Enrichment, isolation and characterization of fungi tolerant to 1-ethyl-3-methylimidazolium acetate

Enrichment, isolation and characterization of fungi tolerant to 1-ethyl-3- methylimidazolium acetate Steven W. Singer 1,2* , Amitha P. Reddy 1,4 , John M. Gladden 1,3 , Hongyun Guo 4 , Terry C. Hazen 1,2 ; Blake A. Simmons 1,5 , Jean S. VanderGheynst ,1,4 Joint BioEnergy Institute, Emeryville, CA 94608; 2 Earth Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720; 3 Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, 94551; 4 Biological and Agricultural Engineering, University of California-Davis, 95616; Biomass Science and Conversion Technology Department, Sandia National Laboratory, Livermore, CA, Corresponding author: Steven W. Singer, Deconstruction Division, Joint Bioenergy Institute, 5885 Hollis Street, Emeryville, CA 94608, Phone: (510) 495-2492,Fax: (510) 486-4252 E-mail- SWSinger@lbl.gov Abstract Aims: This work aims to characterize microbial tolerance to 1-ethyl-3-methylimidazolium acetate ([C2mim][OAc]), ionic liquid that has emerged as a novel biomass pretreatment for lignocellulosic biomass. Methods and Results: Enrichment experiments performed using inocula treated with [C2mim][OAc] under solid and liquid cultivation yielded fungal populationsdominated by Aspergilli. Ionic liquid-tolerant Aspergillus isolates from these enrichments were capable of growing in a radial plate growth assay in the presence of 10% [C2mim][OAc]. When a [C2mim][OAc]-tolerant Aspergillus fumigatus strain was grown in the presence of switchgrass, endoglucanases and xylanases were secreted that retained residual enzymatic activity in the presence of 20% [C2mim][OAc]. Conclusions: The results of the study suggest tolerance to ionic liquids is a general property of Aspergilli. Significance and Impact of the Study: Tolerance to an industrially important ionic liquid was discovered in a fungal genera that is widely used in biotechnology, including biomass deconstruction. Introduction: Lignocellulosic biomass is a potential renewable feedstock that is available for conversion to biofuels that will replace a substantial amount of the petroleum-based transportation fuels (Gomez et al. 2008). Currently, most first-generation biofuels are derived from agricultural food sources such as corn grain and soybeans. Lignocellulosic biomass, which includes agricultural residues (corn stover, rice straw), agricultural by-products (corn fibre, rice hulls and sugarcane bagasse), woody biomass (poplar, pine) and dedicated energy crops (Miscanthus, switchgrass), has a low impact on human food supplies (Blanch et al. 2008). Pretreatment of lignocellulosic biomass is required for efficient saccharification to generate monomeric sugars for biofuel production. Established chemical pretreatment technologies, such as dilute acid and ammonia fibre expansion, greatly improve the yields of sugar from enzymatic hydrolysis, but new advances in pretreatment are needed for industrial production of cellulosic biofuels (Yang and Wyman 2008). Recently, ionic liquids (Fig. 1) have demonstrated excellent promise as solvents for biomass dissolution and cellulose recovery (Swatloski et al. 2002; Turner et al. 2004). The recovered cellulose is enzymatically hydrolyzed to glucose at much faster rates than observed for dilute acid pretreated biomass (Li et al. 2010). Room temperature ionic liquid 1-ethyl-3-methylimidazolium acetate [C2mim][OAc] has proved to be particularly effective at dissolution of cellulose, hemicellulose and lignin from switchgrass, corn stover and eucalyptus (Singh et al. 2009). A combination of electrostatic and hydrogen-bonding interactions between ionic liquid and the plant polymers has been proposed as