CN103846276A - Restoration method for polycyclic aromatic hydrocarbon contaminated soil through immobilized laccase - Google Patents
Restoration method for polycyclic aromatic hydrocarbon contaminated soil through immobilized laccase Download PDFInfo
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- CN103846276A CN103846276A CN201210495811.4A CN201210495811A CN103846276A CN 103846276 A CN103846276 A CN 103846276A CN 201210495811 A CN201210495811 A CN 201210495811A CN 103846276 A CN103846276 A CN 103846276A
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Abstract
The present invention relates to a restoration method for polycyclic aromatic hydrocarbon contaminated soil through immobilized laccase, wherein the immobilized laccase is adopted to restore the polycyclic aromatic hydrocarbon contaminated soil, the soil adopts mud, the water/soil ratio is 3:1, the solution part is a HAc-NaAc buffer solution (the pH value is 4), the initial concentration of the contaminant is 100 mug/ml, the amount of the immobilized laccase poured into the soil is 1 g/50 ml, the temperature is 20-40 DEG C, the pH value is 4-6, and the time is 72 h. According to the present invention, the low concentration or high concentration contaminant can be treated in the wide pH value, temperature and salt concentration range, such that the polycyclic aromatic hydrocarbon contaminated soil can be effectively restored.
Description
Technical field
The present invention relates to the restorative procedure of immobilization laccase to polycyclic aromatic hydrocarbon (PAHs) contaminated soil, adopt specifically two kinds of methods to being fixed of laccase, in different temperature, under different pH, for the reparation of polycyclic aromatic hydrocarbon pollution, object is to the degraded to polycyclic aromatic hydrocarbon by immobilization laccase, for a new approach is opened up in the reparation of organic polluted soil.
Background technology
Laccase (laccase, E.C.I.10.3.2) is the polyphenol oxidase of a kind of discovery cupric early, and as far back as 1883, Japanese Jitian found a kind of protein from the secretion of lacquer tree, and it can make paint solidify rapidly.Laccase can the many phenolic compound generations of catalysis oxidation reaction, and uses molecular oxygen to be reduced into water as electron acceptor.The substrate of laccase effect is quite extensive, comprises that polyphenol, methyl substitute single phenol, aromatic amine, benzenethiol, polymethoxy benzene, and other compound being easily oxidized
[.Owing to having quite wide in range Substratspezifitaet and good stability, laccase has significant application value at aspects such as wastewater treatment, bio-bleaching, aromatic inverting biological sensor structures.
Compared with directly utilizing microbiological treatment pollutant: enzymic catalytic reaction has the following advantages: 1. decomposition efficiency is high.2. toxicity is little, easy to operate.Microorganism usually has pathogenic, and enzyme is very friendly to environment.Because enzyme and product thereof are very easily further biodegradable in environment.3. the scope of application is wide.Enzyme can be processed the pollutant of low concentration or higher concentration within the scope of wider pH, temperature and salinity, and microorganism is generally only applicable to process the pollutant of low concentration.The major defect of enzyme is that price is high, and is subject to the interference deactivation of various factors (as the concentration of salt, pH and organic solvent etc.).But along with the development of modern biotechnology, by separation effectively, purifying, gene expression and recombinant technique, producing a large amount of, cheap enzyme has become possibility.By using novel enzyme immobilization technology, can obviously improve stability in use and the recycling rate of waterused of enzyme, thereby can further reduce its use cost.
Summary of the invention
Goal of the invention:
The present invention relates to the restorative procedure of a kind of immobilization laccase to polycyclic aromatic hydrocarbon pollution, its objective is the polycyclic aromatic hydrocarbon that utilizes immobilization laccase to degrade in soil, by the method for two kinds of different immobilization laccases, seek under different temperature and different pH condition, examine or check the degradation effect of pollutant.
Technical scheme:
The restorative procedure of immobilization laccase to polycyclic aromatic hydrocarbon pollution, is characterized in that: concrete steps are as follows: immobilization laccase is repaired for polycyclic aromatic hydrocarbon pollution; Soil adopts mud, and soil ratio is 3:1, and solution part is HAc-NaAc buffer solution (pH=4), and the initial concentration of pollutant is 100 μ g/ml; Immobilization laccase drops into the amount 1g/50ml in soil, and temperature is 20 ~ 40 DEG C, and pH value is 4 ~ 6, and the time is 72 h.
The method that laccase is fixed is as follows:
Taking nylon wire as carrier, glutaraldehyde as cross linker, is first cross-linked the process for fixation adsorbing afterwards; Concrete operations are: nylon wire is cut into the square of 0.5cm × 0.5cm ~ 1.5cm × 1.5cm size, with containing mass ratio 15 ~ 20%CaCl
2process 15 ~ 20min with the formalin that mass ratio is 15 ~ 20 % water, after cleaning, blot, in 5 ~ 8 mol/L HCl, be hydrolyzed 40 ~ 50 min, be washed till pH neutrality, blot; In the glutaraldehyde solution that nylon wire is 5 ~ 8% in mass concentration, after crosslinked 6 ~ 8h, add 30U laccase solution to fix 8 ~ 10h, with buffer solution for cleaning, being fixed laccase, now the enzyme rate of recovery is 55.56%.
The method that laccase is fixed is as follows:
Taking shitosan as carrier, glutaraldehyde as cross linker has carried out the immobilization of laccase; Concrete operations are: take a certain amount of shitosan and be dissolved in volume ratio 1 ~ 5 % glacial acetic acid, make shitosan precipitation with 2 ~ 5 mol/L NaOH, be washed to neutrality, wet shape chitin carrier is made in suction filtration dehydration, then, after taking 0.1 ~ 0.5g shitosan and 15 ~ 20mL and volumetric concentration and being 5 % glutaraldehyde cross-linking 8 ~ 10h, add 30.0 U enzyme room temperatures absorption to fix 12 ~ 15 h, by buffer solution for cleaning, being fixed laccase, the enzyme rate of recovery is 52.24%.
Advantage and effect:
The present invention proposes the restorative procedure of a kind of immobilization laccase to polycyclic aromatic hydrocarbon pollution, tool has the following advantages:
1. cost is low: selecting shitosan is to enrich, have hydrophily, biocompatibility and Resistance to microbes characteristic etc. based on its source as fixation support.Select its as Laccase Immobilization carrier be based on it at occurring in nature aboundresources, cheap, stable chemical nature, more resist compared with other natural material microorganism decompose, there is the good advantage such as heat endurance and mechanically actuated.Adopt glutaraldehyde to make crosslinking agent, two aldehyde radicals at glutaraldehyde molecule two ends can be respectively with carrier and aglucon on amino be cross-linked with the form of five carbon bridges, this reaction condition gentleness, easy and simple to handle, thereby be widely used.Nylon wire is the hydrophilic carrier being linked up by amido link, and closely similar with peptide bond structure, it is cheap, and stable chemical nature has good operability.
2. effective: enzyme can be processed the pollutant of low concentration or higher concentration within the scope of wider pH, temperature and salinity, and microorganism is generally only applicable to process the pollutant of low concentration.
detailed description of the invention:
The restorative procedure of immobilization laccase to polycyclic aromatic hydrocarbon pollution, is characterized in that: concrete grammar is as follows: concrete steps are as follows: immobilization laccase is repaired for polycyclic aromatic hydrocarbon pollution; Soil adopts mud, and soil ratio is 3:1, and solution part is HAc-NaAc buffer solution (pH=4), and the initial concentration of pollutant is 100 μ g/ml; Immobilization laccase drops into the amount 1g/50ml in soil, and temperature is 20 ~ 40 DEG C, and pH value is 4 ~ 6, and the time is 72 h.
Simultaneously to dissociate laccase in contrast.Consider when immobilization laccase is applied in environment, some Environmental Factors that may run into are studied.First be different temperature, comprise 20 DEG C (room temperatures) and 40 DEG C, now pH is 4; Secondly be different pH values, be set as 4 and 6, temperature is 40 DEG C.Be 72 h the running time of experiment.
The first method that above-mentioned laccase is fixed is as follows:
Taking nylon wire as carrier, glutaraldehyde as cross linker, is first cross-linked the process for fixation adsorbing afterwards; Concrete operations are: nylon wire is cut into the square of 0.5cm × 0.5cm ~ 1.5cm × 1.5cm size, with containing 15 ~ 20%CaCl
2process 15 ~ 20min with the formalin of 15 ~ 20 % water, after cleaning, blot, in 5 ~ 8 mol/L HCl, be hydrolyzed 40 ~ 50 min, be washed till pH neutrality, blot; Nylon wire after crosslinked 6 ~ 8h, adds 30U laccase solution to fix 8 ~ 10h in the glutaraldehyde solution of mass ratio 5 ~ 8%, and with buffer solution for cleaning, being fixed laccase, now the enzyme rate of recovery is 55.56%.
The second method that above-mentioned laccase is fixed is as follows:
Taking shitosan as carrier, glutaraldehyde as cross linker has carried out the immobilization of laccase; Concrete operations are: take a certain amount of shitosan and be dissolved in volume ratio 1 ~ 5 % glacial acetic acid, make shitosan precipitation with 2 ~ 5 mol/L NaOH, be washed to neutrality, wet shape chitin carrier is made in suction filtration dehydration, then take after 0.1 ~ 0.5 g shitosan and 15 ~ 20mL volumetric concentration, 5 % glutaraldehyde cross-linking 8 ~ 10h, add 30.0 U enzyme room temperature absorption to fix 12 ~ 15 h, by buffer solution for cleaning, being fixed laccase, the enzyme rate of recovery is 52.24%
Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not subject to the restriction of embodiment.
Embodiment 1, the impact of temperature on immobilization laccase degradation effect:
Immobilization laccase, after 72h cultivates, all can reach more than 80% and 50% the degradation effect of Pyr and BaP at two kinds of different temperature, and the degradation effect of free laccase is also more satisfactory, and the effect of method two is better than method one.Degradation effect with immobilized microorganism is above compared, and all improves, and this tentatively illustrates, from the angle of degradation rate, laccase is feasible for the reparation of soil.
The relevant parameter of immobilization laccase under table 1 different temperatures
Embodiment 2, the impact of pH on immobilization laccase degradation effect:
Under two condition of different pH, immobilization laccase and free laccase are all obtained good effect to the degraded of pollutant, and this tentatively illustrates that laccase can keep certain activity under the condition that approaches neutrality or meta-acid, can see this point by the rate of recovery of enzyme.
The relevant parameter of the lower immobilization laccase of the different pH values of table 2
Embodiment 3:
Taking nylon wire as carrier, glutaraldehyde as cross linker, is first cross-linked the process for fixation adsorbing afterwards; Concrete operations are: nylon wire is cut into the square of 1.5cm × 1.5cm size, with containing mass ratio 20%CaCl
2process 20min with the formalin that mass ratio is 20 % water, after cleaning, blot, in 8 mol/L HCl, be hydrolyzed 50 min, be washed till pH neutrality, blot; In the glutaraldehyde solution that nylon wire is 8% in mass concentration, after crosslinked 8h, add 30U laccase solution to fix 10h, with buffer solution for cleaning, being fixed laccase, now the enzyme rate of recovery is 55.56%.
Immobilization laccase is repaired for polycyclic aromatic hydrocarbon pollution; Soil adopts mud, and soil ratio is 3:1, and solution part is HAc-NaAc buffer solution (pH=4), and the initial concentration of pollutant is 100 μ g/ml; Immobilization laccase drops into the amount 1g/50ml in soil, and temperature is 40 DEG C, and pH value is 6, and the time is 72 h.
Embodiment 4:
Taking nylon wire as carrier, glutaraldehyde as cross linker, is first cross-linked the process for fixation adsorbing afterwards; Concrete operations are: nylon wire is cut into the square of 0.5cm × 0.5cm size, with containing mass ratio 15%CaCl
2process 15 min with the formalin that mass ratio is 15% water, after cleaning, blot, in 5 mol/L HCl, be hydrolyzed 40 min, be washed till pH neutrality, blot; In the glutaraldehyde solution that nylon wire is 5% in mass concentration, after crosslinked 6h, add 30U laccase solution to fix 8h, with buffer solution for cleaning, being fixed laccase, now the enzyme rate of recovery is 55.56%.
Immobilization laccase is repaired for polycyclic aromatic hydrocarbon pollution; Soil adopts mud, and soil ratio is 3:1, and solution part is HAc-NaAc buffer solution (pH=4), and the initial concentration of pollutant is 100 μ g/ml; Immobilization laccase drops into the amount 1g/50ml in soil, and temperature is 20 DEG C, and pH value is 4, and the time is 72 h.
Embodiment 5:
Taking nylon wire as carrier, glutaraldehyde as cross linker, is first cross-linked the process for fixation adsorbing afterwards; Concrete operations are: nylon wire is cut into the square of 1.0cm × 1.0cm size, with containing mass ratio 18%CaCl
2process 17 min with the formalin that mass ratio is 18% water, after cleaning, blot, in 6 mol/L HCl, be hydrolyzed 45 min, be washed till pH neutrality, blot; In the glutaraldehyde solution that nylon wire is 7% in mass concentration, after crosslinked 7h, add 30U laccase solution to fix 9h, with buffer solution for cleaning, being fixed laccase, now the enzyme rate of recovery is 55.56%.
Immobilization laccase is repaired for polycyclic aromatic hydrocarbon pollution; Soil adopts mud, and soil ratio is 3:1, and solution part is HAc-NaAc buffer solution (pH=4), and the initial concentration of pollutant is 100 μ g/ml; Immobilization laccase drops into the amount 1g/50ml in soil, and temperature is 30 DEG C, and pH value is 5, and the time is 72 h.
Embodiment 6:
Taking shitosan as carrier, glutaraldehyde as cross linker has carried out the immobilization of laccase; Concrete operations are: take a certain amount of shitosan and be dissolved in volume ratio 1% glacial acetic acid, make shitosan precipitation with 2 mol/L NaOH, be washed to neutrality, wet shape chitin carrier is made in suction filtration dehydration, then take after 0.1g shitosan and 15mL volume ratio 5 % glutaraldehyde cross-linking 8h, add the fixing 12h of 30.0 U enzyme room temperature absorption, by buffer solution for cleaning, being fixed laccase, the enzyme rate of recovery is 52.24%.
Immobilization laccase is repaired for polycyclic aromatic hydrocarbon pollution; Soil adopts mud, and soil ratio is 3:1, and solution part is HAc-NaAc buffer solution (pH=4), and the initial concentration of pollutant is 100 μ g/ml; Immobilization laccase drops into the amount 1g/50ml in soil, and temperature is 20 DEG C, and pH value is 4, and the time is 72 h.
Embodiment 7:
Taking shitosan as carrier, glutaraldehyde as cross linker has carried out the immobilization of laccase; Concrete operations are: take a certain amount of shitosan and be dissolved in volume ratio 5 % glacial acetic acid, make shitosan precipitation with 5 mol/L NaOH, be washed to neutrality, wet shape chitin carrier is made in suction filtration dehydration, then take after 0.5g shitosan and 20mL volume ratio 5 % glutaraldehyde cross-linking 10h, add 30.0 U enzyme room temperature absorption to fix 15 h, by buffer solution for cleaning, being fixed laccase, the enzyme rate of recovery is 52.24%.
Immobilization laccase is repaired for polycyclic aromatic hydrocarbon pollution; Soil adopts mud, and soil ratio is 3:1, and solution part is HAc-NaAc buffer solution (pH=4), and the initial concentration of pollutant is 100 μ g/ml; Immobilization laccase drops into the amount 1g/50ml in soil, and temperature is 40 DEG C, and pH value is 6, and the time is 72 h.
Embodiment 8:
Taking shitosan as carrier, glutaraldehyde as cross linker has carried out the immobilization of laccase; Concrete operations are: take a certain amount of shitosan and be dissolved in volume ratio 3 % glacial acetic acid, make shitosan precipitation with 4mol/L NaOH, be washed to neutrality, wet shape chitin carrier is made in suction filtration dehydration, then take after 0.3g shitosan and 18mL volume ratio 5 % glutaraldehyde cross-linking 8 ~ 10h, add 30.0 U enzyme room temperature absorption to fix 12 ~ 15 h, by buffer solution for cleaning, being fixed laccase, the enzyme rate of recovery is 52.24%.
Immobilization laccase is repaired for polycyclic aromatic hydrocarbon pollution; Soil adopts mud, and soil ratio is 3:1, and solution part is HAc-NaAc buffer solution (pH=4), and the initial concentration of pollutant is 100 μ g/ml; Immobilization laccase drops into the amount 1g/50ml in soil, and temperature is 30 DEG C, and pH value is 5, and the time is 72 h.
Conclusion: adopt the restorative procedure of this immobilization laccase of the present invention to polycyclic aromatic hydrocarbon pollution, make organic polluted soil obtain effective reparation.
Claims (3)
1. the restorative procedure of immobilization laccase to polycyclic aromatic hydrocarbon pollution, is characterized in that: concrete steps are as follows: immobilization laccase is repaired for polycyclic aromatic hydrocarbon pollution; Soil adopts mud, and soil ratio is 3:1, and solution part is HAc-NaAc buffer solution (pH=4), and the initial concentration of pollutant is 100 μ g/ml; Immobilization laccase drops into the amount 1g/50ml in soil, and temperature is 20 ~ 40 DEG C, and pH value is 4 ~ 6, and the time is 72 h.
2. by the restorative procedure of immobilization laccase claimed in claim 1 to polycyclic aromatic hydrocarbon pollution, it is characterized in that: the method that laccase is fixed is as follows:
Taking nylon wire as carrier, glutaraldehyde as cross linker, is first cross-linked the process for fixation adsorbing afterwards; Concrete operations are: nylon wire is cut into the square of 0.5cm × 0.5cm ~ 1.5cm × 1.5cm size, with containing mass ratio 15 ~ 20%CaCl
2process 15 ~ 20min with the formalin that mass ratio is 15 ~ 20 % water, after cleaning, blot, in 5 ~ 8 mol/L HCl, be hydrolyzed 40 ~ 50 min, be washed till pH neutrality, blot; In the glutaraldehyde solution that nylon wire is 5 ~ 8% in mass concentration, after crosslinked 6 ~ 8h, add 30U laccase solution to fix 8 ~ 10h, with buffer solution for cleaning, being fixed laccase, now the enzyme rate of recovery is 55.56%.
3. by the restorative procedure of immobilization laccase claimed in claim 1 to polycyclic aromatic hydrocarbon pollution, it is characterized in that: the method that laccase is fixed is as follows:
Taking shitosan as carrier, glutaraldehyde as cross linker has carried out the immobilization of laccase; Concrete operations are: take a certain amount of shitosan and be dissolved in volume ratio 1 ~ 5 % glacial acetic acid, make shitosan precipitation with 2 ~ 5 mol/L NaOH, be washed to neutrality, wet shape chitin carrier is made in suction filtration dehydration, then, after taking 0.1 ~ 0.5g shitosan and 15 ~ 20mL and volumetric concentration and being 5 % glutaraldehyde cross-linking 8 ~ 10h, add 30.0 U enzyme room temperatures absorption to fix 12 ~ 15 h, by buffer solution for cleaning, being fixed laccase, the enzyme rate of recovery is 52.24%.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104209316A (en) * | 2014-09-25 | 2014-12-17 | 沈阳工业大学 | Repairing method for soil polluted by carbofuran and chlorpyrifos through immobilized laccase |
| CN109266641A (en) * | 2018-09-27 | 2019-01-25 | 福建海峡石墨烯产业技术研究院有限公司 | A kind of method and detecting electrode that enzyme is fixed on graphene based on glutaraldehyde |
| CN110846046A (en) * | 2019-11-28 | 2020-02-28 | 浙江海洋大学 | Composite repairing agent for organic contaminated soil, preparation method and application thereof |
| CN113025337A (en) * | 2021-02-04 | 2021-06-25 | 浙江卓锦环保科技股份有限公司 | Preparation method of soil remediation preparation and application of soil remediation preparation in soil remediation |
| CN114477473A (en) * | 2022-03-09 | 2022-05-13 | 南京农业大学 | Method for removing water and soil polycyclic aromatic hydrocarbons by repeatedly using immobilized laccase |
-
2012
- 2012-11-29 CN CN201210495811.4A patent/CN103846276A/en active Pending
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104209316A (en) * | 2014-09-25 | 2014-12-17 | 沈阳工业大学 | Repairing method for soil polluted by carbofuran and chlorpyrifos through immobilized laccase |
| CN109266641A (en) * | 2018-09-27 | 2019-01-25 | 福建海峡石墨烯产业技术研究院有限公司 | A kind of method and detecting electrode that enzyme is fixed on graphene based on glutaraldehyde |
| CN110846046A (en) * | 2019-11-28 | 2020-02-28 | 浙江海洋大学 | Composite repairing agent for organic contaminated soil, preparation method and application thereof |
| CN113025337A (en) * | 2021-02-04 | 2021-06-25 | 浙江卓锦环保科技股份有限公司 | Preparation method of soil remediation preparation and application of soil remediation preparation in soil remediation |
| CN114477473A (en) * | 2022-03-09 | 2022-05-13 | 南京农业大学 | Method for removing water and soil polycyclic aromatic hydrocarbons by repeatedly using immobilized laccase |
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Application publication date: 20140611 |