## 2 Materials and Methods
### Microalgae Cultures
#### 2.3.2 Batch Fermentation Experiments  
Two batch fermentation experiments were carried out using different fermentation media in ASW with the same inoculum and the same conditions of abiotic environment. The experiments were carried out in 500 mL shake flasks (DURAN(r) GLS 80(r) Laboratory Bottle Wide Mouth) containing 400 mL of growing medium and 10% (_v/v_) inoculum of the seed culture of the microorganism. Cultures were incubated in a growth chamber at 25 \({}^{\circ}\)C for 7 days on an orbital shaker set at 120 rpm. Oxygen was sparged into the medium with compressed air at a rate of 150 L h\({}^{-1}\). During the experiment, dissolved oxygen (DO) level was maintained at 50% of saturation by regulating the oxygen supply.  
In order to determine the optimal carbon and nitrogen concentration for growth and lipid accumulation, a screening experiment (experiment 1) was carried out with increasing concentrations of crude glycerol, yeast extract and peptone in ASW. Four crude glycerol concentrations (30, 60, 90, 120 g L\({}^{-1}\)) and five nitrogen concentrations (1.1, 2.2, 3.3, 4.4, 5.5 g L\({}^{-1}\)) were tested in pair combinations. The nitrogen concentration levels derived by combining equal amounts of yeast extract (10, 20, 30, 40, 50 g L\({}^{-1}\)) and peptone (10, 20, 30, 40, 50 g L\({}^{-1}\)). The experiment was carried out with the same inoculum, micronutrients and supplementary minerals at the same conditions of the abiotic environment as described in Section 2.2. According to our findings, the optimal concentration of total nitrogen and crude glycerol was 3.3 g L\({}^{-1}\) and 120 g L\({}^{-1}\), respectively.  
Based on the findings of the screening experiment, experiment 2 was conducted at the same volume of fermentation media that contained crude glycerol (as carbon source) regulated to the optimal carbon concentration (120 g L\({}^{-1}\)) and pre-treated effluent at varying concentrations (0%, 8%, 16%, 32%, 48% _v/v_), replacing partially the relative volume of ASW. Nitrogen was also set to the optimal concentration (3.3 g L\({}^{-1}\)) derived from experiment 1, with the addition of ammonium chloride (NH\({}_{4}\)Cl) at the quantities needed considering the nitrogen levels in the pre-treated effluent and the relevant quantity of the effluent added. Phosphorus, potassium and magnesium concentrations were regulated for comparison reasons to the concentration equal to that of the experiment 1, with the addition of KH\({}_{2}\)PO\({}_{3}\) and MgSO\({}_{4}\).7H\({}_{2}\)O, while micronutrients were added only to the control treatment and not to the media containing the effluent. Thus, the composition of the control fermentation medium (0% effluent) was 120 g L\({}^{-1}\) crude glycerol, 12.5 g L\({}^{-1}\) NH\({}_{4}\)Cl, 1.5 g L\({}^{-1}\) KH\({}_{2}\)PO\({}_{3}\), 0.7 g L\({}^{-1}\) MgSO\({}_{4}\).7H\({}_{2}\)O and micronutrients. The rest of the treatments (8%, 16%, 32%, 48% _v/v_ effluent) contained 120 g L\({}^{-1}\) crude glycerol, 3.3 g L\({}^{-1}\) nitrogen, P, K, 8%, 16%, 32%, 48% _v/v_ effluent and 92%, 84%, 68% and 52% _v/v_ ASW, respectively. Furthermore, the carbon assimilation, the fatty acid content and the proximate composition of the biomass for each treatment were also determined, as described below.