## 2 Materials and Methods  
### The Microorganism, Media Preparation, and Cultivation  
The oleaginous red yeast _Rhodotorula glutinis_ (AS 2.703), obtained from the China General Microbiological Culture Collection Center (CGMCC)(Beijin China), was recultivated on Yeast extract peptone dextrose media (YPD) slants (glucose 20 g/L, peptone 10 g/L, yeast extract 10 g/L, and agar 15 g/L, pH 5) at 30 \({}^{\circ}\)C for three days, then preserved at 4 \({}^{\circ}\)C until used. For the production of lipidsand carotenoids, the described method was used with slight modification [12]. Briefly, two steps were needed: First, seed culture preparation, during which, the 2-day pre-culture yeast cells were cultivated in sterilized 250 mL flasks containing 50 mL YPD broth, then incubated on a rotatory-incubator HZQ-F100 ( Taicang, Jiangsu Province, China) at 28 \({}^{\circ}\)C and 180 rpm for 48 h. Second, the fermentation step involved preparation of the basal fermentation media, which contained (per litre) glucose (60 g), (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) (0.5 g), yeast extract (0.75 g), KH\({}_{2}\)PO\({}_{4}\) (1.5 g), and MgSO\({}_{4}\)\(\overline{\bullet}\)7H\({}_{2}\)O (1 g). This media was used during our initial experiments. For studying effect of different nitrogen sources--peptone (PEP), yeast extract (YE), and ammonium sulfate (Amm)--on lipid and carotenoid production, the nitrogen sources in the basal media were replaced with the selected nitrogen source. Also, three different C/N ratios were prepared using the different nitrogen sources as shown in Table 1. Using ammonium sulfate as a nitrogen source led to changes in both C/S and C/N ratios, so the C/S ratio was calculated and is shown in Table 1. Also, different concentrations of MgSO\({}_{4}\) were added to prepare different C/S ratios using the basal medium as a control (Table 2). The seed cells were centrifuged at 10,000\(\times\)_g_ for 15 min, then washed twice using sterilized 0.9% NaCl solution (same volume) followed by inoculation (10% _v/v_) in 250 mL flasks containing 100 mL of fermentation media. Finally, the cells were incubated under the same condition for six days. Fifty milliliters was withdrawn every three days under the complete aseptic condition and used for dry cell weight, lipid, and carotenoid measurements. All experiments were carried out in three replicas; the mean and the standard deviation were calculated. Effect of Different Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) Concentration on Growth, and Lipid and Carotenoid Production  
After establishing the best conditions for carotenoid enhancement production, a new fermentation medium was prepared (optimized media), and a different amount of Al\({}_{2}\)(SO\({}_{4}\))\({}_{3}\) was added after sterilization to prepare the following final concentrations: 0.1 mM, 0.3 mM, 0.5 mM, 0.7 mM, 1 mM, 2 mM, 4 mM, 6 mM, 8 mM, and 10 mM, and then were treated with the same above-described method. \begin{table}
\begin{tabular}{c c c c c} \hline
**C/N** & **N-Sources** & **g/L** & **C/S** & **C/S –C/N** \\ \hline \multirow{4}{*}{146} & \({}^{*}\) PEP & 1.5 & 240 & 94 \\  & \({}^{*}\) YE & 1.7 & 240 & 94 \\  & (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) & 0.9 & 133 & \(-\)13 \\ \hline \multirow{4}{*}{94} & PEP & 2.4 & 240 & 146 \\  & YE & 2.7 & 240 & 146 \\  & (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) & 1.4 & 116 & 22 \\ \hline \multirow{4}{*}{70} & PEP & 3.2 & 240 & 170 \\  & YE & 3.6 & 240 & 170 \\ \cline{1-1}  & (NH\({}_{4}\))\({}_{2}\)SO\({}_{4}\) & 1.9 & 89 & 19 \\ \hline \end{tabular}  
* \({}^{*}\) PEP: peptone.\({}^{*}\) YE: yeast extract.  
\end{table} 
Table 1: Different nitrogen sources and its calculated carbon to nitrogen ratio (C/N) and carbon to sulfur ratio (C/S). \begin{table}
\begin{tabular}{c c c c} \hline
**C/N** & **MgSO\({}_{4}\) (g/L)** & **C/S** & **C/S –C/N** \\ \hline \multirow{4}{*}{146} & 0.5 & 245 & 108 \\  & 1 & 166 & 20 \\  & 1.5 & 123 & \(-\)23 \\  & 2 & 98 & \(-\)48 \\ \hline \end{tabular}  
\end{table}