CN101429504B - Carrageenin immobilized microorganism and method of producing the same - Google Patents
Carrageenin immobilized microorganism and method of producing the same Download PDFInfo
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- CN101429504B CN101429504B CN2008102260440A CN200810226044A CN101429504B CN 101429504 B CN101429504 B CN 101429504B CN 2008102260440 A CN2008102260440 A CN 2008102260440A CN 200810226044 A CN200810226044 A CN 200810226044A CN 101429504 B CN101429504 B CN 101429504B
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- 244000005700 microbiome Species 0.000 title claims abstract description 43
- 235000010418 carrageenan Nutrition 0.000 title claims abstract description 33
- 229920001525 carrageenan Polymers 0.000 title claims abstract description 33
- 238000000034 method Methods 0.000 title abstract description 14
- 239000000243 solution Substances 0.000 claims abstract description 63
- 239000002245 particle Substances 0.000 claims abstract description 26
- 239000000679 carrageenan Substances 0.000 claims abstract description 19
- 229940113118 carrageenan Drugs 0.000 claims abstract description 19
- UHVMMEOXYDMDKI-JKYCWFKZSA-L zinc;1-(5-cyanopyridin-2-yl)-3-[(1s,2s)-2-(6-fluoro-2-hydroxy-3-propanoylphenyl)cyclopropyl]urea;diacetate Chemical compound [Zn+2].CC([O-])=O.CC([O-])=O.CCC(=O)C1=CC=C(F)C([C@H]2[C@H](C2)NC(=O)NC=2N=CC(=CC=2)C#N)=C1O UHVMMEOXYDMDKI-JKYCWFKZSA-L 0.000 claims abstract description 19
- 238000002360 preparation method Methods 0.000 claims abstract description 16
- 238000002156 mixing Methods 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 15
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 14
- 238000010438 heat treatment Methods 0.000 claims abstract description 8
- 241000589516 Pseudomonas Species 0.000 claims description 13
- 230000000813 microbial effect Effects 0.000 claims description 12
- 239000000725 suspension Substances 0.000 claims description 11
- 229920001661 Chitosan Polymers 0.000 claims description 9
- 229910004298 SiO 2 Inorganic materials 0.000 claims description 6
- 239000002504 physiological saline solution Substances 0.000 claims description 6
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 4
- 239000000203 mixture Substances 0.000 abstract description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract 2
- 239000011259 mixed solution Substances 0.000 abstract 2
- RQFQJYYMBWVMQG-IXDPLRRUSA-N chitotriose Chemical compound O[C@@H]1[C@@H](N)[C@H](O)O[C@H](CO)[C@H]1O[C@H]1[C@H](N)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)N)[C@@H](CO)O1 RQFQJYYMBWVMQG-IXDPLRRUSA-N 0.000 abstract 1
- 229910052681 coesite Inorganic materials 0.000 abstract 1
- 229910052906 cristobalite Inorganic materials 0.000 abstract 1
- 239000007788 liquid Substances 0.000 abstract 1
- 239000000377 silicon dioxide Substances 0.000 abstract 1
- 235000012239 silicon dioxide Nutrition 0.000 abstract 1
- 229910052682 stishovite Inorganic materials 0.000 abstract 1
- 229910052905 tridymite Inorganic materials 0.000 abstract 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 241000589774 Pseudomonas sp. Species 0.000 description 5
- 239000010802 sludge Substances 0.000 description 5
- 229920002521 macromolecule Polymers 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 230000003245 working effect Effects 0.000 description 4
- ISPYQTSUDJAMAB-UHFFFAOYSA-N 2-chlorophenol Chemical compound OC1=CC=CC=C1Cl ISPYQTSUDJAMAB-UHFFFAOYSA-N 0.000 description 3
- 239000008187 granular material Substances 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- 229920000856 Amylose Polymers 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 230000000845 anti-microbial effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000002242 deionisation method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 238000001879 gelation Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920001282 polysaccharide Polymers 0.000 description 2
- 239000005017 polysaccharide Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000005303 weighing Methods 0.000 description 2
- 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 description 1
- 241000206575 Chondrus crispus Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000007863 gel particle Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000010865 sewage Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- -1 sulfate radical polysaccharide Chemical class 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000002351 wastewater Substances 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a carrageenan immobilized microorganism and a preparation method thereof. The method mainly comprises the following steps: mixing a carrageenan solution with water; dissolving the mixture by heating in water bath to obtain the carrageenan solution; then adding SiO2 to the carrageenan solution; mixing microorganism suspending liquid to be immobilized with the carrageenan solution to obtain a mixed solution; adding Chitosan oligosaccharide to KCL solution to obtain a cross-linking agent solution; and dropping the mixed solution to the cross-linking agent solution to formwhite ball-shaped particles, namely the immobilized microorganism. The immobilized microorganism particles have high mechanical strength and long service life.
Description
Technical field
The invention belongs to bioengineering field, relate in particular to a kind of carrageeenen immobilized microbe and preparation method thereof.The present invention can be used for biotechnology, fermentation, environmental pollution monitoring and environmental pollution improvement field.
Background technology
Immobilization technology is to make the more extensive and important means of effective utilization of biological catalyst (enzyme, microorganism cells, animal and plant cell).Immobilized microorganism technique has a wide range of applications in every field such as food and fermentation industries, medicine industry, chemical industry, environment protection and energy developments as an emerging technology in the bioengineering field.
The process for fixation of microorganism cells mainly comprises absorption method, crosslinking and entrapping method.Wherein, the most commonly used with entrapping method.Embedded material can be divided into natural macromolecule amylose class and synthetic macromolecular compound.
As the embedding carrier, the sodium alginate of natural macromolecule amylose class and carrageenin are used at most, and they have curing, are shaped conveniently, and are little to microorganism toxicity, immobilization density advantages of higher.But their physical strength is lower, though available linking agent carries out stabilization treatment, vigor and mass-transfer performance can descend again.
Carrier substance commonly used in the synthetic macromolecular compound has polyacrylamide, light-hardening resin etc.Its advantage is that antimicrobial decomposability is good, physical strength height, stable chemical performance.But the formation condition of polymer network is more violent, and is bigger to the infringement of microorganism cells, and the diversity and the controllability that are shaped are bad.
Carrageenin is to extract a kind of polysaccharide that obtains from carrageen, and it contains many sulfate radical polysaccharide, at K
+Exist down, gelation can take place, form immobilization particle.Carrageenin is as the microorganism entrapped immobilized carrier, and advantage such as biologically active height, good, the antimicrobial capacity of decomposition of chemical stability are strong, cheap is a kind of microorganism embedded immobilization material that has practical potentiality.Carrageenin is water-soluble after heating, behind the adding additive gelation takes place in its aqueous solution, thereby the microorganism embedding is fixed in the gel network.Additive commonly used is KCl.But existingly utilize the immobilized microorganism of carrageenin preparation to exist the particle physical strength relatively poor, easily broken in actual use, cause problems such as work-ing life of immobilization microorganism particles is short, restricted its application immobilized microorganism.
Summary of the invention
Problems such as bad mechanical strength, the work-ing life that exists at present carrageeenen immobilized microbe particle is short, the object of the invention is to provide a kind of carrageeenen immobilized microbe.
Another purpose of the present invention provides the preparation method of above-mentioned carrageeenen immobilized microbe.
Realize that technical scheme of the present invention is as follows:
A kind of carrageeenen immobilized microbe is prepared as follows:
(1) place water to mix carrageenin according to 1.50～2.50% weight percent, heating in water bath leaves standstill then and reduces to normal temperature to dissolving, carrageenan solutions; Adding granularity according to 2～6% weight percent in carrageenan solutions again is 200 purpose SiO
2
(2) solution with microbial suspension and step (1) gained is mixed in proportion, and gets mixing solutions;
(3) in KCl solution, add chitosan according to 0.50～1.50% weight percent, obtain cross-linking agent solution; The weight percent of described KCl solution is 1.5～3%;
(4) be added drop-wise in the cross-linking agent solution that is cooled to 30～45 ℃ with syringe or dropper mixing solutions, form white spheroidal particle, and soaked therein 2～10 hours step (2); Take out white spheroidal particle then, and wash 3～5 times with physiological saline, gained white spheroidal particle is immobilized microorganism.
Microorganism described in the said fixing microbial step (2) is meant the mixture of intending immobilized microorganism or microorganism, as pseudomonas, yeast saccharomyces cerevisiae or active sludge etc.
Microbial suspension described in the said fixing microbial step (2) and carrageenan solutions blended ratio depend on the concentration of microorganism in the concentration of carrageenan solutions and the microbial suspension, also be, the amount that depends on microorganism in the immobilization particle, the weight percent of microorganism is 2～10% in the gained immobilized microorganism of the present invention.
Microbial suspension described in the said fixing microbial step (2), can be according to method culturing micro-organisms known in those skilled in the art, then institute's cultured microorganism is carried out centrifugation, obtain wet thallus, wet thallus is suspended in the NaCl solution gets final product again.
The preparation method of above-mentioned carrageeenen immobilized microbe may further comprise the steps:
(1) place water to mix carrageenin according to 1.50～2.50% weight percent, heating in water bath leaves standstill then and reduces to normal temperature to dissolving, carrageenan solutions; Adding granularity according to 2～6% weight percent in carrageenan solutions again is 200 purpose SiO
2
(2) solution with microbial suspension and step (1) gained is mixed in proportion, and gets mixing solutions;
(3) in KCl solution, add chitosan according to 0.50～1.50% weight percent, obtain cross-linking agent solution; The weight percent of described KCl solution is 1.5～3%;
(4) be added drop-wise in the cross-linking agent solution that is cooled to 30～45 ℃ with syringe or dropper mixing solutions, form white spheroidal particle, and soaked therein 2～10 hours step (2); Take out white spheroidal particle then, and wash 3～5 times with physiological saline, gained white spheroidal particle is immobilized microorganism.
Microorganism described in above-mentioned preparation method's step (2) is meant the mixture of intending immobilized microorganism or microorganism, as pseudomonas, yeast saccharomyces cerevisiae or active sludge etc.
Microbial suspension and carrageenan solutions blending ratio depend on the concentration of microorganism in the concentration of carrageenan solutions and the microbial suspension in above-mentioned preparation method's step (2), also be, the amount that depends on microorganism in the immobilization particle, the weight percent of microorganism is 2～10% in the gained immobilized microorganism of the present invention.
Add SiO among the present invention
2Purpose be in order to regulate the proportion of immobilization microorganism particles.
Adding chitosan among the present invention in gelating agent KCl solution, is in order to improve the physical strength of carrageeenen immobilized microbe gel particle.
Above-mentioned carrageenin, oligochitosan etc. all can obtain by open purchase on market.
The advantage that the present invention has: (1) the inventive method can make the physical strength of immobilization microorganism particles significantly improve, can be by traditional 260g/cm as the physical strength with the immobilization pseudomonas of the inventive method preparation
2Bring up to 470g/cm
2(2) the present invention makes the immobilization microorganism particles long service life, as with work-ing life of the immobilization pseudomonas of the inventive method preparation being increased to 25 batches by 10 batches of traditional method.
Embodiment
The preparation of embodiment 1 immobilization pseudomonas
The preparation method is as follows:
(1) 2.0g carrageenin (Shanghai north connect company produce) is mixed with 100ml deionization water, heating in water bath, and slowly be stirred to and dissolve is cooled to normal temperature then, must carrageenan solutions, and adding the 3g granularity again is 200 purpose SiO
2
(2) taking by weighing 5g pseudomonas (Pseudomonas sp.) thalline (weight in wet base) (cultivates pseudomonas (Pseudomonas sp.) 3 days on 30 ℃, 150 rev/mins shaking bath, centrifugation obtains wet thallus) be suspended among the 10ml0.9%NaCl, mix mutually with 100ml step (1) solution, get mixing solutions;
(3) 1g chitosan (production of Shanghai Acoba L. L. C.) being joined the 100ml weight percent is in 2% the KCl solution, to obtain cross-linking agent solution;
(4) mixing solutions of step (2) is added drop-wise in 40 ℃ the cross-linking agent solution with the syringe of 0.40mm ID, forming diameter is the white ball-type globule of 2mm, and soak 5h therein, wash white ball-type globule with physiological saline then, gained white ball-type globule is immobilization pseudomonas (Pseudomonas sp.).
The proof test of embodiment 2 immobilized microorganism effects
Carry out according to following steps:
(1) immobilization pseudomonas (Pseudomonas sp.) particle and the 50ml chlorophenol concentration of adding 5g embodiment 1 are the synthetic wastewater of 150mg/L in the 250ml triangular flask;
(2) water-bath shaking culture 36h under 25 ℃, 140rpm/min, timing sampling detects the content of chlorophenol;
(3) take out immobilization pseudomonas particle, clean up, carry out the next batch experiment with distilled water;
(4) repeated experiments step (2)～(4).
As a result, compare with the traditional method that does not add oligochitosan, the physical strength of the immobilization pseudomonas (Pseudomonas sp.) of the embodiment of the invention 1 preparation is by the 260g/cm of traditional method
2Bring up to 470g/cm
2, physical strength has improved about 80%; Be increased to 25 batch by original 10 batches duration of service; And obtained effect preferably during the chlorophenol that is carried out degraded test; Illustrate that the present invention has significantly improved the physical strength of immobilization microorganism particles, and prolonged work-ing life greatly.
The preparation of embodiment 3 immobilized active mud
The preparation method is as follows:
(1) 2.0g carrageenin (Shanghai north connect company) is mixed with 100mL deionization water, heating in water bath, slowly be stirred to and dissolve, be cooled to normal temperature then, must carrageenan solutions, adding the 3g granularity to carrageenin is 200 purpose SiO
2
(2) take by weighing 5g active sludge thalline (taking from Gaobeidian City, Beijing sewage work), be suspended among the 10ml0.9%NaCl, get the suspension of active sludge, active sludge suspension is mixed mutually with 100ml step (1) solution, get mixing solutions;
(3) 1g chitosan (Shanghai Acoba L. L. C.) being joined the 100ml weight percent is 2% KCl solution, obtains cross-linking agent solution;
(4) syringe with 0.40mm ID is added drop-wise to the mixing solutions in (2) in the cross-linking agent solution that is cooled to 35 ℃, forming diameter is the white ball-type globule of 2mm, and soak 5h therein, wash white ball-type globule with physiological saline then, gained white ball-type globule is the immobilized active mud granule.
(5) the immobilized active mud granule that step (4) is prepared (is seen the biosensor of patent " biological sensor of oxygen amount needed biochemically " (patent No. is: ZL00132125.0)) as BOD biosensor rapid determination instrument, the result shows, the biological activity of immobilized active mud granule is good, can use more than 3 months continuously.
Claims (2)
1. carrageeenen immobilized microbe is prepared as follows:
(1) place water to mix carrageenin according to 1.50～2.50% weight percent, heating in water bath leaves standstill then and reduces to normal temperature to dissolving, carrageenan solutions; Adding granularity according to 2～6% weight percent in carrageenan solutions again is 200 purpose SiO
2
(2) solution with microbial suspension and step (1) gained is mixed in proportion, and gets mixing solutions; Described microorganism is meant pseudomonas or yeast saccharomyces cerevisiae;
(3) in KCl solution, add chitosan according to 0.50～1.50% weight percent, obtain cross-linking agent solution; The weight percent of described KCl solution is 1.5～3%;
(4) be added drop-wise in the cross-linking agent solution that is cooled to 30～45 ℃ with syringe or dropper mixing solutions, form white spheroidal particle, and soaked therein 2～10 hours step (2); Take out white spheroidal particle then, and wash 3～5 times with physiological saline, gained white spheroidal particle is immobilized microorganism.
2. the preparation method of the described carrageeenen immobilized microbe of claim 1 is characterized in that comprising the steps:
(1) place water to mix carrageenin according to 1.50～2.50% weight percent, heating in water bath leaves standstill then and reduces to normal temperature to dissolving, carrageenan solutions; Adding granularity according to 2～6% weight percent in carrageenan solutions again is 200 purpose SiO
2
(2) solution with microbial suspension and step (1) gained is mixed in proportion, and gets mixing solutions; Described microorganism is meant pseudomonas or yeast saccharomyces cerevisiae;
(3) in KCl solution, add chitosan according to 0.50～1.50% weight percent, obtain cross-linking agent solution; The weight percent of described KCl solution is 1.5～3%;
(4) be added drop-wise in the cross-linking agent solution that is cooled to 30～45 ℃ with syringe or dropper mixing solutions, form white spheroidal particle, and soaked therein 2～10 hours step (2); Take out white spheroidal particle then, and wash 3～5 times with physiological saline, gained white spheroidal particle is immobilized microorganism.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102260440A CN101429504B (en) | 2008-11-04 | 2008-11-04 | Carrageenin immobilized microorganism and method of producing the same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008102260440A CN101429504B (en) | 2008-11-04 | 2008-11-04 | Carrageenin immobilized microorganism and method of producing the same |
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| Publication Number | Publication Date |
|---|---|
| CN101429504A CN101429504A (en) | 2009-05-13 |
| CN101429504B true CN101429504B (en) | 2011-03-30 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2008102260440A Expired - Fee Related CN101429504B (en) | 2008-11-04 | 2008-11-04 | Carrageenin immobilized microorganism and method of producing the same |
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| Country | Link |
|---|---|
| CN (1) | CN101429504B (en) |
-
2008
- 2008-11-04 CN CN2008102260440A patent/CN101429504B/en not_active Expired - Fee Related
Non-Patent Citations (3)
| Title |
|---|
| YAN JIANG等.Biosilica-coated κ-carrageenan microspheres for yeast alcohol dehydrogenase encapsulation.J. Biomater. Sci. Polymer. Edn..2007,18(12),第1517-1526页. * |
| 曹亚莉等.固定化微生物细胞技术在废水处理中的应用.微生物学通报.2003,30(3),第77-81页. * |
| 陈兴国等.头袍氨节的κ-卡拉胶-壳聚糖-海藻酸钠缓释体系研究.南开大学学报（自然科学）.2002,35(2),第72-75页. * |
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