A purple non-sulfur (PNS) photosynthetic bacterium was isolated from an upflow anaerobic sludge blanket (UASB) bioreactor for methane production and was identified as Rhodobacter sphaeroides KKU-PS5 (GenBank Accession no. KC481702) by 16s rRNA gene sequence analysis. Strain KKU-PS5 could utilize glucose, xylose, fructose, arabinose, malate, succinate, acetate, butyrate, lactate and D-mannitol for growth and hydrogen production. Malate was a preferred carbon source while glutamate and Aji-L (waste from the process of crystallizing monosodium glutamate) were the preferred nitrogen sources. The ability to utilize Aji-L as a low-cost nitrogen supplement for photo-biohydrogen production by the strain KKU-PS5 is considered as its desirable characteristic. The threshold substrate concentration of malate was 30 mmol/L. The optimum conditions for hydrogen production from malate were an initial pH of 7.0, FeSO4 concentration of 4 mg/L, temperature of 30 degrees C and light intensity of 6 klux. Under the optimum conditions, the maximum hydrogen production, the hydrogen yield (HY) and the hydrogen production rate (HPR) of 1330 mL-H-2/L, 3.80 mol-H-2/mol-malate, and 11.08 mL-H-2/L h, respectively, were achieved. Hydrogen production under a dark/light cycle led to a decreased HY and HPR in comparison to continuous illumination. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.