Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass
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.
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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)
- et al.
The effect of water-soluble inhibitors from steam-pretreated willow on enzymatic hydrolysis and ethanol fermentation
Enzyme Microb Technol
(1996) - et al.
Reduced inhibition of enzymatic hydrolysis of steam-pretreated softwood
Enzyme Microb Technol
(2001) - et al.
Soluble and insoluble solids contributions to high-solids enzymatic hydrolysis of lignocellulose
Bioresour Technol
(2008) - et al.
Inhibition of cellulases by phenols
Enzyme Microb Technol
(2010) - et al.
Deactivation of cellulases by phenols
Enzyme Microb Technol
(2011) - et al.
Enzyme hydrolysis and ethanol fermentation of liquid hot water (LHW) and AFEX pretreated distiller's grains at high solids loadings
Bioresour Technol
(2008) - et al.
Inhibition of enzymatic cellulolysis by phenolic compounds
Enzyme Microb Technol
(2011) - et al.
Cellulose pretreatments of lignocellulosic substrates
Enzyme Microb Technol
(1994) - et al.
Topographical characterization and surface force spectroscopy of the photochemical lignin model compound
Biophys Chem
(2001) - et al.
Lignin precipitation on the pulp fibers in the ethanol-based organosolv pulping
Colloid Surf A
(2007)
Synthesis of ultrahigh molecular weight phenolic polymers by enzymatic polymerization in the presence of amphiphilic triblock copolymer in water
Polymer
Adsorption of substituted phenols on activated carbon
J Colloid Interface Sci
Adsorption of phenolic compounds by activated carbon—a critical review
Chemosphere
Hydrothermal degradation of polymers derived from plants
Prog Polym Sci
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
Modeling of the process and costs of fuel ethanol production by corn dry grind process
Ind Crop Prod
Inhibition performance of lignocellulose degradation products on industrial cellulase enzymes during cellulose hydrolysis
Appl Biochem Biotechnol
Effect of inhibitors released during steam-explosion treatment of poplar wood on subsequent enzymatic hydrolysis and SSF
Biotechnol Prog
Liquid hot water pretreatment of cellulosic biomass
Enzymatic digestion of liquid hot water pretreated hybrid poplar
Biotechnol Prog
Progress and challenges in enzyme development for biomass utilization
Biofuels
Inhibition of cellulases
Annu Rev Phytopathol
Hydrophobic interaction in tannin protein complexes
J Agric Food Chem
Standard method for the determination of extractives in biomass, chemical analysis and testing task laboratory analytical procedures
NREL Ethanol Project
Standard method for ash in biomass, chemical analysis and testing task laboratory analytical procedures
NREL Ethanol Project
Determination of structural carbohydrates and lignin in biomass. Biomass analysis technology team laboratory analytical procedures
NREL Biomass Program
Determination of sugars, byproducts, and degradation products in liquid fraction process samples. Biomass analysis technology team laboratory analytical procedures (LAP 014)
NREL Biomass Program
Enhanced enzyme activities on hydrated lignocellulosic substrates
Continuous enzymatic saccharification of cellulose with culture filtrates of Trichoderma viride QM6a
Biotechnol Bioeng
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