CN103898005A - Electrochemically-active bacterium and screening method thereof - Google Patents
Electrochemically-active bacterium and screening method thereof Download PDFInfo
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- CN103898005A CN103898005A CN201410010746.0A CN201410010746A CN103898005A CN 103898005 A CN103898005 A CN 103898005A CN 201410010746 A CN201410010746 A CN 201410010746A CN 103898005 A CN103898005 A CN 103898005A
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Abstract
The invention relates to a bacterium variety applied to a bio-electrochemical system, aiming to provide a new variety of electrochemically-active bacterium and a screening method of the electrochemically-active new variety. The 16S rRNA sequence of the electrochemically-active bacterium is shown in SEQ ID NO: 1. The screening method of the electrochemically-active bacterium is characterized in that an anode bio-film enriching electrochemically-active microorganisms is selected as an isolated bacterium source, a micro bio-electrochemical reactor is used as an isolating device, and the electricity generation capacity of the bio-electrochemical reactor is taken as a screening index. The obtained electrochemically-active bacterium, disclosed by the invention, is a new variety of agrobacterium, provides a precious microorganism resource for research on the extracellular electron transfer mechanism of anode microorganisms of the bio-electrochemical system, has the characteristics of high electrochemical activity and salt tolerance, can be used for improving the performance of the bio-electrochemical system, and can be applied to the bio-electrochemical system of a high salt system.
Description
Technical field
The present invention relates to the bacterium kind of applying in a kind of bioelectrochemistry system, particularly the screening method of electrochemical activity bacterium novel species and electrochemical activity novel species thereof (Geobacter anodereducens SD-1).
Background technology
Bioelectrochemistry system (BES) class is utilized the reaction of microorganism anode-catalyzed oxidation, electronics is delivered to negative electrode and carries out reduction reaction, thereby chemical energy is converted into the device of electric energy, mainly comprises microbiological fuel cell (MFC) and microorganism electrolysis cell (MEC).When take waste water as anode substrate, this type systematic can such as, reclaim the energy in abandoned biomass with different energy form (electric energy, Hydrogen Energy etc.) in processing waste water.Therefore, bioelectrochemistry system was widely studied in nearly 10 years, became a new study hotspot.The biological catalyst (anode microorganism) that wherein system relies on, is called electrochemical activity bacterium.Geobacter metallireducens (Geobacter sulfurreducens) is the type strain of electrochemical activity microorganism, its electrochemical activity and mixed culture flora quite, and the dominant population in mixed bacterium anode microbial film often.But this bacterium is to separate and obtain from bed mud with its iron reducing power at first, is used to subsequently in bioelectrochemistry system.Nearest research finds that the process of micro-reduction metal oxide and the behavior that electronics is led electrode by cell are different.Some microorganisms can not reducing metal oxide but can be in BESs electrogenesis separately, vice versa.This shows the electron acceptor(EA) than mineral substance, the characteristic that bacterium growth needs on electrode is different.Therefore, we infer take the electrochemical activity (inoculating the electricity generation ability of the bioelectrochemistry reactor of this bacterium) of bacterium rather than metallic reducing ability as index, take the mixed bacterium anode microbial film of highly enriched electrochemical activity bacterium as isolate source, can obtain electrochemical activity higher, more can adapt to the electrochemical activity bacterium of bioelectrochemistry system.
New electrochemical activity bacterium can be optimized the anode working performance of bioelectrochemistry system on the one hand, can provide new Microbial resources for the research of biological electro-chemical systems anode electronic transfer machine reason on the other hand.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes deficiency of the prior art, and a kind of electrochemical activity bacterium and screening method thereof are provided.
For technical solution problem, solution of the present invention is:
A kind of electrochemical activity bacterium is provided, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation name is called anode reduction earth bacillus SD-1 (Geobacter anodereducens SD-1), preserving number is CGMCC No:8662, preservation date is: 2013.12.30, depositary institution address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.
In the present invention, the 16S rRNA sequence of described this electrochemical activity bacterium is as shown in SEQ ID NO:1.
The present invention also provides the screening method of electrochemical activity bacterium, is to adopt dilution-disappearance method to carry out separation, purifying and the screening of electrochemical activity bacterium; It is characterized in that, the anode microbial film of the electrochemical activity microorganism that is selective enrichment, as isolate source, utilizes miniature organism electrochemical reactor as tripping device, and take the electricity generation ability of bioelectrochemistry reactor as screening index; Specifically comprise:
(1) described enrichment the anode microbial film of electrochemical activity microorganism take from bioelectrochemistry reactor, this bioelectrochemistry reactor is inoculated by the water outlet of urban domestic wastewater treatment plant preliminary sedimentation tank at first, take formic acid as matrix, steady running is half a year at least, so as on its anode material formation of deposits anode microbial film; Colony assay through clone library shows, the dominant population in anode microbial film is the acetic acid bacillus of Geobacter metallireducens and the quantity >20% of quantity >50%; In this bioelectrochemistry reactor, by acetic acid bacillus, formic acid is changed into the acetic acid of concentration <50ppm, then Geobacter metallireducens utilizes this acetic acid to carry out electrogenesis as matrix;
(2) tripping device that screening process is used is miniature organism electrochemical reactor: built by 5mL serum bottle, and the graphite cake that anode material is 1.5cm × 1cm, cathode material is 1.5cm × 1cm stainless (steel) wire; Stainless Steel Wire or two electrodes of titanium silk and negative and positive after sand paper cleaning are connected, as outlet line derived current; The outlet line of electrode is respectively through a diameter 20mm butyl rubber bung; After reactor is sealed by aluminum foil cover, with autoclaving; Get the anode diffraction patterns for biomembrane samples of bioelectrochemistry reactor, with being inoculated in sterilized miniature organism electrochemical reactor after phosphate buffer solution (PBS) doubling dilution;
(3) take the electricity generation ability of miniature organism electrochemical reactor as index, select the microreactor under can the maximum dilution multiple of generation current to carry out next separation cycle, until obtain pure culture microorganism---anode reduction earth bacillus; Described separation cycle refers to: whole process that dilution---inoculate new microreactor---operation microreactor---monitoring current produces.
In the present invention, while moving described miniature organism electrochemical reactor, the composition of the substratum of use is:
In every premium on currency, comprise sodium acetate 1g; NH
4cl0.31g; KCl0.13g; 50mM phosphate buffer solution; Wolfe liquid microelement 12.5mL; Wolfe VITAMIN liquid 5mL;
The composition of described phosphate buffer solution is:
In every premium on currency, comprise NaH
2pO
42H
2o5.618g; Na
2hPO
412H
2o6.155g;
The composition of described Wolfe liquid microelement is:
In every premium on currency, comprise nitrilotriacetic acid(NTA) 1.5g; MgSO
47H
2o3.0g; MnSO
4h
2o0.5g; NaCl1.0g; FeSO
47H
2o0.1g; CoCl
26H
2o0.1g; CaCl
20.1g; ZnSO
47H
2o0.1g; CuSO
45H
2o0.01g; AlK (SO
4)
212H
2o0.01g; H
3bO
30.01g; Na
2moO
42H
2o0.01g;
The composition of described Wolfe VITAMIN liquid is:
In every premium on currency, comprise vitamin H 2.0mg; Folic acid 2.0mg; Vitamin B6 10.0mg; Vitamin B15 .0mg; Riboflavin 5.0mg; Nicotinic acid 5.0mg; D-VB5 calcium 5.0mg; Vitamin B12 0.1mg; Para-amino benzoic acid 5.0mg; Thioctic Acid 5.0mg.
Compared with prior art, beneficial effect of the present invention is:
1, the electrochemical activity bacterium that the present invention obtains is a novel species of earth Bacillaceae, for born of the same parents' exoelectron transport mechanism of studying biological electro-chemical systems anode microorganism provides valuable Microbial resources.
2, electrochemical activity bacterium provided by the invention there is high electrochemical activity with salt tolerant feature, can improve the performance of bioelectrochemistry system, and can in the bioelectrochemistry system of high salt, apply.
Accompanying drawing explanation
Fig. 1 is the transmission electron microscope picture of electrochemical activity bacterium of the present invention in acetic acid-ironic citrate substratum.
Fig. 2 is that electrochemical activity bacterium of the present invention forms the biomembranous scanning electron microscope (SEM) photograph of anode in bioelectrochemistry reactor.
Embodiment
In the present invention, Physiology and biochemistry and molecular biology identification result that preservation name is called the electrochemical activity bacterium of anode reduction earth bacillus (Geobacter anodereducens SD-1) turn out to be a novel species, its biological property:
1, morphological specificity: regular tyrothricin that anode reduction earth bacillus SD-1 is unicellular growth (long 0.8-1.3 μ m, wide 0.3 μ is m); Gram-negative bacteria; Atrichia; Without gemma.
2, physiological and biochemical property: anode reduction earth bacillus SD-1 is oxytolerant anaerobic bacterium, can be at 15-42 ℃, and pH6-8.5, grows under 0-3%NaCl condition, and optimum growth temperature is 30-35 ℃, and optimum growh pH value is 7 left and right; Can be take acetic acid as electron donor, ferriferous oxide is electron acceptor(EA) growth, but can not be take sulphur simple substance and fumarate as electron acceptor(EA) growth.When take acetate as electron donor, take ironic citrate as electron acceptor(EA), under 30 ℃ of optimal growth conditions and pH7, Dai Shiwei 18h.Quite (in 50mM phosphate buffer solution) or 1.5 times are to (in 200mM phosphate buffer solution or salt solution) mixed bacterium for the electricity generation ability of bacterium in bioelectrochemistry system and mixed culture (waste water), and electricity generation ability occupy the prostatitis that obtains pure culture electrochemical activity microorganism.
3, molecular biology identification result: the similarity of kind Geobacter metallireducens the most close to it (Geobacter sulfurreducens) is 98%, but the DNA-DNA hybrid rate of anode reduction earth bacillus SD-1 and Geobacter metallireducens PCA is 61.6%.Therefore, this anode reduction earth bacillus is a novel species.
Specific embodiment:
Anode reduction earth bacillus SD-1 (Geobacter anodereducens SD-1) carries out seed selection according to following step:
One, the separation of bacterial strain, purifying and screening:
Adopt tradition dilution-disappearance method, selective enrichment the anode microbial film of electrochemical activity microorganism as isolate source, use miniature organism electrochemical reactor (mini-MEC) to carry out the separation of electrochemical activity bacterium for tripping device, its concrete steps are as follows:
From enrichment the anode carbon of bioelectrochemistry reactor of electrochemical activity microorganism brush and cut some graphite fibres, transfer in a 5mL serum bottle and (5mL50mM phosphate buffer solution and granulated glass sphere be housed), concussion bottle obtains cell suspending liquid.Then by cell suspending liquid at anaerobism Guan Zhongyong 50mM phosphate buffer solution from 10
– 1-10
– 9doubling dilution continuously.Sample under each extension rate is seeded to miniature organism electrochemical reactor with 10% inoculum size, then at 30 ℃ of operation reactors.Anode microbial growth is by the current monitoring producing.Detect that the reactor that electric current generates runs to this electrogenesis end cycle (20% peak point current).Concussion can detect the reactor under the maximum dilution multiple that electric current generates, and obtains anode microbial cell suspension, then repeats the separating step (dilution, inoculation and operation) of appeal, until obtain pure culture bacterium.The purity of bacterium is by microscopic examination and the monitoring of 16S rRNA cloning and sequencing.
Described enrichment the bioelectrochemistry reactor of anode carbon brush of electrochemical activity microorganism inoculated by sanitary wastewater (the present embodiment has been selected the water outlet of Pennsylvania State Univ-Univ Park USA's waste water treatment plant's preliminary sedimentation tank) at first, take formic acid as matrix, steady running is half a year at least, on anode material formation of deposits anode microbial film; Colony assay through clone library shows, the dominant population in the anode microbial film of this miniature organism electrochemical reactor is the Geobacter metallireducens of quantity >50% and the acetic acid bacillus of > quantity 20%; In this bioelectrochemistry reactor, by acetic acid bacillus, formic acid is changed into the acetic acid (this concentration is much smaller than the substrate concn of the conventional 1g/L acetic acid of reactor) of concentration <50ppm, then Geobacter metallireducens utilizes extremely low concentration acetic acid for the efficient electrogenesis of matrix (electricity generation ability of reactor of 1g/L acetic acid matrix is suitable with adopting).
The tripping device miniature organism electrochemical reactor that screening process is used is: built by 5mL serum bottle; Anode material is the graphite cake of 1.5cm × 1cm; Cathode material is 1.5cm × 1cm stainless (steel) wire; Stainless Steel Wire after sand paper cleaning or titanium silk regulating YIN and YANG two electrodes connect, as outlet line derived current; The outlet line of electrode is respectively through a thick butyl rubber bung (20mm diameter); After reactor is sealed by aluminum foil cover, reactor autoclaving; Reactor moves under 0.7V impressed voltage
Take the electricity generation ability of miniature organism electrochemical reactor as index, select the microreactor under can the maximum dilution multiple of generation current to carry out next separation cycle, until obtain pure culture microorganism---anode reduction earth bacillus; Described separation cycle refers to: whole process that dilution---inoculate new microreactor---operation microreactor---monitoring current produces.
The substratum that operation miniature organism electrochemical reactor uses is: sodium acetate 1g; NH
4cl0.31g; KCl0.13g; 50mM phosphate buffer solution (PBS); Wolfe liquid microelement 12.5mL; Wolfe VITAMIN liquid 5mL.Above-mentioned 50mM phosphate buffer solution (every premium on currency) is: NaH
2pO
42H
2o5.618g and Na
2hPO
412H
2o6.155g.Above-mentioned Wolfe liquid microelement (every premium on currency) is: nitrilotriacetic acid(NTA) 1.5g; MgSO
47H
2o3.0g; MnSO
4h
2o0.5g; NaCl1.0g; FeSO
47H
2o0.1g; CoCl
26H
2o0.1g; CaCl
20.1g; ZnSO
47H
2o0.1g; CuSO
45H
2o0.01g; AlK (SO
4)
212H
2o0.01g; H
3bO
30.01g; Na
2moO
42H
2o0.01g.Above-mentioned Wolfe VITAMIN liquid (every premium on currency) is: vitamin H 2.0mg; Folic acid 2.0mg; Vitamin B6 10.0mg; Vitamin B15 .0mg; Riboflavin 5.0mg; Nicotinic acid 5.0mg; D-VB5 calcium 5.0mg; Vitamin B12 0.1mg; Para-amino benzoic acid 5.0mg; Thioctic Acid 5.0mg.
Two, the Physiology and biochemistry of bacterium and morphological analysis:
The growth of bacterium in ironic citrate-acetic acid substratum detects with acridine orange cell counting; Fe (III) reducing power obtains with the generation that HCl extracts Fe (II) detection method detection Fe (II) ion; Transmission electron microscope is for observing the ne ar of ironic citrate-acetic acid substratum in stationary phase; Scanning electron microscope is for observing the ne ar on bioelectrochemistry reactor Anodic microbial film; The electricity generation ability evaluation of system for the electrochemical activity of bacterium in bioelectrochemistry reactor.Acridine orange cell counting: put backing filter membrane (MF-Millipore Membrane, 5.0 μ m apertures, 25mm diameter) on suction filtration device, add 2-3mL deionized water rinsing suction funnel and moistening backing filter membrane, vacuum filtration.Then, put the polycarbonate leaching film (Millipore of black at backing filter membrane, Isopore Membrane, 0.2 μ m aperture, 25mm diameter), avoid there is bubble between two filter membranes, in suction funnel, add 2mL detect sample, add 200 μ l acridine orange solution (100mg/L), with pipettor repeatedly pressure-vaccum mix sample and acridine orange solution, wait for 2min, vacuum filtration.Black filter membrane is placed on slide glass, adds an immersion oil covered, at the lower counting of fluorescent microscope (Olympus BX61).Precaution: sample needs dilution in advance, makes the number of cells in a visual field in fluorescent microscope between 15-30.HCl extraction Fe (II) detection method is: the nutrient solution sample of 0.1mL joins 5mL0.5N HCl, at room temperature extracts 15min.Then, 0.1mL extraction liquid joins in 50mmol/L HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, N-(2-hydroxyethyl) piperazine-N'-2-ethane sulfonic acid) damping fluid of the luxuriant and rich with fragrance alloxazine of 5mL (1g/L) (pH7).Mix after 15 seconds, measure the absorbance A 562 under 562nm.Morphological Identification reference literature method (eastern elegant pearl, Cai Miaoying. common bacteria system identification handbook. Beijing: Science Press, 2001.)
Three, the classification of bacterium is identified:
Extracting bacterial genomes DNA, is bacterium universal primer for the PCR reaction primer of 16S rDNA amplification: forward primer 27F, 5'-AGAGTTTGATCMTGGCTCAG-3' and reverse primer 1541R, 5'-AAGGAGGTGATCCAGCC-3'.Described pcr amplification system (50 μ L) is: 2 × Taq PCR MasterMix (day root), 25 μ L, the each 2 μ L of primer, water 20 μ L, template 1 μ L.Described pcr amplification method is: 95 ℃ of 5min; 94 ℃ of 1min; 55 ℃ of 1min; 72 ℃ of 1.5min; From second step, totally 30 circulations, finally extend 72 ℃ of 10min, check order.
Claims (4)
1. an electrochemical activity bacterium, be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation name is called anode reduction earth bacillus SD-1(Geobacter anodereducens SD-1), preserving number is CGMCCNo:8662, preservation date is: 2013.12.30.
2. electrochemical activity bacterium according to claim 1, is characterized in that, the 16SrRNA sequence of this electrochemical activity bacterium is as shown in SEQ ID NO:1.
3. the screening method of electrochemical activity bacterium described in claim 1 is to adopt dilution-disappearance method to carry out separation, purifying and the screening of electrochemical activity bacterium; It is characterized in that, the anode microbial film of the described electrochemical activity microorganism that is selective enrichment, as isolate source, utilizes miniature organism electrochemical reactor as tripping device, and take the electricity generation ability of bioelectrochemistry reactor as screening index; Specifically comprise:
(1) described enrichment the anode microbial film of electrochemical activity microorganism take from bioelectrochemistry reactor, this bioelectrochemistry reactor is inoculated by the water outlet of urban domestic wastewater treatment plant preliminary sedimentation tank at first, take formic acid as matrix, steady running is half a year at least, so as on its anode material formation of deposits anode microbial film; Colony assay through clone library shows, the dominant population in anode microbial film is the acetic acid bacillus of Geobacter metallireducens and the quantity >20% of quantity >50%; In this bioelectrochemistry reactor, by acetic acid bacillus, formic acid is changed into the acetic acid of concentration <50ppm, then Geobacter metallireducens utilizes this acetic acid to carry out electrogenesis as matrix;
(2) tripping device that screening process is used is miniature organism electrochemical reactor: built by 5mL serum bottle, and the graphite cake that anode material is 1.5cm × 1cm, cathode material is 1.5cm × 1cm stainless (steel) wire; Stainless Steel Wire or two electrodes of titanium silk and negative and positive after sand paper cleaning are connected, as outlet line derived current; The outlet line of electrode is respectively through a diameter 20mm butyl rubber bung; After reactor is sealed by aluminum foil cover, with autoclaving; Get the anode diffraction patterns for biomembrane samples of bioelectrochemistry reactor, with being inoculated in sterilized miniature organism electrochemical reactor after phosphate buffer solution (PBS) doubling dilution;
(3) take the electricity generation ability of miniature organism electrochemical reactor as index, select the microreactor under can the maximum dilution multiple of generation current to carry out next separation cycle, until obtain pure culture microorganism---anode reduction earth bacillus; Described separation cycle refers to: whole process that dilution---inoculate new microreactor---operation microreactor---monitoring current produces.
4. method according to claim 3, is characterized in that, while moving described miniature organism electrochemical reactor, the composition of the substratum of use is:
In every premium on currency, comprise sodium acetate 1g; NH4Cl0.31g; KCl0.13g; 50mM phosphate buffer solution; Wolfe liquid microelement 12.5mL; Wolfe VITAMIN liquid 5mL;
The composition of described phosphate buffer solution is:
In every premium on currency, comprise NaH
2pO
42H
2o5.618g; Na
2hPO
412H
2o6.155g;
The composition of described Wolfe liquid microelement is:
In every premium on currency, comprise nitrilotriacetic acid(NTA) 1.5g; MgSO
47H
2o3.0g; MnSO
4h
2o0.5g; NaCl1.0g; FeSO
47H
2o0.1g; CoCl
26H
2o0.1g; CaCl
20.1g; ZnSO
47H
2o0.1g; CuSO
45H
2o0.01g; AlK (SO
4)
212H
2o0.01g; H
3bO
30.01g; Na
2moO
42H
2o0.01g;
The composition of described Wolfe VITAMIN liquid is:
In every premium on currency, comprise vitamin H 2.0mg; Folic acid 2.0mg; Vitamin B6 10.0mg; Vitamin B15 .0mg; Riboflavin 5.0mg; Nicotinic acid 5.0mg; D-VB5 calcium 5.0mg; Vitamin B12 0.1mg; Para-amino benzoic acid 5.0mg; Thioctic Acid 5.0mg.
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| CN107555609A (en) * | 2017-08-30 | 2018-01-09 | 中国环境科学研究院 | A kind of method of enhanced biological earth battery electricity generation ability |
| CN108114975A (en) * | 2017-12-07 | 2018-06-05 | 中国科学院地球化学研究所 | The method that micro- aerobic iron-oxidizing bacteria group administers As polluted soil |
| CN108195744A (en) * | 2018-03-27 | 2018-06-22 | 苏州桓晨医疗科技有限公司 | A kind of detection method of particulate matter |
| CN111261220A (en) * | 2018-11-30 | 2020-06-09 | 中国科学院天津工业生物技术研究所 | Implementation method for efficiently screening, designing and modifying electronic carrier |
| CN114836774A (en) * | 2022-06-06 | 2022-08-02 | 北京林业大学 | Method for efficiently recovering phosphorus by using sulfur-reducing bacillus microbial electrolytic cell |
| CN116355791A (en) * | 2022-12-30 | 2023-06-30 | 杭州洛奇亚环保科技有限公司 | Higher-order flora for wastewater multi-biocatalysis degradation treatment and construction method and application thereof |
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| CN107555609A (en) * | 2017-08-30 | 2018-01-09 | 中国环境科学研究院 | A kind of method of enhanced biological earth battery electricity generation ability |
| CN107555609B (en) * | 2017-08-30 | 2021-01-19 | 中国环境科学研究院 | Method for enhancing electric energy production of biogeographic battery |
| CN108114975A (en) * | 2017-12-07 | 2018-06-05 | 中国科学院地球化学研究所 | The method that micro- aerobic iron-oxidizing bacteria group administers As polluted soil |
| CN108195744A (en) * | 2018-03-27 | 2018-06-22 | 苏州桓晨医疗科技有限公司 | A kind of detection method of particulate matter |
| CN111261220A (en) * | 2018-11-30 | 2020-06-09 | 中国科学院天津工业生物技术研究所 | Implementation method for efficiently screening, designing and modifying electronic carrier |
| CN111261220B (en) * | 2018-11-30 | 2023-02-28 | 中国科学院天津工业生物技术研究所 | Implementation method for efficiently screening, designing and modifying electronic carrier |
| CN114836774A (en) * | 2022-06-06 | 2022-08-02 | 北京林业大学 | Method for efficiently recovering phosphorus by using sulfur-reducing bacillus microbial electrolytic cell |
| CN114836774B (en) * | 2022-06-06 | 2023-11-10 | 北京林业大学 | Method for efficiently recycling phosphorus in geobacillus thioreduction microbial electrolytic cell |
| CN116355791A (en) * | 2022-12-30 | 2023-06-30 | 杭州洛奇亚环保科技有限公司 | Higher-order flora for wastewater multi-biocatalysis degradation treatment and construction method and application thereof |
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