Contents lists available at ScienceDirect Industrial Crops & Products journal homepage: www.elsevier.com/locate/indcrop Biodiesel-derived crude glycerol as alternative feedstock for single cell oil production by the oleaginous yeast Candida viswanathii Y-E4 Mohamed Guerfali*, Ines Ayadi, Houssem-Eddine Sassi, Ameni Belhassen, Ali Gargouri, Hafedh Belghith Laboratory of Molecular Biotechnology of Eukaryotes, LMBE, Centre of Biotechnology of Sfax, P.O. Box 1177, TN-3038, Sfax, Tunisia ARTICLE INFO Keywords: Microbial lipids Candida viswanathii Crude glycerol Response surface methodology Biodiesel Linoleic acid ABSTRACT In recent years, much attention has been paid to microbial lipids as promising raw materials for biodiesel production. The use of industrial by-products as carbon sources seems to be a feasible strategy for cost-effective lipid fermentation with oleaginous microorganisms on a large scale. In this work, biodiesel-derived crude gly- cerol was used as a renewable feedstock for microbial lipid production. Chemical characterization has shown that used crude glycerol sample is mainly composed of 64.5 % glycerol, 5.9 % methanol, 11 % water and 5.8 % ash in addition to other impurities. This industrial waste was used as only carbon source in the culture media of 13 oleaginous yeast strains. Candida viswanathii Y-E4 stood out above the other strains, achieving a higher amount of biomass and lipid content (17.2 g/L and 41 %, respectively). Culture conditions were optimized using response surface methodology to enhance the Y-E4 lipid yield. Box-Behnken model was performed to determine the optimum levels of three independent variables and the eventual effect of their interactions on the response. Under optimal conditions, the total biomass of 26.6 g/L with lipid content of 51.9 % (corresponding to a lipid yield of 13.6 g/L) could be achieved after 166 h of culture. The characterization of obtained lipid showed that fatty acids composition and lipid profile were very different between pure and crude glycerol based cultures, with a predominance of long-chain fatty acids, particularly polyunsaturated fatty acids such as linoleic acid (45 %). The predictive determination of biodiesel properties suggests that this oil may effectively be used for bio- diesel manufacture. The current study, therefore, opens new perspectives in relation to the valorization of biodiesel-derived crude glycerol, through completely eco-friendly processes. 1. Introduction The availability of petroleum-based resources has decreased sig- nificantly with the increase of worldwide demand for energy. Indeed, the depletion of fossil reserves associated with serious ecological pro- blems related to greenhouse gas emissions forced researchers to de- velop new energy processes based on renewable and sustainable sources. The use of biofuels such as biodiesel or bioethanol, generated from biological materials is currently a promising alternative. However, biodiesel produced from vegetable oils and animal fat faces several constraints, such as raw material availability, competition with human consumption and exploited farmland (Leung et al., 2010). To overcome this obstacle, many solutions were proposed and one of the most pro- mising consists to produce microbial lipids (to replace conventional oils) through a bioconversion process using oleaginous microorganisms (Ma, 2006). Specifically, microorganisms (microalgae, bacteria, yeasts and fungi) able to accumulate intracellular neutral lipids more than 20 % w/cell dry weight are considered as oleaginous (Ratledge, 2004). Ac- cumulated lipids are mainly composed of triglycerides (80–90 %) and sterol esters (20 %) formed by long-chain fatty acids (C14-C18) showing a highly similar composition to that of vegetable oils (Papanikolaou et al., 2013). Consequently, microbial lipids can in- tegrate several industrial biotechnological transformations especially in the field of bio-energy, oleo-chemistry, cosmetics, etc (Vinarta et al., 2016). Being unicellular, unaffected by the season and climate, char- acterized by a short life cycle, requiring less labor and space for culture as well as being suitable for genetic improvement and large-scale ex- ploitation, all these criteria make yeasts as the best candidates for the development of industrial biotechnological approaches for lipids pro- duction (Vinarta et al., 2016). However, one major problem slows the progress of microbial lipids commercialization, generally related to the high cost of raw material. Using industrial by-products, as renewable and less expensive feedstock in the growth media of oleaginous https://doi.org/10.1016/j.indcrop.2020.112103 Received 14 November 2019; Received in revised form 30 December 2019; Accepted 6 January 2020 ⁎ Corresponding author. E-mail address: mohamed.guerfali@cbs.rnrt.tn (M. Guerfali). Industrial Crops & Products 145 (2020) 112103 0926-6690/ © 2020 Elsevier B.V. All rights reserved. T