CN108285881A - A kind of mycobacterium and its application with synchronous electrogenesis and denitrification activity - Google Patents
A kind of mycobacterium and its application with synchronous electrogenesis and denitrification activity Download PDFInfo
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- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
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Abstract
The present invention relates to a kind of mycobacterium and its applications with synchronous electrogenesis and denitrification activity, belong to microorganisms technical field.The mycobacterium of the present invention is named as Mycobacterium sp.EB 1, is preserved in China typical culture collection center, address on June 26th, 2017：The Chinese Wuhan Wuhan Universitys, postcode：430072, it is referred to as CCTCC, collection number of registering on the books is CCTCC NO：M 2017371.Present invention firstly discovers that mycobacterium (Mycobacterium sp.) EB 1 is one plant of electricity production bacterium with high electro-chemical activity and denitrification activity, the bacterial strain is facultative anaerobic bacteria, and it can be produced electricity using several kinds of carbon source, not only expand the range of electricity-producing microorganism, improve experimental anaerobic condition and substrate broad spectrum activity, while the efficiently nitrate-N pollution in removal waste water；The present invention, which is applied to MFC, realizes nitrogenous effluent synchronization energy recovery and polluted by nitrogen removal, lays a good foundation in the engineer application of actual waste water for MFC.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of branch bar with synchronous electrogenesis and denitrification activity
Bacterium and its application.
Background technology
Microbiological fuel cell (Microbial Fuel Cell, MFC) is as a new generation developed in recent years, green ring
It protects, the electricity production device of the regenerative resource of non-petroleum class, is increasingly becoming the research hotspot in emerging energy field.The work of MFC is former
Reason be anode microorganism under anaerobic by the metabolism catalysis oxidation organic matter of microorganism and discharge electronics and
Proton, electronics are conducted through external circuit to cathode by anode, and receive electronics by catalyst by cathode and reduction reaction occurs,
To realize the removal of organic matter and the generation of electric current, chemical energy is directly converted to electric energy.
Compared with other fuel cells, the power density of MFC is relatively low, it is difficult to substitute other energy to meet the mankind couple
The demand of the energy.Therefore, MFC technologies are more and more applied to sewage treatment field by researchers, that is, solve traditional dirt
Water process high energy consumption and the big disadvantage of sludge yield, and can effectively recycle the potential chemical energy of large amount of organic in sewage.This
It is outer according to national regulation《Integrated wastewater discharge standard》In point out that two key indexes are respectively in sewage discharge primary standard
COD concentration is no more than 50mg/L and total nitrogen concentration is no more than 20mg/L.During traditional biological method treated sewage, nitrate nitrogen
Efficiently removal and qualified discharge are always an important problem of sewage disposal.
For MFC, anode electricity-producing microorganism as organic matter degradation biocatalyst to MFC efficiency of fuel cell generation and energy
The influence of amount recycling plays a key role.Therefore excavation more has the microorganism of this function for enriching electricity-producing microorganism
Diversity, improve efficiency of fuel cell generation be of great significance.Have high yield electroactive by screening and the electricity production of denitrification activity is micro-
Biology is applied in MFC and can reach the purpose of synchronous organics removal and nitrate nitrogen, reduces subsequently to nitrogen source pollution
After-treatment.
Mycobacterium is a kind of Pseudomonas being widely present in environment, and cell dyeing is Gram-positive, amphimicrobian.Mesh
Before, at home and abroad not yet find relevant report about the electricity production characteristic of the bacterium microorganism and its application in MFC.
Invention content
It is a kind of with synchronous electrogenesis and denitrification it is an object of the invention to be provided in place of overcome the deficiencies in the prior art
Active mycobacterium and its application.
To achieve the above object, the technical solution that the present invention takes is：
In a first aspect, the present invention provides a kind of mycobacterium, which is named as Mycobacterium
Sp.EB-1 (hereinafter referred to as bacterial strain EB-1) is preserved in China typical culture collection center, address on June 26th, 2017：
The Chinese Wuhan Wuhan Universitys, postcode：430072, it is referred to as CCTCC, collection number of registering on the books is CCTCC NO：M
2017371。
The bacterial strain EB-1 of the present invention is facultative anaerobic bacteria, rod-shaped, Gram-positive.Under 30 DEG C, aerobic condition, tryptose
Light culture 48h on peptone soy broth tablet, the round easy picking of formation, opaque, neat in edge milky bacterium colony,
Synthetic dyestuff becomes yellow color colonies under illumination condition.
The notable feature of the bacterial strain EB-1 of the present invention is electroactive with high yield, can will be metabolized the electronics that organic matter generates and pass
The anode for passing MFC is realized the degradation of organic matter and is produced electricl energy.When using sodium acetate as electron donor, MFC maximum powers are close
Degree reaches 835.5mW/m2.Another notable feature of the bacterial strain be with synchronous electro catalytic activity and nitrate reduction activity, when
Anolyte with sodium acetate (COD=700mg/L) be electron donor, when nitrate is respectively 20,50,100 and 200mg/L,
MFC still has high yield electroactive and denitrification rate.
Second aspect, the present invention provides above-mentioned mycobacterias in denitrification and/or catalyzing organic degradation/decomposition
Using.The mycobacteria of the present invention can be produced electricity using several kinds of carbon source especially larger molecular organics, and the bacterial strain is real in processing
There is huge application potential in the waste water of border；And the bacterial strain can by anti-nitration reaction by nitrate-nitrogen reduction in waste water be nitrogen,
Efficient denitrification rates and electricity production activity can be kept during processing low ratio of carbon to ammonium waste water.
As the preferred of application of the mycobacteria of the present invention in denitrification and/or catalyzing organic degradation/decomposition
Embodiment, the mycobacteria are used in waste water/sewage disposal.
The third aspect, the present invention provides above-mentioned mycobacterias to carry out electricity production and/or anti-for microbiological fuel cell
Application in nitrification.In microbiological fuel cell, mycobacteria of the invention is used as electricity-producing microorganism.
As application of the mycobacteria of the present invention in carrying out electricity production and/or denitrification for microbiological fuel cell
Preferred embodiment, the electron donor of the mycobacteria is organic matter.
As the preferred embodiment of above application of the present invention, the organic matter be sodium acetate, butyric acid, glucose, sucrose,
At least one of starch.
Fourth aspect, the present invention also provides a kind of microbiological fuel cell, the microbiological fuel cell contains above-mentioned
Mycobacteria.
As the preferred embodiment of microbiological fuel cell of the present invention, the microbiological fuel cell also contains and divides
The electron donor of the electron donor of branch bacillus, the mycobacteria is organic matter；It is highly preferred that the organic matter be sodium acetate,
At least one of butyric acid, glucose, sucrose, starch.
As the preferred embodiment of microbiological fuel cell of the present invention, the microbiological fuel cell includes anode
Room, cathode chamber and the external circuit for jointed anode room and cathode chamber；The anode chamber includes anode, anolyte and as anode
The mycobacteria of catalyst, the anolyte contain the organic matter and minimal medium.
As the preferred embodiment of microbiological fuel cell of the present invention, the anolyte also contains nitrate nitrogen.
As the preferred embodiment of microbiological fuel cell of the present invention, the minimal medium contains following dense
The component of degree：Potassium nitrate 0.144~1.44g/L, two hypophosphite monohydrate 0.8~8.1g/L of sodium dihydrogen, disodium hydrogen phosphate dodecahydrate
2.1~22g/L, Wolfes Mineral Solution 10mL/L, the minimal medium adjust its pH value to 7 by HCl/NaOH solution；
Wherein, Wolfes Mineral Solutions contain the component of following concentration：Amion acetic 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,Na2MoO4·2H2O 0.01g/
L, the Wolfes Mineral Solutions by KOH adjust its to pH value be 7.
As the preferred embodiment of microbiological fuel cell of the present invention, the culture temperature of the microbiological fuel cell
Degree is 30 DEG C.
As the preferred embodiment of microbiological fuel cell of the present invention, the anode is carbon felt.
As the preferred embodiment of microbiological fuel cell of the present invention, the cathode chamber includes cathode and cathode
Liquid；Preferably, the cathode is the air cathode of Pt/C catalysis, the phosphate buffer of the catholyte 0.1mol/L.
As the preferred embodiment of microbiological fuel cell of the present invention, the anode chamber and cathode chamber are by cation
Exchange membrane separates.
Compared with prior art, the beneficial effects of the present invention are：Present invention firstly discovers that mycobacterium
(Mycobacterium sp.) EB-1 is one plant of electricity production bacterium with high electro-chemical activity and denitrification activity, which is
Facultative anaerobic bacteria, and can be produced electricity using several kinds of carbon source, the range of electricity-producing microorganism is not only expanded, experimental anaerobic is improved
Condition and substrate broad spectrum activity, while the efficiently nitrate-N pollution in removal waste water.The present invention, which is applied to MFC, realizes nitrogenous effluent
Synchronous energy recovery and polluted by nitrogen removal, lay a good foundation for MFC in the engineer application of actual waste water.
Description of the drawings
Fig. 1 is flat-plate bacterial colony aspect graphs of the bacterial strain EB-1 of the present invention under light culture；
Fig. 2 is the electron microscope that bacterial strain EB-1 of the present invention adheres in MFC anode surfaces；
Fig. 3 is the electron microscope (enlarged drawing) that bacterial strain EB-1 of the present invention adheres in MFC anode surfaces；
Fig. 4 is voltage output result figures of the bacterial strain EB-1 of the present invention in MFC；
Fig. 5 is polarization curves and power density curve graph of the bacterial strain EB-1 of the present invention in MFC；
Fig. 6 is for bacterial strain EB-1 of the present invention to the reduction result figure of nitrate nitrogen in MFC；
Fig. 7 is for bacterial strain EB-1 of the present invention to the energy regenerating result figure of different electron donors in MFC.
Specific implementation mode
For the object, technical solutions and advantages of the present invention are better described, below in conjunction with the drawings and specific embodiments pair
The present invention is described further.
The present invention mycobacterium be named as Mycobacterium sp.EB-1 (hereinafter referred to as bacterial strain EB-1), in
On June 26th, 2017 is preserved in China typical culture collection center, address：The Chinese Wuhan Wuhan Universitys, postcode：
430072, it is referred to as CCTCC, collection number of registering on the books is CCTCC NO：M 2017371.
The separation and identification of 1 bacterial strain EB-1 of embodiment
(1) screening and enrichment of bacterial strain：Bacterial strain EB-1 screens the dual chamber MFC systems run steadily in the long term to laboratory, takes
Going out anode, there is the active carbon felt of electricity production to be placed in sterilized normal saline solution, and it is attached to anode by ultrasonic disperse
The microorganism on surface.Under aseptic condition, by obtained bacteria suspension by dilution spread in pancreas peptone soybean broth solid culture
On base, aerobic culture 48h according to the otherness of thalline bacterium colony, picking single bacterium colony, and passes through isolated pure bacterium of repeatedly crossing
Strain.Further, by pure inoculation to using sodium acetate in the anolyte solid medium of carbon source, to be placed in anaerobic culture box
In 30 DEG C of several days of culture, observe colonial morphology and its growing state.What picking can be grown under aerobic and anaerobic conditions simultaneously
Bacterial strain is seeded in pancreas peptone soybean broth fluid nutrient medium and expands culture respectively, and thalline were collected by centrifugation.
Anode liquid culture medium at being grouped into：Sodium acetate 1g/L, potassium nitrate 0.38g/L, two hypophosphite monohydrate sodium dihydrogens
6.08g/L, disodium hydrogen phosphate dodecahydrate 21.85g/L, Wolfes Mineral Solution 10mL/L, the minimal medium by
HCl/NaOH solution adjusts its pH value to 7；Wherein, Wolfes Mineral Solutions are grouped as by the group of following concentration：：Amion acetic 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,Na2MoO4·2H20.01 g/L of O, Wolfes Mineral Solutions by KOH adjust its to pH value be 7.
Anolyte solid medium is that agar 15-20g/L is added on above-mentioned anolyte medium component.
(2) pretreatment of MFC components and assembling：Anode chamber, cathode chamber, washer and rubber plug in MFC components are placed in 5% mistake
Soaked overnight in hydrogen peroxide solution, it is spare with sterile water wash.Anode carbon felt impregnates by ethyl alcohol acetone mixture overnight respectively,
Cleaning, ammonium persulfate impregnate 15min, cleaning, high-temperature roasting, 5%NH3After/Ar roastings, it is placed in ultraviolet irradiation in super-clean bench
30min, it is spare.Air cathode passes through the waterproof layer PDMS processing of carbon cloth respectively, the Pt/C of non-waterproof layer loads (0.5mg/cm2)
Afterwards, it is placed in ultraviolet irradiation 30min in super-clean bench, it is spare.Cation-exchange membrane impregnates 2h, nothing by 5% hydrogenperoxide steam generator
Bacterium water clean after, be placed in sterilized 0.1M PBS solutions impregnate it is spare.All components of MFC are in sterile super-clean bench over-assemble
As dual chamber air cathode microbial fuel cell.
(3) inoculation and MFC operations：Anolyte after high-temperature sterilization is aseptically passed through High Purity Nitrogen discharge oxygen, and
It is added to anode chamber；The 0.1M PBS solutions of high-temperature sterilization are added in cathode chamber.The bacterium that centrifugation obtains in transfer needle difference picking (1)
In body to anode chamber, it can be seen that anode chamber is because being inoculated with apparent bacteria suspension.External circuit and anode and cathode, dispatch from foreign news agency are connected by titanium conducting wire
Road connects the resistance of a 1K Ω, and resistance both ends are connected to multichannel voltage analyzer.Computer controls data acquisition modes and preserves
The data of acquisition, the voltage curve changed over time.
(4) identification of bacterium is produced electricity：The inoculating strain corresponding to the highest MFC of voltage output is chosen, EB-1 is named as, to this
Bacterial strain carries out colonial morphology identification, Physiology and biochemistry identification and Molecular Identification respectively, as a result as follows：
Morphological Identification：Tablet is in constant incubator after light culture 48h, and bacterium colony surface is smooth opaque, milky, such as schemes
Shown in 1；Under room temperature light stimulation, bacterium colony becomes yellow, is in rod-shaped under microscope；MFC anode surfaces attachment form such as
Shown in Fig. 2 and Fig. 3.Gram's staining is the positive；Inorganic salts solid medium culture under anaerobic 3-5 days, bacterium colony are
White fine particle.
Physiology and biochemistry is identified：Facultative anaerobic bacteria；It is carbon source that the substances such as glucose, starch, protein, which can be utilized,；Nitrate
Reduction experiment is positive；Methyl red (MR) experiment is positive；V-P reaction tests are feminine gender.
Molecular Identification：After the bacterial strain EB-1 is cultivated under aerobic and anaerobic conditions respectively, picking colony is carried through DNA
It takes, it is consistent to obtain 16s rRNA base sequences after PCR amplification, and homology analysis is carried out in ncbi database, and BLAST retrieves ratio
To showing that the bacterial strain and Mycobacterium fortuitum (NZ_CP011269.1) similitude are up to 99%.Currently, not
See mycobacterium report that can be with electricity generation ability.Therefore, which is named as mycobacterium (Mycobacterium sp.)
EB-1。
According to above qualification result, bacterial strain EB-1 belongs to mycobacterium, is carried out preservation, and depositary institution is Chinese allusion quotation
Type culture collection, address：The Chinese Wuhan Wuhan Universitys, postcode：430072, it is referred to as CCTCC, collection is stepped on
It is CCTCC NO to charge to volume number：M 2017371.
The MFC electricity productions verification of 2 bacterial strain EB-1 of embodiment and electrochemical properties
MFC starts operation according to (2) and (3) in embodiment 1, and voltage output situation is as shown in figure 4, by three periods
Operation, reach stable in the 4th period output voltage, it is meant that bacterial strain EB-1 has reached stabilization in the electro-chemical activity of MFC,
It is considered as and starts successfully.Enter the commencement of commercial operation stage after starting successfully, when the output voltage of detection is less than 20mV, in aseptic condition
Lower replacement anolyte, general a cycle are 3 days or so.Further, after the anolyte more renewed, when output voltage again
When reaching highest and tending towards stability, the test of MFC polarization curves and power density curve is carried out by changing external resistance size, such as
Shown in Fig. 5, maximum power density reaches 835.5mW/m2。
The research of embodiment 3 bacterial strain EB-1 synchronous electrogenesis and denitrification activity
MFC starts operation to voltage output according to (2) and (3) in embodiment 1 and stablizes, and maximum power density reaches embodiment
When 2, it is 1g/L (COD is about 700mg/L) that sodium acetate concentration is fixed in anolyte component, and other inorganic salts ingredients are constant, is led to
The concentration for crossing nitrate nitrogen in change anolyte is respectively 0,20,50,100 and 200mg/L, observes different C/N and compares bacterial strain EB-1
The influence of voltage output and anode anti-nitration reaction in MFC.
The results are shown in Figure 6, and with the raising of nitrate, the electricity production period shortens, but maximum output voltage is almost
It is constant.The result means that the electro-chemical activity of bacterial strain EB-1 is not inhibited by nitrate, the main original that the period shortens
Because being since denitrification process needs organic matter to provide electron donor therefore consumes the sodium acetate in anolyte so that effective
Electronics output reduces, and coulombic efficiency declines.Under different C/N ratios, COD removals, nitrate nitrogen removal rate and energy return bacterial strain EB-1
It is as shown in table 1 to receive situation, when C/N ratios are more than 7, nitrate nitrogen removal rate reaches as high as 98.2%；And when C/N ratios are less than 3.5,
Nitrate nitrogen removal rate drops to 82.8%, while coulombic efficiency (CE) is only 6.5%.Therefore, when bacterial strain EB-1 restores nitrate nitrogen
Compete the electronics of oxidation operation release jointly with anode.
Table 1
4 bacterial strain EB-1 of embodiment utilizes the electricity production characteristic and denitrification activity of different carbon source
MFC starts operation to voltage output according to (2) and (3) in embodiment 1 and stablizes, and maximum power density reaches embodiment
When 2, anolyte is replaced, in addition to changing carbon source, other constituents are constant.Carbon source distinguishes formic acid, lactic acid, butyric acid, Portugal
Grape sugar, sugarcane sugar and starch.The results are shown in Figure 7, and bacterial strain EB-1 can be produced electricity using butyric acid, glucose, sugarcane sugar and starch,
But efficiency of fuel cell generation difference.And bacterial strain is relatively low to the transformation efficiency of small molecule acid such as formic acid and lactic acid, almost without voltage output.This
Electricity production characteristics of the mycobacterium EB-1 disclosed in embodiment in dual chamber air cathode MFC, its purpose is that disclosing bacterial strain utilization
The electricity generation ability of several kinds of carbon source especially larger molecular organics, it is meant that have in handling actual waste water using the bacterial strain huge
Application potential.
Embodiment 5
A kind of embodiment of microbiological fuel cell of the present invention, the microbiological fuel cell of the present embodiment include anode chamber,
Cathode chamber and external circuit for jointed anode room and cathode chamber, the anode chamber and cathode chamber are separated by cation-exchange membrane
It opens；
The anode chamber includes anode, anolyte and the bacterial strain EB-1 as anode catalyst, and the anode is carbon felt, institute
It states anolyte and contains organic matter, minimal medium and nitrate nitrogen；The organic matter is sodium acetate, butyric acid, glucose, sucrose, shallow lake
At least one of powder, the minimal medium are grouped as by the group of following concentration：0.144~1.44g/L of potassium nitrate, two water
Close 0.8~8.1g/L of sodium dihydrogen phosphate, disodium hydrogen phosphate dodecahydrate 2.1~22g/L, Wolfes Mineral Solution 10mL/L, institute
It states minimal medium and its pH value is adjusted to 7 by HCl/NaOH solution；Wherein Wolfes Mineral Solutions by following concentration component
Composition：Amion acetic 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,Na2MoO4·2H2O 0.01g/L, the Wolfes Mineral Solutions are adjusted by KOH
Its to pH value be 7；
The cathode chamber includes cathode and catholyte, and the cathode is the air cathode of Pt/C catalysis, the catholyte
The phosphate buffer of 0.1mol/L.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than is protected to the present invention
The limitation of range is protected, although being explained in detail to the present invention with reference to preferred embodiment, those skilled in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Claims (10)
1. a kind of mycobacterium, which is characterized in that the bacterial strain is mycobacterium (Mycobacterium sp.) EB-1, and preservation is compiled
Number be CCTCC NO：M 2017371.
2. application of the mycobacteria described in claim 1 in denitrification and/or catalyzing organic degradation/decomposition.
3. application as claimed in claim 2, which is characterized in that the mycobacteria is used in waste water/sewage disposal.
4. application of the mycobacteria described in claim 1 in carrying out electricity production and/or denitrification for microbiological fuel cell.
5. application as claimed in claim 4, which is characterized in that the electron donor of the mycobacteria is organic matter.
6. the application as described in claim 2,3 or 5, which is characterized in that the organic matter is sodium acetate, butyric acid, glucose, sugarcane
At least one of sugar, starch.
7. a kind of microbiological fuel cell, which is characterized in that the microbiological fuel cell contains as described in claim 1 point
Branch bacillus.
8. microbiological fuel cell as claimed in claim 7, which is characterized in that the microbiological fuel cell also contains branch
The electron donor of the electron donor of bacillus, the mycobacteria is organic matter；Preferably, the organic matter be sodium acetate, butyric acid,
At least one of glucose, sucrose, starch.
9. microbiological fuel cell as claimed in claim 7 or 8, which is characterized in that including anode chamber, cathode chamber and for connecting
Connect the external circuit of anode chamber and cathode chamber；The anode chamber includes anode, anolyte and the mycobacteria as anode catalyst,
The anolyte contains the organic matter and minimal medium.
10. microbiological fuel cell as claimed in claim 9, which is characterized in that the anolyte also contains nitrate nitrogen.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114381402A (en) * | 2022-01-20 | 2022-04-22 | 广州大学 | Acid-resistant and alkali-resistant aerobic denitrifying bacterium and microbial inoculum for rapid denitrification and application thereof |
| WO2024037604A1 (en) * | 2022-08-17 | 2024-02-22 | 彭光浩 | Mycobacterium sp. based on various carbon-nitrogen metabolic pathways, method for isolating same, and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114381402A (en) * | 2022-01-20 | 2022-04-22 | 广州大学 | Acid-resistant and alkali-resistant aerobic denitrifying bacterium and microbial inoculum for rapid denitrification and application thereof |
| CN114381402B (en) * | 2022-01-20 | 2022-12-16 | 广州大学 | Acid-resistant and alkali-resistant aerobic denitrifying bacterium and microbial inoculum for rapid denitrification and application thereof |
| WO2024037604A1 (en) * | 2022-08-17 | 2024-02-22 | 彭光浩 | Mycobacterium sp. based on various carbon-nitrogen metabolic pathways, method for isolating same, and use thereof |
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