WO2010061987A1

WO2010061987A1 - Method and medium for the production of bioethanol using genus typha l.

Info

Publication number: WO2010061987A1
Application number: PCT/KR2008/006986
Authority: WO
Inventors: Tae Kwon Kim; Sung Eun Lee; Byeoung Soo Park; Wan Jung Kim; Mi Kyung Son
Original assignee: Nanotox Tech
Priority date: 2008-11-27
Filing date: 2008-11-27
Publication date: 2010-06-03

Abstract

The present invention relates to a method and a medium for the production of bioethanol using a perennial herbaceous plant, Genus Typha L. More particularly, the method for the production of bioethanol comprises the steps of a) hydrolyzing Genus Typha L., and b) cultivating an ethanol -producing strain using said hydrolyzed Genus Typha L. solution as a carbon source. Due to the use of Genus Typha L. which is characterized by rigorous development of an aerial part and a rhizome, rapid growth rate and low content of lignin, the method and medium for the production of bioethanol according to the present invention exhibits excellent effects, including a significant increase in the amount of bioethanol produced, decreased cost of production and easy security of resources necessary for production, compared with conventional methods which utilized crops-or wood-based biomass.

Description

METHOD AND MEDIUM FOR THE PRODUCTION OF BIOETHANOL

USING GENUS TYPHA L.

TECHNICAL FIELD

The present invention relates to a method and medium for the production of bioethanol using a perennial herbaceous plant, Genus Typha L. More specifically, the method for the production of bioethanol comprises the steps of a) hydrolyzing Genus Typha L. and b) culturing an ethanol-producing strain using said hydrolyzed Genus Typha L. solution as a carbon source, and the medium for the production of bioethanol contains the hydrolyzed Genus Typha L. solution as a carbon source.

BACKGROUND ART

Recently, as crude oil prices have risen sharply and the concern about energy exhaustion has become ever more serious, attention to alternative energy production has been amplified. Among sources of alternative energy, bioethanol is the most actively raised material. Bioethanol, alone or in mixture with gasoline, can be used as an automobile fuel, and thus it is a representative recycled energy, in addition to biodiesel. Carbon dioxide, which is generated from the combustion of bioethanol, is one of the exceptions to the greenhouse gas calculation provided by the Kyoto Protocols because of its greenhouse gas-reducing effects. In addition, unlike other clean fuels that require the construction of special infrastructure (charging/fueling stations) for their supply, bioethanol can be supplied through existing facilities (gas/filling stations), which enables early commercialization.

Research and development of bioethanol has progressed mainly in the United States and Brazil since the 1970s. Brazil has developed bioethanol using sugar cane as a national project and succeeded in substituting about 30% of the total amount of vehicle fuel consumed with bioethanol in 2004. In the case of the United States, in the 2008 State of the Union Message, the President proclaimed that petroleum consumption should be reduced by 20% until 2017 and that the use of alternative energy such as bioethanol should instead be expanded. Japan, China and other Asian countries are also promoting a policy to increase bioethanol production. As explained above, as the demand and interest in bioethanol increase worldwide, the amount of bioethanol produced is also trending upward.

Research and production of bioethanol was initiated as a part of an effort to develop alternative energy after the 1970s oil crisis, and progress has mainly centered around crop biomass based on corn and sugar cane, and wood biomass based on wood resources. In fact, Brazil has succeeded in fuel commercialization of bioethanol on the basis of its abundant sugar cane plantations. The United States has also accumulated considerable commercialization techniques for the production of bioethanol utilizing abundant corn and wood resources. However, ethical issues have recently been raised regarding crop biomass because it uses valuable food resources as a fuel. Due to a sharp rise of grain price in early 2008, it was analyzed that the production of bioethanol using crop biomass has a disadvantage in terms of supply and demand of raw material, and price competitiveness. In addition, wood biomass has an advantage in that resources are abundant and the amount of ethanol produced is high because the content of cellulose and hemicellulose is about 75% or more. However, because wood biomass has a much sturdier physical and chemical structure than crop biomass, a chemical or enzymatic approach for saccharifϊcation of cellulose and hemicellulose is difficult. Furthermore, wood biomass has a disadvantage because it contains about 15-25% of lignin, which consists of numerous hydrophobic aromatic compounds, and the pre-treatment process for removing such lignin is complicated and expensive. Forest destruction to ensure a biomass supply would also accelerate global warming and environmental destruction, and it would be hard to justify bioethanol as an environment- friendly fuel in light of environmental and ethical issues.

Thus, there is an urgent need to secure a new type of biomass that can replace conventional crop and wood biomass, and to develop techniques for producing bioethanol using the same.

At present, the development of a second-generation bioethanol production technique utilizing herbaceous plants (or non-woody plants) including weeds and reeds has been expedited, primarily in the United States and Spain. Herbaceous plants, which can be used as biomass, include rice straw and bagasse. Herbaceous plants are characterized by relatively high content of cellulose and hemicellulose, and low content of lignin, and thus their pre-treatment is easier than that of conventional wood biomass. In addition, these plants' growth rate is fast, and thus their supply is easy. They also have the advantage of being free of the ethical issues that plague wood biomass. Thus, there is a need to develop a second-generation bioethanol production technique.

Genus Typha L. is a perennial herbaceous plant which belongs to the Typhaceae family, and about 9 to 18 species thereof are distributed worldwide. It has been known that three types of Genus Typha L., i.e., Typha orientalis, Typha angustata and Typha latifolia, grow naturally in Korea (Choi, 2000; Chung, 1957; Im, 1998; Kim and Choi, 2001 ; Lee, 1996; Mori, 1922; Nakai, 1911, 1951). Genus Typha L. is characterized by rigorous development of an aerial part and a rhizome (a root) that results in a great amount of biomass. Genus Typha L. contains a large amount of carbohydrate in its leaf, stem and root, which can be converted into ethanol. Genus Typha L. can fairly compete with conventional crop or wood biomass because its growth rate is much faster compared with wood, and it grows naturally in the swampy land of the temperate regions so that it is easy to cultivate and collect. It has been reported that rice straw and reeds can produce biomass in an amount of 8.5-11.2 ton/ha and 6.4-12.5 ton/ha by dry weight, respectively, whereas Genus Typha L. can produce biomass in an amount of 10.6-14.7 ton/ha and, in case a fertilizer is used, 14.5-22.7 ton/ha (Ja-Hyeong Gu, et al, ARPC Report, 2007, p. 95). Thus, Genus Typha L. merits attention as a future bioenergy crop. Thus, it is understood that developing a method for the production of bioethanol using Genus Typha L., a representative herbaceous plant, would have an important meaning in securing a second-generation bioethanol.

SUMMARY OF THE INVENTION

The present inventors have performed continuous study on new biomasses that can resolve problems caused by conventional methods for the production of bioethanol using crop or wood biomass, such as rapid increase of crop price, weaponization of crop, problems associated with crop ethics, impairment of forest resources and global warming. As a result, they discovered that if an ethanol-producing strain is cultured using as a carbon source a Genus Typha L. solution that has been obtained by hydrolyzing Genus Typha L. with an acid and/or a saccharification enzyme, it exhibits excellent effects, including a significant increase in the amount of bioethanol produced, decreased cost of production and easy security of resources necessary for production, compared with conventional methods that utilized crop or wood biomass, and completed the present invention.

Thus, the first object of the present invention is to provide a method for the production of bioethanol, comprising the steps of a) hydrolyzing Genus Typha L. and b) culturing an ethanol-producing strain using said hydrolyzed Genus Typha L. solution as a carbon source.

The second object of the present invention is to provide a method for the production of bioethanol, comprising the steps of a) hydrolyzing Genus Typha L. with an acid or a saccharification enzyme and b) culturing an ethanol-producing strain using said hydrolyzed Genus Typha L. solution as a carbon source.

The third object of the present invention is to provide a method for the production of bioethanol, comprising the steps of a) first hydrolyzing Genus Typha L. with an acid and second hydrolyzing it with a saccharification enzyme, and b) culturing an ethanol-producing strain using said hydrolyzed Genus Typha L. solution as a carbon source.

The fourth object of the present invention is to provide a medium for the production of bioethanol, containing the hydrolyzed Genus Typha L. solution as a carbon source.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 is a graph that shows the hydrolysis rate when sulfuric acid or hydrochloric acid is used in the method for the production of bioethanol of the present invention.

Fig. 2 is a graph that shows the hydrolysis rate change depending on the concentration of sulfuric acid in the method for the production of bioethanol of the present invention. Fig. 3 is a graph that shows the hydrolysis rate change depending on the amount of

Genus Typha L. added in the method for the production of bioethanol of the present invention.

Fig. 4 is a graph that shows the hydrolysis rate change depending on the amount of a saccharification enzyme, Viscozyme, added in the method for the production of bioethanol of the present invention.

Fig. 5 is a graph that shows the hydrolysis rate change depending on the reaction time of a saccharification enzyme in the method for the production of bioethanol of the present invention.

Fig. 6 shows the results of HPLC confirming the existence of bioethanol that has been distilled according to the method for the production of bioethanol of the present invention. DISCLOSURE OF THE INVENTION

The first aspect of the present invention relates to a method for the production of bioethanol, comprising the steps of a) hydrolyzing Genus Typha L. and b) culturing an ethanol-producing strain using said hydrolyzed Genus Typha L. solution as a carbon source.

The second aspect of the present invention relates to a method for the production of bioethanol, comprising the steps of a) hydrolyzing Genus Typha L. with an acid or a saccharification enzyme and b) culturing an ethanol-producing strain using said hydrolyzed Genus Typha L. solution as a carbon source.

The third aspect of the present invention relates to a method for the production of bioethanol, comprising the steps of a) first hydrolyzing Genus Typha L. with an acid and second hydrolyzing it with a saccharification enzyme, and b) culturing an ethanol-producing strain using said hydrolyzed Genus Typha L. solution as a carbon source.

The fourth aspect of the present invention relates to a medium for the production of bioethanol, containing the hydrolyzed Genus Typha L. solution as a carbon source.

In the method and medium for the production of bioethanol according to the present invention, there is no limit on the kind of Genus Typha L. to be used. Preferably, one or more selected from the group consisting of Typha orientalis, Typha angustata and

Typha latifolia can be used. Any one part selected from leaf, stem and root, or all of them can be used.

Each of leaf, stem and root of Genus Typha L. contains a considerable amount of carbohydrate, which can be converted into ethanol. The composition of leaf, stem and root is shown in Table 1 below.

Table 1

General composition of Genus Typha L. (% d.b)

The first step of the method for the production of bioethanol according to the present invention is hydrolyzing Genus Typha L. At this step, Genus Typha L. is saccharified into a reducing sugar (a fermenting sugar), which is easy for an ethanol-producing strain to use. Because Genus Typha L. contains a relatively small amount of lignin compared with wood biomass, the hydrolysis process itself according to the present invention is sufficient for lignin treatment, and there is no need to additionally use various chemicals that were conventionally required for lignin treatment.

In the method for the production of bioethanol according to the present invention, the hydrolysis of Genus Typha L. can be carried out by using either an acid or a saccharification enzyme, or both.

There is no limit on the kind of acid that can be used for the hydrolysis of the present invention. Preferably, sulfuric acid or hydrochloric acid can be used. There are two major methods for hydrolyzing biomass with an acid: (i) using an acid of weak concentration at high temperature and (ii) using an acid of high concentration at low temperature. The method using an acid of weak concentration at high temperature is used more often because it is easy to neutralize and is safe, due to less addition of the acid. Thus, it is more preferable to adopt the method using an acid of weak concentration at high temperature for the hydrolysis of the present invention.

In order to determine the kind of acid to be used for the hydrolysis of the present invention, 100 mL of 10% (w/w) hydrochloric acid or 100 mL of 6% (w/w) sulfuric acid was added to each sample of crushed Genus Typha L. After treatment at 120 ^°C for 30 min, the concentration of a reducing sugar within the solution was measured to determine the hydrolysis rate. The hydrolysis rate was about 8% when hydrochloric acid was added, whereas it was 20% when sulfuric acid was added (see Fig. 1). It was understood that because sulfuric acid is stronger than hydrochloric acid, it could give a relatively high hydrolysis rate even if a small amount were added. On the other hand, hydrochloric acid, which is weaker than sulfuric acid, is dangerous to use at high temperature due to its high volatility, and thus it is difficult to add 10% or more; thereby, its hydrolysis rate was observed to be low. Therefore, it was concluded that sulfuric acid is more preferable for hydrolysis at high temperature.

In addition, the present invention has established a preferable condition for hydrolyzing Genus Typha L. with an acid. Specifically, in order to determine the concentration of an acid for the hydrolysis of the present invention, the concentration of sulfuric acid was changed from 1% to 6%, and the concentration of a reducing sugar within the solution was measured to determine the hydrolysis rate. As the concentration of sulfuric acid increased, the hydrolysis rate also increased. When the concentration of sulfuric acid was 6%, the hydrolysis rate was about 20% (see Fig. 2). Thus, the concentration of sulfuric acid used for hydrolyzing Genus Typha L. with an acid according to the present invention is preferably 5% to 6%, and more preferably 6%. In addition, the concentration of hydrochloric acid, which is another acid that can be used for the hydrolysis of the present invention, is preferably 10% to 20%.

Furthermore, in order to determine the ratio of Genus Typha L. and an acid for the hydrolysis of the present invention, 100 mL of 6% (w/w) sulfuric acid was added to each 1 g, 1.5 g, 2 g, 2.5 g, 3 g, 3.5 g and 4 g of a sample of crushed Genus Typha L. After reaction at the fixed temperature of 120 ^°C for 1 hour, the concentration of a reducing sugar within the solution was measured to determine the hydrolysis rate. The hydrolysis rate increased to reach about 25% when the amount of Genus Typha L. is around 1.5 g. Thereafter, a similar level was maintained even though the amount of Genus Typha L. increased further (see Fig. 3). Thus, in the present invention, especially when 6% sulfuric acid is used for the hydrolysis at 120 ^°C , the ratio of Genus Typha L. and the acid is preferably 1 : 60-70 (w/v). However, it should be clearly understood that this ratio of Genus Typha L. and the acid is applicable only when 6% sulfuric acid is used at high temperature of 120 ^°C , and the ratio of Genus Typha L. and an acid can vary when the hydrolysis is carried out at low temperature, sulfuric acid of different concentration is used, or an acid other than sulfuric acid is used.

The hydrolysis of Genus Typha L. of the present invention can also be carried by a saccharification enzyme.

As used herein, the term "saccharification" refers to a process of hydrolyzing cellulose by enzymes to produce a sugar and is a different concept from glycosylation, which adds a sugar to a protein. There is no limit on the kind of saccharification enzyme that can be used for the hydrolysis of the present invention. Preferably, one or more selected from the group consisting of carbohydrase, cellulase, amylase and glucosidase can be used. As shown in the following working examples, when used for Genus Typha L., all of the above-mentioned four kinds of saccharification enzymes exhibited a hydrolysis rate up to about 30%. Among them, in particular, a carbohydrase-containing saccharification enzyme showed the highest hydrolysis rate. However, the kind of a saccharification enzyme is not limited thereto.

As a saccharification enzyme to be used for the present invention, commercially available Celluclast 1.5L is preferable as a cellulase, Viscozyme 2L as a carbohydrase, and Fungamyl 800L as an amylase and a glucosidase.

In addition, the present invention has established a preferable condition for hydrolyzing Genus Typha L. with a saccharification enzyme.

Specifically, in order to determine the amount of a saccharification enzyme to be added for the hydrolysis of the present invention, Viscozyme 2L, a carbohydrase-containing saccharification enzyme, was added to a sample of crushed Genus Typha L. The initial amount was 0.1 niL, which was increased by 0.1 mL. After reaction for 24 hours, the concentration of a reducing sugar was measured to determine the hydrolysis rate of Genus Typha L. It was observed that as the amount of added saccharification enzyme increased, the hydrolysis rate of Genus Typha L. increased. When the amount of saccharification enzyme added was 0.5 mL, the hydrolysis rate was about 35%. When more of the saccharification enzyme was added, the hydrolysis rate decreased rather slightly but was maintained at around 35% (see Fig. 4). Thus, the amount of a saccharification enzyme to be used for hydrolysis of a saccharification enzyme according to the present invention is preferably 0.4 to 0.6 mL, and more preferably 0.5 mL per 1 g of Genus Typha L.

Furthermore, the present inventors studied how the reaction time required for hydrolysis influences a hydrolysis rate in the case of hydrolyzing Genus Typha L. with a saccharification enzyme. 0.5 mL of Viscozyme 2L, which is a carbohydrase-containing saccharification enzyme, was added to a sample of crushed Genus Typha L. Every hour, the concentration of a reducing sugar was measured to determine the hydrolysis rate. As time passed, the hydrolysis rate increased and reached about 33% about 18 hours after the reaction was initiated. After 18 hours, the hydrolysis rate increased somewhat slowly (see Fig. 5). Thus, it is preferable to carry out hydrolyzing with a saccharification enzyme of the present invention for 18 to 24 hours.

As explained above, in the present invention, the first hydrolysis with a weak acid or a saccharification enzyme is sufficient for hydrolyzing Genus Typha L. Alternatively, the hydrolysis of Genus Typha L. of the present invention can be carried out by two-step processes that consist of the first hydrolysis by a weak acid followed by the second hydrolysis by a saccharification enzyme.

In the method for the production of bioethanol according to the present invention, the second step is culturing an ethanol-producing strain using the above hydrolyzed Genus Typha L. solution as a carbon source.

There is no limit on the kind of ethanol-producing strain that can be used for the method for the production of bioethanol according to the present invention. Preferably, one or more strains selected from the group consisting of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis can be used. More preferably, the ethanol-producing strain is Saccharomyces cerevisiae.

In addition, the medium for the production of bioethanol of the present invention is one that contains the hydrolyzed Genus Typha L. solution which has been obtained according to the above-described methods. The medium is preferably a complex medium consisting of natural ingredients, such as yeast extract, bean peptone, tripton and the like. Specific examples thereof include YP medium, YM medium and LB medium, which are conventionally used in the art. However, the medium of the present invention is not limited to only a complex medium, and a semi-defined medium can also be used.

The medium for the production of bioethanol of the present invention essentially contains the hydrolyzed Genus Typha L. solution as a carbon source and may additionally contain conventionally used carbon sources.

Due to the use of Genus Typha L. as a carbon source, which is characterized by rigorous development of an aerial part and a rhizome, rapid growth rate and low content of lignin, the method and medium for the production of bioethanol according to the present invention exhibits excellent effects, including a significant increase in the amount of bioethanol produced, decreased cost of production and easy security of resources necessary for production, compared with conventional methods that utilized crop or wood biomass.

In order to confirm the above effects, each of the two hydrolyzed Genus Typha L. solutions, which had been obtained by hydrolysis with an acid or by hydrolysis with a saccharification enzyme, was concentrated and added to a YP medium (yeast extract 1.5%, peptone 1.5%), which was then used as the medium for the production of bioethanol of the present invention. After pre-culturing Saccharomyces cerevisiae as an ethanol-producing strain in a YM medium, it was inoculated into the above medium for the production of bioethanol and shaking-cultured for 80 hours. The concentration of bioethanol was measured using HPLC. The yield (mL) of converted ethanol per 1 g of a reducing sugar added was converted into percentage (%) to give an ethanol conversion rate. It was understood that the ethanol conversion rate for the acid-hydro lyzed Genus Typha L. solution was 30.3% and that for the saccharification enzyme-hydrolyzed Genus Typha L. solution reached 34.5%.

Hereinafter, the present invention will be described in more detail with reference to the following working examples. The working examples are provided only to help understanding of the invention but are not to be construed as limiting the scope of the invention.

EXAMPLES Example 1 : Determination of an acid for the hydrolysis of Genus Typha L.

Genus Typha L. was crushed without sorting leaf, stem or root thereof, and soaked in distilled water at 80 ^°C for 1 hour to give a sample. To each 1 g of the sample was added 100 mL of 10% (w/w) hydrochloric acid or 100 mL of 6% (w/w) sulfuric acid.

After treatment at 120 ^°C for 30 min, the solution was filtered, and the concentration of a reducing sugar within the solution was measured to determine the hydrolysis rate. The results are shown in Fig. 1. As shown in Fig. 1, the hydrolysis rate was about 8% when hydrochloric acid was added, whereas it was 20% when sulfuric acid was added. It was understood that because sulfuric acid is stronger than hydrochloric acid, it could give a relatively high hydrolysis rate even if a small amount were added. On the other hand, hydrochloric acid, which is weaker than sulfuric acid, is dangerous to use at high temperature due to its high volatility, and thus it is difficult to add 10% or more; thereby, its hydrolysis rate was observed to be low. Therefore, it was concluded that sulfuric acid is more preferable for hydrolysis at high temperature.

Example 2: Determination of a concentration of sulfuric acid for the hydrolysis of Genus Typha L.

Genus Typha L. was crushed without sorting leaf, stem or root thereof, and soaked in distilled water at 80 ^°C for 1 hour to give a sample. To each 1 g of the sample was added 100 mL of sulfuric acid whose concentration varied from 1 to 6% (w/w). After treatment at 120 ^°C for 30 min, the solution was filtered, and the concentration of a reducing sugar within the solution was measured to determine the hydrolysis rate. The results are shown in Fig. 2. As shown in Fig. 2, as the concentration of sulfuric acid increased, the hydrolysis rate also increased. When the concentration of sulfuric acid was 6%, the hydrolysis rate was maintained at around 20%. Thus, it was concluded that a proper concentration of sulfuric acid in the hydrolysis step is 5% to 6%. Example 3: Determination of the ratio of Genus Typha L. and an acid for the hydrolysis of Genus Typha L.

Further studies were carried out regarding how the amount of Genus Typha L. added influences a hydrolysis rate in the case of hydrolyzing Genus Typha L. with sulfuric acid. Genus Typha L. was crushed without sorting leaf, stem or root thereof, and soaked in distilled water at 80 ^°C for 1 hour to give a sample. To each 1 g, 1.5 g, 2 g, 2.5 g, 3 g,

3.5 g and 4 g of the sample was added 100 mL of 6% (w/w) sulfuric acid. After reaction at the fixed temperature of 120 "C for 1 hour, the concentration of a reducing sugar within the solution was measured to determine the hydrolysis rate. The results are shown in Fig. 3. As shown in Fig. 3, the hydrolysis rate increased to about 25% when the amount of Genus Typha L. was around 1.5 g. Thereafter, a similar level was maintained even though the amount of Genus Typha L. increased further. It was assumed that the hydrolysis rate did not increase because the hydrolysis reaction to which sulfuric acid may apply is limited when the amount of Genus Typha L. is 2 g or more. Thus, it was concluded that especially when 6% sulfuric acid is used for hydrolysis, the ratio of Genus Typha L. and the acid is 1 : 60-70 (w/v).

Example 4: Determination of a saccharification enzyme for the hydrolysis of Genus Typha L. Further studies were carried out regarding how the addition of a saccharification enzyme, which is able to saccharify a fiber, influences the hydrolysis rate. Genus Typha L. was crushed without sorting leaf, stem or root thereof, and soaked in distilled water at 80 ^°C for 1 hour to give a sample. To each 1 g of the sample was added 0.5 mL of Celluclast 1.5 L, which is a cellulase, Viscozyme 2L, which contains various carbohydrases, or Fungamyl 800L, which contains an amylase and an amyloglucosidase. After reaction for 24 hours, the concentration of a reducing sugar within the solution was measured to determine the hydrolysis rate. The results are shown in Table 2 below. As shown in Table 2, all the saccharification enzymes exhibited a hydrolysis rate up to about 30%, and Viscozyme 2L was the highest at 35%. Thus, it was concluded that Viscozyme 2L is the most preferable saccharification enzyme for optimal saccharification reaction of the pre-treatment solution for the hydrolysis of Genus Typha L.

Table 2

Hydrolysis rate of Genus Typha L. depending on the kind of saccharification enzyme

Example 5: Determination of an amount of a saccharification enzyme added for the hydrolysis of Genus Typha L.

Further studies were carried out regarding how the amount of a saccharification enzyme added influences the hydrolysis rate in the case of hydrolyzing Genus Typha L. with the saccharification enzyme. Under the same condition as Example 4, 0.1 mL of Viscozyme 2L was added and the amount was increased by 0.1 mL. After reaction for 24 hours, the concentration of a reducing sugar was measured to determine the hydrolysis rate. The results are shown in Fig. 4. As shown in Fig. 4, as the amount of the saccharification enzyme added increased, the hydrolysis rate of Genus Typha L. increased. When the amount of the saccharification enzyme added was 0.5 mL, the hydrolysis rate was about 35%. When more of the saccharification enzyme was added, the hydrolysis rate decreased rather slightly but was maintained at around 35%. It was assumed that this happened because the concentration of the enzyme-reactive substrate is fixed even with further addition of the enzyme, and thus additional hydrolysis reaction did not proceed. Thus, it was concluded that the amount of a saccharification enzyme to be used for the hydrolysis of Genus Typha L. is about 0.5 mL per 1 g of Genus Typha L.

Example 6: Determination of a reaction time of a saccharification enzyme for the hydrolysis of Genus Typha L. Further studies were carried out regarding how the reaction time required for hydrolysis influences the hydrolysis rate in the case of hydrolyzing Genus Typha L. with a saccharification enzyme. Under the same condition as Example 5, 0.5 mL of Viscozyme 2L was added and allowed for reaction. Every hour, the concentration of a reducing sugar was measured to determine the hydrolysis rate. The results are shown in Fig. 5. As shown in Fig. 5, as time passed, the hydrolysis rate increased and reached about 33% about 18 hours after the reaction was initiated. After 18 hours, the hydrolysis rate increased somewhat slowly. It was assumed that this happened because the concentration of the enzyme-reactive substrate is fixed and the reversible reaction reached an equilibrium state, and thus the reaction did not proceed further. Thus, it was concluded that in the case of hydrolyzing Genus Typha L. with a saccharification enzyme, the preferable reaction time of the saccharification enzyme is 18 to 24 hours.

Example 7: Production of bioetlianol using the hydrolyzed Genus Typha L. solution as a carbon source

Each of the two hydrolyzed Genus Typha L. solutions (treatment solutions), which had been obtained by hydrolysis with an acid or with a saccharification enzyme, was concentrated and added to a YP medium (yeast extract 1.5%, peptone 1.5%) so that the final concentration of a reducing sugar was 2% (w/w). The medium was then used for the production of bioethanol. After pre-culturing Saccharomyces cerevisiae as an ethanol-producing strain in a YM medium (yeast extract 0.3%, malt extract 0.3%, peptone 0.5%, dextrose 1%) for 18 hours, it was inoculated into 200 mL of the above medium for the production of bioethanol by 5% (v/w) of the volume of the cultured solution. After shaking-culture at 30 ^°C , 150 rpm for 80 hours, the concentration of bioethanol was measured using HPLC. The results are shown in Table 3 below, and the HPLC chromatogram is shown in Fig. 6. The yield (mL) of converted ethanol per 1 g of a reducing sugar added was converted into percentage (%) to give an ethanol conversion rate. As shown in Table 3, the ethanol conversion rate for the acid-hydrolyzed Genus Typha L. solution was 30.3%, and that for the saccharification enzyme-hydrolyzed Genus Typha L. solution reached 34.5%. Table 3 Ethanol conversion rate depending on the hydrolysis method

INDUSTRIAL APPLICABILITY

Genus Typha L. is characterized by rigorous development of an aerial part and a rhizome, which results in a great amount of biomass, and is characterized by a relatively low content of lignin compared with wood biomass. Due to the use of Genus Typha L., the method and medium for the production of bioethanol according to the present invention exhibits a significant increase of bioethanol produced and a decreased cost of production because the survival of ethanol-producing yeasts and microorganisms becomes easy due to the decreased use of various chemicals which were required for lignin treatment. In addition, Genus Typha L. provides easy security of resources because its growth rate is much faster compared with wood biomass and it grows naturally in swampy land in the temperate regions so that it is easy to cultivate and collect. Thus, the method and medium for the production of bioethanol according to the present invention would enable large-scale production of bioethanol in a more economical way, compared with conventional methods which utilized crop or wood biomass.

Claims

WHAT IS CLAIMED IS

1. A method for the production of bioethanol, comprising the steps of a) hydrolyzing Genus Typha L. and b) culturing an ethanol-producing strain using said hydrolyzed Genus Typha L. solution as a carbon source.

2. The method of claim 1, wherein the hydrolysis is carried out by an acid or a saccharification enzyme.

3. The method of claim 1, wherein the hydrolysis consists of the first hydrolysis by an acid followed by the second hydrolysis by a saccharification enzyme.

4. The method of claim 1, wherein the hydrolysis is carried out by sulfuric acid or hydrochloric acid.

5. The method of claim 1, wherein the hydrolysis is carried out by one or more saccharification enzymes selected from the group consisting of carbohydrase, cellulase, amylase and glucosidase.

6. The method of claim 1, wherein the ethanol-producing strain is one or more selected from the group consisting of Saccharomyces cerevisiae, Pichia stipitis and Zymomonas mobilis.

7. The method of claim 1, wherein the ethanol-producing strain is Saccharomyces cerevisiae.

8. A medium for the production of bioethanol, containing as a carbon source the hydrolyzed Genus Typha L. solution according to any one of claims 1 to 7.