Enzymatic Saccharification and Fermentation Technology for ... - J-Stage

Journal of the Japan Petroleum Institute, 58, (3), 128-134 (2015)

128

[Review Paper]

Enzymatic Saccharification and Fermentation Technology for Ethanol Production from Woody Biomass Shinichi YANO＊ Biomass Refinery Research Center, National Institute of Advanced Industrial Science and Technology, 3-11-32 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-0046, JAPAN (Received October 31, 2014)

Fuel ethanol production from biomass is promising technology for alleviation of global warming and reduction of fossil fuel use. In particular, production from non-food resources such as woody biomass is required. However, there are several technological problems such as development of efficient pretreatment technology, reduction of enzyme costs, and inability of conventional yeast to ferment xylose. Our research center has been conducting research to solve these problems and developed our own technology for enhancement of enzyme productivity and development of xylose-fermenting yeast strains. Our technology was effective in bench scale ethanol production experiments from eucalyptus wood using an integrated process from pretreatment to fermentation with high solid saccharification and glucose/xylose co-fermentation using a xylose-fermenting yeast strain. Our results show that ethanol production from woody biomass is feasible. Keywords Woody biomass, Bioethanol, Enzymatic saccharification, Xylose fermenting yeast, Thermotolerant yeast

1.

Introduction

Use of ethanol as a substitute for gasoline may be an effective measure to reduce petroleum consumption and mitigate global warming. Ethanol is already used as an additive or total substitute for gasoline worldwide. In particular, quite large amounts of ethanol for fuel use are produced from corn in the USA and from sugarcane in Brazil. However, those crops are also cultivated for food consumption, so ethanol production from those resources may cause food shortages and price rises. Hence, the use of non-food resources such as woody biomass is expected to provide a new source for ethanol feedstock. Japan relies on imports for the majority of its food supply, so ethanol production from food resources is impractical. In contrast, woody biomass resources such as timber from tree thinning, sawdust, or construction wood wastes are abundant in Japan1),2), so could become a major feedstock for ethanol production. Efficient ethanol production from woody biomass or agricultural residues such as straw presents several technological barriers. These resources consist of three major components, cellulose, hemicellulose, and lignin, and such biomass is called lignocellulosic biomass. Cellulose and hemicellulose can be hydrolyzed to the sugars required as substrates for ethanol fermentation DOI: dx.doi.org/10.1627/jpi.58.128 ＊ E-mail: [email protected]

by either acids or enzymes. At present, hydrolysis by acids such as sulfuric acid is relatively cost-effective but involves several problems, such as generation of substances which inhibit fermentation and decrease sugar yields, due to the excessive decomposition of sugars3). Furthermore, corrosive-resistant equipment and the recovery of used acids are expensive3),4). In contrast, enzymatic hydrolysis, which is also called saccharification, does not require hazardous chemicals and causes no excessive decomposition. However, little enzymatic hydrolysis occurs during direct processing of woody biomass, because lignin and hemicellulose fill in the gaps between cellulose molecules and hinder the approach of the enzyme molecules. Furthermore, cellulose molecules have very rigid structures, which also prevent efficient enzymatic saccharification. Therefore, pretreatment is essential before enzyme application to remove the protection by lignin and hemicellulose and to loosen the rigid structures of the cellulose. Various appropriate pretreatment technologies have been developed for the efficient enzymatic saccharification of lignocellulosic biomass5),6). For example, wet disk milling7), hot-compressed water treatment8), or the combinations of them9) can enhance the digestibility of lignocellulosic biomass by cellulase. However, considerable amounts of enzymes are still required after pretreatment, and generally account for 10 to 20 % of the total ethanol production cost10). Since the ethanol

J. Jpn. Petrol. Inst., Vol. 58,

No. 3, 2015

129

is intended as a substitute for gasoline, low production costs are required according to gasoline prices. Therefore, reduction of enzyme costs is important for enzymatic saccharification. More effective pretreatment methods could lower the amount of enzymes required, but the enzyme cost must also be reduced. Purchase of enzymes from enzyme suppliers is unlikely to result in significant cost reduction. Therefore, ethanol manufacturers should produce the enzymes at the site of ethanol production. This concept is called on-site enzyme production, and its efficacy for cost reduction has been estimated11),12). Cellulase and related enzymes to hydrolyze components of woody biomass can be produced by culture of enzyme-producing microorganisms, generally fungi. Therefore, ethanol manufacturers must have efficient enzyme-producing microorganisms for on-site enzyme production. Cellulose is a polymer of glucose, so glucose is obtained after hydrolysis of cellulose. Fermentation of glucose into ethanol is well-known technology and has been commercialized on the large scale for alcoholicbeverage production and for fuel ethanol production from starch. Although some specific problems present for woody biomass, such as formation of chemicals inhibiting fermentation such as furfural or 5-hydroxymethylfurfural in the pretreatment process, the technology to convert glucose from woody biomass into ethanol by fermentation has been established. The problem for ethanol production from woody biomass is related to hemicellulose. The major components of hemicellulose are sugars, but have different structures from the glucose forming cellulose. Hemicellulose accounts for 20-30 % of lignocellulosic biomass on the dry weight basis13),14), so sugars derived from hemicellulose should also be utilized as substrates for ethanol fermentation to enhance ethanol productivity. The major sugars in hemicellulose vary depending on the source plant15). Mannan is the major component of hemicellulose in softwoods, and mannose obtained by hydrolysis of mannan is easily fermented into ethanol with the conventional yeast for ethanol fermentation, Saccharomyces cerevisiae. However, xylan is the major component of hemicellulose in hardwoods or herbaceous plants, and xylose obtained by hydrolysis of xylan cannot be fermented with S. cerevisiae. Therefore, the most important technological issue in ethanol fermentation from woody biomass is how to convert xylose into ethanol. Several yeast species can ferment xylose into ethanol, but the productivity of ethanol is quite low and not feasible for practical application. Consequently, microorganisms which can ferment xylose must be developed by genetic engineering. The scheme of ethanol production from woody biomass including the major processes described above is shown in Fig. 1. The present review mainly focuses on biological issues: production of cellulase with fungi

Fig. 1

Scheme of Ethanol Production from Woody Biomass

and the development of xylose-fermenting yeast strains with genetic engineering. 2.

Improvement of Cllulase-producing Fungal Srains

Ethanol production from woody biomass will require on-site production of the saccharification enzymes to ensure economic processes, which requires cultivation of cellulase-producing microorganisms. Although both bacteria and fungi can produce cellulase, commercial cellulase production uses fungi which have superior enzyme productivity and secretion of the enzyme into the culture solution16). At present, most commercial cellulase is produced from the fungus Trichoderma reesei, which was isolated in the Solomon Islands during the Second World War, and allowed development of enzyme production technology together with strain improvement to achieve higher enzyme productivity17). Cellulase produced from this fungus is widely used in the textile and food industries. The cellulase-producing fungal strain Y-94 was isolated from soil in northeastern Japan18), and was described as Acremonium cellulolyticus in a patent19). Cellulase produced with this fungal strain is commercialized by Meiji Seika Pharma Co., Ltd. as Acremonium Cellulase, and has a good reputation, especially for the improvement of silage quality20). However, recent phylogenetic analyses using ITS1-5.8S-ITS2 and RNA polymerase II large subunit gene sequences have revealed that this fungus is closely related to genus


No. 3, 2015

130

Ta l a ro m y c e s r a t h e r t h a n g e n u s A c re m o n i u m . Therefore, this species should be described as Talaromyces cellulolyticus21), and this scientific name is used here. Cellulase is not a single enzyme but a mixture of the following three component enzymes: Endoglucanase which forms a nick in the amorphous region of cellulose molecules; cellobiohydrolase which cleaves cellobiose monomer units from the end of the cellulose polymer molecules; and beta-glucosidase which hydrolyzes the cellobiose or cellooligosaccharides to produce glucose. Cellulase produced from T. cellulolyticus culture is characterized by its high beta-glucosidase activity compared with conventional cellulase produced from T. reesei culture18),22). This property is especially important for ethanol production from cellulosic materials because the conventional yeast species used for ethanol production, S. cerevisiae, cannot utilize cellobiose, so that the cellulose must be completely hydrolyzed into glucose. Therefore, cellulase from T. cellulolyticus is considered to be very suitable for ethanol production from woody biomass or cellulosic feedstock. Consequently, research has continued to improve the enzyme productivity to reduce enzyme costs for industrial cellulosic ethanol production. The classical mutation technique was tried to improve cellulase productivity of the fungus. The efficacy of this approach was proved by the history of enzyme productivity improvement in T. reesei17). Improved strains of T. cellulolyticus were obtained as follows: strain TN from Y-94, strain C-1 from TN, and finally strain CF-261223) from C-1, and the last strain had the highest enzyme productivity among T. cellulolyticus strains (Fig. 2). Cellulase activity expressed by the Filter Paper Unit (FPU)24) was enhanced to 18 FPU/mL culture in strain CF-2612 from 2.0 FPU/mL in wild type strain Y-94. The major reason for the classical mutation strategy was that genetic information of this species was not available at that time. Another reason was that no transformation system for this species had been developed. However, we have since developed a transformation system for this species along with the development of a new selection marker system25). A disadvantage of enzymes from T. cellulolyticus is the low activity of beta-xylosidase, which is necessary to obtain xylose from biomass as a substrate for ethanol fermentation. We introduced the beta-xylosidase gene into this species and successfully obtained a strain which produces more beta-xylosidase26). In fact, the saccharification experiments with enzyme solution from this recombinant strain showed higher xylose yield from xylooligosaccharides (Fig. 3). Analyses and characterization of each component enzyme produced by T. cellulolyticus are continuing, so we can improve the enzyme properties by the combina-

Fig. 2●Stain Improvement of Cellulase-producing Fungus, T. cellulolyticus (FPU: Filter Paper Unit)

Fig. 3●Xylose Yields from Saccharification of Xylooligosaccharides with Enzyme Preparation from Wild Type (Y94, white diamonds) and Transformant Strain (YKX1, black squares) of T. cellulolyticus26)


No. 3, 2015

131

Fig. 4

Metabolic Pathways to Convert Xylose into Ethanol via Xylulose

tion of protein engineering and genetic engineering, which greatly increases the potential for use of enzymes from this species. 3.

Development of Xylose-fermenting Yeast Strains

Development of microorganisms which can efficiently ferment xylose is the most important technological issue in the establishment of ethanol production from woody biomass. Yeast species which can naturally ferment xylose have been investigated27)∼29). Such yeasts can ferment xylose into ethanol, but the productivity was low and not feasible for practical application. Genetic engineering for developing efficient xylosefermenting microorganisms offers several approaches. Introduction of the genes for enzymes necessary for ethanol fermentation into microorganisms which can metabolize xylose led to the development of Escherichia coli strains with ethanol fermentation properties30). However, the ethanol productivity was inadequate because the ethanol tolerance of E. coli was not enhanced so that the cultures could not accumulate high concentrations of ethanol. Introduction of xylose fermentation ability into S. cerevisiae is the general approach. S. cerevisiae cannot metabolize xylose, but can metabolize xylulose, a keto sugar and an isomer of xylose, into ethanol via the pentose phosphate pathway. Therefore, problem is to convert xylose into xylulose in S. cerevisiae. In nature, there are two pathways to convert xylose into xylulose (Fig. 4). The direct conversion pathway, which involves xylose isomerase (XI), is common among bacteria. The two-step pathway is common in fungi, in which xylose is first reduced into xylitol with xylose reductase (XR), and then xylitol is converted into xylu-

lose with xylitol dehydrogenase (XDH). However, attempts to express bacterial XI genes in S. cerevisiae have failed for decades31), so the strategy has been to introduce XR and XDH genes from the xylose-fermenting yeast Scheffersomyces stipitis into S. cerevisiae, resulting in many xylose-fermenting S. cerevisiae strains32). Recently, some successful results for functional expression of bacterial XI genes in S. cerevisiae were reported33),34). We adopted the XR-XDH strategy and have obtained xylose-fermenting S. cerevisiae strains. Genes for XR and XDH from S. stipitis and the xylulose kinase gene (Fig. 4) from S. cerevisiae were introduced into the INVSc1 strain35), shochu and bakery yeast36), sake yeast37), and the strain devoid of hexose transporters for research of xylose uptake38). However, the recombinant strains with XR and XDH accumulated quite high concentrations of xylitol, which reduced the ethanol yield from xylose. This problem is considered to be related to the co-enzyme dependence of the enzymes. Reduction of xylose to xylitol with XR utilizes both NADH and NADPH as co-enzymes, whereas XR of S. stipitis preferentially uses NADPH. On the other hand, the co-enzyme for the reaction of XDH is NAD ＋. As XR and XDH require different co-enzymes, imbalance of co-enzymes occurs. In particular, the XDH reaction, which selectively utilizes NAD＋, can suffer deficiency of NAD＋ (oxidized form) resulting in xylitol accumulation. If two enzymes utilize the same coenzyme, the reduced form can be recycled to the oxidized form, and deficiency of co-enzymes will not occur (Fig. 5). XDH which utilizes NADPH rather than NADH has been developed by protein engineering39), and our research group has introduced the gene for this modified XDH together with the XR gene from


No. 3, 2015

132

Fig. 5●Coenzyme Dependence in Natural and Modified Xylose Reductase and Xylitol Dehydrogenase

Fig. 7●Comparison of Separate Hydrolysis and Fermentation (SHF) and Simultaneous Saccharification and Fermentation (SSF)

Fig. 6●Consumption of D-Xylose (circles) and Ethanol Production (squares) in Media Supplemented with D-Xylose as the Sole Carbon Source by Recombinant S. cerevisiae Strains with Natural XDH (open symbols) and Modified XDH (closed symbols)42)

S. stipitis and xylulose kinase gene from S. cerevisiae into the S. cerevisiae strains D452-240), INVSc141), and IR-242). Figure 6 shows the xylose consumption and ethanol production by recombinant strains with the natural and modified XDH42). Although both strains consumed all xylose in the media after 48 hours, the recombinant strain consumed xylose and produced ethanol more rapidly.

at temperatures between the two optima. If fermentation is possible at higher temperatures, higher hydrolysis rates could be achieved and more efficient SSF would be possible. Another advantage of fermentation at higher temperatures is large cost savings for cooling of the fermentation tank in the presence of exothermic fermentation. Prevention of contamination by other microorganisms is also beneficial. Kluyveromyces marxianus is a thermotolerant yeast species generally abundant in fermented foods and recognized as safe. Several strains of K. marxianus were isolated and applied to SSF using cellulosic and lignocellulosic materials43)∼45). We recently isolated a novel strain of K. marxianus, DMB1, from sugarcane bagasse hydrolysate46). This strain shows higher thermotolerance compared with other K. marxianus strain and can grow at 48 ℃ (Fig. 8), so is promising for SSF or fermentation at higher temperatures. K. marxianus can metabolize xylose but not into ethanol. The development of xylose-fermenting K. marxianus strains is underway with the same strategy used for S. cerevisiae. 5.

4.

Ethanol Fermentation with Thermotolerant Yeast Species

The conventional yeast for fermentation, S. cerevisiae, produces ethanol at the optimum temperature of 3035 ℃. However, fermentation at higher temperatures would have several advantages. In the conventional ethanol fermentation process, enzymatic hydrolysis and fermentation occur separately and this process is called separate hydrolysis and fermentation (SHF). In contrast, yeast cultures inoculated with enzymes cause simultaneous saccharification and fermentation (SSF), as shown in Fig. 7. The SSF process can prevent inhibition of enzyme activity by produced glucose, because glucose liberated from cellulose by hydrolysis is immediately consumed by yeast. However, efficient SSF presents a fundamental problem. Generally, optimum temperatures for cellulase are 45-50 ℃, whereas optimum temperatures for conventional yeast fermentation are 30-35 ℃. Therefore, SSF must be performed

Bench Scale Ethanol Production from Woody Biomass

Production of ethanol from woody biomass poses important technological problems in pretreatment, and enzymes for saccharification and xylose fermentation. Our research center has been investigating pretreatment technology based on wet disk milling and hot-compressed water treatment. Combination of our technology for pretreatment, enzymes, and fermentation has been demonstrated on the laboratory scale or bench scale (up to 200 kg biomass per operation). In particular, a bench scale ethanol production experiment from eucalyptus wood by the high solid saccharification and glucose/ xylose co-fermentation method was performed47). In this experiment, a xylose-fermenting recombinant yeast strain based on IR-2 strain36) was used to enhance ethanol yield by glucose/xylose co-fermentation. The obtained ethanol concentration was 5.35 % (w/v), and the ethanol yield was comparable to that on the laboratory scale with synthetic medium (Table 1). As an ethanol


No. 3, 2015

133

Fig. 8●Comparison of Thermotolerance of K. marxianus Strain DMB1, Strain NBRC1777, and S. cerevisiae Strain BY4743 Assessed as Spot Growth on YPD Media at Various Temperatures46)

Table 1

Comparison of Ethanol Yield with a Xylose-fermenting Yeast Strain on Laboratory and Bench Scales

Scale [L]

Substrate

38 0.02

Eucalyptus hydrolysate Complete medium

Initial conc. [g/L]

Produced ethanol

Glucose

Xylose

Galactose

Conc. [g/L]

Yielda) [%]

116.0 45.0

11.0 45.0

0.8 0

53.5 37.1

82.1 80.8

References 47) 36)

a) Yield [%]＝(ethanol [g]/glucose [g]＋xylose [g]＋galactose [g])/0.51×100.

concentration of 4 % (w/v) is considered to be minimal for economic distillation48), saccharification technology of high solid concentration is important to obtain high ethanol concentration in practical production. Considering the general decrease in ethanol yield at high solid operation49), the bench scale yield was satisfactory. This result shows that efficient ethanol production from woody biomass is feasible. 6.

Conclusions

To overcome the technological barriers for efficient production of ethanol from woody biomass, our research center has been conducting research to develop our own technology. We succeeded in the enhancement of enzyme productivity by mutation and genetic engineering, which can reduce enzyme costs in industrial production. To solve the problem that the conventional yeast cannot ferment xylose, we developed xylose-fermenting yeast strains by genetic engineering. We also isolated a novel K. marxianus strain which can grow at 48 ℃ and allow fermentation at higher temperatures, especially in SSF. Finally, we combined our technology for pretreatment, enzymes, and fermentation on the bench scale and achieved high solid saccharification and glucose/xylose co-fermentation with a xylose-fermenting yeast strain. References 1) Yoshioka, T., Hirata, S., Matsumura, Y., Sakanishi, K., Biomass & Bioenergy, 29, 336 (2005). 2) Kinoshita, T., Ohki, T., Yamagata, Y., Appl. Energy, 87, 2923 (2010).

3) Brethauer, S., Wyman, C. E., Bioresource Technol., 101, 4862 (2010). 4) Wijaya, Y. P., Putra, R. D. D., Widyaya, V. T., Ha, J.-M., Suh, D. J., Kim, C. S., Bioresource Technol., 164, 221 (2014). 5) Brodeur, G., Yau, E., Badal, K., Collier, J., Ramachandran, K. B., Ramakrishnan, S., Enzyme Res., Article ID 787532 (2011). 6) Mood, S. H., Golfeshan, A. H., Tabatabaei, M., Jouzani, G. S., Najafi, G. H., Gholami, M., Ardjmand, M., Renew. Sustain. Energy Rev., 27, 77 (2013). 7) Hideno, A., Inoue, H., Tsukahara, K., Fujimoto, S., Minowa, T., Inoue, S., Endo, T., Sawayama, S., Bioresource Technol., 100, 2706 (2009). 8) Yu, G., Yano, S., Inoue, H., Inoue, S., Endo, T., Sawayama, S., Appl. Biochem. Biotechnol., 160, 539 (2010). 9) Hideno, A., Inoue, H., Yanagida, T., Tsukahara, K., Endo, T., Sawayama, S., Bioresource Technol., 104, 743 (2012). 10) Sanchez, A., Gomez, D., Fuel, 130, 100 (2014). 11) Barta, Z., Kovacs, K., Reczey, K., Zacchi, G., Enzyme Res., Article ID 734182, (2010). 12) Takimura, O., Yanagida, T., Fujimoto, S., Minowa, T., J. Jpn. Petrol. Inst., 56, (3), 150 (2013). 13) Chandel, A. K., Chandrasekhar, G., Radhika, K., Ravinder, R., Ravindra, P., Biotehchnol. Mol. Biol. Rev., 6, (1), 8 (2011). 14) Editorial Committee for Handbook of Woody New Materials, “Handbook of Woody New Materials,” Gihoudo Shuppan Co., Ltd., Tokyo (1996), p. 641. 木質新素材ハンドブック編集委員会編，“木質新素材ハンドブック，”技報堂出版，東京 (1996), p. 641. 15) Girio, F. M., Fonseca, C., Carvalheiro, F., Duarte, L. C., Marques, S., Bogel-Lukasik, R., Bioresource Technol., 101, 4775 (2010). 16) Sharada, R., Venkateswarlu, G., Venkateshwar, S., Rao, M. A., Intl. J. Pharm. Chem. Biol. Sci., 3, 1070 (2013). 17) Peterson, R., Nevalainen, H., Microbiology, 158, 58 (2012). 18) Yamanobe, T., Mitsuishi, Y., Takasaki, Y., Agric. Biol. Chem., 51, 65 (1987). 19) Yamanobe, T., Mitsuishi, Y., Takasaki, Y., JP 13177660 (1985). 20) Zhang, J. G., Kumai, S., Fukumi, R., Ueda, H., Kobayashi, R., Grassland Science, 45, 20 (1999). 21) Fujii, T., Hoshino, T., Inoue, H., Yano, S., FEMS Microbiol.


No. 3, 2015

134 Lett., 351, 32 (2014). 22) Fujii, T., Fang, X., Inoue, H., Murakami, K., Sawayama, S., Biotechnol. Biofuels, 2, 24 (2009). 23) Fang, X., Yano, S., Inoue, H., Sawayama, S., J. Biosci. Bioeng., 107, 256 (2009). 24) Ghose, T. K., Pure Appl. Chem., 59, 257 (1987). 25) Fujii, T., Iwata, K., Murakami, K., Yano, S., Sawayama, S., Biosci. Biotechnol. Biochem., 76, 245 (2012). 26) Kanna, M., Yano, S., Inoue, H., Fujii, T., Sawayama, S., AMB Express, 1, 15 (2011). 27) du Preez, J. C., Bosch, M., Prior, B. A., Enzyme Microb. Technol., 8, 360 (1986). 28) Ryabova, O. B., Chmil, O. M., Sibirny, A. A., FEMS Yeast Res., 4, 157 (2003). 29) Morais, C. G., Cadete, R. M., Uetanabaro, A. P. T., Rosa, L. H., Lachance, M-A., Rosa, C. A., Fungal Genet. Biol., 60, 19 (2013). 30) Ohta, K., Beall, D. S., Mejia, J. P., Shanmugam, K. T., Ingram, L. O., Appl. Environ. Microbiol., 57, 893 (1991) . 31) van Maris, A. J. A., Winkler, A. A., Kuyper, M., de Laat, W. T. A. M., van Dijken, J. P., Pronk, J. T., Adv. Biochem. Eng./ Biotechnol., 108, 179 (2007). 32) Matsushika, A., Inoue, H., Kodaki, T., Sawayama, S., Appl. Microbiol. Biotechnol., 84, 37 (2009). 33) Brat, D., Boles, E., Wiedemann, B., Appl. Environ. Microbiol., 75, 2304 (2009). 34) Hector, R. E., Dien, B. S., Cotta, M. A., Mertens, J. A., Biotechnol. Biofuels, 6, 84 (2013). 35) Matsushika, A., Sawayama, S., J. Biosci. Bioeng., 106, 306 (2008).

36) Matsushika, A., Inoue, H., Murakami, K., Takimura, O., Sawayama, S., Bioresource Technol., 100, 2392 (2009). 37) Fujii, T., Matsushika, A., Goshima, T., Murakami, K., Yano, S., Biosci. Biotechnol. Biochem., 77, 1579 (2013). 38) Goncalves, D. L., Matsushika, A., de Sales, B. B., Goshima, T., Bon, E. P. S., Stambuk, B. U., Enzyme Microb. Technol., 63, 13 (2014). 39) Watanabe, S., Kodaki, T., Makino, K., J. Biol. Chem., 280, 10340 (2005). 40) Matsushika, A., Watanabe, S., Kodaki, T., Makino, K., Sawayama, S., J. Biosci. Bioeng., 105, 296 (2008). 41) Matsushika, A., Watanabe, S., Kodaki, T., Makino, K., Inoue, H., Murakami, K., Takimura, O., Sawayama, S., Appl. Microbiol. Biotechnol., 81, 243 (2008). 42) Matsushika, A., Inoue, H., Watanabe, S., Kodaki, T., Makino, K., Sawayama, S., Appl. Environ. Microbiol., 75, 3818 (2009). 43) Ballesteros, I., Ballesteros, M., Negro, M. J., Appl. Biochem. Biotechnol., 28-29, 307 (1991). 44) Banat, I. M., Nigam, P., Marchant, R., World J. Microbiol. Biotechnol., 8, 259 (1992). 45) Cunha, R., Castro, A., Roberto, I. C., Appl. Biochem. Biotechnol., 172, 1553 (2014). 46) Goshima, T., Tsuji, M., Inoue, H., Yano, S., Hoshino, T., Matsushika, A., Biosci. Biotechnol. Biochem., 77, 1505 (2013). 47) Fujii, T., Murakami, K., Endo, T., Fujimoto, S., Minowa, T., Matsushika, A., Yano, S., Sawayama, S., Bioprocess Biosyst. Eng., 37, 749 (2014). 48) Koppram, R., Olsson, L., Biotechnol. Biofuel, 7, 54 (2014). 49) Palmqvist, B., Liden, G., AMB Express, 4, 41 (2014).

要旨木質系バイオマスからのエタノール生産のための酵素糖化および発酵技術矢野伸一（独）産業技術総合研究所バイオマスリファイナリー研究センター，739-0046 広島県東広島市鏡山3-11-32 地球温暖化の緩和と化石燃料の使用削減のために，バイオマ

酵，などの技術課題が存在する。我々の研究センターでは，酵

スから生産されるエタノールの燃料としての使用が期待されて

素生産糸状菌の改良による酵素生産性の向上，遺伝子組換えに

いる。資源の食用との競合を考えると，木質系バイオマスなど

よるキシロース発酵性酵母の開発など，これらの課題を解決で

の非食用バイオマスからの生産が特に望まれる。しかし，木質

きる技術を開発し，それらを統合することで，キシロースの発

からのエタノール生産には，効率的な前処理技術の開発，糖化

酵を含む木質からのエタノール生産をベンチ規模で実証するこ

酵素コストの低減，通常の酵母ではできないキシロースの発

とができた。


No. 3, 2015