Biohydrogen is one of the most potential alternative fuels for the future. Among various substances from agricultural factory waste, molasses and wastewater from ethanol distillation plants are the main focus of this study. To overcome the limitation of bio-H-2 production, the direct screening of waste substrates for an efficient producer using coculture free technique is an interesting choice. Competent species with H-2-producing ability and valuable volatile acid such as acetic acid were selected. Several bacteria were isolated and had their species identified by microbial 16SrDNA sequence analysis and phylogenetic analysis by Maximum Likelihood method. Bacillus coaglulans, B. circulans, Sporolactobacillus inulinus, Lactobacillus spp., Pediococcus acidilactici, Paenibacillus thermophilus, Chronobacter sakazaki and Clostridium beijerinckii were found in both reducing-sugar rich waste and wastewater. For bio-H-2 application in this criteria, potent natural hydrogen producer B. coagulans MO11 and C. beijerinckii CN were picked to test the growth ability on different substrates (carbon-dioxide, L-arabinose, D-xylose, D-glucose, D-fructose, sucrose, maltose, cellobiose, starch and others). Wastewater from sugarcane factories and other agricultural factories in Thailand were tested for physical and biochemical characteristics which would make them suitable renewable substrates. Among all, Bacillus coagulans MO11 could produce hydrogen gas using molasses and ethanol refinery wastewater effectively (1.634 mol(H2)/mol (hexose)) as detected by Drager tube, which was the maximum yield in this study. In comparison, the experiment performed by using B. coagulans MO11 and C. beijerinckii CN fed with 40X diluted molasses wastewater showed better production of H-2 gas in Clostridium beijerinckii CN during the final 72-hr gas collection (79 mL/L and 125 mL/L respectively). The results pointed in the same direction when replacing 40X diluted molasses with 5X diluted refinery wastewater supplemented with trace elements. (C) 2020 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.