The effects of light dark duration alternation on bacterial growth and hydrogen (H-2) production performance of Rhodobacter sphaeroides MDC 6522 from Armenian mineral springs were firstly investigated in batch culture. Five types of light dark duration were applied: 1st culture was illuminated continuously (control); 2nd and 3rd cultures were illuminated after inoculation during 24-48 h, then dark conditions were applied; 4th and 5th cultures were illuminated after 24-48 h dark period. R. sphaeroides did not grow well in the dark conditions, but growth and H-2 production were restored upon illumination. When culture was illuminated after 24 h dark period, H-2 yield was maintained at a constant high level during 96-144 h, and duration of photo-fermentation was delayed up to 144 h, compared to the control. When culture was illuminated till H-2 production started (48 h), and then dark conditions were applied, H-2 production ability of R. sphaeroides persisted during the growth up to 120 h, whereas in the control cells H-2 production was not observed. Moreover, the results indicated that light dark alternation affected the formation of photosynthetic pigments and membrane H+ conductance. This study is of significant for understanding of bacterial metabolism regulation mechanisms and for application of purple bacteria in H-2 production biotechnology. Copyright (C) 2015, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.