LPMO-supported saccharification of biomass: effects of continuous aeration of reaction mixtures with variable fractions of water-insoluble solids and cellulolytic enzymes

BACKGROUND: High substrate concentrations and high sugar yields are important aspects of enzymatic saccharification of lignocellulosic substrates. The benefit of supporting the catalytic action of lytic polysaccharide monooxygenase (LPMO) through continuous aeration of slurries of pretreated softwood was weighed against problems associated with increasing substrate content (quantitated as WIS, water-insoluble solids, in the range 12.5–17.5%), and was compared to the beneficial effect on the saccharification reaction achieved by increasing the enzyme preparation (Cellic CTec3) loadings. Aerated reactions were compared to reactions supplied with N(2) to assess the contribution of LPMO to the saccharification reactions. Analysis using (13)C NMR spectroscopy, XRD, Simons’ staining, BET analysis, and SEM analysis was used to gain further insights into the effects of the cellulolytic enzymes on the substrate under different reaction conditions. RESULTS: Although glucose production after 72 h was higher at 17.5% WIS than at 12.5% WIS, glucan conversion decreased with 24% (air) and 17% (N(2)). Compared to reactions with N(2), the average increases in glucose production for aerated reactions were 91% (12.5% WIS), 70% (15.0% WIS), and 67% (17.5% WIS). Improvements in glucan conversion through aeration were larger (55–86%) than the negative effects of increasing WIS content. For reactions with 12.5% WIS, increased enzyme dosage with 50% improved glucan conversion with 25–30% for air and N(2), whereas improvements with double enzyme dosage were 30% (N(2)) and 39% (air). Structural analyses of the solid fractions revealed that the enzymatic reaction, particularly with aeration, created increased surface area (BET analysis), increased disorder (SEM analysis), decreased crystallinity (XRD), and increased dye adsorption based on the cellulose content (Simons' staining). CONCLUSIONS: The gains in glucan conversion with aeration were larger than the decreases observed due to increased substrate content, resulting in higher glucan conversion when using aeration at the highest WIS value than when using N(2) at the lowest WIS value. The increase in glucan conversion with double enzyme preparation dosage was smaller than the increase achieved with aeration. The results demonstrate the potential in using proper aeration to exploit the inherent capacity of LPMO in enzymatic saccharification of lignocellulosic substrates and provide detailed information about the characteristics of the substrate after interaction with cellulolytic enzymes.