CN105233458A - Method for degrading bisphenol A through white-rot fungus crude enzyme - Google Patents
Method for degrading bisphenol A through white-rot fungus crude enzyme Download PDFInfo
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- CN105233458A CN105233458A CN201510669380.2A CN201510669380A CN105233458A CN 105233458 A CN105233458 A CN 105233458A CN 201510669380 A CN201510669380 A CN 201510669380A CN 105233458 A CN105233458 A CN 105233458A
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Abstract
The invention discloses a method for degrading bisphenol A through a white-rot fungus crude enzyme. The white-rot fungi crude enzyme is prepared, a tartaric acid buffer solution, a bisphenol A solution, a hydrogen peroxide solution and veratryl alcohol are added, the pH value of a reaction system is controlled to range from 2.5 to 3, the concentration of the veratryl alcohol is 10 mmol/L, the temperature is kept between 20 DEG C and 50 DEG C, the reaction lasts for 30 minutes to 50 minutes, 1.0 mL-3.0 mL methyl alcohol is added for stopping the reaction, and therefore the bisphenol A is degraded through the white-rot fungi crude enzyme. A bio-enzyme catalytic process is adopted in the method, and the method has the beneficial effects of being high in removing efficiency, convenient, safe, free of pollution and the like; and as amboceptor matter is added, the biocatalysis reaction efficiency can be improved, the reaction time can be shortened, the enzyme using amount is reduced, and the running cost is reduced.
Description
Technical field
The invention belongs to environment remediation technical field, particularly one utilizes microorganism---the method for white-rot fungi crude enzyme liquid degraded bisphenol-A.
Background technology
Bisphenol-A (bisphenolA, BPA), chemical formula is C
15h
16o
2, one of persistence organic pollutant, has " three cause " effect.UNEP (United Nations Environment Program) it can be used as continue ozone hole and global warm after need the third generation environmental problem of urgently administering to bring into schedule.How to reduce this harm, by which type of method to be degraded this pollutant, or to be translated into that other is pollution-free, to endanger little compound be one of sciences problems anxious to be resolved.Physical absorption and the chemical degradation method of constantly discovery are in recent years good to the removal effect of BPA in environment, but its drawback is also apparent.Physical adsorption process does not relate to the degraded of BPA, effect is simple and adsorption time is short, but because physical absorption has invertibity, possibility back suction is attached under certain conditions for the BPA removed, BPA gets back among reactant system again, so physical adsorption process is insecure to a certain extent, it does not reach the real object removed.Chemical method degraded BPA shows quite high efficiency, even may permineralization BPA under catalytic condition.But consider that BPA concentration is lower than other common interference substrate concentrations, removes to safe range cost high, complicated operation, may produce the environmental problems such as secondary pollution in addition in processing procedure.Therefore seek a kind of removal efficiency high, convenient, safety and free of contamination minimizing technology is imperative.
White-rot fungi be a class can cause wood rot decompose fungi, be also a kind of important environmental contaminants degradative fungi.Different from other fungi, the metabolism of white-rot fungi to BPA depends on the lignin-degrading enzymes (lignin peroxidase of a series of exocytosis, manganese peroxidase and laccase) effect (AsgherM, BhattiHN, AshrafMandLeggeRL.Recentdevelopmentsinbiodegradationofin dustrialpollutantsbywhiterotfungiandtheirenzymesystemBio degradation.2008, 19:771-783), therefore the crude enzyme liquid of its secretion can the degraded of apparent white-rot fungi to bisphenol-A itself to bisphenol-A degraded.
In the method for traditional decomposition bisphenol-A, it is physical adsorption process that the bisphenol-A of high concentration uses many, but there is invertibity in absorption, and bisphenol-A has low concentration benefit, still damaging effect is being had far below other common interference substrate concentrations, therefore low concentration bisphenol-A can not be absorbed completely, it can not reach the real object removing harm; Usually using chemical method for removing the low bisphenol-A of concentration, in chemical method, removing bisphenol-A to the chemical reagent amount of safe range not only demand is large, operating procedure is more loaded down with trivial details, and easily may produce the environmental problems such as secondary pollution in processing procedure.
Depending on based on white-rot fungi the principle that ectoenzyme carrys out degradation of contaminant, propose a kind of utilization microorganism---white-rot fungi decomposes the new method of bisphenol-A.White-rot fungi growth and breeding is fast, cultivation operation is comparatively easy.People obtain white-rot fungi than being easier in life, the cheap and green safety of cost compare.The ectoenzyme relative amount of white rot fungus secretion is abundanter.Enzymatic reaction has very high catalysis special efficacy, green safety and wide to concentration of substrate claimed range, low concentration material can be removed, use white-rot fungi decomposition bisphenol-A greatly can improve the effect of resolution process bisphenol-A class material, it has outstanding advantage on the wastewater processing technology of catalytic elimination containing bisphenol-A class material, can reach the object effectively removing environmental contaminants repairing environment.
Summary of the invention
Problems such as the object of the invention is the invertibity for existing in prior art is to BPA degradation process, workload is large, cost is high, complex steps, the degree of accuracy are low, provides a kind of white-rot fungi crude enzyme liquid that utilizes to degrade the method for bisphenol-A.
Concrete steps are:
(1) culture medium is prepared
The glucose of 10.0 ~ 20.0 grams, 1.0 ~ 2.0 grams of Tween 80s, 2.0 ~ 3.0 grams of potassium dihydrogen phosphates, 0.5 ~ 1.0 gram of magnesium sulfate, 1.0 ~ 2.0 grams of calcium chloride, 0.001 gram of vitamin B1,0.5 ~ 2.0 gram of sodium chloride, 0.2 ~ 1.0 gram of manganese sulfate and 1.0 ~ 3.0mL trace element mixed solution is added in triangular flask, be settled to 0.05 ~ 1.5 liter, sterilizing 20 minutes under 1.103MPa, 121 DEG C of conditions, obtained limit nitrogen fluid nutrient medium is for subsequent use.
(2) preparation of white-rot fungi crude enzyme liquid
Be that 1:9 measures the obtained limit nitrogen fluid nutrient medium of spore suspension and step (1) according to volume ratio, spore suspension is inoculated in limit nitrogen fluid nutrient medium, at 25 ~ 40 DEG C, be placed in shaken cultivation case under the condition of 100 ~ 200 revs/min to suspend cultivation 5 ~ 7 days, gained nutrient solution slowly grinds in beaker, the cell homogenates nutrient solution obtained after fragmentation through refrigerated centrifuge at 4 DEG C, under the condition of 8000 revs/min centrifugal 5 ~ 15 minutes, get supernatant, then with aseptic 0.45 μm of membrane filtration, filtrate after filtration is placed in pellicle, room temperature is got circulating water and is dialysed 12 hours, then molecular weight 10-kDa ultrafiltration concentration, the dislysate obtained is white-rot fungi crude enzyme liquid, the refrigerator being placed in 4 DEG C is preserved, for subsequent use.
(3) degraded of bisphenol-A
Taking 1.0 ~ 3.0mL concentration is that the tartaric acid buffer of 250mmol/L adds in ready sterilized conical flask, then 20 ~ 50 DEG C of waters bath with thermostatic control are placed in, add the white-rot fungi crude enzyme liquid that 1.0 ~ 3.0mL step (2) is obtained again, 5mL concentration is the bisphenol-A solution of 0.20mmol/L, 0.5 ~ 2.0mL concentration is hydrogen peroxide and the veratryl alcohol of 0.3 ~ 0.5mmol/L, the pH value controlling reaction system is 2.5 ~ 3, in reaction system, veratryl alcohol concentration is 10mmol/L, react 30 ~ 50 minutes, add 1.0 ~ 3.0mL methyl alcohol cessation reaction, namely realize utilizing white-rot fungi crude enzyme liquid degraded bisphenol-A.
The present invention is according to the means of prior art process bisphenol-A and effect, and white-rot fungi relies on the feature of ectoenzyme degraded environmental contaminants, devises a kind of method using white rot fungus secretion enzyme to degrade to bisphenol-A.In the method, selected process material is only microorganism, and growth and breeding is taken soon, conveniently, with low cost, and biology enzyme has efficient catalytic ability.In addition, operate comparatively easy, and cost compare is low, not easily causes the pollution of secondary.
Accompanying drawing explanation
Fig. 1 is the degradation rate variation with temperature situation of white-rot fungi crude enzyme liquid catalytic degradation bisphenol-A in the embodiment of the present invention.
Fig. 2 is the situation of change of degradation rate with pH value of white-rot fungi crude enzyme liquid catalytic degradation bisphenol-A in the embodiment of the present invention.
Fig. 3 is that the degradation rate of white-rot fungi crude enzyme liquid catalytic degradation bisphenol-A in the embodiment of the present invention is with the situation of change starting agent hydrogen peroxide concentration.
Fig. 4 be veratryl alcohol in the embodiment of the present invention interpolation on white-rot fungi crude enzyme liquid degraded bisphenol-A affect situation.
Fig. 5 is the comparison diagram of degradation rate when adding veratryl alcohol in white-rot fungi crude enzyme liquid catalytic degradation bisphenol-A in the embodiment of the present invention and do not add veratryl alcohol.
Detailed description of the invention
The technological means realized to make this patent, creation characteristic, reach object and effect clearly and be easy to understand, be further elaborated this patent below in conjunction with the drawings and specific embodiments, listed embodiment is only the present invention is described and does not limit the present invention.
The micro-mixed solution used in the present embodiment contains: 3.0gMgSO
47H
20,1.0gNaCl, 0.1gCaCI
22H
2o, 0.1gFeSO
47H
2o, 0.1gCoSO
4, 0.01gAlK (S04)
212H
2o, 0.1gZnSO
47H
2o, 0.01gCuSO
45H
2o, 0.01gH
3bO
3and 0.01gNa
2moO
42H
2o.
(1) culture medium is prepared
The glucose of 10 grams, 1.0 grams of Tween 80s, 2.56 grams of potassium dihydrogen phosphates, 0.71 gram of magnesium sulfate, 1.56 grams of calcium chloride, 0.001 gram of Cobastab are added in triangular flask
1, 1.0 grams of sodium chloride, 0.5 gram of manganese sulfate and 2.0mL trace element mixed solutions, be settled to 1.0 liters, sterilizing 20 minutes under 1.103MPa, 121 DEG C of conditions, obtained limit nitrogen fluid nutrient medium is for subsequent use.
(2) preparation of white-rot fungi crude enzyme liquid
Be that 1:9 measures the obtained limit nitrogen fluid nutrient medium of spore suspension and step (1) according to volume ratio, spore suspension is inoculated in limit nitrogen fluid nutrient medium, at 37 DEG C, be placed in shaken cultivation case under the condition of 150 revs/min to suspend cultivation 6 days, gained nutrient solution slowly grinds in beaker, the cell homogenates nutrient solution obtained after fragmentation through refrigerated centrifuge at 4 DEG C, under the condition of 8000 revs/min centrifugal 10 minutes, get supernatant, then with aseptic 0.45 μm of membrane filtration, filtrate after filtration is placed in pellicle, room temperature is got circulating water and is dialysed 12 hours, then molecular weight 10-kDa ultrafiltration concentration, the dislysate obtained is white-rot fungi crude enzyme liquid, the refrigerator being placed in 4 DEG C is preserved, for subsequent use.
Adopt following methods to measure enzyme to live: get teat glass 1, add veratryl alcohol solution, tartaric acid buffer, crude enzyme liquid, mixing, adds 0.1mLH
2o
2reaction, recorded once at the absorbance at 310nm place every 15 seconds after reaction starts, until absorbance maintains steady state.Each reaction system does three parallel tests.1 Ge Meihuo unit (U) is defined as the enzyme amount needed for oxidation per minute 1 μm of ol Li Lu alcohol.Formulae discovery enzyme is adopted to live:
Wherein: 9300mol
-1lcm
-1for the molar absorption coefficient of product veratraldehyde under 310nm that lignin peroxidase catalysis veratryl alcohol is formed; V is reactant liquor cumulative volume; X is the volume of the enzyme liquid added.
(3) degraded of bisphenol-A
Taking 2mL concentration is respectively that the tartaric acid buffer of 250mmol/L adds in the conical flasks of ready sterilized 7 numberings, then 20 are placed in respectively, 25,30,35,40, in 45 and 50 DEG C of waters bath with thermostatic control, add the obtained white-rot fungi crude enzyme liquid of 2mL step (2) more respectively, bisphenol-A solution that 5mL concentration is 0.20mmol/L, 1.0mL concentration is the hydrogen peroxide of 0.4mmol/L, the pH value controlling reaction system is 3, react 50 minutes, add 2.0mL methyl alcohol cessation reaction, namely realize utilizing white-rot fungi crude enzyme liquid degraded bisphenol-A.
Replace hydrogen peroxide with deionized water again, repeat above-mentioned steps, as the control group of the present embodiment.
By reactant liquor centrifugal 15min under the speed of 4500 revs/min respectively obtained after the degraded of the present embodiment bisphenol-A, get supernatant and carry out the mensuration of content of bisphenol A and calculate degradation rate (computing formula is: degradation rate=(C-B) × 100%/A; Wherein A is reaction system bisphenol-A initial concentration; B is process bisphenol-A ultimate density; C is the bisphenol-A ultimate density in contrast; Adopt 4-antipyrine method to measure content of bisphenol A), result as shown in Figure 1, along with the rising degraded of temperature take the lead in increasing gradually after reduce gradually.When reaction temperature is 30 DEG C, degradation rate reaches and is 74.70% to the maximum.
Repeat step (1) ~ (3) of the present embodiment, only change pH value=2 of reaction system, 3,4,5,6,7 and 8, by reactant liquor centrifugal 15min under the speed of 4500 revs/min, get supernatant and carry out the mensuration of content of bisphenol A and calculate degradation rate.As shown in Figure 2, when pH value is 4.0, degradation rate is 75.09% to result.
Repeat step (1) ~ (3) of the present embodiment, only change the concentration of the hydrogen peroxide solution added, be respectively 0.1,0.2,0.3,0.4,0.5,0.6 and 0.7mmol/L, by reactant liquor centrifugal 15min under the speed of 4500 revs/min, get supernatant and carry out the mensuration of content of bisphenol A and calculate degradation rate.The concrete outcome obtained as shown in Figure 3, degrade and take the lead in increasing rear reduction gradually by the increase along with hydrogen peroxide concentration.When hydrogen peroxide concentration is 0.4mmol/L, degradation rate reaches maximum, and its value is 75.14%.
White-rot fungi, while secretion peroxidase, also secretes another kind of compound veratryl alcohol (VA).This compound is a kind of natural substrate of enzyme, is also the catalyst of enzyme simultaneously, and in order to improve the degradation rate of bisphenol-A further, experiment has also investigated the interpolation of veratryl alcohol to the impact of white-rot fungi crude enzyme liquid degraded bisphenol-A.
Experiment gets 2mL crude enzyme liquid, 2mL tartaric acid buffer, 1mL veratryl alcohol solution, 0.1mL bisphenol-A (0.24mmol/L) solution, under oscillating condition, starts reaction with hydrogen peroxide.This reaction system is designed to 6 groups, respectively the VA solution of correspondence 1,2,4,6,8 and 10mmol/L, and each reaction group sets up 7 test tubes, respectively corresponding reaction time 0.5,3,5,10,20,30 and 60min.Extract reaction solution mensuration bisphenol A concentration at special time, as shown in Figure 4, as can be seen from the figure each group reaction substantially all reached balance in 30 minutes to the concrete outcome obtained, and along with the increase of veratryl alcohol concentration, the bisphenol A concentration recorded is lower, and namely degradation rate is higher.When the addition of veratryl alcohol is 10mmol/L, its degradation effect is best, be 97.09% by calculating the degradation rate of bisphenol-A, 21.95% is exceeded than the most high degradation rate 75.14% when not adding veratryl alcohol, namely, when having under veratryl alcohol existent condition and veratryl alcohol concentration is 10mmol/L, white-rot fungi crude enzyme liquid substantially can degradable bisphenol-A.
The method is simple to operate, with low cost, and green safety is pollution-free, and degradation rate is high, and the degradation rate of bisphenol-A can reach 97.09%.
Claims (1)
1. utilize a method for white-rot fungi crude enzyme liquid degraded bisphenol-A, it is characterized in that concrete steps are:
(1) culture medium is prepared
The glucose of 10.0 ~ 20.0 grams, 1.0 ~ 2.0 grams of Tween 80s, 2.0 ~ 3.0 grams of potassium dihydrogen phosphates, 0.5 ~ 1.0 gram of magnesium sulfate, 1.0 ~ 2.0 grams of calcium chloride, 0.001 gram of vitamin B1,0.5 ~ 2.0 gram of sodium chloride, 0.2 ~ 1.0 gram of manganese sulfate and 1.0 ~ 3.0mL trace element mixed solution is added in triangular flask, be settled to 0.05 ~ 1.5 liter, sterilizing 20 minutes under 1.103MPa, 121 DEG C of conditions, obtained limit nitrogen fluid nutrient medium is for subsequent use;
(2) preparation of white-rot fungi crude enzyme liquid
Be that 1:9 measures the obtained limit nitrogen fluid nutrient medium of spore suspension and step (1) according to volume ratio, spore suspension is inoculated in limit nitrogen fluid nutrient medium, at 25 ~ 40 DEG C, be placed in shaken cultivation case under the condition of 100 ~ 200 revs/min to suspend cultivation 5 ~ 7 days, gained nutrient solution slowly grinds in beaker, the cell homogenates nutrient solution obtained after fragmentation through refrigerated centrifuge at 4 DEG C, under the condition of 8000 revs/min centrifugal 5 ~ 15 minutes, get supernatant, then with aseptic 0.45 μm of membrane filtration, filtrate after filtration is placed in pellicle, room temperature is got circulating water and is dialysed 12 hours, then molecular weight 10-kDa ultrafiltration concentration, the dislysate obtained is white-rot fungi crude enzyme liquid, the refrigerator being placed in 4 DEG C is preserved, for subsequent use,
(3) degraded of bisphenol-A
Taking 1.0 ~ 3.0mL concentration is that the tartaric acid buffer of 250mmol/L adds in ready sterilized conical flask, then 20 ~ 50 DEG C of waters bath with thermostatic control are placed in, add the white-rot fungi crude enzyme liquid that 1.0 ~ 3.0mL step (2) is obtained again, 5m concentration is the bisphenol-A solution of 0.20mmol/L, 0.5 ~ 2.0mL concentration is hydrogen peroxide and the veratryl alcohol of 0.3 ~ 0.5mmol/L, the pH value controlling reaction system is 2.5 ~ 3, in reaction system, veratryl alcohol concentration is 10mmol/L, react 30 ~ 50 minutes, add 1.0 ~ 3.0mL methyl alcohol cessation reaction, namely realize utilizing white-rot fungi crude enzyme liquid degraded bisphenol-A.
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Cited By (11)
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| CN105753173A (en) * | 2016-04-09 | 2016-07-13 | 广州聚注专利研发有限公司 | Method for degrading bisphenol A in sewage by virtue of biological activated carbon prepared from oyster mushroom strain |
| CN106006994A (en) * | 2016-05-20 | 2016-10-12 | 佛山市聚成生化技术研发有限公司 | Method for degrading bisphenol A by virtue of crude enzyme liquid of lentinula edodes |
| CN107029379A (en) * | 2017-04-18 | 2017-08-11 | 桂林理工大学 | A kind of method of utilization white-rot fungi crude enzyme liquid degraded bisphenol b |
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| CN105753173A (en) * | 2016-04-09 | 2016-07-13 | 广州聚注专利研发有限公司 | Method for degrading bisphenol A in sewage by virtue of biological activated carbon prepared from oyster mushroom strain |
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| CN107412999A (en) * | 2017-04-18 | 2017-12-01 | 桂林理工大学 | A kind of method for bisphenol-c of being degraded using white-rot fungi crude enzyme liquid |
| CN108163961A (en) * | 2018-01-15 | 2018-06-15 | 湖北民族学院 | A kind of method and system that bisphenol-A is removed using Machilus nanmu crude enzyme liquid |
| CN109354309A (en) * | 2018-11-07 | 2019-02-19 | 环境保护部华南环境科学研究所 | Heavy metal MULTIPLE COMPOSITE treatment process for industrial wastewater |
| CN109384277A (en) * | 2018-11-07 | 2019-02-26 | 环境保护部华南环境科学研究所 | The composite treating agent of comprehensive removal heavy metal out of electroplating wastewater |
| CN109354309B (en) * | 2018-11-07 | 2021-11-12 | 环境保护部华南环境科学研究所 | Heavy metal multiple composite treatment process for industrial wastewater |
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