CN102093948A - Immobilization bioreactor with enhanced oxygen transfer and application thereof - Google Patents
Immobilization bioreactor with enhanced oxygen transfer and application thereof Download PDFInfo
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- CN102093948A CN102093948A CN2010105786915A CN201010578691A CN102093948A CN 102093948 A CN102093948 A CN 102093948A CN 2010105786915 A CN2010105786915 A CN 2010105786915A CN 201010578691 A CN201010578691 A CN 201010578691A CN 102093948 A CN102093948 A CN 102093948A
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- immobilization
- oxygen transfer
- chitin
- reactor
- bioreactor
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 239000001301 oxygen Substances 0.000 title claims abstract description 27
- 229910052760 oxygen Inorganic materials 0.000 title claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 17
- 229920001542 oligosaccharide Polymers 0.000 claims abstract description 15
- 150000002482 oligosaccharides Chemical class 0.000 claims abstract description 15
- 239000000463 material Substances 0.000 claims abstract description 13
- 229920002101 Chitin Polymers 0.000 claims description 41
- 108090000790 Enzymes Proteins 0.000 claims description 21
- 102000004190 Enzymes Human genes 0.000 claims description 21
- 230000002787 reinforcement Effects 0.000 claims description 10
- 244000144725 Amygdalus communis Species 0.000 claims description 7
- 239000011573 trace mineral Substances 0.000 claims description 7
- 235000013619 trace mineral Nutrition 0.000 claims description 7
- 239000000919 ceramic Substances 0.000 claims description 5
- 229920005830 Polyurethane Foam Polymers 0.000 claims description 4
- 229920000053 polysorbate 80 Polymers 0.000 claims description 4
- 239000011496 polyurethane foam Substances 0.000 claims description 4
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 claims description 3
- 238000007789 sealing Methods 0.000 claims description 3
- 241001557886 Trichoderma sp. Species 0.000 claims description 2
- 238000009826 distribution Methods 0.000 claims description 2
- 230000015556 catabolic process Effects 0.000 abstract description 13
- 238000006731 degradation reaction Methods 0.000 abstract description 13
- 230000012010 growth Effects 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 5
- 230000000593 degrading effect Effects 0.000 abstract description 3
- 238000010008 shearing Methods 0.000 abstract description 3
- 229920001661 Chitosan Polymers 0.000 abstract 3
- 238000005192 partition Methods 0.000 abstract 3
- 230000014759 maintenance of location Effects 0.000 abstract 1
- 241000233866 Fungi Species 0.000 description 5
- 238000004519 manufacturing process Methods 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 4
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 4
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 229950006780 n-acetylglucosamine Drugs 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 239000005862 Whey Substances 0.000 description 3
- 102000007544 Whey Proteins Human genes 0.000 description 3
- 108010046377 Whey Proteins Proteins 0.000 description 3
- 239000007979 citrate buffer Substances 0.000 description 3
- 238000000855 fermentation Methods 0.000 description 3
- 230000004151 fermentation Effects 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000010865 sewage Substances 0.000 description 3
- 238000003756 stirring Methods 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- XBDQKXXYIPTUBI-UHFFFAOYSA-N dimethylselenoniopropionate Natural products CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000005227 gel permeation chromatography Methods 0.000 description 2
- 210000001822 immobilized cell Anatomy 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- -1 polypropylene Polymers 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 description 1
- HLLSOEKIMZEGFV-UHFFFAOYSA-N 4-(dibutylsulfamoyl)benzoic acid Chemical compound CCCCN(CCCC)S(=O)(=O)C1=CC=C(C(O)=O)C=C1 HLLSOEKIMZEGFV-UHFFFAOYSA-N 0.000 description 1
- 241000186426 Acidipropionibacterium acidipropionici Species 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 241000228245 Aspergillus niger Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 102000012286 Chitinases Human genes 0.000 description 1
- 108010022172 Chitinases Proteins 0.000 description 1
- 241000193403 Clostridium Species 0.000 description 1
- 241000238424 Crustacea Species 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 101000925662 Enterobacteria phage PRD1 Endolysin Proteins 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 206010020880 Hypertrophy Diseases 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 239000004743 Polypropylene Substances 0.000 description 1
- 241000235527 Rhizopus Species 0.000 description 1
- 240000005384 Rhizopus oryzae Species 0.000 description 1
- 235000013752 Rhizopus oryzae Nutrition 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000223259 Trichoderma Species 0.000 description 1
- 241000223260 Trichoderma harzianum Species 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000008351 acetate buffer Substances 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 238000010564 aerobic fermentation Methods 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000001857 anti-mycotic effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000002543 antimycotic Substances 0.000 description 1
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 235000011089 carbon dioxide Nutrition 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000006196 deacetylation Effects 0.000 description 1
- 238000003381 deacetylation reaction Methods 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
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- 230000002255 enzymatic effect Effects 0.000 description 1
- 239000003925 fat Substances 0.000 description 1
- 235000019197 fats Nutrition 0.000 description 1
- 229960002442 glucosamine Drugs 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
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- 230000003308 immunostimulating effect Effects 0.000 description 1
- 229910052500 inorganic mineral Inorganic materials 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
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- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- XONPDZSGENTBNJ-UHFFFAOYSA-N molecular hydrogen;sodium Chemical compound [Na].[H][H] XONPDZSGENTBNJ-UHFFFAOYSA-N 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 230000000050 nutritive effect Effects 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 235000019260 propionic acid Nutrition 0.000 description 1
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- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- IUVKMZGDUIUOCP-BTNSXGMBSA-N quinbolone Chemical compound O([C@H]1CC[C@H]2[C@H]3[C@@H]([C@]4(C=CC(=O)C=C4CC3)C)CC[C@@]21C)C1=CCCC1 IUVKMZGDUIUOCP-BTNSXGMBSA-N 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
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- 238000005728 strengthening Methods 0.000 description 1
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- 239000000725 suspension Substances 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
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- 238000005406 washing Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
Images
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses an immobilization bioreactor with enhanced oxygen transfer. The bioreactor provided by the invention comprises a tank body, wherein a partition plate is arranged at the bottom of the tank body, a columnar reaction bed is arranged on the partition plate and comprises an inner tube and an outer tube which are vertically sheathed with each other, the inner tube and the outer tube are both made of porous carrier, the top of the gap between the inner tube and the outer tube and the top of the hollow cavity of the inner tube are sealed by seal covers, a plurality of vent holes are arranged on the partition plate at which the bottom of the gap is located and are distributed surrounding the inner tube, and the inner wall of the inner tube and the outer wall of the outer tube are both attached with immobilization materials. The invention also discloses a method for degrading chitosan by using the reactor to product chitosan oligosaccharides. The immobilization bioreactor disclosed by the invention has the advantages of enhanced oxygen transfer, small shearing force, good oxygen supply conditions, high mycelia immobilization capacity and long activity retention time. According to the method disclosed by the invention, the chitosan degrading efficiency is improved by using the above reactor through optimized growth conditions and a degradation medium.
Description
Technical field
The present invention relates to technical field of bioengineering, relate in particular to a kind of reinforcement oxygen transfer immobilization bioreactor and application thereof.
Background technology
Continuously stirring jar (CSTR) is because the high speed rotating of blade is bigger to the growth effect of filamentous fungus, and free mycelia easily is wound into and makes on the stirring rake that stirring resistance increases, and such reactor is unsuitable for the immobilization of filamentous fungus and cultivates.Immobilization reactor at present relatively more commonly used comprise three-phase fluid bed reactor (Three-phase fluidized bedreactor, TFBR), airlift reactor (Air-lift reactor) and rotating disk reactor (Rotatingdisc reactor) etc.
Airlift reactor because simple in structure, shearing force is little, nutritive substance mixes, oxygen is in liberal supply and have very big advantage in the filamentous fungus immobilization is cultivated.But cause the hypertrophy of mycelia easily, result in blockage, make to cultivate and be forced to stop so that being full of reactor.
At present oneself is widely used in the sewage disposal rotating disk reactor, is one of biomembrance process important device of disposing of sewage.Blain etc. have reported with unrooted rhizopus, aspergillus niger and have been fixed in the polypropylene disc surfaces, form immobilized cell reactor and are used for aerobic fermentation.Rotating bio-disc reactor with its oxygen supply abundance, less energy consumption, occupation of land is little, running cost is low etc., and the application of characteristics in the mould immobilization fermentation has great potential.
That three-phase fluid bed reactor has is simple in structure, power consumption is low, mix, for characteristics such as amount abundances, oneself is applied to aspects such as Rhizopus oryzae immobilized production L-lactic acid at present.But be not suitable for the reaction that product suppresses.In order to make reactor be in fluidized state, mycelia fixed density is generally little than packed bed reactor and stirred pot in the reactor, and throughput is had certain influence.
Yang etc. have developed a kind of spiral fiber bed bio-reactor (Convolutedfi borusbed bioreactor, CFBB), in fact this reactor combines reaction bed and two kinds of immobilization reactors of fluidized-bed advantage separately, it has the big advantage of immobilized cell density that packed bed reactor has concurrently, have the mass transfer that three-phase fluid bed reactor has again and the advantage such as good of conducting heat, overcome in common packed bed reactor and the membrane reactor because the cell growth causes bed to expand caused reactor plugs; It has been avoided again in the three-phase fluidized bed because the phenomenon of the reactor fluctuation of service that biomembranous growth causes simultaneously.Because carrier is fixed in the reactor with volution, therefore provide maximum surface-area for thalline absorption; Simultaneously again can be by changing the thickness of carrier, control mass transfer situation.Slit between the volution carrier comes off the thalline of excess growth and falls to the bottom of reactor with liquid stream, and the carbonic acid gas that air and metabolism simultaneously produces freely upwards by reactor, is on good terms thalline, substratum and air three and is in contact with one another well.
Oneself has carried out a series of researchs with this reactor Yang etc., and co-immobilization Streptococus lactis and Clostridium formicoacetium are converted into acetone with whey (lactose); The whey permeate (de-lactosew heyp ermeate) of immobilization Propionibacterium acidipropionici fermentation demulsification acid is a propionic acid, and yield is 85% of a theoretical value; It is homotype lactic acid that immobilization S.la ctics transforms lactose, and the yield of lactic acid is up to more than 95%; Immobilization Kluvoromyces agifs extractive fermentation whey is an ethanol, and this method can be removed the influence of alcohol to yeast growth effectively.Certain trial has also been done in this external aspect of setting up kinetic model.The shortcoming of this reactor maximum is that air is passed through between the carrier by the bottom of reactor, the bubble residence time is short, the mycelia that therefore can only be carrier surface can obtain oxygen supply preferably, the mycelium of carrier inside is not owing to there is bigger Turbulent Kinetic, so the oxygen transfer resistance is big, can not get sufficient oxygen supply, upgrowth situation is poor.Because the reactor mass transfer is in poor shape, the immobilization mycelia can not get sufficient oxygen supply, keeps secular vigor so have a strong impact on the immobilized bacterium filament.
Chitin (Chitin) is the natural macromolecule amylose compounds that mainly is formed by connecting by β-1,4 glycosidic link by the N-acetylglucosamine.Chitin mainly is present in the cell walls of the shell of Crustacean and some fungi at occurring in nature.Occurring in nature chitin year biosynthesizing amount has 10,000,000,000 tons approximately, wherein 10% derives from the ocean, is to be only second to cellulosic second largest renewable resources on the earth.But these resource long-terms are not fully utilized and run off naturally as refuse regrettably, and the whole world is in order to the amount of chitin extraction also only about 6000 tons.
Chitin is nitrogenous polyose natural bioactivity substance, human body is had crucial physiological function, in the international chitin conference in 1991 they are defined as necessary the 6th biological key element except that sugar, protein, fat, VITAMIN and mineral substance of human body.Chitin can not be water-soluble, needs the effect by chitin enzyme in the human body etc., just can be absorbed through being converted into low-molecular-weight material, and this has limited their effect greatly.In recent years along with the going deep into of research, it is found that crust oligosaccharides that chitin generates through incomplete hydrolysis not only solubleness significantly improve, be easy to be absorbed by the body.The crust oligosaccharides that studies show that different polymerization degree in recent years has a series of important physical effects such as antibacterium, antimycotic, antitumor and immuno-stimulating, causes that day by day people pay attention to.
The crust oligosaccharides can obtain by the method for acid hydrolysis or enzyme liberating.Nineteen fifty-seven, Hoorwitz just begins with the concentrated hydrochloric acid chitin of degrading, preparation crust poly oligosaccharide and glucosamine, but acid system degraded yield is low, and product separation difficulty, environmental pollution are serious, are eliminated gradually in recent years.Since finding chitin enzyme (chitinase), adopted the enzymic degradation chitin to become the focus of research.Enzymic degradation has reaction conditions gentleness, degraded yield advantages of higher, is the developing direction of current degradation of chitin.It is found that at present a lot of microorganisms can produce the degradation of chitin enzyme.There is multiple filamentous fungus in ocean and the land, wherein much has the ability that produces the chitin lytic enzyme, wherein the wooden mould particularly, many chitin lytic enzymes that can produce high vigor.
But traditional chitin enzymic degradation comprises the production of chitin enzyme and two steps of degraded of chitin, owing to carry out under each comfortable different equipment and the reaction conditions, total production cycle is longer, and facility investment is big, and Financial cost is higher.Shortcomings such as but also existence is low such as enzyme activity, produces enzymatic process condition control complexity, and degradation cycle is long.The research work of current enzymic degradation mainly concentrates on the exploitation of producing the enzyme novel bacterial, the separation purification of enzyme and the preliminary study of enzyme liberating mechanism, and is also fewer to the research of the production of chitin enzyme and chitin enzymolysis process.The present invention has developed a kind of new enhanced oxygen transfer immobilization bioreactor and has been used for aerobic hyphomycetic high-density immobilization; And utilize this reactor to develop a kind of novel process that chitin enzyme and crust oligosaccharides production original position are carried out simultaneously of producing, and can reduce the degraded and the extraction cost of producing the crust oligosaccharides, improve the degradation efficiency of chitin, and can reduce sewage discharge.
Summary of the invention
The invention provides a kind of immobilization bioreactor, this reactor is strengthened oxygen transfer, and shearing force is little, and the oxygen supply situation is good, and thalline immobilization amount is big and the time that maintains vigour is long.
A kind of reinforcement oxygen transfer immobilization bioreactor, comprise tank body, tank base is provided with dividing plate, dividing plate is provided with the reaction bed of column, described reaction bed comprises the inner core and the urceolus that are made of porous support that vertically wears, the capped sealing in top of top, gap between inner core and the urceolus and inner core cavity, the dividing plate at place, bottom, gap are provided with some ventilating pits around the inner core distribution, and the inwall of inner core and the outer wall of urceolus all are attached with immobilization material.
The porosity of described porous support is 50～95%, can select materials such as porous ceramic plate for use.
The porosity of described immobilization material is 50～95%, can select hydrophilic polyurethane foam for use, and molecular weight is 20～40KD.
Described ventilating pit quantity is 1000～2000, and the aperture is 0.5～1.0mm.
The present invention also provides above-mentioned bio-reactor to produce the method for crust oligosaccharides, comprise: wooden mould (the Trichoderma sp.) that can produce the chitin enzyme is fixed on the reaction bed, in tank body, add the degraded substratum that contains chitin, cultivated 60～100 hours down at 26～30 ℃, air flow is 2～5vvm.
The prescription of described degraded substratum is:
Wherein, the Mandels trace element formula is: MnSO
4H
2O 1.8g/L, FeSO
47H
2O 5.5g/L, ZnSO
47H
2O 1.8g/L, CoCl
26H
2O 3.5g/L.
Described chitin molecule amount is 100KD～1000KD.
Apparatus of the present invention feed pressurized air by the gap between reaction bed two annular carriers, the top, gap is sealed, make air see through carrier and immobilization material enters tank body, make the mycelia on the immobilization material can access competent oxygen, and can grow and extend in the hole of immobilization material, increase the immobilization amount of mycelia, thereby improved product enzyme efficient.
The growth conditions after the inventive method is passed through to optimize and the prescription of degraded substratum, and utilize above-mentioned reactor, strengthened oxygen mass transfer, degradation of chitin efficient height.
Description of drawings
Fig. 1 is the axial section of reactor of the present invention;
Fig. 2 is the radial section figure of reactor of the present invention;
Fig. 3 is the axial section of reaction bed.
Embodiment
As shown in Figure 1, a kind of reinforcement oxygen transfer immobilization bioreactor comprises tank body 1, and tank body 1 middle part is cylindrical, and top and bottom are roughly tapered, and blade diameter length ratio is 1: 2～1: 8, and volume is 20L, and the bottom is provided with dividing plate 3.
Urceolus 21 outer walls and inner core 22 outer walls all are attached with porous immobilization material 24, and the present embodiment immobilization material adopts hydrophilic polyurethane foam, and porosity is 80%, and molecular weight is 30.3KD.The porosity of above-mentioned porous ceramic plate is 60%.During use, tank body 1 bottom connects pneumatic pump 4, the input sterile air.
Substratum
(1) growth medium (%): glucose 15, peptone 1, urea 0.24, (NH
4)
2SO
40.48, K
2SO
40.1, MgCl
20.12, MgSO
47H
2O 0.08, tween (80) 0.08, NaCl 0.4, KBr0.05, Mandels trace element 0.6Ml/L.PH withers to 4.5 with the 1mol/L citrate buffer solution.
Mandels trace element: MnSO
4H
2O 1.8g/L, FeSO
47H
2O 5.5g/L, ZnSO
47H
2O1.8g/L, CoCl
26H
2O 3.5g/L.
(2) degraded substratum (%): soluble chitin (200KD, deacetylation 30%) 5%, KH
2PO
40.5%, CaCl
2H
2O 0.05%, MgSO
47H
2O 0.05%, tween 80 0.12%, Mandels trace element 2mL/L.PH transfers to 4.5 with the 1mol/L citrate buffer solution.Above substratum is all at 121 ℃ of 30min that sterilize down.PH transfers to 4.5 with the 1mol/L citrate buffer solution.
Above substratum is all at 121 ℃ of 30min that sterilize down.
The immobilization that wood is mould
Grown cultures is imported in the tank body 1, and be inoculated in the growth medium, make the spore concentration in the growth medium reach 106/mL in the ratio of 6% (v/v) spore suspension with trichoderma harziarum (Trichoderma harzianum) ATCC 48131.At 30 ℃, to cultivate under the air flow 2.5vvm condition, mycelia is fixed on the hydrophilic polyurethane foam.
Continuous degradation
At first behind usefulness pH4.50.05M acetate buffer solution washing reaction bed 2 times, remove unnecessary growth medium, and then in tank body 1, add 12L degraded substratum, 28 ℃, cultivate after 80 hours under the air flow 2.5vvm condition, the degraded substratum that adds the soluble chitin that contains same concentrations with certain flow carries out continuous degradation, and wherein thinning ratio (D) is defined as the volumetric flow rate of substratum adding and the ratio of reactor volume.
Test result
Choose the degraded substratum of the chitin that contains same molecular quality (500KD) same concentrations (2.25%), utilize reactor of the present invention and conventional helical fibre bed bio-reactor to produce the crust oligosaccharides under the same conditions respectively, the tank body volume of two kinds of reactors, reaction bed surface-area and size, bacterial classification immobilization time are all identical, test result such as following table 1 (a, b, c) shown in:
The comparison of two kinds of bio-reactor parameters of table 1 (a)
The comparison of two kinds of bio-reactor parameters of table 1 (b)
The comparison of two kinds of bio-reactor parameters of table 1 (c)
As seen from table: obtaining under the identical condition of crust oligosaccharides mean polymerisation degree, the chitin enzyme chitin enzyme of strengthening in the oxygen transfer immobilization bioreactor that is higher than far away in the spiral fiber bed bio-reactor alive is lived; Thinning ratio (D) is compared also and has been increased, and degradation efficiency greatly improves.
The measuring method that the chitin enzyme is lived
Adopt Monreal and Reese method, the phosphoric acid expansion chitin of 1mL 1% (with 0.05M pH5.0 Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution preparation), the enzyme liquid of an amount of dilution of 1mL, 50 ℃ are incubated 60 minutes, drip the sodium hydroxide of 1 droplet 10N, centrifugal 10 minutes, get supernatant liquor, measure the content of N-acetylglucosamine in the supernatant liquor with the Reissig method.(enzyme liquid is measured NAG content in contrast with same procedure).A chitin enzyme international unit (IU) is expressed as the N-acetylglucosamine amount that per minute under these conditions discharges 1 μ mol.
Crust oligosaccharides mean polymerisation degree measuring method
Crust oligosaccharides mean polymerisation degree (DP) equals the molecular weight of crust oligosaccharides weight-average molecular weight (Mw) divided by N-acetylglucosamine glycosides, the mensuration of crust oligosaccharides weight-average molecular weight Mw adopts gel permeation chromatography (GPC) method, serves as with reference to product with the dextran standard specimen of known molecular amount.
Claims (9)
1. strengthen the oxygen transfer immobilization bioreactor for one kind, comprise tank body, tank base is provided with dividing plate, dividing plate is provided with the reaction bed of column, it is characterized in that: described reaction bed comprises the inner core and the urceolus that are made of porous support that vertically wears, the capped sealing in top of top, gap between inner core and the urceolus and inner core cavity, the dividing plate at place, bottom, gap are provided with some ventilating pits around the inner core distribution, and the inwall of inner core and the outer wall of urceolus all are attached with immobilization material.
2. reinforcement oxygen transfer immobilization bioreactor according to claim 1 is characterized in that the porosity of described porous support is 50～95%.
3. reinforcement oxygen transfer immobilization bioreactor according to claim 1 is characterized in that described porous support is a porous ceramic plate.
4. reinforcement oxygen transfer immobilization bioreactor according to claim 1 is characterized in that the porosity of described immobilization material is 50～95%.
5. reinforcement oxygen transfer immobilization bioreactor according to claim 1 is characterized in that described immobilization material is a hydrophilic polyurethane foam, and molecular weight is 20～40KD.
6. reinforcement oxygen transfer immobilization bioreactor according to claim 1 is characterized in that described ventilating pit quantity is 1000～2000, and the aperture is 0.5～1.0mm.
7. method of utilizing the described reinforcement oxygen transfer of claim 1 immobilization bioreactor to produce the crust oligosaccharides, comprise: wooden mould (the Trichoderma sp.) that can produce the chitin enzyme is fixed on the reaction bed, in tank body, add the degraded substratum that contains chitin, cultivated 60～100 hours down at 26～30 ℃, air flow is 2～5vvm.
8. method according to claim 1 is characterized in that, the prescription of described degraded substratum is:
Chitin 1%～5%, KH
2PO
40.1～0.5%, CaCl
2H
2O 0.01～0.08%, MgSO
47H
2O0.01～0.05%, tween 80 0.08～0.12%, Mandels trace element 0.5～5mL/L;
Wherein, the Mandels trace element formula is: MnSO
4H
2O 1.8g/L, FeSO
47H
2O 5.5g/L, ZnSO
47H
2O 1.8g/L, CoCl
26H
2O 3.5g/L.
9. method according to claim 8 is characterized in that: described chitin molecule amount is 100KD～1000KD.
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| CN103865958A (en) * | 2012-12-13 | 2014-06-18 | 南京工业大学 | Method for producing ethanol by adopting immobilized yeast cells for continuous fermentation |
| CN105255731A (en) * | 2015-11-02 | 2016-01-20 | 南方医科大学珠江医院 | Circulation filling type cell culture system and bioreactor of circulation filling type cell culture system |
| CN114250147A (en) * | 2021-12-29 | 2022-03-29 | 上海日泰医药设备工程有限公司 | Biological reaction device |
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| CN114276927A (en) * | 2021-12-29 | 2022-04-05 | 上海日泰医药设备工程有限公司 | Folding carrier column for bioreactor |
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