CN108676788A - A kind of fixed yeast continuously ferments technology - Google Patents
A kind of fixed yeast continuously ferments technology Download PDFInfo
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- CN108676788A CN108676788A CN201810321243.3A CN201810321243A CN108676788A CN 108676788 A CN108676788 A CN 108676788A CN 201810321243 A CN201810321243 A CN 201810321243A CN 108676788 A CN108676788 A CN 108676788A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
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- C12P7/08—Ethanol, i.e. non-beverage produced as by-product or from waste or cellulosic material substrate
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Abstract
Continuously ferment technology the invention discloses a kind of fixed yeast, fibrous material is fixed in spherical grid by it is used as entrapment media, entrapment media is filled in 15 concatenated fermentation tanks simultaneously, the effect that yeast passes through absorption is set to be fixed on fiber carrier and ferment, in order to maintain fixed yeast activity in terminal fermentation tank, fixed yeast lasting stability continuous ferment is realized by switching fermentation tank sequence.Not only input cost is low for the process for fixation, easy to operate, process stabilizing, adsorption effect are good, but also fixed yeast can be made to have lasting, higher activity.
Description
Technical field
The invention belongs to industrial biotechnology fields, and in particular to a kind of fixed yeast continuously ferments technology.
Background technology
Due to the excessive exploitation of fossil energy and other non-renewable energy resources, energy crisis has become world today's development
Primarily solve the problems, such as.Find the emphasis that new alternative energy source has become in these years energy strategy.Ethyl alcohol is not only one kind can
Generative fuel, and can be used in mixed way with gasoline and mix in any proportion, it is good always to alleviate the pressure of oil demand
Energy substitution product.Ethyl alcohol production at present depends on biofermentation, because of raw material sources extensive (such as corn, cassava, stalk
Deng), therefore development potentiality is larger.
The big inoculum concentration continuous flow that traditional ethanol fermentation uses adds (feed supplement formula) mode to carry out free fermentation, fermentation period
Long, wine degree is not high, and the fermentation death rate is higher, and microbiological contamination phenomenon easily occurs.Fixed yeast research history is more long, advantage
It is also obvious that such as fermenting speed is fast, high conversion rate, and can recycle, it is convenient for continuous ferment.However the case to put into production
Arranging few the problem of lacking, being primarily present has：Entrapment media is of high cost, mechanical strength is low, adsorption effect and mass transfer effect are poor
And continuous ferment yeast activity the problems such as easily degenerating.
Invention content
Continuously ferment technology the technical problem to be solved in the present invention is to provide a kind of fixed yeast, 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 fixed yeast continuously ferments technology, it includes the following steps：
(1) ball grid equipped with adsorbing medium is filled in 2~5 Tandem-type fermentors；
(2) it is pumped into seed liquor into the Tandem-type fermentor 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；It repeats
It ferments to fermentation stability；
(4) after fermentation stability, it is continuously pumped into new zymotic fluid, realization is continuously fermented；
(5) when the yeast death rate reaches 25~35% in the fermentation tank of end, change the position of end fermentation tank, restore end
The activity of yeast in fermentation tank is held, and continues to be continuously pumped into new zymotic fluid.
In step (1), the adsorbing medium is any one in plant fiber, animal origin and chemical synthetic fiber
Or several combination.
Wherein, the adsorbing medium can be handled as follows in advance：Adsorbing medium is first placed on to a concentration of 1
In surface modifier (such as polyethyleneimine and succimide) aqueous solution of~100g/L and the crosslinking agent (penta of 1~100g/L
Dialdehyde, 2- methyl-1s, 3- malonaldehyde and (2S, 3R) -2,3- dihydroxy butanedial) 1~40 hour (temperature is impregnated in aqueous solution
It is room 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 polytetrafluoroethylene
Or polyethylene.
In step (1), the adsorbing medium is square sheet, and the ratio of the length of side and ball grid diameter is 0.5
~0.8：1；Wherein, 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；In each fermentation tank, adsorbing medium over dry
Amount be 3~15g/L.
In step (2), the seed liquor is human configuration culture medium, the saccharified liquid of corn, cassava or grain in storage for years, fiber
Any one or the combination of several of them in cellulose hydrolysate and molasses, yeast quantity are 1.5~3.5 hundred million/ml；
Wherein,
The formula of the human configuration culture medium is 30~100g/L of glucose, 10~20g/L of peptone, yeast extract 10
~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 cellulose, feed liquid mass ratio is 1：2~3, pH are 4.5~5.2；Hydrolysising condition is rotating speed
100~250r/min, 45~60 DEG C of temperature digest 1~2.5 hour；
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 seed, which is fixed, to be terminated to refer to that yeast thalline adsorbance reaches 1.0~3.0 hundred million/ml.
In step (3), the zymotic fluid is human configuration culture medium, the saccharified liquid of corn, cassava or grain in storage for years, fiber
Any one or the combination of several of them in cellulose hydrolysate and molasses；
Wherein,
The formula of the human configuration culture medium is 200~270g/L of glucose, 3~10g/L of peptone, yeast extract 3
~10g/L, 0.1~1g/L of magnesium sulfate, Ph4.0~4.5；
In the saccharified liquid of the corn, cassava or grain in storage for years, material water quality is frequently 2.5~3：1；
In the hydrolyzate of the cellulose, feed liquid mass ratio is 1：2~3, pH are 4.5~5.2；Hydrolysising condition is rotating speed
100~250r/min, 45~60 DEG C of temperature digest 1~2.5 hour；
The initial total sugar of the molasses is 20~180g/L.
In step (3), single batch fermentation terminates to be down to 0.5 ° of Bx hereinafter, reduced sugar is down to 0.2~0.4g/ with appearance sugar
Subject to 100mL.
In step (4), fermentation stability refers to that continuous 3 batches of fermentation periods difference is no more than 6 hours, sugar alcohol conversion ratio error
No more than 5%.
In step (4), when being continuously pumped into new zymotic fluid, feed flow rate is single batch fermentation time reciprocal 1~8 times, single
Position is h-1。
In step (5), the position of the change end fermentation tank refers to the flow direction for changing zymotic fluid so that zymotic fluid is not
It is finally flowed out from end fermentation tank again；It is preferred that referring to that (i.e. zymotic fluid ferments terminal fermentation tank from terminal with the exchange of starting fermentation tank
Tank starts to feed, then is flowed out from original starting tank), or fed since the 2nd fermentation tank, successively from the 3rd, the streams such as the 4th
Into, and flowed out from former first fermentation tank.
In step (5), the yeast death rate refers to the ratio of free yeast death concentration and free yeast total concentration
Value.
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；Adsorbing medium surface area is big, absorption effect
Fruit is good；
2, the immobilization way loading density that the present invention uses is low, and gap is big between carrier, compared to other absorption or cross-linking method
Mass transfer effect is preferable, and fermenting speed is fast, and substrate conversion efficiency is high；
3, entrapment media filling is simple, convenient for taking out and overhauling, is suitable for industrial applications；
4, in continuous ferment, by switching fermentation tank sequence (or fermentation broth stream to), restore end fermentation tank immobilization
Yeast activity makes fixed yeast remain higher fermentability.
Description of the drawings
Fig. 1 is the structural schematic diagram of ball grid；
Fig. 2 is the flow diagram of the present invention；
Reference numeral：1 sour tank, 2 pumps, 3 feed supplement tanks, 4 carbohydrase tanks, 5 fermentation tanks, 6 motors, 7 thermostatted waters.
Specific implementation mode
Embodiment 1 is continuously fermented as fermentation raw material using bamboo fibre fixed yeast using synthetic media produces alcohol fuel
First, the cotton fiber (thickness 0.2cm) for being cut into length of side 8cm is fixed on straight as shown in Figure 1 made of polyvinyl chloride
On diameter 8cm ball grid axis (ball length of side 1cm), it is prepared into entrapment media；Then by entrapment media with 80cm2/ L's
Loading is put into ratio of height to diameter 2:In 1 20L reactors.Then cultured strain is added in fermentation tank, is 15L/h with flow velocity
The fixed 42h of cycle.Until 2.0 hundred million/ml of thalline adsorption concentration.It is then discharged out waste liquid.(glucose is dense for fed-batch cultivation culture medium again
Degree is 200g/L), liquid amount 72% carries out circulating fermentation, and wherein temperature is 34 DEG C, zymotic fluid pH4.2, flow velocity 15L/h.5
The equal fermentation period of criticize flat is 12h, and reaction density reaches 94.2g/L, and sugar alcohol conversion ratio and yield are respectively 92.2% and 7.85g/
L/h after fermentation stability, is discharged zymotic fluid, 4 20L tanks is cascaded, with dilution rate 0.135h-1Continuous feed (glucose
Concentration 220.5g/L) and it discharges, ethyl alcohol reaches 105.2g/L after fermentation stability, sugar alcohol conversion ratio 93.4%, yield 14.20g/L/h,
The 6 days cell mortalities that ferment are more than 30%, change feed liquid flow direction (negative direction), and carrying out stream with identical dilution rate adds, and fermentation 20 is small
Shi Hou, tank discharging sugar in end is dense to be down to 2 hereinafter, concentration of alcohol reaches 104.8g/L, conversion ratio 93%.Pass through switching mode, hair
Ferment can stable operation 2 months as long as.
Embodiment 2 is continuously fermented as fermentation raw material using bamboo fibre fixed yeast using cassava liquefied fermented glutinous rice culture medium produces fuel
Ethyl alcohol
First, the column fiber (thickness 0.2cm) for being cut into length of side 4cm is fixed on made of polytetrafluoroethylene (PTFE) as shown in Figure 1
On diameter 3cm ball grid axis (ball length of side 1cm), it is prepared into entrapment media；Then by entrapment media with 90cm2/L
Loading be put into ratio of height to diameter 2.5:In 1 10L reactors.Then cultured strain is added in fermentation tank, is with flow velocity
The fixed 48h of 10L/h cycles.Until 2.2 hundred million/ml of thalline adsorption concentration.Then cassava liquefied fermented glutinous rice is prepared：Solid-liquid ratio 1:2.5, add
Press filtration after the liquefaction 2 hours of 85 DEG C of α-amylase (15U/g cassavas dry weight), is discharged immobilization seed waste liquid.It flows again and adds cassava liquefied fermented glutinous rice,
And carbohydrase (180U/g cassavas dry weight) is added, liquid amount 75% carries out circulating fermentation, and wherein temperature is 35 DEG C, zymotic fluid
PH4.4, flow velocity 10L/h.The equal fermentation period of 7 criticize flats is 28h, and reaction density reaches 103.0g/L, sugar alcohol conversion ratio and yield
Respectively 91.6% and 3.68g/L/h after fermentation stability, is discharged zymotic fluid, 3 10L tanks is cascaded (Fig. 2), with dilute
Release rate 0.12h-1Continuous feed (sugared concentration 215.8g/L) simultaneously discharges, and ethyl alcohol reaches 102.6g/L, sugar alcohol conversion ratio after fermentation stability
93.0%, yield 12.3g/L/h.Feed liquid is fed from the 2nd fermentation tank after continuously fermenting 6 days, and from former 1st tank discharging, is trained
After supporting 28 hours, concentration of alcohol restores to 103.0g/L, and conversion ratio is about 93.4.
It can stable operation 40 days or more.
Embodiment 3 is continuously fermented as fermentation raw material using activated carbon fibre fixed yeast using molasses culture medium produces fuel second
Alcohol
First, the activated carbon fibre for being cut into diameter 5cm is fixed in diameter 4cm ball grids made of polytetrafluoroethylene (PTFE)
(mesh length of side 0.5cm), is prepared into entrapment media；Then by entrapment media with 100cm2The loading of/L is put into ratio of height to diameter
1.8:In 1 20L reactors.Then cultured strain is added in fermentation tank, is the fixed 32h of 30L/h cycles with flow velocity.Directly
To 2.0 hundred million/ml of thalline adsorption concentration.Then the molasses being acidified are diluted to the dense 250g/L of sugar, and immobilization seed waste liquid is discharged.
Sugaring honey is flowed again, and liquid amount 75% carries out circulating fermentation, and wherein temperature is 35 DEG C, zymotic fluid pH4.4, flow velocity 30L/h.6 batches
The average fermentation period is 48h, and reaction density reaches 118.8g/L.Alcohol yied reaches 2.48gL-1h-1, average conversion
93.0%.After fermentation stability, zymotic fluid is discharged, 3 20L tanks are cascaded, with dilution rate 0.07h-1(sugar is dense for continuous feed
Degree 246.8g/L) and discharge, ethyl alcohol reaches 118.6g/L, sugar alcohol conversion ratio 94.0%, yield 8.3g/L/h after fermentation stability.Continuously
Feed liquid is fed from the 3rd fermentation tank after fermentation 2 days, and after former first tank discharging, fermentation stability, concentration of alcohol reaches
118.0g/L or more, conversion ratio are more than 93.8%.
Embodiment 4 is continuously fermented production using straw 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 5cm, polytetrafluoroethylene (PTFE) is put into and is made
Diameter 5cm ball grids in (mesh length of side 4cm), be prepared into entrapment media；Then by entrapment media with 50cm2/L's
Loading is put into ratio of height to diameter 1.5:In 1 50L reactors.Then cultured strain is added in fermentation tank, is with flow velocity
The fixed 48h of 40L/h cycles.Until 1.8 hundred million/ml of thalline adsorption concentration.Then stalk hydrolyzate is prepared：It will take off entirely (de- wooden
Element and hemicellulose) rich in cellulosic material press solid-liquid ratio 1:4, add cellulase (35U/g dry weights), 50 DEG C of 2 hydrolysis 36 small
When after press filtration, be discharged immobilization seed waste liquid.Hydrolyzate is flowed again, and liquid amount 75% carries out circulating fermentation, and wherein temperature is 33
DEG C, zymotic fluid pH4.2, flow velocity 40L/h.The equal fermentation period of 4 criticize flats is 36h (initial total sugar 135g/L), and reaction density reaches
62.2g/L.Alcohol yied reaches 1.73gL-1h-1, average conversion 90.2%.After fermentation stability, zymotic fluid, series connection 2 is discharged
Platform fermentation tank, with the stream of dilution rate 0.056 plus new culture medium.Ethyl alcohol 63.2g/L (conversion ratio 91.6%) after culture 28 hours, even
Feed liquid is fed from the 2nd fermentation tank after supervention ferment 5 days, and is discharged from former first tank, after cultivating 32 hours, fermenting alcohol concentration
Restore to 63g/L or more, conversion ratio is more than 91%.
Claims (12)
- The technology 1. a kind of fixed yeast continuously ferments, which is characterized in that it includes the following steps：(1) ball grid equipped with adsorbing medium is filled in 2~5 Tandem-type fermentors；(2) it is pumped into seed liquor into the Tandem-type fermentor 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；It repeats to ferment To fermentation stability；(4) after fermentation stability, it is continuously pumped into new zymotic fluid, realization is continuously fermented；(5) when the yeast death rate reaches 25~35% in the fermentation tank of end, change the position of end fermentation tank, restore end hair The activity of yeast in fermentation tank, and continue to be continuously pumped into new zymotic fluid.
- The technology 2. fixed yeast according to claim 1 continuously ferments, which is characterized in that in step (1), the suction Attached medium is any one or the combination of several of them in plant fiber, animal origin and chemical synthetic fiber.
- The technology 3. fixed yeast according to claim 1 continuously ferments, which is characterized in that in step (1), the ball Type grid is made of polytetrafluoroethylene (PTFE) or polyethylene.
- The technology 4. fixed yeast according to claim 1 continuously ferments, which is characterized in that in step (1), the suction Attached medium is square sheet, and the ratio of the length of side and ball grid diameter is 0.5~0.8：1；Wherein, in a Ball Grid Equipped with 2~4 adsorbing mediums.
- The technology 5. fixed yeast according to claim 1 continuously ferments, 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；In each fermentation tank, the amount of adsorbing medium over dry is 3~15g/L.
- The technology 6. fixed yeast according to claim 1 continuously ferments, which is characterized in that in step (2), the kind Sub- liquid is human configuration culture medium, the saccharified liquid of corn, cassava or grain in storage for years, any one in cellulosic hydrolysate and molasses Or several combinations, yeast quantity are 1.5~3.5 hundred million/ml；Wherein,The formula of the human configuration culture medium be 30~100g/L of glucose, 10~20g/L of peptone, yeast extract 10~ 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 cellulose, feed liquid mass ratio is 1：2~3, pH are 4.5~5.2；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 is 0.5~4BV.
- The technology 7. fixed yeast according to claim 1 continuously ferments, which is characterized in that in step (3), the kind Son is fixed to be terminated to refer to that yeast thalline adsorbance reaches 1.0~3.0 hundred million/ml.
- The technology 8. fixed yeast according to claim 1 continuously ferments, 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, any one in cellulosic hydrolysate and molasses Or several combination；Wherein,The formula of the human configuration culture medium be 200~270g/L of glucose, 3~10g/L of peptone, yeast extract 3~ 10g/L, 0.1~1g/L of magnesium sulfate, Ph4.0~4.5；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 cellulose, feed liquid mass ratio is 1：2~3, pH are 4.5~5.2；The initial total sugar of the molasses is 20~180g/L.
- The technology 9. fixed yeast according to claim 1 continuously ferments, which is characterized in that in step (3), single batch fermentation End is subject to appearance sugar and is down to 0.5 ° of Bx hereinafter, reduced sugar is down to 0.2~0.4g/100mL.
- The technology 10. fixed yeast according to claim 1 continuously ferments, which is characterized in that in step (4), fermentation is steady Surely refer to that continuous 3 batches of fermentation periods difference is no more than 6 hours, sugar alcohol conversion ratio error is no more than 5%.
- The technology 11. fixed yeast according to claim 1 continuously ferments, which is characterized in that continuous to pump in step (4) When entering new zymotic fluid, feed flow rate is single batch fermentation time reciprocal 1~8 times, unit h-1。
- The technology 12. fixed yeast according to claim 1 continuously ferments, which is characterized in that described in step (5) The position for changing end fermentation tank refers to the flow direction for changing zymotic fluid so that zymotic fluid is no longer finally flowed out from end fermentation tank.
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| CN109852596A (en) * | 2019-04-04 | 2019-06-07 | 江苏省生产力促进中心 | A method of nuclease P 1 is prepared using immobilization Penicillium citrinum fermentation |
| 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 |
| CN110117526A (en) * | 2019-05-06 | 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 device and its application in ethyl alcohol is prepared in Immobilized yeast |
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| 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 |
| CN109852596A (en) * | 2019-04-04 | 2019-06-07 | 江苏省生产力促进中心 | A method of nuclease P 1 is prepared using immobilization Penicillium citrinum fermentation |
| CN110117526A (en) * | 2019-05-06 | 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 device and its application in ethyl alcohol is prepared in Immobilized yeast |
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