CN101358175B - Virus-free in situ and alcohol fermentation method of composite bacteria for lignocellulose hydrolysis product - Google Patents
Virus-free in situ and alcohol fermentation method of composite bacteria for lignocellulose hydrolysis product Download PDFInfo
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- CN101358175B CN101358175B CN2008102228977A CN200810222897A CN101358175B CN 101358175 B CN101358175 B CN 101358175B CN 2008102228977 A CN2008102228977 A CN 2008102228977A CN 200810222897 A CN200810222897 A CN 200810222897A CN 101358175 B CN101358175 B CN 101358175B
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Abstract
The present invention relates to an in-situ detoxification ethanol fermentation method of lignocellulose hydrolysate, which respectively inoculates Y5 and CBS6054 into fermentation culture medium containing dilute acid lignocellulose hydrolysate to carry out in-situ detoxification alcoholic fermentation. The method can efficiently metabolize glucose and xylose in the dilute acid lignocellulose hydrolysate to produce ethanol, and rapidly metabolize furfural and 5-hydroxymethylfurfural. The yield of ethanol is 0.44g/g, reaching 87.5 percent of the theoretical value. Moreover, any detoxification processing is not needed, the requirement on equipment is low, the operation is convenient, and the cost is low.
Description
Technical field
The present invention relates to a kind of original position detoxication alcohol fermentation method of ligno-cellulose hydrolysate using, specifically, be with the glucose in the efficient metabolism lignocellulose of a kind of energy dilute acid hydrolysis liquid and the composite bacteria of wood sugar producing and ethanol and tachymetabolism supressor furfural and 5 hydroxymethyl furfural, be inoculated in the fermention medium of lignocellulose-containing dilute acid hydrolysis liquid and carry out the original position detoxication alcohol fermentation.
Background technology
Be that the lignocellulose dilute acid hydrolysis is considered to the business-like production technique of the easiest realization in the raw material production alcoholic acid research and development field with the lignocellulose, carrying out a large amount of deep researchs both at home and abroad in this field.But up to the present, also existing some problems aspect the dilute acid hydrolysis lignocellulosic materials for fuel ethanol both at home and abroad, wherein one of major technology bottleneck is the efficient ethanol fermentation of hydrolysis sugar liquid, comprises the microbial strains that efficiently utilizes wood sugar producing and ethanol, anti-/ decomposing inhibitor.
The natural bacterial classification that can carry out the wood sugar ethanol fermentation seldom, study comparatively deep Pichiastipitis energy xylose-fermenting at present and have very high alcohol yied, people such as Lohmeier-Vogel discover that P.stipitis is relatively more responsive to the supressors such as furfural, hydroxymethylfurfural and acetate in the lignocellulose dilute acid hydrolysis product.Have only the minority yeast can tolerate the supressor of higher concentration in the lignocellulose dilute acid hydrolysis product, as Saccharomyces cerevisiae TMB3400, S.cerevisiae TMB3006, Coniochaeta ligniaria NRRL30616 etc., but their but energy metabolism wood sugar producing and ethanols not.Supressor in the hydrolysate can influence microbial growth and fermentation capacity, and wherein furfural and 5 hydroxymethyl furfural (5-HMF) are main supressors.Usually will carry out detoxification treatment to hydrolyzed solution before the fermentation, this just needs to add treatment facility, and will make detoxification equipment and the fine integration of zymotechnique, thereby has improved the complicacy of whole ethanol fermentation technology and improved production cost.
(promptly this fermented bacterium can efficient metabolism fermented liquid toxic material when carrying out ethanol fermentation for lignocellulose dilute acid hydrolysis product being carried out the original position detoxification, need not carry out the fermenting process that any detoxification pre-treatment ethanol fermentation just can normally carry out) and the ethanol fermentation of glucose and wood sugar, researchist of the present invention utilizes efficient metabolizable glucose producing and ethanol of a kind of energy and efficient metabolic poison, but the Wine brewing yeast strain S.cerevisiae Y5 of energy metabolism wood sugar producing and ethanol not, form composite bacteria with efficient metabolism wood sugar producing and ethanol and to the bacterium P.stipitisCBS6054 of inhibitor sensitivity, lignocellulose dilute acid hydrolysis product is carried out the original position detoxication alcohol fermentation.
Summary of the invention
The original position detoxication alcohol fermentation method that the purpose of this invention is to provide a kind of ligno-cellulose hydrolysate using.
For achieving the above object, researchist of the present invention at first works out a kind of S. cervisiae S.cerevisiae Y5, this yeast is metabolizable glucose producing and ethanol and efficient metabolic poison efficiently, and then form composite bacteria with S. cervisiae Y5 and efficient metabolism wood sugar producing and ethanol and to the bacterium P.stipitisCBS6054 of inhibitor sensitivity, ligno-cellulose hydrolysate using is carried out the original position detoxication alcohol fermentation.
S. cervisiae Y5 separates acquisition by this laboratory from grain distillery's downflow sludge, this bacterial strain has been deposited in China Committee for Culture Collection of Microorganisms common micro-organisms center on September 4th, 2008, Datun Road, Chaoyang District, Beijing City, address institute of microbiology of the Chinese Academy of Sciences, its classification called after Saccharomyces cerevisiae Y5, deposit number CGMCC NO.2660.
Above-mentioned S. cervisiae Y5, it has following characteristics:
1, can the glucose fermentation producing and ethanol;
2, can degrade furfural and 5-HMF;
3, can flocculate, can in fermentor tank, realize immobilization, reach the purpose of high-density culture and zymamsis, the average fermentation time of zymamsis is shortened;
4, fermenting speed is fast, can tolerate the ethanol of the highest 50-130g/L;
5, has good mixed fermentation compatibility with pichia spp.
S. cervisiae Y5 can efficient metabolizable glucose producing and ethanol and efficient metabolic poison, but its energy metabolism wood sugar producing and ethanol not.
Composite bacteria provided by the invention, it contains S.cerevisiae Y5 and P.stipitisCBS6054.S.cerevisiae Y5 is 1:5-5:1, preferred 1:1 with the bacterium number ratio of P.stipitisCBS6054 in every by weight gram composite bacteria.
Wherein, bacterial classification S.cerevisiaeY5 is by this laboratory screening and preservation.Bacterial classification P.stipitisCBS6054 is available from U.S. ATCC.
This composite bacteria can be carried out the original position detoxication alcohol fermentation to lignocellulose dilute acid hydrolysis product, that is, and and glucose and wood sugar producing and ethanol and tachymetabolism supressor furfural and 5 hydroxymethyl furfural in the efficient metabolism lignocellulose dilute acid hydrolysis liquid.
Y5 provided by the invention and CBS6054 composite bacteria can be widely used in the original position detoxication alcohol fermentation of lignocellulose dilute acid hydrolysis product.
The original position detoxication alcohol fermentation method of lignocellulose dilute acid hydrolysis product of the present invention, it is inoculated in Y5 and CBS6054 respectively in the fermention medium that contains lignocellulose dilute acid hydrolysis liquid, at 25 ℃-38 ℃, preferred 30 ℃, 0rpm-500rpm, preferred 80rpm, cultivate 8-72h, preferred 72h.
Wherein, according to every ml fermention medium 10
6Cfu-10
10The amount of cfu inoculation Y5 and CBS6054, described fermention medium is with yeast extract paste 10g/L, peptone 20g/L, lignocellulose dilute acid hydrolysis liquid 100ml, at pH5.0-5.5, under 121 ℃, makes behind the sterilization 20min.
Y5 and CBS6054 be respectively at glucose 20g/L, yeast extract paste 10g/L, and peptone 20g/L, pH5.0-5.5 under 121 ℃, under the substratum of sterilization 20min, cultivates propagation.
The original position detoxication alcohol fermentation method for brewing of ligno-cellulose hydrolysate using of the present invention, it has following advantage:
1, S. cervisiae Y5 and CBS6054 composite bacteria, efficiently glucose and the wood sugar in the metabolism lignocellulose dilute acid hydrolysis product, and energy tachymetabolism thank to ethanol fermentation supressors such as furfural and 5 hydroxymethyl furfural.
2, carry out ethanol fermentation with S. cervisiae Y5 and CBS6054 composite bacteria, do not need to carry out any detoxification treatment, equipment requirements is simple, and is easy to operate, and cost is low.
3, can solve the not defective of ability poison or unfermentable wood sugar producing and ethanol of single bacterial strain.Improve commercial productivity, reduce industrial cost.
Description of drawings
Fig. 1 (A) is a Y5 glucose ethanol fermentation;
Fig. 1 (B) is a Y4 glucose ethanol fermentation;
Fig. 1 (C) is a CBS6054 glucose ethanol fermentation;
Fig. 2 is a CBS6054 wood sugar ethanol fermentation;
Fig. 3 (A) is a Y5 mixing sugar ethanol fermentation;
Fig. 3 (B) is a Y4 mixing sugar ethanol fermentation;
Fig. 3 (C) is a CBS6054 glucose ethanol fermentation;
Fig. 4 is a composite bacteria Y5+CBS6054 mixing sugar ethanol fermentation;
Fig. 5 is a composite bacteria Y4+CBS6054 mixing sugar ethanol fermentation;
Fig. 6 is the original position detoxication alcohol fermentation of the lignocellulose dilute acid hydrolysis liquid of composite bacteria Y5+CBS6054;
Among the figure: "+" expression adds furfural 1.37g/L and 5-HMF0.47g/L.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Embodiment 1 composite bacteria of the present invention is to the original position detoxication alcohol fermentation of ligno-cellulose hydrolysate using
Bacterial classification S.cerevisiaeY5 (Y5) and I.orientalis Y4 (Y4) are by this laboratory screening and preservation.Bacterial classification P.stipitis CBS6054 (CBS6054) is available from U.S. ATCC.
The preparation of proliferated culture medium: glucose 20g/L, yeast extract paste 10g/L, peptone 20g/L, pH5.0-5.5,121 ℃ of sterilization 20min.
The preparation of fermention medium: (wherein glucose is 15.4g/L, wood sugar 6.6g/L, furfural 1.37g/L, 5-HMF0.47g/L, acetate 10g/LCa (OH) for yeast extract paste 10g/L, peptone 20g/L, lignocellulose dilute acid hydrolysis liquid 100ml
2Regulate hydrolyzed solution pH to 5.0), 115 ℃ of sterilization 30min.
Bacterial classification Y5 and CBS6054 are inoculated in respectively in the 100ml proliferated culture medium, and 30 ℃, 150rpm are cultivated 12-14h, and the viable count that obtains bacterial classification is respectively 10
7Cfu/L, 10
8Cfu/L.
In the 100ml fermention medium, 30 ℃, 80rpm are cultivated 72h with CBS6054, each 5ml co-inoculation of Y5 seed liquor.
The ethanol fermentation of experimental example 1 single culture Y5, Y4, CBS6054
1, test materials
Bacterial classification S.cerevisiaeY5 (Y5) and I.orientalis Y4 (Y4) are by this laboratory screening and preservation.Bacterial classification P.stipitis CBS6054 (CBS6054) is available from U.S. ATCC.
2, test method
Bacterial classification S.cerevisiaeY5, I.orientalisY4 and P.stipitisCBS6054 are inoculated in respectively in the proliferated culture medium among the 100ml embodiment 1,30 ℃, 150rpm are cultivated 12h, and the dry cell weight that obtains bacterial classification is respectively 2.7g/L, 3.0g/L, 3.3g/L.
Get Y5, Y4 and each 5ml of CBS6054 bacterium liquid be inoculated in respectively 100ml glucose, glucose+, wood sugar, wood sugar+and the liquid nutrient medium of mixing sugar, mixing sugar+(concentration of glucose and wood sugar is respectively 24g/l, 13g/l) in, wherein "+" expression adds furfural 1.37g/L and 5-HMF0.47g/L, ethanol and sugared concentration are measured in 30 ℃, 80rpm cultivation.
3, analytical procedure
(Waters2690) measures sugared concentration with high performance liquid chromatograph, chromatographic condition: and the ammonia post (200 * 4.6mm), 40 ℃ of column temperatures, waters410 differential detector, moving phase: V (acetonitrile): V (water)=80:20, flow velocity 1mL/min, sample size 20 μ L.
Measure the content of ethanol and furfural and 5-HMF with gas chromatograph (SP-3420).Alcohol determining condition: 80 ℃ of column temperatures, 150 ℃ of injection compartment temperature, 50 ℃ of detector temperatures, sample size 0.5 μ L.Furfural and 5-HMF condition determination: 120 ℃ of column temperatures, 200 ℃ of injection compartment temperature, 50 ℃ of detector temperatures, sample size 0.5 μ L.
The bacterium flow measurement be with sample after the dyeing of 0.1% Lv Shi alkalescence methylene blue, measure bacterium amount in the fermented liquid with blood cell counting.
4, method of calculation
Ethanol content (g/L)=(sample peak area * concentration of standard solution)/standardized solution peak area * 100%;
Alcohol yied (g/g)=record ethanol content/(amount of consumption sugar * 0.51)
Furfural and the same ethanol content of 5-HMF cubage.
5, test-results
As Fig. 1 (a, b, c) as can be known, 80rpm, with glucose be unique substrate, when the unrestraint agent exists, Y5 and Y4 are at the intact glucose of 12h metabolism, CBS6054 then needs 24h.When adding 1.37g/L furfural and 0.47g/L5-HMF, to the not influence of glucose metabolism speed of Y5 and Y4, and very big to the negatively influencing of CBS6054 glucose metabolism speed, the metabolism time lengthening is to 36h.As seen, bacterial strain Y5 and Y4 have very strong glucose metabolism ability and the ability of metabolism fermentation inhibitor furfural and 5-HMF, but can not utilize the wood sugar producing and ethanol, and by contrast, bacterial strain CBS6054 metabolizable glucose speed is slower than preceding 2 bacterial strains.
As shown in Figure 2,80rpm is being unique substrate with wood sugar, and in the substratum of xylose concentration 11.4g/L and unrestraint agent, the xylose utilization rate of P.stipitisCBS6054 fermentation 72h is 100%, and alcohol yied 5.3g/L reaches 91.3% of theoretical yield.And Y5 and Y4 can not utilize the wood sugar producing and ethanol.When being added with 1.37g/L furfural and 0.47g/L5-HMF, CBS6054 metabolism wood sugar is subjected to suppressing fully.
As Fig. 3 (a, b, c) as can be known, Y5, Y4 can not metabolism wood sugar producing and ethanols, but in containing the mixing sugar substratum of 1.37g/L furfural and 0.47g/L5-HMF, 12h can the intact whole glucose of metabolism.CBS6054 accretion rate to sugar when inhibitor exists obviously reduces.Through the lag phase of 72h, the utilization ratio of glucose is 100% when 96h.The utilization of wood sugar just begins to start at 96h, and the utilization ratio of 144h only is 45%.
6, conclusion
Y4 and Y5 have the characteristic of efficient metabolism fermentation inhibitor furfural and 5-HMF and efficient metabolizable glucose producing and ethanol, but can not utilize the wood sugar producing and ethanol, CBS6054 has the ability of better metabolism wood sugar producing and ethanol, the xylose utilization rate of 72h is 100%, alcohol yied reaches 91.3% of theoretical yield, but to the fermentation inhibitor sensitivity.
The mixing sugar ethanol fermentation of experimental example 2 mixed strains Y5+CBS6054 and Y4+CBS6054
1, test materials
Bacterial classification, proliferated culture medium are all with experimental example 1.
The preparation of fermention medium: yeast extract paste 10g/L, peptone 20g/L, glucose 24g/L, wood sugar 13g/L, furfural 1.37g/L, 5-HMF0.47g/L regulates hydrolyzed solution pH to 5.0 with calcium hydroxide, 115 ℃ of sterilization 30min.
2, test method
CBS6054, each 5ml co-inoculation of Y5 seed liquor are in the 100ml fermention medium, and CBS6054, each 5ml co-inoculation of Y4 seed liquor are in the 100ml fermention medium, and 30 ℃, 80rpm are cultivated, and measure the concentration of ethanol, sugar, furfural and 5-HMF.
3, analytical procedure (with experimental example 1)
4, method of calculation (with experimental example 1)
5, test-results
(1) the mixing sugar ethanol fermentation of composite bacteria Y5+CBS6054
As shown in Figure 4,80rpm1.37g/L furfural, 0.47g/L5-HMF the mixing sugar substratum in, glucose metabolism is not affected, and the utilization ratio of 12h is 100%, and furfural and 5-HMF can not measure in the residual quantity of 12h, the utilization ratio of wood sugar reaches 100% at 144h, ethanol production is 16.6g/L, and alcohol yied 0.46g/g reaches 91.2% of theoretical yield.And in whole fermenting process altogether, the well-grown of Y5 and CBS6054, fermentation 0h and 144h, Y5 and CBS6054 bacterium amount ratio in dilute acid hydrolysis liquid is respectively 4:5,2:3, illustrates that they have stronger compatibility.
(2) composite bacteria Y4+CBS6054 mixing sugar ethanol fermentation
As shown in Figure 5, although Y4 all degrades furfural and 5-HMF at 12h, the utilization ratio of wood sugar only is 26.8% at 144h, and alcohol yied is 3.3g/g.Fermentation 0h and 144h, CBS6054 and the Y4 relative proportion in dilute acid hydrolysis liquid is respectively 4:5,1:8, and Y4 becomes the dominant microflora in the mixed culture, and the growth of CBS6054 is subjected to severe inhibition, shows that Y4 and CBS6054's is compatible relatively poor.
6, conclusion
When adding with the furfural of dilute acid hydrolysis liquid equivalent and 5-HMF inhibitor in the mixing sugar substratum, compound culture Y5+CBS6054 shows well blend sugar ethanol fermentation characteristic.Y5 is 100% in the glucose utilization rate of 12h, and the fermentation inhibitor of metabolism has simultaneously been removed CBS6054 because of there is the lag phase that occurs in inhibitor, and makes the CBS6054 well-grown and can make full use of wood sugar.The ethanol production of compound culture Y5+CBS6054 mixing sugar fermentation is 16.6g/L, and alcohol yied 0.46g/g has reached 91.2% of theoretical value.
Composite bacteria Y4+CBS6054 ethanol fermentation ability is relatively poor, Y4 becomes the absolute predominance flora during the fermentation, and the growth of CBS6054 is subjected to severe inhibition, the utilization ratio of wood sugar only is 26.8% at 144h, alcohol yied is 3.3g/g, may be that some mesostate that Y4 produces has suppressed the growth of CBS6054, and then has influenced the metabolism of wood sugar because during the fermentation.
The dilute acid hydrolysis liquid original position detoxication alcohol fermentation of embodiment 3 mixed strains Y5+CBS6054
1, test materials
Bacterial classification: bacterial classification S.cerevisiaeY5 (Y5) is by this laboratory screening and preservation; Bacterial classification P.stipitis CBS6054 (CBS6054) is available from U.S. ATCC.
Proliferated culture medium, fermention medium are all with embodiment 1
2, test method
CBS6054, each 5ml of Y5 seed liquor are inoculated in the fermention medium of 100ml without any detoxification treatment, 30 ℃, 80rpm cultivation, the concentration of mensuration ethanol, sugar, furfural and 5-HMF.
3, analytical procedure (with experimental example 1)
4, method of calculation (with experimental example 1)
5, test-results
As shown in Figure 6, composite bacteria Y5+CBS6054 is to the original position detoxication alcohol fermentation of lignocellulose dilute acid hydrolysis liquid, 80rpm, and 16h glucose utilization rate is 100%, furfural and 5-HMF are degraded fully at 16h.The utilization of wood sugar begins later on to start at 16h, and the utilization ratio of 144h is 67.8%, and the utilization ratio of 168h reaches 100%, and alcohol yied is 0.44g/g, reaches 87.5% of theoretical value.Two bacterial strains well-grown in whole fermentation process, Y5 and CBS6054 are respectively 3:4,2:3 in the relative proportion of fermentation 0h and 168h, and the compatible fine of them is described.In the glucose utilization stage of initial 16h, the Y5 speed of growth is very fast, in case glucose utilization is intact, CBS6054 begins to utilize wood sugar growth and producing and ethanol.
6, conclusion
With the compound culture of S.cerevisiaeY5+P.stipitisCBS6054 the ethanol fermentation of lignocellulose dilute acid hydrolysis liquid original position detoxification has been obtained satisfied result, alcohol yied 0.44g/g reaches 87.5% of theoretical value.Therefore adopting Y5 and the compound cultivation of CBS6054 is a kind of feasible method to the not detoxication alcohol fermentation of lignocellulose dilute acid hydrolysis product.Can do further research to shorten the xylose metabolism time by methods such as this compound culture immobilization or employing feed supplement batch fermentations, further improve alcohol yied.
Claims (9)
1. a S. cervisiae (Saccharomyces cerevisiae) Y5, preserving number CGMCC No.2660.
2. composite bacteria, it contains S.cerevisiae Y5 and P.stipitisCBS6054.
3. composite bacteria as claimed in claim 2 is characterized in that, to count ratio with the bacterium of P.stipitisCBS6054 be 1 to S.cerevisiae Y5 in every by weight gram composite bacteria: 5-5: 1.
4. composite bacteria as claimed in claim 3 is characterized in that, to count ratio with the bacterium of P.stipitisCBS6054 be 1: 1 to S.cerevisiae Y5 in every by weight gram composite bacteria.
5. as the application of the arbitrary described composite bacteria of claim 2-4 in the original position detoxication alcohol fermentation of lignocellulose dilute acid hydrolysis product.
6. the original position detoxication alcohol fermentation method of a lignocellulose dilute acid hydrolysis product, it is characterized in that, S.cerevisiae Y5 and P.stipitis CBS6054 are inoculated in respectively in the fermention medium that contains lignocellulose dilute acid hydrolysis liquid, cultivate 8-72h at 25 ℃-38 ℃, 0rpm-500rpm.
7. method as claimed in claim 6 is characterized in that, S.cerevisiae Y5 and P.stipitis CBS6054 are inoculated into fermention medium after, cultivate 72h at 30 ℃, 80rpm.
8. as claim 6 or 7 described methods, it is characterized in that, according to every ml fermention medium 10
6Cfu-10
10Amount inoculation S.cerevisiae Y5 and the P.stipitis CBS6054 of cfu.
9. as claim 6 or 7 described methods, it is characterized in that described fermention medium is with yeast extract paste 10g/L, peptone 20g/L, lignocellulose dilute acid hydrolysis liquid 100ml,, under 121 ℃, make behind the sterilization 20min at pH5.0-5.5.
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| CN102277390B (en) * | 2011-08-02 | 2014-11-05 | 首都师范大学 | Method for producing ethyl alcohol by utilizing non-detoxicated enzymatic hydrolysate during lignocellulose steam blasting pretreatment |
| CN103184169B (en) * | 2011-12-31 | 2014-10-29 | 安琪酵母股份有限公司 | Cellulose yeast, double-strain saccharomyces cerevisiae composition and fermentation method of cellulosic ethanol |
| CN102899379B (en) * | 2012-11-05 | 2014-06-04 | 江苏科技大学 | Method for degrading furfuraldehyde in process for preparing biomass fuel by dilute acid method |
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| CN104673689A (en) * | 2013-11-27 | 2015-06-03 | 首都师范大学 | Method for producing ethanol by fermentation with Saccharomyces cerevisiae flora for showing cellulases |
| CN107916279A (en) * | 2016-10-11 | 2018-04-17 | 中国科学院大连化学物理研究所 | A kind of detoxification fermentation method in situ of ligno-cellulose hydrolysate |
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| CN102424808A (en) * | 2011-12-26 | 2012-04-25 | 南开大学 | Straw-degrading composite microbial inoculum and application thereof in pretreatment of ethanol production |
| CN102424808B (en) * | 2011-12-26 | 2013-04-24 | 南开大学 | Preparation method of straw-degrading composite microbial inoculum and application thereof |
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