CN103215201B - Klebsiella oxytoca and its application in bioelectricity production - Google Patents
Klebsiella oxytoca and its application in bioelectricity production Download PDFInfo
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- 241000588749 Klebsiella oxytoca Species 0.000 title claims abstract description 10
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- 241000894006 Bacteria Species 0.000 claims description 49
- 230000001580 bacterial effect Effects 0.000 claims description 31
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- 241000588748 Klebsiella Species 0.000 claims description 12
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- 241000588754 Klebsiella sp. Species 0.000 description 3
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- 241000588747 Klebsiella pneumoniae Species 0.000 description 2
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- 235000007164 Oryza sativa Nutrition 0.000 description 2
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Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
- Y02E60/00—Enabling technologies; Technologies with a potential or indirect contribution to GHG emissions mitigation
- Y02E60/30—Hydrogen technology
- Y02E60/50—Fuel cells
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P70/00—Climate change mitigation technologies in the production process for final industrial or consumer products
- Y02P70/50—Manufacturing or production processes characterised by the final manufactured product
Abstract
The invention discloses a Klebsiella oxytoca strain and its application in bioelectricity production. The strain is characterized in that it is Klebsiella oxytoca Z6, which is preserved in China Center for Type Culture Collection (called CCTCC for short) on November 6, 2012 and has a preservation number of CCTCC NO: M 2012446. The strain can transmit electrons to an extracellular electron acceptor under an anaerobic condition. When the strain is inoculated into a microbial fuel cell, it can degrade organic matters and generate electricity simultaneously. Compared with other strains, the Klebsiella oxytoca Z6 strain not only has very strong electrochemical activity, but also can make use of various organic matters as the sole carbon source for electricity generation. Based on the above characteristics, the strain has very good application in environmental pollution remediation and bio-energy recovery.
Description
Technical field
The invention belongs to environmental pollution biological treatment and bioenergy technical field, be specifically related to a strain acid-producing Klebsiella bacterium and produce bioelectric application.
Background technology
In recent years, microbiological fuel cell (Microbial Fuel Cell, MFC) technology has been subject to paying close attention to widely as a kind of emerging wastewater treatment and pollution remediation technology.It is directly oxidized and produces electronics and proton by microorganism by organism in fuel or inorganics, the proton and the electronics that produce are passed to negative electrode through proton exchange membrane and external circuit respectively, react with final electron acceptor(EA) and produce water, the most at last the chemical energy of organism or inorganics transform electric energy in fuel.This technology reclaims the biological treatment of pollution to combine with bioenergy, when disposing of sewage, can also obtain electric energy.
In microbiological fuel cell, electrogenesis microorganism (Exoelectrogens) is being brought into play critical effect.Electrogenesis microorganism is those microorganisms that can carry out the transmission of born of the same parents' exoelectron, they can form microbial film in anode enrichment, can pass through metabolism degradation of organic substances, produce electronics simultaneously and be passed to outside born of the same parents, be delivered on electrode directly or indirectly again, thereby realize biocatalysis and drive the operation of MFC.Along with the difference of electrogenesis microbe species, its mechanism that produces electronics and transmission electronics is also different, and the electrochemical activity showing exists notable difference.
At present, the electrogenesis microorganism that Chinese scholars has been found that focuses mostly at Proteobacteria, and also having a small amount of microorganism that is positioned at Firmicutes, acidfast bacilli door and Bacteroidetes to be proved can electrogenesis.But in general, known electrogenesis microbe species is still very limited.Meanwhile, aspect electrogenesis characteristic, known most of electrogenesis bacterium still exist electricity generation performance lower, utilize the defects such as substrate type is more single, can not meet and repair polytype environmental pollution.Therefore, be necessary screening electrogenesis microorganism more efficiently.
Klebsiella (Klebsiella sp.) belongs to γ-distortion Gammaproteobacteria, does not move, and has pod membrane, is a kind of Gram-negative, the amphimicrobian iron-reducing bacterium of heterotrophism.Existing some reports about Klebsiella sp. bacterium catalyzing organic electrogenesis in MFC.The Klebsiella sp.ME17 being separated in the Klebsiella pneumoniae bacterial strain L17 being separated to from soil and rice soil is successively tested it and is utilized glucose electricity generation performance in MFC.Wherein, the 16S rDNA sequence of Klebsiella sp.ME17 and Klebsiella trevisanii ATCC33558T (AF129444) and Klebsiella planticolaATCC33531T (Y17659) are the most approaching, but Physiology and biochemistry experimental result shows that it is the novel species of klebsiella.From above-mentioned report, the Klebsiella bacterium Klebsiella pneumonia L17 and the Klebsiella sp.ME17 that have reported are respectively separating obtained from soil and rice soil, through MFC test, show that it has electrochemical activity, meanwhile, the substrate type of test is only glucose.Can find out up to the present, also do not have directly from the MFC anode microbial film of enrichment, to be separated to the play-by-play of Klebsiella sp. electrogenesis bacterium, utilize different electron donor electrogenesis characteristics to need to be verified.
Summary of the invention
The present invention has overcome above-mentioned defect, a strain acid-producing Klebsiella bacterium is provided and produces bioelectric application.
A strain acid-producing Klebsiella bacterium provided by the present invention, derive from the mud mixture of the bright sewage work of Guangzhou West second pond, through the electrochemistry enrichment culture of microbiological fuel cell, artificial separation and purification, obtain, this bacterium is acid-producing Klebsiella bacterium (Klebsiella oxytoca) Z6, by the center preservation of Chinese Typical Representative culture collection, be called for short CCTCC, deposit number is: CCTCC NO:M 2012446, preservation date is on November 6th, 2012, and preservation address is China. Wuhan. and Wuhan University.The bacterium colony of this bacterium on ironic citrate substratum is circle, black, the smooth of the edge is glossy, diameter 2mm.The form of observing this bacterium under transmission electron microscope is shaft-like, and width is 0.5 μ m-0.7 μ m, and length is 0.6 μ m-1.7 μ m, peritrichous, and growth logarithmic phase is 2～20h.Biological characteristics is: Gram-negative, amphimicrobian, has iron reductibility, can utilize trisodium citrate, glucose, glycerol, Sodium.alpha.-hydroxypropionate, L-glutamic acid.This bacterium can under anaerobic be transmitted electronics to extracellular electron acceptor.The accession number of the 16S rDNA of bacterial strain Z6 is JX185133.
The application of above-mentioned bacterial strains in producing bioelectricity, specifically comprises the steps:
(1) by inoculation claimed in claim 1 to ironic citrate substratum, 30 ± 5 ℃ of anaerobism are cultivated; The phase growth bacterium liquid in mid-term of taking the logarithm, centrifugal, abandon supernatant liquor, add PBS damping fluid and shake up, by above-mentioned steps, by gained bacteria suspension recentrifuge, so repeatedly clean 2 times, interpolation PBS damping fluid is made bacteria suspension;
(2) start single chamber air cathode microbial fuel cell, after the bacteria suspension obtaining is mixed with nutritive medium, be inoculated in microbiological fuel cell, temperature-constant operation under the culture temperature of step (1) in step (1).More than the output voltage of MFC reaches 100mV, be considered as starting successfully, after this, only change the nutritive medium containing electron donor.
Preferably, the described ironic citrate nutrient media components of step (1) is as follows: Tryptones 10gL
-1, yeast extract 5gL
-1, sodium-chlor 10gL
-1, add 20mmolL
-1ironic citrate be electron donor; The described nutrient composition of step (2) is 30mmolL
-1electron donor, 50mmolL
-1pBS damping fluid and a small amount of VITAMIN and trace element.
Preferably, in described nutritive medium, electron donor is one or more in Citrate trianion, glucose, glycerol, Sodium.alpha.-hydroxypropionate and L-glutamic acid; The formula of described PBS damping fluid is NH
4cl 0.31gL
-1, NaH
2pO
4h
2o 2.452gL
-1, Na
2hPO
44.576gL
-1, KCl 0.13gL
-1, pH 7.0.
The OD of the bacteria suspension of making preferably,
600be 0.6; The pH of described substratum and nutritive medium is 7.0～7.4.
Preferably, microbiological fuel cell described in step (2) is that cylindrical (diameter of section is 2cm, useful volume 6.28cm
3), material is polycarbonate.Air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is Nitric Acid Modified carbon felt, and negative electrode inner side is one deck cationic exchange membrane, and interelectrode distance is 2cm, with titanium silk, does electronics collector.The concrete method of modifying of Nitric Acid Modified carbon felt is: by pretreated carbon for felt concentrated nitric acid (65%) soak 5h, with deionized water, repeatedly rinse to neutrality, put into 120 ℃ of baking ovens and dry after 2h, be placed in moisture eliminator standby.Carbon felt pretreatment process is: with acetone, soak after 3h, take out and wash, to remove surperficial oil-soluble substance through vacuum pump; With deionized water rinsing and soak, boil, every 0.5h, change water 1 time again, boil altogether 3h, electrode materials is put into 120 ℃ of baking ovens and dry after 2h, be placed in moisture eliminator standby.
Preferably, the output voltage (U) of MFC described in step (2) adopts Keithley 2700 to gather
Preferably, described in step (2), cationic exchange membrane soaks 24h with 5%NaCl in advance.
Preferably, until step (2) cell voltage, there is stable can, after the repeat cycle, interpolation external source electronics in anode chamber and transmit intermediate A QDS.
Preferably, the described culture temperature of step (1) is 30 ± 1 ℃, and incubation time is 16-18h, and centrifugal condition is centrifugal 10 minutes of 5000r/min.
Preferably, the volume ratio of described bacteria suspension and nutritive medium is 1:5.
The application of described bacterial strain CCTCC M 2012446 in environmental pollution biological treatment and bioenergy technical field.Take Citrate trianion during as matrix, and the single chamber air cathode MFC of this strain construction can produce maximum output voltage 0.31V, and maximum power density is 287.4mWm
-2.Except Citrate trianion, this bacterium can also utilize glucose, Sodium.alpha.-hydroxypropionate, glycerol, L-glutamic acid electrogenesis.This explanation bacterial strain Klebsiella oxytoca Z6 not only has stronger electrochemical activity, can also utilize polytype organism to carry out electrogenesis as sole carbon source.Add external source electronics transmission intermediate (AQDS) electricity generation process of this bacterial strain is had to certain promoter action.
The present invention compared with prior art advantage is that this bacterial strain CCTCC M 2012446 shows stronger electrochemical activity, compares with other bacterial strains, can produce comparatively significant electric energy, take Citrate trianion during as matrix maximum power density be 287.4mWm
-2.This bacterial strain CCTCC M 2012446 can also utilize polytype organism electrogenesis, this means this bacterium will various industrial sewage process and environmental pollution reparation in play a significant role.Meanwhile, this bacterial strain is amphimicrobian type, does not need strict anaerobic environment in electricity generation process, has reduced the requirement to waste water and environmental pollution treatment device in practical application.
Accompanying drawing explanation
Fig. 1 is that embodiment 1 be take the MFC trigger voltage figure that second pond mud is inoculum.
Fig. 2 is the bacterial strain cyclic voltammetry curve figure of embodiment 2.
Fig. 3 is that the bacterial strain of embodiment 3 be take MFC voltage, power density and coulomb efficiency diagram that gas chromatography is matrix.
Fig. 4 is that the bacterial strain of embodiment 4 be take MFC voltage (a), polarization curve (b) and the electrode potential figure (c) that Citrate trianion is matrix.
Fig. 5 is MFC anode and microbial film microscopic examination figure in embodiment 4, (a) is blank anode, * 500 times; (b) be anode microbial film, * 500 times; (c) be anode microbial film, * 5000 times; (d) for to be attached to the thalline on single carbon fiber, * 40000 times.
Fig. 6 is the original position cyclic voltammetry curve figure of MFC anode in embodiment 5, (a) is different times schematic diagram, is (b) the CV curve of different times.
Fig. 7 adds the influence curve figure of external source AQDS to MFC output voltage in embodiment 6.
Embodiment
Below in conjunction with specific embodiment, the present invention is more specifically described in detail, but embodiments of the present invention are not limited to this, for not dated especially processing parameter, can carry out with reference to routine techniques.In the embodiment of the present invention, biochemistry pool mud used is from Guangzhou City Silang Wastewater Treatment Plant.
Embodiment 1
Screening and the separation of bacterial strain CCTCC M 2012446
Get the second pond mud 300mL of sewage work and transfer in 500mL beaker, add glucose nutritive medium 200mL, stir, use NaHCO
3solution is adjusted pH value to 7.0 left and right (6.7-7.2), is placed in 30 ℃ of incubators and cultivates 1 day.Measure pH value, and use NaHCO in good time
3solution is adjusted pH value to 6.7-7.2, stirs, and after its precipitation, abandoning supernatant.So repeat to cultivate 3 days standby.
Start single chamber air cathode microbial fuel cell, described MFC is rectangular parallelepiped (length * wide * height=8.5 * 7 * 6.5cm
3, useful volume 200mL), the synthetic glass that material is 0.5cm, one end is sealed with blind plate, and the other end is placed negative electrode with clamping plate.Air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is Nitric Acid Modified carbon felt (BET 1200m
2g
-1, 2mm), interelectrode distance is 3cm, with titanium silk, does electronics collector.During startup, get the mud after preculture, inoculation MFC after mixing in the ratio of 1:1 with nutritive medium, extrernal resistance is 1000 Ω, under (30 ± 1) ℃ constant temperature, moves.Nutritive medium is for containing 1gL
-1the PBS(pH 7.0 of glucose) and a small amount of VITAMIN and trace element, after 121 ℃ of sterilizing 20min, use.More than the output voltage of MFC reaches 100mV, be considered as starting successfully.After this, only change the nutritive medium containing electron donor.By Keithley 2700, gather the output voltage (U) of MFC.As shown in Figure 1, start battery 180 as a child output voltage reaches stable to result substantially, and maximum output voltage is 0.37V.At MFC anode surface, can observe one deck microbial film.
Until this MFC steady running, after 1 month, taking out length has biomembranous anode, scrapes anode microbial film some with blade, and inoculation ironic citrate liquid nutrient medium is placed in anaerobism under (30 ± 1) ℃ condition and cultivates 1 day.Then, get culture coating ironic citrate solid medium.Cultivate after one day, according to features such as colonial morphology, color, the transparencys, the bacterium colony of picking surface characteristic obvious difference, is seeded to respectively and in ironic citrate liquid nutrient medium, carries out anaerobism cultivation.So repeatedly go down to posterity 7-8 time, obtain many strains pure culture bacterial strain.Pure bacterium is carried out to its electrochemical activity of cyclic voltammetric analysis verification, finally obtain a strain electrochemical activity bacterium Z6.Through Molecular Identification, morphologic observation and utilization of carbon source analysis, this dientification of bacteria is acid-producing Klebsiella bacterium (Klebsiella oxytoca).Described liquid culture based formulas is: Tryptones 10gL
-1, yeast extract 5gL
-1, sodium-chlor 10gL
-1, ironic citrate 20mmolL
-1, pH is 7.4; Described solid culture based formulas is that liquid nutrient medium adds 23gL
-1agar powder.
Embodiment 2
The cyclic voltammetric analysis of bacterial strain CCTCC M 2012446
Klebsiella oxytoca Z6 is seeded to (Tryptones 10gL in ironic citrate substratum
-1, yeast extract 5gL
-1, sodium-chlor 10gL
-1, ironic citrate 20mmolL
-1, pH is 7.4), (30 ± 1) ℃ anaerobism is cultivated 18h.The phase growth bacterium liquid in mid-term of taking the logarithm, centrifugal 10 minutes of 5000r/min, abandons supernatant liquor, adds PBS damping fluid and shakes up, and by above-mentioned steps, by gained bacteria suspension recentrifuge, so repeatedly cleans 2 times, and interpolation PBS damping fluid is made bacteria suspension, OD
600it is 0.6 left and right.With electrochemical workstation, gained bacteria suspension is done to cyclic voltammetry, test adopts three-electrode system, take glass-carbon electrode as working electrode, and Ag/AgCl electrode is reference electrode, and platinized platinum is to electrode, sweeps speed for 100mVs
-1, sweep limit is-0.8V～0.8V.Experimental result is shown in Fig. 2, bacterial strain Klebsiella oxytoca Z6 cultivates after 1d in LB substratum, obtain cyclic voltammetry curve have a pair of obvious redox peak, wherein oxidation peak is between-50mV～130mV, reduction peak is between 210mV～360mV.
The bacterial strain CCTCC M 2012446 of the present embodiment explanation resulting separation can carry out the transmission of born of the same parents' exoelectron, has stronger electrochemical activity.
Embodiment 3
The application of bacterial strain CCTCC M 2012446 in processing kinds of artificial waste water
Described bacterial strain CCTCC M 2012446 is seeded to LB substratum, and (30 ± 1) ℃ anaerobism is cultivated 18h.The phase growth bacterium liquid in mid-term of taking the logarithm, centrifugal 10 minutes of 5000r/min, abandons supernatant liquor, adds PBS damping fluid and shakes up, and by above-mentioned steps, by gained bacteria suspension recentrifuge, so repeatedly cleans 2 times, and interpolation PBS damping fluid is made bacteria suspension, OD
600it is 0.6 left and right.
Start single chamber air cathode microbial fuel cell, described microbiological fuel cell is that cylindrical (diameter of section is 2cm, useful volume 6.28cm
3), material is polycarbonate.Air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is Nitric Acid Modified carbon felt, and negative electrode inner side is one deck cationic exchange membrane, and interelectrode distance is 2cm, with titanium silk, does electronics collector.During startup, the bacterium liquid that the phase of taking the logarithm grows mid-term, inoculation MFC after mixing in the ratio of 1:5 with nutritive medium, extrernal resistance is 1000 Ω, under (30 ± 1) ℃ constant temperature, moves.Described nutrient composition is: 30mmolL
-1electron donor (Citrate trianion, glucose, glycerol, Sodium.alpha.-hydroxypropionate or L-glutamic acid), 50mmolL
-1pBS damping fluid and a small amount of VITAMIN and trace element, used after 121 ℃ of sterilizing 20min.More than the output voltage of MFC reaches 100mV, be considered as starting successfully.After this, only change the nutritive medium containing electron donor.The output voltage of MFC (U) adopts Keithley 2700 to gather;
calculate.M is the molecular weight of oxygen, 32; F is the uncommon constant in not bright Delhi; B is every mole of available amount of electrons of oxygen, 4; υ
anfor anolyte compartment's volume; Δ COD is t
bthe amount of the COD of time internal consumption.
As shown in Figure 3, under 30 ℃, the condition of extrernal resistance 1000 Ω, this bacterium is respectively with 30mmolL for experimental result
-1when Trisodium Citrate, glucose, glycerol, Sodium.alpha.-hydroxypropionate, L-glutamic acid are electron donor, the maximum output voltage of generation is respectively 0.31V, 0.213V, 0.207V, 0.26V, 0.107V.Maximum power density and coulomb efficiency that the Citrate trianion of take obtains during as electron donor are respectively 287.4mWm
-2, 37.73%; Take glucose during as electron donor, and the maximum power density of generation and coulomb efficiency are respectively 140.4mWm
-2, 3.7%; Take glycerol during as electron donor, and the maximum power density of generation and coulomb efficiency are respectively 140mWm
-2, 15.47%; Take Sodium.alpha.-hydroxypropionate during as electron donor, and the maximum power density of generation and coulomb efficiency are respectively 215mWm
-2, 2.28%; Take L-glutamic acid during as electron donor, and the maximum power density of generation and coulomb efficiency are respectively 36.4mWm
-2, 6%.
Known by the present embodiment, bacterial strain CCTCC M 2012446 has can utilize polytype organism electrogenesis, for the application of this bacterium in repairing broad variety environmental pollution and bioenergy recovery laid a good foundation.
Embodiment 4
The application of bacterial strain CCTCC M 2012446 in processing artificial simulated wastewater
Described bacterial strain CCTCC M 2012446 is seeded to ironic citrate substratum (Tryptones 10gL
-1, yeast extract 5gL
-1, sodium-chlor 10gL
-1, ironic citrate 20mmolL
-1, pH is 7.4), (30 ± 1) ℃ anaerobism is cultivated 18h.The phase growth bacterium liquid in mid-term of taking the logarithm, centrifugal 10 minutes of 5000r/min, abandons supernatant liquor, adds PBS damping fluid and shakes up, and by above-mentioned steps, by gained bacteria suspension recentrifuge, so repeatedly cleans 2 times, and interpolation PBS damping fluid is made bacteria suspension, OD
600it is 0.6 left and right.
Start single chamber air cathode microbial fuel cell, described MFC is that cylindrical (diameter of section is 2cm, useful volume 6.28cm
3), material is polycarbonate.Air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is Nitric Acid Modified carbon felt, and negative electrode inner side is one deck cationic exchange membrane, and interelectrode distance is 2cm, with titanium silk, does electronics collector.During startup, inoculation MFC after bacteria suspension is mixed in the ratio of 1:5 with nutritive medium, extrernal resistance is 1000 Ω, under (30 ± 1) ℃ constant temperature, moves.Described nutrient composition is: 30mmolL
-1trisodium Citrate, 50mmolL
-1pBS damping fluid and a small amount of VITAMIN and trace element, used after 121 ℃ of sterilizing 20min.More than the output voltage of MFC reaches 100mV, be considered as starting successfully.After this, only change the nutritive medium containing electron donor.
The output voltage (U) that gathers MFC by Keithley 2700, reference electrode during potential electrode electromotive force is Ag/AgCl electrode.Electric current (I) is by Ohm's law I=U/R
extcalculate and obtain, R
extfor external loop resistance.The drafting of polarization curve and power density curve, by regulating external loop resistance, is measured after corresponding output voltage, calculates and curve plotting.Current density I
sutilize respectively formula I with volumetric power density P
s=U/ (R
exta
an) and P=U
2/ (R
extv) calculate, wherein, A
anbe respectively annode area and the useful volume of MFC with V.
Experimental result as shown in Figure 4, be take Citrate trianion during as matrix, and first MFC has experienced the lag phase of about 20h after inoculating strain Z6, and then voltage starts progressively to rise, change matrix after rate of voltage rise obviously accelerate, and start successfully at 50h.Move 6 all after date maximum output voltages and reach 0.31V(Fig. 4 a), occur stable repeatably output voltage, when extrernal resistance is 400 Ω, obtaining maximum power density is 287.4mWm
-2(Fig. 4 b).Along with the increase of current density, anode potential raises gradually, and cathode potential reduces (Fig. 4 c) gradually.To MFC anode microbial film electron microscopic observation as shown in Figure 5, bacterial strain Z6 can be attached on anode and a large amount of enrichments form microbial films, and all has filament to be connected between thalline and between thalline and electrode.
The present embodiment shows, the running status of bacterial strain CCTCC M 2012446 in MFC is good, can under the condition of not adding external source electronics transmission intermediate, utilize Citrate trianion to produce higher electric energy, there is stronger electrochemical activity, for its application in environmental pollution reparation and bioenergy recovery provides assurance.
Embodiment 5
The anode characteristic of bacterial strain CCTCC M 2012446 in processing artificial simulated wastewater process
When MFC obtains stable can repetition after output voltage, utilize electrochemical workstation (CHI700, Shanghai occasion China) to carry out original position cyclic voltammetry to MFC anode.Described test adopts three-electrode system, take MFC anode as working electrode, and negative electrode is to electrode, inserts Ag/AgCl electrode as reference electrode in anolyte compartment.In 3 stages in an electrogenesis cycle, (Fig. 6 a), changes voltage rising stage (A), plateau (B) and decrement phase (C) after matrix, respectively MFC is carried out to cyclic voltammetry scan.Sweep speed for 5mVs
-1, the voltage range of scanning is-0.4V～0.4V.
The cyclic voltammetry curve of MFC anode as shown in Figure 6 b, the peak value at the CV curve redox peak of voltage during the rising stage is all lower, the peak value at the CV curve redox peak of voltage platform phase all presents increase tendency, and the peak value at the redox peak of the CV curve of voltage drop phase is obviously greater than the above two.This may be because along with the going deep into of electrogenesis cycle, and the issuable electronics of bacterial strain Z6 transmits intermediate and accumulates gradually.In addition, the spike potential difference of the CV curve of different times is little, and wherein oxidation peak is between-160mV～-200mV, and reduction peak is between 20mV～-20mV.
The present embodiment explanation bacterial strain CCTCC M 2012446 can transmit electronics by the mode of secretion electronics transmission intermediate, for this bacterium efficient electronics that transmits under the condition of not adding external source electronics transmission intermediate provides possibility, reduced the practical application cost of this bacterium in environmental pollution reparation and bioenergy recovery.
Embodiment 6
External source AQDS is the impact on artificial wastewater's treatment effect on bacterial strain CCTCC M 2012446
Described bacterial strain CCTCC M 2012446 is seeded to LB substratum, and (30 ± 1) ℃ anaerobism is cultivated 18h.The phase growth bacterium liquid in mid-term of taking the logarithm, centrifugal 10 minutes of 5000r/min, abandons supernatant liquor, adds PBS damping fluid and shakes up, and by above-mentioned steps, by gained bacteria suspension recentrifuge, so repeatedly cleans 2 times, and interpolation PBS damping fluid is made bacteria suspension, OD
600it is 0.6 left and right.
Described microbiological fuel cell is that cylindrical (diameter of section is 2cm, useful volume 6.28cm
3), material is polycarbonate.Air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is Nitric Acid Modified carbon felt, and negative electrode inner side is one deck cationic exchange membrane, and interelectrode distance is 2cm, with titanium silk, does electronics collector.
During startup, inoculation MFC after bacteria suspension is mixed in the ratio of 1:5 with nutritive medium, extrernal resistance is 1000 Ω, under (30 ± 1) ℃ constant temperature, moves.Nutritive medium is for containing 30mmolL
-1trisodium Citrate, 50mmolL
-1pBS damping fluid (pH 7.0) and a small amount of VITAMIN and trace element, after 121 ℃ of sterilizing 20min, use.More than the output voltage of MFC reaches 100mV, be considered as starting successfully.After this, only change the nutritive medium containing electron donor.
Until cell voltage, occur stablizing and can, after the repeat cycle, in anode chamber, add 50 μ mol L
-1external source AQDS.Result as shown in Figure 7, is added after external source AQDS, and the output voltage of MFC rises to 0.41V, during before without interpolation AQDS, improves 0.1V, is stopping adding after AQDS, and the output voltage of battery returns to rapidly the level before interpolation.
This example explanation external source electronics transmits intermediate A QDS this bacterium electricity generation process is had to obvious promoter action, for improving this bacterium electricity generation performance, efficient recovery bioenergy provides possibility.
Above-described embodiment is preferably embodiment of the present invention; but embodiments of the present invention are not restricted to the described embodiments; other any do not deviate from change, the modification done under spirit of the present invention and principle, substitutes, combination, simplify; all should be equivalent substitute mode, within being included in protection scope of the present invention.
Claims (10)
1. a strain acid-producing Klebsiella bacterium, is characterized in that, this bacterium is acid-producing Klebsiella bacterium (Klebsiella oxytoca) Z6, by the center preservation of Chinese Typical Representative culture collection, be called for short CCTCC, deposit number is: CCTCC NO:M 2012446, preservation date is on November 6th, 2012.
2. the application of bacterial strain in producing bioelectricity described in claim 1.
3. application according to claim 2, is characterized in that, comprises the steps:
(1) by inoculation described in claim 1 to ironic citrate substratum, 30 ± 5 ℃ of anaerobism are cultivated; The phase growth bacterium liquid in mid-term of taking the logarithm, centrifugal, abandon supernatant liquor, add PBS damping fluid and shake up, by above-mentioned steps, by gained bacteria suspension recentrifuge, so repeatedly clean 2 times, interpolation PBS damping fluid is made bacteria suspension;
(2) start single chamber air cathode microbial fuel cell, after the bacteria suspension obtaining is mixed with nutritive medium, be inoculated in microbiological fuel cell, temperature-constant operation under the culture temperature of step (1) in step (1).
4. application according to claim 3, is characterized in that, the described ironic citrate nutrient media components of step (1) is as follows: Tryptones 10gL
-1, yeast extract 5gL
-1, sodium-chlor 10gL
-1, add 20mmolL
-1ironic citrate be electron donor; The described nutrient composition of step (2) is 30mmolL
-1electron donor, 50mmolL
-1pBS damping fluid and a small amount of VITAMIN and trace element.
5. application according to claim 4, is characterized in that, in described nutritive medium, electron donor is one or more in Citrate trianion, glucose, glycerol, Sodium.alpha.-hydroxypropionate and L-glutamic acid; The formula of described PBS damping fluid is NH
4cl 0.31gL
-1, NaH
2pO
4h
2o 2.452gL
-1, Na
2hPO
44.576gL
-1, KCl 0.13gL
-1, pH 7.0.
6. according to the application described in claim 3 or 4 or 5, it is characterized in that, described in the OD of the bacteria suspension made
600be 0.6; The pH of described substratum and nutritive medium is 7.0～7.4.
7. according to the application described in claim 3 or 4 or 5, it is characterized in that, described in step (2), microbiological fuel cell is cylindrical, and material is polycarbonate; Air cathode is for carrying platinum carbon paper, and anode is Nitric Acid Modified carbon felt, and negative electrode inner side is one deck cationic exchange membrane, and interelectrode distance is 2cm, with titanium silk, does electronics collector.
8. according to the application described in claim 3 or 4 or 5, it is characterized in that, until step (2) cell voltage, occur stable can, after the repeat cycle, interpolation external source electronics in anode chamber and transmit intermediate A QDS.
9. according to the application described in claim 3 or 4 or 5, it is characterized in that, the described culture temperature of step (1) is 30 ± 1 ℃, and incubation time is 16-18h, and centrifugal condition is centrifugal 10 minutes of 5000r/min.
10. according to the application described in claim 3 or 4 or 5, it is characterized in that, the volume ratio of described bacteria suspension and nutritive medium is 1:5.
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