Microorganism and growth conditions
In this study, R. oryzae (ATCC 9363) was used. It was sporulated on streaked agar plates containing Potato Dextrose Agar for 4-5 days at 30 °C. After sporulation, the plates were stored at 4 °C until the preparation of spore suspension. The spore suspension was obtained by washing the agar plates containing sporulated fungus with sterile water. Spore concentration in the suspension was determined by counting the spores on a hemocytometer.
For the fermentation study, 250 ml Erlenmeyer flasks containing 100 ml of medium were used. The liquid medium is composed of (all w/v) 0.2% glucose; 0.2% (NH4)2SO4; 0.065% KH2PO4; 0.025% MgSO4.7H2O; 0.005% ZnSO4.7H2O and PPW.
PPW samples were collected from the household disposals and kept frozen (at -20 °C) until used. After thawing, PPW biomass was dried in an incubator at 75 °C until it reached a constant weight. The dried biomass was grounded in a blender to a granular form and sieved to separate into size fractions: smaller than 0.125, 0.125-0.250, 0.250-0.500, 0.500-1.000, 1.000-2.000, 2.000-4.000 (all in mm).
These six PPW size fractions were added to nutritional media at a loading rate of 2% in order to prepare the medium for the PPW particle size research and to prepare the nutritional media for the PPW loading rate investigation. The sizes (0.125-0.250 mm) and (1-2 mm) of PPW were added at loading rates of 2, 4, and 8%.
Flasks were inoculated with spore suspension to a final concentration of 105 spores/ml. After the inoculation, the cultures were incubated in a shaker incubator at 35 °C and 150 rpm. The total incubation time was 96 hours for the PPW particle size study and 144 or 192 hours (depending on particle size) for the PPW loading rate study.