a b s t r a c tIncreasing microalgal starch content by nutrient limitation has been regarded as an affordable approach for the production of third generation bioethanol. This work evaluated starch accumulation in Chlorella vulgaris P12 under different initial concentrations of nitrogen (0-2.2 g urea L À1 ) and iron (0-0.08 g FeNa-EDTA L À1 ) sources, using a central composite design (CCD) for two factors. The obtained model: Starch content (%) = 8.220 À 16.133X 1 + 13.850X 2 1 , relating starch accumulation in microalgae with the coded level for initial urea concentration in the growth medium (X 1 ) presented a good concordance between the predicted and experimental values (R 2 = 0.94). Since accumulation of starch occurred at nitrogen depletion conditions under which the cell growth was much slower than that observed during nitrogen supplemented cultivations, a two-stage cultivation process for high starch accumulation (>40%) and cell growth of C. vulgaris was proposed: a first cultivation stage using nitrogen-and iron-supplemented medium (initial urea and FeNa-EDTA concentrations of 1.1 and 0.08 g L À1 , respectively), followed by a second cultivation stage in a nitrogen-and iron-free medium. The high starch content obtained suggests C. vulgaris P12 as a very promising feedstock for bioethanol production.

Growth parameters and biochemical composition of the green microalga Chlorella vulgaris cultivated under different mixotrophic conditions were determined and compared to those obtained from a photoautotrophic control culture. Mixotrophic microalgae showed higher specific growth rate, final biomass concentration and productivities of lipids, starch and proteins than microalgae cultivated under photoautotrophic conditions. Moreover, supplementation of the inorganic culture medium with hydrolyzed cheese whey powder solution led to a significant improvement in microalgal biomass production and carbohydrate utilization when compared with the culture enriched with a mixture of pure glucose and galactose, due to the presence of growth promoting nutrients in cheese whey. Mixotrophic cultivation of C. vulgaris using the main dairy industry by-product could be considered a feasible alternative to reduce the costs of microalgal biomass production, since it does not require the addition of expensive carbohydrates to the culture medium.

Different methods for estimating starch in Chlorella vulgaris were compared with the view of establishing a procedure suitable for rapid and accurate determination of starch content in this microalgal species. A close agreement was observed between methods that use perchloric acid and enzymatic methods that use α-amylase and amyloglucosidase to hydrolyze the starch of microalgae grown under different nitrogen culture conditions. Starch values obtained by these methods were significantly higher than those estimated by using hydrochloric acid as solubilizing and hydrolyzing agent. The enzymatic method (EM1) proved to be the most rapid and precise method for microalgal starch quantification. Furthermore, the evaluation of resistant starch by enzymatic methods assayed in nitrogen-sufficient and nitrogen-starved cells showed that no formation of this type of starch occurred in microalgae, meaning that this should not interfere with starch content determinations.