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(eng): High concentration of dissolved oxygen within microalgae cultures reduces the performance of corresponding microalgae cultivation system (MCS). The main aim of this study is to provide a reliable computational fluid dynamics (CFD)-based methodology enabling to simulate two relevant phenomena governing the distribution of dissolved oxygen within MCS: (i) mass transfer through the liquid–air interface and (ii) oxygen evolution due to microalgae photosynthesis including the inhibition by the same dissolved oxygen. On an open thin-layer cascade (TLC) reactor, a benchmark numerical study to assess the oxygen distribution was conducted. While the mass transfer phenomenon is embedded within CFD code ANSYS Fluent, the oxygen evolution rate has to be implemented via user-defined function (UDF). To validate our methodology, experimental data for dissolved oxygen distribution within the 80 meter long open thin-layer cascade reactor are compared against numerical results. Moreover, the consistency of numerical results with theoretical expectations has been shown on the newly derived differential equation describing the balance of dissolved oxygen along the longitudinal direction of TLC.We argue that employing our methodology, the dissolved oxygen distribution within any MCS can be reliably determined in silico, and eventually optimized or/and controlled.