Rapid Evaluation of Amine-Functionalized Solvents for Biomass Deconstruction Using High-Throughput Screening and One-Pot Enzymatic Saccharification

Efficient and sustainable pretreatment of lignocellulosic biomass is critical for biofuel and biochemical production, yet its optimization is often hindered by slow, labor-intensive experimental methods. Here, we report the first demonstration of a custom-built, miniaturized, high-throughput screening platform integrated with one-pot enzymatic saccharification, enabling parallel evaluation of solvent type, feedstock, and temperature with minimal material use and high reproducibility. As a proof-of-concept, the HTX platform was used to screen five amine-functionalized solvents, including isopropanolamine, butylamine, <i>N</i>-methylbutylamine, ethanolamine, and ethanolamine acetate across three bioenergy crops (sorghum, poplar, and switchgrass) and pretreatment temperatures ranging from 80 to 140 °C. Vacuum drying successfully removed more than 99% of the solvents from the pretreated biomass, eliminating the need for water washing prior to saccharification. Isopropanolamine and <i>N</i>-methylbutylamine yielded the highest glucose (70-80%) and xylose (58-67%) release, with trends reflecting feedstock recalcitrance. The produced hydrolysates supported robust growth of an engineered strain of the yeast <i>Rhodosporidium toruloides</i>, confirming biocompatibility. This high-throughput platform provides a scalable, feedstock-agnostic framework for rapid pretreatment screening, accelerating solvent-feedstock pairing and process optimization. Its ability to integrate pretreatment, solvent removal, saccharification, and microbial conversion in a miniaturized format offers significant advantages for cost-competitive biorefinery development.