Abstract
In this study, a two-stage fermentation strategy using Saccharomyces cerevisiae and Escherichia coli was developed for spent coffee grounds (SCG) hydrolysate valorization. In the first stage, S. cerevisiae ATCC 13,007 was cultivated in thermally and acid-hydrolyzed SCG hydrolysates at different substrate concentrations. Acid hydrolysis significantly improved yeast growth compared to thermal hydrolysis, with 10% acid-hydrolyzed non-diluted SCG hydrolysate selected as the optimal substrate. Complete glucose consumption (~ 150 mM) and high ethanol production (~ 240 mM) were observed after 24 h, indicating strong fermentative metabolism and efficient carbon conversion. Yeast cultivation substantially modified the physicochemical composition of the hydrolysate, increasing total carbon from 0.1 to 4.4–5.0 g L⁻¹ and shifting the C/N ratio from 1:7 in the initial hydrolysate to 16:1 in cell-free supernatant (CFS) and 4:1 in lysed cell supernatant (LCS). In the second stage, these residual media were used for E. coli BW25113 cultivation and hydrogenase activity analysis. Twice diluted LCS medium supported the highest biomass formation (0.58 g CDW L⁻¹) and the highest hydrogenase activity (110–120 mU mg⁻¹ after dilution), while CFS showed lower bacterial growth and enzyme activity. The superior performance of LCS was associated with its more favorable C/N ratio and improved nutrient availability resulting from yeast cell disruption. Measurable hydrogen-oxidizing activity confirmed the formation of catalytically active [NiFe]-hydrogenases. These results demonstrate that two-stage fermentation using yeast-pretreated SCG hydrolysate provides an effective strategy for waste valorization, microbial biomass production, and sustainable hydrogenase generation with potential applications in bioenergy and aerobic biocatalysis.