CN108285880B

CN108285880B - Bacillus with electrogenesis characteristic and denitrification activity and application thereof

Info

Publication number: CN108285880B
Application number: CN201810013536.5A
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: 2021-03-30
Anticipated expiration: 2038-01-04
Also published as: CN108285880A

Abstract

The invention relates to bacillus with electrogenesis characteristic and denitrification activity and application thereof, belonging to the technical field of microbial technology and batteries. The Bacillus of the invention is named as Bacillus sp.EM-1, which is preserved in China center for type culture Collection in 2017, 6 and 26 months, and the preservation number is CCTCC NO: m2017372. The Bacillus (Bacillus sp.) EM-1 is an electrogenesis bacterium with efficient electrogenesis characteristics and denitrification activity, the strain is facultative anaerobe, and can utilize multiple carbon sources to generate electricity, thereby not only expanding the range of electrogenesis microbes, improving anaerobic experimental conditions and substrate broad spectrum, but also efficiently removing nitrate nitrogen pollution in wastewater.

Description

Bacillus with electrogenesis characteristic and denitrification activity and application thereof

Technical Field

The invention belongs to the technical field of microbial technology and batteries, and particularly relates to bacillus with electrogenesis characteristics and denitrification activity and application thereof.

Background

With the increasing severity of global warming and fossil fuel exhaustion, environmental protection and renewable energy research are receiving more and more attention and attention. Microbial Fuel Cells (MFCs) are becoming a research hotspot in the field of new energy sources as new-generation, green, pollution-free, non-petroleum renewable energy sources for recent development. MFC combines microbial technology and battery technology, utilizes the microorganism as biocatalyst and directly converts the chemical energy in the organic matter into electric energy, and has double effects of power generation and waste treatment.

Compared to other fuel cells, the power density of MFCs is relatively low, and it is temporarily 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. Therefore, the realization of simultaneous energy recovery and removal of carbon and nitrogen pollutants by MFC technology is an urgent problem to be solved.

For MFC, the influence of anode electrogenic microorganisms as catalysts for organic matter degradation plays a key role in MFC electrogenesis 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.

Bacillus is an important group of denitrifying bacteria present in conventional bioreactors. At present, a plurality of species such as thermophilic bacillus, giant bacillus, paenibacillus and the like in the genus all have denitrification functions, and are an important genus in denitrifying bacteria. In addition, researchers screen and obtain a bacillus subtilis MMR-1 with electrogenesis performance, and prove that the bacillus has electrogenesis performance. However, no relevant reports have been found at home and abroad on the study of the simultaneous power generation characteristics and denitrification activity of the genus in the MFC field.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide bacillus with electrogenesis characteristics and denitrification activity and application thereof.

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

in a first aspect, the invention provides a Bacillus, named Bacillus sp.EM-1 (hereinafter referred to as strain EM-1), which has been deposited in China center for type culture Collection in 26 months 6 and 2017, with the address: china, wuhan university, zip code: 430072, abbreviated as CCTCC, registration number of preservation center is CCTCC NO: m2017372.

The strain EM-1 is facultative anaerobe, rod-shaped and gram-positive. Culturing for 48h on tryptone soybean broth culture medium to form milky white colonies with rough colony surface, irregular edge, opacity and fold protrusion.

The bacillus of the invention is characterized by high yield of electrical activity, and can transfer electrons generated by metabolic organic matters to the anode of the MFC, realize the degradation of the organic matters and generate electric energy. When sodium acetate is used as an electron donor of the bacillus, the maximum power density of the MFC is as high as 684.0mW/m². The strain has another remarkable characteristic of synchronous electrocatalytic activity and nitrate reduction activity (denitrification activity), and when the anolyte takes sodium acetate (COD is 700mg/L) as an electron donor of the bacillus and the nitrate nitrogen concentrations are 20mg/L, 50mg/L, 100 mg/L 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 the above-described bacillus for denitrification and/or catalysing the degradation/decomposition of organic matter. The bacillus can utilize various carbon sources, particularly macromolecular organic matters, to generate electricity, and has huge application potential in treating actual wastewater; the bacillus can reduce nitrate nitrogen into harmless nitrogen through denitrification reaction, and has potential application value in treatment of nitrogen-containing wastewater.

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

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

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

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

In a fourth aspect, the present invention provides a microbial fuel cell comprising the bacillus 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 bacillus, wherein the electron donor of bacillus is an organic substance; more preferably, the organic substance is at least one of formic acid, sodium acetate, lactic acid, 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 the bacillus adsorbed on the surface of the anode, wherein the anolyte contains the organic matter and the inorganic salt culture medium.

In a more preferred embodiment of the microbial fuel cell of the present invention, the anolyte further comprises 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: 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 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 its pH value of 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 Bacillus (Bacillus sp.) EM-1 for the first time, which is an electrogenesis bacterium with high-efficiency electrogenesis characteristic and denitrification activity, the strain is 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 experiment 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 functions of synchronous energy recovery and nitrogen pollution removal when the MFC is used for treating nitrogen-containing wastewater, and lays a foundation for the engineering application of MFC in actual wastewater.

Drawings

FIG. 1 is a colony morphology of the strain EM-1 of the present invention cultured on a plate;

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

FIG. 3 is an electron micrograph (enlarged view) of the strain EM-1 of the present invention attached to the anode surface of MFC;

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

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

FIG. 6 is a graph showing the results of the reducing ability of the strain EM-1 of the present invention to nitrate nitrogen in MFC;

FIG. 7 is a graph showing the results of energy recovery of the strain EM-1 of the present invention in MFC for various 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 Bacillus of the invention is named as Bacillus sp.EM-1 (hereinafter referred to as strain EM-1), and has been preserved in China center for type culture Collection in 26 months 6 in 2017, with the address: china, wuhan university, zip code: 430072, abbreviated as CCTCC, registration number of preservation center is CCTCC NO: m2017372.

EXAMPLE 1 isolation and characterization of Strain EM-1

(1) And (3) screening and enriching strains, namely screening the strain EM-1 to a double-chamber MFC system which stably runs in a laboratory for a long time, taking out a carbon felt with an anode and electrogenesis activity, placing the carbon felt into a sterilized normal saline solution, and dispersing microorganisms attached to the surface of the anode through 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.

Wherein, the anolyte culture medium contains the following components in concentration: 1g/L of sodium acetate, 0.38g/L of potassium nitrate, 6.08g/L of sodium dihydrogen phosphate dihydrate,21.85g/L disodium hydrogen phosphate dodecahydrate, 10mL/L Wolfes mineral solution and 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 0.1 g/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 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 EM-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: culturing for 48h on tryptone soy broth medium to form milky white colonies with rough colony surface, irregular edge, opaque colony surface and fold protrusion, as shown in FIG. 1. The rod-shaped MFC is microscopically positive in gram stain, and the form of attachment to the MFC anode surface is shown in FIGS. 2 and 3.

Physiological and biochemical identification: facultative anaerobes; various substances such as formic acid, lactic acid, butyric acid, glucose, sucrose, starch and the like can be used as carbon sources; positive in nitrate reduction experiment; negative in Methyl Red (MR) test; the V-P reaction test is positive.

And (3) molecular identification: after the strain EM-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 Bacillus licheniformis (KM492826.1) is up to 99 percent by BLAST retrieval comparison. Therefore, this bacterium was named Bacillus (Bacillus sp.) EM-1.

According to the identification result, the strain EM-1 belongs to bacillus, 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: m2017372.

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

MFC was started up according to (2) and (3) in example 1, the voltage output condition is as shown in FIG. 4, and after three cycles of operation, the output voltage was stabilized in the fourth cycle, which means that the electrochemical activity of the strain EM-1 in MFC was stabilized, and the start-up was regarded as 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. Furthermore, after the anode liquid is replaced by new anode liquid,when the output voltage reaches the maximum and tends to be stable again, the MFC polarization curve and the power density curve are tested by changing the external resistance, and the result is shown in FIG. 5, wherein the maximum power density reaches 684.0mW/m²。

EXAMPLE 3 investigation of Simultaneous Electricity production and Denitrification Activity of Strain EM-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 strain EM-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 EM-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 electron donors in the denitrification process, so that the effective electron output is reduced, and the coulombic efficiency is reduced. The COD removal, nitrate nitrogen removal and energy recovery conditions of the strain EM-1 under different C/N ratios are shown in Table 1, and when the C/N ratio is more than 7, the nitrate nitrogen removal rate can reach 97.9 +/-0.1 percent; when the C/N ratio is less than 3.5, the removal rate of nitrate nitrogen is reduced to 80.0 +/-0.1%, and the Coulombic Efficiency (CE) is only 7.4 +/-1.1%.

TABLE 1

EXAMPLE 4 Electricity production characteristics and Denitrification Activity of Strain EM-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 shown in FIG. 7, the strain EM-1 was able to generate electricity using the above carbon source, but the difference in the power generation efficiency was large. Research shows that the strain has low conversion efficiency on small molecular acids such as formic acid and lactic acid, the energy recovery is only 4-5%, and the voltage output is also low. The utilization efficiency of organic matters with relatively long carbon chains, such as glucose, sucrose or starch, is high, and the coulombic efficiency can reach more than 40%. The electricity generation characteristics of the bacillus EM-1 disclosed in the embodiment in the double-chamber air cathode MFC aim to disclose the electricity generation capability of the strain by 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 EM-1 adsorbed on the surface of the anode, wherein the anode is a carbon felt, and the anolyte contains an organic matter, an inorganic salt culture medium and nitrate nitrogen; the organic matter is at least one of formic acid, sodium acetate, lactic acid, butyric acid, glucose, sucrose and starch, and the inorganic salt culture medium contains 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 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,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 pH 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 bacillus is characterized in that the strain is bacillusBacillus sp. EM-1, the preservation number is CCTCC NO: m2017372.

2. Use of the bacillus according to claim 1 for denitrification and/or for catalyzing the degradation/decomposition of organic material, wherein the organic material is at least one of formic acid, sodium acetate, lactic acid, butyric acid, glucose, sucrose, starch.

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

4. The use of the bacillus of claim 1, wherein the electron donor of the bacillus is an organic substance, and the organic substance is at least one of formic acid, sodium acetate, lactic acid, butyric acid, glucose, sucrose and starch.

5. A microbial fuel cell comprising the bacillus of claim 1; the microbial fuel cell also comprises an electron donor of bacillus, wherein the electron donor of the bacillus is an organic matter; the organic matter is at least one of formic acid, sodium acetate, lactic acid, butyric acid, glucose, sucrose and starch;

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 the bacillus adsorbed on the surface of the anode, wherein the anolyte contains the organic matter and the inorganic salt culture medium; the anolyte also contains nitrate nitrogen.