Clostridium butyricum TM-9A strain was employed as pure strain for scale up of fermentative hydrogen production in batch mode. In laboratory scale batch fermentations, 46 mmol/L hydrogen produced from molasses at optimum pH, molasses concentration and C/N ratio of; 7.5, 2.5%, and 10, respectively. Scale up of molasses fermentation by TM-9A strain in 13.5 L scale bioreactor under decreased partial pressure at regulated pH, enhnaced the hydrogen titer titer from 46 mmol H-2/L (in laboratory scale) to 73 mmol H-2/L (1.58 fold increase). Hydrogen productivity was 1650 mL/L. Scale up of glucose fermentation by TM-9A in 13.5 L scale bioreactor at optimumcondition produced; 71.9 mmol H-2/L (1634 mL/L). The biogas was composed of 65-60% H-2 and 35-40% CO2. Hydrogen yield of TM-9A from glucose and molasses in the proto scale fermentation process was, 3.335 mole H-2/mole glucose and 73 mmol H-2/9 g of COD reduced, respectively. These studies imply that Clostridum butyricum TM-9A strain has significant potential for hydrogen production from spent organic matter in pilot scale. (C) 2021 Elsevier Ltd. All rights reserved.