CN103215201A - Klebsiella oxytoca and its application in bioelectricity production - Google Patents
Klebsiella oxytoca and its application in bioelectricity production Download PDFInfo
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- 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, (Microbial Fuel Cell, MFC) technology has been subjected to paying close attention to widely as a kind of emerging wastewater treatment and pollution remediation technology microbiological fuel cell.It directly by microorganism with organism in the fuel or inorganics oxidation and produce electronics and proton, the proton and the electronics that produce are passed to negative electrode through proton exchange membrane and external circuit respectively, produce water with the reaction of final electron acceptor(EA), the most at last the chemical energy of organism or inorganics and transform electric energy in the fuel.The biological treatment that this technology will be polluted is reclaimed with bioenergy and is combined, and when disposing of sewage, can also obtain electric energy.
In microbiological fuel cell, electrogenesis microorganism (Exoelectrogens) is being brought into play critical effect.The electrogenesis microorganism is that those can carry out the microorganism that born of the same parents' exoelectron transmits, they can form microbial film in the anode enrichment, can pass through the metabolism degradation of organic substances, produce electronics simultaneously and be passed to outside the born of the same parents, directly or indirectly it is delivered on the electrode again, thereby realizes biocatalysis and drive the operation of MFC.Along with the difference of electrogenesis microbe species, it is also different with the mechanism of transmitting electronics that it produces electronics, and there is notable difference in the electrochemical activity that shows.
At present, the electrogenesis microorganism that Chinese scholars has been found that focuses mostly at Proteobacteria, and also having a spot 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.Simultaneously, aspect the electrogenesis characteristic, known most of electrogenesis bacterium still exist electricity generation performance lower, utilize the first-class defective of substrate type competitive list, can not satisfy 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 pod membrane is arranged, and 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.Successively all it utilizes the glucose electricity generation performance to the Klebsiella sp.ME17 that is separated in Klebsiella pneumoniae bacterial strain L17 that is separated to from soil and the rice soil by test 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 the 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, show that through the MFC test it has electrochemical activity, simultaneously, the substrate type of test only is a glucose.As can be seen, 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 remain to be verified.
Summary of the invention
The present invention has overcome above-mentioned defective, 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 Guangzhou Xi Lang sewage work second pond, obtain through the electrochemistry enrichment culture of microbiological fuel cell, artificial separation and purification, this bacterium is acid-producing Klebsiella bacterium (Klebsiella oxytoca) Z6, by China's typical culture collection center preservation, be called for short CCTCC, deposit number is: CCTCC NO:M 2012446, preservation date is on November 6th, 2012, and the preservation address is a China. Wuhan. and Wuhan University.The bacterium colony of this bacterium on the ironic citrate substratum is circle, black, the edge is smooth glossy, the about 2mm of diameter.The form that transmission electron microscope is observed this bacterium down 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 the growth logarithmic phase is 2～20h.Biological characteristics is: Gram-negative, amphimicrobian has the iron reductibility, can utilize trisodium citrate, glucose, glycerol, Sodium.alpha.-hydroxypropionate, L-glutamic acid.This bacterium can under anaerobic be transmitted electronics to born of the same parents' exoelectron 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) with the described inoculation of claim 1 to the 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 the PBS damping fluid and shake up,, clean so repeatedly 2 times gained bacteria suspension recentrifuge by above-mentioned steps, add the PBS damping fluid and make bacteria suspension;
(2) start the single chamber air cathode microbial fuel cell, with the bacteria suspension that obtains in the step (1) be inoculated in microbiological fuel cell after nutritive medium mixes, temperature-constant operation gets final product under the culture temperature of step (1).When the output voltage of MFC reaches more than the 100mV, be considered as starting successfully, after this, only change the nutritive medium that contains 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, electron donor is in Citrate trianion, glucose, glycerol, Sodium.alpha.-hydroxypropionate and the L-glutamic acid one or more in the described nutritive medium; The prescription 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.
Preferably, the OD of the described bacteria suspension of making
600Be 0.6; The pH of described substratum and nutritive medium is 7.0～7.4.
Preferably, microbiological fuel cell described in the step (2) is that cylindrical (diameter of section is 2cm, useful volume 6.28cm
3), material is a polycarbonate.Air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is a Nitric Acid Modified carbon felt, and the negative electrode inboard is one deck cationic exchange membrane, and interelectrode distance is 2cm, does the electronics collector with the titanium silk.The concrete method of modifying of Nitric Acid Modified carbon felt is: pretreated carbon felt is soaked 5h with concentrated nitric acid (65%), washes repeatedly to neutrality with deionized water, put into 120 ℃ of baking ovens oven dry 2h after, place in the moisture eliminator standby.Carbon felt pretreatment process is: after soaking 3h with acetone, take out through vacuum pump and to wash, to remove the oil-soluble substance on surface; With deionized water rinsing and soak, boil, change water 1 time again, boil 3h altogether every 0.5h, electrode materials is put into 120 ℃ of baking ovens oven dry 2h after, place in the moisture eliminator standby.
Preferably, the output voltage (U) of MFC described in the step (2) adopts Keithley 2700 to gather
Preferably, cationic exchange membrane soaks 24h with 5%NaCl in advance described in the step (2).
Preferably, treat step (2) but after the stable repeat cycle appears in cell voltage, add external source electron transport intermediate A QDS in the anode chamber.
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.When being matrix with the Citrate trianion, the single chamber air cathode MFC of this strain construction can produce maximum output voltage 0.31V, and maximum power density is 287.4mWm
-2Except that 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.Interpolation external source electron transport intermediate (AQDS) has certain promoter action to the electricity generation process of this bacterial strain.
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, and maximum power density is 287.4mWm when being matrix with the Citrate trianion
-2This bacterial strain CCTCC M 2012446 can also utilize polytype organism electrogenesis, this means this bacterium will various industrial sewage handle and the environmental pollution reparation in play a significant role.Simultaneously, this bacterial strain is the amphimicrobian type, do not need strict anaerobic environment in the electricity generation process, has reduced in the practical application requirement to waste water and environmental pollution treatment device.
Description of drawings
Fig. 1 is the MFC trigger voltage figure of inoculum with second pond mud for embodiment 1.
Fig. 2 is the bacterial strain cyclic voltammetry curve figure of embodiment 2.
Fig. 3 is MFC voltage, power density and the enclosed pasture efficiency diagram of matrix with multiple organism for the bacterial strain of embodiment 3.
Fig. 4 is MFC voltage (a), polarization curve (b) and the electrode potential figure (c) of matrix with the Citrate trianion for the bacterial strain of embodiment 4.
Fig. 5 is MFC anode and microbial film microscopic examination figure among the embodiment 4, (a) is blank anode, * 500 times; (b) be the anode microbial film, * 500 times; (c) be the anode microbial film, * 5000 times; (d) be attached to the thalline on the single carbon fiber, * 40000 times.
Fig. 6 is MFC anodic original position cyclic voltammetry curve figure among the embodiment 5, (a) is the different times synoptic diagram, (b) is the CV curve of different times.
Fig. 7 adds external source AQDS to MFC output voltage influence graphic representation among the embodiment 6.
Embodiment
Below in conjunction with specific embodiment the present invention is done further concrete detailed description the in detail, but embodiments of the present invention are not limited thereto, the processing parameter for not indicating especially can carry out with reference to routine techniques.Used biochemistry pool mud is from Guangzhou Xi Lang sewage work in the embodiment of the invention.
Embodiment 1
Screening and the separation of bacterial strain CCTCC M 2012446
Get the second pond mud 300mL of sewage work and transfer in the 500mL beaker, add glucose nutritive medium 200mL, stir, use NaHCO
3Solution is adjusted about pH value to 7.0 (6.7-7.2), places 30 ℃ of incubators to cultivate 1 day.Measure the pH value, and use NaHCO in good time
3Solution is adjusted the pH value to 6.7-7.2, stirs, and treats its post precipitation, abandoning supernatant.So repeat to cultivate 3 days standby.
Start the single chamber air cathode microbial fuel cell, described MFC is rectangular parallelepiped (length * wide * height=8.5 * 7 * 6.5cm
3, useful volume 200mL), material is the synthetic glass of 0.5cm, and an end seals 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, does the electronics collector with the titanium silk.During startup, get the mud after the pre-cultivation, and inoculate MFC after the mixed of nutritive medium by 1:1, extrernal resistance is 1000 Ω, operation under (30 ± 1) ℃ constant temperature.Nutritive medium is for containing 1gL
-1The PBS(pH 7.0 of glucose) and a small amount of VITAMIN and trace element, use behind 121 ℃ of sterilization 20min.When the output voltage of MFC reaches more than the 100mV, be considered as starting successfully.After this, only change the nutritive medium that contains electron donor.Gather the output voltage (U) of MFC by Keithley 2700.The result as shown in Figure 1, start battery 180 as a child output voltage reaches stable substantially, maximum output voltage is 0.37V.Can observe one deck microbial film at the MFC anode surface.
Treat this MFC steady running after 1 month, take out the long biomembranous anode that has, it is some to scrape the anode microbial film with blade, and inoculation ironic citrate liquid nutrient medium places under (30 ± 1) ℃ condition anaerobism to cultivate 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 carries out the anaerobism cultivation in the ironic citrate liquid nutrient medium.Go down to posterity 7-8 time so repeatedly, obtain many strains pure culture bacterial strain.Pure bacterium is carried out its electrochemical activity of cyclic voltammetric analysis verification, obtain a strain electrochemical activity bacterium Z6 at last.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 the 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 the PBS damping fluid and shakes up, and, cleans so repeatedly 2 times gained bacteria suspension recentrifuge by above-mentioned steps, adds the PBS damping fluid and makes bacteria suspension, OD
600Be about 0.6.With electrochemical workstation the gained bacteria suspension is done the cyclic voltammetric test, three-electrode system is adopted in test, is working electrode with the glass-carbon electrode, and the Ag/AgCl electrode is a reference electrode, and platinized platinum is a counter electrode, sweeps speed and is 100mVs
-1, sweep limit is-0.8V～0.8V.Experimental result is seen Fig. 2, bacterial strain Klebsiella oxytoca Z6 cultivates 1d in the LB substratum after, obtain cyclic voltammetry curve a pair of tangible redox peak is arranged, wherein oxidation peak is between-50mV～130mV, reduction peak is between 210mV～360mV.
The bacterial strain CCTCC M 2012446 of 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 handling kinds of artificial waste water
Described bacterial strain CCTCC M 2012446 is seeded to the 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 the PBS damping fluid and shakes up, and, cleans so repeatedly 2 times gained bacteria suspension recentrifuge by above-mentioned steps, adds the PBS damping fluid and makes bacteria suspension, OD
600Be about 0.6.
Start the 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 a polycarbonate.Air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is a Nitric Acid Modified carbon felt, and the negative electrode inboard is one deck cationic exchange membrane, and interelectrode distance is 2cm, does the electronics collector with the titanium silk.During startup, the phase growth bacterium liquid in mid-term of taking the logarithm and is inoculated MFC after the mixed of nutritive medium by 1:5, and extrernal resistance is 1000 Ω, moves under (30 ± 1) ℃ constant temperature.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 use behind 121 ℃ of sterilization 20min.When the output voltage of MFC reaches more than the 100mV, be considered as starting successfully.After this, only change the nutritive medium that contains 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
AnBe anolyte compartment's volume; Δ COD is t
bThe amount of the COD of time internal consumption.
Experimental result as shown in Figure 3, under 30 ℃, the condition of extrernal resistance 1000 Ω, this bacterium is respectively with 30mmolL
-1Trisodium Citrate, glucose, glycerol, Sodium.alpha.-hydroxypropionate, when L-glutamic acid is electron donor, the maximum output voltage of generation is respectively 0.31V, 0.213V, 0.207V, 0.26V, 0.107V.Maximum power density that obtains during for electron donor with the Citrate trianion and enclosed pasture efficient are respectively 287.4mWm
-2, 37.73%; When being electron donor with glucose, the maximum power density of generation and enclosed pasture efficient are respectively 140.4mWm
-2, 3.7%; When being electron donor with the glycerol, the maximum power density of generation and enclosed pasture efficient are respectively 140mWm
-2, 15.47%; When being electron donor with the Sodium.alpha.-hydroxypropionate, the maximum power density of generation and enclosed pasture efficient are respectively 215mWm
-2, 2.28%; When being electron donor with L-glutamic acid, the maximum power density of generation and enclosed pasture efficient are respectively 36.4mWm
-2, 6%.
By present embodiment as can be known, bacterial strain CCTCC M 2012446 has can utilize polytype organism electrogenesis, for the application of this bacterium in repairing the recovery of broad variety environmental pollution and bioenergy laid a good foundation.
Embodiment 4
The application of bacterial strain CCTCC M 2012446 in handling 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 the PBS damping fluid and shakes up, and, cleans so repeatedly 2 times gained bacteria suspension recentrifuge by above-mentioned steps, adds the PBS damping fluid and makes bacteria suspension, OD
600Be about 0.6.
Start the single chamber air cathode microbial fuel cell, described MFC is that cylindrical (diameter of section is 2cm, useful volume 6.28cm
3), material is a polycarbonate.Air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is a Nitric Acid Modified carbon felt, and the negative electrode inboard is one deck cationic exchange membrane, and interelectrode distance is 2cm, does the electronics collector with the titanium silk.During startup, bacteria suspension and nutritive medium are inoculated MFC after the mixed by 1:5, extrernal resistance is 1000 Ω, operation under (30 ± 1) ℃ constant temperature.Described nutrient composition is: 30mmolL
-1Trisodium Citrate, 50mmolL
-1PBS damping fluid and a small amount of VITAMIN and trace element use behind 121 ℃ of sterilization 20min.When the output voltage of MFC reaches more than the 100mV, be considered as starting successfully.After this, only change the nutritive medium that contains electron donor.
By the output voltage (U) of Keithley 2700 collection MFC, the reference electrode during the potential electrode electromotive force is the Ag/AgCl electrode.Electric current (I) is by Ohm's law I=U/R
ExtCalculate and obtain R
ExtBe external loop resistance.The drafting of polarization curve and power density curve, is calculated and curve plotting behind the output voltage of measurement correspondence by regulating external loop resistance.Current density I
sUtilize formula I respectively 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, when being matrix with the Citrate trianion, MFC has at first experienced the lag phase of about 20h behind inoculating strain Z6, voltage begins progressively to rise then, 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), stable repeatably output voltage occurs, obtaining maximum power density when extrernal resistance is 400 Ω 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 attached on the anode and a large amount of enrichments form microbial films, and all have filament to be connected between the thalline and between thalline and the electrode.
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 electron transport intermediate, utilize Citrate trianion to produce higher electric energy, has stronger electrochemical activity, for its application in environmental pollution reparation and bioenergy recovery provides assurance.
The anode characteristic of bacterial strain CCTCC M 2012446 in handling artificial simulated wastewater process
When MFC obtain stable can repeat output voltage after, utilize electrochemical workstation (CHI700, Shanghai occasion China) that the MFC anode is carried out the test of original position cyclic voltammetric.Three-electrode system is adopted in described test, is working electrode with the MFC anode, and negative electrode is a counter electrode, inserts the Ag/AgCl electrode in the anolyte compartment as reference electrode.(Fig. 6 a), promptly change voltage rising stage (A), plateau (B) and decrement phase (C) after the matrix, respectively MFC is carried out cyclic voltammetry scan in 3 stages in an electrogenesis cycle.Sweep speed and be 5mVs
-1, the voltage range of scanning is-0.4V～0.4V.
MFC anodic cyclic voltammetry curve is shown in Fig. 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 decrement phase is obviously greater than the above two.This may be because along with the going deep into of electrogenesis cycle, the issuable electron transport intermediate of bacterial strain Z6 accumulates gradually.In addition, the spike potential difference of the CV curve of different times is little, wherein oxidation peak-160mV～-200mV between, reduction peak 20mV～-20mV between.
Present embodiment explanation bacterial strain CCTCC M 2012446 can transmit electronics by the mode of secretion electron transport intermediate, for this bacterium efficient transfer electronics under the condition of not adding external source electron transport intermediate provides possibility, reduced the practical application cost of this bacterium in environmental pollution reparation and bioenergy recovery.
Embodiment 6
External source AQDS is to the influence of 2012446 pairs of artificial wastewater's treatment effects of bacterial strain CCTCC M
Described bacterial strain CCTCC M 2012446 is seeded to the 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 the PBS damping fluid and shakes up, and, cleans so repeatedly 2 times gained bacteria suspension recentrifuge by above-mentioned steps, adds the PBS damping fluid and makes bacteria suspension, OD
600Be about 0.6.
Described microbiological fuel cell is that cylindrical (diameter of section is 2cm, useful volume 6.28cm
3), material is a polycarbonate.Air cathode is for carrying platinum carbon paper (0.5mgcm
-2), anode is a Nitric Acid Modified carbon felt, and the negative electrode inboard is one deck cationic exchange membrane, and interelectrode distance is 2cm, does the electronics collector with the titanium silk.
During startup, bacteria suspension and nutritive medium are inoculated MFC after the mixed by 1:5, extrernal resistance is 1000 Ω, operation under (30 ± 1) ℃ constant temperature.Nutritive medium is for containing 30mmolL
-1Trisodium Citrate, 50mmolL
-1PBS damping fluid (pH 7.0) and a small amount of VITAMIN and trace element, use behind 121 ℃ of sterilization 20min.When the output voltage of MFC reaches more than the 100mV, be considered as starting successfully.After this, only change the nutritive medium that contains electron donor.
After treating that but the stable repeat cycle appears in cell voltage, add 50 μ mol L in the anode chamber
-1External source AQDS.The result as shown in Figure 7, add external source AQDS after, the output voltage of MFC rises to 0.41V, does not improve 0.1V when having the AQDS of interpolation before, after stopping to add AQDS, the output voltage of battery returns to the level before adding rapidly.
This example explanation external source electron transport intermediate A QDS has obvious facilitation to this bacterium electricity generation process, for improve this bacterium electricity generation performance, the efficient recovery bioenergy provides possibility.
The foregoing description is a preferred implementation 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 the principle, substitutes, combination, simplify; all should be the substitute mode of equivalence, be included within 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 China's typical culture collection center preservation, be called for short CCTCC, deposit number is: CCTCC NO:M 2012446, preservation date are on November 6th, 2012.
2. the application of the described bacterial strain of claim 1 in producing bioelectricity.
3. application according to claim 2 is characterized in that, comprises the steps:
(1) with the described inoculation of claim 1 to the 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 the PBS damping fluid and shake up,, clean so repeatedly 2 times gained bacteria suspension recentrifuge by above-mentioned steps, add the PBS damping fluid and make bacteria suspension;
(2) start the single chamber air cathode microbial fuel cell, with the bacteria suspension that obtains in the step (1) be inoculated in microbiological fuel cell after nutritive medium mixes, temperature-constant operation gets final product under the culture temperature of 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, electron donor is one or more in Citrate trianion, glucose, glycerol, Sodium.alpha.-hydroxypropionate and the L-glutamic acid in the described nutritive medium; The prescription 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 claim 3 or 4 or 5 described application, it is characterized in that the OD of the described bacteria suspension of making
600Be 0.6; The pH of described substratum and nutritive medium is 7.0～7.4.
7. according to claim 3 or 4 or 5 described application, it is characterized in that microbiological fuel cell is cylindrical described in the step (2), material is a polycarbonate; Air cathode is for carrying the platinum carbon paper, and anode is a Nitric Acid Modified carbon felt, and the negative electrode inboard is one deck cationic exchange membrane, and interelectrode distance is 2cm, does the electronics collector with the titanium silk.
8. according to claim 3 or 4 or 5 described application, it is characterized in that, treat step (2) but after the stable repeat cycle appears in cell voltage, add external source electron transport intermediate A QDS in the anode chamber.
9. according to claim 3 or 4 or 5 described application, it is characterized in that the described culture temperature of step (1) is 30 ± 1 ℃, incubation time is 16-18h, and centrifugal condition is centrifugal 10 minutes of 5000r/min.
10. according to claim 3 or 4 or 5 described application, it is characterized in that the volume ratio of described bacteria suspension and nutritive medium is 1:5.
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| CN108866038A (en) * | 2018-06-15 | 2018-11-23 | 武汉理工大学 | Electrically-active bacterium isolation and purification method based on gradient dilution and constant potential culture |
| CN109796024A (en) * | 2019-03-11 | 2019-05-24 | 福建师范大学 | A method of kaolin is brightened using composite nutrient culture microorganism |
| CN109796025A (en) * | 2019-03-11 | 2019-05-24 | 福建师范大学 | A method of utilizing di-iron trioxide in Mixed Microbes also protokaolin |
| CN109796026A (en) * | 2019-03-11 | 2019-05-24 | 福建师范大学 | A kind of kaolin bleaching method of low cost |
| CN109796023A (en) * | 2019-03-11 | 2019-05-24 | 福建师范大学 | A kind of method of kaolin microorganism iron-removal and whitening |
| CN112063550A (en) * | 2020-08-24 | 2020-12-11 | 郑州轻工业大学 | For reducing Fe in complex stateⅢAnaerobic strain, culture method and application thereof |
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