CN107328636A - A kind of method of high efficiency extraction micropopulation induction signal molecule - Google Patents
A kind of method of high efficiency extraction micropopulation induction signal molecule Download PDFInfo
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- CN107328636A CN107328636A CN201710572159.4A CN201710572159A CN107328636A CN 107328636 A CN107328636 A CN 107328636A CN 201710572159 A CN201710572159 A CN 201710572159A CN 107328636 A CN107328636 A CN 107328636A
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- extraction
- high efficiency
- nitrogen
- induction signal
- signal molecule
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/40—Concentrating samples
Abstract
The invention discloses a kind of method of high efficiency extraction micropopulation induction signal molecule, efficient concentration is realized to the colony induction signaling molecule in aerobic granular sludge reactor water outlet.This method filters reactor water outlet through 0.45 μm of glass fiber filter, then with dichloromethane, chloroform, the organic solvents such as ethyl acetate are mixed according to a certain volume, by signaling molecule from aqueous phase is transferred to organic phase, realize signaling molecule preliminary concentration, solution after extraction passes through Rotary Evaporators vacuum distillation, realize the initial concentration of signaling molecule, evaporative flask used in vacuum distillation is dried up after fully being washed through corresponding organic solvent with distillation surplus solution through nitrogen, and the solid constant volume after being blown according to test condition using corresponding organic solvent nitrogen, realize the efficiently concentrating of signaling molecule.This method is easy to operate, the signaling molecule rate of recovery is high, can be a kind of improved efficient signal molecule extracting method the problem of vacuum distillation solution hardly possible is collected during effective address signal molecular concentration.
Description
Technical field
The present invention relates to a kind of method of high efficiency extraction micropopulation induction signal molecule, belong to biological wastewater treatment neck
Domain.
Background technology
Quorum sensing is the main mode of intelligence transmission between microbial cell, and microorganism is according to its population density, environment
The one or more signaling molecules of factor variations secretion, enter cell by diffusion, transport protein effect and combine differential protein, enter
And regulate and control downstream functional gene expression and realize the vital movements such as biofilm formation, virulence factor secretion, bioluminescence.Signaling molecule
Three classes are broadly divided into, the N- acyl homoserine lactones (Acyl-Homoserine respectively acted between Gram-negative bacteria
Lactone, AHL), the polypeptides matter that acts between gram-positive bacteria and while act on gram-positive bacteria and feminine gender
AI-2 materials (pentanone of 4,5- dihydroxy -2,3- bis- is precursor substance, occurs to reset and spontaneous reaction is formed) between bacterium, this three
Class signaling molecule can be acted, it is necessary to meet three conditions：1 zero signaling molecules reach critical concentration；2 zero signals point
Sub- synzyme high activity；The receptor protein concentration of 3 zero signaling molecules is high.There is shape as the special of biomembrane in aerobic particle mud
Formula, its functional flora majority is Gram-negative bacteria, therefore AHL classes signaling molecule is to Granular sludge formation and stable system operation
There is important regulative, it is most extensive in aerobic particle mud area research.It is many at present using sense microorganism, TLC,
The methods such as HPLC-MS/MS carry out qualitative, sxemiquantitative, quantitative analysis to system AHL.It is most micro- on aerobic particle mud system
The research of biocenose sensing shows that AHL mainly influences the form of aerobic particle mud by the composition and secretion that influence EPS
And physiological characteristic.
But aerobic particle mud system AHL contents are substantially at the ppb-ppt orders of magnitude at present, it is difficult to meet quantitative, fixed
Property the required detection limit of analysis, it is therefore necessary to AHL contents are improved in solution by way of concentration to reach that detection is required.
It is generally at present to take a large amount of water outlets applied to the aerobic granular sludge reactor water outlet AHL methods concentrated, through decompression after liquid-liquid extraction
Distillation is settled to required volume, but for small size water outlet and the relatively low system of AHL contents to reach more highly enriched ratio, it is difficult to
The product after vacuum distillation is fully dissolved using trace solvent, causes extraction efficiency relatively low.Therefore, efficient AHL extractions are researched and developed
Method for concentration is taken, is research aerobic particle mud body to meet small size water outlet and the concentration requirement of the relatively low system of AHL contents
The important prerequisite of the quorum sensing regulatory mechanism of system.
The content of the invention
It is an object of the invention to provide a kind of method of high efficiency extraction micropopulation induction signal molecule, in traditional AHL
Increase nitrogen on the basis of concentration and blow constant volume step, to meet small size water outlet and the concentration requirement of the relatively low system of AHL contents.
The method of high efficiency extraction micropopulation induction signal molecule, is mainly included the following steps that, (1) reactor water outlet is received
Collection and filtering；(2) liquid-liquid extraction；(3) it is concentrated under reduced pressure；(4) nitrogen blows constant volume：This method is by reactor water outlet through 0.45 μm of glass fibers
Membrane filtration is tieed up, is then mixed according to a certain volume with the organic solvent such as dichloromethane, chloroform, ethyl acetate, makes signal
Molecule realizes signaling molecule preliminary concentration from aqueous phase is transferred to organic phase, and the solution after extraction is in certain temperature and pressure strip
By Rotary Evaporators vacuum distillation under part, the initial concentration of signaling molecule is realized, evaporative flask used in vacuum distillation is through should mutually have
Machine solvent is dried up after fully washing with distillation surplus solution through nitrogen, and nitrogen is blown using corresponding organic solvent according to test condition
Solid constant volume afterwards, realizes the efficiently concentrating of signaling molecule.
The technical scheme that the present invention is specifically used is as follows：
The method of high efficiency extraction micropopulation induction signal molecule, comprises the following steps：
Step 1：Whole mud mixtures of water outlet stage in the sbr reactor device cycle of operation are collected, are led to again after precipitation
Cross the glass core filter filtering equipped with 0.45 μm of glass fiber filter；
Step 2：The liquid filtered of learning from else's experience is placed in pear shape separatory funnel, is added organic solvent and is used as extractant, profit
Vibrated with separating funnel oscillator, extract repeated several times, collect organic phase；
Step 3：It will be evaporated by Rotary Evaporators continuous feed mouthful in the organic phase suction evaporative flask of collection to organic phase,
Extractant is taken fully to wash evaporative flask several times, wash liquid is collected into centrifuge tube；
Step 4：The centrifuge tube is placed on nitrogen evaporator, constantly regulate nitrogen blow needle height, makes centrifuge tube during nitrogen blows
Interior liquid level is in vibrating state, until centrifugation liquid in pipe drying；Then the solid after being blown using solvent nitrogen carries out constant volume, complete
The extraction and enrichment of colony induction signaling molecule into sbr reactor device water outlet.
Based on such scheme, parameter and material in each step can use following preferred scheme：
Preferably, in described step 1 mud mixture first pass through in advance 1-2min precipitation after be reloaded into glass core filter
Filtered in device.
Preferably, the organic solvent described in described step 2 includes dichloromethane, chloroform or ethyl acetate.
Preferably, the mixed volume ratio of centrifuged supernatant and organic solvent is 1 in described step 2:(1-2).
Preferably, in each extraction process of described step 2, separating funnel oscillator vibrates 5- with 200-300rpm
10min, extraction is repeated 2-5 times.
Preferably, in described step 3, the vacuum distillation temperature of Rotary Evaporators is 30-40 DEG C, and pressure is 500-
800mbar。
Preferably, during the nitrogen of described step 4 blows, nitrogen flow is controlled in 1.0-3.0L/min.
Beneficial effects of the present invention：
Constant volume step is blown because the present invention sets up nitrogen on the basis of classical signal molecule extracting process, is made in evaporative flask
Solid matter is fully washed and accurate constant volume, small size water outlet and the concentration requirement of the relatively low system of AHL contents is met, with effective
Study the quorum sensing regulatory mechanism of aerobic particle mud system.
Brief description of the drawings
Fig. 1 is the flow chart of high efficiency extraction micropopulation induction signal molecule.
Fig. 2 is AHL recovery of standard addition under embodiment and reference examples different operating flow.
Embodiment
Below by way of the drawings and specific embodiments, the present invention is described further.
As shown in figure 1, a kind of generally including for method of high efficiency extraction micropopulation induction signal molecule is following basic
Step：(1) reactor effluent collection and filtering；(2) liquid-liquid extraction；(3) it is concentrated under reduced pressure；(4) nitrogen blows constant volume.
As shown in figure 1, each step specific implementation is as follows：
(1) step 1, reactor effluent collection and filtering
According to the sbr reactor device cycle of operation, the whole mud mixtures of reactor water outlet stage are collected using vial, through 1-
2min precipitations are filtered by the glass core filter equipped with 0.45 μm of glass fiber filter, are accurately pipetted 50-100mL and are filtered out
Water, for next step extraction.
(2) step 2, liquid-liquid extraction
Step 1 filtered fluid 50-100mL is taken in 200-500mL pear shape separatory funnels, according to 1:1-2 volume ratios add two
The organic solvents such as chloromethanes, chloroform or ethyl acetate are as extractant, using separating funnel oscillator with 200-300rpm
5-10min is vibrated, extraction is repeated 2-5 times, collects organic phase.
(3) step 3, vacuum distillation
The organic phase of collection step 2, sets 30-40 DEG C of vacuum distillation temperature, according to Clausius-Clapeyron equations such as
Under：
Wherein：T1, T2Temperature residing for-liquid；
p1, p2—T1, T2Correspondence hold-up vapour pressure；
ΔvapHm- molar heat of vaporization.
Calculate organic phase and reach that corresponding pressure is 500-800mbar during boiling point, temperature, pressure condition according to obtained by calculating
Rotary evaporation instrument parameter is set.The organic phase of collection is sucked into 50mL by Buchi R210 Rotary Evaporators continuous feed mouthful to steam
It is evaporated in hair bottle to organic phase.The corresponding extractants of 0.5-1.0mL are taken fully to wash evaporative flask 2-3 times, wash liquid careful collection
Into 1.5-2.0mL centrifuge tubes.
(4) step 4, nitrogen blows constant volume
Centrifuge tube containing organic solution obtained by step 3 is placed on nitrogen evaporator, regulation high pure nitrogen flow 1.0-3.0L/
Min, regulation nitrogen blow needle height, makes liquid level in centrifuge tube be in micro, slight vibrating state, constantly regulate nitrogen blow needle position to centrifuge tube
Interior liquid drying, according to subsequent detection needs, takes 50-200 μ L coordinative solvent constant volumes, may finally be by the letter in reactor water outlet
Number 500-2000 times of molecular concentration.
Embodiment
The purpose of the present embodiment is the high efficiency extraction colony induction signaling molecule from sbr reactor device water outlet, specifically according to
Lower step is carried out：
Step 1, reactor effluent collection and filtering
According to the sbr reactor device cycle of operation, the whole mud mixtures of reactor water outlet stage, warp are collected using vial
1min precipitations are filtered by the glass core filter equipped with 0.45 μm of glass fiber filter, are accurately pipetted 100mL filtering water outlets, are used
In next step extraction, the present embodiment chooses bytyry homoserine lactone (C4-HSL), caproyl homoserine lactone (C6-
HSL), 3- oxohexanoyls homoserine lactone (3OC6-HSL), 3- oxo caprylyl homoserine lactones (3OC8-HSL), ten
Dialkyl group homoserine lactone (C12-HSL) is studied as typical case AHL (blows) sample mark-on time with/without nitrogen under different operating flow
Yield.
Step 2, liquid-liquid extraction
Step 1 filtered fluid 100mL is taken in 500mL pear shape separatory funnels, according to 1:1 volume ratio adds dichloromethane conduct
Extractant, vibrates 10min with 300rpm using separating funnel oscillator, extraction is repeated 3 times, collects organic phase.
Step 3, vacuum distillation
The organic phase of collection step 2, sets 40 DEG C of vacuum distillation temperature, according to Clausius-Clapeyron equations：
Wherein：Temperature residing for T1, T2-liquid；
p1, p2- T1, T2 correspondence hold-up vapour pressure；
ΔvapHm- molar heat of vaporization.
Calculate dichloromethane and reach that corresponding pressure is 800mbar during boiling point, set according to gained temperature, pressure condition is calculated
Put rotary evaporation instrument parameter.The organic phase of collection is sucked by 50mL evaporations by Buchi R210 Rotary Evaporators continuous feed mouthful
It is evaporated in bottle to organic phase.Take the corresponding extractants of 1.0mL fully to wash evaporative flask 3 times, wash liquid careful collection to 2.0mL from
In heart pipe.
Step 4, nitrogen blows constant volume
Centrifuge tube containing organic solution obtained by step 3 is placed on nitrogen evaporator, high pure nitrogen flow 2.0L/min is adjusted,
Nitrogen blow needle height is adjusted, liquid level is in micro, slight vibrating state, constantly regulate nitrogen blow needle position is to centrifuging intraluminal fluid
Body is dried up, according to subsequent detection needs, takes 100 μ L coordinative solvents to carry out constant volume to the solid matter in centrifuge tube, most anti-at last
The signaling molecule in device water outlet is answered to concentrate 1000 times.
Synchronous to set reference examples in order to contrast the effect of the present invention, the difference of reference examples and above-described embodiment is not set
The nitrogen for putting step 4 blows constant volume.As shown in Fig. 2 as a result showing due to setting up nitrogen on the basis of classical signal molecule extracting process
Constant volume step is blown, the solid matter in evaporative flask is fully washed and accurate constant volume, meets small size water outlet and AHL contents are relatively low
System concentration requirement, effectively to study the quorum sensing regulatory mechanism of aerobic particle mud system.
Claims (7)
1. a kind of method of high efficiency extraction micropopulation induction signal molecule, it is characterised in that comprise the following steps：
Step 1：Whole mud mixtures of water outlet stage in the sbr reactor device cycle of operation are collected, pass through dress again after precipitation
There is the glass core filter filtering of 0.45 μm of glass fiber filter；
Step 2：The liquid of filtering of learning from else's experience is placed in pear shape separatory funnel, adds organic solvent as extractant, using point
Liquid funnel oscillator is vibrated, and extracts repeated several times, collects organic phase；
Step 3：The organic phase of collection is sucked by Rotary Evaporators continuous feed mouthful and is evaporated in evaporative flask to organic phase, extraction is taken
Agent is taken fully to wash evaporative flask several times, wash liquid is collected into centrifuge tube；
Step 4：The centrifuge tube is placed on nitrogen evaporator, constantly regulate nitrogen blow needle height, makes centrifugation intraluminal fluid during nitrogen blows
Face is in vibrating state, until centrifugation liquid in pipe drying；Then the solid after being blown using solvent nitrogen carries out constant volume, completes
The extraction and enrichment of colony induction signaling molecule in sbr reactor device water outlet.
2. the method for high efficiency extraction micropopulation induction signal molecule as claimed in claim 1, it is characterised in that described
Mud mixture is first passed through to be reloaded into glass core filter after 1-2min precipitations in advance and filtered in step 1.
3. the method for high efficiency extraction micropopulation induction signal molecule as claimed in claim 1, it is characterised in that described
Organic solvent described in step 2 includes dichloromethane, chloroform or ethyl acetate.
4. the method for high efficiency extraction micropopulation induction signal molecule as claimed in claim 1, it is characterised in that described
The mixed volume ratio of centrifuged supernatant and organic solvent is 1 in step 2:(1-2).
5. the method for high efficiency extraction micropopulation induction signal molecule as claimed in claim 1, it is characterised in that described
In each extraction process of step 2, separating funnel oscillator vibrates 5-10min with 200-300rpm, and extraction is repeated 2-5 times.
6. the method for high efficiency extraction micropopulation induction signal molecule as claimed in claim 1, it is characterised in that described
In step 3, the vacuum distillation temperature of Rotary Evaporators is 30-40 DEG C, and pressure is 500-800mbar.
7. the method for high efficiency extraction micropopulation induction signal molecule as claimed in claim 1, it is characterised in that described
During the nitrogen of step 4 blows, nitrogen flow is controlled in 1.0-3.0L/min.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1330590C (en) * | 2001-10-17 | 2007-08-08 | 先进环境技术有限公司 | Organic waste treatment |
| CN106318990A (en) * | 2016-08-19 | 2017-01-11 | 浙江工商大学 | Preparation for accelerating methane oxidation of refuse landfill and preparation method and application of preparation |
-
2017
- 2017-07-13 CN CN201710572159.4A patent/CN107328636A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1330590C (en) * | 2001-10-17 | 2007-08-08 | 先进环境技术有限公司 | Organic waste treatment |
| CN106318990A (en) * | 2016-08-19 | 2017-01-11 | 浙江工商大学 | Preparation for accelerating methane oxidation of refuse landfill and preparation method and application of preparation |
Non-Patent Citations (7)
| Title |
|---|
| MATTHEW FLETCHER等: "Biosensors for Qualitative and Semiquantitative Analysis of Quorum Sensing Signal Molecules", 《PSEUDOMONAS: METHODS AND PROTOCOLS》 * |
| MEI-ZHEN WANG等: "Effect of Pseudomonas sp. HF-1 inoculum on construction of a bioaugmented system for tobacco wastewater treatment:analysis from quorum sensing", 《ENVIRON SCI POLLUT RES》 * |
| RODRIGO H. GONZALEZ等: "Detection and characterization of quorum sensing signal molecules in Acinetobacter strains", 《MICROBIOL. RES. 》 * |
| 于多: "群体感应（QS）对MBR膜污染影响分析", 《中国优秀硕士学位论文全文数据库 工程科技I辑》 * |
| 森夏因主编: "《毒物分析方法学》", 28 February 1986, 群众出版社 * |
| 陈峰: "微生物的群体感应信号分子(综述)", 《上海农业学报》 * |
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