The information on continuous operation and the use of actual waste as a feedstock are essential for the practical application of photo-fermentative H-2 production. For the first 200 days, continuous H-2 production from lactate was attempted using purple non-sulfur (PNS) bacteria, Rhodobacter sphaeroides KD131, under an illumination of 110 W/m(2). During the continuous operation, 30% of the fermenter volume was replaced by fresh feedstock once a day, and substrate concentration was gradually increased from 5 mM to 30 mM. H-2 production was negligible at 5 mM, which was ascribed to the fact that the electrons contained in lactate were mostly consumed for cell growth and soluble microbial products (SMPs) production. As lactate concentration increased, H-2 production gradually increased and reached a maximum at 20 mM, showing a substrate conversion efficiency (SCE) of 38%, a H-2 yield of 2.3 mol H-2/mol lactateadded, and a H-2 production rate of 309 mL H-2/L-fermenter/d. Further increases of lactate concentration resulted in a drop of H-2 production (<1.0 mol H-2/mol lactate(added)). When the feedstock was changed to actual waste obtained from a 1-day lactate fermentation of food waste, stable H-2 production was maintained, but showed a decreased SCE of 24%. It was speculated that the low performance was due to the fact that actual waste contained not only pure lactate but also other organic compounds that could not be utilized by PNS bacteria. In addition, compared to feeding with pure lactate, the electron consumption to the cell growth was higher in feeding with actual waste, which led to the lower performance. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.