## 3 Results  
### The Optimization of the Fermentation Culture Conditions  
Under the primary fermentation conditions, the Se content and biomass of the Se-enriched DB-yeast were 6.91 mg/L and 7.1 g/L, respectively. A single-factor experiment and \(L_{9}\)(\({}^{4}\))-orthogonal test for optimization of fermentation conditions were used to improve the Se-enriched capacity of the DB-yeast. Single-factor experiments for optimization of the fermentation conditions are shown in Figures 1 and 2. As shown in Figure 1A, both the Se content and biomass of the Se-enriched DB-yeast firstly increased and then decreased as the temperature increased from 24 to 32 \({}^{\circ}\)C. The optimal temperature was 28 \({}^{\circ}\)C. As shown in Figure 1B, as the initial pH increased (from 3.0 to 7.0), the biomass of the Se-enriched DB-yeast continuously increased; however, the Se content of the Se-enrichedDB-yeast firstly increased and then decreased as initial pH increased. These data indicate that a slightly acidic environment is good for industrial discarded _S. cerevisiae_ growth and Se fermentation accumulation. The optimal initial pH was 4.0 under overall consideration. As shown in Figure 1C, the biomass of the Se-enriched DB-yeast slightly decreased but, overall, it was maintained at a high level over a culture time of 24 to 32 h. The Se content of the Se-enriched DB-yeast firstly increased and then decreased as the cultured time increased. The optimal cultured time was 30 h. As shown in Figure 1D, the biomass of the Se-enriched DB-yeast increased as the inoculation volume increased from 4.0% to 15.0%. The Se content of the Se-enriched DB-yeast firstly increased and then decreased, and the optimal inoculation volume was 12.0%. As shown in Figure 2A, the Se content of the Se-enriched DB-yeast obviously increased as the concentration of added Se increased from 10 to 50 mg/mL. However, the biomass of the Se-enriched DB-yeast showed the opposite trend, which was probably related to the cytotoxicity of inorganic Se as its concentration increased. The optimal Se adding concentration was 30 mg/mL under overall consideration. As shown in Figure 2B, the biomass of the Se-enriched DB-yeast increased as the time of Se addition increased. The Se content of the Se-enriched DB-yeast firstly increased and then decreased, and the optimal Se addition time was 9 h.  
To further improve the fermentation conditions, the initial pH (3.0, 4.0, and 5.0), inoculation volume (9.0%, 12.0%, and 15.0%), added Se (10, 20, and 30 mg/mL), and Se addition time (6, 9, and 12 h) were used for the \(L_{9}\)(3\({}^{4}\))-orthogonal test. The parameter design, extreme difference analysis, and variance analysis results are shown in Tables S1-S3 (Supplementary Materials). The computed _F_-value of Se added (A) was much greater than that of the tabular _F0.01_ value, suggesting that it has an extremely significant effect on the Se content of the Se-enriched DB-yeast. In addition, the computed _F_-value of the Se addition time (D) was between the tabular _F0.01_ and _F0.05_ value, suggesting that it has a significant effect on the Se content of the Se-enriched DB-yeast. By contrast, the effects of pH (B) and inoculation volume (C) on the Se content of the Se-enriched DB-yeast were not significant. Figure 1: Effects of (**A**) temperature, (**B**) pH, (**C**) culture time, and (**D**) inoculation volume on Se content and biomass of Se-enriched discarded beer (DB)-yeast fermentation. Hence, the effect sizes of the significant variables were as follows: added Se (A) \(>\) Se addition time (D) \(>\) inoculation volume (C) \(>\) pH (B). The combination of the optimization conditions was A\({}_{3}\)B\({}_{1}\)C\({}_{3}\)D\({}_{2}\).