CN101728544B - Application of citrobacter freundii in electricity generation by microorganism and electricity generation method - Google Patents
Application of citrobacter freundii in electricity generation by microorganism and electricity generation method Download PDFInfo
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- CN101728544B CN101728544B CN2009101936395A CN200910193639A CN101728544B CN 101728544 B CN101728544 B CN 101728544B CN 2009101936395 A CN2009101936395 A CN 2009101936395A CN 200910193639 A CN200910193639 A CN 200910193639A CN 101728544 B CN101728544 B CN 101728544B
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- citrobacter freundii
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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
Abstract
The invention discloses an application of citrobacter freundii in electricity generation by microorganism and an electricity generation method. The citrobacter freundii is taken as anode catalyst of the microbiological fuel cell to be applied to electricity generation by microorganism. The electricity generation method comprises preparing citrobacter freundii to be as inoculum for microbiological fuel cell; preparing catholyte and anolyte containing fuel; adding the inoculum for microbiological fuel cell into the anode chamber of the microbiological fuel cell, standing for culturing, and carrying out electricity generation detection. The invention opens up a new application field for citrobacter freundii and provides a new highly adaptable electrogenesis microorganism. The citrobacter freundii LY-3 is easy to culture, and the electrogenesis is high with various organism electrogenesis utilized.
Description
Technical field
The invention belongs to environment and technical field of new energies, specifically, relate to application and the electricity-generating method thereof of citrobacter freundii (Citrobacter freundii) aspect electricity generation by microorganism.
Background technology
In recent years, in the face of the dual crisis of energy shortage and environmental pollution, (MicrobialFuel Cell MFC) with its unique character, demonstrates great research and using value to microbiological fuel cell.Microbiological fuel cell is a kind ofly to utilize microbe as catalyst, the chemical energy in the fuel is converted into the device of electric energy.As far back as 1911, Britain botanist Potter just found that microbe can produce electric current.But subsequently, the research of related microorganism fuel cell is made slow progress always, and up to the eighties in 20th century, the extensive use of redox mediators makes the power output of MFC be greatly improved, and has promoted the development of MFC thus.But redox mediators costs an arm and a leg, part is poisonous, has restricted it and has further developed.Subsequently, the researcher finds that in succession electron transport that certain micro-organisms can be under the condition of no amboceptor produces in directly with body is to electrode.So far, the research of MFC has obtained breakthrough.At present, the improvement of to the effect that not having amboceptor MFC electricity generation performance of MFC research, and in sewage disposal, produce the application of aspects such as hydrogen, bio-sensing, biological restoration.
The electrogenesis bacterium is the key element of MFC electrogenesis, is the important content of MFC research.At present, if the electrogenesis microbial host Proteobacteria (Proteobacteria) that separates and the bacterium of Firmicutes (Firmicutes), mostly be facultative anaerobe, have metabolism modes such as anaerobic respiration and fermentation, acquisition energy such as oxidable carbohydrate, organic acid are kept growth.The electrogenesis microbe of having reported has Rhodopseudomonas palustris (Rhodopseudomonaspalustris) and human pallid bacillus (Ochrobactrum anthropi); The iron vat red is educated bacterium (Rhodofoferaxferrireducens); Aeromonas hydrophila (Aeromonas hydrophilia), pseudomonas aeruginosa (Pseudomonas aeruginosa) and uncommon ten thousand Salmonellas (Shewanella putrefactions), S.oneidensis; Sulphur is original place bacillus (Geobacter sulfurreducens), metallic reducing ground bacillus (G.metallireducens), propionic acid sulfolobus (Desulfoblbus propionicus) also, clostridium butyricum (Clostridium butyricum) and Bai Shi clostridium (Clostridium beijerinckii) etc.
Citrobacter freundii (Citrobacter freundii) is mainly used in the correlative study of clinical bacteria and enzyme engineering at present, and relevant citrobacter freundii has the electrogenesis activity and still finds no report at home and abroad.
Summary of the invention
The object of the present invention is to provide the application of citrobacter freundii (Citrobacter freundii) aspect electricity generation by microorganism.
To achieve these goals, the present invention has taked following technical scheme:
Citrobacter freundii (Citrobacter freundii) is applied to electricity generation by microorganism as the anode catalyst of microbiological fuel cell.
Described citrobacter freundii (Citrobacter freundii) is LY-3, and deposit number is CGMCCNo.3246.
Citrobacter freundii bacterium colony smooth surface, translucent, neat in edge, surperficial glossy can be electron acceptor with the anode, utilize organic substance electrogenesis such as glucose, sodium acetate.
The 16SrDNA sequence of bacterial strain of the present invention is shown in SEQ ID NO:1.
Measure and 16S rDNA gene sequencing by morphological feature observation, Physiology and biochemistry, bacterial strain of the present invention is the new bacterial strain of citrobacter freundii, called after LY-3.
The present invention also provides citrobacter freundii (Citrobacter freundii) to be applied to the electricity-generating method of electricity generation by microorganism, may further comprise the steps:
(1) preparation citrobacter freundii (Citrobacter freundii) is as the microbe fuel cell inoculation thing;
(2) preparation catholyte and the anolyte that contains fuel;
(3) in the anode of microbial fuel cell chamber, add the microbe fuel cell inoculation thing, connect external circuit, carry out electrogenesis and detect.
The preparation method of microbe fuel cell inoculation thing is in the described step (1): citrobacter freundii is inoculated in the LB fluid nutrient medium, and 30 ℃, 100rpm cultivates 24h, and the centrifugal 4min of 4000rpm collects thalline, obtains the microbe fuel cell inoculation thing.
Fuel in the described step (2) is glucose or sodium acetate.Anolyte in the described step (2) is the mixed solution of the PBS of the glucose of 2g/L and 100mM/L.Catholyte in the described step (2) is 0.05M FeCN.
The amount of microbe fuel cell inoculation thing is the 0.2g/mL anolyte in the described step (3).
Compared with prior art, the present invention has following beneficial effect:
The present invention has opened up new application for citrobacter freundii, for microbiological fuel cell provides a kind of new adaptable electrogenesis microbe.Citrobacter freundii LY-3 is easy to cultivate, and can utilize multiple organic substance electrogenesis, and electrogenesis voltage is big.
Bacterial strain citrobacter freundii Citrobacter freundii LY-3 of the present invention has been preserved in China Committee for Culture Collection of Microorganisms of the depositary institution common micro-organisms center preservation of State Intellectual Property Office's appointment on August 21st, 2009, deposit number is CGMCC No.3246.
Description of drawings
Fig. 1 is the 16S rDNA phylogenetic tree of bacterial strain Citrobacter freundii LY-3 of the present invention;
Fig. 2 is the CV scintigram of bacterial strain Citrobacter freundii LY-3 bacterium liquid of the present invention;
Fig. 3 utilizes voltage that glucose and sodium acetate produce for fuel curve chart over time for bacterial strain Citrobacter freundii LY-3 of the present invention.
Embodiment
Further illustrate the present invention below in conjunction with specific embodiment.These embodiment only are used to illustrate the present invention, and can not limit protection scope of the present invention.
Embodiment 1: the screening of bacterial strain Citrobacter freundiiLY-3 of the present invention
1. bacterial strain separates
The mud sample is gathered at dark 10cm place under (Guangzhou) dry lakebed surface in Agricultural University Of South China, transfers to fast in the anaerobic operation case that has reached anaerobic condition, and sample thief adds in the test tube on a small quantity, the dilution of 10ml sterilized water, vibration mixing.Sample thief dilution 1ml is expelled in the 10ml IRM fluid nutrient medium, 30 ℃ of enrichment culture three generations.Then, in LB solid medium upper flat plate line, 30 ℃ of anaerobism are cultivated 3d, observe colony characteristics, and the bacterium line that the picking colony form is different respectively separates, and cultivate for 3 generations to obtain preserving behind the pure bacterial strain, treat that electrochemistry investigates.
Wherein, the component of IRM culture medium is: NaHCO
32.5g/L, KCl 0.1g/L, NH
4Cl 1.5g/L, NaH
2PO
40.6g/L, NaAc 20mM, ironic citrate 20mM, yeast extract 0.5g/L.
The component of LB culture medium is: peptone 10g/L, and yeast extract 5g/L, NaCl 10g/L adds 2% agar on the solid medium.
2. the screening of electrogenesis bacterium
The bacterium liquid of the pure bacterial strain that separation is obtained carries out cyclic voltammetric (CV) scanning and determines whether the bacterial strain of surveying has electro-chemical activity.Adopt three electrodes (the graphite work electrode of electrochemical workstation, platinum is to electrode, saturated calomel electrode) method is carried out CV scanning to 10ml bacterium liquid, scans between-0.8～0.8V with the speed of 100mV/s, and the 10ml culture medium is done contrast (CK), screening obtains the bacterial strain that a strain has electro-chemical activity, be LY-3, scanning result as shown in Figure 1, reduction peak has appearred in the CV of LY-3 bacterial strain scanning, and redox is irreversible, and it is electroactive to illustrate that bacterial strain LY-3 has.
Embodiment 2: the evaluation of bacterial strain Citrobacter freundiiLY-3 of the present invention
By morphological feature observation, Physiology and biochemistry mensuration and 16S rDNA gene sequencing the LY-3 bacterial strain that embodiment 1 obtains is identified.
1. morphological feature is observed
LY-3 bacterial strain bacterium colony smooth surface, translucent, neat in edge, surperficial glossy.
2. Physiology and biochemistry is measured
Bacterial strain LY-3 is as shown in table 1 with the Physiology and biochemistry character contrast of " common bacteria system identification handbook " middle Citrobacter bacterial classification.
The Physiology and biochemistry character contrast of table 1 bacterial strain LY-3 and citrobacter freundii
Annotate :+: the positive;-: feminine gender; ND: do not survey; D: difference
3.16S rDNA phylogenetic analysis
Extracting the full gene that kit extracts LY-3 with DNA of bacteria, is template with the gene that extracts, with universal primer 63f:5 '-AGAGTTTGATCCTGGCTCAG-3 ' then; 1387r:5 '-ACGGCTACCTTGTTACGACTT-3 ' carries out pcr amplification.
The PCR reaction system is: dna profiling 1 μ L, 10 * PCR buffer solution, 2 μ L, each 1 μ L of upstream and downstream primer, Taq archaeal dna polymerase 1 μ L, 10 * dNTP, 2 μ L, moisturizing to 20 μ L.
The pcr amplification program is: 94 ℃ of pre-sex change 5min; 94 ℃ of 40s, 42 ℃ of 1min, 72 ℃ of 1min, 30 circulations are extended 10min, 4 ℃ of preservations in 72 ℃ at last.
Amplified production carries out the analysis of 10g/L agarose gel electrophoresis, and purifying and recovery PCR product send biotech firm to carry out sequencing, and the 16SrDNA sequence of LY-3 is shown in SEQ ID NO:1.Submit to NCBI (www.ncbi.nlm.nih.gov) to carry out Megablast the sequence that obtains, obtain the high correlated series of homology, compare with ClustalX software, Mega software carries out Phylogenetic Analysis, adopts the adjacent method constructing system to grow tree.Fig. 2 is the phylogenetic tree of being done according to 16S rDNA sequence.
Amplification obtains the gene order of 1.6kbp, in GenBank nucleic acid sequence data storehouse, carry out the homologous sequence search, the homology of LY-3 and Citrobacter freundii (AB210978) is 99%, LY-3 is the new bacterial strain of citrobacter freundii, it number is GQ465944 that called after Citrobacter freundiiLY-3, GenBank accept.
Embodiment 3: the electrogenesis that bacterial strain Citrobacter freundii LY-3 of the present invention is fuel with glucose is investigated
Present embodiment makes up the microbiological fuel cell that utilizes the LY-3 generating according to prior art and method, and microbiological fuel cell adopts two Room, comprises anode chamber and cathode chamber, exchange membrane and external circuit.Electrode material is the carbon felt, connects extrernal resistance 1000 Ω.
The electrogenesis principle of MFC is: organic substance acts as a fuel in the anaerobism anode chamber by the citrobacter freundii oxidation, produce electronics and proton, wherein, electronics is caught and passes to galvanic anode by citrobacter freundii, electronics arrives negative electrode by external circuit, thereby form the loop and produce electric current, and proton arrives negative electrode by exchange membrane, with the oxygen water generation reaction.
The electrogenesis of present embodiment is investigated step:
(1) LY-3 is inoculated in the LB fluid nutrient medium, 30 ℃, 100rpm cultivates 24h, and the centrifugal 4min of 4000rpm collects thalline, as the microbe fuel cell inoculation thing;
(2) anode chamber adds the mixed solution of the PBS of glucose that anode reaction liquid is 2g/L and 100mM/L, and it is 0.05M FeCN that cathode chamber adds cathode reaction liquid; Wherein the prescription of PBS is: KCl 0.26g/L, NH
4Cl 0.62g/L, NaH
2PO
44.9g/L, Na
2HPO
49.15g/L.
(3) inoculate LY-3 bacterial strain (inoculum concentration is the 0.2g/mL anolyte) in the anode chamber, 30 ℃ leave standstill cultivation.
(4) anode, negative electrode are connected by 1000 Ω extrernal resistances, the every 1s record of data collecting card primary voltage, the output voltage result is as shown in Figure 3.
As shown in Figure 3, LY-3 may be used on electrogenesis in the microbiological fuel cell, is that the microbiological fuel cell maximum voltage of fuel reaches 0.52V with glucose.
Embodiment 4: the electrogenesis that bacterial strain Citrobacter freundii LY-3 of the present invention is fuel with the sodium acetate is investigated
Experimental technique and experimental procedure be with embodiment 3, only anode reactant liquor in the step (2) changed into the mixed solution of the PBS of the sodium acetate of 2g/L and 100mM/L.The output voltage result as shown in Figure 3.
As shown in Figure 3, LY-3 may be used on electrogenesis in the microbiological fuel cell, and the microbiological fuel cell maximum voltage that with the sodium acetate is fuel is 0.39V.
Above-mentioned detailed description is at the specifying of possible embodiments of the present invention, and this embodiment is not in order to limiting claim of the present invention, does not allly break away from equivalence of the present invention and implements or change, all should be contained in the claim of the present invention.
Sequence table
＜110〉Guangzhou Energy Resource Inst., Chinese Academy of Sciences
＜120〉application and the electricity-generating method of citrobacter freundii in electricity generation by microorganism
<160>1
<170>Patent In version 3.1
<210>1
<211>1419bp
<212>rDNA
<213>Citrobacter freundii
<400>1
catgcagtcg aacggtagca cagagagctt gctctcgggt gacgagtggc 50
ggacgggtga gtaatgtctg ggaaactgcc cgatggaggg ggataactac 100
tggaaacggt agctaatacc gcataatgtc gcaagaccaa agagggggac 150
cttcgggcct cttgccatcg gatgtgccca gatgggatta gcttgtaggt 200
ggggtaacgg ctcacctagg cgacgatccc tagctggtct gagaggatga 250
ccagccacac tggaactgag acacggtcca gactcctacg ggaggcagca 300
gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcc atgccgcgtg 350
tatgaagaag gccttcgggt tgtaaagtac tttcagcgag gaggaaggcg 400
ttgtggttaa taaccttagt gattgacgtt actcgcagaa gaagcaccgg 450
ctaactccgt gccagcagcc gcggtaatac ggagggtgca agcgttaatc 500
ggaattactg ggcgtaaagc gcacgcaggc ggtctgtcaa gtcggatgtg 550
aaatccccgg gctcaacctg ggaactgcat ccgaaactgg caggctagag 600
tcttgtagag gggggtagaa ttccaggtgt agcggtgaaa tgcgtagaga 650
tctggaggaa taccgggtgg cgaaggcggc cccctggaca aagactgacg 700
ctcaggtgcg aaagcgtggg ggagcaaaca ggattagata ccctggtagt 750
ccacgccgta aacgatgtcg acttggaggt tgtgcccttg aggcgtggct 800
tccggagcta acgcgttaag tcgaccgcct ggggagtacg gccgcaaggt 850
taaaactcaa atgaattgac gggggcccgc acaagcggtg ggagcatgtg 900
gtttaattcg atgcaacgcg aagaacctta cctactcttg acatcccgag 950
gaatttagca gagatgcttt ggtgccttcg ggaactgtga gacaggtgct 1000
gcatggctgt cgtcagctcg tgttgtgaaa tgttgggtta agtcccgcaa 1050
cgagcgcaac ccttatcctt tgttgccagc ggtccggccg ggaactcaaa 1100
ggagactgcc agtgataaac tggaggaagg tggggatgac gtcaagtcat 1150
catggccctt acgagtaggg ctacacacgt gctacaatgg catatacaaa 1200
gagaagcgac ctcgcgagag caagcggacc tcataaagta tgtcgtagtc 1250
cggattggag tctgcaactc gactccatga agtcggaatc gctagtaatc 1300
gtggatcaga atgccacggt gaatacgttc ccgggccttg tacacaccgc 1350
ccgtcacacc atgggagtgg gttgcaaaag aagtaggtag cttaaccttc 1400
gggagggcgc tacc acttt 1419
Claims (7)
1. the application of citrobacter freundii (Citrobacter freundii) in electricity generation by microorganism, it is characterized in that: citrobacter freundii is applied to electricity generation by microorganism as the anode catalyst of microbiological fuel cell, and the deposit number of described citrobacter freundii is CGMCC No.3246.
2. the described citrobacter freundii of claim 1 (Citrobacter freundii) is applied to the electricity-generating method of electricity generation by microorganism, it is characterized in that: may further comprise the steps:
(1) preparation citrobacter freundii (Citrobacter freundii) is as the microbe fuel cell inoculation thing;
(2) preparation catholyte and the anolyte that contains fuel;
(3) in the anode of microbial fuel cell chamber, add the microbe fuel cell inoculation thing, connect external circuit, carry out electrogenesis and detect.
3. citrobacter freundii according to claim 2 (Citrobacter freundii) is applied to the electricity-generating method of electricity generation by microorganism, it is characterized in that: the preparation method of microbe fuel cell inoculation thing is in the described step (1): (Citrobacter freundii) is inoculated in the LB fluid nutrient medium with citrobacter freundii, 30 ℃, 100rpm cultivates 24h, the centrifugal 4min of 4000rpm collects thalline, obtains the microbe fuel cell inoculation thing.
4. citrobacter freundii according to claim 2 (Citrobacter freundii) is applied to the electricity-generating method of electricity generation by microorganism, it is characterized in that: the fuel in the described step (2) is glucose or sodium acetate.
5. citrobacter freundii according to claim 2 (Citrobacter freundii) is applied to the electricity-generating method of electricity generation by microorganism, it is characterized in that: the anolyte in the described step (2) is the mixed solution of the PBS of the glucose of 2g/L and 100mM/L.
6. citrobacter freundii according to claim 2 (Citrobacter freundii) is applied to the electricity-generating method of electricity generation by microorganism, it is characterized in that: the catholyte in the described step (2) is 0.05M FeCN.
7. citrobacter freundii according to claim 2 (Citrobacter freundii) is applied to the electricity-generating method of electricity generation by microorganism, it is characterized in that: the addition of microbe fuel cell inoculation thing is the 0.2g/mL anolyte in the described step (3).
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| CN109321496B (en) * | 2018-09-30 | 2020-06-23 | 浙江工业大学 | Citrobacter freundii ZJB-17010 and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6454944B1 (en) * | 2000-11-08 | 2002-09-24 | Larry J. Raven | Process and apparatus for conversion of biodegradable organic materials into product gas |
| CN1949577A (en) * | 2005-10-14 | 2007-04-18 | 中国科学院过程工程研究所 | Bioreactor-direct microbe fuel cell and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US6454944B1 (en) * | 2000-11-08 | 2002-09-24 | Larry J. Raven | Process and apparatus for conversion of biodegradable organic materials into product gas |
| CN1949577A (en) * | 2005-10-14 | 2007-04-18 | 中国科学院过程工程研究所 | Bioreactor-direct microbe fuel cell and use thereof |
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