KR101630832B1 - Novel strain for efficient simultaneous saccharification and fermentation at high temperature and Bio-ethanol production method using the novel strain - Google Patents

Abstract

The present invention relates to a novel high temperature tolerant strain having excellent simultaneous saccharification fermentation efficiency and a method for producing bioethanol using the same. The present invention relates to the use of Saccharomyces cerevisiae S. cerevisiae . The isolated strain has excellent growth rate and ethanol production rate at a high temperature of 40 to 45 ° C. Therefore, it can be applied to the production of bioethanol using the simultaneous saccharification and fermentation method at high temperature have.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel high-temperature resistant strain having excellent simultaneous saccharification fermentation efficiency and a method for producing bioethanol using the same,

The present invention relates to a novel high temperature tolerant strain having excellent simultaneous saccharification fermentation efficiency and a method for producing bioethanol using the same.

The production of bioethanol from lignocellulosic biomass has recently attracted attention particularly from the environmental and economic standpoints (Non-Patent Document 1). Lignocellulosic biomass is composed mainly of 40-50% cellulose, 25-30% hemicellulose and 15-20% lignin (Non-Patent Document 2). Cellulose is a linear polymer of glucose, and hemicellulose is a branched heteropolymer such as glucose, xylose, arabinose and galactose. Lignin is a hydrophobic complex of cross-linked aromatic polymers. Since the cellulose polymer is firmly embedded in the complex of hemicellulose and lignin, pretreatment must be carried out in order to obtain the cellulose polymer from the complex complex (Non-Patent Documents 3 and 4). Therefore, the result of the pretreatment is determined by effective elimination by physical, chemical, and thermal hydrolysis of lignin and hemicellulose (Non-Patent Documents 5 and 6). After the pretreatment, cellulose and hemicellulose are converted into fermentable sugars through enzymatic hydrolysis (Non-Patent Documents 7 and 8). Finally, microbial fermentation using a fermentable sugar is performed for ethanol production. This process is called separate hydrolysis and fermentation (SHF) (Non-Patent Document 9).

In contrast to the isolated saccharification fermentation method, the simultaneous saccharification and fermentation (SSF) is called microbial fermentation and enzyme hydrolysis together (Non-Patent Document 10). The simultaneous saccharification fermentation method has advantages in cost reduction (at least 20%) since the fermentation time is short and the equipment to be used is reduced as compared with the separation saccharification fermentation method (Non-Patent Documents 11 and 12). However, the main difficulties in simultaneous saccharification fermentation are that the enzyme reaction is not performed at the optimum enzyme temperature condition (Non-Patent Documents 13 and 14). For this reason, the simultaneous saccharification fermentation method requires a long reaction time, and the yield of sugar required for fermentation is low, and when the simultaneous saccharification fermentation method is applied, the yield of ethanol is low unless a large amount of enzyme is added (Non-Patent Document 15). Therefore, the development of thermostable or high-temperature resistant microorganisms has been continuously required to overcome the limitations of the conventional simultaneous saccharification fermentation technology (Non-Patent Document 16). Although the optimum temperature for the enzymatic hydrolysis is 50 占 폚, the range of 40 to 50 占 폚 does not greatly affect the sugar yield (Non-Patent Document 11). In particular, several potential advantages of high temperature fermentation have been reported, such as minimization of microbial contamination (non-patent document 17), the need for additional cooling devices (non-patent document 18), and convenience of conventional ethanol extraction (non-patent document 17). Nonklang et al. (Non-patent document 19) predicted that the cost of producing ethanol would be greatly reduced if the fermentation temperature increased by 5 ° C ($ 30,000 annually in a 30,000 kL ethanol plant).

Simultaneous regulation of multiple genes (non-patent documents 23 and 24), evolutionary engineering (non-patent document 25), and genome manipulation (including non-patent documents 20 to 22) and global transcription machinery engineering (gTME) Several methods for stress-resistant yeast strains suitable for bioethanol production including genome shuffling (non-patent document 26) have been proposed. In particular, three studies (non-patent references 27-29) have reported that reactive oxygen species (ROS) levels are reduced in yeast strains with ethanol tolerance, inhibitor resistance and high temperature tolerance for bioethanol production. A major target for environmental stresses such as oxidative stress and thermal shock is mitochondria (Non-Patent Document 30). In particular, induction of active oxygen by mitochondrial damage destroys the balance of redox reactions and kills yeast cells (Non-Patent Document 30). Therefore, when improving yeast strains through mitochondrial mutagenesis, it is necessary to consider reducing the levels of active oxygen associated with resistance to most environmental stresses.

Accordingly, the present inventors have continued the present invention and developed a novel strain having excellent simultaneous saccharification fermentation efficiency at high temperature, and completed the present invention.

Limayene, Ricke SC. (2012) Lignocellulosic biomass for bioethanol production: current perspectives, potential issues and future prospects. Progress in Energy and Combustion Science. 38, 449-467. Halherbe S, Cloete TE. (2002) Lignocellulose biodegradation: fundamentals and applications. Review in Environmental Science and BioTechnology 1, 105-114. Gong CS, Cao NJ, Du J, Tsao GT. (1999) Ethanol production from renewable resources. Advances in Biochemical Engineering / Biotechnology 65, 207-241. Sanchez OJ, Cardona CA. (2008) Trends in biotechnological production of fuel ethanol from different feedstocks. Bioresource Technology 99, 1270-5295. Agbor VB, Cicek N, Sparling R, Berlin A, Levin DB. (2011) Biomass pretreatment: fundamentals toward application. Biotechnology Advances 29, 675-685. Kumar P, Barrett DM, Delwiche MJ, Stroeve P. (2009) Methods for pretreatment of lignocellulosic biomass for efficient hydrolysis and biofuels production. Industrial and Engineering Chemical Research 48, 3713-3729. Chandra RP, Bura R, Mabee WE, Berlin A, Pan X, Saddler JN. (2007) Substrate pretreatment: the key to effective enzymatic hydrolysis of lignocellulosics? Advances in Biochemical Engineering / Biotechnology 108, 67-93. Mosier N, Wyman C, Dale B, Elander R, Lee Y Y, Holtzapple M, Ladisch M. (2005). Bioresource Technology 96, 673-686. Saha BC, Nichols NN, Qureshi N, Cotta MA. (2011) Comparison of separate hydrolysis and fermentation and simultaneous saccharification and fermentation processes for ethanol production from wheat straw by recombinant Escherichia coli strain FBR5. Applied Microbiology and Biotechnology 92, 865-874. Olofsson K, Bertilsson M, Liden G. (2008) A short review on SSF an interesting process option for ethanol production from lignocellulosic feedstocks. Biotechnology for Biofuels 1, 7 Kishna SH, Reddy TJ, Chowdary GV. (2001) Simultaneous saccharification and fermentation of lignocellulosic wastes to ethanol using a thermotolerant yeast. Bioresource Technology 77, 193-196. Nguyen QA, Saddler JN. (1991) An integrated model for the technical and economic evaluation of an enzymatic biomass conversion process. Bioresource Technology 35, 275-282. Bhallae, Bansal N, Kumar S, Bischoff KM, Sani RK. (2013) Improved lignocellulosic conversion to biofuels with thermophilic bacteria and thermostable enzymes. Bioresource Technology 128, 751-759. Sun Y, Cheng J. (2002) Hydrolysis of lignocellulosic materials for ethanol production: a review. Bioresource Technology 83, 1-11. Suyawati L, Wilkins MR, Bellmer DD, Huhnke RK, Maness NO, Banat IM. (2008) Simultaneous saccharification and fermentation of kanlow switchglass pretreated by hydrothermolysis using Kluyveromyces marxianus IMB4. Biotechnology and Bioengineering 101, 894-902. Castro RCA, Roberto IC. (2013) Selection of thermotolerant Kluyveromyces marxianus strain with potential application for cellulosic ethanol production by simultaneous saccharification and fermentation. Applied Biochemistry and Biotechnology (In press) Abdel-Banat BMA, Hoshida H, Ano A, Nonklang S, and Akada R. (2010) High-temperature fermentation: how can processes for ethanol production? Applied Microbiology and Biotechnology 85, 861-867. Kumar S, Dheeran P, Singh SP, Mishra IM, Adhikari DK. (2013) Cooling system economy in ethanol production using thermotolerant yeast Kluyveromyces sp. IIPE453. American Journal of Microbiological Research 1, 39-44. Nonklang S, Abdel-Banat BMA, Cha-aim K, Moonjai N, Hoshida H, Limtong S, Yamada M, . Applied Microbiology and Biotechnology 74, 7514-7521. Fujita K, Matsuyama A, Kobayashi Y, Iwahashi H. (2006) The genome-wide screening of yeast deletion mutants to identify the genes required for tolerance to ethanol and other alchohols. FEMS Yeast Research 6, 744-750. Gorsich SE, Dien BS, Nichols NN, Slinginger PJ, Lui ZL, Skory CD. (2006) Tolerance to furfural-induced stress associated with pentose phosphate pathway genes ZWF1, GND1, RPE1 and TLK1 in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology 71, 339-349. Kim HS, Kim NR, Yang J, Choi W. (2011) Identification of novel genes responsible for ethanol and / or thermotolerance by transposon mutagenesis in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology 91, 1159-72. Alper H, Moxley J, Nevoigt E, Fink GR, Stephanopoulos G. (2006) Engineering yeast transcription machinery for improved ethanol tolerance and production. Science 314, 1565-1568. (2011) Construction of Saccharomyces cerevisiae strains with enhanced ethanol tolerance by mutagenesis of TATA-binding protein gene and identification. Kim, WY; Kim, of novel genes associated with ethanol tolerance. Biotechnology and Bioengineering. 108, 1776-1787. Cakar ZP, Seker UO, Tamerler C, Sonderegger M, Sauer U. (2005) Evolutionary engineering of multiple-stress resistant Saccharomyces cerevisiae. FEMS Yeast research 5, 569-578. Shi DJ, Wang CL, Wang KM. (2009) Genome shuffling to improve thermotolerance, ethanol tolerance, ethanol tolerance and ethanol productivity of Saccharomyces cerevisiae. Journal of Industrial Microbiology and Biotechnology. 36,139-147. Kim, HS, Kim, W, Choi, W. (2012) Insertion of transposon in the vicinity of SSK2 confers enhanced tolerance to furfural in Saccharomyces cerevisiae. Applied Microbiology and Biotechnology 95, 531-540. Mutation of the TATA-binding protein confer enhanced tolerance to hyperosmotic stress in Saccharomyces cerevisiae. Kim, JH, Yang J, Kwon H, An J, Choi W, Kim W. Applied Microbiology and Biotechnology 97, 8227-8238. Yang KM, Lee NR, Woo JM, Choi W, Zimmerman M, Blank LM, Park JB. (2012) Ethanol reduced mitochondrial membrane integrity and thereby impacts carbon metabolism of Saccharomyces cerevisiae. FEMS Yeast Research 12, 675-684. Perrone GG, Tan S, Dawes IW. (2008) Reactive oxygen species and yeast apoptosis. Biochimica et Biophysica Acta 1783, 1354-1368.

It is an object of the present invention to provide a process for the production of ethanolic saccharomyces cerevisiae cerevisiae ) variant strains.

Another object of the present invention is to provide a method for producing ethanol using Saccharomyces cerevisiae mutant strain.

The invention saccharide as MY unrestricted access serenity (Saccharomyces cerevisiae) ability saccharide as MY unrestricted access serenity (Saccharomyces that the ethanol produced by causing a defect mutation in the cerevisiae mutant strains.

The mutant strain was deposited with the accession number KACC93194P, and the growth temperature or ethanol fermentation temperature of the mutant strains may be 40 to 45 캜.

The present invention also relates to the aforementioned Saccharomyces cerevisiae cerevisiae mutant strain to biomass and saccharification and fermentation.

The biomass may be a fibrous biomass including a large anthracite after an alkali pretreatment.

The fibrous biomass may be selected from herbaceous plants including herbaceous plants, giant aphids, caterpillars, reeds, rice straw and litters.

The saccharification and fermentation may be performed simultaneously at 40 ° C to 45 ° C for 24 to 72 hours.

The amount of glucan in the biomass may be 3 to 20 wt%.

The ethanol production process may be selected from the group consisting of a batch process, a semi-continuous process or a continuous process.

The strain Saccharomyces cerevisiae according to the present invention exhibits an excellent growth rate and an ethanol production rate at a high temperature of 40 to 45 DEG C and can be applied to the production of bioethanol using a simultaneous saccharification and fermentation method at a high temperature have.

Figure 1 is a graph showing the effect of saccharomyces cerevisiae ( Saccharomyces < RTI ID = 0.0 > cerevisiae ) mutation.
Figure 2 is a saccharide at the 30 ℃ and 42 ℃ My process serenity non-zero (Saccharomyces cerevisiae) and KCTC 7928 saccharose as MY unrestricted access serenity (Saccharomyces cerevisiae ) mbc 1 to 4 and culture (b) in a glycerol-based agar medium.
3 is 30 ℃ (a) and 42 ℃ (b) in the complex media in my process to a non-zero celebrity Saccharomyces (S. cerevisiae) KCTC 7928 and the saccharide in my process serenity non-zero (in Saccharomyces cerevisiae ) mbc 1 to 4. (Drawing Description - Saccharomyces cerevisiae) The error bars represent the standard deviations of two independent experiments: KCTC 7928 (), mbc 1 (x), mbc 2 (), mbc 3 (), mbc 4
FIG. 4 is a graph showing changes in composition (a), conversion of fermentable sugars (b), and SEM photograph (x100) (c) (before and after pretreatment (gray) and after pretreatment Indicating the standard deviation of the experiment).
FIG. 5 is a graph showing the activity of Saccharomyces < RTI ID = 0.0 > S. cerevisiae ) KCTC 7928 (a) and Saccharomyces cerevisiae cerevisiae) mbc 2 (b) shows the bio-ethanol production results through the Simultaneous Saccharification and Fermentation from pretreated thatched it is also due to (drawing Description ethanol (●), glucose (▲), xylene as agarose (■), arabinose The error bars represent the standard deviation of two independent experiments).
Figure 6 shows that the pretreated solids are fed at high concentrations and are incubated at 42 < 0 > C for 48 hours with Saccharomyces cerevisiae (final ethanol concentration (bar) and ethanol yield (●), error bars represent the standard deviations of two independent experiments). Fig. 2 shows the results of the ethanol production of the simultaneous saccharification fermentation using S. cerevisiae mbc 2.
Figure 7 is a graph showing the effect of saccharomyces cerevisiae cerevisiae ) KCTC 7928 and Saccharomyces cerevisiae cerevisiae ) mbc 2 at 30 < 0 > C and 42 < 0 > C after 6 hours. Active oxygen in the cells was confirmed by fluorescence microscopy as shown in FIG. 7 (a), and was expressed by relative DCF fluorescence relative to the number of cells (b). At 30 < 0 > C, saccharomyces cerevisiae cerevisiae) were not significantly different from KCTC 7928 and mbc2, in stress conditions of 42 ℃ radicals of mbc2 was confirmed that relatively low. The bar in the graph represents the mean value of two independent experiments.

Hereinafter, the present invention will be described in detail.

The present invention is a capability that is manufactured by causing a respiratory deficient mutation in the non-restricting my process celebrity in Saccharomyces (Saccharomyces cerevisiae) Ethanol production Saccharomyces My process serenity non-zero (Saccharomyces cerevisiae mutant strains.

The mutant strain was deposited with the National Center for Agricultural Genetic Resources under accession number KACC93194P.

Saccharomyces cerevisiae is a representative yeast belonging to the carrot fungus. Most of the yeasts used for brewing, including baker's yeast and brewery yeast, are closely related to this species.

The term "respiratory deficit mutation" refers to a mutant of yeast that has a relatively small or almost no respiratory capacity and is smaller than a normal colony, and is classified as a cytosolic respiratory deficit mutation and a nuclear respiratory deficit mutation. The cytosolic respiratory deficit mutation is caused by the deficiency of mitochondrial DNA, which is classified as a mode in which the mitochondrial DNA is completely lost or a large deletion of mitochondrial DNA and the subsequent residue are overlapped. The cytosolic respiratory deficit mutation occurs naturally at a rate of about 1 in 500 cells in culture and can be artificially induced by proliferation in medium containing acriflavine or ethidium bromide.

In one embodiment of the present invention, induction of respiratory deficit mutation was induced with ethidium bromide. However, mutation induction is not limited by the above-mentioned method, and mutation induction methods widely used in this technical field can be used.

The growth temperature or the ethanol fermentation temperature of the mutant strain is 40 to 42 캜.

In addition, the present invention provides a method for producing ethanol, comprising saccharifying and fermenting Saccharomyces cerevisiae mutant strains by treating biomass.

The method for producing ethanol in the biomass of the present invention is an alkaline pretreatment process for producing ethanol in a fibrous biomass and participating in the saccharification and fermentation step to remarkably improve the production yield of ethanol.

The fibrous biomass may be selected from herbaceous crops including but not limited to herbaceous plants, giant aquifers, caterpillars, reeds, rice straw and litters, but is not limited to and includes all fibrous biomass.

The fiber-based biomass may include Miscanthus composed of cellulose, hemicellulose and lignin, and the content of glucan in the fibrous biomass is preferably 20 wt% or less, more preferably 3 to 20 wt% desirable.

The saccharification and fermentation step is performed simultaneously at 40 ° C to 45 ° C for 12 to 72 hours.

The ethanol production process is selected from the group consisting of a batch process, a semi-continuous process, or a continuous process. Such methods are well known in the art.

If necessary, those skilled in the art can perform other additional steps and / or processes. For example, the fermentation liquid obtained by the fermentation process may be purified according to a method known in the art, .

In one embodiment of the present invention, we have shown that respiratory deficit mutations induced by 0.5 μg / ml of ethidium bromide (EtBr) lead to high temperature resistant Saccharomyces cerevisiae cerevisiae mbc strains 1 to 4 were selected. This strain has an increased growth rate and fermentability in YPD medium (1% yeast extract, 2% peptone 2% glucose, 1.5% agar) at 42 ° C compared to the host S. cerevisiae KCTC 7928 Respectively. Saka access to my serenity Wiese (Saccharomyces cerevisiae mbc 2 (KACC93194P) was found to be most suitable for bioethanol production at 42 ° C.

Through a batch high temperature / high pressure reactor, Miscanthus was pretreated in a 1.5M sodium hydroxide vessel at 150 占 폚 for a residence time of 30 minutes, resulting in 42.6% pretreated solids containing 77.9% cellulose. Then, a saccharide through a simultaneous saccharification fermentation process using a solid component containing a 3% glucan at 42 ℃ My process serenity non-zero (Saccharomyces cerevisiae mbc 2 and the results of the control. The ethanol concentration and yields from Saccharomyces cerevisiae mbc 2 were 15.3 ± 0.223 g / L and 0.46 ± 0.007 g / g (90.1%) for 48 hours, respectively, and the control was 8.3 ± 0.90 g / L and 0.25 ± 0.006 g / g (49.0%), respectively. In addition, 6% by weight of Miscanthu pretreated after incubation at 42 [deg.] C for 48 hours, and Saccharomyces cerevisiae at 9% The final ethanol concentration by cerevisiae mbc 2 was 29.5 ± 1.991 g / L and 41.9 ± 0.462 g / L. The ethanol yields were 0.44 ± 0.029 g / g (86.3%) and 0.42 ± 0.005 g / g (82.4%), respectively.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

< Example  1> High Temperature Resistance Sakaromayses Serebiza ( Saccharomyces cerevisiae ) mbc 1 to 4 strain selection

<1-1> Sakaromayses Serebiza ( Saccharomyces cerevisiae ) KCTC  7928 Culture

Saka access to my serenity Wiese (Saccharomyces cerevisiae), KCTC 7928 was cultured in YPD medium (1% yeast extract, 2% peptone, 2% glucose, 1.5% agar) of 30 ℃ was used as a host strain for respiratory deficient mutation induction. Host strains and mutants were cultured in glycerol-based agar (GBA) at 30 ° C to identify respiratory defects. The glycerol-based agar medium contained 2% glycerol, 2% yeast extract, 1.5% agar.

<1-2> Random breathing deficiency mutation induction and high temperature resistant mutation selection

Ethidium bromide (EtBr) was used to induce a randomized respiratory deficit mutation. As shown in Fig. 1, saccharomyces cerevisiae S. cerevisiae KCTC 7928 was transferred to fresh YPD medium and the initial cell density was adjusted to 0.2 OD ₆₀₀ . When the cell density reached 1.0 OD ₆₀₀ , 0.5 μg / ml of ethidium bromide was added to the medium. Then, 100 μl of the inoculum was put into a YPD culture dish and cultured at 42 ° C. (FIG. 1).

High-temperature tolerance mutations were initially screened at 42 ° C along a large size colony scale, and high-temperature tolerance mutants were selected using spot assays. Briefly, 4-μl aliquots of mutants cultured to 1.0 OD ₆₀₀ and control cells were serially diluted 10-fold and plated onto solid YPD culture dishes. Cultures were incubated at 30 ° C and 42 ° C for 2 to 3 days.

Thus, a total of 81 mutants were isolated, and finally, four large colonies grown at 42 ° C were separated into high temperature resistant mutants. The selected Saccharomyces cerevisiae To determine if the S. cerevisiae mutants were resistant to high temperatures, spot assays were performed. As a result, all of the four strains showed a growth rate superior to the control strain at 42 ° C (FIG. 2, (a)). Because respiratory deficit mutations can not grow in glycerol-based media, the growth of mutants to determine if the selected mutation is a respiratory deficit could be assessed through glycerol, and all mutants were found to have respiratory defects and high temperature tolerance (Figure 2, (b).

The major concern of the inventors was whether the improvement of ethanol yield of the high temperature resistant mutants and the applicability to the simultaneous saccharification fermentation method using the pretreated wheat germ were possible. Therefore, the four isolated strains were selected by the growth ability and the glucose efficacy in the 42 ° C YPD medium .

As expected, the overall growth rate and ethanol production of the mutants at 42 캜 were clearly higher than in the control (Fig. 3, (b)). The mutants produced an average of 9.7 g / L ethanol 2.2 times higher than the ethanol concentration in the control. The maximum ethanol concentration was 10.2 g / L, and saccharomyces cerevisiae cerevisiae mbc 2 strain (Fig. 3, (b)). Thus, my process to the saccharide of the mutant non-limiting serenity (Saccharomyces cerevisiae mbc2 was selected as the target strain.

< Example  2> Miscanthus ) Alkali Pretreatment and Composition Analysis

Pretreatment was carried out using an 800-ml high temperature / high pressure reactor equipped with temperature and pressure sensors. Miscanthus was mixed with 1.5 M NaOH (1: 9, 500 ml working volume) and then added to 800-ml equipment and reacted at 150 ° C for 30 minutes at 120 rpm. Before the pretreated Miscanthus was collected in the separator through the cyclone system, nitrogen gas was introduced into the reactor to a pressure of 6 MPa. Solids were obtained using a Buchner funnel equipped with a 10-μm nylon filter and neutralized with tap water. Structural carbohydrate composition analysis of pretreated Miscanthus was performed using high performance liquid chromatography (HPLC, Waters, USA). The sample was loaded onto a HPX-87H column (Bio-Rad, Hercules, CA, USA) set at 65 ° C. The sugar was extracted from 5 mM sulfuric acid at a constant flow rate of 0.5 ml / min. The peaks were identified by the refractory index (RI) identified as retention time and quantified according to the calibration curve for each sugar. After that, it was appropriately determined according to the sugar concentration of pretreated Miscanthus solids according to the concentration of each sugar and used for simultaneous saccharification and fermentation (SSF).

The raw sample, which consists of 40.5% cellulose, 23.8% hemicellulose and 24.1% lignin (Fig. 4 (a)).

Analysis of the composition of the alkali pretreated soleus resulted in 42.6% of pretreated solids containing 66.6% of water. Cellulosic content of pretreated soymilk rapidly increased from 40.5% to 77.9%. By contrast, hemicelluloses were seen to be slightly degraded and removed from the supernovae. Lignin content was significantly decreased from 24.1% to 8.0% due to pretreatment. It was judged that the degree of delignification in the pretreated lettuce was determined by the alkaline pretreatment and the enzymatic hydrolysis thereof (FIG. 4, (a)). In addition, the apparent increase in cellulose content suggests that pretreatment was effective, since the high temperature resistant mutant mbc2 could only consume the same six sugar groups as glucose.

< Example  3> Preprocessed  The Miscanthus ) &Lt; / RTI &gt;

The results of the pretreatment are determined by enzymatic hydrolysis, in which the fermentable carbohydrate monomer is released from cellulose and hemicellulose in the pretreated biomass. Therefore, enzyme pretreatment using 3% glucose solids (g / L) was carried out at 50 ° C for 48 hours and the pretreatment effect was measured using a 20FPU enzyme cocktail. As a result, a glucose yield of 92.4% was obtained from the pretreated herringbone, and the glucose concentration was 30.7 g / L, whereas the 3.9 g / L glucose was converted from the untreated control group (FIG. 4, . Thus, the preprocessed crude hydrolyzate is saccharomyces cerevisiae ( Saccharomyces &lt; RTI ID = 0.0 &gt; cerevisiae mbc2. &lt; / RTI &gt;

< Example  4> Preprocessed  The Miscanthus ) Scanning electron microscopy

Scanning electron microscope (SEM) images were analyzed (TM-100, Hitachi, Tokyo, Japan) to examine the structural changes of pretreated Miscanthus . The sample was mounted on an aluminum stub and imaged under vacuum at an accelerating voltage of 15 kV.

In FIG. 4 (c), the scanning electron microscope image shows the untreated or treated untreated sample. The dense and intact structure was visible in the raw sample, and the particles were broken and the pores were exposed. In contrast, there were twisted and disjointed fibers and bundles due to the destruction of the cell wall complex in pretreated itch. Therefore, it was concluded that the accessibility of glycosylation enzyme was increased with the increase of external surface area in pretreated itch.

< Example  5> Preprocessed  The Miscanthus ) To produce bioethanol

<5-1> Sakaromayses Serebiza ( Saccharomyces cerevisiae ) KCTC  7928 and saccharomyces Serebiza ( Saccharomyces cerevisiae ) mbc  Comparison of ethanol concentration produced by 2

Prior to experimenting with the realization of the simultaneous saccharification fermentation method, fermentation was carried out in an artificial medium at 42 ° C. The healthy yeast cells that reached the exponential growth period were harvested and transferred to 200 ml YPD medium supplemented with 5% glucose. The initial cell density was adjusted to 0.6 OD ₆₀₀ . The cells were shaken at 30 ° C and 42 ° C at 150 rpm. Samples were collected at intervals of 12 hours and then the cell density was measured at 600 nm and analyzed by gas chromatography (GC (Agilent 6980N, Wilmington, DE, USA) using a carrier helium gas at a flow rate of 15 ml / min and HP-INNOWaX 19091N- ) Was used to determine cell growth rate and ethanol concentration.

Simultaneous saccharification fermentation was performed by adding 10 ml of 1 M citric acid buffer (pH 4.8), 20 FPU / g cellulase (Novozymes, Cellic Ctec II), and 10 ml of pre-treated Miscanthus containing 3-9% 10 ml of YP (20% peptone, 10% yeast extract 1 L) was added and sterile water was added to make the fermentation mixture 90 ml. At the same time, the yeast cells were harvested by centrifugation at 15,000 rpm for 10 minutes at 4 占 폚. The harvested cells were washed with sterile water and cell pellets were obtained from the centrifugation. The concentrated cells were resuspended in 10 ml of sterile water. Cells were added to the 90 ml mixture resulting in a working volume of 100 ml. Finally, simultaneous saccharification fermentation was carried out by stirring at 150 rpm for 72 hours under the condition of 42 캜. Then, the ethanol and sugar concentration of the sample were analyzed periodically using gas chromatography and high performance liquid chromatography.

As a result, the glucose conversion was higher in Saccharomyces cerevisiae mbc 2 and in the control than in glucose fermentation until 12 hours (Fig. 5b). Glucose concentration was determined for 48 hours using saccharomyces cerevisiae cerevisi ae) mbc 2, but increased continuously up to 48 hours in the control (Fig. 5A). At this time, the residual glucose of the control was 14.52 ± 0.406 g / L, and it was judged that the control lost the fermentability after 12 hours due to temperature - sensitivity. In Saccharomyces cerevisiae mbc 2, ethanol production persisted for more than 36 hours, and saccharification and fermentation almost stopped after 48 hours (FIG. 5b). Accordingly, the glucose concentration was almost always close to 0 (0.73 ± 0.320 g / L). Saka access to my serenity Wiese (Saccharomyces The concentration and yield of ethanol produced by S. cerevisiae mbc 2 were 15.3 ± 0.223 g / L and 0.46 ± 0.007 g / g, respectively. In contrast, the maximum ethanol concentration and yield of the control were 8.3 ± 0.190 g / L and 0.25 ± 0.006 g / g, respectively. Thus, my process as Saccharomyces celebrity non-zero (Saccharomyces cerevisiae mbc 2 can be mass-produced from ethanol at high temperatures.

<5-2> High concentration of substrate Sakaromayses Serevise ( Saccharomyces cerevisiae ) of mbc 2 At the pitching rate   The same time Glycation  Fermentation

For practical simultaneous saccharification fermentation, the substrate and enzyme concentrations consistent with the amount of the inoculum should be determined in view of the critical conditions, but in this example Saccharomyces cerevisiae According to the amount of mbc 2 inoculum, a simultaneous saccharification fermentation was conducted in which a large amount of solids was added at 42 ° C. 20 FPU cellulase was used and Saccharomyces cerevisiae The initial pitching rate of S. cerevisiae mbc 2 was adjusted on the basis of cell volume (g / L): 1.0-4.0 g / L and a solid containing 6% by weight and 9% by weight glucan was used. Based on 66 wt% water and 78 wt% cellulose, the total wet weight of the 9 wt% glucan containing solids was about 36 g. The solids containing 9 wt% glucan were almost full in one flask, so that the enzymes and yeast cells could not mix properly. Thus, solids containing 9 wt% glucan were considered to be the appropriate concentration for determining the likelihood of high temperature simultaneous saccharification fermentation with high solids loading in the present invention.

As shown in Fig. 6, simultaneous saccharification fermentation was carried out by Saccharomyces cerevisiae mbc 2 at 42 ° C for 48 hours. From the solids containing 6 wt.% And 9 wt.% Glucan at 4.11 g / L cell weight, a maximum ethanol concentration of 29.5. + -. 1.991 g / L and 41.9. 0.462 g / L respectively was obtained. The ethanol yields were 0.44 ± 0.029 g / g (86.1%) and 0.42 ± 0.005 g / g (82.1%), respectively. However, fermentation with a cell weight of 3.08 g / L or less exhibited a low ethanol concentration (7.3 to 36.9 g / L) and a relatively low ethanol yield (0.1 to 0.38 g / g). The result is a saccharide at 42 ℃ My process serenity non-zero (Saccharomyces suggesting that the fermentation capacity of S. cerevisiae mbc 2 is limited by the massive addition of solids unless the cell weight exceeds 4.0 g / L. The ethanol productivity (g / L / h) was 0.87 g / L / h in 9% initial glucan for 48 hours.

Accordingly, high temperature resistant Saccharomyces cerevisiae cerevisiae mbc 2 can be considered as a strain for the production of bioethanol from pretreated wheat germ at high temperature.

< Example  6> Intracellular  Measurement of residual free oxygen

FIG radicals in the cell based on the results of 1 is the most that would My process serenity of non-zero indices in Saccharomyces growth phase is determined to be distinct differences (Saccharomyces cerevisiae) and KCTC 7928 Saccharomyces after 6 hours My process serenity non-zero (Saccharomyces cerevisiae mbc 2 were cultured at 30 ° C and 42 ° C, respectively, and the concentrations of active oxygen were compared after 6 hours. The cell numbers of the first two yeasts were the same at a cell density of 0.4 OD ₆₀₀ . 5,6-Chloromethyl-2'7'-dichlorodihydrofluorescein diacetate (CM-H ₂ DCFDA) was used as a reagent for measuring reactive oxygen species. The excitation of green fluorescence was 493 nanometers (nm) in ultraviolet wavelength and 521 nm in wavelength of emission. Two yeasts were incubated for 6 hours at 30 ° C and 42 ° C for 6 hrs. After harvesting, the yeast cells were harvested by centrifugation and then suspended in 100 μl of PBS (phosphate buffered saline). A final concentration of 10 μM of CM-H ₂ DCFDA was added to the cell suspension , And incubated at 30 ° C for 30 minutes. Then, active oxygen was measured. As a result, the relative fluorescence value (active oxygen amount) did not differ by 36% in the initial culture (0 hour), and it was increased after 6 hours culture. The increased amount of active oxygen was measured at 30 ° C., which was not stressed, by using Saccharomyces cerevisiae cerevisiae ) KCTC 7928 and Saccharomyces cerevisiae cerevisiae ) mbc 2, respectively, the relative amounts of active oxygen were 63% and 60%, respectively. However, the two yeasts cultured at 42 &lt; 0 &gt; C were 100% and 71%, respectively. Saccharomyces cerevisiae cerevisiae ) mbc 2 showed about 30% lower accumulation of active oxygen. This demonstrates that the high temperature tolerance of mbc is an accumulation of low active oxygen by the mutation of mitochondria by EtBr.

National Institute of Agricultural Science KACC93194P 20140214

Claims (14)

In my process to the non-zero in serenity (Saccharomyces cerevisiae) (Saccharomyces cerevisiae) the deposit number in the saccharide entrusted to KACC93194P My process serenity non-limiting capability in the ethanol produced by causing the defect mutation in the mutant strain Saccharomyces,
Wherein said mutant strains have an appropriate temperature for ethanol fermentation of 40 to 45 DEG C for Saccharomyces cerevisiae mutant strain. delete 2. The method according to claim 1, wherein the growth temperature of the mutant strains is 40-45 DEG C, saccharomyces cerevisiae S. cerevisiae mutant strains. delete A process for producing ethanol comprising treating saccharomyces cerevisiae mutant strain according to claim 1 with biomass and saccharifying and fermenting at 40 to 45 ° C for 12 to 72 hours. 6. The method of claim 5, wherein the biomass is fibrous biomass. 7. The method according to claim 6, wherein the fibrous biomass is selected from herbaceous plants including herbaceous plants, giant aphids, caterpillars, reed, rice straw and litter. 8. The method according to claim 7, wherein the giant aqueducts are pretreated. The method according to claim 8, wherein the pretreatment is an alkaline pretreatment. 6. The method according to claim 5, wherein the saccharification and fermentation are performed simultaneously. delete delete 6. The method of claim 5, wherein the biomass has a glucan content of 3 to 20% by weight. 6. The method according to claim 5, wherein the ethanol production process is carried out by a process selected from the group consisting of a batch process, a semi-continuous process or a continuous process.

Images