CN109097351A - A kind of preparation method of the novel immobilised enzymes for oil spilling reparation - Google Patents
A kind of preparation method of the novel immobilised enzymes for oil spilling reparation Download PDFInfo
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- CN109097351A CN109097351A CN201810866034.7A CN201810866034A CN109097351A CN 109097351 A CN109097351 A CN 109097351A CN 201810866034 A CN201810866034 A CN 201810866034A CN 109097351 A CN109097351 A CN 109097351A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- 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
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
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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
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/344—Biological treatment of water, waste water, or sewage characterised by the microorganisms used for digestion of mineral oil
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/32—Hydrocarbons, e.g. oil
Abstract
The present invention relates to field of environmental biotechnology more particularly to a kind of preparation methods of the novel immobilised enzymes for oil spilling reparation, comprising the following steps: (1) screens microalgae, enrichment culture；(2) it grinds, is cleaned with phosphate buffer solution, resuspension, obtain enzymolysis liquid；(3) konjaku glucomannan airsetting sol solution, freeze-drying is added in enzymolysis liquid；(4) it is freeze-dried；(5) it crushes to get the novel immobilised enzymes for oil spilling reparation.The mechanical strength of immobilised enzymes oil spilling degradation agent of the invention is higher, and resistance to physical impact, mass-transfer performance is higher, while the stability of immobilised enzymes oil spilling degradation agent is very high, is not easily broken decomposition, can rapidly, expeditiously degraded oil, nontoxic secondary residual；Aerogel material adsorption capacity is good, porous, and porosity is high, and aperture is big；Cost of manufacture is lower, and preparation process is simple and easy, is suitble to large-scale production and application.
Description
Technical field
The present invention relates to field of environmental biotechnology more particularly to a kind of systems of the novel immobilised enzymes for oil spilling reparation
Preparation Method.
Background technique
Oil pollution refers to that during exploitation, refining, storing, use, crude oil and various petroleum products enter environment
It is polluted caused by and.It is currently mostly pollution of the petroleum to ocean, it has also become global serious problems.Oil pollution is not single
One the problem of, can cause the chain pollution problem such as soil, water body, air by it.Petroleum pollution in ocean harm be various such as
Form oil film in the water surface and hinder the gas exchanges between water body and atmosphere: oils is adhered to fish, algae and planktonic organism
On, it is dead to cause marine organisms, and destroy seabird living environment, seabird death and population quantity is caused to decline.Oil pollution is also
Aquatic products quality decline can be made to cause economic loss.
Administers petroleum hydrocarbon contaminated soil and is also only limitted to physics and chemical method before the 1980s, i.e., heat treatment and
Chemical Leaching method, but soil texture and component can be destroyed, it is expensive and be difficult to carry out.Currently, the biology of oil pollution is repaired
Multiple approach is mainly based on microorganism remediation.Biological prosthetic is toxic in the vital metabolic activity reduction soil environment using biology
The concentration of nuisance makes contaminated soil be restored to the process of health status.Biological treating be it is universally acknowledged, have it is safe and non-toxic,
Efficiently remedying oil-polluted feature is the optimal path of Revegetation of Eco-environment using the oil spilling that technical treatment degradation is removed,
To protection China's marine eco-environment, the sustainable development of marine resources is maintained to be of great significance and application value.And
In microorganism remediation technology, enzyme degradation technique accounts for very big specific gravity.As a kind of biodegradation agent, enzyme has than generalization
The features such as chemical catalyst is high-efficient, environmentally protective.But during marine oil spill is biological prosthetic, there is enzymes to be lost seriously,
The problems such as enzymatic activity sharp fall, remediation efficiency is lower.In addition, marine oil spill multidigit needs enzyme preparation to float in extra large body surface face
On bubbling through the water column.Therefore, finding a kind of novel enzyme for oil spilling reparation is skilled person's technical problem urgently to be resolved.
Summary of the invention
The present invention is reduced, is repaired to overcome in oil spilling reparation traditional enzyme degradation technique to be lost serious, enzymatic activity there is enzyme
The low problem of multiple rate, provides that a kind of mechanical strength is higher, mass-transfer performance is higher, repair rate is high, is suitble to large-scale production and answers
The preparation method of novel immobilised enzymes for oil spilling reparation.
To achieve the goals above, the invention adopts the following technical scheme:
A kind of preparation method of the novel immobilised enzymes for oil spilling reparation, comprising the following steps:
(1) microbial identification separation will be carried out from the water sample of oil spill accident collection in worksite, microalgae is screened out from it, to the microalgae
It carries out enrichment culture 7~15 days, obtains algae solution；
(2) algae solution that step (1) obtains is filtered to the filter membrane for being enriched with frond, the filter membrane of frond will be enriched in liquid nitrogen
It is taken out after cooling 30~60s, 3~5g quartz sand is added and is ground in mortar, wash out algae solution with phosphate buffer solution, in
Centrifuge 10000r/min is centrifuged 15min, takes supernatant；Sediment fraction is cleaned 2 times with phosphate buffer solution again, is merged 3 times
The supernatant of centrifugally operated recycles above-mentioned phosphate buffer solution to carry out resuspension, obtains enzymolysis liquid；
(3) konjaku glucomannan airsetting sol solution is added in the resulting enzymolysis liquid of step (2), stirred evenly, in -10~-30 DEG C
Under the conditions of temperature, 8~16h of precooling obtains freezing inoculation liquid；Konjaku glucomannan is connected by glucose and β -1,4 glycosidic bond
The macromolecule polysaccharide come has high viscosity, good film forming and gelation, has certain bioactivity；
(4) step (3) is obtained into freezing inoculation liquid freeze-drying process, obtains the konjaku glucomannan aeroge containing enzyme；
(5) the konjaku glucomannan aeroge obtained in step (4) containing enzyme little particle is ground into repair to get for oil spilling
Multiple novel immobilised enzymes.
Novel immobilised enzymes prepared by the present invention for oil spilling reparation plays catalytic action in certain spatial dimension, and
Can repeatedly with the enzyme of continuous use.Enzyme immobilization technology is able to maintain the stabilization of enzymatic activity compared with other bioremediation technologies,
Increasing enzyme density, improves degradation rate, treatment effeciency is high, and it is strong to the tolerance of environment, and can recycle, the rate of recovery is high, energy
It enough effectively improves target area inland sea and is permeated with oily pollution.
Preferably, in step (1), the process conditions of enrichment culture are as follows: culture medium is boiled into 10~20min with seawater,
Nutritive salt is added after suction filtration, the concentration of each component is NH in the nutritive salt4Cl 0.1~0.3g/L, K2HPO40.05~
0.005~0.02g/L of 0.1g/L and ironic citrate.
Chlorella crude enzyme liquid to carry out the later period extracts experiment and algae passage, carries out the enrichment culture of chlorella.It is described
Enrichment culture are as follows: culture medium is boiled into 12~18min with seawater, is the supplement necessary nutriment of chlorella growth after suction filtration,
Corresponding nutritive salt is added, NH is added4Cl is supplement nitrogen source, K2HPO4Can supplement the nutrients while supplementing phosphorus source Element Potassium,
The addition of ironic citrate can then meet chlorella during the growth process to the needs of ferro element, therefore each group in these three nutritive salt
The concentration divided is respectively NH4Cl 0.2g/L, K2HPO40.05g/L and ironic citrate 0.01g/L.
Preferably, grinding temperature is 0~4 DEG C in step (2).
Preferably, the concentration of the phosphate buffer solution is 1.0~1.5mmol/L in step (2).
Preferably, the pH of the phosphate buffer solution is 7.0~8.2 in step (2).
Preferably, the konjaku glucomannan aeroge is cross-linking modified by nano-photocatalyst in step (3)
Konjaku glucomannan aeroge.
By the cross-linking modified konjaku glucomannan aeroge of nano-photocatalyst, konjaku glucomannan aeroge is being kept
On the basis of network structure, nano-photocatalyst is loaded, so that degradation of the present invention for the novel immobilised enzymes of oil spilling reparation
During, the light of nature is made full use of, the effect with photocatalytic redox degradation of organic substances is synergistic, improves molten oil
Degradation rate.
Preferably, the nano-photocatalyst is carbon nanotube/g-C3N4Composite catalyst has preferable biofacies
Capacitive and biggish specific surface area, while belonging to visible light catalyst, light utilization efficiency, the molten oil of photocatalytic degradation have been expanded significantly
Organic matter it is high-efficient.
Therefore, the invention has the following beneficial effects:
(1) mechanical strength of immobilised enzymes oil spilling degradation agent is higher, and resistance to physical impact, mass-transfer performance is higher, while immobilised enzymes
The stability of oil spilling degradation agent is very high, is not easily broken decomposition, can rapidly, expeditiously degraded oil, nontoxic secondary residual；Airsetting
Glue material adsorption capacity is good, porous, and porosity is high, and aperture is big；Cost of manufacture is lower, and preparation process is simple and easy, is suitble to scale
Production and application；
(2) chlorella is as the microorganism being widely present, have the characteristics that easily to cultivate, breed it is very fast.Chlorella is in the present invention
From oil spill accident collection in worksite, separation, culture and obtain, by domestication after, have efficient degradation rate, on this basis, cooperate gas
Gel rubber material adsorption effect, degradation effect are obvious；
(3) aerogel material for using Photocatalyst further increases the effect that oil spilling is removed in degradation using photocatalysis
Fruit.
Specific embodiment
Below by specific embodiment, the technical solutions of the present invention will be further described.
In the present invention, if not refering in particular to, all devices and raw material is commercially available or the industry is common are following
Method in embodiment is unless otherwise instructed conventional method in that art.
Embodiment 1
(1) microbial identification separation will be carried out from the water sample of oil spill accident collection in worksite, microalgae is screened out from it, to the microalgae
It carries out enrichment culture 15 days, obtains algae solution；The process conditions of enrichment culture are as follows: culture medium is boiled into 10min with seawater, after suction filtration
Nutritive salt is added, the concentration of each component is NH in the nutritive salt4Cl 0.1g/L, K2HPO40.1g/L and ironic citrate
0.02g/L；
(2) algae solution for obtaining step (1) filters, and the filter membrane for being enriched with frond is wrapped up with masking foil, places cooling in liquid nitrogen
It is taken out after 30s, 3g quartz sand is added and is ground in mortar, grinding temperature is 0 DEG C；The phosphate-buffered for being 7.0 with pH is molten
Liquid washes out algae solution, is centrifuged 15min in centrifuge 10000r/min, takes supernatant；Sediment fraction is clear with phosphate buffer solution again
It washes 2 times, merges the supernatant of 3 centrifugally operateds, recycle above-mentioned phosphate buffer solution to carry out resuspension, obtain enzymolysis liquid；
(3) konjaku glucomannan airsetting sol solution is added in the resulting enzymolysis liquid of step (2), stirs evenly, is put into -10 DEG C of ice
Case, precooling 8h；
(4) step (3) is obtained into freezing inoculation liquid and is put into freeze drier, freeze-drying process obtains the konjaku Portugal containing enzyme
Sweet glycan aeroge；
(5) aeroge containing enzyme will be obtained in step (4) and is ground into little particle, obtain the novel immobilised enzymes for oil spilling reparation.
Embodiment 2
(1) microbial identification separation will be carried out from the water sample of oil spill accident collection in worksite, microalgae is screened out from it, to the microalgae
It carries out enrichment culture 20 days, obtains algae solution；The process conditions of enrichment culture are as follows: culture medium is boiled into 20min with seawater, after suction filtration
Nutritive salt is added, the concentration of each component is NH in the nutritive salt4Cl 0.3g/L, K2HPO40.05g/L and ironic citrate
0.005g/L；
(2) algae solution for obtaining step (1) filters, and the filter membrane for being enriched with frond is wrapped up with masking foil, places cooling in liquid nitrogen
It is taken out after 60s, 4g quartz sand is added and is ground in mortar, grinding temperature is 4 DEG C；The phosphate-buffered for being 8.2 with pH is molten
Liquid washes out algae solution, is centrifuged 15min in centrifuge 10000r/min, takes supernatant；Sediment fraction is clear with phosphate buffer solution again
It washes 2 times, merges the supernatant of 3 centrifugally operateds, recycle above-mentioned phosphate buffer solution to carry out resuspension, obtain enzymolysis liquid；
(3) konjaku glucomannan airsetting sol solution is added in the resulting enzymolysis liquid of step (2), stirs evenly, is put into -20 DEG C of ice
Case, precooling 12h；
(4) step (3) is obtained into freezing inoculation liquid and is put into freeze drier, freeze-drying process obtains the konjaku Portugal containing enzyme
Sweet glycan aeroge；
(5) aeroge containing enzyme will be obtained in step (4) and is ground into little particle, obtain the novel immobilised enzymes for oil spilling reparation.
Embodiment 3
(1) microbial identification separation will be carried out from the water sample of oil spill accident collection in worksite, microalgae is screened out from it, to the microalgae
It carries out enrichment culture 20 days, obtains algae solution；The process conditions of enrichment culture are as follows: culture medium is boiled into 10~20min with seawater, is taken out
Nutritive salt is added after filter, the concentration of each component is NH in the nutritive salt4Cl 00.2g/L, K2HPO40.08g/L and citric acid
Iron 0.01g/L；
(2) algae solution for obtaining step (1) filters, and the filter membrane for being enriched with frond is wrapped up with masking foil, places cooling in liquid nitrogen
It is taken out after 90s, 4g quartz sand is added and is ground in mortar, grinding temperature is 3 DEG C；Algae is washed out with phosphate buffer solution
Liquid is centrifuged 15min in centrifuge 10000r/min, takes supernatant；Sediment fraction is cleaned 3 times with phosphate buffer solution again, is closed
And the supernatant of 3 centrifugally operateds, it recycles above-mentioned phosphate buffer solution to carry out resuspension, obtains enzymolysis liquid；
(3) konjaku glucomannan airsetting sol solution is added in the resulting enzymolysis liquid of step (2), stirs evenly, is put into -25 DEG C of ice
Case, precooling 18h；
(4) step (3) is obtained into freezing inoculation liquid and is put into freeze drier, freeze-drying process obtains the konjaku Portugal containing enzyme
Sweet glycan aeroge；
(5) aeroge containing enzyme will be obtained in step (4) and is ground into little particle, obtain the novel immobilised enzymes for oil spilling reparation.
Embodiment 4
Embodiment 4 the difference from embodiment 1 is that, in step (3), taro Glucomannan aeroge used be by carbon nanotube/
g-C3N4The cross-linking modified konjaku glucomannan aeroge of composite catalyst.Wherein, carbon nanotube/g-C3N4Composite catalyst by
Commercially available technical grade carbon nanotube and g-C3N4After aqueous solution is blended according to the amount 1:1 of substance, it is made in 150 DEG C of hydro-thermal reaction 6h.
Embodiment 1-4 is made and is used for the novel immobilised enzymes of oil spilling reparation and carries out being applied to simulation oil spilling environment respectively,
Detect oil spilling degradation effect.Fuel Oil Remaining in culture medium in embodiment 1-4 is measured using spectrophotometry.It is filtered to remove airsetting
Glue uses the de- fragrant petroleum ether of 30ml to clean remaining culture medium in culture bottle as solvent, and is transferred to separatory funnel, is added anhydrous
Sodium sulphate water suction, removes water layer, draws the upper layer 0.5ml oil, is settled to 10ml with petroleum ether.Light absorption value is measured under the conditions of 420nm,
Degradation property is determined referring to crude oil standard curve, and the results are shown in Table 1.
1. measurement result of table
| Performance indicator | Embodiment 1 | Embodiment 2 | Embodiment 3 | Embodiment 4 |
| Oil absorbency | 14.46g/g | 15.24g/g | 17.95g/g | 20.15g/g |
| Degradation rate | 75.40% | 82.12% | 85.78% | 90.25% |
As can be seen from Table 1, using present invention process preparation the novel immobilised enzymes for oil spilling reparation can effective immobilized enzyme,
And its growth activity is kept, crude oil adsorption effect and microalgae degradation effect are preferable.Wherein, by Photocatalyst for overflowing
The microalgae degradation effect for the novel immobilised enzymes that oil is repaired is more preferably.
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the present invention in any form, without departing from power
There are also other variations and modifications under the premise of technical solution documented by benefit requirement.
Claims (7)
1. a kind of preparation method of the novel immobilised enzymes for oil spilling reparation, which comprises the following steps:
(1) microbial identification separation will be carried out from the water sample of oil spill accident collection in worksite, microalgae is screened out from it, to the microalgae
It carries out enrichment culture 7~15 days, obtains algae solution；
(2) algae solution that step (1) obtains is filtered to the filter membrane for being enriched with frond, the filter membrane of frond will be enriched in liquid nitrogen
It is taken out after cooling 30~60s, 3~5g quartz sand is added and is ground in mortar, wash out algae solution with phosphate buffer solution, in
Centrifuge 10000r/min is centrifuged 15min, takes supernatant；Sediment fraction is cleaned 2 times with phosphate buffer solution again, is merged 3 times
The supernatant of centrifugally operated recycles above-mentioned phosphate buffer solution to carry out resuspension, obtains enzymolysis liquid；
(3) konjaku glucomannan airsetting sol solution is added in the resulting enzymolysis liquid of step (2), stirred evenly, in -10~-30 DEG C
Under the conditions of temperature, 8~16h of precooling obtains freezing inoculation liquid；
(4) step (3) is obtained into freezing inoculation liquid freeze-drying process, obtains the konjaku glucomannan aeroge containing enzyme；
(5) the konjaku glucomannan aeroge obtained in step (4) containing enzyme little particle is ground into repair to get for oil spilling
Multiple novel immobilised enzymes.
2. a kind of preparation method of novel immobilised enzymes for oil spilling reparation according to claim 1, which is characterized in that
In step (1), the process conditions of enrichment culture are as follows: culture medium is boiled into 10~20min with seawater, adds nutritive salt after suction filtration,
The concentration of each component is NH in the nutritive salt4Cl 0.1~0.3g/L, K2HPO40.05~0.1g/L and ironic citrate 0.005
~0.02g/L.
3. a kind of preparation method of novel immobilised enzymes for oil spilling reparation according to claim 1, which is characterized in that
In step (2), grinding temperature is 0~4 DEG C.
4. a kind of preparation method of novel immobilised enzymes for oil spilling reparation according to claim 1, which is characterized in that
In step (2), the concentration of the phosphate buffer solution is 1.0~1.5mmol/L.
5. a kind of preparation method of novel immobilised enzymes for oil spilling reparation according to claim 1, which is characterized in that
In step (2), the pH of the phosphate buffer solution is 7.0~8.2.
6. a kind of preparation side of the novel immobilised enzymes for oil spilling reparation described according to claim 1 or 2 or 3 or 4 or 5
Method, which is characterized in that in step (3), the konjaku glucomannan aeroge is the evil spirit cross-linking modified by nano-photocatalyst
Taro Glucomannan aeroge.
7. a kind of preparation method of novel immobilised enzymes for oil spilling reparation according to claim 6 pair, feature exist
In the nano-photocatalyst is carbon nanotube/g-C3N4Composite catalyst.
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Application publication date: 20181228 |