The deployment of lignocellulosic biomass in biorefineries is often limited by the chemical complexity of hydrolysates generated during pretreatment and enzymatic saccharification. These hydrolysates exhibit significant variability in sugar composition and inhibitor content depending on the feedstock and processing conditions, frequently creating microbial bottlenecks that hinder fermentation and bioconversion. In this study, we systematically evaluate the chemical and biological profiles of twelve hydrolysates derived from three representative biomass types—sorghum (grass), poplar (hardwood), and pine (softwood)—processed using four distinct pretreatment strategies: an aqueous ionic liquid (cholinium lysinate), a distillable protic amine (butylamine), dilute sulfuric acid, and liquid hot water. Each hydrolysate was characterized for monomeric sugars (glucose, xylose, mannose, arabinose), oligosaccharides, common fermentation inhibitors (furfural, HMF, organic acids, phenolics), and physicochemical properties such as conductivity. To assess biological compatibility, we conducted parallel microbial growth studies using four biotechnologically relevant organisms: two bacteria (Escherichia coli, Pseudomonas putida) and two yeasts (Rhodosporidium toruloides, Saccharomyces cerevisiae). Distinct strain-specific tolerance patterns were observed, and hydrolysates were clustered based on their fermentation compatibility profiles using multivariate analysis. Our findings provide a mechanistic link between hydrolysate chemistry and microbial performance, offering a platform to guide rational pretreatment selection and microbial engineering strategies in feedstock-agnostic lignocellulosic biorefineries.
