Characteristics and identification of Aureobasidium pullulans isolated in Thailand

Abstract

Aureobasidium pullulans, a black yeast with industrial significance because of its production of extracellular polysaccharides, was studied with the ultimate aim to enhance pullulan yield. New strains were isolated from nature in Thailand. Its identification was performed by Diazonium Blue B staining, morphology with the light microscopy, scanning and transmission electron microscopy, and sequencing of the Internal Transcribed Spacer domain of the rDNA gene. For the latter technique a large set of reference strains was founded, allowing the reconstruction of a phylogenetic tree of Dothideales. A. pullulans formed a well-delimited cluster, while the ITS was found to be heterogeneous within the species. A phenomenon could as yet not be explained and requires further study. Our strains were identical to the neotype culture of A. pullulans, and thus their correct identification was confirmed. Subsequently, the structure of pullulan was analyzed by IR-spectrophotometry. Pullulan production of our strains was measured by ethanol precipitation. Highest harvest was obtained when glucose and ammonium sulphate were used as Carbon and Nitrogen sources, respectively. Optimal pullulan production was achieved at a pH varying between 6.5-7.5, and at the temperature of 25-30 ํC. A. pullulans CHULA-SU showed optimal production of pullulan at pH 6.5, 25 ํC, with glucose and ammonium sulphate as a carbon and nitrogen sources, respectively. A. pullulans CHULA-PR produced most pullulan at pH 7.5, 30 ํC, using the same carbon and nitrogen sources. A. pullulans CHULA-CU produced pullulan optimally at pH 6.5, 30 ํC, using the same media.