The loss of carbon dioxide (CO ) to the environment during microalgae cultivation is undesirable for both environmental and economic reasons. In this study, a phototrophic biofilm growth model was developed and validated with the objective to maximize both CO utilization efficiency and production of microalgae in biofilms. The model was validated in growth experiments with CO as the limiting substrate. The CO utilization and biomass productivity were maximized by changing the gas flow rate, the number of biofilm reactors in series and gas composition. Based on simulations, the maximum CO utilization efficiency that was reached was 96% based on a process employing flue gas. The corresponding drop in productivity was only 2% in comparison to the non-CO limited reference situation. In order to achieve this, 25 biofilm reactors units, or more, must be operated in series. Based on these results, it was concluded that concentrated CO streams and plug flow behavior of the gaseous phase over the biofilm surface are essential for high productivity and CO utilization efficiency. Biotechnol. Bioeng. 2017;114: 769-776. © 2016 Wiley Periodicals, Inc.