CN106047849A - Immobilized microbial system as well as preparation method thereof and water toxicity detection method - Google Patents
Immobilized microbial system as well as preparation method thereof and water toxicity detection method Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
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- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/025—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
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- G01N33/1866—Water using one or more living organisms, e.g. a fish using microorganisms
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- G—PHYSICS
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Abstract
The invention provides an immobilized microbial system, which comprises a microbial carrier and microorganisms attached to the surface of the microbial carrier, wherein the microbial carrier is prepared by carbonizing plant tissues. The invention also provides a preparation method of the immobilized microbial system and a water toxicity detection method. The preparation method disclosed by the invention, which prepares the immobilized microbial system from the plant carbonized tissues by virtue of a one-step method, is simple to implement; moreover, according to the immobilized microbial system provided by the invention, the microorganisms are immobilized by virtue of an attachment process, so that the microorganisms naturally deposit on pore surfaces of a carrier material, and a cross-linking curing process is avoided; and in addition, the adopted microbial carrier, which is of a three-dimensional spatial reticular structure, is relatively good in mass transfer performance. Therefore, the immobilized microbial system provided by the invention is relatively good in activity in a process of detecting the water toxicity; the immobilized microbial system can be repeatedly applicable to water toxicity detection; and the immobilized microbial system is relatively good in detection effect.
Description
Technical field
The present invention relates to water body detection technique field, particularly relate to a kind of fixing microorganism system and preparation method thereof and
The detection method of water body toxicity.
Background technology
Along with the progress of society, the mankind are also constantly deepening the impact on surrounding while self promotion, and
The environmental pollution thus resulted in reacts on again the mankind, becomes the obstacle of restriction human social development.At present, strengthen environment to protect
Protect, prevent and administer the problems such as the most contaminated environment and have become as researcher and the problem of environmentalist primary concern,
Also it is whole mankind's huge challenge of needing facing.In field of environment protection, environmental monitoring is as studying, analyze, evaluating
And assurance environmental quality, detect, predict, judge the important technology of environmental pollution development trend, have become as promotion many
Subject and the necessary power-assisted of research direction development, be also to provide the main of environmental information for governments at all levels, administration section and the common people
Means.
Toxicity is as a relative concept, and no matter its result is the method by actually detected or founding mathematical models
Obtain and all can be affected by biological subject body individual factors.The research of Martinez et al. shows, tropical fish Prochilodus
Lineatus is far above Oncorhynchus mykiss and Salmo salar to the sensitivity of glyphosate-class herbicides.Capkin
Et al. show for the research of 5a,6,9,9a-hexahydro-6,9-methano-2,4, when the concentration of poisonous substance is constant, build its survival rate the biggest of tested salmon parr is more
High；And with the increase of time of contact, the dosis tolerata of exogenous polyamines is substantially reduced by Fish.Pablo and Bl á ha is respectively with shellfish
And the Toxic test results that the germling of batrachia is study subject also demonstrates the opposing that the teenage germling of biology stimulates to external world
Ability is relatively weak, and sensitivity is higher.Therefore, the most also need to take into full account the feature of monitoring object and investigation
Emphasis, the proceed from the reality suitable subject kind of selection and state.
In the face of the environmental problem day by day complicated, simple environmental monitoring and theory analysis purely the most do not adapt to institute
Have the needs of task, also require researcher grasp Environmental Analytical Chemistry ABC while, according to modern analysis
Detailed programs in environmental monitoring are done and are studied targetedly by the structure of chemistry.Fish are that the biological environment applied the earliest is dirty
Dye monitoring subject, but its growth cycle is longer, it is impossible to reflection water quality toxicity pollution situation in time.In general, the lowest
Deng biology quantity is the hugest, kind is the most present in the nature, its subject being expected to become immediate toxicities detection.
So, microorganism is of universal significance as subject.
Utilizing in the water body toxicity detection method that microorganism is subject, the microorganism used is suspended state, i.e. divides
Dissipate microorganism in aqueous.Using the shortcoming of microorganism of suspended state is first to carry out the training of microorganism before experiment every time
Support work, add workload, improve testing cost.But fixing microorganism is difficult to be applied to the detection of water body toxicity
, after reacting with toxicant due to fixing microorganism, there will be two kinds of situations, and one is that microorganism can be made by toxicant
Become damage, so that microorganism deactivated；Another kind is that microorganism produces tolerance to toxicant, thus again meets toxicant
Suppression can not be embodied.The Chinese patent of Application No. CN201410020376, it is provided that a kind of for toxicity detection bio-sensing
The biomembranous preparation method of device, comprises the following steps: takes activated sludge and inoculates in culture medium, obtains bacterium solution；By polyethylene
Alcohol and sodium alginate join in distilled water, and heating in water bath is then cooled to room temperature, obtains polyvinyl alcohol gel；By bacterium solution from
After the heart, outwelling supernatant, obtain the precipitate arrived and mix with polyvinyl alcohol gel, mixture is uniformly painted by recycling surface plate
Membranaceous；Membranaceous mixture is dried so that it is after film forming, film is put in the crosslinking curing solution prepared in advance, crosslinking
After solidification, take out the film solidified, i.e. obtain the biomembrane for toxicity detection biosensor.
This biomembrane for toxicity detection biosensor that prior art provides uses the method for embedding to be prepared into
Arriving, preparation method is complicated；And the crosslinking curing in this method preparation process can affect the activity of microorganism；It addition, it is this
The material that method is wrapped up outside microorganism hinders the infiltration mass transfer of material in aqueous solution.Therefore.Prior art provide this
Activity is relatively low, Detection results is poor when water body toxicity detects to plant fixing biosensor organisms film.
Summary of the invention
In view of this, it is an object of the invention to provide a kind of fixing microorganism system and preparation method thereof and water body poison
Property detection method, during the fixing microorganism system detection water body toxicity using that the present invention provides, the activity of microorganism is higher,
Detection results is preferable.
The invention provides a kind of fixing microorganism system, including:
Microbe carrier, described microbe carrier is prepared by plant tissue carbonization；
It is attached to the microorganism on described microbe carrier surface.
Preferably, described plant tissue includes chalina, Bulbus Allii Fistulosi or Bulbus Allii Cepae.
Preferably, the microorganism that described microorganism obtains after being BODseed multi strain co cultivation.
The invention provides the preparation method of a kind of fixing microorganism system, including:
Microorganism is attached to microbe carrier surface, obtains the microorganism system fixed；Or
In microbe carrier surface in situ cultivating microorganism, obtain the microorganism system fixed；
Described microbe carrier is prepared by plant tissue carbonization.
Preferably, the temperature of described carbonization is 150 DEG C～250 DEG C；
The time of described carbonization is 3 hours～9 hours.
Preferably, the temperature of described Culture in situ microorganism is 20 DEG C～55 DEG C；
Time≤200 hour of described Culture in situ microorganism.
The present invention uses plant carbide tissue one-step method to prepare fixing microorganism system, and preparation method is simple；And
And the fixing microorganism system that the present invention provides uses attachment method by Microorganism incubation, make microorganism natural sediment in carrier material
The surface of material hole, it is not necessary to carry out crosslinking curing process；It addition, the microbe carrier in the present invention is the netted knot of three dimensions
Structure, has preferably infiltration mass transfer ability.Therefore, during the fixing microorganism system detection water body toxicity that the present invention provides
Activity preferably, can be repeatedly applied to the detection of water body toxicity, and Detection results is preferable.
The invention provides the detection method of a kind of water body toxicity, it is characterised in that use fixing microorganism system to enter
Row water body toxicity detects；
Described fixing microorganism system is the fixing microorganism system described in technique scheme.
Preferably, described water body toxicity detection method particularly as follows:
Background solution, nutritional solution and toxicity solution are circulated successively and is passed through fixing microorganism system, described background solution
The time that is passed through more than time that is passed through of nutritional solution, the time that is passed through of described nutritional solution and toxicity solution to be passed through the time identical；
Electrochemical method testing background solution, nutritional solution and toxicity solution is used to each lead into fixing microorganism system
After, the electrochemical signals that fixing microorganism system produces, it is designated as background solution electrochemical signals, nutritional solution electrochemistry letter respectively
Number and toxicity solution electrochemistry signal；
The toxicity of toxicity solution is obtained according to the difference between nutritional solution electrochemical signals and toxicity solution electrochemistry signal；
Described background solution is the activity of microorganism in stable and that recovery is fixing microorganism system；
Described nutritional solution provides nutrient substance for the microorganism in fixing microorganism system；
Described toxicity solution is the nutritional solution containing toxicant.
Preferably, the time that is passed through of described background solution is 1 minute～200 minutes；
The time that is passed through of described nutritional solution and toxicity solution is 1 minute～30 minutes.
Preferably, the toxicant in described toxicity solution is toxic heavy metal ion.
The detection method of the water body toxicity that the present invention provides uses the fixing microorganism system described in technique scheme
Detect, it is not necessary to carrying out microorganism culturing can detect, and detection method is simple；And the work of microorganism during detecting
Property is higher, may be repeated detection, and Detection results is good.
Accompanying drawing explanation
In order to be illustrated more clearly that the embodiment of the present invention or technical scheme of the prior art, below will be to embodiment or existing
In having technology to describe, the required accompanying drawing used is briefly described, it should be apparent that, the accompanying drawing in describing below is only this
Inventive embodiment, for those of ordinary skill in the art, on the premise of not paying creative work, it is also possible to according to
The accompanying drawing provided obtains other accompanying drawing.
Fig. 1 is the SEM figure of the microbe carrier that the embodiment of the present invention 1 prepares；
Fig. 2 is the SEM figure of the microbe carrier that the embodiment of the present invention 2 prepares；
Fig. 3 is the SEM figure of the microbe carrier that the embodiment of the present invention 3 prepares；
Fig. 4 is the CLSM detection figure of the microbe carrier that the embodiment of the present invention 4 prepares；
Fig. 5 is the CLSM detection figure of the microbe carrier that the embodiment of the present invention 5 prepares；
Fig. 6 is the CLSM detection figure of the microbe carrier that the embodiment of the present invention 6 prepares；
Fig. 7 is the signal of telecommunication record figure that the embodiment of the present invention 8 circulation measures water body toxicity；
Fig. 8 is the amplification signal graph that the embodiment of the present invention 8 detects water body toxicity one the measurement cycle；
Fig. 9 is for measuring Cr6+The suppression ratio in continuous 6 cycles；
Figure 10 is Cr6+Suppression ratio and concentration map；
Figure 11 is for measuring variable concentrations Cr6+The suppression ratio of continuous 1 month；
Figure 12 is for measuring Cu2+The suppression ratio in continuous 5 cycles；
Figure 13 is Cu2+Suppression ratio and concentration map；
Figure 14 is for measuring variable concentrations Cu2+The suppression ratio of continuous 1 month；
Figure 15 is Bi3+、Cd2+、Zn2+、Pb2+Concentration and suppression ratio figure；
Figure 16 is concentration and the suppression ratio figure of DCP.
Detailed description of the invention
Technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described enforcement
Example is only a part of embodiment of the present invention rather than whole embodiments.Based on the embodiment in the present invention, this area is common
Technical staff improved or retouching other examples all, broadly fall into the scope of protection of the invention.
The invention provides a kind of fixing microorganism system, including:
Microbe carrier, described microbe carrier is prepared by plant tissue carbonization；
It is attached to the microorganism on described microbe carrier surface.
The fixing microorganism system that the present invention provides includes microbe carrier, and described microbe carrier is by plant tissue carbon
Change prepares.In the present invention, described plant tissue is preferably chalina, Bulbus Allii Fistulosi or Bulbus Allii Cepae.In the present invention, described micro-life
The preparation method of thing carrier is preferably:
Plant tissue is carried out carbonization and prepares microbe carrier.
In the present invention, the temperature of described carbonization is preferably 150 DEG C～250 DEG C, more preferably 160 DEG C～220 DEG C, optimum
Elect 180 DEG C～220 DEG C as.In the present invention, the time of described carbonization is preferably 3 hours～9 hours, more preferably 5 hours～8
Hour, most preferably 6 hours～7 hours.Plant tissue is preferably carried out carbonization by the present invention in a kettle..In the present invention,
Described plant tissue is consistent with plant tissue described in technique scheme, does not repeats them here.
In the present invention, the good biocompatibility of described microbe carrier, without to carrier while fixation of microbe
The pre-treatments such as surface carries out modifying, etching, simple to operate, beneficially fast monitored water body；And microbe carrier is loose
Network structure, has good mass transfer ability after loading microorganisms, fixing microorganism has higher activity, and can bear
Carrying more microorganism, the testing result obtained when aquatic monitoring is the most accurate.
In the present invention, required size is cut into after preferably being cleaned by plant tissue before plant tissue being carried out carbonization.
In the present invention, wash after the carbonized product organics removal that described carbonization preferably will obtain after completing, be dried, obtain microorganism
Carrier.In the present invention, the reagent used by organics removal is preferably alcohol compound, and more preferably carbon number is 1～5
Alcohols chemical combination, most preferably ethanol.In the present invention, the reagent of described washing is preferably water, more preferably secondary water.At this
In bright, described dry method is preferably lyophilizing.
In the present invention, described microorganism is to cultivate the microorganism that BODseed mixed vaccine obtains.In the present invention, described
Microorganism is that multi strain co cultivation obtains, and can be used for the monitoring of variety classes environmental water sample, has universality.BODseed mixes
Representative in environmental monitoring of bacterium, is made up of seven kinds of bacterium, is shown in Table 1 in detail, and table 1 is the group of BODseed mixed vaccine
Becoming, these strains are representative in environment measuring:
The composition of table 1 BODseed mixed vaccine
The invention provides the preparation method of a kind of fixing microorganism system, including:
Microorganism is attached to microbe carrier surface, obtains the microorganism system fixed；Or
In microbe carrier surface in situ cultivating microorganism, obtain the microorganism system fixed；
Described microbe carrier is prepared by plant tissue carbonization.
In the present invention, described microorganism and microbe carrier and microorganism and microbe carrier described in technique scheme
Unanimously, do not repeat them here.
The present invention does not has special restriction, people in the art to the method that microorganism is attached to microbe carrier surface
Microorganism can be transferred to microbe carrier surface according to practical operation condition by member.
In the present invention, the temperature of described Culture in situ microorganism is preferably 20 DEG C～55 DEG C, more preferably 25 DEG C～50
DEG C, most preferably 30 DEG C～40 DEG C.In the present invention, the time of described Culture in situ microorganism preferably≤200 hours, more preferably
It is 1 hour～150 hours, more preferably 10 hours～100 hours, most preferably 30 hours～60 hours.In the present invention, institute
The method stating microorganism culturing in situ is preferably:
Microbe carrier is put into microbial culture medium carries out concussion cultivation.
In the present invention, described microbial culture medium includes thalline and culture fluid.In the present invention, described thalline is preferably
BODseed mixed vaccine.In the present invention, described culture fluid is preferably Luria-Bertani (LB) culture fluid (peptone 10g, ferment
Female cream 5g, distilled water 1000mL, pH 7.0), beef-protein medium (cultivate antibacterial with) (Carnis Bovis seu Bubali cream 3g, peptone
10g, NaCl 5g, water 1000mL, pH7.0-7.2) or YEPD culture fluid (Carnis Bovis seu Bubali cream 3g, yeast extract 3g, tryptone
3g, glucose 10g, water 1000mL, pH5.5).If desired solid medium carries out microorganism culturing in the above-mentioned culture fluid mistake of preparation
Journey is added the agar of 1.5%～2.0%.Culture fluid and culture medium are in preparation sterilizing on the same day, and sterilising conditions is 121 DEG C,
20min。
The invention provides the detection method of a kind of water body toxicity, use fixing microorganism system to carry out water body toxicity inspection
Survey；Described fixing microorganism system is the fixing microorganism system described in technique scheme.
In the present invention, described water body toxicity detection method particularly as follows:
Background solution, nutritional solution and toxicity solution are circulated successively and is passed through fixing microorganism system, described background solution
The time that is passed through more than time that is passed through of nutritional solution, the time that is passed through of described nutritional solution and toxicity solution to be passed through the time identical；
Electrochemical method testing background solution, nutritional solution and toxicity solution is used to each lead into fixing microorganism system
After, the electrochemical signals that fixing microorganism system produces, it is designated as background solution electrochemical signals, nutritional solution electrochemistry letter respectively
Number and toxicity solution electrochemistry signal；
The toxicity of toxicity solution is obtained according to the difference between nutritional solution electrochemical signals and toxicity solution electrochemistry signal；
Described background solution is the activity of microorganism in stable and that recovery is fixing microorganism system；
Described nutritional solution provides nutrient substance for the microorganism in fixing microorganism system；
Described toxicity solution is the nutritional solution containing toxicant.
Water body toxic material can be made the principle of quickly response based on microorganism by the present invention, is examined by electrochemical method
Survey signal and set up the quantization method of signal, thus monitoring aqueous bio toxicant and pollute.The water body toxicity that the present invention provides
Detection method can overcome the defects such as tradition toxicity assay is time-consuming, expensive, range of application is narrow, it is adaptable to the most online water
Body Toxicity Monitoring.
Background solution, nutritional solution and toxicity solution are circulated and are passed through fixing microorganism system by the present invention successively.At this
In bright, described background solution is the activity of microorganism in stable and that recovery is fixing microorganism system, and preferably PBS buffers
Liquid.In the present invention, the pH value of described PBS is preferably 6.5～7.5, more preferably 7.In the present invention, described nutrition
Liquid provides nutrient substance, preferably glucose-glutamic acid (GGA) solution for the microorganism in fixing microorganism system.At this
In invention, the concentration of described GGA solution is preferably 15mg/L～25mg/L, more preferably 20mg/L.In the present invention, described poison
Property solution is the nutritional solution containing toxicant.In the present invention, described toxicant is preferably toxic heavy metal ion, as
Cr6+。
In the present invention, the time that is passed through of described background solution is more than the time that is passed through of nutritional solution, leading to of described nutritional solution
The angle of incidence and toxicity solution to be passed through the time identical.In the present invention, the time that is passed through of described background solution be preferably 1 minute～
200 minutes, more preferably 10 minutes～150 minutes, most preferably 400 minutes～100 minutes.In the present invention, described nutrition
The time that is passed through of liquid and toxicity solution is preferably 1 minute～30 minutes, more preferably 5 minutes～20 minutes, most preferably 10 points
Clock～15 minutes.
In the present invention, described background solution, nutritional solution and toxicity solution circulate successively and are passed through fixing microorganism system,
After i.e. background solution is passed through the fixing microorganism system background solution time, nutritional solution is passed through fixing microorganism system nutritional solution
Time, then toxicity solution is passed through the time that fixing microorganism system is identical with nutritional solution again, and then background solution is led to again
Enter the fixing microorganism system background solution time, the like, in such a manner by background solution, nutritional solution and toxicity
Solution circulation is passed through fixing microorganism system.The present invention can use timing controller and constant flow pump composition sampling system, uses
Fixing microorganism system is entered in conveying background solution, nutritional solution and toxicity solution.In the present invention, described background is molten
The sample introduction speed of liquid, nutritional solution and toxicity solution is preferably 1mL/min～4mL/min, more preferably 2mL/min～3mL/min.
The present invention can use microorganism reactor and constant incubator anabolic reaction device, in the constant incubator of 37 DEG C, uses micro-
Bioreactor puts fixing microorganism system.
In the present invention, method testing background solution, nutritional solution and the toxicity solution of electrochemistry is used to each lead into fixing
Microorganism system after, the electrochemical signals that fixing microorganism system produces, be designated as background solution electrochemical signals, battalion respectively
Nutrient solution electrochemical signals and toxicity solution electrochemistry signal.In the present invention, electrode and electrochemical workstation can be used as number
The electrochemical signals produced according to the microorganism system that acquisition system record is fixing, fixing to be passed through after different liquids with electrode
Microorganism system is tested, and measures current signal value by the electrochemical analyser of electrochemical workstation.
The present invention obtains toxicity solution according to the difference between nutritional solution electrochemical signals and toxicity solution electrochemistry signal
Toxicity.The present invention is by detecting content or the character of the change-detection noxious substance of active microorganism metabolic function.At this
In bright, noxious substance can suppress the activity of microorganism in fixing microorganism system, and the present invention is preferably with solution of different nature
The electric current produced that reacts after being passed through fixing microorganism system weighs the respiration capability of microorganism, in reaction by microorganism also
Former amount can be measured by the chronoamperometry in electrochemical method, and nutritional solution is passed through fixing microorganism system and produces
Electric current and toxicity solution be passed through the difference of the electric current that fixing microorganism system produces and be noxious substance to microbial respiratory
The impact of effect.
In the present invention, by Formulas I, current value can be converted into the toxicant suppression hundred to Microorganism respiration
Point rate, i.e. represents toxicity:
In Formulas I, I represents toxicant suppression ratio respiratory to challenge organisms, is expressed as a percentage, represents toxicity
Size；icontrolRepresent the current value obtained after nutritional solution is passed through fixing microorganism system, isampleRepresent toxicity solution to lead to
The current value obtained after entering fixing microorganism system.The application is with " the suppression respiratory degree of organism " for " toxicity "
Biological indicator, when being poisoned according to microorganism, the electron transport rate of its respiratory chain is directly affected, by calculating suppression ratio
The toxicity characterizing different toxicant is strong and weak, establishes the biological method that fixation of microbe is subject detection toxicity.
Without carrying out the training of microorganism before the water body toxicity detection method detection water body that the present invention provides, and
The physiologically active of microorganism can be kept during detection, the detection method of the water body toxicity that the present invention provides can accurately with
Being repeatedly used for a long, the method has advantage simple to operate, easy to use, with low cost.
Chemical reagent used by following example of the present invention unless otherwise mentioned, all uses during analysis and meets national standard
Analytical pure chemical reagent, is to be the ultra-pure water that Millipore Mlli-Q was purified with water.
The preparation of phosphate buffer:
Phosphate buffered solution (PBS, 0.12mol/L Na2HPO4/0.08mol/L KH2PO4/ 0.1mol/L KCl, pH
7.0): by 10.8872g potassium dihydrogen phosphate (KH2PO4), 42.976g disodium hydrogen phosphate (Na2HPO4·12H2O), dilute with secondary water
Release to 5L.This solution ph 7.0.
GGA standard solution is prepared according to APHA method:
Weigh glucose (C6H12O6) and glutamic acid (C5H9NO4) each 150mg, use secondary water dissolution, at 100mL volumetric flask
In be diluted to the graticule (BOD of this solution5Value is about 2000mg O L-1, it is designated as GGA2000 for distinguishing).It is stored in refrigerator, root
Obtain by GGA2000 dilution according to the BOD standard solution of other concentration of requirement of experiment.
Toxic metal ions Cr6+Solution: by potassium dichromate (K2Cr2O7, M=294.18) prepare, take 0.735g K2Cr2O7
Being dissolved in bis-water of 5mL, concentration is 0.5mol L-1.It is calculated 4mg L-1Cr6+, take 4 μ L 500mmol L-1Cr6+, it is dissolved in
50mL PBS solution, calculates the Cr of differently configured concentration the most again6+Solution.
Toxic metal ions Cu2+Solution: by Cu (NO3)2Solution preparation obtains the Cu of 1g/L2+, calculate the most again and join
Put the Cu of variable concentrations2+Solution.
BODseed mixed vaccine is the BODseed mixed vaccine of Cole Parmer.
Fluorescein isothiocyanate (Fluorescein isothiocyanate, FITC) solution: be formulated as 1mg with secondary water
mL-1。
Constant-temperature shaking incubator: ZHWY-2102C type, Shanghai ZHICHENG Anaiytical Instrument Manufacturing Co., Ltd., Shanghai, China.
Vacuum drying oven: DZF-6020 type, upper Nereid grand experimental facilities company limited, Shanghai, China.
Constant incubator: Stettlen DH2500A type, Tianjin, China.
Freezer dryer: Christ laboratory freezer dryer ALPHA 1-2LD plus, Germany.
Dissolved oxygen sensing uses Song Baotong oxygen pump (SB-648) of business.
Constant flow pump is Baoding Lange peristaltic pump BT100-1J (Baoding, China).
Electrochemical measurement use Shanghai occasion China produce CHI 830B electrochemical analyser (CHI Co., Shanghai,
China), potentiostatic mode detection is used.Electrode is three-electrode system, and including metal working electrode, electrode and Ag/AgCl are joined by platinum
Ratio electrode, electrolyte is the KCl of 0.1mol/L.
Scanning electron Electronic Speculum (scanning electron microscope, SEM): XL30ESEM scanning electron microscopy
Mirror.
Laser-induction fluorescence co-focusing microscope (confocal laser scanning microscope, CLSM): Lay
Card TCS SP2 (Lycra microscopic system Heidelberg GmbH, Mannheim, Germany) subsidiary argon ion laser (457/488/
514nm)。
Embodiment 1
Chalina is cleaned, cuts into and need size.Load reactor, carry out 6h carbonization under the conditions of 180 DEG C, by obtain
Carbonized product is put in ethanol, removes organic residue；Then after rinsing with secondary water, under the conditions of 0.08mpar, lyophilizing is standby,
Obtain microbe carrier.
The microbe carrier preparing embodiment 1 carries out SEM sign, and testing result is as it is shown in figure 1, Fig. 1 is this
The SEM figure of the microbe carrier that bright embodiment 1 prepares.
Embodiment 2
Prepare microbe carrier according to the method described in embodiment 1, use Bulbus Allii Fistulosi to replace Fructus Luffae as different from Example 1
Flesh.
The microbe carrier preparing embodiment 2 carries out SEM sign, and testing result is as in figure 2 it is shown, Fig. 2 is this
The SEM figure of the microbe carrier that bright embodiment 2 prepares.
Embodiment 3
Prepare microbe carrier according to the method described in embodiment 1, use Bulbus Allii Cepae to replace Fructus Luffae as different from Example 1
Flesh.
The microbe carrier preparing the embodiment of the present invention 3 carries out SEM sign, and testing result is as it is shown on figure 3, Fig. 3
SEM figure for the microbe carrier that the embodiment of the present invention 3 prepares.
From Fig. 1～Fig. 3 it can be seen that microbe carrier prepared by the embodiment of the present invention remains the physics of protozoa tissue
Structure, has good hole mass transfer characteristic good；Meanwhile, loose structure makes microbe carrier have the highest specific surface area, favorably
In a large amount of loading microorganisms.
Embodiment 4
The microbe carrier embodiment of the present invention 1 prepared puts in microbial culture medium, in 37 DEG C of isothermal vibrations
Cultivate 48 hours, note reducing concussion frequency, in order to avoid the microorganism being attached to microbe carrier surface is come off by concussion, consolidate
Fixed microorganism system；
Described microbial culture medium includes BODseed mixed vaccine and LB culture fluid (peptone 10g, yeast extract 5g, distilled water
1000mL, pH 7.0).
Fixing microorganism system PBS embodiment 4 prepared washes except unnecessary microbe carrier, with different
Hydrogen thiocyanate luciferin solution carries out 30min dyeing, removes unattached dyestuff with PBS cleaning down, is 488nm in excitation wavelength
Under carry out CLSM detection.
As shown in Figure 4, Fig. 4 is the CLSM's of the microbe carrier that the embodiment of the present invention 4 prepares to CLSM testing result
Detection figure.
Embodiment 5
Prepare fixing microorganism system according to the method described in embodiment 4, use embodiment as different from Example 4
The microbe carrier that 2 prepare replaces the microbe carrier that embodiment 1 prepares.
The fixing microorganism system prepared embodiment 5 according to the method described in embodiment 4 carries out CLSM sign,
Testing result is as it is shown in figure 5, CLSM detection figure that Fig. 5 is the microbe carrier that the embodiment of the present invention 5 prepares.
Embodiment 6
Prepare fixing microorganism system according to the method described in embodiment 4, use embodiment as different from Example 4
The microbe carrier that 3 prepare replaces the microbe carrier that embodiment 1 prepares.
The fixing microorganism system prepared embodiment 6 according to the method described in embodiment 4 carries out CLSM sign,
As shown in Figure 6, Fig. 6 is the CLSM detection figure of the microbe carrier that the embodiment of the present invention 6 prepares to testing result.
From Fig. 4～Fig. 6, microorganism is uniform in microbe carrier surface attachment, and load capacity is big.It is micro-that the present invention provides
Bio-carrier may be used for Microorganism incubation.And for the microbe carrier that raw material prepares, there is more preferable micro-life with Bulbus Allii Fistulosi
Thing fixed performance.
The microbe carrier that the present invention provides shows good bio-compatible in terms of keeping the long period of activity of microorganism
Property, by SEM figure and the sign of CLAM figure, the spacial framework feature of microbe carrier makes the microorganism system fixed be formed
One three-dimensional " microbial cell set ", can good mass transfer, loading microorganisms；And this microbe carrier has
The strongest biodegradability, insensitive to pH, there is stronger adaptation ability.
Embodiment 7
The system for water body toxicity detection that the embodiment of the present invention provides, including:
One timing controller (TPC8-8TD model, Beijing, China) and constant flow pump (Baoding Lange peristaltic pump
BT100-1J, Baoding, China) sampling system that forms；One microorganism reactor and constant incubator (Stettlen DH2500A
Type, Tianjin, China) reactor that forms；And electrode and electrochemical workstation (Shanghai occasion China CHI 830B, Shanghai, China)
The data collecting system of composition.
Timing controller is the TPC8-8 timing routine controller that multidimensional fine control computer tech development center, Beijing is developed
(Beijing, China).Important technological parameters is as follows: power supply is direct current 24V/2A switching power supply, and controller terminal EG is
Ground wire, terminal 24V is positive pole；The way of output is the output of NPN transistor open collector, and output is effective: turn on over the ground, output
Invalid: open circuit；Output voltage is for connecting under loading condition, when exporting effective: close to 0V；When exporting invalid: 24V；The output of every road
Being less than 7W for electric current less than 300mA, power, resistance is more than 80 Ω；Input voltage is the on-off model in the range of 0-24V；Defeated
Entering signal is level signal；Sensor is power supply 15～24V, the NPN type Boolean value output sensor of Low level effective.With USB interface
Connect computer to drive.
Peristaltic pump supports the use the silica gel tube into diameter 2.5mm.At 11.5rpm (～3mL min-1) under flow velocity, by set
The program sample introduction reserved, PBS, as background solution, maintains the stability of microorganism.In 37 DEG C of calorstats, embodiment 5 is made
After the standby fixing microflora obtained loads microorganism reactor, with electrode, the reaction of toxicant is tested, logical
Cross electrochemical analyser and measure current signal value.
Selecting gold electrode as working electrode, Ag/AgCl is as reference electrode, and platinum filament is to electrode, and this electrode makes institute
The instrument set up has higher sensitivity.Use chrono-amperometric method to measure electrochemical signals, voltage setting-700mV, use
Instrument is CHI830B type electrochemical workstation.
Embodiment 8
The water body toxicity detecting system using embodiment 7 to provide carries out water body toxicity detection:
PBS buffer solution is background solution；
Take GGA standard solution 10mL with liquid-transfering gun, be diluted to 1000mL with PBS, take 500mL and be placed in conical flask, as
Nutritional solution；
Take Cr6+20 μ l are (by calculating 20 μ l Cr6+It is i.e. 20mg L-1Cr6+) and the mixing of above-mentioned other 500mL nutritional solution,
As toxicity solution；
Need logical oxygen instrument blowing air oxygen 20 minutes saturated to oxygen before sample introduction.
The system detected for water body toxicity is carried out program setting, and the flow velocity of pump is 3mL min-1, background solution sample introduction
40 minutes, nutritional solution sample introduction 10 minutes, toxicity solution sample introduction 10 minutes, background solution sample introduction 40 minutes again the like circulation
Sample introduction, each cycle period is 100 minutes.
As shown in Figure 7 and Figure 8, Fig. 7 is that the embodiment of the present invention 8 circulation measures water body to the testing result that test obtains after terminating
The signal of telecommunication record figure of toxicity, Fig. 8 is the amplification signal graph that the embodiment of the present invention 8 detects water body toxicity one the measurement cycle.From
Fig. 7 and Fig. 8, it can be seen that the signal value that PBS buffer solution produces can reach about 530nA, is passed through GGA standard solution, micro-
The biological Repiration that carried out in nutritional solution, oxygen content in water minimizing, signal decreases, for about 370nA.And in micro-life
When the signal of thing reaches stable, add 20mg L-1Cr6+With 20mg L-1The mixed solution (toxicity solution) of GGA, signal value reduces
Amplitude substantially diminish, for about 420nA.Heavy metal ion Cr6+The Repiration of microorganism is had suppression, and respiratory intensity becomes
Weak, the oxygen of consumption reduces, and current value reduces amplitude and diminishes, and the method that the present invention provides can measure the poisonous huge sum of money in water body
Belong to ion.
Embodiment 9
According to the water body toxicity detection method of embodiment 8 to Cr6+Toxicity solution is carried out continuously the toxicity detection in 6 cycles,
Calculating suppression ratio according to the signal of telecommunication figure that record obtains according to the formula of Formulas I, result is as it is shown in figure 9, Fig. 9 is for measuring Cr6+Continuous 6
The suppression ratio in individual cycle, as shown in Figure 9, the repeated experiment of many groups, the datagram obtained is basically identical, the water that the present invention provides
Body detection method of toxicity has preferable repeatability.
Embodiment 10
Take GGA2000 solution 5mL PBS and be diluted to 500mL, take 300mL as nutritional solution (GGA20 solution)；
Remaining GGA solution is divided into four parts, every part of 50mL, adds 1 μ L, 5 μ L, 10 μ L, 20 μ L respectively in every part
1mg L-1Cr6+, obtain Cr6+Concentration is 1mg L-1、5mg L-1、10mg L-1、20mg L-1Toxicity solution.
Detecting toxicity solution according to the method described in embodiment 8, the toxicity that each cycle changes variable concentrations is molten
Liquid.
Calculate suppression ratio according to the signal of telecommunication figure that obtains of record according to the formula of Formulas I, testing result as shown in Figure 10, Figure 10
For Cr6+Suppression ratio and concentration map, it can be seen from fig. 10 that add Cr during the detection in four cycles6+Current signal value drops
Low less, along with Cr6+The increase of concentration, current value reduction tapers into, and inhibitory action is obvious, can be seen that in suppression ratio figure
Suppression ratio and concentration are some linear, and the water body toxicity detection method that the present invention provides can detect a huge sum of money for variable concentrations
Belong to ion.
Embodiment 11
According to the water body toxicity detection method of embodiment 10 Cr to variable concentrations6+Toxicity solution is carried out continuously 1 month
Toxicity detection, calculates suppression ratio according to the signal of telecommunication figure that obtains of record according to the formula of Formulas I, result as shown in figure 11, Tu11Wei
Measure variable concentrations Cr6+The suppression ratio of continuous 1 month, as shown in Figure 11, the signal value obtained during detection is basicly stable, no
Different with control of the concentration effect, increase with the increase of concentration in certain limit.The water body toxicity detection method that the present invention provides
Metal ion in water body can be measured for a long time, there is certain stability and the suitability.
Embodiment 12
Detect water body toxicity according to the method for embodiment 9, use the Cu of 5mg/L2+Toxicity solution is carried out continuously 5 cycles
Toxicity detection.
Calculating suppression ratio according to the signal of telecommunication figure that record obtains according to the formula of Formulas I, as shown in figure 12, Figure 12 is for surveying for result
Amount Cu2+The suppression ratio in continuous 5 cycles, as shown in Figure 12, the repeated experiment of many groups, the datagram obtained is basically identical, this
The water body toxicity detection method that invention provides has preferable repeatability.
Embodiment 13
Detect water body toxicity according to the method described in embodiment 10, use Cu2+Concentration is 1mg L-1、2mg L-1、5mg L-1、10mg L-1Toxicity solution.
Calculate suppression ratio according to the signal of telecommunication figure that obtains of record according to the formula of Formulas I, testing result as shown in figure 13, Figure 13
For Cu2+Suppression ratio and concentration map, it can be observed from fig. 13 that along with Cu during the detection in four cycles2+Concentration increases, and presses down
Making of gradually strengthening, but suppression ratio is relatively low, suppression ratio and concentration are good linear relationship.The water body toxicity that the present invention provides
Detection method can detect the heavy metal ion of variable concentrations.
Embodiment 14
According to the water body toxicity detection method of embodiment 13 Cu to variable concentrations2+Toxicity solution is carried out continuously 1 month
Toxicity detection.
Calculating suppression ratio according to the signal of telecommunication figure that record obtains according to the formula of Formulas I, as shown in figure 14, Figure 14 is for surveying for result
Amount variable concentrations Cu2+The suppression ratio of continuous 1 month, as shown in Figure 14, the signal value obtained during detection is basicly stable, different
Control of the concentration effect is different, increases with the increase of concentration in certain limit.The water body toxicity detection method energy that the present invention provides
Enough long-term metal ions measured in water body, have certain stability and the suitability.
The present invention is by Cr6+With Cu2+Carry out repeatability experiment, Concentraton gradient experiment and long-time stability experiment, can
To find out, adding poisonous ion, the respiratory activity of microorganism is suppressed, and when adding PBS, activity can be recovered again, and the present invention provides
The good stability of fixing microorganism system, the poisonous of detectable substance and nothing can be characterized the most intuitively by the size of current value
Poison, the detection method of the water body toxicity that the present invention provides can be used to the heavy metal ion detecting in water body.
Embodiment 15
Preparation Bi3+Concentration is the aqueous solution of 50mg/L；
Take GGA2000 solution 5mL PBS and be diluted to 500mL, take 300mL as nutritional solution (GGA20 solution)；
Remaining GGA solution is divided into four parts, every part of 50mL, adds 5 μ L, 10 μ L, 20 μ L, 30 μ L respectively in every part
50mg L-1Bi3+, obtain Bi3+Concentration is 5mg L-1、10mg L-1、20mg L-1、30mg L-1Toxicity solution.
According to the method described in embodiment 10, above-mentioned toxicity solution is detected.
Calculate suppression ratio according to the signal of telecommunication figure that obtains of record according to the formula of Formulas I, testing result as shown in figure 15, Figure 15
For Bi3+、Cd2+、Zn2+、Pb2+Concentration and suppression ratio figure.
Embodiment 16
Preparation Cd2+Concentration is the aqueous solution of 50mg/L；
Take GGA2000 solution 5mL PBS and be diluted to 500mL, take 300mL as nutritional solution (GGA20 solution)；
Remaining GGA solution is divided into four parts, every part of 50mL, adds 5 μ L, 10 μ L, 20 μ L, 30 μ L respectively in every part
50mg L-1Cd2+, obtain Cd2+Concentration is 5mg L-1、10mg L-1、20mg L-1、30mg L-1Toxicity solution.
According to the method described in embodiment 10, above-mentioned toxicity solution is detected.
Calculating suppression ratio according to the signal of telecommunication figure that record obtains according to the formula of Formulas I, testing result is as shown in figure 15.
Embodiment 17
Preparation Zn2+Concentration is the aqueous solution of 50mg/L；
Take GGA2000 solution 5mL PBS and be diluted to 500mL, take 300mL as nutritional solution (GGA20 solution)；
Remaining GGA solution is divided into four parts, every part of 50mL, adds 5 μ L, 10 μ L, 20 μ L, 30 μ L respectively in every part
50mg L-1Zn2+, obtain Zn2+Concentration is 5mg L-1、10mg L-1、20mg L-1、30mg L-1Toxicity solution.
According to the method described in embodiment 10, above-mentioned toxicity solution is detected.
Calculating suppression ratio according to the signal of telecommunication figure that record obtains according to the formula of Formulas I, testing result is as shown in figure 15.
Embodiment 18
Preparation Pb2+Concentration is the aqueous solution of 50mg/L；
Take GGA2000 solution 5mL PBS and be diluted to 500mL, take 300mL as nutritional solution (GGA20 solution)；
Remaining GGA solution is divided into four parts, every part of 50mL, adds 5 μ L, 10 μ L, 20 μ L, 30 μ L respectively in every part
50mg L-1Pb2+, obtain Pb2+Concentration is 5mg L-1、10mg L-1、20mg L-1、30mg L-1Toxicity solution.
According to the method described in embodiment 10, above-mentioned toxicity solution is detected.
Calculating suppression ratio according to the signal of telecommunication figure that record obtains according to the formula of Formulas I, testing result is as shown in figure 15.
By Figure 15 Bi3+、Cd2+、Zn2+、Pb2+Relation curve between concentration and the suppression ratio of four metal ion species, can obtain
Strong and weak to the toxicity of each ion corresponding, their size order is Cd2+(20mg L-1)>Pb2+(20mg L-1)>Bi3+(20mg
L-1)>Zn2+(20mg L-1).The metal ion that the result that present invention detection obtains and prior art are reported is in toxicity trend
Consistent.The toxicity detection of the water body toxicity detection method heavy metal ion that the present invention provides has universality.
Embodiment 19
Preparation 3,5-chlorophenesic acid (DCP) concentration is respectively 5mgL-1、10mgL-1、15mgL-1、20mgL-1Toxicity solution
(0.1g DCP is dissolved in 100mL water, obtains the DCP mother solution of 1000mg/L；Then with water mother solution is diluted to required molten
Degree)；
According to the method described in embodiment 10, above-mentioned toxicity solution is detected.
Calculate suppression ratio according to the signal of telecommunication figure that obtains of record according to the formula of Formulas I, testing result as shown in figure 16, Figure 16
Concentration and suppression ratio figure for DCP.As shown in Figure 16, DCP does not the most suppress the Repiration of microorganism, promotes micro-life on the contrary
The Repiration of thing, this is owing to DCP itself is the least to the toxicity of microorganism, simultaneously as Organic substance, is added into fixing
In microflora, add the substrate of bacteria metabolism effect, thus add the consumption of oxygen in solution, occur in that
The result of negative suppression.The water body toxicity detection method that the present invention provides also can carry out toxicity detection to organic toxic pollutant.
As seen from the above embodiment, the invention provides a kind of fixing microorganism system, including: microbe carrier, institute
State microbe carrier to be prepared by plant tissue carbonization；It is attached to the microorganism on described microbe carrier surface.The present invention adopts
Preparing fixing microorganism system by plant carbide tissue one-step method, preparation method is simple；And the present invention provide consolidate
Fixed microorganism system uses attachment method by Microorganism incubation, makes microorganism natural sediment in the surface of carrier material hole, nothing
Crosslinking curing process need to be carried out；It addition, the microbe carrier that the present invention uses is three-dimensional space net structure, there is preferably biography
Matter performance.Therefore, during the fixing microorganism system detection water body toxicity that the present invention provides, activity is preferably, and can repeat should
Detecting for water body toxicity, Detection results is preferable.
Above-described is only the preferred embodiment of the present invention, it is noted that for the ordinary skill of the art
For personnel, under the premise without departing from the principles of the invention, it is also possible to make some improvements and modifications, these improvements and modifications are also
Should be regarded as protection scope of the present invention.
Claims (10)
1. a fixing microorganism system, including:
Microbe carrier, described microbe carrier is prepared by plant tissue carbonization；
It is attached to the microorganism on described microbe carrier surface.
Fixing microorganism system the most according to claim 1, it is characterised in that described plant tissue include chalina,
Bulbus Allii Fistulosi or Bulbus Allii Cepae.
Fixing microorganism system the most according to claim 1, it is characterised in that described microorganism is BODseed mixing
The microorganism that bacterium obtains after cultivating.
4. a preparation method for fixing microorganism system, including:
Microorganism is attached to microbe carrier surface, obtains the microorganism system fixed；Or
In microbe carrier surface in situ cultivating microorganism, obtain the microorganism system fixed；
Described microbe carrier is prepared by plant tissue carbonization.
Method the most according to claim 4, it is characterised in that the temperature of described carbonization is 150 DEG C～250 DEG C；
The time of described carbonization is 3 hours～9 hours.
Method the most according to claim 4, it is characterised in that the temperature of described Culture in situ microorganism is 20 DEG C～55
℃；
Time≤200 hour of described Culture in situ microorganism.
7. the detection method of a water body toxicity, it is characterised in that use fixing microorganism system to carry out water body toxicity detection；
Described fixing microorganism system is the fixing microorganism system in claims 1 to 3 described in any one.
Method the most according to claim 7, it is characterised in that the detection method of described water body toxicity particularly as follows:
Background solution, nutritional solution and toxicity solution are circulated successively and is passed through fixing microorganism system, leading to of described background solution
The angle of incidence more than time that is passed through of nutritional solution, the time that is passed through of described nutritional solution and toxicity solution to be passed through the time identical；
After using electrochemical method testing background solution, nutritional solution and toxicity solution to each lead into fixing microorganism system, Gu
The electrochemical signals that fixed microorganism system produces, be designated as respectively background solution electrochemical signals, nutritional solution electrochemical signals and
Toxicity solution electrochemistry signal；
The toxicity of toxicity solution is obtained according to the difference between nutritional solution electrochemical signals and toxicity solution electrochemistry signal；
Described background solution is the activity of microorganism in stable and that recovery is fixing microorganism system；
Described nutritional solution provides nutrient substance for the microorganism in fixing microorganism system；
Described toxicity solution is the nutritional solution containing toxicant.
Method the most according to claim 8, it is characterised in that the time that is passed through of described background solution is 1 minute～200 points
Clock；
The time that is passed through of described nutritional solution and toxicity solution is 1 minute～30 minutes.
Method the most according to claim 8, it is characterised in that the toxicant in described toxicity solution is a poisonous huge sum of money
Belong to ion.
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