Gluconate and glucose were selected as the carbon substrates in the production of poly-3-hydroxybutyrate (PHB). Gluconate was utilized to maximize the specific growth rate during the first stage of cell growth, whereas glucose was used to maximize PHB biosynthesis during the second stage of PHB accumulation. The sequential feeding of gluconate and glucose resulted in a 50% enhancement of PHB productivity as compared to the cultures cultivated on glucose alone. In conjunction with secondary glucose uptake, the presence of a trace amount of ammonium increased the rate of PHB biosynthesis during the stage of PHB accumulation. Via the feeding of 0.03 mmol/h of $NH_{4}Cl$ solution prior to the exhaustion of the initial amount of $NH_{4}Cl$, PHB productivity was significantly enhanced as compared to the cultures raised on glucose alone. The glucose-grown culture evidenced a higher level of NADPH during the $NH_{4}Cl$-exausted PHS accumulation stage than was observed in the gluconate-grown culture, which reflects that the reason of higher PHB production observed when glucose was used as a carbon source. $NH_{4}Cl$ feeding following the depletion of initial $NH_{4}Cl$ resulted in elevated levels of both ATP and NADPH, which increased the PHB biosynthesis rate, and also in a decrease in the level of NADH, which reflected the alleviation of the inhibitory effects on the cells caused by nitrogen depletion.