In this study, exergy analysis of batch biohydrogen production through WGS (water gas shift) reaction using an anaerobic photosynthetic bacteria Rhodospirillum rubrum was carried out for the first time. Various carbon sources including formate, acetate, malate, glucose, fructose, and sucrose were applied to support microbial growth in the presence of CO-rich syngas. The microorganisms utilized carbon monoxide and produced molecular hydrogen concurrently. The process was analyzed based on both conventional exergy and eco-exergy concepts for determining the exergetic parameters i.e., exergy destruction and exergy efficiency. Unlike the exergy efficiency, the exergy destruction based on the eco-exergy concept was remarkably lower than what obtained via the conventional exergy theory. Minimum normalized exergy destruction values of 189.67 and 181.40 kJ/kJ H-2 were achieved for acetate as substrate using the exergy and eco-exergy approaches, respectively. In better words, acetate was identified as the most appropriate carbon source for biohydrogen production from the exergy point of view. Finally, the findings of this study confirmed that exergy analysis could be employed as an adaptable framework to determine and compare the renewability of biological hydrogen production using different routes in order to decide on the most suitable approach and conditions. (C) 2015 Elsevier Ltd. All rights reserved.