## 2 Glycerol-Based Biomanufacturing Using _Yarrowia_
### Bioconversion of Glycerol to Lipids
#### 2.1.2 Pure and Crude Glycerol as Carbon Source for Lipid Production by _Yarrowia lipolytica_
The authors showed that minimization of citric acid synthesis via appropriate medium development based on nitrogen starvation and supplementation (thiamine and uracil) could increase lipid accumulation in mere batch cultivation without any genetic modification [42]. Similarly, using _Y. lipolytica_ SKY7, [PERSON] et al., searched for the optimal glycerol concentration (34.4 to 168.2 g L\({}^{-1}\)) and C/N ratio (25 to 150) to maximize lipid production. An initial glycerol concentration of 112.5 g L\({}^{-1}\), C/N molar ratio of 100, and 5% \(v/v\) inoculum supplementation were optimal for biomass and lipid production. Based on the above optimal parameters, lipid concentration of 43.8% \(w/w\) with a biomass concentration of 14.8 g L\({}^{-1}\) was achieved. The glycerol uptake also increased with the increase in glycerol concentration. At low C/N ratio, glycerol consumption was found to be high (79.43 g L\({}^{-1}\) on C/N 25) whereas consumption decreased when the C/N ratio was raised to 150 (40.8 g L\({}^{-1}\)) [63]. In _Y. lipolytica_ fermentations, the use of biodiesel-derived crude glycerol without purification was considered tedious due to the impurities contained therein which could influence biochemical pathways and limit the production [64]. Only a few published works describe its use as a sole carbon source and the initial studied products were citric acid and lipids [53; 65]. Instead of pure glycerol, [PERSON] et al., used samples without any prior purification from five different waste products; each contained various concentrations of crude glycerol (42-87%). The best results for lipid production were obtained using glycerol from fat saponification that reached 1.69 g L\({}^{-1}\) (25% \(w/w\)) with a biomass yield of 0.17 g g\({}^{-1}\) in flasks. Batch cultivation in a bioreactor resulted in enhanced lipid production of 4.72 g L\({}^{-1}\) with a biomass yield 0.21 g g\({}^{-1}\), showing that crude glycerol from soap production could be efficiently converted to SCO by _Y. lipolytica_ without any prior purification [54]. In another study that also used crude glycerol, the lipid production by _Y. lipolytica_ SKY7 was investigated with and without pH control [55]. The pH influences the cell membrane and concomitantly the carbon assimilation mechanisms. Lipid and citric acid production improved with pH control (pH 6.5) and biomass concentration between the two tested conditions were similar. In the pH-controlled experiments, the biomass and lipid concentration reached 18 and 7.78 g L\({}^{-1}\) (45.5% \(w/w\)), respectively, with a lipid yield of 0.179 g g\({}^{-1}\) after 60 h. At this time point, lipid degradation was observed in the pH-controlled reactor, whereas this phenomenon occurred after 84 h with uncontrolled pH. Apart from lipids, citric acid was also produced in both fermentations but at a lower concentration in uncontrolled pH. The authors stated that controlling the pH enhances the lipid production by 15% compared to pH-uncontrolled fermentation and favors citric acid production in late fermentation stage [55]. Interestingly, [PERSON] et al., reported that the inhibition by crude glycerol compared to pure glycerol was negligible when they optimized the concentration of wastewater sludge solids mixed with crude glycerol in fermentation media to produce lipids. Their results indicated that 20 g L\({}^{-1}\) of sludge solids with 40 g L\({}^{-1}\) of crude glycerol resulted in the highest lipid content, 29.35% \(w/w\), after 96 h [56]. Similarly, a lipid content of 35% \(w/w\) was achieved when crude glycerol was supplemented with olive oil in a study that used various surfactants and oils as inducers to enhance lipid production by _Y. lipolytica_[50]. It seems that the utilization of crude glycerol when mixed with other waste materials overcomes the negative effects exerted by the contained impurities and alleviates the scale-up of these fermentation processes [64]. Mixtures of crude glycerol with co-substrates such as saturated FFA also altered the profile of lipids produced by _Y.