Improvement of Bioethanol Production from Sweet Sorghum Juice under Very High Gravity Fermentation: Effect of Nitrogen, Osmoprotectant, and Aeration

To improve ethanol production fermentation efficiency from sweet sorghum juice under a very high gravity (VHG, 280 g/L of total sugar) condition by Saccharomyces cerevisiae NP01, dried spent yeast (DSY), yeast extract, and glycine concentrations were optimized using an L9 (34) orthogonal array design. The results showed that the order of influence on the ethanol concentration (PE) was yeast extract > glycine > DSY. The optimal nutrient concentrations for ethanol production were determined as follows: yeast extract, 3; DSY, 4; and glycine, 5 g/L. When a verification experiment under the projected optimal conditions was done, the P, ethanol yield (Yp/s), and ethanol productivity (Qp) values were 120.1 g/L, 0.47, and 2.50 g/L·h, respectively. These values were similar to those of the positive control experiment with yeast extract supplementation at 9 g/L. The yeast viability under the optimal condition was higher than that of the control experiment. To improve sugar utilization and ethanol production, aeration at 2.5 vvm for 4 h was applied under the optimal nutrient supplementation. The P, Yp/s, and Qp values were significantly increased to 134.3 g/L, 0.50, and 2.80 g/L·h, respectively.


Introduction
Bioethanol is an alternative fuel that has been of much interest for most of the past 50 years [1].Ethanol can be blended with gasoline or can be used alone in dedicated engines.Additionally, it can be used in advanced flexi-fuel hybrid engines [2].Thus, a strong need exists for efficient ethanol production using low-cost raw materials and production processes.Ethanol can be produced from numerous biomass feedstocks.In addition to the main raw materials, i.e., sugarcane, corn grain, tapioca starch, and sugarcane molasses, sweet sorghum (Sorghum bicolor (L.) Moench) juice is a promising alternative feedstock for bioethanol production.Its stalks contain high levels of fermentable sugars.This hardy crop can be cultivated in nearly all regions of the world, including tropical areas [3].
The very high gravity (VHG) fermentation process has been introduced as a technology for ethanol production to improve its cost-effectiveness.This process involves the preparation of a mash and its fermentation to completion.The mash contains at least 270 g/L of dissolved solids [4].Normally, ethanol fermentations at high sugar levels or under VHG conditions are sluggish, and sugar is rarely completely fermented.This may be due to an increase in the osmotic pressure and fermentation time, which has an adverse effect on microbial cells.It has been reported that Saccharomyces cerevisiae, can better utilize sugars in broth culture, as well as tolerate higher ethanol concentrations with adequate nutrient supplementation [4][5][6][7].VHG fermentations use a yeast strain that can withstand greater osmotic stresses and higher ethanol concentrations.Therefore, supplementation of the media with an osmoprotectant can improve the prospects for cell survival.It was reported that glycine (a poor nitrogen source) might serve as an osmoprotectant.It indeed has a high stimulatory effect on yeast growth and fermentation [8].Additionally, sugar consumption increased when glycine was added to a VHG ethanolic fermentation employing S. cerevisiae NCYC 1324.
Laopaiboon et al. [4] and Nuanpeng et al. [9] showed that sweet sorghum juice strain KKU 40 was suitable raw material for ethanol production.VHG fermentation from sweet sorghum juice required only nitrogen supplementation to improve ethanol production.Nitrogen sources, such as yeast extract, are widely used at laboratory scale to promote yeast growth, ethanol yield, and fermentation efficiency for VHG fermentation [4,[9][10][11].Gomez-Flores et al. [12] reported that yeast extract could increase sugar consumption because it consisted of adenine, lactose, and trehalose, that improve protein synthesis and cell growth [13].However, yeast extract is relatively expensive and thus are not suitable for large scale fermentations.Suwanapong et al. [14] showed that dried spent yeast (DSY) is a suitable nitrogen source for VHG ethanol fermentations from sweet sorghum juice.However, in fermentation with DSY supplementation (21 g/L), the resulting ethanol yield and productivity were 7% and 27% lower than when 9 g/L of yeast extract was added to the medium.Therefore, incorporating DSY with a small amount of yeast extract in the presence of an osmoprotectant should result in equivalent ethanol yield and substrate utilization to a fermentation supplemented with yeast extract at a level of 9 g/L under VHG conditions.
In this research, an orthogonal array design (OAD) is applied to study the interactions of parameters that play fundamental roles in these types of fermentations [15,16].The number of test conditions required can be sizably reduced using OAD.The results of the OAD produce an optimal set of conditions, and it arranges the various factors in the order of their influence for effective optimization of experimental conditions [17,18].
Numerous research studies reported the optimum aeration rate during fermentation to promote yeast growth and ethanol production.Arshad et al. [19] indicated that aeration (0.2-0.6 vvm) improved viable cell growth.However, the final ethanol concentration (P E ) decreased at higher aeration rates.Jayus et al. [20] found that an aeration rate of 0.3 vvm for 4 h increased the number of yeast cells and the P E values simultaneously increased from 102.9 g/L to 120.9 g/L compared with an unaerated culture.Khongsay et al. [7] found that an aeration rate of 2.5 vvm for 4 h during VHG fermentation from sweet sorghum juice increased the yeast cell population and the P E value, as well as reduced the fermentation time.
In the present study, the main factors affecting a VHG ethanol fermentation from sweet sorghum juice by S. cerevisiae NP01 were investigated.First, the yeast extract, DSY, and glycine levels in the sweet sorghum juice were optimized using L 9 (3 4 ) OAD.Then, verification experiments with these parameters at the optimal conditions were done.Finally, an aeration rate of 2.5 vvm, aeration time of 4 h, and an agitation rate of 200 rpm [7] were used to improve ethanol production in a batch VHG fermentation under the optimal conditions.

Microorganism and Inoculum Preparation
S. cerevisiae NP01 was inoculated into yeast extract malt extract (YM) medium and incubated at 30 • C with an agitation rate of 150 rpm for 15 h [21].Then, the yeast was transferred into a medium containing 360 mL of sweet sorghum juice (total sugar concentration of 150 g/L) and incubated under the same conditions for 12 h.

Raw Materials and Nutrient Supplements
Sweet sorghum juice (cv.KKU 40) extracted from the plant stalks was obtained from the Faculty of Agriculture, Khon Kean University, Thailand.The juice contained total soluble solids of 17 • Bx.Due to storage problems and the need to prevent bacterial contamination, the sweet sorghum juice was concentrated to a high sugar concentration of 68 • Bx and stored at 4 • C for further experiments.The compositions of the sweet sorghum juice were analyzed as previously reported [22].It contained 160.56 g/L of total sugar, which consisted of sucrose, 112.39; glucose, 24.08, and fructose, 22.48 g/L.
DSY was donated by the Beerthip Brewery (1991) Co., Ltd., Bang Baan, Phra Nakhon Sri Ayutthaya, Thailand.The protein content of this DSY was 50.12% dry weight [21].The yeast extract used as a nitrogen source (protein content, 74.50% DCW) was purchased from HiMedia Laboratories Pvt. Ltd., Mumbai, India [21], and the glycine used as an osmoprotectant was purchased from BDH, England.

Ethanol Production Medium
An ethanol production (EP) medium was made from the concentrated sweet sorghum juice in Section 2.2 by diluting with distilled water to obtain 280 g/L of total sugar.Yeast extract, DSY, and glycine were added into the EP medium, as shown in Table 1.The medium was transferred into a 500 mL air-locked Erlenmeyer flask (working volume of 400 mL) before sterilization by autoclave [4].

Orthogonal Experiment Design
To investigate the influence of yeast extract (A), DSY (B), and glycine (C) levels on ethanol fermentation, the L 9 (3 4 ) orthogonal design in Table 1 was used.Three levels of each parameter, A (1, 3, and 5 g/L), B (4, 12, and 20 g/L), and C (1, 3, and 5 g/L), were set.The effects of each parameter on the characteristic properties were estimated using ANOVA [23,24].

Fermentation Conditions
Batch ethanol fermentations under various conditions were performed in 500-mL air-locked flasks.The initial yeast cell concentration in the sterile EP medium was 5 × 10 7 cells/mL [25].Temperature and agitation rate were controlled at 30 • C and 100 rpm, respectively.Samples were collected for analysis at regular time intervals.Additional experiments under optimal conditions were done to validate the results of the orthogonal tests.These fermentations were done in a 2-L bioreactor (Biostat ® B, B. Braun Biotech, Germany) with a final working volume of 1 L. It was autoclaved at 110 • C for 40 min [4].
Additionally, EP media containing 9 g/L yeast extract and without nutrient supplementation were used for ethanol fermentation as positive and negative control treatments, respectively.

Effect of Aeration on Ethanol Production
Ethanol fermentation under optimal conditions (Section 2.4) was done as previously described (Section 2.5).The agitation rate, aeration rate, and aeration time were 200 rpm, 2.5 vvm, and 4 h, respectively [7].Samples were taken at regular time intervals for chemical analyses.

Analytical Methods
The number of viable yeast cells was determined using a hemacytometer with methylene blue staining [26], and level of total sugar in the fermentation broth was determined in terms of total carbohydrates using a phenol sulfuric acid method [27].Ethanol (P E , g/L) and glycerol concentrations (P G , g/L) were analyzed using gas chromatography and high performance liquid chromatography (HPLC), respectively [4,28].The ethanol yield (Y p/s ) and volumetric ethanol productivity (Q p , g/L•h) were determined, according to Laopaiboon et al. [29].Fermentable nitrogen was measured by formol titration (modified from Zoecklein et al., [26]).

The Results of Orthogonal Experiments
Figure 1 shows the main parameter changes during ethanol fermentation of Run 1 (Table 1).No lag phase was observed, indicating that the yeast cells were very active.Total sugar concentration decreased from 273.6 to 38.0 g/L after 56 h, whereas viable cells increased from 4.1 × 10 7 to 3.1 × 10 8 cells/mL in 12 h and slightly decreased after 36 h to 2.1 × 10 8 cells/mL at 72 h.The lower cell concentrations might have been due to high ethanol concentrations.This is in agreement with Bai et al. [30], who reported that during ethanol fermentation, yeast cells were negatively impacted by high ethanol concentrations and its corresponding inhibition of yeast cell growth, particularly under VHG conditions.

The Results of Orthogonal Experiments
Figure 1 shows the main parameter changes during ethanol fermentation of Run 1 (Table 1).No lag phase was observed, indicating that the yeast cells were very active.Total sugar concentration decreased from 273.6 to 38.0 g/L after 56 h, whereas viable cells increased from 4.1 × 10 7 to 3.1 × 10 8 cells/mL in 12 h and slightly decreased after 36 h to 2.1 × 10 8 cells/mL at 72 h.The lower cell concentrations might have been due to high ethanol concentrations.This is in agreement with Bai et al. [30], who reported that during ethanol fermentation, yeast cells were negatively impacted by high ethanol concentrations and its corresponding inhibition of yeast cell growth, particularly under VHG conditions.
The pH of the broth slightly decreased from 4.7 to 4.4 over the first 12 h.The ethanol concentration (PE) significantly increased to 107.5 g/L at 48 h, while the ethanol volumetric productivity (Qp) and ethanol yield (Yp/s) were 2.24 g/L•h and 0.46 g/g, respectively.The main parameter changes during the ethanol fermentation of Runs 2 to 9 were similar to those of Run 1 (data not shown).The PE, Qp, and Yp/s values of Runs 1 to 9 are shown in Table 1.= total sugar and = ethanol).
The pH of the broth slightly decreased from 4.7 to 4.4 over the first 12 h.The ethanol concentration (P E ) significantly increased to 107.5 g/L at 48 h, while the ethanol volumetric productivity (Q p ) and ethanol yield (Y p/s ) were 2.24 g/L•h and 0.46 g/g, respectively.The main parameter changes during the ethanol fermentation of Runs 2 to 9 were similar to those of Run 1 (data not shown).The P E , Q p , and Y p/s values of Runs 1 to 9 are shown in Table 1.

Impact of Multi-Factors on Ethanol Concentration
The P E values of Runs 1 to 9 range from 105.5 to 119.8 g/L (Table 1).Range analysis was used to elucidate the importance of factors (yeast extract, factor A; DSY, factor B; and glycine, factor C) in the orthogonal experimental runs (Table 2).A larger range (R) value of a factor represents a greater effect on the final P E value.The highest (7.74) and lowest range values (1.67) were found on factors A and B, respectively.This indicates that yeast extract had a greater influence on ethanol production than glycine and DSY, respectively.The optimal concentrations of yeast extract, DSY, and glycine for improving the P E value were 3, 4 and 5 g/L, respectively (A 2 B 1 C 3 ).ANOVA was applied to determine the order of influence of the three parameters under study on the final P level.An F-value of 178.95 implies model significance, as there is only a 4.66% likelihood that these values could arise due to noise.The prob (F < 0.05) value indicates significance.According to the data, the order of influence (F yeast extract = 364.03,F glycine = 108.64,and F DSY = 64.18) is the same as the magnitude of the R values.The correlation between the predicted values and experimental data resulted in an R 2 value of 0.998 (99.8%), confirming an excellent fit [31].
Figure 2 shows the P E values using these three factors at various levels ranging from 107.5 (k 1 ) to 115.3 (k 2 ) g/L as the yeast extract increased from 1 to 5 g/L.A maximal P E value, 115.3 g/L (k 2 ), was obtained using 3 g/L of yeast extract.A higher yeast extract concentration, 5 g/L, did not improve ethanol production owing to limitations of yeast metabolism.For the DSY concentrations tested, S. cerevisiae NP01 showed the highest P E value, 111.6 g/L (k 1 ), at a concentration of 4 g/L.The ethanol concentration did not increase with increasing levels of DSY in the medium.The results using DSY are similar to those of yeast extract, indicating a limitation in the yeast metabolism.Bafrncová et al. [6] showed that excess assimilable nitrogen did not result in increased rates of ethanol production or reduce fermentation time.Additionally, a maximal P E value, 113.1 g/L (k 3 ), was obtained at a glycine concentration of 5 g/L.Thomas et al. [8] showed that the stimulatory effect of glycine on yeast growth increased with concentrations up to 40 mM or 0.53 mg/L under VHG condition using S. cerevisiae (uvarum) NCYC 1324.However, they reported that further increases in the glycine concentration did not improve fermentation or growth.Thomas et al. [8] reported that under VHG fermentation, glycine was not used as a nitrogen source for yeast growth, but rather, it acted as an osmoprotectant.

The verification experiments
From the results of the multi-factors on the PE values, the optimal condition for improving PE values from fermentation of sweet sorghum juice by S. cerevisiae NP01 under VHG conditions was determined as A2B1C3, or yeast extract at 3 g/L, DSY at 4 g/L, and glycine at 5 g/L.The model was used to predict the results of experiments in a 2-L bioreactor under optimal conditions.The results of these verification experiments are shown in Figure 3.A maximal PE value, 120.0 g/L, was obtained at 48 h.At this time, the viable cell numbers were 3.0 × 10 8 cells/mL, and 18.6 g/L of total sugar remained.The calculated Qp and Yp/s values were 2.50 g/L•h and 0.47 g/g, respectively.The results of PE, Qp, and Yp/s in the 2-L bioreactor were similar to those in the flask culture under optimal conditions (Run 4, Table 1), indicating that the fermentations are comparable.

Impact of Multi-Factors on Ethanol Concentration
The PE values of Runs 1 to 9 range from 105.5 to 119.8 g/L (Table 1).Range analysis was us elucidate the importance of factors (yeast extract, factor A; DSY, factor B; and glycine, factor C) i orthogonal experimental runs (Table 2).A larger range (R) value of a factor represents a greater on the final PE value.The highest (7.74) and lowest range values (1.67) were found on factors A B, respectively.This indicates that yeast extract had a greater influence on ethanol production glycine and DSY, respectively.The optimal concentrations of yeast extract, DSY, and glycin improving the PE value were 3, 4 and 5 g/L, respectively (A2B1C3).ANOVA was applied to deter the order of influence of the three parameters under study on the final P level.An F-value of 1 implies model significance, as there is only a 4.66% likelihood that these values could arise d noise.The prob (F < 0.05) value indicates significance.According to the data, the order of influ (Fyeast extract = 364.03,Fglycine = 108.64,and FDSY = 64.18) is the same as the magnitude of the R values correlation between the predicted values and experimental data resulted in an R 2 value of (99.8%), confirming an excellent fit [31].= glycine, DSY = dried spent yeast).

The Verification Experiments
From the results of the multi-factors on the P E values, the optimal condition for improving P E values from fermentation of sweet sorghum juice by S. cerevisiae NP01 under VHG conditions was determined as A 2 B 1 C 3 , or yeast extract at 3 g/L, DSY at 4 g/L, and glycine at 5 g/L.The model was used to predict the results of experiments in a 2-L bioreactor under optimal conditions.The results of these verification experiments are shown in Figure 3.A maximal P E value, 120.0 g/L, was obtained at 48 h.At this time, the viable cell numbers were 3.0 × 10 8 cells/mL, and 18.6 g/L of total sugar remained.The calculated Q p and Y p/s values were 2.50 g/L•h and 0.47 g/g, respectively.The results of P E , Q p , and Y p/s in the 2-L bioreactor were similar to those in the flask culture under optimal conditions (Run 4, Table 1), indicating that the fermentations are comparable.

The verification experiments
From the results of the multi-factors on the PE values, the optimal condition for improving PE values from fermentation of sweet sorghum juice by S. cerevisiae NP01 under VHG conditions was determined as A2B1C3, or yeast extract at 3 g/L, DSY at 4 g/L, and glycine at 5 g/L.The model was used to predict the results of experiments in a 2-L bioreactor under optimal conditions.The results of these verification experiments are shown in Figure 3.A maximal PE value, 120.0 g/L, was obtained at 48 h.At this time, the viable cell numbers were 3.0 × 10 8 cells/mL, and 18.6 g/L of total sugar remained.The calculated Qp and Yp/s values were 2.50 g/L•h and 0.47 g/g, respectively.The results of PE, Qp, and Yp/s in the 2-L bioreactor were similar to those in the flask culture under optimal conditions (Run 4, Table 1), indicating that the fermentations are comparable.

Comparison of Ethanol Fermentation from Sweet Sorghum Juice with and without Nutrient Supplementation
The control treatments under the same condition without nutrient supplementation (negative control experiment) and with 9 g/L of yeast extract (positive control experiment) were also determined in a 2-L bioreactor (Figure 4).Table 3 summarizes the important parameters of the batch ethanol fermentation from sweet sorghum juice under no supplementation, 9 g/L of yeast extract, and the optimal nutrient conditions.The P E and Y p/s values under the optimal conditions were similar to those with yeast extract supplementation, but the fermentation time of the former was shorter than the latter, resulting in a higher Q p under the optimal conditions.However, these values were significantly higher than those under no nutrient supplementation.The percentage of sugar consumed under the optimal conditions and yeast extract supplementation were also similar.They were approximately 25% to 27% higher than with no supplementation.These results indicate that supplementation with a combination yeast extract and DSY (as a nitrogen source) and glycine (as an osmoprotectant) significantly promoted sugar consumption and ethanol production efficiency (P E and Q p ) from the sweet sorghum juice under VHG fermentation.This might have resulted from better yeast cell growth, cell viability, and/or adequate nutrients under optimal supplementation (Figure 4).These results show that the optimal condition, A 2 B 1 C 3 , is best for ethanol The Y p/s values under all conditions tested did not markedly differ, implying that the metabolic pathway of ethanol conversion by S. cerevisiae NP01 was the same.The Y p/s values ranged from 0.47 to 0.49.This implied that by-products, such as glycerol, were produced under these conditions.By-product determination showed a lower glycerol concentration under optimal conditions.The yeast viability under all conditions tested gradually diminished after 36 h (Figure 4), presumably due to ethanol accumulation in the broth [32].However, the yeast viability under optimal conditions was higher than with yeast extract and without nutrient supplementation.These results might be due to the presence of glycine and its role as an osmoprotectant [8].

Comparison of Ethanol Fermentation from Sweet Sorghum Juice with and without Nutrient Supplementation
The control treatments under the same condition without nutrient supplementation (negative control experiment) and with 9 g/L of yeast extract (positive control experiment) were also determined in a 2-L bioreactor (Figure 4).Table 3 summarizes the important parameters of the batch ethanol fermentation from sweet sorghum juice under no supplementation, 9 g/L of yeast extract, and the optimal nutrient conditions.The PE and Yp/s values under the optimal conditions were similar to those with yeast extract supplementation, but the fermentation time of the former was shorter than the latter, resulting in a higher Qp under the optimal conditions.However, these values were significantly higher than those under no nutrient supplementation.The percentage of sugar consumed under the optimal conditions and yeast extract supplementation were also similar.They were approximately 25% to 27% higher than with no supplementation.These results indicate that supplementation with a combination yeast extract and DSY (as a nitrogen source) and glycine (as an osmoprotectant) significantly promoted sugar consumption and ethanol production efficiency (PE and Qp) from the sweet sorghum juice under VHG fermentation.This might have resulted from better yeast cell growth, cell viability, and/or adequate nutrients under optimal supplementation (Figure 4).These results show that the optimal condition, A2B1C3, is best for ethanol fermentation.The Yp/s values under all conditions tested did not markedly differ, implying that the metabolic pathway of ethanol conversion by S. cerevisiae NP01 was the same.The Yp/s values ranged from 0.47 to 0.49.This implied that by-products, such as glycerol, were produced under these conditions.By-product determination showed a lower glycerol concentration under optimal conditions.The yeast viability under all conditions tested gradually diminished after 36 h (Figure 4), presumably due to ethanol accumulation in the broth [32].However, the yeast viability under optimal conditions was higher than with yeast extract and without nutrient supplementation.These results might be due to the presence of glycine and its role as an osmoprotectant [8].).Table 4 shows the amount of fermentable nitrogen utilized in the experiments as well as in the positive and negative control experiments.In the orthogonal experiments, utilization of fermentable nitrogen in the medium increased with the ethanol concentration.The fermentable nitrogen concentrations of the nine runs in the orthogonal array design were much lower when supplementing with yeast extract.Even though the P E values of the juice supplemented with 9 g/L of yeast extract (the control, 121.1 g/L) and R4 (119.8 g/L) were very close, the fermentable nitrogen utilized in the control (317.5 mg/L) was much higher than of R4 (249.3 mg/L), suggesting the fermentation was not limited by the availability of fermentable nitrogen, even with no nutrient supplementation.This implies that nitrogen utilization by the yeast under these conditions depends on yet unknown factors.

Effects of Aeration on Ethanol Fermentation
Under optimal nutrient conditions (A 2 B 1 C 3 ) for ethanol production from sweet sorghum juice, the sugar in the fermentation was not completely utilized with 18.6 g/L of total sugar remaining in the broth at the end of the process.Aeration was applied to improve sugar utilization.Khongsay et al. [7] reported that the highest ethanol production efficiency from sweet sorghum juice supplemented with 9 g/L yeast extract under a VHG condition (290 g/L of total sugar) was achieved with agitation at 200 rpm, aeration at 2.5 vvm, and an aeration time of 4 h.This optimal aeration condition was applied with optimal nutrient conditions (A 2 B 1 C 3 ).
Figure 5 shows the profiles of viable yeast cells, pH, residual total sugar, and ethanol levels during ethanol production from sweet sorghum juice by S. cerevisiae NP01 in a medium containing 280 g/L of total sugar using optimal aeration [7] and nutrient conditions.The initial total sugar level was 277.0 g/L with an initial cell concentration of 2.0 × 10 7 cells/mL.The P E (134.3 g/L), Y p/s (0.50), and Q p (2.80 g/L•h) values were obtained after 48 h (including 4 h of aeration) (Table 5).The results showed that under optimal nutrient conditions with appropriate agitation, aeration, and aeration time, the efficiencies of ethanol production in terms of P E , Y p/s , and Q p values were approximately 6% to 12% higher than those under the same conditions with no aeration.The highest viable yeast cell numbers under aeration were approximately 14% higher than those with no aeration (Figure 6).These results indicated that aeration for only 4 h could significantly promote yeast growth and markedly improve the fermentation efficiencies.Hammond et al. [33] showed that the oxygen present earlier in the fermentation was rapidly utilized for membrane synthesis.The glycerol concentration or P G under aeration was 2 g/L lower than that with no aeration.Alfenore et al. [34] reported the beneficial effects of aeration in the form of better control of by-product production.Glycerol was the primary by-product.It was greatly reduced from 12 g/L (no aeration) to 4 g/L (aeration at 0.2 vvm throughout the fermentation).Reddy and Reddy [10] reported that higher glycerol content indicates more microbial stress.Cot et al. [35] reported that high ethanol concentrations resulted in the loss of membrane integrity, denaturation of membrane proteins, and inactivation of some intracellular metabolic products.Aeration may protect the cells from the unfavorable effects of high ethanol concentrations, improving membrane functionality, resulting in higher viable yeast cell counts, P E , and Q p values.Additionally, Yue et al. [32] reported that large amounts of residual fermentable sugars often remain at the end of fermentation.Some of them were converted into glycerol, which is involved in the osmotic regulation of the cells.The results of our study indicated that the aeration rate had a positive effect on sugar utilization and ethanol production in terms of P E , Q p , and Y p/s (Table 5).Adequate amounts of essential nutrients, appropriate aeration, and agitation time promoted sugar utilization of S. cerevisiae NP01, leading to an improvement of ethanol production [7].
Energies 2019, 12, x FOR PEER REVIEW 9 of 13 was 277.0 g/L with an initial cell concentration of 2.0 × 10 7 cells/mL.The PE (134.3 g/L), Yp/s (0.50), and Qp (2.80 g/L•h) values were obtained after 48 h (including 4 h of aeration) (Table 5).The results showed that under optimal nutrient conditions with appropriate agitation, aeration, and aeration time, the efficiencies of ethanol production in terms of PE, Yp/s, and Qp values were approximately 6% to 12% higher than those under the same conditions with no aeration.The highest viable yeast cell numbers under aeration were approximately 14% higher than those with no aeration (Figure 6).These results indicated that aeration for only 4 h could significantly promote yeast growth and markedly improve the fermentation efficiencies.Hammond et al. [33] showed that the oxygen present earlier in the fermentation was rapidly utilized for membrane synthesis.The glycerol concentration or PG under aeration was 2 g/L lower than that with no aeration.Alfenore et al. [34] reported the beneficial effects of aeration in the form of better control of by-product production.Glycerol was the primary byproduct.It was greatly reduced from 12 g/L (no aeration) to 4 g/L (aeration at 0.2 vvm throughout the fermentation).Reddy and Reddy [10] reported that higher glycerol content indicates more microbial stress.Cot et al. [35] reported that high ethanol concentrations resulted in the loss of membrane integrity, denaturation of membrane proteins, and inactivation of some intracellular metabolic products.Aeration may protect the cells from the unfavorable effects of high ethanol concentrations, improving membrane functionality, resulting in higher viable yeast cell counts, PE, and Qp values.Additionally, Yue et al. [32] reported that large amounts of residual fermentable sugars often remain at the end of fermentation.Some of them were converted into glycerol, which is involved in the osmotic regulation of the cells.The results of our study indicated that the aeration rate had a positive effect on sugar utilization and ethanol production in terms of PE, Qp, and Yp/s (Table 5).Adequate amounts of essential nutrients, appropriate aeration, and agitation time promoted sugar utilization of S. cerevisiae NP01, leading to an improvement of ethanol production [7].

Impact of Multi-Factors on Ethanol Concentration
= total sugar, = ethanol).
Table 5. Fermentation parameters of ethanol production from the sweet sorghum juice under optimal nutrient conditions (A 2 B 1 C 3 ) with and without aeration.Table 5. Fermentation parameters of ethanol production from the sweet sorghum juice under optimal nutrient conditions (A2B1C3) with and without aeration.

Aeration PE (g/L)
Yp/s (g/g) Qp (g/L•h) PG (g/L) SC (%) t (h) No 120.1 ± 0.1 a 0.47 ± 0.00 a 2.50 ± 0.00 a 10.8 ± 0.2 a 93.1 ± 1.9 a The effects of aeration on ethanol fermentation have been reported in serval studies.Table 6 compares the ethanol production efficiency under VHG fermentations (280 to 300 g/L of sugar) with various aerated controls.The PE and Qp values ranged from 120.9 to 140.2 g/L and 1.77 to 3.36 g/L h, respectively.The differing results might have been due to variations in the raw materials, yeast strain, and operating conditions.The PE and Qp values in our study were relatively high compared to similar research studies, indicating that optimal nutrient and aeration conditions were achieved.However, the cost-effectiveness of the process should be further studied.The effects of aeration on ethanol fermentation have been reported in serval studies.Table 6 compares the ethanol production efficiency under VHG fermentations (280 to 300 g/L of sugar) with various aerated controls.The P E and Q p values ranged from 120.9 to 140.2 g/L and 1.77 to 3.36 g/L h, respectively.The differing results might have been due to variations in the raw materials, yeast strain, and operating conditions.The P E and Q p values in our study were relatively high compared to similar research studies, indicating that optimal nutrient and aeration conditions were achieved.However, the cost-effectiveness of the process should be further studied.

Figure 5 .
Figure 5. Fermentation profiles during ethanol production by S. cerevisiae NP01 from the sweet sorghum juice supplemented with yeast extract (3 g/L), DSY (4 g/L), and glycine (5 g/L) at an agitation rate, 200 rpm; aeration rate, 2.5 vvm, and aeration time, 4 h.The arrows indicate the start and the end of aeration.( = log viable cells,  = pH,  = total sugar,  = ethanol).

Figure 5 .Figure 1 .
Figure 5. Fermentation profiles during ethanol production by S. cerevisiae NP01 from the sweet sorghum juice supplemented with yeast extract (3 g/L), DSY (4 g/L), and glycine (5 g/L) at an agitation rate, 200 rpm; aeration rate, 2.5 vvm, and aeration time, 4 h.The arrows indicate the start and the end of aeration.( = log viable cells, = pH, Energies 2019, 12, x FOR PEER REVIEW

48 2 .
5 vvm for 4 h at agitation rate of 200 rpm 134.3 ± 0.2 b 0.50 ± 0.00 b 2.80 ± 0.00 b 8.7 ± 0.1 b 96.8 ± 0.7 b 48 PE: ethanol concentration; Yp/s: ethanol yield Qp: ethanol productivity; PG: glycerol concentration; SC: sugar consumption, and t: fermentation time.a, b : Means superscripted with the same letter within the same column are not significantly different using Duncan's multiple range test at a 0.05 level.The experiments were performed in triplicate, and the results are expressed as mean ±SD.

Table 1 .
The L 9(3 4) orthogonal test and the experimental results.
E (g/L) Q p (g/L•h) Y p/s (g/g) Data superscripted by the same letter within the same column are not significantly different using Duncan's multiple range test at a 0.05 level.The experiments were performed in triplicate, and the results are expressed as mean ±SD.

Table 2 .
The range analysis of L 9(3 4) orthogonal experimental runs of ethanol concentration.

Table 2 .
The range analysis of L9 (3 4 ) orthogonal experimental runs of ethanol concentration.
A: Yeast ExtractB: DSY a Blank C: Glycine

Table 3 .
Fermentation parameters of ethanol fermentation from the sweet sorghum juice with and without nutrient supplements.E : ethanol concentration; Q p : ethanol productivity; Y p/s : ethanol yield; P G : glycerol concentration; sugar consumption and t: fermentation time.a, b, c : Data superscripted with the same letter within the same column are not significantly different using Duncan's multiple range test at the level of 0.05.The experiments were performed in triplicate, and the results are expressed as mean ±SD. P

Table 4 .
Fermentable nitrogen during ethanol production from the sweet sorghum juice under various nutrient supplements.E : ethanol concentration, a R i = no. of run.The experiments were performed in triplicate, and the results were expressed as mean ±SD. P

Table 6 .
Comparison of very high gravity (VHG) ethanol fermentation under aeration in various studies.