Isolation and identification of thermophilic filamentous fungi from hot spring waters and researching of their phytase production

This study focused on the isolation of thermophilic filamentous fungi from hot spring waters, characterization of morphologic and molecular tecnique and researching of their phytase production capabilities. The sampling was performed one time of year 2008 and 2009 from 12 hot spring in Kütahya and 4 hot spring in Afyon. Totally 137 fungus isolates were obtained as the results of the isolation procedures from 47 different hot water samples taken from hot spring waters. It was determined by temperature tests that 25 of them from these isolates were thermophilic. Morphologic and molecular identification based on ITS, D1/D2 region of 26-28S rDNA of thermophilic isolates was carried out. Phytase enzyme production by using Thermomyces lanuginosus which is selected by phytase screening studies was carried out by solid state fermentation (SSF). Classical approach and Central composite design (CCD) of response surface methodology were applied for the optimization of phytase production. As a result of the morphologic and molecular identifications it was determined that thermophilic fungi isolates were the members of Myceliophthora thermophila, Myceliophthora hinnulea, Melanocarpus albomyces, Rhizomucor pusillus, Thermomyces lanuginosus, Humicola grisea, Humicola insolens, Scytalidium thermophilum, Talaromyces thermophilus, Thielavia terrestris ve Malbranchea cinnamomea species. Thermophilic fungi of hot spring waters of Kütahya and Afyon provinces were exhibited with these identification studies for the first time. Optimization strategy which was used to production of phytase was constituted a combination of traditional and statistical methods. Maximum pyhtase activity was achieved by using rice bran. The optimum levels of variables that supported maximum enzyme activity were moisture 70%, culture time 7 days, inoculum size 40%, culture temperature 55 0C, initial pH 7,5, aeration area 30%, age of seeding culture 5 days, sucrose 1%, ZnSO4 2,5 mM. An overall 10,83-fold enhancement in phytase activity (0,30 to 3,248 U) was attained due to the optimization and spesific phytase activity was determined as 8,97 U/mg. Molecular weight of T. lanuginosus phytase enzyme was determined as ~30 kDa by SDS-PAGE and zymogram analysis proved that this band belong to phytase enzyme. Key words: Thermophilic fungus, molecular identification, hot spring water, phytase, response surface methodology.