Improvement of multiple stress tolerance in yeast strain by sequential mutagenesis for enhanced bioethanol production Rajni Kumari and Krishna Pramanik * Department of Biotechnology and Medical Engineering, National Institute of Technology, Rourkela 769008, Orissa, India Received 27 April 2012; accepted 2 July 2012 Available online 4 August 2012 The present work deals with the improvement of multiple stress tolerance in a glucoseexylose co-fermenting hybrid yeast strain RPR39 by sequential mutagenesis using ethyl methane sulfonate, N-methyl-N 0 -nitro-N-nitrosoguanidine, near and far ultraviolet radiations. The mutants were evaluated for their tolerance to ethanol, temperature and fermentation inhibitors. Among these mutants, mutant RPRT90 exhibited highest tolerance to 10% initial ethanol concentration, 2 g L L1 furfural and 8 g L L1 acetic acid. The mutant also showed good growth at high temperature (39e40  C). A study on the combined effect of multiple stresses during fermentation of glucoseexylose mixture (3:1 ratio) was performed using mutant RPRT90. Under the combined effect of thermal (39  C) and inhibitor stress (0.25 g L L1 vanillin, 0.5 g L L1 furfural and 4 g L L1 acetic acid), the mutant produced ethanol with a yield of 0.379 g g L1 , while under combined effect of ethanol (7% v/v) and inhibitor stress the ethanol yield obtained was 0.43 g g L1 . Further, under the synergistic effect of sugar (250 g L L1 ), thermal (39  C), ethanol (7% v/v) and inhibitors stress, the strain produced a maximum of 47.93 g L L1 ethanol by utilizing 162.42 g L L1 of glucoseexylose mixture giving an ethanol yield of 0.295 g g L1 and productivity of 0.57 g L L1 h L1 . Under same condition the fusant RPR39 produced a maximum of 30.0 g L L1 ethanol giving a yield and productivity of 0.21 g g L1 and 0.42 g L L1 h L1 respectively. The molecular char- acterization of mutant showed considerable difference in its genetic profile from hybrid RPR39. Thus, sequential mutagenesis was found to be effective to improve the stress tolerance properties in yeast. Ó 2012, The Society for Biotechnology, Japan. All rights reserved. [Key words: Mutagenesis; Stress tolerance; Ethanol fermentation; Mutants; Fermentation inhibitors] Lignocellulosic biomass has been considered as a potential substrate for bioethanol production. In this context, the recent research focuses on the fermentation of hexose and pentose sugar components present in lignocellulosic biomass to ethanol using hybrid strains that are developed by combining genes of Saccharo- myces cerevisiae and xylose fermenting yeasts (1,2). However, during fermentation, yeast cells are subjected to multiple stresses that lead to adverse effect on the bioethanol production. The stresses such as high temperature, high ethanol concentration and toxic inhibitors seem to have individual as well as synergistic effect on the viability and efficiency of yeast to produce ethanol (3). As per published literature, the ethanol stress reduces the cell metabolic activities and solute transport across the plasma membrane by increasing membrane fluidity whereas the thermal stress enhances the lag phase and affects the membrane functions (4e6). Furthermore, the release of a variety of toxic inhibitors during the pretreatment of biomass also affects the fermentation performance. As for example, phenolic compounds cause the loss of membrane integrity while organic acid and aldehyde inhibitors result in accumulation of reactive oxygen species (7e9). Therefore, the yeast strains used for ethanol fermentation of lignocellulosic substrates must have the important physiological properties like tolerance to high tempera- ture, high ethanol concentration and toxic inhibitors (10). Hence, the improvement of the stress tolerance of the hybrid strains is of paramount importance to make them much more effective in producing bioethanol at an industrial scale. It is reported that the stress tolerance in yeast strains is regu- lated by complex gene interactions (11). Therefore, a number of methods have been the choice of researchers to improve the genetic constitution of industrially important microorganisms. Mutation is one of such method that can induce change in the genomic composition of any microorganism. Induced mutagenesis using physical and chemical mutagens has been considered as a simple and rational approach for yeast strain improvement (6). Researchers have reported the improvement of yeast strains by inducing mutation using ultraviolet (UV) radiation (12,13) and chemical agents (14,15). UV radiations, ethyl methane sulfonate (EMS) and N-methyl-N 0 -nitro-N-nitrosoguanidine (MNNG) treat- ment has been found to influence the different metabolic activities in yeasts. EMS alkylates DNA base pairs and thus brings out tran- sition of AeT to GeC base pairs causing point mutagenesis (16). MNNG produces a variety of lesions by the reaction with DNA (17) while UV radiations induce mitotic crossing over, mitotic gene conversion and reverse mutation by formation of cyclobutane dimers in S. cerevisiae (18,19). * Corresponding author. Tel.: þ91 0661 2462283; fax: þ91 0661 2472926. E-mail addresses: rajni.nitrkl@gmail.com (R. Kumari), kpr@nitrkl.ac.in (K. Pramanik). www.elsevier.com/locate/jbiosc Journal of Bioscience and Bioengineering VOL. 114 No. 6, 622e629, 2012 1389-1723/$ e see front matter Ó 2012, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2012.07.007