This study investigated the growth physiology and polyhydroxybutyrate (PHB) storage potential of Paracoccus pantotrophus from glucose and mixed organic substrate both under aerobic conditions and with dissolved oxygen limitation in a sequence of anaerobic conditions. The first part of the experiments involved full aerobic batch cultivations with glucose as the sole carbon source. In the next part, a batch growth system with anaerobic-aerobic cycles was employed, fed either with glucose or a substrate mixture of peptone and meat extract supplemented with varying concentrations of casein. In fully aerobic batch experiments, substrate storage was observed as an auxiliary microbial process along with growth, converting 20% of the glucose in the feed to PHB. PHB accumulation reached 27% of the cell dry weight after nine hours of incubation. This is an original observation revealing, the significant feature of the metabolism of P. pantotrophus generating PHB from glucose rather than commonly observed glycogen accumulation. Introduction of an anaerobic phase with an operation involving a sequence of anaerobic/aerobic periods induced a significant increase in PHB accumulation to a level corresponding to around 50% of the cell dry weight. In the experiments with the same anaerobic/aerobic sequence and utilizing an organic carbon mixture with increasing doses of casein, substrate removal remained limited but it always resulted in PHB accumulation. Further studies need to be conducted in a way to optimize the PHB generation potential of the same microorganism from different substrate mixtures.