A novel fed-batch strategy based on carbon/nitrogen (C/N) ratio in a microbial co-culture production medium broth was carried out in a biocalorimeter for improved production of poly (3-hydroxybutyrate) (PHB). Shake flask study suggested that the C/N ratio of 10 increased the yield of PHB by 2.8 times. Online parameters monitored during the C/N ratio of 10 in biocalorimeter (BioRC1e) indicated that the heat profile was maintained in the fed-batch mode resulting in a PHB yield of 30.3 +/- 1.5 g/L. The oxy-calorific heat yield coefficient during the fed-batch strategy was found to be 394.24 +/- 18.71 kJ/O-2 due to the oxidative metabolism of glucose. The reported heat-based model adapted for PHB concentration prediction in the present fed-batch mode. The heat-based model has a Nash-Sutcliffe efficiency of 0.9758 for PHB prediction. PHB obtained by fed-batch-mode was characterized using gas chromatography-mass spectrometry (GC-MS) for the monomer-acid analysis, Thermogravimetric analysis (TGA) for thermal stability of PHB, and Fourier transform infrared spectroscopy (FT-IR) for confirmation of functional groups. Here, we establish a favorable C/N ratio for achieving optimal PHB yield and a predictive heat-based model to monitor its production.