Abstract
Coffee, one of the most widely consumed beverages worldwide, generates significant lignocellulosic waste. This study investigates the use of spent coffee grounds (SCGs) as a substrate for biomass and biohydrogen (H₂) production in pure cultures of obligate anaerobic Clostridium species ( C. pasteurianum , C. beijerinckii , C. acetobutylicum , and C. intestinale ), providing an environmentally friendly bioenergy solution. Bacteria were grown on the physicochemically pre-treated SCGs (4 % w/v) hydrolysate at pH of 5.5–7.5. Kinetics of H₂ production, oxidation-reduction potential (ORP) and pH were evaluated over 168 h, with standard media serving as a control. Quantitative analyses of total carbohydrates (TCs) and volatile solids (VSs) showed a strong correlation between nutrient consumption and microbial activity. Results revealed that, compared with controls and pH 5.5, all four strains demonstrated enhanced growth, H₂ yield and rate on SCG hydrolysate, at an initial pH 7.5. C. beijerinckii and C. pasteurianum exhibited the best growth (OD₆₀₀ of 1.5). With C. pasteurianum , the peak of H 2 yield was 1621 ± 10 ml L −1 g −1 TCs when utilizing SCGs. As bacterial growth progressed and pH decreased, ORP also decreased, reaching a value of −460 ± 10 mV. C․ beijerinckii exhibited the highest carbohydrate-removal efficiency of 73 % at 168 h, whereas C. acetobutylicum demonstrated the lowest at 44 % under the same conditions. The results highlight the potential of SCGs as a cost-effective and renewable feedstock for biomass and bio-hydrogen production, underscoring the ability of Clostridium species to utilize lignocellulose coffee waste, paving the way for sustainable bioenergy applications.