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Renewable Energy 34 (2009) 1354–1358

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Renewable Energy

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Production of ethanol from cassava pulp via fermentation with a surface-engineered yeast strain displaying glucoamylase

Akihiko Kosugi a, Akihiko Kondo b, Mitsuyoshi Ueda c, Yoshinori Murata a, Pilanee Vaithanomsat d,Warunee Thanapase d, Takamitsu Arai a, Yutaka Mori a,*

a Post-harvest Science and Technology Division, Japan International Research Center for Agricultural Sciences (JIRCAS), 1-1 Ohwashi, Tsukuba, Ibaraki 305-8686, Japanb Department of Chemical Science and Engineering, Faculty of Engineering, Kobe University, Nada-ku, Kobe, 657-8501, Japanc Department of Applied Biochemistry, Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japand Nanotechnology and Biotechnology Division, Kasetsart Agricultural and Agro-Industrial Product Improvement Institute (KAPI), Kasetsart University, 50 Chatuchak, Ladyao, Bangkok10900, Thailand

a r t i c l e i n f o

Article history:Received 6 February 2008Accepted 6 September 2008Available online 26 October 2008

Keywords:Cassava pulpGlucoamylaseSaccharomycescerevisiaeArming yeastsEthanolSurface-engineering

* Corresponding author. Tel./fax: þ81 29 838 6623E-mail address: ymori@affrc.go.jp (Y. Mori).

0960-1481/$ – see front matter � 2008 Elsevier Ltd.doi:10.1016/j.renene.2008.09.002

a b s t r a c t

Cassava (Manihot esculenta Crantz) pulp, produced in large amounts as a by-product of starchmanufacturing, is a major biomass resource in Southeast Asian countries. It contains abundant starch(approximately 60%) and cellulose fiber (approximately 20%). To effectively utilize the cassava pulp, anattempt was made to convert its components to ethanol using a sake-brewing yeast displaying glu-coamylase on the cell surface. Saccharomyces cerevisiae Kyokai no. 7 (strain K7) displaying Rhizopusoryzae glucoamylase, designated strain K7G, was constructed using the C-terminal-half region ofa-agglutinin. A sample of cassava pulp was pretreated with a hydrothermal reaction (140 �C for 1 h),followed by treatment with a Trichoderma reesei cellulase to hydrolyze the cellulose in the sample. TheK7G strain fermented starch and glucose in pretreated samples without addition of amylolytic enzymes,and produced ethanol in 91% and 80% of theoretical yield from 5% and 10% cassava pulp, respectively.

� 2008 Elsevier Ltd. All rights reserved.

1. Introduction

Cassava (Manihot esculenta Crantz) is widely cultivated intropical areas and used as food and animal fodder. In Thailand,approximately 10 million tons of fresh cassava tubers are consumedannually as a starch staple. When starch is extracted from cassavatubers during manufacturing, grated cassava tubers are separatedinto starch granules and fibrous residual materials by waterextraction followed by centrifugation. The fibrous residual material,called cassava pulp, accounts for approximately 10–30% by weight(wet) of the original tubers. Therefore, the tapioca starch industryin Thailand is estimated to generate at least one million ton ofcassava pulp annually from 10 million tons of fresh tubers.According to reports [1] and processing practices in Thailand,a large amount of starch (up to 60%, on a dry weight basis) togetherwith cellulosic fiber is contained in the cassava pulp.

Ethanol is increasingly used as an alternative fuel in the trans-portation sector. In general, fuel ethanol is produced mainly fromsugar cane, corn, and, in Thailand, cassava. However, a dramaticincrease in ethanol production using the crops mentioned earlier

.

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may not be practical, because these same crops are importantsources of food and feed, and expansion of fuel ethanol productionusing these crops could lead to shortages and price increase in foodand feed. Using agricultural wastes as a source for ethanolproduction is an effective alternative. In this context, cassava pulphas great potential as a raw material for ethanol productionbecause it contains large amount of starch and cellulosic substancesthat can be hydrolyzed and fermented to make ethanol.

Ethanol production from starchy materials by conventionalfermentation requires saccharification with amylolytic enzymesand subsequent fermentation using the yeast Saccharomyces cer-evisiae, because this yeast cannot utilize starchy materials. Thetwo-step process results in high production costs and lowproductivity of ethanol. Many reports have been published on thedevelopment of S. cerevisiae strains capable of secreting amylo-lytic enzymes [2]. Although yeast strains capable of utilizingstarch have been developed, their ethanol-producing abilityremains unsatisfactory. Recently, yeast strains displaying variousproteins on their cell surface have been developed using geneticengineering [3]. Laboratory yeast strains displaying Rhizopusoryzae glucoamylase (EC 3.2.1.3) [4], which cleaves glucose froma-1,4-linked and a-1,6-linked polysaccharides, have producedethanol directly from soluble and cooked corn starch [5,6].

A. Kosugi et al. / Renewable Energy 34 (2009) 1354–1358 1355

However, these surface-engineered yeast stains, because of thenature of the parent laboratory strain, cannot produce and accu-mulate high concentrations of ethanol at a rapid rate. Therefore, itis necessary to apply the surface-engineering technology toa high-ethanol-yielding and ethanol-tolerant industrial strain toproduce a practically useful yeast strain for ethanol production.

This report describes the development of a surface-engineeredyeast strain displaying R. oryzae glucoamylase using an industrialethanol-producing yeast, S. cerevisiae Kyokai no. 7, and itsfermentation properties on cassava pulp.

2. Materials and methods

2.1. Substrates, enzymes, media, and strains

Cassava pulp was obtained from Sanguan Wongse StarchIndustries (Thailand). Dry pulp was prepared by heating 1 kg wetcassava pulp at 90 �C for 24 h followed by grinding and sievingthrough a 0.5-mm mesh screen (ZM-100; Retsch, Haan, Germany).The dry pulp was stored in plastic bags at 4 �C. The enzymes usedfor starch hydrolysis, a-amylase from Aspergillus oryzae and glu-coamylase from Aspergillus niger, were obtained from MegazymeCo, Ltd. Cellulase from Trichoderma reesei (Sigma) was used tohydrolyze the cellulosic fiber in the cassava pulp. The sake-brewingyeast, S. cerevisiae Kyokai no. 7 (K7), was obtained from the NationalResearch Institute of Brewing (NRIB). S. cerevisiae MT8-1/pGA11 [5]displaying R. oryzae glucoamylase was used as a source for plasmidextraction. The yeasts were grown aerobically at 30 �C withcomplete medium (YPD) containing 20 g peptone, 10 g yeast extract(Difco Laboratories, Detroit, Mich.), and 20 g glucose per liter.Escherichia coli DH5a (TaKaRa) was used for genetic manipulation.E. coli was grown at 37 �C in Luria–Bertani medium containing100 mg of ampicillin per millilitre.

2.2. Hydrothermal treatment of cassava pulp

Cassava pulp suspended in water at concentrations of 5–30%was put in a pressure tube (Ace Glass Inc. USA) and heated attemperatures from 120 to 180 �C for 1 h. To examine the effects ofacid, H2SO4 was added to the pulp suspensions at final concentra-tions of 0.1 or 2.0% before heating.

2.3. Enzymatic hydrolysis

The pretreated pulp slurries were enzymatically hydrolyzed todetermine the maximum obtainable sugar. The pH of the slurrieswas preadjusted to 5.0 with sodium hydroxide, and 3 M sodiumacetate buffer at pH 5.0 was added at a final concentration of50 mM. Cellulase was used in the ratio of 3 U/g dry pulp. Hydrolysisof cellulosic fiber in the slurries was conducted at 50 �C for 72 hbefore subsequent starch hydrolysis. Samples were collected after24, 48, and 72 h for analysis of released glucose. After the slurry wastreated with cellulase, a-amylase (300 U/g dry pulp) and glucoa-mylase (100 U/g dry pulp) were added to hydrolyze the starchcompletely. Starch hydrolysis in the slurry was conducted at 50 �Cfor 48 h. The amount of released glucose was determined using theWako glucose CII test (Wako Pure Chemicals Co., Osaka, Japan).Clear supernatants were obtained from the hydrolyzed slurries bycentrifugation at 7000 rpm for 20 min.

2.4. Fermentation by strain K7

The hydrolyzed slurries (pulp hydrolysates) were supplementedwith yeast extract and yeast nitrogen base with amino acids (DifcoLaboratories, Detroit, MI) at final concentrations of 5 g/L and 7 g/L,respectively. The pH was adjusted to 6.0 with calcium hydroxide

and the media were filtered with aseptic filter discs (0.22-mm,Advantec, Tokyo, Japan). Precultured strain K7 was inoculated intothe medium at 5% (v/v) and incubated at 30 �C. Samples werecollected every 24 h from fermentation broths and analyzed forethanol. A gas chromatograph (model GC-2014: Shimadzu, Kyoto,Japan) with a flame-ionization detector (FID) was used to measureethanol concentration in samples under the following conditions:glass column (8.0 mm by 3.2 m) packed with Chromosorb 103 (60/80 mesh); temperatures of column, injector, and detector, 185, 175,and 250 �C, respectively; helium carrier gas flow rate, 20 ml/min. n-Propanol was used as an internal standard.

2.5. Construction of plasmids for displaying R. oryzae glucoamylaseon strain K7

Plasmid pGA11 for cell surface display of R. oryzae glucoamylasewas constructed as described previously in Ref. [5]. To amplifya DNA fragment between glyceraldehydes-3-phosphate dehydro-genase (GAPDH) promoter and GAPDH terminator containing thecoding region of the glucoamylase with the 30 half of a-agglutiningene, two primers containing artificial SphI and Sse837 I sites(underlined) (50-ACATGCATGCACCAGTTCTCACACGGAACA-30 and50-TCCTGCAGGTCAATCAATGAATCG AAAATG-30) were designed anda 4.3-kbp fragment was amplified by PCR using Ex Taq polymerase(TaKaRa). The amplified fragments were digested with the cognaterestriction enzymes and inserted between the SphI and Sse837 Isites of pAUR101 (TaKaRa) to generate pAU101RGA. To integratepAU101RGA into the S. cerevisiae K7 genomic DNA, the plasmid wasdigested with StuI to generate a linear DNA fragment. This DNAfragment was transformed into the strain K7 by the lithium acetatemethod following manufacturer’s instructions (TaKaRa). Trans-formants were obtained on YPD plates containing 1.0 mg/ml Aur-eobasidin A (TaKaRa).

2.6. Glucoamylase assay

After aerobic cultivation of transformed strains in YPD con-taining Aureobasidin A at 30 �C for 24 h, cells were collected bycentrifugation for 5 min at 8000 rpm, resuspended in distilledwater, and used for the enzyme assay. The substrate solution forglucoamylase assay was prepared by adding soluble starch to theboiling 20 mM sodium acetate buffer (pH 5.0) to give a concentra-tion of 1%. The sample was mixed with the substrate solution andincubated at 30 �C for 30 min. Reaction was stopped by boiling themixture for 10 min. Released glucose was determined using theWako glucose CII test. One unit of glucoamylase was defined asthe amount of enzyme required to release 1 mmol of glucose perminute from starch [11].

2.7. Fermentation by stain K7G

Slurries of pulp pretreated with hydrothermal reaction andcellulase were supplemented with yeast extract and yeast nitrogenbase with amino acids at final concentrations of 5 g/L and 7 g/L,respectively. The pH was adjusted to 6.0 with calcium hydroxide.The slurries were inoculated with the strain K7G and fermented at30 �C. Samples were collected every 24 h from fermentation brothsand analyzed for ethanol by gas chromatography.

2.8. Composition of cassava pulp

Starch content in dry cassava pulp was estimated using the totalstarch assay kit following manufacturer’s instructions (Megazyme,Ireland). Reducing sugar and glucose concentrations in dry cassavapulp were measured by the Somogyi Nelson method and the Wakoglucose CII test, respectively, using 70% ethanol extracts. The

Cassava pulp

Hydrothermal reactionTemp: 120ºC to 180ºCTime: 60 minH2SO4: 0% to 2%

Hydrolysis of fiberCellulase: 3U/g pulpTemp: 50ºCTime: 72 h

Hydrolysis of starchα-Amylase: 300U/g pulpGlucoamylase: 100U/g pulpTemp: 50ºCTime: 48 h

Fermentation testYeast strain: K7Temp: 30ºCTime: 2 to 7 days

Fermentation testYeast strain: K7GTemp: 30ºCTime: 5 to 7 days

Pulp hydrolysates

Pretreatedpulp

Fig. 1. Experimental procedures used to evaluate the hydrolysis of cassava pulp.

A. Kosugi et al. / Renewable Energy 34 (2009) 1354–13581356

starch-free fiber was obtained by centrifugation at 10,000 rpm for20 min after starch hydrolysis using a-amylase and amyloglucosi-dase for 2 days at 50 �C. Monosaccharide analysis of the fiber wasperformed with high performance anion-exchange chromatog-raphy using CarboPac PA (Dionex Corporation, Sunnyvale, CA, USA)with pulsed amperometric detection (HPAEC-PAD). The mobilephase was 2% NaOH at a flow rate of 0.6 ml/min at 28 �C. The totalnitrogen content of cassava pulp was estimated using a modifiedDuma’s method (Sumigraph NC-900, Shimadzu, Kyoto, Japan).Klason lignin was determined using the Hagglund method [7].

3. Results and discussion

3.1. Components of cassava pulp

Table 1 shows the composition of cassava pulp produced afterstarch extraction at a starch factory in Thailand. Significantamounts of starch (60.6%) and non-starch polysaccharide (29% asfiber) were detected in the pulp. Results are similar to thosereported by Srioth et al. [1]. Monosaccharide analysis of the non-starch polysaccharides indicated that glucans, such as cellulose,were the major polysaccharide. The analyzed compounds accoun-ted for 94.7% of the weight of the total dry pulp.

3.2. Optimization of pretreatment conditions for cassava pulp

To determine optimal pretreatment methods for ethanolproduction from cassava pulp, hydrothermal reaction with andwithout dilute sulfuric acid was examined. The experimentalprocedure is summarized in Fig. 1. First, to test the effects ofa combination of hydrothermal reaction (120–180 �C for 60 min)and addition of H2SO4 (0.1% and 2%), glucose yield in the pulphydrolysates was measured after enzyme treatments of the cassavapulp (Fig. 1).

The greatest glucose yield (0.75 g/g pulp) was obtained underhydrothermal conditions (140 �C for 1 h) without addition of H2SO4

(Fig. 2). Yields were equivalent to approximately 90% of the theo-retical value according to the composition shown in Table 1. Nomajor differences in glucose yields between 120 �C and 160 �C werefound with or without addition of 0.1% H2SO4 (Fig 2). Glucose yieldsfrom hydrothermal reaction with 2% H2SO4 were lower than thosewith or without 0.1% H2SO4 (Fig. 2), suggesting formation of by-products with increased H2SO4 concentration. Reports have indi-cated that by-products, such as hydroxylmethyl-furfural (HMF),furfural, furoic acid, and phenol, are produced from irreversibleconversion of pentoses, hexoses, and lignin during pretreatmentwith acid [8]. Based on these results, hydrothermal reaction at140 �C without H2SO4 was used to pretreat cassava pulp.

Table 1Composition of cassava pulp

Components g/100 g Dry pulp

Starch 60.6Reducing sugars (glucose)a 4.7 (0.09)a

Nitrogen 0.4

Non-starch polysaccharidesGlucan 19.1Xylan 4.2Arabinan 1.4Galactan 0.5Mannan 0.7Others 0.9Klason lignin 2.2

Total 94.7

Analyses were conducted according to Section 2.a Free glucose in the dry pulp.

However, these conditions are not optimal for energy balance.Assuming that thermal efficiency is 100% and the specific heat forcassava pulp is 1.25 kJ/kg k [9], the energy consumed in heating1 ml of 5% pulp suspension from 25 �C to 140 �C and keeping it at140 �C was 488.6 J. Because the glucose generated (0.744 g/g drypulp) provides 580.3 J, the energy balance was þ91.7 J. Similarly,the energy balance with no heating and at 120, 160, and 180 �Cwithout H2SO4 was þ241.8, þ154.1, �4.1, and �143.7 J, respectively,indicating lowering treatment temperature is important to improveenergy balance. In practice, where complete insulation is impos-sible, it is also necessary to shorten treatment time to reduce theenergy required to maintain reaction temperature.

To examine the effects of pulp concentration on glucose yield,5%, 10%, 20%, and 30% suspensions of pulp were hydrolyzed withenzymes after hydrothermal reaction at 140 �C for 1 h. Glucoseyield was highest with 5% pulp, and it decreased as pulp concen-tration increased (Fig. 3). The low hydrolysis efficiencies withhigher concentrations of pulp probably are due to inhibition ofenzymes by increasing amounts of end-products, by-products,and/or insufficiency of hydrothermal reaction by substrate over-load [10]. The energy balance with 5, 10, 20, and 30% cassava pulp

0.0

0.2

0.4

0.6

0.8

1.0

No thermaltreatment

120ºC60 min

140ºC60 min

160ºC60 min

180ºC60 min

Glu

cose

yie

ld(g

glu

cose

/g d

ry p

ulp)

Treatment

: No acid, : 0.1 %H2SO4, : 2.0 % H2SO4,

Fig. 2. Effects of hydrothermal reaction with and without addition of H2SO4 onenzyme hydrolysis. Concentration of cassava pulp was 5% (w/v) for all conditions.Enzyme hydrolysis was conducted after the hydrothermal reaction.

Table 3Glucoamylase activity of transformed yeast strain K7G

Strains Activity (U/g [wet wt.] of cells)

K7 NDa

K7Ab NDa

K7G 60.2

Values are averages of three independent experiments.a Not detectable.b K7A is yeast strain K7 harboring pAUR112 as control against K7G.

0.0

0.2

0.4

0.6

0.8

1.0

5 % 30 %Cassava pulp concentration (w/v)

Glu

cose

yie

ld(g

glu

cose

/g d

ry p

ulp)

20 %10 %

Fig. 3. Effects of increasing pulp concentration on enzyme hydrolysis. The hydro-thermal reaction was conducted at 140 �C for 60 min without addition of H2SO4.Enzyme treatment was performed after the hydrothermal reaction.

A. Kosugi et al. / Renewable Energy 34 (2009) 1354–1358 1357

was þ91.7, þ488.6, þ1140.3, and þ1901.1 J, respectively. Thus, pulpconcentration is significant for achieving high hydrolysis efficiencyand energy gain.

3.3. Fermentation of pulp hydrolysates using strain K7

Strain K7, which is used for industrial brewing of Japanesesake, is capable of high-ethanol productivity and tolerancecompared with laboratory strains. To determine whether thepulp hydrolysates inhibit yeast growth, fermentation tests wereperformed using strain K7. High-ethanol yields and productivitieswere observed when 5% and 10% pulp hydrolysates were fer-mented with strain K7, while ethanol yield and productivity from20% and 30% pulp hydrolysates were significantly lower (Table 2).In contrast, all reference fermentations, where glucose was usedas substrate instead of cassava pulp hydrolysates, showed high-ethanol yield and productivity even with high substrateconcentrations (Table 2). Thus, low performance from fermen-tation with 20% and 30% pulp hydrolysates probably is due toaccumulation of inhibitory by-products from the hydrothermaltreatment [8].

30

40

50

ucti

on)

3.4. Fermentation of pretreated pulp using the yeast transformantK7G displaying R. oryzae glucoamylase

PCR testing confirmed that the linearized plasmid pAU101RGAwas integrated into the genomic DNA of strain K7, and that thetransformant strain K7G successfully integrated one copy of theR. oryzae glucoamylase gene with a-agglutinin into the AUR1allele of strain K7 genomic DNA [11]. The glucoamylase activity ofthe strain K7G was 60.2 U/g [wet weight] of cells (Table 3). Noactivity was detected with wild-type strain K7 and control strain

Table 2Fermentation of cassava pulp hydrolysates by strain K7

Cassavapulp(w/v %)

Initialsubstratea

(g glucose/L)

Ethanolproduced(g/L)

Ethanol yieldon substrateb

(g ethanol/gsubstrate)

Ethanolproductivityc

(g ethanol/L/h)

Yield(%)

5 37.2 (33) 18.6 (17.1) 0.50 (0.51) 0.77 (0.71) 98 (100)10 63.1 (65) 29.6 (32.2) 0.47 (0.49) 0.62 (0.67) 92 (96)20 105.8 (108) 32.9 (48.5) 0.31 (0.45) 0.27 (0.67) 61 (88)30 155.5 (165) 46.0 (65.6) 0.30 (0.40) 0.27 (0.68) 58 (78)

a Initial substrate is the amount of glucose contained in the pulp hydrolysates.b Ethanol produced [in g/L] divided by initial substrate [in g glucose/L].c Ethanol produced [in g/L] divided by total fermentation time [in hours]. The valuesin parentheses are the results for reference fermentation, where cassava pulphydrolysates were replaced by glucose.

K7A harboring pAUR112. These results indicate that K7G dis-played R. oryzae glucoamylase on its cell surface by the anchoringfunction of the C-terminal a-agglutinin.

Cassava pulp treated with cellulase following hydrothermalreaction (140 �C for 1 h) was fermented using K7G and K7Awithout addition of amylolytic enzymes. As shown in Fig. 4, K7Gproduced significant amounts of ethanol from 5%, 10%, 20%, and30% pulp, while K7A produced very little ethanol (Fig 4). Ethanolproduction rate from cassava pulp by K7G without amylasetreatment was slower, especially during the early phase offermentation compared to that by K7 with cassava pulp treatedwith a-amylase and glucoamylase. The slow ethanol production byK7G presumably is due to the time required for saccharification ofstarch by the glucoamylase displayed on the cell surface. Thefermentation rate by K7G could be easily improved by increasinginitial cell density [12] and by codisplaying a-amylases with glu-coamylase [6].

Strain K7G effectively fermented cellulase-pretreated cassavapulp to produce ethanol in high yields: 91% and 80% of the theo-retical value with 5% and 10% of cassava pulp, respectively, (Table 4).The maximum ethanol concentration accumulated by K7G wascomparable to those attained by K7 with cassava pulp pretreatedwith amylase in addition to cellulase.

The results obtained in this study show the effectiveness ofsurface-engineered yeast displaying amylase on the surface for theproduction of ethanol from cassava pulp. Research continues intothe construction of a yeast strain codisplaying cellulases andamylases, which could fully utilize cassava pulp and directlyproduce ethanol without addition of enzymes.

0

10

20

0 1 2 3 4 5 6 7

Eth

anol

pro

d(g

/lite

r

Time (days)

Fig. 4. Time course of ethanol production on pretreated cassava pulp using yeast strainK7G displaying glucoamylase. Open circles represent 5% pretreated pulp, closed circlesrepresent 10% pretreated pulp, open squares represent 20% pretreated pulp, closedsquares represent 30% pretreated pulp. Closed triangles indicate the fermentationprofile of control strain K7 harboring pAUR112 (K7A).

Table 4Fermentation of pretreated cassava pulp by strain K7G

Cassavapulp(w/v %)

Total sugara

(g glucose/L)Ethanolproduced(g/L)

Ethanol yieldon substrateb

(g ethanol/gtotal sugar)

Ethanolproductivityc

(g ethanol/L/h)

Yield(%)

5 36.5 16.9 0.46 0.18 9110 61.1 25.2 0.41 0.26 8020 116.0 34.9 0.30 0.24 5930 151.4 42.1 0.28 0.25 55

a Total sugar is indicated as the amount of glucose plus glucose moiety in the starchcontained in the pretreated pulp.b Ethanol produced [in g/L] divided by total sugar [in g glucose/L].c Ethanol produced [in g/L] divided by total fermentation time [in hours].

A. Kosugi et al. / Renewable Energy 34 (2009) 1354–13581358

4. Conclusions

To utilize cassava pulp containing abundant starch and cellulosicfiber, its components were converted to ethanol using S. cerevisiaestrain K7G displaying glucoamylase on its cell surface, which wasconstructed using a cell surface-engineering system based on a-agglutinin. The strain K7G effectively produced high yields ofethanol from cassava pulp pretreated by hydrothermal reaction(140 �C for 1 h) and then by cellulase to hydrolyze the cellulose inthe pulp.

Acknowledgments

The authors are grateful to Mr. Thosapol Tantiwong and Mr.Achcha Leeungkul (Sanguan Wongse Industries Co., Ltd) forproviding cassava pulp. The authors wish to thank Dr. Shingo

Magara and Dr. Ryohei Tanaka (Forestry and Forest ProductsResearch Institute) for technical support in carbohydrate analysis.

References

[1] Sriroth K, Chollakup R, Chotineeranat S, Piyachomkwan K, Oates CG. Pro-cessing of cassava waste for improved biomass utilization. Bioresour Technol2000;71:63–9.

[2] Briol G, Onsan I, Kirdar B, Oliver SG. Ethanol production and fermentationcharacteristics of recombinant Saccharomyces cerevisiae strains grown onstarch. Enzyme Microb Technol 1998;22:672–7.

[3] Ueda M, Tanaka A. Cell surface engineering of yeast: construction of armingyeast with biocatalyst. J Biosci Bioeng 2000;90:125–36.

[4] Ashikari T, Kiuchi-Goto N, Tanaka Y, Shibano Y, Amachi T, Yoshizumi H. Highexpression and efficient secretion of Rhizopus oryzae glucoamylase in the yeastSaccharomyces cerevisiae. Appl Environ Microbiol 1989;30:515–20.

[5] Murai T, Ueda M, Yamamura M, Atomi H, Shibasaki Y, Kamasawa N, et al.Construction of a starch-utilizing yeast by cell surface engineering. ApplEnviron Microbiol 1997;63:1362–6.

[6] Shigechi H, Jun Koh J, Fujita Y, Matsumoto T, Bito Y, Ueda M, et al. Directproduction of ethanol from raw corn starch via fermentation by use of a novelsurface-engineered yeast strain codisplaying glucoamylase and a-Amylase.Appl Environ Microbiol 2004;70:5037–40.

[7] Hagglund E. The determination of lignin. In: Chemistry of wood. New York:Academic Press; 1951. p. 324–32.

[8] Klinke HB, Thomsen AB, Ahring BK. Inhibition of ethanol-producing yeast andbacteria by degradation products produced during pre-treatment of biomass.Appl Microbiol Biotechnol 2004;66:10–26.

[9] Minowa T. Hydrothermal liquefaction. In: Biomass handbook. Tokyo: Ohmsha;2002. p. 125–43.

[10] Sun Y, Cheng J. Hydrolysis of lignocellulosic materials for ethanol production:a review. Bioresour Technol 2002;83:1–11.

[11] Akada R, Murakane T, Nishizawa Y. DNA extraction method for screening yeastclones by PCR. Biotechniques 2000;28:668–74.

[12] Kondo A, Shigechi H, Abe M, Uyama K, Matsumoto T, Takahashi S, et al. High-level ethanol production from starch by a flocculent Saccharomyces cerevisiaestrain displaying cell-surface glucoamylase. Appl Microbiol Biotechnol2002;58:291–6.