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Continuous glycerol production in a packed-bed bioreactor with immobilized cells of Saccharomyces cerevisiae
Bioresource Technology 49 (1994) 209-212 © 1994 Elsevier Science Limited Printed in Great Britain. All rights reserved 0260-8774/94/$7.00 0960-8524(94)00029-8
ELSEVIER
CONTINUOUS GLYCEROL PRODUCTION IN A PACKED-BED BIOREACTOR WITH IMMOBILIZED CELLS OF SA CCHAR OMYCES CERE VISIAE G. Gonzfilez Benito,* M. Ozores & M. Pefia Department of Chemical Engineering, Faculty of Sciences, Universityof Valladolid, Prado de la Magdalena s/n, 47005-Valladolid, Spain (Received 15 April 1994; revised version received 10 May 1994; accepted 5 June 1994)
Abstract Batch and continuous glycerol fermentations of sugar beet molasses have been studied using sulphite as a combining agent with the aldehyde function. The processes were carried out in a vertical packed-bed reactor with Saccharomyces cerevisiae yeast cells immobilized in sintered glass rings. The influence of sulphite dosage and the dilution rate on the performance of the bioreactor was studied. A maximum glycerol concentration of 30 g dm -3 and a productivity of 36"6 g dm-3 d -t at T=28°C, p H = 6"9 with 25 g dm -3 sodium sulphite, for a dilution rate of 1"22 d-i, was achieved.
The aim of this work was the study of a fermentation process for the production of glycerol from sugar beet molasses in a vertical packed-bed reactor with Saccharomyces cerevisiae yeast cells immobilized in sintered glass rings. Sodium sulphite was used as steering agent. Initial batch experiments were carried out in order to determine the optimum dosage of sulphite. Afterwards, the experimentation was completed working in a continuous mode, and the influence of the dilution rate on the yield of glycerol was studied.
Key words: glycerol, Saccharomyces cerevisiae, immobilized cells.
Microorganism Saccharomyces cerevisiae, provided by the sugarfactory ACOR-Valladolid, was used. The microorganism was maintained in Petri plates as described elsewhere (Bravo & Gonzalez Benito, 1991). The yeasts were precultured at 30°C under aerobic conditions, continuously shaken (250 rpm) in a rotary shaker. The composition of this preculture medium, previously sterilized at 120°C for 15 min, was (dm-3): 20 g sucrose equivalent from molasses, 5 g soya peptone, 3 g yeast extract, and pH = 3.9.
METHODS
INTRODUCTION Glycerol can be obtained from anaerobic ethanol fermentation. The glycerol is a subproduct of the reaction, produced in a concentration of 2-5 g d m - 3. Enhancement of the yield of glycerol is attained by modifying the fermentation process by means of the addition of sodium sulphite, which reacts with the acetaldehyde formed as an intermediate during the normal course of the fermentation (Freeman & Donald, 1956; Harris & Hajny, 1960; Maier et al., 1986). Vacuum fermentation (Virkar & Panesar, 1987) and the application of CO2 gassing (Kalle et al., 1985; Bisping et al., 1989) are other ways of increasing the glycerol yield and/or reducing the sulphite requirement. In the literature there have been several reports about the glycerol fermentation: use of different substrates with free cells (Vijaikishore & Karanth, 1984; Vijaikishore & Karanth, 1986; Virkar & Panesar, 1987); working in batch or continuous methods and the application of supports for the immobilization of the yeast while using sucrose as substrate (Bisping & Rehm, 1986; Hecker et al., 1990).
Bioreactor The batch and continuous processes were carried out in a jacketed tubular reactor, 34-5 cm length and 5-2 cm internal diameter, made of methyl methacrylate. The top of the column reactor was fitted with a decanter to allow removal of CO2 gas from the liquid phase and to permit entrapment of possible fragments from the sintered glass, free yeasts, etc. The total volume of the fermentator was 3-5 dm 3, the liquid volume was 2.3 dm 3 and the available volume of the bed was 0"658 dm 3. The fermentation medium was recirculated through the column reactor by a centrifugal pump. The reactor temperature was maintained at 28°C. A peristaltic pump was used for feeding the reactor.
*To whom correspondence should be addressed. 209
210
G. Gonzflez Benito, M. Ozores, M. Pefia
Immobilization Immobilization was carried out on sintered glass rings; Siran, 0'9 × 0"9 cm, provided by S.A. Schott Iberica (Avda. de Roma 2, Barcelona, Spain). The fixed-bed column (Fig. 1) was filled with glass rings previously sterilized (120°C for 15 min). Cells harvested from a preculture medium were suspended in 500 ml of a solution whose concentration ~vas (dm-3): MgSO4, 0.55 g; (NH4)2SO4, 5.10 g; KH2PO4, 1.53 g. This solution was made to circulate downstream through the column reactor by means of a peristaltic pump (flow rate; 5 dm 3 h -1 ) for 72 h at ambient temperature (Bisping & Rehm, 1986). Feedstock medium The feedstock medium was made from crude sugar beet molasses and diluted to obtain a suitable concentration of sucrose. Then, the molasses was roughly filtered to avoid the solids settled over the pores of the glass support and pasteurized at 85°C for 10 min before the nutrients were added non-aseptically. The composition of the fermentation medium was (dm-3): equivalent sucrose from molasses, 120g; (NH4)CI, 4.90g; KH2PO 4, 1"53g and MgSO 4, 0"55g as nutrients. Batch and continuous fermentations After the immobilization of the yeast, the saline medium was replaced by fresh feedstock medium. The reactor was placed on recycle mode during the whole time of the batch and continuous fermentations. The pH level and the temperature inside the reactor were maintained at 6.9 and 28°C, respectively. Different sodium sulphite concentrations in powder form (not sterilized) were added in two equal portions as indicated in the literature. The first portion was added on starting the fermentation and the other portion 2 h later. The batch period was 40 h. Continuous fermentation began after an initial batch phase, feeding the reactor with the feedstock solution when the sucrose from the molasses medium was almost consumed. Sodium sulphite was added during
~luent
temperature controller
feed
recycle
pump
pump
Fig. 1. Schematic diagram of the fixed-bed bioreactor experimental system.
continuous fermentation in order to maintain the initial concentration. Different experiments were conducted operating with different dilution rates. The dilution rate was based on the volume of the bed. The reactor pH and temperature were maintained at 6"9 and 28°C, respectively throughout the experiments. For each new value of the dilution rate the experimentation was allowed a run-time of approximately 8 days. For each run, samples of the effluent were analyzed daily for suspended yeast cells, carbohydrates, ethanol and glycerol. The immobilized yeast-cells' concentration was analyzed weekly. Analytical methods Concentrations of carbohydrates, glycerol and ethanol were determined by HPLC using a Waters Sugar PAK TM 1 column and 50 mg dm -3 sodium calcium A E D T salt solution at 75°C as mobile phase and a flow rate of 0.7 ml min-1. Eluted materials were detected using a Waters differential refractometer detector 410 (Waters Chromatography Division, Millipore Corporation, 34 Maple Street, Milford, MA 01757). Determinations of pH were made by a glass electrode with an Aqualytic pH meter 21. A platinum electrode (Ingold) was used for the redox potential determinations. Oxygen determinations were carried out with a probe (Aqualytic oxi 921 ). The number of adsorbed cells per gram of sintered glass rings was determined by releasing the cells from the support in 100 ml of sterilized water by use of sonication. Cells were then counted in a Thoma-hematocytometer using a Nikon microscope. This counting method was also employed to determine the yeast cell concentration in the bulk liquid. The methylene blue staining method was used to determine the cell viability (Bravo & Gonzfilez Benito, 1991 ). RESULTS AND DISCUSSION In order to work under completely mixed conditions, the reactor was always operating in recycle mode with a high recirculation flow, about 40 times the feed flow. The sucrose concentration in the molasses of the feedstock medium was maintained at 120 g dm -3. Batch experiments were carried out in order to analyze the influence of the concentration of sulphite on the yield of glycerol. The sulphite was dosed as mentioned previously. Then, starting from the results of the batch experiments, it was operated in continuous form, working with different dilution rates. Finally, a continuous fermentation was developed using the best operating conditions. Batch experiments Different fermentation runs were carried out adding different doses of sodium sulphite (20, 25, 30 and 40 g dm -3) to the medium in order to analyze the influence of the sulphite concentration over the fermentation process. The experiment was repeated three times for each dose of sulphite. Figure 2 shows the
Glycerol production in a packed-bed reactor 50-
211
60
50
40
0.76 ~,
_J
~=
4O
J,
1.22 cl"
~
J,
¢
106d,
o
o~20-
§ ao
8 10
10
0
O2O
25
30
0
4O
i
i
1O0
200
i
300
Batch fermentation. Influence of sulphite concentration: [] glycerol, ~ ethanol.
average concentrations of glycerol and ethanol obtained versus the sulphite dosage after 40 h of fermentation. The results indicate that there was a range between 25-30 g 1-J of sulphite where the glycerol concentration was highest. Less ethanol was obtained when the sulphite dose was 25 g 1-1. Therefore, the following experiments were carried out with this dosage of sulphite.
Continuous experimentation Continuous experimentation was carried out starting from the results of the batch experiments cited above. The initial dosage of sulphite used was 25 g dm -3 added to the molasses feedstock medium in the usual form, and after 40 h the bioreactor was continuously fed. Temperature and pH were maintained in all experiments at 28°C and 6.9, respectively, and sodium sulphite was added throughout the process in order to maintain the initial sulphite concentration and the redox potential. It was operated with various dilution rates (D = 0"76, 1"06, 1-22 and 1"52 d-t). Figure 3 shows the behaviour of the continuous fermentation, i.e. evolution of the product concentrations throughout experimentation. These experimentation runs were repeated twice• In this figure, the arrows indicate a new value of the dilution rate in the bioreactor. It is seen that as the dilution rate was raised the glycerol concentration gradually decreased. When the dilution rate was higher than 1-22 d - 1 the glycerol concentration was drastically reduced. With a dilution rate of 1"52 d -~ the lowest glycerol concentration was obtained. However, the bioreactor was able to recover its glycerol production capacity when the dilution rate was lowered. The dependence of the glycerol production on dilution rate is shown in Fig. 4. The maximum productivity, 36.6 g dm -3 d t, was attained for a dilution rate of 1.22 d- 1. For this case, the glycerol concentration was 3 0 g l -l Finally, a new, continuous experiment was carried out, working with the experimental conditions that pro-
=
500
600
700
800
T i m e (h)
s u l p h i t e c o n c e n t r a t i o n (g/L)
Fig. 2.
400
Fig. 3.
Continuous glycerol production. Alteration of dilution rate; • glycerol, ~ ethanol.
50
~
50
40
-~
30
/N
40
•
\
30
._~ ._
10
0,5
10
1
1,5
;0 2
D(d ~)
Fig. 4. Continuous glycerol production. Influence of dilution rate: • glycerol concentration, ~ glycerol productivity.
vided the maximum glycerol productivity in order to maintain the fermentation for a long period of time. The starting conditions were: sucrose as molasses, 120gdm-3; sulphite, 2 5 g d m - 3 ; D=1.22d-1; T = 28°C; and pH = 6.9. For these operating conditions, the redox potential value remained essentially constant (between - 1 4 0 and - 1 6 0 mV) due to the constant presence in the medium of sodium sulphite. The oxygen concentration was less than 0.2 ppm. A suitable behaviour of the bioreactor could be achieved for a total experimentation period of 700 h. The productivity was maintained at about 36 _+ 1 g dm -3 d - ~ with a glycerol yield of 25% (based on consumed sucrose from molasses). Through the entire run, few yeasts were released into the medium. The cells of Saccharomyces cerevisiae were perfectly immobilized in the sintered glass rings and were arranged in clusters in the centre of the glass pores, forming large ag~egatiQns, as was shown by scanmng electron n u c r o g r a p ~ T h e same pattern of growth has been noted by other authors (Hecker et al., 1990). Nevertheless, the cell viability at the end of the fermentation period (700 h) was about 60% due to the prolonged presence of
212
G. Gonz6lez Benito, M. Ozores, M. Pefta
sulphite in the medium. When the experimentation run was prolonged more than approximately 700 h, the cells' viability decreased and a large amount of free yeast cells began to be released in to the culture medium. Moreover, contamination appeared in the bioreactor. In conclusion, the sintered-glass rings were suitable as a matrix for the immobilization of yeast cells in a fermentation process for glycerol production from sugarbeet molasses without treatment (only rough filtration and pasteurization) attaining a good glycerol productivity.
REFERENCES Bisping, B. & Rehm, H. J. (1986). Glycerol production by cells of Saccharomyces cerevisiae, immobilized in sintered glass. Microbiol. Biotechnol., 23, 174-9. Bisping, B., Hecker, D. & Rehm, H. J. (1989). Glycerol production by semicontinuous fed-batch fermentation with immobilized cells of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 32, 119-23. Bravo, P. & Gonz~ilez Benito, G. (1991). Continuous ethanol
fermentation by immobilized yeast cells in a fluidized-bed reactor. J. Chem. Tech. Biotechnol., 52, 127-34. Freeman, G. G. & Donald, G. M. S. (1956). The influence of certain variables on glycerol formation in the presence of sulfites. Appl. Microbiol., 5, 197-210. Harris, J. F. & Hajny, G. J. (1960). Glycerol production: a pilot plant investigation for continuous fermentation and recovery. Biochem. Microbiol. Technol. Eng., 2, 9-24. Hecker, D., Bisping, B. & Rehm, H. J. (1990). Continuous glycerol production by the sulphite process with immobilized cells of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 32,627-32. Kalle, G. P., Naik, S. C. & Lashkari, B. Z. (1985). Improved glycerol production from cane molasses by the sulfite process with vacuum or continuous carbon dioxide sparging during fermentation. J. Ferment. Technol., 63, 231-7. Maier, K., Hinze, H. & Leuschel, L. (1986). Mechanism of sulfite action on the energy metabolism of Saccharomyces cerevisiae. Biochim. Biophys. Acta, 848, 120-30. Vijaikishore, P. & Karanth, N. G. (1984). Glycerol production from glucose in alkaline medium. Biotechnol. Lett., 6, 103-8. Vijaikishore, P. & Karanth, N. G. (1986). Glycerol production by immobilized cells of Pichia farinosa. Biotechnol. Lett., 8, 257-60. Virkar, P. D. & Panesar, M. S. (1987). Glycerol production by anaerobic vacuum fermentation of molasses on pilot scale. Biotechnol. Bioeng., 29, 773-4.
ELSEVIER
CONTINUOUS GLYCEROL PRODUCTION IN A PACKED-BED BIOREACTOR WITH IMMOBILIZED CELLS OF SA CCHAR OMYCES CERE VISIAE G. Gonzfilez Benito,* M. Ozores & M. Pefia Department of Chemical Engineering, Faculty of Sciences, Universityof Valladolid, Prado de la Magdalena s/n, 47005-Valladolid, Spain (Received 15 April 1994; revised version received 10 May 1994; accepted 5 June 1994)
Abstract Batch and continuous glycerol fermentations of sugar beet molasses have been studied using sulphite as a combining agent with the aldehyde function. The processes were carried out in a vertical packed-bed reactor with Saccharomyces cerevisiae yeast cells immobilized in sintered glass rings. The influence of sulphite dosage and the dilution rate on the performance of the bioreactor was studied. A maximum glycerol concentration of 30 g dm -3 and a productivity of 36"6 g dm-3 d -t at T=28°C, p H = 6"9 with 25 g dm -3 sodium sulphite, for a dilution rate of 1"22 d-i, was achieved.
The aim of this work was the study of a fermentation process for the production of glycerol from sugar beet molasses in a vertical packed-bed reactor with Saccharomyces cerevisiae yeast cells immobilized in sintered glass rings. Sodium sulphite was used as steering agent. Initial batch experiments were carried out in order to determine the optimum dosage of sulphite. Afterwards, the experimentation was completed working in a continuous mode, and the influence of the dilution rate on the yield of glycerol was studied.
Key words: glycerol, Saccharomyces cerevisiae, immobilized cells.
Microorganism Saccharomyces cerevisiae, provided by the sugarfactory ACOR-Valladolid, was used. The microorganism was maintained in Petri plates as described elsewhere (Bravo & Gonzalez Benito, 1991). The yeasts were precultured at 30°C under aerobic conditions, continuously shaken (250 rpm) in a rotary shaker. The composition of this preculture medium, previously sterilized at 120°C for 15 min, was (dm-3): 20 g sucrose equivalent from molasses, 5 g soya peptone, 3 g yeast extract, and pH = 3.9.
METHODS
INTRODUCTION Glycerol can be obtained from anaerobic ethanol fermentation. The glycerol is a subproduct of the reaction, produced in a concentration of 2-5 g d m - 3. Enhancement of the yield of glycerol is attained by modifying the fermentation process by means of the addition of sodium sulphite, which reacts with the acetaldehyde formed as an intermediate during the normal course of the fermentation (Freeman & Donald, 1956; Harris & Hajny, 1960; Maier et al., 1986). Vacuum fermentation (Virkar & Panesar, 1987) and the application of CO2 gassing (Kalle et al., 1985; Bisping et al., 1989) are other ways of increasing the glycerol yield and/or reducing the sulphite requirement. In the literature there have been several reports about the glycerol fermentation: use of different substrates with free cells (Vijaikishore & Karanth, 1984; Vijaikishore & Karanth, 1986; Virkar & Panesar, 1987); working in batch or continuous methods and the application of supports for the immobilization of the yeast while using sucrose as substrate (Bisping & Rehm, 1986; Hecker et al., 1990).
Bioreactor The batch and continuous processes were carried out in a jacketed tubular reactor, 34-5 cm length and 5-2 cm internal diameter, made of methyl methacrylate. The top of the column reactor was fitted with a decanter to allow removal of CO2 gas from the liquid phase and to permit entrapment of possible fragments from the sintered glass, free yeasts, etc. The total volume of the fermentator was 3-5 dm 3, the liquid volume was 2.3 dm 3 and the available volume of the bed was 0"658 dm 3. The fermentation medium was recirculated through the column reactor by a centrifugal pump. The reactor temperature was maintained at 28°C. A peristaltic pump was used for feeding the reactor.
*To whom correspondence should be addressed. 209
210
G. Gonzflez Benito, M. Ozores, M. Pefia
Immobilization Immobilization was carried out on sintered glass rings; Siran, 0'9 × 0"9 cm, provided by S.A. Schott Iberica (Avda. de Roma 2, Barcelona, Spain). The fixed-bed column (Fig. 1) was filled with glass rings previously sterilized (120°C for 15 min). Cells harvested from a preculture medium were suspended in 500 ml of a solution whose concentration ~vas (dm-3): MgSO4, 0.55 g; (NH4)2SO4, 5.10 g; KH2PO4, 1.53 g. This solution was made to circulate downstream through the column reactor by means of a peristaltic pump (flow rate; 5 dm 3 h -1 ) for 72 h at ambient temperature (Bisping & Rehm, 1986). Feedstock medium The feedstock medium was made from crude sugar beet molasses and diluted to obtain a suitable concentration of sucrose. Then, the molasses was roughly filtered to avoid the solids settled over the pores of the glass support and pasteurized at 85°C for 10 min before the nutrients were added non-aseptically. The composition of the fermentation medium was (dm-3): equivalent sucrose from molasses, 120g; (NH4)CI, 4.90g; KH2PO 4, 1"53g and MgSO 4, 0"55g as nutrients. Batch and continuous fermentations After the immobilization of the yeast, the saline medium was replaced by fresh feedstock medium. The reactor was placed on recycle mode during the whole time of the batch and continuous fermentations. The pH level and the temperature inside the reactor were maintained at 6.9 and 28°C, respectively. Different sodium sulphite concentrations in powder form (not sterilized) were added in two equal portions as indicated in the literature. The first portion was added on starting the fermentation and the other portion 2 h later. The batch period was 40 h. Continuous fermentation began after an initial batch phase, feeding the reactor with the feedstock solution when the sucrose from the molasses medium was almost consumed. Sodium sulphite was added during
~luent
temperature controller
feed
recycle
pump
pump
Fig. 1. Schematic diagram of the fixed-bed bioreactor experimental system.
continuous fermentation in order to maintain the initial concentration. Different experiments were conducted operating with different dilution rates. The dilution rate was based on the volume of the bed. The reactor pH and temperature were maintained at 6"9 and 28°C, respectively throughout the experiments. For each new value of the dilution rate the experimentation was allowed a run-time of approximately 8 days. For each run, samples of the effluent were analyzed daily for suspended yeast cells, carbohydrates, ethanol and glycerol. The immobilized yeast-cells' concentration was analyzed weekly. Analytical methods Concentrations of carbohydrates, glycerol and ethanol were determined by HPLC using a Waters Sugar PAK TM 1 column and 50 mg dm -3 sodium calcium A E D T salt solution at 75°C as mobile phase and a flow rate of 0.7 ml min-1. Eluted materials were detected using a Waters differential refractometer detector 410 (Waters Chromatography Division, Millipore Corporation, 34 Maple Street, Milford, MA 01757). Determinations of pH were made by a glass electrode with an Aqualytic pH meter 21. A platinum electrode (Ingold) was used for the redox potential determinations. Oxygen determinations were carried out with a probe (Aqualytic oxi 921 ). The number of adsorbed cells per gram of sintered glass rings was determined by releasing the cells from the support in 100 ml of sterilized water by use of sonication. Cells were then counted in a Thoma-hematocytometer using a Nikon microscope. This counting method was also employed to determine the yeast cell concentration in the bulk liquid. The methylene blue staining method was used to determine the cell viability (Bravo & Gonzfilez Benito, 1991 ). RESULTS AND DISCUSSION In order to work under completely mixed conditions, the reactor was always operating in recycle mode with a high recirculation flow, about 40 times the feed flow. The sucrose concentration in the molasses of the feedstock medium was maintained at 120 g dm -3. Batch experiments were carried out in order to analyze the influence of the concentration of sulphite on the yield of glycerol. The sulphite was dosed as mentioned previously. Then, starting from the results of the batch experiments, it was operated in continuous form, working with different dilution rates. Finally, a continuous fermentation was developed using the best operating conditions. Batch experiments Different fermentation runs were carried out adding different doses of sodium sulphite (20, 25, 30 and 40 g dm -3) to the medium in order to analyze the influence of the sulphite concentration over the fermentation process. The experiment was repeated three times for each dose of sulphite. Figure 2 shows the
Glycerol production in a packed-bed reactor 50-
211
60
50
40
0.76 ~,
_J
~=
4O
J,
1.22 cl"
~
J,
¢
106d,
o
o~20-
§ ao
8 10
10
0
O2O
25
30
0
4O
i
i
1O0
200
i
300
Batch fermentation. Influence of sulphite concentration: [] glycerol, ~ ethanol.
average concentrations of glycerol and ethanol obtained versus the sulphite dosage after 40 h of fermentation. The results indicate that there was a range between 25-30 g 1-J of sulphite where the glycerol concentration was highest. Less ethanol was obtained when the sulphite dose was 25 g 1-1. Therefore, the following experiments were carried out with this dosage of sulphite.
Continuous experimentation Continuous experimentation was carried out starting from the results of the batch experiments cited above. The initial dosage of sulphite used was 25 g dm -3 added to the molasses feedstock medium in the usual form, and after 40 h the bioreactor was continuously fed. Temperature and pH were maintained in all experiments at 28°C and 6.9, respectively, and sodium sulphite was added throughout the process in order to maintain the initial sulphite concentration and the redox potential. It was operated with various dilution rates (D = 0"76, 1"06, 1-22 and 1"52 d-t). Figure 3 shows the behaviour of the continuous fermentation, i.e. evolution of the product concentrations throughout experimentation. These experimentation runs were repeated twice• In this figure, the arrows indicate a new value of the dilution rate in the bioreactor. It is seen that as the dilution rate was raised the glycerol concentration gradually decreased. When the dilution rate was higher than 1-22 d - 1 the glycerol concentration was drastically reduced. With a dilution rate of 1"52 d -~ the lowest glycerol concentration was obtained. However, the bioreactor was able to recover its glycerol production capacity when the dilution rate was lowered. The dependence of the glycerol production on dilution rate is shown in Fig. 4. The maximum productivity, 36.6 g dm -3 d t, was attained for a dilution rate of 1.22 d- 1. For this case, the glycerol concentration was 3 0 g l -l Finally, a new, continuous experiment was carried out, working with the experimental conditions that pro-
=
500
600
700
800
T i m e (h)
s u l p h i t e c o n c e n t r a t i o n (g/L)
Fig. 2.
400
Fig. 3.
Continuous glycerol production. Alteration of dilution rate; • glycerol, ~ ethanol.
50
~
50
40
-~
30
/N
40
•
\
30
._~ ._
10
0,5
10
1
1,5
;0 2
D(d ~)
Fig. 4. Continuous glycerol production. Influence of dilution rate: • glycerol concentration, ~ glycerol productivity.
vided the maximum glycerol productivity in order to maintain the fermentation for a long period of time. The starting conditions were: sucrose as molasses, 120gdm-3; sulphite, 2 5 g d m - 3 ; D=1.22d-1; T = 28°C; and pH = 6.9. For these operating conditions, the redox potential value remained essentially constant (between - 1 4 0 and - 1 6 0 mV) due to the constant presence in the medium of sodium sulphite. The oxygen concentration was less than 0.2 ppm. A suitable behaviour of the bioreactor could be achieved for a total experimentation period of 700 h. The productivity was maintained at about 36 _+ 1 g dm -3 d - ~ with a glycerol yield of 25% (based on consumed sucrose from molasses). Through the entire run, few yeasts were released into the medium. The cells of Saccharomyces cerevisiae were perfectly immobilized in the sintered glass rings and were arranged in clusters in the centre of the glass pores, forming large ag~egatiQns, as was shown by scanmng electron n u c r o g r a p ~ T h e same pattern of growth has been noted by other authors (Hecker et al., 1990). Nevertheless, the cell viability at the end of the fermentation period (700 h) was about 60% due to the prolonged presence of
212
G. Gonz6lez Benito, M. Ozores, M. Pefta
sulphite in the medium. When the experimentation run was prolonged more than approximately 700 h, the cells' viability decreased and a large amount of free yeast cells began to be released in to the culture medium. Moreover, contamination appeared in the bioreactor. In conclusion, the sintered-glass rings were suitable as a matrix for the immobilization of yeast cells in a fermentation process for glycerol production from sugarbeet molasses without treatment (only rough filtration and pasteurization) attaining a good glycerol productivity.
REFERENCES Bisping, B. & Rehm, H. J. (1986). Glycerol production by cells of Saccharomyces cerevisiae, immobilized in sintered glass. Microbiol. Biotechnol., 23, 174-9. Bisping, B., Hecker, D. & Rehm, H. J. (1989). Glycerol production by semicontinuous fed-batch fermentation with immobilized cells of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 32, 119-23. Bravo, P. & Gonz~ilez Benito, G. (1991). Continuous ethanol
fermentation by immobilized yeast cells in a fluidized-bed reactor. J. Chem. Tech. Biotechnol., 52, 127-34. Freeman, G. G. & Donald, G. M. S. (1956). The influence of certain variables on glycerol formation in the presence of sulfites. Appl. Microbiol., 5, 197-210. Harris, J. F. & Hajny, G. J. (1960). Glycerol production: a pilot plant investigation for continuous fermentation and recovery. Biochem. Microbiol. Technol. Eng., 2, 9-24. Hecker, D., Bisping, B. & Rehm, H. J. (1990). Continuous glycerol production by the sulphite process with immobilized cells of Saccharomyces cerevisiae. Appl. Microbiol. Biotechnol., 32,627-32. Kalle, G. P., Naik, S. C. & Lashkari, B. Z. (1985). Improved glycerol production from cane molasses by the sulfite process with vacuum or continuous carbon dioxide sparging during fermentation. J. Ferment. Technol., 63, 231-7. Maier, K., Hinze, H. & Leuschel, L. (1986). Mechanism of sulfite action on the energy metabolism of Saccharomyces cerevisiae. Biochim. Biophys. Acta, 848, 120-30. Vijaikishore, P. & Karanth, N. G. (1984). Glycerol production from glucose in alkaline medium. Biotechnol. Lett., 6, 103-8. Vijaikishore, P. & Karanth, N. G. (1986). Glycerol production by immobilized cells of Pichia farinosa. Biotechnol. Lett., 8, 257-60. Virkar, P. D. & Panesar, M. S. (1987). Glycerol production by anaerobic vacuum fermentation of molasses on pilot scale. Biotechnol. Bioeng., 29, 773-4.