Title of Invention

A METHOD FOR PRODUCING CELLULOSIC ETHANOL

Abstract

A method for producing cellulosic ethanol is disclosed, which comprises the following steps: 1) adding the medium containing the source materials of cellulose and/or hemicellulose to fermentation reaction vessel; 2) adding cellulase to fermentation reaction vessel, and inoculating Candida Lusitaniae; 3) running simultaneous saccharification fermentation (SSFs) with the combined action of cellulose and Candida lusitaniae, and obtaining cellulosic ethanol by separation. Candida lusitaniae used in SSFs in present invention has higher ethanol tolerance and a higher ethanol production rate, it can use cellulose and hemicellulose as source materials and effectively produce cellulosic ethanol with cellulase in SSFs.

Full Text

FORM 2 THE PATENTS ACT 1970 (39 of 1970) & The Patents Rules, 2003 COMPLETE SPECIFICATION (See section 10 and rule 13) 1. ' A method for producing cellulosic ethanol ' 2. 1. (A) ANGELYEAST CO., LTD. (B) China (C) 168 Chengdong Road, Yichang, Hubei 443003 China. The following specification particularly describes the invention and the manner in which it is to be performed. Technical field The invention involves the technical field of exploitation of bioenergy, specially involves a method for producing cellulosic ethanol. Background art As petroleum resources exhausting, the oil price rising and the environment deteriorating, to look for renewable resources to replace fossil resource and to exploit clean energy resource has been a focus in the industrial biotechnology field. The exploitation of biomass energy can difuse the dependence on petroleum resources,control carbon dioxide emissions, and promote the development of new industry chains in agriculture, which plays an important role in promoting new country construction in our country. Fuel ethanol,as a renewable clean resource, has been developing in America and Brazil from 70’s last century. The productivity of ethanol has been reached more than 5,000,000 tons per year in our country, wherein the annual yield of fuel ethanol remains about 1,100,000 tons, only second to Brazil, America, ranking the third in the world. A great deal of maize has been used as a resource to exploit bioenergy, which causes the maize price increase continually, so , after the upsurge of turning foodstuff into ethanol, the exploitation of cellulosic ethanol techniques has become a direction for large-scale commercialization of cellulosic ethanol. Lignocellulos material is a largest renewable resource on the earth, including forest, crop straw, the processing remains of agricultural by-products etc., which enters on the important place in the energy flows and material recycle flows of natural ecosystem. It is a potential resource to produce fuel ethanol, thus the possible answer of demand of energy in the future. Currently, it has prominent issue for the techniques in pilot-scale of cellulosic ethanol. Firstly, the commercialized industrial yeast on the market can ferment glucose and maltose, but not cellulosic disaccharide. The expensiveß- glucosidase, besides cellulase, needs to be added in the production process, in order to avoid the inhibition of cellulosic disaccharide. Cellulase is needed to hydrolyze cellulose into oligosaccharide. One of these oligosaccharides is cellulosic disaccharide, which includes 2 glucose molecules which can be further disassembled into glucose. However, cellulose does not have enough ß- glucosidase to turn cellulosic disaccharide disassembled from the hydrolysis of cellulose into glucose. Cellulosic disaccharide can inhibit endoglucanase and exoglucanase, and reduce the production rate and yield of the whole ethanol in Simultaneous Saccharification and Fermentation (SSFs). Furthermore, a great deal of the expensive ß- glucosidase need to be supplemented, which results in the remarkably high cost. On the other hand, the yeast to ferment cellulosic disaccharide has been found, but it has a very low ethanol tolerance. Secondly, SSFs can not guarantee that enzymolysis temperature harmonizes with the fermentation temperature. Generally, the enzymolysis temperature is 40-50?, while the fermentation temperature of Saccharomyces cerevisiae is 28-35?,thus it has to address the compromise in the process, resulting in excessive amounts of enzyme or yeast, generally with the time in SSFs more than 100 hours, lower fermentation efficiency. Thirdly, under the terms of high content of substrate, a large quantity of cellulose is still not converted, the effect of ezymolysis decreases remarkably, and the yield of ethanol is also too low, generally only around 3-5%(v/v). The preparation rate and the yield of ethanol fermentation in present can not economically meet the requirements of SSFs. A national patent (CN 101130792A) reported a method to prepare fuel ethanol by ezymolyzing straw, wherein, fermentation time lasted 10-15days, the efficiency of ezymolysis was remarkably low. Therefore, there is no feasibility of industrialization. Another national patent(CN101270372A)reported a method to concentrate sugar solution in fermentation liquid of cellulosic ethanol which aimed to resolve the problem of too low sugar concentration after enzymolysis. This shows the current enzymolysis technology can not meet the requirements of industrialization. Although this patent technology involving concentration can be utilized to change the enzymolyzed sugar concentration, the problem of the low enzymolysis rate of cellulose is not resolved yet, which can cause problems of low utilization rate of raw materials and high production cost. A patent CN 101230359 reported a method to concentrate cellulosic ethanol by a modified polydimethyl siloxane polymer membrane, which also wanted to resolve the problem of too low concentration of fermentation ethanol and high cost of distillation, but no way to avoid low efficiency of ezymolysis and fermentation. Contents of the invention The aim of the present invention is to provide a method to produce cellulosic ethanol, by increasing the efficiency of SSFs, thus increasing the production rate and the yield of cellulosic ethanol. To achieve the aim of the present invention, the technical solutions are supplied as follows: A method for producing cellulosic ethanol, which comprises the following steps: 1) adding the medium containing the source materials of cellulose and/or hemicelluloses to fermentation reaction vessel; 2) adding cellulase to fermentation reaction vessel, and inoculating Candida lusitaniae; 3) conducting simultaneous saccharification fermentation(SSFs) with the combined action of cellulose and Candida lusitaniae, and obtaining cellulosic ethanol by separation. Wherein, the origin of the source materials of cellulose and/or hemicelluloses in step 1) are renewable lignocellulose biomass. Such as wheat straw, rice straw and fast growing forest etc. are used as the source materials of cellulosic ethanol. This biomass contains 20-70% cellulose and 10-40% hemicelluloses of plants dry weight, and partial lignin. At the same time, it also includes all kinds of biomass which are rich in cellulose and discarded from the environments and living goods. Wherein, the said medium in step 1) contains 15%-30% cellulose and/or hemicelluloses source materials, still contains a little yeast extracts, peptone, antibiotics. The pH value is controlled to 3.5-6.0. The medium can be added Tween 80 and ergosterol etc according to the properties of liquids. Wherein, cellulase in step 2) contains endoglucanase , exoglucanase and a little ß- glucosidase. Wherein, Candida lusitaniae in step 2) prefers the yeast which is domesticated by ethanol and cellulosic disaccharide. The medium used for domestication contains cellulosic disaccharide and proper amounts of ethanol. 5%?10%?15% three concentration grades of cellulosic disaccharide are used for domestication, separated by underlines generation after generation, and then 1%,2%,3% ethanol grades are used for domestication, obtaining advantageous strains by separation. Wherein, pH value in SSFs is 3.5-6.0, the reaction temperature is 35-45?, preferred 40-45?. Candida lusitaniae used in SSFs in the present invention has higher ethanol tolerance and a higher ethanol production rate, which can use cellulose and hemicelluloses as source materials and effectively produce cellulosic ethanol with cellulase in SSFs. The tests show that Candida lusitaniae has a higher ethanol production rate and a higher yield by the comparison of the fermentation of other yeast using glucose and cellulosic disaccharide; in addition, compared with other yeasts, Candida lusitaniae has higher ethanol tolerance. Description of figures Fig.1 is the comparison of productivity of ethanol in SSFs between different kinds of yeast and Candida lusitaniae. Fig.2 is the comparison of ethanol tolerance of different kinds of yeast. Mode of Carrying out the Invention The present invention is detailed by the following modes of carrying out the invention. The fermentation source materials involved in the present invention contain cellulose and/or hemicelluloses. The origin is renewable lignocellulose biomass, such as wheat straw, rice straw and fast growing forest etc., which are all used as the source materials of cellulosic ethanol. This biomass contains 20-70% cellulose and 10-40% hemicelluloses by plants dry weight, and partial lignin. At the same time, it also includes all kinds of biomass which are rich in cellulose and discarded from the environments and living goods. cellulase used in the present invention contains endoglucanase , exoglucanase and a littleß- glucosidase. Cellulase is used to hydrolyze cellulose into oligosaccharides, such as glucose. One of these oligosaccharides is cellulosic disacchariede, including 2 molecules glucose, which can dissemble into glucose. ß- glucosidase can turn cellulosic disaccharide hydrolyzed from cellulose into glucose, but cellulase only contains a little ß- glucosidase, thus cellulase itself can not utilize cellulosic disaccharide sufficiently. Candida lusitaniae used in the present invention can utilize cellulose hydrolysis products, cellulosic disaccharide and glucose, also can utilize mannose and galactose hydrolyzed from hemicelluloses, and the above combined sugar and other related sugar also can utilize it to ferment. Wherein, said disaccharides can be sucrose, maltose, lactose and cellulosic disaccharide, but not including melibiose and fucose. The monosaccharide hydrolyzed from polysaccharide can also be utilized, for example starch and glucose hydrolyzed from cellulose and other monosaccharides, such as fructose, sorbose, mannose and galactose. The following illustrates concretely the steps and processes to effectively produce cellulosic ethanol in SSFs involved in the present invention. 1.The cultivation and domestication of Candida lusitaniae Candida lusitaniae separated from soil samples enriches in potato agar medium. This yeast strain is purebred in biology, which is appraised by Candida lusitaniae (CLAS 5566 ). One sample is conserved in the conservation centre of Alan G. Mac Diarmid Research Institute of Renewable Energy, Three Gorges University. The medium used for domestication contains cellulosic disaccharide and proper amounts of ethanol. 5%?10%?15% three concentration grades are used for domestication, separated by underlines generation after generation, and then 1%,2%,3% ethanol grades are used for domestication, obtaining advantageous strains by separation. 2.Fermentation conditions 1)Dissolved Oxygen Control of fermentation process The required scope in the fermentation process is wide. Dissolved oxygen can be either micro-oxygenation in interrupted fermentation, or a little air ventilated through the substrate inoculated in continuous fermentation. Furthermore, it can also be anaerobic fermentation. The required technique will depend on the initial cell density, substrate concentration and inoculation conditions. 2)The formula of fermentation medium The medium used for fermentation is normal medium, which contains proper nitrogen source, minerals, vitamin and carbon source. These carbon sources include hexoses (glucose, galactose and mannose) and disaccharides(cellulosic disaccharide). The medium used for fermentation and inoculation mainly includes 18% cellulose, 1% Angel yeast extract,2% peptone and 2mg/L antibiotics. pH value is 3.5-6.0. The medium can be added Tween 80 and ergosterol etc. according to the properties of liquids. 3)The temperature control of fermentation process The temperature of this fermentation process can range from 28?to 45?, however the optimum temperature is 40-45?. 3 Simultaneous saccharification fermentation(SSFs) In the process in SSFs, the saccharification involves the hydrolysis process of cellulose. Cellulase is utilized to hydrolyze cellulose into oligosaccharides. One of these oligosaccharides is cellulosic disaccharide, which includes 2 glucose molecules, and can be disassembled into glucose. The yeast strain used in the present invention is Candida lusitaniae, which can ferment cellulosic disaccharides very well at 42?, can be used in SSFs to provide faster production rate of wine and higher ethanol yield,superior to the known other yeasts used for the fermentation of cellulosic disaccharide. By comparing the effect of SSFs on the final wine production with that of interrupted saccharification fermentations(ISFs), the results in Table 1 show that the utilization rate of cellulose in SSFs has been increased by 50% when compared with that in ISFs, and yeast efficiency has been increased twice. Table 1 The effects of SSFs and ISFs on the final wine production Number SSFs process ISFs process saccharification rate 85.1% 58.5% residual reducing sugar (v/v) 0.15% 0.235% alcohol degree (v/v,20?) 10.2% 5.3% Figure 1 illustrates the data in SSFs of different yeast strains. CLAS5566 is compared with other yeast strains, Saccharomyces cerevisiae, and high temperature resistant active dry yeast after 6 days later, the concentration of ethanol utilizing yeast strain CLAS 5566 attained about 90g/L. it is clear to see that it is superior to other yeast strains in rates and yields by comparing with other yeast strains. Figure 2 shows that at 40?, CLAS 5566 obtained from 25% cellulose has a ethanol resistant concentration attaining to 100g/L. The ethanol tolerance is remarkably superior to that of Saccharomyces cerevisiae and high temperature resistant active dry yeast, a little lower than that of super wine yeast from Angel company. Scale preparation example 1 Adding the medium containing cellulose to a 6L fermentation reaction vessel filled with 2L water, the sample amount reaches 2.5L, to ensure the enough space while inoculating. The medium mixes in the fermenter containing lipids(30ml/L oleic acid), sterilizes for 30-40 mins at 120?. Antibiotics contains 500mg penicillin with the concentration of 10mg/L and 500mg(10mg/L)streptomcycin. pH value is 4.5-5.0. In succession, cellulase is added and CLAS 5566 special yeast for cellulosic ethanol is inoculated. Cell density is 2X107, the solution is diluted to 3L with water. Enzymes destroy cellulose into glucose and cellulosic disaccharide, and the yeast ferments into ethanol. The temperature is controlled at 38?, simultaneous saccharification ferments for 72 hours, to obtain 89g/L ethanol, and then ethanol is fractionated from fermentation substrates, to obtain cellulosic ethanol. Scale preparation example 2 Adding the medium containing cellulose to a 20L fermentation reaction vessel filled with 4L water, the sample amount reaches 5.5L, to ensure the enough space while inoculating. The medium mixes in the fermenter, containing lipids(ergosterol 5mg/L)sterilizes for 30-40 min at 120?. Antibiotics contains 500mg penicillin with the concentration of 10mg/L and 500mg(10mg/L)streptomcycin. pH value is 4.5-5.0. In succession, cellulase is added, and CLAS 5566 special yeast for cellulosic ethanol is inoculated. Cell density is 2X108, and the solution is dilutee to 5L with water. Enzymes destroy cellulose into glucose and cellulosic disaccharide, and the yeast ferments into ethanol. The temperature is controlled at 42? and simultaneous saccharification ferments for 96 hours, to obtain 98g/L ethanol, and then ethanol is fractionated from fermentation substrates, to obtain cellulosic ethanol. The method for producing cellulosic ethanol provided by the present invention is introduced in detail above. It needs to point out that the content described in the modes of carrying out the invention are preferred modes for better implementation of the invention. The protection scope is not limited to the technical solutions described in the above modes, while the essential content in claims should be the standard, any possible modification in the process belongs to the protection scope of the invention as long as it deviates from the essential content of the claims in the invention. We Claim 1. A method for producing cellulosic ethanol, comprises the following steps: 1) adding the medium containing the source materials of cellulose and/or hemicelluloses to fermentation reaction vessel; 2) adding cellulase to fermentation reaction vessel, and inoculating Candida lusitaniae; 3) conducting simultaneous saccharification fermentation(SSFs) with the combined action of cellulose and Candida lusitaniae, and obtaining cellulosic ethanol by separation. 2. The method according to claim 1, wherein, the origin of the source materials of cellulose and/or hemicelluloses in step 1) is renewable lignocellulose biomass. 3. The method according to claim 1, wherein, said medium in step 1) contains 15%-30% cellulose and/or hemicelluloses source materials. 4. The method according to claim 3, wherein, said medium in step 1) still contains a little yeast extracts, peptone and antibiotics. 5. The method according to claim 1, wherein, the pH value of said medium in step 1) is controlled within the scope of 3.5-6.0. 6. The method according to claim 1, wherein, cellulase in step 2) contains endoglucanase , exoglucanase and a little ß- glucosidase. 7. The method according to claim 1, wherein, Candida lusitaniae in step 2) is the yeast which is domesticated by ethanol and cellulosic disaccharide. 8. The method according to claim 7, wherein, 5%?10%?15% three concentration grades of cellulosic disaccharide are used for domestication, separated by underlines generation after generation, and then 1%,2%,3% ethanol grades are used for domestication, obtaining advantageous strains by separation. 9. The method according to claim 1, wherein, the reaction temperature of simultaneous saccharification fermentation(SSFs) in step 3) is 35-45?. 10. The method according to claim 9, wherein, the reaction temperature of SSFs in step 3) is 40-45?. Dated this 6th Day of January, 2011.

Documents:

31-MUMNP-2011-CORRESPONDENCE(10-1-2011).pdf

31-MUMNP-2011-CORRESPONDENCE(16-4-2013).pdf

31-MUMNP-2011-CORRESPONDENCE(28-2-2013).pdf

31-MUMNP-2011-CORRESPONDENCE(28-4-2011).pdf

31-MUMNP-2011-DECLARATION(28-4-2011).pdf

31-MUMNP-2011-ENGLISH TRANSLATION(16-4-2013).pdf

31-MUMNP-2011-FORM 1(28-4-2011).pdf

31-MUMNP-2011-FORM 18(10-1-2011).pdf

31-MUMNP-2011-FORM 26(28-4-2011).pdf

31-MUMNP-2011-FORM 3(18-7-2013).pdf

31-MUMNP-2011-FORM 3(28-2-2013).pdf

31-MUMNP-2011-FORM 3(28-4-2011).pdf

31-MUMNP-2011-FORM 5(28-4-2011).pdf

31-MUMNP-2011-OTHER DOCUMENT(18-7-2013).pdf

31-MUMNP-2011-OTHER DOCUMENT(28-2-2013).pdf

31-MUMNP-2011-REPLY TO EXAMINATION REPORT(18-7-2013).pdf

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Claims.pdf

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FER Reply.pdf

Form-1.doc

Form-3.doc

Form-5.doc

Priority Document.pdf