Anaerobic valorization of tobacco waste into high-value aromas via integrated substrate and biocatalyst co-optimization

Tobacco waste, a major byproduct of the tobacco industry, represents a challenging lignocellulosic resource for valorization. Its efficient conversion into high-value chemicals is often hampered by a metabolic mismatch between pretreated substrates and biocatalysts. This study establishes an integrated co-optimization (ICO) strategy to address this bottleneck. A potent anaerobic aroma-producing yeast, Saccharomyces cerevisiae G5, was first isolated from a native tobacco-aging microbiome via a niche-targeted bioprospecting strategy. Subsequently, a multi-objective optimization model guided hydrothermal pretreatment (HTP) to generate a metabolically-tailored hydrolysate specifically for G5, rationally balancing the release of key aroma precursors against the formation of inhibitors. This approach pinpointed optimal conditions at 100℃, 30 min, and a 1:8 solid-liquid ratio. The ICO strategy resulted in a 4.5-fold increase in the final relative aboundance of target aroma compounds compared to the unoptimized control (Group D) and boosted Relative Aroma Selectivity (RAS) from 57.2 ± 3.4% to 91.5 ± 1.2%. Absolute quantification of the fungal community revealed the core mechanism: the optimized HTP created niche availability that enabled subsequent competitive exclusion by the elite strain G5, thereby redirecting metabolic flux efficiently towards desired aromas. This work provides a robust and transferable framework for the high-value, biocatalyst-centric valorization of tobacco and other complex industrial crops.