Effect of heat pre-treatment temperature on isolation of hydrogen producing functional consortium from soil Anita Ravindran a , Sunil Adav a , Shang-Shyng Yang a, b, c, * a Institute of Microbiology and Biochemistry, National Taiwan University, Taipei 10617, Taiwan b Department of Biochemical Science and Technology, National Taiwan University, Taipei 10617, Taiwan c Department of Food Science, China University of Science and Technology, Taipei 11581, Taiwan article info Article history: Received 30 August 2009 Accepted 10 April 2010 Available online 23 May 2010 Keywords: Hydrogen production Functional consortium Heat pre-treatment Forest soil abstract A functional hydrogen producing consortium was isolated from soil by heat pre-treatment technique and hydrogen production at different substrate concentration was evaluated. The forest soil was heat pre- treated at 65, 80, 95, 105 and 120  C temperature for 1 h. As revealed by PCR-DGGE analysis and hydrogen yield, the hydrogen producing microbial community changed with increase in heat pre- treatment temperatures giving potential hydrogen producing consortium at 95e105  C soil pre-treat- ment. The maximum hydrogen production rate, hydrogen yield and cumulative hydrogen with 15e20 g glucose were 1390e1576 mL/L/day, 1.83e1.93 mol H 2 /mol glucose, and 2966e3146 mL H 2 /L, respectively. The metabolic pathways shifted from ethanol-type to acetateeformate type as soil pre-treatment temperature increased from 65 to 120  C. The soil heat pre-treatment approach is effective for isolating hydrogen producing natural Clostridium consortium from the soil as enumerations of the functional strains need specific temperature range to florish. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Most of the current energy sources mainly come from fossil fuels. However, excessive fossil fuel utilization causes emissions of green- house gases (CO 2 , CH 4 and CO) resulting in global warming and acidic rain that affects the earth’s climate, weather conditions, vegetation and aquatic ecosystems [1]. Considering the global warming and environmental pollution, there is increasing interest in hydrogen which promises to be a good substitute for fossil fuels. Hydrogen is one of the most promising alternative energy for sustainable green energy production because of its clean, efficient, renewable, and non-polluting characteristics [2e4]. The conversion of poly- saccharides or organic wastes to hydrogen has been performed via either fermentation [5,6] or photosynthesis [7,8]. The former is generally preferred, because it does not rely on the availability of light sources and the transparency of mixed liquor [9]. Fermentative hydrogen has been studied for a large group of pure fermentative bacteria, such as Bacillus [10,11], Clostridium [5,6,12e15], Enterobacter [16], Thermotoga [14,17], and Actinomycetes [18]. The hydrogen yield with these pure cultures ranged from 0.53 to 2.52 mol H 2 /mol substrate. However, maintaining the pure cultures under reactor operating conditions are very challenging, therefore, for industrial applications the mixed cultures are preferred as they are more prac- tical to use, easier to control and may have a broader choice of feed- stocks [19]. The major challenge for biological hydrogen production is the preparation of large amount of active, stable and efficient hydrogen producing inoculum. For efficient performance, the functional natural microflora is the greatest interest in practice [20,21]. The inoculum preparation by microbial isolation with conventional plating technique is complicated, time consuming and only a small fraction of naturally occurring microflora could be cultivated. In certain cases, the key functional strains are unculturable by conventional plating technique therefore the symbiotic association and/or mechanism involved in naturally co-existing microbes cor- responding to the biological hydrogen production cannot be detailed. In the literature, dilution to extinction approach has been used for the isolation of pure culture and functional consortia and the success depends upon retaining the functional strains in used dilution. In addition, enrichment by acid incubation is the commonly used technique for screening hydrogen producing bacteria [22,23]. Recently, Adav et al. [24] employed concentration to extinction approach to isolate functional hydrogen producing consortium from lignocellulosic biomass. Oh et al. [18] tested the * Corresponding author. Department of Biochemical Science and Technology, National Taiwan University, Taipei 10617, Taiwan. Tel.: þ86 886 2 33664456; fax: þ86 886 2 23679827. E-mail address: ssyang@ntu.edu.tw (S.-S. Yang). Contents lists available at ScienceDirect Renewable Energy journal homepage: www.elsevier.com/locate/renene 0960-1481/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.renene.2010.04.010 Renewable Energy 35 (2010) 2649e2655