Bioethanol is considered the most environmentally friendly as renewable fuels. Indonesia has abundant microbe diversity which is potential for bioprospecting such as fermenting agents using agriculture product as raw materials for producing bioethanol. This study aims to isolate, characterise and molecular identify of 15 isolates of bioethanol-producing yeasts from various sources. Characterisation based on ethanol production, cell morphology and various substrate utilisation has been carried out. Molecular characterisation of 15 yeast isolates using tree sets of primers had been carried out. Amplification in the internal area of transcribe spacers (ITS) was successfully carried out with an amplitude of 400 bp-900 bp. Amplifiers in the D1/D2 26s rDNA domain are 250 bp. Amplification with ScerF2 and ScerR2 specific primers was carried out successfully and proved that there were two isolates which were not Saccharomyces cerevisiae analysis of yeast genetic diversity showed 12 yeast isolates classified as S. cerevisiae and the rest belonged to the genus Clavispora, Candida and Kodamaea (Pichia).
Increased consumption of xylose-glucose and yeast tolerance to lignocellulosic hydrolysate are the keys to the success of second-generation bioethanol production. Candida tropicalis KBKTI 10.5.1 is a new isolated strain that has the ability to ferment xylose. In contrast to Saccharomyces cerevisiae DBY1 which only can produce ethanol from glucose fermentation. The research objective is the application of the genome shuffling method to increase the performance of ethanol production using lignocellulosic hydrolysate. Mutants were selected on xylose and glucose substrates separately and using random amplified polymorphic DNA (RAPD) analysis. The ethanol production using lignocellulosic hydrolysate by parents and mutants was evaluated using a batch fermentation system. Concentrations of ethanol, residual sugars, and by-products such as glycerol, lactate and acetate were measured using HPLC machine equipped with Hiplex H for carbohydrate column and a refraction index detector (RID). Ethanol produced by Fcs1 and Fcs4 mutants on acid hydrolysate increased by 26.58% and 24.17% from parent DBY1, by 14.94% and 21.84% from parent KBKTI 10.5.1. In contrast to the increase in ethanol production on alkaline hydrolysate, Fcs1 and Fcs4 mutants only experienced an increase in ethanol production by 1.35% from the parent KBKTI 10.5.1. Ethanol productivity by Fcs1 and Fcs4 mutants on acid hydrolysate reached 0.042 g/L/h and 0.044 g/L/h. The recombination of the genomes of different yeast species resulted in novel yeast strains that improved resistance performance and ethanol production on lignocellulosic hydrolysates.