Species Identification of Stress Resistance Yeasts Isolated from Banana Waste for Ethanol Production

Abstract

Banana waste putrefaction was naturally shown the potential activities of microorganisms in fermenting ethanol. Yeasts have been known as one of the potential microorganisms that wildly grown in banana waste that has the ability in producing ethanol. The objectives of the research were to isolate and identify yeasts with stress resistance ability towards high ethanol and glucose in the production of ethanol. Yeasts isolation has been done by using Potato Dextrose Agar/PDA (Oxoid Ltd.) that was modified with 3% yeast extract/YE (Kraft Inc.) and 10 ppm Amoxicillin, then incubated for 48h at room temperature. The yeast-like isolates were identified microscopic and macroscopically then cultured on Nutrient Broth/NB (Oxoid Ltd.) with the addition of 3% yeast extract/YE (Kraft Inc.), 10 ppm Amoxicillin and 30% glucose or ethanol for stress resistance ability test. The resistance ability of yeasts toward high glucose and ethanol media was determined by measuring optical density (OD) on UV-Vis spectrophotometer λ=600nm. The isolates species identified using sequence analysis of the rRNA gene internal transcribed spacer (ITS) region with the primers of ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′), the sequences compared with the GenBank database using the BLAST algorithm. The isolates were mixed and inoculated into banana wastes for 72h then the ethanol contents measured by chromium dichromate oxidation methods every 24h. The results showed that three wild yeasts (B1, B2, B3) were isolated from banana wastes, with the ability to produce ethanol with a concentration of 10.72±2.53% at 48h. Species identification showed that isolate B1, B2, and B3 were 98.99% (295/298), 100% (330/330), 99.71% (695/597) identical with Pichia bruneiensis strain CLIB 1453, Kodamaea ohmeri F3 and Hanseniaspora sp. R2 respectively.