CN101134955A - Solid composite micro-organism micro-balloon for organic contaminant biodegradation and method for preparing the same - Google Patents
Solid composite micro-organism micro-balloon for organic contaminant biodegradation and method for preparing the same Download PDFInfo
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- 239000007787 solid Substances 0.000 title claims abstract description 42
- 239000002131 composite material Substances 0.000 title claims abstract description 25
- 238000006065 biodegradation reaction Methods 0.000 title abstract description 4
- 238000000034 method Methods 0.000 title description 17
- 244000005700 microbiome Species 0.000 title description 17
- 239000000356 contaminant Substances 0.000 title 1
- 238000002360 preparation method Methods 0.000 claims abstract description 33
- 239000004005 microsphere Substances 0.000 claims abstract description 27
- 239000002957 persistent organic pollutant Substances 0.000 claims abstract description 26
- 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
- 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
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- 229910000019 calcium carbonate Inorganic materials 0.000 claims abstract description 4
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- 241000894006 Bacteria Species 0.000 claims description 17
- 239000002594 sorbent Substances 0.000 claims description 16
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- 238000006731 degradation reaction Methods 0.000 claims description 9
- 230000002538 fungal effect Effects 0.000 claims description 8
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
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- 235000002595 Solanum tuberosum Nutrition 0.000 claims description 3
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- HVUMOYIDDBPOLL-XWVZOOPGSA-N Sorbitan monostearate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O HVUMOYIDDBPOLL-XWVZOOPGSA-N 0.000 claims description 2
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The present invention discloses one kind of solid composite microbe microsphere for biodegrading organic pollutant and its preparation process. The macroporous microbe-embedding microsphere is prepared with sodium alginate as the embedding material, solid calcium carbonate as the pore creating agent, and adsorbent capable of adsorbing organic pollutant and thallus for degrading other pollutant added into the embedding suspension. It combines adsorption on the pollutant and biodegradation, and has high pollutant eliminating efficiency and high pollutant eliminating speed.
Description
Technical field
The invention belongs to the biodegradation method of Organic pollutants, relate in particular to a kind of biodegradable solid composite microbe microsphere of organic pollutant and preparation method thereof that is used for.
Background technology
Utilizing the microorganism biodegrade is to administer the effective ways of organic pollutant environmental pollution, but because the toxicity of organic pollutant, in the biological treating of organic pollutant, microorganism is difficult to effectively grow, thereby causes degradation efficiency low etc.Improvement with petroleum hydrocarbon contaminated soil is example, and petroleum hydrocarbon is formed complicated, and bio-toxicity is strong, but its biology availability is low simultaneously, and therefore adding bacterial classification is difficult to accumulate biomass in abominable petroleum hydrocarbon contaminated soil, and Degradation and Transformation efficient is low.At present, abroad developed at the biodegradable microbial preparation of petroleum hydrocarbon, this class preparation mainly exists with bacterium liquid and dry powder form, in use be unfavorable for storage and transport, influenced greatly by soil ecosystem, the activity inducement phase is long, and with the indigenous microorganism interaction process in shortcoming such as active easily forfeiture, need use in a large number and repeatedly add, cause that governance efficiency is low, cost is high.
Cell fixation (immobilization) technology is to grow up from immobilized enzyme the seventies in 20th century.It is a kind of space field that free cell is positioned to limit with the means of chemistry or physics, and makes it keep active, the method for recycling.Concentration and purity, maintenance high-efficiency strain, the microorganism that immobilized cell technique helps improving the microorganism cells in the bio-reactor run off less, tenable environment impact strong, operational stability is good, be beneficial to and denitrogenate and remove high-enriched organics or some hard-degraded substance.The relative free cell of immobilized cell has many advantages, and immobilized cell technique has been obtained certain progress aspect environmental improvement.People such as Pometto in 1997 have announced that synthesized polymer materials such as utilizing polyethylene or polypropylene and vegetable material prepare the patent of microorganism cells fixation support, the carrier that obtains can be used for biofilm reactor and handles waste gas and wastewater treatment (US5595893), but this carrier is the organic polymer material synthetic vectors, its biocompatibility and biodegradability are relatively poor, easily environment are caused secondary pollution.People such as Wang Xin, Li Peijun will move the glue bacillus and be embedded in polyvinyl alcohol and the boric acid complex carrier, and the soil China and Philippines that are used to degrade, pyrene pollutent, and the result shows that in containing the natural soils of indigenous bacterium immobilized microorganism has very big advantage to the degraded of pollutent.In the cell fixation application process, because embedded material is to a certain degree hindering the diffusion of substrate and oxygen, to the biological degradation generation inhibition of macromolecule substrate.People such as Liu Zheng are at patent application " a kind of solid composite microbe microsphere for soil rehabilitation and preparation method thereof " (number of patent application: disclose 200510130675.9) and a kind ofly utilized alginate calcium to prepare the oversized hole embedded material to carry out microbial immobilized a kind of novel method, developed preparation alginate calcium oversized hole microballoon technology, on traditional microorganism embedding carrier calcium alginate microsphere, introduce macropore, solve mass transfer problem, can improve oxygen transmission and substrate transmittance process in the microballoon.But for the organic pollutant of indissoluble in the environment, substrate has problems with contacting still of imbedded microbe.Therefore how to improve in the contaminate environment, the efficient that immobilized microorganism panning pollutes substrate is the biodegradable deciding step of petroleum hydrocarbon.
Summary of the invention
At the substrate that exists in the existing microorganism embedding preparation and the contact problems of imbedded microbe, the object of the invention is to provide a kind of biodegradable solid composite microbe microsphere of organic pollutant and preparation method thereof that is used for, the oversized hole microorganism embedding carrier that this method produces can be enriched with organic pollutants, promote the growth of imbedded microbe, thereby reach the high and fireballing purpose to organic pollutant biological degradation efficient.
The present invention can be realized by following proposal.
A kind of biodegradable solid composite microbe microsphere of organic pollutant that is used for is to be embedded material with the sodium alginate, and solid carbonic acid calcium is pore-creating agent, and embedding has the sorbent material of adsorptive power to organic pollutant and has the fungi and the bacteria culture of biodegradability; This solid composite microbe microsphere diameter is 3～10mm, the radial duct of inner 10～80 μ m that distribute.
A kind of preparation method who is used for the biodegradable solid composite microbe microsphere of organic pollutant comprises the steps:
1) cultivate on the plane: bacterium is adopted the LB solid medium, and fungi adopts solid potato culture medium to cultivate in advance in culture dish, and culture temperature is 25 ℃～30 ℃, cultivates 2～10 days, preserves standby;
2) the dull and stereotyped preparation of the fungus culture fungal spore liquid that obtains fungal spore liquid preparation: utilize 1) utilizes sterilization 5～50mM (pH=7.0) PBS damping fluid flushing spore liquid, and 4 ℃ of preservations of preparation spore suspension are standby;
3) bacterium shake-flask culture: the microbionation that step (1) middle plateform is cultivated shaking culture in the LB liquid nutrient medium, 20～30 ℃ of culture temperature, rotating speed 130～180rpm; Cultivate to collect in 15～30 hours and be in the thalline of plateau and get inoculum, 4 ℃ of preservations are standby;
4) preparation sodium alginate one sorbent material solution: sodium alginate is dissolved in the water, and wherein sodium alginate concentration is 1～4%; After treating that sodium alginate dissolves fully, add sorbent material, addition is preferably 1～10% (w/v), mixes;
5) under the normal temperature, 500～1500 purpose calcium carbonate granules are joined in 0.1%～0.6% the span60 solution, ultra-sonic dispersion evenly after, add 4 to) in the solution of preparation, lime carbonate concentration is 1%～10%; According to the degraded needs of different organic pollutants, 4) in add 2) in the fungal spore suspension or 3 for preparing) in obtain inoculum or 2) and 3) mixed solution behind the mixing according to a certain percentage, finely dispersed suspension liquid is made in stirring;
6) draw the suspension liquid that obtains through step 5) with dropper, be added dropwise to concentration and be in 1%～3% the calcium chloride solution, drop contact formation solid spherical solids precipitation afterwards with calcium chloride solution;
7) spheroidal particle that obtains collection 6), and in 0.1～0.5M hydrochloric acid, soak 10～60min, after waiting not have tangible bubble and generating, filter the solid spherical particle that obtains pore, aseptic water washing 1～5 time, the organic pollutant that promptly obtains being coupled absorption and biodegradable microorganism embedding microballoon.
Above-mentioned fungi and bacterium are fungi and the bacterium with decomposing petroleum hydrocarbon ability.
Above-mentioned fungi is a kind of for fungies such as thorn little Ke Yinhan bacterium of spore and Phanerochaete chrysosporium.
Above-mentioned bacterium is from Rhodopseudomonas (Newcombe D A etc., AppliedMicrobiology and Biotechnology, 1999,51:877-82), Rhod (Newcombe D A etc., Applied Microbiology and Biotechnology, 1999,51:877-82), micrococcus sp (Torres LG etc., Process Biochemistry 40 (10): 3296-3302 OCT 2005), enterobacter (Katsivela E etc., Biodegradation 16 (2): 169-180 MAR 2005), corynebacterium (RahmanKSM etc., Bioresource Technology 85 (3): 257-261 DEC 2002), flavobacterium (Rahman KSM etc., Bioresource Technology 85 (3): 257-261DEC 2002) and bacillus (Das K etc., Bioresource Technology 98 (7): 1339-1345 MAY 2007).
Above-mentioned fungi and bacterium all can be buied from Chinese common micro-organisms DSMZ.
Above-mentioned steps 5) 2 described in) and 3) according to a certain percentage the mixed solution behind the mixing by application number be: the described method preparation of 200510130676.3 patent.
Above-mentioned sorbent material is 1～3 kind in the sorbent materials such as xylogen, Mierocrystalline cellulose, gac.
Advantage and beneficial effect that the inventive method gained microbial inoculum is had are: (1), not only have protectiveness but also have under the prerequisite of high mass transfer performances having kept the microsphere immobilized cell of oversized hole; utilize the effectively pollutent in the enrichment environment of the pollutant absorbent that adds in the material; slow down the migration of pollutent in environment; avoid the diffusion of environmental pollution, can improve the efficient of imbedded microbe bio-transformation pollutent simultaneously again.(2), the embedding carrier and the pollutant absorbent that use have environmental friendliness, advantages such as good biocompatibility do not need to carry out other processing.(3) the present invention has broad application prospects in the biological restoration process of oil-polluted soils and sudden control of environmental pollution process.
Description of drawings
Fig. 1 is the photo of the novel oversized hole microorganism embedding carrier of the present invention's preparation.
Fig. 2 is the photo (lime carbonate has removed, and magnification is 4 times) of the oversized hole solid microbe microballoon of the present invention's preparation under the opticmicroscope.
The embedding of Fig. 3 oversized hole is to the promotion column diagram of fungal growth
1: embedding fungi not;
2: calcium alginate embedded;
3: oversized hole is calcium alginate embedded
Fig. 4 is the oversized hole solid microbe microballoon of the present invention's preparation under the opticmicroscope photo (magnification is 10 times) through cultivating in 48 hours under 25 ℃～30 ℃.
Fig. 5 carrier of the present invention is to the adsorpting characteristic curve figure of phenanthrene.
Embodiment
Following example gives further instruction at the luxuriant and rich with fragrance preparation of organic pollutant novel microorganism embedding preparation to method provided by the invention, but does not limit the present invention.
Embodiment 1
(1) cultivate on the plane: adopt the fungi of Phanerochaete chrysosporium (phanerochaetechrysosoporium is available from Chinese common micro-organisms DSMZ) as petroleum hydrocarbon degradation.Preparation potato plating medium (PDA) is inoculated into Phanerochaete chrysosporium on the PDA plating medium, cultivates 6 days at 28 ℃, preserves standby;
(2) utilize the cultured PDA flat board of sterilization 20mMPBS (pH=7.0) flushing, make spore liquid, spore concentration is 10/ml;
(3) preparation sodium alginate one sorbent material solution, wherein sodium alginate concentration is 2%, and sorbent material selects that phenanthrene is had the strong lignin biopolymer that absorbs, and content is 5%;
(4) at normal temperatures, 800 order calcium carbonate microparticles are dispersed in (3) described solution, lime carbonate concentration is 5%; Add the spore suspension that obtains in (2) then in solution, making the spore concentration in the suspension liquid is 10
6Individual/ml, fully stir and make finely dispersed suspension.
(5) draw the suspension that obtains in (4) with dropper, be added dropwise in the calcium chloride solution that concentration is 1.1 %, drop with form solid spherical solids precipitation (as shown in Figure 1) immediately after calcium chloride solution contacts.
(6) collect spheroidal particle, in 0.5M HCl solution, soak 20min, wait not have tangible bubble and generate, collect the spheroidal particle of removing the lime carbonate solia particle, and, promptly make solid composite microbe microsphere (as shown in Figure 2) with aseptic water washing 2 times.
Embodiment 2
Place 30 ℃ of cultivations after 24 hours the solid composite microbe microsphere of embodiment 1 gained, the fungal biomass that has compared the prepared solid composite microbe microsphere of free spore (sample one), atresia sodium alginate to embed spore (sample two) and embodiment 1 by weighting method, (see figure 3) shows as a result, the prepared solid composite microbe microsphere of the present invention can well promote fungi growth, and the 24h fungal biomass is respectively sample one and sample two 28.6 and 2.5 times.Cultivate after 48 hours the fungi form (as shown in Figure 4) in the prepared solid composite microbe microsphere of microscopic examination embodiment 1.
Embodiment 3
The The adsorption kinetic data of organic pollutant phenanthrene
Place 20ml to contain 5mg/L phenanthrene, 200mg/LTween80 and 200mg/L NaN the solid composite microbe microsphere 0.4g (weight in wet base) of embodiment 1 gained
3In 5mM PBS (pH=7.0) damping fluid of (suppressing the imbedded microbe activity), investigate the kinetics of adsorption of carrier to the organic pollutant phenanthrene, contrast liquid-phase system when not adding any sorbent material wherein, also having according to what make in the patent 200510130675.9 (CN) is oversized hole only, does not contain the carrier of organic pollutant class sorbent material.(see figure 5) is added in the solution of the prepared composite microbe microsphere of embodiment 1 as a result, during remaining luxuriant and rich with fragrance content 10h is 12% of initial luxuriant and rich with fragrance content, illustrate that the prepared carrier of the present invention has good adsorptive power to phenanthrene, and contrast and added the almost not variation of the Sino-Philippines content of solution of the carrier for preparing according to patent 200510130675.9 (CN).
Claims (8)
1. one kind is used for biodegradable solid composite microbe microsphere of organic pollutant and preparation method thereof, be to be embedded material with the sodium alginate, solid carbonic acid calcium is pore-creating agent, and embedding has the sorbent material of adsorptive power to organic pollutant and has the fungi and the bacteria culture of biodegradability; This solid composite microbe microsphere diameter is 3～10mm, the radial duct of inner 10～80 μ m that distribute.
2. according to the described solid composite microbe microsphere of claim 1, it is characterized in that described sorbent material is 1～3 kind of sorbent materials such as xylogen, Mierocrystalline cellulose, gac.
3. according to claim 1 or 2 described solid composite microbe microspheres, it is characterized in that described fungi with petroleum hydrocarbon degradation ability is a kind of of fungies such as thorn little Ke Yinhan bacterium of spore and Phanerochaete chrysosporium.
4. according to claim 1 or 2 described solid composite microbe microspheres, it is characterized in that described bacterium with petroleum hydrocarbon degradation ability is from one or more of Rhodopseudomonas, Rhod, micrococcus sp, enterobacter, corynebacterium, flavobacterium and bacillus etc.
5. the described preparation method who is used for the biodegradable solid composite microbe microsphere of organic pollutant of claim 1 comprises the steps:
1) cultivate on the plane: bacterium is adopted the LB solid medium, and fungi adopts solid potato culture medium to cultivate in advance in culture dish, and culture temperature is 25 ℃～30 ℃, cultivates 2～10 days, preserves standby;
2) the dull and stereotyped preparation of the fungus culture fungal spore liquid that obtains fungal spore liquid preparation: utilize 1) utilizes sterilization 5～50mM (pH=7.0) PBS damping fluid flushing spore liquid, and 4 ℃ of preservations of preparation spore suspension are standby;
3) bacterium shake-flask culture: the microbionation that step (1) middle plateform is cultivated shaking culture in the LB liquid nutrient medium, 20～30 ℃ of culture temperature, rotating speed 130～180rpm; Cultivate to collect in 15～30 hours and be in the thalline of plateau and get inoculum, 4 ℃ of preservations are standby;
4) preparation sodium alginate one sorbent material solution: sodium alginate is dissolved in the water, and wherein sodium alginate concentration is 1～4%; After treating that sodium alginate dissolves fully, add sorbent material, addition is preferably 1～10% (w/v), mixes;
5) under the normal temperature, 500～1500 purpose calcium carbonate granules join in 0.1%～0.6% the span60 solution, ultra-sonic dispersion evenly after, add 4 to) in the solution of preparation, lime carbonate concentration is 1%～10%; According to the degraded needs of different organic pollutants, 4) in add 2) in the spore suspension or 3 for preparing) in obtain inoculum or 2) and 3) mixed solution behind the mixing according to a certain percentage, finely dispersed suspension liquid is made in stirring;
6) draw the suspension liquid that obtains through step (5) with dropper, be added dropwise to concentration and be in 1%～3% the calcium chloride solution, drop contact formation solid spherical solids precipitation afterwards with calcium chloride solution;
7) spheroidal particle that obtains collection 5), and in 0.1～0.5M hydrochloric acid, soak 10～60min, after waiting not have tangible bubble generation, filtration obtains the solid spherical particle of pore, aseptic water washing 1～5 time, the organic pollutant that promptly obtains being coupled absorption and biodegradable solid microbe embedding microballoon.
6. according to the preparation method of the described solid composite microbe microsphere of claim 5, it is characterized in that described sorbent material is 1～3 kind of sorbent materials such as xylogen, Mierocrystalline cellulose, gac.
7. according to the preparation method of the described solid composite microbe microsphere of claim 5, it is characterized in that described fungi with petroleum hydrocarbon degradation ability is a kind of of fungies such as thorn little Ke Yinhan bacterium of spore and Phanerochaete chrysosporium.
8. according to the preparation method of the described solid composite microbe microsphere of claim 5, it is characterized in that described bacterium with petroleum hydrocarbon degradation ability is from one or more of Rhodopseudomonas, Rhod, micrococcus sp, enterobacter, corynebacterium, flavobacterium and bacillus etc.
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