Carbon concentration and oxygen availability affect lipid and carotenoid production by carob pulp syrup‐grown Rhodosporidium toruloides NCYC 921 Eng. Life Sci. 2015, 15, 815–823 www.els-journal.com Teresa Margarida Parreira Claudia Freitas Alberto Reis José Roseiro Teresa Lopes da Silva Unidade de Bioenergia, Laboratório Nacional de Energia e Geologia, Lisboa, Portugal Research Article Carbon concentration and oxygen availability affect lipid and carotenoid production by carob pulp syrup-grown Rhodosporidium toruloides NCYC 921 The simultaneous effect of oxygen availability and carbon source concentration on yeast lipid and carotenoid production has never been studied before. In this work, a Doehlert distribution design was used to study the simultaneous effect of carbon concentration and oxygen availability on Rhodosporidium toruloides NCYC 921 carotenoid and lipid production. A cheap industrial byproduct was used as carbon source (carob pulp syrup). A total sugar concentration of 106.3 g/L and a medium volume of 0.120 L induced the highest total carotenoid and total fatty acid productivities (4.60 μg/Lh and 0.029 g/Lh, respectively). Flow cytometry was used to assess yeast stress response under different cultivation conditions. The highest proportion of cells with permeabilised membrane (>20%) was induced when the cultivations were carried out at the highest sugar concentration studied (130.0 g/L) or when the culture reached the minimum final medium pH (4.60). The results showed that the total sugar concentration had a positive influence on the yeast biomass and carotenoid content, while the oxygen availability had little influence on the biomass concentration, but had a slight positive influence on the carotenoid content. Regarding the fatty acids, the two factors had a negative impact on the synthesis of these compounds. Keywords: Carbon concentration / Carotenoids / Lipids / Oxygen availability / Yeast � Additional supporting information may be found in the online version of this article atthe publisher’s web-site Received: January 8, 2015; revised: March 25, 2015; accepted: May 27, 2015 DOI: 10.1002/elsc.201500002 1 Introduction In the midst of the energy crisis, third generation biofuels (de- rived from microalgae and other microbes) are considered to be viable fuel alternatives [1, 2]. However, to be economically sustainable, the microbial biofuel production process must val- orize the whole biomass fractions to be converted into biofuels and high value added products under the frame of an integrated biorefinery concept. Correspondence: Dr. Teresa Lopes da Silva (teresa.lopessilva@ lneg.pt), Unidade de Bioenergia, Laboratório Nacional de Energia e Geologia, I.P., Estrada do Paço do Lumiar 22, 1649-038 Lisboa, Portugal Abbreviations: DiOC6 (3), 3,3-dihexylocarbocyanine iodide; MUFAs, monounsaturated fatty acids; PI, propidium iodide; PUFAs, polyunsat- urated fatty acids; SATs, saturated fatty acids; TFAs, total fatty acids The oleaginous yeast Rhodosporidium toruloides NCYC 921 (which species is an anamorph of Rhodotorula glutinis species) has been widely reported as a potential oil producer yeast [3, 4]. However, the yeast biomass, beyond its high lipid content (that can be converted into biodiesel), is rich in high value added products such carotenoids with commercial interest [5] their commercialization may contribute to reduce the overall process cost of biofuels and carotenoid production [6]. The use of low-cost substrates in media formulations may also reduce the costs of the microbial biofuels production [7]. Carob pulp syrup has been used in media formulations for micro- bial lipid production [8]. The carob tree (Ceratonia siliqua L.) is native to the Mediterranean region, including southern Eu- ropean countries. Carob seeds comprise 10% of the fruit dry weight and are used for gum production for food industry. The remaining 90% of the fruit dry weight (the pulp) contains high content of sugar (sucrose, glucose and fructose). The easily C© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim 815 Parreira et al 2015