ISOLATION AND EVALUATION OF XYLOSE-FERMENTING THERMOTOLERANT YEASTS FOR BIOETHANOL PRODUCTION
Xylose is the main fermentable sugar obtained by hydrolysis of hemicellulosic fraction of lignocellulosic materials. Xylose-fermenting microorganisms are essential for the economic conversion of lignocellulose to ethanol. The aim of this work was therefore to isolate and evaluate thermotolerant xylose fermenting yeasts. Natural habitats of yeasts were examined for the presence of thermotolerant strains able to produce ethanol from xylose. Soil, wood and fruit (pawpaw, orange, mango, pineapple and cashew) samples were screened by enrichment in 2% xylose-yeast extract liquid medium. Among the 320 thermotolerant yeasts isolated, 45 produced more than 1g/l of ethanol from 20g/l of xylose. When their fermentation ability was tested in 3% xylose, only three isolates (Pa27, Ma9, and Pi131) produced more than 7g/l of ethanol after 72 hours and were selected for further studies. Molecular identification was carried out using Internal Transcribed Spacer (ITS) resulting in determination of the species. Isolate Pa27 was Pichia kudriavzevii strain DBMY82, Ma9 was Candida tropicalis strain m56a and Pi131 was Pichia kudriavzeviistrain H156A. Optimization studies were carried out to check the effects of different process parameters such as initial pH, inoculum size, temperature, concentration of xylose, and xylose-glucose ratio on ethanol production and yield. The optimal conditions were: pH 5.5 for Pa27 and Pi13, and 4.5 for Ma91, inoculum size r1.2-1.5 O.D (600nm), temperature 35-380C and xylose concentration 70g/l. Under these conditions, isolate Pa27, Ma9 and Pi131 recorded maximum ethanol concentration of 24.30 Â± 0.17 (g/l), 22.61 Â± 0.18 (g/l) and 25.70 Â± 0.17 (g/l) after 120 hours, and maximum ethanol yield of 0.35 Â± 0.002 (g/g) after 120 hours, 0.35 Â± 0.007 (g/g) and 0.39 Â± 0.002 (g/g) after 96 hours, respectively. At incubation temperature of 420C and other optimum operating conditions, the maximum ethanol concentrations were 14.60 Â± 0.28 (g/l), 14.07 Â± 0.28 (g/l) and 15.70 Â± 0.28 (g/l) after 168 hours, while maximum ethanol yields were 0.27 Â± 0.005 (g/g), 0.27 Â± 0.005 (g/g) and 0.29 Â± 0.006 (g/g) after 96 hours for isolate Pa27, Ma9 and Pi131, respectively. The isolates co-fermented glucose and xylose to ethanol and the presence of small amount of glucose improved the xylose utilization rate. When the total sugar was 50 g/l, 8:2 (xylose 40 g/l and glucose 10 g/l) gave maximum ethanol concentrations of 18.50 ± 0.24 (g/l) and 18.77 ± 0.38 after 96 hours, and ethanol yield of 0.45 ± 0.024 (g/g) and 0.46 ± 0.018 (g/g) after 24 hours by isolate Pa27 and Pi131, respectively. Isolate Ma9 had maximum ethanol concentration of 18.47 ± 0.25 (g/l) after 120 hours and yield 0.45 ± 0.018 (g/g) after 48 hours. At 420C and under the same condition, isolate Pa27, Ma9 and Pi131 had .nmaximum ethanol concentrations of 15.30 ± 0.28 (g/l), 12.63 ± 0.09 (g/l) and 15.97 ± 0.39 (g/l) respectively after 120 hours. Isolate Pa27 and Ma9 recorded ethanol yields of 0.33 ± 0.013 (g/g) and 0.31 ± 0.013 (g/g) after 48 hours while isolate Pi131 recorded 0.34 ± 0.004 (g/g) after 72hours. The performance of these yeasts compared favorably with those reported for some other xylose-fermenting yeasts.