Elsevier

Bioresource Technology

Volume 100, Issue 18, September 2009, Pages 4203-4213
Bioresource Technology

Effect of xylanase supplementation of cellulase on digestion of corn stover solids prepared by leading pretreatment technologies

https://doi.org/10.1016/j.biortech.2008.11.057Get rights and content

Abstract

Solids resulting from pretreatment of corn stover by ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), controlled pH, dilute acid, lime, and sulfur dioxide (SO2) technologies were hydrolyzed by enzyme cocktails based on cellulase supplemented with β-glucosidase at an activity ratio of 1:2, respectively, and augmented with up to 11.0 g xylanase protein/g cellulase protein for combined cellulase and β-glucosidase mass loadings of 14.5 and 29.0 mg protein (about 7.5 and 15 FPU, respectively)/g of original potential glucose. It was found that glucose release increased nearly linearly with residual xylose removal by enzymes for all pretreatments despite substantial differences in their relative yields. The ratio of the fraction of glucan removed by enzymes to that for xylose was defined as leverage and correlated statistically at two combined cellulase and β-glucosidase mass loadings with pretreatment type. However, no direct relationship was found between leverage and solid features following different pretreatments such as residual xylan or acetyl content. However, acetyl content not only affected how xylanase impacted cellulase action but also enhanced accessibility of cellulose and/or cellulase effectiveness, as determined by hydrolysis with purified CBHI (Cel7A). Statistical modeling showed that cellulose crystallinity, among the main substrate features, played a vital role in cellulase–xylanase interactions, and a mechanism is suggested to explain the incremental increase in glucose release with xylanase supplementation.

Introduction

Highly efficient conversion of carbohydrates in biomass to fermentable sugars is essential to commercially competitive biological processes for making cellulosic ethanol (Wyman, 2007, Yang and Wyman, 2008), and although enzymes realize the high yields required, the corresponding high doses are very expensive (Merino Sandra and Cherry, 2007, Tu et al., 2007, Wingren et al., 2005). Furthermore, current commercial enzymes are mainly intended for pulp and paper and food industries and still lack the proportions of different enzymes and their components required for effective biological production of fermentable sugars from cellulosic biomass (Hespell et al., 1997, Merino Sandra and Cherry, 2007). Typically cellulase, β-glucosidase, xylanase, β-xylosidase, and some accessory activities are required to hydrolyze sugar polymers effectively (Bisaria and Ghose, 1981, Kuhad et al., 1997, Saha and Bothast, 1997), with the proportions of each depending on the type of biomass and pretreatment used. Among leading thermochemical pretreatments, those at low pH hydrolyze xylan to xylose and xylooligomers (Allen et al., 2001, Kabel et al., 2007a, Vazquez et al., 2002) but can also degrade both of these (Kumar and Wyman, 2008d, Lloyd and Wyman, 2005, Saeman, 1945). Furthermore, because the resulting degradation products are strong inhibitors to cellulase and fermenting microorganisms (Kumar and Wyman, 2008e, Larsson et al., 1999, Palmqvist et al., 1996), the pretreatment liquor must be detoxified prior to fermentation (Lynd et al., 2008, Palmqvist and Hahn-Hagerdal, 2000, Weil et al., 2002). On the other hand, high pH alkaline pretreatments leave most of the xylan in the solids, with the result that xylanase as well as possibly other accessory enzymes are needed in addition to cellulase to realize high sugar yields (Chandra et al., 2007, Kumar and Wyman, in press, Mosier et al., 2005, Yang and Wyman, 2008). It has been reported that thermochemical or enzymatic removal of xylan enhances cellulose digestion by reducing the xylan coating and linkages to cellulose (Allen et al., 2001, Ishizawa et al., 2007), but the mechanism of why xylan impacts cellulose digestion is still not entirely clear. Furthermore, data has not been developed to compare the effect of supplementing cellulase with xylanase on sugar release from solids prepared by different promising pretreatments.

In this study, baseline sugar release data was developed for a cellulase plus β-glucosidase mass loading of 29.0 mg/g glucan in unpretreated2 corn stover for solids produced by the leading pretreatment technologies of ammonia fiber expansion (AFEX), ammonia recycled percolation (ARP), dilute sulfuric acid, lime, controlled pH, and sulfur dioxide (SO2). Then, xylanase and β-xylosidase supplementation were employed to determine whether these two activities enhanced performance at the same cellulase loading plus a lower cellulase loading of 14.5 mg/g glucan in unpretreated corn stover. In addition, the influence of acetyl content on cellulase–xylanase interactions was studied, and factors and possible mechanisms for enhancement of glucan digestion by xylanase supplementation were also determined.

Section snippets

Materials

Pure cellulose, Avicel PH-101, was purchased from FMC Corporation, Philadelphia, PA (Cat 11365, Lot 1094627). Birchwood xylan was purchased from Sigma Chemicals, St. Louis, MO. Corn stover was generously provided by the National Renewable Energy Laboratory (NREL, Golden, CO) from a lot that they had collected at the Kramer farm in nearby Wray, CO. Solids prepared by corn stover pretreatment were generously given to us by our CAFI partners from Auburn University, Michigan State University, NREL,

Effect of xylanase supplementation on glucose and xylose release

Pretreated corn stover solids were enzymatically hydrolyzed at fixed CTB mass loadings of 14.5 and 29.0 mg/g glucan in unpretreated corn stover with xylanase supplementation to various degrees for a total of 72 h to establish trends in enzyme effectiveness. However, longer hydrolysis times (e.g., 7 days) would likely be employed commercially to capitalize on the additional sugar release expected.

Conclusions

Glucan and xylan digestion data for cellulase plus β-glucosidase mass loadings of 14.5 and 29.0 mg/g glucan in raw corn stover with xylanase mass supplementation ratios of 1 and 5 are summarized in Table 6. Xylanase supplementation substantially increased xylose release from solids resulting from all pretreatments but had less effect on glucose release. Overall, xylanase supplementation enhanced pretreatment performance at a given enzyme loading in the following order of increasing impact with

Acknowledgements

Support from the US Department of Energy Office of the Biomass Program (contract DE-FG36-04GO14017) and the National Institute of Standards and Technology (award 60NANB1D0064) made this research possible. We are also grateful to the Center for Environmental Research and Technology of the Bourns College of Engineering at the University of California, Riverside and the Thayer School of Engineering at Dartmouth College for providing key equipment and facilities. Auburn, Michigan State, Purdue, and

References (74)

  • S. Larsson et al.

    The generation of fermentation inhibitors during dilute acid hydrolysis of softwood

    Enzyme and Microbial Technology

    (1999)
  • T.A. Lloyd et al.

    Combined sugar yields for dilute sulfuric acid pretreatment of corn stover followed by enzymatic hydrolysis of the remaining solids

    Bioresource Technology

    (2005)
  • N. Mosier et al.

    Features of promising technologies for pretreatment of lignocellulosic biomass

    Bioresource Technology

    (2005)
  • E. Palmqvist et al.

    Fermentation of lignocellulosic hydrolysates I: inhibition and detoxification

    Bioresource Technology

    (2000)
  • E. Palmqvist et al.

    The effect of water-soluble inhibitors from steam-pretreated willow on enzymic hydrolysis and ethanol fermentation

    Enzyme and Microbial Technology

    (1996)
  • M.J. Selig et al.

    Synergistic enhancement of cellobiohydrolase performance on pretreated corn stover by addition of xylanase and esterase activities

    Bioresource Technology

    (2008)
  • P.K. Smith et al.

    Measurement of protein using bicinchoninic acid

    Analytical Biochemistry

    (1985)
  • F. Teymouri et al.

    Optimization of the ammonia fiber explosion (AFEX) treatment parameters for enzymatic hydrolysis of corn stover

    Bioresource Technology

    (2005)
  • T.M. Wood et al.

    The effect of acetyl groups on the hydrolysis of ryegrass cell walls by xylanase and cellulase from Trichoderma koningii

    Phytochemistry

    (1986)
  • C.E. Wyman

    What is (and is not) vital to advancing cellulosic ethanol

    Trends in Biotechnology

    (2007)
  • H. Alizadeh et al.

    Pretreatment of switchgrass by ammonia fiber explosion (AFEX)

    Applied Biochemistry and Biotechnology

    (2005)
  • S.G. Allen et al.

    A comparison of aqueous and dilute-acid single-temperature pretreatment of yellow poplar sawdust

    Industrial and Engineering Chemistry Research

    (2001)
  • G. Chambat et al.

    Variation of xyloglucan substitution pattern affects the sorption on celluloses with different degrees of crystallinity

    Cellulose (Dordrecht, Netherlands)

    (2005)
  • R.P. Chandra et al.

    Substrate pretreatment: the key to effective enzymatic hydrolysis of lignocellulosics?

    Advances in Biochemical Engineering/Biotechnology

    (2007)
  • V.S. Chang et al.

    Fundamental factors affecting biomass enzymatic reactivity

    Applied Biochemistry and Biotechnology

    (2000)
  • V.S. Chang et al.

    Lime pretreatment of switchgrass

    Applied Biochemistry and Biotechnology

    (1997)
  • A.C. Fernandes et al.

    Homologous xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes

    Biochemical Journal

    (1999)
  • M. García-Aparicio et al.

    Xylanase contribution to the efficiency of cellulose enzymatic hydrolysis of barley straw

    Applied Biochemistry and Biotechnology

    (2007)
  • M.C. Gray et al.

    Solubilities of oligomer mixtures produced by the hydrolysis of xylans and corn stover in water at 180 °C

    Industrial and Engineering Chemistry Research

    (2007)
  • K. Grohmann et al.

    The role of ester groups in resistance of plant cell wall polysaccharides to enzymic hydrolysis

    Applied Biochemistry and Biotechnology

    (1989)
  • R. Gupta et al.

    Substrate dependency and effect of xylanase supplementation on enzymatic hydrolysis of ammonia-treated biomass

    Applied Biochemistry and Biotechnology

    (2008)
  • J.A. Hansson et al.

    Sorption of hemicelluloses on cellulose fibers I. Sorption of xylans

    Svensk Papperstidning

    (1969)
  • J. Hanus et al.

    The xyloglucan-cellulose assembly at the atomic scale

    Biopolymers

    (2006)
  • A.B.B.C.B.S. Heiko Winter

    Preparation of arabinoxylan and its sorption on bacterial cellulose during cultivation

    Macromolecular Symposia

    (2005)
  • R.B. Hespell et al.

    Hydrolysis by commercial enzyme mixtures of AFEX-treated corn fiber and isolated xylans

    Applied Biochemistry and Biotechnology

    (1997)
  • C.I. Ishizawa et al.

    Porosity and its effect on the digestibility of dilute sulfuric acid pretreated corn stover

    Journal of Agricultural and Food Chemistry

    (2007)
  • Kaya, A., Gradwell, S., Glasser, W.G., Esker, A.R., 2005. Studies of xylan self-assembly on cellulose via surface...
  • Cited by (0)

    1

    Currently with Zymetis, Inc., College park, MD 20742, United States.

    View full text