Bioethanol production from Lantana camara (red sage): Pretreatment, saccharification and fermentation Ramesh Chander Kuhad a, * , Rishi Gupta a , Yogender Pal Khasa a , Ajay Singh b a Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India b Lystek International Incorporation, 107-279 Weber Street North, Waterloo, Ontario, Canada N2J3H8 article info Article history: Received 18 March 2010 Received in revised form 26 May 2010 Accepted 7 June 2010 Available online 2 July 2010 Keywords: Lantana camara Cellulosic ethanol Dilute acid hydrolysis Enzymatic hydrolysis Fermentation abstract Lantana camara contains 61.1% (w/w) holocellulose and can serve as a low-cost feedstock for bioethanol production. Acid hydrolysis (3.0%, v/v H 2 SO 4 , 120 °C for 45 min) of L. camara produced 187.14 mg/g total sugars along with fermentation inhibitors such as phenolics (8.2 mg/g), furfurals (5.1 mg/g) and hydroxy methyl furfurals (6.7 mg/g). Sequential application of overliming (pH 10.0) and activated charcoal (1.5%, w/v) adsorption was used to remove these toxic compounds from the acid hydrolysate. The acid-pre- treated biomass of L. camara was further delignified through combined pretreatment of sodium sulphite (5.0% w/v) and sodium chlorite (3.0% w/v), which resulted in about 87.2% lignin removal. The enzymatic hydrolysis of delignified cellulosic substrate showed 80.0% saccharification after 28 h incubation at 50 °C and pH 5.0. Fermentation of acid and enzymatic hydrolysates with Pichia stipitis and Saccharomyces cere- visiae gave rise to 5.16 and 17.7 g/L of ethanol with corresponding yields of 0.32 and 0.48 g/g after 24 and 16 h, respectively. Ó 2010 Elsevier Ltd. All rights reserved. 1. Introduction Lantana camara, commonly known as red sage, is one of the world’s top 100 worst invasive species. It has invaded millions of hectares of grazing land globally (Day et al., 2003). In Australia alone, since its introduction as an ornamental plant in the 1840’s it has spread to infest four million hectares (www.weeds.org.au). While in India, the weed has invaded most of the tropical and sub- tropical parts and is found in areas from the seacoast to 5000 ft in altitude (Sankaran, 2007). The approximate total biomass pro- duced by L. camara per year ranges from 15 to 17 tonnes/ha (Bhatt et al., 1994), which projects the availability of Lantana biomass in huge quantity. Therefore, the abundance of L. camara biomass (lig- nocellulosic material) is likely to offer a potential feed stock for ethanol production. Currently, the biomass to ethanol conversion technology relies mainly on chemical and enzymatic treatments. Chemical hydroly- sis of biomass with dilute sulphuric acid has long been recognized as a critical step for removing the hemicellulosic fraction from the lignocellulosic substrate to economize the biological conversion of cellulosic biomass to ethanol. However, the pentose sugar-rich acid hydrolysate also contains toxic byproducts such as furfural, hydroxy methyl furfural (HMF) and phenolics, which significantly affect yeast cell metabolism during fermentation (Palmqvist and Hahn-Hagerdal, 2000; Chandel et al., 2007). Although various detoxification methods have been investigated for the removal of fermentation inhibitory compounds (Palmqvist and Hahn-Hager- dal, 2000; Chandel et al., 2007), of which overliming and activated charcoal adsorption methods are widely used (Miyafuji et al., 2003; Gupta et al., 2009). An appropriate delignification strategy is essential for the effi- cient enzyme hydrolysis of cellulosic biomass as lignin hinders the saccharification process. The cellulase components such as b-glucosidase and endoglucanase showed higher binding affinity towards lignin compared to carbohydrates, which in turn lowered the saccharification efficiency (Kaya et al., 2000). Various delignifi- cation approaches have been exploited in the past such as alkali pretreatment (Carillo et al., 2005), hydrogen peroxide pretreat- ment (Saha and Cotta, 2007), sulphite pretreatment (Kuhad et al., 1999), ammonia fiber expansion pretreatment (Teymouri et al., 2005) and sodium chlorite pretreatment (Gupta et al., 2009). The efficient conversion of biomass into ethanol requires opti- mum utilization of both pentose and hexose sugars. The widely studied yeast for hexose fermentation, Saccharomyces cerevisiae, is unable to utilize pentose sugars while, among the pentose fer- menting yeasts only Pichia stipitis, Pachysolen tannophilus and Can- dida shehatae are proven to be highly efficient in the conversion of xylose to ethanol (Abbi et al., 1996a,b). The fermentation of both types of sugars can be carried out either by co-cultivation or 0960-8524/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.06.043 * Corresponding author. Address: Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India. Tel.: +91 124 24112062; fax: +91 11 24115270. E-mail address: kuhad85@gmail.com (R.C. Kuhad). Bioresource Technology 101 (2010) 8348–8354 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech