8-50: Isolation of fungal mutants exhibiting elevated pullulan production on hydrolyzed prairie cordgrass

Pullulan is a commercially available polysaccharide gum that has several industrial applications. Pullulan can be synthesized by the fungus Aureobasidium pullulans from glucose released from hydrolyzed prairie cordgrass since it contains about 30% cellulose. Currently, prairie cordgrass is primarily utilized to feed livestock. In this study, mutants of A. pullulans ATCC 42023 were isolated that exhibited enhanced pullulan production on a prairie cordgrass hydrolysate. Both mutants were isolated following chemical mutagenesis using 1% methyl methanesulfonate. One mutant was isolated by screening colonies on a solid prairie cordgrass hydrolysate-containing medium for an increased mucoid phenotype. The second mutant was isolated by its resistance to the glucose analogue 2-deoxy-D-glucose. Both mutants were tested for their ability to produce the polysaccharide on an acid-treated cordgrass hydrolysate. The hydrolysate was prepared by adding 0.1% sulfuric acid to dried, ground grass with the mixture (10% solids) being autoclaved at 121^oC for 30 minutes. The acid-treated cordgrass was filtered and the filtrate was used in a phosphate-buffered medium (pH 6.0) to grow the fungus. The fungal cells were grown for 168 hours at 30^oC in shake flask cultures. After samples of the culture medium were collected and subjected to centrifugation, the pullulan concentration in the resultant supernatants was determined gravimetrically. Both isolated mutants produced higher pullulan levels than A. pullulans ATCC 42023 after 168 hours of growth on the acid hydrolysate-containing medium. In conclusion, it was possible to isolate mutants of A. pullulans ATCC 42023 capable of elevated polysaccharide production from hydrolyzed prairie cordgrass.