Oleaginous fungal lipid fermentation on combined acid- and alkali-pretreated corn stover hydrolysate for advanced biofuel production Zhenhua Ruan, Michael Zanotti, Steven Archer, Wei Liao, Yan Liu ⇑ Department of Biosystems and Agricultural Engineering, Michigan State University, East Lansing, MI 48824, USA highlights  A novel combined hydrolysis was developed for fungal lipid fermentation.  Detoxification and pH adjustment were minimized for lignocellulosic biofuel production.  Lipid accumulation on hydrolysate had a comparable performance with the control. article info Article history: Received 19 December 2013 Received in revised form 17 March 2014 Accepted 19 March 2014 Available online 27 March 2014 Keywords: Biodiesel Combined hydrolysis Lignocellulosic biomass Oleaginous fungus Lipid accumulation abstract A combined hydrolysis process, which first mixed dilute acid- and alkali-pretreated corn stover at a 1:1 (w/w) ratio, directly followed by enzymatic saccharification without pH adjustment, has been developed in this study in order to minimize the need of neutralization, detoxification, and washing during the process of lignocellulosic biofuel production. The oleaginous fungus Mortierella isabellina was selected and applied to the combined hydrolysate as well as a synthetic medium to compare fungal lipid accumu- lation and biodiesel production in both shake flask and 7.5 L fermentor. Fungal cultivation on combined hydrolysate exhibited comparable cell mass and lipid yield with those from synthetic medium, indicating that the integration of combined hydrolysis with oleaginous fungal lipid fermentation has great potential to improve performance of advanced lignocellulosic biofuel production. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Lignocellulosic biomass is one of the most abundant and renewable sources in nature. Many studies have explored the pos- sibility of using it as a feedstock for advanced biofuels, particularly bioethanol production (Lynd et al., 2005). However, investigations of utilizing lignocellulose for microbial lipid production are still rel- atively limited. The ability to convert fermentable sugars from lig- nocellulosic material to lipid in a cost-effective manner is a key technological challenge to fully unlocking the commercial potential for such a process (Lynd et al., 2008). Three major steps identified in the production of microbial oil from lignocellulosic biomass include: hydrolyzing the lignocellulose into fermentable sugars; metabolizing those sugars by oleaginous microorganisms into microbial lipid; and finally generating biodiesel from the microbial lipid (Zhao, 2005). Unfortunately, fermentation of lignocellulosic hydrolysate is often preceded by a washing and detoxification steps that require a large amount of water and chemicals (Wooley et al., 1999). In order to develop a technically and economically feasible process for lignocellulosic biofuel production, feedstock pretreat- ment and enzymatic hydrolysis need to be optimized according to physiological characteristics of the target microorganism. Lignocellulose is a naturally recalcitrant material consisting of a heterogeneous matrix of three macromolecules: cellulose, hemi- cellulose and lignin. Physico-chemical pretreatment disrupts the structure of lignocellulosic biomass, removes substrate-specific barriers to enzymatic hydrolysis, and thus improves its digestibil- ity. While pretreatment is a crucial step in the biological conversion of lignocellulose to biofuels, it has likewise been identified as the second most expensive unit in the production of lignocellulosic ethanol preceded by feedstock cost (Mosier et al., 2005). Several thermochemical pretreatment methods have been employed to overcome the recalcitrance nature of lignocellulose, including dilute acid, ammonia fiber expansion, hot water, dilute alkali and organo-solvent methods (Alvira et al., 2010). Among these pretreatment methods, dilute sulfuric acid pretreatment http://dx.doi.org/10.1016/j.biortech.2014.03.095 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Address: 524 S. Shaw Ln., Room 203, East Lansing, MI 48824, USA. Tel.: +1 517 432 7387; fax: +1 517 432 2892. E-mail address: liuyan6@msu.edu (Y. Liu). Bioresource Technology 163 (2014) 12–17 Contents lists available at ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech