Sulphite addition during steam pretreatment enhanced both enzyme-mediated cellulose hydrolysis and ethanol production

Abstract

Sulphite addition during steam pretreatment of softwoods under acidic, neutral and alkaline conditions was assessed to try to minimize lignin condensation. Although pretreatment under neutral/alkaline conditions resulted in effective lignin sulphonation, non-uniform size reduction was observed. In contrast, acidic sulphite steam treatment at 210 °C for 10 min resulted in homogenous particle size reduction and water-insoluble component that was 62% carbohydrate and 33% lignin. This carbohydrate-rich substrate was readily hydrolyzed and fermented which indicated the lack of fermentation inhibitors in the steam-pretreated whole slurry. The use of high solid loading (25% w/v) resulted in a hydrolysis yield of 58% at an enzyme loading of 40 mg protein/g glucan and efficient fermentation (46.6 g/L of ethanol). This indicated that the addition of acidic sulphite at the steam pretreatment of softwoods improved both the enzymatic hydrolysis and fermentation of steam-pretreated whole slurries.

Keywords

Sulphite steam pretreatment / Acid steam pretreatment / High consistency hydrolysis / Fermentation / Detoxification / Whole slurry

References

[1] Alriksson B, Cavka A, Jönsson LJ. Improving the fermentability of enzymatic hydrolysates of lignocellulose through chemical in-situ detoxification with reducing agents. Bioresour Technol, 2011, 102(2): 1254-1263.

[2] Baig KS. Interaction of enzymes with lignocellulosic materials: causes, mechanism and influencing factors. Bioresour Bioprocess, 2020

[3] Bura R, Chandra R, Saddler J. Influence of xylan on the enzymatic hydrolysis of steam-pretreated corn stover and hybrid poplar. Biotechnol Prog, 2009, 25(2): 315-322.

[4] Cavka A, Alriksson B, Ahnlund M, Jönsson LJ. Effect of sulfur oxyanions on lignocellulose-derived fermentation inhibitors. Biotechnol Bioeng, 2011, 108(11): 2592-2599.

[5] Chandra R, Ewanick S, Hsieh C, Saddler JN. The characterization of pretreated lignocellulosic substrates prior to enzymatic hydrolysis, part 1: a modified Simons’ staining technique. Biotechnol Prog, 2008, 24: 1178-1185.

[6] Chandra RP, Ewanick SM, Chung PA, Au-Yeung K, Rio LD, Mabee W, Saddler JN. Comparison of methods to assess the enzyme accessibility and hydrolysis of pretreated lignocellulosic substrates. Biotechnol Lett, 2009

[7] Chandra RP, Chu QL, Hu J, Zhong N, Lin M, Lee JS, Saddler J. The influence of lignin on steam pretreatment and mechanical pulping of poplar to achieve high sugar recovery and ease of enzymatic hydrolysis. Biores Technol, 2016, 199: 135-141.

[8] Cullis IF. The effect of steam explosion pretreatment parameters on softwood delignification efficiency. Master Thesis, 2003, Vancouver: The University of British Columbia.

[9] Cullis IF, Saddler JN, Mansfield SD. Effect of initial moisture content and chip size on the bioconversion efficiency of softwood lignocellulosics. Biotechnol Bioeng, 2004, 85(4): 413-421.

[10] Del Rio LF, Chandra RP, Saddler JN. The effects of increasing swelling and anionic charges on the enzymatic hydrolysis of organosolv-pretreated softwoods at low enzyme loadings. Biotechnol Bioeng, 2011, 108(7): 1549-1558.

[11] Ji H, Wang L, Tao F, Yao Z, Li X, Dong C, Pang Z. A hydrotrope pretreatment for stabilized lignin extraction and high titer ethanol production. Bioresour Bioprocess, 2022, 9(1): 40.

[12] Jönsson LJ, Martín C. Pretreatment of lignocellulose: formation of inhibitory by-products and strategies for minimizing their effects. Biores Technol, 2016, 199: 103-112.

[13] Jönsson LJ, Alriksson B, Nilvebrant N-O. Bioconversion of lignocellulose: inhibitors and detoxification. Biotechnol Biofuels, 2013, 6(1): 16.

[14] Kapu NS, Piddocke M, Saddler JJN. High gravity and high cell density mitigate some of the fermentation inhibitory effects of softwood hydrolysates. AMB Express, 2013, 3(1): 15.

[15] Katz S, Beatson RP, Anthony MS. The determination of strong and weak acidic groups in sulphite pulps. Sven Papperstidning Nord Cellul, 1984, 87: 48.

[16] Kokta BV, Ahmed A. Steam explosion pulping. Young, 1998, New York: Wiley, 191-211.

[17] Kumar AK, Sharma S. Recent updates on different methods of pretreatment of lignocellulosic feedstocks: a review. Bioresour Bioprocess, 2017

[18] Kumar L, Arantes V, Chandra R, Saddler J. The lignin present in steam pretreated softwood binds enzymes and limits cellulose accessibility. Bioresour Technol, 2012, 103(1): 201-208.

[19] Lan TQ, Gleisner R, Zhu JY, Dien BS, Hector RE. High titer ethanol production from SPORL-pretreated lodgepole pine by simultaneous enzymatic saccharification and combined fermentation. Bioresour Technol, 2013, 127: 291-297.

[20] Li J, Henriksson G, Gellerstedt G. Lignin depolymerization/repolymerization and its critical role for delignification of aspen wood by steam explosion. Biores Technol, 2007, 98(16): 3061-3068.

[21] Lin M. Investigation of a two-stage steam/organosolv pretreatment approach for the fractionation of softwood biomass. Master Thesis, 2016, Vancouver: The University of British Columbia.

[22] Mok YK, Arantes V, Saddler JN. A NaBH4 coupled ninhydrin-based assay for the quantification of protein/enzymes during the enzymatic hydrolysis of pretreated lignocellulosic biomass. Appl Biochem Biotechnol, 2015, 176(6): 1564-1580.

[23] Mooney CA, Mansfield SD, Beatson RP, Saddler JN. The effect of fiber characteristics on hydrolysis and cellulase accessibility to softwood substrates. Enzyme Microb Technol, 1999, 25(8): 644-650.

[24] Nakagame S, Chandra RP, Kadla JF, Saddler JN. The isolation, characterization and effect of lignin isolated from steam pretreated Douglas-fir on the enzymatic hydrolysis of cellulose. Biores Technol, 2011, 102(6): 4507-4517.

[25] Palmqvist E, Hahn-Hägerdal B. Fermentation of lignocellulosic hydrolysates. II: Inhibitors and mechanisms of inhibition. Bioresour Technol, 2000, 74(1): 25-33.

[26] Robertson G, Olson J, Allen P, Chan BEN, Seth R. Measurement of fiber length, coarseness, and shape with the fiber quality analyzer. Tappi J, 1999, 82(10): 93-98.

[27] Singleton VL, Rossi RA Jr. Colorimetry to total phenolics with phosphomolybdic acid reagents. Am J Enol Viniculture, 1965, 16(48): 144-158.

[28] Sjöström E. The structure of wood. Wood chemistry, 1993, Amsterdam: Elsevier, 1-20.

[29] Sluiter A, Hames B, Ruiz R, Scarlata C, Sluiter J, Templeton D. Determination of sugars, byproducts, and degradation products in liquid fraction process samples. Laboratory analytical procedure (LAP), 2006, Colorado: NREL, 08.

[30] Soudham VP, Alriksson B, Jönsson LJ. Reducing agents improve enzymatic hydrolysis of cellulosic substrates in the presence of pretreatment liquid. J Biotechnol, 2011, 155(2): 244-250.

[31] van der Zwan T, Hu J, Saddler JN. Mechanistic insights into the liquefaction stage of enzyme-mediated biomass deconstruction. Biotechnol Bioeng, 2017, 114(11): 2489-2496.

[32] Zhong N. Sulphite treatment to enhance the ethanol production of softwood whole slurries. PhD Thesis, 2018, Vancouver: The University of British Columbia.

[33] Zhong N, Chandra R, Saddler JJN. Sulfite post-treatment to simultaneously detoxify and improve the enzymatic hydrolysis and fermentation of a steam-pretreated softwood lodgepole pine whole slurry. ACS Sustain Chem Eng, 2019, 7(5): 5192-5199.

[34] Zhou H, Zhu JY, Luo X, Leu SY, Wu X, Gleisner R, Dien BS, Hector RE, Yang D, Qiu X, Horn E, Negron J. Bioconversion of beetle-killed lodgepole pine using SPORL: process scale-up design, lignin coproduct, and high solids fermentation without detoxification. Ind Eng Chem Res, 2013, 52(45): 16057-16065.