## 3 Results
### Impact of Aluminium Sulfate on Lipogenesis and Caroenoid Production  
Herein, a new optimized medium was prepared to maximize the carotenoid production under a high C/N ratio, and its effect on the growth and lipid production was also investigated. The optimum conditions can be summarised as follows: using Amm as a sole nitrogen source, keeping the C/N ratio at 149, and adjust the C/S ratio with MgSO\({}_{4}\) to 120. By using this medium as a control, different Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) concentrations were added after medium sterilization then its effects on growth, lipid, and carotenoid production by _R. glutinis_ were studied. First, the Nile red treated cells were examined under fluorescence microscopy, as shown in Figure 3. In the optimized media (control group), the size of the cell was reduced, and the number of golden fluorescent lipid bodies was also reduced compared with the same C/N ratio using Amm as a nitrogen source (C/S ratio 133); this indicated that reducing the C/S ratio led to a slight decrease in lipid production. When adding 0.1 mM Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\), no significant difference was observed between this treatment and the control one. The lipid bodies were obviously decreased, starting from 0.3 mM while the cells remained enlarged. With a higher concentration of Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\), the lipid bodies were greatly reduced to be scarcely observed at 1 mM. \begin{table}
\begin{tabular}{l c c c c c c c c c c c c c} \hline  & **YE-446** & **YE-94** & **YE-70** & **PIF-146** & **PIF-94** & **PIF-70** & **Amin-146** & **Amin-94** & **Amin-70** & **C/S-254** & **C/S-166** & **C/S-123** & **C/S-96** \\ \hline Toroh & 33.3 \(\pm\) 3.3 & 67.2 \(\pm\) 2.8 & 53.4 \(\pm\) 9 & 62.2 \(\pm\) 2.1 & 12.05 \(\pm\) 3.96 & 36.6 \(\pm\) 1.9 & 10.5 \(\pm\) 0.49 & 17.6 \(\pm\) 6.8 & 0.78 \(\pm\) 5 & 0 & 0 & 0 & 0 & 0 \\ Torun & 52.5 \(\pm\) 6.1 & 52.4 \(\pm\) 3.0 & 37.8 \(\pm\) 10 & 57.3 \(\pm\) 7.4 & 58.4 \(\pm\) 9.7 & 50.5 \(\pm\) 3.5 & 62.8 \(\pm\) 8.5 & 61.1 \(\pm\) 5.4 & 57.4 \(\pm\) 6.9 & 50.1 \(\pm\) 0.71 & 53.4 \(\pm\) 5.9 & 54.7 \(\pm\) 3.8 & 54.8 \(\pm\) 1 \\ \(\gamma\)-car & 44.2 \(\pm\) 4.2 & 38.6 \(\pm\) 2.5 & 52.3 \(\pm\) 8.8 & 33.6 \(\pm\) 3.6 & 28.3 \(\pm\) 3.6 & 44.4 \(\pm\) 5.6 & 11.1 \(\pm\) 4.4 & 24 \(\pm\) 10 & 32.1 \(\pm\) 3.1 & 21.4 \(\pm\) 2.3 & 33.6 \(\pm\) 4.5 & 40.3 \(\pm\) 4.3 & 41.5 \(\pm\) 3.2 \\ \(\beta\)-car & 0 & 0 & 0 & 0 & 0 & 0 & 0 & 5.4 \(\pm\) 4.8 & 7.6 \(\pm\) 2.9 & 8.9 \(\pm\) 4.1 & 12.2 \(\pm\) 4.2 & 0 & 0 & 0 & 0 \\ \hline \end{tabular}  
* Toroh: torularhodene. Toru: torulene. \(\gamma\)-car: \(\gamma\)-carotene. \(\beta\)- car: \(\beta\)-carotene. Yeast cells were grown under different nitrogen sources—peptoine (PEP), yeast extract (YE), and ammonium sulfate (Amm)—with different C/N ratios (146, 94, and 7), and different C/S ratios (254, 166, 123, and 98) with the same C/N ratio (146). HPLC was used for separation and identification. The data are the mean of two replicate tests, and the standard deviation was calculated and represented as (\(\pm\)SD).  
\end{table} 
Table 5: The percentage of individual carotenoids, \(\gamma\)-carotene, \(\beta\)-carotene, torulene, and torularhodene in the carotenoid profile extracted from _Rhodotorula glutinis_. The effect of different Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) on DCW, TL, and TP production by _R. glutinis_ after six days is described in Figure 4. First, the growth in the control was around 10.6 g/L and remained almost steady above 10 g/L till 0.7 mM, after which, the growth gradually decreased with increasing Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) concentrations up to 10 mM, at which no growth was observed, and the effect of aluminium was statistically significant at 4 mM and highly significant at 6 and 8 mM compared with the control (Figure 4a). Contrary to the studied yeast strain, another study carried out on _Rhodotorula taivanensis_ RS1 found that RS1 is a high-aluminium (Al)-tolerant yeast that can survive in Al concentrations up to 200 mM [52].