Demonstration‐scale enzymatic saccharification of sulfite‐pulped spruce with addition of hydrogen peroxide for LPMO activation

Abstract

The saccharification of lignocellulosic materials like Norway spruce is challenging due to the recalcitrant nature of the biomass, and it requires optimized and efficient pretreatment and enzymatic hydrolysis processes to make it industrially feasible. In this study, we report successful enzymatic saccharification of sulfite‐pulped spruce (Borregaard's BALI™ process) at demonstration scale, achieved through the controlled delivery of hydrogen peroxide (H2O2) for the activation of lytic polysaccharide monooxygenases (LPMOs) present in the cellulolytic enzyme preparation. We achieved 85% saccharification yield in 4 days using industrially relevant conditions – that is, an enzyme dose of 4% (w/w dry matter of substrate) of the commercial cellulase cocktail Cellic CTec3 and a substrate loading of 12% (w/w). Addition of H2O2 and the resulting controlled and high LPMO activity had a positive effect on the rate of saccharification and the final sugar titer. Clearly, the high LPMO activity was dependent on feeding the reactors with the LPMO co‐substrate H2O2, as in situ generation of H2O2 from molecular oxygen was limited. These demonstration‐scale experiments provide a solid basis for the use of H2O2 to improve enzymatic saccharification of lignocellulosic biomass at large industrial scale.© 2020 The Authors. Biofuels, Bioproducts, and Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd.

Enzymatic processing of lignocellulosic biomass: principles, recent advances and perspectives

“…As lignin constitutes nearly a third of plant biomass, the fate of the lignin fraction will need to be considered in the further development of biorefining processes for efficient and economic processing of lignocellulosic feedstocks [24,285,311]. A good example for the way forward is the so-called BALI process, where sulfite pretreatment generates both valuable carbohydrate and lignin streams which can be turned into valuable products [65,301].…”

“…3) and may only be feasible when the feedstock is relatively rich in lignin. For substrates with low lignin content, direct supply of H 2 O 2 works extremely well [248], also at demonstration scale [65]. For lignin-rich substrates, however, the benefits of direct addition of external H 2 O 2 are less clear [248], presumably due to side-reactions occurring between added H 2 O 2 and lignin [185].…”

“…To employ LPMOs in the degradation of lignin-poor cellulosic substrates, it is necessary to supply the saccharification reaction with external reducing agents like ascorbic acid to activate the LPMOs [250]. For saccharification of cellulose-rich sulfite-pulped spruce, it has been shown that lignin-containing spent sulfite liquor can work as an electron donor [62,65]. On the other hand, accumulating data confirm that the LPMO reaction can be driven by lignin remaining in the biomass after various pretreatments, including dilute-sulfuric acid pretreatment [134], steam explosion [250] or hydrothermal pretreatment [48,185], although to varying extents [296].…”

“…Similarly to BGs, GH61s, today called LPMOs, have been incorporated in the Cellic CTec series [48,62,135,160,250]. Of note, while the contribution of LPMOs to the efficiency of today's cellulase cocktails is clear and important [49,65,146,167,[248][249][250], optimizing this impact is not easy and requires careful consideration of reaction conditions [60], as discussed below.…”

“…It has been shown for various reaction setups that too high feeding rates of externally added H 2 O 2 [200,248] or too high levels of in situ production of H 2 O 2 [185,269] lead to LPMO inactivation. Recent studies following the accumulation of LPMO products over the course of H 2 O 2 -assisted saccharification of industrial feedstocks [37,65,167,248] clearly indicate that LPMO inactivation occurs presumably due to the accumulation of H 2 O 2 in the reaction mixture, although the extent and rate of inactivation over time remain to be elucidated. Notably, there is a clear difference between LPMOs in terms of redox stability [66,272], partly due to the presence or absence of CBMs (discussed below).…”