© 2018, Springer Science+Business Media, LLC, part of Springer Nature.The polymers/enzymes produced by the microorganisms isolated from the various thermal sources have stability at different levels and their usage areas can be improved according to this characteristic. In this study, prokaryotic diversity of water sample taken from Çitgöl thermal spring in Kütahya-Simav, has been investigated by cultivation techniques. In the result of this study, Geobacillus thermoparaffinivorans, Geobacillus kaustophilus, Geobacillus sp., Geobacillus thermoleovorans and Thermus thermophilus were isolated from water sample. Then, the scanning studies were performed to choose an effective PHB producer with thermal stability. Designing of experiments, analyzing of experimental results, creating the mathematical models and finding optimization conditions were performed using Box-Behnken experimental design method. PHB yield of G. kaustophilus C4-2 increased to 8.66% PHB under the determined conditions which were found as 35 °C, 3.75 days and 10 g/L sucrose. The validity and practicability of this statistical optimization strategy was confirmed relation between predicted and experimental values. The characterization of obtained PHB was performed through analyses including FTIR, H-NMR, TG/DTA and viscosity. According to the result of TG/DTA, PHB was thermal resistant and could remain 14.3% without decomposition at 600 °C.