Skip to main content
SpringerLink
Log in

Simultaneous improvement of saccharification and ethanol production from crystalline cellulose by alleviation of irreversible adsorption of cellulase with a cell surface-engineered yeast strain

  • Bioenergy and biofuels
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Enzymatic hydrolysis of cellulosic material is an essential step in the bioethanol production process. However, complete cellulose hydrolysis by cellulase is difficult due to the irreversible adsorption of cellulase onto cellulose. Thus, part of the cellulose remains in crystalline form after hydrolysis. In this study, after 96-h hydrolysis of Avicel crystalline cellulose, 47.1 % of the cellulase was adsorbed on the cellulose surface with 10.8 % crystalline cellulose remaining. In simultaneous saccharification and fermentation of 100 g/L Avicel with 1.0 filter paper unit/mL cellulase, a wild-type yeast strain produced 44.7 g/L ethanol after 96 h. The yield of ethanol was 79.7 % of the theoretical yield. On the other hand, a recombinant yeast strain displaying various cellulases, such as β-glucosidase, cellobiohydrolase, and endoglucanase, produced 48.9 g/L ethanol, which corresponds to 87.3 % of the theoretical yield. Higher ethanol production appears to be attributable to higher efficiency of cellulase displayed on the cell surface. These results suggest that cellulases displayed on the yeast cell surface improve hydrolysis of Avicel crystalline cellulose. Indeed, after the 96-h simultaneous saccharification and fermentation using the cellulase-displaying yeast, the amount of residual cellulose was 1.5 g/L, one quarter of the cellulose remaining using the wild-type strain, a result of the alleviation of irreversible adsorption of cellulases on the crystalline cellulose.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Adney B, Baker J (1996) Measurement of cellulase activities (LAP). NREL TP-510-42628. National Renewable Energy Laboratory, Golden CO

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Desai SG, Converse AO (1997) Substrate reactivity as a function of the extent of reaction in the enzymatic hydrolysis of lignocellulose. Biotechnol Bioeng 56:650–655

    Article  CAS  Google Scholar 

  • Eriksson T, Karlsson J, Tjerneld F (2002) A model explaining declining rate in hydrolysis of lignocellulose substrates with cellobiohydrolase I (Cel7A) and endoglucanase I (Cel7B) of Trichoderma reesei. Appl Biochem Biotechnol 101:41–60

    Article  CAS  Google Scholar 

  • Fujita Y, Ito J, Ueda M, Fukuda H, Kondo A (2004) Synergistic saccharification, and direct fermentation to ethanol, of amorphous cellulose by use of an engineered yeast strain codisplaying three types of cellulolytic enzyme. Appl Environ Microbiol 70:1207–1212

    Article  CAS  Google Scholar 

  • Galbe M, Zacchi G (2007) Pretreatment of lignocellulosic materials for efficient bioethanol production. Adv Biochem Eng Biotechnol 108:41–65

    CAS  Google Scholar 

  • Gao D, Chundawat SPS, Uppugundla N, Balan V, Dale BE (2011) Binding characteristics of Trichoderma reesei cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass. Biotechnol Bioeng 108:1788–1800

    Article  CAS  Google Scholar 

  • Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem 59:257–268

    Article  CAS  Google Scholar 

  • Gole A, Vyas S, Sainkar SR, Lachke A, Sastry M (2001) Enhanced temperature and pH stability of fatty amine-endoglucanase composites: fabrication, substrate protection, and biological activity. Langmuir 17:5964–5970

    Article  CAS  Google Scholar 

  • Hasunuma T, Kondo A (2012) Consolidated bioprocessing and simultaneous saccharification and fermentation of lignocellulose to ethanol with thermotolerant yeast strains. Process Biochem 47:1287–1294

    Article  CAS  Google Scholar 

  • Hasunuma T, Sung KM, Sanda T, Yoshimura K, Matsuda F, Kondo A (2011) Efficient fermentation of xylose to ethanol at high formic acid concentrations by metabolically engineered Saccharomyces cerevisiae. Appl Microbiol Biotechnol 90:997–1004

    Article  CAS  Google Scholar 

  • Karlsson M, Ekeroth J, Elwing H, Carlsson U (2005) Reduction of irreversible protein adsorption on solid surfaces by protein engineering for increased stability. J Biol Chem 280:25558–25564

    Article  CAS  Google Scholar 

  • Kondo A, Shigechi H, Abe M, Uyama K, Matsumoto T, Takahashi S, Ueda M, Tanaka A, Kishimoto M, Fukuda H (2002) High-level ethanol production from starch by a flocculent Saccharomyces cerevisiae strain displaying cell-surface glucoamylase. Appl Microbiol Biotechnol 58:291–296

    Article  CAS  Google Scholar 

  • Kumar R, Wyman CE (2009) Cellulase adsorption and relationship to features of corn stover solids produced by leading pretreatments. Biotechnol Bioeng 103:252–267

    Article  CAS  Google Scholar 

  • Lupoi JS, Smith EA (2011) Evaluation of nanoparticle-immobilized cellulase for improved ethanol yield in simultaneous saccharification and fermentation reactions. Biotechnol Bioeng 108:2835–2843

    Article  CAS  Google Scholar 

  • Ma A, Hu Q, Qu Y, Bai Z, Liu W, Zhuang G (2008) The enzymatic hydrolysis rate of cellulose decreases with irreversible adsorption of cellobiohydrolase I. Enzyme Microb Technol 42:543–547

    Article  CAS  Google Scholar 

  • Matano Y, Hasunuma T, Kondo A (2012a) Display of cellulases on the cell surface of Saccharomyces cerevisiae for high yield ethanol production from high-solid lignocellulosic biomass. Bioresour Technol 108:128–133

    Article  CAS  Google Scholar 

  • Matano Y, Hasunuma T, Kondo A (2012b) Cell recycle batch fermentation of high-solid lignocellulose using a recombinant cellulase-displaying yeast strain for high yield ethanol production in consolidated bioprocessing. Bioresour Technol (in press)

  • Nakamura N, Yamada R, Katahira S, Tanaka T, Fukuda H, Kondo A (2008) Effective xylose/cellobiose co-fermentation and ethanol production by xylose-assimilating S. cerevisiae via expression of β-glucosidase on its cell surface. Enzyme Microb Technol 43:233–236

    Article  CAS  Google Scholar 

  • Otter DE, Munro PA, Scott GK, Geddes R (1989) Desorption of Trichoderma reesei cellulase from cellulose by a range of desorbents. Biotechnol Bioeng 34:291–298

    Article  CAS  Google Scholar 

  • Palonen H, Tenkanen M, Linder M (1999) Dynamic interaction of Trichoderma reesei cellobiohydrolases Cel6A and Cel7A and cellulose at equilibrium and during hydrolysis. Appl Environ Microbiol 65:6–11

    Google Scholar 

  • Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton (2008) Determination of structural carbohydrates and lignin in biomass: laboratory 476 analytical procedure (LAP). NREL/TP-510-42618. National Renewable Energy Laboratory, Golden, CO

  • Taherzadeh MJ, Karimi K (2008) Pretreatment of lignocellulosic wastes to improve ethanol and biogas production: a review. Int J Mol Sci 9:1621–1651

    Article  CAS  Google Scholar 

  • Ueda M, Tanaka A (2000) Cell surface engineering of yeast: construction of arming yeast with biocatalyst. J Biosci Bioeng 90:125–136

    CAS  Google Scholar 

  • Väljamäe P, Sild V, Pettersson G, Johansson G (1998) The initial kinetics of hydrolysis by cellobiohydrolases I and II is consistent with a cellulose surface-erosion model. Eur J Biochem 253:469–475

    Google Scholar 

  • Xiao Z, Zhang X, Gregg DJ, Saddler JN (2004) Effects of sugar inhibition on cellulases and beta-glucosidase during enzymatic hydrolysis of softwood substrates. Appl Biochem Biotechnol 113–116:1115–1126

    Article  Google Scholar 

  • Yamada R, Taniguchi N, Tanaka T, Ogino C, Fukuda H, Kondo A (2011) Direct ethanol production from cellulosic materials using a diploid strain of Saccharomyces cerevisiae with optimized cellulase expression. Biotechnol Biofuels 4:8

    Article  CAS  Google Scholar 

  • Yang B, Wyman CE (2006) BSA treatment to enhance enzymatic hydrolysis of cellulose in lignin containing substrates. Biotechnol Bioeng 94:611–617

    Article  CAS  Google Scholar 

  • Yang B, Willies DM, Wyman CE (2006) Changes in the enzymatic hydrolysis rate of avicel cellulose with conversion. Biotechnol Bioeng 94:1122–1128

    Article  CAS  Google Scholar 

  • Yang J, Zhang X, Yong Q, Yu S (2010) Three-stage hydrolysis to enhance enzymatic saccharification of steam-exploded corn stover. Bioresour Technol 101:4930–4935

    Article  CAS  Google Scholar 

  • Zhang YP, Lynd LR (2004) Toward an aggregated understanding of enzymatic hydrolysis of cellulose: noncomplexed cellulase systems. Biotechnol Bioeng 88:797–824

    Article  CAS  Google Scholar 

  • Zhu Z, Sathitsuksanoh N, Percival Zhang Y-H (2009) Direct quantitative determination of adsorbed cellulase on lignocellulosic biomass with its application to study cellulase desorption for potential recycling. Analyst 134:2267–2272

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This work was supported through the project P07015 by the New Energy and Industrial Technology Development Organization (NEDO) under the sponsorship of the Ministry of Economy, Trade, and Industry (METI) of Japan. This work was also supported in part by Grants-in-Aid for Young Scientists (B) to TH from the Ministry of Education, Culture, Sports and Technology (MEXT) of Japan and a Special Coordination Fund for Promoting Science and Technology, Creation of Innovative Centers for Advanced Interdisciplinary Research Areas (Innovative Bioproduction Kobe) from MEXT, Japan.

Conflict of interest

None.

Author information

Authors and Affiliations

  1. Department of Chemical Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan

    Yuki Matano & Akihiko Kondo

  2. Organization of Advanced Science and Technology, Kobe University, 1-1 Rokkodai, Nada, Kobe, 657-8501, Japan

    Tomohisa Hasunuma

Authors
  1. Yuki Matano
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tomohisa Hasunuma
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Akihiko Kondo
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Akihiko Kondo.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Matano, Y., Hasunuma, T. & Kondo, A. Simultaneous improvement of saccharification and ethanol production from crystalline cellulose by alleviation of irreversible adsorption of cellulase with a cell surface-engineered yeast strain. Appl Microbiol Biotechnol 97, 2231–2237 (2013). https://doi.org/10.1007/s00253-012-4587-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-012-4587-x

Keywords

Search

Navigation