Investigating the role of AA9 LPMOs in enzymatic hydrolysis of differentially steam-pretreated spruce

Background To realize the full potential of softwood-based forest biorefineries, the bottlenecks of enzymatic saccharification of softwood need to be better understood. Here, we investigated the potential of lytic polysaccharide monooxygenases (LPMO9s) in softwood saccharification. Norway spruce was steam-pretreated at three different severities, leading to varying hemicellulose retention, lignin condensation, and cellulose ultrastructure. Hydrolyzability of the three substrates was assessed after pretreatment and after an additional knife-milling step, comparing the efficiency of cellulolytic Celluclast + Novozym 188 and LPMO-containing Cellic CTec2 cocktails. The role of Thermoascus aurantiacus Ta LPMO9 in saccharification was assessed through time-course analysis of sugar release and accumulation of oxidized sugars, as well as wide-angle X-ray scattering analysis of cellulose ultrastructural changes. Results Glucose yield was 6% ( w / w ) with the mildest pretreatment (steam pretreatment at 210 °C without catalyst) and 66% ( w / w ) with the harshest (steam pretreatment at 210 °C with 3%( w / w ) SO 2 ) when using Celluclast + Novozym 188. Surprisingly, the yield was lower with all substrates when Cellic CTec2 was used. Therefore, the conditions for optimal LPMO activity were tested and it was found that enough O 2 was present over the headspace and that the reducing power of the lignin of all three substrates was sufficient for the LPMOs in Cellic CTec2 to be active. Supplementation of Celluclast + Novozym 188 with Ta LPMO9 increased the conversion of glucan by 1.6-fold and xylan by 1.5-fold, which was evident primarily in the later stages of saccharification (24–72 h). Improved glucan conversion could be explained by drastically reduced cellulose crystallinity of spruce substrates upon Ta LPMO9 supplementation. Conclusion Our study demonstrated that LPMO addition to hydrolytic enzymes improves the release of glucose and xylose from steam-pretreated softwood substrates. Furthermore, softwood lignin provides enough reducing power for LPMOs, irrespective of pretreatment severity. These results provided new insights into the potential role of LPMOs in saccharification of industrially relevant softwood substrates.