In the present work, a continuous photo-fermentative hydrogen (H-2) production from lactate was attempted at various hydraulic retention times (HRTs) (48-120 h). Electron balance was made at each operating conditions to elucidate different performances. At 120 h HRT, H-2 production was negligible, while 42% and 52% of substrate electrons diverted towards cell growth and soluble microbial products (SMPs), respectively. After changing HRT to 96 h, H-2 yield jumped to 2.3 mol-H-2/mol-lactateadded with less SMPs production and minimal cell growth. The highest H-2 production rate of 260 mL-H-2/L-fermenter/d was also achieved at 96 h HRT. When HRT was further shortened to 72 and 48 h, H-2 yield dropped to 1.4 and 0.2 mol-H-2/mol-lactate(added), respectively. While almost all of the lactate was degraded at <72 h HRT, only 65% of the lactate was consumed at 48 h HRT. From 200th day, the feedstock was changed to lactate-rich acidified food waste, which was obtained via one-day fermentation of food waste and subsequent centrifugation. At 2 g chemical oxygen demand/L, substrate conversion efficiency reached 35%, which was slightly lower than that of feeding pure lactate. SMPs were found to be mainly consisted of low molecular weight compounds (<500 Da), and the majority of organic matters were aromatic proteins at 120 h HRT and it was shifted to humic-like region in 96 h HRT. Copyright (C) 2013, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.