BackgroundMiscanthus is a leading bioenergy crop with enormous lignocellulose production potential for biofuels and chemicals. However, lignocellulose recalcitrance leads to biomass process difficulty for an efficient bioethanol production. Hence, it becomes essential to identify the integrative impact of lignocellulose recalcitrant factors on cellulose accessibility for biomass enzymatic hydrolysis. In this study, we analyzed four typical pairs of Miscanthus accessions that showed distinct cell wall compositions and sorted out three major factors that affected biomass saccharification for maximum bioethanol production.ResultsAmong the three optimal (i.e., liquid hot water, H2SO4 and NaOH) pretreatments performed, mild alkali pretreatment (4\% NaOH at 50 degrees C) led to almost complete biomass saccharification when 1\% Tween-80 was co-supplied into enzymatic hydrolysis in the desirable Miscanthus accessions. Consequently, the highest bioethanol yields were obtained at 19\% (\% dry matter) from yeast fermentation, with much higher sugar-ethanol conversion rates by 94-98\%, compared to the other Miscanthus species subjected to stronger pretreatments as reported in previous studies. By comparison, three optimized pretreatments distinctively extracted wall polymers and specifically altered polymer features and inter-linkage styles, but the alkali pretreatment caused much increased biomass porosity than that of the other pretreatments. Based on integrative analyses, excellent equations were generated to precisely estimate hexoses and ethanol yields under various pretreatments and a hypothetical model was proposed to outline an integrative impact on biomass saccharification and bioethanol production subjective to a predominate factor (CR stain) of biomass porosity and four additional minor factors (DY stain, cellulose DP, hemicellulose X/A, lignin G-monomer).ConclusionUsing four pairs of Miscanthus samples with distinct cell wall composition and varied biomass saccharification, this study has determined three main factors of lignocellulose recalcitrance that could be significantly reduced for much-increased biomass porosity upon optimal pretreatments. It has also established a novel standard that should be applicable to judge any types of biomass process technology for high biofuel production in distinct lignocellulose substrates. Hence, this study provides a potential strategy for precise genetic modification of lignocellulose in all bioenergy crops.