## Materials and Methods  
### Microorganism, Media and Culture Conditions  
The yeast strain used in the current study was a new indigenous wild-type _Y. lipolytica_ strain, coded LMBF Y-46, isolated from gilt-head (sea) bream (_Sparus aurata_) fish [17], having been previously demonstrated as capable of producing significant amounts of yeast biomass and mannitol in a first screening-type study [16]. The maintenance of the strain as well as the salt composition of the basal medium used for the fermentations, have both previously been described [18; 19]. The nitrogen sourcesemployed were (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) (containing _c._ 21%, _w/w_, of nitrogen) and yeast extract (containing _c._ 7%, _w/w_, of nitrogen and _c._ 8%, _w/w_, of carbon). Initially (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) at 1.5 g/L and yeast extract at 1.0 g/L were employed; at this stage, a relatively low initial glycerol concentration (_Gly\({}_{0}\)_) was employed (\(\approx\)40 g/L). Thereafter, and in order to enhance the production of yeast biomass, in addition to potentially enhancing glycerol assimilation, higher initial nitrogen concentrations were employed; in this second set of cultures, (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) at an initial concentration of 2.0 g/L and yeast extract at an initial concentration of 3.0 g/L were employed [18]. Additionally, higher initial concentrations of glycerol were used (up to _c._ 150 g/L). The carbon sources used in all trials were either industrial (biodiesel-derived) crude glycerol (Agroinvest SA; Achladi, Fthiotis prefecture, Greece) with a purity of _c._ 81% _w/w_[18] or pure glycerol (Prolabo Chemicals, Fontenaysous bois, France, purity _c._ 98% _w/w_). The impurities in industrial glycerol were composed of 11-12% _w/w_ water, 5-6% _w/w_ potassium salts, 1% _w/w_ free-fatty acids and \(<\)0.2% _w/w_ methanol. The initial pH of all media was \(6.0\pm 0.1\). Shake-flask trials were conducted batch-wise in Erlenmeyer non-baffled flasks of 250 mL filled with \(50\pm 1\) mL of culture medium, previously sterilized (at \(T\) = 115 \({}^{\circ}\)C; 20 min) and inoculated with 1 mL of exponential pre-culture containing 1-3 \(\times\) 10\({}^{6}\) cells (the initial cell mass concentration--\(X_{0}\) was \(\approx\)0.10 g/L) [18; 19]. The pre-culture was carried out in liquid-modified YPD medium, in which dextrose had been replaced by glycerol (the pre-culture medium contained yeast extract at 10.0 g/L, peptone at 10.0 g/L and pure glycerol at 20.0 g/L). Pre-cultures were incubated in an orbital shaker (for \(24\pm 2\) h at \(180\pm 5\) rpm, \(T\) = 28 \(\pm\) 1 \({}^{\circ}\)C) after inoculation of a loop by a freshly prepared slant of the strain. After inoculation by the pre-culture, flasks were incubated at \(T\) = \(28\pm 1\)\({}^{\circ}\)C at \(180\pm 5\) rpm in a rotary shaker (New Brunswick Innova 43 Incubator Shaker; NH, USA). In the experiments performed in shake-flask mode, a constant drop in the pH value of the medium was observed throughout the culture time, and the pH value was always maintained in the flasks at values of \(\geq\)4.5 \(\pm\) 0.1 by aseptically adding 2M NaOH (for more details see: [PERSON] et al. [19]). In most of the culture steps and for most of the shake-flask experiments performed, the medium pH remained at \(>\)4.8, being found within the range 4.8-5.8. Batch and pulsed fed-batch bioreactor experiments were also performed, in a bench-top 3 L bioreactor (Applikon Biotechnology; Delft, The Netherlands), containing 1.9 L of previously sterilized fermentation medium (at \(T\) = 115 \({}^{\circ}\)C; 45 min) and inoculated with 100 mL of 24 h exponential _Y. lipolytica_ pre-culture (see previously for the pre-culture details). Culture conditions in the bioreactor experiments were as in [PERSON] et al.