Microbial community analysis of thermophilic mixed culture sludge for biohydrogen production from palm oil mill effluent Supachai Nitipan a,b , Chonticha Mamimin c , Nugul Intrasungkha a,b , Nils Kåre Birkeland d , Sompong O-Thong a,b,* a Department of Biology, Faculty of Science, Thaksin University, Patthalung 93110, Thailand b Microbial Resource Management Research Unit, Faculty of Science, Thaksin University, Patthalung 93110, Thailand c Biotechnology Program, Department of Biology, Faculty of Science, Thaksin University, Patthalung 93110, Thailand d Department of Biology and Centre for Geobiology, University of Bergen, P.O. Box 7800, N-5020, Bergen, Norway article info Article history: Received 16 March 2014 Received in revised form 19 May 2014 Accepted 20 May 2014 Available online 18 June 2014 Keywords: Biohydrogen production Hydrogen-producing bacteria Microbial community Thermophilic fermentation Palm oil mill effluent abstract The microbial community structure of thermophilic mixed culture sludge used for bio- hydrogen production from palm oil mill effluent was analyzed by fluorescence in situ hy- bridization (FISH) and 16S rRNA gene clone library techniques. The hydrogen-producing bacteria were isolated and their ability to produce hydrogen was confirmed. The microbial community was dominated by Thermoanaerobacterium species (~66%). The remaining mi- croorganisms belonged to Clostridium and Desulfotomaculum spp. (~28% and ~6%, respec- tively). Three hydrogen-producing strains, namely HPB-1, HPB-2, and HPB-3, were isolated. 16S rRNA gene sequence analysis of HPB-1 and HPB-2 revealed a high similarity to Ther- moanaerobacterium thermosaccharolyticum (98.6% and 99.0%, respectively). The Thermoa- naerobacterium HPB-2 strain was a promising candidate for thermophilic fermentative hydrogen production with a hydrogen yield of 2.53 mol H 2 mol 1 hexose from organic waste and wastewater containing a mixture of hexose and pentose sugars. Thermoanaerobacterium species play a major role in thermophilic hydrogen production as confirmed both by mo- lecular and cultivation-based analyses. Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved. Introduction Currently, the disposal of organic wastes and wastewater are an economic burden on communities and industries. Creating a marketable product from these waste products would provide numerous benefits by reducing treatment costs. The production of biohydrogen by dark hydrogen fermentation is a process that can achieve two simultaneous objectives: the production of bioenergy and reduction of pollution [1]. Dark fermentation appears to be an attractive strategy because it can continually produce hydrogen without the need for light, * Corresponding author. Department of Biology, Faculty of Science, Thaksin University, Patthalung 93110, Thailand. Tel./fax: þ66 074 693992. E-mail address: sompong.o@gmail.com (S. O-Thong). Available online at www.sciencedirect.com ScienceDirect journal homepage: www.elsevier.com/locate/he international journal of hydrogen energy 39 (2014) 19285 e19293 http://dx.doi.org/10.1016/j.ijhydene.2014.05.139 0360-3199/Copyright © 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.