CN109111499A - A method of extracting ground bacillus Extracellular polymers - Google Patents
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- CN109111499A CN109111499A CN201810966285.2A CN201810966285A CN109111499A CN 109111499 A CN109111499 A CN 109111499A CN 201810966285 A CN201810966285 A CN 201810966285A CN 109111499 A CN109111499 A CN 109111499A
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- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
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Abstract
The invention discloses a kind of methods for extracting ground bacillus Extracellular polymers, using microbiological fuel cell culture biomembrane, it is combined by ultrasound and centrifugation, Multi-layer technology is carried out to ground bacillus Extracellular polymers, extracts soluble S-EPS, loose combination EPS (LB-EPS) and the EPS that combines closely (TB-EPS).The bacterium damage in extraction process is effectively reduced, the extracted amount of Extracellular polymers is improved.This is also to characterize for the first time to the characteristic of ground bacillus Extracellular polymers, and the ground bacillus Extracellular polymers of extraction are conducive to environmental pollution reparation, and has certain application prospect on extracellular electron transport route and repairing environment.
Description
Technical field
The present invention relates to a kind of methods for extracting ground bacillus Extracellular polymers, belong to microorganism field.
Background technique
Microorganism Extracellular polymers EPS is mainly made of polysaccharide, albumen and humic acid, is glued by one kind of microorganism secretion
Property organic polymer polymer, be attached to bacterium surface or be centered around around bacterium, formed a protective layer, to make microorganism
It can also survive under conditions of the environmental factors such as drying, temperature and PH are unfavorable.Extracellular polymers are divided into solubility by research at present
Extracellular polymers (S-EPS), loose mating type Extracellular polymers (LB-EPS), type of combining closely Extracellular polymers (TB-EPS),
By it is outer and in be distributed in surface and the surrounding of bacterium.There are many kinds of extracting methods for Extracellular polymers, but its extracting method is not yet
Unified standard, the characteristic of Extracellular polymers can also change with the change of extracting method.
Ground bacillus Geobacter sulfurreducens PCA and Geobacter soli GSS01 has efficient born of the same parents
Exoelectron transmission capacity can be used as the mode species of ground bacillus research.Ground bacillus is as the weight for studying extracellular electron transport route
Bacterial strain is wanted, there are three types of electron transport route classification is currently known, direct electron transfer, the transmitting of electron shuttle body and nanometer are led
Line electron transmission.It in the prior art, is that Extracellular polymers are extracted to aerobic particle mud mostly.Studies have shown that Extracellular polymers
In there is the redox mediators for participating in extracellular electron transport route, therefore find a kind of the extracellular more of high efficiency extraction ground bacillus
Polymers be it is extremely urgent, this can provide technical support for the relationship research of Extracellular polymers EPS and extracellular electron transport route.
Summary of the invention
The purpose of the present invention is to provide a kind of methods for extracting ground bacillus Extracellular polymers.
The technical solution used in the present invention is:
A method of ground bacillus Extracellular polymers being extracted, the step is as follows:
1) soluble S-EPS in the electrolyte of microbiological fuel cell is collected；By microbiological fuel cell graphite plate electrode
On biomembrane scrape, biomembrane is suspended to obtain biomembrane suspension with solution；
2) biomembrane suspension is ultrasonic, concussion, is centrifuged to obtain supernatant and precipitating at ultrasound, and supernatant liquid filtering takes filtrate to obtain
LB-EPS；
3) precipitating after step 2) centrifugation is suspended in the solution, ultrasound is centrifugally separating to obtain supernatant and precipitating, repeats
2~3 times, by the supernatant hybrid filtering film of all acquisitions, filtrate is taken to obtain TB-EPS.
Further, solution described in step 1) is 0.9%NaCl solution.
Further, ultrasound described in step 2) is ultrasound 1~5 minute in ice-water bath.
Further, concussion condition described in step 2) is 100~200 revs/min of concussions 7~15 minutes.
Further, step 2), 3) centrifugal condition be in 4000~7000g, 4~6 DEG C of 10~20min of centrifugation.
Further, step 2), 3) described in ultrasonic power be 300~500W, ultrasonic time be 5~12 minutes.
Further, step 2), 3) use 0.22 μm of aperture membrane filtration.
The Extracellular polymers prepared according to method described in any of the above embodiments.
Further, the Extracellular polymers are soluble Extracellular polymers, loose combination Extracellular polymers and close
In conjunction at least one of Extracellular polymers.
The beneficial effects of the present invention are:
The present invention is to provide a kind of method of high efficiency extraction ground bacillus Extracellular polymers, using microbiological fuel cell culture
Ultrasound is combined with centrifugal method, carries out the Multi-layer technology of ground bacillus Extracellular polymers, extract solubility respectively by biomembrane
S-EPS, loose combination EPS (LB-EPS) and the EPS that combines closely (TB-EPS).The present invention carries out Extracellular polymers to ground bacillus
Multi-layer technology, it is possible to reduce the damage of bacterium in extraction process, and the extracted amount of Extracellular polymers is improved as much as possible.This hair
The redox active albumen that the Extracellular polymers of bright extraction contain is more, and for the first time to the characteristic of ground bacillus Extracellular polymers
It is characterized, this can provide technical support for the research between Extracellular polymers EPS and extracellular electron transport route.
Detailed description of the invention
Fig. 1 is the polysaccharide (A) for the EPS that PCA and GSS01 is extracted in distinct methods, albumen (B) content；
Fig. 2 is bacterial strain PCA after EDTA method (B), ultrasonic method (C) and heating (D) three kinds of Extracellular polymers of extraction
Scanning electron microscope (SEM) photograph；
Fig. 3 is bacterial strain GSS01 after EDTA method (B), ultrasonic method (C) and heating (D) are extracted three kinds of Extracellular polymers
Scanning electron microscope (SEM) photograph；
Fig. 4 is shows fluorescent microscopy images of the bacterial strain PCA after EDTA method (B), ultrasonic method (C) and heating (D) extract EPS；
Fig. 5 is fluorescence microscope of the bacterial strain GSS01 after EDTA method (B), ultrasonic method (C) and heating (D) extract EPS
Figure；
Fig. 6 is using distinct methods to the fluorescence spectra of bacterial strain PCA S-EPS, LB-EPS and TB-EPS extracted；
Fig. 7 is using distinct methods to the fluorescence spectra of bacterial strain GSS01 S-EPS, LB-EPS and TB-EPS extracted；
The CV figure of LB-EPS (A), TB-EPS (B) that Fig. 8 extracts for bacterial strain PCA in distinct methods；
The CV figure of LB-EPS (A), TB-EPS (B) that Fig. 9 extracts for bacterial strain GSS01 in distinct methods.
Specific embodiment
Below with reference to embodiment, the invention will be further described.
Embodiment 1
1) CHI1010C electrochemical workstation culture anode biomembrane is utilized, microbiological fuel cell uses three electrode bodies
System, wherein saturated calomel electrode is reference electrode, and 15mm*10mm*5mm parallelepiped graphite slab is for working electrode and to electrode, electricity
Chem workstation runs Amperometric i-t Curve mode, when electric current is run to period 3 highest point, electrochemistry
Work station is out of service.Battery is dismantled, working electrode graphite plate is removed, electrolyte is taken to obtain soluble E PS (S-EPS), is used
0.9%NaCl solution rinses graphite plate electrode, to remove the soluble E PS (S-EPS) speckled on graphite plate.It will be on graphite plate
Biomembrane scrapes, and is suspended in 0.9%NaCl solution, obtains biological suspensions, takes certain volume suspension to measure OD600 value, again
It takes certain volume suspension to dry, calculates its density, and calculate cell concentration；
2) the biological suspensions 1ml in step 1) is taken to be placed in centrifuge tube, separately plus 0.9%NaCl solution is to 7ml, adjusts super
Sound cell crushing instrument (II D of SCIENTZ-) power is to 480W, by biological suspensions ice-water bath ultrasound 2min, then at 150 revs/min
Horizontal concussion 10min under the conditions of clock, then ultrasound 2min, then 5000g, 4 DEG C of centrifugation 15min will be precipitated and supernatant separation, on
Clear liquid uses the membrane filtration in 0.22 μm of aperture, and filtrate is taken to obtain loose combination EPS (LB-EPS)；
3) by the precipitating after step 2) centrifugation, 4mL is complemented to the resuspension of 0.9%NaCl solution, adjusts ultrasonic cell disintegration
Instrument (II D of SCIENTZ-) power collects supernatant to 480W, ice-water bath ultrasound 10min, 5000g, 4 DEG C of centrifugation 20min；Make again
The precipitating after centrifugation is resuspended with 1mL 0.9%NaCl solution, 5000g, 4 DEG C of centrifugation 15min collect supernatant；Reuse 1mL
The precipitating after centrifugation is resuspended in 0.9%NaCl solution, and 5000g, 4 DEG C of centrifugation 15min collect supernatant；Finally 3 supernatants are mixed
It closes, using the membrane filtration in 0.22 μm of aperture, filtrate is taken to obtain combine closely EPS (TB-EPS).
Embodiment 2
1) CHI1010C electrochemical workstation culture anode biomembrane is utilized, microbiological fuel cell uses three electrode bodies
System, wherein saturated calomel electrode is reference electrode, and 15mm*10mm*5mm parallelepiped graphite slab is for working electrode and to electrode, electricity
Chem workstation runs Amperometric i-t Curve mode, when electric current is run to period 3 highest point, electrochemistry
Work station is out of service.Battery is dismantled, working electrode graphite plate is removed, electrolyte is taken to obtain soluble E PS (S-EPS) S-EPS,
Graphite plate electrode is rinsed with 0.9%NaCl solution, to remove the soluble E PS (S-EPS) speckled on graphite plate.It will be on graphite plate
Biomembrane scrape, be suspended in 0.9%NaCl solution, obtain biological suspensions, certain volume suspension is taken to measure OD600 value,
It takes certain volume suspension to dry again, calculates its density, and calculate cell concentration；
2) the biological suspensions 1ml in step 1) is taken to be placed in centrifuge tube, separately plus 0.9%NaCl solution is to 7ml, adjusts super
Sound cell crushing instrument (II D of SCIENTZ-) power is to 500W, by biological suspensions ice-water bath ultrasound 5min, then at 200 revs/min
Horizontal concussion 10min, ultrasonic 2min under the conditions of clock, then 5000g, 6 DEG C of centrifugation 20min will be precipitated and supernatant separation, supernatant
Liquid uses the membrane filtration in 0.22 μm of aperture, and filtrate is taken to obtain loose combination EPS (LB-EPS)；
3) by the precipitating after step 2) centrifugation, 4mL is complemented to the resuspension of 0.9%NaCl solution, adjusts ultrasonic cell disintegration
Instrument (II D of SCIENTZ-) power is to 500W, and after ultrasonic 12min, 7000g, 6 DEG C of centrifugation 20min collect supernatant；Reuse 1mL
The precipitating after centrifugation is resuspended in 0.9%NaCl solution, and 7000g, 6 DEG C of centrifugation 20min collect supernatant；Reuse 1mL 0.9%
The precipitating after centrifugation is resuspended in NaCl solution, and 7000g, 4 DEG C of centrifugation 15min collect supernatant；Finally 3 supernatants are mixed, are made
With the membrane filtration in 0.22 μm of aperture, filtrate is taken to obtain combine closely EPS (TB-EPS).
Embodiment 3
1) CHI1010C electrochemical workstation culture anode biomembrane is utilized, microbiological fuel cell uses three electrode bodies
System, wherein saturated calomel electrode is reference electrode, and 15mm*10mm*5mm parallelepiped graphite slab is for working electrode and to electrode, electricity
Chem workstation runs Amperometric i-t Curve mode, when electric current is run to period 3 highest point, electrochemistry
Work station is out of service.Battery is dismantled, working electrode graphite plate is removed, electrolyte is taken to obtain soluble E PS (S-EPS) S-EPS,
Graphite plate electrode is rinsed with 0.9%NaCl solution, to remove the soluble E PS (S-EPS) speckled on graphite plate.It will be on graphite plate
Biomembrane scrape, be suspended in 0.9%NaCl solution, obtain biological suspensions, certain volume suspension is taken to measure OD600 value,
It takes certain volume suspension to dry again, calculates its density, and calculate cell concentration；
2) the biological suspensions 1ml in step 1) is taken to be placed in centrifuge tube, separately plus 0.9%NaCl solution is to 7ml, adjusts super
Sound cell crushing instrument (II D of SCIENTZ-) power is to 500W, by biomembrane suspension ice-water bath ultrasound 1min, then 200 turns/
Horizontal concussion 7min, ultrasonic 2min under the conditions of minute, then 4000g, 4 DEG C of centrifugation 10min will be precipitated and supernatant separation, on
Clear liquid uses the membrane filtration in 0.22 μm of aperture, and filtrate is taken to obtain loose combination EPS (LB-EPS)；
3) by the precipitating after step 2) centrifugation, 4mL is complemented to the resuspension of 0.9%NaCl solution, adjusts ultrasonic cell disintegration
Instrument (II D of SCIENTZ-) power is to 300W, and after ultrasonic 5min, 4000g, 4 DEG C of centrifugation 10min collect supernatant；Reuse 1mL
The precipitating after centrifugation is resuspended in 0.9%NaCl solution, and 4000g, 4 DEG C of centrifugation 10min collect supernatant；Reuse 1mL0.9%
The precipitating after centrifugation is resuspended in NaCl solution, and 4000g, 4 DEG C of centrifugation 15min collect supernatant；Finally 3 supernatants are mixed, are made
With the membrane filtration in 0.22 μm of aperture, filtrate is taken to obtain combine closely EPS (TB-EPS).
Further effect detection is done to the present invention.
1. albumen, the polysaccharide of the EPS that distinct methods extract compare
The polysaccharide of S-EPS, LB-EPS and TB-EPS, albumen are detected, steps are as follows for detection method:
Utilize the polyoses content of Phenol sulfuric acid procedure detection EPS, the specific steps are as follows: 1) take 1mL sample, 2.5mL is added
96% sulphuric acid hydrolysis；2) the phenol solution colour developing of 500 μ L 4% is added；3) develop the color 30min, uses ultraviolet specrophotometer
(UV2600) test sample, Detection wavelength 490nm.
Protein Detection is carried out using Bradford agent, specific steps are as follows: 1) taking 20 μ L samples and BSA that enzyme is added
200 μ L Bradrod solution are added in target hole；2) 25-30 DEG C of reaction 5min of room temperature, microplate reader (BIO-Rad iMark,
TY0608 the absorbance value that wavelength is A595) is surveyed.
As a result: as seen from Figure 1, bacterial strain GSS01 and PCA extracts the polysaccharide of Extracellular polymers, protein content with ultrasonic method most
It is more, and Extracellular polymers are mainly made of polysaccharide and albumen, it is possible to it is inferred to containing for the Extracellular polymers of ultrasonic method extraction
Amount is most.
2. the shape characteristic that difference extracts EPS method
Cell morphology is observed using scanning electron microscope (ULTRA 55), scanning electron microscope example preparation step is as follows:
1) the fixed 5h of 2.5% glutaraldehyde；
2) distilled water immersion 6 times, each 10-20min；
3) Gradient elution using ethanol is used, 30% ethyl alcohol impregnates twice, each 10min；
4) 50%% ethyl alcohol impregnates twice, each 10min；
5) 70% ethyl alcohol impregnates twice, each 15min；
6) 90% ethyl alcohol impregnates twice, each 15min；
7) anhydrous % ethyl alcohol impregnates twice, each 15min；
8) lyophilizer (SCIENTZ-10N) freeze-drying 12h is let cool；
9) platform is pasted after sample drying, sputters gold, and sample making is completed.
As a result: by Fig. 2 and Fig. 3 scanning electron microscope (SEM) photograph as it can be seen that in terms of bacterium breakage rate, relative to control group, either PCA
(Fig. 2) or GSS01 (Fig. 3), bacterium breakage rate caused by heating is relatively more, EDTA method and ultrasonic method bacterium breakage compared with
It is few.Either GSS01 or PCA, compared with the control group, bacterium surface is more smooth after ultrasonic method is extracted, and shows to remain in bacterium
The EPS content shown is reduced, and the EPS content extracted which show ultrasonic method is most.
3. the cellular damage rate that difference extracts EPS method
It is right using LIVE/DEAD BacLight Bacterial Viability Kit kit (Invitrogen, CA)
The cell membrane scraped from graphite plate is dyed, and flow cytometer (BD FACSCanto) and inverted fluorescence microscope are then passed through
(OLYMPUS IX53) determines that (flow cytomery the results are shown in Table 1 to ultrasonic extraction, be inverted fluorescence to cellular damage rate minimum
Microscopy Results are shown in Fig. 4 and Fig. 5).
Specific staining procedure is as follows: 1) biomembrane being suspended in 0.9%NaCl solution, sample to be contaminated: dyestuff mixed liquor
It is dyed according to 1000:3；2) dyestuff mixed liquor is mixed by syto9.0 and PI equal proportion；3) dyeing is grasped in dark situation
Make, 15min is protected from light after dyeing, is cleaned after the completion of dyeing with sterile saline, extra dyeing liquor is removed.Dyeing is completed
Observe result immediately afterwards.The green portion of biomembrane is more, then living cells is more, otherwise lower.
The loss cell rate of 1 Different Extraction Method of table
As a result: by Fig. 4 (PCA) and Fig. 5 (GSS01) it is found that the red fluorescence of heating is most, cellular damage rate is highest
, ultrasonic method and EDTA method RED sector are all relatively fewer, and cellular damage rate is lower.And the fluorescence intensity of PI is stronger, indicates thin
Born of the same parents' death rate is higher, and the PI value after GSS01 ultrasonic extraction is that PI value is 28628 after 95463, PCA ultrasonic extraction as shown in Table 1,
Either PCA or GSS01, the fluorescence intensity after ultrasonic method is extracted is minimum, the cellular damage after showing ultrasonic extraction
Rate is minimum.
4. the EPS component characteristics that Different Extraction Method obtains
Three-dimensional fluorescence spectrum is by excitation wavelength (y-axis), launch wavelength (x-axis), fluorescence intensity (z-axis) three-dimensional coordinate institute table
The matrix spectrum of sign.Three-dimensional fluorescence spectrum figure generally has three-dimensional projection and contour fluorescence spectra both representations.
The fluorescence intensity of substance is related with the wavelength of exciting light and the measured transmitting wavelength of light, it is possible to utilize three-dimensional fluorescence spectrum
Determine the component of unknown materials.
In this experiment, the three-dimensional fluorescence spectrum of sepectrophotofluorometer (Hitachi F-7000) measurement EPS, three-dimensional fluorescence are utilized
The excitation wavelength range of spectrum is 200-450nm, increment 5nm, launch wavelength 200-600nm, increment 5nm.Fig. 6 is bacterium
The fluorescence spectra for the EPS that strain PCA is extracted in distinct methods, Fig. 7 are the fluorescence for the EPS that bacterial strain GSS01 is extracted in distinct methods
Spectrogram.
As a result: by Fig. 6 and Fig. 7 it is found that the three-dimensional fluorescence spectrum of S-EPS finds three absorption peaks: EX/EM=275/ altogether
350nm, 315/400nm, 230/380nm, 275/350nm are tryptophan absorbence peak, generally represent protein, 315/400nm is
Humic acid material absorption peak, 230/380nm are fulvic acid class material absorption peak；The three-dimensional fluorescence spectrum of LB-EPS and TB-EPS
Two absorption peaks: EX/EM=275/350nm, 225/350nm are found altogether, and wherein 225/350nm is aromatic amino acid absorption
Peak.
To sum up, in EPS component other than containing protein, polysaccharide and nucleic acid, there is also fulvic acid and humic acid.This is first
The secondary characteristic to ground bacillus Extracellular polymers characterizes.
5. the volt-ampere analysis for the EPS that Different Extraction Method obtains
Oxidation using cyclic voltammetry (Cyclic Voltammetry, CV) to the Extracellular polymers of Different Extraction Method
Reproducibility is studied.CV scanning is completed on electrochemical workstation CHI660E, and CV sweep parameter is as follows: Init E (V)=
0.2V, High E (V)=0.2V, Low E (V)=- 0.8V, Final E (V)=0.2V, scanning speed=5mV/s.
As a result: by Fig. 8 (PCA) and Fig. 9 (GSS01) it is found that the CV scanning figure of LB-TBS and TB-EPS has two pairs of oxidations
Reduction peak, formal potential are -0.5V or so and 0V or so, and it is identical that this illustrates that the LB-EPS that three kinds of methods are extracted and TB-EPS have
Redox active albumen.From the point of view of peak value size, peak value size is directly proportional to redox active albumen, ultrasonic method and heating
The peak value of method is greater than the peak value of EDTA, illustrates that the EPS redox active protein content of the extraction of ultrasonic method and heating is wanted
Than EDTA fado.(see Fig. 8 (B) and Fig. 9 (B)) in the TB-EPS CV scanning figure of PCA and GSS01, the peak value ratio of ultrasonic method
EDTA method and heating are high, illustrate that the redox active albumen contained in the TB-EPS of ultrasonic method extraction is most.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Claims (9)
1. a kind of method for extracting ground bacillus Extracellular polymers, which is characterized in that the step is as follows:
1) soluble S-EPS in the electrolyte of microbiological fuel cell is collected；It will be on microbiological fuel cell graphite plate electrode
Biomembrane scrapes, and biomembrane is suspended to obtain biomembrane suspension with solution；
2) biomembrane suspension is ultrasonic, concussion, is centrifuged to obtain supernatant and precipitating at ultrasound, and supernatant liquid filtering takes filtrate to obtain LB-
EPS；
3) precipitating after step 2) centrifugation is suspended in the solution, ultrasound is centrifugally separating to obtain supernatant and precipitating, repeats 2~3
It is secondary, by the supernatant hybrid filtering of all acquisitions, filtrate is taken to obtain TB-EPS.
2. the method according to claim 1, wherein solution described in step 1) is 0.9%NaCl solution.
3. the method according to claim 1, wherein ultrasound described in step 2) is the ultrasound 1~5 in ice-water bath
Minute.
4. the method according to claim 1, wherein concussion condition described in step 2) is 100~200 revs/min
Clock shakes 7~15 minutes.
5. the method according to claim 1, wherein step 2), 3) centrifugal condition be 4000~7000g, 4~
6 DEG C of 10~20min of centrifugation.
6. the method according to claim 1, wherein step 2), 3) described in ultrasonic power be 300~500W,
Ultrasonic time is 5~12 minutes.
7. the method according to claim 1, wherein step 2), 3) use 0.22 μm of aperture membrane filtration.
8. the Extracellular polymers of described in any item method preparations according to claim 1~7.
9. Extracellular polymers according to claim 8, which is characterized in that the Extracellular polymers are more outside extracellular soluble
At least one of polymers, loose combination Extracellular polymers and Extracellular polymers of combining closely.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112062290A (en) * | 2020-08-07 | 2020-12-11 | 光大水务科技发展（南京）有限公司 | Method for improving biofilm formation efficiency of biological filter for treating high-nitrate wastewater |
| CN114316079A (en) * | 2021-01-11 | 2022-04-12 | 暨南大学 | Extraction and purification method of extracellular polysaccharide of thioredoxin |
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