Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass

https://doi.org/10.1016/j.enzmictec.2011.01.007Get rights and content

Abstract

Liquid hot water, steam explosion, and dilute acid pretreatments of lignocellulose generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds. Their effect is seen when an increase in the concentration of pretreated biomass in a hydrolysis slurry results in decreased cellulose conversion, even though the ratio of enzyme to cellulose is kept constant. We used lignin-free cellulose, Solka Floc, combined with mixtures of soluble components released during pretreatment of wood, to prove that the decrease in the rate and extent of cellulose hydrolysis is due to a combination of enzyme inhibition and deactivation. The causative agents were extracted from wood pretreatment liquid using PEG surfactant, activated charcoal or ethyl acetate and then desorbed, recovered, and added back to a mixture of enzyme and cellulose. At enzyme loadings of either 1 or 25 mg protein/g glucan, the most inhibitory components, later identified as phenolics, decreased the rate and extent of cellulose hydrolysis by half due to both inhibition and precipitation of the enzymes. Full enzyme activity occurred when the phenols were removed. Hence detoxification of pretreated woods through phenol removal is expected to reduce enzyme loadings, and therefore reduce enzyme costs, for a given level of cellulose conversion.

Introduction

Liquid hot water, steam explosion, and dilute acid pretreatments generate soluble inhibitors which hamper enzymatic hydrolysis as well as fermentation of sugars to ethanol [1], [2], [3], [4], [5], [6]. Toxic and inhibitory compounds will vary with pretreatment and include soluble sugars, furan derivatives (hydroxymethyl fulfural, furfural), organic acids (acetic, formic and, levulinic acid), and phenolic compounds [3], [5], [6], [7]. The amounts of these soluble inhibitors and their distribution depend on type and severity of pretreatment, concentration of lignocellulosic solids during pretreatment and hydrolysis, and biomass type. They become more pronounced as the biomass concentration in the hydrolysis slurry increases [1], [2], [8], [9]. Washing the pretreated solids with hot water improved enzymatic digestibility of various pretreated lignocellulose feedstocks, thus indicating that at least some of the formed inhibitors are water soluble [2], [3], [8], [9], [10], [11].

Phenolic acids, and particularly, tannic and gallic acid, inhibit β-glucosidase from Trichoderma reesei about twice as much as β-glucosidase from Aspergillus niger and cause deactivation as well as inhibition [5], [6]. Phenol concentrations that result in pronounced decreases in enzyme activity correspond to soluble components associated with 50 g/L or more of pretreated wood. Similar observations have been reported for acid pretreated corn stover [12]. The action of complex mixtures of phenolic compounds has resulted in diverse conclusions regarding their effect on cellulases [3], [5], [6], [7], [13], [14].

The purpose of this study was to identify major soluble inhibitors released during LHW (liquid hot water) pretreatment and to assess the extent of inhibition in high-solids pretreatment slurries. The effect of removing individual inhibitors from pretreatment liquid on cellulose hydrolysis was examined. Inhibitory effects of phenolic compounds were also examined by removing soluble phenolics from pretreatment liquid using Pluronic L62D, activated carbon, or ethyl acetate, recovering the inhibitors, and then adding them back to cellulase in measured amounts. The impact of different inhibitor fractions, and the manner in which they inhibit or precipitate cellulase proteins was determined. This study gave insights into the contribution of the identified inhibitors on enzyme inhibition and deactivation as well as approaches for overcoming inhibition and deactivation to improve cellulose conversion.

Section snippets

Materials

Hammer-milled maple pinchips were provided by Mascoma Corporation (Lebanon, NH). Corn stover milled to 1 in. particle size was obtained from the Agronomy Department at Purdue University. Solka Floc® 300FCC was purchased from International Fiber Corporation (Urbana, Ohio). Spezyme CP (cellulase) and Multifect Pectinase (xylanase) were provided by Genencor, a Danisco Division (Palo Alto, CA). Novozym 188 (β-glucosidase, Novo Nordisk, Novo Allé, Denmark) was purchased from Sigma (Cat. No. C6150).

Composition of maple LHW pretreatment liquid

Liquid hot water (LHW) pretreatment of 230 g/L red maple (Table 1(A)) at 200 °C for 20 min (severity of 4.25), solubilizes 80% of the xylan, 95% of acetyl and arabinan, and less than 1% of the cellulose and lignin initially present, and results in the solid and liquid compositions given in Table 1. Oligomeric and monomeric pentoses prevail in the pretreatment liquid while glucose oligomers and monomers were less than 2 g/L. The LHW pretreatment conditions used here minimize cellulose solubilization

Conclusions

Economical processing of lignocellulose at high-solid concentrations is challenging due to mass transfer limitations, low water activities, insufficient mixing, and soluble inhibitors that fall into four categories: soluble sugars, organic acids, furans, and phenolic compounds. Phenolic compounds and xylo-oligosaccharides were found to be the most important causes of decreased cellulase activity. Soluble xylose sugars, both in oligomeric and monomeric forms, inhibit cellulases instantaneously,

Acknowledgements

The material in this work was supported by DOE Grant #DE-FC36-08G018103 and DE-FG02-06ER64301, and a Mascoma Sponsored Research Agreement (#203081). The authors wish to thank Xingya (Linda) Liu, Thomas Kreke, Marilyn Slininger for their excellent technical assistance, and Miroslav Sedlak, David Hogsett, and Kevin Wenger for their internal review of this article. We thank Genencor for their gift of enzymes.

Statement of competing interest: Michael Ladisch is Chief Technology Officer at Mascoma

References (63)

  • Y.J. Kim et al.

    Synthesis of ultrahigh molecular weight phenolic polymers by enzymatic polymerization in the presence of amphiphilic triblock copolymer in water

    Polymer

    (2008)
  • F. Caturla et al.

    Adsorption of substituted phenols on activated carbon

    J Colloid Interface Sci

    (1988)
  • A. Dąbrowski et al.

    Adsorption of phenolic compounds by activated carbon—a critical review

    Chemosphere

    (2005)
  • O. Bobleter

    Hydrothermal degradation of polymers derived from plants

    Prog Polym Sci

    (1994)
  • J.P. Delgenes et al.

    Effects of lignocellulose degradation products on ethanol fermentations of glucose and xylose by Saccharomyces cerevisiae, Zymomonas mobilis, Pichia stipitis, and Candida shehatae

    Enzyme Microb Technol

    (1996)
  • J.R. Kwiatkowski et al.

    Modeling of the process and costs of fuel ethanol production by corn dry grind process

    Ind Crop Prod

    (2006)
  • X. Jing et al.

    Inhibition performance of lignocellulose degradation products on industrial cellulase enzymes during cellulose hydrolysis

    Appl Biochem Biotechnol

    (2009)
  • M. Cantarella et al.

    Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF

    Biotechnol Prog

    (2004)
  • Y. Kim et al.

    Liquid hot water pretreatment of cellulosic biomass

  • Y. Kim et al.

    Enzymatic digestion of liquid hot water pretreated hybrid poplar

    Biotechnol Prog

    (2009)
  • S.T. Merino et al.

    Progress and challenges in enzyme development for biomass utilization

    Biofuels

    (2007)
  • M. Mandels et al.

    Inhibition of cellulases

    Annu Rev Phytopathol

    (1965)
  • H.I. Oh et al.

    Hydrophobic interaction in tannin protein complexes

    J Agric Food Chem

    (1980)
  • B.D. Hames
  • T. Ehrman

    Standard method for the determination of extractives in biomass, chemical analysis and testing task laboratory analytical procedures

    NREL Ethanol Project

    (1994)
  • T. Ehrman

    Standard method for ash in biomass, chemical analysis and testing task laboratory analytical procedures

    NREL Ethanol Project

    (1994)
  • A. Sluiter et al.

    Determination of structural carbohydrates and lignin in biomass. Biomass analysis technology team laboratory analytical procedures

    NREL Biomass Program

    (2006)
  • A. Sluiter et al.

    Determination of sugars, byproducts, and degradation products in liquid fraction process samples. Biomass analysis technology team laboratory analytical procedures (LAP 014)

    NREL Biomass Program

    (2005)
  • Ladisch MR, Kohlmann K, Westgate P, Weil J, Yang Y. Processes for treating cellulosic material. US Patent 5,846,787...
  • K.L. Kohlmann et al.

    Enhanced enzyme activities on hydrated lignocellulosic substrates

  • T.K. Ghose

    Continuous enzymatic saccharification of cellulose with culture filtrates of Trichoderma viride QM6a

    Biotechnol Bioeng

    (1969)
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