CN106754589B - Mixed flora and application thereof, and microbial power generation system and microbial fuel cell containing mixed flora - Google Patents

Abstract

The present invention relates to the field of microbial electricity generation, in particular to a mixed bacterium group and its use, a microbial electricity generation system and a microbial fuel cell containing the mixed microbial group. In the present invention, by constructing an engineered mixed bacteria group, it has the characteristics of clear division of labor. The use of electrogenic bacteria expands the carbon source spectrum of electrogenic bacteria. The present invention selects Escherichia coli or Bacillus subtilis, the model strains of prokaryotic gram-negative bacteria and gram-positive bacteria, and constructs two kinds of fermentation bacteria with different purposes by means of genetic engineering modification and the like, and expands the microbial fuel cell. The carbon source spectrum enhanced the electron transfer efficiency of electrogenic bacteria. At the same time, from the three important perspectives of material flow, energy flow and information flow, a stable, reasonable and efficient functional hybrid bacteria co-production system is constructed.

Description

Mixed flora and use thereof, microbial electricity-generating system and microbe containing the mixed flora biofuel cell

technical field

The present invention relates to the field of microbial electricity generation, in particular to a mixed bacterium group and its use, a microbial electricity generation system and a microbial fuel cell containing the mixed microbial group.

Background technique

Microbial Fuel Cell (MFC) is a device that uses microorganisms to directly convert chemical energy in organic matter into electrical energy. Its basic working principle is: in the anaerobic environment of the anode chamber, the organic matter is decomposed under the action of microorganisms and releases electrons and protons. It is effectively transferred between the anode and the anode electrode, and is transferred to the cathode through an external circuit to form a current, while the proton is transferred to the cathode through the proton exchange membrane, and the oxidant gets electrons at the cathode and is reduced and combined with protons to form water.

Compared with other existing technologies that utilize organic matter for energy production, microbial fuel cells have operational and functional advantages: first, it directly converts the substrate into electricity, ensuring high energy conversion efficiency; second, different from the current For all biological energy treatments, microbial fuel cells can operate effectively under ambient conditions at room temperature; thirdly, microbial fuel cells do not require exhaust gas treatment, because the main component of the exhaust gas it produces is carbon dioxide, which does not have the potential to be used under normal conditions. Reused energy; fourth, microbial fuel cells do not need to input large energy, because if a single-chamber microbial fuel cell only needs to be ventilated, the cathode gas can be passively supplemented; fifth, in local areas lacking power infrastructure, microbial fuel Batteries have the potential for a wide range of applications, while also expanding the diversity of fuels used to meet our energy needs.

In summary, microbial fuel cells are devices that convert chemical energy into electrical energy by using electricity-producing microorganisms that can transport electrons generated by metabolic reactions in cells out of cells through unique cell membrane proteins, and can overcome the environmental impact of renewable energy such as wind energy and solar energy. It plays an important role in the field of environmental treatment and new energy, and has a good development prospect. However, due to the harsh growth environment of electrogenic bacteria, low power generation efficiency and slow electron transfer process, industrial application has not been realized so far.

Electricity-producing bacteria dominated by Shewanella have been widely studied by scientists, and people have focused on the transformation of electricity-producing bacteria and the optimization of electrode materials, but the effect is not significant. For example: (1) Extensive and cheap substrates cannot be used as carbon sources (narrow carbon source spectrum); (2) In order to maintain electricity production, a large amount of riboflavin needs to be added externally, and the cost is high; (3) Genetically modified The electricity-producing bacteria grow more slowly, and the electricity-generating activity is not as good as that of the wild bacteria; (4) the duration is short and the electricity-producing is unstable; (5) the electricity-producing bacteria are relatively fragile, and increasing the metabolic burden of riboflavin will affect their production. (6) The multi-task of "fermentation-electricity production" is completed by a single electrogenic bacteria; (7) Complex mineral solution and multivitamin solution need to be added to the medium, which is costly and complicated to prepare. Existing microbial fuel cells need to continuously sample during the power generation process to detect the content of key substances in the system. If it is not enough, it needs to be added in time to maintain the activity of the bacteria and the stability of the system. The stability and repeatability of the system are poor, the duration of electricity generation is short, dozens of mineral and vitamin solutions need to be added externally, the cost is high, and the system is complex.

Therefore, it is of great practical significance to provide an efficient and stable microbial fuel cell.

SUMMARY OF THE INVENTION

In view of this, the present invention provides a mixed bacterial flora and its use, a microbial power generation system and a microbial fuel cell containing the mixed bacterial flora. The present invention has the characteristics of clear division of labor by constructing a mixed bacterial colony. Fermentation bacteria 1 provides the carrier riboflavin for electron transfer for the system, and fermentation bacteria 2 converts the most commonly used and low-cost glucose into small molecular acids and provides them to the electrogenic bacteria for use. . The invention selects Escherichia coli-prokaryotic model strain, constructs two kinds of fermentation bacteria with different purposes by means of genetic engineering and other means, expands the carbon source spectrum of the microbial fuel cell, and enhances the electron transfer efficiency of the electricity-producing bacteria. At the same time, from the three important perspectives of material flow, energy flow and information flow, a stable, reasonable and efficient functional mixed bacteria symbiosis system is constructed.

In order to achieve the above-mentioned purpose of the invention, the present invention provides the following technical solutions:

The invention provides a mixed bacteria group, which is characterized in that it includes fermentation bacteria and electrogenic bacteria.

In some specific embodiments of the present invention, the fermenting bacteria in the mixed flora include bacteria with high riboflavin production.

In some specific embodiments of the present invention, the fermenting bacteria in the mixed flora include bacteria that use penta-carbon sugar, hexa-carbon sugar, and cellobiose as carbon sources to produce small-molecule acids.

In the present invention, the small molecular acid is selected from lactic acid, formic acid, amino acid and the like. Any small molecule acid known to those skilled in the art falls within the protection scope of the present invention, which is not limited herein.

In the present invention, the process of generating electricity does not require external substances, using glucose as the carbon source, and the substrate can be extended to xylose, cellobiose, or even the treatment solution of cellulose, all within the protection scope of the present invention. The invention is not limited here.

In some specific embodiments of the present invention, the high riboflavin-producing bacteria in the mixed flora is Bacillus subtilis; the bacteria producing small molecular acids using penta-carbon sugar and hexa-carbon sugar as carbon sources are large intestine bacilli;

The electrogenic bacteria is Shewanella.

In some specific embodiments of the present invention, the inoculation ratio of the high riboflavin-producing bacteria to the electrogenic bacteria in the mixed flora is not greater than 1:1;

The inoculation ratio of the bacteria producing small-molecule acids with the five-carbon sugar and the six-carbon sugar as the carbon source and the electricity-generating bacteria is not more than 1:20.

In other specific embodiments of the present invention, the inoculation ratio of the fermenting bacteria and the electrogenic bacteria in the mixed flora is 1:20.

In some specific embodiments of the present invention, the inoculation ratio of the high riboflavin-producing bacteria to the electrogenic bacteria in the mixed flora is not less than 1:10000;

The inoculation ratio of the bacteria producing small-molecule acids with the five-carbon sugar and the six-carbon sugar as the carbon source and the electricity-generating bacteria is 1:20.

The present invention also provides the application of the mixed flora in converting chemical energy into electrical energy.

The present invention also provides a microbial electricity-generating system, including the mixed flora.

In some specific embodiments of the present invention, the inoculation amount of the mixed bacteria group in the microbial electricity generating system in the microbial electricity generating system is such that the electricity generating bacteria does not exceed 4OD.

In some specific embodiments of the present invention, the microbial electricity generation system further comprises nitrate.

In some specific embodiments of the present invention, the final concentration of the nitrate in the microbial electricity generation system in the microbial electricity generation system is not greater than 10 mM.

In some specific embodiments of the present invention, the pH value of the microbial electricity generating system is 6.2-7.2.

In some specific embodiments of the present invention, the microbial electricity generation system further comprises a buffer, and the buffer is HEPES. Preferably it is 1×HEPES.

In some specific embodiments of the present invention, the concentration of the carbon source in the microbial power generation system is 10 g/L.

In some specific embodiments of the present invention, 500 mL of the microbial power generation system includes 1.5 g of potassium dihydrogen phosphate, 8.55 g of potassium monohydrogen phosphate dodecahydrate, 0.25 g of sodium chloride, 0.5 g of ammonium chloride, and magnesium sulfate. 0.12g, calcium chloride 5.5mg.

The present invention also provides a microbial fuel cell, comprising the mixed bacteria group or the microbial power generation system.

The present invention has the characteristics of clear division of labor by constructing a mixed bacterial colony. Fermentation bacteria 1 provides the carrier riboflavin for electron transfer for the system, and fermentation bacteria 2 converts the most commonly used and low-cost glucose into small molecular acids and provides them to the electrogenic bacteria for use. . The invention selects Escherichia coli or Bacillus subtilis, and constructs two kinds of fermentation bacteria with different purposes by means of genetic engineering transformation, which expands the carbon source spectrum of the microbial fuel cell and enhances the electron transfer efficiency of the electrogenic bacteria. At the same time, from the three important perspectives of material flow, energy flow and information flow, a stable, reasonable and efficient functional mixed bacteria symbiosis system is constructed.

The microbial power generation system and the microbial fuel cell provided by the present invention solve the following problems:

(1) The microbial fuel cell itself has a short duration and unstable electricity production. It needs to add expensive substances such as substrates and electron carriers regularly to maintain a high electricity production;

(2) The substrate spectrum and carbon source spectrum of the electrogenic bacteria Shewanella are narrow, and can only use small molecules of lactic acid, formic acid, amino acids and other substances, but cannot use a wide range of carbon sources - five- and six-carbon sugars, such as glucose, Xylose, etc., cannot convert the chemical energy abundant in cellulose or the extensive and cheap glucose conversion into electricity;

(3) From the perspective of "system self-supply", develop an independent, stable and efficient microbial fuel cell system;

(4) The problem that different strains cannot be co-cultivated in mixed-bacteria fuel cells. How to make the synergistic effect between the electricity-producing bacteria and the fermenting bacteria to improve the electricity production and electricity production time of the system;

(5) How to select and engineer fermentation bacteria as the input of important substances.

The present invention can realize that only a certain amount of glucose is added at the beginning, and no external substances are added in the middle, and high-efficiency power generation can be realized for more than 100 hours.

The microbial electricity-generating system of the invention is simple, only needs a solution composed of no more than 10 basic and cheap salts, and the system does not need to add complex mineral salt solution and complex vitamin solution.

This patent breaks through the traditional single-bacteria transformation thinking in the field of microbial fuel cells, and uses the advantages and characteristics of the clear division of labor of mixed bacteria to construct a mixed power generation system of Escherichia coli-Bacillus subtilis-Shewanella. Compared with other fuels The battery has the advantages of high power, long duration, low cost, and no need for refilling in the middle.

In the construction process, two kinds of genetically modified Escherichia coli with different functions were used as fermentation bacteria to provide key substances for the system, which became the important material, energy and information driving force for the mutual communication, mutual symbiosis and driving functions between bacteria.

One of the Escherichia coli uses glucose to metabolize small molecular acids (lactic acid, formic acid, amino acids, etc.) for the electricity-producing bacteria to use, and the other engineered Bacillus subtilis produces riboflavin, which is used as the carrier of electron transfer for the electricity-producing bacteria. Improving the electron transfer efficiency can effectively increase the power production. At the same time, under the special conditions of electricity production, the electricity-producing bacteria can also promote the metabolic rate of the fermentation bacteria, so that the electricity output of the system lasts longer. In addition, the control of conditions such as mixed culture conditions and inoculation ratio also has an important impact on the power generation effect.

为在葡萄糖为碳源，单独希瓦氏菌的产电图，产电量极低，48小时候基本没电。

为三种菌混合的结果，效果明显。After a lot of groping, as shown in Figure 2: under the condition of mixed bacteria fuel cell culture,

For the electricity production diagram of Shewanella alone when glucose is the carbon source, the electricity production is extremely low, and there is basically no electricity in 48 hours.

For the result of mixing the three kinds of bacteria, the effect is obvious.

Our mixed bacteria system uses three species and three bacteria to achieve their co-energy.

(1) The entry points of material flow, energy and information flow riboflavin and small molecular acid are distributed into two kinds of fermentation bacteria. (The concept of thorough division of labor for mixed bacteria microbial fuel cells)

(2) Escherichia coli + Bacillus subtilis were selected as fermentation bacteria. The large intestine has its own unique advantages such as simple genetic manipulation, fast reproduction, and simple culture conditions. widely used engineering bacteria. Bacillus subtilis itself is a riboflavin-producing bacterium (it can be produced without genetic modification, but the highest riboflavin-producing subtilis in the world is used here), and it is a model bacterium of Gram-positive bacteria , the research is more extensive and thorough.

(3) The effect of the battery: the output power exceeds 520mV, and the power generation time exceeds 100 hours.

(4) The power generation process does not require external substances, uses glucose as the carbon source, and the substrate can be extended to the treatment solution of xylose, cellobiose, and even cellulose.

Compared with Escherichia coli, which produces high riboflavin, (1) functionally, Bacillus subtilis can provide more riboflavin; (2) from the perspective of system: the complexity of mixed flora is higher , more difficult to control; (3) the effect is better.

Description of drawings

In order to illustrate the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that are required in the description of the embodiments or the prior art.

示希瓦氏菌；

示希瓦氏菌+大肠杆菌；

示希瓦氏菌+大肠杆菌+大肠杆菌；Fig. 1 shows the power generation effect comparison diagram of Embodiment 5; wherein,

Shewanella;

Shewanella + Escherichia coli;

Shewanella + Escherichia coli + Escherichia coli;

示希瓦氏菌；

示希瓦氏菌+枯草芽孢杆菌+大肠杆菌；FIG. 2 shows a comparison chart of the power generation effect of Example 6; wherein,

Shewanella;

Shewanella + Bacillus subtilis + Escherichia coli;

Fig. 3 shows the pH value test paper diagram; wherein Fig. 3(A) refers to the pH value test paper control diagram; Fig. 3(B) refers to the pH value test paper diagram of Example 7;

示希瓦氏菌+小分子酸；

示希瓦氏菌+大肠杆菌；

示希瓦氏菌+大肠杆菌+大肠杆菌(产点菌比发酵菌＝20:1)；

示希瓦氏菌+大肠杆菌+大肠杆菌(产点菌比发酵菌＝10:1)；Figure 4 shows the effect of inoculation ratio and pH on the system in Example 7; wherein,

Shewanella + small molecule acid;

Shewanella + Escherichia coli;

Show Shewanella + Escherichia coli + Escherichia coli (the ratio of point-producing bacteria to fermenting bacteria = 20:1);

Show Shewanella + Escherichia coli + Escherichia coli (the ratio of point-producing bacteria to fermenting bacteria = 10:1);

FIG. 5 shows a diagram of a fuel cell device.

Detailed ways

The present invention discloses a mixed bacteria group and its use, a microbial power generation system and a microbial fuel cell containing the mixed bacteria group, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments, and it is obvious that relevant persons can make changes or appropriate changes and combinations of the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The mixed bacterial flora and its application provided by the present invention, as well as the raw materials and reagents used in the microbial power generation system and the microbial fuel cell containing the mixed bacterial flora can be purchased from the market.

Wherein, the construction method of the bacteria producing small molecular acids with penta-carbon sugar and six-carbon sugar as carbon sources is as follows: in order to enhance the ability of fermentation bacteria 2 to produce small molecular acids under anaerobic conditions, the present invention introduces ldhE into Escherichia coli Gene (lactic acid production gene, Lactobacillus origin), the pflB gene was knocked out by λ-Red homologous recombination technology;

The construction method of the high riboflavin-producing Escherichia coli is: introducing the ribABDEC gene cluster into the Escherichia coli;

The construction method of the high riboflavin-producing Bacillus subtilis is: overexpressing the prs and ywlF genes in the Bacillus subtilis, down-regulating the Pur operon and PurR regulatory genes (glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, andpbuO). The strains were constructed according to the article: Shi S, Chen T, Zhang Z, et al. Transcriptome analysisguided metabolic engineering of Bacillus subtilis for riboflavin production[J].Metabolic engineering,2009,11(4):243-252.

Below in conjunction with embodiment, the present invention is further elaborated:

Example 1 Mixed flora

Construction of Bacillus subtilis with high riboflavin production: through genetic manipulation, the prs and ywlF genes in Bacillus subtilis were overexpressed, and the Pur operon and PurR regulatory genes (glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO) were down-regulated. ).

Constructing Escherichia coli producing small molecular acids with five-carbon sugars and six-carbon sugars as carbon sources: using λ-Red homologous recombination technology to knock out the pflB gene in E. GENWIZE company), the method is: cut by EcoRI and PstI, connect to the pSB1C plasmid and then import the plasmid with the ldhE gene into the above-mentioned Escherichia coli with the pflB gene knocked out, screen for chloramphenicol resistance, and screen out the correct the transformant. Take the constructed Bacillus subtilis with high riboflavin production, and the constructed Escherichia coli and Shewanella that produce small molecular acids with five-carbon sugars and six-carbon sugars as carbon sources, according to:

The high riboflavin-producing Bacillus subtilis and the electrogenic bacteria Shewanella are mixed according to the inoculation ratio of not more than 1:1;

Escherichia coli producing small molecular acids using penta-carbon sugar and six-carbon sugar as carbon sources and the electrogenic bacteria Shewanella were mixed according to the inoculation ratio of no more than 1:20.

Example 2 Mixed flora

Construction of Bacillus subtilis with high riboflavin production: through genetic manipulation, the prs and ywlF genes in Bacillus subtilis were overexpressed, and the Pur operon and PurR regulatory genes (glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO) were down-regulated. ).

Constructing Escherichia coli producing small molecular acids with five-carbon sugars and six-carbon sugars as carbon sources: using λ-Red homologous recombination technology to knock out the pflB gene in E. GENWIZE company), the method is: cut by EcoRI and PstI, connect to the pSB1C plasmid and then import the plasmid with the ldhE gene into the above-mentioned Escherichia coli with the pflB gene knocked out, screen for chloramphenicol resistance, and screen out the correct the transformant.

Take the constructed Bacillus subtilis with high riboflavin production, and the constructed Escherichia coli and Shewanella that produce small molecular acids with five-carbon sugars and six-carbon sugars as carbon sources, according to:

The high riboflavin-producing Bacillus subtilis and the electrogenic bacterium Shewanella were mixed in an inoculation ratio of 1:20;

The Escherichia coli and the electrogenic bacteria Shewanella were mixed according to the inoculation ratio of 1:200 using penta-carbon sugar and hexa-carbon sugar as carbon source to produce small molecular acid.

Example 3 Mixed flora

Construction of Escherichia coli 1 with high riboflavin production: Introducing the ribABDEC gene cluster into Escherichia coli;

Construction of Escherichia coli 2 producing small molecular acids using penta- and six-carbon sugars as carbon sources: using λ-Red homologous recombination technology to knock out the pflB gene in E. , synthesized by GENWIZE company), the method is: cut by EcoRI and PstI, connect to the pSB1C plasmid and then import the plasmid with the ldhE gene into the above-mentioned Escherichia coli with the pflB gene knocked out, and screen for chloramphenicol resistance. correct transformant.

Take the constructed Escherichia coli 1 that produces high riboflavin, and the constructed Escherichia coli 2 that uses five-carbon sugars and six-carbon sugars as carbon sources to produce small molecular acids and mixes with Shewanella, according to:

The high riboflavin-producing Escherichia coli 1 and the electrogenic bacteria Shewanella are mixed in an inoculation ratio not exceeding 1:1;

Escherichia coli 2, which uses penta-carbon sugar and hexa-carbon sugar as carbon sources to produce small molecular acids, was mixed with the electrogenic bacteria Shewanella in an inoculation ratio of 1:20.

Example 4 Mixed flora

Construction of Escherichia coli 1 with high riboflavin production: Introducing the ribABDEC gene cluster into Escherichia coli;

Construction of Escherichia coli 2 producing small molecular acids using penta- and six-carbon sugars as carbon sources: using λ-Red homologous recombination technology to knock out the pflB gene in E. , synthesized by GENWIZE company), the method is: cut by EcoRI and PstI, connect to the pSB1C plasmid and then import the plasmid with the ldhE gene into the above-mentioned Escherichia coli with the pflB gene knocked out, and screen for chloramphenicol resistance. correct transformant.

Take the constructed Escherichia coli 1 that produces high riboflavin, and the constructed Escherichia coli 2 that uses five-carbon sugars and six-carbon sugars as carbon sources to produce small molecular acids and mixes with Shewanella, according to:

The high riboflavin-producing Escherichia coli 1 and the electrogenic bacteria Shewanella were mixed according to the inoculation ratio of 1:20;

Escherichia coli 2, which uses penta-carbon sugar and hexa-carbon sugar as the carbon source to produce small molecular acid, and the electrogenic bacteria Shewanella are mixed according to the inoculation ratio of no more than 1:200.

Example 5 Microbial power generation system

Cathode: potassium ferricyanide (purchased by sigma) solution;

Anode: respectively take Shewanella, Shewanella+Escherichia coli 2 (Escherichia coli producing small molecular acid with penta-carbon sugar and six-carbon sugar as carbon source), Shewanella+Escherichia coli prepared in Example 3 2 (Escherichia coli producing small molecular acids with five-carbon sugar and six-carbon sugar as carbon source) + Escherichia coli 1 (Escherichia coli with high riboflavin production);

A layer of proton exchange membrane (purchased by DuPont) is sandwiched between the two stages;

The wire in the middle is connected to a 2k resistor, and the porous carbon cloth is connected to the wire inside the bottle as an electrode (2.5cm x 2.5cm for the anode and 2.5cm x 3cm for the cathode).

Experiment sequence: (1) Assemble the anode and the cathode first (no proton exchange membrane is added in the middle), insert the electrode without connecting the resistance, sterilize (121 ℃ 15min) and dry after the assembly, add the prepared catholyte, and then Add the mixed culture solution to the anode (add HEPES, nitrate, and glucose to the prepared basic medium), calculate the concentration of the cultured bacteria, centrifuge, resuspend the bacteria with the culture solution in the anode, and add it to In the battery, the order of addition is Shevards-Escherichia coli-Escherichia coli, and finally the resistor is installed. Put it into a 30°C incubator for cultivation, take it out every 2-15 hours, and measure the voltage with a multimeter. In the process of exploring the conditions, take about 500uL of liquid from the bottle every 4 hours, remove the bacteria, filter, and use HPLC (high performance liquid chromatography) to detect the content of key metabolites, and use pH test paper to detect Whether the pH of the system is in a neutral state.

Among them, the culture medium includes:

Result: After the battery is connected, put it into a 30°C incubator, take it out every 4 or 8 hours, record the data with a multimeter, record the voltage value recorded by the multimeter each time, and draw a time-voltage curve.

See Table 1 and Figure 1.

Table 1 Data Results

It can be seen from Figure 1 that the electricity-producing bacteria cannot utilize glucose, the electricity production is very low, no more than 100mV, and the electricity production basically stops after 48 hours. Under the co-cultivation conditions of Shewanella and Escherichia coli, the maximum power output reaches 250mV, and it is stable for nearly 100 hours at 200mV. ) of the microbial fuel cell has a good increase in electricity production (over 350mV), and the duration is more than 100 hours.

Example 6 Microbial power generation system

Cathode: potassium ferricyanide (purchased by sigma) solution;

Anode: respectively take Shewanella, Shewanella+Escherichia coli (Escherichia coli (Escherichia coli producing small molecular acid with penta-carbon sugar and six-carbon sugar as carbon source), Shewanella+Escherichia coli (Escherichia coli) prepared in Example 1 Escherichia coli producing small molecular acids with five-carbon sugar and six-carbon sugar as carbon source) + Bacillus subtilis (Bacillus subtilis with high riboflavin production);

A layer of proton exchange membrane (purchased by DuPont) is sandwiched between the two stages;

The lead wire in the middle is connected to a 2k resistor, and the porous carbon cloth is connected to the lead wire in the bottle (the anode is 2.5cm×2.5cm, and the cathode is 2.5cm×3cm).

Experiment sequence: (1) Assemble the anode and the cathode first (no proton exchange membrane is added in the middle), insert the electrode without connecting the resistance, sterilize (121 ℃ 15min) and dry after the assembly, add the prepared catholyte, and then Add the mixed culture solution to the anode (add HEPES, nitrate, and glucose to the prepared basic medium), calculate the concentration of the cultured bacteria, centrifuge, resuspend the bacteria with the culture solution in the anode, and add it to In the battery, the order of addition is Shewanella-Escherichia coli-Bacillus subtilis, and finally the resistor is installed. Put it into a 30°C incubator for cultivation, take it out every 2-15 hours, and measure the voltage with a multimeter. In the process of exploring the conditions, take about 500uL of liquid from the bottle every 4 hours, remove the bacteria, filter, and use HPLC (high performance liquid chromatography) to detect the content of key metabolites, and use pH test paper to detect Whether the pH of the system is in a neutral state.

Among them, the culture medium includes:

Result: After the battery is connected, put it into a 30°C incubator, take it out every 4 or 8 hours, record the data with a multimeter, record the voltage value recorded by the multimeter each time, and draw a time-voltage curve.

See Table 2 and Figure 2.

Table 2 Data Results

As shown in Figure 2, the electricity-producing bacteria could not utilize glucose, the electricity production was very low, no more than 100mV, and the electricity production basically stopped after 48 hours. The microbial fuel cells of the three bacteria (fermenting bacteria are Escherichia coli and Bacillus subtilis, and electricity-producing bacteria are Shewanella) invented by this patent have a good improvement in electricity production (the maximum can reach 520mV), and the duration exceeds 520mV. 100 hours.

Example 7 Optimization of fuel cell system

The fuel cell consists of two bottles of yin and yang (see Figure 5 for the device diagram), a solution of potassium ferricyanide (purchased by Sigma) as a cathode, a proton exchange membrane (purchased by DuPont) sandwiched between the two bottles, and a bacterial solution as the anode. The lead wire in the middle is connected to a 2k resistor, and the porous carbon cloth is connected to the lead wire in the bottle (the anode is 2.5cm×2.5cm, and the cathode is 2.5cm×3cm). Experiment sequence: (1) Assemble the bottle first (no proton exchange membrane is added in the middle), insert the electrode without connecting the resistance, sterilize (121 ℃ 15min) and dry after the assembly, add the prepared catholyte, and then put the anode on the anode. Add the mixed culture solution (add HEPES, nitrate, and glucose to the prepared basic medium), calculate the concentration of the cultured cells, centrifuge, resuspend the cells with the culture solution in the anode, and add it to the battery , the order of addition is Shewanella - Escherichia coli - Escherichia coli (or Bacillus subtilis), and finally a resistor is installed. Put it into a 30°C incubator for cultivation, take it out at regular intervals, and measure the voltage with a multimeter. In the process of exploring the conditions, about 500uL of liquid was taken from the bottle every 4h, the bacteria were removed, filtered, and the content of key metabolites was detected by HPLC (high performance liquid chromatography), and the pH test paper detection system was used at the same time. Whether the pH is in a neutral state (because the tolerance range of electrogenic bacteria is pH: 6.2-7, the pH should generally be controlled above 6.5-6.8).

Among them, the culture medium includes:

Bacteria liquid:

1# is Shewanella + small molecule acid (externally added sodium lactate);

2# is Shewanella + Escherichia coli (fermentation bacteria 1, producing small molecular acid)

3# is Shewanella+Escherichia coli (fermentation bacteria 1, producing small molecular acid)+Escherichia coli (fermentation bacteria 2 producing riboflavin)—preparation in Example 3;

4# is Shewanella+Escherichia coli (fermentation bacteria 1, producing small molecular acid)+Escherichia coli (fermentation bacteria 2 producing riboflavin)—prepared in Example 1;

Result: Record the voltage value recorded by the multimeter each time, and draw it into a time-voltage curve. The results are shown in Figure 4.

Determine the effect of inoculation ratio and pH value on fuel cell electricity production:

Measure the pH of the system, as shown in Figure 3:

The pH of the battery anode was detected. The results are shown in Figure 3. Shewanella No. 1 + small molecular acid, the system is basically neutral, and the pH of the Shewanella No. 2 + Escherichia coli system is compared with the standard pH test paper, and the pH is considered to be low. In 5.4, No. 3 Shewanella Shewanella + Escherichia coli (fermentation bacteria 1, producing small molecular acids) + Escherichia coli (fermentation bacteria 2 producing riboflavin) (electricity bacteria: fermentation bacteria = (1～10 ):1), compared with the standard pH test paper, the pH is close to 5.4, which is more acidic. 4# is Shewanella + Escherichia coli (fermentation bacteria 1, producing small molecular acid) + Escherichia coli (fermentation bacteria 2 producing riboflavin) (electricity bacteria: fermentation bacteria greater than 20:1), and the standard pH test paper The pH is close to 6.2, which is still slightly acidic.

in conclusion:

1# is Shewanella + small molecule acid (externally added sodium lactate), and the pH of the system is neutral;

2# is Shewanella + Escherichia coli (fermentation bacteria 1, producing small molecular acid), the pH is low, which affects electricity production;

3# is Shewanella + Escherichia coli (fermentation bacteria 1, producing small molecular acid) + Escherichia coli (fermentation bacteria 2 producing riboflavin) (electrogenic bacteria: fermentation bacteria = (1～10):1), pH It is better than 2#, but it is still more acidic, which causes the power of the system to rise rapidly at the beginning. However, due to the accumulation of small molecular acids produced by fermentation bacteria, it is not the environment for the survival and production of electricity, resulting in the failure of electricity-producing bacteria to produce electricity or even die.

4# is Shewanella + Escherichia coli (fermentation bacteria 1, producing small molecular acid) + Escherichia coli (fermentation bacteria 2 producing riboflavin) (electric bacteria: fermentation bacteria greater than 20:1), the proportion of fermentation bacteria is small , the acid production is less, the impact on the system is smaller than that of 3#, and the electricity is higher. After optimization, 1 × HEPES was added to ensure that the pH of the system was in the neutral range, which was suitable for the survival of electrogenic bacteria. Slow consumption), prolonging the power generation time, the results are shown in Figure 2.

Regarding the glucose concentration, it is mainly the effect on the amount of acid production. The total amount of small molecular acid required by the electrogenic bacteria is very small. The initial concentration of glucose of 10g/L was tried, but the small molecular acid produced was too much, and the experiment could not be continued. Gradually decreased to below 10g/L. (The curve of glucose consumption and lactic acid production is measured by sampling in the middle. The basic situation is that lactic acid is always available and sufficient), so reducing the amount of glucose is to control the amount of acid production and prevent the electricity-producing bacteria from metabolizing too much. Acid accumulation affects system pH.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

Claims (7)

1. a mixed bacterial group, is characterized in that, comprises fermentation bacteria and electrogenic bacteria; The fermentation bacteria include bacteria that produce high riboflavin; The fermentation bacteria also include bacteria capable of using penta-carbon sugar, hexa-carbon sugar and cellobiose as carbon sources to produce small molecular acids; The bacterium that produces high riboflavin is Bacillus subtilis; the bacterium that uses penta-carbon sugar and hexa-carbon sugar as carbon source to produce small molecular acid is Escherichia coli; The electrogenic bacteria is Shewanella; The construction method of the Escherichia coli producing small molecular acids using five-carbon sugars and six-carbon sugars as carbon sources is as follows: using λ-Red homologous recombination technology in Escherichia coli to knock out the pflB gene, and then introducing lactobacillus-derived lactic acid producing gene ldhE gene; The construction method of the high riboflavin-producing Bacillus subtilis is: overexpressing the prs and ywlF genes in the Bacillus subtilis, down-regulating the Pur operon and PurR regulatory genes glyA, guaC, pbuG, xpt-pbuX, yqhZ-folD, and pbuO . 2. mixed flora according to claim 1, is characterized in that, the inoculation ratio of the bacteria of described high riboflavin production and described electrogenic bacteria is not more than 1: 1. 3. mixed bacterial colony as claimed in claim 2, is characterized in that, the bacteria inoculation ratio of described taking five-carbon sugar, six-carbon sugar as carbon source to produce small molecule acid and described electrogenic bacteria is not more than 1: 20. 4. The application of the mixed flora according to any one of claims 1 to 3 in converting chemical energy into electrical energy. 5 . A microbial electricity-generating system, characterized in that, comprising the mixed flora as claimed in any one of claims 1 to 3. 6 . 6 . The microbial electricity-generating system according to claim 5 , wherein the inoculation amount of the mixed bacteria group in the microbial electricity-generating system is that the electricity-producing bacteria does not exceed 4OD. 7 . 7 . A microbial fuel cell, characterized in that it comprises the mixed flora as claimed in any one of claims 1 to 3 or the microbial power generation system as claimed in claim 5 or 6 .

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