CN110028577A - A kind of immobilized spherule and the preparation method and application thereof containing metallothionein - Google Patents
A kind of immobilized spherule and the preparation method and application thereof containing metallothionein Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
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Abstract
The invention discloses a kind of immobilized spherules containing metallothionein, the preparation method comprises the following steps: the bacterium of (1) culture expression metallothionein；(2) culture solution or thallus PBS buffer solution are hanged, more than three times, ultrasonication must be crushed bacterium solution to multigelation；(3) broken bacterium solution, silica are added in sodium alginate-polyvinyl alcohol aqueous solution, obtain mixed liquor；(4) mixed liquor is added drop-wise in boric acid-barium chloride solution, 4 DEG C of fixed 4h to get.Immobilized spherule containing metallothionein of the invention, containing a large amount of cell fragments, protein etc. in sphere, heavy metal ion adsorbed site is more, is conducive to contact and combination of the adsorption site with heavy metal ion, with preferable absorption property, it can be used for handling the waste water containing heavy metal；With good mechanical strength, there is good reusing, and not will cause secondary pollution；Technology of the invention has important application value in heavy metal containing wastewater treatment.
Description
Technical field
The present invention relates to a kind of immobilized spherule and the preparation method and application thereof containing metallothionein, belongs to heavy metal
Technical field of waste water processing.
Background technique
Heavy metal wastewater thereby pollution is one of the main reason for causing environmental pollution.Untreated industrial wastewater discharge is to ring
In border, polluted-water and soil are main channel of the food by heavy metal pollution.China's industrialization development is rapid, but ring
But development relatively lags behind for guarantor's technology and consciousness.A large amount of untreated industrial wastewaters are discharged directly into environment, lead to China
Heavy metal pollution problem is serious.It is shown according to relevant survey data, the pollution rate of China's river,lake and reservior has been over 80%.
The content of beary metal of the big river basins in the Yellow River, Song Hua River etc. ten severely exceeds, and reaches V class as defined in China.Urban river has
There is total mercury more than III class water body standard of surface water in 35.11% section, and the total cadmium of 18.46% stream cross section is more than III class water body mark
It is quasi-.In the big water system of the seven of China, ChangJiang River cadmium pollution situation is only second to COD, BOD and mercury pollution；The total cadmium of Yellow River System contains
It measures exceeded section and reaches 16.7%.The heavy metal wastewater thereby pollution condition in China is very serious, the moment threaten ecological environment and
People's health.
Currently, common heavy metal containing wastewater treatment technology has chemical precipitation, ion exchange, electrochemical process, membrane separation process etc.,
It is described as follows:
(1) chemical precipitation method: most popular from the method for removing removing heavy metals in inorganic wastewater is chemical precipitation.Change
Learning the precipitation method is mainly to form the hydroxide precipitating of heavy metal ion.Lime (calcium hydroxide), potassium hydroxide or sodium hydroxide
It is the reagent of most common precipitating weight, because they are easily obtained and cost is relatively low.But chemical precipitation is needed using a large amount of
Chemical substance, to reduce metal to acceptable emission level.In addition, sludge is excessive, need to be further processed, precipitated metal
Process is slow, easily occurs to precipitate bad, long-term influence of the aggregation and sludge handling process of metal precipitates on environment.
(2) ion-exchange: ion-exchange is to remove removing heavy metals in another slave waste water industrially successfully used
Method, principle is the charge-exchange using between exchanger and heavy metal ion, to realize the removal of heavy metal ion.This
The shortcomings that kind method is higher cost, limits application.
(3) electrochemical process: using the principle of electrolysis, electrochemical treatment is that move to the heavy metal ion in waste water negative
Pole is restored, and then is precipitated, and to achieve the purpose that removing heavy metals, and effectively recycles heavy metal.But electrochemical treatments
Method needs power supply cost high, puts into biggish fund, thus, suitable for the weight high with high recovery value, concentration of metal ions
Metallic wastewater processing.
(4) membrane separation process: membrane separation technique is high-efficient, easy to operate, saves space, without secondary pollution, is applied to weight
Metallic pollution processing has broad prospects.In simple terms, membrane separation technique is exactly to utilize inside and outside the film under the pressure of concentration difference
Solvent and solute are separated or are concentrated, and during the separation process, do not change the chemical form of solute by semi-permeable membrane.But UF membrane
The cost of technology is excessively high, needs largely to invest, therefore, it is impossible to be widely used.
(5) immobilization technology: immobilized microorganism technique is a kind of new bio technology developed using immobilization technology,
Grow up on the basis of being the enzyme immobilization technology in the biochemical sixties.Immobilized microorganism technique is logical
Crossing either physically or chemically makes microorganism keep technology that is active and being capable of Reusability in region in a limited space.It is fixed
After changing microorganism, the adsorption capacity to heavy metal ion can be obviously improved.Research and application show immobilized microorganism technique
It is that a kind of microbe density is high, reaction speed is fast, nontoxic, product is easily separated, reaction is easy to control, the skill of process equipment miniaturization
Art, therefore, immobilization technology have a wide range of applications potentiality and development prospect in terms of heavy metal containing wastewater treatment.
River intelligent equal (2016) Cd is adsorbed using sodium alginate-polyvinyl alcohol-gelatin embedding immobilized yeast2+、Pb2+
The mixed solution of ion, discovery is when the mass fraction of sodium alginate is 2%, the mass fraction of polyvinyl alcohol is 2%, the matter of gelatin
When amount score is 0.5%, immobilization sphere has preferable mechanical performance and mass-transfer performance；In 120mg/ml metal ion solution
In, when pH is 5, Cd2+、Pb2+The adsorption rate of ion reaches maximum, ionic adsorption amount respectively reached 65.78mg/g and
79.87mg/g.Lee main forces, Zhou Li et al. (2017) are useless from mine using one plant of the immobilization of sodium alginate-polyvinyl alcohol investment
In slag accumulation area soil screening separation obtain to heavy metal ion Pb2+And Cd2+Function stem with efficient tolerance, will
Immobilization sphere contains Pb for adsorbing2+And Cd2+Effluent containing heavy metal ions, pH be 6 when, Pb2+And Cd2+Adsorbance reach
114.36mg/g and 82.12mg/g are arrived.
Currently, immobilized in immobilized microorganism technique frequently with complete bacterial strain, be unfavorable for intracellular protein with it is molten
The contact and combination of heavy metal ion, cause adsorption efficiency not high in liquid.
Summary of the invention
For the above-mentioned prior art, the present invention provides a kind of immobilized spherules containing metallothionein, and its preparation
Method.The present invention prepares immobilization sphere using the method for embedding broken bacterium solution, keeps metallothionein abundant with heavy metal ion
In conjunction with raising absorption property.
The present invention is achieved by the following technical solutions:
A kind of preparation method of the immobilized spherule containing metallothionein, comprising the following steps:
(1) bacterium of culture expression metallothionein or recombination metallothionein, obtains culture solution；
The expression metallothionein or the bacterium for recombinating metallothionein, can be wild strain, are also possible to pass through base
Because of the engineering bacteria that recombinant technique obtains, such as: by gene recombination technology, the plasmid containing metallothionein gene is transferred to large intestine
Bacillus obtains the recombination bacillus coli that can express metallothionein；It, can be by specific metallothionein by gene recombination technology
It is white that (different metallothioneins is different to the sensibility of different heavy metals, therefore, can be directed to a certain specific heavy metal, needle
To the building engineering bacteria of property) it is transferred to engineering bacteria；
Further, specific condition of culture is as follows: recombination bacillus coli is taken, is added in the LB liquid medium of the benzyl containing ammonia,
37 DEG C of 8~14h of culture；1ml bacterium solution is taken, is added in 100ml LB liquid medium, 37 DEG C of cultures to OD600It is 0.6~1.0,
The IPTG (isopropylthiogalactoside) of final concentration of 0.5mmol/ml is added, Fiber differentiation 4h obtains culture solution, 10000r/
Min, 4 DEG C be centrifuged to obtain thallus；
(2) culture solution or thallus PBS buffer solution are hanged, prepares bacteria suspension；More than three times by bacteria suspension multigelation,
Ultrasonication thallus (uses ultrasonic cell disruptor), obtains broken bacterium solution；
Further, the ratio between thallus weight in wet base and PBS buffer solution volume are 0.04~0.08g/ml；
Further, the multigelation refers in -80 DEG C and 37 DEG C of multigelations；
(3) broken bacterium solution, silica are added in sodium alginate-polyvinyl alcohol aqueous solution, mix, obtains mixed liquor；
Further, in the mixed liquor, the concentration of sodium alginate is 1%~3% (mass volume ratio, unit g/ml),
The concentration of polyvinyl alcohol is 1%~3% (mass volume ratio, unit g/ml), and the concentration of silica is 0.5% (quality volume
Than unit g/ml), cell concentration is 10~20mg/ml；
The sodium alginate-polyvinyl alcohol aqueous solution can be prepared by the following method to obtain: by sodium alginate and polyethylene
Alcohol mixing, be dissolved in distilled water in 90 DEG C or more heating water baths, be cooled to room temperature to get；
Further, the molecular weight of the polyvinyl alcohol is 15~200,000, alcoholysis degree 99.8%~100%, and viscosity 33~
44mpa*s；
Under the conditions of (4) 4 DEG C (ice bath), mixed liquor is added drop-wise in boric acid-barium chloride solution, 4 DEG C of fixed 4h, is taken out,
It washes with water to get the immobilized spherule containing metallothionein, bead partial size can be used in adsorbent solution in 4.5~4.7mm
Heavy metal ion；
Further, in the boric acid-barium chloride solution, the concentration of boric acid is 1% (mass volume ratio, unit g/
Ml), the concentration of barium chloride is 3% (mass volume ratio, unit g/ml).
The immobilized spherule containing metallothionein being prepared using the above method, it is broken containing a large amount of cells in sphere
Piece, protein etc., heavy metal ion adsorbed site is more, is conducive to adsorption site (such as recombination metallothionein) and heavy metal ion
Contact and combination, have preferable absorption property, can be used for handle contain heavy metal Cd2+And Cu2+Waste water.
Immobilized spherule containing metallothionein of the invention, using sodium alginate, polyvinyl alcohol, silicon dioxide embedded
Broken bacterium solution has good mechanical strength, and shape specification (size shape is similar, and forming situation is stablized), is easy to solid-liquid
Separation has good absorption property and reusing, and not will cause secondary pollution and (embed broken thallus, prevent it
Secondary pollution is caused to environment), without extracting albumen, (directly broken thallus, eliminates the step of mentioning power albumen, reduces into
This)；Technology of the invention has important application value in heavy metal containing wastewater treatment.
All documents recited in the present invention, their full content are incorporated herein by reference, and if these are literary
When offering expressed meaning and the inconsistent present invention, it is subject to statement of the invention.In addition, the various terms that use of the present invention and
Phrase is with well known to a person skilled in the art general senses.The term and phrase referred to if any inconsistent with common art-recognized meanings,
The meaning that the present invention of being subject to is stated.
Detailed description of the invention
Fig. 1: the expression (electrophoretogram) of metallothionein is recombinated.
Fig. 2: BLB, WM1, BM1 bead are to Cd2+Absorption result figure.
Fig. 3: BLB, WM2, BM2 bead are to Cu2+Absorption result figure.
Fig. 4: solution ph is to absorption Cd2+Influence diagram.
Fig. 5: solution ph is to Adsorption of Cu2+Influence diagram.
Fig. 6: adsorption time is to absorption Cd2+Influence diagram.
Fig. 7: adsorption time is to Adsorption of Cu2+Influence diagram.
Fig. 8: Cd2+Initial concentration is to absorption Cd2+Influence diagram.
Fig. 9: Cu2+Initial concentration is to Adsorption of Cu2+Influence diagram.
Figure 10: cadmium ion desorption and sorption cycle test result schematic diagram again.
Figure 11: copper ion parsing and sorption cycle test result schematic diagram again.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated.However, the scope of the present invention is not limited to following realities
Apply example.One of skill in the art, can be to the present invention it is understood that under the premise of without departing substantially from the spirit and scope of the present invention
Carry out various change and modification.
The present invention carries out general and/or specific description to the material and test method arrived used in test.Though
So many materials and operating method used in purpose are it is known in the art that still the present invention is still herein to realize the present invention
It is described in detail as far as possible.
Instrument involved in following embodiments, reagent, material etc. are unless otherwise noted existing in the prior art
Conventional instrument, reagent, material etc., can be obtained by regular commercial sources.Experimental method involved in following embodiments, inspection
Survey method etc. is unless otherwise noted existing routine experiment method in the prior art, detection method etc..
Test the preparation and application of the immobilized spherule containing metallothionein
1. key instrument and reagent
(1) reagent: sodium alginate is that commercially available chemistry is pure, and polyvinyl alcohol, silica, boric acid, barium chloride are that analysis is pure.
(2) instrument: pH meter (ORION STAR A211, Thermo Scientific), ultrasonic cell disruptor
(II D of SCIENTZ-, NingBo XinZhi Biology Science Co., Ltd), inductively coupled plasma body Atomic Absorption Spectrometer
(iCAP 7000 SERIES, Thermo Scientific), -80 DEG C of refrigerators (Ultra-Low Temperature Freezer,
New Brunswick), shaking table (Orbital Shaker TS-100, its woods Bell's instrument manufacturing Co., Ltd of Jiangsu Haimen).
2. recombinating the expression of tetrahymena thermophila's metallothionein
The conversion of 2.1 recombination metallothionein plasmids
(1) by recombinant plasmid, (the present embodiment has carried out two groups of parallel tests and one group of check experiment, the difference being transferred to
It is: recombination GST tagged-plasmid, recombinant plasmid pGEX-MTT1, recombinant plasmid pGEX-MTT2) (recombinant plasmid pGEX-MTT1,
PGEX-MTT2 is that building obtains by conventional method, and MTT1, MTT2 are tetrahymena thermophila's metallothionein gene) it is transferred to
E. coli bl21 is applied to LB solid plate, 37 DEG C of culture 12h in constant incubator.
(2) 5ml LB liquid medium is taken, ammonia benzyl is added.Single colonie is picked them separately, the LB Liquid Culture of the benzyl containing ammonia is added
In base, 37 DEG C of shaken cultivations are to OD in constant-temperature shaking incubator600It is 1.0, is added, Fiber differentiation 4h；
(3) 10000r/min, 4 DEG C of centrifugations are received bacterium, are then resuspended with PBS buffer solution, ultrasonication.
(4) 12%SDS-PAGE electrophoresis runs glue detection；Testing result as shown in Figure 1, the full bacterium in Fig. 1 refer to it is broken
Bacteria suspension；Supernatant refers to the supernatant for obtaining broken bacteria suspension centrifugation (10000r/min, 4 DEG C of centrifugations)；Precipitating refers to
After removing supernatant, the liquid hanged will be precipitated with 1ml PBS buffer solution.
As seen from Figure 1, recombination metallothionein MTT1 and MTT2 (weight is expressed to this success of the test in Escherichia coli body
The size of group metallothionein MTT1 is about 44KD, and the size of recombination metallothionein MTT2 is about 38KD).
The Fiber differentiation of 2.2 recombination bacillus colis
Recombination bacillus coli is taken, 5ml LB liquid medium (ammonia benzyl is added) is added, 37 DEG C are incubated overnight.1ml bacterium solution is taken,
It is added in 100ml LB liquid medium, 37 DEG C of cultures to OD600It is 0.6~1.0, IPTG Fiber differentiation is added.
3. the preparation of immobilization sphere
The preparation of 3.1 blank spheres
(1) by 0.4g sodium alginate and 0.4g polyvinyl alcohol, 90 DEG C or more heating water baths are dissolved in 15ml distilled water
In.After room temperature is cooling, 0.1g silica, 5mlPBS buffer is added, stirs evenly, mixed liquor (20ml) is made, wherein sea
The concentration of mosanom is 2%, and the concentration of polyvinyl alcohol is 2%, and the concentration of silica is 0.5%.
Polyvinyl alcohol used in the present embodiment, molecular weight is 15~200,000, alcoholysis degree 99.8%~100%, and viscosity 33~
44mpa*s；
Under the conditions of (2) 4 DEG C (ice bath), mixed liquor is added drop-wise to 50ml boric acid-barium chloride solution (boric acid-barium chloride water
In solution, the concentration of boric acid is 1%, the concentration of barium chloride be 3%) in, 4 DEG C of fixed 4h, taking-up (filtering) washes with water, i.e.,
Immobilization sphere is obtained, bead partial size is in 4.5~4.7mm.
The preparation of the complete thallus sphere of 3.2 immobilizations
(1) collect recombination metallothionein Escherichia coli, 10000r/min, 4 DEG C be centrifuged to obtain thallus, it is outstanding with PBS buffer solution
It rises, prepares bacteria suspension, the ratio between thallus weight in wet base and PBS buffer solution volume are 0.06g/ml.
(2) method in 3.1 is pressed, replaces PBS buffer solution in 3.1 with bacteria suspension, preparation is embedded with the immobilization of complete thallus
Sphere.
3.3 immobilizations are crushed the preparation of thallus sphere
(1) collect recombination metallothionein Escherichia coli, 10000r/min, 4 DEG C be centrifuged to obtain thallus, PBS buffer solution is hanged,
Bacteria suspension is prepared, the ratio between thallus weight in wet base and PBS buffer solution volume are 0.06g/ml.Bacteria suspension is frozen repeatedly in -80 DEG C and 37 DEG C
Melt three times.With ultrasonication machine ultrasonication thallus, broken bacterium solution is obtained.
(2) method in 3.1 is pressed, replaces PBS buffer solution in 3.1 with broken bacterium solution, preparation is embedded with the fixation of broken thallus
Change sphere.
4. adsorption test
4.1 3 kinds of beads are to Cd2+Absorption
(1) according to the method described above, blank bead (abbreviation BLB), the embedding recombination complete thallus of metallothionein MTT1 are prepared
Immobilized spherule (abbreviation WM1), embedding recombination metallothionein MTT1 be crushed bacterium solution immobilized spherule (abbreviation BM1).
(2) BLB, WM1, BM1 are added separately to 250 μ g/ml Cd of 50ml2+Solution (Cd2+Anion in solution is
Cl-) (total amount of cadmium ion is 12.5mg), 4h is adsorbed in oscillation, takes out immobilization sphere, measures Cd in solution2+Concentration is calculated and is inhaled
Attached amount.
As a result as shown in Figure 2 (BLB adsorbance 6.08mg, WM1 adsorbance 6.61mg, BM1 adsorbance 7.45mg), by Fig. 2
It is found that the cadmium sorption amount that immobilization is crushed bacterium solution improves nearly 0.8mg compared with the complete bacterium solution of immobilization, the 6.4% of total amount is accounted for；
Compared with the cadmium sorption amount that blank sphere improves about 1.4mg, account for total amount 11.2% (this experiment only used 50mL solution as
Subjects, but in actual application, liquor capacity to be treated is very big, is better able to embody fixation after amplification
Change the superiority of broken bacterium solution).
As it can be seen that immobilization is crushed bacterium sphere compared to immobilization viable bacteria sphere, can significantly improve to Cd2+Absorption property
(SPSS single factor test ANOVA analysis, S-N-K, LSD, DUNCAN are analyzed, and are had between the cadmium sorption amount of three kinds of beads significant
Sex differernce；In addition one-way analysis of variance shows p < 0.01, and to be extremely significant, the adsorbance of BM1 bead is significantly higher than remaining two kinds
Bead), this may be since more adsorption sites, such as cell fragment, intracellular protein after bacterial cell disruption, will can be exposed, and
The albumen such as metallothionein can be contacted directly with metal ion, be conducive to adsorb, therefore immobilization is crushed bacterium and can significantly mention
High absorption property.
4.2 3 kinds of beads are to Cu2+Absorption
(1) according to the method described above, the immobilized spherule (letter of BLB, the embedding recombination complete thallus of metallothionein MTT2 are prepared
Claim WM2), embedding recombination metallothionein MTT2 be crushed bacterium solution immobilized spherule (abbreviation BM2).
(2) BLB, WM2, BM2 are added separately to 250 μ g/ml Cu of 50ml2+Solution (Cu2+Anion in solution is
SO4 2-) (total amount of copper ion is 12.5mg), 4h is adsorbed in oscillation, takes out immobilization sphere, measures Cu in solution2+Concentration calculates
Adsorbance.
As a result as shown in Figure 3 (BLB adsorbance 5.99mg, WM2 adsorbance 6.55mg, BM2 adsorbance 7.15mg), by Fig. 3
It is found that the cupric ion adsorption that immobilization is crushed bacterium solution improves nearly 0.6mg compared with the complete bacterium solution of immobilization, the 4.5% of total amount is accounted for；
The immobilization sphere for less embedding bacterium solution improves about 1.16mg, account for total amount 9.28% (50mL solution has only been used in this experiment
As subjects, but in actual application, liquor capacity to be treated is very big, is better able to embody after amplification
Immobilization is crushed the superiority of bacterium solution).
As it can be seen that immobilization is crushed bacterium sphere compared to immobilization viable bacteria sphere, can significantly improve to Cu2+Absorption property
(SPSS single factor test ANOVA analysis, S-N-K, LSD, DUNCAN analysis, three kinds of beads have significant difference；In addition, Dan Yin
Plain variance analysis shows p < 0.01, and to be extremely significant, the cupric ion adsorption conspicuousness of BM2 bead is higher than other two kinds of beads),
This may be due to that after bacterial cell disruption, will can expose more adsorption sites, such as cell fragment, intracellular protein, and metal sulphur
The albumen such as albumen can be contacted directly with metal ion, be conducive to adsorb, therefore the broken bacterium of immobilization can significantly improve absorption
Performance.
4.3 orthogonal test
4.3.1 immobilization sphere adsorbs Cd2+Orthogonal test
According to orthogonal test table (table 1 is orthogonal experimental factor water-glass, and table 2 is the orthogonal arrage of orthogonal test), 9 groups are prepared
Embedding recombination metallothionein MTT1 is crushed the immobilized spherule of bacterium solution, and preparation method is crushed thallus sphere with reference to 3.3 immobilizations
Preparation, factor A bacteria concentration refers to bacteria concentration (bacteria concentration 20mg/ml, 15mg/ml, 10mg/ml, corresponding bacterium in mixed liquor
The ratio between body weight in wet base and PBS buffer solution volume are respectively 0.08g/ml, 0.06g/ml, 0.04g/ml), factor B PVA concentration refers to
The concentration of polyvinyl alcohol in mixed liquor, factor C boric acid concentration refer to the concentration of boric acid in boric acid-barium chloride solution.
Table 1
Table 2
9 groups of embedding recombination metallothionein MTT1 immobilized spherule for being crushed bacterium solution is added separately to 250 μ g/ of 50ml
ml Cd2+Solution, oscillation absorption 4h, takes out immobilization sphere, measures Cd in solution2+Concentration calculates adsorbance, as a result such as table 3
It is shown.
Table 3 is that immobilization sphere adsorbs Cd2+Orthogonal experiments
Influence size order by the available each factor of table 3 to experimental result are as follows: A (bacteria concentration) > C (boric acid concentration) > B
(polyvinyl alcohol concentration).This shows that bacteria concentration influences the most significant, poly- second to the absorption property of immobilization sphere Adsorption of Cadmium
Enol concentration influences the absorption property of bead Adsorption of Cadmium minimum.In addition, comparison K value size, it can be deduced that influence because
The superiority and inferiority of each level of element, it can be deduced that A is best with level 1, and B is best with level 1, and C is best with level 2, i.e. optimum condition
For A1B1C2.
4.3.2 immobilization sphere Adsorption of Cu2+Orthogonal test
According to orthogonal test table (table 1 is orthogonal experimental factor water-glass, and table 2 is the orthogonal arrage of orthogonal test), 9 groups are prepared
Embedding recombination metallothionein MTT2 is crushed the immobilized spherule of bacterium solution, and preparation method is crushed thallus sphere with reference to 3.3 immobilizations
Preparation, factor A bacteria concentration refers to bacteria concentration (bacteria concentration 20mg/ml, 15mg/ml, 10mg/ml, corresponding bacterium in mixed liquor
The ratio between body weight in wet base and PBS buffer solution volume are respectively 0.08g/ml, 0.06g/ml, 0.04g/ml), factor B PVA concentration refers to
The concentration of polyvinyl alcohol in mixed liquor, factor C boric acid concentration refer to the concentration of boric acid in boric acid-barium chloride solution.
9 groups of embedding recombination metallothionein MTT2 immobilized spherule for being crushed bacterium solution is added separately to 250 μ g/ of 50ml
ml Cu2+Solution, 120r/min oscillation absorption 4h, takes out immobilization sphere, measures Cu in solution2+Concentration calculates adsorbance, knot
Fruit is as shown in table 4.
Influence size order by the available each factor of table 4 to experimental result are as follows: A (bacteria concentration) > C (boric acid concentration) > B
(polyvinyl alcohol concentration).This shows that bacteria concentration influences the most significant, poly- second to the absorption property of immobilization sphere absorption copper ion
Enol concentration influences the absorption property of bead absorption copper ion minimum.In addition, according to the size of K value, it can be deduced that A is with level
1 is best, and B is best with level 2, and C is best with level 2, i.e., optimum condition is A1B2C2.By confirmatory experiment, with A1B2C2 condition
The immobilization sphere of preparation exceeds the cupric ion adsorption of experimental group to the adsorbance of copper ion, thus immobilization sphere adsorbs
The optimum condition of copper ion is A1B2C2.
Table 4 is immobilization sphere Adsorption of Cu2+Orthogonal experiments
4.4 solution ph is to absorption Cd2+Influence
(1) according to orthogonal experiments, MTT1 cell concentration is prepared as the immobilization of 20mg/ml with optimum condition A1B1C2
Broken thallus bead (abbreviation 20-BM1).
(2) 250 μ g/ml Cd of 50ml is adjusted2+The pH value of solution, is separately added into 20-BM1, and oscillation absorption 4h takes out and fixes
Change sphere, measures Cd in solution2+Concentration calculates adsorbance, as a result as shown in Figure 4.
As shown in Figure 4, during pH value is increased to 7 from 1, immobilization recombinates tetrahymena thermophila's metallothionein MTT1
Escherichia coli sphere first increases the adsorbance of cadmium ion and declines afterwards.The cadmium sorption amount of immobilization sphere reaches maximum value and is
7.86mg (pH value is 4 at this time).
4.5 solution ph are to Adsorption of Cu2+Influence
(1) according to orthogonal experiments, MTT2 cell concentration is prepared as the immobilization of 20mg/ml with optimum condition A1B2C2
Broken thallus bead (abbreviation 20-BM2).
(2) 250 μ g/ml Cu of 50ml is adjusted2+The pH value of solution, is separately added into 20-BM2, and oscillation absorption 4h takes out and fixes
Change sphere, measures Cu in solution2+Concentration calculates adsorbance, as a result as shown in Figure 5.
As shown in Figure 5, pH value recombinates tetrahymena thermophila's metallothionein MTT2 Escherichia coli sphere absorbing copper to immobilization
The influence of ion is similar to immobilization sphere Adsorption of Cadmium.With pH value from 2 be raised to 6 during, the copper of immobilization sphere
Ionic adsorption amount first increases to be declined afterwards, and it is 7.45mg that adsorbance, which reaches maximum value, (pH value is 4 at this time).
4.6 adsorption times are to absorption Cd2+Influence
According in 4.4 as a result, adjust Cd2+The pH value of solution is to 4, to 250 μ g/ml Cu of 50ml2+20- is added in solution
Adsorption time 1,2,4,6,8,10,12h is arranged in BM1, takes out immobilization sphere, measures Cd in solution2+Concentration calculates adsorbance,
As a result as shown in Figure 6.
It will be appreciated from fig. 6 that immobilization recombinates absorption of tetrahymena thermophila's metallothionein MTT1 Escherichia coli for cadmium ion
Pass through quick adsorption, the last adsorption process for slowly reaching adsorption equilibrium for one.
4.7 adsorption time is to Adsorption of Cu2+Influence
According in 4.5 as a result, adjust Cu2+The pH value of solution is to 4, to 250 μ g/ml Cu of 50ml2+20- is added in solution
BM2, adsorption time 1,2,4,6,8,10,12h take out immobilization sphere, measure Cu in solution2+Concentration calculates adsorbance, knot
Fruit is as shown in Figure 7.
As shown in Figure 7, similar to the process of immobilization sphere Adsorption of Cadmium, immobilization recombinates tetrahymena thermophila's metal sulphur
It is a quick adsorption that albumen MTT2 Escherichia coli sphere, which adsorbs copper ion, and final slowly absorption reaches the process of balance.
4.8 metal ion initial concentrations are to absorption Cd2+Influence
Cd is prepared respectively2+Concentration is each 50ml of metal ion solution of 50,100,150,200,250,300 μ g/ml, is adjusted
PH value is added 20-BM1, after adsorbing 4h, takes out immobilization sphere, measure Cd in solution to 42+Concentration calculates adsorbance, as a result
As shown in Figure 8.
As shown in Figure 8, with the increase of cadmium ion initial concentration, the cadmium sorption amount of immobilized spherule increases, finally,
The absorption of cadmium ion tends to be saturated.When metal ion initial concentration is lower, the diffusion rate of ion is low, and adsorbance is lower, with
The raising of concentration, diffusion rate increase, adsorbance also increases with it, and finally, adsorption site tends to be saturated, and absorption reaches flat
Weighing apparatus.
4.9 metal ion initial concentrations are to Adsorption of Cu2+Influence
Cu is prepared respectively2+Concentration is each 50ml of metal ion solution of 50,100,150,200,250,300 μ g/ml, is adjusted
PH value is added 20-BM2, after adsorbing 4h, takes out immobilization sphere, measure Cu in solution to 42+Concentration calculates adsorbance, as a result
As shown in Figure 9.
As shown in Figure 9, as copper ion concentration increases, the adsorbance of copper ion is consequently increased, the suction of final copper ion
It is attached to tend to be saturated.Diffusion plays an important role in adsorption process, reaches a timing in the initial concentration of metal ion, inhales
Attached site tends to be saturated, and absorption reaches equilibrium state.
4.10 desorptions and adsorption test again
(1) adsorption test: preparation 20-BM1 and 20-BM2 takes 200 μ g/ml cadmium-ion solution of 50ml and copper ion respectively
Solution adjusts pH value to 4, immobilized spherule is added, adsorbs 4h, takes out, distilled water cleaning is spare.
(2) desorption experiment: immobilization sphere in (1) is added in 50ml 10mmol hydrochloric acid, and oscillation desorption is taken out, steamed
Distilled water cleaning is spare.
(3) adsorption test again: taking immobilization sphere in (2), repeats step (1).
(4) three circulations are repeated, as a result as shown in Figure 10,11.
By Figure 10,11 it is found that second of absorption is lower than the amount of metal ion of first time absorption, and third time is adsorbed and second
It is secondary close, it is believed that in multiple desorption with sorption cycle again, immobilization sphere still maintains certain heavy metal adsorption energy
Power.It can be inquired into subsequent research and how improve desorption effect, such as attempt new strippant, enhance reusing.
5. experiment conclusion
The present invention is carrier material using sodium alginate, polyvinyl alcohol, silica, using the side for embedding broken bacterium solution
Method is prepared for a kind of containing recombination metallothionein immobilized spherule.Immobilized spherule has good absorption property and repetition
Usability.Broken bacterium solution is embedded, enhances immobilization sphere to the absorption property of heavy metal ion, and enhances recombination thermophilic four
The application of film worm metallothionein.It is prepared by the present invention containing recombination metallothionein immobilization sphere, material source is wide, at
This is cheap, has important application value in heavy metal containing wastewater treatment.
Above-described embodiment is provided to those skilled in the art, how to implement and use to be advocated with full disclosure and description
Embodiment, rather than for limiting range disclosed herein.Obvious modification will to those skilled in the art
Within the scope of the appended claims.The all publications, patents and patent applications of this specification citation are incorporated by reference into this
Text, as these publications, patents and patent applications respectively show particularly and individually to be incorporated herein by reference.
Claims (10)
1. a kind of preparation method of the immobilized spherule containing metallothionein, it is characterised in that: the following steps are included:
(1) bacterium of culture expression metallothionein or recombination metallothionein, obtains culture solution；
(2) culture solution or thallus PBS buffer solution are hanged, prepares bacteria suspension；More than three times by bacteria suspension multigelation, ultrasonic
Broken thallus obtains broken bacterium solution；
(3) broken bacterium solution, silica are added in sodium alginate-polyvinyl alcohol aqueous solution, mix, obtains mixed liquor；
Under the conditions of (4) 4 DEG C, mixed liquor is added drop-wise in boric acid-barium chloride solution, 4 DEG C of fixed 4h, takes out, wash with water, i.e.,
Obtain the immobilized spherule containing metallothionein.
2. the preparation method of the immobilized spherule according to claim 1 containing metallothionein, it is characterised in that: described
It expresses metallothionein or recombinates the bacterium of metallothionein, be selected from wild strain, or the engineering obtained by gene recombination technology
Bacterium.
3. the preparation method of the immobilized spherule according to claim 1 containing metallothionein, it is characterised in that: described
In step (1), specific condition of culture is as follows: recombination bacillus coli is taken, is added in the LB liquid medium of the benzyl containing ammonia, 37 DEG C of cultures
8~14h；1ml bacterium solution is taken, is added in 100ml LB liquid medium, 37 DEG C of cultures to OD600It is 0.6~1.0, is added dense eventually
Degree is IPTG, the Fiber differentiation 4h of 0.5mmol/ml, obtains culture solution, 10000r/min, 4 DEG C be centrifuged to obtain thallus.
4. the preparation method of the immobilized spherule according to claim 1 containing metallothionein, it is characterised in that: described
In step (2), the ratio between thallus weight in wet base and PBS buffer solution volume are 0.04~0.08g/ml.
5. the preparation method of the immobilized spherule according to claim 1 containing metallothionein, it is characterised in that: described
In step (2), the multigelation refers in -80 DEG C and 37 DEG C of multigelations.
6. the preparation method of the immobilized spherule according to claim 1 containing metallothionein, it is characterised in that: described
In step (3), in the mixed liquor, the concentration of sodium alginate is 1%~3%, and the concentration of polyvinyl alcohol is 1%~3%, dioxy
The concentration of SiClx is 0.5%, and cell concentration is 10~20mg/ml.
7. the preparation method of the immobilized spherule according to claim 1 containing metallothionein, it is characterised in that: described
In step (3), the molecular weight of the polyvinyl alcohol is 15~200,000.
8. the preparation method of the immobilized spherule according to claim 1 containing metallothionein, it is characterised in that: described
In step (4), in the boric acid-barium chloride solution, the concentration of boric acid is 1%, and the concentration of barium chloride is 3%.
9. being prepared using the preparation method of the immobilized spherule containing metallothionein described in any one of claim 1~8
The obtained immobilized spherule containing metallothionein.
10. the immobilized spherule as claimed in claim 9 containing metallothionein answering in waste water of the processing containing heavy metal
With.
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| CN101298614A (en) * | 2008-03-05 | 2008-11-05 | 山西九龙湾农林科技开发有限公司 | Potamon metallothionin gene, transgenic blue algae thereof and preparation of metal adsorption material |
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