CN104498470B - A kind of co-immobilization whiterot fungi gel particle - Google Patents
A kind of co-immobilization whiterot fungi gel particle Download PDFInfo
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- CN104498470B CN104498470B CN201510022825.8A CN201510022825A CN104498470B CN 104498470 B CN104498470 B CN 104498470B CN 201510022825 A CN201510022825 A CN 201510022825A CN 104498470 B CN104498470 B CN 104498470B
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Abstract
The invention belongs to modifying porcelain field, and in particular to a kind of co-immobilization whiterot fungi gel particle.It is of the invention the ectoenzyme in crude enzyme liquid is assembled crosslinking using ammonium sulfate and glutaraldehyde, cross-linked enzyme aggregate is formed, then scattered fixation is divided into the free mycelia of whiterot fungi, co-immobilization whiterot fungi gel particle is made.White rot enzyme of the present invention exists with enzyme aggregate form; improve its resistivity to proteolytic enzyme; so as to protect cross-linked enzyme aggregate not decomposed by whiterot fungi B in gel particle; ensure that the ectoenzyme of whiterot fungi A secretions maintains certain enzyme activity for a long time; the startup time is shorter; adaptability to dyeing waste-water environment is stronger, can be directly used for the fluidized-bed reactor of wastewater treatment, the processing for all dyeing waste-waters.
Description
Technical field
The invention belongs to modifying porcelain technical field, and in particular to a kind of co-immobilization whiterot fungi gel particle.
Background technology
Synthetic dyestuffs are the aromatic series macromolecular compounds of first stability, with high bio-toxicity, difficult degradation and high chroma
The features such as, serious harm can be produced to aquatile system, human body health is directly or indirectly influenceed, needed in industrial wastewater
The important pollutant administered.In view of processing cost, the factors such as secondary pollution are produced, it is main that biological treatment is still dyeing waste-water
Governing measure.Biological treatment dye techniques more ripe at present are to carry out non-specific idol using anaerobic bacteria flora azo dyes
Nitrogen reduction-decolor, but the technique is difficult to the other structures type dye such as triphenylmethane and Anthraquinones of degrading, and dyestuff is taken off
Toxic effect can also produce the higher amine phenolic compounds of bio-toxicity sometimes with also poor.
In the biosystem being currently known, whiterot fungi can under aerobic condition non-specific degradation of dye, and have aobvious to it
The detoxification of work.Whiterot fungi be a class based on Basidiomycetes, the fungi that can become white rot of wood, its degraded to dyestuff
It is essentially relying on the lignin degradation ectoenzyme system of secondary metabolism stage secretion.The ectoenzyme system is main by lignin peroxide
Enzyme, manganese peroxidase and the similar work enzyme system composition of laccase three, they, which can be utilized respectively in the middle of different electronics, passs body oxidation
Xenobiotics, has necessarily because these ectoenzymes need not directly be contacted with degradation product, therefore to the bio-toxicity of dyestuff
Resistivity.
Whiterot fungi is that mechanical force too conference destroys its filament in a class filamentous fungi, degradation reaction device, so as to destroy
Mycelia secretes lignin degradation extracellular enzyme, and mechanical force is too small and influences the effect of mass transmitting of dyeing waste water water body oxygen, so as to reduce
The degradation capability of whiterot fungi, while the harsh temperature of its degradation reaction, pH conditions, have also been significantly greatly increased industrial practical operation difficult
Degree.Whiterot fungi immobilization technology can effectively improve the disadvantages mentioned above of whiterot fungi commercial Application, and the embedding bead of whiterot fungi also makes it
It is easy to apply in the flowing bed apparatus of wastewater treatment.But simple immobilized white rot fungus cell can not solve whiterot fungi industrial treatment
All technical problems of dyeing waste-water.
Whiterot fungi secretion synthesis lignin degradation ectoenzyme stability of a system extreme difference, under optimal culture conditions, water body
In the enzyme activity of the extracellular enzyme be also at big ups and downs state, it is difficult to stable, which results in prolonging for waste water water body residence time
It is long.Meanwhile, secretion secondary metabolism stage of the synthesis in whiterot fungi of lignin biodegrading enzyme system, it is meant that needed in industrial practical application
Very important person is that Additional carbon sources are added into waste water water body, adjusts carbon-nitrogen ratio.This not only brings the increase of processing cost, also greatly carries
The high control difficulty of reaction system.Research also confirms that some organic pollutants can suppress the synthesis of lignin biodegrading enzyme system, and water
The presence of heavy metal is then more in body can suppress the enzyme activity of lignin biodegrading enzyme system, especially influence aobvious to the enzyme activity of manganese peroxidase
Write, presence of these materials in waste water water body limits the application of traditional whiterot fungi reactor.
Although industrially simple can be solved the above problems to a certain degree using immobilization lignin biodegrading enzyme system system, but be drawn
The problem of having entered new, lignin peroxidase and manganese peroxidase are, using hydrogen peroxide as final electron acceptor, directly to use
Fixed ferment treatment waste water needs to add hydrogen peroxide to water body or enzymatic reaction produces the material of hydrogen peroxide.And due to natural ring
The need for border is evolved, while larger to specific dyeing waste water lignin biodegrading enzyme system oxidability, mycelial growth ability is also stronger
Strain of the whiterot fungi strain in natural environment find that difficulty is larger, this also turns into the problem of having to be solved.
The content of the invention
The object of the present invention is to overcome the problems of the prior art, and there is provided a kind of efficient place for dyeing waste-water
Reason, the extensive co-immobilization whiterot fungi gel particle of condition of compatibility.
What above-mentioned purpose was achieved through the following technical solutions：
A kind of co-immobilization whiterot fungi gel particle is through the following steps that prepare：
1）The preparation of whiterot fungi A ectoenzyme cross-linked aggregates：After whiterot fungi A is cultivated 6-20 days in nutrient solution, training is collected
Nutrient solution, dialysis removes inorganic ions, and concentration is made in crude enzyme liquid, crude enzyme liquid and adds ammonium sulfate, stirs, and reaction solution is made；To anti-
The glutaraldehyde for being added dropwise that concentration is 25% in liquid is answered, a few hours are crosslinked under the conditions of isothermal vibration, is centrifuged, repeatedly washing is heavy for distilled water
Form sediment, prepare enzyme crosslinking aggregation, add distilled water and enzyme aggregate suspension is made；
Step 1）Cross-linked enzyme aggregate is made in the middle ectoenzyme by whiterot fungi A, and increase ectoenzyme is to proteolytic enzyme
Resistance, so that it can maintain enzymatic activity for a long time in gel particle.
2）The preparation of whiterot fungi B cell homogenate：Whiterot fungi B is cultivated 3-10 days in nutrient solution, and filter cloth collects mycelia, is placed in
In distilled water, bead breaks up mycelia, and whiterot fungi B cell homogenates is made.
3）The preparation of co-immobilization gel particle：By step 1）Obtained enzyme aggregate suspension and step 2）It is obtained white
Rotten bacterium B cell homogenates mixing adds sodium alginate, and homogeneous mixture is made, homogeneous mixture is instilled to 2% CaCl2Solution
In, fixation is stored at room temperature, by gained particulate matter distillation water washing, co-immobilization whiterot fungi gel particle is made.
Step 3）In, whiterot fungi A ectoenzyme cross-linked aggregates and whiterot fungi B hyphal cell co-immobilization are eliminated
The competitive inhibitory effects of two kinds of whiterot fungis.
Described, the component of nutrient solution：2g/LKH2PO4、0.25g/LMgSO4、0.1g/LCaCl2、5mg/LMnSO4、5mg/
The micro- storing solution of LVB1,0.2g/L ammonium tartrate, 20g/L glucose and 150ml/L.
Described, the component of micro- storing solution：NaCl1.0g/L、MgSO4•7H2O3.0g/L、FeSO4•
7H2O100mg/L、CoSO4•7H2O100mg/L、CaCl2100mg/L、ZnSO4•7H2O100mg/L、CuSO4•5H2O10mg/L、
KAl(SO4)2100mg/L、H3BO310mg/L and NaMoO410mg/L。
Described, step 1）The addition of middle ammonium sulfate is the 60%-90% of crude enzyme liquid quality, stirs 10-60min.
Described, step 1）Mass concentration of the middle glutaraldehyde in reaction solution is 0.2-0.4%, and crosslinking temperature is 20-30
DEG C, the concussion frequency of crosslinking is 100rpm, and crosslinking time is 20-30h, and centrifugal speed is 14000r/min, and centrifugation time is
10min。
Described, step 3）The lignin degradation extracellular enzyme total enzyme activity of middle homogeneous mixture is more than 50U/ml, what it was included
Hyphal cell dry weight is more than 5mg/ml.
Described, step 3）The mass concentration of middle sodium alginate in the mixture is 2%, and the set time is 1-3h.
Crude enzyme liquid includes the one or more in lignin peroxidase, manganese peroxidase and laccase.
The co-immobilization whiterot fungi gel particle of the present invention, its preparation technology route is shown in Fig. 1.
In above-mentioned co-immobilization whiterot fungi gel particle, whiterot fungi A and whiterot fungi B can be any identical or different white
Rotten bacterium strain.According to applicable wastewater environment condition, it is whiterot fungi A to select the strain stronger to heteroplasia matter degradation capability, prepares and hands over
Join enzyme aggregate, the faster strain of growth rate be whiterot fungi B, prepare cell homogenates, can be made adaptability it is stronger, grow compared with
Hurry up, the gel particle that treatment efficiency is higher.
Beneficial effects of the present invention：
1. the present invention is before embedding white rot enzyme, the formation of whiterot fungi A ectoenzymes is crosslinked using crosslinking agent glutaraldehyde
Enzyme aggregate.Compared with resolvase, the hydrophobic effect increase between enzyme aggregate electrostatic force and enzyme molecule is conducive to preventing enzyme molecule
Generation unfolding or dissociation, so as to enhance stability of the protein to hot and other denaturants.Meanwhile, cross-linked enzyme aggregate phase
To orderly three-dimensional structure, the resistivity to proteolytic enzyme is improved, so as to protect cross-linked enzyme aggregate not by gel
Intragranular whiterot fungi B is decomposed, it is ensured that the ectoenzyme of whiterot fungi A secretions maintains certain enzyme activity for a long time.And cross-linked enzyme aggregate
This carrier-free is fixed, and it is made full use of the confined space inside gel particle, each gel particle is accommodated more
The white rot enzyme of volume.
2. the co-immobilization whiterot fungi gel particle of the present invention has contained lignin biodegrading enzyme system before starting, effectively shorten and fix
Change the startup time of whiterot fungi.Simultaneously in gel particle ectoenzyme cross-linked aggregates presence, reduce and wood secreted to whiterot fungi B
The demand of plain degradation enzyme system, can be applied to the presence of the wastewater treatment for suppressing whiterot fungi secretion lignin biodegrading enzyme system pollutant, enhancing
Adaptability of the gel particle system to dyeing waste-water environment.
3. in the gel particle of the present invention, while except embedding white rot enzyme aggregation, also embed whiterot fungi mycelia
Cell.These mycelia directly can generate hydrogen peroxide using Pollutants in Wastewater and its catabolite, be the oxidation drop of enzyme system
Solution provides electron acceptor terminal.
4. the fluidized-bed reactor in wastewater treatment can be used directly in the co-immobilization whiterot fungi gel particle of the present invention, use
In the processing of all dyeing waste-waters.
Brief description of the drawings
Fig. 1 is the preparation technology route of the co-immobilization whiterot fungi gel particle of the present invention；
Fig. 2 is the degradation efficiency of the co-immobilization whiterot fungi gel particle azo dyes of the present invention；
In figure, a is the degradation efficiency of the azo dyes of embodiment 1, and b is the degradation efficiency of the azo dyes of embodiment 2；
Fig. 3 is the degradation efficiency of co-immobilization whiterot fungi gel particle of the invention to triphenylmethane dye；
In figure, a is degradation efficiency of the embodiment 1 to triphenylmethane dye, and b is embodiment 2 to triphenylmethane
The degradation efficiency of dyestuff；
Fig. 4 is the co-immobilization whiterot fungi gel particle of embodiment 1 to denaturant Cr（VI）Stability test；
Fig. 5 is the co-immobilization whiterot fungi gel particle of embodiment 2 to denaturant Cr（VI）Stability test.
Embodiment
Below by specific embodiment, the advantage to the present invention is described in further detail, but the present invention is not limited
In following examples.
Embodiment 1
Whiterot fungi A selects Trametes versicolor（Purchased from Chinese industrial Culture Collection, bacterial strain is protected
It is CICC50001 to hide numbering）, whiterot fungi B is from Phanerochaete chrysosporium（Purchased from Guangdong microorganism fungus kind
Collection, bacterial strain deposit number is GIM3.383）.
Whiterot fungi A is cultivated after 10 days in nutrient solution, 4.5m filter paper collect nutrient solution, dialysis remove matrix in it is inorganic from
Son, film filter concentrates 10 times of acquisition crude enzyme liquids.Into crude enzyme liquid, addition accounts for the ammonium sulfate of crude enzyme liquid quality 60%, stirring
10min, is made reaction solution；0.2% is slowly added dropwise into reaction solution（w/w）Concentration be 25% glutaraldehyde, 30 DEG C and 100rpm
Under the conditions of isothermal vibration crosslinking 20h after, 14000r/min centrifugation 10min remove supernatant, precipitation is repeatedly washed with distilled water, remove
Excessive glutaraldehyde and ammonium sulfate, are made cross-linked enzyme aggregate, add distilled water and enzyme aggregate suspension is made, be placed in 4 DEG C of refrigerators
In save backup.
After whiterot fungi B is cultivated 3 days, filter cloth collects mycelia, is placed in distilled water, bead breaks up mycelia, and whiterot fungi B is made
Cell homogenates.Enzyme aggregate suspension and cell homogenates are well mixed, and mixture is made, 2% alginic acid is added into mixture
Sodium (w/w), is made homogeneous mixture.12.5mg/ml hyphal cell and 2.34 × 10 is included in homogeneous mixture6U/ml enzyme
Cross-linked aggregates.By the CaCl of homogeneous mixture instillation 2%2It is stored at room temperature in solution after fixed 2h, distilled water flushing obtained 3 ±
The co-immobilization whiterot fungi gel particle of 0.2mm diameters.
The component of nutrient solution：2g/LKH2PO4、0.25g/LMgSO4、0.1g/LCaCl2、5mg/LMnSO4、5mg/LVB1、
The micro- storing solution of 0.2g/L ammonium tartrates, 20g/L glucose and 150ml/L.
The component of micro- storing solution：NaCl1.0g/L、MgSO4•7H2O3.0g/L、FeSO4•7H2O100mg/L、
CoSO4•7H2O100mg/L、CaCl2100mg/L、ZnSO4•7H2O100mg/L、CuSO4•5H2O10mg/L、KAl(SO4)2100mg/L、H3BO310mg/L and NaMoO410mg/L。
Embodiment 2
Whiterot fungi A and whiterot fungi B select whiterot fungi Phanerochaete chrysosporium（Purchased from the micro- life in Guangdong
Thing DSMZ, bacterial strain deposit number is GIM3.383）.
Whiterot fungi A is cultivated after 20 days in nutrient solution, 4.5m filter paper collect nutrient solution, dialysis remove matrix in it is inorganic from
Son, film filter concentrates 10 times of acquisition crude enzyme liquids.Into crude enzyme liquid, addition accounts for the ammonium sulfate of crude enzyme liquid quality 90%, stirring
60min, is made reaction solution；0.4% is slowly added dropwise into reaction solution（w/w）Concentration be 25% glutaraldehyde, 20 DEG C and 100rpm
Under the conditions of isothermal vibration crosslinking 30h after, 14000r/min centrifugation 10min remove supernatant, precipitation is repeatedly washed with distilled water, remove
Excessive glutaraldehyde and ammonium sulfate, are made cross-linked enzyme aggregate, add distilled water and enzyme aggregate suspension is made, be placed in 4 DEG C of refrigerators
In save backup.
After whiterot fungi B is cultivated 10 days, filter cloth collects mycelia, is placed in distilled water, bead breaks up mycelia, and whiterot fungi is made
B cell homogenates.Enzyme aggregate suspension and cell homogenates are well mixed, and mixture is made, 2% marine alga is added into mixture
Sour sodium (w/w), is made homogeneous mixture.20.7mg/ml hyphal cell and 1.73 × 10 is included in homogeneous mixture7U/ml's
Enzyme crosslinking aggregation.By the CaCl of homogeneous mixture instillation 2%2It is stored at room temperature in solution after fixed 3h, distilled water flushing is made 3
The co-immobilization whiterot fungi gel particle of ± 0.2mm diameters.
The component be the same as Example 1 of the component of nutrient solution and micro- storing solution.
New type gel particle is tested the degradation efficiency of azo dyes
The Acid Brilliant Scarlet GR for weighing 40mg is dissolved in 1L white rot bacteria culture fluid, and artificially colored waste water, acid scarlet is made
GR is the conventional azo dyes of industry.40ml waste water and the co-immobilization of 0.36 ± 0.03g dry weights are added in 100ml conical flasks
Whiterot fungi gel particle.With distilled water substitute enzyme aggregate suspension, other steps with correspondence embodiment, respectively be made two it is right
According to the whiterot fungi gel particle-control group 1 and control group 2 of group, the quality controls gel particle such as addition.In 25 DEG C and 100rpm
Under the conditions of constant-temperature shaking culture 2.5 days, artificially colored waste water absorbance before and after culture is determined at 510nm, dye is calculated as follows
The decolored degradation rate of material, degradation efficiency of the reflection gel particle to dyestuff.
Decolorization and gegradation rate（%）=（A0-At）/A0*100%
Wherein, A0And AtAbsorbance before and after respectively artificially colored waste water culture.
Parallel laboratory test 10 times, experimental result is shown in Fig. 2.Experiment shows that co-immobilization whiterot fungi gel particle is relative to tradition
Immobilized white rot fungus start the time it is shorter, there is stronger degradation capability to dyestuff.
New type gel particle is tested the degradation efficiency of triphenylmethane dye
By the similar method of testing of above-mentioned degraded azo dyes, Acid Brilliant Scarlet GR is substituted with basic fuchsin artificial dye is made
Color waste water, basic fuchsin is the conventional triphenylmethane dye of industry.Constant-temperature shaking culture 7 days, before determining culture at 540nm
Artificial wastewater's absorbance, calculates decolorization and gegradation rate afterwards.Parallel laboratory test 10 times, experimental result is shown in Fig. 3.It is demonstrated experimentally that right
In triphenylmethane dye, co-immobilization whiterot fungi gel particle of the invention has stronger degradation capability.Meanwhile, contrast
Fig. 2 and Fig. 3, can may be selected to use different whiterot fungi extracellular enzyme and hyphal cell according to the difference of waste water component.
To denaturant Cr（VI）Stability test
Forefathers' research shows Cr（VI）There is obvious inhibiting effect to the enzyme activity of lignin biodegrading enzyme system, with various concentrations Cr（VI）
Characterize the severe degree of wastewater environment.Enzyme aggregate suspension is substituted with free enzyme liquid, other steps are with correspondence embodiment, difference
The common fixed white rot bacterium gel particle-control group 1 and control group 2 of two control groups is made.40ml is added in 100ml conical flasks
White rot bacteria culture fluid, 40mg/L Acid Brilliant Scarlet GRs, the co-immobilization gel particle and the Cr of various concentrations of 0.48 ± 0.02g dry weights
（VI）.Constant-temperature shaking culture determines water body decolouring degree before and after culture, in fact after 3 days 510nm under the conditions of 25 DEG C and the 100rpm
Test result and see Fig. 4 and Fig. 5.As can be seen from Figure, the gel particle comprising cross-linked enzyme aggregate is substantially than containing resolvase（Be free of
Cross-linked enzyme aggregate）Gel particle to Cr（VI）Inhibitory action has stronger resistivity.Even in 180mg/L high concentrations
Cr（VI）Under the conditions of, the co-immobilization whiterot fungi gel particle of the invention can still maintain 50% or so to Acid Brilliant Scarlet GR
Decolored degradation degree.
Claims (2)
1. a kind of co-immobilization whiterot fungi gel particle, it is characterised in that through the following steps that preparing：
1）The preparation of whiterot fungi A ectoenzyme cross-linked aggregates：After whiterot fungi A is cultivated 10-20 days in nutrient solution, culture is collected
Liquid, dialysis removes inorganic ions, and concentration is made in crude enzyme liquid, crude enzyme liquid and adds ammonium sulfate, stirs, obtain reaction solution；To reaction solution
It is middle that the glutaraldehyde that concentration is 25% is added dropwise, it is crosslinked under the conditions of isothermal vibration, centrifugation, distillation water washing precipitation is prepared into enzyme crosslinking
Aggregation, adds distilled water and enzyme aggregate suspension is made；
The component of the nutrient solution：2g/LKH2PO4、0.25g/LMgSO4、0.1g/LCaCl2、5mg/LMnSO4、5mg/LVB1、
The micro- storing solution of 0.2g/L ammonium tartrates, 20g/L glucose and 150ml/L；
The step 1）The addition of middle ammonium sulfate is the 60%-90% of crude enzyme liquid quality, stirs 10-60min；
The step 1）Mass concentration of the middle glutaraldehyde in reaction solution is 0.2-0.4%, and crosslinking temperature is 20-30 DEG C, crosslinking
Concussion frequency is 100rpm, and crosslinking time is 20-30h, and centrifugal speed is 14000r/min, and centrifugation time is 10min；
2）The preparation of whiterot fungi B cell homogenate：Whiterot fungi B is cultivated 3-10 days in nutrient solution, and filter cloth collects mycelia, is placed in distillation
In water, bead breaks up mycelia, and whiterot fungi B cell homogenates is made；
3）The preparation of co-immobilization gel particle：By step 1）Obtained enzyme aggregate suspension and step 2）Obtained whiterot fungi
B cell homogenates mixing, obtains mixture；Sodium alginate is added into mixture, homogeneous mixture is made, homogeneous mixture is dripped
Enter 2% CaCl2In solution, fixation is stored at room temperature, by gained particulate matter distillation water washing, co-immobilization whiterot fungi is made and coagulates
Glue particle；
The whiterot fungi A is Trametes versicolor, purchased from Chinese industrial Culture Collection, bacterial strain preservation
Numbering is CICC50001 or whiterot fungi Phanerochaete chrysosporium, purchased from the Microbiological Culture Collection of Guangdong
The heart, bacterial strain deposit number is GIM3.383；
The whiterot fungi B is whiterot fungi Phanerochaete chrysosporium, purchased from the Microbiological Culture Collection of Guangdong
The heart, bacterial strain deposit number is GIM3.383；
The step 3）The lignin degradation extracellular enzyme total enzyme activity of middle homogeneous mixture is more than 50U/ml, its hyphal cell included
Dry weight is more than 5mg/ml；
The step 3）The mass concentration of middle sodium alginate in the mixture is 2%, and the set time is 1-3h.
2. whiterot fungi gel particle according to claim 1, it is characterised in that the component of the micro- storing solution：
NaCl1.0g/L、MgSO4•7H2O3.0g/L、FeSO4•7H2O100mg/L、CoSO4•7H2O100mg/L、CaCl2100mg/L、
ZnSO4•7H2O100mg/L、CuSO4•5H2O10mg/L、KAl(SO4)2100mg/L、H3BO310mg/L and NaMoO410mg/L。
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| CN104787901A (en) * | 2015-04-21 | 2015-07-22 | 浙江工商大学 | Method for decolorizing printing and dyeing wastewater by adopting co-immobilized white-rot fungi |
| CN105176963B (en) * | 2015-09-16 | 2018-10-26 | 齐鲁工业大学 | The bio-microcapsule and preparation method of a kind of calcium alginate-sodium carboxymethylcellulose co-immobilization whiterot fungi and its ectoenzyme |
| CN106011119B (en) * | 2016-05-23 | 2020-03-24 | 电子科技大学 | Preparation method of crosslinked β -lactamase aggregate for pharmaceutical wastewater treatment |
| CN106011124B (en) * | 2016-08-05 | 2019-03-26 | 齐鲁工业大学 | A kind of whiterot fungi biological microsphere and preparation method |
| CN109136213A (en) * | 2018-08-22 | 2019-01-04 | 贵州大学 | A method of utilizing the black dyestuff of immobilized white rot fungus degrading activity |
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| Title |
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| A Ca-alginate particle co-immobilized with Phanerochaete chrysosporium cells and the combined cross-linked enzyme aggregates from Trametes versicolor;Yanchun Li et al.;《Bioresource Technology》;20150921;第198卷;464-469 * |
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