Consolidated Bioprocessing Ethanol Production by Using a Mushroom

Plant cell walls are the most abundant biomass source in nature and are of increasing importance because worldwide attention has now focused on bioethanol production to combat global warming and to safeguard global energy. Because of competition between food and fuel production, lignocelluloses are expected to be utilized for future fuel ethanol production. One of the major problems in producing ethanol from lignocellulosic biomass is the expensive production cost. Consolidated bioprocessing (CBP) is gaining recognition as a potential breakthrough for low-cost biomass processing (Lynd, 1996; Lynd et al., 2002; Lynd et al., 2005; Van Zyl et al., 2007; Xu et al. 2009). CBP of lignocellulose to bioethanol refers to the combination of the 4 biological events required for this conversion process (production of lignocellulose-degrading enzymes, hydrolysis of polysaccharides present in pre-treated biomass and fermentation of hexose and pentose sugars) in one reactor. However, no natural microorganism exhibits all the features desired for CBP. Bacteria and yeast have been the primary candidates for CBP research and some progress has been made in this regard. Traditionally, proponents of CBP processes have identified two primary developmental pathways capable of producing industrially viable CBP microbial strains. These are category I, engineering a cellulase producer, such as Clostridium thermocellum, to be ethanologenic; and category II, engineering an ethanologen, such as Saccharomyces cerevisiae or Zymomonas mobilis, to be cellulolytic (Lynd, 1996; Lynd et al., 2002; Lynd et al., 2005; Van Zyl et al., 2007; Xu et al., 2009). However, the both categories have advantages and disadvantages. Cellulase producer lacks ethanol tolerance, and it is very difficult to coexpress of multiple saccharification enzyme genes in ethanol producer. Especially, heterologous expression of Trichoderma reesei cellobiohydrolases (cellobiohydrolase I and cellobiohydrolase II), which play the crucial role in cellulose degradation, are generally poor. Basidiomycetes, also known as wood-rotting fungi, can achieve the complete breakdown of lignins (Cooke & Rayner, 1984; Cullen, 1997), and are considered primary agents of plant litter decomposition in terrestrial ecosystems (Thorn et al., 1996). Furthermore, some basidiomycetes produce alcohol dehydrogenases, thus allowing the production of wine using a mushroom (Okamura et al., 2000; Okamura et al., 2001). These properties of basidiomycetes appear suitable for use in CBP. In a preliminary study, we screened some edible mushrooms for their ability to produce ethanol and found that Flammulina velutipes is a good producer of ethanol. F. velutipes is a white-rot fungus that grows from spring through


Introduction
Plant cell walls are the most abundant biomass source in nature and are of increasing importance because worldwide attention has now focused on bioethanol production to combat global warming and to safeguard global energy. Because of competition between food and fuel production, lignocelluloses are expected to be utilized for future fuel ethanol production. One of the major problems in producing ethanol from lignocellulosic biomass is the expensive production cost. Consolidated bioprocessing (CBP) is gaining recognition as a potential breakthrough for low-cost biomass processing (Lynd, 1996;Lynd et al., 2002;Lynd et al., 2005;Van Zyl et al., 2007;Xu et al. 2009). CBP of lignocellulose to bioethanol refers to the combination of the 4 biological events required for this conversion process (production of lignocellulose-degrading enzymes, hydrolysis of polysaccharides present in pre-treated biomass and fermentation of hexose and pentose sugars) in one reactor. However, no natural microorganism exhibits all the features desired for CBP. Bacteria and yeast have been the primary candidates for CBP research and some progress has been made in this regard. Traditionally, proponents of CBP processes have identified two primary developmental pathways capable of producing industrially viable CBP microbial strains. These are category I, engineering a cellulase producer, such as Clostridium thermocellum, to be ethanologenic; and category II, engineering an ethanologen, such as Saccharomyces cerevisiae or Zymomonas mobilis, to be cellulolytic (Lynd, 1996;Lynd et al., 2002;Lynd et al., 2005;Van Zyl et al., 2007;Xu et al., 2009). However, the both categories have advantages and disadvantages. Cellulase producer lacks ethanol tolerance, and it is very difficult to coexpress of multiple saccharification enzyme genes in ethanol producer. Especially, heterologous expression of Trichoderma reesei cellobiohydrolases (cellobiohydrolase I and cellobiohydrolase II), which play the crucial role in cellulose degradation, are generally poor. Basidiomycetes, also known as wood-rotting fungi, can achieve the complete breakdown of lignins (Cooke & Rayner, 1984;Cullen, 1997), and are considered primary agents of plant litter decomposition in terrestrial ecosystems (Thorn et al., 1996). Furthermore, some basidiomycetes produce alcohol dehydrogenases, thus allowing the production of wine using a mushroom (Okamura et al., 2000;Okamura et al., 2001). These properties of basidiomycetes appear suitable for use in CBP. In a preliminary study, we screened some edible mushrooms for their ability to produce ethanol and found that Flammulina velutipes is a good producer of ethanol. F. velutipes is a white-rot fungus that grows from spring through www.intechopen.com late autumn on a variety of hardwood tree stubs and dead stems and is widely distributed in temperate to subarctic regions. Currently, F. velutipes is the most produced mushroom in bed cultivation in Japan, the annual production being 130,000 tons/year. Artificial cultivation of mushrooms in polypropylene bottles is popular in Japan. F. velutipes has been characterized as wide adapted strain for various kinds of substance of artificial cultivation media, thus suggesting that the strain may be useful in the conversion of a wide variety of biomass types. In this study, we investigated the properties of ethanol fermentation by F. velutipes to determine its suitability for CBP, because the use of basidiomycetes for bioethanol production is not common and the ethanol fermentation abilities of basidiomycetes are not well characterized. Furthermore, several biomass such as sorghums and rice straw were used as raw material to evaluate the detail conversion from biomass to ethanol by F. velutipes.

Properties of ethanol fermentation by F. velutipes
Because the use of basidiomycetes in bioethanol production is not common, and the ethanol fermentation abilities of basidiomycetes are not well characterized, we investigated the properties of ethanol fermentation by F. velutipes to determine its suitability for CBP (Mizuno et al., 2009b). Before the experiment, to obtain a suitable strain for CBP, 10 F. velutipes strains, culture stock of the Forest Institute of Toyama Prefectural Agricultural, Forestry, and Fisheries Research Center, were screened for cellulase production and ethanol fermentation. The Fv-1 strain was selected for further study because it not only produces high levels of cellulases, but also because its ability to ferment ethanol is superior to the other strains. Firstly, fermentation of D-glucose was done by F. velutipes Fv-1. Figure 1A shows a conversion of 1% w/v of D-glucose to ethanol by F. velutipes. The consumption of D-glucose started gradually after incubation, and it was depleted after 6 d. Ethanol production correlated with sugar consumption, and it reached a maximum after 6 d. Thereafter, the amount of ethanol decreased gradually. Finally, F. velutipes converted 10 g/l of D-glucose to 4.5 g/l of ethanol, equivalent to a theoretical ethanol recovery rate of 88%. In the case of ethanol production from 5% w/v D-glucose, ethanol production reached a maximum, and all of the D-glucose was consumed after 18 d of incubation (Fig. 1B), and 50 g/l of D-glucose was converted to 22.4 g/l of ethanol, equivalent to a theoretical ethanol recovery rate of 87%. The conversion rate was the same as the case of 1% w/v of D-glucose. Because the incubation time to ferment 1% w/v sugar is shorter than the case of 5% w/v, we employed 1% w/v of sugar concentration in subsequent experiments. Secondary, determination of the fermentation specificity of sugars by F. velutipes Fv-1 was done using various monosaccharides. As shown in Fig. 2, both D-mannose and D-fructose were converted to ethanol by F. velutipes. Consumption of D-mannose occurred slightly faster than that of D-glucose; it started immediately after incubation and was completely depleted after 5 d. Ethanol production from D-mannose was similar to that from D-glucose. It started during the first day of incubation and reached a maximum after 6 d. Furthermore, 4.4 g/l of ethanol was produced from 10 g/l of D-mannose, equivalent to a theoretical ethanol recovery rate of 86% ( Fig. 2A). In contrast, consumption of D-fructose was slower than that of D-mannose. It started slowly after incubation and took 7 d to completely consume the D-fructose. Production of ethanol correlated with sugar consumption, and Symbols: closed circle, sugar; closed square, ethanol. The initial D-glucose concentration was (A) 1% w/v and (B) 5% w/v. (Reproduced from Mizuno et al., 2009b) Fig. 1. Ethanol production from fermentation of D-glucose by F. velutipes maximum conversion of D-fructose to ethanol was observed after 6 d. Upon completion of incubation, 4.0 g/l of ethanol was obtained from 10 g/l of D-fructose ( Fig. 2B), yielding a theoretical conversion rate of 77%. In contrast to these sugars, F. velutipes did not convert Larabinose, D-xylose, or D-galactose to ethanol (Figs. 2C, 2D, and 2E). Although there was slight consumption of D-xylose and D-galactose during incubation, ethanol production was not observed. In the case of L-arabinose, little sugar consumption was observed. Next, we examined the fermentation specificity of F. velutipes F v -1 t o w a r d v a r i o u s disaccharides. As shown in Fig. 3, F. velutipes possibly converted these sugars to ethanol and produced high yields. The theoretical conversion rates of these sugars were 83% and 77% from sucrose and maltose respectively. Degradation of sucrose was observed immediately after the incubation to import the sugar. The amount of reducing sugars was maximum on day 3 and was completely consumed after 7 d of incubation. Ethanol production was observed 1 d after incubation, and the amount of ethanol reached a maximum after 6 d. Finally, 4.5 g/l of ethanol was produced from 10 g/l of sucrose (Fig. 3A). In the case of maltose, degradation was observed on the first day of incubation, and the amount of reducing sugars reached a maximum after 2 d. Furthermore, the reducing sugars were completely depleted after 7 d of incubation. Ethanol production started during the first day of incubation and reached a maximum after 7 d. At the end of incubation, 10 g/l of maltose was converted to 3.8 g/l of ethanol (Fig. 3B). No conversion of xylobiose to ethanol was detected (data not shown), but a significant amount of ethanol production was observed when cellobiose was used as the carbon source (Fig. 4A). Cellobiose began degrading during the first day of incubation, and both D-glucose and cellobiose were completely depleted after 8 d. -Glucosidase activity increased gradually during incubation. Ethanol production started after 1 d of incubation, and the amount of ethanol reached a maximum after 8 d. Upon completion of incubation, 10 g/l of cellobiose was converted to 4.5 g/l of ethanol (Fig.  4A). The theoretical conversion rate was 83%, a value similar to that of glucose and significantly higher than that of maltose. A high yield of ethanol was observed also in the higher concentration of cellobiose (Fig. 4D). Finally, 25 g/l of ethanol was produced from 50 g/l of D-glucose, and the theoretical conversion rate was 91%. Since cellobiose was converted to ethanol at a relatively high rate, the conversions of cellooligosaccharides to ethanol by F. velutipes were also investigated. Figures 4B and 4C show Symbols: closed circle, sugar; closed square, ethanol. The initial sugar concentration was 1% w/v. (Reproduced from Mizuno et al., 2009b)  the results of the conversion of cellotriose and cellotetraose to ethanol. Both cellooligosaccharides were effectively converted to ethanol by F. velutipes. During incubation, cellotriose was initially hydrolyzed to D-glucose and cellobiose, and almost 80% of the initial amount of cellotriose was hydrolyzed by 2 d. Cellotriose was not detected after 5 d of incubation, and D-glucose and cellobiose were completely depleted after 7 d. -Glucosidase Symbols: closed circle, reducing sugar; closed square, ethanol. The initial sugar concentration was 1% w/v. (Reproduced from Mizuno et al., 2009b)

Fig. 3. Ethanol fermentation from (A) sucrose and (B) maltose by F. velutipes
was slightly induced by 6 d, and the activity gradually increased after 6 d. The amount of ethanol increased during incubation and reached a maximum after 7 d of incubation. F. velutipes produced 4.2 g/l of ethanol from 10 g/l of cellotriose, equivalent to a theoretical conversion rate of 76% (Fig. 4B). In the case of cellotetraose, it was initially hydrolyzed to cellotriose, cellobiose, and D-glucose, and more than 90% of the cellotetraose was hydrolyzed by 2 d. Cellotetraose was not detected after 3 d of incubation, and cellotriose, cellobiose, and D-glucose were completely depleted after 4, 6 and 7 d respectively. -Glucosidase activity increased rapidly over 2 d then decreased gradually from 2 d to 5 d, and stabilized at an activity level of about 30 mU/ml. The amount of ethanol increased after incubation, and 4.4 g/l of ethanol was produced from 10 g/l of cellotetraose after 7 d of incubation (Fig. 4C). The ethanol recovery for the theoretical conversion value was 78%. To date, many microorganisms, including Saccharomyces cerevisiae, Zymmonas mobilis, Pichia stipitis, Rhizopus oryzae, and Clostridium thermocellum, have been reported to produce ethanol (DeMoss & Gibb, 1951;Maas et al., 2006;Ng et al., 1981;Parekh & Wayman, 1986;Weimer & Zeikus, 1977). In general, S. cerevisiae is the most widely used microorganism in the industry and is popular in bioethanol production, because it has high efficiency of ethanol production and high ethanol tolerance. However, we focused on basidiomycetes to develop CBP because these microorganisms have both lignocellulose degradation and ethanol fermentation abilities. Here, we characterized properties of ethanol fermentation by F. velutipes Fv-1. The strain converted D-glucose to ethanol at a theoretical conversion rate of 88%, comparable to those of S. cerevisiae and Zymomonas (Swings & DeLey, 1977). On the other hand, F. velutipes scarcely converted pentose and D-galactose to ethanol (Fig. 2). These properties of F. velutipes are similar to those of S. cerevisiae (Barnett, 1976). Moreover, F. velutipes demonstrated the preferable features for CBP when oligosaccharides were used as starting materials (Figs. 3 and 4). The tested oligosaccharides were converted to ethanol at almost the same rate as that of D-glucose, and -glucosidase activity increased during fermentation. These features are indispensable in CBP, which requires saccharification and fermentation of cellulose contained in the cell wall. It has been reported that C. thermocellum and P. stipitis can ferment cellobiose (Parekh & Wayman, 1986). Furthermore, C. thermocellum can also convert cellulose to ethanol directly (Ng et al., 1981;Lynd et al., 1989  1997). However, this species cannot be used at the scene of ethanol production because fermentation of C. thermocellum is strongly inhibited at relatively low ethanol concentrations (5 g/l) (Herrero & Gomez, 1980). In contrast, it has been reported that basidiomycetes have tolerance of up to 120 g/l of ethanol (Okamura et al., 2001), and therefore basidiomycetes are more suitable for CBP than Clostridium strains. From these results, we concluded that F. velutipes possesses advantageous characteristics for use in CBP.

Use of whole crop sorghums as a raw material in consolidated bioprocessing bioethanol production using Flammulina velutipes
The ethanol fermentation abilities of basidiomycetes have not been well characterized, we evaluated the ability of F. velutipes in CBP. Preliminary fermentation experiments indicate that F. velutipes convert sugars to ethanol much more under the high concentration of biomass which close to solid state cultivation than liquid cultivation condition. Therefore, we employed solid state cultivation which usually performed in artificial cultivation of mushrooms for the coversion of biomass to produce bioethanol. Sorghum is selected as a possible raw material to produce bioethanol by CBP using F. velutipes. Sorghum is a C4 crop of the grass family belonging to the genus Sorghum bicolor L. It is well adapted to temperate climates and can be cultured from Kyushu to Tohoku area in Japan. The plant grows to a height from about 120 to above 400 cm, depending on the variety and growing conditions, and can be an annual or a short perennial crop. Sorghum is considered to be one of the most drought resistant agricultural crops, as it is able to remain dormant during the driest periods (Xu et al., 2000). These properties of sorghum are suitable as raw material for the ethanol production. We evaluated the ability of F. velutipes in CBP using sorghum strains as a raw material, and solid-state CBP of ground sorghum strains (SIL-05 and Kyushukou No. 4) using F. velutipes was investigated. The possibility of sorghum strains as a raw material in the CBP ethanol production by F. velutipes is also discussed below. We selected grinding for the pretreatment of sorghum strains. This can be used on both dry and wet materials, and the cost of grinding is one of the cheapest compared to other methods used for milling biomass. The grinding of sorghum was carried out with an ultrafine friction grinder. Grinding was performed at room temperature, and was repeated twice. To examine the efficiency of grinding as a pretreatment, the degree of saccharification was tested using commercially available enzymes Celluclast 1.5L (Sigma, St. Louis, MO), Novozyme 188 (Sigma) and Multifect xylanase (Genencor Kyowa, Tokyo). The saccharification yields of SIL-05 and Kyushukou No. 4 by the enzymes were 30.1% and 51.7% respectively (Fig. 5A). Kyushukou No. 4 is one of the sorghum brown mid-rib (bmr) mutants in which cafferic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, activity is reduced as compared to the wild type (Bout & Vermerris, 2003). This property of bmr significantly affected the hydrolysis of polysaccharides in the biomass, but there were no significant differences in the proportions of hydrolysis of the components such as cellulose and hemicellulose (Fig. 5). When the saccharification yields of cellulose and hemicelluloses were compared, degradation of hemicelluloses was slightly higher than for cellulose in both types of sorghum. Next, solid-state ethanol fermentation by F. velutipes was performed for both sorghum strains. Solid-state fermentation is advantageous because it carries a low ethanol production cost. Generally, sorghums contain 70-80% v/v water, corresponding to 43-25% w/v. These concentrations are necessary to obtain relatively high final ethanol concentrations. Furthermore, it is possible to reduce the costs of many procedures, such as amount of water, concentration of biomass, treatment of waste water, and so forth, if the water concentration of the raw materials in the all ethanol production procedures is retained. The Fv-1 strain was selected for further study because it not only produces high levels of cellulases, but also because its ability to ferment ethanol is superior to the other strains. Mycelia of Fv-1 were harvested in the late exponential growth phase by centrifugation at 3,000  g and washed with sterile water. The prepared wet mycelia (20 mg of dry weight) were mixed with 100 mg of ground sorghum for solid-state fermentation. A larger amount of ethanol was produced from SIL-05 than from Kyushukou No. 4 (Fig. 6). Because SIL-05 contained a larger amount of soluble sugars than Kyushukou No. 4 (Table 1), it should be advantageous for total ethanol fermentation. The ethanol conversion rates for the soluble sugars contained in SIL-05 and Kyushukou No. 4 were 57.2% and 38.9% respectively. The addition of saccharification enzymes was not effective for SIL-05 (Fig. 6A). This corresponded with the results of enzymatic hydrolysis (SIL-05 just hydrolyzed almost 30%) (Fig. 6). However, the ethanol conversion rate for the degraded cellulose was 85.6%, significantly higher than that for soluble sugars. In contrast, although total ethanol production was not high, ethanol production from Kyushukou No. 4 significantly increased when saccharification enzymes were added to the culture (Fig. 6B). Because the cellulose and hemicellulose in Kyushukou No. 4 were more easily hydrolyzed than SIL-05 by cellulases, significantly more ethanol was produced by the addition of the saccharification enzymes. The ethanol conversion rate for the degraded cellulose of Kyushukou No. 4 (98.3%) was much higher than that of SIL-05 (85.6%). Thus, the bmr mutation appears to be useful for CBP because it gives a high yield of glucose from biomass without acid or alkali pretreatment. However, the results indicate that the production of cellulases by F. velutipes is not sufficient for CBP, or that the saccharification enzymes are suppressed by carbon  catabolites due to the existence of soluble sugars. Therefore, an effective saccharification enzyme inducing method for F. velutipes in the CBP is required.
In this work, we demonstrated CBP ethanol fermentation of sorghum strains by F. velutipes Fv-1. The procedure is quite simple and cost effective, and can reduce energy consumption, because the raw material is simply ground and then mixed with mycelia. We demonstrated the merit of high concentrations of soluble sugars and the bmr mutation in sorghums. Both sorghum strains can be used in CBP. The bmr mutation is only found in sorghums, corn, and pearl millet, giving sorghum an advantage over many other crops for ethanol production. Future studies should focus on the improvement of CBP using F. velutipes and the selective breeding of novel types of sorghums with high concentrations of soluble sugars and the bmr mutation.

Solid state fermentation of rice straw by F. velutipes
The solid state ethanol fermentation by F. velutipes was performed for ammonia treated rice straw. Solid state cultivation has a large merit to decrease the ethanol production cost. But it has demerit on the saccharification of biomass. As shown in Fig. 7A, saccharification of biomass at high concentration is quite difficult. Significant amount of cellulase is necessary to obtain enough level of saccharification, and saccharification yield do not increase in proportion to the amount of cellulase if increased the amount of cellulase. Furthermore, saccharification yield will be significantly decreased under the high substrate condition. The hydrolysis rate of 30% w/v biomass was very low (less than 10%). In contrast, ethanol yield was equivalent to 80-90% of hydrolysis rate so that the merit of our process using F. velutipes was proven (Fig. 7B). In the case that enzymes were not added, ethanol production by F. velutipes was only 0.026 l/kg of dry biomass, equivalent to a theoretical ethanol recovery rate of 5.9% from total hexose. In contrast to no enzymes addition, in the case that 1 and 5 mg/g product of enzymes were added to the fermentation, ethanol production after 15 d by F. velutipes was 0.26 and 0.34 l/kg of dry biomass, respectively. The ethanol conversion rates of 1 and 5 mg/g product enzymes addition were 61.6% and 77.8% for total hexose, respectively. The maximum weight loss was approximately 70% in the case that no enzymes were added to the fermentation, while the maximum weight loss for enzyme addition of 1 and 5 mg/g product were approximately 90% and 96% respectively (data not shown). These results suggest F. velutipes has favourable properties for CBP. It could be expected that development of novel bioethanol production process by using F. velutipes.

Development of a gene transfer system for the mycelia of F. velutipes
As shown in above, we found the edible mushroom F. velutipes Fv-1 strain to be an efficient ethanol producer, and, we demonstrated its preferable properties of ethanol fermentation from various sugars (Mizuno et al., 2009b), whole crop sorghums and rice straw (Mizuno et al., 2009a). However, the strain can only slightly convert pentoses, which account for approximately 20-30% of plant cell walls, into ethanol (Mizuno et al., 2009a). Therefore, genetic engineering of the pentose metabolism is necessary to make possible the ethanol fermentation from pentose. Furthermore, more efficient (low cost) conversion of biomass to ethanol could be expected if saccharification ability was strengthened by expressing cellulases. A transformation method of F. velutipes by the electroporation protocol for basidiospores has been reported (Kuo et al., 2004), but it requires a long period to produce basidiospores because it must go through fruiting body formation, and cannot eliminate the risk of contamination in the process of spore harvest. Since screening of many transformants is needed for improvement of the metabolic pathway by genetic engineering, the development of a simpler transformation method is desired to obtain high numbers of transformants. Therefore, an adequate condition for protoplast preparation from mycelia of F. velutipes Fv-1 strain was investigated, and simpler a transformation protocol for this fungus was developed by the calcium-PEG method and the restriction enzyme-mediated-integration (REMI) method. First, we constructed a pFvT vector for transformation of the F. velutipes Fv-1 strain (Fig.  8A). The vector possessed a F. velutipes tryptophan synthetase gene promoter and terminator (GenBank no. AB028647) to regulate expression of the constructed genes, and the hygromycin phosphotransferase gene (hph) from Escherichia coli as selection marker. The hph gene was obtained from pCAMBIA1201 vector (CAMBIA; http://www.cambia.org/). Next, conditions to prepare protoplast from the mycelia of F. velutipes were optimized by modifying a method for Phanerochaete sordida (Yamagishi et al., 2007  Genetic transformation was investigated using the pFvT vector and the protoplasts prepared as described above. The transformation procedures for Lentinus edodes (Sato et al., 1998) and Schizophyllum commune (Van Peer et al., 2009) were modified for the transformation of F. velutipes Fv-1. In the course of the transformation process, the effect of the structure of the plasmid DNA on transformation was evaluated using circular and linear pFvT plasmids. Approximately 6-fold transformants were obtained when the plasmid DNA was linearized (Table 2). Because the REMI method is a popular transformation tool for fungi (Hirano et al., 2000;Maier & Schäfer, 1999;Riggle & Kumamoto, 1998;Sato et al., 1998), we evaluated the effect of REMI on the transformation for F. velutipes Fv-1. The F. velutipes Fv-1 strain was transformed by pFvT with a restriction enzyme, BglI, KpnI, or PstI. The addition of the restriction enzymes increased the number of transformants by about 1.6-to 5.8-fold (Table 2). The suggests that the addition of restriction enzymes enhanced the transformation efficiency of F. velutipes. Therefore, to find the optimum enzyme concentration for REMI, we  Table 2. Numbers of transformants obtained by the REMI method performed transformation using circular pFvT plasmid with the presence of various concentrations of PstI (Fig. 9). As for the results, the number of transformants obtained was affected by the amount of restriction enzyme. The efficiency was significantly increased by the addition of PstI at 25 units, by it gradually decreased when the PstI amount was over 25 units, suggesting that the optimal value for transformation mediated by the PstI is 25 units.
In conclusion, we found a simple transformation procedure for the mycelia of F. velutipes Fv-1 strain by the calcium-PEG method combined with REMI. The transformation method of F. velutipes Fv-1 strain does not require a process of spore formation, because the mycelia could be used as starting material. Moreover, a high efficiency of transformation was obtained by the adoption of REMI.

Number of transformants
Amount of PstI(units) Fig. 9. Effects of the amount of PstI on transformation by REMI method (Reproduced from Maehara et al., 2010a)

Improvement of the transformation efficiency of Flammulina velutipes Fv-1 using the glyceraldehydes-3-phosphate dehydrogenase gene promoter
To make possible genetic manipulation in F. velutipes, we constructed the pFvT plasmid containing the hygromycin phosphotransferase gene (hph) under the control of the www.intechopen.com tryptophan synthetase gene (trp1) promoter, and developed an easy transformation method for F. velutipes by the REMI method (Maehara et al., 2010a). Here, we focused on the promoter of the glyceraldehyde-3-phosphate dehydrogenase (gpd) gene because many tools such as promoters and selection markers are desirable for effective metabolic pathway engineering of F. velutipes Fv-1. The gpd promoters are the most frequently used constitutive promoters in basidiomycetes. GPD constitutes up to 5% of the soluble protein in Saccharomyces cerevisiae and other higher eukaryotic organisms (Piechaczyk et al., 1984;Punt et al., 1990), and gpd mRNA accounts for 2-5% of the poly (A)+ RNA in yeast (Holland & Holland, 1978).
In this section, we described that construction of new plasmids having the hph gene from Escherichia coli as a selection marker, which regulated by the gpd promoter and the potency of the gpd promoter from F. velutipes were evaluated. First we constructed three vectors, pFvG, pFvTgh, and pFvGgh, by modification of pFvT. The pFvT vector possessed a trp1 promoter and terminator regulating the expression of the constructed genes, and the hph gene as selection marker (Fig. 8A, Maehara et al., 2010a). Vectors pFvG (Fig. 8B) and pFvGgh (Fig. 8D) contained the gpd promoter and the terminator of F. velutipes (Kuo et al., 2004) located upstream and downstream of a multiple cloning site (MCS), and both pFvTgh (Fig. 8C) and pFvGgh (Fig. 8D) contained the gpd promoter and the terminator located upstream and downstream of the hph gene (Maehara et al., 2010b).
To determine the potency of the gpd promoter, we compared transformation efficiency by the gpd promoter with that by the trp1 promoter. Gene integrations were performed by the REMI method. Protoplasts were prepared from mycelia of the F. velutipes Fv-1 strain, and then plasmids were transformed into the protoplasts with PstI (25 U). As shown in Table 2, about 10 transformants (10.7 to 12.3) were obtained by the transformation of pFvT and of pFvG, which contain the hph gene controlled by the trp1 promoter. In contrast, as for the results of the transformation of pFvTgh and pFvGgh, the numbers of transformants were significantly increased and about 24.7 to 33.3 transformants were obtained, suggesting that the activity of the gpd promoter was higher than that of the trp1 promoter in F. velutipes Fv-1. There is a difference of about 500-bp in the length of pFvT and pFvG, or pFvTgh and pFvGgh, but no significant difference in the number of transformants obtained by pFvT and by pFvG, and by pFvTgh and pFvGgh was not observed. It might suggest, that the difference of the sizes of these plasmids was not affected on transformation efficiency. To compare the activity of the gpd and the trp1 promoter, the expression levels of the hph gene in each transformant were examined by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from each set of three transformants and equal amounts of RNAs from each set of three clones were mixed and used as template for RT-PCR. As shown in Fig. 10A, the intensities of the bands of the pFvTgh and pFvGgh transformants were stronger than that of the pFvT and pFvG transformants (upper panel), suggesting that the expression level of the hph gene in the pFvTgh and pFvGgh transformants was higher than that in the pFvT and pFvG transformants. The results were corresponded to the transformation efficiency presented in Table 2, and strongly suggest that the gpd promoter is functional in the heterologous gene expression system in F. velutipes Fv-1 to improve the expression level of the target gene. Finally, in order to determine whether the plasmid vector was integrated into the genomic DNA by the REMI method, the genomic DNAs of 10 randomly selected pFvGgh transformants were analyzed by Southern blot using the digoxigenin-labeled hph gene as a probe (Fig. 10B). Hybridization signals were detected in all the transformants, and multiple  hybridization signals were also detected in some transformants. There was no signal from the genomic DNA of wild-type Fv-1 as a negative control (data not shown). These results indicate that at least a single hph gene was introduced into all the transformants, and the hph gene is thought to exist as a multicopy in the genomic DNAs of many transformants (Fig. 10B,lanes 3,4,5,6,8,9 and 11). The same size bands were detected between 2,027 and 3,530bp in four transformants (Fig. 10B, lanes 3, 4, 8 and 9). These bands might represent about 2,700-bp of the full-length gpd promoter-hph-gpd terminator region. A 6.9-kb DNA fragment, corresponding to the size of the pFvGgh plasmid, was observed in the genome of only one clone (Fig. 10B, lane 6), indicating that the full length of the plasmid was successfully introduced into the transformant. Consequently, we estimate the probability of integration of full-length pFvGgh vector by the REMI method to be approximately 10%. In our previous study, the probability of integration of the full-length vector was 30% so that the frequency of REMI events of Fv-1 was 10-30% (Maehara et al., 2010a). This value seems to be the comparable level in the case of model mushroom, Coprinus cinereus (8-56%) (Granado et al., 1997).
In conclusion, we demonstrated that the gpd promoter from F. velutipes Fv-1 would be a useful in the transformation system of the strain. The transformation efficiency was about 3 times improved by the use of the gpd promoter. The vectors constructed in this study will be available to improve the genetic engineering of F. velutipes Fv-1 for ethanol fermentation from pentose.

Conclusion
In spite of CBP is gaining recognition of a low-cost biomass processing as it involves enzyme production, completely no enzyme process which does not add the saccharification enzymes have not been established. In this study, we demonstrated that F. velutipes can highly convert biomass to ethanol using only small amount of saccharification enzyme even in the quite high concentration of biomass such as 30% w/v. These results suggest F. velutipes has favorable properties for CBP. Generally, artificial cultivation of mushrooms in polypropylene bottles is performed under the condition of water content 70 to 80%. The condition must be most suitable condition to cultivate the mushrooms. Therefore, F. velutipes will be especially effective in situations that CBP performed under the high concentration of biomass. We believe that this point would be advantage of F. velutipes compared with the other microorganisms engineered for CBP and even for fungus which is possible to ferment the both pentose and hexose. In the future, we would like to develop a novel bioethanol production process by using F. velutipes.

Acknowledgment
This work was financially supported by a grant-in-aid (Development of Biomass Utilization Technologies for Revitalizing Rural Areas) from the Ministry of Agriculture, Forestry, and Fisheries of Japan. Recent studies have shown strong evidence of human activity impact on the climate of the planet. Higher temperatures and intensification of extreme weather events such as hurricanes are among the consequences. This scenario opens up several possibilities for what is now called "green" or low carbon economy. We are talking about creating new businesses and industries geared to develop products and services with low consumption of natural resources and reduced greenhouse gases emission. Within this category of business, biofuels is a highlight and the central theme of this book. The first section presents some research results for first generation ethanol production from starch and sugar raw materials. Chapters in the second section present results on some efforts around the world to develop an efficient technology for producing secondgeneration ethanol from different types of lignocellulosic materials. While these production technologies are being developed, different uses for ethanol could also be studied. The chapter in the third section points to the use of hydrogen in fuel cells, where this hydrogen could be produced from ethanol.