Production of recombinant Oryza sativa non-symbiotic hemoglobin 1 (OsHb1) by Escherichia coli was maximized in shake-flask cultures in media containing tryptone, yeast extract, sodium chloride and byproduct glycerol from biodiesel production. Response surface methodology (RSM) and artificial neural networks (ANNs), followed by multiple response optimization through a desirability function were applied to evaluate the amount of OsHb1 produced. The results obtained by the application of ANNs were more reliable since better statistical parameters were obtained. The optimal conditions were (g L(-1)), tryptone, 42.69; yeast extract, 20.11; sodium chloride, 17.77; and byproduct glycerol, 0.33. A maximum recombinant protein concentration of 3.50g L(-1) and a minimum biomass concentration of 18.48 g L(-1) were obtained under these conditions. Although the concentrations of tryptone, yeast extract and sodium chloride are relatively high, the increase in the yield with respect to biomass formed (Y(P/X)) overcomes this disadvantage. (C) 2010 Elsevier Ltd. All rights reserved.