Biohydrogen production from sugarcane bagasse by integrating dark- and photo-fermentation Pankaj K. Rai, S.P. Singh ⇑ , R.K. Asthana, Shweta Singh Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221005, India highlights  Acid and enzyme pretreated SCB was utilized to produce H 2 .  Effective removal of fermentation inhibitors by adsorbent resin.  SSFF and two-step fermentation was effectively utilized for H 2 production. article info Article history: Received 16 September 2013 Received in revised form 19 October 2013 Accepted 23 October 2013 Available online 12 November 2013 Keywords: Sugarcane bagasse Acid hydrolysis SSFF H 2 Production abstract Hydrogen production from sugarcane bagasse (SCB) by integrating dark-fermentation by Enterobacter aerogenes MTCC 2822 and photo-fermentation by Rhodopseudomonas BHU 01 was investigated. The SCB was hydrolysed by sulphuric acid and the hydrolysate detoxified by passing through adsorbent resin column (Amberlite XAD-4) to remove the inhibitory furfural, and subjected to dark-fermentation. The cellulosic residue from acid hydrolysis was hydrolysed by the new isolate Cellulomonas fimi to release sugars for H 2 production by E. aerogenes, through simultaneous saccharification, filtration and fermenta- tion (SSFF). Cumulative H 2 production during dark-fermentation and SSFF was 1000 and 613 ml/L, respectively. The spent media of dark-fermentation and SSFF were utilized for photo-fermentation by Rhodopseudomonas BHU 01. The cumulative H 2 production was 755 ml/L for dark-fermentation and 351 ml/L for SSFF spent medium. Ó 2013 Elsevier Ltd. All rights reserved. 1. Introduction Hydrogen, a promising alternative to fossil fuels and a clean en- ergy carrier (Das and Veziroglu, 2001), is produced biologically via biophotolysis, dark-fermentation and photo-fermentation. Sugars such as glucose, fructose, galactose, arabinose, lactose and sucrose are the widely studied substrates for biohydrogen production (Kumar and Das, 2000; Lin and Jo, 2003; Rai et al., 2012). As the cost of the substrate is of prime concern for the economics of bio- hydrogen production, there is need to go for cheaper and abundant feedstocks for making the process cost-effective. The use of organic wastes from industries and agriculture not only supports green energy generation but also helps in bioremediation (Venkata Mohan, 2009). A large fraction of wastes from municipal, industrial and agricultural sector comprising lignocellulosic biomass, is renew- able and inexpensive, and thus well suited for biofuel production. One of the major lignocellulosic material found in large quantities especially in a tropical country like India, is sugarcane bagasse (SCB) produced during cane milling (Singh et al., 2007). The bagasse is composed of cellulose (40–45%), hemicellulose (30–35%), lignin (20–30%). In general, 1 metric ton of sugarcane generates 280 kg of bagasse, about 50% of this residue is used in su- gar mills/distilleries as the energy source while the remaining is composted or used in paper industry (Peng et al., 2009). Cellulose in bagasse is a highly ordered molecule consisting of linear insolu- ble polymers of repeating b-1, 4 linked b-D glucopyranose units. The main problem in the use of cellulose is its crystalline structure that makes it difficult to hydrolyze. This necessitates use of novel techniques to achieve optimum hydrolysis and the extraction of fermentable sugars from the SCB cellulose. Hydrogen production from lignocellulosic feedstock involves pre-treatment by acid/alka- li or by other means to open up its crystalline structure, followed by enzymatic hydrolysis of cellulose and hemicellulose(s) to yield sugars that can be utilized for fermentative H 2 production (Panagiotopoulos et al., 2013a). For enzymatic hydrolysis of the lignocellulosic material, most studies used the expensive commer- cial enzymes which add to the cost of fermentation and thus limit commercialization of cellulose-based hydrogen production (Stork et al., 2009). The enzymatic hydrolysis and fermentation can be 0960-8524/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biortech.2013.10.117 ⇑ Corresponding author. Tel.: +91 0542 2307146 47 (O)/2414560 (R); fax: +91 0542 2366402. E-mail address: spsinghbhu@rediffmail.com (S.P. Singh). Bioresource Technology 152 (2014) 140–146 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech