Photofermentation and lipid accumulation by the purple non-sulfur photosynthetic bacterium, Rhodobacter sp. KKU-PS1, from malic acid was investigated in batch fermentation. Media compositions including malic acid concentration, glutamate concentration, amount of vitamin addition and Fe concentration were optimized by response surface methodology with central composite design in order to achieve a maximum hydrogen production rate (R-m). The predicted R-m of 11.7 mL H-2/L. h was obtained under optimal media compositions of 2.36 g/L malic acid, 224 mg/L glutamate, and 13.5 mL/L of vitamin solution addition along with 64 mg/L of Fe. The cellular lipid content in Rhodobacter sp. KKU-PS1 was checked at the end of photo-fermentative process. A nitrogen limited condition was suitable for lipid accumulation by Rhodobacter sp. KKU-PS1. A maximum lipid production of approximately 592 mg/L was obtained with 4.7 g/L malic acid, 159 mg/L glutamate, 21.7 mL/L of vitamin solution along with 330 mg/L Fe (equivalent to C/N ratio of 132). Triacylglycerol (TAG) was the major component of the lipid in which the predominant fatty acid was octadecanoic acid (C18:1) in the form of oleic acid (C18:1n-9). The physicochemical properties of biodiesel predicted from fatty acid methyl esters (FAMEs) compositions of lipid produced by Rhodobacter sp. KKU-PS1 under the suitable conditions indicated that the lipid produced by the strain KKU-PS1 has a fairly good potential for use as biodiesel. Copyright (C) 2016, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.