## 3 Results
### Impact of Aluminium Sulfate on Lipogenesis and Caroenoid Production
Total lipid and L-RP of the control reached 4.68 g/L and 44.1%, respectively (decreased by around 8.2% compared with Amm at C/N 146 and C/S ratio 133). After adding 0.1 mM from Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\), the lipid production was slightly enhanced to 4.74 g/L and 44.55%, respectively. However, gradually increasing Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) in the growth medium sharply reduced TL and L-RP to 2.6 g/L and 24.8%, respectively, at 0.7 mM, which is an approximately 50% decrease compared to the control, respectively. The further increasing of Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) concentrations in the growth medium kept reducing the lipid accumulation inside the cells to being scarcely produced at 8 mM (0.61 g/L and 13.2%, respectively). The statistical analysis showed the significant difference of lipid production at 0.3 mM aluminium and there was a highly significance difference with further higher concentrations of aluminium sulfate compared with the control (Figure 4b). The remarkable reduction in lipid production after adding aluminium sulfate to the medium may be related to its effect on the phospholipid pathway and increased lipid peroxidation, as reported in References [37; 38], when the effect of aluminium ions studied regarding lipid peroxidation in the root tips of soybean and on ox-brain phospholipid liposomes after inducing by iron(II) salts at acidic pH values. On the other hand, recent studies that were carried out on algae point to the role of metal stress in enhancing lipid production by algae for iron, magnesium, and calcium under dark condition [53], and copper and cadmium ions under heterotrophic culture conditions [54]. In contrast to lipids, carotenoid production was stimulated with the new optimized medium to 0.95 mg/L with cellular carotenoids around 89.04 ug/g, compared with Amm with C/N 146 and C/S 133 (increased around 1.2- and 1.1-fold, respectively). Surprisingly, the gradual adding of Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) to the growth medium led to a dramatic increase of total pigment and cellular carotenoid to 2.21 mg/L and 212.9 ug/g, respectively, at 0.7 mM, which is an increase of around 2.3- and 2.4-fold, respectively, compared with the control (Figure 4c). Aluminium sulfate showed statistical significance regarding pigment production by _R. glutinis_ at 0.3 and 4 mM, and was highly significant at the in-between studied concentrations. All the reports studying the effect of metal on carotenoid production confirmed its  
Figure 3: Microscopic examination of yeast cells stained with Nile red fluorescence dye. Yeast cells were grown in a shaken flask under different Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) concentrations using the optimized medium as a control, with C/N equal to 146 (using ammonium sulfate as a sole nitrogen source) and C/S equal to 120, at 180 rpm and at 28 \({}^{\circ}\)C for six days. (**a**) Control. The Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) concentrations were (**b**) 0.1 mM, (**c**) 0.3 mM, (**d**) 0.7 mM, and (**e**) 1 mM. The magnification bar is equal to 10 \(\mu\)m.  
stimulatory role on carotenoid production, which is explained by hypothesising a possible activation or inhibition mechanism of selected metal ions on specific carotenoid enzymes, in particular, on specific desaturase involved in carotenoid biosynthesis [14]. In contrast to lipids, carotenoid production was stimulated with the new optimized medium to 0.95 mg/L with cellular carotenoids around 89.04 ug/g, compared with Amm at C/N 146 and C/S 133 (increased around 1.2- and 1.1-fold, respectively). Surprisingly, the gradual adding of Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) to the growth medium led to a dramatic increase of total pigment and cellular carotenoid to 2.21 mg/L and 212.9 ug/g, respectively, at 0.7 mM, which was an increase of around 2.3- and 2.4-fold, respectively, compared with the control (Figure 4c). Aluminium sulfate showed statistical significance on pigment production by _R. glutinis_ at 0.3 and 4 mM, and was highly significant at the in-between studied concentrations.