Background A major obstacle to the application of microalgae for bio-fuel production is light intensity reduction in high density cultures. Mixotrophic operation can solve this problem by exploiting the ability of microalgae to grow on organic carbon in dark conditions. In this article, mixotrophic growth of Nannochloropsis oculata and Chlorella vulgaris is analyzed. The influence of nitrate and glucose concentration on cell growth in 300 mL Erlenmeyer flasks was investigated using an experimental factorial design. The analysis was extended and validated by analyzing mixotrophic growth in a 6 L bubble column. Results The addition of glucose at concentration 0.1 g L-1 inhibited or enhanced the growth of Chlorella depending on nitrate concentration, while it increased the growth rate of Nannochloropsis at all nitrate concentrations tested. Statistically significant interaction between glucose and nitrate concentration was revealed for Chlorella; negligible interaction was found for Nannochloropsis. A kinetic characterization, shedding light on the influence of glucose on microalgal growth, was conducted through identification of a Monod-like model. Experimental tests in 6 L bubble column confirmed for both Chlorella and Nannochloropsis the possibility to significantly enhance growth in the presence of light deficiency through mixotrophic operation. Conclusions Mixotrophic growth is governed by the interaction between organic carbon and nitrogen. This can have a profound impact on kinetic modeling of microalgal growth and on the operation of cultivation systems. Controlling the C/N ratio is in particular fundamental to optimize reactor operation. (c) 2013 Society of Chemical Industry