Clostridium spp. holds significant potential hydrogen-producing bacteria for biohydrogen production which can be used to treat a wide range of carbon sources, including biomass waste. Previous studies have given considerable attention to C. acetobutylicum, C. beijerinckii, and C. butyricum, which have efficiently converted sugar into biohydrogen. This study aims to isolate the potent Clostridia genera and investigate optimal conditions for cell growth and volatile fatty acids production, such as temperature, initial pH, and medium compositions. This study also examined the effect of different biochar (10 g/L), magnetite (magnetic nanoparticles, 100 mg/L), and magnetic fields on biohydrogen production performances. The results revealed that Clostridium tertium IGP01 was isolated with optimal growth conditions when sucrose was utilized as a carbon source with an organic nitrogen source at 37 °C with an initial pH of 5.5. In this condition, C. tertium IGP01 achieves the highest biohydrogen production yield of 4.21 ± 0.19 mmol H