CN101948737A - Acetone-butanol in-situ extraction continuous fermentation device and technology - Google Patents
Acetone-butanol in-situ extraction continuous fermentation device and technology Download PDFInfo
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Abstract
The invention provides an acetone-butanol in-situ extraction continuous fermentation device and technology. The device comprises an No.1 seeding tank and an No.2 seeding tank which are alternately inoculated. Acetone-butanol fermentation is divided into two stages to continuously operate, wherein the first stage is conventional acid production period fermentation, and the second stage is in-situ extraction product synthesis period fermentation; the second period adopts a method of coupling extraction and fermentation and is matched with low-speed stirring to furthest lower product inhibiting effect and greatly improve butanol fermentation efficiency; and finally, static phase processing realizes the initial separation of mash and fermentation products. The invention has the advantages that the device and the technology have good extraction effect in production, can obviously improve butanol ratio in the fermentation product, can greatly lower production cost, and have obvious economic benefit. The technology is scientific and reasonable. The flow device is simple and easy to realize, can realize the new fermentation technology provided by the invention by slightly reforming the traditional butanol fermentation flow, and has wide application prospect.
Description
Technical field
The present invention relates to the acetone butanol fermentation technology, particularly a kind of acetone-butanol original position extracts continuously ferment device and technology.
Background technology
Butanols is a kind of big power fuel of combining of alternative gasoline, and its physico-chemical property is compared more near gasoline with ethanol.At present the industrial process of butanols mainly contains three kinds of propylene oxo synthesis, aldol condensation method and fermentation methods.Preceding two kinds mainly is raw material with petroleum resources, but exhausted day by day along with petroleum resources, this method no longer is suitable for big industrial production.The fermentative Production butanols mainly is to produce by the clostridium acetobutylicum anaerobically fermenting, with cereal (corn, corn cob, rye, wheat) starch is raw material, add water and be mixed into mash, through cooking disinfection, add the pure acetone Clostridium acetobutylicum, ferment at 36～37 ℃, fermentation liquid obtains butanols, acetone and ethanol through rectifying separation.Acetone, butanols, alcoholic acid ratio are 3: 6: 1 in the product.Butanols only accounts for 60% of total solvent output as main products, and the raising ratio of butanol is the important topic in this industry.Domestic many scientific research institutions and fermentation enterprise all take up the exploitation of high butanols than bacterial strain, for example: Institute of Microorganism, Academia Sinica, Shanghai industrial microorganism institute, Institute of Nuclear and New Energy Technology, Tsing University etc.Alleviated product inhibition problem to a certain extent though improve production of butanol bacterial strain butanols tolerance, still difficult 75% of the total solvent output that surpasses of butanols fermentation yield.Must from zymotechnique, further improve, occur some gas at present and proposed fermentation, the liquid-liquid extraction fermentation, the infiltration extractive fermentation, technology such as pervaporation fermentation realize reducing the purpose that product suppresses by the instantaneous separation of butanols in fermentation.But, in continuously fermenting,, be difficult to be widely used in scale operation because these technology all exist poor stability, equipment price higher.
The patent and the achievement of the U.S. relevant biological butanol aspect are maximum, 2004 USDA Agricultural Research Institute (USDA-ARS) research project " utilize the Bai Shi clostridium to transform cellulose biomass and produce biological butanol " project verification, and finished in 2009, U.S. green bio company limited (GBL) and EKB biotech firm invest 85.5 ten thousand Euros and have set up fuel butylic fermentation factory.U.S. EGI company utilizes the genetic engineering technique improved strain, by metabolic engineering regulation and control and patented technologies such as immobilization reactor, film recovery continuously, makes that butanols accounts for 90% of total solvent output in the fermented liquid, and this is present hi-tech level.The acetone butanol fermentation original position extraction and separation technology of institute of microbiology of China Chinese Academy of Sciences exploitation wins initial success in batch fermentation.The present invention has proposed conventional product acid phase fermentation and original position extraction product synthesis phase fermentation two-stage operate continuously technology on this basis, has significantly improved the ratio of butanols in the product.
Summary of the invention
The objective of the invention is at the product inhibition that exists in the acetone butanol fermentation process (especially butanols), on interrupted extraction fermentation basis, provide a kind of original position extraction to continuously ferment and prepare the method for acetone-butanol.
Technical scheme of the present invention:
A kind of acetone-butanol original position extracts the device that continuously ferments, comprise the one grade fermemtation jar, second order fermentation jar and quiet jar mutually, the one grade fermemtation jar, the second order fermentation jar is connected in series by pipeline with quiet jar mutually, seeding tank of one grade fermemtation jar configuration and second seed jar, seeding tank is connected with the one grade fermemtation jar by pipeline with the second seed jar and is provided with switching valve, second order fermentation jar configuration alkaline stream adds jar and extraction agent stream adds jar, alkaline stream adds jar and extraction agent stream adds jar and is connected with the second order fermentation jar by pipeline respectively, be provided with agitator in the second order fermentation jar, the material inlet pipeline is provided with two feedstock pumps, two feedstock pumps are connected with the second order fermentation jar with the one grade fermemtation jar respectively by pipeline, be provided with baffle plate in the quiet phase jar, the baffle plate both sides are respectively equipped with mash delivery pipe and tunning delivery pipe.
Described one grade fermemtation jar and/or second order fermentation jar are two in parallel or series connection of above fermentor tank.
A kind of acetone-butanol original position extraction continuous fermentation process adopts conventional fermentation of acid phase and the original position extraction product synthesis phase fermentation two-stage continuous fermentation method of producing, and implementation step is:
1) fermentation seed liquid is cultivated in advance: in operate continuously, the hocket pre-cultivation of seed liquor of two seeding tanks, and method is with after the seed culture medium deoxygenation, adopts the clostridium acetobutylicum bacterial suspension inoculation, anaerobic environment is cultured to logarithmic phase;
2) conventional product acid phase fermentation: before the inoculation of one grade fermemtation jar, open one grade fermemtation jar feedstock pump, raw material is added in the one grade fermemtation jar, lead to N
2The gas deoxygenation is also carried out temperature and is bathed, seed liquor in the seeding tank is inoculated in the one grade fermemtation jar by switching valve, the static logarithmic growth after date that is cultured to, replenish charging, continue to cultivate until producing overflow, overflowing liquid enters the second order fermentation jar, open switching valve simultaneously, switch to the second seed jar, continue stream seed addition liquid, be 6-8 hour the switching time of two seeding tanks;
3) original position extraction product synthesis phase fermentation: before overflowing liquid enters the second order fermentation jar, open second order fermentation jar feedstock pump, raw material is added in the second order fermentation jar, logical N
2The gas deoxygenation is also carried out temperature and is bathed, and adds alkali lye by stream and keeps potential of hydrogen in the substratum, and stream adds extraction agent simultaneously, opens the second order fermentation jar agitator, and cultured continuously 8～12 hours obtains tunning;
4) second order fermentation jar fermented liquid enters quiet phase jar in the overflow mode, and after the static layering, the upper strata is extraction agent and tunning, and lower floor is a mash, goes to follow-up unit to isolate tunning after the discharge respectively and reclaims extraction agent.
The component of described seed culture medium is that every liter of substratum contains: glucose 40g, Tryptones 6g, yeast extract 2g, beef extract 2g, (NH
4)
2SO
43g, MgSO
47H
2O 0.2g, KH
2PO
40.5g and FeSO
47H
2O 0.01g.
The concentration of described clostridium acetobutylicum bacteria suspension is 1 * 10
6～1 * 10
7Individual cell/mL, inoculum size volume percent are 3～20%, culture temperature is 30 ℃～37 ℃.
Described raw material is corn steep liquor or tapioca syrup, and its content in substratum is 20～40g/L; Nutraceutical addition is: 0.3g/L CaCl
2, 0.2g/L MgSO
47H
2O, 0.5g/L KH
2PO4,0.5g/L urea.
The warm bath temperature of raw material is under 30 ℃～37 ℃ in the described one grade fermemtation jar, and the warm bath time is 2～3 hours; The inoculum size of seed liquor is 3%～20% (v/v), and the stream rate of acceleration of inoculum size is 5%～8% (v/v) per hour, and the thinning ratio of replenishing charging is 0.02h
-1～0.06h
-1
The warm bath temperature of raw material is under 30 ℃～37 ℃ in the described second order fermentation jar, and the warm bath time is 2～3 hours; The alkali lye that stream adds is 0.5M NaOH solution, keeps medium pH=6.0～6.5; The extraction agent that stream adds be oleyl alcohol (suitable-the 9-octadecenyl alcohol, molecular formula C
18H
36O) and the mixture of one or both arbitrary proportions in the decyl alcohol, the extraction agent of adding and the ratio of substratum are per hour 1: 2～1: 10; The stir speed (S.S.) of agitator is 10～125r/min; The thinning ratio that stream adds raw material is 0.02h
-1～0.06h
-1
Technological principle of the present invention: adopt product acid phase fermentation and original position extraction product synthesis phase fermentation two-stage to continuously ferment and produce the method for butanols, can in former butylic fermentation factory flow process, transform a little and can realize the novel fermentation technology that this patent provides, easy to implement, have a extensive future; The two-stage fermentation mode will be produced acid phase and product synthesis phase separate operation, and only stream adds extraction agent in the second order fermentation jar, can effectively reduce the extraction agent consumption; Stream adds alkali lye and keeps potential of hydrogen in the fermented liquid in the second order fermentation jar, quickens thalline and enters the product synthesis phase; Adopt the original position extractive fermentation in the second order fermentation jar, add extraction agent and stirring at low speed by stream, the effect of tunning such as butanols in the strengthening extraction agent extractive fermentation liquid, alleviated the toxic side effect of butanols to greatest extent to fermentation reaction, even adopt the original fermentation strain of butylic fermentation factory, output also can improve greatly; Original position extractive fermentation liquid enters quiet phase jar through overflow in the second order fermentation jar, finish the initial gross separation of mash and extraction agent (containing tunning) by quiet effect mutually, rather than the directly quiet phase layering of wait in fermentor tank, can in fermentation, bring into play maximum effect of extracting, realize that simultaneously continuous in-situ extractive fermentation technology is connected with the tight of follow-up tunning sepn process.
Advantage of the present invention is: the novel process that adopts the extraction of acetone-butanol original position to continuously ferment, effect of extracting is good, can significantly improve the ratio of butanol in the tunning, compare with traditional acetone butylic fermentation technology, butanols ratio in the product can improve 20%～30%, reduce production costs greatly, have tangible economic benefit; This craft science is reasonable, and flow path device is simple and easy to implement, can transform a little in traditional butylic fermentation technical process and can realize the new process for fermenting that this patent provides, and has a extensive future.
Description of drawings
Accompanying drawing extracts the device schema that continuously ferments for this acetone-butanol original position.
Concrete embodiment:
Specific embodiments of the present invention is described in detail as follows with reference to accompanying drawing, but for illustrative purposes only rather than the restriction the present invention.
Embodiment 1:
1) with the corn steep liquor be raw material, its content in substratum is 20g/L, and the additional nutrient thing is 0.3g/LCaCl
2, 0.2g/L MgSO
47H
2O, 0.5g/L KH
2PO4,0.5g/L urea; Configuration 0.5M NaOH injects alkali liquid tank; It is stand-by that oleyl alcohol stoste is injected the extraction agent conservation tank.Fermentation begins equipment is carried out the situ steam sterilization.
2) preparation seed liquor: (Clostridium acetobutylicun CGMCCAS1.132) is fermented bacterium with clostridium acetobutylicum, (available from Chinese microbial preservation management committee common micro-organisms preservation center), the component of every liter of seed culture medium is: 40g glucose, the 6g Tryptones, the 2g yeast extract, the 2g beef extract, 3g (NH
4)
2SO
4, 0.2g MgSO
47H
2O, 0.5g KH
2PO
4, 0.01g FeSO
47H
2O regulates pH=6.0～6.5.Seed culture medium is injected a seed fermentation jar, and injection rate is that 2/3 volume of fermentor tank gets final product, logical N
2Deoxygenation 2～3 hours.With concentration is 1 * 10
6～1 * 10
7The CGMCC AS1.132 bacteria suspension of individual cell/mL inserts a seeding tank by 8% inoculum size, is cultured to logarithmic phase under 33 ℃ of anaerobic conditions; The second seed jar was by carrying out seed liquor with quadrat method after 8 hours, and two seeding tanks were alternately cultivated seed liquor every 6～8 hours.
3) seed liquor was cultivated after 5 hours, opened one grade fermemtation jar raw material recycle pump, made raw material enter fermentor tank 2/3 volume place, closed pump, fed N
2After the deoxygenation, 33 ℃ of temperature were bathed 2～3 hours.Insert the seed liquor in the seeding tank No. one by 5% in the one grade fermemtation jar, 33 ℃ of static cultivations 8 hours reach logarithmic phase, open the recycle feed of raw material recycle pump, and thinning ratio is 0.03h
-1Open second seed jar inoculation valve simultaneously, per hour press the inoculum size stream seed addition liquid of 5% (v/v), continue to cultivate after 4 hours, begin to produce overflow, overflowing liquid enters the second order fermentation jar.During operate continuously, seeding tank of switching in 7 hours.
4) the second order fermentation jar shifts to an earlier date charging 2/3 volume, and 33 ℃ of temperature were bathed after 2～3 hours, logical N
2Deoxygenation.One grade fermemtation jar overflowing liquid enters the second order fermentation jar from the bottom, monitor fermentation process pH drips 0.5M NaOH and keeps medium pH=6.0～6.5, beginning stirring at low speed (125r/min).The pure and mild raw material of stream oiling simultaneously, per hour the ratio of oleyl alcohol of Jia Ruing and substratum is 1: 6; The thinning ratio that adds feed liquid is 0.03h
-1, cultured continuously 8 hours, overflowing liquid enters quiet phase jar.
Get quiet phase jar at the middle and upper levels organism carry out proximate analysis, used test set is that the Sp-34 gas chromatograph is divided in Beijing north, chromatographic column is capillary column 0V-17 (30m * 0.25mm * 0.25 μ m), adopts fid detector.Column temperature is 50 ℃, and syringe, detector temperature are 260 ℃.The 1st secondary program initial temperature is 50 ℃, keep 3 minutes after, rise to 160 ℃ with the speed of 15 ℃/min, the 2nd secondary program 20 ℃/min to 260 ℃ that heats up keeps 2min.Carrier gas is a high-purity N
2, nebulizer gas pressure is 0.12MPa.Table 1 is the example one each component content in 200 hours organic phases that continuously ferments.
The quiet phase jar of table 1 organic phase is at the middle and upper levels formed
As can be seen from Table 1, quiet phase jar organic phase at the middle and upper levels mainly contains tunning and extraction agent oleyl alcohol such as acetone-butanol, and the content of water only accounts for 3.02%.After the separation of extraction agent oleyl alcohol, the output of butanols accounts for 87.2% of tunning output, than adopting traditional zymotic technology to carry out having improved 27% when CGMCC AS1.132 ferments.Separate the oleyl alcohol reusable edible that obtains.
Embodiment 2:
1) with the tapioca syrup be raw material, its content in substratum is 40g/L, and the additional nutrient thing is 0.3g/LCaCl
2, 0.2g/L MgSO
47H
2O, 0.5g/L KH
2PO4,0.5g/L urea; Configuration 0.5M NaOH injects alkali liquid tank; It is stand-by that oleyl alcohol stoste is injected the extraction agent conservation tank.Fermentation begins equipment is carried out the situ steam sterilization.
2) preparation seed liquor: (Clostridium acetobutylicunm CGMCCAS1.244) is fermented bacterium with clostridium acetobutylicum, (available from Chinese microbial preservation management committee common micro-organisms preservation center).
Step is with example one, and culture temperature is set at 35 ℃.
3) seed liquor was cultivated after 7 hours, opened one grade fermemtation jar raw material recycle pump, made raw material enter fermentor tank 2/3 volume place, closed pump, inserted the seed liquor in the seeding tank No. one by 8% in the one grade fermemtation jar, fed N
2After the deoxygenation, 35 ℃ of static cultivations, monitor fermentation process pH drips 0.5M NaOH and keeps medium pH=6.0～6.5.Cultivate after 12 hours, open the recycle feed of raw material recycle pump, thinning ratio is 0.05h
-1Open second seed jar inoculation valve simultaneously, per hour press the inoculum size stream seed addition liquid of 8% (v/v), continue to cultivate after 6 hours, begin to produce overflow, overflowing liquid enters the second order fermentation jar.During operate continuously, seeding tank of switching in 8 hours.
4) during second order fermentation, 35 ℃ of cultivations, per hour the ratio of extraction agent of Jia Ruing and substratum is 1: 4; The thinning ratio that adds raw material is 0.05h
-1, cultured continuously 12 hours, overflowing liquid enters quiet phase jar.
Adopt the same detection method of example one to quiet phase jar at the middle and upper levels organism detect, table 2 is the example two 200 hours organic phase component tables that continuously ferment.
Organic phase is formed in the quiet phase jar of table 2
As can be seen from Table 2, adopt processing method of the present invention to carry out CGMCC AS1.244 fermentation, after the extraction agent oleyl alcohol was separated, ratio of butanol reached 85.6% in the tunning, than adopting traditional zymotic technology to carry out having improved 25% when CGMCCAS1.244 ferments.
More than the explanation of two examples, the novel process that acetone-butanol original position extraction provided by the invention is continuously fermented can significantly improve the ratio of butanol in the tunning, has certain economic benefits.The personnel of association area can suitably change or change and make up fully according to method provided by the invention, realize this technology.Of particular note, all these are by technical process provided by the invention being carried out similar change or change and being reconfigured, and are apparent to those skilled in the art, all are regarded as in technical characterictic of the present invention, scope and content.
Claims (8)
1. an acetone-butanol original position extracts the device that continuously ferments, it is characterized in that: comprise the one grade fermemtation jar, second order fermentation jar and quiet jar mutually, the one grade fermemtation jar, the second order fermentation jar is connected in series by pipeline with quiet jar mutually, seeding tank of one grade fermemtation jar configuration and second seed jar, seeding tank is connected with the one grade fermemtation jar by pipeline with the second seed jar and is provided with switching valve, second order fermentation jar configuration alkaline stream adds jar and extraction agent stream adds jar, alkaline stream adds jar and extraction agent stream adds jar and is connected with the second order fermentation jar by pipeline respectively, be provided with agitator in the second order fermentation jar, the material inlet pipeline is provided with two feedstock pumps, two feedstock pumps are connected with the second order fermentation jar with the one grade fermemtation jar respectively by pipeline, be provided with baffle plate in the quiet phase jar, the baffle plate both sides are respectively equipped with mash delivery pipe and tunning delivery pipe.
2. extract the device that continuously ferments according to the described acetone-butanol original position of claim 1, it is characterized in that: described one grade fermemtation jar and/or second order fermentation jar are two in parallel or series connection of above fermentor tank.
3. one kind according to the described acetone-butanol original position of claim 1 extraction continuous fermentation process, it is characterized in that: adopt the conventional fermentation of acid phase and the original position extraction product synthesis phase two-stage continuous fermentation method that ferments that produces, implementation step is:
1) fermentation seed liquid is cultivated in advance: in operate continuously, the hocket pre-cultivation of seed liquor of two seeding tanks, and method is with after the seed culture medium deoxygenation, adopts the clostridium acetobutylicum bacterial suspension inoculation, anaerobic environment is cultured to logarithmic phase;
2) conventional product acid phase fermentation: before the inoculation of one grade fermemtation jar, open one grade fermemtation jar feedstock pump, raw material is added in the one grade fermemtation jar, lead to N
2The gas deoxygenation is also carried out temperature and is bathed, seed liquor in the seeding tank is inoculated in the one grade fermemtation jar by switching valve, the static logarithmic growth after date that is cultured to, replenish charging, continue to cultivate until producing overflow, overflowing liquid enters the second order fermentation jar, open switching valve simultaneously, switch to the second seed jar, continue stream seed addition liquid, be 6-8 hour the switching time of two seeding tanks;
3) original position extraction product synthesis phase fermentation: before overflowing liquid enters the second order fermentation jar, open second order fermentation jar feedstock pump, raw material is added in the second order fermentation jar, logical N
2The gas deoxygenation is also carried out temperature and is bathed, and adds alkali lye by stream and keeps potential of hydrogen in the substratum, and stream adds extraction agent simultaneously, opens the second order fermentation jar agitator, and cultured continuously 8～12 hours obtains tunning;
4) second order fermentation jar fermented liquid enters quiet phase jar in the overflow mode, and after the static layering, the upper strata is extraction agent and tunning, and lower floor is a mash, goes to follow-up unit to isolate tunning after the discharge respectively and reclaims extraction agent.
4. according to the described acetone-butanol original position extraction of claim 3 continuous fermentation process, it is characterized in that: the component of described seed culture medium is that every liter of substratum contains: glucose 40g, Tryptones 6g, yeast extract 2g, beef extract 2g, (NH
4)
2SO
43g, MgSO
47H
2O 0.2g, KH
2PO
40.5g and FeSO
47H
2O 0.01g.
5. according to the described acetone-butanol original position extraction of claim 3 continuous fermentation process, it is characterized in that: the concentration of described clostridium acetobutylicum bacteria suspension is 1 * 10
6～1 * 10
7Individual cell/mL, inoculum size volume percent are 3～20%, culture temperature is 30 ℃～37 ℃.
6. according to the described acetone-butanol original position extraction of claim 3 continuous fermentation process, it is characterized in that: described raw material is corn steep liquor or tapioca syrup, and its content in substratum is 20～40g/L; Nutraceutical addition is: 0.3g/L CaCl
2, 0.2g/L MgSO
47H
2O, 0.5g/L KH
2PO4,0.5g/L urea.
7. according to the described acetone-butanol original position extraction of claim 3 continuous fermentation process, it is characterized in that: the warm bath temperature of raw material is under 30 ℃～37 ℃ in the described one grade fermemtation jar, and the warm bath time is 2～3 hours; The inoculum size of seed liquor is 3%～20% (v/v), and the stream rate of acceleration of inoculum size is 5%～8% (v/v) per hour, and the thinning ratio of replenishing charging is 0.02h
-1～0.06h
-1
8. according to the described acetone-butanol original position extraction of claim 3 continuous fermentation process, it is characterized in that: the warm bath temperature of raw material is under 30 ℃～37 ℃ in the described second order fermentation jar, and the warm bath time is 2～3 hours; The alkali lye that stream adds is 0.5M NaOH solution, keeps medium pH=6.0～6.5; The extraction agent that stream adds be oleyl alcohol (suitable-the 9-octadecenyl alcohol, molecular formula C
18H
36O) and the mixture of one or both arbitrary proportions in the decyl alcohol, the extraction agent of adding and the ratio of substratum are per hour 1: 2～1: 10; The stir speed (S.S.) of agitator is 10～125r/min; The thinning ratio that stream adds raw material is 0.02h
-1～0.06h
-1
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| CN104946506A (en) * | 2014-12-05 | 2015-09-30 | 中国石油化工股份有限公司 | Device and method for producing butanol by taking lignocellulose as raw material through fermentation |
| CN104946516A (en) * | 2014-12-05 | 2015-09-30 | 中国石油化工股份有限公司 | Device and method for producing butyl alcohol through continuous fermentation of lignocellulose |
| CN105999763A (en) * | 2016-05-20 | 2016-10-12 | 天津大学 | Sophorolipid fermentation and biological separation system and method |
| CN108192817A (en) * | 2018-02-13 | 2018-06-22 | 大庆天之草生物新材料科技有限公司 | A kind of production technology and equipment of serialization supply exponential phase bacterium solution |
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