CN101353629B - In situ detoxication alcohol fermentation method of ligno-cellulose hydrolysate using single strain - Google Patents
In situ detoxication alcohol fermentation method of ligno-cellulose hydrolysate using single strain Download PDFInfo
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Abstract
The invention provides a method for adopting single strains to carry out fermentation to in-situ virus-eliminated alcohol of hydrolyzate of lignocellulose; therefore, the invention firstly provides a new strain of pichia stipitis of trunks and the preservation number is CGMCC No.2661; the experiment proves that the strain can carry out detoxification in situ to lignocellulose diluted acid hydrolyzate, can transform glucose and xylose in lignocellulose diluted acid hydrolysate into ethanol effectively and reach 92.4 percent of the highest theoretical value of the ethanol. The use of the straincan simplify the technique that the lignocellulose is used as materials to produce the ethanol, reduces the production cost of the ethanol and has significant theoretical and practical significance to the commercialization of the production of lignocellulose ethanol.
Description
Technical field
The present invention relates to a kind of alcohol fermentation method, specifically a kind of original position detoxication alcohol fermentation method that adopts single culture to ligno-cellulose hydrolysate using, the invention still further relates to the new bacterial strain of the microorganism that is used for ethanol fermentation, the new bacterial strain of specifically a kind of pichia stipitis (Pichia.Stipitis).
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
[1], carried out a large amount of and deep research in this field both at home and abroad.But up to the present, also existing bottleneck problem aspect the dilute acid hydrolysis lignocellulosic materials for fuel ethanol both at home and abroad, solving this technical bottleneck and be the wood sugar producing and ethanol that will efficiently be fermented in the hydrolysis sugar liquid and want the bacterial classification of the fermentation inhibitor in the efficient metabolic water hydrolysis products.
The bacterial classification that can carry out the wood sugar ethanol fermentation is less, studies comparatively deep Pichiastipitis energy xylose-fermenting at present and has very high alcohol yied
[2], 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
[3]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
[4,5]Supressor in the hydrolysate can influence microbial growth and fermentation capacity, and wherein furfural and 5 hydroxymethyl furfural (5-HMF) are main supressors
[6]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
[7]
For lignocellulose dilute acid hydrolysis product being carried out original position detoxification (the i.e. toxicant of this fermented bacterium in can efficient metabolism fermented liquid when carrying out ethanol fermentation, need not carry out any extra detoxification treatment, the fermenting process that ethanol fermentation just can efficiently carry out) and the ethanol fermentation of glucose and wood sugar, the inventor obtains the bacterial strain Pichia stipitis Y7 of a kind of efficient metabolizable glucose and wood sugar producing and ethanol and efficient metabolic poison by a large amount of screening operations, the contriver attempts using this bacterial strain that lignocellulose dilute acid hydrolysis product is carried out the original position detoxication alcohol fermentation, to understand the efficient ethanol fermentation that this bacterium carries out glucose and the detoxification of wood sugar original position to lignocellulose dilute acid hydrolysis product, provide basic data for further using.
Summary of the invention
The object of the present invention is to provide the new bacterial strain of a kind of pichia stipitis (Pichia.Stipitis) Y7 preserving number is CGMCC No.2661.
Another object of the present invention is to provide the application of this bacterial strain in the original position detoxication alcohol fermentation of lignocellulose dilute acid hydrolysis liquid.
Bacterial strain is found in secular ligno-cellulose hydrolysate experimentation among the present invention, and this bacterium metabolism is vigorous, can efficiently utilize glucose and wood sugar in the hydrolyzed solution, can tolerate the inhibitor of high density, is the core of fermentation technique flow process.This bacterial strain on September 4th, 2008 in (address: Datun Road, Chaoyang District, Beijing City, China Committee for Culture Collection of Microorganisms common micro-organisms center, Institute of Microorganism, Academia Sinica, postcode 100101) preservation, classification called after pichia stipitis (Pichia.Stipitis), preserving number is CGMCC No.2661.
In an embodiment, bacterial strain of the present invention is cultivated in containing the YPD substratum of glucose, the result shows that ethanol yield can reach 0.486g/g sugar, reaches 95.3% of theoretical value; Cultivate in containing the YPD substratum of wood sugar, the result shows that ethanol yield can reach 0.42g/g sugar, reaches 83.2% of theoretical value.
In an embodiment, bacterial strain of the present invention is cultivated in containing the YPD substratum of furfural inhibitor, the result shows that the concentration of 4g/L does not play the obvious suppression effect to bacterial strain of the present invention; When cultivating in the substratum that is containing 5 hydroxymethyl furfural, the result shows, the concentration of this 4g/L to the growth of Y7 without any influence.Further, these two kinds of inhibitor are added in the YPD substratum simultaneously, investigate its restraining effect to bacterial strain of the present invention, the result shows that when adding 3g/L furfural and 3g/L hydroxymethylfurfural in the YPD substratum, the delayed growth phase of a 24h has only appearred in Y7.
On the basis of above-mentioned research, Y7 seed liquor 10ml is inoculated in the dilute acid hydrolysis liquid of 100ml without any detoxification treatment 30 ℃, 150rpm cultivation, the concentration of mensuration ethanol, sugar.Glucose 36h in the lignocellulose dilute acid hydrolysis liquid has utilized, and wood sugar 72h has utilized, and ethanol production is 32.53g/L, and alcohol yied is a 0.47g/g sugar, reaches 92.4% of theoretical value.
The fermentation condition that Y7 is used for the original position detoxication alcohol fermentation of lignocellulose dilute acid hydrolysis liquid is: 25～35 ℃, (0～300) rpm, fermented 24～96 hours; Fermention medium is: lignocellulose dilute acid hydrolysis liquid, yeast extract paste an amount of (other somatomedins also can replace), peptone an amount of (other can utilize nitrogenous source also can replace), Ca (OH)
2Regulating pH is 4.5～6.0.Get the adding of 10mL Y7 seed liquor 100mL hydrolyzed solution (adding yeast extract paste 10g/L, peptone 10g/L, Ca (OH) are housed
2Adjusting pH5.0) in the triangular flask, optimal conditions of fermentation is 150rpm, 30 ℃ of cultivations, pH5.0-5.5.During inoculation fermentation, the viable count of seed liquor should be 10
5-10
10Individual/ml, inoculum size should be at 1～20% (W/W).
The lignocellulose dilute acid hydrolysis is considered to the business-like production technique of the easiest realization, but this hydrolysis process is when producing fermentable sugars, also producing a large amount of has strong inhibiting inhibitor to the ethanol fermentation microorganism, so the sugar in the hydrolyzed solution is converted into the bottleneck that alcoholic acid original position poison-removing method becomes this alcohol production technology.The present invention carries out the original position detoxication alcohol fermentation with Pichia.Stipitis Y7 to lignocellulose dilute acid hydrolysis liquid, has obtained good result, and having solved with the lignocellulose is raw material production alcoholic acid bottleneck problem.
Bacterial strain of the present invention can efficiently be converted into ethanol with glucose in the lignocellulose dilute acid hydrolysis liquid and wood sugar, reach ethanol the highest theoretical value 92.4%, this result yet there are no report at home and abroad.Low alcohol production cost.Commercialization to the lignocellulose alcohol production has important theory and practical significance.
Description of drawings
What Fig. 1 showed is the ethanol fermentation of Y7 to glucose;
What Fig. 2 showed is the ethanol fermentation of Y7 to wood sugar;
What Fig. 3 showed is the ethanol fermentation of Y7 to glucose and wood sugar mixing sugar;
What Fig. 4 showed is the tolerance of Y7 to anti-furfural;
What Fig. 5 showed is the tolerance of Y7 to 5 hydroxymethyl furfural;
What Fig. 6 showed is the tolerance of Y7 to furfural and 5 hydroxymethyl furfural;
What Fig. 7 showed is the ethanol fermentation of Y7 to lignocellulose dilute acid hydrolysis liquid.
Embodiment
Following examples further specify content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, modification or replacement to the inventive method, step or condition are done all belong to scope of the present invention.
If do not specialize the conventional means that used technique means is well known to those skilled in the art among the embodiment.
The isolation identification of embodiment 1 Y7
1 screening process
From grain distillery's downflow sludge, separate acquisition by this laboratory, in being added with the YPD liquid nutrient medium of furfural and hydroxymethylfurfural, carry out enrichment culture, isolate single bacterium colony containing on the solid plate of wood sugar coating then, after a large amount of screenings, find, wherein a strain metabolism is vigorous, can efficiently utilize glucose and wood sugar in the hydrolyzed solution, can tolerate the inhibitor of high density, with its called after Y7, carry out slant preservation at last.
2 morphological specificitys
Bacterium colony is smooth, and thalline spheroidal is less, breeds in the binary fission mode, does not produce false silk.
3 cultural characteristics
After cultivating for some time in the liquid medium within, bacterium liquid muddiness does not have the flocculation phenomenon.
4 metabolic characteristicss
Wood sugar and glucose producing and ethanol can be utilized fast, the high density inhibitor can be tolerated simultaneously.
1 material and method
1.1 bacterial classification
Bacterial classification Pichia.Stipitis Y7
1.2 substratum (g/L)
YPD substratum: glucose 20, yeast extract paste 10, peptone 20, pH5.0～5.5.
Fermention medium: yeast extract paste 10, peptone 20.In addition, add glucose respectively, wood sugar (concentration is as the criterion with practical measurement) pH5.0～5.5 according to different needs.
1.3 lignocellulose dilute acid hydrolysis liquid (g/L)
This institute is provided by East China University of Science with lignocellulose dilute acid hydrolysis liquid, and wherein glucose is 44.678, wood sugar 24.35, furfural 1.37,5-HMF0.47, acetate 10, Ca (OH)
2Regulate hydrolyzed solution pH to 5.0.
1.4 culture of strains
Bacterial classification Y7 is inoculated in the 100ml YPD substratum, and 30 ℃, 150rpm are cultivated 12h.
1.5 ethanol fermentation
Y7 seed liquor (viable count 8 * 10
8Individual/ml～9 * 10
8Individual/ml) 10ml is inoculated in 100ml fermention medium and the dilute acid hydrolysis liquid of 100ml without any detoxification treatment, 30 ℃, 150rpm cultivation, the concentration of mensuration ethanol, sugar.
1.6 anti-inhibitor experiment
The furfural that adds different gradients in the YPD substratum respectively, the mixture of hydroxymethylfurfural and furfural and hydroxymethylfurfural (ratio is 1:1), timing sampling, sample is through dilution metering bacterium liquid OD value.
1.7 analytical procedure
1.7.1 sugared concentration determination
High performance liquid chromatograph (Waters 2690) is measured sugared concentration, 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.
1.7.2 ethanol content is measured
Gas chromatograph (SP-3420) is measured the content of ethanol and furfural and 5-HMF.Alcohol determining condition: 80 ℃ of column temperatures, 150 ℃ of injection compartment temperature, 50 ℃ of detector temperatures, sample size 0.5 μ L.
2 results and discussion
2.1 the fermentation of glucose, wood sugar, mixing sugar
2.1.1Y7 glucose fermentation characteristic
As shown in Figure 1, in containing the dextrose culture-medium of 24.86g/L, glucose 12h has utilized, and maximum ethanol production is 12.08g/L, and alcohol yied is a 0.486g/g sugar, reaches 95.3% of theoretical value.
2.1.2Y7 wood-sugar fermentation characteristic:
As Fig. 2, in containing 23.81g/L wood sugar substratum, wood sugar 72h has utilized, and maximum ethanol production is 10.10g/L, and alcohol yied is a 0.424g/g sugar, reaches 83.2% of theoretical value.
Fig. 1 and Fig. 2 as can be seen, this bacterium is metabolizable glucose and wood sugar producing and ethanol efficiently, 12 hours glucose ethanol fermentation and 72 hours wood sugar ethanol fermentation have reached advanced international standard.
2.1.3Y7 mixing sugar fermentation character:
As Fig. 3, in the mixing sugar substratum that contains 16.29g/L glucose 8.15g/L wood sugar, glucose 12h has utilized, and wood sugar 36h has utilized, and maximum ethanol production is 11.36g/L, and alcohol yied is a 0.465g/g sugar, reaches 91.2% of theoretical value.
As seen from Figure 3, when glucose and wood sugar existed simultaneously, glucose had promoted the conversion of wood sugar, only needed 48 hours wood sugar can be converted into ethanol.Transformed wood sugar fully in 48 hours and become ethanol, at home and abroad also do not reach and appear in the newspapers.
2.2Y7 tolerance to inhibitor furfural and 5 hydroxymethyl furfural
Utilize on the basis of glucose, wood sugar, mixing sugar producing and ethanol characteristic having understood this bacterium, further illustrate the ability that this bacterium is an inhibitor, mainly investigated the degradation capability of this bacterium furfural and 5 methyl furfural.As shown in Figure 4, Y7 is in the YPD substratum, and the furfural amount that can tolerate reaches 4g/L, but the lag period of 24h occurred, and when 3g/L, only be 12h lag period.
When Fig. 5 has shown in substratum the 5 hydroxymethyl furfural that adds 4g/L, the concentration of this inhibitor to the growth of Y7 without any influence.
As shown in Figure 6, in the YPD substratum, when adding 4g/L furfural and 4g/L5-hydroxymethylfurfural simultaneously, thalli growth has been subjected to suppressing fully, but when adding 3g/L furfural and 3g/L hydroxymethylfurfural in the YPD substratum, the delayed growth phase of a 24h has only appearred in Y7.
2.3Y7 original position detoxication alcohol fermentation to lignocellulose dilute acid hydrolysis liquid
Understood this bacterium can tachymetabolism glucose and wood sugar producing and ethanol, again efficiently on the basis of metabolism fermentation inhibitor, then, with lignocellulose dilute acid hydrolysis liquid is the ethanol fermentation substrate, illustrates the characteristic of the practical application of glucose in this bacterium metabolism lignocellulose dilute acid hydrolysis liquid and wood sugar and metabolism fermentation inhibitor wherein.As shown in Figure 7, the glucose 36h in the lignocellulose dilute acid hydrolysis liquid has utilized, and wood sugar 72h has utilized, and ethanol production is 32.53g/L, and alcohol yied is a 0.471g/g sugar, reaches 92.4% of theoretical value.
The preparation of 1 seed liquor
In the 250mL triangular flask, add 100mL YPD liquid nutrient medium (glucose 20g/L, peptone 20g/L, yeast extract paste 10g/L), put into 115 ℃ of Autoclave sterilizations 25 minutes.The single bacterium colony of picking one ring inserts triangular flask, 150rpm, and shaking table was cultivated 12 hours for 30 ℃.
The processing of 2 hydrolyzed solutions
In hydrolyzed solution, add peptone (20g/L), yeast extract paste (10g/L), Ca (OH)
2Regulating pH is 5.0, puts into 115 ℃ of Autoclave sterilizations 25 minutes.
The fermentation of 3 lignocellulose dilute acid hydrolysis liquid
Get 10mL Y7 seed liquor (viable count 8 * 10
8Individual/ml～9 * 10
8Individual/ml) 150rpm, 30 ℃ of cultivations of shaking table are equipped with in the triangular flask of hydrolyzed solution in adding.
4 analytical procedures
4.1 sugared concentration determination
High performance liquid chromatograph (Waters 2690) is measured sugared concentration, 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.
4.2 ethanol content is measured
Gas chromatograph (SP-3420) is measured the content of ethanol and furfural and 5-HMF.Alcohol determining condition: 80 ℃ of column temperatures, 150 ℃ of injection compartment temperature, 50 ℃ of detector temperatures, sample size 0.5 μ L.
5 fermentation results
As shown in Figure 7, the glucose 36h in the lignocellulose dilute acid hydrolysis liquid has utilized, and wood sugar 72h has utilized, and ethanol production is 32.53g/L, and alcohol yied is a 0.471g/g sugar, reaches 92.4% of theoretical value.
Reference
[1]Tania?I.Georgieva，Birgitte?K.Ahring.Evaluation?of?continuous?ethanol?fermentationof?dilute-acid?corn?stover?hydrolysate?using?thermophilic?anaerobic?bacteriumThermoanaerobacter?BG1L1，[J].Appl?Microbiol?Biotechnol，2007，77：61-68.
[2]du?Preez?JC，Prior?BA.A?quantitative?screening?of?some?xylose?fermenting?yeastisolates[J].Biotechnol?Lett，1985，7：241-248.
[3]Lohmeier-Vogel，E.M，Sopher，et?al.Intracellular?acidification?as?mechanism?for?theinhibition?by?Acid?hydrolysis-derived?inhibitors?of?xylose?fermentation?by?yeasts.Journal?of?Industrial?Microbiology?Biotechnology，1998，20：75-80.
[4]B
rbel?Hahn-H
gerdal，Kaisa?Karhumaa，César?Fonseca，etal.Industrialpentose-fermenting?yeast?strains[J].Appl?Microbiol?Biotechnol，2007，74:937-953.
[5]Nancy?N.Nichols，Bruce?S.Dienr，Gema?M.Guisaoo，etal.Bioabatement?to?RemoveInhibitors?from?Biomass-Derived?Sugar?Hydrolysates[J].Applied?Biochemistry?andBiotechnology，2005，121-124：379-390.
[6]Michiel?J.Boonstra，B^oke?Tjeerdsma.Chemical?analysis?of?heat?treated?softwoods.Holz?als?Roh-und?Werkstoff，2006，64：204-211.
[7]Frank?K?Agbogbo，Kevin?S.Wenger.Production?of?ethanol?from?corn?stoverhemicellulose?hydrolyzate?using?Pichia?stipitis[J].J?Ind?Microbiol?Biotechnol，2007，34：723-727.
Claims (6)
1. pichia stipitis (Pichia stipitis) Y7 CGMCC No.2661.
2. the application of the described bacterial strain of claim 1 in the original position detoxication alcohol fermentation of lignocellulose dilute acid hydrolysis liquid.
3. the original position detoxication alcohol fermentation method of a lignocellulose dilute acid hydrolysis liquid is characterized in that using the described bacterial strain of claim 1 to ferment.
4. method as claimed in claim 3, it is characterized in that the described bacterial strain of claim 1 is made seed liquor, the fermention medium of inoculation lignocellulose-containing dilute acid hydrolysis liquid ferments, fermentation condition is: 25～35 ℃, 0～300rpm, fermented 24～96 hours; Fermention medium is: lignocellulose dilute acid hydrolysis liquid, and yeast extract paste is an amount of, and peptone is an amount of, Ca (OH)
2Regulating pH is 4.5～6.0.
5. method as claimed in claim 4 is characterized in that fermentation condition is: 30 ℃, 150rpm, fermented 72 hours; Fermention medium is: lignocellulose dilute acid hydrolysis liquid, yeast extract paste 10g/L, peptone 20g/L.
6. as claim 4 or 5 described methods, it is characterized in that the viable count of seed liquor is 10
5-10
10Individual/ml, inoculum size is 1%～20%.
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Cited By (1)
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| CN102010833A (en) * | 2010-06-21 | 2011-04-13 | 唐传生物科技（厦门）有限公司 | Novel pichia strain and method for mixing and culturing same to biologically detoxify hemicellulose hydrolysate |
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| CN103898166A (en) * | 2012-12-25 | 2014-07-02 | 中粮营养健康研究院有限公司 | Method of producing ethanol |
| CN105420291A (en) * | 2015-12-18 | 2016-03-23 | 清华大学 | Method of using lignocellulose to produce butyl alcohol |
| CN108823112B (en) * | 2018-06-15 | 2023-11-17 | 首都师范大学 | Method for improving ethanol yield by directionally optimizing xylose metabolic pathway and engineering saccharomyces cerevisiae |
| CN114107093B (en) * | 2021-11-03 | 2023-10-27 | 浙大宁波理工学院 | Cellulose degrading bacterium for high-yield cellulase and application thereof |
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| CN102010833A (en) * | 2010-06-21 | 2011-04-13 | 唐传生物科技（厦门）有限公司 | Novel pichia strain and method for mixing and culturing same to biologically detoxify hemicellulose hydrolysate |
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