A mesophilic alkaline tolerant fermentative microbe was isolated from estuarine sediment samples and designated as Clostridium butyricum TM-9A, based on 16S rRNA gene sequence. Batch experiments were conducted for investigation of TM-9A strain for its growth and hydrogen productivity from glucose, in an iron containing basal solution supplemented with yeast extract as organic nitrogen source. Hydrogen production started to evolve when cell growth entered exponential phase and reached maximum production rate at late exponential phase. Maximum hydrogen production was observed at 37 °C, initial pH of 8.0 in the presence of 1% glucose. Optimization of process parameters resulted in increase in hydrogen yield from 1.64 to 2.67 mol of H2/mol glucose. Molar yield of H2 increased further from 2.67 to 3.1 mol of H2/mol of glucose with the decrease in hydrogen partial pressure, obtained by lowering the total pressure in the head space of the batch reactor. Acetate and butyrate were the measure volatile fatty acids generated during hydrogen fermentation. TM-9A strain produced hydrogen efficiently from a range of pentose and hexose sugars including di-, tri and poly-saccharides like; xylose, ribose, glucose, rhamnose, galactose, fructose, mannose, sucrose, arabinose, raffinose, cellulose, cellobiose and starch. ► A new hydrogen producing fermentative anaerobe was isolated from estuarine sediment. ► 16S rDNA sequence analysis identified the microbe as; Clostridium butyricum TM-9A. ► Process parameters were optimized for TM-9A for hydrogen production from glucose. ► Optimization of process parameters improved molar hydrogen yield of TM-9A strain. ► TM 9A strain utilized various mono-, di- and ploy-saccharides, to produce hydrogen.