By optimizing concentrations of key nutrients in the media of Synechocystis sp. PCC 6803, we achieved nearly 150-fold greater photofermentative hydrogen (H(2)) production than was achieved by analogous, sulfur-deprived cultures, which are well known to produce much more H(2) than cultures grown on complete media. This was associated with a 44-fold increase in glycogen concentration. Using response surface methodology to determine optimum conditions, we found that, instead of completely starving cells of sulfur or another essential nutrient, the highest H(2) production (0.81 +/- 0.36 mu mol H(2) mg Chl(-1) h(-1)) occurred with 0.52 mM NH(4)(+), 20.1 mu M SO(4)(2-), and 46 mM HCO(3)(-). H(2) profiling experiments provided initial screening of NH(4)(+), HCO(3)(-), SO(4)(2-), and PO(4)(3-) concentrations and identified the significant variables in H(2) production to be NH(4)(+), SO(4)(2-), and the interactions of both NH(4)(+) and SO(4)(2-) with HCO(3)(-). Our results indicate that optimized amounts of nitrogen and sulfur in the nutrient media are superior to total deprivation of these nutrients for H(2) production. (C) 2008 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.