Improving enzymatic saccharification on lignocellulosic biomass is crucial for reducing the cost of bioethanol [1]. Norway spruce (Picea abies) was pretreated with steam explosion without addition of acid catalyst (autocatalysis).   The pretreated spruce contained 50 %(w/w) glucan and lignin 39% %(w/w). The xylan and mannan content were reduced from 20% (w/w) to 5 %(w/w) during the pretreatment (Figure 1).Lytic polysaccharide monooxygenases (LPMOs), specifically an LPMO from Thermoascus aurantiacus (TaLPMO9A), have been shown to improve the saccharification yield when supplementing Celluclast® 1.5 L and Novozyme 188.  However, the saccharification yield (7.6 ± 1.1% g/g (available glucose)) was still significantly lower than harshly pretreated spruce (69.6 ± 0.7% (w/w)) at 72 h [2]. Thus, further optimisation is needed to utilise mildly pretreated spruce.TaLPMO9A have been shown to bind to amorphous cellulose unproductively, resulting in lower overall efficiency [3]. Residual hemicellulose coating the cellulose might also hinder enzymes from accessing the cellulose, as has been shown in a study applying a functionally similar LPMO to TaLPMO9A [4]. In this study, we compared an enzyme loading strategy where Celluclast® 1.5 L, Novozyme 188,  and TaLPMO9A were added in 2 steps, and adding all enzymes in one step. The rationale was to allow amorphous cellulose and hemicellulose coating be removed to expose more cellulose for the second enzyme addition. The glucose release of this strategy (5.8 ± 0.9% (w/w)) is not significantly different from adding all enzymes at 0 h (6.3 ± 0.6% (w/w)).A previous study has shown that the effect of LPMO on saccharification became more pronounced after 48 h [2]. This indicates that the effect of the second TaLPMO9A addition might only become measurable after 72 h. Prolonging the saccharification could help to better understand the effect of the LPMO addition.