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The goal of the proposed project was to develop an anaerobic fermentation process that converts negative-value organic wastes into hydrogen-rich gas in a continuous-flow reactor under different opening conditions, such as hydraulic retention time (HRTL), heat treatment, pH, and substrates. A series of batch tests were also conducted in parallel to the continuous study to evaluate the hydrogen conversion efficiency of two different organic substrates, namely sucrose and starch. A heat shock (at 90°C for 15 minutes) was applied to the sludge in an external heating chamber known as a sludge activation chamber, as a method to impose a selection pressure to eliminate non spore-forming, hydrogen-consuming bacteria and to activate spore germination. The experimental result showed that the heat activation of biomass enhanced hydrogen production by selecting for hydrogen-producing, spore-forming bacteria. The batch feeding at a shorter HRT of 20 hours (or higher organic loading rate) favored hydrogen production, whereas, at a longer HRT of 30 hours, methane was detected in the gas phase. The major organic acids of hydrogen fermentation were acetate, butyrate, and propionate. Up to 23.1% of influent chemical oxygen demand was consumed in biomass synthesis. Batch tests showed that the hydrogen-production potential of starch was lower than sucrose, and better conversion efficiency from starch was obtained at a lower pH of 4.5. However, addition of sucrose to starch improved the overall hydrogen-production potential and hydrogen-production tale. This study showed that sustainable biohydrogen production from carbohydrate-rich substrates is possible through heat activation of settled sludge. [ABSTRACT FROM AUTHOR]