Background/Aims: The influence of different parameters such as temperature, irradiance, nitrate concentration, pH, and an external carbon source on Synechocystis PCC6803 growth was evaluated. Methods: 4.5-ml cuvettes containing 2 ml of culture, a high-throughput system equivalent to batch cultures, were used with gas exchange ensured by the use of a Parafilmâ„¢ cover. The effect of the different variables on maximum growth was assessed by a multi-way statistical analysis. Results: Temperature and pH were identified as the key factors. It was observed that Synechocystis cells have a strong influence on the external pH. The optimal growth temperature was 33°C while light-saturating conditions were reached at 40 µE·m(-2)·s(-1). Conclusion: It was demonstrated that Synechocystis exhibits a marked difference in behavior between autotrophic and glucose-based mixotrophic conditions, and that nitrate concentrations did not have a significant influence, probably due to endogenous nitrogen reserves. Furthermore, a dynamic metabolic model of Synechocystis photosynthesis was developed to gain insights on the underlying mechanism enabling this cyanobacterium to control the levels of external pH. The model showed a coupled effect between the increase of the pH and ATP production which in turn allows a higher carbon fixation rate.