Sugar loss and enzyme inhibition due to oligosaccharide accumulation during high solids-loading enzymatic hydrolysis

Xue, Saisi; Uppugundla, Nirmal; Bowman, Michael J.; Cavalier, David; Da Costa Sousa, Leonardo; E Dale, Bruce.; Balan, Venkatesh

doi:10.1186/s13068-015-0378-9

Title: Sugar loss and enzyme inhibition due to oligosaccharide accumulation during high solids-loading enzymatic hydrolysis

Journal Article · Thu Nov 26 00:00:00 EST 2015 · Biotechnology for Biofuels

DOI:https://doi.org/10.1186/s13068-015-0378-9· OSTI ID:1618625

; Uppugundla, Nirmal; Bowman, Michael J.; Cavalier, David; Da Costa Sousa, Leonardo; E Dale, Bruce.; Balan, Venkatesh

Accumulation of recalcitrant oligosaccharides during high-solids loading enzymatic hydrolysis of cellulosic biomass reduces biofuel yields and increases processing costs for a cellulosic biorefinery. Recalcitrant oligosaccharides in AFEX-pretreated corn stover hydrolysate accumulate to the extent of about 18–25 % of the total soluble sugars in the hydrolysate and 12–18 % of the total polysaccharides in the inlet biomass (untreated), equivalent to a yield loss of about 7–9 kg of monomeric sugars per 100 kg of inlet dry biomass (untreated). These oligosaccharides represent a yield loss and also inhibit commercial hydrolytic enzymes, with both being serious bottlenecks for economical biofuel production from cellulosic biomass. Very little is understood about the nature of these oligomers and why they are recalcitrant to commercial enzymes. This work presents a robust method for separating recalcitrant oligosaccharides from high solid loading hydrolysate in gramme quantities. Composition analysis, recalcitrance study and enzyme inhibition study were performed to understand their chemical nature. Results indicate that, oligosaccharide accumulation occurs during high solid loading enzymatic hydrolysis of corn stover (CS) irrespective of using different pretreated corn stover (dilute acid: DA, ionic liquids: IL, and ammonia fibre expansion: AFEX). The methodology for large-scale separation of recalcitrant oligosaccharides from 25 % solids-loading AFEXcorn stover hydrolysate using charcoal fractionation and size exclusion chromatography is reported for the first time. Oligosaccharides with higher degree of polymerization (DP) were recalcitrant towards commercial enzyme mixtures [Ctec2, Htec2 and Multifect pectinase (MP)] compared to lower DP oligosaccharides. Enzyme inhibition studies using processed substrates (Avicel and xylan) showed that low DP oligosaccharides also inhibit commercial enzymes. Addition of monomeric sugars to oligosaccharides increases the inhibitory effects of oligosaccharides on commercial enzymes. In conclusion, the carbohydrate composition of the recalcitrant oligosaccharides, ratios of different DP oligomers and their distribution profiles were determined. Recalcitrance and enzyme inhibition studies help determine whether the commercial enzyme mixtures lack the enzyme activities required to completely de-polymerize the plant cell wall. Such studies clarify the reasons for oligosaccharide accumulation and contribute to strategies by which oligosaccharides can be converted into fermentable sugars and provide higher biofuel yields with less enzyme.

View Journal Article

Cite

Export

Save

Research Organization:: Wisconsin Alumni Research Foundation, Madison, WI (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER)

Grant/Contract Number:: DE‐FC02‐07ER64494; FC02-07ER64494

OSTI ID:: 1618625

Alternate ID(s):: OSTI ID: 1240995

Journal Information:: Biotechnology for Biofuels, Journal Name: Biotechnology for Biofuels Vol. 8 Journal Issue: 1; ISSN 1754-6834

Publisher:: Springer Science + Business MediaCopyright Statement

Country of Publication:: Netherlands

Language:: English

Citation Metrics:

Cited by: 50 works

Citation information provided by
Web of Science

References (50)

Yield-determining factors in high-solids enzymatic hydrolysis of lignocellulose Kristensen, Jan B.; Felby, Claus; Jørgensen, Henning Biotechnology for Biofuels, Vol. 2, Issue 1 https://doi.org/10.1186/1754-6834-2-11	journal	January 2009
A novel integrated biological process for cellulosic ethanol production featuring high ethanol productivity, enzyme recycling and yeast cells reuse Jin, Mingjie; Gunawan, Christa; Uppugundla, Nirmal Energy & Environmental Science, Vol. 5, Issue 5 https://doi.org/10.1039/c2ee03058f	journal	January 2012
Synergistic action of enzyme preparations towards recalcitrant corn silage polysaccharides Neumüller, Klaus G.; Streekstra, Hugo; Schols, Henk A. Biomass and Bioenergy, Vol. 60 https://doi.org/10.1016/j.biombioe.2013.11.004	journal	January 2014
Xylooligomers are strong inhibitors of cellulose hydrolysis by enzymes Qing, Qing; Yang, Bin; Wyman, Charles E. Bioresource Technology, Vol. 101, Issue 24, p. 9624-9630 https://doi.org/10.1016/j.biortech.2010.06.137	journal	December 2010
Economic evaluation of preconcentration in production of ethanol from dilute sugar solutions Zacchi, G.; Axelsson, A. Biotechnology and Bioengineering, Vol. 34, Issue 2 https://doi.org/10.1002/bit.260340211	journal	June 1989
Profiling of soluble neutral oligosaccharides from treated biomass using solid phase extraction and LC–TOF MS Vismeh, Ramin; Humpula, James F.; Chundawat, Shishir P. S. Carbohydrate Polymers, Vol. 94, Issue 2 https://doi.org/10.1016/j.carbpol.2013.02.005	journal	May 2013
‘Cradle-to-grave’ assessment of existing lignocellulose pretreatment technologies da Costa Sousa, Leonardo; Chundawat, Shishir PS; Balan, Venkatesh Current Opinion in Biotechnology, Vol. 20, Issue 3 https://doi.org/10.1016/j.copbio.2009.05.003	journal	June 2009
Energy, wealth, and human development: Why and how biomass pretreatment research must improve Dale, Bruce E.; Ong, Rebecca G. Biotechnology Progress, Vol. 28, Issue 4 https://doi.org/10.1002/btpr.1575	journal	July 2012
Multi-scale visualization and characterization of lignocellulosic plant cell wall deconstruction during thermochemical pretreatment Chundawat, Shishir P. S.; Donohoe, Bryon S.; da Costa Sousa, Leonardo Energy & Environmental Science, Vol. 4, Issue 3, 973 https://doi.org/10.1039/c0ee00574f	journal	January 2011
Enzymatic conversion of lignocellulose into fermentable sugars: challenges and opportunities Jørgensen, Henning; Kristensen, Jan Bach; Felby, Claus Biofuels, Bioproducts and Biorefining, Vol. 1, Issue 2 https://doi.org/10.1002/bbb.4	journal	October 2007
Enzyme resistant feruloylated xylooligomer analogues from thermochemically treated corn fiber contain large side chains, ethyl glycosides and novel sites of acetylation Appeldoorn, Maaike M.; de Waard, Pieter; Kabel, Mirjam A. Carbohydrate Research, Vol. 381 https://doi.org/10.1016/j.carres.2013.08.024	journal	November 2013
Reactor design for minimizing product inhibition during enzymatic lignocellulose hydrolysis: I. Significance and mechanism of cellobiose and glucose inhibition on cellulolytic enzymes Andrić, Pavle; Meyer, Anne S.; Jensen, Peter A. Biotechnology Advances, Vol. 28, Issue 3, p. 308-324 https://doi.org/10.1016/j.biotechadv.2010.01.003	journal	May 2010
Analysis of oligosaccharides in lignocellulosic biomass hydrolysates by high-performance anion-exchange chromatography coupled with mass spectrometry (HPAEC–MS) Coulier, Leon; Zha, Ying; Bas, Richard Bioresource Technology, Vol. 133 https://doi.org/10.1016/j.biortech.2013.01.085	journal	April 2013
β-Xylosidases and α-l-arabinofuranosidases: Accessory enzymes for arabinoxylan degradation Lagaert, Stijn; Pollet, Annick; Courtin, Christophe M. Biotechnology Advances, Vol. 32, Issue 2 https://doi.org/10.1016/j.biotechadv.2013.11.005	journal	March 2014
LC/CE–MS tools for the analysis of complex arabino-oligosaccharides Westphal, Yvonne; Kühnel, Stefan; Schols, Henk A. Carbohydrate Research, Vol. 345, Issue 15 https://doi.org/10.1016/j.carres.2010.07.011	journal	October 2010
Inhibition of the Trichoderma reesei cellulases by cellobiose is strongly dependent on the nature of the substrate : Product Inhibition in Cellulose Hydrolysis Gruno, Marju; Väljamäe, Priit; Pettersson, Göran Biotechnology and Bioengineering, Vol. 86, Issue 5 https://doi.org/10.1002/bit.10838	journal	March 2004
Current Challenges in Commercially Producing Biofuels from Lignocellulosic Biomass Balan, Venkatesh ISRN Biotechnology, Vol. 2014 https://doi.org/10.1155/2014/463074	journal	January 2014
Gradient Elution Analysis. II. Oligosaccharides. Alm, R. S.; Sörensen, Nils Andreas; Nestvold, Magne Acta Chemica Scandinavica, Vol. 6 https://doi.org/10.3891/acta.chem.scand.06-1186	journal	January 1952
Economic evaluation of alternative ethanol fermentation processes Maiorella, B. L.; Blanch, H. W.; Wilke, C. R. Biotechnology and Bioengineering, Vol. 26, Issue 9 https://doi.org/10.1002/bit.260260902	journal	September 1984
Xylan oligosaccharides and cellobiohydrolase I (TrCel7A) interaction and effect on activity Baumann, Martin J.; Borch, Kim; Westh, Peter Biotechnology for Biofuels, Vol. 4, Issue 1 https://doi.org/10.1186/1754-6834-4-45	journal	January 2011
How biotech can transform biofuels Lynd, Lee R.; Laser, Mark S.; Bransby, David Nature Biotechnology, Vol. 26, Issue 2, p. 169-172 https://doi.org/10.1038/nbt0208-169	journal	February 2008
Comparative material balances around pretreatment technologies for the conversion of switchgrass to soluble sugars Garlock, Rebecca J.; Balan, Venkatesh; Dale, Bruce E. Bioresource Technology, Vol. 102, Issue 24 https://doi.org/10.1016/j.biortech.2011.04.002	journal	December 2011
Comparison of enzymatic reactivity of corn stover solids prepared by dilute acid, AFEX™, and ionic liquid pretreatments Gao, Xiadi; Kumar, Rajeev; Singh, Seema Biotechnology for Biofuels, Vol. 7, Issue 1 https://doi.org/10.1186/1754-6834-7-71	journal	January 2014
Deconstruction of Lignocellulosic Biomass to Fuels and Chemicals Chundawat, Shishir P. S.; Beckham, Gregg T.; Himmel, Michael E. Annual Review of Chemical and Biomolecular Engineering, Vol. 2, Issue 1, p. 121-145 https://doi.org/10.1146/annurev-chembioeng-061010-114205	journal	July 2011
Studying the rapid bioconversion of lignocellulosic sugars into ethanol using high cell density fermentations with cell recycle Sarks, Cory; Jin, Mingjie; Sato, Trey K. Biotechnology for Biofuels, Vol. 7, Issue 1 https://doi.org/10.1186/1754-6834-7-73	journal	January 2014
A comparative study of ethanol production using dilute acid, ionic liquid and AFEX™ pretreated corn stover Uppugundla, Nirmal; da Costa Sousa, Leonardo; Chundawat, Shishir PS Biotechnology for Biofuels, Vol. 7, Issue 1 https://doi.org/10.1186/1754-6834-7-72	journal	January 2014
Mixture optimization of six core glycosyl hydrolases for maximizing saccharification of ammonia fiber expansion (AFEX) pretreated corn stover Gao, Dahai; Chundawat, Shishir P. S.; Krishnan, Chandraraj Bioresource Technology, Vol. 101, Issue 8, p. 2770-2781 https://doi.org/10.1016/j.biortech.2009.10.056	journal	April 2010
Fungal enzyme sets for plant polysaccharide degradation van den Brink, Joost; de Vries, Ronald P. Applied Microbiology and Biotechnology, Vol. 91, Issue 6, p. 1477-1492 https://doi.org/10.1007/s00253-011-3473-2	journal	July 2011
Effect of enzyme supplementation at moderate cellulase loadings on initial glucose and xylose release from corn stover solids pretreated by leading technologies Kumar, Rajeev; Wyman, C. E. Biotechnology and Bioengineering, Vol. 102, Issue 2, p. 457-467 https://doi.org/10.1002/bit.22068	journal	February 2009
Microfluidic Glycosyl Hydrolase Screening for Biomass-to-Biofuel Conversion Bharadwaj, Rajiv; Chen, Zhiwei; Datta, Supratim Analytical Chemistry, Vol. 82, Issue 22 https://doi.org/10.1021/ac102243f	journal	November 2010
Chromatographic determination of 1, 4-β-xylooligosaccharides of different chain lengths to follow xylan deconstruction in biomass conversion Li, Hongjia; Qing, Qing; Kumar, Rajeev Journal of Industrial Microbiology & Biotechnology, Vol. 40, Issue 6 https://doi.org/10.1007/s10295-013-1254-x	journal	March 2013
Isolation and characterization of unhydrolyzed oligosaccharides from switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with commercial enzyme preparations Bowman, Michael J.; Dien, Bruce S.; Vermillion, Karl E. Carbohydrate Research, Vol. 407 https://doi.org/10.1016/j.carres.2015.01.018	journal	April 2015
Soluble inhibitors/deactivators of cellulase enzymes from lignocellulosic biomass Kim, Youngmi; Ximenes, Eduardo; Mosier, Nathan S. Enzyme and Microbial Technology, Vol. 48, Issue 4-5 https://doi.org/10.1016/j.enzmictec.2011.01.007	journal	April 2011
Xylo-oligosaccharides are competitive inhibitors of cellobiohydrolase I from Thermoascus aurantiacus Zhang, Junhua; Viikari, Liisa Bioresource Technology, Vol. 117 https://doi.org/10.1016/j.biortech.2012.04.072	journal	August 2012
Structural characterization of (1→2)-β-xylose-(1→3)-α-arabinose-containing oligosaccharide products of extracted switchgrass (Panicum virgatum, L.) xylan after exhaustive enzymatic treatment with α-arabinofuranosidase and β-endo-xylanase Bowman, Michael J.; Dien, Bruce S.; Vermillion, Karl E. Carbohydrate Research, Vol. 398 https://doi.org/10.1016/j.carres.2014.08.006	journal	October 2014
Chemical and Physicochemical Pretreatment of Lignocellulosic Biomass: A Review Brodeur, Gary; Yau, Elizabeth; Badal, Kimberly Enzyme Research, Vol. 2011 https://doi.org/10.4061/2011/787532	journal	January 2011
Ethanol Production from Xylo-oligosaccharides by Xylose-Fermenting Saccharomyces cerevisiae Expressing β-Xylosidase Fujii, Tatsuya; Yu, Guoce; Matsushika, Akinori Bioscience, Biotechnology, and Biochemistry, Vol. 75, Issue 6 https://doi.org/10.1271/bbb.110043	journal	June 2011
Liquid chromatography–mass spectrometry investigation of enzyme-resistant xylooligosaccharide structures of switchgrass associated with ammonia pretreatment, enzymatic saccharification, and fermentation Bowman, Michael J.; Dien, Bruce S.; Hector, Ronald E. Bioresource Technology, Vol. 110 https://doi.org/10.1016/j.biortech.2012.01.062	journal	April 2012
Hydrolysis of different chain length xylooliogmers by cellulase and hemicellulase Qing, Qing; Wyman, Charles E. Bioresource Technology, Vol. 102, Issue 2 https://doi.org/10.1016/j.biortech.2010.09.001	journal	January 2011
Hemicellulases and auxiliary enzymes for improved conversion of lignocellulosic biomass to monosaccharides Gao, Dahai; Uppugundla, Nirmal; Chundawat, Shishir PS Biotechnology for Biofuels, Vol. 4, Issue 1 https://doi.org/10.1186/1754-6834-4-5	journal	January 2011
A study of methods for preparing oligosaccharides from cellulose Miller, Gail Lorenz; Dean, John; Blum, Robert Archives of Biochemistry and Biophysics, Vol. 91, Issue 1 https://doi.org/10.1016/0003-9861(60)90448-3	journal	November 1960
Binding characteristics of Trichoderma reesei cellulases on untreated, ammonia fiber expansion (AFEX), and dilute-acid pretreated lignocellulosic biomass Gao, Dahai; Chundawat, Shishir P. S.; Uppugundla, Nirmal Biotechnology and Bioengineering, Vol. 108, Issue 8 https://doi.org/10.1002/bit.23140	journal	April 2011
Multifaceted characterization of cell wall decomposition products formed during ammonia fiber expansion (AFEX) and dilute acid based pretreatments Chundawat, Shishir P. S.; Vismeh, Ramin; Sharma, Lekh N. Bioresource Technology, Vol. 101, Issue 21 https://doi.org/10.1016/j.biortech.2010.06.027	journal	November 2010
Fermentation of xylo-oligosaccharides by Bifidobacterium adolescentis DSMZ 18350: kinetics, metabolism, and β-xylosidase activities Amaretti, Alberto; Bernardi, Tatiana; Leonardi, Alan Applied Microbiology and Biotechnology, Vol. 97, Issue 7 https://doi.org/10.1007/s00253-012-4509-y	journal	October 2012
Strong cellulase inhibitors from the hydrothermal pretreatment of wheat straw Kont, Riin; Kurašin, Mihhail; Teugjas, Hele Biotechnology for Biofuels, Vol. 6, Issue 1 https://doi.org/10.1186/1754-6834-6-135	journal	January 2013
Comparative Analysis of End Point Enzymatic Digests of Arabino-Xylan Isolated from Switchgrass (Panicum virgatum L) of Varying Maturities using LC-MSn Bowman, Michael; Dien, Bruce; O'Bryan, Patricia Metabolites, Vol. 2, Issue 4 https://doi.org/10.3390/metabo2040959	journal	November 2012
Liquefaction of lignocellulose at high-solids concentrations Jørgensen, Henning; Vibe-Pedersen, Jakob; Larsen, Jan Biotechnology and Bioengineering, Vol. 96, Issue 5 https://doi.org/10.1002/bit.21115	journal	January 2007
Optimization of enzyme complexes for lignocellulose hydrolysis Berlin, Alex; Maximenko, Vera; Gilkes, Neil Biotechnology and Bioengineering, Vol. 97, Issue 2, p. 287-296 https://doi.org/10.1002/bit.21238	journal	January 2007
The use of high-solids loadings in biomass pretreatment-a review Modenbach, Alicia A.; Nokes, Sue E. Biotechnology and Bioengineering, Vol. 109, Issue 6 https://doi.org/10.1002/bit.24464	journal	February 2012
Conversion of biomass-derived oligosaccharides into lipids Gong, Zhiwei; Wang, Qian; Shen, Hongwei Biotechnology for Biofuels, Vol. 7, Issue 1 https://doi.org/10.1186/1754-6834-7-13	journal	January 2014

Cited By (8)

Comprehensive characterization of non-cellulosic recalcitrant cell wall carbohydrates in unhydrolyzed solids from AFEX-pretreated corn stover Gunawan, Christa; Xue, Saisi; Pattathil, Sivakumar Biotechnology for Biofuels, Vol. 10, Issue 1 https://doi.org/10.1186/s13068-017-0757-5	journal	March 2017
Insight into the role of α-arabinofuranosidase in biomass hydrolysis: cellulose digestibility and inhibition by xylooligomers Xin, Donglin; Chen, Xiang; Wen, Peiyao Biotechnology for Biofuels, Vol. 12, Issue 1 https://doi.org/10.1186/s13068-019-1412-0	journal	March 2019
Some critical aspects of the enzymatic hydrolysis at high dry-matter content: a review: BAD MIXING AND ENZYMES DEACTIVATION AT HIGH DM CONCENTRATION Battista, Federico; Bolzonella, David Biofuels, Bioproducts and Biorefining, Vol. 12, Issue 4 https://doi.org/10.1002/bbb.1883	journal	May 2018
Heterologous Expression and Characterization of an Acidic GH11 Family Xylanase from Hypocrea orientalis Li, Hailong; Wu, Hongli; Jiang, Fengjiao Applied Biochemistry and Biotechnology, Vol. 184, Issue 1 https://doi.org/10.1007/s12010-017-2532-2	journal	July 2017
Cellulose solvent-based pretreatment for enhanced second-generation biofuel production: a review Satari, Behzad; Karimi, Keikhosro; Kumar, Rajeev Sustainable Energy & Fuels, Vol. 3, Issue 1 https://doi.org/10.1039/c8se00287h	journal	January 2019
Fed-batch enzymatic hydrolysis of alkaline organosolv-pretreated corn stover facilitating high concentrations and yields of fermentable sugars for microbial lipid production Gong, Zhiwei; Wang, Xuemin; Yuan, Wei Biotechnology for Biofuels, Vol. 13, Issue 1 https://doi.org/10.1186/s13068-019-1639-9	journal	January 2020
Characterization of a thermophilic cellulase from Geobacillus sp. HTA426, an efficient cellulase-producer on alkali pretreated of lignocellulosic biomass Potprommanee, Laddawan; Wang, Xiao-Qin; Han, Ye-Ju PLOS ONE, Vol. 12, Issue 4 https://doi.org/10.1371/journal.pone.0175004	journal	April 2017
In Situ Rheological Method to Evaluate Feedstock Physical Properties Throughout Enzymatic Deconstruction Coffman, Philip; McCaffrey, Nicole; Gardner, James Frontiers in Energy Research, Vol. 6 https://doi.org/10.3389/fenrg.2018.00053	journal	July 2018

Similar Records

A comparative study of ethanol production using dilute acid, ionic liquid and AFEX™ pretreated corn stover

Journal Article · Tue May 13 00:00:00 EDT 2014 · Biotechnology for Biofuels · OSTI ID:1618625

Uppugundla, Nirmal; da Costa Sousa, Leonardo; Chundawat, Shishir P. S.; +8 more

Understanding the structure and composition of recalcitrant oligosaccharides in hydrolysate using high-throughput biotin-based glycome profiling and mass spectrometry

Journal Article · Tue Feb 15 00:00:00 EST 2022 · Scientific Reports · OSTI ID:1618625

Xue, Saisi; Pattathil, Sivakumar; da Costa Sousa, Leonardo; +4 more

Comparison of enzymatic reactivity of corn stover solids prepared by dilute acid, AFEX™, and ionic liquid pretreatments

Journal Article · Wed Jan 01 00:00:00 EST 2014 · Biotechnology for Biofuels · OSTI ID:1618625

Gao, Xiadi; Kumar, Rajeev; Singh, Seema; +4 more

Related Subjects

09 BIOMASS FUELS
recalcitrant oligosaccharides
high solids-loading
commercial enzymes
AFEX-CS hydrolysate
charcoal fractionation
size exclusion chromatography
enzyme inhibition

Title: Sugar loss and enzyme inhibition due to oligosaccharide accumulation during high solids-loading enzymatic hydrolysis

Citation Formats

References (50)

Cited By (8)

Similar Records

Related Subjects