Biohythane produced via dark fermentation is much greener than hythane that is generated using natural gas. Biohythane production using a single-stage system has potential to increase the economic viability since it requires fewer controls than a two-stage system that has individual acidogenic and methanogenic reactors. This single-stage system is an innovative method in producing biohythane. The present work investigated the performance of a mesophilic single-stage system with a batch mode operation to generate biohythane. The reactor was seeded with hydrogenic and methanogenic bacteria (HB and MB), which were entrapped in κ-carrageenan/ gelatin beads (2%/2% w/w) using the dripping method. The energy yield of 0.41 to 1.48 kJ g–1 glucose and the hydrogen content in biohythane (H2/(H2 + CH4)) of 0.35 to 0.69 were obtained. These results indicate that different biohythane compositions would be obtained by regulating the HB/MB bacteria concentration ratio, substrate concentration and cultivation pH. Moreover, a comparison of two-stages and single-stage systems as well as the challenges were also elucidated.