CN103290092B - Simple and efficient method for evaluating electricity generating capacity of microorganisms - Google Patents
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- 230000009514 concussion Effects 0.000 claims description 5
- 238000004042 decolorization Methods 0.000 claims description 5
- 229940012189 methyl orange Drugs 0.000 claims description 5
- 231100000219 mutagenic Toxicity 0.000 claims description 5
- 230000003505 mutagenic effect Effects 0.000 claims description 5
- 239000001888 Peptone Substances 0.000 claims description 4
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 4
- 229940041514 candida albicans extract Drugs 0.000 claims description 4
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- 239000012138 yeast extract Substances 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 150000001875 compounds Chemical class 0.000 claims description 3
- 238000002474 experimental method Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- AAAQKTZKLRYKHR-UHFFFAOYSA-N triphenylmethane Chemical compound C1=CC=CC=C1C(C=1C=CC=CC=1)C1=CC=CC=C1 AAAQKTZKLRYKHR-UHFFFAOYSA-N 0.000 claims description 3
- QBZIEGUIYWGBMY-FUZXWUMZSA-N (5Z)-5-hydroxyimino-6-oxonaphthalene-2-sulfonic acid iron Chemical compound [Fe].O\N=C1/C(=O)C=Cc2cc(ccc12)S(O)(=O)=O.O\N=C1/C(=O)C=Cc2cc(ccc12)S(O)(=O)=O.O\N=C1/C(=O)C=Cc2cc(ccc12)S(O)(=O)=O QBZIEGUIYWGBMY-FUZXWUMZSA-N 0.000 claims description 2
- 238000010790 dilution Methods 0.000 claims description 2
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- 239000002184 metal Substances 0.000 claims description 2
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- KCXFHTAICRTXLI-UHFFFAOYSA-N propane-1-sulfonic acid Chemical class CCCS(O)(=O)=O KCXFHTAICRTXLI-UHFFFAOYSA-N 0.000 claims 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a simple and efficient method for evaluating electricity generating capacity of microorganisms, belonging to the field of bacteriology and molecular biology. Electricity generating microorganisms subjected to chemical or physical mutagenesis are subjected to flat coating after being diluted and subjected to anaerobic culture, and the electricity generating microorganisms with excellent electricity generating efficiency are screened according to the size of a decoloring transparent ring on a flat plate and the size and morphology of monoclone. The novel screening method is novel in concept, is capable of remarkably improving the screening efficiency and shortening the screening time, and is high in accuracy rate. Therefore, the simple and efficient method has the characteristics of convenience, rapidness, wide application range and the like, and has strong actual application value in the field of research of the electricity generating microorganisms.
Description
Technical field
The present invention relates to a kind of simple and efficient method assessing microorganism electricity generation ability.Belong to bacteriology and biology field.
Background technology
Electrogenesis microorganism (Exoelectrogens) is that a class can utilize gas chromatography for carbon source, and by self respiration, transfer transport metabolism produced, to extracellular electron acceptor, carries out the environmental microorganism of alienation anaerobic respiration.Current electrogenesis microorganism has been widely used in the research and apply of microbiological fuel cell (Microbial Fuel Cells, MFC).But natural microorganism electricity generation ability is lower, become a technical bottleneck of restriction MFC development.In addition, studies have found that electrogenesis microorganism can carry out biological degradation to a series of environmental organic pollutants such as comprising azoic dyestuff, metal complex dye, triphenylmethane dye, oil of mirbane.And the repair ability further studying discovery electrogenesis microbe depends on its electricity generation performance.Its degradation mechanism discharges by the non-specific electronics of cell surface the non-specific deoxidization, degradation mediated.But the electronics that electrogenesis microorganism produces is when being delivered to born of the same parents and being outer by Mtr approach, the electrical power discharged is lower, thus constrains the degradation rate of organic pollutant.How to improve the electricity generation ability of microorganism thus improve the study hotspot that its biological restoration usefulness is also current environmental area.
Thus, the focus of current research has been become by the electricity generation ability of the method such as strain mutagenesis and genetically engineered raising microorganism.But microorganism electricity generation ability is mainly by assembling MFC battery at present, then detects the methods such as current density and assess in actual motion, its cycle length, complex operation; And by flow cell sorter, the difference according to cell surface membrane potential is screened, required equipment is complicated, fluorescent probe costly, and requires higher to appointed condition.How simplifying appraisal procedure, improving assess effectiveness is the important prerequisite optimizing electrogenesis production by biological electrical characteristic.Therefore a kind of more fast and convenient detection method is needed badly.
Summary of the invention
The present invention is directed to the evaluation and test of current microorganism electricity generation ability and lack predicament that is efficient, simple and easy method, by flat band method and anaerobic jar coupling, using Azo Dye-Methyl Orange as indicator, provide a kind of high-efficient simple appraisal procedure of electrogenesis microorganism electricity generation ability.
In order to achieve the above object, technical scheme of the present invention is as follows:
A kind of simple and efficient method assessing microorganism electricity generation ability, the bacterial strain adopted is pattern electrogenesis microorganism Shewanellaoneidensis MR-1, mutagenic compound are ethylmethane sulfonate (EMS), indicator can not enter cell and can by the dyestuff of Shewanella oneidensis MR-1 degradation and decolorization, and appraisal procedure is carried out according to following step:
(1) picking Shewanella oneidensis MR-1 mono-clonal, access 50ml yeast extract paste peptone liquid nutrient medium (also claims LB substratum, containing yeast extract 5g/l, peptone 10g/l, NaCl l0g/l), in 30 DEG C under aerobic condition, 180rpm concussion is cultured to late log phase;
(2) get the thalline 1ml of step (1) gained late log phase, add EMS20 μ l in Bechtop, mixing of turning upside down is placed in 30 DEG C of shaking tables, and 1h is cultivated in 180rpm concussion;
(3) by mycelium dilution good for step (2) mutagenesis to 1 × 10
3cFU/ml, gets the previously prepared good solid plate of bacterium liquid coating of 100 μ l; Solid plate used is the indicator of agar B and 200mg/l adding 1.8% in Shewanella oneidensis MR-1 mineral salts medium, the pH=7.0 of agar B;
(4) solid plate of bacterium liquid coated in step (3) is inverted in anaerobic jar, is full of high pure nitrogen by anaerobic jar, cultivate in 30 DEG C of lucifuges, carry out the decolouring of electrogenesis microorganism to solid-state dye, form decolouring transparent circle not of uniform size;
(5) according to size and the monoclonal size and form of decolouring circle transparent around mono-clonal on solid plate, the electrogenesis microorganism that electricity generation ability is high is filtered out;
(6) the electrogenesis microorganism that step (5) is selected is carried out liquid nutrient medium decolorization experiment, further the decoloring ability of each bacterium of checking.
Indicator specifically adopts Azo Dye-Methyl Orange, azoic dyestuff, triphenylmethane dye or non-azo metal dye naphthol green B containing sulfanilamide (SN) group.
The consistence of the electricity generation ability that the present invention utilizes confirm early stage and dye decolored speed, establish a kind of using Azo Dye-Methyl Orange as indicator, the method of decolouring transparent circle is detected by anaerobism flat board, efficiently can assess the easy technique of the electricity generation ability of electrogenesis microorganism, thus significantly shorten screening time, improve screening efficiency.Present method has easy and simple to handle, and the advantage such as convenient and swift, wide accommodation, has stronger actual application value.
Accompanying drawing explanation
Fig. 1 is that the bacterial strain that the present invention has a different electricity generation ability forms decolouring transparent circle not of uniform size.
Embodiment
Thalline S.oneidensis MR-1 of the present invention is a kind of electrogenesis microorganism (Genome sequence of thedissimilatory metal ion – reducing bacterium Shewanella oneidensis.Nature Biotechnology.2002 of pattern, 20:1118-1123), given by California, USA university professor Nelson.This bacterium is stored in US mode typical case thing and collects center (ATCC), and strain number is ATCC700550
tM.This bacterial classification can directly be bought from this center, but the method is also applicable to the electrogenesis microorganism of other kinds.Indicator used is azo dyes tropeolin-D.Mutagenic compound used are chemical mutagen ethylmethane sulfonate EMS.
Novelty of the present invention is:
(1) microorganism electricity generation ability assessment principle
Be different from the approach such as the current density of conventional microbiological fuel cell, electrochemical analysis and cell membrane potential, the present invention be build on electrogenesis microorganism electrogenesis mechanism and it is on the machine-processed consistent basis of dye degrades, by the dye degrades measures of effectiveness to electrogenesis microorganism, indirectly reflect the power of its electricity generation ability, thus establish a kind of simple and easy appraisal procedure for microorganism electricity generation ability.
(2) appraisal procedure of electrogenesis microorganism electricity generation ability
By physics or chemomorphosis method by after electrogenesis microorganism Shewanella mutagenesis, be diluted to certain multiple, coat on the solid medium containing indicator tropeolin-D, solid medium is inverted in lucifuge in anaerobic jar and cultivates.The decoloring ability of electrogenesis microorganism to dyestuff depends on its electricity generation performance, and dye decolored degradation is under anaerobic formed decolouring transparent circle by the electrogenesis microorganism after mutagenesis.
(3) examination of different electricity generation ability:
As shown in Figure 1,1 represents the mono-clonal that S.oneidensis MR-1 is formed at solid culture primary surface, 2 represent the decolouring transparent circle that S.oneidensisMR-1 is formed methyl orange, and 3 represent the S.oneidensis MR-1 mineral salt solid medium containing indicating dye.According to size and the monoclonal size and form of the transparent circle that decolours around mono-clonal different on solid medium, preliminary judgement has the bacterial strain of higher electricity generation ability.There is the mutagenic fungi of higher electricity generation ability, there is larger decolouring transparent circle and (or) mono-clonal diameter larger.
Mutagenesis and the electricity generation ability evaluation process of the present embodiment S.oneidensis MR-1 are specific as follows:
(1) the S.oneidensis MR-1 be kept in glycerine is lined on LB flat board, 30 DEG C of incubated overnight;
(2) with single bacterium colony that toothpick picking activates, be inoculated in 50ml LB liquid nutrient medium, 30 DEG C, 180rpm is cultured to late log phase;
(3) get bacterium liquid 1ml in (2) and in 1.5ml centrifuge tube, add 20 μ l EMS, fully mixing of turning upside down, in 30 DEG C, 180rpm concussion cultivation 1h.Period takes out centrifuge tube every 15min, turns upside down 3 ~ 4 times;
(4) bacterium liquid good for mutagenesis in (3) is diluted to about 1 × 10
3cFU/ml, gets 100 μ l and is coated with previously prepared good solid plate substratum.The S.oneidensis MR-1 mineral salts medium (Bretschger et al.Appl Environ Microbiol, 2007,73 (21): 7003-12) of formula for the addition of 1.8% agar B and 200mg/l tropeolin-D;
(5) coated flat-plate inverted is placed in anaerobic jar, and makes to be full of high pure nitrogen in anaerobic jar, in 30 DEG C of lucifuge quiescent culture, until flat board grows mono-clonal; Shewanella take lactic acid as electron donor, tropeolin-D is that electron acceptor(EA) carries out anaerobic growth, so in thalli growth process, the tropeolin-D around each mono-clonal is constantly consumed, due to the monoclonal electricity generation ability difference of difference, so form decolouring transparent circle not of uniform size.
(6) as figure, according to size and the monoclonal size and form of the transparent circle that decolours around mono-clonal, select the mono-clonal with larger decolouring transparent circle and (or) the larger bacterial strain of mono-clonal diameter carries out next step checking, these bacterial strains of preliminary judgement have higher decoloring ability than starting strain;
(7) mono-clonal selected in (6) is carried out liquid nutrient medium decolorization experiment, further the decoloring ability of these mutagenic strains of checking and starting strain S.oneidensis MR-1.Concrete operations are as follows: these mono-clonals are inoculated in respectively in 50ml liquid LB that enlarged culturing is to late log phase, and the centrifugal 10min of 5000rpm, removes supernatant, and with S.oneidensis MR-1 mineral salts medium resuspension, adjusting bacterium dense is 4 ~ 6 × 10
6cFU/ml, the S.oneidensisMR-1 mineral salts medium containing 200mg/l tropeolin-D that access prepares in advance, 30 DEG C, 180rpm, lucifuge anaerobic shake is cultivated until decolouring.
Claims (2)
1. assess the simple and efficient method of microorganism electricity generation ability for one kind, the bacterial strain adopted is pattern electrogenesis microorganism Shewanellaoneidensis MR-1, mutagenic compound are ethylmethane sulfonates, indicator can not enter cell and can by the dyestuff of Shewanellaoneidensis MR-1 degradation and decolorization, it is characterized in that, appraisal procedure is carried out according to following step:
(1) picking Shewanella oneidensis MR-1 mono-clonal, access 50ml yeast extract paste peptone liquid nutrient medium, substratum is containing yeast extract 5g/l, peptone 10g/l and NaCl l0g/l, and in 30 DEG C under aerobic condition, 180rpm concussion is cultured to late log phase;
(2) get the thalline 1ml of step (1) gained late log phase, add EMS20 μ l in Bechtop, mixing of turning upside down is placed in 30 DEG C of shaking tables, and 1h is cultivated in 180rpm concussion;
(3) by mycelium dilution good for step (2) mutagenesis to 1 × 10
3cFU/ml, gets the previously prepared good solid plate of bacterium liquid coating of 100 μ l; Solid plate used is the indicator of agar B and 200mg/l adding 1.8% in Shewanella oneidensis MR-1 mineral salts medium, the pH=7.0 of agar B;
(4) solid plate of bacterium liquid coated in step (3) is inverted in anaerobic jar, is full of high pure nitrogen by anaerobic jar, cultivate in 30 DEG C of lucifuges, carry out the decolouring of electrogenesis microorganism to solid-state dye, form decolouring transparent circle not of uniform size;
(5) according to size and the monoclonal size and form of the transparent circle that decolours around mono-clonal on solid plate, the electrogenesis microorganism that electricity generation ability is high is filtered out;
(6) the electrogenesis microorganism that step (5) is selected is carried out liquid nutrient medium decolorization experiment, further the decoloring ability of each bacterium of checking.
2. a kind of simple and efficient method assessing microorganism electricity generation ability according to claim 1, it is characterized in that, described indicator is Azo Dye-Methyl Orange, azoic dyestuff, triphenylmethane dye or non-azo metal dye naphthol green B containing sulfanilamide (SN) group.
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Non-Patent Citations (3)
| Title |
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| Amanda N. Payne & Thomas J. DiChristina.Arapid mutant screening technique for detection of technetium [Tc(VII)] reduction-de¢ * |
| cient mutants of Shewanella oneidensis MR-1.《FEMS》.2006,第259卷282-287. * |
| 田启建等.小溪自然保护区甲基橙降解真菌的筛选及其脱色能力分析.《贵州农业科学》.2011,第39卷(第11期),129-132. * |
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