The production of high-value bioproducts from lignocellulosic residues is an attractive strategy to enhance the economics of emerging biorefineries. This study evaluates the feasibility of producing astaxanthin (AXT) with Xanthophyllomyces dendrorhous using enzymatic hydrolysates obtained from steam-exploded Eucalyptus grandis wood, a species associated with large volumes of industrial processing residues. Five strains (Y-17810, Y-17268, Y-17832, Y-27179 and DSM 5626) were screened in glucose, xylose and hydrolysate media. Among them, DSM 5626 exhibited the highest production, achieving AXT titers of up to 1.7 mg L−1 that were statistically comparable to those obtained with synthetic glucose, indicating that eucalyptus-derived substrates can support carotenoid biosynthesis. Xylose supported lower AXT titers, reflecting metabolic and redox constraints inherent to pentose utilization. Glutamate supplementation enhanced carotenoid formation, whereas excessive oxidative stress reduced AXT accumulation, suggesting that oxidative stress may exert both stimulatory and inhibitory effects depending on its intensity. HPLC–MS–ESI profiling revealed shifts in carotenoid intermediates consistent with changes in metabolic flux through the CrtS-dependent oxidation pathway. A simplified mass balance was used to explore the potential for AXT co-production within lignocellulosic biorefinery schemes. Overall, these results identify DSM 5626 as a robust AXT-producing strain and highlight eucalyptus hydrolysates as a renewable substrate with potential for pigment bioproduction.
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