Similar comparisons following SPORL pretreatment were 43% versus 55% and 0.11 versus 0.20 L kg−1 of wood for NM6 and aspen, respectively. Bioconversion performance of NE222 and DN5 fell between that of aspen and NM6. While substrate lignin content and lignin removal by pretreatments did not affect substrate enzymatic digestibility, the wood lignin content was found to negatively affect xylan or hemicellulose removal using both DA and SPORL pretreatments. The ability of lignin protecting hemicellulose removal dictates PBR through affecting disk milling energy for size reduction of pretreated wood chips, substrate enzymatic digestibility. The SPORL pretreatment not only improved sugar and ethanol yields over DA for all four poplars, but also better dealt with the differences among them, suggesting better tolerance to feedstock variability.