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.

(A) Closed circle, SIL-05; closed square, Kyusyukou No. 4. Broken lines were drawn by roughly following the experimental data points. (B) White, cellulose; black, hemicelluloses. (Reproduced from Mizuno et al., 2009a)

Fig. 5. (A) Time course of sorghum hydrolysis and (B) saccharification yield of cellulose and hemicellulose incubated for 72 h

Consolidated Bioprocessing Ethanol Production by Using a Mushroom 199

catabolites due to the existence of soluble sugars. Therefore, an effective saccharification

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*

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

These results suggest *F. velutipes* has favourable properties for CBP. It could be expected that

Water content (%) 78 (± 0.7) 80 (± 1.4) Soluble sugar (%) 12 (± 0.1) 4.5 (± 0.0) Cellulose content (%)a 5.1 (± 0.3) 7.4 (± 0.8) Hemicellulose content (%)b 3.6 (± 0.1) 5.2 (± 0.5) Other content (%)c 1.2 (± 0.2) 2.4 (± 0.5)

a The amount of hexose was determined by the anthrone-sulfuric acid method. b The amount of pentose was determined by the orcin-Fe3+-hydrochloric acid method.

c All components except for sugars. (Reproduced from Mizuno et al., 2009a)

enzyme inducing method for *F. velutipes* in the CBP is required.

**3.2 Solid state fermentation of rice straw by** *F. velutipes*

approximately 90% and 96% respectively (data not shown).

development of novel bioethanol production process by using *F. velutipes*.

Table 1. Compositions of SIL-05 and Kyushukou No. 4

mutation.

SIL-05 Kyushukou No. 4

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

White, no added enzymes; black, 15 μl Celluclast 1.5 L and 10 μl Multifect xylanase added.6 Reproduced from Mizuno et al., 2009a)

Fig. 6. Solid-state ethanol fermentation of (A) SIL-05 and (B) Kyushukou No. 4 by *Flammulina velutipes*

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

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

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

concentrations are necessary to obtain relatively high final ethanol concentrations.

White, no added enzymes; black, 15 μl Celluclast 1.5 L and 10 μl Multifect xylanase added.6

Fig. 6. Solid-state ethanol fermentation of (A) SIL-05 and (B) Kyushukou No. 4 by

Reproduced from Mizuno et al., 2009a)

*Flammulina velutipes*

of ground sorghum for solid-state fermentation.

a The amount of hexose was determined by the anthrone-sulfuric acid method.

b The amount of pentose was determined by the orcin-Fe3+-hydrochloric acid method.

c All components except for sugars. (Reproduced from Mizuno et al., 2009a)

Table 1. Compositions of SIL-05 and Kyushukou No. 4

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.

#### **3.2 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*.

Consolidated Bioprocessing Ethanol Production by Using a Mushroom 201

B

*Bgl*I

*Bgl*I

D

*ampr*

E*ori*

(6461 bp)

Ftrp-t *hph* Ftrp-p

pFvGgh (6958 bp)

*hph*

, ampicilin resistance gene; E*ori*, pUC19 ori. (Reproduced

Fgpd-t Fgpd-p

E*ori*

*ampr* pFvG

MCS

Fgpd-t

*Pst*I

MCS

Fgpd-t

*Pst*I

Fgpd-p

*Nco*I *Sal*I *Cla*I *Hind*III

Fgpd-p

*Kpn*I

*Nco*I *Sal*I *Cla*I *Hind*III

*Nco*I *Sal*I *Cla*I *Hind*III *Eco*RI

MCS

Ftrp-t

MCS

Ftrp-t

*Pst*I

Ftrp-p

*Nco*I *Sal*I *Cla*I *Hind*III *Eco*RI

MCS, multiple cloning sites; Ftrp-p, *trp1* promoter from *F. velutipes*; Ftrp-t, *trp1* terminator from *F. velutipes*; Fgpd-p, *gpd* promoter from *F. velutipes*; Fgpd-t, *gpd* terminator from *F. velutipes*; *hph*,

strain was grown in PCMY (1% polypeptone, 0.2% casamino acid, 1% malt extract, and 0.4% yeast extract) medium at 25C for 3 d. Then the mycelia were collected and incubated in enzyme solution [1.5% cellulase Onozuka-RS (Yakult Pharmaceutical, Tokyo) and 1.5% lysis enzyme (Sigma, St. Louis, MO) in 0.75 M MgOsm (0.75 M MgSO4, 20 mM MES, pH 6.3)] at 30C for 5 h. The protoplasts were filtered through Miracloth (Cosmo Bio, Tokyo), washed at twice with 1 M SorbOsm (1.0 M sorbitol, 10 mM MES, pH 6.3), and suspended in SorbOsm plus 40 mM CaCl2 solution to a final concentration of approximately 108 protoplasts ml-1. 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

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, *Bgl*I, *Kpn*I, or *Pst*I. 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

*Pst*I

Ftrp-p

*Kpn*I

E*ori*

pFvT (5962 bp)

*hph*

pFvTgh (6459 bp)

*hph* Fgpd-t Fgpd-p

hygromycin B phosphotransferase gene; *amp*r

Fig. 8. Structures of the plasmids used in this study

from Maehara et al., 2010b)

was linearized (Table 2).

E*ori*

*ampr*

*Bgl*I

*ampr*

C

*Bgl*I

A

(A) Saccharification yield of ammonia treated rice straw by enzymes. (B) Solid-state ethanol fermentation of ammonia treated rice straw by *F. velutipes*. Right gray, no added enzymes; gray, 1 mg/g product enzymes added; black, 5 mg/g product enzymes added.

Fig. 7. Saccharification and ethanol production at high biomass concentration

#### **4. Development of a gene transfer system for** *F. velutipes*

#### **4.1 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). The *F. velutipes* Fv-1

(A) Saccharification yield of ammonia treated rice straw by enzymes. (B) Solid-state ethanol

Fig. 7. Saccharification and ethanol production at high biomass concentration

**4.1 Development of a gene transfer system for the mycelia of** *F. velutipes*

transformation method is desired to obtain high numbers of transformants.

(REMI) method.

**4. Development of a gene transfer system for** *F. velutipes*

product enzymes added; black, 5 mg/g product enzymes added.

fermentation of ammonia treated rice straw by *F. velutipes*. Right gray, no added enzymes; gray, 1 mg/g

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

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

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). The *F. velutipes* Fv-1

MCS, multiple cloning sites; Ftrp-p, *trp1* promoter from *F. velutipes*; Ftrp-t, *trp1* terminator from *F. velutipes*; Fgpd-p, *gpd* promoter from *F. velutipes*; Fgpd-t, *gpd* terminator from *F. velutipes*; *hph*, hygromycin B phosphotransferase gene; *amp*r, ampicilin resistance gene; E*ori*, pUC19 ori. (Reproduced from Maehara et al., 2010b)

Fig. 8. Structures of the plasmids used in this study

strain was grown in PCMY (1% polypeptone, 0.2% casamino acid, 1% malt extract, and 0.4% yeast extract) medium at 25C for 3 d. Then the mycelia were collected and incubated in enzyme solution [1.5% cellulase Onozuka-RS (Yakult Pharmaceutical, Tokyo) and 1.5% lysis enzyme (Sigma, St. Louis, MO) in 0.75 M MgOsm (0.75 M MgSO4, 20 mM MES, pH 6.3)] at 30C for 5 h. The protoplasts were filtered through Miracloth (Cosmo Bio, Tokyo), washed at twice with 1 M SorbOsm (1.0 M sorbitol, 10 mM MES, pH 6.3), and suspended in SorbOsm plus 40 mM CaCl2 solution to a final concentration of approximately 108 protoplasts ml-1.

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, *Bgl*I, *Kpn*I, or *Pst*I. 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

Consolidated Bioprocessing Ethanol Production by Using a Mushroom 203

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 &

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

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 *Pst*I (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*

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

Holland, 1978).

of the *gpd* promoter from *F. velutipes* were evaluated.

Fv-1 to improve the expression level of the target gene.

\*The values represent the average and standard deviation of triplicate. (Reproduced from Maehara et al., 2010a & 2010b)

Table 2. Numbers of transformants obtained by the REMI method

performed transformation using circular pFvT plasmid with the presence of various concentrations of *Pst*I (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 *Pst*I at 25 units, by it gradually decreased when the *Pst*I amount was over 25 units, suggesting that the optimal value for transformation mediated by the *Pst*I 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.

Fig. 9. Effects of the amount of *Pst*I on transformation by REMI method (Reproduced from Maehara et al., 2010a)

#### **4.2 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

No. of transformants\*

Circular 0.7 ± 1.2 7.3 ± 1.2 18.3 ± 6.1 25.7 ± 7.1 Linear 4.4 ± 0.6 12.7 ± 6.4 20.0 ± 8.7 21.7 ± 7.1

Circular 11.3 ± 2.6 12.3 ± 2.5 28.3 ± 1.3 33.3 ± 2.9 Linear 11.3 ±6.6 10.7 ± 6.9 24.7 ± 8.4 27.7 ± 11

\*The values represent the average and standard deviation of triplicate. (Reproduced from Maehara et

performed transformation using circular pFvT plasmid with the presence of various concentrations of *Pst*I (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 *Pst*I at 25 units, by it gradually decreased when the *Pst*I amount was over 25 units, suggesting that the optimal value for transformation mediated by the *Pst*I 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

Amount of *Pst*I (units)

Fig. 9. Effects of the amount of *Pst*I on transformation by REMI method (Reproduced from

**4.2 Improvement of the transformation efficiency of** *Flammulina velutipes* **Fv-1 using**

To make possible genetic manipulation in *F. velutipes*, we constructed the pFvT plasmid containing the hygromycin phosphotransferase gene (*hph*) under the control of the

**the glyceraldehydes-3-phosphate dehydrogenase gene promoter**

none *Bgl*I *Kpn*I *Pst*I

pFvT pFvG pFvTgh pFvGgh

**Form of pFvT Restriction enzyme (50 U)**

**Form of DNA Plasmid**

Table 2. Numbers of transformants obtained by the REMI method

Number of transformants

al., 2010a & 2010b)

the adoption of REMI.

Maehara et al., 2010a)

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 *Pst*I (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

Consolidated Bioprocessing Ethanol Production by Using a Mushroom 205

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

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

Barnett, JA. (1976). The utilization of sugars by Yeasts. *Advances in Carbohydrate Chemistry*

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**5. Conclusion**

production process by using *F. velutipes*.

**6. Acknowledgment**

Fisheries of Japan.

**7. References**

(A) RT-PCR-based evaluation of the expression of the hygromycin phosphotransferase gene: Upper panel, *hph* gene; Lower panel, *-tubulin* gene as a control. (B) Southern blotting of *Pst*I digested genomic DNA using the *hph* gene probe: Lane M, DNA molecular size markers (values on left); lane 1, pFvGgh as a positive control; lanes 2-11, genomic DNA from transformants. (Reproduced from Maehara et al., 2010b)

Fig. 10. Analysis of the transformants obtained by REMI method

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 3,530 bp 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.

#### **5. Conclusion**

204 Bioethanol

**Fv-1 (pFvG)**

**M 1 2 3 4 5 6 7 8 9 10 11**

*-tubulin* gene as a control. (B) Southern blotting of *Pst*I digested genomic

**Marker (pFvGgh)**

**Fv-1 (pFvTgh)**

F*trp-*p*-hph* F*gpd-*p-*hph*

**Fv-1**

**Fv-1 WT**

(A) RT-PCR-based evaluation of the expression of the hygromycin phosphotransferase gene: Upper

DNA using the *hph* gene probe: Lane M, DNA molecular size markers (values on left); lane 1, pFvGgh as a positive control; lanes 2-11, genomic DNA from transformants. (Reproduced from Maehara et al.,

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 3,530 bp 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.,

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

*hph*

*-tubulin*

A

B

panel, *hph* gene; Lower panel,

2010b)

1997).

from pentose.

21,226 bp

5,148

6,958 3,530

2,027

Fig. 10. Analysis of the transformants obtained by REMI method

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*.

#### **6. 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.

#### **7. References**

Consolidated Bioprocessing Ethanol Production by Using a Mushroom 207

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**10**

**SSF Fermentation of Rape Straw**

William Linnane2 and Mette H. Thomsen3

*3Chemical Engineering Program, Madsar Institute*

*Technical University of Denmark*

*2Roskilde University*

*3United Arab Emirates*

*1,2Denmark*

**and the Effects of Inhibitory Stress on Yeast**

In 2003 R. E. Smalley (Smalley 2003) made a list of the top 10 problems mankind was going

With the declining amount of fossil fuels, and the increasing energy demand, it is not possible to satisfy our large energy consumption without alternative energy sources, especially sustainable ones that also take care of problem number 4, (Environment). One of the possible ways to produce a liquid sustainable energy source is to replace our large gasoline demand with fermented biomass (bioethanol). To ensure that the food availability does not decrease (problem 3), the biomass for bioethanol fermentation is only gathered from waste materials of the food production, such as the straw of cereals. The fermentation

Biomass of interest for cellulosic produced ethanol includes wheat straw, rape straw and macro algae. In 2009 the production of oilseed rape in EU was 21×106 ton together with an even larger amount of rape straw (Eurostat 2010). The most commonly used rape in Europe is a winter rape with a low erucic acid and glucosinolate content (Wittkop et. al. 2009). The rape straw is composed of 32% cellulose and 22% hemicelluloses. In this chapter the focus

on waste products is commonly referred to as 2nd generation bioethanol.

will be on the fermentation of sugars derived from cellulose.

**1. Introduction**

4. Environment 5. Poverty

6. Terrorism and War

1. Energy 2. Water 3. Food

7. Disease 8. Education 9. Democracy 10. Population

to face in the next 50 years.

Anders Thygesen1, Lasse Vahlgren1, Jens Heller Frederiksen1,

*1Biosystems Division, Risø National Laboratory for Sustainable Energy,*

### **SSF Fermentation of Rape Straw and the Effects of Inhibitory Stress on Yeast**

Anders Thygesen1, Lasse Vahlgren1, Jens Heller Frederiksen1, William Linnane2 and Mette H. Thomsen3

*1Biosystems Division, Risø National Laboratory for Sustainable Energy, Technical University of Denmark 2Roskilde University 3Chemical Engineering Program, Madsar Institute 1,2Denmark 3United Arab Emirates*

#### **1. Introduction**

In 2003 R. E. Smalley (Smalley 2003) made a list of the top 10 problems mankind was going to face in the next 50 years.

1. Energy

208 Bioethanol

Xu, Q.; Singh, A. & Himmel, ME. (2009). Perspectives and new directions for the production

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*bicolor L*. Moench). *Genome*, Vol. 43, pp. 461-469, 0831-2796

of bioethanol using consolidated bioprocessing of lignocellulose. *Current Opinion in*

Molecular mapping of QTLs conferring stay-green in grain sorghum (*Sorghum*

complementation of a uracil auxotroph of the hyper lignin-degrading basidiomycete *Phanerochaete sordida* YK-624. *Applied Microbiology and Biotechnology,*

With the declining amount of fossil fuels, and the increasing energy demand, it is not possible to satisfy our large energy consumption without alternative energy sources, especially sustainable ones that also take care of problem number 4, (Environment). One of the possible ways to produce a liquid sustainable energy source is to replace our large gasoline demand with fermented biomass (bioethanol). To ensure that the food availability does not decrease (problem 3), the biomass for bioethanol fermentation is only gathered from waste materials of the food production, such as the straw of cereals. The fermentation on waste products is commonly referred to as 2nd generation bioethanol.

Biomass of interest for cellulosic produced ethanol includes wheat straw, rape straw and macro algae. In 2009 the production of oilseed rape in EU was 21×106 ton together with an even larger amount of rape straw (Eurostat 2010). The most commonly used rape in Europe is a winter rape with a low erucic acid and glucosinolate content (Wittkop et. al. 2009). The rape straw is composed of 32% cellulose and 22% hemicelluloses. In this chapter the focus will be on the fermentation of sugars derived from cellulose.

was added.

**2.2 Wet oxidation of rape straw**

reaction products (Thomsen et. al. 2009).

**2.3 SSF fermentation and enzymatic hydrolysis**

SSF Fermentation of Rape Straw and the Effects of Inhibitory Stress on Yeast 211

In order to verify that erioglaucine is equal in quality to methylene blue, a dose response curve for the toxic compound fufuryl alcohol is produced, and the mortality is then examined with the two color agents. 6 fermentations of 100 ml are started with different levels of furfuryl alcohol (0.0, 1.5, 3.5, 6.0, 9.0 and 15.0) (mL/L) and 2 g/L Turbo yeast (from AlcoTec). All the fermentations are completed in blue cap flasks in Millipore water and with 100 g/L glucose monohydrate. The yeast is left to ferment at 25, 32, 40 and 45C, using 100 rpm of stirring for 24 hours. Weight loss is measured during working hours to monitor the fermentation. After 24 hours samples are taken for HPLC analysis as described by Thomsen et al. (2009). A methylene blue and an erioglaucine solution are produced each of 0.29 g/L. The 6 samples are divided into 12 portions to perform double repetition. Six portions were mixed with methylene blue in a 9:1 ratio and six portions with erioglaucine in a 1:1 ratio. The samples are examined with microscopy at 1000x magnification just after the color agent

Rape straw was produced after harvest during cultivation of *Brassica napus Linnaeus* commonly known as rape in Denmark in 2007. The straw was milled to a particle size of 2 mm using a knife mill. The milled straw was then soaked in 80°C hot water for 20 min before wet oxidation in a 2 L loop autoclave (Bjerre et al., 1996). The autoclave setup includes 1 L water, 60 g dry milled rape straw with the canister pressurized to 12 bar oxygen during the reaction. The mixing of oxygen and liquid is obtained by a pumping wheel. The wet oxidation was performed at 205°C for 3 min (Arvaniti et. al 2011). Following the wet oxidation, pressure is released and the prehydrolysate is cooled to room temperature. The

During wet oxidation, oxygen is introduced in the pre-treatment phase at high pressure and temperature. This causes 50 % (w/w) of the lignin to oxidize into CO2, H2O, carboxylic acids and phenolic compounds. It is important that the lignin fraction is low since lignin can denaturize the enzymes involved in the subsequent hydrolysis of cellulose. The majority of the hemicellulose (80 %) is dissolved while 10 % is oxidized to CO2, H2O and carboxylic acids. During this process, carboxylic acids, phenolic compounds, and furans are produced, which act as inhibitors in the fermentation process. However, the concentrations are too low to fully hinder microbial growth as wet oxidation also degrades the toxic intermediate

The dry matter content (DM) of the solid straw fraction after wet oxidation (filtercake) is measured by drying at 105°C overnight. The procedure for SSF is as follows: 250 mL flasks are loaded with 100 ml substrate with either 17 g DM or with 8 g DM. The substrate is adjusted to pH 4.8 with 6 mol/L of NaOH. After pH adjustment, 15 FPU/g DM of Celluclast (Novozymes mixture of endo- and exo-glucanase) is added together with 0.20 ml of Novozym 188 (βglycosidase) per ml Celluclast using sterile conditions. The flasks are shaken at 50 C with 120 rpm for 24 hours during the pre-hydrolysis. After the pre-hydrolysis, 10FPU/g DM of Celluclast and 0.20 ml of Novozym 188 (β-glycosidase) per ml Celluclast are added together with 1-4 g/L of Turbo yeast and 0.8 g/L of urea and the pH value re-adjusted to 4.8 with addition of NaOH. The flasks are sealed with glycerol yeast locks and incubated at 37°C with shaking at 120 rpm. During SSF, the flasks are weighed to measure ethanol production with respect to time, and after the SSF termination a sample of the supernatant is analyzed by

filter cake and filtrate are separated and stored at -18° C (Thygesen et. al. 2003).

The enzymatic hydrolysis can be described in three steps: Endoglucanase separates chains of cellulose by breaking down the bonds in amorphous regions of the crystalline cellulose, thereby creating more free ends in the cellulose. Exoglucanase breaks the cellulose down from the non-reducing end into cellubiose (the disaccharide derived from cellobiose) (Teeri and Koivula 1995). This explains the importance of endoglucanase as it creates more "attack points" for exoglucanase. β-glycosidase breaks down cellubiose into glucose. Cellulase enzymes are commonly produced by the fungus *Trichoderma reesei* (Busto et al. 1996). This process has two functions, first it produces the glucose needed for the fermentation, and second it turns the non-soluble cellulose into soluble sugars, which provides the liquid medium required for fermentation.

The fiber structure consists of cellulose microfibrils, bound to each other with hemicellulose and lignin. A model of a plant fibre is shown in Fig. 1.

The hemicellulose content consists of branched and acetylated carbohydrates. These molecules consist of 90% xylan and 10% arabinan in wheat straw (Puls and Schuseil 1993). The lignin content of the straw consists of polymerized molecules with a phenolic structure. The ethanol production process was conducted by simultaneous saccharification of cellulose with cellulase enzymes, and fermentation of the produced glucose with Turbo yeast (*Saccharomyces cerevisiae, Brewer's yeast*) (SSF) (Thomsen et al. 2009).

The production efficiency of the fermentation is strongly dependant on the wellbeing of the yeast. To visualize the health of the yeast, microscopic tests using blue staining were conducted. The bioethanol is produced from wet oxidized rape straw and the effect of the important fermentation inhibitor furfuryl alcohol is tested.

#### **2. Materials and methods**

#### **2.1 Furfuryl alcohol effect on glucose fermentation and microscopy**

To assess the number of inactive yeast cells "blue staining" is usually done. Methylene blue is the most commonly used color agent. It is a redox indicator that turns colorless in the presence of the active enzymes produced by the yeast *Saccharomyces cerevisiae*. For verification of the results this compound is compared with erioglaucine (E133) (Brilliant blue No. 1).

In order to verify that erioglaucine is equal in quality to methylene blue, a dose response curve for the toxic compound fufuryl alcohol is produced, and the mortality is then examined with the two color agents. 6 fermentations of 100 ml are started with different levels of furfuryl alcohol (0.0, 1.5, 3.5, 6.0, 9.0 and 15.0) (mL/L) and 2 g/L Turbo yeast (from AlcoTec). All the fermentations are completed in blue cap flasks in Millipore water and with 100 g/L glucose monohydrate. The yeast is left to ferment at 25, 32, 40 and 45C, using 100 rpm of stirring for 24 hours. Weight loss is measured during working hours to monitor the fermentation. After 24 hours samples are taken for HPLC analysis as described by Thomsen et al. (2009). A methylene blue and an erioglaucine solution are produced each of 0.29 g/L. The 6 samples are divided into 12 portions to perform double repetition. Six portions were mixed with methylene blue in a 9:1 ratio and six portions with erioglaucine in a 1:1 ratio. The samples are examined with microscopy at 1000x magnification just after the color agent was added.

#### **2.2 Wet oxidation of rape straw**

210 Bioethanol

The enzymatic hydrolysis can be described in three steps: Endoglucanase separates chains of cellulose by breaking down the bonds in amorphous regions of the crystalline cellulose, thereby creating more free ends in the cellulose. Exoglucanase breaks the cellulose down from the non-reducing end into cellubiose (the disaccharide derived from cellobiose) (Teeri and Koivula 1995). This explains the importance of endoglucanase as it creates more "attack points" for exoglucanase. β-glycosidase breaks down cellubiose into glucose. Cellulase enzymes are commonly produced by the fungus *Trichoderma reesei* (Busto et al. 1996). This process has two functions, first it produces the glucose needed for the fermentation, and second it turns the non-soluble cellulose into soluble sugars, which provides the liquid

The fiber structure consists of cellulose microfibrils, bound to each other with hemicellulose

Fig. 1. Structure of cellulosic fibers in e.g. rape straw (adapted from Bjerre & Schmidt 1997) The hemicellulose content consists of branched and acetylated carbohydrates. These molecules consist of 90% xylan and 10% arabinan in wheat straw (Puls and Schuseil 1993). The lignin content of the straw consists of polymerized molecules with a phenolic structure. The ethanol production process was conducted by simultaneous saccharification of cellulose with cellulase enzymes, and fermentation of the produced glucose with Turbo yeast

The production efficiency of the fermentation is strongly dependant on the wellbeing of the yeast. To visualize the health of the yeast, microscopic tests using blue staining were conducted. The bioethanol is produced from wet oxidized rape straw and the effect of the

To assess the number of inactive yeast cells "blue staining" is usually done. Methylene blue is the most commonly used color agent. It is a redox indicator that turns colorless in the presence of the active enzymes produced by the yeast *Saccharomyces cerevisiae*. For verification of the results this compound is compared with erioglaucine (E133) (Brilliant

(*Saccharomyces cerevisiae, Brewer's yeast*) (SSF) (Thomsen et al. 2009).

**2.1 Furfuryl alcohol effect on glucose fermentation and microscopy**

important fermentation inhibitor furfuryl alcohol is tested.

**2. Materials and methods**

blue No. 1).

medium required for fermentation.

and lignin. A model of a plant fibre is shown in Fig. 1.

Rape straw was produced after harvest during cultivation of *Brassica napus Linnaeus* commonly known as rape in Denmark in 2007. The straw was milled to a particle size of 2 mm using a knife mill. The milled straw was then soaked in 80°C hot water for 20 min before wet oxidation in a 2 L loop autoclave (Bjerre et al., 1996). The autoclave setup includes 1 L water, 60 g dry milled rape straw with the canister pressurized to 12 bar oxygen during the reaction. The mixing of oxygen and liquid is obtained by a pumping wheel. The wet oxidation was performed at 205°C for 3 min (Arvaniti et. al 2011). Following the wet oxidation, pressure is released and the prehydrolysate is cooled to room temperature. The filter cake and filtrate are separated and stored at -18° C (Thygesen et. al. 2003).

During wet oxidation, oxygen is introduced in the pre-treatment phase at high pressure and temperature. This causes 50 % (w/w) of the lignin to oxidize into CO2, H2O, carboxylic acids and phenolic compounds. It is important that the lignin fraction is low since lignin can denaturize the enzymes involved in the subsequent hydrolysis of cellulose. The majority of the hemicellulose (80 %) is dissolved while 10 % is oxidized to CO2, H2O and carboxylic acids. During this process, carboxylic acids, phenolic compounds, and furans are produced, which act as inhibitors in the fermentation process. However, the concentrations are too low to fully hinder microbial growth as wet oxidation also degrades the toxic intermediate reaction products (Thomsen et. al. 2009).

#### **2.3 SSF fermentation and enzymatic hydrolysis**

The dry matter content (DM) of the solid straw fraction after wet oxidation (filtercake) is measured by drying at 105°C overnight. The procedure for SSF is as follows: 250 mL flasks are loaded with 100 ml substrate with either 17 g DM or with 8 g DM. The substrate is adjusted to pH 4.8 with 6 mol/L of NaOH. After pH adjustment, 15 FPU/g DM of Celluclast (Novozymes mixture of endo- and exo-glucanase) is added together with 0.20 ml of Novozym 188 (βglycosidase) per ml Celluclast using sterile conditions. The flasks are shaken at 50 C with 120 rpm for 24 hours during the pre-hydrolysis. After the pre-hydrolysis, 10FPU/g DM of Celluclast and 0.20 ml of Novozym 188 (β-glycosidase) per ml Celluclast are added together with 1-4 g/L of Turbo yeast and 0.8 g/L of urea and the pH value re-adjusted to 4.8 with addition of NaOH. The flasks are sealed with glycerol yeast locks and incubated at 37°C with shaking at 120 rpm. During SSF, the flasks are weighed to measure ethanol production with respect to time, and after the SSF termination a sample of the supernatant is analyzed by

$$\rm{C}\_{6}H\_{12}O\_{6} \to \rm{2C}\_{2}H\_{5}OH + \rm{2CO}\_{2} \tag{1}$$

$$\text{Weight of } ethanol = weightloss \times \frac{\text{MW} \{CH}\_3CH\_2OH\}}{\text{MW} \{CO\_2\}} \tag{2}$$

$$ECE\% = \frac{E thanol\ concentration\ after\ SSF}{E thanol\ potential\ of\ biomass\ in\ SSF} \tag{3}$$

$$n\left(\mathbb{C}\_6H\_{10}O\_5\right)\_nH\_2O + (n-1)H\_2O \to n\mathbb{C}\_6H\_{12}O\_6\tag{4}$$

$$Yield\_{ethanol\,from\,cell\,luse} = \frac{2\,\text{M}\,\text{w}\_{\{C\_2H\_6O\}}}{\text{M}\,\text{w}\_{\{C\_6H\_{10}O\_5\}}} = \frac{2 \times 46.1}{162} = 0.57\,\text{g/g}\tag{5}$$

SSF Fermentation of Rape Straw and the Effects of Inhibitory Stress on Yeast 215

Fig. 4. The amount of ethanol produced during SSF of wet oxidized rape straw using 170

Yeast CO2 Ethanol Ethanol potential ECE g/L g/L g/L g/L % **170 g/L straw DM 34.9 51.9 67.2**  2 32.3 33.8 51.9 65.2 4 30.3 31.7 51.9 61.1 6 33.7 35.3 51.9 68.0 8.2 33.9 35.5 51.9 68.4 **80 g/L straw DM** 18.4 24.5 75.2 2 16.8 17.6 24.5 71.8 4 17.8 18.6 24.5 76.1 6 17.8 18.6 24.5 76.1 8.2 16.8 17.6 24.5 71.8

Table 2. Ethanol production and ECE (ethanol conversion efficiency) for different DM contents As shown in table 2, the high DM content result in low ECE% of 67% compared to 75% when using low DM content. This is an essential problem in ethanol production since industrial distillation works best with more than 50 g/L ethanol, which could potentially be achieved with 75% ECE and 220 g/L of rape straw. However, in reality this is a challenge since increased DM contents result in reduced ECE% due to increasing viscosity and difficulties in mixing and higher concentrations of inhibitors. No direct correspondence between the final ECE% and the initial amount of yeast is found in this study. This indicates that the drop in ECE% at high DM can be due to decreasing enzyme performance in high

The rate of fermentation is calculated for the time period with the highest fermentation rate and the results are shown in Fig. 5. For 170 g/L DM content that is between 10 and 52 hours, and for 80 g/L DM content it is between 2 and 4 hours. This time period covers the phase

g/L DM and initial yeast concentrations of (2.0 to 8.2) g/L.

DM pre-hyrolysates of rape straw.

Table 1. Components of rape straw before and after the wet oxidation pretreatment resulting in a solid and a liquid phase. \*NCWM is none cell wall material

Fig. 3. The amount of ethanol produced on wet oxidized rape straw using 80 g/L DM and yeast concentrations of (2.0 to 8.2) g/L.

(rate of hydrolysis). Since the enzyme loading was the same in all the samples the fermentations produce ethanol at roughly the same rate once the excess sugar has been used. In experiments with high dry matter content such as 170 g/L the difference between the yeast dependant and the enzyme dependant phase is more pronounced than with low DM content (Fig. 4). The viscosity of the sample changes drastically during SSF since hydrolysis changes insoluble cellulose to soluble glucose. This means that the rate of both the yeasts and the enzymes production increases over time, as the production rate of both the enzymes and the yeast is related to the viscosity of the medium. An explanation can be that a high concentration of dry matter gives higher inhibitor concentrations and a larger yeast concentration can make faster detoxification.

Rape straw Solid phase Liquid phase Compound g/100 g DM Compound g/100 g DM Compound g/L Cellulose 32 Cellulose 54 Glucose 1.1 Hemicellulose 16 Xylan 13 Xylose 6.7

Lignin 18 Lignin 23 Acetic acid 0.9 Ash 5 Ash 3 Formic acid 0.9 NCWM 20 NCWM 13 Furfural 0.1 Phenolics 1.3

Table 1. Components of rape straw before and after the wet oxidation pretreatment resulting

Fig. 3. The amount of ethanol produced on wet oxidized rape straw using 80 g/L DM and

(rate of hydrolysis). Since the enzyme loading was the same in all the samples the fermentations produce ethanol at roughly the same rate once the excess sugar has been used. In experiments with high dry matter content such as 170 g/L the difference between the yeast dependant and the enzyme dependant phase is more pronounced than with low DM content (Fig. 4). The viscosity of the sample changes drastically during SSF since hydrolysis changes insoluble cellulose to soluble glucose. This means that the rate of both the yeasts and the enzymes production increases over time, as the production rate of both the enzymes and the yeast is related to the viscosity of the medium. An explanation can be that a high concentration of dry matter gives higher inhibitor concentrations and a larger yeast

in a solid and a liquid phase. \*NCWM is none cell wall material

yeast concentrations of (2.0 to 8.2) g/L.

concentration can make faster detoxification.

Arabinan 1 Arabinose 0.5

pH 3.9

Fig. 4. The amount of ethanol produced during SSF of wet oxidized rape straw using 170 g/L DM and initial yeast concentrations of (2.0 to 8.2) g/L.

Table 2. Ethanol production and ECE (ethanol conversion efficiency) for different DM contents

As shown in table 2, the high DM content result in low ECE% of 67% compared to 75% when using low DM content. This is an essential problem in ethanol production since industrial distillation works best with more than 50 g/L ethanol, which could potentially be achieved with 75% ECE and 220 g/L of rape straw. However, in reality this is a challenge since increased DM contents result in reduced ECE% due to increasing viscosity and difficulties in mixing and higher concentrations of inhibitors. No direct correspondence between the final ECE% and the initial amount of yeast is found in this study. This indicates that the drop in ECE% at high DM can be due to decreasing enzyme performance in high DM pre-hyrolysates of rape straw.

The rate of fermentation is calculated for the time period with the highest fermentation rate and the results are shown in Fig. 5. For 170 g/L DM content that is between 10 and 52 hours, and for 80 g/L DM content it is between 2 and 4 hours. This time period covers the phase

SSF Fermentation of Rape Straw and the Effects of Inhibitory Stress on Yeast 217

Fig. 6. Relative correspondence between added furfuryl alcohol and ethanol production

**Ethanol conc.(g/L)**

0.0 15.0 42.4 0.5 21.0 38.7 1.0 25.7 32.4 1.5 27.2 33.4 2.0 25.0 37.2 2.5 21.6 45.2 Table 3. Final ethanol and glucose concentrations in yeast fermentations with added furfuryl

to approximately 80% increase in ethanol production given that sufficient amounts of glucose are available. For SSF the amount of glucose needed for this effect to be visible is only present in the start of the SSF which is also where the positive feedback effect of the

To describe the effect of lacking inhibitory stress on the yeast the maximum fermentation rate with 2 g/L yeast of both 80 g/L DM and 170 g/L DM in the SSF experiments with rape

The rate of fermentation for the furfuryl alcohol experiment is calculated for the period of 5 to 19 hours, which is estimated to be the highest rate of fermentation for the experiment. As shown in Fig. 7, it seems that even though microbial inhibitors are generally restricting the fermentation process, a medium completely without inhibitors (such as the control from Fig. 8) will have a decreased fermentation rate compared to medium with a small dose of

**Glucose (g/L)**

during 24h of fermentation in a glucose medium.

yeasts fermentation rate is mainly present.

straw are compared and shown in Fig. 7.

microbial inhibitors.

alcohol

**Dose (mL/L)**

Fig. 5. Rate of ethanol fermentation with respect to the concentration of yeast

where the excess sugars from the hydrolysis is fermented and the positive feedback effect seen in high DM contents is also expressed. As shown in Fig. 5 generally the maximum rate of fermentation increases as a function of the yeast concentration. The slope coefficient of 80 g/L DM was found by linear regression to be 0.48 h-1 while it was found to be 0.20 h-1 at 170 g/L DM. The amount of yeast therefore contributes strongly to the positive feedback effect as explained in Fig. 4 and in this time period the dependency is also due to the fact that high yeast content can simply ferment the excess sugar from the hydrolysis faster than low contents of yeast.

#### **3.4 The effect of furfuryl alcohol on yeast**

The obtained concentration of ethanol versus the concentration of furfuryl alcohol is shown in Fig. 6. The amount of produced ethanol increased from 10 g/L with pure glucose to 24 g/ L at the dose of 1.5 mL/L furfuryl alcohol. At higher concentrations a decrease in ethanol is observed with almost no ethanol being produced at the dose of 15 mL/L. This indicates that with the presence of small doses of furfuryl alcohol, ethanol production can increase.

To provide information about the sharp increase between measurement one and two, another experiment was conducted with the same method as the previous, except the dose of furfuryl alcohol were (0.0, 0.5, 1.0, 1.5, 2.0 and 2.5) mL/L. Samples of this experiment were analyzed by HPLC and the results are shown in table 3.

As shown in table 3, the fermentation will peak with a dose of 1.5 mL/L and at levels above that the amount of ethanol produced will decrease versus the concentration of furfuryl alcohol. It was further shown that there was plenty of leftover glucose for the yeast to ferment. Therefore in none of the cases the loss in fermentation rate was due to lack of glucose.

As shown in Table 3 there is a strong correspondence between the amount of ethanol produced and the dose of furfuryl alcohol. Furfuryl alcohol is a microbial growth inhibitor, but when added in small concentrations it will force the yeast cells to increase their metabolism to survive. This effect should be present for the norm of microbial inhibitors. Despite of the fact that furfuryl alcohol is toxic to the yeast, the stress that it causes can lead

Fig. 5. Rate of ethanol fermentation with respect to the concentration of yeast

contents of yeast.

glucose.

**3.4 The effect of furfuryl alcohol on yeast**

were analyzed by HPLC and the results are shown in table 3.

where the excess sugars from the hydrolysis is fermented and the positive feedback effect seen in high DM contents is also expressed. As shown in Fig. 5 generally the maximum rate of fermentation increases as a function of the yeast concentration. The slope coefficient of 80 g/L DM was found by linear regression to be 0.48 h-1 while it was found to be 0.20 h-1 at 170 g/L DM. The amount of yeast therefore contributes strongly to the positive feedback effect as explained in Fig. 4 and in this time period the dependency is also due to the fact that high yeast content can simply ferment the excess sugar from the hydrolysis faster than low

The obtained concentration of ethanol versus the concentration of furfuryl alcohol is shown in Fig. 6. The amount of produced ethanol increased from 10 g/L with pure glucose to 24 g/ L at the dose of 1.5 mL/L furfuryl alcohol. At higher concentrations a decrease in ethanol is observed with almost no ethanol being produced at the dose of 15 mL/L. This indicates that

To provide information about the sharp increase between measurement one and two, another experiment was conducted with the same method as the previous, except the dose of furfuryl alcohol were (0.0, 0.5, 1.0, 1.5, 2.0 and 2.5) mL/L. Samples of this experiment

As shown in table 3, the fermentation will peak with a dose of 1.5 mL/L and at levels above that the amount of ethanol produced will decrease versus the concentration of furfuryl alcohol. It was further shown that there was plenty of leftover glucose for the yeast to ferment. Therefore in none of the cases the loss in fermentation rate was due to lack of

As shown in Table 3 there is a strong correspondence between the amount of ethanol produced and the dose of furfuryl alcohol. Furfuryl alcohol is a microbial growth inhibitor, but when added in small concentrations it will force the yeast cells to increase their metabolism to survive. This effect should be present for the norm of microbial inhibitors. Despite of the fact that furfuryl alcohol is toxic to the yeast, the stress that it causes can lead

with the presence of small doses of furfuryl alcohol, ethanol production can increase.

Fig. 6. Relative correspondence between added furfuryl alcohol and ethanol production during 24h of fermentation in a glucose medium.

Table 3. Final ethanol and glucose concentrations in yeast fermentations with added furfuryl alcohol

to approximately 80% increase in ethanol production given that sufficient amounts of glucose are available. For SSF the amount of glucose needed for this effect to be visible is only present in the start of the SSF which is also where the positive feedback effect of the yeasts fermentation rate is mainly present.

To describe the effect of lacking inhibitory stress on the yeast the maximum fermentation rate with 2 g/L yeast of both 80 g/L DM and 170 g/L DM in the SSF experiments with rape straw are compared and shown in Fig. 7.

The rate of fermentation for the furfuryl alcohol experiment is calculated for the period of 5 to 19 hours, which is estimated to be the highest rate of fermentation for the experiment. As shown in Fig. 7, it seems that even though microbial inhibitors are generally restricting the fermentation process, a medium completely without inhibitors (such as the control from Fig. 8) will have a decreased fermentation rate compared to medium with a small dose of microbial inhibitors.

SSF Fermentation of Rape Straw and the Effects of Inhibitory Stress on Yeast 219

During the experiments with dose response on furfuryl alcohol in glucose fermentation, a relation was found between cell death and the concentration of furfuryl alcohol (Fig. 8). Yet at this writing, there still exist some inconsistencies between erioglaucine and methylene blue. Overall the results produced by methylene blue appeared slightly more consistent and

In this study the main focus was on production of bioethanol from rape straw, but there are a lot of other possible feedstocks suitable for bioethanol production in general, all cellulosic material can be converted by physical/chemical pretreatment followed by enzymatic hydrolysis into glucose. It is also possible to produce bioethanol from sugar and starch from crops such as corn, wheat, sugarcane, and sugar beet*,* but since sugar is a food source, using it could decrease food availability for future generations. Using food sources or available agricultural land for pure energy production is generally classified as a 1st generation technology, and is not normally regarded as a sustainable energy source. To compare the different feedstocks Table 4 is produced. However, there are numerous ways to co-produce food and feedstocks for bioenergy when utilizing the lignocellulosic residues from

As table 4 shows, bast fibers have a very high cellulose content (60-63%) and a low lignin content (3-4 %) which should make them ideal for producing bioethanol, but bast fibers as a feedstock would fall under the category of 1st generation bioethanol, because the production of bast fibers requires land that could otherwise be used for food production. Rape straw has a low cellulose content compared to other straw fibers (32%) but in return the hemicellulose (14%) and lignin (18%) content is also low compared to wheat straw (20%) and corn stover (19 – 21%). Low lignin content is good for the enzymatic hydrolysis, since lignin can denaturize cellulase enzymes (Thygesen et. al 2003). Low hemicellulose content will result in a slightly lower concentration of microbial growth inhibitors derived from oxidation of the hemicellulose. Sugarcane bagasse seems to be the ideal 2nd generation feedstock with its high cellulose content (43%) and low lignin content (11%) but sugarcanes require high temperatures and a lot of rainfall to grow and are therefore only energy efficient when grown in tropical regions, which limits the amount of ethanol produced from sugarcane bagasse worldwide. It is possible to produce bioethanol from wood fibers, like waste wood from carpentry or

Fig. 9. Methylene blue stained yeast cells after heating to 100 ºC.

matching with the data for ethanol production.

agricultural production as shown in table 4.

**3.6 Comparison of feed stocks**

Fig. 7. Fermentation rate with respect to dose of furfuryl alcohol from the glucose experiment from Fig 6. For comparison the fermentation rate with rape straw of both 80 g/L DM and 170 g/L DM content from Fig. 5 is investigated for similar yeast concentrations (2g/L).

#### **3.5 Vitality of the yeast cells**

Fig. 8 shows the yeast cells after a glucose fermentation. A large part of the yeast cells have become inactive, even though there is still enough sugar in the substrate to sustain living cells.

Fig. 8. Methylene blue stained yeast cells after glucose fermentation.

Fig. 9. Methylene blue stained yeast cells after heating to 100 ºC.

During the experiments with dose response on furfuryl alcohol in glucose fermentation, a relation was found between cell death and the concentration of furfuryl alcohol (Fig. 8). Yet at this writing, there still exist some inconsistencies between erioglaucine and methylene blue. Overall the results produced by methylene blue appeared slightly more consistent and matching with the data for ethanol production.

#### **3.6 Comparison of feed stocks**

218 Bioethanol

Fig. 7. Fermentation rate with respect to dose of furfuryl alcohol from the glucose experiment from Fig 6. For comparison the fermentation rate with rape straw of both 80 g/L DM and 170

Fig. 8 shows the yeast cells after a glucose fermentation. A large part of the yeast cells have become inactive, even though there is still enough sugar in the substrate to sustain living

g/L DM content from Fig. 5 is investigated for similar yeast concentrations (2g/L).

Fig. 8. Methylene blue stained yeast cells after glucose fermentation.

**3.5 Vitality of the yeast cells**

cells.

In this study the main focus was on production of bioethanol from rape straw, but there are a lot of other possible feedstocks suitable for bioethanol production in general, all cellulosic material can be converted by physical/chemical pretreatment followed by enzymatic hydrolysis into glucose. It is also possible to produce bioethanol from sugar and starch from crops such as corn, wheat, sugarcane, and sugar beet*,* but since sugar is a food source, using it could decrease food availability for future generations. Using food sources or available agricultural land for pure energy production is generally classified as a 1st generation technology, and is not normally regarded as a sustainable energy source. To compare the different feedstocks Table 4 is produced. However, there are numerous ways to co-produce food and feedstocks for bioenergy when utilizing the lignocellulosic residues from agricultural production as shown in table 4.

As table 4 shows, bast fibers have a very high cellulose content (60-63%) and a low lignin content (3-4 %) which should make them ideal for producing bioethanol, but bast fibers as a feedstock would fall under the category of 1st generation bioethanol, because the production of bast fibers requires land that could otherwise be used for food production. Rape straw has a low cellulose content compared to other straw fibers (32%) but in return the hemicellulose (14%) and lignin (18%) content is also low compared to wheat straw (20%) and corn stover (19 – 21%). Low lignin content is good for the enzymatic hydrolysis, since lignin can denaturize cellulase enzymes (Thygesen et. al 2003). Low hemicellulose content will result in a slightly lower concentration of microbial growth inhibitors derived from oxidation of the hemicellulose. Sugarcane bagasse seems to be the ideal 2nd generation feedstock with its high cellulose content (43%) and low lignin content (11%) but sugarcanes require high temperatures and a lot of rainfall to grow and are therefore only energy efficient when grown in tropical regions, which limits the amount of ethanol produced from sugarcane bagasse worldwide. It is possible to produce bioethanol from wood fibers, like waste wood from carpentry or

SSF Fermentation of Rape Straw and the Effects of Inhibitory Stress on Yeast 221

relevance when running SSF with high DM content. After prolonged testing of Turbo yeast, the optimal temperature of the SSF is found to be 37°C. Furfuryl alcohol and possibly other growth inhibitors as well, show a positive effect on the rate of fermentation when added in small dosages, since yeast will increase its metabolism under stress. The positive effect of growth inhibitors is so strong that the fermentation rate in sugar media is lower than the fermentation rates in a medium produced from wet oxidized rape straw (filter cake), given

The Danish Research Council, DSF is gratefully acknowledged for supporting the research project: Biorefinery for sustainable reliable economical fuel production from energy crops (2104-06-0004). The European Union is acknowledged for supporting the EU-project: Integration of biology and engineering into an economical and energy-efficient 2G bioethanol biorefinery (Proethanol nr. 251151). Efthalia Arvaniti is acknowledged for academic advice. Tomas Fernqvist, Ingelis Larsen and Annette Eva Jensen are thanked for

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EUROSTAT, 2010. *Agricultural statistics - Main results 2008-09.* Eurostat. pp. 83-92. ISBN: 978-

http://epp.eurostat.ec.europa.eu/portal/page/portal/product\_details/publicatio

Hänninen, T.; Thygesen, A.; Mehmood, S.; Madsen, B. & Hughes, M. (2011). Effect of

Kaar, W.E.; Cool, L.G.; Merriman, M.M. & Brink, D.L. (1991). The complete analysis of wood

Martin, C.; Klinke, H.B. & Thomsen, A.B. (2007). Wet oxidation as a pretreatment method

saccharification and fermentation conditions for ethanol production from pre-

*production of bioethanol: optimization of the wet oxidation process and characterization of products.* Technical university of Denmark, Risø-R-967, ISBN 87-550-2279-0,

cellulases induced in *Trichoderma reesei* cultures. *Bioresource Technology,* Vol.57,

mechanical damage on the susceptibility of flax and hemp fibres to chemical

polysaccharides using HPLC. *Journal of Wood Chemistry and Technology*, Vol.11,

for enhancing the enzymatic convertibility of sugarcane bagasse. *Enzyme and Microbial Technology*, Vol.40, No.3, (February 2007), pp. 426-432, ISSN 01410229. Fai, P.B. & Grant, A. (2009). A comparative study of *Saccharomyces cerevisae* sensitivity

against eight yest species sensitivities to a range of toxicants. *Chemosphere*, Vol.75,

technical assistance and HTX Roskilde for providence of microscope cameras.

the DM concentration does not exceed critical levels.

treated rape straw. *Unpublished data*.

of John Wiley & Sons, New York, USA.

n?p\_product\_code=KS-ED-10-001

No.4, pp. 447-463, ISSN 02773813.

No.3, (April 2009), pp. 289-296, ISSN 00456535.

degradation. *Unpublished data*.

No.2, (August 1996), pp. 187-192, ISSN 09608524.

92-79-15246-7 ISSN: 1830-463X doi:10.2785/44845. Available:

Roskilde, Denmark.

**5. Acknowledgement**

**6. References**

Table 4. The composition of cellulose containing and plant -based raw materials including straw, wood, marine biomass and bast fibers. The individual data comes from the following sources: 1. Thygesen et. al. 2005, 2. Petersson et. al. 2007, 3. Martin et. al. 2007, 4. Schultz-Jensen et. al. 2010, 5. Schultz-Jensen et. al. 2011, 6. Hänninen et. al. 2011.

willow, which can grow on land not suitable for agriculture, using pretreatment methods such as steam explosion (Söderström et. al. 2002). The high lignin content in wood fibers increases the amount of enzymes needed and the time period of the fermentation. Furthermore wood fibers have other uses and can easily be burned to produce electricity and heat in a cogeneration plant.

Marine biomass has the advantage that it does not use the same space as agriculture and even though it is not a waste product from food production it is still a viable feedstock because it does not reduce food availability. *Chaetomorpha linum* has very low lignin content (6 – 8 %) and cellulose content similar to straw fibers (34 – 40 %). *C. linum* and other types of useable macroalgae are easy to grow in most of the world, and is therefore a suitable candidate for expanding the bioethanol production to more than what can be obtained from waste products (Schultz-Jensen et. al. 2011).

#### **4. Conclusion**

The amount of yeast needed for SSF of pretreated rape straw is dependent on the DM content, despite the fact that enzymes continue to be the primary rate-determining factor. The positive feedback effect from the yeast lowering the sugar concentration can have high relevance when running SSF with high DM content. After prolonged testing of Turbo yeast, the optimal temperature of the SSF is found to be 37°C. Furfuryl alcohol and possibly other growth inhibitors as well, show a positive effect on the rate of fermentation when added in small dosages, since yeast will increase its metabolism under stress. The positive effect of growth inhibitors is so strong that the fermentation rate in sugar media is lower than the fermentation rates in a medium produced from wet oxidized rape straw (filter cake), given the DM concentration does not exceed critical levels.

#### **5. Acknowledgement**

220 Bioethanol

Feedstock Cellulose Xylose Arabinose Lignin Ash Ref.

*mays*) 33 Hemicellulose = 21 19 7 1

*napus*) 32 14 2 18 5 2

*cereal*) 41 22 3 16 5 2

(*Triticum*) 39 20 2 20 7 4

(*Picea abies*) 49 Hemicellulose = 20 30 0 1

*usitatissimum)* 60 8 1 3 4 6 Hemp (*Cannabis*) 63 9 1 4 4 1 Table 4. The composition of cellulose containing and plant -based raw materials including straw, wood, marine biomass and bast fibers. The individual data comes from the following sources: 1. Thygesen et. al. 2005, 2. Petersson et. al. 2007, 3. Martin et. al. 2007, 4. Schultz-

willow, which can grow on land not suitable for agriculture, using pretreatment methods such as steam explosion (Söderström et. al. 2002). The high lignin content in wood fibers increases the amount of enzymes needed and the time period of the fermentation. Furthermore wood fibers have other uses and can easily be burned to produce electricity

Marine biomass has the advantage that it does not use the same space as agriculture and even though it is not a waste product from food production it is still a viable feedstock because it does not reduce food availability. *Chaetomorpha linum* has very low lignin content (6 – 8 %) and cellulose content similar to straw fibers (34 – 40 %). *C. linum* and other types of useable macroalgae are easy to grow in most of the world, and is therefore a suitable candidate for expanding the bioethanol production to more than what can be obtained from

The amount of yeast needed for SSF of pretreated rape straw is dependent on the DM content, despite the fact that enzymes continue to be the primary rate-determining factor. The positive feedback effect from the yeast lowering the sugar concentration can have high

Jensen et. al. 2010, 5. Schultz-Jensen et. al. 2011, 6. Hänninen et. al. 2011.

34 - 40 4 - 7 8 - 13 6 - 8 8 - 24 5

(*Saccharum*) 43 Hemicellulose = 31 11 6 3

**Straw fibres**  Corn stover (*Zea*

Rape straw (*Brassica*

Sugarcane Bagasse

Winter rye (*Secale*

Wheat straw

**Wood fibres**  Norway spruce

**Marine biomass**  *Green hairweed (Chaetomorpha linum)*

> **Bast fibres**  Flax (*Linum*

and heat in a cogeneration plant.

**4. Conclusion**

waste products (Schultz-Jensen et. al. 2011).

% w/w % w/w % w/w % w/w % w/w

The Danish Research Council, DSF is gratefully acknowledged for supporting the research project: Biorefinery for sustainable reliable economical fuel production from energy crops (2104-06-0004). The European Union is acknowledged for supporting the EU-project: Integration of biology and engineering into an economical and energy-efficient 2G bioethanol biorefinery (Proethanol nr. 251151). Efthalia Arvaniti is acknowledged for academic advice. Tomas Fernqvist, Ingelis Larsen and Annette Eva Jensen are thanked for technical assistance and HTX Roskilde for providence of microscope cameras.

#### **6. References**

**11**

*France*

**Competing Plant Cell Wall**

*University of Strasbourg*

**Digestion Recalcitrance by Using**

Vincent Phalip, Philippe Debeire and Jean-Marc Jeltsch

**Fungal Substrate- Adapted Enzyme Cocktails**

General needs for energy are still increasing. In 2000, the energy provided worldwide was 10 Gt of oil equivalent (Gtoe) and the demand is forecasted to be around 15 Gtoe for 2020 (source: Energy Information Administration [EIA], 2002, as cited in Scragg, 2005). During the 20th century, coal proportion in energy supply decreased whereas oil and gas increased drastically. First after the 1973 oil crisis and afterwards periodically depending on oil prices, developments for producing energy by new ways were considered. In the last decade, the depletion of fossil energy sources appeared as a reality although exhaustion time remains highly controversial. Currently, it is clear that considerable efforts to promote alternative sources of energy are driven by both environmental concern (limiting fuel by-products

Ethanol from biomass (Bioethanol) is one of these alternative sources. Despite polemics for biomass uses i.e. biofuels vs food and some alarmist politicians' declarations, this alternative is really promising considering (i) the ability to satisfy a significant part of the demand for energy and (ii) biomass renewability. Polemics and limitations could have been almost rational when first generation of biofuels was concerned, "noble parts" of plants, the same used for food, being transformed. It is not the case anymore for any modern project. Another problem raised is the part of the cultivated surfaces to be reserved to biofuels, but in fact, realistic scenario is not to replace all fossil fuels volumes, but only part of them by

The general scheme of "Biomass to ethanol" process is presented elsewhere in this book. Our purpose in this section is to highlight the numerous and various ways to optimize the whole process from biomass to ethanol at different steps: choice of the biomass, pretreatment, enzyme productions, enzymatic hydrolysis, and ethanol fermentation. First of all, as discussed earlier, in the second generation of biofuels, biomass collection should not compete with food plants. Biomass should be abundant and cultural practices as

emissions) and economic necessity linked to the fossil fuel depletion.

**1.3 "Biomass to ethanol" process and review of improvements**

**1.2 Bioethanol among other alternative sources**

**1. Introduction**

**1.1 General needs for energy**

using wastes preferentially.

## **Competing Plant Cell Wall Digestion Recalcitrance by Using Fungal Substrate- Adapted Enzyme Cocktails**

Vincent Phalip, Philippe Debeire and Jean-Marc Jeltsch *University of Strasbourg France*

#### **1. Introduction**

222 Bioethanol

Petersen, M.Ø.; Larsen, J. & Thomsen, M.H. (2009). Optimization of hydrothermal

Petersson, A.; Thomsen, M.H.; Hauggaard-Nielsen, H. & Thomsen, A.B. (2007). Potential

Puls, J. & Schuseil, J. (1993). Chemistry of hemicelluloses: Relationship between

Schultz-Jensen, N.; Leipold, F.; Bindslev, H. & Thomsen, A.B. (2011). Plasma assisted

Schultz-Jensen, N.; Thygesen, A.; Thomsen, S.T.; Leipold, F.; Roslander, C. & Thomsen, A.B.

Söderström, J.; Pilcher, L.; Galbe, M. & Zacchi, G. (2002). Two step steam pretreatment of

Teeri, T.,T. & Koivula, A. (1995). Cellulose degradation by native and engineered fungal

Thomsen, M.H.; Thygesen, A. & Thomsen, A.B. (2009). Identification and characterization of

Thomsen, M.H.; Thygesen, A. & Thomsen, A.B. (2008). Hydrothermal treatment of wheat

Thygesen, A.; Thomsen, A.B.; Schmidt, A.S.; Jørgensen, H.; Ahring, B.K. & Olsson, L. (2003).

Thygesen, A.; Possemiers, S.; Thomsen, A. & Verstraete, W. (2010). Intergration of microbial

*Waste and Biomass Valorization*, Vol.1, No.1, pp. 9-20, ISSN 18772641. Wittkop, B.; Snowdon, R.J. & Friedt, W. (2009). Status and perspectives of breeding for

*Biotechnology*, Vol.98-100, (Spring 2002), pp. 5-21, ISSN 02732289.

cellulases. *Carbohydrates in Europe*, Vol.12, pp.28-33, ISSN 13850040.

*Chaetomorpha Linum* for the production of bioethanol. *Unpublished data*. Smalley, R.E., 2003-last update, Top Ten Problems of Humanity for Next 50 Years. Energy &

(May 2009), pp. 834-840, ISSN 09619534.

December 2007), pp. 812-819, ISSN 09619534.

(February 2011), pp. 558-572, ISSN 02732289.

447–455, ISSN 01757598.

No.6, pp. 563-576, ISSN 09690239.

pp. 131-140, ISSN 00142336.

No.5, (April 2003), pp. 606-615, ISSN 01410229.

Research Monograph, ISBN<PHONE_NUMBER>, Great Britain.

pretreatment of wheat straw for production of bioethanol at low water consumption without addition of chemicals. *Biomass and Bioenergy*, Vol.33, No.5,

bioethanol and biogas production using lignocellulosic biomass from winter rye, oilseed rape and faba bean. *Biomass and Bioenergy*, Vol.31, No.11-12, (November-

hemicellulose structure and enzymes required for hydrolysis, In: *Hemicellulose and Hemicellulases*, M.P. Coughlan & G.P. Hazlewood, (ed.), pp. 1-27, Portland Press

pretreatment of wheat straw. *Applied Biochemistry and Biotechnology*, Vol.163, No.4,

(2011). Investigation of different pretreatment technologies for the macroalgae

NanoTechnology Conference, Rice University, May 3, 2003. Available at http://www.scribd.com/doc/2962283/Richard-Smalleys-energy-talk [15/8-2011, .

softwood with SO2 impregnation for ethanol production. *Applied Biochemistry and*

fermentation inhibitors formed during hydrothermal treatment and following SSF of wheat straw. *Applied Microbiology and Biotechnology*, Vol.83, No.3, (June 2009), pp.

straw at pilot plant scale using a three-step reactor system aiming at high hemicellulose recovery, high cellulose digestibility and low lignin hydrolysis. *Bioresource Technology*, Vol.99, No.10, (July 2008), pp. 4221-4228, ISSN 09608524. Thygesen, A.; Oddershede, J.; Lilholt, H.; Thomsen, A.B. & Ståhl, K. (2005). On the

determination of crystallinity and cellulose content in plant fibres. *Cellulose*, Vol. 12,

Production of cellulose and hemicellulose-degrading enzymes by filamentous fungi cultivated on wet-oxidised wheat straw. *Enzyme and Microbial Technology*, Vol.32,

electrolysis (MECs) in the biorefinery for production of ethanol, H2 and phenolics.

enhanced yield and quality of oilseed crops for Europe. *Euphytica*, Vol.170, No 1-2,

#### **1.1 General needs for energy**

General needs for energy are still increasing. In 2000, the energy provided worldwide was 10 Gt of oil equivalent (Gtoe) and the demand is forecasted to be around 15 Gtoe for 2020 (source: Energy Information Administration [EIA], 2002, as cited in Scragg, 2005). During the 20th century, coal proportion in energy supply decreased whereas oil and gas increased drastically. First after the 1973 oil crisis and afterwards periodically depending on oil prices, developments for producing energy by new ways were considered. In the last decade, the depletion of fossil energy sources appeared as a reality although exhaustion time remains highly controversial. Currently, it is clear that considerable efforts to promote alternative sources of energy are driven by both environmental concern (limiting fuel by-products emissions) and economic necessity linked to the fossil fuel depletion.

#### **1.2 Bioethanol among other alternative sources**

Ethanol from biomass (Bioethanol) is one of these alternative sources. Despite polemics for biomass uses i.e. biofuels vs food and some alarmist politicians' declarations, this alternative is really promising considering (i) the ability to satisfy a significant part of the demand for energy and (ii) biomass renewability. Polemics and limitations could have been almost rational when first generation of biofuels was concerned, "noble parts" of plants, the same used for food, being transformed. It is not the case anymore for any modern project. Another problem raised is the part of the cultivated surfaces to be reserved to biofuels, but in fact, realistic scenario is not to replace all fossil fuels volumes, but only part of them by using wastes preferentially.

#### **1.3 "Biomass to ethanol" process and review of improvements**

The general scheme of "Biomass to ethanol" process is presented elsewhere in this book. Our purpose in this section is to highlight the numerous and various ways to optimize the whole process from biomass to ethanol at different steps: choice of the biomass, pretreatment, enzyme productions, enzymatic hydrolysis, and ethanol fermentation. First of all, as discussed earlier, in the second generation of biofuels, biomass collection should not compete with food plants. Biomass should be abundant and cultural practices as

Competing Plant Cell Wall Digestion Recalcitrance

**2. Diversity of plant cell wall structures**

and variable structure. Adapted from Dalboge (1997).

is shown in Fig. 1.

by Using Fungal Substrate- Adapted Enzyme Cocktails 225

The plant cell wall structures are highly diverse. Various lignocellulosic species have been used for biofuels production, woods, crop by-products, herbaceous plants, beet pulp, municipal and paper industry wastes. Although all these different biomasses contain typically four major components (i.e. cellulose, hemicelluloses, pectin and lignin), the architecture of the cell wall, the fine biochemical structures of these components and their interactions into the cell wall could be quite different. Nevertheless, cellulose and hemicelluloses leading, with lignins, to the formation of an insoluble, tridimensional network is a constant behavior. A schematic drawing of the plant cell wall polysaccharides

> Cellulose (-1,4 glucan)

Rhamnogalacturonan

arabinogalactan

Fig. 1. Schematic representation of plant cell wall polysaccharides. Cellulose and -1,3/1,4 glucan are composed of glucose residues (red). -1,4-xyloglucan is a glucose-based polymer substituted by xylose residues (green) themselves possibly linked to galactose (blue) and fucose (black). -1,4-arabinoxylan is a xylose backbone linked with arabinose (orange), and/or with glucuronic acid acid (white). Xylose could be substituted by acetyl groups (blue circle). -1,4-mannan is a mannose polymer (dark red) linked to some galactose residues and sometimes acetylated. -1,4-polygalacturonan is formed of linear chains of galacturonic acid (pale yellow) linked by rhamnose (pink). Galacturonic residues could be either methylated (red circle) or linked to xylose residues. Rhamnogalacturonan is highly ramified and is also called "hairy region" for this reason. The basic backbone is a rhamnose-galacturonic acid motif. Side chain of arabinose, galactose, mixed or not, linear or not, forms a very complex

sustainable as possible. Interest was recently focused on plants providing good yields of biomass for a given surface as the tall Miscanthus. Reduction of lignin cell wall content is another interesting approach to enhance sugar recovery from biomass, lignin being an abundant and resistant polymer limiting the digestion of biomass in biofuels processes. With anti-sense technology, tobacco plants lines were obtained with 20% lower lignin content (Kavousi et al., 2010). The modified lines displayed a threefold increase of saccharification efficiency compared to wild type. Of course, the application of such studies in larger scales depends on the acceptance of transgenic plants by the society. Decision to use these plants has to be supported by studies of environmental risks and potential benefits (Talukder, 2006). Literature about pretreatment is very abundant, describing various methods: physical, chemical or combination of both (Soccol et al. 2010). Fine optimization of conditions should be performed individually depending on biomass. Among innovative method proposed, dry wheat straw has been treated successfully with supercritical CO2. After treatment, 1kg biomass yields to 149 g sugars (Alinia et al., 2010). Another currently emerging feature for bioethanol process amelioration is protein engineering. For instance, a cellulase from the filamentous bacterium *Thermobifida fusca* has been modified both in its catalytic domain and in its carbohydrate binding module (Li et al., 2010). A mutant enzyme displays a two fold increase activity, and a better synergy with other enzymes, leading it to be very useful for biomass digestion. At the next step, i.e. sugar fermentation to ethanol, many efforts have been run to allow yeast to perform both hexoses and pentoses fermentations. Industrial yeast *Saccharomyces cerevisiae* strains, fermenting only hexoses have been modified by addition of xylose degradation enzymes (Hector et al., 2010). Finally the outcome of engineering could be the use of synthetic biology, which is creating cell systems able to convert biomass to sugars and also to ferment them to ethanol. This strategy needs better fundamental knowledge to be developed (Elkins et al., 2010).

As discussed above, the step following the pretreatment of the biomass could be performed via the enzymatic hydrolysis of the cell wall polysaccharides into fermentescible, monomeric sugars. Unfortunately, it is well known that recalcitrance of plant cell wall to enzymatic digestion impairs the process. The behavior and the efficiency of the cell wall degrading enzymes (CWDE) *in situ* and *in vitro* with isolated polysaccharides are completely different. The properties of the CWDE, as conformation, hydrophobicity, capacity of adsorption onto the cell wall, interaction with the lignins, and catalytic efficiency in heterogeneous catalysis, are major parameters which should be considered and studied.

This chapter focuses on biomass degradation enzymes. What is the best strategy to produce the most efficient enzymes? What is the best choice depending of up- and downstream steps: commercial enzyme cocktail, enzymes produced by a given microorganism or heterologous production of individual enzymes? Efficiencies and cost of enzymes, two bottlenecks in the process, will be discussed. For some authors, the improvements of the conversion of biomass to sugar offer larger cost-saving potential than those concerning the step from sugar to biofuels (Lynd et al., 2008). These authors evaluated two scenarios; the first based on current technology and the second one including advanced nonbiological steps. In both cases, conversion of polysaccharides from biomass could be improved by increasing polysaccharides hydrolysis yields combined by lowered enzyme inputs. On-site enzyme production was also identified as beneficial for cost of the whole ethanol production process.

#### **2. Diversity of plant cell wall structures**

224 Bioethanol

sustainable as possible. Interest was recently focused on plants providing good yields of biomass for a given surface as the tall Miscanthus. Reduction of lignin cell wall content is another interesting approach to enhance sugar recovery from biomass, lignin being an abundant and resistant polymer limiting the digestion of biomass in biofuels processes. With anti-sense technology, tobacco plants lines were obtained with 20% lower lignin content (Kavousi et al., 2010). The modified lines displayed a threefold increase of saccharification efficiency compared to wild type. Of course, the application of such studies in larger scales depends on the acceptance of transgenic plants by the society. Decision to use these plants has to be supported by studies of environmental risks and potential benefits (Talukder, 2006). Literature about pretreatment is very abundant, describing various methods: physical, chemical or combination of both (Soccol et al. 2010). Fine optimization of conditions should be performed individually depending on biomass. Among innovative method proposed, dry wheat straw has been treated successfully with supercritical CO2. After treatment, 1kg biomass yields to 149 g sugars (Alinia et al., 2010). Another currently emerging feature for bioethanol process amelioration is protein engineering. For instance, a cellulase from the filamentous bacterium *Thermobifida fusca* has been modified both in its catalytic domain and in its carbohydrate binding module (Li et al., 2010). A mutant enzyme displays a two fold increase activity, and a better synergy with other enzymes, leading it to be very useful for biomass digestion. At the next step, i.e. sugar fermentation to ethanol, many efforts have been run to allow yeast to perform both hexoses and pentoses fermentations. Industrial yeast *Saccharomyces cerevisiae* strains, fermenting only hexoses have been modified by addition of xylose degradation enzymes (Hector et al., 2010). Finally the outcome of engineering could be the use of synthetic biology, which is creating cell systems able to convert biomass to sugars and also to ferment them to ethanol. This strategy needs better fundamental knowledge to be

As discussed above, the step following the pretreatment of the biomass could be performed via the enzymatic hydrolysis of the cell wall polysaccharides into fermentescible, monomeric sugars. Unfortunately, it is well known that recalcitrance of plant cell wall to enzymatic digestion impairs the process. The behavior and the efficiency of the cell wall degrading enzymes (CWDE) *in situ* and *in vitro* with isolated polysaccharides are completely different. The properties of the CWDE, as conformation, hydrophobicity, capacity of adsorption onto the cell wall, interaction with the lignins, and catalytic efficiency in heterogeneous catalysis, are major parameters which should be

This chapter focuses on biomass degradation enzymes. What is the best strategy to produce the most efficient enzymes? What is the best choice depending of up- and downstream steps: commercial enzyme cocktail, enzymes produced by a given microorganism or heterologous production of individual enzymes? Efficiencies and cost of enzymes, two bottlenecks in the process, will be discussed. For some authors, the improvements of the conversion of biomass to sugar offer larger cost-saving potential than those concerning the step from sugar to biofuels (Lynd et al., 2008). These authors evaluated two scenarios; the first based on current technology and the second one including advanced nonbiological steps. In both cases, conversion of polysaccharides from biomass could be improved by increasing polysaccharides hydrolysis yields combined by lowered enzyme inputs. On-site enzyme production was also identified as beneficial for cost of the whole ethanol production

developed (Elkins et al., 2010).

considered and studied.

process.

The plant cell wall structures are highly diverse. Various lignocellulosic species have been used for biofuels production, woods, crop by-products, herbaceous plants, beet pulp, municipal and paper industry wastes. Although all these different biomasses contain typically four major components (i.e. cellulose, hemicelluloses, pectin and lignin), the architecture of the cell wall, the fine biochemical structures of these components and their interactions into the cell wall could be quite different. Nevertheless, cellulose and hemicelluloses leading, with lignins, to the formation of an insoluble, tridimensional network is a constant behavior. A schematic drawing of the plant cell wall polysaccharides is shown in Fig. 1.

Fig. 1. Schematic representation of plant cell wall polysaccharides. Cellulose and -1,3/1,4 glucan are composed of glucose residues (red). -1,4-xyloglucan is a glucose-based polymer substituted by xylose residues (green) themselves possibly linked to galactose (blue) and fucose (black). -1,4-arabinoxylan is a xylose backbone linked with arabinose (orange), and/or with glucuronic acid acid (white). Xylose could be substituted by acetyl groups (blue circle). -1,4-mannan is a mannose polymer (dark red) linked to some galactose residues and sometimes acetylated. -1,4-polygalacturonan is formed of linear chains of galacturonic acid (pale yellow) linked by rhamnose (pink). Galacturonic residues could be either methylated (red circle) or linked to xylose residues. Rhamnogalacturonan is highly ramified and is also called "hairy region" for this reason. The basic backbone is a rhamnose-galacturonic acid motif. Side chain of arabinose, galactose, mixed or not, linear or not, forms a very complex and variable structure. Adapted from Dalboge (1997).

Competing Plant Cell Wall Digestion Recalcitrance

by Using Fungal Substrate- Adapted Enzyme Cocktails 227

fields to be prospected rather than being focalized on a single system. As an example for biomass degradation, many efforts have been directed though *Trichoderma reesei* due to historical reasons (discovered during World War II because it degraded uniforms and cotton tents) and to its capacity to produce cell wall degrading enzymes, specially cellulases. But, as it will be described later in this section, this fungus is not –by far for some categories of enzymes-

Genome sequence availability offers the scientific community the opportunity to analyze them, deciphering their metabolism, in order to find response to fundamental or applied questions. As valorization of plant biomass arise as an important question to be addressed, several studies attempt to describe the fungal polysaccharide degradation potential. An extensive and complete work leads to a comparison of the genome of 13 fungi (Martinez et al., 2008). The first observation is that the yeast model *Saccharomyces cerevisiae* is poorer in CWDE than filamentous fungi (Fig. 2). This is not surprising regarding their respective lifestyles; all the filamentous fungi shown in Fig. 2 are saprotrophs or pathogens in the opposite of *S. cerevisiae*. This is a first argument for considering the natural habitat of a fungus when examining it for a peculiar application. Here, clearly, fungi living in plant environment displayed many more genes encoding CWDE or associated activities. Note that the model for plant polysaccharide degradation, *Trichoderma reesei*, displays fewer putative glycosyl hydrolases (200) than the pathogens *Magnaporthe grisea* (231) and *Fusarium graminearum* (243). Perhaps even more important is the number of cellulose binding modules (CBM), allowing a better enzyme-substrate binding and then a better efficiency in natural cellulose hydrolysis. *T. reesei* was predicted to have half CBM than the two pathogens (Fig. 2). In the same study, *T. reesei* is shown to be poorer than *M. grisea* and *F.*

the most equipped in CWDE. Other fungi have to be considered then.

*graminearum* in cellulases, hemicellulases and pectinases (Martinez et al., 2008).

300 GH

GT CBM CE PL

Fig. 2. Number of predicted glycosyl hydrolases (GH), glucosyl transferase (GT), cellulose binding modules (CBM), carbohydrate esterase (CE) and polysaccharide lyases (PL) in the genome of *Saccharomyces cerevisiae*, *Aspergillus oryzae*, *Neurospora crassa*, *Trichoderma reesei*, :

*Magnaporthe grisea* and *Fusarium graminearum* (data from Martinez et al., 2008).

0

50

100

150

200

250

As another example of variable composition of plant cell wall, lignins and sugars in cell walls of different origins were quantified (Table 1). Although this study is not exhaustive, it is clear that every plant has its own characteristic. These biomasses have been used as growth substrates for *Fusarium graminearum* (see paragraph 4).

Regarding cell wall composition variation, it could be postulated that cell wall degradation recalcitrance could be related to cell wall structure and ultra structure variability.

Table 1. Lignin, glucose and xylose contents of hop, destarched wheat bran, corn cops and birch. Results are expressed as % of dry matter (unpublished results from the laboratory). Klason lignin was quantified as the acid-soluble residue after sulfuric acid hydrolysis (Rémond et al. 2010) and sugar contents were estimated by enzymatic methods as described in Phalip et al. (2009).

#### **3. A strategy for improving biomass hydrolysis: Studying (and using afterwards) fungi able to degrade plant cell wall components**

#### **3.1 Introduction on phytopathogenicity, saprophytism**

The primary choice of a microorganism (fungus) potentially providing cell wall degrading enzymes should be directed toward one naturally present in plant environment i.e. a phytopathogen or a saprophyte. Considering the ecology, fungi are qualified as "decomposers" in the opposite to plants, the producers and animals, the consumers. Some fungi, called saprotrophs, get nutrients from dead organisms, especially plants. Some other are pathogen, attacking living organisms. Invasive growth thanks to hyphae gets fungi very adapted to penetrate plants. Hyphae diameter (2–10 µm) permit cell penetration and their hyphal growth in several directions allow them to colonize quickly the plant material with very close contact. Many saprotrophs, phytopathogens and other fungi living in plant environment developed tools for gaining energy from plants during their evolution. Cell wall degrading enzymes (CWDE) are one of these tools which are also efficient for bioethanol production. This is the reason why this chapter focuses on CWDE produced and secreted by some fungi.

#### **3.2 Genome level studies**

After the completion of the project of the backer's yeast *Saccharomyces cerevisiae* genome sequencing in 1996, genomes of some fungi have been decrypted. Many reasons drive the decision to sequence one genome and not the other one: some fungi being for a long time scientific models, others displaying industrial relevance, and others acting as saprophytes or pathogens. Backer et al. (2008) propose an interesting concept: let's change our way of thinking and let's consider microorganisms (especially fungi) as reservoirs for sustainable answers to environmental concerns. This point of view fits well with the directing idea of this chapter. To improve the "biomass to ethanol" process, we have to consider many options and enlarge the

As another example of variable composition of plant cell wall, lignins and sugars in cell walls of different origins were quantified (Table 1). Although this study is not exhaustive, it is clear that every plant has its own characteristic. These biomasses have been used as

Regarding cell wall composition variation, it could be postulated that cell wall degradation

Hop Wheat bran Corn cops Birch

recalcitrance could be related to cell wall structure and ultra structure variability.

Lignin 30.9 17.3 6.5 18.1 Total neutral sugars 35.2 50.1 63.5 57.6 Glucose (i.e. glucans) 20.1 14.8 28.8 40.0 Xylose (i.e. xylans) 2.7 15.0 21.0 15.6

Table 1. Lignin, glucose and xylose contents of hop, destarched wheat bran, corn cops and birch. Results are expressed as % of dry matter (unpublished results from the laboratory). Klason lignin was quantified as the acid-soluble residue after sulfuric acid hydrolysis (Rémond et al. 2010) and sugar contents were estimated by enzymatic methods as described

The primary choice of a microorganism (fungus) potentially providing cell wall degrading enzymes should be directed toward one naturally present in plant environment i.e. a phytopathogen or a saprophyte. Considering the ecology, fungi are qualified as "decomposers" in the opposite to plants, the producers and animals, the consumers. Some fungi, called saprotrophs, get nutrients from dead organisms, especially plants. Some other are pathogen, attacking living organisms. Invasive growth thanks to hyphae gets fungi very adapted to penetrate plants. Hyphae diameter (2–10 µm) permit cell penetration and their hyphal growth in several directions allow them to colonize quickly the plant material with very close contact. Many saprotrophs, phytopathogens and other fungi living in plant environment developed tools for gaining energy from plants during their evolution. Cell wall degrading enzymes (CWDE) are one of these tools which are also efficient for bioethanol production. This is the reason why this chapter focuses on CWDE produced and

After the completion of the project of the backer's yeast *Saccharomyces cerevisiae* genome sequencing in 1996, genomes of some fungi have been decrypted. Many reasons drive the decision to sequence one genome and not the other one: some fungi being for a long time scientific models, others displaying industrial relevance, and others acting as saprophytes or pathogens. Backer et al. (2008) propose an interesting concept: let's change our way of thinking and let's consider microorganisms (especially fungi) as reservoirs for sustainable answers to environmental concerns. This point of view fits well with the directing idea of this chapter. To improve the "biomass to ethanol" process, we have to consider many options and enlarge the

**3. A strategy for improving biomass hydrolysis: Studying (and using**

**afterwards) fungi able to degrade plant cell wall components**

**3.1 Introduction on phytopathogenicity, saprophytism**

growth substrates for *Fusarium graminearum* (see paragraph 4).

in Phalip et al. (2009).

secreted by some fungi.

**3.2 Genome level studies**