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Achieving high yields in enzymatic saccharification of cellulose is a critical step in biochemical conversion of pretreated lignocellulosic biomass. Sugar formed during saccharification serves as substrate for fermenting microorganisms producing bio-based fuels and chemicals. An oxidoreductase, lytic polysaccharide monooxygenase (LPMO), has recently gained attention for its potential to act synergistically with conventional hydrolytic enzymes catalyzing the deconstruction of cellulose. This investigation has focused on LPMO-supported enzymatic saccharification of cellulose, exploring the process conditions, particularly the redox environment, affecting LPMO-supported saccharification of biomass. The involvement of LPMO necessitates reevaluation of industrial process configurations, especially in terms of aeration strategies. The impact of aeration on saccharification and fermentation, for example through potential side effects on fermentation inhibitors generated during the pretreatment, is not well understood, and the aim of the investigations has been to shed light on that gap of knowledge.

The role of lignin as a reductant in LPMO-supported enzymatic saccharification was investigated, focusing on both lignin in the solid fraction and water-soluble lignin degradation products in the liquid fraction. A novel experimental set-up with controlled gas addition (six parallel reactions, three with air and three with N₂) was used to regulate the redox environment. Glucose production was consistently higher in reactions with air. Both lignin in the solid fraction and degradation products in the liquid fraction efficiently supported LPMO catalysis.

The benefits of continuous aeration in LPMO-supported enzymatic saccharification were weighed against the negative effects associated with high solids loadings in reaction mixtures. Studies in the range 12.5% to 17.5% water-insoluble solids (WIS) showed that the positive effects of aeration to support LPMO were larger than the negative effects of high solids loadings. Notably, glucan conversion with aeration at 17.5% WIS exceeded that obtained with N₂ at 12.5% WIS. Additionally, doubling the enzyme dosage was less effective in enhancing glucan conversion than using aeration rather than N₂. These findings demonstrate the significant potential of continuous aeration to boost LPMO activity when using high solids loadings in biomass conversion.

A hybrid hydrolysis and fermentation (HHF) process, incorporating an initial pre-hydrolysis phase with aeration at a relatively high temperature, was compared to simultaneous saccharification and fermentation (SSF). Using steam-exploded softwood as substrate, pre-hydrolysis with aeration improved glucan conversion in HHF, but the overall conversion remained modest. Extending the aeration period from 24 h to 48 h slightly enhanced saccharification but had a negative impact on the subsequent fermentation with Saccharomyces cerevisiae yeast. Thus, under the experimental conditions used, HHF with aeration led to increased glucan conversion, but the benefits were not sufficient to achieve an ethanol yield and productivity that was comparable to those achieved using SSF.

The potential negative impact of aeration on subsequent fermentation was investigated further in studies of the liquid phase of steam-exploded softwood. Compared to parallel N₂ control reactions, aeration caused a more inhibitory environment for S. cerevisiae yeast. Although the concentrations of some inhibitors, such as furfural, decreased during aeration, there was a slight but consistent increase in the concentrations of formaldehyde, a phenomenon that could, at least partially, explain increased inhibition. Sulfite detoxification was effective regardless of aeration. Laccase treatment showed mixed effects on fermentability, which could be attributed to the treatment causing an overall decrease invthe content of phenolic inhibitors, but also formation of more toxic substances from relatively harmless precursors.