Thirty-nine purple non-sulfur bacteria (PNSB) were isolated from 162 environmental samples. These isolates of PNSB were tested for their ability to produce biohydrogen (Bio-H-2) at 40 degrees C and only 14 isolates were noted to possess such ability, which were considered as thermotolerant photosynthetic Bio-H-2 producing bacteria (tt-PHPB). Of 14 isolates of tt-PHPB, KKU-SN1/1 was observed to have the highest Bio-H-2 productivity. Central composite design was employed to optimize the operating conditions for maximal Bio-H-2 production by the strain KKU-SN1/1. Under optimal conditions (7.6 g/L malic acid, 11 g/L glutamic acid, pH 6.7, at 40 degrees C) the Bio-H-2 production was increased by 68.28%. The purity of Bio-H-2 produced was 92.22%, as suggested by gas chromatography (GC-TCD). Based on morphological characteristics and 16S rDNA sequence analysis, the strain KKU-SN1/1 was identified as Rhodopseudomonas pentothenatexigens, with 99.7% similarity. To our knowledge, this is the first report on Bio-H-2 production by R. pentothenatexigens KKU-SN1/1. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.