CN108660132A - A kind of surface immobilized yeast fermentation technique - Google Patents

A kind of surface immobilized yeast fermentation technique Download PDF

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Publication number
CN108660132A
CN108660132A CN201810321244.8A CN201810321244A CN108660132A CN 108660132 A CN108660132 A CN 108660132A CN 201810321244 A CN201810321244 A CN 201810321244A CN 108660132 A CN108660132 A CN 108660132A
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Prior art keywords
fermentation
immobilized yeast
fermentation technique
yeast
yeast fermentation
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CN201810321244.8A
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Chinese (zh)
Inventor
陈勇
刘庆国
应汉杰
邹亚男
赵南
刘桂文
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Nanjing Hi Tech Institute Of Biotechnology Research Co Ltd
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Nanjing Hi Tech Institute Of Biotechnology Research Co Ltd
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N11/00Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/02Enzymes or microbial cells immobilised on or in an organic carrier
    • C12N11/10Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
    • C12N11/12Cellulose or derivatives thereof
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12PFERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00Preparation of oxygen-containing organic compounds
    • C12P7/02Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • C12P7/06Ethanol, i.e. non-beverage
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02EREDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00Technologies for the production of fuel of non-fossil origin
    • Y02E50/10Biofuels, e.g. bio-diesel

Abstract

The invention discloses a kind of surface immobilized yeast fermentation techniques, and fibrous material is fixed in spherical grid by it, are then fixed on yeast on fiber carrier by the effect of absorption.The technology overcomes the deficiencies in the prior art, and not only input cost is low for the process for fixation, easy to operate, process stabilizing, adsorption effect are good, but also has protection and facilitation well to cell activity and growth.Using the fixed yeast cells fermentative production of ethanol of the medium, the conversion ratio that the long-term efficient stable fermentation of yeast can not only be effectively ensured, substrate can also be improved.

Description

A kind of surface immobilized yeast fermentation technique
Technical field
The invention belongs to industrial biotechnology fields, and in particular to a kind of surface immobilized yeast fermentation technique.
Background technology
With natural resources scarcity and environmental problem it is outstanding day by day, the status of biofermentation technique is also increasingly heavier It wants, also achieves fast development in recent years.However conventional liquid submerged fermentation still remains natural defect, such as due to mechanicalness Damage and discomfort liquid environment, the stability of cell growth is poor, easily occur significant apoptosis and autolysis and Spatiotemporal efficiency is low etc., causes the maturity of biofermentation technique to be difficult to comparable with chemical catalyst, this is also biology manufacture mesh Before be difficult to substitute one of the main bottleneck of chemistry manufacture.
For the deficiency during traditional zymotic, much scholars have studied work of the fixed yeast to alcohol fermentation at present With.Immobilized cell technology essence is that free cell is positioned to the area of space of restriction with means physically or chemically, makes it Keep catalytic activity, and can Reusability, reduce cell be lost in.Immobilized cell have that fermentation period is short, tolerance is strong and The advantages that high conversion rate.Traditional process for fixation mainly uses gel embedding technology, that is, uses carrageenan, carragheen, seaweed Fermentation is fixed to cell in sour calcium, sodium alginate etc..Although such occlusion vehicle has convenient formation and immobilization density high The advantages that, but its mechanical strength is low, and mass transfer effect is poor.We have applied for surface immobilized yeast fermentation technique (such as patent before Number ZL201210538977.X), i.e., the fibrous material of specially treated is attached to support frame, and be filled into bioreactor In, then yeast cells is fixed on the fibrous material in bioreactor, to realize the purpose of Immobilized yeast. The technology used carrier prolonged and repeated can utilize, and fermentation efficiency significantly improves, and stability is very strong, but this method exists centainly Defect, i.e. early investment cost is higher, and repair is relatively difficult.
Invention content
The technical problem to be solved in the present invention is to provide a kind of surface immobilized yeast fermentation techniques, to solve the prior art The problems such as existing ineffective.
In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:
A kind of surface immobilized yeast fermentation technique, it includes the following steps:
(1) adsorbing medium is fixed in Ball Grid, and Ball Grid is filled in fermentation tank;
(2) it is pumped into seed liquor into the fermentation tank described in step (1), cultivates and recycles fixation;
(3) after seed is fixed, seed wine with dregs is discharged, is pumped into zymotic fluid, the zymotic fluid more renewed after fermentation.
In step (1), the adsorbing medium is fiber, polyurethane foam, in wooden slag and stalk any one or it is several The combination of kind.
Wherein, the adsorbing medium can also first pass through the processing of following mode in advance:
Adsorbing medium is first placed on a concentration of 1~100g/L surface modifier (such as polyetherimide, Diethylenetriamine, Dimethylamine propylamine) impregnating 1~40 hour in aqueous solution and in the cross-linking agent aqueous solution of 1~100g/L, (temperature is normal Temperature), then dried after fibrous material is placed in deionized water fully rinsing.
In step (1), the Ball Grid is made of the material of corrosion-and high-temp-resistant acid and alkali-resistance, preferably polytetrafluoroethyl-ne Alkene, polyvinyl chloride or polyethylene.
In step (1), the adsorbing medium is round sheet, a diameter of 6~12cm;The Ball Grid is a diameter of 15~25cm.
In step (1), 2~4 adsorbing mediums are housed in a Ball Grid.
In step (1), the ratio of height to diameter of the fermentation tank is 1.2~2.2:1, sheet adsorbing medium loading be 50~ 200cm2/L。
In step (2), the formula of the seed liquor is human configuration culture medium, the saccharification of corn, cassava or grain in storage for years One kind in liquid (clear liquid after press filtration) and molasses, yeast quantity are 2.0~3.5 hundred million/ml;
Wherein,
The formula of the human configuration culture medium be 20~100g/L of glucose, 5~20g/L of peptone, yeast extract 5~ 20g/L, 0.1~1g/L of magnesium sulfate, Ph4.0~5.0;
In the saccharified liquid of the corn, cassava or grain in storage for years, material water quality ratio is 2.5~3:1;
The initial total sugar of the molasses is 20~180g/L;
Wherein, the culture and to recycle fixation refer to that (pot bottom comes out Cyclic culture at 30~35 DEG C, and tank upper end enters Material), internal circulating load be 0.5~4BV (1BV refer to 1 hour recycle 1 time, i.e. 1v/v/h).
In step (3), the zymotic fluid is human configuration culture medium, the saccharified liquid (press filtration of corn, cassava or grain in storage for years Clear liquid afterwards), any one or the combination of several of them in the hydrolyzate and molasses of stalk or cellulose;
Wherein,
The formula of the human configuration culture medium be 20~100g/L of glucose, 5~20g/L of peptone, yeast extract 5~ 20g/L, 0.1~1g/L of magnesium sulfate, Ph4.0~5.0;
In the saccharified liquid of the corn, cassava or grain in storage for years, material water quality ratio is 2.5~3:1;
In the hydrolyzate of the stalk or cellulose, feed liquid mass ratio is 1:2~3, pH are 4.5~5.2;Hydrolysising condition For 100~250r/min of rotating speed, 45~60 DEG C of temperature digests 1~2.5 hour;
The initial total sugar of the molasses is 20~180g/L.
Wherein, seed liquor volume accounts for the 5~20% of the volume of the zymotic fluid used in one time fermentation.
In step (3), the seed, which is fixed, to be terminated to refer to that yeast thalline adsorbance reaches 1.0~3.0 hundred million/ml, institute The fermentation ends stated refer to that the concentration of reduced sugar in fermentation tank is reduced to 0.5~4g/L.
Advantageous effect:
Compared with prior art, the present invention has following advantage:
1, the immobilization technology material therefor that the present invention uses is at low cost, non-toxic;Physico-chemical property is stablized, can be in acid item Non-degradable up to 1 year under part, adsorbing medium surface area is big, and adsorption effect is good;Moreover, dead cell can voluntarily fall off, living cells energy It enough realizes own amplification, maintains very high production efficiency always;
2, gap is big between the immobilization way carrier that uses of the present invention, compared to other absorption or cross-linking method mass transfer effect compared with Good, fermenting speed is fast, and substrate conversion efficiency is high;
3, absorption carrier input cost is low, and filling and taking-up are simple, and easy access is suitable for industrial applications.
Description of the drawings
Figure 1A is the vertical view of sorbing material;
Figure 1B is the vertical view of the holder of fixed sorbing material;
Fig. 2A is the schematic diagram of Ball Grid;
Fig. 2 B are the internal structure front view of Ball Grid;
The unit of size is cm in figure.
Specific implementation mode
The present invention is described below with reference to specific embodiments.It will be appreciated by those skilled in the art that these embodiments are only For illustrating the present invention, do not limit the scope of the invention in any way.
Experimental method in following embodiments is unless otherwise specified conventional method.Original as used in the following examples Material, reagent etc. are commercially available products unless otherwise specified.
Embodiment 1 prepares alcohol fuel using synthetic media as fermentation raw material using bamboo fibre fixed yeast
First, the bamboo fibre (thickness 0.15cm) for being cut into diameter 15cm is put into made of polytetrafluoroethylene (PTFE) (PTFE) as schemed In the ball grid of diameter 20cm shown in 1, it is prepared into fixation support (Fig. 2);Then by entrapment media with 150cm2/ L's fills out Charge is put into ratio of height to diameter 2:In 1 15L reactors.Then cultured strain is added in fermentation tank, is followed for 30L/h with flow velocity Ring fixes 36h.Until 2.0 hundred million/ml of thalline adsorption concentration.It is then discharged out waste liquid.Fed-batch cultivation culture medium (concentration of glucose again For 220g/L), liquid amount 75% carries out circulating fermentation, and wherein temperature is 33 DEG C, zymotic fluid pH4.5, flow velocity 30L/h.5 batches The average fermentation period is 20h, and reaction density reaches 103.5g/L, and specific ionization fermentation shortens 8h.Alcohol yied reaches 5.2gL- 1h-1, average conversion 92.1%, specific ionization fermentation raising 20% and 6.5%, ferments than Immobilization in Sodium Alginate carrier and carries respectively High 35% and 3.5%.
Embodiment 2 prepares alcohol fuel using cassava liquefied fermented glutinous rice culture medium as fermentation raw material using cotton fiber fixed yeast
First, the cotton fiber for being cut into diameter 20cm is put into diameter 25cm ball grids made of polyethylene, is prepared into Entrapment media;Then by entrapment media with 120cm2The loading of/L is put into ratio of height to diameter 1.8:In 1 15L reactors.So Cultured strain is added in fermentation tank afterwards, is the fixed 48h of 15L/h cycles with flow velocity.Until thalline adsorption concentration 2.5 hundred million A/ml.Then cassava liquefied fermented glutinous rice is prepared:Solid-liquid ratio 1:2.5, add 85 DEG C of α-amylase (15U/g cassavas dry weight) to liquefy 2 hours, it is cold But immobilization seed waste liquid is discharged afterwards.It flows again and adds cassava liquefied fermented glutinous rice, and carbohydrase (180U/g cassavas dry weight), liquid amount is added 75%, circulating fermentation is carried out, wherein temperature is 35 DEG C, zymotic fluid pH4.5, flow velocity 15L/h.The equal fermentation period of 10 criticize flats is For 24 hours, reaction density reaches 102.8g/L, and specific ionization fermentation shortens 4h.Alcohol yied reaches 4.28gL-1h-1, average conversion 91.8%, respectively 20% and 3% are improved than stalk absorption carrier.
Embodiment 3 prepares fuel using high concentration cassava liquefied fermented glutinous rice culture medium as fermentation raw material using cotton fiber fixed yeast Ethyl alcohol
First, the cotton fiber for being cut into diameter 8cm is put into diameter 12cm ball grids made of polyvinyl fluoride, is prepared into Entrapment media;Then by entrapment media with 160cm2The loading of/L is put into ratio of height to diameter 1.8:In 1 50L reactors.So Cultured strain is added in fermentation tank afterwards, is the fixed 48h of 30L/h cycles with flow velocity.Until thalline adsorption concentration 2.0 hundred million A/ml.Then cassava liquefied fermented glutinous rice is prepared:Solid-liquid ratio 1:2, add 90 DEG C of α-amylase (18U/g cassavas dry weight) to liquefy 1.5 hours, it is cold But immobilization seed waste liquid is discharged afterwards.It flows again and adds cassava liquefied fermented glutinous rice, and carbohydrase (200U/g cassavas dry weight), liquid amount is added 75%, circulating fermentation is carried out, wherein temperature is 36 DEG C, zymotic fluid pH4.2, flow velocity 30L/h.The equal fermentation period of 6 criticize flats is 44h (initial total sugar 263g/L), reaction density reach 125.2g/L, and specific ionization fermentation shortens 6h.Alcohol yied reaches 2.85gL- 1h-1, average conversion 93.1%, under identical fermentation condition, than patent (patent No. ZL201210538977.X) institute before us Process for fixation (same sorbing material is attached to steel wire net surface) yield improves 2.5%, than the support used in it Material cost reduces 80% or more, and picks and places more convenient, it is easier to be applied to production.
Embodiment 4 is prepared using corn liquefied fermented glutinous rice culture medium as fermentation raw material using the cotton fiber fixed yeast of chemical modification Alcohol fuel
First, by cotton fiber after glutaraldehyde is chemically crosslinked, it is cut into the size of diameter 15cm, polytetrafluoroethylene (PTFE) is put into and is made Diameter 20cm ball grids in, be prepared into entrapment media;Then by entrapment media with 120cm2The loading of/L is put into Ratio of height to diameter 1.6:In 1 50L reactors.Then cultured strain is added in fermentation tank, is that 45L/h cycles are fixed with flow velocity 48h.Until 2.2 hundred million/ml of thalline adsorption concentration.Then corn liquefied fermented glutinous rice is prepared:Solid-liquid ratio 1:2.3, add α-amylase (15U/g Cassava dry weight) 86 DEG C of liquefaction 1.5 hours, immobilization seed waste liquid is discharged after cooling.It flows again and adds cassava liquefied fermented glutinous rice, and saccharification is added Enzyme (200U/g cassavas dry weight), liquid amount 75% carry out circulating fermentation, and wherein temperature is 34 DEG C, zymotic fluid pH4.2, and flow velocity is 45L/h.The equal fermentation period of 8 criticize flats is 20h (initial total sugar 216.7g/L), and reaction density reaches 102.5g/L, specific ionization fermentation Shorten 6h.Alcohol yied reaches 5.13gL-1h-1, average conversion 92.6%, specific ionization fermentation raising 6.0%.
Embodiment 5 prepares alcohol fuel using molasses as fermentation raw material using activated carbon fibre fixed yeast
First, activated carbon fiber is cut into the size of diameter 12cm, it is spherical to be put into diameter 16cm made of polytetrafluoroethylene (PTFE) In grid, it is prepared into entrapment media;Then by entrapment media with 200cm2The loading of/L is put into ratio of height to diameter 1.5:1 In 50L reactors.Then cultured strain is added in fermentation tank, is the fixed 48h of 60L/h cycles with flow velocity.Until thalline 2.0 hundred million/ml of adsorption concentration.Then molasses culture medium is prepared:Molasses and water 1:1.2 dilutions adjust acid to 3.0, and heating acidification 1 is small When, it stands and removes precipitation, immobilization seed waste liquid is discharged after cooling.Sugaring honey is flowed again, liquid amount 75% carries out circulating fermentation, Wherein temperature is 35 DEG C, zymotic fluid pH4.0, flow velocity 60L/h.The equal fermentation period of 20 criticize flats is 28h (initial total sugar 230g/L), Reaction density reaches 108.3g/L, and specific ionization fermentation shortens 6h.Alcohol yied reaches 3.87gL-1h-1, average conversion 92.1%, specific ionization fermentation improves 3.8%.

Claims (10)

1. a kind of surface immobilized yeast fermentation technique, which is characterized in that it includes the following steps:
(1) adsorbing medium is fixed in Ball Grid, and Ball Grid is filled in fermentation tank;
(2) it is pumped into seed liquor into the fermentation tank described in step (1), cultivates and recycles fixation;
(3) after seed is fixed, seed wine with dregs is discharged, is pumped into zymotic fluid, the zymotic fluid more renewed after fermentation.
2. surface immobilized yeast fermentation technique according to claim 1, which is characterized in that in step (1), the suction Attached medium is fiber, polyurethane foam, any one or the combination of several of them in wooden slag and stalk.
3. surface immobilized yeast fermentation technique according to claim 1, which is characterized in that in step (1), the ball The material of type grid is polytetrafluoroethylene (PTFE), polyvinyl chloride or polyethylene.
4. surface immobilized yeast fermentation technique according to claim 1, which is characterized in that in step (1), the suction Attached medium is round sheet, a diameter of 6~12cm;A diameter of 15~the 25cm of the Ball Grid.
5. surface immobilized yeast fermentation technique according to claim 1, which is characterized in that in step (1), a ball-type 2~4 adsorbing mediums are housed in grid.
6. surface immobilized yeast fermentation technique according to claim 1, which is characterized in that in step (1), the hair The ratio of height to diameter of fermentation tank is 1.2~2.2:1, sheet adsorbing medium loading is 50~200cm2/L。
7. surface immobilized yeast fermentation technique according to claim 1, which is characterized in that in step (2), the kind The formula of sub- liquid is human configuration culture medium, one kind in the saccharified liquid and molasses of corn, cassava or grain in storage for years, and yeast quantity is 2.0~3.5 hundred million/ml;
Wherein,
The formula of the human configuration culture medium is 20~100g/L of glucose, 5~20g/L of peptone, 5~20g/ of yeast extract L, 0.1~1g/L of magnesium sulfate, Ph4.0~5.0;
In the saccharified liquid of the corn, cassava or grain in storage for years, material water quality ratio is 2.5~3:1;
The initial total sugar of the molasses is 20~180g/L;
Wherein, the culture and to recycle fixation refer to Cyclic culture at 30~35 DEG C, internal circulating load 0.5-4BV.
8. surface immobilized yeast fermentation technique according to claim 1, which is characterized in that in step (3), the hair Zymotic fluid is human configuration culture medium, the saccharified liquid of corn, cassava or grain in storage for years, in the hydrolyzate and molasses of stalk or cellulose Any one or the combination of several of them;
Wherein,
The formula of the human configuration culture medium is 20~100g/L of glucose, 5~20g/L of peptone, 5~20g/ of yeast extract L, 0.1~1g/L of magnesium sulfate, Ph4.0~5.0;
In the saccharified liquid of the corn, cassava or grain in storage for years, material water quality ratio is 2.5~3:1;
In the hydrolyzate of the stalk or cellulose, feed liquid mass ratio is 1:2~3, pH are 4.5~5.2;
1:3-1:2, pH4.5-5.2, hydrolysising condition is rotating speed 100-250r/min, and 45-60 DEG C of temperature digests 1-2.5 hours;
The initial total sugar of the molasses is 20~180g/L.
9. surface immobilized yeast fermentation technique according to claim 1, which is characterized in that seed liquor volume accounts for primary hair The 5~20% of the volume of zymotic fluid used in ferment.
10. surface immobilized yeast fermentation technique according to claim 1, which is characterized in that described in step (3) Seed, which is fixed, to be terminated to refer to that yeast thalline adsorbance reaches 1.0~3.0 hundred million/ml, and the fermentation ends refer in fermentation tank The concentration of reduced sugar is reduced to 0.5~4g/L.
CN201810321244.8A 2018-04-11 2018-04-11 A kind of surface immobilized yeast fermentation technique Pending CN108660132A (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109943555A (en) * 2018-12-29 2019-06-28 南京工业大学 A method of distiller's yeast, which is continuously prepared, using surface immobilized technology carries out alcoholic fermentation
CN109988757A (en) * 2018-12-29 2019-07-09 江苏省生产力促进中心 A kind of method that surface immobilized yeast semicontinuous fermentation prepares ethyl alcohol
CN110117588A (en) * 2018-12-29 2019-08-13 南京高新工大生物技术研究院有限公司 A kind of Novel fermentation tank and its application in ethyl alcohol is prepared in Immobilized yeast

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1083861A (en) * 1992-09-09 1994-03-16 中国科学院沈阳应用生态研究所 Multi-layer separated biological device and this reactor are used for production of alcohol with fermented mellow corn using
CN103865958A (en) * 2012-12-13 2014-06-18 南京工业大学 Method for production of ethanol by continuous fermentation of immobilized yeast cells
CN105018328A (en) * 2015-08-12 2015-11-04 广西壮族自治区农业科学院农产品加工研究所 Method for making banana fruit vinegar through corncob immobilized acetobacter

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1083861A (en) * 1992-09-09 1994-03-16 中国科学院沈阳应用生态研究所 Multi-layer separated biological device and this reactor are used for production of alcohol with fermented mellow corn using
CN103865958A (en) * 2012-12-13 2014-06-18 南京工业大学 Method for production of ethanol by continuous fermentation of immobilized yeast cells
CN105018328A (en) * 2015-08-12 2015-11-04 广西壮族自治区农业科学院农产品加工研究所 Method for making banana fruit vinegar through corncob immobilized acetobacter

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109943555A (en) * 2018-12-29 2019-06-28 南京工业大学 A method of distiller's yeast, which is continuously prepared, using surface immobilized technology carries out alcoholic fermentation
CN109988757A (en) * 2018-12-29 2019-07-09 江苏省生产力促进中心 A kind of method that surface immobilized yeast semicontinuous fermentation prepares ethyl alcohol
CN110117588A (en) * 2018-12-29 2019-08-13 南京高新工大生物技术研究院有限公司 A kind of Novel fermentation tank and its application in ethyl alcohol is prepared in Immobilized yeast
CN109988757B (en) * 2018-12-29 2021-04-27 江苏省生产力促进中心 Method for preparing ethanol by semi-continuous fermentation of surface immobilized yeast

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