CN102093948B - 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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- CN102093948B CN102093948B CN 201010578691 CN201010578691A CN102093948B CN 102093948 B CN102093948 B CN 102093948B CN 201010578691 CN201010578691 CN 201010578691 CN 201010578691 A CN201010578691 A CN 201010578691A CN 102093948 B CN102093948 B CN 102093948B
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- immobilization
- chitin
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- oxygen transfer
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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 is prone to be wound into and makes on the stirring rake that stirring resistance increases, and such reactor drum is inappropriate 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 in the filamentous fungus immobilization is cultivated, have very big advantage.But cause the hypertrophy of mycelia easily, result in blockage, make and cultivate compelled stopping so that being full of reactor drum.
At present oneself is widely used in the WWT rotating disk reactor, is one of biomembrance process important device of disposing of sewage.Blain etc. have reported with unrooted rhizopus, black mold and have been fixed in the Vestolen PP 7052 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.
Characteristics such as that three-phase fluid bed reactor has is simple in structure, power consumption is low, mix, confession amount abundance, 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 drum be in fluidized state, mycelia fixed density is generally little than packed bed reactor and stirred pot in the reactor drum, 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 drum has combined 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 and the advantage such as good of conducting heat again, overcome in common packed bed reactor and the membrane reactor because cell is grown and caused bed to expand caused reactor plugs; It has been avoided again in the three-phase fluidized bed because the phenomenon of the reactor drum fluctuation of service that biomembranous growth causes simultaneously.Because carrier is fixed in the reactor drum with volution, therefore provide maximum surface-area to supply thalline absorption; Simultaneously again can be through 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 drum with flow, and the carbonic acid gas that air and metabolism simultaneously produces freely upwards through reactor drum, 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 drum 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 maximum shortcoming of this reactor drum is that air is passed through between the carrier by the bottom of reactor drum; The bubble residence time is short; The mycelia that therefore can only be carrier surface can obtain oxygen supply preferably, and the mycelium of carrier inside is not owing to there being bigger Turbulent Kinetic, so the oxygen transfer resistance is big; Can not get sufficient oxygen supply, upgrowth situation is poor.Because the reactor drum 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 through β-1,4 glycosidic link by the N-acetylglucosamine.Chitin mainly is present in the cell walls of shell and some fungi of Crustacean 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, and it is necessary except that sugar, protein, fat, VITAMINs and the 6th biological key element the mineral substance to confirm as human body to them in the international chitin conference in 1991.Chitin can not be water-soluble, needs the effect through chitin enzyme in the human body etc., just can be absorbed through being converted into low-molecular-weight material, this big limitations their effect.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.Research in recent years shows that the crust oligosaccharides of different polymerization degree 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 through 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 GS, 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 mikrobes 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 complicacy, 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 drum 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 drum 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, and dividing plate is provided with the reaction bed of column; Described reaction bed comprises the inner core and the urceolus that are made up 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 is 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.
Said 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 through the gap between reaction bed two annular carriers; The top, gap is sealed; Make air permeation carrier and immobilization material get into tank body, make that 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 drum, strengthened oxygen mass transfer, degradation of chitin efficient is high.
Description of drawings
Fig. 1 is the axial section of reactor drum of the present invention;
Fig. 2 is the radial section figure of reactor drum 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.
Dividing plate 3 is provided with the reaction bed 2 of cylindricality; Reaction bed 2 is made up of with inner core 22 the vertical urceolus of being processed by porous ceramic plate 21 that wears each other; The top, gap between urceolus 21 and the inner core 22 and the top of inner core 21 are by ceramic capping 23 sealings; The dividing plate 3 at place, bottom, gap is provided with the ventilating pit 31 of a row around inner core 22, roughly 1000～2000 of quantity, and the aperture is 0.5mm.
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 through after with 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 drum 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 reactor drums, 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)
Can be known by 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, measure the content of N-acetylglucosamine in the supernatant with the Reissig method.(enzyme liquid is measured NAG content as contrast with same procedure).A chitin enzyme iu (IU) is expressed as the N-acetylglucosamine amount that PM 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 article with the VISOSE standard specimen of known molecular amount.
Claims (5)
1. strengthen the oxygen transfer immobilization bioreactor for one kind, comprise tank body, tank base is provided with dividing plate, and 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 up 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 is 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;
Described porous support is a porous ceramic plate, and the porosity of described porous support is 50 ~ 95%;
Described immobilization material is a hydrophilic polyurethane foam, and molecular weight is 20 ~ 40KD, and the porosity of described immobilization material is 50 ~ 95%.
2. reinforcement oxygen transfer immobilization bioreactor according to claim 1 is characterized in that said ventilating pit quantity is 1000 ~ 2000, and the aperture is 0.5 ~ 1.0mm.
3. 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.
4. method according to claim 3 is characterized in that, the prescription of described degraded substratum is:
Chitin 1% ~ 5%, KH
2P0
40.1 ~ 0.5%, CaCl
2H
2O 0.01 ~ 0.08%, MgS0
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: MnS0
4H
2O 1.8g/L, FeS0
47H
2O 5.5g/L, ZnS0
47H
2O 1.8g/L, CoCl
26H
2O 3.5g/L.
5. method according to claim 4 is characterized in that: described chitin molecule amount is 100KD ~ 1000KD.
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| CN103865958B (en) * | 2012-12-13 | 2016-06-29 | 南京工业大学 | A kind of method adopting fixed yeast cell continuous fermentation to produce ethanol |
| CN105255731B (en) * | 2015-11-02 | 2016-11-09 | 南方医科大学珠江医院 | Circumfusion formula cell culture system and bioreactor thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064069A (en) * | 1991-02-05 | 1992-09-02 | 景德镇陶瓷学院 | Ceramic carrier for fixed cells and preparation method |
| CN101215519A (en) * | 2007-01-05 | 2008-07-09 | 中国科学院过程工程研究所 | Fixation cell (or enzyme) internal circulation fluidized bed reactor and application thereof in organic phase biological catalysis |
| CN101402910A (en) * | 2008-03-04 | 2009-04-08 | 周剑平 | Reaction still for producing high-viscosity cell immobilization carrier |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS57127432A (en) * | 1981-01-30 | 1982-08-07 | Mitsubishi Heavy Ind Ltd | Ventilating and stirring device with incorporated heat exchanger |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1064069A (en) * | 1991-02-05 | 1992-09-02 | 景德镇陶瓷学院 | Ceramic carrier for fixed cells and preparation method |
| CN101215519A (en) * | 2007-01-05 | 2008-07-09 | 中国科学院过程工程研究所 | Fixation cell (or enzyme) internal circulation fluidized bed reactor and application thereof in organic phase biological catalysis |
| CN101402910A (en) * | 2008-03-04 | 2009-04-08 | 周剑平 | Reaction still for producing high-viscosity cell immobilization carrier |
Non-Patent Citations (1)
| Title |
|---|
| JP昭57-127432A 1982.08.07 |
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