In order to ensure efficient functioning of hydrogen fermentation systems that use Clostridium as the dominant hydrogen producer, energy-intensive process such as heat pretreatment of inoculum and/or substrate, continuous injection, and control of anaerobic conditions are required. Here, we describe a simple hydrogen fermentation system designed using microflora from leaf-litter cattle-waste compost. Hydrogen and volatile fatty acid production was measured at various hydraulic retention times, and bacterial genera were determined by PCR amplification and sequencing. Although hydrogen fermentation yield was approximately one-third of values reported in previous studies, this system requires no additional treatment and thus may be advantageous in terms of cost and operational control. Interestingly, Clostridium was absent from this system. Instead, Megasphaera elsdenii was the dominant hydrogen-producing bacterium, and lactic acid-producing bacteria (LAB) were prevalent. This study is the first to characterize M. elsdenii as a useful hydrogen producer in hydrogen fermentation systems. These results demonstrate that pretreatment is not necessary for stable hydrogen fermentation using food waste. Crown Copyright (C) 2010 Published by Elsevier Ltd on behalf of Professor T. Nejat Veziroglu. All rights reserved.