Optimization of four parameters, i.e., zinc (Zn²⁺), magnesium (Mg²⁺), manganese (Mn²⁺) and yeast extract for bioethanol production from sweet sorghum juice by Saccharomyces cerevisiae NP 01 under very high gravity (VHG, 270 g·L⁻¹ of total sugar) conditions was performed using an L₉ (3⁴) orthogonal array design. The fermentation was carried out at 30 °C in 500-mL air-locked Erlenmeyer flasks at the agitation rate of 100 rpm and the initial yeast cell concentration in the juice was approximately 5 × 10⁷ cells·mL⁻¹. The results showed that the order of influence was yeast extract > Mn²⁺ > Zn²⁺ > Mg²⁺ and the optimum nutrient concentrations for the ethanol fermentation were Zn²⁺, 0.01; Mg²⁺, 0.05; Mn²⁺, 0.04; and yeast extract, 9 g·L⁻¹. The verification experiments under the optimum condition clearly indicated that the metals and nitrogen supplementation improved ethanol production efficiency under the VHG fermentation conditions. The ethanol concentration (P), yield (Y_p/s) and productivity (Q_p) were 120.58 ± 0.26 g·L⁻¹, 0.49 ± 0.01 and 2.51 ± 0.01 g·L⁻¹·h⁻¹, respectively, while in the control treatment (without nutrient supplement) P, Y_p/s and Q_p were only 93.45 ± 0.45 g·L⁻¹, 0.49 ± 0.00 and 1.30 ± 0.01 g·L⁻¹·h⁻¹, respectively.

Optimization of three parameters: agitation rate (A; 100, 200 and 300 rpm), aeration rate (B; 0.5, 1.5 and 2.5 vvm) and aeration timing (C; 2, 4 and 6 h), for ethanol production from sweet sorghum juice under very high gravity (VHG, 290 g L⁻¹ of total sugar) conditions by Saccharomyces cerevisiae NP 01 was attempted using an L₉ (3⁴) orthogonal array design. The fermentation was carried out at 30 °C in a 2-L bioreactor and the initial yeast cell concentration was approximately 2 × 10⁷ cells mL⁻¹. The results showed that the optimum condition for ethanol fermentation should be A₂B₃C₂ corresponding to agitation rate, 200 rpm; aeration rate, 2.5 vvm and aeration timing, 4 h. The verification experiments under the optimum condition clearly indicated that the aeration and agitation strategies improved ethanol production. The ethanol concentration (P), productivity (Q_p) and ethanol yield (Y_p/s) were 132.82 ± 1.06 g L⁻¹, 2.55 ± 0.00 g L⁻¹h⁻¹ and 0.50 ± 0.00, respectively. Under the same condition without aeration (agitation rate at 200 rpm), P and Q_p were only 118.02 ± 1.19 g L⁻¹ and 2.19 ± 0.04 g L⁻¹h⁻¹, respectively while Y_p/s was not different from that under the optimum condition.

Sweet sorghum stalks were used as a low cost carrier for immobilization of Saccharomyces cerevisiae NP 01 to produce ethanol from sweet sorghum juice. The effects on ethanol production of carrier size (6 × 6 × 6 to 20 × 20 × 20 mm³) and initial cell concentrations (5 × 10⁷ to 2 × 10⁸ cells mL⁻¹) for cell immobilization were investigated. The ethanol production medium was the juice containing 230 g L⁻¹ of total sugar without nutrient supplementation. The fermentations were carried out under static conditions in 500-mL air-locked Erlenmeyer flasks at 30 °C. The results showed that the optimum size of sorghum stalk pieces for repeated-batch ethanol production was 6 × 6 × 6 mm³, while the optimum initial cell concentration for the immobilization was 1.0 × 10⁸ cells mL⁻¹. The immobilized yeast under these conditions could be used for at least eight successive batches without any losses of ethanol production efficiencies. The average ethanol concentration, productivity and yield of the eight successive batches were 99.28 ± 3.53 g L⁻¹, 1.36 ± 0.05 g L⁻¹ h⁻¹ and 0.47 ± 0.03 g g⁻¹, respectively.

Optimization of nutrient supplements i.e., yeast extract (1, 3 and 5 g·L⁻¹), dried spent yeast (DSY: 4, 12 and 20 g·L⁻¹) and osmoprotectant (glycine: 1, 3 and 5 g·L⁻¹) to improve the efficiency of ethanol production from a synthetic medium under very high gravity (VHG) fermentation by Saccharomyces cerevisiae NP 01 was performed using a statistical method, an L₉ (3⁴) orthogonal array design. The synthetic medium contained 280 g·L⁻¹ of sucrose as a sole carbon source. When the fermentation was carried out at 30 °C, the ethanol concentration (P), yield (Y_p/s) and productivity (Q_p) without supplementation were 95.3 g·L⁻¹, 0.49 g·g⁻¹ and 1.70 g·L⁻¹·h⁻¹, respectively. According to the orthogonal results, the order of influence on the P and Q_p values were yeast extract > glycine > DSY, and the optimum nutrient concentrations were yeast extract, 3; DSY, 4 and glycine, 5 g·L⁻¹, respectively. The verification experiment using these parameters found that the P, Y_p/s and Q_p values were 119.9 g·L⁻¹, 0.49 g g⁻¹ and 2.14 g·L⁻¹·h⁻¹, respectively. These values were not different from those of the synthetic medium supplemented with 9 g·L⁻¹ of yeast extract, indicating that DSY could be used to replace some amount of yeast extract. When sweet sorghum juice cv. KKU40 containing 280 g·L⁻¹ of total sugar supplemented with the three nutrients at the optimum concentrations was used as the ethanol production medium, the P value (120.0 g·L⁻¹) was not changed, but the Q_p value was increased to 2.50 g·L⁻¹·h⁻¹.