Excess CO 2 supply inhibits mixotrophic growth of Chlorella protothecoides and Nannochloropsis salina Eleonora Sforza a,⇑ , Renato Cipriani a , Tomas Morosinotto b , Alberto Bertucco a , Giorgio M. Giacometti b a Department of Chemical Engineering Principles and Practice ‘‘I. Sorgato’’, University of Padova, Via Marzolo 9, 35131 Padova, Italy b Department of Biology, University of Padova, Via U. Bassi 58/B, 35121 Padova, Italy article info Article history: Received 28 July 2011 Received in revised form 6 October 2011 Accepted 8 October 2011 Available online 28 October 2011 Keywords: Mixotrophy Glycerol Carbon dioxide Biodiesel Microalgae abstract Mixotrophy can be exploited to support algal growth over night or in dark-zones of a photobioreactor. In order to achieve the maximal productivity, however, it is fundamental also to provide CO 2 in excess to maximize photosynthetic activity and phototropic biomass production. The aim of this paper is to verify the possibility of exploiting mixotrophy in combination with excess CO 2 . Two species with high biomass productivity were selected, Nannochloropsis salina and Chlorella protothecoides. Different organic sub- strates available at industrial scale were tested, and glycerol chosen for its ability to support growth of both species. In mixotrophic conditions, excess CO 2 stimulated photosynthesis but blocked the metabolization of the organic substrate, thus canceling the advantages of mixotrophy. By cultivating mic- roalgae under day–night cycle, organic substrate supported growth during the night, but only if CO 2 sup- ply was not provided. This represents thus a possible method to reconcile CO 2 stimulation of photosynthesis with mixotrophy. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Oil production from microalgae is one promising alternative to complement and eventually replace fossil fuels in next decades. Microalgae, in fact, are photosynthetic unicellular organisms which can grow at a much faster rate than plants and reach higher pro- ductivities (Chisti, 2008). Microalgal cultures have a number of additional advantages when compared with other bio-based renewable sources. First of all, unlike first-generation biofuels, extensive microalgal cultivations do not compete with food crops for arable land, since they can be grown on marginal areas or in aquatic systems (Chisti, 2008). In addition, microalgae can reduce carbon dioxide emissions, by adsorbing CO 2 from combustion gases. In fact, atmospheric CO 2 concentration is limiting for algal growth and flue gas can be used as a cheap source with the double goal of supporting algal growth and reducing carbon dioxide re- leased into the atmosphere, although SO 2 and NO x might cause microalgae growth inhibition (Lee et al., 2002). When algae are grown with CO 2 as the unique carbon source, light provides all the energy required for biomass production. Under autotrophic conditions, however, growth is limited by light availability and, during the night, productivity is further reduced because of respiration losses. However, some phototrophic algae can also use organic carbon sources to support their growth and photoheterotrophy, or mixotrophy, is broadly defined as a growth regime in which CO 2 and organic carbon are simultaneously assim- ilated, with both respiratory and photosynthetic metabolism oper- ating concurrently. An organic carbon source can in principle support active growth within a photobioreactor, during night time, or in dark zones of the process increasing the overall biomass pro- ductivity. If organic compounds supporting growth are derived from industrial or agricultural wastes, an increased productivity is also achieved at low cost. Additional potential environmental benefits are also to be considered if wastewaters from municipal, agricultural or industrial activities are exploited as sources for or- ganic molecules (Pittman et al., 2011). Beside the benefits in terms of biomass accumulation, the addition of an organic carbon source was also reported to stimulate lipids accumulation (Heredia-Arroyo et al., 2010). It is worth men- tioning, however, that all studies analysing mixotrophic biomass and lipid production were performed in laboratory conditions and the possibility that the reported biomass yield can be main- tained over long cultivation periods remains undemonstrated (Pitt- man et al., 2011). Especially at the industrial scale, mixotrophy is likely to increase bacterial and fungal contaminations, a problem that can be controlled only in closed and strongly controlled sys- tems such as photobioreactors (Lee and Zhang, 1999). The use of 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.10.025 ⇑ Corresponding author. Tel.: +39 0498275462; fax: +39 0498275461. E-mail address: eleonora.sforza@unipd.it (E. Sforza). Bioresource Technology 104 (2012) 523–529 Contents lists available at SciVerse ScienceDirect Bioresource Technology journal homepage: www.elsevier.com/locate/biortech