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Reuse of thin stillage from rice spirit for the culture of the yeast Saccharomyces cerevisiae
Process Biochemistry, Vol. 31, No. 6, pp. 617-620, 1996 Copyright © 1996 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0032-9592/96 $15.00 +0.00 ELSEVIER
S0032-9592(96)00011-8
Reuse of Thin Stillage from Rice Spirit for the Culture of the Yeast Saccharomyces cerevisiae Fan-Chaing Yang* & Han-Lin Tung Department of Chemical Engineering,Tunghai University,Taichung, Taiwan 40704, R.O.C. (Received 28 September 1995; revised manuscript received and accepted 21 January 1996)
Distillery effluent from rice spirit production was utilized for high cell density cultivation of Saccharomyces cerevisiae. Different modes of yeast culture, including batch, fed-batch and continuous, were carried out. When pretreated stillage was used as the medium in batch culture, the yeast concentration reached 6 g (dry wt)/litre. Since the pH increased with growth during batch culture, a fed-batch technique with pH control was developed and cell concentration increased to 20 g (dry wt)/litre. In continuous culture, when the dilution rate was maintained at 0"016-0"036/h, cell concentration reached 70 g (dry wt)/litre and the average residual sugar remained below 0.4 g/litre. The results proved the feasibility of the use of thin stillage for yeast culture.
INTRODUCTION
and microbial biomass. With the view of resource recovery, if the organic components in stillage can be reused to produce yeast cells, which can be employed directly as the inoculum in the brewing or wine-making process, not only could the loading of wastewater treatment be diminished, but the utilization efficiency of raw materials would be increased. In this study the stillage was reused for yeast culture and different operation modes were compared to develop an ideal method for high cell density cultivation. 4
Rice spirit is a distilled alcoholic beverage in Taiwan, which is mainly used for Chinese cooking. Rice distilleries produce large volumes of wastewater, known as 'thin stillage', which has a low pH and a high organic content. The stillage generated by distilleries in Taiwan is stated to be around 2 x 106 litre/day with a pollution load up to 25 000 ppm BOD. Although treatments of distillery wastewater have been achieved by activated sludge or anaerobic digestion methods, from the point of view of resource recovery, these methods are not economical. Therefore, the development of an effective system for the utilization of distillery wastewater is highly desirable. Many investigations are underway to discover suitable methods for the utilization of similar types of distillery wastewater, including the production of alcoholic drinks, 1 ethanol, 2 enzymes 3
MATERIALS AND METHODS Yeast strain Saccharomyces cerevisiae THB002, which had been used for wine production, was obtained from Taichung Winery (Taichung, Taiwan). The organism was maintained on slants of YM-agar at 4°C. YM broth (Difco Laboratories) contained the following components (g/litre): yeast
*To whom correspondence should be addressed. 617
618
Fan-Chiang Yang, Han-Lin Tung
Table 1. Compositionof thin stillagefrom rice spirits pH Residual starch (w/v) Total sugars (w/v) Reducing sugar (w/v) Total phosphates (mg/litre) COD (ppm) BOD (ppm)
3-5 0.1% 0.26% 0.112% 128.65 50 920 25 000
extract, 3; malt extract, 3; peptone, 5; dextrose, 10; and for YM-agar, 20 g agar. Media Thin stillage was provided by Taichung Winery (Taichung, Taiwan) and the composition of the stillage is shown in Table 1. As the thin stillage contained some suspended particles, the determination of dry well weight by centrifuging and drying was impossible. In order to overcome this problem, prior to use, the thin stillage was pretreated by filtration using a 1.2 #m filter. YM broth or pretreated stillage were sterilized at 120°C for 20 min before use as growth media. Analytical methods Biomass (cell dry weight) was measured by centrifuging a 10 ml sample at 3000 rpm for 15 min. After discharging the upper clear liquid, the preweighed centrifuge tube containing yeast slurry was dried overnight in an oven at 90°C. The contents of reducing sugar in the media were determined by Bertrand's method. Total acids were determined by titration with 0-1 N NaOH. Experimental methods
Shake flask culture YM broth was used in preliminary tests to study the effect of pH on the growth of S. cerevisiae THB002. The pretreated stillage without any additives or pH adjustment was used directly as the medium in flask culture. 300 ml of YM broth or the pretreated stillage was prepared in a 500-ml Erlenmeyer flask and after sterilization and inoculation, the flask was shaken (Model 902, Hotech Co.) at 110 rpm and 32°C. A 24 h flask culture using the stillage was employed for the preparation of the inoculum in the fermenter culture. Batch fermenter Batch culture was carried out in a 2-1itre jar
fermenter (NBS BIOFLO Model C32) containing 0.9 litre of the pretreated stillage without any additives or pH adjustment. After autoclave sterilization and cooling, 100 ml of a 24-h old culture was inoculated into the fermenter. Air flow rate and temperature were controlled at 1 vvm and 32°C, respectively. The agitation speed of the impeller was controlled at 400 rpm. Samples were taken at regular intervals to determine cell weight and pH.
Fed-batch and continuous cultures Fed-batch and continuous cultures were carried out in a 10-1itre bench-scale fermenter (B. Braun BIOMATE E) which contained initially 4.5 litre pretreated stillage without any additives or pH adjustment. After autoclave sterilization and cooling, 500 ml of a 24-h old culture was inoculated into the fermenter. The fermenter propagation was conducted at 32°C, with an agitation rate of 600 rpm and an aeration rate at 1 vvm. For use in fed-batch culture the pretreated stillage was supplemented with 125 g/litre glucose, 10 g/litre KH2PO4, 10 g/litre (NH4)2SO4and the pH was adjusted to 2.0 with HCI. Since the pH of the broth increased during the batch culture, the feed medium was added via the pH controller. This pH-stat feeding strategy was therefore used both for pH control and nutrient provision. Samples were taken at regular intervals to determine cell weight and reducing sugar. For use in continuous culture the pretreated stillage was enriched with 125 g/litre glucose, 10 g/litre KH2PO 4 and 10 g/litre (NH4)2SO4, but without pH adjustment. The medium and the effluent were pumped into or out of the fermenter simultaneously with a multi-tube peristaltic pump. The dilution rate was controlled at the level of 0.016-0.036/h. Samples were taken at regular intervals to determine cell weight and reducing sugar. The pH was controlled at 4 with HCI and NaOH.
RESULTS AND DISCUSSION Effect of initial pH on cell growth The effect of initial pH on cell growth in shake flask culture is indicated in Fig. 1. The optimum initial pH for the growth of S. cerevisiae THB002 is around 3.5.
Cultivation of Saccharomyces cerevisiae using thin stillage
619
8
7 4
~6
3
~4
i
2
2
,/
11~.5
I
I
2.5
I
3.5
I
4.5
• Cell density o pH
•
•
5.5
I 48
6.5
I
I
I
96
144
192
Time (hr)
pH
Fig. 1. Effect of initial p H on cell growth in shake flask culture of Saccharomyces cerevisiae T H B 0 0 2 in YM
Fig. 3. T i m e course of batch fermenter culture using thin stillage as the medium.
media.
Shake flask The time course of shake flask culture is shown in Fig. 2. Cell mass increased with a decrease of total acids. Since only a little reducing sugar remained in thin stillage, some other components must be used as carbon sources for yeast growth, these were assumed to be organic acids. Batch fermenter culture The variations of cell mass and pH during batch culture using a 2-1itre jar fermenter are shown in Fig. 3. The cell concentration in batch culture reached 6 g dry wt. The pH rose along with cell mass and the increase of cell mass and pH seemed to be strongly associated. As described earlier, this phenomenon could be attributed to the fact that organic acids or amino acids in thin stillage were consumed as C-sources or N-
sources in the culture. These characteristics were applied to design a pH-stat feeding strategy in the following fed-batch culture. Fed-batch culture The peristaltic pump for pH control of acid feeding was also used as a nutrient feeding pump. The pretreated stillage was enriched with glucose to study the effects of glucose concentrations on yeast growth and cell density. 4 Higher concentrations of glucose enhanced cell density but did not achieve effective utilisation of raw materials due to the Crabtree effect. The concentration of glucose added to the feed medium was thus adjusted to 125 g/litre in fedbatch culture (Fig. 4). Since the total amount of glucose fed by the pH controller system was small, the cell concentration achieved was
20
18 16
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0.3 ~
~
8 ~ 10 e~
~-o_o_.~. ~_o___, ~
"~ 6 0~
4
Cell
0 Total acids
/
I
I
I
0
40
S0
120
2
0 160
/
/
/
0
I
I
I
I
I
I
24
48
72
96
120
144
Time (hr)
Time (hour)
Fig. 2. T i m e course of shake flask culture using thin
Fig. 4. T i m e course of fed-batch culture with the pH-
stillage as the medium.
stat method.
Fan-Chiang Yang, Han-Lin Tung
620
limited to 20 g (dry wt)/litre at the stage of fedbatch culture. Some limitations, of the pH-stat control strategy were discovered. Thus during the final stage of fed-batch culture the variations of pH values became less sensitive to the increase of yeast cells and the supply of thin stillage from the acid pump was insufficient to meet the demand of yeast growth. The final concentration of cell mass was therefore restricted to around 20 g (dry wt)/litre.
8C
7O
t-
60
~ 5o
4O 0.01
Continuous culture In order to improve the situation mentioned previously, a continuous culture was carried out at the end of the fed-batch culture. The dilution rate was initially set at 0.016/h. The results shown in Fig. 5 are the whole time course of fed-batch and continuous cultures. Cell mass increased rapidly to 44 g (dry wt)/litre in 32 h and finally reached 70 g (dry wt)/litre at steady state. Residual sugar concentration remained below 0-4 g/litre and demonstrated a high utilization of carbon source. The effect of dilution rate on cell density is indicated in Fig. 6. Cell concentrations were maintained at 70 g (dry wt)/litre. Since the aeration rate was maintained at the same level of 1 vvm throughout, the con-
I 0.02
I 0.03 Dilution rate (l/hr)
0.04
Fig. 6. Effect of dilution rate on cell density in continuous culture.
centration of dissolved oxygen fell to less than 10% at a dilution rate of 0.036 h - l . CONCLUSION These results indicate the feasibility of using thin stillage for yeast culture. Although the suspended solids remaining in thin stillage were removed by filtration in this study, these were presumed to have no adverse effect on yeast culture. Therefore, removal of suspended solids should be unnecessary in practice.
ACKNOWLEDGEMENT
8O 70
o
00
/°°
°e°%o
/
The authors wish to thank the National Science Council of R.O.C. for financial support (NSC 82-0410-E-029003).
~ 50 ~" 40 "~
Fed-batch process
/ f
-7 30
REFERENCES
Continuous process
]
!-
20
]0 /f'--'--''''''" 0
[ 48
I 96
I 144 Time (hr)
I 192
I 240
288
Fig. 5. Time course of fed-batch culture with the pHstar method and continuous culture at the dilution rate of 0.016-0"036/h.
1. Ronkainen, P., Leppanen, O. & Harju, K., The reuse of stillage water in the mashing of grain as a means of energy conservation. J. Inst. Brew., 84 (1978) 115-17. 2. Ueda, S., Teramoto, Y., Ohba, R., Ueki, T., Kimura, K. & Shiota, S., Batchwise ethanol fermentation with shochu distillery waste. J. Ferment. Bioeng., 72 (1991) 270-3. 3. Morimura, S., Kida, K., Yakita, Y., Sondon, Y. & Myoga, H . , Production of saccharifying enzyme using the wastewater of a shochu distillery. J. Ferment. Bioeng., 71 (1991) 329-34. 4. Tung, H. L. M., Sci. Thesis, Department of Chemical Engineering, Tunghai University, Taiwan, 1993.
S0032-9592(96)00011-8
Reuse of Thin Stillage from Rice Spirit for the Culture of the Yeast Saccharomyces cerevisiae Fan-Chaing Yang* & Han-Lin Tung Department of Chemical Engineering,Tunghai University,Taichung, Taiwan 40704, R.O.C. (Received 28 September 1995; revised manuscript received and accepted 21 January 1996)
Distillery effluent from rice spirit production was utilized for high cell density cultivation of Saccharomyces cerevisiae. Different modes of yeast culture, including batch, fed-batch and continuous, were carried out. When pretreated stillage was used as the medium in batch culture, the yeast concentration reached 6 g (dry wt)/litre. Since the pH increased with growth during batch culture, a fed-batch technique with pH control was developed and cell concentration increased to 20 g (dry wt)/litre. In continuous culture, when the dilution rate was maintained at 0"016-0"036/h, cell concentration reached 70 g (dry wt)/litre and the average residual sugar remained below 0.4 g/litre. The results proved the feasibility of the use of thin stillage for yeast culture.
INTRODUCTION
and microbial biomass. With the view of resource recovery, if the organic components in stillage can be reused to produce yeast cells, which can be employed directly as the inoculum in the brewing or wine-making process, not only could the loading of wastewater treatment be diminished, but the utilization efficiency of raw materials would be increased. In this study the stillage was reused for yeast culture and different operation modes were compared to develop an ideal method for high cell density cultivation. 4
Rice spirit is a distilled alcoholic beverage in Taiwan, which is mainly used for Chinese cooking. Rice distilleries produce large volumes of wastewater, known as 'thin stillage', which has a low pH and a high organic content. The stillage generated by distilleries in Taiwan is stated to be around 2 x 106 litre/day with a pollution load up to 25 000 ppm BOD. Although treatments of distillery wastewater have been achieved by activated sludge or anaerobic digestion methods, from the point of view of resource recovery, these methods are not economical. Therefore, the development of an effective system for the utilization of distillery wastewater is highly desirable. Many investigations are underway to discover suitable methods for the utilization of similar types of distillery wastewater, including the production of alcoholic drinks, 1 ethanol, 2 enzymes 3
MATERIALS AND METHODS Yeast strain Saccharomyces cerevisiae THB002, which had been used for wine production, was obtained from Taichung Winery (Taichung, Taiwan). The organism was maintained on slants of YM-agar at 4°C. YM broth (Difco Laboratories) contained the following components (g/litre): yeast
*To whom correspondence should be addressed. 617
618
Fan-Chiang Yang, Han-Lin Tung
Table 1. Compositionof thin stillagefrom rice spirits pH Residual starch (w/v) Total sugars (w/v) Reducing sugar (w/v) Total phosphates (mg/litre) COD (ppm) BOD (ppm)
3-5 0.1% 0.26% 0.112% 128.65 50 920 25 000
extract, 3; malt extract, 3; peptone, 5; dextrose, 10; and for YM-agar, 20 g agar. Media Thin stillage was provided by Taichung Winery (Taichung, Taiwan) and the composition of the stillage is shown in Table 1. As the thin stillage contained some suspended particles, the determination of dry well weight by centrifuging and drying was impossible. In order to overcome this problem, prior to use, the thin stillage was pretreated by filtration using a 1.2 #m filter. YM broth or pretreated stillage were sterilized at 120°C for 20 min before use as growth media. Analytical methods Biomass (cell dry weight) was measured by centrifuging a 10 ml sample at 3000 rpm for 15 min. After discharging the upper clear liquid, the preweighed centrifuge tube containing yeast slurry was dried overnight in an oven at 90°C. The contents of reducing sugar in the media were determined by Bertrand's method. Total acids were determined by titration with 0-1 N NaOH. Experimental methods
Shake flask culture YM broth was used in preliminary tests to study the effect of pH on the growth of S. cerevisiae THB002. The pretreated stillage without any additives or pH adjustment was used directly as the medium in flask culture. 300 ml of YM broth or the pretreated stillage was prepared in a 500-ml Erlenmeyer flask and after sterilization and inoculation, the flask was shaken (Model 902, Hotech Co.) at 110 rpm and 32°C. A 24 h flask culture using the stillage was employed for the preparation of the inoculum in the fermenter culture. Batch fermenter Batch culture was carried out in a 2-1itre jar
fermenter (NBS BIOFLO Model C32) containing 0.9 litre of the pretreated stillage without any additives or pH adjustment. After autoclave sterilization and cooling, 100 ml of a 24-h old culture was inoculated into the fermenter. Air flow rate and temperature were controlled at 1 vvm and 32°C, respectively. The agitation speed of the impeller was controlled at 400 rpm. Samples were taken at regular intervals to determine cell weight and pH.
Fed-batch and continuous cultures Fed-batch and continuous cultures were carried out in a 10-1itre bench-scale fermenter (B. Braun BIOMATE E) which contained initially 4.5 litre pretreated stillage without any additives or pH adjustment. After autoclave sterilization and cooling, 500 ml of a 24-h old culture was inoculated into the fermenter. The fermenter propagation was conducted at 32°C, with an agitation rate of 600 rpm and an aeration rate at 1 vvm. For use in fed-batch culture the pretreated stillage was supplemented with 125 g/litre glucose, 10 g/litre KH2PO4, 10 g/litre (NH4)2SO4and the pH was adjusted to 2.0 with HCI. Since the pH of the broth increased during the batch culture, the feed medium was added via the pH controller. This pH-stat feeding strategy was therefore used both for pH control and nutrient provision. Samples were taken at regular intervals to determine cell weight and reducing sugar. For use in continuous culture the pretreated stillage was enriched with 125 g/litre glucose, 10 g/litre KH2PO 4 and 10 g/litre (NH4)2SO4, but without pH adjustment. The medium and the effluent were pumped into or out of the fermenter simultaneously with a multi-tube peristaltic pump. The dilution rate was controlled at the level of 0.016-0.036/h. Samples were taken at regular intervals to determine cell weight and reducing sugar. The pH was controlled at 4 with HCI and NaOH.
RESULTS AND DISCUSSION Effect of initial pH on cell growth The effect of initial pH on cell growth in shake flask culture is indicated in Fig. 1. The optimum initial pH for the growth of S. cerevisiae THB002 is around 3.5.
Cultivation of Saccharomyces cerevisiae using thin stillage
619
8
7 4
~6
3
~4
i
2
2
,/
11~.5
I
I
2.5
I
3.5
I
4.5
• Cell density o pH
•
•
5.5
I 48
6.5
I
I
I
96
144
192
Time (hr)
pH
Fig. 1. Effect of initial p H on cell growth in shake flask culture of Saccharomyces cerevisiae T H B 0 0 2 in YM
Fig. 3. T i m e course of batch fermenter culture using thin stillage as the medium.
media.
Shake flask The time course of shake flask culture is shown in Fig. 2. Cell mass increased with a decrease of total acids. Since only a little reducing sugar remained in thin stillage, some other components must be used as carbon sources for yeast growth, these were assumed to be organic acids. Batch fermenter culture The variations of cell mass and pH during batch culture using a 2-1itre jar fermenter are shown in Fig. 3. The cell concentration in batch culture reached 6 g dry wt. The pH rose along with cell mass and the increase of cell mass and pH seemed to be strongly associated. As described earlier, this phenomenon could be attributed to the fact that organic acids or amino acids in thin stillage were consumed as C-sources or N-
sources in the culture. These characteristics were applied to design a pH-stat feeding strategy in the following fed-batch culture. Fed-batch culture The peristaltic pump for pH control of acid feeding was also used as a nutrient feeding pump. The pretreated stillage was enriched with glucose to study the effects of glucose concentrations on yeast growth and cell density. 4 Higher concentrations of glucose enhanced cell density but did not achieve effective utilisation of raw materials due to the Crabtree effect. The concentration of glucose added to the feed medium was thus adjusted to 125 g/litre in fedbatch culture (Fig. 4). Since the total amount of glucose fed by the pH controller system was small, the cell concentration achieved was
20
18 16
j..--./
0.3 ~
~
8 ~ 10 e~
~-o_o_.~. ~_o___, ~
"~ 6 0~
4
Cell
0 Total acids
/
I
I
I
0
40
S0
120
2
0 160
/
/
/
0
I
I
I
I
I
I
24
48
72
96
120
144
Time (hr)
Time (hour)
Fig. 2. T i m e course of shake flask culture using thin
Fig. 4. T i m e course of fed-batch culture with the pH-
stillage as the medium.
stat method.
Fan-Chiang Yang, Han-Lin Tung
620
limited to 20 g (dry wt)/litre at the stage of fedbatch culture. Some limitations, of the pH-stat control strategy were discovered. Thus during the final stage of fed-batch culture the variations of pH values became less sensitive to the increase of yeast cells and the supply of thin stillage from the acid pump was insufficient to meet the demand of yeast growth. The final concentration of cell mass was therefore restricted to around 20 g (dry wt)/litre.
8C
7O
t-
60
~ 5o
4O 0.01
Continuous culture In order to improve the situation mentioned previously, a continuous culture was carried out at the end of the fed-batch culture. The dilution rate was initially set at 0.016/h. The results shown in Fig. 5 are the whole time course of fed-batch and continuous cultures. Cell mass increased rapidly to 44 g (dry wt)/litre in 32 h and finally reached 70 g (dry wt)/litre at steady state. Residual sugar concentration remained below 0-4 g/litre and demonstrated a high utilization of carbon source. The effect of dilution rate on cell density is indicated in Fig. 6. Cell concentrations were maintained at 70 g (dry wt)/litre. Since the aeration rate was maintained at the same level of 1 vvm throughout, the con-
I 0.02
I 0.03 Dilution rate (l/hr)
0.04
Fig. 6. Effect of dilution rate on cell density in continuous culture.
centration of dissolved oxygen fell to less than 10% at a dilution rate of 0.036 h - l . CONCLUSION These results indicate the feasibility of using thin stillage for yeast culture. Although the suspended solids remaining in thin stillage were removed by filtration in this study, these were presumed to have no adverse effect on yeast culture. Therefore, removal of suspended solids should be unnecessary in practice.
ACKNOWLEDGEMENT
8O 70
o
00
/°°
°e°%o
/
The authors wish to thank the National Science Council of R.O.C. for financial support (NSC 82-0410-E-029003).
~ 50 ~" 40 "~
Fed-batch process
/ f
-7 30
REFERENCES
Continuous process
]
!-
20
]0 /f'--'--''''''" 0
[ 48
I 96
I 144 Time (hr)
I 192
I 240
288
Fig. 5. Time course of fed-batch culture with the pHstar method and continuous culture at the dilution rate of 0.016-0"036/h.
1. Ronkainen, P., Leppanen, O. & Harju, K., The reuse of stillage water in the mashing of grain as a means of energy conservation. J. Inst. Brew., 84 (1978) 115-17. 2. Ueda, S., Teramoto, Y., Ohba, R., Ueki, T., Kimura, K. & Shiota, S., Batchwise ethanol fermentation with shochu distillery waste. J. Ferment. Bioeng., 72 (1991) 270-3. 3. Morimura, S., Kida, K., Yakita, Y., Sondon, Y. & Myoga, H . , Production of saccharifying enzyme using the wastewater of a shochu distillery. J. Ferment. Bioeng., 71 (1991) 329-34. 4. Tung, H. L. M., Sci. Thesis, Department of Chemical Engineering, Tunghai University, Taiwan, 1993.