CN102268382A - High temperature resistant Issatchenkia orientalis, application thereof and method for producing ethanol through fermentation - Google Patents
High temperature resistant Issatchenkia orientalis, application thereof and method for producing ethanol through fermentation Download PDFInfo
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Abstract
The invention relates to the field of fermentation, in particular to a strain of high temperature resistant Issatchenkia orientalis, application thereof and a method for producing ethanol through fermentation. The strain of high temperature resistant Issatchenkia orientalis IPE100 is separated and screened from farmland crop waste, has a number of CGMCC (China General Microbiological Culture Collection Center) No.4927, has the capacity of fermenting ethanol at high temperature, and can be applied to high temperature fermentation of ethanol by using lignocelluloses hydrolysis products and high temperature solid fermentation of ethanol by using sweet sorghum.
Description
Technical field
The present invention relates to the fermentation field, particularly, relate to the method for the high temperature resistant Issatchenkia orientalis of a strain and application and fermentative production of ethanol.
Background technology
Because the enhancing of the reserves finite sum people environmental consciousness of the mineral substance energy utilizes reproducible biomass to carry out the attention that Energy production more and more causes people.Therefore, seek and a kind ofly can guarantee energy regular supply and to pollute little fuel extremely urgent.Ethanol is a kind of renewable energy source, also is the most promising substitute products of fossil fuel.Method ethanol production is based on saccharomycetes to make fermentation, and the suitableeest leavening temperature of industrial yeast bacterium generally should be above 36 ℃ at 30 ℃～33 ℃.Temperature further raising can cause yeast aging and dead, makes its ethanol fermentation ability and alcoholic acid patience is reduced, and fermenting process can not normally carry out, and production efficiency is low, is unsuitable for industrial production.Therefore, need take a series of cooling measures aborning, very expensive as with phreatic water spray etc., summer high temperature season especially, more difficult temperature control.Thermotolerant yeast is applied to ethanol fermentation produces, can save the required a large amount of water coolants of traditional zymotic,, improve fermentation efficiency and separate all favourable with alcoholic acid for reducing production costs.
The application prospect of Issatchenkia orientalis (Issatchenkia orientalis) is extensive, can be used for dye decolored, poisonous inhibition detoxification and ethanol fermentation etc., wherein Issatchenkia orientalis is subjected to researchist's extensive concern to the detoxification of inhibition in hemicellulose and the ligno-cellulose hydrolysate using (as furfural, Vanillin, P-hydroxybenzoic acid, methyl catechol etc.), this bacterial classification can be cultivated altogether with other bacterial classifications, realizes the biosynthesizing of detoxification and purpose product.Chinese patent (publication number: CN 101914592 A, CN 101805701 A, CN 101805761 A, CN 101735958 A) disclose a strain Issatchenkia orientalis (Issatchenkia orientalis) S-7CCTCC NO:M206098 in and had the ability of microbial metabolism inhibition in the degradation of hemicellulose hydrolysate, can be used with other microorganism strains, be used for the production of Xylitol, ethanol, pectinose, Issatchenkia orientalis mainly plays detoxification in its process.Itself just has the ability of producing and ethanol some Issatchenkia orientalis, Chinese patent (publication number: disclose a strain Issatchenkia orientalis (Issatchenkia orientalis) xlc6CCTCC NO:M207003 CN 101029300A) and the citric acid in the fruit juice can be degraded to ethanol; Yang Xiushan etc. disclose a strain Issatchenkia orientalis (Issatchenkia orientalis) Y4CGMCC No.2159 can utilize ligno-cellulose hydrolysate using fermentative production of ethanol (Chinese patent publication number: CN 101165165 A; Shen, field, Zhou Guixiong, Zhang Lanbo, Yang Xiushan. solar energy journal, 2010,31:1-4), yet its appropriate incubation temperature of Issatchenkia orientalis that more than is used for ethanol fermentation all is no more than 35 ℃.
Summary of the invention
Propose and finished the present invention in order to address the above problem.
The purpose of this invention is to provide the high temperature resistant Issatchenkia orientalis of a strain (Issatchenkia orientalis) IPE100.
A further object of the present invention provides above-mentioned high temperature resistant Issatchenkia orientalis and is used for the application of biological fermentation alcoholic acid.
A further object of the present invention provides a kind of above-mentioned high temperature resistant Issatchenkia orientalis biological fermentation production alcoholic acid method of utilizing.
The present invention separates the high temperature resistant Issatchenkia orientalis bacterium of a strain (Issatchenkia orientalis) IPE100 bacterial strain from the agricultural waste material of farmland, be numbered CGMCC No.4927, has the ability of at high temperature carrying out ethanol fermentation.
The present invention also provides above-mentioned high temperature resistant Issatchenkia orientalis bacterium (Issatchenkia orientalis IPE100) to be used to produce alcoholic acid and has used.
The present invention also provides and has utilized above-mentioned high temperature resistant Issatchenkia orientalis bacterium (Issatchenkia orientalis IPE100) biological fermentation to produce the alcoholic acid method, and leavening temperature is 38～45 ℃.
Above-mentioned high temperature resistant Issatchenkia orientalis bacterium (the Issatchenkia orientalis IPE100) biological fermentation that utilizes according to the present invention is produced the alcoholic acid method, and it can utilize ligno-cellulose hydrolysate using high temperature ethanol fermentation and sweet sorghum high temperature ethanol solid state fermentation.
Particularly,, utilize above-mentioned high temperature resistant Issatchenkia orientalis bacterium (Issatchenkia orientalis) IPE100 biological fermentation to produce the alcoholic acid method according to embodiments of the invention, wherein,
1, culture presevation
Bacterial classification is high temperature resistant, and Issatchenkia orientalis IPE100 bacterial strain is preserved in alkaline nutrient agar inclined-plane in 4 ℃ of refrigerators, and switching in 3 months once.Substratum is formed: Tryptones 5g/L, and yeast extract 3g/L, glucose 20g/L, agar 15g/L was in 120 ℃ of sterilizations 15 minutes.
2, seed culture
Liquid nutrient medium is formed: Tryptones 5g/L, and yeast extract 3g/L, glucose 20g/L; In 120 ℃ of sterilizations 15 minutes.Inoculation, 42 ℃, 150rpm shake-flask culture 16h had both got liquid seeds.
3, ligno-cellulose hydrolysate using high temperature ethanol fermentation
Fermention medium is formed: (NH
4)
2SO
40.8-1.2g/L, MgSO
40.01-0.03g/L, K
2HPO
40.02-0.04g/L all the other are ligno-cellulose hydrolysate using stoste, regulate pH to 5-7.Preferred fermention medium is formed: (NH
4)
2SO
41g/L, MgSO
40.02g/L, K
2HPO
40.03g/L all the other are ligno-cellulose hydrolysate using stoste, regulate pH to 6.Fermentation condition: 38-45 ℃, 120-180rpm, inoculum size 3-6% (v/v).Preferred fermentation condition: 42 ℃, 150rpm, inoculum size 4% (v/v).
4, sweet sorghum high temperature ethanol solid state fermentation
Fermention medium is formed: nutrient salt solution and sweet broomcorn straw dry powder are pressed 2-4: the mixed of 1 (w/w), wherein nutrient salt solution is formed: (NH
4)
2SO
40.8-1.2g/L, MgSO
40.01-0.03g/L, K
2HPO
40.01-0.04g/L.Preferred fermention medium is formed: nutrient salt solution and sweet broomcorn straw dry powder are by the mixed of 3: 1 (w/w), and wherein nutrient salt solution is formed: (NH
4)
2SO
41g/L, MgSO
40.02g/L, K
2HPO
40.025g/L.Fermentation condition: 38-45 ℃, inoculum size 2-5% (v/w), static cultivation.Preferred fermentation condition: 42 ℃, inoculum size 3% (v/w).
The invention provides the high temperature resistant Issatchenkia orientalis bacterium of strain IPE100 bacterial strain, it has the high temperature bottom fermentation and produces the alcoholic acid ability; And provide a kind of method of utilizing this bacterial strain to carry out ligno-cellulose hydrolysate using high temperature ethanol fermentation and sweet sorghum high temperature ethanol solid state fermentation.
Advantage of the present invention is:
1. the present invention finds and the high temperature resistant Issatchenkia orientalis bacterium (Issatchenkia orientalis IPE100) that provides can directly utilize the lignocellulose hydrolyzate to carry out the high temperature ethanol fermentation.Because the biological detoxication characteristic that had of bacterial strain itself need not any detoxification treatment; Because bacterial strain has resistant to elevated temperatures characteristic, fermenting process need not be taked cooling measure, and zymotechnique is simple, has reduced fermentation costs; Because it is higher that thermophilic fermentation finishes secondary fermentation wine with dregs temperature, carry out ethanol distillation institute energy requirement and reduce, help reducing energy consumption and production costs;
2. the present invention utilizes high temperature resistant Issatchenkia orientalis bacterium (Issatchenkia orientalis IPE100) to carry out sweet sorghum high temperature ethanol solid state fermentation, can well overcome solid ferment process thermal accumlation and the difficult shortcoming of heat radiation, solve the problem that the culture medium temperature raises and causes microbial fermentation efficient to reduce, can carry out the ethanol solid state fermentation of high bed height, improve fermentation efficiency, be convenient to realize suitability for industrialized production.
(microbial preservation number is high temperature resistant Issatchenkia orientalis bacterium (Issatchenkia orientalis IPE100): CGMCC No.4927; The preservation time is: 2011 06 month No. 8; Depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center; Preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; 100101)
Description of drawings
Fig. 1 is the stereoscan photograph of high temperature resistant Issatchenkia orientalis bacterium (Issatchenkia orientalis IPE100).
Embodiment
The screening of embodiment 1 high temperature resistant Issatchenkia orientalis IPE100
The separation of IPE100 and screening: collected specimens from farmland farm crop (maize straw) waste of Daxing, Beijing, and with after the sample activation that collects, getting wherein, 1g adds 100mL in the YPG substratum that adds penbritin, 250mL triangular flask shaking table shaking culture, 42 ℃, 150rpm.Behind the enrichment culture 48h with nutrient solution through behind certain gradient dilution, be applied on the solid plate of same medium, behind 42 ℃ of cultivation 48h, single bacterium colony of picking products of typical yeast form, again behind the purifying three times of repeating to rule on the same medium solid plate, 4 ℃ of preservations.By its alcohol yied of ethanol fermentation measuring and fermentation period under the differing temps, detect the detoxification ability of bacterial strain then, filter out effect bacterial strain preferably according to the fermenting experiment result at last inhibition by the inhibition degradation experiment
The evaluation of IPE100: observation, Physiology and biochemistry mensuration and 18S rDNA gene sequencing result by morphological specificity identify the IPE100 bacterial strain.
1, morphological specificity is observed: bacterium colony creamy white, rounded, smooth, the butter shape of shape; After cultivating the long period, the bacterium colony drying has the mycelium extension, and pseudohypha branch distributes; It is avette or oval that cell is, and the cell after the propagation is multipole to sprout (as shown in Figure 1).
2, Physiology and biochemistry measurement result:
3,18S rDNA gene sequencing:
Extracting the full DNA that test kit (having TIANGEN Biotech (Beijing) Co., Ltd. to provide) extracts IPE100 with pastoris genomic dna, is template with the full DNA that extracts then, carries out pcr amplification.Compare measuring the 18S rDNA gene order of the IPE100 that comes out and the sequence in the GenBank nucleic acid sequence data storehouse, adopt ClustalW software to mate arrangement, grow tree with the neighbor-joining analytical method constructing system in MEGA version 4.0 softwares, TreeView software indicating system tree is used in the check back.The result shows that the homology of IPE100 and Issatchenkia orientalis (AB 160862) is higher.
Embodiment 2 usefulness high temperature resistant Issatchenkia orientalis IPE100 of the present invention carries out ligno-cellulose hydrolysate using high temperature ethanol fermentation
Adopt liquid seed culture medium,, obtain liquid seeds high temperature resistant Issatchenkia orientalis IPE100 shake-flask culture 16h under 42 ℃, 150rpm condition.Liquid seeds is inoculated into according to the inoculum size of 4% (v/v) contains (NH
4)
2SO
41g/L, MgSO
40.02g/L, K
2HPO
40.03g/L, ligno-cellulose hydrolysate using, in the fermention medium of pH 6.0,42 ℃, 150rpm cultivates 48h, and alcohol yied reaches maximum value, is 93.8% of theoretical value (0.51g/g).Used ligno-cellulose hydrolysate using is composed as follows: glucose 95.70g/L, furfural 0.05g/L, 5 hydroxymethyl furfural 0.15g/L.
Embodiment 3 usefulness high temperature resistant Issatchenkia orientalis IPE100 of the present invention carries out ligno-cellulose hydrolysate using high temperature ethanol fermentation
Adopt liquid seed culture medium,, obtain liquid seeds high temperature resistant Issatchenkia orientalis IPE100 shake-flask culture 16h under 42 ℃, 150rpm condition.Liquid seeds is inoculated into according to the inoculum size of 3% (v/v) contains (NH
4)
2SO
41.2g/L, MgSO
40.03g/L, K
2HPO
40.04g/L, ligno-cellulose hydrolysate using, in the fermention medium of pH 7.0,38 ℃, 180rpm cultivates 48h, and alcohol yied reaches maximum value, is 90.3% of theoretical value (0.51g/g).Used ligno-cellulose hydrolysate using is formed with embodiment 1.
Embodiment 4 usefulness high temperature resistant Issatchenkia orientalis IPE100 of the present invention carries out ligno-cellulose hydrolysate using high temperature ethanol fermentation
Adopt liquid seed culture medium,, obtain liquid seeds high temperature resistant Issatchenkia orientalis IPE100 shake-flask culture 16h under 42 ℃, 150rpm condition.Liquid seeds is inoculated into according to the inoculum size of 6% (v/v) contains (NH
4)
2SO
40.8g/L, MgSO
40.01g/L, K
2HPO
40.02g/L, ligno-cellulose hydrolysate using, in the fermention medium of pH 5.0,45 ℃, 120rpm cultivates 48h, and alcohol yied reaches maximum value, is 91.6% of theoretical value (0.51g/g).Used ligno-cellulose hydrolysate using is formed with embodiment 1.
Embodiment 5 usefulness high temperature resistant Issatchenkia orientalis IPE100 of the present invention carries out sweet sorghum high temperature ethanol solid state fermentation
Adopt liquid seed culture medium,, obtain liquid seeds high temperature resistant Issatchenkia orientalis IPE100 shake-flask culture 16h under 42 ℃, 150rpm condition.To contain (NH
4)
2SO
41g/L, MgSO
40.01g/L, K
2HPO
40.025g/L nutrient salt solution and sweet broomcorn straw dry powder according to the ratio mixing of 3: 1 (w/w), insert liquid seeds, mixing by the inoculum size of 3% (v/w), 42 ℃, static cultivation 36h, alcohol yied reaches maximum value, is 91.2% of theoretical value (0.51g/g).Used sweet broomcorn straw dry powder sugar degree 51.3% (w/w).
Embodiment 6 usefulness high temperature resistant Issatchenkia orientalis IPE100 of the present invention carries out sweet sorghum high temperature ethanol solid state fermentation
Adopt liquid seed culture medium,, obtain liquid seeds high temperature resistant Issatchenkia orientalis IPE100 shake-flask culture 16h under 42 ℃, 150rpm condition.To contain (NH
4)
2SO
40.8g/L, MgSO
40.01g/L, K
2HPO
40.01g/L nutrient salt solution and sweet broomcorn straw dry powder according to the ratio mixing of 4: 1 (w/w), insert liquid seeds, mixing by the inoculum size of 5% (v/w), 45 ℃, static cultivation 36h, alcohol yied reaches maximum value, is 90.3% of theoretical value (0.51g/g).Used sweet broomcorn straw dry powder sugar degree 51.3% (w/w).
Embodiment 7 usefulness high temperature resistant Issatchenkia orientalis IPE100 of the present invention carries out sweet sorghum high temperature ethanol solid state fermentation
Adopt liquid seed culture medium,, obtain liquid seeds high temperature resistant Issatchenkia orientalis IPE100 shake-flask culture 16h under 42 ℃, 150rpm condition.To contain (NH
4)
2SO
41.2g/L, MgSO
40.03g/L, K
2HPO
40.04g/L nutrient salt solution and sweet broomcorn straw dry powder according to the ratio mixing of 2: 1 (w/w), insert liquid seeds, mixing by the inoculum size of 2% (v/w), 42 ℃, static cultivation 36h, alcohol yied reaches maximum value, is 90.8% of theoretical value (0.51g/g).Used sweet broomcorn straw dry powder sugar degree 51.3% (w/w).
Claims (8)
1. the high temperature resistant Issatchenkia orientalis of a strain is characterized in that, its deposit number is CGMCC No.4927.
2. the described Issatchenkia orientalis of claim 1 is used to produce the alcoholic acid application.
3. a biological fermentation is produced the alcoholic acid method, it is characterized in that, uses the described Issatchenkia orientalis of claim 1 to ferment, and wherein, leavening temperature is 38～45 ℃.
4. method according to claim 3 is characterized in that, utilizes the solid-state thermophilic fermentation of ligno-cellulose hydrolysate using thermophilic fermentation or sweet sorghum to produce ethanol.
5. method according to claim 4 is characterized in that, utilizes the ligno-cellulose hydrolysate using thermophilic fermentation to produce ethanol, and wherein, fermention medium is formed: (NH
4)
2SO
40.8-1.2g/L, MgSO
40.01-0.03g/L, K
2HPO
40.02-0.04g/L all the other are ligno-cellulose hydrolysate using stoste, regulate pH to 5-7; Fermentation condition: 120-180rpm, inoculum size 3-6% (v/v).
6. method according to claim 5 is characterized in that, fermention medium is formed: (NH
4)
2SO
41g/L, MgSO
40.02g/L, K
2HPO
40.03g/L all the other are ligno-cellulose hydrolysate using stoste, regulate pH to 6; The ferment condition: 42 ℃, 150rpm, inoculum size 4% (v/v).
7. method according to claim 4, it is characterized in that, utilize the solid-state thermophilic fermentation of sweet sorghum to produce ethanol, wherein, fermention medium is formed: nutrient salt solution and sweet broomcorn straw dry powder are pressed 2-4: the mixed of 1 (w/w), wherein nutrient salt solution is formed: (NH
4)
2SO
40.8-1.2g/L, MgSO
40.01-0.03g/L, K
2HPO
40.01-0.04g/L; Fermentation condition: 38-45 ℃, inoculum size 2-5% (v/w), static cultivation.
8. method according to claim 7 is characterized in that, fermention medium is formed: nutrient salt solution and sweet broomcorn straw dry powder are by the mixed of 3: 1 (w/w), and wherein nutrient salt solution is formed: (NH
4)
2SO
41g/L, MgSO
40.02g/L, K
2HPO
40.025g/L; Fermentation condition: 42 ℃, inoculum size 3% (v/w).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586125A (en) * | 2012-02-21 | 2012-07-18 | 湖北白云边酒业股份有限公司 | Highly ester producing issatchenkia orientalis yeast and composition as well as application thereof |
| CN113122460A (en) * | 2021-04-25 | 2021-07-16 | 天津科技大学 | Rice straw degrading bacterium and screening method and application thereof |
| CN115772477A (en) * | 2022-07-25 | 2023-03-10 | 西北农林科技大学 | Issatchenkia orientalis strain and application thereof |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146590B (en) * | 2013-01-30 | 2014-10-08 | 中国科学院成都生物研究所 | Yeast strain and application thereof |
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| CN101165165A (en) * | 2007-10-10 | 2008-04-23 | 首都师范大学 | Novel use for issatchenkia orientalis |
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| CN101165165A (en) * | 2007-10-10 | 2008-04-23 | 首都师范大学 | Novel use for issatchenkia orientalis |
Non-Patent Citations (2)
| Title |
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| 《Biotechnology Letter》 20080415 Dong-Hwan Kim等 Co-fermentation of grape must by Issatchenkia orientalis and Saccharomyces cerevisiae reduces the malic acid content in wine 1633-1638 1-8 第30卷, 第9期 * |
| 《中国优秀硕士学位论文全文数据库》 20100215 宋瑶 耐高温酒精酵母菌株的筛选及其发酵特性研究 B024-27 1-8 , 第2期 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102586125A (en) * | 2012-02-21 | 2012-07-18 | 湖北白云边酒业股份有限公司 | Highly ester producing issatchenkia orientalis yeast and composition as well as application thereof |
| CN102586125B (en) * | 2012-02-21 | 2013-08-14 | 湖北白云边酒业股份有限公司 | Highly ester producing issatchenkia orientalis yeast and composition as well as application thereof |
| CN113122460A (en) * | 2021-04-25 | 2021-07-16 | 天津科技大学 | Rice straw degrading bacterium and screening method and application thereof |
| CN113122460B (en) * | 2021-04-25 | 2023-08-08 | 天津科技大学 | Rice straw degrading bacterium and screening method and application thereof |
| CN115772477A (en) * | 2022-07-25 | 2023-03-10 | 西北农林科技大学 | Issatchenkia orientalis strain and application thereof |
| CN115772477B (en) * | 2022-07-25 | 2024-05-03 | 西北农林科技大学 | Issatchenkia orientalis strain and application thereof |
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