Evaluation of different strains of Saccharomyces cerevisiae for ethanol production from high-amylopectin BRS AG rice (Oryza sativa L.)

Optimization of the enzymatic hydrolysis

A study of the reaction parameters during the enzymatic hydrolysis contained in the BRS PAMPA rice was carried out. The enzymatic action time was evaluated during the starch liquefaction and saccharification. The best reaction condition obtained was used in the hydrolysis reactions of BRS PAMPA rice and BRB AG rice for fermentation reactions with the S. cerevisiae strain.

During the starch liquefaction step, the action of Termamyl 2X enzyme at different reaction times was analyzed, as well as the absence of this enzyme in the process. In addition, the amyloglucosidase AMG 300L action during the saccharification step was also studied at different reaction times. Aliquots were collected and analyzed by HPLC for glucose quantification. The glucose concentration (g L⁻¹) in function of the time reaction is shown in Fig. 1.

Based on the results shown in Fig. 1, it was observed that the hydrolysis 13 resulted in the highest final glucose concentration (about 130 g L⁻¹). This hydrolysis was carried out with Termamyl 2X for 1-h and with AMG 300 L for 3-h. Furthermore, regardless of Termamyl 2X reaction time in the liquefaction step, a higher glucose concentration was obtained after 3 h of reaction with amyloglucosidase AMG 300 L.

Analyzing the behavior of Termamyl 2X, in the reactions without this α-amylase acting at the liquefying step, the AMG 300 L enzyme does not act efficiently in the saccharification step to obtain the glucose (Fig. 1—hydrolysis 1, 6 and 11). As the internal bonds of amylose and amylopectin were not cleaved to dextrins, the amyloglucosidase could not disrupt the α-1,4 and α-1,6-glycosidic bonds at the saccharification step, leading to a lower final concentration of glucose. Moreover, based on the bar graph, the lowest glucose concentration was obtained for hydrolysis 1, in which the AMG 300 L enzyme acted for 1 h. In the hydrolyses represented by bars 6 and 11, in which the AMG 300L reacted for 2 h and 3 h respectively, the final glucose concentration was similar, approximately 48 g L⁻¹.

For the reactions in which the Termamyl 2X reacted for 30 min (Fig. 1—hydrolysis 2, 7 and 12), the lowest glucose concentration was obtained for the hydrolysis 2, in which the AMG 300 L enzyme reacted for 1 h. In the hydrolysis 7 and 12, the amyloglucosidase AMG 300L had acted for 2 h and 3 h respectively, and the final glucose concentration was similar in both cases, about 103 g L⁻¹. According to Fig. 1, the reactions carried out in absence of Termamyl 2X led to the lowest glucose concentrations compared to the enzymatic reactions performed with Termamyl 2X, indicating that this enzyme plays an important role in the process related to the cleavage of α-1,4-glycosidic bonds.

The reactions with Termamyl 2X that lasted for 1 h (Fig. 1, hydrolysis 3, 8 and 13) led to the highest final glucose concentration. In hydrolysis 3, the amyloglucosidase AMG 300L has acted for 1 h, and the final glucose concentration was 107 g L⁻¹. In hydrolysis 8 the AMG 300L has acted for 2 h and the final glucose concentration was about 120 g L⁻¹.

The final glucose concentration obtained in hydrolysis 13 was approximately 130 g L⁻¹ (Fig. 1). In this reaction, the AMG 300 L has acted for 3 h, leading to the highest yield among all evaluated reactions. This result proves that the highest glucose concentration is obtained when the time of α-amylase reaction is fixed to 1 h, and the longer is the reaction time of amyloglucosidase in the saccharification step.

The final glucose concentrations obtained in the reactions of Termamyl 2X carried out for 2 h (Fig. 1—hydrolysis 4, 9 and 14) and 3 h (Fig. 1—hydrolysis 5, 10 and 15) were much lower than the conversions found for the reactions of Termamyl 2X carried out for 1 h. Thus, it is observed that long reaction times for the liquefaction step (Termamyl 2X acting) does not lead to higher yields of glucose conversion.

This behavior is probably due to the formation of both the maltotriose and the maltose. After 3 h of Termamyl reaction, there was possibly a greater formation of maltotriose, which possibly hampers the action of AMG 300 L during the saccharification step. It is expected that the maltotriose and the maltose are hydrolyzed at a lower rate compared to larger oligosaccharide chains²⁶. Figure 2 shows the chromatograms obtained for the samples withdrawn after the action of the Termamyl 2X enzyme in different reaction times, showing a change in the behavior at the peaks with a retention time of approximately 6.5 min and 7.6 min. For 1 h of Termamyl 2X, it was observed an increase in the peak with a retention time of 7.6 min and a division or supposed overlap in the peak with a retention time of 6 min, compared to the chromatogram referring to the action of Termamyl 2X for 30 min. In particular for the reactions with Termamyl 2X acting for 2 h and 3 h, there was a significant increase in these peaks. Based on the information provided by Aminex HPLC Columns, for the HPX87H column, the compounds that are identified in these respective retention times can be maltotriose (three molecules of glucose) and maltose (two molecules of glucose)²⁷.

Fermentation of BRS PAMPA rice with different strains of Saccharomyces cerevisiae

The glucose fermentation to obtain ethanol was carried out in triplicate for each strain of S. cerevisiae: BG-1, CAT-1, FT-858, JP-1, PE-2, SA-1. Figure 3 shows the glucose concentrations throughout the fermentations. All glucose was consumed within 48 h after the experiment, thus the reactions were carried out for 48 h.

Based on the presented glucose concentration profiles, at the beginning of the fermentation, the glucose concentration was 124 g L⁻¹. The strain JP-1 appears to be the one with the fastest kinetic reaction. This strain had a lower glucose concentration than the others, with the same reaction times, despite the values of the other strains being within the error bar of standard deviation.

The strain PE-2 had slower reaction kinetics because, after 32 h of fermentation, the glucose concentration was 45.0 ± 0.48 g L⁻¹ while the other strains had a concentration around 20 g L⁻¹ or below that value. At 48 h of fermentation, the strains CAT-1, BG-1, FT-858 had already consumed all the glucose. The other strains also consumed all the glucose, considering the values of the standard deviation chosen by the triplicates. The glucose concentration at 48 h of fermentation was approximately 0.17 g L⁻¹ for strain JP-1, 0.20 g L⁻¹ for strain PE-2 and 0.51 g L⁻¹ for strain SA-1.

The ethanol concentration profiles are shown in Fig. 4. It was observed that after 4 h of fermentation, the strain SA-1 produced the largest amount of ethanol, 13 g L⁻¹. In contrast, PE-2 led to the lowest ethanol concentration for the same time of reaction, approximately 2 g L⁻¹. The ethanol concentration profile of BG-1 shows that this strain had more difficulty in converting glucose into ethanol compared with the other strains since the ethanol concentration was the lowest. After 48 h of fermentation, the ethanol final concentration for BG-1 was about 50 g L⁻¹. The strains that produced a greater amount of ethanol were CAT-1 and SA-1, with a final concentration of approximately 59 g L⁻¹. The strains FT-858 and PE-2 produced about 54 g L⁻¹ and 55 g L⁻¹ ethanol after 48 h of fermentation, respectively. The strain JP-1 produced 53 g L⁻¹ of ethanol after 48 h of fermentation. Besides ethanol, which was the product of interest, all strains produced glycerol as a co-product during the fermentations, as shown in Fig. 5.

The glycerol concentration during the fermentative reactions shows a similarity for all strains of S. cerevisiae. However, throughout the fermentation, strain SA-1 had produced higher concentrations of glycerol. In this way, this strain was in conditions of greater stress compared to the others since the reaction conditions are determinants for the cellular metabolism of the strains.

There exist many stressful yeast conditions that provide a lower yield and higher formation of co-product. Some of them are fermentation temperature that usually varies between 32 and 35 °C; ethanol concentration that reaches 8%–11% (v/v) towards the end of each fermentation cycle and bacterial contamination.

In addition to osmotic stress, substrate concentration, agitation rate, time of fermentation reaction and fermentative medium pH are factors that can cause yeast stress, leading to a higher production of co-products. At the end of fermentation, which occurred after 48 h, the glycerol concentration produced by strain SA-1 was 6.7 g L⁻¹. The strain, which produced the second-highest glycerol concentration, was CAT-1, with approximately 5.2 g L⁻¹. In contrast, strain PE-2 was the one that produced the lowest amount of glycerol. After 48 h the glycerol concentration was 3.1 g L⁻¹ for this strain. The strains FT-858, JP-1, BG-1 yielded 4.8 g L⁻¹, 4.3 g L⁻¹ and 3.9 g L⁻¹, respectively.

After analyzing the concentration profiles for glucose consumption, ethanol production and glycerol, we calculated the fermentation yield, glucose (substrate) conversion factors in ethanol (Y_E/S)_, glucose conversion factors in glycerol (Y_G/S), productivity and rates of substrate consumption. The results obtained are shown in Table 1.

Based on the parameters obtained and shown in Table 1, the maximum yield was obtained by using strain SA-1 in fermentation reaction. This strain had a 93.0% yield, and 58.92 ± 10.83 g L⁻¹ as final ethanol concentration. However, after 48 h of fermentation, the substrate had not been fully consumed, leaving 0.51 ± 0.23 g L⁻¹ as residual glucose concentration. The strain CAT-1 presented the second-highest yield with 92.7% and had consumed at all the glucose. The final ethanol concentration obtained was 58.93 ± 12.85 g L⁻¹.

Based on ethanol production curves for the fermentations carried out with these two strains (SA-1 and CAT-1) presented in Fig. 4, the final ethanol concentration can be considered statistically equivalent. However, the glycerol concentration was lower for CAT-1, with a concentration of 5.17 ± 1.04 g L⁻¹, while for SA-1 the glycerol concentration was 6.67 ± 0.72 g L⁻¹. Thus, the yield of the SA-1 strain was slightly higher and formed more glycerol as a fermentation co-product than CAT-1.

The strains FT-858, PE-2 and JP-1 showed similar fermentation parameters. The yield of FT-858 was 85.3% and the final ethanol concentration was 54.22 ± 4.12 g L⁻¹. The yield of PE-2 was 86.6% and the final ethanol concentration was 55.0 ± 2.67 g L⁻¹. The final glycerol concentration was higher for FT-858, 4.84 ± 0.62 g L⁻¹ while the glycerol concentration for PE-2 was 3.12 ± 0.24 g L⁻¹. The total substrate consumption occurred for the strain FT-858. Considering that the residual glucose for PE-2 was 0.20 ± 0.35 g L⁻¹, it can be concluded that this strain also consumed all the glucose during the fermentation. The process productivity and the rate of substrate consumption were the same for these two strains, 1.12 g L⁻¹ and 2.59 g L⁻¹, respectively. Strain JP-1 had an 84.1% as fermentation yield. The final ethanol concentration was 53.43 ± 2.57 g L⁻¹and the final glycerol concentration was 4.33 ± 0.27 g L⁻¹. As with PE-2, it can be assumed that this strain consumed all the substrate since the residual glucose concentration was 0.17 ± 0.30 g L⁻¹.

The fermentation carried with strain BG-1 showed the lowest yield, 78.6%, despite all the glucose being consumed and high concentrations of glycerol, 3.91 ± 0.76 g L⁻¹, were not formed. As shown in Fig. 4, throughout the fermentation this strain produced lower concentrations of ethanol compared to the other strains. Therefore, this strain had obtained the lowest productivity value of 1.04 g_ethanol·L⁻¹·h⁻¹.

The BRS PAMPA rice fermentations that presented the highest yield and highest ethanol concentration were those carried out with strains CAT-1 and SA-1. However, SA-1 had not consumed the substrate at all and had led to the highest glycerol concentration, while strain CAT-1 consumed the glucose at all and had led to a lower concentration of glycerol as a co-product. High yield is also observed in industrial-scale fermentations when strain CAT-1 is used²⁴.

Each S. cerevisiae commercial strain has specific characteristics that are considered when choosing the fermentation process. Brazilian distilleries have many strains on the market to use in industrial fermentation processes, however, the strains most used at fermentations to obtain ethanol are CAT-1 and PE-2. These two strains represent 70% of the Brazilian market for fuel ethanol production²³. The strain CAT-1 was isolated from the Catanduva and the strain PE-2 from Pedra plants⁷. These strains are more adapted to industrial conditions and provide higher ethanol yield and lower amount of residual sugar. In addition, they produce little foam during fermentation and do not flocculate²⁸.

It is generally agreed that statistical evaluation of the experimental data provides fundamental insight into the fermentation behavior as well as the comparative performance of S. cerevisiae commercial strain. It is important to bear in mind that from a statistical point of view the ability of all strains, evaluated in the present work, to convert BRS PAMPA rice into ethanol can be considered equivalent, as depicted in Fig. 6A. Although CAT-1 and SA-1 exhibit the highest average value for ethanol concentration (approximately 59 g L⁻¹) compared to the other strains, based on the ANOVA statistical test carried out with a significance level of 0.05, the mean values of ethanol concentration determined for all evaluated strains are not significantly different. On the other hand, when the concentration of produced glycerol is considered, ANOVA results come back with a different outcome (Fig. 6B), showing that the mean value obtained of the glycerol production by strain SA-1 t (with the highest mean value, approximately 7 g L⁻¹) is statistically different from the one determined for PE-2, JP-1 and BG-1, and PE-2 is significantly different from CAT-1. Based on glucose consumption and glycerol formation (Table 1), it is reasonable to assume the CAT-1 may be considered as the best strain for the ethanol production from high-amylopectin BRS PAMPA rice.

Fermentation of BRS AG rice with different strains of Saccharomyces cerevisiae

The glucose fermentation with BRS AG rice to obtain ethanol was carried out in triplicate for each strain of S. cerevisiae: BG-1, CAT-1, FT-858, JP-1, PE-2, SA-1. Figure 7 shows the glucose concentrations throughout the fermentations. All glucose was consumed within 48 h after the experiment, thus the reactions were carried out for 48 h.

Based on the presented glucose concentration profiles (Fig. 7), at the beginning of the fermentation, the glucose concentration was 136.5 g L⁻¹. The strains CAT-1, JP-1 and SA-1 presented faster reaction kinetics compared to the other strains. These strains along with FT-858 had lower glucose concentrations with the same reaction times. In contrast, PE-2 was the slowest reaction kinetics with the lowest glucose consumption. After 48 h of fermentation, the glucose concentration in the fermentation medium was about 12.26 ± 2.02 g L⁻¹ for this strain. The strain BG-1 also had slower reaction kinetics in comparison to the strains CAT-1, FT-858, JP-1 and SA-1 (Fig. 7), however considering the residual glucose concentration of 1.03 ± 1.79 g L⁻¹ after 48 h of reaction, it is reasonable to assume that substrate was significantly consumed. The strain FT-858 had already consumed all the glucose after 48 h of fermentation. The strains JP-1 and SA-1 had not consumed the glucose at all, leaving residual glucose in the fermentative medium with a concentration near zero. The glucose concentration with 48 h of fermentation was 0.53 ± 0.09 g L⁻¹ for strain JP-1 and 0.72 ± 0.21 g L⁻¹ for strain SA-1.

The ethanol concentration profiles during the fermentations are shown in Fig. 8. It was observed that at 4 h of fermentation the strains that produced the highest amounts of ethanol were JP-1, with a concentration of 12.56 ± 0.74 g L⁻¹, and SA-1, with a concentration of 13.09 ± 0.32 g L⁻¹. On the other hand, BG-1 and PE-2 were the strains that had the lowest ethanol concentration for the same reaction time, 5.54 ± 1.17 g L⁻¹ and 5.73 ± 0.83 g L⁻¹, respectively. The ethanol concentration profile of PE-2 demonstrated a greater difficulty of this strain in the conversion of glucose into ethanol, compared with the other strains. This is because strain PE-2 obtained the lowest ethanol concentrations during the fermentation and had not consumed all the glucose present in the fermentative medium. After 48 h of fermentation, the final ethanol concentration for this strain was about 54.10 ± 6.67 g L⁻¹. The strains that produced a greater amount of ethanol were FT-858 with a concentration of 62.45 ± 6.43 g L⁻¹ and CAT-1, with a concentration of 61.25 ± 9.02 g L⁻¹. The strain JP-1 produced 58.56 ± 11.52 g L⁻¹ of ethanol after 48 h of fermentation. The final ethanol concentration for strain BG-1 was 54.67 ± 4.36 g L⁻¹, and for strain SA-1 was 57.80 ± 12.61 g L⁻¹.

The glycerol concentration profiles obtained as co-product in all fermentations performed with BRS AG rice is shown in Fig. 9. As with fermentations carried out with BRS PAMPA rice, during the whole fermentation process, the strain SA-1 produced higher concentrations of glycerol, demonstrating that this strain was in conditions of greater stress compared to the others. This condition may be related to the high osmotic pressure of the fermentation medium, as well as adverse conditions such as acid pH, high temperature, high substrate concentration and even the presence of contaminants in the medium fermentative. These factors corroborate in the production of fermentation co-products, such as glycerol^18,29.

The strain that led to the highest glycerol concentration after 48 h of fermentation was SA-1, with 7.20 ± 0.54 g L⁻¹. The strain that produced the second-highest glycerol concentration was CAT-1, with 5.82 ± 0.80 g L⁻¹. In contrast, strain PE-2 was the that produced the lowest amount of glycerol. After 48 h the glycerol concentration was 3.31 ± 0.21 g L⁻¹ for this strain. The strains FT-858, BG-1 and JP-1 produced 5.34 ± 0.68 g L⁻¹, 5.21 ± 1.03 g L⁻¹ and 4.15 ± 0.59 g L⁻¹, respectively.

Based on the results, a similarity in the curves of glycerol production is observed in the fermentations with the BRS PAMPA rice and BRS AG rice. For both cases, the strains that led to a higher formation of glycerol, as a co-product, was SA-1, followed by CAT-1 and FT-858. The only difference for the fermentations of these two types of rice in relation to the final glycerol concentration was that in the BRS PAMPA rice case, the strain that led to the fourth-largest concentration of this co-product was JP-1, and in the case of BRS AG rice, was strain BG-1. The lowest glycerol concentration obtained for both was for fermentation carried out with the strain PE-2.

After 48 h of reaction, the glucose consumption, ethanol and glycerol production were analyzed for the fermentation of BRS AG rice. The fermentations yield was calculated, glucose (substrate) conversion factors in ethanol (Y_E/S), glucose conversion factors in glycerol (Y_G/S), productivity and consumption substrate rates. The results obtained are shown in Table 2.

Based on the parameters obtained for the fermentations with different strains of S. cerevisiae presented in Table 2, the strain that presented the highest yield was FT-858, with 89.6%. The strain CAT-1 presented the second-highest yield, with 87.9% and had consumed all the glucose. In addition, productivity was similar for these two strains, 1.30 g_ethanol L⁻¹ h⁻¹ for strain FT-858 and 1.28 g_ethanol L⁻¹ h⁻¹ for CAT-1, with equal rate of substrate consumption of 2.84 g_glucose L⁻¹ h⁻¹.

Compared to the fermentation carried out with strain BG-1, the parameters obtained for the fermentation with JP-1 were better. The yield obtained for strain JP-1 was 84.3% and for strain BG-1 was 79.0%. The process productivity was lower for BG-1 (1.14 g_ethanol L⁻¹ h⁻¹) and JP-1 (1.22 g_ethanol L⁻¹ h⁻¹). The rate of substrate consumption was similar, 2.82 g_glucose L⁻¹ h⁻¹ for strain BG-1 and 2.83 g_glucose L⁻¹ h⁻¹ for strain JP-1. The strain PE-2 showed 85.2% of yield with the lowest productivity value (1.13 g_ethanol L⁻¹ h⁻¹) and a rate of substrate consumption of 2.59 g_glucose L⁻¹ h⁻¹. The fermentation carried out with strain SA-1 showed a 83.3% yield, exhibiting a substrate consumption rate equal to 2.83 g_glucose L⁻¹ h⁻¹ and productivity of 1.20 g_ethanol L⁻¹ h⁻¹.

Thus, for both fermentations carried out with BRS PAMPA and BRS AG rice, the strain CAT-1 led to the second-highest yield and final ethanol concentration. Industrially, this strain is the most used in fermentations of sugarcane juice. One reason is the aluminum tolerance of cane juice. The strain FT-858 is also tolerant to aluminum and is used to reduce the problems caused by this metal during fermentation^23,28.

The reaction conditions were determinant in the final concentration of ethanol and glycerol production. Stressful conditions for the yeast provide a lower yield and higher formation of this co-product. Higher amounts of glycerol are obtained when the yeast is under osmotic stress since in this condition the yeast loses water and initiates the synthesis of glycerol to protect the cells from dehydration and protect them from the effects arising from the stress condition²⁴. In addition to osmotic stress, substrate concentration, agitation rate, presence of contaminants, fermentation time, pH and temperature of the fermentative medium are factors that can cause yeast stress and lead to a higher formation of co-products and lower yield of the product of interest, ethanol^17,18,23.

In fermentations carried out for 48 h with the different strains of S. cerevisiae, both rice cultivars led to high ethanol yields. However, BRS AG rice led to yields close to 90%, with four strains (CAT-1 and FT-858) and yields between 80 and 85% (BG-1, JP-1, PE-2 and SA-1). On the other hand, the yields obtained for BRS PAMPA rice were above 90% for CAT-1 and SA-1 strains. The yield was around 85% for three strains (FT-858, JP-1, PE-2). The lowest yield was obtained for the fermentation using BRS PAMPA rice and the BG-1 strain and was 78.6%. With these results, it can be said that the rice developed by Embrapa Clima Temperado proved to be an excellent alternative for ethanol production by fermentation with different S. cerevisiae strains compared to conventional rice BRS PAMPA.

Although the profiles of ethanol and glycerol production seem to be relatively different throughout the fermentation time (Figs. 8 and 9), it also seems that the final ethanol concentration was the statistically same. For this reason, a statistical analysis considering the mean values at the end of the fermentation (48 h) was performed to give a piece of comparative information about the ability of the strains to produce ethanol and glycerol, as shown in Fig. 10. According to ANOVA statistical test, mean values of the ethanol concentration at the end of the BRS AG rice fermentations carried out with all strains cannot be considered different, as illustrated in Fig. 10A. However, when the final glycerol concentration at 48 h fermentation is considered in the statistical analysis, the comparative result from the Tukey test showed that performance of SA-1, which is the strain that led to the highest mean value of glycerol concentration (7.2 g L⁻¹) is statistically different from the other strains PE-2, JP-1 and BG-1, while the strain led to the lowest glycerol concentration (3.3 g L⁻¹), PE-2 is significantly different from FT-858, CAT-1 and BG-1. It is worth mentioning that both CAT-1 and FT-858 have very similar mean values of ethanol concentration (61 g L⁻¹ and 62 g L⁻¹, respectively) and glycerol concentration (5.8 g L⁻¹ and 5.3 g L⁻¹, respectively), which is also not statistically different according to ANOVA assessment (Fig. 10). Based on this analysis, CAT-1 and FT-858 might be considered as the most preferred strains to produce ethanol from high-amylopectin BRS AG rice.