PHB production occurs only under nitrogen limiting conditions (Takahiro,1986),it was carried out in two steps: i) PHB producers were grown in 500 mL Erlenmeyer flask containing 100 mL carbon rich growth medium.(Jung et al.,1997).Flask were inoculated at the rate of 1% inoculums (v/v)and incubated at 30ºC for 24 hrs with constant shaking at 120 rpm and ii) after getting optimal cell concentration ,cell mass was centrifuged at 10000 rpm for 10 min, washed with sterile distilled water, inoculated in nitrogen deficient minimal medium and incubated for 48 hrs at 37ºC on a rotatory shaker(120 rpm).
Optimization of cheap carbon source (Glycerol) and various