Efficient Pretreatment for Bioethanol Production from Water Hyacinth (Eichhornia crassipes) Involving Naturally Isolated and Recombinant Enzymes and Its Recovery Saprativ P. Das, a Rajeev Ravindran, a Arabinda Ghosh, a Deepmoni Deka, b Debasish Das, a Mohammad Jawed, b Carlos M.G.A. Fontes, c and Arun Goyal a a Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati-781 039, Assam, India; arungoyl@iitg.ernet.in (or) debasishd@iitg.ernet.in (for correspondence) b Centre for Environment, Indian Institute of Technology Guwahati, Guwahati-781 039, Assam, India c CIISA--Centro de Investigac¸ ~ ao Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinaria, Avenida da Universidade T  ecnica, 1300 477, Lisboa, Portugal Published online 00 Month 2013 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/ep.11885 Simultaneous saccharification and fermentation (SSF) experiments involving water hyacinth (Eichhornia crassipes), an abundantly available renewable bioresource, using hydro- lytic enzymes, and fermentative microbes were investigated. Water hyacinth containing 30.01 (%, w/w) cellulose, 44.49 (%, w/w) hemicellulose, and 20.04 (%, w/w) lignin was subjected to three different pretreatments, namely, wet oxidation, phos- phoric acid (H 3 PO 4 )-acetone, and ammonia fiber explosion (AFEX). Hydrolytic enzymes, namely, recombinant Clostridium thermocellum cellulase (GH5) and hemicellulase (GH43), Tri- choderma reesei and Bacillus subtilis AS3 cellulases were employed separately for saccharification. Saccharomyces cerevi- siae and Candida shehatae were used for fermentation. The AFEX pretreated 1% (w/v) water hyacinth along with recombi- nant cellulase (GH5)-hemicellulase (GH43) consortium gave the highest ethanol titer of 1.52 g/L as compared with wet oxida- tion (1.23 g/L) and phosphoric acid-acetone pretreatments (1.31 g/L). The best SSF combination with 5% (w/v) substrate at shake flask contributed an ethanol titer and yield of 7.83 g/L, 0.266 (g of ethanol/g of substrate) and its scale up at bioreactor level resulted in significantly higher ethanol titer and yield of 14.39 g/L and 0.489 (g/g), respectively. 93.0 (%, v/v) ethanol from bioreactor was recovered by rotary evaporator with 20.4% purification efficiency. V C 2013 American Institute of Chemical Engineers Environ Prog, 00: 000–000, 2013 Keywords: water hyacinth, GH5 cellulase, GH43 hemicel- lulase, Bioethanol, HPAEC-PAD INTRODUCTION Lignocellulosic ethanol production has gained tremendous significance in recent years due to its projection as a feasible alternative to petroleum based fuels. Water hyacinth (Eichhornia crassipes) is a monocotyledonous, freshwater noxious weed inhabiting water bodies in an uncontrolled manner [1]. E. crassipes is a typical menace infesting large areas of water bodies causing ecological and socioeconomic problems. The possibility of converting water hyacinth to biogas or fuel ethanol is currently an area of great research interest in India [1]. Under the climatic conditions of Assam, a daily average water hyacinth biomass productivity of 0.26 ton of dry biomass per hectare has been reported [2]. Owing to its high hemicellulose content and advantage of a non- competitor to food crops, water hyacinth can be a potential source for bioethanol production [3, 4]. The major rate limit- ing step in economically efficient conversion of lignocellulo- sic substrates to liquid biofuels is efficacy of hydrolytic enzymes and degree of crystallinity of the lignocellulosic bio- mass. The different pretreatments, namely, wet oxidation [5], phosphoric acid (H 3 PO 4 )-acetone treatment [6], and ammo- nia fiber explosion (AFEX) [7] breakdown the lignin seal from hemicellulose, reduce cellulose crystallinity, and increase the porosity for enzymatic hydrolysis [8]. The thermophilic bacterium Clostridium thermocellum contains genes coding for exocellular multienzyme com- plexes called cellulosomes exhibiting endoglucanase and exoglucanase activity [9]. C. thermocellum cellulosome dis- plays 50-fold higher specific activity than the corresponding Trichoderma reesei system against crystalline cellulose [10]. Glycoside hydrolase family 5 (GH5) and family 43 (GH43) are enzymes with varying substrate specificity including cel- lulase [9] and hemicellulase activity [11], respectively that can be used for deriving monomeric sugars out of lignocellulosic biomass. Bacillus subtilis (AS3) produces a thermostable cel- lulase system exhibiting activity fairly close to fungal cellu- lases and can remain active in a wide range of pH conditions [12]. Simultaneous saccharification and fermenta- tion (SSF) is a promising single step modus operandi of enzymatic hydrolysis of cellulose and the fermentation of Additional Supporting Information may be found in the online ver- sion of this article. V C 2013 American Institute of Chemical Engineers Environmental Progress & Sustainable Energy (Vol.00, No.00) DOI 10.1002/ep Month 2013 1