CN102520040B - Microbial reactor for detecting water toxicity and method for detecting water toxicity - Google Patents
Microbial reactor for detecting water toxicity and method for detecting water toxicity Download PDFInfo
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Abstract
The invention provides a microbial reactor for detecting water toxicity, which comprises a carrier and microbes immobilized on the carrier. The invention also provides a method for detecting the water toxicity, which comprises the following steps of: (a) adding an electron mediator and a water sample to be detected into the microbial reactor of the technical scheme, and culturing to obtain a mixed solution; and (b) detecting current of the mixed solution obtained in the step (a) by adopting a three-electrode system. According to the microbial reactor and the method, the microbes which are immobilized on the carrier and serve as a tested organism can keep activity for a long time and can be recycled, relative to suspended microbes for detecting water toxicity, repeated culture, centrifuging, cleaning and other processes are not required each time, so that the operation time is saved, the operation complexity is reduced, the activity of the microbes on the carrier can be maintained for a long time, and the sensitivity and stability of detection cannot be influenced.
Description
Technical field
The invention belongs to water body detection technique field, relate in particular to the detection method of a kind of microorganism reactor for detection of water body toxicity and the total toxicity of water body.
Background technology
Along with human production activity's development, a large amount of chemicals constantly enter in water environment, become the primary pollution source of water pollution.These Pollution by Chemicals things are scarcely Body composition compositions, neither needed by human body nutriment or maintain the necessary material of normal physiological function, but it can contact and enter with human body human body, produce certain biological action, as entered by the amplification of food web the even life that may be detrimental to health in human body when poisonous and harmful chemicals.In addition, in water body, Pollution by Chemicals also can make the biology in water body receive harm, and therefore, water body toxicity is detected and monitored is to evaluate water body whether to receive pollution and judge the important means of pollution level.
In water body toxicity detection and monitoring method, biological detection is because the advantages such as visual result are subject to extensive concern, the biologies such as batrachia, fish, mouse, planktonic organism, marine alga have been used to water body toxicity and have detected, but, carry out when water body toxicity detects, thering is the shortcomings such as long, cost is high, complicated operation of test period with above-mentioned biology, not only be difficult to promote, and can not meet field quick detection needs.Microbial population is large, growth cycle is short, high to the susceptibility of environmental change, have and the similar physicochemical characteristics of higher mammal and enzyme mechanism, therefore be applicable to that exploitation saves time, the quick bio of low consumption, amoral dispute is learned toxotest method, be especially applicable to exploitation small portable water body toxicity checkout equipment.In Mixed bacteria method, the detection technique based on luminescent microorganism is most widely used.International ISO11348-3 regulation is used deep-sea photobacteria V.fischeri as biological subject, for equilibrium osmotic pressure, test must be carried out under high salinity condition, may cause the change of some chemicals character in sample, as, for low-concentration metallic sample, salt correction can cause false-negative result; And for the low sample of some solubleness, as phenol etc., can be because separating out of poisonous substance causes false positive results.And the method adopts fluoroscopic examination, detection signal is subject to water turbidity impact, is unsuitable for muddy and coloured sample.In addition, photobacteria is not ubiquitous microorganism at occurring in nature, obtains and preserve comparatively difficulty, and commodity photobacteria price is comparatively expensive, causes testing cost higher.
Electric analysis Instrument has the advantages such as detection sensitivity is high, cost is low, applicable miniaturization, is widely used in the fields such as environmental monitoring, bioanalysis, medical science detection.In the metabolic processes of microorganism, microorganism can pass to electron mediator by electronics by the respiration of self, in the time that toxicant exists, the respiration of microorganism is suppressed, and then hinder the electronics transmission between microorganism and electron mediator, therefore, can develop the electrochemical method and the instrument that detect and monitor for water body toxicity based on microorganism.As U.S. Lincon Technology has developed Micredox system, it can be for detection of the BOD of water body; The potassium ferricyanide is introduced Micredox system by the people such as Zelanian Pasco, by being determined under different toxicant existence conditions microorganism and as the electronics transfer rate between the potassium ferricyanide molecule of electron mediator, confirming that this system can be used for water body toxicity and detects.But, this system is directly taking suspension microorganism as biological subject, the processes such as suspension microorganism all needs again to cultivate while use at every turn, centrifugal, cleaning, running time and Operating Complexity are not only increased, and because each suspension microorganism state of cultivating can not reach in full accord, can affect sensitivity and the stability of detection.
Summary of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of detection method of microorganism reactor and water body toxicity for detection of water body toxicity, detection method provided by the invention is highly sensitive, good stability, simple to operate, is suitable for online water body toxicity and detects and monitor.
The invention provides a kind of microorganism reactor for detection of water body toxicity, comprising: carrier and be fixed on the microorganism on described carrier.
Preferably, described carrier is activated charcoal, porous potter's clay, micropore glass, alginate, carragheen, agar, polyacrylamide gel, polyvinyl alcohol gel or isocyanurate foam.
Preferably, described microorganism is one or more in Escherichia coli, fluorescent pseudomonas, pseudomonasputida and enterobacter cloacae.
Compared with prior art, microorganism reactor provided by the invention comprises carrier and is fixed on the microorganism on described carrier.In this microorganism reactor, microorganism is fixed in carrier, can not move, but the carrier inside of can freely coming in and going out such as little molecule substrate and reaction metabolic product, not only can keep active and can reuse, during for detection of water body toxicity without all repeating to cultivate at every turn, the process such as centrifugal, cleaning, not only save the running time, reduced Operating Complexity, and because microorganism can keep for a long time active constant on described carrier, can not affect sensitivity and the stability of detection.
The present invention also provides a kind of detection method of water body toxicity, comprises the following steps:
A) in the microorganism reactor described in technique scheme, add electron mediator and water sample to be measured, after cultivation, obtain mixed solution;
B) adopt three-electrode system to detect the electric current of the mixed solution that described step obtains in a).
Preferably, a) also comprise before in described step:
In the microorganism reactor described in technique scheme, add electron mediator and standard water sample, after cultivation, obtain the first mixed solution;
Adopt three-electrode system to detect the electric current of described the first mixed solution.
Preferably, the temperature of described cultivation is 35 DEG C～38 DEG C, and the time of described cultivation is 0.5h～2h.
Preferably, described step a) in, described electron mediator is the derivant of the potassium ferricyanide, dimethyl diaminophenazine chloride, ferrocene or ferrocene.
Preferably, described step a) in, the concentration of described electron mediator in described water sample to be measured is 40mmol/L～50mmol/L.
Preferably, described step b) in, the working electrode in described three-electrode system is tiny array electrode.
Preferably, described tiny array electrode is platinum tiny array electrode.
The present invention, to be fixed on microorganism on carrier as biological subject, adds after electron mediator and water body to be measured in described microorganism reactor, can adopt three-electrode system to detect the electric current of water sample to be measured, thereby obtain water body toxicity testing result.The present invention not only can keep active and can reuse as biological subject using the microorganism of being fixed on carrier, during for detection of water body toxicity without all repeating to cultivate at every turn, the process such as centrifugal, cleaning, not only save the running time, reduced Operating Complexity, and because microorganism can keep for a long time active constant on described carrier, can not affect sensitivity and the stability of detection; The present invention detects the toxicity of water body to be measured by electrochemical method, be not subject to the impact of water turbidity and colourity, simultaneously can be using multiple-microorganism as biological subject, avoid the selectivity to toxicant, there is higher sensitivity and stability, be more applicable to the detection demand of water body toxicity.Experimental result shows, adopts IC50 value that method provided by the invention detects the toxicant obtaining and the IC50 value that adopts MICROTOX detector to detect to obtain quite, lower than the IC50 value that detection obtains taking suspension microorganism as biological subject.
Brief description of the drawings
Fig. 1 is the electromicroscopic photograph that is embedded with colibacillary calcium alginate filament prepared by the embodiment of the present invention 1;
Fig. 2 is the electromicroscopic photograph that is not embedded with colibacillary calcium alginate filament prepared by the embodiment of the present invention;
The concentration that Fig. 3 provides for the embodiment of the present invention 21 and the curve map of electric current inhibiting rate;
The concentration that Fig. 4 provides for the embodiment of the present invention 22 and the curve map of electric current inhibiting rate;
The concentration that Fig. 5 provides for the embodiment of the present invention 23 and the curve map of electric current inhibiting rate;
The concentration that Fig. 6 provides for the embodiment of the present invention 24 and the curve map of electric current inhibiting rate;
The fixation of microbe weight that Fig. 7 provides for the embodiment of the present invention 26 and the curve map of electric current inhibiting rate.
Embodiment
The invention provides a kind of microorganism reactor for detection of water body toxicity, comprising: carrier and be fixed on the microorganism on described carrier.
In microorganism reactor provided by the invention, microorganism is fixed in carrier, can not move, but the carrier inside of can freely coming in and going out such as little molecule substrate and reaction metabolic product, not only can keep active and can reuse, during for detection of water body toxicity without all repeating to cultivate at every turn, the process such as centrifugal, cleaning, not only save the running time, reduced Operating Complexity, and because microorganism can keep for a long time active constant on described carrier, can not affect sensitivity and the stability of detection.
In the present invention, the effect of described carrier is fixation of microbe, and it need to meet following characteristic: operating process is simple; To cell nonhazardous effect; Mass-transfer performance is good; Be difficult for being decomposed by microorganism; Stable in properties; Intensity is high, the life-span is long etc.In the present invention, described carrier can be inorganic material, natural organic or synthetic organic material etc., wherein, inorganic material includes but not limited to activated charcoal, porous potter's clay, micropore glass etc., be preferably activated charcoal, have that intensity is large, mass transfer performances good, to the advantage such as cell nonhazardous, preparation process be simple; Natural organic includes but not limited to alginate, carragheen, agar etc., is preferably alginate, has the advantage such as microorganism nonhazardous effect, mass transfer performances be good; Synthetic organic material includes but not limited to polyacrylamide gel, polyvinyl alcohol gel, isocyanurate foam etc., is preferably polyvinyl alcohol gel, has that intensity is high, good chemical stability.
In the present invention, described microorganism exists as the biological subject that detects water body toxicity, and the present invention there is no particular restriction to it, can be single culture, can be also mixed bacteria, and those skilled in the art can select as required or determine.Described microorganism is preferably one or more in Escherichia coli, fluorescent pseudomonas, pseudomonasputida and enterobacter cloacae, more preferably Escherichia coli or fluorescent pseudomonas.
The mode that the present invention is fixed on described carrier described microorganism is not particularly limited, and can be the methods such as absorption, embedding, crosslinked, chemical covalency or self immobilization.The number of the present invention to described microorganism and the weight of described carrier are not particularly limited, and those skilled in the art can determine voluntarily according to the microbe species of selecting, kind of carrier and fixed form.
The present invention is not particularly limited the preparation method of described microorganism reactor, can prepare in accordance with the following methods:
(1) absorption method: according to the effect of static, surface tension and adhesion between charged microbial cell and carrier, make microorganism be fixed on carrier surface and the inner method that forms microorganism reactor, specifically comprise the following steps:
Carrier drying is placed in the nutrient solution of inoculation thalline, after cultivation, obtains microorganism reactor.
Adopting absorption method while preparing microorganism reactor, described carrier is the porous class carrier with adsorption function, as activated charcoal etc.
First the present invention will cultivate after carrier drying in inoculation has the nutrient solution of thalline, and in incubation, microorganism is adsorbed to carrier inside or surface, forms microorganism reactor.The present invention is all not particularly limited described parameter dry and that cultivate, and those skilled in the art can select and determine according to kind of carrier, the microbe species etc. selected.If, carrier is carbon felt, microorganism while being fluorescent pseudomonas, dry temperature is preferably 60 DEG C～100 DEG C, and the time is preferably 3h～8h; Cultivation temperature is preferably 28 DEG C～32 DEG C, and the time is preferably 20h～25h, preferably under the rotating speed of 80r/min～120r/min, cultivates.
(2) investment: be again embedded immobilization, be that microbial cell is embedded in polymkeric substance or film, or make microbial cell diffuse into porous carrier inside, little molecule substrate and reaction metabolic product can freely be come in and gone out, and microorganism maintains static.
Taking calcium alginate as example, the method that employing investment is prepared microorganism reactor is as follows:
Cultivate microorganism, obtain microbial suspension;
Sodium alginate is soluble in water, obtain sodium alginate soln;
Described microbial suspension and described sodium alginate soln are mixed, obtain mixed solution;
Adopt syringe that described mixed solution is injected in calcium chloride solution, obtain being embedded with the calcium alginate silk of microorganism;
After the described calcium alginate silk that is embedded with microorganism is solidified, obtain microorganism reactor.
The present invention is not particularly limited described cultivation method of microorganism, expands cultivation according to different types of microorganism of selecting.The number of the present invention to described microorganism and the weight ratio of described calcium alginate are not particularly limited, and those skilled in the art can determine voluntarily.
(3) cross-linking method: utilize the functional group of the amino in enzyme molecule in microorganism or hydroxyl and crosslinking chemical to react, be cross-linked to form covalent bond, make microbial cells mutually form reticulate texture, realize microbial immobilization.
(4) chemical covalent method: non aqueous carrier is combined with the form of covalent bond with microorganism.
(5) self immobilization method: rely on flocculation immobilization on carrier of microorganism self to form.
Obtain after microorganism reactor, can use it for water body toxicity detects and monitors, and while use without all repeating to cultivate at every turn, the process such as centrifugal, cleaning, not only save the running time, reduced Operating Complexity, and because microorganism can keep for a long time active constant on described carrier, can not affect sensitivity and the stability of detection.
The present invention also provides a kind of detection method of water body toxicity, comprises the following steps:
A) in the microorganism reactor described in technique scheme, add electron mediator and water sample to be measured, obtain mixed solution;
B) adopt three-electrode system to detect the electric current of the mixed solution that described step obtains in a).
The present invention is to be fixed on microorganism on carrier as biological subject, in described microorganism reactor, add after water sample to be measured and electron mediator, can adopt three-electrode system to detect water sample electric current to be measured, the present invention detects the toxicity of water sample to be measured by electrochemical method, be not subject to the impact of water turbidity and colourity, simultaneously can be using multiple-microorganism as biological subject, avoid the selectivity to toxicant, there is higher sensitivity and stability, be more applicable to the detection demand of water body toxicity.
Method provided by the invention can be used for water body acute toxicity and detects, also can be for the daily monitoring of water body toxicity, during for the daily monitoring of water body toxicity, electron mediator and water body to be measured are joined in the microorganism reactor described in technique scheme and cultivated, adopt three-electrode system to detect the electric current of described water body to be measured simultaneously, can judge the daily toxicity of water body according to curent change; While detection for water body acute toxicity, using unpolluted water or tap water as standard water body, after standard water sample and water sample to be measured are mixed with electron mediator respectively, join in the microorganism reactor described in technique scheme and cultivate, adopt three-electrode system to detect respectively the electric current of described standard water sample and water sample to be measured simultaneously, after contrasting, can judge water body acute toxicity situation to be measured.
In the present invention, described water body acute toxicity detection method specifically comprises the following steps:
In the microorganism reactor described in technique scheme, add standard water sample and electron mediator, obtain the first mixed solution;
Adopt three-electrode system to detect the current value of described the first mixed solution;
In the microorganism reactor described in technique scheme, add electron mediator and water sample to be measured, obtain the second mixed solution;
Adopt three-electrode system to detect the current value of described the second mixed solution;
Judge water body toxicity pollution condition according to the current value of the current value of described the first mixed solution and described the second mixed solution.
The described daily monitoring method to water body specifically comprises the following steps:
In the microorganism reactor described in technique scheme, add electron mediator and water sample to be measured, obtain mixed solution;
Adopt three-electrode system to detect the current value of described mixed solution;
Can judge water body toxicity situation of change according to the variation of current value.
In the present invention, the microorganism reactor adopting when described standard water sample and water sample to be measured are detected or monitored, kind, the consumption etc. of electron mediator are all identical, and the present invention describes as an example of water body to be measured example.
When water body toxicity to be measured is detected, first in the microorganism reactor described in technique scheme, add electron mediator and water sample to be measured, after cultivating, obtain mixed solution.Temperature, time and the mode of the present invention to described cultivation is not particularly limited, and can determine according to the kind of the microorganism of selecting, while being Escherichia coli, is preferably heat insulating culture 0.5h～2h at 35 DEG C～38 DEG C as microorganism.
Carrying out in incubation, microorganism carries out metabolism, and consume oxygen decomposing organic matter, and be translated into simple inorganics, shown in (1), formula (2):
O
2(aq)+4H
++4e
-→2H
2O
(I) (2)
In this process, organism is degraded by microorganisms, and generates CO
2, and produce 4 electronics; Oxygen molecule in water is accepted 4 electronics, generates two hydrones; Because the solubleness of oxygen in water is lower, under the condition of anaerobic, electron mediator, as the potassium ferricyanide etc. can replace oxygen as artificial electron accepter, generates CO
2and potassium ferrocyanide, shown in (3):
From formula (1), formula (2) and formula (3), the organic process of microbial degradation can be reduced to the process that electronics transmits, be that microbial degradation speed and electronics transfer rate are proportional, and then cause respiratory rate and electronics to be transmitted into ratio.
In the time that toxicant is invaded water body, the eupnea effect of mentioned microorganism will be suppressed, and even cause the death of microorganism, and microorganism is because the quantity of the electronics that respiration is outwards transmitted also can reduce thereupon.According to this process, can adopt water body to be measured to enter the inhibition degree sign water body toxicity that microorganism reactor front and back microbial respiratory speed is subject to, be specially the relation (IC adopting between electric current inhibiting rate and concentration, Inhibitory Concentration) characterize water body toxicity, can pass through formula (I) and calculate electric current inhibiting rate:
In formula (I), I is electric current inhibiting rate, represents with the form of percentage;
I
lim controlfor the limiting current value of standard water sample;
I
lim samplefor the limiting current value of water sample to be measured;
IC50 value is the concentration of electric current inhibiting rate toxicant while reaching 50%.
In the present invention, described electron mediator is electron accepter, is preferably the derivant of the potassium ferricyanide, dimethyl diaminophenazine chloride, ferrocene or ferrocene, more preferably the potassium ferricyanide.The concentration of described electron mediator in described water sample to be measured is preferably 40mmol/L～50mmol/L, more preferably 43mmol/L～48mmol/L.
In the present invention, the volume of described water sample to be measured and the weight ratio of described fixation of microbe are preferably 0.5mL～5mL: 0.01g～0.10g, more preferably 1mL～3mL: 0.01g～0.05g.It should be noted that, in microorganism reactor, the quantity of microorganism and the quantity of carrier can be determined voluntarily by those skilled in the art, therefore, those skilled in the art can be according to the weight ratio of the ratio-dependent of micro organism quantity in microorganism reactor and vehicle weight volume of water sample to be measured and fixation of microbe.
Cultivation obtains after mixed solution, adopts three-electrode system to measure the current value of described mixed solution.Described three-electrode system is the three-electrode system forming by working electrode, to electrode and contrast electrode, wherein, electrode is preferably to Pt electrode, contrast electrode is preferably Ag/AgCl (saturated KCl), working electrode can be the single electrode of single electrode, tiny array electrode, process modification or the tiny array electrode through modifying, be preferably tiny array electrode or the tiny array electrode through modifying, more preferably Pt tiny array electrode.In order to improve the sensitivity of detection, the present invention before use preferably carries out surface treatment to described working electrode, preferably adopts 0.05 μ m α-Al
2o
3described working electrode is carried out to polishing.
The present invention preferably adopts CHI832B type galvanochemistry instrument to measure the timing electric current of described mixed solution, and measuring voltage is preferably 400mV～500mV, more preferably 420mV～480mV.
Obtain respectively after the limiting current value of standard water sample and water sample to be measured, these two limiting current values are compared, can qualitatively judge water body toxicity to be measured; Obtain respectively, after the limiting current value of standard water sample and water sample to be measured, can calculating the IC50 value of each toxicant according to formula (I), thereby can judge the toxicity size of toxicant.Experimental result shows, adopts IC50 value that method provided by the invention detects the toxicant obtaining and the IC50 value that adopts MICROTOX detector to detect to obtain quite, lower than the IC50 value that detection obtains taking suspension microorganism as biological subject.
The present invention, to be fixed on microorganism on carrier as biological subject, adds after water body to be measured and electron mediator in described microorganism reactor, can adopt three-electrode system to detect the electric current of water sample to be measured, thereby obtain water body toxicity testing result.The present invention not only can keep active and can reuse as biological subject using the microorganism of being fixed on carrier, during for detection of water body toxicity without all repeating to cultivate at every turn, the process such as centrifugal, cleaning, not only save the running time, reduced Operating Complexity, and because microorganism can keep for a long time active constant on described carrier, can not affect sensitivity and the stability of detection; The present invention detects the toxicity of water body to be measured by electrochemical method, be not subject to the impact of water turbidity and colourity, simultaneously can be using multiple-microorganism as biological subject, avoid the selectivity to toxicant, there is higher sensitivity and stability, be more applicable to the detection demand of water body toxicity.
In order to further illustrate the present invention, be described in detail for detection of the microorganism reactor of water body toxicity and the detection method of water body toxicity provided by the invention below in conjunction with embodiment.
Embodiment 1
In 37 DEG C, the shaking table of 200r/min, Escherichia coli are cultivated in concussion, cultivate 10h left and right, when Escherichia coli Growth is during to stationary phase, take out and centrifugal, and the physiological saline with 0.7% is clean, obtains Escherichia coli suspending liquid; Sodium alginate is dissolved in the water of 70 DEG C, heating, obtains sodium alginate soln while stirring; Escherichia coli suspending liquid is mixed with sodium alginate soln, obtain the mixed liquor of sodium alginate and thalline, wherein, the concentration of sodium alginate is 2%; It is CaCl 5%, that magneton stirs that the mixed liquor of described sodium alginate and thalline is pushed to concentration with syringe
2in solution, obtain being embedded with colibacillary calcium alginate filament; Described calcium alginate filament is solidified after 10h at 4 DEG C, and the physiological saline with 0.7% cleans, and obtains microorganism reactor.
Described calcium alginate silk is carried out to electron-microscope scanning, result is referring to Fig. 1 and Fig. 2, Fig. 1 is the electromicroscopic photograph that is embedded with colibacillary calcium alginate filament prepared by the embodiment of the present invention 1, and Fig. 2 is the electromicroscopic photograph that is not embedded with colibacillary calcium alginate filament prepared by the embodiment of the present invention.From Fig. 1 and Fig. 2, the present invention has realized the embedding of Escherichia coli in calcium alginate.
Embodiment 2
Compound concentration is the potassium ferricyanide aqueous solution of 45mmol/L, in the microorganism reactor that contains immobilized microorganism prepared by 0.01g embodiment 1, adds 1mL potassium ferricyanide aqueous solution, and heat insulating culture 1h at 37 DEG C, obtains mixed solution; With 25 through 0.05 μ m α-Al
2o
3the tiny array electrode that the list of 25 μ m of polishing props up Pt electrode composition is that working electrode, Ag/AgCl (saturated KCl) electrode are contrast electrode, Pt silk as to electrode, adopt CHI 832B type galvanochemistry instrument to carry out cyclic voltammetry scan and timing current detecting to described mixed solution: cyclic voltammetry scan scope is 0～600mV, and sweep velocity is made as 50mV/s; Timing current/voltage is set as 450mV, the limiting current value i getting standard samples
lim control;
Compound concentration is the Cr of 5mg/L, 10mg/L, 15mg/L, 20mg/L, 30mg/L, 50mg/L and 100mg/L respectively
6+solution, respectively to described Cr
6+solution adds the potassium ferricyanide, and to make the concentration of the described potassium ferricyanide be 45mmol/L; Continue respectively solution described in 1mL to be joined and contain in immobilized microorganism reactor prepared by 0.01g embodiment 1, heat insulating culture 1h at 37 DEG C, obtains mixed solution; With 25 through 0.05 μ m α-Al
2o
3the tiny array electrode that the list of 25 μ m of polishing props up Pt electrode composition is that working electrode, Ag/AgCl (saturated KCl) electrode are contrast electrode, Pt silk as to electrode, adopt CHI 832B type galvanochemistry instrument to carry out cyclic voltammetry scan and timing current detecting to described mixed solution: cyclic voltammetry scan scope is 0～600mV, and sweep velocity is made as 50mV/s; Timing current/voltage is set as 450mV, obtains respectively the limiting current value i of testing sample
lim sample;
Calculate respectively the IC50 value of each water sample to be measured according to formula (I) formula, result is referring to table 1, the IC50 value of the inorganic pollutant that table 1 provides for the embodiment of the present invention and comparative example.
Comparative example 1
Step and method according to embodiment 2 is tested, and difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 1, the IC50 value of the inorganic pollutant that table 1 provides for the embodiment of the present invention and comparative example.
Embodiment 3
Step and method according to embodiment 2 is tested, and difference is, compound concentration is the Hg of 1mg/L, 3mg/L, 5mg/L, 7mg/L, 10mg/L, 15mg/L and 20mg/L respectively
2+solution, result is referring to table 1, the IC50 value of the inorganic pollutant that table 1 provides for the embodiment of the present invention and comparative example.
Embodiment 4
Step and method according to embodiment 2 is tested, and difference is, compound concentration is the Cd of 10mg/L, 20mg/L, 30mg/L, 40mg/L, 50mg/L, 60mg/L and 70mg/L respectively
2+solution, result is referring to table 1, the IC50 value of the inorganic pollutant that table 1 provides for the embodiment of the present invention and comparative example.
Comparative example 3
Step and method according to embodiment 3 is tested, and difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 1, the IC50 value of the inorganic pollutant that table 1 provides for the embodiment of the present invention and comparative example.
Embodiment 5
Step and method according to embodiment 2 is tested, and difference is, compound concentration is the Bi of 20mg/L, 30mg/L, 50mg/L, 60mg/L, 70mg/L, 80mg/L and 100mg/L respectively
3+solution, result is referring to table 1, the IC50 value of the inorganic pollutant that table 1 provides for the embodiment of the present invention and comparative example.
Comparative example 4
Step and method according to embodiment 5 is tested, and difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 1, the IC50 value of the inorganic pollutant that table 1 provides for the embodiment of the present invention and comparative example.
Embodiment 6
Step and method according to embodiment 2 is tested, and difference is, compound concentration is the Pb of 5mg/L, 15mg/L, 20mg/L, 30mg/L, 50mg/L, 60mg/L and 70mg/L respectively
2+solution, result is referring to table 1, the IC50 value of the inorganic pollutant that table 1 provides for the embodiment of the present invention and comparative example.
Comparative example 5
Step and method according to embodiment 6 is tested, and difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 1, the IC50 value of the inorganic pollutant that table 1 provides for the embodiment of the present invention and comparative example.
Embodiment 7
Step and method according to embodiment 2 is tested, and difference is, compound concentration is the Zn of 5mg/L, 15mg/L, 20mg/L, 30mg/L, 50mg/L, 60mg/L and 70mg/L respectively
2+solution, result is referring to table 1, the IC50 value of the inorganic pollutant that table 1 provides for the embodiment of the present invention and comparative example.
Comparative example 6
Step and method according to embodiment 7 is tested, and difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 1, the IC50 value of the inorganic pollutant that table 1 provides for the embodiment of the present invention and comparative example.
The IC50 value of the inorganic pollutant that table 1 embodiment of the present invention and comparative example provide
In table 1, described standard value is to adopt MICROTOX detector to detect the IC50 value obtaining.
As shown in Table 1, compared with suspension microorganism detection method, detection method result provided by the invention is more sensitive, accurate.
Embodiment 8
Prepare microorganism reactor according to the method for embodiment 1 and step, difference is, described CaCl
2concentration be 1%, be 24h set time.
Embodiment 9
Compound concentration is the potassium ferricyanide aqueous solution of 45mmol/L, adds 1mL potassium ferricyanide aqueous solution to containing in immobilized microorganism reactor prepared by 0.01g embodiment 8, and heat insulating culture 1h at 37 DEG C, obtains mixed solution; With 5 through 0.05 μ m α-Al
2o
3the tiny array electrode that the list of 25 μ m of polishing props up Pt electrode composition is that working electrode, Ag/AgCl (saturated KCl) electrode are contrast electrode, Pt silk as to electrode, adopt CHI 832B type galvanochemistry instrument to carry out cyclic voltammetry scan and timing current detecting to described mixed solution: cyclic voltammetry scan scope is 0～600mV, and sweep velocity is made as 50mV/s; Timing current/voltage is set as 450mV, the limiting current value i getting standard samples
lim control;
Compound concentration is 3 of 1mg/L, 3mg/L, 5mg/L, 7mg/L, 8mg/L, 9mg/L and 10mg/L respectively, 5-chlorophenesic acid solution, and to described 3,5-chlorophenesic acid solution adds the potassium ferricyanide respectively, and to make the concentration of the described potassium ferricyanide be 45mmol/L; Continue respectively in immobilized microorganism reactor prepared by 0.01g embodiment 8, to add solution described in 1mL to containing, heat insulating culture 1h at 37 DEG C, obtains mixed solution; With 5 through 0.05 μ m α-Al
2o
3the tiny array electrode that the list of 25 μ m of polishing props up Pt electrode composition is that working electrode, Ag/AgCl (saturated KCl) electrode are contrast electrode, Pt silk as to electrode, adopt CHI 832B type galvanochemistry instrument to carry out cyclic voltammetry scan and timing current detecting to described mixed solution: cyclic voltammetry scan scope is 0～600mV, and sweep velocity is made as 50mV/s; Timing current/voltage is set as 450mV, obtains respectively the limiting current value i of testing sample
lim sample;
Calculate respectively the IC50 value of each water sample to be measured according to formula (I) formula, result is referring to table 2, the IC50 value of the organic contaminant that table 2 provides for the embodiment of the present invention and comparative example.
Comparative example 7
Step and method according to embodiment 9 is tested, and difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 2, the IC50 value of the organic contaminant that table 2 provides for the embodiment of the present invention and comparative example.
Embodiment 10
Step and method according to embodiment 9 is tested, difference is, compound concentration is the phenol solution of 50mg/L, 80mg/L, 100mg/L, 150mg/L, 170mg/L, 175mg/L and 180mg/L respectively, result is referring to table 2, the IC50 value of the organic contaminant that table 2 provides for the embodiment of the present invention and comparative example.
The IC50 value of the organic contaminant that table 2 embodiment of the present invention and comparative example provide
In table 2, described standard value is to adopt MICROTOX detector to detect the IC50 value obtaining.
As shown in Table 2, compared with suspension microorganism detection method, detection method result provided by the invention is more sensitive, accurate.
Embodiment 11
Prepare microorganism reactor according to the method for embodiment 1 and step, difference is, described CaCl
2concentration be 10%, be 8h set time.
Embodiment 12
Compound concentration is the potassium ferricyanide aqueous solution of 45mmol/L, adds 1mL potassium ferricyanide aqueous solution to containing in immobilized microorganism reactor prepared by 0.01g embodiment 11, and heat insulating culture 1h at 37 DEG C, obtains mixed solution; With 30 through 0.05 μ m α-Al
2o
3the tiny array electrode that the list of 25 μ m of polishing props up Pt electrode composition is that working electrode, Ag/AgCl (saturated KCl) electrode are contrast electrode, Pt silk as to electrode, adopt CHI 832B type galvanochemistry instrument to carry out cyclic voltammetry scan and timing current detecting to described mixed solution: cyclic voltammetry scan scope is 0～600mV, and sweep velocity is made as 50mV/s; Timing current/voltage is set as 450mV, the limiting current value i getting standard samples
lim controi;
Compound concentration is the fenamiphos solution of 1mg/L, 3mg/L, 5mg/L, 10mg/L, 15mg/L, 20mg/L and 30mg/L respectively, adds the potassium ferricyanide respectively to described fenamiphos solution, and to make the concentration of the described potassium ferricyanide be 45mmol/L; Continue respectively in immobilized microorganism reactor prepared by 0.01g embodiment 11, to add solution described in 1mL to containing, heat insulating culture 1h at 37 DEG C, obtains mixed solution; With 30 through 0.05 μ m α-Al
2o
3the tiny array electrode that the list of 25 μ m of polishing props up Pt electrode composition is that working electrode, Ag/AgCl (saturated KCl) electrode are contrast electrode, Pt silk as to electrode, adopt CHI 832B type galvanochemistry instrument to carry out cyclic voltammetry scan and timing current detecting to described mixed solution: cyclic voltammetry scan scope is 0～600mV, and sweep velocity is made as 50mV/s; Timing current/voltage is set as 450mV, obtains respectively the limiting current value i of testing sample
lim sample;
Calculate respectively the IC50 value of each water sample to be measured according to formula (I) formula, result is referring to table 3, the IC50 value of the agricultural chemicals that table 3 provides for the embodiment of the present invention and comparative example.
Comparative example 8
Test according to implementing 11 step and method, difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 3, the IC50 value of the agricultural chemicals that table 3 provides for the embodiment of the present invention and comparative example.
Embodiment 13
Step and method according to embodiment 13 is tested, difference is, compound concentration is the ametryn solution of 1mg/L, 3mg/L, 5mg/L, 7mg/L, 10mg/L, 15mg/L and 20mg/L respectively, and result is referring to table 3, the IC50 value of the agricultural chemicals that table 3 provides for the embodiment of the present invention and comparative example.
Comparative example 9
Test according to implementing 13 step and method, difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 3, the IC50 value of the agricultural chemicals that table 3 provides for the embodiment of the present invention and comparative example.
Embodiment 14
Step and method according to embodiment 12 is tested, difference is, compound concentration is the 5a,6,9,9a-hexahydro-6,9-methano-2,4 solution of 1mg/L, 3mg/L, 5mg/L, 7mg/L, 10mg/L, 15mg/L and 20mg/L respectively, and result is referring to table 3, the IC50 value of the agricultural chemicals that table 3 provides for the embodiment of the present invention and comparative example.
Comparative example 10
Test according to implementing 14 step and method, difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 3, the IC50 value of the agricultural chemicals that table 3 provides for the embodiment of the present invention and comparative example.
The IC50 value of the agricultural chemicals that table 3 embodiment of the present invention and comparative example provide
In table 3, described standard value is to adopt MICROTOX detector to detect the IC50 value obtaining.
As shown in Table 3, compared with suspension microorganism detection method, detection method result provided by the invention is more sensitive, accurate.
Embodiment 15
Prepare microorganism reactor according to the method for embodiment 1 and step, difference is, described CaCl
2concentration be 15%, be 6h set time.
Embodiment 16
Compound concentration is the potassium ferricyanide aqueous solution of 45mmol/L, adds 1mL potassium ferricyanide aqueous solution to containing in immobilized microorganism reactor prepared by 0.01g embodiment 15, and heat insulating culture 1h at 37 DEG C, obtains mixed solution; With 50 through 0.05 μ m α-Al
2o
3the tiny array electrode that the list of 25 μ m of polishing props up Pt electrode composition is that working electrode, Ag/AgCl (saturated KCl) electrode are contrast electrode, Pt silk as to electrode, adopt CHI 832B type galvanochemistry instrument to carry out cyclic voltammetry scan and timing current detecting to described mixed solution: cyclic voltammetry scan scope is 0～600mV, and sweep velocity is made as 50mV/s; Timing current/voltage is set as 450mV, the limiting current value i getting standard samples
lim control;
Compound concentration is the arsenic trioxide solution of 1mg/L, 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L and 30mg/L respectively, adds the potassium ferricyanide respectively to described arsenic trioxide solution, and to make the concentration of the described potassium ferricyanide be 45mmol/L; Continue respectively in immobilized microorganism reactor prepared by 0.01g embodiment 15, to add solution described in 1mL to containing, heat insulating culture 1h at 37 DEG C, obtains mixed solution; With 50 through 0.05 μ m α-Al
2o
3the tiny array electrode that the list of 25 μ m of polishing props up Pt electrode composition is that working electrode, Ag/AgCl (saturated KCl) electrode are contrast electrode, Pt silk as to electrode, adopt CHI 832B type galvanochemistry instrument to carry out cyclic voltammetry scan and timing current detecting to described mixed solution: cyclic voltammetry scan scope is 0～600mV, and sweep velocity is made as 50mV/s; Timing current/voltage is set as 450mV, obtains respectively the limiting current value i of testing sample
lim samlpe;
Calculate respectively the IC50 value of each water sample to be measured according to formula (I) formula, result is referring to table 4, the IC50 value of the respiration inhibitor that table 4 provides for the embodiment of the present invention and comparative example.
Comparative example 11
Step and method according to embodiment 16 is tested, and difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 4, the IC50 value of the respiration inhibitor that table 4 provides for the embodiment of the present invention and comparative example.
Embodiment 17
Step and method according to embodiment 16 is tested, difference is, compound concentration is the potassium cyanide solution of 1mg/L, 2mg/L, 3mg/L, 4mg/L, 5mg/L, 6mg/L and 7mg/L respectively, and result is referring to table 4, the IC50 value of the respiration inhibitor that table 4 provides for the embodiment of the present invention and comparative example.
Comparative example 12
Step and method according to embodiment 17 is tested, and difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 4, the IC50 value of the respiration inhibitor that table 4 provides for the embodiment of the present invention and comparative example.
Embodiment 18
Step and method according to embodiment 16 is tested, difference is, compound concentration is the salicylic acid solution of 1mg/L, 5mg/L, 10mg/L, 12mg/L, 15mg/L, 20mg/L and 25mg/L respectively, result is referring to table 4, the IC50 value of the respiration inhibitor that table 4 provides for the embodiment of the present invention and comparative example.
Comparative example 13
Step and method according to embodiment 18 is tested, and difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 4, the IC50 value of the respiration inhibitor that table 4 provides for the embodiment of the present invention and comparative example.
Embodiment 19
Step and method according to embodiment 16 is tested, difference is, compound concentration is the dinitrophenol dinitrophenolate solution of 5mg/L, 10mg/L, 15mg/L, 20mg/L, 25mg/L, 30mg/L and 35mg/L respectively, result is referring to table 4, the IC50 value of the respiration inhibitor that table 4 provides for the embodiment of the present invention and comparative example.
Comparative example 14
Step and method according to embodiment 19 is tested, and difference is, using the Escherichia coli that suspend as biological subject, result is referring to table 4, the IC50 value of the respiration inhibitor that table 4 provides for the embodiment of the present invention and comparative example.
The IC50 value of the respiration inhibitor that table 4 embodiment of the present invention and comparative example provide
In table 4, described standard value is to adopt MICROTOX detector to detect the IC50 value obtaining.
As shown in Table 4, compared with suspension microorganism detection method, detection method result provided by the invention is more sensitive, accurate.
Embodiment 20
Carbon felt is placed in to 80 DEG C of baking ovens and toasts 6h, the carbon felt after baking is placed in to the nutrient solution of inoculating fluorescent pseudomonas, in 30 DEG C, the shaking table of 100r/min, 24h is cultivated in concussion, and obtaining absorption has the carbon felt of fluorescent pseudomonas; After having the carbon felt of fluorescent pseudomonas to clean absorption with phosphate buffer solution (PBS) buffer solution, obtain microorganism reactor.
Embodiment 21
Step and method according to embodiment 2 is tested, difference is, the microorganism reactor that the microorganism reactor of preparing with embodiment 20 replaces embodiment 1 to prepare, and compound concentration is the Cr of 10mg/L, 20mg/L, 30mg/L, 50mg/L, 60mg/L and 100mg/L
6+solution, result is referring to Fig. 3, the concentration that Fig. 3 provides for the embodiment of the present invention 21 and the curve map of electric current inhibiting rate, as shown in Figure 3, Cr
6+iC50 value be 51mg/L.
Embodiment 22
Step and method according to embodiment 21 is tested, and difference is, compound concentration is the Hg of 25mg/L, 50mg/L, 60mg/L, 100mg/L, 150mg/L and 200mg/L
2+solution, result is referring to Fig. 4, the concentration that Fig. 4 provides for the embodiment of the present invention 22 and the curve map of electric current inhibiting rate, as shown in Figure 4, Hg
2+iC50 value be 97.85mg/L.
Embodiment 23
Step and method according to embodiment 21 is tested, and difference is, compound concentration is the Pb of 0mg/L, 50mg/L, 100mg/L, 200mg/L, 300mg/L and 400mg/L
2+solution, result is referring to Fig. 5, the concentration that Fig. 5 provides for the embodiment of the present invention 23 and the curve map of electric current inhibiting rate, as shown in Figure 5, Pb
2+iC50 value be 178mg/L.
Embodiment 24
Step and method according to embodiment 21 is tested, and difference is, compound concentration is the Cd of 0mg/L, 50mg/L, 100mg/L, 150mg/L, 200mg/L, 300mg/L and 400mg/L
2+solution, result is referring to Fig. 6, the concentration that Fig. 6 provides for the embodiment of the present invention 24 and the curve map of electric current inhibiting rate, as shown in Figure 6, Cd
2+iC50 value be 178mg/L.
Embodiment 25
Step and method according to embodiment 21 is tested, and difference is, 3 of 1mg/L, 3mg/L, 5mg/L, 7mg/L, 8mg/L, 9mg/L and 10mg/L, and 5-chlorophenesic acid solution, result shows, the IC50 value of 3,5-chlorophenesic acid is 14mg/L.
Embodiment 26
The potassium ferricyanide aqueous solution that compound concentration is 45mmol/L respectively, add 1mL potassium ferricyanide aqueous solution to containing in immobilized microorganism reactor prepared by 0.01g, 0.02g, 0.03g, 0.06g and 1.0g embodiment 11 respectively, at 37 DEG C, heat insulating culture 1h, obtains mixed solution; With 30 through 0.05 μ m α-Al
2o
3the tiny array electrode that the list of 25 μ m of polishing props up Pt electrode composition is that working electrode, Ag/AgCl (saturated KCl) electrode are contrast electrode, Pt silk as to electrode, adopt CHI 832B type galvanochemistry instrument to carry out cyclic voltammetry scan and timing current detecting to described mixed solution: cyclic voltammetry scan scope is 0～600mV, and sweep velocity is made as 50mV/s; Timing current/voltage is set as 450mV, the limiting current value i getting standard samples
lim control;
Compound concentration is 3 of 16mg/L, 5-chlorophenesic acid solution, and to described 3,5-chlorophenesic acid solution adds the potassium ferricyanide, and to make the concentration of the described potassium ferricyanide be 45mmol/L; Continue respectively in immobilized microorganism reactor prepared by 0.01g, 0.02g, 0.03g, 0.06g and 1.0g embodiment 11, to add solution described in 1mL to containing, heat insulating culture 1h at 37 DEG C, obtains mixed solution; With 30 through 0.05 μ m α-Al
2o
3the tiny array electrode that the list of 25 μ m of polishing props up Pt electrode composition is that working electrode, Ag/AgCl (saturated KCl) electrode are contrast electrode, Pt silk as to electrode, adopt CHI 832B type galvanochemistry instrument to carry out cyclic voltammetry scan and timing current detecting to described mixed solution: cyclic voltammetry scan scope is 0～600mV, and sweep velocity is made as 50mV/s; Timing current/voltage is set as 450mV, obtains respectively the limiting current value i of testing sample
lim sample; Calculate respectively the electric current inhibiting rate value of each water sample to be measured according to formula (I) formula, result is referring to Fig. 7, the fixation of microbe weight that Fig. 7 provides for the embodiment of the present invention 26 and the curve map of electric current inhibiting rate, as shown in Figure 7, in the time that fixation of microbe is 0.01g～0.10g, the sensitivity detecting is all higher, and sensitivity is the highest when weight is 0.01g, and the result obtaining with this understanding has good reappearance simultaneously.
From above-described embodiment and comparative example, when microorganism reactor provided by the invention detects for water body toxicity, can improve sensitivity and the stability of detection, not only can pollute by toxicant by qualitative detection water body, and can judge the toxicity size of the toxicant of polluted-water.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (9)
1. for detection of a microorganism reactor for water body toxicity, comprising: carrier and be fixed on the microorganism on described carrier, described carrier is alginate;
Described microorganism is fixed on described carrier by the method for embedding;
Described embedding is: cultivate microorganism, obtain microbial suspension;
Sodium alginate is soluble in water, obtain sodium alginate soln;
Described microbial suspension and described sodium alginate soln are mixed, obtain mixed solution;
Adopt syringe that described mixed solution is injected in calcium chloride solution, obtain being embedded with the calcium alginate silk of microorganism;
After the described calcium alginate silk that is embedded with microorganism is fixed, obtain microorganism reactor.
2. microorganism reactor according to claim 1, is characterized in that, described microorganism is one or more in Escherichia coli, fluorescent pseudomonas, pseudomonasputida and enterobacter cloacae.
3. a detection method for water body toxicity, comprises the following steps:
A) in the microorganism reactor described in claim 1～2 any one, add water sample to be measured and electron mediator, after cultivation, obtain mixed solution;
B) adopt three-electrode system to detect the electric current of the mixed solution that described step obtains in a).
4. detection method according to claim 3, is characterized in that, a) also comprises before in described step:
To the microorganism reactor adding in standard water sample described in electron mediator and claim 1～2 any one, after cultivation, obtain the first mixed solution;
Adopt three-electrode system to detect the electric current of described the first mixed solution.
5. detection method according to claim 3, is characterized in that, the temperature of described cultivation is 30 DEG C～40 DEG C, and the time of described cultivation is 0.5h～2h.
6. according to the detection method described in claim 3～5 any one, it is characterized in that, described step a) in, described electron mediator is the derivant of the potassium ferricyanide, dimethyl diaminophenazine chloride, ferrocene or ferrocene.
7. detection method according to claim 6, is characterized in that, described step a) in, the concentration of described electron mediator in described water sample to be measured is 40mmol/L～50mmol/L.
8. detection method according to claim 3, is characterized in that, described step b) in, the working electrode in described three-electrode system is tiny array electrode.
9. detection method according to claim 8, is characterized in that, described tiny array electrode is platinum tiny array electrode.
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| CN104535632B (en) * | 2015-01-28 | 2017-01-25 | 哈尔滨工业大学宜兴环保研究院 | Method for determining IC50 value in water body employing combination of mixed bacteria and electrochemical technology |
| CN104914152B (en) * | 2015-05-22 | 2017-09-22 | 中国科学院长春应用化学研究所 | The cleaning method of the microelectrode of water body toxicity detection means |
| CN107703201A (en) * | 2017-09-28 | 2018-02-16 | 韦彩霞 | A kind of aquaculture persticide residue early warning system |
| CN108107102B (en) * | 2017-11-24 | 2019-11-22 | 中国科学院理化技术研究所 | A kind of electrochemistry water body acute biological toxicity evaluation method based on isolation method |
| CN109097591B (en) * | 2018-08-02 | 2021-03-23 | 中南大学 | Calcium alginate immobilized microbial adsorbent, preparation method thereof and application thereof in recycling platinum group metal secondary resources |
| CN109142491B (en) * | 2018-08-20 | 2020-09-15 | 北京航空航天大学 | Water quality monitoring method based on continuous flow membrane-free biological cathode microbial fuel cell |
| CN112205399B (en) * | 2020-10-22 | 2022-03-04 | 中国科学院合肥物质科学研究院 | Construction method of electrically-driven controlled-release and migrating gel-based pesticide system, constructed pesticide system and application thereof |
| CN112305029A (en) * | 2020-10-23 | 2021-02-02 | 清华大学 | Electrode biological film rapid forming method and microbial electrochemical sensor |
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