In some embodiments of the invention, 500mL of the microbial power generation system comprises 1.5g of monopotassium phosphate, 8.55g of monopotassium phosphate dodecahydrate, 0.25g of sodium chloride, 0.5g of ammonium chloride, 0.12g of magnesium sulfate and 5.5mg of calcium chloride.
The invention also provides a microbial fuel cell which comprises the mixed flora or the microbial power generation system.
The invention constructs the mixed flora to ensure the clear division of the work, the zymocyte 1 provides the carrier riboflavin for electron transfer for the system, and the zymocyte 2 converts the most common and cheap glucose into micromolecular acid to be provided for the electrogenesis bacteria. According to the invention, escherichia coli or bacillus subtilis is selected, and 2 kinds of fermentation bacteria with different purposes are constructed by means of genetic engineering transformation and the like, so that the carbon source spectrum of the microbial fuel cell is expanded, and the electron transfer efficiency of the electrogenic bacteria is enhanced. Meanwhile, a stable, reasonable and efficient functional mixed bacteria symbiotic system is constructed from three important angles of material flow, energy flow and information flow.
The microbial power generation system and the microbial fuel cell provided by the invention solve the following problems:
(1) the microbial fuel cell has short duration and unstable electricity generation, and expensive substances such as substrates, electronic carriers and the like are required to be added regularly to maintain higher electricity generation;
(2) the Shewanella as an electrogenic bacterium has narrow substrate spectrum and carbon source spectrum, can only utilize substances such as micromolecular lactic acid, formic acid, amino acid and the like, can not utilize wide carbon sources such as five-carbon sugar and six-carbon sugar, such as glucose, xylose and the like, and can not convert cellulose or rich chemical energy in wide and cheap glucose conversion into electric energy;
(3) from the perspective of 'self supply of the system', an independent, stable and efficient microbial fuel cell system is developed;
(4) the problem that different strains in the mixed bacteria fuel cell can not be co-cultured. The problem of how to make the electricity-producing bacteria and the zymocyte have synergistic effect and improve the electricity-producing quantity and the electricity-producing time of the system;
(5) how to select and engineer zymophyte is used as the input of important substances.
The invention can realize high-efficiency power generation for over 100 hours by only adding certain glucose at the beginning and adding no substances at the middle.
The microbial electrogenesis system is simple, only needs solution consisting of no more than 10 basic and cheap salts, and does not need to add complex mineral salt solution and vitamin complex solution.
The patent breaks through the traditional single-bacterium transformation thinking in the field of microbial fuel cells, uses the advantages and the characteristics of the mixed bacterium group with clear division of labor, constructs a mixed power generation system of escherichia coli-shewanella, and has the advantages of higher electric quantity, longer duration, very low cost, no need of material supplement in the middle and the like compared with other fuel cells.
In the construction process, two genetically modified escherichia coli with different functions are used as zymophyte to provide key substances for a system, and become important substance, energy and information driving force for mutual communication, mutual benefit symbiosis and driving functions among bacteria.
One of the escherichia coli uses glucose to metabolize small molecular acid (lactic acid, formic acid, amino acid and the like) for the electricity-producing bacteria to use, and the other engineering escherichia coli produces riboflavin which is used as a carrier for electron transfer and is used for the electricity-producing bacteria to improve the electron transfer efficiency and effectively improve the electricity production quantity. Meanwhile, under the special condition of electricity generation, the electricity generation bacteria can also promote the metabolism speed of the zymophyte, so that the electricity output duration of the system is longer. In addition, the control of conditions such as mixed culture conditions, inoculation ratio and the like also has important influence on the electricity generation effect.
After a large number of conditions are groped for,as shown in fig. 1:the electrogenesis diagram of Shewanella alone is that the electrogenesis quantity is extremely low and basically no electricity exists in 48 hours when glucose is used as a carbon source.The two bacteria are mixed without feeding in the middle, and the electricity generation amount is far lower than a green line (system) under the condition.Three bacteria of the system constructed by the people cooperate with each other to synergistically promote the improvement of the electricity production quantity.
2 species and three bacteria are used in the mixed bacteria system to realize the co-energy.
(1) Cut-in points of matter flow, energy and information flow riboflavin and small acid were distributed to 2 species of ferments. (concept of thorough division of microbial fuel cell)
(2) Escherichia coli and Escherichia coli are selected as zymocyte, the large intestine has the unique advantages of simple gene operation, high propagation speed, simple culture condition and the like, and the primary metabolite has strong production and secretion capacity and is the engineering bacterium which is most widely used in the biochemical engineering.
(3) The effect of the battery is as follows: the electricity generation quantity exceeds 350mV, and the electricity generation time exceeds 100 hours;
(4) the electricity generation process does not need to add substances, glucose is used as a carbon source, and the substrate can be extended to be xylose, cellobiose and even cellulose treatment solution.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 is a graph showing the comparison of the electricity generation effect of example 5; wherein,shewanella sp;shewanella sp + E.coli;shewanella sp, Escherichia coli, and Escherichia coli;
FIG. 2 is a graph showing the comparison of the electric power generation effect of example 6; wherein,shewanella sp;shewanella sp, Bacillus subtilis, and Escherichia coli;
FIG. 3 shows a pH indicator strip; wherein FIG. 3(A) is a comparison of pH paper; FIG. 3(B) is a chart of pH indicator paper of example 7;
FIG. 4 shows the effect of inoculation ratio and pH on the system of example 7; wherein,shewanella + small molecule acid;shewanella sp + E.coli;shewanella sp. + escherichia coli (dotrophic bacteria: zymogen 20: 1);shewanella sp. + escherichia coli (dotrophic bacteria: zymogen 10: 1);
fig. 5 shows a diagram of a fuel cell apparatus.
Detailed Description
The invention discloses a mixed flora and application thereof, a microbial power generation system containing the mixed flora and a microbial fuel cell. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The mixed flora provided by the invention and the application thereof, and raw materials and reagents used in a microbial power generation system and a microbial fuel cell containing the mixed flora can be purchased from the market.
The construction method of the bacteria for producing the small molecular acid by taking the pentose and the hexose as carbon sources comprises the following steps: in order to enhance the capability of zymocyte 2 to generate small molecular acid under the anaerobic condition, the invention introduces an ldhE gene (a lactic acid-producing gene, a lactobacillus source) into escherichia coli, and uses a lambda-Red homologous recombination technology to knock out a pflB gene
The construction method of the escherichia coli with high riboflavin production comprises the following steps: introducing ribABDEC gene cluster into escherichia coli;
the construction method of the bacillus subtilis with high riboflavin production comprises the following steps: overexpression of the prs and ywlF genes in B.subtilis down-regulated the Pur operon and PurR regulatory genes (glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, andpbuO). The strain was constructed as per the article: shi S, Chen T, Zhang Z, et al, transformed and analyzed mutation engineering of Bacillus subtilis for riboflavin production [ J ]. Metabolic engineering,2009,11(4):243- & ltu/& gt 252.
The invention is further illustrated by the following examples:
example 1 Mixed flora
Constructing bacillus subtilis with high riboflavin production: through gene manipulation, prs and ywlF genes in the bacillus subtilis are over-expressed, and the Pur operon and PurR regulatory genes (glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, andpbuO) are down-regulated.
Constructing escherichia coli for producing small molecular acid by taking pentose and hexose as carbon sources: the pflB gene was knocked out in E.coli by the lambda-Red homologous recombination technique (see the attached schematic), and then the ldhE gene (lactic acid-producing gene, Lactobacillus origin, synthesized by GENWIZE) was introduced by: the plasmid ligated with the ldhE gene was introduced into the above-mentioned E.coli knocked out the pflB gene by digesting with EcoRI and PstI, ligating the digested plasmid to the pSB1C plasmid, and then, chloramphenicol resistance was selected to select the correct transformant. Mixing the constructed bacillus subtilis with high riboflavin yield, the constructed escherichia coli for producing small molecular acid by taking pentose and hexose as carbon sources and the shewanella, and mixing the bacillus subtilis with the shewanella according to the following steps:
mixing bacillus subtilis with high riboflavin production and Shewanella as an electrogenic bacterium according to a bacterium inoculation ratio not more than 1: 1;
escherichia coli which produces small molecular acid by taking pentose and hexose as carbon sources is mixed with Shewanella as an electrogenic bacterium according to a inoculation ratio of not more than 1: 20.
Example 2 Mixed flora
Constructing bacillus subtilis with high riboflavin production: through gene manipulation, prs and ywlF genes in the bacillus subtilis are over-expressed, and the Pur operon and PurR regulatory genes (glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, andpbuO) are down-regulated.
Constructing escherichia coli for producing small molecular acid by taking pentose and hexose as carbon sources: the pflB gene was knocked out in E.coli by the lambda-Red homologous recombination technique (see the attached schematic), and then the ldhE gene (lactic acid-producing gene, Lactobacillus origin, synthesized by GENWIZE) was introduced by: the plasmid ligated with the ldhE gene was introduced into the above-mentioned E.coli knocked out the pflB gene by digesting with EcoRI and PstI, ligating the digested plasmid to the pSB1C plasmid, and then, chloramphenicol resistance was selected to select the correct transformant.
Mixing the constructed bacillus subtilis with high riboflavin yield, the constructed escherichia coli for producing small molecular acid by taking pentose and hexose as carbon sources and the shewanella, and mixing the bacillus subtilis with the shewanella according to the following steps:
mixing bacillus subtilis with high riboflavin production and Shewanella as an electrogenic bacterium according to the inoculation ratio of 1: 20;
escherichia coli which produces small molecular acid by taking pentose and hexose as carbon sources is mixed with Shewanella as an electricigen according to the inoculation ratio of 1: 200.
Example 3 Mixed flora
Constructing Escherichia coli 1 with high riboflavin production: introducing ribABDEC gene cluster into escherichia coli;
constructing escherichia coli 2 for producing small molecular acid by taking pentose and hexose as carbon sources: the pflB gene was knocked out in E.coli by the lambda-Red homologous recombination technique (see the attached schematic), and then the ldhE gene (lactic acid-producing gene, Lactobacillus origin, synthesized by GENWIZE) was introduced by: the plasmid ligated with the ldhE gene was introduced into the above-mentioned E.coli knocked out the pflB gene by digesting with EcoRI and PstI, ligating the digested plasmid to the pSB1C plasmid, and then, chloramphenicol resistance was selected to select the correct transformant.
Mixing the constructed Escherichia coli 1 with high riboflavin yield, the constructed Escherichia coli 2 which takes pentose and hexose as carbon sources to produce micromolecule acid and Shewanella, and mixing the Escherichia coli 1, the Escherichia coli 2 and the Shewanella according to the following steps:
mixing the escherichia coli 1 with high riboflavin production with Shewanella as an electricigen according to a inoculation ratio not more than 1: 1;
escherichia coli 2 for producing small molecular acid by using pentose and hexose as carbon sources is mixed with Shewanella as an electrogenic bacterium according to the inoculation ratio of 1: 20.
Example 4 Mixed flora
Constructing Escherichia coli 1 with high riboflavin production: introducing ribABDEC gene cluster into escherichia coli;
constructing escherichia coli 2 for producing small molecular acid by taking pentose and hexose as carbon sources: the pflB gene was deleted in E.coli by the lambda-Red homologous recombination technique, and then the ldhE gene (lactic acid-producing gene, Lactobacillus-derived, synthesized by GENWIZE) was introduced by: the plasmid ligated with the ldhE gene was introduced into the above-mentioned E.coli knocked out the pflB gene by digesting with EcoRI and PstI, ligating the digested plasmid to the pSB1C plasmid, and then, chloramphenicol resistance was selected to select the correct transformant.
Mixing the constructed Escherichia coli 1 with high riboflavin yield, the constructed Escherichia coli 2 which takes pentose and hexose as carbon sources to produce micromolecule acid and Shewanella, and mixing the Escherichia coli 1, the Escherichia coli 2 and the Shewanella according to the following steps:
mixing the escherichia coli 1 with high riboflavin production with Shewanella as an electricigen according to the inoculation ratio of 1: 20;
escherichia coli 2 for producing small molecular acid by using pentose and hexose as carbon sources is mixed with Shewanella as an electrogenic bacterium according to a inoculation ratio of not more than 1: 200.
EXAMPLE 5 microbial electrogenesis System
Cathode: potassium ferricyanide (purchased by sigma corporation) solution;
anode: shewanella, Shewanella + Escherichia coli 2 (Escherichia coli producing small molecular acid by using pentose and hexose as carbon sources) prepared in example 3 and Escherichia coli 1 (Escherichia coli highly producing riboflavin) were respectively taken;
a layer of proton exchange membrane (purchased from dupont) is sandwiched between the two stages;
the middle wire was connected to a 2k resistor, and porous carbon cloth was connected to the wire in the bottle as an electrode (anode 2.5cm x2.5cm, cathode 2.5cm x 3 cm).
The experimental sequence is as follows: (1) assembling an anode and a cathode (without adding a proton exchange membrane in the middle), inserting an electrode, connecting no resistor, sterilizing (15 min at 121 ℃) after the assembly is finished, drying, adding prepared catholyte, adding mixed culture solution (adding HEPES, nitrate and glucose into prepared basic culture medium) into the anode, calculating the concentration of cultured thalli, centrifuging, resuspending thalli by using the culture solution in the anode, adding the thalli into a battery, adding Shewanella-Escherichia coli in sequence, and finally installing the resistor. Culturing in 30 deg.C incubator, taking out every 2-15 hr, and measuring voltage with multimeter. In the process of groping conditions, about 500uL of liquid is taken out from a bottle every 4 hours, thalli in the liquid are separated, the liquid is filtered, the content of key metabolites in the liquid is detected by HPLC (high performance liquid chromatography), and meanwhile, whether the pH of a system is in a neutral state is detected by using a pH test paper.
Wherein, the culture solution includes:
as a result: after the cell is connected, the cell is put into an incubator at 30 ℃, taken out every 4 or 8 hours, data is recorded by a universal meter, the voltage value recorded by the universal meter every time is recorded, and a time-voltage curve is drawn.
See table 1 and figure 1.
Table 1 data results
As can be seen from FIG. 1, the electrogenic bacteria cannot utilize glucose, the electricity production is very low, not exceeding 100mV and the electricity production substantially stops after 48 hours. The electricity generation amount of the microbial fuel cell of the three bacteria (the zymocyte is escherichia coli, and the electricity generation bacteria is Shewanella) is improved well (more than 350mV) on the electricity generation amount, and the duration time is more than 100 hours.
EXAMPLE 6 microbial electrogenesis System
Cathode: potassium ferricyanide (purchased by sigma corporation) solution;
anode: shewanella, Shewanella + Escherichia coli (Escherichia coli producing small molecular acid by using pentose and hexose as carbon sources) prepared in example 1, and Bacillus subtilis (Bacillus subtilis highly producing riboflavin) were respectively taken;
a layer of proton exchange membrane (purchased from dupont) is sandwiched between the two stages;
the middle wire was connected to a 2k resistor and the inside of the bottle was connected to a porous carbon cloth (anode 2.5 cm. times.2.5 cm, cathode 2.5 cm. times.3 cm).
The experimental sequence is as follows: (1) assembling an anode and a cathode (without adding a proton exchange membrane in the middle), inserting an electrode, connecting no resistor, sterilizing (15 min at 121 ℃) after the assembly, drying, adding prepared catholyte, adding mixed culture solution (adding HEPES, nitrate and glucose into prepared basic culture medium) into the anode, calculating the concentration of cultured thalli, centrifuging, resuspending thalli by using the culture solution in the anode, adding the thalli into a battery, adding Shewanella-Escherichia coli-bacillus subtilis in sequence, and finally installing the resistor. Culturing in 30 deg.C incubator, taking out every 2-15 hr, and measuring voltage with multimeter. In the process of groping conditions, about 500uL of liquid is taken out from a bottle every 4 hours, thalli in the liquid are separated, the liquid is filtered, the content of key metabolites in the liquid is detected by HPLC (high performance liquid chromatography), and meanwhile, whether the pH of a system is in a neutral state is detected by using a pH test paper.
Wherein, the culture solution includes:
as a result: after the cell is connected, the cell is put into an incubator at 30 ℃, taken out every 4 or 8 hours, data is recorded by a universal meter, the voltage value recorded by the universal meter every time is recorded, and a time-voltage curve is drawn.
See table 2 and figure 2.
Table 2 data results
As shown in FIG. 2, the electrogenic bacteria cannot utilize glucose, the electricity generation amount is low, not exceeding 100mV, and the electricity generation basically stops after 48 hours. The microbial fuel cell of the three bacteria (the zymocyte is escherichia coli and bacillus subtilis, and the electrogenesis bacteria are Shewanella) has good improvement (the highest electricity generation amount can reach 520mV) on the electricity generation amount, and the duration time is more than 100 hours.
Example 7 optimization of Fuel cell systems
The fuel cell comprises a cathode and a anode of two bottles (shown in figure 5), a cathode of potassium ferricyanide (purchased from sigma company) solution, a layer of proton exchange membrane (purchased from DuPont company) sandwiched between the two bottles, an anode of bacteria solution, a middle lead connected with a 2k resistor, and a porous carbon cloth (the anode is 2.5cm multiplied by 2.5cm, and the cathode is 2.5cm multiplied by 3cm) connected with the lead in the bottle. The experimental sequence is as follows: (1) firstly, assembling a bottle (without adding a proton exchange membrane in the middle), inserting an electrode, connecting no resistor, sterilizing (15 min at 121 ℃) after the bottle is assembled, drying, adding prepared catholyte, then adding mixed culture solution (adding HEPES, nitrate and glucose into prepared basic culture medium) into an anode, calculating the concentration of cultured thalli, centrifuging, resuspending thalli by using the culture solution in the anode, adding the thalli into a battery, adding Shewanella-Escherichia coli (or Bacillus subtilis) in sequence, and finally installing the resistor. Culturing in 30 deg.C incubator, taking out at regular intervals, and measuring voltage with multimeter. In the process of searching conditions, about 500uL of liquid is taken out from a bottle every 4h, thalli in the liquid are separated, the liquid is filtered, the content of key metabolites in the liquid is detected by HPLC (high performance liquid chromatography), and meanwhile, pH test paper is used for detecting whether the pH of a system is in a neutral state (because the tolerance range of the electrogenic bacteria is 6.2-7, the pH is generally controlled to be more than 6.5-6.8).
Wherein, the culture solution includes:
bacterial liquid:
the No. 1 is Shewanella and small molecular acid (sodium lactate is added externally);
2# Shewanella and Escherichia coli (zymogen 1, producing small molecule acid)
3# is Shewanella and Escherichia coli (zymogen 1, producing small molecule acid) and Escherichia coli (zymogen 2 producing riboflavin) -prepared in example 3;
the 4# is Shewanella and Escherichia coli (zymogen 1, producing small molecule acid) and Escherichia coli (zymogen 2 producing riboflavin) -prepared in example 1;
as a result: the voltage values recorded by the multimeter were recorded each time and plotted as a time-voltage curve, the results being shown in FIG. 4.
Determining the influence of the inoculation ratio and the pH value on the electricity generation of the fuel cell:
the system pH was measured as shown in FIG. 3:
the result of detecting the pH of the battery anode is shown in fig. 3, the system is maintained to be basically neutral by the Shewanella # 1 + the small molecular acid, and the pH of the Shewanella # 2 + the escherichia coli system is compared with the standard pH test paper, and the pH is considered to be lower than 5.4, and the Shewanella # 3 + the escherichia coli (zymogen 1, producing the small molecular acid) + the escherichia coli (zymogen 2 producing riboflavin) (electrogen: zymogen ═ 1 (1-10): 1), and the pH is close to 5.4 and is slightly acidic compared with the standard pH test paper. The No. 4 is Shewanella, Escherichia coli (zymogen 1, producing small molecular acid), Escherichia coli (zymogen 2 producing riboflavin) (electrogenesis bacteria: zymogen more than 20:1), and compared with standard pH test paper, the pH is close to 6.2 and is still acidic.
And (4) conclusion:
the No. 1 is Shewanella and small molecular acid (sodium lactate is added externally), and the pH of the system is neutral;
the 2# is Shewanella and Escherichia coli (zymocyte 1, producing small molecular acid), the pH is lower, and the electricity generation is influenced;
the 3# is Shewanella, Escherichia coli (zymogen 1, which produces micromolecular acid), Escherichia coli (zymogen 2 which produces riboflavin) (electrogenic bacteria, wherein the pH value of the electrogenic bacteria is better than that of the 2# and is 1:1, but the pH value is still slightly acidic, so that the initial electric quantity of a system is increased quickly, but the electrogenic bacteria cannot produce electricity or even die due to accumulation of micromolecular acid produced by the zymogen and not the living and electricity-producing environment of the electrogenic bacteria.
The No. 4 is Shewanella, Escherichia coli (zymogen 1, producing small molecular acid) and Escherichia coli (zymogen 2 producing riboflavin) (electrogenic bacteria: zymogen more than 20:1), the proportion of the zymogen is small, the acid production amount is small, the influence on the system is small compared with that of No. 3, and the electric quantity is higher. Through optimization, 1 XHEPES is added, the pH value of the system is ensured to be about a neutral range, the system is suitable for the survival of the electrogenic bacteria, the proportion of zymogens in the system is reduced, the excessive accumulation (slow generation and slow consumption) of the micromolecule acid at the beginning is avoided, the electrogenic time is prolonged, and the result is shown in figure 2.
The glucose concentration is mainly influenced by the amount of acid produced, the total amount of small molecular acid required by the electrogenic bacteria is small, and the initial concentration of glucose of 10g/L is too high to continue the test. Gradually reduced to below 10 g/L. (the curve of the consumption of glucose and the lactic acid production by intermediate sampling is basically that lactic acid is always available and sufficient), so that the reduction of the glucose amount is to control the acid production amount and prevent the pH of the system from being influenced by acid accumulation caused by over metabolism of the electrogenic bacteria.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The mixed flora is characterized by comprising zymocyte and electrogenesis bacteria.
2. The mixed consortium of claim 1, wherein the fermentation tubes comprise high riboflavin-producing bacteria.
3. The mixed population of claim 1 or 2, wherein the fermentation tubes comprise tubes capable of producing small acid molecules using pentose, hexose, cellobiose as a carbon source.
4. The mixed population according to any one of claims 1 to 3, wherein said fermentation tubes are E.coli;
the electrogenic bacteria are Shewanella.
5. The mixed consortium of any one of claims 1 to 4, wherein the inoculation ratio of said riboflavin-highly producing bacteria to said electricity-producing bacteria is not more than 1: 1.
6. The mixed flora according to claim 3, wherein the ratio of inoculation of the bacteria for producing the small molecular acid by using the pentose and the hexose as carbon sources to inoculation of the electrogenic bacteria is not more than 1: 20.
7. use of a mixed flora according to any of claims 1 to 6 for converting chemical energy into electrical energy.
8. A microbial power generation system comprising a mixed population according to any one of claims 1 to 6.
9. The microbial electrogenesis system of claim 8, wherein the mixed population has an inoculum size of electrogenesis bacteria of no more than 4OD in the microbial electrogenesis system.
10. A microbial fuel cell comprising a mixed population according to any one of claims 1 to 6 or a microbial power generation system according to claim 8 or 9.
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