Storage and handling of pretreated lignocellulose affects the redox chemistry during subsequent enzymatic saccharification

Abstract

The decomposition of lignocellulose in nature, as well as when used as feedstock in industrial settings, takes place in a dynamic system of biotic and abiotic reactions. In the present study, the impact of abiotic reactions during the storage of pretreated lignocellulose on the efficiency of subsequent saccharification was investigated. Abiotic decarboxylation was higher in steam-pretreated wheat straw (SWS, up till 1.5% CO₂) than in dilute-acid-catalysed steam-pretreated forestry residue (SFR, up till 3.2% CO₂) which could be due to higher iron content in SFR and there was no significant CO₂ production in warm-water-washed slurries. Unwashed slurries rapidly consumed O₂ during incubation at 50 °C; the behaviour was more dependent on storage conditions in case of SWS than SFR slurries. There was a pH drop in the slurries which did not correlate with acetic acid release. Storage of SWS under aerobic conditions led to oxidation of the substrate and reduced the extent of enzymatic saccharification by Cellic®  CTec3. Catalase had no effect on the fractional conversion of the aerobically stored substrate, suggesting that the lower fractional conversion was due to reduced activity of the lytic polysaccharide monooxygenase component during saccharification. The fractional conversion of SFR was low in all cases, and cellulose hydrolysis ceased before the first sampling point. This was possibly due to excessive pretreatment of the forest residues. The conditions at which pretreated lignocellulose are stored after pretreatment significantly influenced the extent and kind of abiotic reactions that take place during storage. This in turn influenced the efficiency of subsequent saccharification. Pretreated substrates for laboratory testing must, therefore, be stored in a manner that minimizes abiotic oxidation to ensure that the properties of the substrate resemble those in an industrial setting, where pretreated lignocellulose is fed almost directly into the saccharification vessel.

Keywords

Biotic / Abiotic / LPMO / Catalase / Decarboxylation / O₂ consumption

References

[1] Belt T, Mollerup F, Hanninen T, Rautkari L. Inhibitory effects of Scots pine heartwood extractives on enzymatic holocellulose hydrolysis by wood decaying fungi. Int Biodeterior Biodegradation, 2018, 132: 150-156.

[2] Bondesson P-M, Galbe M, Zacchi G. Ethanol and biogas production after steam pretreatment of corn stover with or without the addition of sulphuric acid. Biotechnol Biofuels, 2013, 6: 11.

[3] Eibinger M, Sattelkow J, Ganner T, Plank H, Nidetzky B. Single-molecule study of oxidative enzymatic deconstruction of cellulose. Nature Communications, 2017, 8(1): 894.

[4] Franko B, Galbe M, Wallberg O. Influence of bark on fuel ethanol production from steam-pretreated spruce. Biotechnol Biofuels, 2015, 8: 15.

[5] Hall SJ, Silver WL. Iron oxidation stimulates organic matter decomposition in humid tropical forest soils. Glob Change Biol, 2013, 19(9): 2804-2813.

[6] Johansen KS. Discovery and industrial applications of lytic polysaccharide mono-oxygenases. Biochem Soc Trans, 2016, 44(1): 143-149.

[7] Kellock M, Maaheimo H, Marjamaa K, Rahikainen J, Zhang H, Holopainen-Mantila U, Ralph J, Tamminen T, Felby C, Kruus K. Effect of hydrothermal pretreatment severity on lignin inhibition in enzymatic hydrolysis. Biores Technol, 2019, 280: 303-312.

[8] Müller G, Várnai A, Johansen KS, Eijsink VG, Horn SJ. Harnessing the potential of LPMO-containing cellulase cocktails poses new demands on processing conditions. Biotechnol Biofuels, 2015, 8: 187.

[9] Page SE, Kling GW, Sander M, Harrold KH, Logan JR, McNeill K, Cory RM. Dark formation of hydroxyl radical in arctic soil and surface waters. Environ Sci Technol, 2013, 47(22): 12860-12867.

[10] Peciulyte A, Samuelsson L, Olsson L, McFarland K, Frickmann J, Østergård L, Halvorsen R, Scott B, Johansen K. Redox processes acidify and decarboxylate steam-pretreated lignocellulosic biomass and are modulated by LPMO and catalase. Biotechnol Biofuels, 2018, 11(1): 165.

[11] Quinlan RJ, Sweeney MD, Leggio LL, Otten H, Poulsen J-CN, Johansen KS, Krogh KB, Jørgensen CI, Tovborg M, Anthonsen A. Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components. Proc Natl Acad Sci, 2011, 108(37): 15079-15084.

[12] Scott BR, Huang HZ, Frickman J, Halvorsen R, Johansen KS. Catalase improves saccharification of lignocellulose by reducing lytic polysaccharide monooxygenase-associated enzyme inactivation. Biotech Lett, 2016, 38(3): 425-434.

[13] Trusiak A, Treibergs LA, Kling GW, Cory RM. The role of iron and reactive oxygen species in the production of CO₂ in arctic soil waters. Geochim Cosmochim Acta, 2018, 224: 80-95.

[14] Vaaje-Kolstad G, Westereng B, Horn SJ, Liu Z, Zhai H, Sorlie M, Eijsink VG. An oxidative enzyme boosting the enzymatic conversion of recalcitrant polysaccharides. Science, 2010, 330(6001): 219-222.

[15] Walpen N, Getzinger GJ, Schroth MH, Sander M. Electron-donating phenolic and electron-accepting quinone moieties in peat dissolved organic matter: quantities and redox transformations in the context of peat biogeochemistry. Environ Sci Technol, 2018, 52(9): 5236-5245.

[16] Walpen N, Lau MP, Fiskal A, Getzinger GJ, Meyer SA, Nelson TF, Lever MA, Schroth MH, Sander M. Oxidation of reduced peat particulate organic matter by dissolved oxygen: quantification of apparent rate constants in the field. Environ Sci Technol, 2018, 52(19): 11151-11160.

[17] Zhang J, Shao S, Bao J. Long term storage of dilute acid pretreated corn stover feedstock and ethanol fermentability evaluation. Biores Technol, 2016, 201: 355-359.

Funding

Novo Nordisk Fonden(NNF17SA0027704)

Energimyndigheten (SE)(41259-1)