1 Introduction

Due to rising focuses on the environmental problems and the periodic crises in some of the larger oil exporting countries, bioethanol has becomes a viable and realistic alternative energy in the global market [1–4]. Highly concentrated bioethanol production means less volume in fermentation tanks and consumes less distillery energy. The very high gravity (VHG) technology is attracting more and more interests due to its economic value [5–8].

The analog fermentation technique is a diminution of the current ethanol industry production processes. In this process, the incubate temperature rise when fermentation enter the peak period. Judging from the cost of production, raw material fermentation technique is minimal in all of the techniques. However, since the starch material has not been liquefied and saccharified, the free glucose concentration is low in the mash and this would be an inhibition to the ethanol fermentation. Now the ripe fermentation process is the most sophisticated fermentation process, but at the same time, the energy cost is very high because of the large amount of cooling water which is used to maintain the temperature of the mash. In simultaneous saccharification and fermentation, two different processes (saccharification and fermentation) were carried on in the same bioreactor at the same time [9, 10]. As the single saccharification step is saved, the cost of production can be significantly reduced. Considering about the large scale of the bioethanol industry, simultaneous saccharification and fermentation is more suitable to the actual production. At the same time, the cooling energy consumption will significantly reduce if thermotolerant strain which can work normally at high temperature in the fermentation process is used.

Due to the advantages of thermotolerant strain, it was broadly studied in recent years. Several breeding methods such as physical and chemical mutagenesis [11, 12], adaptation [13], protoplast fusion [14], evolution engineering [15], global transcription machinery engineering [16], and genome shuffling [17], had been previously used to improve the thermotolerance and ethanol production of yeast. D’Amore et al. screened numerous yeast strains for glucose fermentation at 40 °C [18]. In another study, Banat’s yeast (Saccharomyces cerevisiae) showed a maximum ethanol production of 7.0 % and 6.9 % (w/v) at 37 °C and 40 °C, when 14 % (w/v) glucose used as substrate [19]. S. cerevisiae F111, which can grow at temperatures up to 50 °C, has been isolated and applied to industrial scale fermentation. S. cerevisiae NPO1 produced 11.3 % of ethanol from sweet sorghum juice medium containing 24.8 % of total sugar (89 % theoretical ethanol yield) [6, 20].As the properties of yeast thermotolerance are controlled by so many genes and mediated through so many pathways, the achievements of previous studies were not efficient adequately.

In this study, the ethanol production of thermotolerant yeast F3 constructed through genome shuffling and its control strain AY12 was measured by four techniques at different temperature to verify the advantages of F3. Then the fermentation conditions were optimized by orthogonal test.

2 Material and Method

2.1 Strain and Incubate Conditions

In this study, S. cerevisiae AY12 used is an industrial diploid strain. S. cerevisiae F3 is a thermotolerant yeast constructed through genome shuffling based on AY12.

The first stage seed medium: 0.5 % yeast extract is added to 8°Brix corn hydrolyzate. It is sterilized at 100 °C for 15 min and then cooled to room temperature before used.

The second stage seed medium: 0.5 % yeast extract is added to 12°Brix corn hydrolyzate. It is sterilized at 100 °C for 15 min and then cooled to room temperature before used.

Raw material fermentation medium: 60 g corn flour is in a 250 ml flask, then add 130 ml water (60−70 °C), 20 min later, 30 μl (10 U/g) amylase, 90 μl (150 U/g) glucoamylase, 1.2 ml acid protease (15 U/g), 1 ml nutrient solution (150 g MgSO4/L, 75 g KH2PO4/L, 81 g CON2H4/L) is added. The mash is cooled to room temperature before used.

The ripe material fermentation medium: 60 g corn flour is in a 250 ml flask, then add 130 ml water (60−70 °C), 20 min later, 30 μl (10 U/g) amylase is added and incubated at 85−90 °C for 1.5 h in a water bath incubator, 90 μl (150 U/g) glucoamylase is added and incubated at 55−60 °C for 20 min in a water bath incubator, 1.2 ml acid protease (15 U/g), 1 ml nutrient solution is added and incubated at 55−60 °C for 20 min in a water bath incubator. The mash is cooled to room temperature before used.

Analog technique fermentation medium: It is the same as the ripe material fermentation medium. Simultaneous saccharification and fermentation medium: 60 g corn flour is in a 250 ml flask, then add 130 ml water (60−70 °C), 20 min later, 30 μl (10 U/g) amylase is added and incubated at 85−90 °C for 1.5 h in a water bath incubator, 90 μl (150 U/g) glucoamylase, 1.2 ml acid protease (15 U/g), 1 ml nutrient solution is added. The mash is cooled to room temperature before used.

2.2 Fermentation Technique

The seed culture process of four kinds of techniques in our study is same. First, the strain is inoculated into tube with 4 ml sterilized first stage seed medium, static culture for about 24 h. Then put it all into 250 ml flask with 36 ml sterilized second stage seed medium, static culture for 16−17 h. Then the inoculum is added to fermentation medium by size of 10 %. The mash is incubated at the given temperature. In the process of analog technique, the mash is first incubated at 30 °C during the initial 11 h, then at 40 °C for the rest time (about 61 h). Ethanol was removed from the fermented mash via distilling apparatus at the end of fermentation.

3 Result and Discussion

3.1 Effect of Different Temperature on the Ethanol Production of F3 and AY12

In order to investigate the effect of different temperature on the ethanol production of AY12 and F3 which was constructed through genome shuffling in our previous work, we separately tested their ethanol productions in temperatures of 30 °C, 35 °C, 38 °C, 40 °C, 42 °C, respectively, and the results are shown in Fig. 106.1.

Fig. 106.1
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Effect of ethanol production of AY12 and F3 at different incubate temperature

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As can be seen from Fig. 106.1, the ethanol production of AY12 and F3 were almost the same at 30 °C. While the performance of F3 was better than AY12 at 35 °C, 38 °C, 40 °C, when the incubate temperature rise to 42 °C, the ethanol production of these two strains was almost identical. Though the ethanol production of both strains decreased as the increase of the incubate temperature, it is obvious that the ethanol production of F3 was higher than AY12 at almost all the temperatures. According to the results above, it is obvious that the thermotolerance of the strain was increased compared with the control strain AY12. In conclusion, the strain F3 constructed through genome shuffling displays a higher thermotolerance compared with strain AY12.

3.2 Effect of Fermentation Technique and Temperature on the Ethanol Production of F3 and AY12

In order to determine the most appropriate fermentation technique of the thermotolerant strain F3, experiments were carried on through different techniques (raw material fermentation technique, ripe material fermentation technique, analog fermentation technique, simultaneous saccharification, and fermentation technique) at different temperatures. 30 °C, 38 °C, 40 °C had been selected as the fermentation temperatures. The results are shown in Tables 106.1 and 106.2.

Table 106.1 Fermentation result of AY12
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Table 106.2 Fermentation result of F3
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According to the results above, the ethanol production of ripe material fermentation technique (15, 14.3, 10.2) was highest of all four fermentation techniques, and that of raw material fermentation technique (9.5, 10.1, 8.8) was the lowest of all these technique at temperature of 30 °C, 38 °C, 40 °C, respectively. At 38 °C, the ethanol production of raw material fermentation technique increased first compared with that at 30 °C, and then decreased when the temperature rise to 40 °C. But we all know that the production cost of ripe material fermentation technique is very high and this is not suitable to actual production. Therefore, considering both of the production cost and fermentation results, simultaneous saccharification, and fermentation is the best technique for the ethanol production of F3.

3.3 Optimization of the Fermentation Conditions of F3 Through Simultaneous Saccharification and Fermentation Technique

In order to save the material and shorten the fermentation time, here we studied the optimum fermentation conditions of F3. We tested three factors that influence simultaneous saccharification and fermentation condition for ethanol production via single-factor test. The three factors are ratios of material to water, inoculum concentration and fermentation time. And then, we optimized the fermentation process through orthogonal test.

3.3.1 Effect of Ratios of Water to Material on Alcohol Production

Ratios of water to material were adjusted to 2.4, 2.6, 2.8, 3.0, and 3.2; the experimental data is shown in Fig. 106.2. As presented, the yield of ethanol exhibits a basically stable situation when ratio of water to material is in the range of 2.2–2.8. However, as the ratios of water to material rise above 2.8, the ethanol production was significantly decreased.

Fig. 106.2
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Effect of ratios of water to material on alcohol production

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3.3.2 Effect of Inoculum Concentrations on Alcohol Production

Inoculum concentration also plays an important role in ethanol production. Fermentation time will be extended if the inoculum concentration is too small. However, large inoculum concentration leads to a high inoculum cost. 10 ml, 15 ml, 20 ml, 25 ml inoculum were added to the mash. Results are shown in Fig. 106.3. As we can see, the ethanol production will not be increased when the inoculum concentration rise above 15 ml.

Fig. 106.3
figure 3

Effect of inoculum concentrations on alcohol production

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3.3.3 Effect of Fermentation Time on Alcohol Production

Shorter fermentation time means lower cooling cost, higher equipment utilization, and business profits, so the fermentation time was optimized. As results presented in Fig. 106.4, the ethanol production rise slower than previous time after 48 h. The production would not increase if fermentation time was prolonged.

Fig. 106.4
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Effect of fermentation time on alcohol production

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3.3.4 Orthogonal Test

According to the results of the single-factor tests, ratios of water to material, inoculum concentration, and fermentation time were optimized by orthogonal experiment. Ratios of water to material 2.8, inoculum concentration 15 ml, fermentation time 48 h were used as intermediate level in the orthogonal tests. The experimental design and results of analysis are shown in Tables 106.3 and 106.4.

Table 106.3 Factors and levels of orthogonal test
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Table 106.4 Results of orthogonal test
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Based on the results of experiments, the influence of factors on the ethanol production was ordered from larger to little as the amount of. As can be seen from Table 106.4, the best combination was A1B3C3, revealing that the optimized fermentation conditions are as follow: ratios of water to material 2.6, inoculum concentration 25 ml, fermentation time 56 h.

At last, three parallel experiments which have been carried out on ethanol fermentation by the optimal technique to verify the orthogonal test result (Table 106.5). The ethanol productions of them were 12.1, 12.1, 12.2, respectively, and the average was 12.1.

Table 106.5 Results of verification testing
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4 Conclusion

From all the experiments above, we have verified the advantage of the thermotolerant strain F3 compared with its control strain AY12. Simultaneous saccharification and fermentation technique has been identified to be the proper technique for F3 through the ethanol production comparison of four techniques. The fermentation conditions were optimized via single-factor test and orthogonal test. The optimal fermentation conditions are as follows, the ratio of nutriment and water 1:2.6, inoculum size 25 ml, fermentation period 56 h.