An online estimator for the Oxygen Transfer Rate OTR for Escherichia coli cultured in bioreactors was developed, which allowed for improved culture outcomes. E. coli are used to manufacture recombinant proteins used as therapeutics, such as insulin and human growth hormone. E. coli cultures require high levels of oxygen in order to produce the therapeutics efficiently. Previous methods to estimate OTR used values for the volumetric mass transfer coefficient, kLa, which had been determined from separate experiments and thus set to a constant value or by stopping oxygen flow periodically to the bioreactor to update the kLa value. In this work, the kLa value was estimated in real-time and continuously from the on-line dissolved oxygen concentration and off-gas measurements. The gas phase mixing in the head space and time response of the off-gas sensor were accounted for in the model. The improved estimates of OTR were incorporated into a model of E. coli metabolism to better predict the metabolic state of E. coli, such that glucose could be fed to E. coli at near optimal rates. Additionally, the effects of enriching the air with pure oxygen were accounted for in the estimator model, as this enrichment is necessary to reach final cell densities representative of the industrial process.