A solid YM plate containing 2%
agar was used to examine cell growth and viability. Selleckchem SNX-5422 All experiments were LEE011 order carried out with two replications. Yeast adaptation and mutation selection Adaptation procedures were developed based on procedures by Wei et al.  and Dinh et al.  with modifications. Briefly, inhibitor-tolerant strain NRRL Y-50049 was cultured on a YM with 10% glucose containing ethanol in designated concentrations. Cultures were treated with a quick freeze at -80°C at the mid-log phase and thawed at 30°C in a water-bath. The treatment procedures were repeated. Incubations were continued at 30°C until a stationary phase was reached. Surviving cultures were sequentially transferred to fresh medium containing higher ethanol concentrations. These procedures were repetitively carried out until a target tolerance level reached. Tolerant mutants were selected from at least 40 complete cycles using a medium containing no less than 8% ethanol. Culture characteristics were confirmed by cell morphology, growth rate, metabolic
profiling, and sequence verification of its identity using nuclear large subunit ribosomal RNA gene . Assays for tolerance and viability Cells were selleck chemical grown at 30°C and 250 rpm into the late exponential growth phase at OD600 reading of 1.0 when cultures contained approximately 1×107 cells/ml. An assay using serial dilutions of the culture was applied onto an YM plate of 2% glucose containing 8% (v/v) ethanol for ethanol tolerance test using 10-fold serial dilutions of
cell suspension. The culture plates were incubated at 30°C and examined 4 days after incubation. Tolerance to inhibitors furfural and HMF were examined in a similar manner on YM plates of 2% glucose containing 10 mM each of furfural and HMF 7 days for after incubation. Cell viability was examined for cultures grown under a challenge with 8% of ethanol over time. The time point after 6-h pre-culture when ethanol was added into the culture was designated as 0 h. Samples were taken starting at 24 h after the ethanol challenge until 168 h with a 24-h interval. Cell growth was examined on a solid YM using an assay similar as described above. Sample collection and HPLC analysis Cell growth was monitored by absorbance at OD600 under ethanol stress. Samples were taken and cells harvested at 0, 1, 6, 24, and 48 h after the 8% ethanol addition for mRNA expression analysis using procedures as previous described . Yeast cells were immediately frozen on dry ice and then stored at -80°C until use. Samples of culture supernatants were taken periodically from 0 h to 120 h after the ethanol challenge for metabolic profiling analysis.