Using coffee waste as feedstock for continuous cultivation of Sulfolobus acidocaldarius

Sulfolobus acidocaldarius is a thermoacidophilic archaeon with high potential for robust continuous bioprocesses under extreme conditions. In this study, a continuous cultivation process using spent coffee grounds (SCGs) as a lignocellulosic feedstock was systematically developed. To enable substrate utilisation, dilute acid hydrolysis of SCGs was optimised, balancing sugar release, inhibitor formation, and process compatibility. Hydrolysis at 100 °C for 1 h using 20 g SCGs per 100 mL 5 % H₂SO₄ resulted in a hydrolysate that supported microbial growth comparable to defined medium conditions. Subsequent shake flask experiments confirmed the uptake of hydrolysate-derived sugars and demonstrated tolerance of S. acidocaldarius to co-extracted inhibitors. Building on these results, continuous bioreactor cultivations were established and progressively adapted to SCG hydrolysate. Following substrate preparation and process optimisation, stable continuous cultivation on SCG hydrolysate was successfully established. After medium optimisation to mitigate osmotic stress, stable operation was achieved for 900 h. An optimal dilution rate of 0.036 h^-1 was identified, while operation at 0.044 h^-1 resulted in cell washout and substrate accumulation. Notably, cultivation on SCG hydrolysate at optimal conditions resulted in an 11 % increase in carbon-specific biomass production rate compared to continuous cultivation on defined sugars, indicating efficient substrate utilisation. Overall, this work demonstrates the feasibility of integrating waste-derived substrates into continuous cultivation of S. acidocaldarius and provides key process parameters for the development of more sustainable and scalable archaeal bioprocesses. These findings establish SCG hydrolysate as a viable alternative feedstock for continuous extremophile bioprocessing, highlighting its industrial potential.