CN107937388A - A kind of immobilised enzymes oil spilling degradation agent - Google Patents
A kind of immobilised enzymes oil spilling degradation agent Download PDFInfo
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- CN107937388A CN107937388A CN201711182088.3A CN201711182088A CN107937388A CN 107937388 A CN107937388 A CN 107937388A CN 201711182088 A CN201711182088 A CN 201711182088A CN 107937388 A CN107937388 A CN 107937388A
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- immobilised enzymes
- oil spilling
- degradation agent
- activation
- enzyme liquid
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 121
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 121
- 230000015556 catabolic process Effects 0.000 title claims abstract description 84
- 238000006731 degradation reaction Methods 0.000 title claims abstract description 84
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 64
- 239000007788 liquid Substances 0.000 claims abstract description 57
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 54
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims abstract description 54
- 230000004913 activation Effects 0.000 claims abstract description 45
- 238000001994 activation Methods 0.000 claims abstract description 44
- 240000006394 Sorghum bicolor Species 0.000 claims abstract description 33
- 235000011684 Sorghum saccharatum Nutrition 0.000 claims abstract description 33
- 235000015505 Sorghum bicolor subsp. bicolor Nutrition 0.000 claims abstract description 31
- 239000010902 straw Substances 0.000 claims abstract description 31
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims abstract description 27
- 238000010521 absorption reaction Methods 0.000 claims abstract description 18
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 claims abstract description 14
- 238000004132 cross linking Methods 0.000 claims abstract description 14
- 239000000661 sodium alginate Substances 0.000 claims abstract description 14
- 235000010413 sodium alginate Nutrition 0.000 claims abstract description 14
- 229940005550 sodium alginate Drugs 0.000 claims abstract description 14
- 238000002360 preparation method Methods 0.000 claims abstract description 12
- 150000001875 compounds Chemical class 0.000 claims abstract description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- 239000012046 mixed solvent Substances 0.000 claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 9
- 239000003431 cross linking reagent Substances 0.000 claims description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- 239000005457 ice water Substances 0.000 claims description 8
- 239000012528 membrane Substances 0.000 claims description 8
- 241000195649 Chlorella <Chlorellales> Species 0.000 claims description 7
- 239000002202 Polyethylene glycol Substances 0.000 claims description 7
- 239000008367 deionised water Substances 0.000 claims description 7
- 229910021641 deionized water Inorganic materials 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- 229920001223 polyethylene glycol Polymers 0.000 claims description 7
- 239000000843 powder Substances 0.000 claims description 7
- 238000001179 sorption measurement Methods 0.000 claims description 7
- 239000000203 mixture Substances 0.000 claims description 6
- BDKKLAMRIATMPA-UHFFFAOYSA-N C[SiH3].[O] Chemical compound C[SiH3].[O] BDKKLAMRIATMPA-UHFFFAOYSA-N 0.000 claims description 4
- 241000195493 Cryptophyta Species 0.000 claims description 4
- 239000011324 bead Substances 0.000 claims description 4
- 239000003610 charcoal Substances 0.000 claims description 4
- 238000004898 kneading Methods 0.000 claims description 4
- 230000001404 mediated effect Effects 0.000 claims description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 239000002245 particle Substances 0.000 claims description 4
- 238000004321 preservation Methods 0.000 claims description 4
- 230000007420 reactivation Effects 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 3
- 230000008859 change Effects 0.000 claims description 3
- 238000011010 flushing procedure Methods 0.000 claims description 3
- LFQCEHFDDXELDD-UHFFFAOYSA-N tetramethyl orthosilicate Chemical compound CO[Si](OC)(OC)OC LFQCEHFDDXELDD-UHFFFAOYSA-N 0.000 claims description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims 3
- 244000062793 Sorghum vulgare Species 0.000 claims 1
- 238000002242 deionisation method Methods 0.000 claims 1
- 235000019713 millet Nutrition 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 17
- 244000005700 microbiome Species 0.000 abstract description 8
- 239000003209 petroleum derivative Substances 0.000 abstract description 7
- 230000009286 beneficial effect Effects 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 4
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 3
- 238000000354 decomposition reaction Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 229910052740 iodine Inorganic materials 0.000 description 3
- 239000011630 iodine Substances 0.000 description 3
- 238000011031 large-scale manufacturing process Methods 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 2
- 230000001684 chronic effect Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 239000007789 gas Substances 0.000 description 2
- 238000000227 grinding Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000001301 oxygen Substances 0.000 description 2
- 229910052760 oxygen Inorganic materials 0.000 description 2
- 230000002093 peripheral effect Effects 0.000 description 2
- 238000005067 remediation Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- GONAVIHGXFBTOZ-UHFFFAOYSA-N 3,5-difluorobenzoic acid Chemical class OC(=O)C1=CC(F)=CC(F)=C1 GONAVIHGXFBTOZ-UHFFFAOYSA-N 0.000 description 1
- JRLTTZUODKEYDH-UHFFFAOYSA-N 8-methylquinoline Chemical group C1=CN=C2C(C)=CC=CC2=C1 JRLTTZUODKEYDH-UHFFFAOYSA-N 0.000 description 1
- 239000005711 Benzoic acid Substances 0.000 description 1
- 208000005623 Carcinogenesis Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 235000010233 benzoic acid Nutrition 0.000 description 1
- 230000036952 cancer formation Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 231100000504 carcinogenesis Toxicity 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000003344 environmental pollutant Substances 0.000 description 1
- 229910052731 fluorine Inorganic materials 0.000 description 1
- 125000001153 fluoro group Chemical group F* 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000004435 hydrogen atom Chemical group [H]* 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000003534 oscillatory effect Effects 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 230000029553 photosynthesis Effects 0.000 description 1
- 238000010672 photosynthesis Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 125000005575 polycyclic aromatic hydrocarbon group Chemical group 0.000 description 1
- 231100000004 severe toxicity Toxicity 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 239000004575 stone Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
Classifications
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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
- C12N11/18—Multi-enzyme systems
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B09—DISPOSAL OF SOLID WASTE; RECLAMATION OF CONTAMINATED SOIL
- B09C—RECLAMATION OF CONTAMINATED SOIL
- B09C1/00—Reclamation of contaminated soil
- B09C1/10—Reclamation of contaminated soil microbiologically, biologically or by using enzymes
-
- 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
-
- 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
- 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
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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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
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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
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- 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
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
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- Organic Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Environmental & Geological Engineering (AREA)
- Hydrology & Water Resources (AREA)
- Biodiversity & Conservation Biology (AREA)
- Water Supply & Treatment (AREA)
- Molecular Biology (AREA)
- Oil, Petroleum & Natural Gas (AREA)
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- Mycology (AREA)
- Soil Sciences (AREA)
- Enzymes And Modification Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a kind of immobilised enzymes oil spilling degradation agent, mass percent concentration shared by broomcorn straw activated carbon is 1.5 2.2% in immobilised enzymes oil spilling degradation agent, and mass percent concentration shared by sodium alginate is 6.8 7.9%, and compound enzyme concentration is 6 × 106‑8×106Cell/g, immobilised enzymes oil spilling degradation agent can the salinity of working environment be 5 55 ‰, temperature is 10 30 DEG C, and pH is 6.5 9.0, and to the degradation rate of petroleum hydrocarbon up to 70 90%, its preparation method includes：Enzyme liquid preparation, phosphoric acid activation, steam activation, absorption, crosslinking.Have the beneficial effect that：Immobilised enzymes oil spilling degradation agent is stronger to tolerance, the mechanicalness of environment, and the mass transfer performances of immobilised enzymes oil spilling degradation agent are greatly improved in broomcorn straw activated carbon, enzyme liquid is increased with oil contact area, greatly improves the activity and degradation efficiency of microorganism.
Description
Technical field
The present invention relates to oily degradation agent field, more particularly, to a kind of immobilised enzymes oil spilling degradation agent.
Technical background
Oil pollution substantially has two types：Sudden input and chronic long input.Sudden input includes oil tanker thing
Therefore with the leakage of offshore oil exploitation.Just such as harbour and vessel oily water are discharged for chronic long input, and natural seabed is oozed
Leakage etc., petroleum pollution in ocean seems remote, in fact closely bound up with our life.Ecological hazard caused by oil pollution meeting
Including：Oil covers across the sea, has blocked the gas exchanges of oxygen and carbon dioxide, has seriously caused water hypoxia；Oil
Opaqueness can hinder sunlight to inject ocean, be reduced plus dissolved oxygen, hinder the photosynthesis of plant under ocean, indirect stimulation
The generation of red tide；Contained polycyclic aromatic hydrocarbon has severe toxicity to organism in oil, is transmitted most by biological concentration and food chain
Transmit into the human body eventually, there is very strong carcinogenesis to people.
Oil degradation refers to be made oil degradation using means such as peripheral doses, chemical remediation, bioremediation technologies and divided
Dissipate, so as to achieve the purpose that to administer oil pollution.Since peripheral doses (as being heat-treated) are while pollutant in soil is destroyed
Also the component and structure of soil are destroyed, and it is expensive；Chemical remediation effect is preferable, but used chemical reagent can produce
Raw secondary pollution, limits its application range；But be rapid, no residual hazard with biodegradable method advantage, low cost, therefore
Receive significant attention.
The content of the invention
It is an object of the invention to provide a kind of immobilised enzymes oil spilling degradation agent, the mechanical strength of degradation agent, mass transfer performances compared with
Height, balling-up is preferable, and degradation agent has good degradation effect to petroleum hydrocarbon.
The present invention is directed to the problem of being mentioned in background technology, and the technical solution taken is：A kind of immobilised enzymes oil spilling degraded
Agent, mass percent concentration shared by broomcorn straw activated carbon is 1.5-2.2% in immobilised enzymes oil spilling degradation agent, sodium alginate institute
It is 6.8-7.9% to account for mass percent concentration, and compound enzyme concentration is 6 × 106-8×106Cell/g, immobilised enzymes oil spilling degradation agent
Can the salinity of working environment be 5-55 ‰, temperature is 10-30 DEG C, pH 6.5-9.0, to the degradation rate of petroleum hydrocarbon up to 70-
90%, compared with free enzyme liquid, its degradation rate improves 35%-50%, and tolerance, the mechanicalness to environment are stronger, sorghum stalks
The mass transfer performances of immobilised enzymes oil spilling degradation agent are greatly improved in stalk activated carbon, enzyme liquid is increased with oil contact area, greatly
The activity and degradation efficiency that improve microorganism.
Preferably, the preparation method of immobilised enzymes oil spilling degradation agent includes：Enzyme liquid preparation, phosphoric acid activation, steam activation,
Absorption, crosslinking, specifically include following steps：
It is prepared by enzyme liquid：After cultivating 7-15 days and the bead algae solution in exponential phase is filtered, and filter membrane selects 0.55-
0.60 μm, remove the filter membrane containing chlorella, liquid nitrogen frozen, adds 2.8-3.5 times of PBS buffer afterwards, PBS buffer
PH is 6.8-7.5, concentration 0.35-0.55mol/L；It is ground at a temperature of 0-4 DEG C, lapping liquid pours into centrifuge tube and in 0-
4 DEG C, centrifuge 30-45 minutes under conditions of 8500-9500r/min, take out supernatant up to enzyme liquid, be put in 0-4 DEG C of preservation；Take pair
The chlorella in number growth period obtains enzyme liquid after filtering, grind, centrifuge, and preparation is simple for enzyme liquid, and under low temperature
Extraction will not destroy the activity of enzyme liquid；
Phosphoric acid activation：Dried broomcorn straw powder is weighed with phosphoric acid according to 1:0.95-1.1 is uniformly mixed, and after being sufficiently stirred, is made
Mediated 50-60 minutes at 115-120 DEG C with kneader, sample is granulated into the cylindrical particle of 2.0-2.5mm, sample is hard after kneading
After change, phosphoric acid activation is carried out in high temperature furnace, sample is put into when furnace temperature rises to 480-490 DEG C, keeps the temperature 15-18 minutes, activation
Sample is cooled to room temperature afterwards, is washed with deionized to neutrality, is phosphoric acid activation sample after drying；Phosphoric acid high-temperature activation can be with
Broomcorn straw powder surface substantial amounts of micropore is occurred, prepare for further activation；
Steam activation：70-100g phosphoric acid activation samples are taken to be replaced in high temperature furnace, with the inflow of 4.0-6.5mL/min,
When re-activation 1.5-2.0 is small under 785-805 DEG C of steam atmosphere, sample is cooled to room temperature after activation, crushed 180-220
Mesh sieve is up to broomcorn straw activated carbon；After steam activation, the micropore of broomcorn straw activated carbon is more flourishing, and average pore size is more
Small, total hole volume, iodine sorption value higher, are more conducive to the absorption of activated carbon and enzyme liquid；
Absorption：By broomcorn straw activated carbon and enzyme liquid according to mass ratio 1:7-8 is uniformly mixed into capable absorption, adsorption temp 30-
33 DEG C, thermostatic absorption 60-90 minutes；Broomcorn straw activated carbon is loose structure, its mass-transfer performance is stronger, can be produced with enzyme liquid
The contact of larger area, can greatly improve the activity and degradation efficiency of attached microorganism；
Crosslinking：Polyethylene glycol, sodium alginate, deionized water, complex enzyme liquid are mixed into obtain mixed solvent, then according to weight ratio 1:
Mixed solvent is added dropwise in crosslinking agent by 1.1-1.2, controlled at 0-2 DEG C, stirring crosslinking 20-30 minutes in ice-water bath,
Then move to drying at room temperature and obtain immobilised enzymes microballoon in 45-60 minutes, obtained immobilised enzymes microballoon is rushed with physiological saline
2-3 is washed all over as immobilised enzymes oil spilling degradation agent；The mechanical strength of immobilised enzymes oil spilling degradation agent is higher, resistance to physical impact, passes
Matter performance and balling property are higher, while the stability of immobilised enzymes oil spilling degradation agent is very high, is not easily broken decomposition, Ke Yixun
Speed, expeditiously degraded oil, nontoxic secondary residual, cost is relatively low, is adapted to large-scale production and application, is a kind of safe efficient
Oil spilling degradation agent.
Preferably, in the mixed solvent polyethylene glycol, sodium alginate, deionized water, the mass ratio of complex enzyme liquid are 1:8-9:
12-15:18-20；Polyethylene glycol, sodium alginate, the appropriate proportioning of enzyme liquid help to be prepared mechanical strength, mass-transfer performance,
The preferable immobilised enzymes microballoon of balling property, helps to improve the application range of oil spilling degradation agent and applies timeliness, improve degraded
Efficiency, it is cost-effective.
Preferably, crosslinking agent is that mass ratio is 1:0.88-1.25:Methyl orthosilicate, the three oxygen methyl-monosilane of methyl of 8-10
With mixture of the methanol in ice-water bath；The crosslinking agent suitably matched can improve the weatherability, corrosion-resistant of immobilised enzymes microballoon
Property with resistance to physical impact, extend oil spilling degradation agent service life.
Compared with prior art, the advantage of the invention is that：Immobilised enzymes oil spilling degradation agent has more broad work
Environment, its suitable services salinity are 5-55 ‰, and temperature is 10-30 DEG C, pH 6.5-9.0, to the degradation rate of petroleum hydrocarbon up to 70-
90%, compared with free enzyme liquid, its degradation rate improves 35%-50%, and tolerance, the mechanicalness to environment are stronger, sorghum stalks
The mass transfer performances of immobilised enzymes oil spilling degradation agent are greatly improved in stalk activated carbon, enzyme liquid is increased with oil contact area, greatly
The activity and degradation efficiency that improve microorganism.
Embodiment
The present invention program is described further below by embodiment：
Embodiment 1：
A kind of immobilised enzymes oil spilling degradation agent, mass percent shared by broomcorn straw activated carbon is dense in immobilised enzymes oil spilling degradation agent
Spend for 1.5-2.2%, mass percent concentration shared by sodium alginate is 6.8-7.9%, and compound enzyme concentration is 6 × 106-8×
106Cell/g, immobilised enzymes oil spilling degradation agent can the salinity of working environment be 5-55 ‰, temperature is 10-30 DEG C, pH 6.5-
9.0, to the degradation rate of petroleum hydrocarbon up to 70-90%, compared with free enzyme liquid, its degradation rate improves 35%-50%, and to ring
Tolerance, the mechanicalness in border are stronger, and the mass transfer performances of immobilised enzymes oil spilling degradation agent are greatly improved in broomcorn straw activated carbon, make
Enzyme liquid increases with oil contact area, greatly improves the activity and degradation efficiency of microorganism.
Embodiment 2：
The preparation method of immobilised enzymes oil spilling degradation agent comprises the following steps：
1）It is prepared by enzyme liquid：After cultivating 7 days and the bead algae solution in exponential phase is filtered, and filter membrane selects 0.55 μ
M, removes the filter membrane containing chlorella, and the PBS buffer of liquid nitrogen frozen, afterwards 2.8 times of addition, the pH of PBS buffer is 6.8,
Concentration is 0.35mol/L；It is ground at a temperature of 0 DEG C, lapping liquid pours into centrifuge tube and under conditions of 0 DEG C, 8500r/min
Centrifugation 30 minutes, takes out supernatant up to enzyme liquid, is put in 0 DEG C of preservation；Take the logarithm growth period chlorella through filtering, grinding, from
Enzyme liquid is obtained after the heart, preparation is simple for enzyme liquid, and extraction will not destroy the activity of enzyme liquid under low temperature；
Phosphoric acid activation：Dried broomcorn straw powder is weighed with phosphoric acid according to 1:0.95 uniformly mixing, after being sufficiently stirred, using pinching
Conjunction machine is mediated 50 minutes at 115 DEG C, sample is granulated into the cylindrical particle of 2.0mm, after sample hardening, in high temperature furnace after kneading
Phosphoric acid activation is carried out, sample is put into when furnace temperature rises to 480 DEG C, 15 minutes is kept the temperature, sample is cooled to room temperature after activation, is spent
Ion water washing is phosphoric acid activation sample after drying to neutrality；It is big that phosphoric acid high-temperature activation can occur broomcorn straw powder surface
The micropore of amount, prepares for further activation；
Steam activation：Take 70g phosphoric acid activation samples to be replaced in high temperature furnace, with the inflow of 4.0mL/min, steamed in 785 DEG C of water
When re-activation 1.5 is small under gas atmosphere, sample is cooled to room temperature after activation, crushed 180 mesh sieves up to broomcorn straw activity
Charcoal；After steam activation, the micropore of broomcorn straw activated carbon is more flourishing, average pore size smaller, total hole volume, iodine sorption value
Higher, is more conducive to the absorption of activated carbon and enzyme liquid；
Absorption：By broomcorn straw activated carbon and enzyme liquid according to mass ratio 1:7 are uniformly mixed into capable absorption, and adsorption temp is 30 DEG C,
Thermostatic absorption 60 minutes；Broomcorn straw activated carbon is loose structure, its mass-transfer performance is stronger, and larger area can be produced with enzyme liquid
Contact, can greatly improve the activity and degradation efficiency of attached microorganism；
Crosslinking：Polyethylene glycol, sodium alginate, deionized water, complex enzyme liquid are mixed into obtain mixed solvent, the poly- second two of in the mixed solvent
Alcohol, sodium alginate, deionized water, the mass ratio of complex enzyme liquid are 1:8:12:18, then according to weight ratio 1:1.1 will mix it is molten
Agent is added dropwise in crosslinking agent, and crosslinking agent is that mass ratio is 1:0.88:8 methyl orthosilicate, three oxygen methyl-monosilane of methyl and methanol
Mixture in ice-water bath, controlled at 0 DEG C, stirring crosslinking 20 minutes in ice-water bath, then move to drying at room temperature 45
Minute immobilised enzymes microballoon is obtained, 2 times are immobilised enzymes oil spilling by obtained immobilised enzymes microballoon normal saline flushing
Degradation agent；The mechanical strength of immobilised enzymes oil spilling degradation agent is higher, and resistance to physical impact, mass-transfer performance is higher with balling property, together
When immobilised enzymes oil spilling degradation agent stability it is very high, be not easily broken decomposition, can rapidly, expeditiously degraded oil, it is nontoxic
Pair residual, cost is relatively low, is adapted to large-scale production and application, is a kind of safe and efficient oil spilling degradation agent.
Embodiment 3：
A kind of immobilised enzymes oil spilling degradation agent, mass percent shared by broomcorn straw activated carbon is dense in immobilised enzymes oil spilling degradation agent
Spend for 1.5-2.2%, mass percent concentration shared by sodium alginate is 6.8-7.9%, and compound enzyme concentration is 6 × 106-8×
106Cell/g, immobilised enzymes oil spilling degradation agent can the salinity of working environment be 5-55 ‰, temperature is 10-30 DEG C, pH 6.5-
9.0, to the degradation rate of petroleum hydrocarbon up to 70-90%, compared with free enzyme liquid, its degradation rate improves 35%-50%, and to ring
Tolerance, the mechanicalness in border are stronger, and the mass transfer performances of immobilised enzymes oil spilling degradation agent are greatly improved in broomcorn straw activated carbon, make
Enzyme liquid increases with oil contact area, greatly improves the activity and degradation efficiency of microorganism.
The preparation method of immobilised enzymes oil spilling degradation agent includes：Enzyme liquid preparation, phosphoric acid activation, steam activation, absorption, friendship
Connection, specifically includes following steps：
It is prepared by enzyme liquid：After cultivating 15 days and the bead algae solution in exponential phase is filtered, and filter membrane selects 0.60 μm,
The filter membrane containing chlorella is removed, the PBS buffer of liquid nitrogen frozen, afterwards 3.5 times of addition, the pH of PBS buffer is 7.5, dense
Spend for 0.55mol/L；Be ground at a temperature of 4 DEG C, lapping liquid pour into centrifuge tube and under conditions of 4 DEG C, 9500r/min from
The heart 45 minutes, takes out supernatant up to enzyme liquid, is put in 4 DEG C of preservations；Take the logarithm growth period chlorella through filtering, grinding, centrifuging
After obtain enzyme liquid, preparation is simple for enzyme liquid, and extraction will not destroy the activity of enzyme liquid under low temperature；
Phosphoric acid activation：Dried broomcorn straw powder is weighed with phosphoric acid according to 1:1.1 uniformly mixing, after being sufficiently stirred, using pinching
Conjunction machine is mediated 60 minutes at 120 DEG C, sample is granulated into the cylindrical particle of 2.5mm, after sample hardening, in high temperature furnace after kneading
Phosphoric acid activation is carried out, sample is put into when furnace temperature rises to 490 DEG C, 18 minutes is kept the temperature, sample is cooled to room temperature after activation, is spent
Ion water washing is phosphoric acid activation sample after drying to neutrality；It is big that phosphoric acid high-temperature activation can occur broomcorn straw powder surface
The micropore of amount, prepares for further activation；
Steam activation：100g phosphoric acid activation samples are taken to be replaced in high temperature furnace, with the inflow of 6.5mL/min, in 805 DEG C of water
When re-activation 2.0 is small under vapor atmosphere, sample is cooled to room temperature after activation, 220 mesh sieves is crushed and lives up to broomcorn straw
Property charcoal；After steam activation, the micropore of broomcorn straw activated carbon is more flourishing, average pore size smaller, total hole volume, iodine absorption
It is worth higher, is more conducive to the absorption of activated carbon and enzyme liquid；
Absorption：By broomcorn straw activated carbon and enzyme liquid according to mass ratio 1:8 are uniformly mixed into capable absorption, and adsorption temp is 33 DEG C,
Thermostatic absorption 90 minutes；Broomcorn straw activated carbon is loose structure, its mass-transfer performance is stronger, and larger area can be produced with enzyme liquid
Contact, can greatly improve the activity and degradation efficiency of attached microorganism；
Crosslinking：Polyethylene glycol, sodium alginate, deionized water, complex enzyme liquid are mixed into obtain mixed solvent, then according to weight ratio 1:
1.2 are added dropwise to mixed solvent in crosslinking agent, and controlled at 2 DEG C, stirring crosslinking 30 minutes, then move to room in ice-water bath
Temperature obtains immobilised enzymes microballoon in dry 60 minutes, the 3 times as fixations of immobilised enzymes microballoon normal saline flushing that will be obtained
Change enzyme oil spilling degradation agent；The mechanical strength of immobilised enzymes oil spilling degradation agent is higher, resistance to physical impact, mass-transfer performance and balling property
It is higher, while the stability of immobilised enzymes oil spilling degradation agent is very high, is not easily broken decomposition, can rapidly, stone of expeditiously degrading
Oil, nontoxic secondary residual, cost is relatively low, is adapted to large-scale production and application, is a kind of safe and efficient oil spilling degradation agent.
In the mixed solvent polyethylene glycol, sodium alginate, deionized water, the mass ratio of complex enzyme liquid are 1:9:15:20.Crosslinking
Agent is that mass ratio is 1:1.25: 0.016:10 methyl orthosilicate, 3,5- difluoro-benzoic acids, three oxygen methyl-monosilane of methyl and first
Mixture of the alcohol in ice-water bath；3,5- difluoro-benzoic acids are added in crosslinking agent can greatly improve cross-linking effect, 3,5- difluoros
The fluorine atom of benzoic acid has larger elecrtonegativity, can form hydrogen bond with hydrogen atom, realize that small range is handed on molecular scale
Join the base of a fruit to close, so as to increase bridging property, improve the mechanical strength and stability of immobilised enzymes microballoon, strengthen impact resistance, protect
Protect immobilised enzymes to overflow from oscillatory surge, extend the service life of immobilization oil spilling degradation agent, reduce cost.
By the immobilised enzymes oil spilling degradation agent for adding modified broomcorn straw activated carbon, it is not added with modified broomcorn straw activated carbon
Immobilised enzymes oil spilling degradation agent and simple activity charcoal carry out physicochemical property contrast, comparing result is as shown in table 1.
The performance of the different immobilised enzymes oil spilling degradation agents of table 1. compares
As can be drawn from Table 1, the immobilization complex enzyme microballoon of the modified broomcorn straw activated carbon of addition of the invention is in mechanical strength
Improve a lot with mass-transfer performance, the cell of embedding has higher activity, to the degradation rate of petroleum hydrocarbon up to 78-89%.
Immobilised enzymes oil spilling degradation agent in embodiment 1-3 is subjected to physicochemical property comparison, comparative result is as shown in table 2.
The performance of immobilised enzymes oil spilling degradation agent compares in 2. embodiment 1-3 of table
As shown in Table 2, the mechanical strength of immobilised enzymes oil spilling degradation agent, mass-transfer performance, balling-up, immobilization efficiency preferably and
And it is relatively stable, its degradation rate to oil is higher, and especially, the items of the immobilised enzymes oil spilling degradation agent in embodiment 3 refer to
Mark is better than embodiment 1, embodiment 2.
Routine operation in operating procedure of the present invention is well known to those skilled in the art, herein without repeating.
Technical scheme is described in detail in embodiment described above, it should be understood that the above is only
For the specific embodiment of the present invention, it is not intended to limit the invention, all any modifications made in the spirit of the present invention,
Supplement or similar fashion replacement etc., should all be included in the protection scope of the present invention.
Claims (8)
- A kind of 1. immobilised enzymes oil spilling degradation agent, it is characterised in that：A kind of immobilised enzymes oil spilling degradation agent, immobilised enzymes overflow Mass percent concentration shared by broomcorn straw activated carbon is 1.5-2.2% in oily degradation agent, and mass percent shared by sodium alginate is dense It is 6 × 10 to spend for 6.8-7.9%, compound enzyme concentration6-8×106Cell/g, immobilised enzymes oil spilling degradation agent can working environment salt It is 10-30 DEG C to spend for 5-55 ‰, temperature, pH 6.5-9.0.
- 2. a kind of immobilised enzymes oil spilling degradation agent according to claim 1, its preparation method include：Prepared by enzyme liquid, phosphoric acid Activation, steam activation, absorption, crosslinking, it is characterised in that：The cross-linking step is：By polyethylene glycol, sodium alginate, deionization Water, complex enzyme liquid mix to obtain mixed solvent, then according to weight ratio 1:Mixed solvent is added dropwise in crosslinking agent by 1.1-1.2, control Temperature processed is 0-2 DEG C, stirring crosslinking 20-30 minutes in ice-water bath, then moves to drying at room temperature and is fixed for 45-60 minutes Change enzyme microballoon, by obtained immobilised enzymes microballoon normal saline flushing 2-3 all over as immobilised enzymes oil spilling degradation agent.
- A kind of 3. immobilised enzymes oil spilling degradation agent according to claim 2, it is characterised in that：It is poly- in the cross-linking step Ethylene glycol, sodium alginate, deionized water, the mass ratio of complex enzyme liquid are 1:8-9:12-15:18-20.
- A kind of 4. immobilised enzymes oil spilling degradation agent according to claim 2, it is characterised in that：Friendship in the cross-linking step It is that mass ratio is 1 to join agent:0.88-1.25:Methyl orthosilicate, three oxygen methyl-monosilane of methyl and the methanol of 8-10 is in ice-water bath Mixture.
- A kind of 5. immobilised enzymes oil spilling degradation agent according to claim 2, it is characterised in that：The enzyme liquid preparation process For：After cultivating 7-15 days and the bead algae solution in exponential phase is filtered, and filter membrane selects 0.55-0.60 μm, takes Under the filter membrane containing chlorella, liquid nitrogen frozen, adds 2.8-3.5 times of PBS buffer, the pH of PBS buffer is 6.8- afterwards 7.5, concentration 0.35-0.55mol/L；Be ground at a temperature of 0-4 DEG C, lapping liquid pour into centrifuge tube and 0-4 DEG C, Centrifuged 30-45 minutes under conditions of 8500-9500r/min, take out supernatant up to enzyme liquid, be put in 0-4 DEG C of preservation.
- A kind of 6. immobilised enzymes oil spilling degradation agent according to claim 2, it is characterised in that：The phosphoric acid activation step For：Dried broomcorn straw powder is weighed with phosphoric acid according to 1:0.95-1.1 is uniformly mixed, and after being sufficiently stirred, is existed using kneader 115-120 DEG C is mediated 50-60 minutes, sample is granulated into the cylindrical particle of 2.0-2.5mm, after sample hardening, in height after kneading Phosphoric acid activation is carried out in warm stove, sample is put into when furnace temperature rises to 480-490 DEG C, keeps the temperature 15-18 minutes, it is after activation that sample is cold But to room temperature, it is washed with deionized to neutrality, is phosphoric acid activation sample after drying.
- A kind of 7. immobilised enzymes oil spilling degradation agent according to claim 2, it is characterised in that：The steam activation step For：70-100g phosphoric acid activation samples are taken to be replaced in high temperature furnace, with the inflow of 4.0-6.5mL/min, in 785-805 DEG C of water When re-activation 1.5-2.0 is small under vapor atmosphere, sample is cooled to room temperature after activation, crushed 180-220 mesh sieves up to high Fine strain of millet straw-stem active charcoal.
- A kind of 8. immobilised enzymes oil spilling degradation agent according to claim 2, it is characterised in that：The adsorption step is：Will Broomcorn straw activated carbon is with enzyme liquid according to mass ratio 1:7-8 is uniformly mixed into capable absorption, and adsorption temp is 30-33 DEG C, and constant temperature is inhaled It is 60-90 minutes attached.
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