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Effects of yeast invertase on ethanol production in molasses
JOURNAL OF FERMENTATION AND BIOENGINEERING Vol. 79, No. 5, 513-515. 1995
Effects of Yeast Invertase on Ethanol Production KAZUHIKO Tokyo Research Laboratories,
Kyowa Received
TAKESHIGE
Hakko Kogyo 15 November
AND
Co. Ltd., 1994IAccepted
in Molasses
KOZO OUCHI* 3-6-6, Asahimachi, 20 February
Machida,
Tokyo 194, Japan
1995
Ethanol fermentation by an alcohol yeast, YOY655, was slower in molasses than in a nutrition rich medium with the same sugar content. Osmolality was much higher in the molasses, and the slower fermentation in the molasses was ascribed to depressed fermentation under the high osmotic pressure. Yeast invertase was an important factor in regulating the osmolality and the fermentation rate in the molasses. [Key words:
Saccharomyces cerevisiae, invertase,
ethanol
production,
molasses]
mosm/kg. Mo130 contained 17.6% sucrose, 6.2% fructose and 4.7% glucose. The portion of the osmolality deduced to be from these sugar components was 1117 mosm/kg; it was therefore considered that about half (995 mosm/kg) of the osmolality in Mo130 resulted from the contribution of molecules other than sugars. KC1 was added to Mo120 (molasses medium containing 20% sugar) at varying concentrations to adjust the osmolality in the fermentation medium. The rate of ethanol fermentation decreased as the concentration of KC1 increased (Fig. 2). A similar result was obtained when sorbitol was added as an osmoticum (data not shown). Additionally, a reverse relationship was found between the molasses concentration and the fermentation rate (Fig. 3). These findings indicate that high osmolality is an important factor in depressing the ethanol fermentation in Mo130. Several studies have reported that high concentrations of substrates inhibit growth and fermentation of yeast in industrial ethanol production as the result of high osmotic pressure (2-4). Since two-thirds of sugars in molasses are sucrose, hydrolysis of sucrose into glucose and fructose increases osmolality in a molasses medium. The effects of invertase activity on ethanol fermentation were examined using X2180-lB, which lost almost all invertase activity in Mo130 (1). A commercial yeast invertase was added ex-
For economic reasons, molasses is widely used as a raw material for industrial alcohol production. We previously reported that ethanol fermentation in molasses by the laboratory yeast X2180-IB is much weaker than that by the alcohol yeast YOY655, although both strains can produce comparable amounts of ethanol in a nutrient medium, and that the weaker fermentation of X2180-1B can be mostly ascribed to its weaker invertase activity which is almost completely inhibited by certain factors in the molasses (1). These findings indicate that one of the characteristics required of an industrial alcohol yeast is strong invertase activity for the hydrolysis of sucrose even under the inhibitory conditions existing in molasses. In this report, we describe the negative effect of osmolality on ethanol fermentation, and the important role of invertase in osmotic regulation in molasses. If the invertase activity is too weak, the supply of hydrolyzates for fermentation will be insufficient, but if it is too strong, rapid hydrolysis of sucrose increases the ethanol production is osmolality, and as a result, depressed. YPS30 contained 1% yeast extract, 2% Polypepton and 30% sucrose. Mo120 and Mo130 contained 0.2% ammonium sulfate and concentrated molasses to make the total sugar content either 20 or 3O%(w/v), respectively. Osmolality was measured by an osmometer (Osmette Model 2007, Precision Systems Inc., Massachusetts, USA) from the freezing points of the media. The osmolality in Fig. 4b was calculated from the sugar analysis data by dividing the concentration (g/f) of each component by its molecular weight. All the other materials and methods were as described previously (1). When ethanol fermentation of YOY655 was compared in Mo130 and YPS30, the fermentation rate and ethanol yieId were apparently lower in the former medium (Fig. 1). Since molasses contains fewer amino acids and nucleic bases than YPS30, one possible reason for the slower fermentation in Mo130 is a deficiency of nutrition. However, because supplementing sufficient amounts of casamino acids, adenine and uracil to Mo130 showed no positive effect on either the fermentation rate or the ethanol yield (data not shown), this is not regarded as plausible. Another possibility is the high osmolality in molasses, which contains much larger amounts of salts than the nutrient media. The osmolality of Mo130 was 2112 mosm/kg, while that of YPS30 was 1011 ~_.~ ~~ * Corresponding author.
otc’0
20
40 Time(h)
60
80
FIG. 1. Time courses of ethanol production in YPS30 (open circles) and in Mo130 (filled circles). These media were fermented by YOY655 with shaking at 30°C.
513
514
TAKESHIGE
J. FERMENT. BIOENG..
AND OUCH1
0
20 Time
0’
60
40
-1
0
(h)
10 Molasses
FIG. 2.
Effect of KC1 on ethanol production. KC1 was added to Mo120 as an osmoticum at 0.1 M (filled circles), 0.2M (open triangles), 0.4M (filled triangles), or 0.6M (open squares). These media and Mo120 without KC1 (open circles) were fermented as in Fig. 1.
ternally to the culture in Mo130 after fermentation for 16 h, when most of the pre-existing monosaccharides had been exhausted. The addition of invertase at 0.05 and 0.5 U/ml raised the levels of ethanol production in that order. However, the addition of lOU/ml invertase did not raise the level much higher, but rather reduced it lower than that attained at 0.5 U/ml (Fig. 4a). The increase of osmolality appeared soon after the addition of invertase and it was highest at 10 U/ml (Fig. 4b). Ethanol production was highest at 0.5 U/ml, but this amount of activity might be still too strong because such a transient increase also appeared after the addition of invertase. Ideally, the supply of monosaccharides by invertase should be well balanced with their consumption by the yeast SO that the osmolality in medium remains at a minimum during fermentation. In this respect, the invertase activity of YOY655 is possibly too
20 concentration
30 (“h)
FIG. 3. Relationship between ethanol production rate and molasses concentration. Different concentrations of molasses media were fermented as in Fig. 1. The rate of ethanol production was calculated from the quantity of ethanol produced between 2 and 4 h fermentation.
strong for the fermentation in Mo130, because the rapid hydrolysis of sucrose accumulated fructose and resulted in an increase of osmolality (1). If it was possible to regulate the invertase activity, yeasts such as YOY655 could perform more efficient fermentation in Mol30. We are grateful to Miss M. Umeda for her technical assistance. This work was supported by the project of “Research and Development Concerning Highly Efficient Ethanol Fermentation Techniques by High Productivity Yeasts.” Japanese Ministry of International Trade and Industry’s,
REFERENCES 1. Takeshige, K. and Ouchi, K.: Factors affecting the ethanol productivity of yeast in molasses. J. Ferment. Bioeng., 79, 449452 (1995). 2. Beuchat, L. R.: Influence of water activity on growth, meta-
(a)
(b) 1.2 y
_I
0
20
40 Time
60
80
1.”
(h)
FIG. 4. Effects of invertase activity on (a) ethanol production and (b) osmolality 1B in Mo130 at 0.05 (filled circles), 0.5 U (open triangles), or 10 U/ml (filled triangles) (open circles) was also fermented by X2180-1B with shaking at 30°C.
0
20
40
80
80
Time (h) in molasses. Invertase was added to the culture of X2180after 16 h fermentation. Mo130 without invertase addition
VOL. 79, 1995
bolic activities and survival of yeasts and molds. J. Food Prot., 46, 135-141 (1983). 3. Hahn-Haegerdal, B., Larrson, M., and Mattiasson, B.: Shift in metabolism towards ethanol production in Saccharomyces cerevisiae using alterations of the physical-chemical microen-
NOTES
515
vironment. Biotecbnol. Bioeng. Symp., 12, 199-202 (1982). 4. Jones, R. P., Pamment, N., and Freenfield, P. F.: Alcohol fermentation by yeasts-the effect of environmental and other variables. Process Biochem., 16, 42-49 (1981).
Effects of Yeast Invertase on Ethanol Production KAZUHIKO Tokyo Research Laboratories,
Kyowa Received
TAKESHIGE
Hakko Kogyo 15 November
AND
Co. Ltd., 1994IAccepted
in Molasses
KOZO OUCHI* 3-6-6, Asahimachi, 20 February
Machida,
Tokyo 194, Japan
1995
Ethanol fermentation by an alcohol yeast, YOY655, was slower in molasses than in a nutrition rich medium with the same sugar content. Osmolality was much higher in the molasses, and the slower fermentation in the molasses was ascribed to depressed fermentation under the high osmotic pressure. Yeast invertase was an important factor in regulating the osmolality and the fermentation rate in the molasses. [Key words:
Saccharomyces cerevisiae, invertase,
ethanol
production,
molasses]
mosm/kg. Mo130 contained 17.6% sucrose, 6.2% fructose and 4.7% glucose. The portion of the osmolality deduced to be from these sugar components was 1117 mosm/kg; it was therefore considered that about half (995 mosm/kg) of the osmolality in Mo130 resulted from the contribution of molecules other than sugars. KC1 was added to Mo120 (molasses medium containing 20% sugar) at varying concentrations to adjust the osmolality in the fermentation medium. The rate of ethanol fermentation decreased as the concentration of KC1 increased (Fig. 2). A similar result was obtained when sorbitol was added as an osmoticum (data not shown). Additionally, a reverse relationship was found between the molasses concentration and the fermentation rate (Fig. 3). These findings indicate that high osmolality is an important factor in depressing the ethanol fermentation in Mo130. Several studies have reported that high concentrations of substrates inhibit growth and fermentation of yeast in industrial ethanol production as the result of high osmotic pressure (2-4). Since two-thirds of sugars in molasses are sucrose, hydrolysis of sucrose into glucose and fructose increases osmolality in a molasses medium. The effects of invertase activity on ethanol fermentation were examined using X2180-lB, which lost almost all invertase activity in Mo130 (1). A commercial yeast invertase was added ex-
For economic reasons, molasses is widely used as a raw material for industrial alcohol production. We previously reported that ethanol fermentation in molasses by the laboratory yeast X2180-IB is much weaker than that by the alcohol yeast YOY655, although both strains can produce comparable amounts of ethanol in a nutrient medium, and that the weaker fermentation of X2180-1B can be mostly ascribed to its weaker invertase activity which is almost completely inhibited by certain factors in the molasses (1). These findings indicate that one of the characteristics required of an industrial alcohol yeast is strong invertase activity for the hydrolysis of sucrose even under the inhibitory conditions existing in molasses. In this report, we describe the negative effect of osmolality on ethanol fermentation, and the important role of invertase in osmotic regulation in molasses. If the invertase activity is too weak, the supply of hydrolyzates for fermentation will be insufficient, but if it is too strong, rapid hydrolysis of sucrose increases the ethanol production is osmolality, and as a result, depressed. YPS30 contained 1% yeast extract, 2% Polypepton and 30% sucrose. Mo120 and Mo130 contained 0.2% ammonium sulfate and concentrated molasses to make the total sugar content either 20 or 3O%(w/v), respectively. Osmolality was measured by an osmometer (Osmette Model 2007, Precision Systems Inc., Massachusetts, USA) from the freezing points of the media. The osmolality in Fig. 4b was calculated from the sugar analysis data by dividing the concentration (g/f) of each component by its molecular weight. All the other materials and methods were as described previously (1). When ethanol fermentation of YOY655 was compared in Mo130 and YPS30, the fermentation rate and ethanol yieId were apparently lower in the former medium (Fig. 1). Since molasses contains fewer amino acids and nucleic bases than YPS30, one possible reason for the slower fermentation in Mo130 is a deficiency of nutrition. However, because supplementing sufficient amounts of casamino acids, adenine and uracil to Mo130 showed no positive effect on either the fermentation rate or the ethanol yield (data not shown), this is not regarded as plausible. Another possibility is the high osmolality in molasses, which contains much larger amounts of salts than the nutrient media. The osmolality of Mo130 was 2112 mosm/kg, while that of YPS30 was 1011 ~_.~ ~~ * Corresponding author.
otc’0
20
40 Time(h)
60
80
FIG. 1. Time courses of ethanol production in YPS30 (open circles) and in Mo130 (filled circles). These media were fermented by YOY655 with shaking at 30°C.
513
514
TAKESHIGE
J. FERMENT. BIOENG..
AND OUCH1
0
20 Time
0’
60
40
-1
0
(h)
10 Molasses
FIG. 2.
Effect of KC1 on ethanol production. KC1 was added to Mo120 as an osmoticum at 0.1 M (filled circles), 0.2M (open triangles), 0.4M (filled triangles), or 0.6M (open squares). These media and Mo120 without KC1 (open circles) were fermented as in Fig. 1.
ternally to the culture in Mo130 after fermentation for 16 h, when most of the pre-existing monosaccharides had been exhausted. The addition of invertase at 0.05 and 0.5 U/ml raised the levels of ethanol production in that order. However, the addition of lOU/ml invertase did not raise the level much higher, but rather reduced it lower than that attained at 0.5 U/ml (Fig. 4a). The increase of osmolality appeared soon after the addition of invertase and it was highest at 10 U/ml (Fig. 4b). Ethanol production was highest at 0.5 U/ml, but this amount of activity might be still too strong because such a transient increase also appeared after the addition of invertase. Ideally, the supply of monosaccharides by invertase should be well balanced with their consumption by the yeast SO that the osmolality in medium remains at a minimum during fermentation. In this respect, the invertase activity of YOY655 is possibly too
20 concentration
30 (“h)
FIG. 3. Relationship between ethanol production rate and molasses concentration. Different concentrations of molasses media were fermented as in Fig. 1. The rate of ethanol production was calculated from the quantity of ethanol produced between 2 and 4 h fermentation.
strong for the fermentation in Mo130, because the rapid hydrolysis of sucrose accumulated fructose and resulted in an increase of osmolality (1). If it was possible to regulate the invertase activity, yeasts such as YOY655 could perform more efficient fermentation in Mol30. We are grateful to Miss M. Umeda for her technical assistance. This work was supported by the project of “Research and Development Concerning Highly Efficient Ethanol Fermentation Techniques by High Productivity Yeasts.” Japanese Ministry of International Trade and Industry’s,
REFERENCES 1. Takeshige, K. and Ouchi, K.: Factors affecting the ethanol productivity of yeast in molasses. J. Ferment. Bioeng., 79, 449452 (1995). 2. Beuchat, L. R.: Influence of water activity on growth, meta-
(a)
(b) 1.2 y
_I
0
20
40 Time
60
80
1.”
(h)
FIG. 4. Effects of invertase activity on (a) ethanol production and (b) osmolality 1B in Mo130 at 0.05 (filled circles), 0.5 U (open triangles), or 10 U/ml (filled triangles) (open circles) was also fermented by X2180-1B with shaking at 30°C.
0
20
40
80
80
Time (h) in molasses. Invertase was added to the culture of X2180after 16 h fermentation. Mo130 without invertase addition
VOL. 79, 1995
bolic activities and survival of yeasts and molds. J. Food Prot., 46, 135-141 (1983). 3. Hahn-Haegerdal, B., Larrson, M., and Mattiasson, B.: Shift in metabolism towards ethanol production in Saccharomyces cerevisiae using alterations of the physical-chemical microen-
NOTES
515
vironment. Biotecbnol. Bioeng. Symp., 12, 199-202 (1982). 4. Jones, R. P., Pamment, N., and Freenfield, P. F.: Alcohol fermentation by yeasts-the effect of environmental and other variables. Process Biochem., 16, 42-49 (1981).