Four automatic substrate feeding strategies were developed and investigated in this study to obtain rapid, repeatable, and reliable high cell densities of Pseudomonas putida KT2440 from glucose. Growth yield data of the key nutrients, Y (X/Glucose), Y (X/NH4), Y (X/PO4), Y (X/Mg), and Y (CO2/Glucose), were determined to be 0.41, 5.44, 13.70, 236, and 0.65 g g(-1), respectively. Although standard exponential feeding strategy worked well when the predetermined mu was set at 0.25 h(-1), an exponential glucose feeding strategy with online mu (max) estimation resulted in a higher average biomass productivity (3.4 vs 2.8 g l(-1) h(-1)). A CO2 production rate based pulse glucose feeding strategy also resulted in good overall productivity (3.0 g l(-1) h(-1)) and can be used as an alternative to pH-stat or DO-stat feeding. A cumulative CO2 production based continuous feed with real-time cumulative glucose consumption estimation resulted in much higher biomass productivity (4.3 g l(-1) h(-1)) and appears to be an excellent and reliable approach to fully automating high-cell-density fed-batch cultivation of P. putida.