Tsinghua Science and Technology Tsinghua University, China ISSN: 1007-0212 Vol. 6, Num. 3, 2001, pp. 225-230

Tsinghua Science and Technology, Vol. 6, No. 3, August 2001 pp. 225-230

Hyperproduction of Alcohol Using Yeast Fermentation in Highly Concentrated Molasses Medium

GU Yansong¹, QIAO Min ², ZHOU Quan ³,  ZHOU Zhengmao ¹ , CHEN Guoqiang ³

1. School of Chemical Engineering and Materials, Beijing Institute of Technology, Beijing 100081, China;
2. Laboratory of Microbial Fermentation Engineering, Yunnan University, Kunming 650091, China;
3. Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China

Received: 2000-11-12

Code Number: ts01071

Abstract:   

Cane molasses, a major byproduct in the sugar industry, is generally consumed for alcohol production. However, the alcohol production process needs to overcome three major challenges including increasing the productivity of alcohol fermentation, lowering the energy consumption for alcohol conversion and decreasing the environmental pollution caused by the alcoholic yeast fermentation process. To meet these challenges, a screening process was conducted using 13 high osmotic tolerant yeast strains. Among the strains, a Saccharomyces cerevisiae strain 1912 was found to produce high alcohol concentrations during fermentation with high starting molasses concentrations such as 50% (W/V) molasses. In the test, 13.6% (V/V) alcohol was produced in the molasses fermentation broth after 72 h of incubation with an initial Yunnan molasses concentration of 50% in a 5 L fermentor. 15.0% (V/V) alcohol was obtained after 48 h of fermentation in shaking flasks containing 30% (W/V) initial total sugar concentration in diluted molasses. The performance of this strain in the shaking flasks was successfully scaled up to a 5-L fermentor vessel. Strain 1912 seems to be a better alcohol producer than the currently used alcohol production strain 2.1190.

Key  words:  alcohol fermentation; molasses; Saccharomyces cerevisiae

Introduction   

Cane molasses is a major byproduct of the sugar industry. Most sugar refineries use yeast fermentation in molasses for alcohol production. However, traditional alcohol fermentation adds little value to the economy since only low alcohol content is produced in the fermentation broth[1, 2]. This has caused various problems including poor process economics and significant wastewater pollution (high biochemical oxygen demand (BOD)). Many attempts have been made to increase the alcohol concentration in the broth and to achieve high conversion efficiency of sugar to alcohol[1-4].

However, a high alcohol concentration in the fermentation broth is toxic to most yeast. To achieve high alcohol concentration in production, it is important to have a yeast strain capable of resisting the high alcohol concentration and, ideally, also capable of resisting the high sugar concentration substrate. Therefore, the worldwide alcohol fermentation industry is seeking to develop processes using high initial sugar concentration and high temperature to achieve high final alcohol concentration and low residual sugar concentration[1-5].

Most strains currently in use are incapable of tolerating high sugar osmotic pressure and high alcohol concentration, thus, a low initial sugar concentration solution is used to start the fermentation and this is easily contaminated by other microbes, especially bacteria. The final alcohol concentration usually fluctuates between  7%-10% . Although immobilization techniques have increased the final alcohol concentration to around 10%[6] , frequent contamination and alcohol inhibition destabilize the process, causing decreased alcohol yield, increased power consumption and wastewater containing high BOD[7].

Many studies intending to reduce alcohol inhibition and increase alcohol fermentation productivity have been carried out in four areas, including removal of alcohol during fermentation, which is difficult for large scale processes[8, 9] ; application of recombinant strains[10] ; application of wild type or mutated strains able to resist high alcohol concentrations; and the addition of nutrients to improve the viability of the yeast strains during fermentation[2, 5, 11]. The later two methods produce fast results and are low cost.

In this paper, we carried out a screening process to identify suitable strains and suitable processes for hyperproduction of alcohol using molasses as a substrate.

1 Materials and Methods   

1.1 Substrates and yeast strains

Cane molasses from different areas contains different components. Molasses from two sources were used in these tests. One from Yuanjiang of Yunnan province contained 60% (W/V) total sugar and few insoluble components after heating at  100 7°C  and acidification using sulfuric acid. The second source, molasses obtained from Zhaoqing in Guangdong province, contained large amounts of insoluble substances after the heat and acid treatments, and its total sugar content was only 42% (W/V).

Thirteen Saccharomyces cerevisiae strains Nos.  2.178 , 2.1460, 2.1635, 2.214, 1625, 1306, 1912, 1373, 1408, 1488, 1384, 1190, and 4608 that were kindly supplied by the China Food Fermentation Research Institute were employed for this study. All the strains have been documented to show tolerance to high alcohol concentration.

1.2 Molasses pretreatment

1.2.1 Molasses pretreatment for fermentation

Method A: The raw molasses was diluted to contain  21%-25%  total sugar (Guangdong molasses). The diluted molasses was heated at  80 7°C  for 1 h followed by 6 h of sedimentation during the molasses cooling process. The supernatant was then taken and (NH₄)₂SO₄ 1.5 g/L, MgSO₄  0.5 g/L  and KH₂PO₄  0.5 g/L  were dissolved in the supernatant. The solution was centrifuged at 3000 g for 15 min. This supernatant was sterilized for 30 min at  121 7°C .

Method B: The raw molasses was diluted to contain 21%-25% sugar (Guangdong molasses). The diluted molasses was mixed with  1.5 g/L  NAH₂PO₄ followed by heating at  100 7°C  for 15 min with precipitation during the cooling process lasting 6 h. The supernatant was then mixed with (NH₄)₂SO₄ 1.5 g/L, MgSO₄ 0.5 g/L and KH₂PO₄  0.5 g/L . This solution was centrifuged at 3000 g for 15 min and sterilized for 30 min at 121 °C.

Method C: The raw molasses was diluted to contain 30% total sugar (Yunnan molasses), followed by the addition of (NH₄)₂SO₄  1.5 g/L , MgSO₄ 0.5 g/L and KH₂PO₄ 0.5 g/L. Finally, the solution was sterilized for 30 min at  121 7°C .

1.2.2 Molasses pretreatment for seed culture

Molasses pretreated using method C was diluted to contain 12%-15% total sugar.

1.3 Study of tolerance to various sugar concentrations

Thirteen strains were grown on diluted molasses containing 30% total sugar prepared using the gradient agar petri dish. Among them, 6 strains including Nos.1912, 1306, 1384, 1190, 1408, and 4608 which showed better growth at high sugar concentrations were collected and cultured in a  500-mL  shaking flask containing molasses medium with 30% total sugar to test their ability to produce alcohol at high sugar concentrations.

1.4 Seed culture

1.4.1 Culturing in wort liquid

Six selected strains were cultured in 150 mL wort medium (7-8 °Bx) in 500-mL shaking flasks at  30 7°C  and 200 r/min for 24 h. The cell density was approximately 2x10⁸ -3x10⁸ /mL after 24 h of growth.  

1.4.2 Culturing in molasses

Six strains were cultured in 150 mL molasses medium (15-20 °Bx) in 500-mL shaking flasks at  30 7°C  and 200 r/min. After approximately 36 h, the cell density reached 2x10⁸ -3x10⁸ /mL.

1.5 Growth in shaking flasks

Six strains were cultured in 120 mL molasses medium containing  22%-30%  total sugar (inoculum size was 1 : 10 V/V) in 500-mL shaking flasks. Incubation was carried out at  30 7°C  and 100 r/min for 72 h.

1.6 Growth and alcohol production in a  controlled fermentor

Among the six strains, Saccharomyces cerevisiae strains Nos. 2.1190, 4608,  and 1912 that showed better alcohol production ability, were grown in a 5 L molasses medium containing 25%-30% total sugar (inoculum size was 1 : 10 V/V) in a 5 L fermentor (TOKYO KEISO P-115-V) at  30 °C  for 72-80 h. The agitation rate was low (100 r/min for 20 min every 4 h without compressed air supply) to produce a  weak aerobic condition. With anaerobic fermentation, no agitation or air flow was provided to the cells.

1.7 Adaptation study of strain Saccharomyces cerevisiae No.1912 for increasing molasses and alcohol concentrations

Strain No.1912 was found to have the strongest ability to produce high alcohol concentrations when grown in high sugar concentrations. The strain was cultured at  30 7°C  for  24-36 h  in diluted Guangdong molasses containing a total sugar content of  12%-15% . The liquid culture was taken using an inoculum loop and streak spread on petri plates containing agar molasses medium with  12%-15%  total sugar.  The 20 most obvious single colonies were selected and cultured separately in wort medium containing 8% (V/V) ethanol at  30 °C  for 24 h. The colonies grown out of the 8% alcohol solution were selected and again spread on molasses petri plates containing 12%-15% total sugar. The 40 most obvious colonies were selected and cultured separately in diluted Guangdong molasses containing 20%-25% total sugar. The cultures and transfers were carried out three times. The 10 strains showing the most active growth after this adaptation were selected for the fermentation study.

1.8 Analytical methods

1.8.1 Alcohol concentration

The alcohol concentration was measured according to the Chinese alcohol industry standard procedure using the hydrometer method[12].

1.8.2 Total sugar concentration

The total sugar concentration was analyzed using the sulfuric acid-phenol method[13].

2 Results and Discussion   

2.1 Fermentation in shaking flasks

2.1.1 Fermentation in diluted Yunnan molasses 

Six yeast strains showing tolerance to high sugar concentrations were selected from the 13 strains used in this study. The 6 strains were then tested in shaking flasks to measure their alcohol fermentation capability and alcohol tolerance (Table 1). Among them, strain 1912 produced the most alcohol (16%) and had the highest yield. The standard industrial alcohol production strain 2.1190 was used as a control for comparison with other strains. The industrial strain had the lowest alcohol production ability and lowest alcohol yield.

The lag phases for the three strains Nos.  2.1190 , 4608, and 1912 were short due to the inoculum size and the characteristics of these strains (Fig. 1). The initial inocula were 3x10⁸ cells/mL in wort medium. The alcohol concentration rose approximately 2.5% from 36 h to 72 h, indicating that the alcohol strongly inhibits cell growth and fermentation. The residual sugar was more than  80 g/L  and the alcohol concentration reached  11.7%-13.7%  after 36 h. Therefore, the decrease of fermentation productivity was related to ethanol inhibition rather than exhaustion of the carbon resource. At high alcohol concentrations, the yeast must consume more sugar to obtain the same amount of alcohol compared with low alcohol concentrations because the yeast cells require more energy to modify their cellular compositions, especially the cell membrane, to survive in the toxic environment resulting from the higher alcohol concentration[1, 14].

Strain 1912 showed the strongest alcohol production ability among the three yeast strains, with an alcohol content reaching 16% (V/V) after 72 h of fermentation in the Yunnan molasses, while strains 4608 and 2.1190 produced 15% and 13.7% alcohol for the same conditions (Fig. 1).

2.1.2 Fermentation in diluted Guangdong  molasses

The six strains produced less alcohol in the Guangdong molasses compared with that in the Yunnan molasses (Table 2) mainly because the Guangdong molasses contained less total sugar (23%) after the one-fold dilution, compared with 30% for the Yunnan molasses. Although the fermentation time was 80 h, significantly longer than the 72 h for the Yunnan molasses, the alcohol concentration could not be increased. When the fermentation was carried out for 36 h, only less than 2.8% alcohol was produced, compared with 11.7%-13.7% produced when Yunnan molasses was used.

Strains  1912 and 4608 were capable of fermenting molasses at high alcohol concentration, with strain 1912 producing more alcohol than strain 4608 (Table 2, Fig. 2). Strain 1912 also showed that it produced more alcohol than strains 4608 and 2.1190 between 24 h to 80 h although the three strains all had a relatively long lag phase lasting approximately 24 h with the Guangdong molasses.

These results suggest that the molasses quality strongly affected the fermentation results. The total sugar concentration of the Guangdong molasses was 23% after one-fold dilution compared with 30% for the Yunnan molasses. Therefore, the sugar osmotic pressure seems to not significantly affect the cell behavior. The Guangdong molasses also contained many more impurities than the Yunnan molasses (data not shown). Therefore, two processes were adopted to improve the molasses quality, including pretreating the Guangdong molasses to remove as many of the impurities as possible[15]  and domesticating the strains in the Guangdong molasses[11].

The lag phase for strain 1912 was observed to decrease from 24 h to 10 h after the domestication process when fermented in the Guangdong molasses (Fig.3). The domesticated strain 1912 produced 13% alcohol after 72 h of fermentation compared with  11.2%  alcohol by the non-domesticated strain after 80 h.

The domesticated strain 1912 quickly lost its ability when cultured 3 times in malt wort, with re-fermentation in Guangdong molasses leading to a long lag phase similar to Fig. 2. This phenomenon has been reported previously[11]. Therefore, a stable domesticated strain must be properly maintained under domesticated conditions for industrial application to avoid loss of the desired properties[16]. Our results showed that the domesticated strain 1912 tolerated up to 27% total sugar when grown in pretreated Guangdong molasses.

2.2 Fermentor study

Fermentor experiments were carried out in two 5-L fermentors using the Yunnan molasses with strains Nos. 1912 and 4608. The industrial strain 2.1190 was employed as a control. The strain 2.1190 showed similar behaviour in the fermentor as in the shaking flask except for a shorter lag phase observed in the fermentor study (Fig. 4). The fermentation efficiency in the fermentation phase was much the same as in the shaking flasks, with the cell growth and alcohol production inhibited when the alcohol concentration reached about 12% in both the fermentor and in the shaking flasks. The results showed that the fermentation process using strain  2.1190  could be easily scaled up from the shaking flask to the fermentor. Similar scale results should be expected with the other strains.

Strain 1912 produced more alcohol than strain 4608 after 72 h of fermentation in Yunnan molasses in the 5-L fermentor (Table 3) which further confirms that strain 1912 was the best alcohol producing strain grown in the two types of molasses.

Fermentor  studies were also carried out with an initial total sugar concentration of 25%. Strain 1912 was grown at pH  4.5  and  30 7°C . Two fermentation processes were run with the weak aerobic condition and with the anaerobic condition. The lag phase and the initial main fermentation phase were similar in the two fermentation processes. However, the fermentation productivity with the weak aerobic condition was much higher than that with the anaerobic condition after 20 h of fermentation. The final alcohol concentration produced by strain 1912 with weak aeration after 72 h of fermentation was  13.6% , much higher than the 11.4% with the anaerobic condition (Fig. 5)

3 Conclusions   

Strain 1912 was found to have higher alcohol productivity in two types of molasses, Yunnan molasses and Guangdong molasses, compared with strain 4608 and  2.1190  that are the industrial alcohol production strains currently in use. Strain 1912 produced over 13% alcohol (V/V, %), much higher than the  7%-10%  commonly produced in the alcohol industry. The process was scaled up from a shaking flask to a 5-L fermentor with the same results demonstrating the possibility of using strain 1912 to scale the process to industrial alcohol production. Tests at a factory in Yunnan province showed that strain 1912 produced similar results in a 120-L fermentor and a 90-m³ industrial alcohol fermentor. The application of strain 1912 to replace strain  2.1190  will significantly improve alcohol production economy.

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