ARTICLE High-Efficiency Hydrogen Production by an Anaerobic, Thermophilic Enrichment Culture From an Icelandic Hot Spring Perttu E.P. Koskinen, 1 Chyi-How Lay, 2 Jaakko A. Puhakka, 1 Ping-Jei Lin, 3 Shu-Yii Wu, 3 Jo ´hann O ¨ rlygsson, 4 Chiu-Yue Lin 2 1 Department of Chemistry and Bioengineering, Tampere University of Technology, P.O. Box 541, FIN-33101 Tampere, Finland; telephone: þ358-3-3115-11; fax: þ358-3-3115-2869; e-mail: perttu.koskinen@tut.fi 2 Department of Environmental Engineering and Science, Feng Chia University, Taichung, Taiwan 3 Department of Chemical Engineering, Feng Chia University, Taichung, Taiwan 4 Faculty of Natural Resource Sciences, University of Akureyri, Borgir Nordurslod, Akureyri, Iceland Received 14 January 2008; revision received 12 March 2008; accepted 14 April 2008 Published online 24 April 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21948 ABSTRACT: Dark fermentative hydrogen production from glucose by a thermophilic culture (33HL), enriched from an Icelandic hot spring sediment sample, was studied in two continuous-flow, completely stirred tank reactors (CSTR1, CSTR2) and in one semi-continuous, anaerobic sequencing batch reactor (ASBR) at 588C. The 33HL produced H 2 yield (HY) of up to 3.2 mol-H 2 /mol-glucose along with acetate in batch assay. In the CSTR1 with 33HL inoculum, H 2 produc- tion was unstable. In the ASBR, maintained with 33HL, the H 2 production enhanced after the addition of 6 mg/L of FeSO 4  7H 2 O resulting in HY up to 2.51 mol-H 2 /mol- glucose (H 2 production rate (HPR) of 7.85 mmol/h/L). The H 2 production increase was associated with an increase in butyrate production. In the CSTR2, with ASBR inoculum and FeSO 4 supplementation, stable, high-rate H 2 produc- tion was obtained with HPR up to 45.8 mmol/h/L (1.1 L/h/ L) and HY of 1.54 mol-H 2 /mol-glucose. The 33HL batch enrichment was dominated by bacterial strains closely affi- liated with Thermobrachium celere (99.8–100%). T. celere affiliated strains, however, did not thrive in the three open system bioreactors. Instead, Thermoanaerobacterium aotear- oense (98.5–99.6%) affiliated strains, producing H 2 along with butyrate and acetate, dominated the reactor cultures. This culture had higher H 2 production efficiency (HY and specific HPR) than reported for mesophilic mixed cultures. Further, the thermophilic culture readily formed granules in CSTR and ASBR systems. In summary, the thermophilic culture as characterized by high H 2 production efficiency and ready granulation is considered very promising for H 2 fermentation from carbohydrates. Biotechnol. Bioeng. 2008;101: 665–678. ß 2008 Wiley Periodicals, Inc. KEYWORDS: biohydrogen; biological hydrogen produc- tion; thermophilic microorganisms; dark fermentation; Thermobrachium celere; Thermoanaerobacterium aotear- oense Introduction Hydrogen is seen as an ideal energy carrier of the future (Bockris, 2002; Turner, 2004). Today, H 2 is used as reductant in many industrial processes (Das and Vezirog ˘lu, 2001; Nath and Das, 2003). In general, hydrogen can be produced from renewable energy sources by thermo-, photo-, and electro-chemical, radiolytic and biological processes (for reviews, see Dincer, 2002; Ni et al., 2006a). The biological H 2 production occurs via indirect/direct photolysis or photo/dark fermentations (Das and Vezirog ˘lu, 2001). Out of the biological processes, in the near future, dark fermentations are seen as the most economically feasible because of their highest H 2 production rates (Levin et al., 2004) and the potential of integrating waste treatment with simultaneous H 2 production (Kapdan and Kargi, 2006). Correspondence to: P.E.P. Koskinen Contract grant sponsor: Academy of Finland Contract grant number: 107425; 122065 Contract grant sponsor: Nordic Energy Research Contract grant number: 28-02 Contract grant sponsor: Tampere University of Technology Graduate School Contract grant sponsor: Feng Chia University Contract grant number: 94GB66; 06G27023 Contract grant sponsor: Taiwan’s Bureau of Energy Contract grant number: 96-D0137-2 ß 2008 Wiley Periodicals, Inc. Biotechnology and Bioengineering, Vol. 101, No. 4, November 1, 2008 665