CN108285881B

CN108285881B - Mycobacterium with synchronous electricity generation and denitrification activity and application thereof

Info

Publication number: CN108285881B
Application number: CN201810013537.XA
Authority: CN
Inventors: 刘鸿; 金小君; 王川
Original assignee: Guangzhou University; Chongqing Institute of Green and Intelligent Technology of CAS
Current assignee: Guangzhou University; Chongqing Institute of Green and Intelligent Technology of CAS
Priority date: 2018-01-04
Filing date: 2018-01-04
Publication date: 2021-06-08
Anticipated expiration: 2038-01-04
Also published as: CN108285881A

Abstract

The invention relates to a mycobacterium with synchronous electricity generation and denitrification activity and application thereof, belonging to the technical field of microorganisms. The Mycobacterium of the invention is named as Mycobacterium sp.eb-1, which has been deposited in the chinese type culture collection at 26 months 6 and 2017, address: china, wuhan university, zip code: 430072, abbreviated as CCTCC, registration number of preservation center is CCTCC NO: m2017371. The invention discovers for the first time that the Mycobacterium (Mycobacterium sp) EB-1 is an electrogenesis bacterium with high electrochemical activity and denitrification activity, the strain is a facultative anaerobe, and can utilize a plurality of carbon sources to generate electricity, thereby not only expanding the range of electrogenesis microbes, improving the anaerobic experimental conditions and the substrate broad spectrum, but also efficiently removing the nitrate nitrogen pollution in the wastewater; the invention is applied to MFC to realize the synchronous energy recovery and nitrogen pollution removal of nitrogen-containing wastewater, and lays a foundation for the engineering application of MFC in actual wastewater.

Description

Mycobacterium with synchronous electricity generation and denitrification activity and application thereof

Technical Field

The invention belongs to the technical field of microorganisms, and particularly relates to a mycobacterium with synchronous electricity generation and denitrification activity and application thereof.

Background

Microbial Fuel Cells (MFCs) are becoming a research hotspot in the field of new energy sources as new-generation, green and environmentally-friendly electricity generation devices for non-petroleum renewable energy sources developed in recent years. The MFC has the working principle that anode microorganisms catalyze and oxidize organic matters and release electrons and protons under the metabolism action of the microorganisms under the anaerobic condition, the electrons are conducted to a cathode from an anode through an external circuit, and the electrons are received by the cathode through catalysis of a catalyst to generate a reduction reaction, so that the removal of the organic matters and the generation of current are realized, and chemical energy is directly converted into electric energy.

Compared to other fuel cells, MFC has a relatively low power density and is difficult to replace other energy sources to meet human demand for energy. Therefore, more and more researchers of MFC technology are applied to the field of sewage treatment, the defects of high energy consumption and large sludge yield of traditional sewage treatment are overcome, and potential chemical energy of a large number of organic matters in sewage can be effectively recovered. In addition, according to the national regulation of the integrated wastewater discharge standard, two key indexes in the primary wastewater discharge standard are that the COD concentration does not exceed 50mg/L and the total nitrogen concentration does not exceed 20 mg/L. In the process of treating sewage by the traditional biological method, the efficient removal and standard discharge of nitrate nitrogen are always an important problem in sewage treatment.

For MFC, the influence of anode electrogenic microorganisms as biocatalysts for organic matter degradation plays a key role in MFC electrogenic efficiency and energy recovery. Therefore, the mining of more microorganisms with the function has important significance for enriching the diversity of the electricity-generating microorganisms and improving the electricity-generating efficiency. By screening the electrogenesis microorganisms with high electrogenesis activity and denitrification activity and applying the electrogenesis microorganisms to the MFC, the aim of synchronously removing organic matters and nitrate nitrogen can be fulfilled, and the subsequent secondary treatment on nitrogen source pollution is reduced.

Mycobacteria are a genus of bacteria that are widely present in the environment, and the cells stain gram-positive, facultative anaerobic. At present, no relevant report is found about the electricity generation characteristics of the microorganism of the genus and the application of the microorganism in MFC at home and abroad.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a mycobacterium with synchronous electricity generation and denitrification activity and application thereof.

In order to achieve the purpose, the invention adopts the technical scheme that:

in a first aspect, the present invention provides a Mycobacterium named Mycobacterium sp.eb-1 (hereinafter referred to as strain EB-1), which has been deposited at the chinese collection of type cultures at 26 months 6 and 2017, address: china, wuhan university, zip code: 430072, abbreviated as CCTCC, registration number of preservation center is CCTCC NO: m2017371.

The bacterial strain EB-1 of the invention is facultative anaerobe, rod-shaped and gram-positive. Culturing in dark on tryptone soybean broth culture medium plate at 30 deg.C under aerobic condition for 48h to form round milky colony which is easy to pick, opaque, and uniform in edge, and synthesizing pigment under illumination condition to obtain yellow colony.

The strain EB-1 of the invention has the obvious characteristics of high electroproduction activity, and can transfer electrons generated by metabolic organic matters to the anode of MFC, realize the degradation of the organic matters and generate electric energy. When sodium acetate is used as an electron donor, the maximum power density of the MFC reaches 835.5mW/m². The strain has another remarkable characteristic of synchronous electrocatalytic activity and nitrate reduction activity, and when the anolyte takes sodium acetate (COD is 700mg/L) as an electron donor and the nitrate nitrogen concentration is 20, 50, 100 and 200mg/L respectively, the MFC still has high electroproduction activity and denitrification rate.

In a second aspect, the present invention provides the use of a mycobacterium as described above for denitrification and/or catalysing the degradation/decomposition of organic matter. The mycobacterium of the invention can utilize a plurality of carbon sources, especially macromolecular organic matters, to generate electricity, and the strain has huge application potential in treating actual wastewater; the bacterial strain can reduce nitrate nitrogen in the wastewater into nitrogen through denitrification reaction, and can keep high-efficiency denitrification rate and electrogenesis activity in the process of treating the wastewater with low carbon-nitrogen ratio.

As a preferred embodiment of the use of the Mycobacterium of the present invention in denitrification and/or catalytic degradation/decomposition of organic matter, the Mycobacterium is used in wastewater/sewage treatment.

In a third aspect, the present invention provides the use of a mycobacterium as described above for electricity generation and/or denitrification in a microbial fuel cell. In the microbial fuel cell, the mycobacterium of the present invention is used as an electricity-producing microorganism.

As a preferred embodiment of the application of the mycobacterium in power generation and/or denitrification of a microbial fuel cell, the electron donor of the mycobacterium is an organic substance.

In a preferred embodiment of the above application of the present invention, the organic substance is at least one of sodium acetate, butyric acid, glucose, sucrose and starch.

In a fourth aspect, the invention also provides a microbial fuel cell comprising the mycobacterium as described above.

In a preferred embodiment of the microbial fuel cell of the present invention, the microbial fuel cell further comprises an electron donor of a mycobacterium, wherein the electron donor of the mycobacterium is an organic substance; more preferably, the organic substance is at least one of sodium acetate, butyric acid, glucose, sucrose and starch.

As a preferred embodiment of the microbial fuel cell of the present invention, the microbial fuel cell comprises an anode chamber, a cathode chamber and an external circuit for connecting the anode chamber and the cathode chamber; the anode chamber comprises an anode, anolyte and mycobacteria serving as an anode catalyst, wherein the anolyte contains the organic matter and an inorganic salt culture medium.

In a preferred embodiment of the microbial fuel cell of the present invention, the anolyte further contains nitrate nitrogen.

As a preferred embodiment of the microbial fuel cell of the present invention, the inorganic salt medium contains the following components in concentrations: potassium nitrate 0.144-1.44 g/ml/or pH valueL, 0.8-8.1 g/L of sodium dihydrogen phosphate dihydrate, 2.1-22 g/L of disodium hydrogen phosphate dodecahydrate and 10mL/L of Wolfes mineral solution, wherein the pH value of the inorganic salt culture medium is adjusted to 7 by HCl/NaOH solution; wherein the Wolfes mineral solution contains the following components in concentration: aminoacetic acid 1.5g/L, MgSO₄·7H₂O 3g/L，MnSO₄·2H₂O 0.5g/L,NaCl 1.0g/L, FeSO₄·7H₂O 0.1g/L,CoCl₂ 0.1g/L,CaCl₂ 0.1g/L,ZnSO₄0.1g/L,CuSO₄·5H₂O 0.01g/L,AlK(SO₄)₂ 0.01g/L,H₃BO₃ 0.01g/L,Na₂MoO₄·2H₂O0.01 g/L, the Wolfes mineral solution is adjusted to pH 7 by KOH.

In a preferred embodiment of the microbial fuel cell of the present invention, the culture temperature of the microbial fuel cell is 30 ℃.

As a preferred embodiment of the microbial fuel cell of the present invention, the anode is a carbon felt.

As a preferred embodiment of the microbial fuel cell of the present invention, the cathode compartment comprises a cathode and a catholyte; preferably, the cathode is a Pt/C catalyzed air cathode, and the catholyte is 0.1mol/L phosphate buffer.

As a preferred embodiment of the microbial fuel cell of the invention, the anode and cathode compartments are separated by a cation exchange membrane.

Compared with the prior art, the invention has the beneficial effects that: the invention discovers for the first time that the Mycobacterium (Mycobacterium sp) EB-1 is an electrogenesis bacterium with high electrochemical activity and denitrification activity, the strain is a facultative anaerobe, and can utilize a plurality of carbon sources to generate electricity, thereby not only expanding the range of electrogenesis microorganisms, improving the anaerobic experimental conditions and the substrate broad spectrum, but also efficiently removing the nitrate nitrogen pollution in the wastewater. The invention is applied to MFC to realize the synchronous energy recovery and nitrogen pollution removal of nitrogen-containing wastewater, and lays a foundation for the engineering application of MFC in actual wastewater.

Drawings

FIG. 1 is a plate colony morphology diagram of strain EB-1 of the present invention under dark culture;

FIG. 2 is an electron microscope image of the attachment of the bacterial strain EB-1 of the present invention on the MFC anode surface;

FIG. 3 is an electron microscope image (enlarged) of the attachment of the bacterial strain EB-1 of the present invention on the MFC anode surface;

FIG. 4 is a graph showing the voltage output of the strain EB-1 of the present invention in MFC;

FIG. 5 is a plot of the polarization curve and power density of the strain EB-1 of the present invention in MFC;

FIG. 6 is a graph showing the results of reduction of nitrate nitrogen in MFC by the strain EB-1 of the present invention;

FIG. 7 is a graph showing the results of energy recovery of the strain EB-1 of the present invention in MFC for different electron donors.

Detailed Description

To better illustrate the objects, aspects and advantages of the present invention, the present invention will be further described with reference to the accompanying drawings and specific embodiments.

The Mycobacterium of the invention is named as Mycobacterium sp. EB-1 (hereinafter referred to as strain EB-1), and has been deposited in China center for type culture Collection in 6 month and 26 month in 2017, with the address: china, wuhan university, zip code: 430072, abbreviated as CCTCC, registration number of preservation center is CCTCC NO: m2017371.

EXAMPLE 1 isolation and characterization of Strain EB-1

(1) Screening and enriching of strains: strain EB-1 is screened to a double-chamber MFC system which runs stably for a long time in a laboratory, a carbon felt with an anode and electrogenesis activity is taken out and placed in a sterilized normal saline solution, and microorganisms attached to the surface of the anode are dispersed by ultrasound. And (3) under an aseptic condition, diluting the obtained bacterial suspension, coating the diluted bacterial suspension on a tryptone soybean broth solid culture medium, carrying out aerobic culture for 48h, picking a single bacterial colony according to the difference of bacterial colonies of the thalli, and carrying out streaking separation for multiple times to obtain a pure bacterial strain. Further, inoculating the pure strain into an anolyte solid culture medium taking sodium acetate as a carbon source, placing the pure strain into an anaerobic incubator for culturing for a plurality of days at 30 ℃, and observing the colony morphology and the growth condition thereof. Selecting strains which can grow under aerobic and anaerobic conditions, respectively inoculating the strains into tryptone soybean broth liquid culture medium for enlarged culture, and centrifugally collecting thalli.

The anode liquid culture medium comprises the following components: 1g/L of sodium acetate, 0.38g/L of potassium nitrate, 6.08g/L of sodium dihydrogen phosphate dihydrate, 21.85g/L of disodium hydrogen phosphate dodecahydrate and 10mL/L of Wolfes mineral solution, wherein the pH value of the inorganic salt culture medium is adjusted to 7 by HCl/NaOH solution; wherein the Wolfes mineral solution consists of the following components in concentration: : aminoacetic acid 1.5g/L, MgSO₄·7H₂O 3g/L，MnSO₄·2H₂O 0.5g/L, NaCl 1.0g/L,FeSO₄·7H₂O 0.1g/L,CoCl₂ 0.1g/L,CaCl₂ 0.1g/L,ZnSO₄ 0.1g/L, CuSO₄·5H₂O 0.01g/L,AlK(SO₄)₂ 0.01g/L,H₃BO₃ 0.01g/L,Na₂MoO₄·2H₂O0.01 g/L, Wolfes mineral solution was adjusted to pH 7 by KOH.

The solid culture medium of the anolyte is prepared by adding agar 15-20 g/L.

(2) MFC parts pre-treatment and assembly: the anode chamber, cathode chamber, gasket and plug in the MFC components were soaked overnight in 5% hydrogen peroxide solution and rinsed with sterile water for future use. Respectively soaking the anode carbon felt in an ethanol-acetone mixed solution overnight, cleaning, soaking ammonium persulfate for 15min, cleaning, roasting at high temperature, and carrying out 5% NH treatment₃And after the/Ar roasting, placing the mixture in a super clean bench for ultraviolet irradiation for 30min for later use. The air cathode is respectively treated by a waterproof layer PDMS of carbon cloth and a Pt/C load (0.5 mg/cm) of a non-waterproof layer²) And then placing the mixture in a super clean bench for ultraviolet irradiation for 30min for later use. Soaking the cation exchange membrane in 5% hydrogen peroxide solution for 2h, washing with sterile water, and soaking in sterilized 0.1M PBS solution for use. All the components of the MFC are assembled on a sterile super clean bench to form a double-chamber air cathode microbial fuel cell.

(3) Inoculation and MFC run: introducing high-purity nitrogen into the anode solution after high-temperature sterilization under the aseptic condition to discharge oxygen, and adding the anode solution into the anode chamber; the cathode compartment was charged with a autoclaved 0.1M PBS solution. Respectively picking the thalli obtained by centrifugation in the step (1) by an inoculating needle into an anode chamber, and observing that the anode chamber is inoculated with obvious bacterial suspension. The titanium wire is connected with an external circuit and a cathode and an anode, the external circuit is connected with a 1K omega resistor, and two ends of the resistor are connected to a multi-channel voltage tester. The computer controls the data acquisition mode and stores the acquired data to obtain a voltage curve which changes along with time.

(4) Identification of the electrogenic bacteria: selecting an inoculation strain corresponding to the MFC with the highest voltage output, named as EB-1, and performing colony morphology identification, physiological and biochemical identification and molecular identification on the strain respectively, wherein the results are as follows:

and (3) morphological identification: after the plate is cultured in a constant temperature incubator for 48 hours in the dark, the surface of the colony is smooth and opaque, and is milk white, as shown in figure 1; under the stimulation of illumination at room temperature, the bacterial colony becomes yellow and takes the shape of a rod under a microscope; the form of the attachment to the anode surface of the MFC is shown in fig. 2 and 3. Gram staining positive; culturing in inorganic salt solid culture medium under anaerobic condition for 3-5 days to obtain white fine particles.

Physiological and biochemical identification: facultative anaerobes; glucose, starch, protein and other substances can be used as carbon sources; positive in nitrate reduction experiment; positive in Methyl Red (MR) test; the V-P reaction test is negative.

And (3) molecular identification: after the strain EB-1 is cultured under aerobic and anaerobic conditions, colonies are picked, DNA extraction and PCR amplification are carried out to obtain 16s rRNA base sequences which are consistent, homology analysis is carried out in an NCBI database, and the similarity of the strain and Mycobacterium fortuitum (NZ _ CP011269.1) is up to 99 percent by BLAST retrieval comparison. At present, no mycobacterium with the ability to produce electricity is reported. Therefore, this bacterium was named as Mycobacterium sp EB-1.

According to the identification result, the strain EB-1 belongs to the mycobacterium and is preserved, wherein the preservation unit is China center for type culture Collection, address: china, wuhan university, zip code: 430072, abbreviated as CCTCC, registration number of preservation center is CCTCC NO: m2017371.

EXAMPLE 2 verification of MFC Electricity Generation and electrochemical Properties of Strain EB-1

MFC was started up to run as in (2) and (3) in example 1, and electricity was suppliedThe voltage output situation is shown in fig. 4, after three cycles of operation, the output voltage is stabilized in the fourth cycle, which means that the electrochemical activity of the strain EB-1 in MFC is stabilized, and the start is considered to be successful. And entering a formal operation stage after successful start, and replacing the anolyte under an aseptic condition when the detected output voltage is lower than 20mV, wherein one cycle is about 3 days generally. Further, after replacing the new anolyte, when the output voltage reaches the maximum and tends to be stable again, the test of the MFC polarization curve and the power density curve is carried out by changing the external resistance, as shown in FIG. 5, the maximum power density reaches 835.5mW/m²。

EXAMPLE 3 study of Simultaneous Electricity production and Denitrification Activity of Strain EB-1

MFC was started up according to (2) and (3) in example 1 until the voltage output was stable and the maximum power density reached example 2, the fixed sodium acetate concentration in the anolyte composition was 1g/L (COD was about 700mg/L), the other inorganic salt components were unchanged, and the effect of different C/N ratios on the voltage output and anode denitrification reaction of the strain EB-1 in MFC was observed by changing the nitrate nitrogen concentrations in the anolyte to 0, 20, 50, 100 and 200mg/L, respectively.

As a result, as shown in fig. 6, the power generation cycle becomes shorter as the nitrate nitrogen concentration increases, but the maximum output voltage hardly changes. The result means that the electrochemical activity of the strain EB-1 is not inhibited by the concentration of nitrate nitrogen, and the main reason for shortening the period is that sodium acetate in the anolyte is consumed because organic matters are required to provide an electron donor in the denitrification process, so that the effective electron output is reduced, and the coulombic efficiency is reduced. Under different C/N ratios of the strain EB-1, COD removal, nitrate nitrogen removal rate and energy recovery conditions are shown in table 1, and when the C/N ratio is more than 7, the nitrate nitrogen removal rate can reach 98.2 percent at most; when the C/N ratio is less than 3.5, the nitrate nitrogen removal rate is reduced to 82.8%, and the Coulombic Efficiency (CE) is only 6.5%. Therefore, when the bacterial strain EB-1 reduces nitrate nitrogen, the bacterial strain EB-1 and the anode compete together for electrons released by organic matter oxidation.

TABLE 1

Example 4 Electricity production characteristics and Denitrification Activity of Strain EB-1 Using different carbon sources

And (3) starting the MFC according to the (2) and (3) in the example 1 until the voltage output is stable and the maximum power density reaches the maximum power density of the MFC in the example 2, replacing the anolyte, and changing other components except the carbon source. The carbon source is formic acid, lactic acid, butyric acid, glucose, sucrose and starch. As a result, as shown in FIG. 7, the strain EB-1 can generate electricity by using butyric acid, glucose, sucrose and starch, but the efficiency of electricity generation is different. And the strain has low conversion efficiency on small molecular acid such as formic acid and lactic acid, and almost has no voltage output. The electricity generation characteristics of the mycobacterium EB-1 disclosed in the embodiment in the double-chamber air cathode MFC aim to disclose the electricity generation capability of the strain in utilizing various carbon sources, particularly macromolecular organic matters, and mean that the strain has great application potential in treating actual wastewater.

Example 5

In one embodiment of the microbial fuel cell of the present invention, the microbial fuel cell of this embodiment comprises an anode chamber, a cathode chamber and an external circuit for connecting the anode chamber and the cathode chamber, wherein the anode chamber and the cathode chamber are separated by a cation exchange membrane;

the anode chamber comprises an anode, anolyte and a strain EB-1 serving as an anode catalyst, wherein the anode is a carbon felt, and the anolyte contains organic matters, an inorganic salt culture medium and nitrate nitrogen; the organic matter is at least one of sodium acetate, butyric acid, glucose, sucrose and starch, and the inorganic salt culture medium comprises the following components in concentration: 0.144-1.44 g/L of potassium nitrate, 0.8-8.1 g/L of sodium dihydrogen phosphate dihydrate, 2.1-22 g/L of disodium hydrogen phosphate dodecahydrate and 10mL/L of Wolfes mineral solution, wherein the pH value of the inorganic salt culture medium is adjusted to 7 by HCl/NaOH solution; wherein the Wolfes mineral solution consists of the following components in concentration: aminoacetic acid 1.5g/L, MgSO₄·7H₂O 3g/L，MnSO₄·2H₂O 0.5g/L,NaCl 1.0g/L,FeSO₄·7H₂O 0.1 g/L,CoCl₂ 0.1g/L,CaCl₂ 0.1g/L,ZnSO₄ 0.1g/L,CuSO₄·5H₂O 0.01g/L,AlK(SO₄)₂ 0.01g/L,H₃BO₃ 0.01g/L,Na₂MoO₄·2H₂O0.01 g/L, the Wolfes mineral solution is adjusted to a pH value of 7 by KOH;

the cathode chamber comprises a cathode and catholyte, wherein the cathode is a Pt/C catalyzed air cathode, and the catholyte is 0.1mol/L phosphate buffer.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and not for limiting the protection scope of the present invention, and although the present invention is described in detail with reference to the preferred embodiments, it should be understood by those skilled in the art that modifications or equivalent substitutions can be made on the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention.

Claims

1. A mycobacterium, wherein the strain is a mycobacterium (M.) (Mycobacterium sp.) EB-1, with a preservation number of CCTCC NO: m2017371.

2. Use of the mycobacterium of claim 1 to catalyze the degradation of organic matter.

3. The use according to claim 2, wherein the mycobacterium is used in wastewater treatment.

4. Use of the mycobacterium of claim 1 for power generation and/or denitrification in a microbial fuel cell.

5. The use of claim 2, wherein the organic substance is at least one of sodium acetate, butyric acid, glucose, sucrose, starch.

6. A microbial fuel cell comprising the mycobacterium of claim 1.

7. The microbial fuel cell of claim 6, further comprising an electron donor for mycobacteria, wherein the electron donor for mycobacteria is an organic substance; the organic matter is at least one of sodium acetate, butyric acid, glucose, sucrose and starch.

8. The microbial fuel cell of claim 7, comprising an anode compartment, a cathode compartment, and an external circuit for connecting the anode compartment to the cathode compartment; the anode chamber comprises an anode, anolyte and mycobacteria serving as an anode catalyst, wherein the anolyte contains the organic matter and an inorganic salt culture medium.

9. The microbial fuel cell of claim 8, wherein the anolyte further comprises nitrate nitrogen.