Abstract: Bacillus megaterium SW1-2 showed enhanced growth and polyhydroxybutyrate (PHB) production during cultivation on date palm syrup (DEPS) or sugar cane molasses. FT-IR and NMR spectroscopic analyses of the polymer accumulated during growth on DEPS revealed specific absorption peaks characteristic for PHB. 1.65 g/L of PHB (56.9% CDW) was produced during growth on medium supplemented with 2 g/L of DEPS. Approximately, 36.1% CDW of PHB were recorded during growth on sugar cane molasses. Six runs of different fed-batch cultivation strategies were tested, the optimal run showed approximately 6.87-fold increase. Modified E2 medium was prefered recording 10.11 and 11.34 g/L of total PHB produced for runs 1 and 2, at the end of 96 h incubation period, respectively. Decrease in PHB was recorded during growth on complex medium (run 3 and run 4). In another independent optimization strategy, ten variables were concurrently examined for their significance on PHB production by Plackett-Burman statistical design for the first time. Among variables, DEPS-II and inoculum concentration followed by KH2PO4 and (NH4)2SO4 were found to be the most significant variables encourage PHB production. Indeed, DEPS-II or Fresh syrup is more significant than commercial syrup DEPS-I (p-value= 0.05). RPM, incubation period have highly negative effect on PHB production. Role of ago-industrial wastes, especially DEPS, in enhancement of PHB production was closely discussed.