CN108251307A - A kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in anaerobic activated sludge - Google Patents

A kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in anaerobic activated sludge Download PDF

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CN108251307A
CN108251307A CN201711317275.8A CN201711317275A CN108251307A CN 108251307 A CN108251307 A CN 108251307A CN 201711317275 A CN201711317275 A CN 201711317275A CN 108251307 A CN108251307 A CN 108251307A
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extracellular
generation ability
electricity generation
production bacterium
electricity production
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温洪宇
王国振
王梦影
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Jiangsu Normal University
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Abstract

The present invention provides a kind of methods that the extracellular electricity production bacterium of high electricity generation ability is screened in anaerobic activated sludge.It can be by Fe in ironic citrate according to extracellular electricity production bacterium3+It is reduced into this principle of Fe, it is detached by ironic citrate solid medium, the anaerobic bacteria in purifying anaerobic activated sludge, and the size for becoming black scope according to periphery of bacterial colonies culture medium tentatively selectes out some high extracellular electricity production bacterium of electricity generation ability, the tentatively selected extracellular electricity production bacterium of high electricity generation ability is measured and compared by cyclic voltammetry again, finishing screen selects the extracellular electricity production bacterium of optimal high electricity generation ability, and the inducing and acclimating for subsequent electricity production bacterium lays the foundation.This method is simple and practicable, can be with the extracellular electricity production bacterium of the high electricity generation ability filtered out in anaerobic activated sludge of efficiently and accurately.

Description

A kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in anaerobic activated sludge
Technical field
The present invention relates to extracellular electricity production bacterium screening fields more particularly to one kind to screen high yield electric energy in anaerobic activated sludge The method of the extracellular electricity production bacterium of power.
Background technology
The organic substance and the general name of inorganic substances that anaerobic activated sludge is anaerobe group and they are depended on.Detest Comprising abundant anaerobe in oxygen activity sludge, it is suitble to the extracellular electricity production bacterium of screening.Extracellular electricity production bacterium has directly by electronics Pass to compound or the ability of indirect electron receptor, mostly anaerobic bacteria or facultative anaerobic bacteria.Many anaerobic bacterias can only be by electronics Soluble xenobiontics are passed to, such as nitrate or sulfate, they can be entered by diffusion inside cell membrane, extracellular Electricity production bacterium has is different from other anaerobes by electron transmission to extracellular ability, and this ability may act on microorganism combustion Expect battery electricity production.Extracellular electricity production bacterium generates electronics in galvanic anode oxidation of organic compounds, cathode is transmitted to by external circuit, with cathode Oxygen, proton reduction reaction generation water occurs, complete entire oxidation-reduction process and generate electric current.The born of the same parents in entire electricity generation process Outer electricity production bacterium plays an important role as biocatalyst.The extracellular electricity production bacterium of high electricity generation ability is screened to research single bacterium to microorganism The electricity production of fuel cell influences and its electronics metastasis important in inhibiting.Current only two quasi-mode alienation metal reducing miroorganisms quilts It isolates and purifies out and has carried out systematic research, simple effective method there is no to the screening of other extracellular electricity production bacterium, and sieve The electricity production bacterium for selecting high electricity generation ability is the key that carry out follow-up expand to study.Patent CN201210059296 discloses a kind of sieve The method for selecting electricity-producing microorganism, but the microorganism that this method filters out is not electricity production bacterium entirely, can not accurately screen very much and produce The higher electricity production bacterium of electric energy power works in order to subsequent inducing and acclimating.
Invention content
In view of above-mentioned deficiencies of the prior art, high electricity generation ability is screened in anaerobic activated sludge the present invention provides one kind The method of extracellular electricity production bacterium.Under anaerobic, the anaerobic bacteria being enriched in anaerobic activated sludge.By the bacterium solution dilution of anaerobic bacteria simultaneously It is coated on ironic citrate solid medium；It can be by Fe3 in ironic citrate according to extracellular electricity production bacterium+This principle of Fe is reduced into, It checks whether the single bacterium colony that ironic citrate solid medium is made to become black from brown, makes ironic citrate solid medium by brown Discoloration is purified to obtain pure bacterial strain for the single bacterium colony of black.Comparing each pure bacterial strain makes ironic citrate solid medium become black scope Size, become black scope it is maximum is tentatively chosen to be the extracellular electricity production bacterium of high electricity generation ability.By cyclic voltammetry and compare The electro-chemical activity of multiple extracellular electricity production bacterium of tentatively selected high electricity generation ability, the highest bacterial strain of electro-chemical activity is optimal high yield The extracellular electricity production bacterium of electric energy power.
Technical scheme is as follows：
A kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in anaerobic activated sludge, including step：
S1：The anaerobic bacteria being enriched in LB fluid nutrient mediums in anaerobic activated sludge；
S2：It dilutes the bacterium solution of anaerobic bacteria and bacterium solution is coated on ironic citrate solid medium；
S3：The single bacterium colony for whether having and culture medium being made to become black from brown is checked on ironic citrate solid medium, if nothing Step S1 and S2 are then repeated, by plate streak to culture medium is made to be purified to obtain by the single bacterium colony of brown blackening if having Pure bacterial strain；
S4：It repeats step S3 and obtains more than one pure bacterial strain, under equal conditions comparing each pure bacterial strain consolidates ironic citrate Body culture medium becomes the size of black scope, and the pure bacterial strain for becoming black scope maximum is tentatively chosen to be the extracellular electricity production bacterium of high electricity generation ability；
S5：It repeats step S4 and obtains more than one tentatively selected extracellular electricity production bacterium of high electricity generation ability；
S6：Under equal conditions by cyclic voltammetry and compare each tentatively selected extracellular production of high electricity generation ability The electro-chemical activity of electric bacterium, the highest bacterial strain of electro-chemical activity are the optimal high extracellular electricity production bacterium of electricity generation ability.
Preferably, the formula of ironic citrate solid medium is：
Tryptone：10g/L
Yeast extract：5g/L,
Sodium chloride：10g/L
Ironic citrate：20mmol/L
Agar powder：18g/L
PH=7 ± 0.3 is adjusted with NaOH
Preferably, it is carried out under anaerobic conditions of the step S1 to S5 in anaerobism work station, temperature is protected in anaerobism work station It holds at 30 DEG C.
Preferably, cyclic voltammetry is three electrode cycle voltammetries, and for glass-carbon electrode as working electrode, Ti electrode is to electricity Pole, Ag/AgCl electrodes are as reference electrode.
Preferably, step S6 is specifically included：
S61：Sterile LB fluid nutrient mediums are measured by three electrode cycle voltammetries and obtain curve 1；
S62：It is measured by three electrode cycle voltammetries to being inoculated with the extracellular electricity production bacterium of tentatively selected high electricity generation ability Obtain curve 2；
S63：Correlation curve 1 and the curve 2 obtain the reduction peak of the extracellular electricity production bacterium of tentatively selected high electricity generation ability；
S64：Repeat the reduction that step S61, S62 and S63 obtain multiple extracellular electricity production bacterium of tentatively selected high electricity generation ability Peak, comparison compare each reduction peak, and highest reduction peak is the optimal high extracellular electricity production bacterium of electricity generation ability.
Compared with prior art, beneficial effects of the present invention：
A kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in anaerobic activated sludge that this method provides, according to born of the same parents Outer electricity production bacterium can be by Fe3 in ironic citrate+This principle of Fe is reduced into, is detached by ironic citrate solid medium, purifying is detested Anaerobic bacteria in oxygen sludge, and the size of black scope become according to periphery of bacterial colonies culture medium tentatively to select several high electricity generation abilities extracellular Bacterium is produced electricity, then is measured and compares one by one by cyclic voltammetry electricity production bacterium extracellular to the high electricity generation ability tentatively filtered out, So as to filter out the higher extracellular electricity production bacterium of a collection of electricity generation ability, it is ready for subsequent inducing and acclimating, this method is simple It is easy, the extracellular electricity production bacterium of high electricity generation ability in anaerobic activated sludge can be filtered out with efficiently and accurately.
Description of the drawings
Fig. 1 is that one kind described in the embodiment of the present invention 1 screens the extracellular electricity production of high electricity generation ability in anaerobic activated sludge The flow diagram of the method for bacterium；
Fig. 2 is that one kind described in the embodiment of the present invention 1 screens the extracellular electricity production of high electricity generation ability in anaerobic activated sludge Three-electrode system of the method for bacterium carries out bacterium solution and blank cultures the experimental result picture of CV scannings；
Specific embodiment：
The present invention is described further with reference to the accompanying drawings.
Embodiment 1
As shown in Figure 1, a kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in anaerobic activated sludge,
S1：Anaerobic activated sludge is taken to be stored in 4 DEG C of refrigerator in the PE pipes after sterilizing in sewage treatment plants, For use.200ml routine LB fluid nutrient mediums are prepared, lead to nitrogen 15 minutes, remove oxygen in culture medium.It is taken in anaerobism work station 0.2g activated sludge is added in LB fluid nutrient mediums, is placed on the shaking table in anaerobism work station and is carried out Anaerobic culturel.Shaking table turns Number 190rpm, 30 DEG C of cultivation temperature, incubation time 48h.After culture, LB fluid nutrient mediums are become cloudy by clarifying.
S2：Gradient dilution is carried out to bacterium solution in anaerobism work station, is diluted to 10-4、10-5、10-6Gradient, by three dilutions The bacterium solution of gradient is coated on ironic citrate solid medium, and each gradient is coated with two tablets.By the flat of three dilution gradients Plate, which is put in anaerobic culture box, can cultivate 48h, 30 DEG C of temperature.
S3：The single bacterium colony that culture medium is made to become black is found on ironic citrate solid medium after incubation, if without if Repeat the above steps S1 and S2, will become the bigger single bacterium colony oese of black scope if having and chooses, uses plate streak It is purified.It crosses after 3-4 generations, wherein pure bacterial strain is obtained, and be named as B-1 bacterial strains, using Gram's stain to B-1 bacterial strains It is dyed, Gram-negative bacteria is viewed as under microscope.Whole operation process carries out in anaerobism work station.
S4：After multiple pure bacterial strains to be obtained, the size that each pure bacterial strain makes culture medium become black scope is compared, becomes black scope most Big person is the tentatively selected extracellular electricity production bacterium of high electricity generation ability.
S5：Step S4 is repeated to obtain more tentatively selected extracellular electricity production bacterium of high electricity generation ability.
S6：Bacterial strain B-1 is seeded in LB fluid nutrient mediums, while makes LB fluid nutrient mediums after blank sterilizing.By 2 parts After fluid nutrient medium is put into the interior culture of anaerobic culture box 48 hours.CV scannings are carried out using three-electrode system, as shown in Fig. 2, bacterium There is apparent oxidation peak in the position of 0.9V in liquid CV, blank cultures are then without apparent redox peaks.
S7：Continue under equal conditions to measure the tentatively selected extracellular electricity production bacterium of high electricity generation ability, and compare each preliminary The oxidation peak of the extracellular electricity production bacterium of selected high electricity generation ability, and then the relatively high extracellular electricity production bacterium of a collection of electricity generation ability is selected, Element task is carried out for the work of subsequent inducing and acclimating.
A kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in anaerobic activated sludge that this method provides, according to born of the same parents Outer electricity production bacterium can be by Fe3 in ironic citrate+This principle of Fe is reduced into, is detached by ironic citrate solid medium, purifying is detested Anaerobic bacteria in oxygen activity sludge, the size for becoming black scope according to periphery of bacterial colonies culture medium tentatively select the extracellular production of high electricity generation ability Electric bacterium simultaneously passes through the further comparison screening of cyclic voltammetry electricity production bacterium progress extracellular to the high electricity generation ability tentatively filtered out, from And finishing screen selects the extracellular electricity production bacterium of optimal high electricity generation ability, to make further research, this method is simple and practicable, can be efficient Accurately filter out the extracellular electricity production bacterium of high electricity generation ability in anaerobic activated sludge.
It should be understood that the application of the present invention is not limited to the above, it, can for the normal technician of this field To be improved or converted according to the above description, all these modifications and variations should all belong to the guarantor of appended claims of the present invention Protect range.

Claims (5)

  1. A kind of 1. method that the extracellular electricity production bacterium of high electricity generation ability is screened in anaerobic activated sludge, which is characterized in that including step：
    S1：The anaerobic bacteria being enriched in LB fluid nutrient mediums in anaerobic activated sludge；
    S2：It dilutes the bacterium solution of the anaerobic bacteria and the bacterium solution is coated on ironic citrate solid medium；
    S3：Whether check on the ironic citrate solid medium to have makes the ironic citrate solid medium become black from brown The single bacterium colony of color, if repeating said steps S1 and S2 without if, if having through plate streak to making the ironic citrate solid The single bacterium colony that culture medium is become black from brown is purified to obtain pure bacterial strain；
    S4：Repeating said steps S3 obtains more than one described pure bacterial strain, and under equal conditions comparing each pure bacterial strain makes institute The size that ironic citrate solid medium becomes black scope is stated, the ironic citrate solid medium is made to become the described of black scope maximum Pure bacterial strain is the tentatively selected extracellular electricity production bacterium of high electricity generation ability；
    S5：Repeating said steps S4 obtains more than one described tentatively selected extracellular electricity production bacterium of high electricity generation ability,
    S6：Under equal conditions pass through the electrification of the extracellular electricity production bacterium of high electricity generation ability tentatively selected described in cyclic voltammetry Learn activity, the electro-chemical activity of the comparison extracellular electricity production bacterium of tentatively selected high electricity generation ability, the electro-chemical activity highest Bacterial strain be the optimal high extracellular electricity production bacterium of electricity generation ability.
  2. 2. a kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in activated sludge according to claim 1, special Sign is that the formula of the ironic citrate solid medium is：
    Tryptone：10g/L
    Yeast extract：5g/L,
    Sodium chloride：10g/L
    Ironic citrate：20mmol/L
    Agar powder：18g/L
    NaOH adjusts pH=7 ± 0.3.
  3. 3. a kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in activated sludge according to claim 1, special Sign is that the step S1 to S5 is carried out in anaerobism work station, and temperature is 30 DEG C in the anaerobism work station.
  4. 4. a kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in activated sludge according to claim 1, special Sign is that the cyclic voltammetry in the step S6 is three electrode cycle voltammetries, and three electrode is glass-carbon electrode, titanium Electrode, Ag/AgCl electrodes, for the glass-carbon electrode as working electrode, the Ti electrode is to electrode, the Ag/AgCl electrodes As reference electrode.
  5. 5. a kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in activated sludge according to claim 1, special Sign is that the step S6 is specifically included：
    S61：Sterile LB fluid nutrient mediums are measured by the three electrode cycles voltammetry and obtain curve 1；
    S62：It is carried out by the three electrode cycles voltammetry to being inoculated with the extracellular electricity production bacterium of tentatively selected high electricity generation ability It measures and obtains curve 2；
    S63：The curve 1 and the curve 2 are compared, obtains the reduction of the extracellular electricity production bacterium of tentatively selected high electricity generation ability Peak；
    S64：Repeating said steps S61, S62 and S63 obtain more than one described tentatively selected extracellular electricity production of high electricity generation ability The reduction peak of bacterium compares the reduction peak of more than one extracellular electricity production bacterium of tentatively selected high electricity generation ability, is gone back described in highest The extracellular electricity production bacterium of tentatively selected high electricity generation ability corresponding to parent peak is the optimal high extracellular electricity production bacterium of electricity generation ability.
CN201711317275.8A 2017-12-12 2017-12-12 A kind of method that the extracellular electricity production bacterium of high electricity generation ability is screened in anaerobic activated sludge Pending CN108251307A (en)

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Patent Citations (3)

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Publication number Priority date Publication date Assignee Title
CN101407763A (en) * 2008-11-28 2009-04-15 哈尔滨工业大学 Method for separating electrochemical activity bacterial strain and detecting electrochemical activity
CN103215205A (en) * 2013-04-08 2013-07-24 华南理工大学 Citrobacter freundii and application thereof to production of bioelectricity
CN104277991A (en) * 2013-07-08 2015-01-14 中国科学院城市环境研究所 Anaerobic electrogenesis bacterium separation and electrochemical activity identification method

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Application publication date: 20180706