- Email: [email protected]
Influence of Osmoregulators on Osmotolerant Yeast Candida krusei for the Production of Glycerol1
Chinese J. Chem. Eng., 14(3) 371-376
(2006)
Influence of Osmoregulators on OsmotolerantYeast Candida krusei for the Production of Glycerol* CHEN Guo( f%H),YAO Shading(#k % %)** and GUAN Yixin( %
%)
Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 3 10027, China
Abstract Cundidu knrsei was osmotolerant yeast for the production of glycerol. Extracellular osmotic pressure was one of the key factors to induce the enzyme activity of glycerol-3-phosphate dehydrogenase(GPDH) which severely affected the glycerol productivity. Three osmoregulators such as NaCl, PEG4OOO and glycerol were used to investigate their effects on yeast growth, glucose consumption and glycerol production. In order to determine the effect of extracellular glycerol concentration, different amounts of glycerol were initially supplemented as an osmoregulator to increase glycerol production. The maximum glycerol concentration attained 179g.L-' with initial glycerol concentration of 8Og.L-' in medium, compared with 41g.L-' in the control experiment. These results were successfully reproduced for fed-batch process in an air-lift reactor. Keywords Cnndida knrsei, glycerol, osmotolerant yeast, osmoregulator,air-lift reactor
1 INTRODUCTION Microbial fermentation is a feasible way for the production of glycerol. In recent years, glycerol production by osmotolerant yeasts such as Cundidu glycerinogenes"], Pichiu furinose'21 and Cundiu kr~sei'~], has received much attention. In these processes, glycerol was produced as a compatible solute to respond the extracellular high osmotic stress. Remize et uL.'~] reported that glycerol export and glycerol-3-phosphate dehydrogenase(GPDH) were rate-limiting for glycerol production in Succhuromyces cerevisiue. According to glycerol metabolism in yeast, the first step of glycerol formation was the synthesis of dihydroxyacetone phosphate catalyzed by GPDH which included two isoenzymes, i.e. the osmotically induced Gpdlp and the constitutive Gpd2p"'. High activity of Gpdlp could be obtained by adding osmoregulators such as carbohydrates and inorganic salts into medium. Liu et ul. P.61 and Blomberg and Adler"] used NaCl as an osmoregulator to improve glycerol production by C. krusei. But the addition of osmoregulators such as NaCl would cause burden in the downstream purification of glycerol. In traditional opinion, the product of fermentation inhibited the cell growth and the product accumulation. But in these yeasts it was quite different because glycerol was produced as a compatible solute to impede the intracellular water losing under the condition of extracellular high osmotic stress.
In this work, C. krusei will be used as model yeast. The cell growth, glucose consumption and glycerol production will be studied under the conditions that three different osmoregulators are added into medium. The glycerol will be initially added into the medium as an osmoregulator to provide cells extracellular hyperosmotic environment. The effects of different initial glycerol concentrations on yeast growth, glucose consumption and glycerol production will be also performed in shaking flasks and in an &-lift reactor. It will be proven that the initial addition of glycerol into medium can be used as a considerable method for osmotolerant yeast in glycerol fermentation industry.
2 MATERIALS AND METHODS 2.1 Microorganism and medium In all experiments, the strain used was osmotolerant yeast Candida krusei (ICM-Y-05) purchased from the Institute of Process Engineering, Chinese Academy of Sciences (Beijing, China). The agar slant medium contained ~ o o ~ Lglucose, -' 3 g ~ urea, ' 3 g . ~ - 'corn steep liquor and 2OgL-' agar. For seed culture, the medium contained lOOgL-' glucose, 3gL-' urea and 3 g G ' corn steep liquor. For fermentation, the medium composition was 200g.L-' glucose, 2.5g.L-' urea, 3g-L-' corn steep liquor and 3.5g*L-'K&m4.
Received 2005-09-20, accepted 2006-01- 18.
* Supported by the National Natural Science Foundation of China (No.20576118). ** To whom correspondence should be addressed. E-mail: [email protected]
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372
2.2 Microorganism culture The seed slant was inoculated and incubated at 35°C for 48h, and then stored at 4°C. Seed was pre-cultured aerobically in 500ml shaking flask containing lOOml seed medium at 35"C, rotating speed 150r.min-' for 24h. For fermentation, a volume of 5ml pre-cultured seed was transferred into 500ml shaking flask containing 50ml fermentation medium, and then followed by 5-day incubation at 35"C, 150r.min-I. Fig. 1 is the sketch of an air-lift loop reactor which was used in the experiment for fed-batch culture. For batches of fermentation, 50ml pre-cultured seed was added into reactor contained 450ml fermentation medium. In the process, the aeration rate was maintained by 0.3ml.ml - '.min (volume of air per volume of medium per min) and the pH of medium was about 5-6. The dissolved oxygen was not controlled.
Medium
Figure 1 A sketch of 500ml air-lift internal loop reactor
2.3 Assay of dry cell weight, residual glucose and glycerol concentration Biomass concentration was estimated from the absorbance of appropriately diluted culture medium at 600nm (Ultrospec33OOpro WNisible, Amersham Biosciences) according to the predetermined correlation between OD (optical density) and dry cell mass m of biomass. Glucose concentration was determined by the dinitro salicylic acid (DNS) method[*].Glycerol concentration was determined by periodate-chromotropic acid analysis method"], where the supernatant of centrifuged samples was used for glycerol analysis spectrophqtometrically at 575nm. Average growth rate was calculated as June, 2006
mrn -"z1 m, . t
Average glucose consumption rate was calculated with
- 'glu
'g1u.i
t
Glycerolhiomass yield was calculated using Cg1y.m
- 'g1y.i
mm
GlyceroVglucoseyield 'glym 'g1u.i
- 'g1y.i -'glu
Glycerol productivity was 'g1y.m
- 'g1y.i t
3 RESULTS AND DISCUSSION 3.1 Effects of different osmoregulators on glycerol production Extracellular hyperosmotic pressure was very important for osmotolerant yeast of C. krusei to produce compatible intracellular glycerol. Generally high glucose concentration was used to provide extracellular hyperosmotic environment. But it would cause higher residual glucose in the end which troubled the purification of glycerol. Addition of other osmoregulators was an alternative. Different osmoregulators such as NaC1, PEG4OOO and glycerol were studied in this work. The time courses of biomass, residual glucose and glycerol concentrations in shaking flasks by C. krusei were depicted in Fig.2. As shown in Fig.2(a), the final cell densities with the addition of three osmoregulators were much lower than that without osmoregulators. It indicated that the growth of yeast would be restrained in some extent by osmoregulators. The biomass concentration in medium with initial NaCl concentration of 4Og.L-' was (4.74 & 0.24)g-L-' which was much lower than (7.49kO.37)g.L-I in medium without osmoregulators. The addition of glycerol resulted in the lowest negative effect on biomass concentration compared with another two osmoregulators. Fig.2(b) showed that glucose was almost exhausted at lO8h in medium without existing of PEG4OOO. The residual glucose was still as high as 42.4g.L-' after 120h when PEG4000 was added into medium as osmoregulator. It meant that PEG4000 inhibited the glucose consumption. In medium without
Influence of Osmoregulatorson OsmotolerantYeast Cundida krusei for the Production of Glycerol
osmoregulators, more energy was provided for cell leading to faster growth thus decreasing productivity of glycerol. As shown in Fig.2(c), a maximum glycerol concentration of 85g.L-I (excluding initial glycerol concentration of 2Og.L-') and a maximum glyceroVglucose yield of 46.4% were obtained under
373
2 0 g . ~ - ' glycerol as osmoregulator. The results indicated that glycerol was a very good osmoregulator which induced high activity of critical enzyme Gpdlp. From Fig.3, glyceroVglucose yield was 29.4%, 26.5%, 46.4% by addition of PEG4000, NaCl and glycerol in medium, respectively, compared with 21.8% without osmoregulators.
=
Ep;3glycerol concentration
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100
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(b) 120 I 7
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A
B C different osmoregulators
D
3.2 Effects of initial glycerol concentration on glycerol production
M
7
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Figure 3 Maximum glycerol concentration, yield and productivity with different osmoregulators in medium (A- no osrnoregulators; B-20g.L-' glycerol; C- 4Og.L-' NaCl; D- 2Og.L-' PEG4000)
200 160
60
0
cultivation time, h
5
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-g a2 4g M
0 productivity
.u-
;8 ay M.$
0
glycerollglucose yield
I00
I
100
Since the addition of glycerol as osmoregulator could greatly enhance the glycerol concentration and glyceroVglucose yield from the aforementioned results, it is necessary to study the effects of different initial glycerol concentrations on glycerol production. As shown in Fig.4(a), the final biomass concentration decreased with the increasing of initial glycerol concentration. If the initial glycerol concentration exceeded 20g-L-', the growth of cell would be severely inhibited. The final biomass concentration was 6.72g.L-' and 5.56g.L-' with initial glycerol concentration 2Og.L-' and 40g.L-', respectively. But the final biomass concentration was nearly the same when initial glycerol concentration was beyond 40g.L . The cell growth rate was decreased with increasing initial glycerol concentration especially in the earlier stage of cell growth. From the Fig.4(b), at initial 24h, the glucose consumption rate was equivalent for all experiments. Subsequently, the glucose consumption rate was quite different and increasing the initial glycerol concentration slowed the glucose consumption. From Fig.4(c), glycerol producing rate enhanced with the addition of initial glycerol. Glycerol concentration decreased somewhat at the earlier stage with initial ~
0
20
40
60
80
100
120
cultivation time, h (C)
Mgure 2 Time profdes of cell growth(a), residual glucose level (b) and glycerol concentration(c) with different osmoregulators in medium by C. krusei at 35C 0 no osmoregulator; 0 2%(mass concentration) glycerol; W 2%(mass concentration) PEG4000, 0 4%(massconcentration ) NaCl
'
Chinese J. Ch. E. 14(3) 371 (2006)
Chinese J. Ch. E. (Vol. 14, N0.3)
374
glycerol in medium, which was not observed without initial glycerol in medium. That means glycerol is not only an osmoregulator, but also incorporated into metabolic pathway. Glycerol concentration decreased at intermediate period, which possibly was caused by competition between production and glycerol trans-
20
0
40
60
80
100
120
cultivation time, h
(4 250 I
I
20
0
40
60
80
100
port. Different glycerol concentration would induce corresponding regulation mechanism. On one hand high activity of enzyme Gpdlp was induced, on the other hand tunnel of glycerol transport was activated. At the earlier stage, glycerol was transferred into cells and accumulated in the cells to balance the extracellular hyperosmotic pressure. If the glycerol accumulated in cell to a certain value, the glycerol began to permeate out of cell. It was a dynamic equilibrium of extracellular and intracellular osmotic pressure. The better glycerol production was obtained at initial glycerol concentration of 2OgL-' and 80 gL-'. From the Fig.5, a maximum glycerol concentration of 99g.L-l (excluding initial sglycerol concentration of 80gL-'), a maximum glycerol yield of 55% and a maximum glycerol productivity of 22g.L-'.d-' were obtained at initial glycerol concentration of 8Og.L-' . Table 1 was the comparison of average cell growth rate, glucose consumption rate and glycerol producing rate with initial addition of different osmoregulators and different concentration of glycerol.
120
cultivation time, h (b) 200
,
I
A
B
C D cultivation time, h
E
Figure 5 Maximum glycerol Concentration, yield and productivity with different initial glycerol concentration in medium (A-no glycerol; B-20g.L-I glycerol; C-4Og.L-' glycerol; D-6Og.L-l glycerol;E-8Og.L-' glycerol)
0
20
40
60
80
100
120
cultivation time, h (c)
Figure 4 Time profiles of cell growtb(a), residual sugar level (b) and glycerol concentration (c) with different initial glycerol concentrationin medium by C. krusei at 3572 W no glycerol; 0 2Og.L-' glycerol; A 4Og.L-' glycerol; 0 6Og.L-' glycerol; 0 8OgL-' glycerol
June, 2006
3.3 Fed-batch culture in an air-lift internal loop reactor To investigate the effects of initial glycerol as osmoregulator on the production of glycerol, in this part five fed-batches of fermentation were carried out in an air-lift internal loop reactor. The initial glucose concentration was 200g.~-'.The initial glycerol concentration was 8Og.L-'. The cultivation time of each batch was 96h. At the end of each batch, 200ml fermentation medium was taken out and 2OOml fresh fer-
Influence of Osmoregulatorson Osmotolerant Yeast Cundida krusei for the Production of Glycerol Table 1 Comparison of average cell growth, glucose consumption and glycerol producing rate with the addition of different osmoregulatorsin medium Average cell growth rate, h-' A
Glucose consumption rate, g.L-'.h-'
0.065
Glycerolhiomass yield
1.812
5.74
B
0.038
1.821
11.2
C
0.05 1
1.242
7.58
D
0.057
1.625
12.63
E
0.046
1.518
12.95
F
0.044
1.646
8.99
G
0.044
1.593
18.68
8 I
250
6
200
UF 65
150
7 cl
2 4
100
E 3
',
4 CONCLUSIONS Different osmoregulators were added into the ini-
.?
y T+
'5g Mp 8 C
s2
50
1
0 100
0
200 300 cultivation time, h
400
500
-
M
$ 3
6
e 9
-g E M
Figure 6 Fed-batch fermentation for production of glycerol using C. krusei in air-lift reactor W biomass; 0 residual glucose concentration; A glycerol concentration 6o I
Note: A-no osmoregulatom; B--40g.L-' NaC1; C-2OgL-' PEG~OOO,D - ~ O ~ L -giyami; ~ E-IO~L-' glycerol; F - - 6 o g P g l y m d ; G8Og.L-l g l y m d in initial medim.
mentation medium including 50% glucose was added into reactor. Fig6 showed the time course of glycerol fermentation in air-lift reactor by C . krusei. In the first batch, glucose was consumed gradually for the low concentration of initial biomass growth. In the following batches, glucose was consumed rapidly and exhausted in 72h. Final biomass concentration was compared in five batches, with the final glycerol concentration of each batch 154 g.L-', 186 g.L-', 203 g.L-', 217g.L-' and 233g.L-' correspondingly. Fig.7 showed the glycerol yield and productivity for each batch of fermentation in air-lift reactor. The average yield and productivity of glycerol for five batches are 45.7% and 22.85g.L- 'd- respectively. From the results, initial addition of glycerol as osmoregulator was an effective method to increase the production of glycerol. Table 2 compared our results with the literature results.
375
0
=
glyceroVglucose yield
1
2
3 batch
4
5
6
Figure 7 The glycerol yield and productivity for 5 batches of fermentation in an air-lift reactor
tial medium to understand the effects of extracellular osmotic pressure on glycerol production. The results showed that osmoregulators actually could increase the synthesis of glycerol by yeast C. krusei. Compared with other osmoregulators, glycerol as an osmoregulator was more effective for production of glycerol and did not burden the downstream processing of glycerol. Glycerol was initially added into medium to obtain high extracellular osmotic pressure which could induce glycerol-3-phosphate dehydrogenase. High productivity of glycerol was obtained under different initial glycerol concentration. The maximal glycerol
Table 2 Comparison of some representative microorganismsused for glycerol production Yeast S. cerevisiae S. cerevisiae
Oxygen
anaerobic anaerobic C. glycerinogenes aerobic f! farinose aerobic C. krusei aerobic C. krusei aerobic
Fermentation time, d 4-5 5-7 3-5 4-5 3-5 3
in broth, GlyceroVglucose Average g b c erol productivRef. yield, g.L-l % ity, g.L-'.d-' - 80 25% 30 [I01 45 23% 9 [Ill 11-130 52%63% 28-32 [I1 300 NIA 75 PI 60 25% [ 12,131 19.4 90-120 45.7% this work 22.85
cgly
Processes fed-batch sulfite batch alkali batch osmotolerant fed-batch osmotolerant multistage osmotolerant fed-batch osmotolerant
Chinese J. Ch. E. 14(3) 371 (2006)
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yield was 55% at initial concentration of glycerol 8% in shaking flasks. For fed-batch fermentation in 550ml air-lift loop reactor, the average yield and productivity of glycerol for five batches attained 45.7 % and 22.85 g.L-’.d-’. Glycerol played an essential role as compatible solute during osmoregulation in yeast^[^*'^] and glycerol permeated through the Fps 1p-channel from the cells with the absence of extracellular hyperosmotic tress['^''^'. According to our results, it was taken granted that initial addition of glycerol not only induced the high enzyme activity of GPDH, but also activated the other diffusion channels of glycerol. So initially addition of glycerol into medium was an effective way to increase glycerol production for the osmotolerant yeast C. krusei.
NOMENCLATURE Cglu Cglu(i) Cgly(i) Cgly(m)
mi mm t
residual glucose concentration, g.L-l initial glucose concentration,g.L-’ initial glycerol concentration, g ~ - ’ maximum glycerol concentration, g.L-’ initial dry cell mass, g.L-’ maximum dry cell mass, g.L-’ cultivation time, h
REFERENCES Wang, Z.X., Zhuge, J., Fang, H.Y., Prior, B.A., “Glycerol production by microbial fermentation: A review”, Biotechnol. Adv., 19.201-223(2001). Vijaikishore. P., Karanth, N.G, “Glycerol production by immobilized cells of Pichia farinose”, Biotechnol. Lett., 8,257-260( 1986). Liu, H.J., Liu, D.H., Zhong, J.J., “Novel fermentation strategy for enhancing glycerol production by Candida krusei“,Biotechnol.Pro&.,19, 1615-1619(2003). Remize, F., Barnavon, L., Dequin, S., “Glycerol export and glycerol-3-phosphate dehydrogenase, but not glycerol phosphatase, are rate limiting for glycerol production in Saccharomyces cerevisiae”, Metab. Eng., 3,
June, 2006
301-3 12(2001). Liu, Y.Q., Liu, D.H., Su, Q., Xie, D.M., “Glycerol production by Candida krusei employing NaCl as an osmoregulator in batch and continuous fermentations”, Biotechnol. IRtt., 24, 1137-1 140(2002). 6 Liu, Y.Q., Liu, D.H., Su, Q., “Glycerol production by batch and continuous fermentation with NaCl as osmoregulator”, J. Chem. Ind. Eng. (China)., 54, 259263(2003). (in Chinese) 7 Blomberg, A., Adler, L., “Physiology of osmotolerance in fungi”, Adv. Microbiol. Physiol., 33, 145-212(1992). 8 Miller, GL., “Use of dinitrosalicylic acid reagent for determination of reducing sugars”, Anal. Chem., 31, 426428( 1959). 9 Ashwath, M.F.R., Newman, A.A., Analytical Methods for Glycerol, Academic Press, New York, 19-2 1( 1979). 10 Kalle, GP., Naik, S.C., “Continuous fed-batch vacuum fermentation system for glycerol from molasses by sulfite process”, J. Ferment. Technol.,63,411-414(1985). 11 Schade, A.L., Farber, E., “Glycerol”, U. S. Pat., 2414838( 1947). 12 Liu, H.J., Liu, D.H., Zhong, J.J., “Oxygen limitation improves glycerol production by Candih krusei in a bioreactor”, Process Biochem., 39, 1899-1902(2004). 13 Liu, Y.Q., Liu, D.H., Su, Q., Liu, J.R., Xie, D.M., “Critical influence of osmotic pressure on continuous production of glycerol by an osmophilic strain of Candida krusei in a multistage cascade bioreactor”, Process Biochem., 38,427-432(2002). 14 Blomberg, A., “Metabolic surprises in Saccharomyces cerevisiae during adaption to saline conditions: Question, some answers and a model”, FEMS Microbial. Lett., 182, 86-97(2000). 15 Tamas, M.J., Luyten, K., Sutherland, F.C.W., Hernandez, A., Albertyn, J., Valadi, H., Prior, B.A., Kilian, S.G. Ramos, J., Gustafsson, L., Thevelein, J.M., Hohmann, S., “Fpslp controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation”, Mol. Microbiol., 31,1087-1 104(1999). 16 Hohmann, S., Bill, R.M., Kayingo, G, prior, B.A., “Microbial MIP channels”, TrendsMicrobiol.,8,33-38(2O00). 5
(2006)
Influence of Osmoregulators on OsmotolerantYeast Candida krusei for the Production of Glycerol* CHEN Guo( f%H),YAO Shading(#k % %)** and GUAN Yixin( %
%)
Department of Chemical and Biochemical Engineering, Zhejiang University, Hangzhou 3 10027, China
Abstract Cundidu knrsei was osmotolerant yeast for the production of glycerol. Extracellular osmotic pressure was one of the key factors to induce the enzyme activity of glycerol-3-phosphate dehydrogenase(GPDH) which severely affected the glycerol productivity. Three osmoregulators such as NaCl, PEG4OOO and glycerol were used to investigate their effects on yeast growth, glucose consumption and glycerol production. In order to determine the effect of extracellular glycerol concentration, different amounts of glycerol were initially supplemented as an osmoregulator to increase glycerol production. The maximum glycerol concentration attained 179g.L-' with initial glycerol concentration of 8Og.L-' in medium, compared with 41g.L-' in the control experiment. These results were successfully reproduced for fed-batch process in an air-lift reactor. Keywords Cnndida knrsei, glycerol, osmotolerant yeast, osmoregulator,air-lift reactor
1 INTRODUCTION Microbial fermentation is a feasible way for the production of glycerol. In recent years, glycerol production by osmotolerant yeasts such as Cundidu glycerinogenes"], Pichiu furinose'21 and Cundiu kr~sei'~], has received much attention. In these processes, glycerol was produced as a compatible solute to respond the extracellular high osmotic stress. Remize et uL.'~] reported that glycerol export and glycerol-3-phosphate dehydrogenase(GPDH) were rate-limiting for glycerol production in Succhuromyces cerevisiue. According to glycerol metabolism in yeast, the first step of glycerol formation was the synthesis of dihydroxyacetone phosphate catalyzed by GPDH which included two isoenzymes, i.e. the osmotically induced Gpdlp and the constitutive Gpd2p"'. High activity of Gpdlp could be obtained by adding osmoregulators such as carbohydrates and inorganic salts into medium. Liu et ul. P.61 and Blomberg and Adler"] used NaCl as an osmoregulator to improve glycerol production by C. krusei. But the addition of osmoregulators such as NaCl would cause burden in the downstream purification of glycerol. In traditional opinion, the product of fermentation inhibited the cell growth and the product accumulation. But in these yeasts it was quite different because glycerol was produced as a compatible solute to impede the intracellular water losing under the condition of extracellular high osmotic stress.
In this work, C. krusei will be used as model yeast. The cell growth, glucose consumption and glycerol production will be studied under the conditions that three different osmoregulators are added into medium. The glycerol will be initially added into the medium as an osmoregulator to provide cells extracellular hyperosmotic environment. The effects of different initial glycerol concentrations on yeast growth, glucose consumption and glycerol production will be also performed in shaking flasks and in an &-lift reactor. It will be proven that the initial addition of glycerol into medium can be used as a considerable method for osmotolerant yeast in glycerol fermentation industry.
2 MATERIALS AND METHODS 2.1 Microorganism and medium In all experiments, the strain used was osmotolerant yeast Candida krusei (ICM-Y-05) purchased from the Institute of Process Engineering, Chinese Academy of Sciences (Beijing, China). The agar slant medium contained ~ o o ~ Lglucose, -' 3 g ~ urea, ' 3 g . ~ - 'corn steep liquor and 2OgL-' agar. For seed culture, the medium contained lOOgL-' glucose, 3gL-' urea and 3 g G ' corn steep liquor. For fermentation, the medium composition was 200g.L-' glucose, 2.5g.L-' urea, 3g-L-' corn steep liquor and 3.5g*L-'K&m4.
Received 2005-09-20, accepted 2006-01- 18.
* Supported by the National Natural Science Foundation of China (No.20576118). ** To whom correspondence should be addressed. E-mail: [email protected]
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2.2 Microorganism culture The seed slant was inoculated and incubated at 35°C for 48h, and then stored at 4°C. Seed was pre-cultured aerobically in 500ml shaking flask containing lOOml seed medium at 35"C, rotating speed 150r.min-' for 24h. For fermentation, a volume of 5ml pre-cultured seed was transferred into 500ml shaking flask containing 50ml fermentation medium, and then followed by 5-day incubation at 35"C, 150r.min-I. Fig. 1 is the sketch of an air-lift loop reactor which was used in the experiment for fed-batch culture. For batches of fermentation, 50ml pre-cultured seed was added into reactor contained 450ml fermentation medium. In the process, the aeration rate was maintained by 0.3ml.ml - '.min (volume of air per volume of medium per min) and the pH of medium was about 5-6. The dissolved oxygen was not controlled.
Medium
Figure 1 A sketch of 500ml air-lift internal loop reactor
2.3 Assay of dry cell weight, residual glucose and glycerol concentration Biomass concentration was estimated from the absorbance of appropriately diluted culture medium at 600nm (Ultrospec33OOpro WNisible, Amersham Biosciences) according to the predetermined correlation between OD (optical density) and dry cell mass m of biomass. Glucose concentration was determined by the dinitro salicylic acid (DNS) method[*].Glycerol concentration was determined by periodate-chromotropic acid analysis method"], where the supernatant of centrifuged samples was used for glycerol analysis spectrophqtometrically at 575nm. Average growth rate was calculated as June, 2006
mrn -"z1 m, . t
Average glucose consumption rate was calculated with
- 'glu
'g1u.i
t
Glycerolhiomass yield was calculated using Cg1y.m
- 'g1y.i
mm
GlyceroVglucoseyield 'glym 'g1u.i
- 'g1y.i -'glu
Glycerol productivity was 'g1y.m
- 'g1y.i t
3 RESULTS AND DISCUSSION 3.1 Effects of different osmoregulators on glycerol production Extracellular hyperosmotic pressure was very important for osmotolerant yeast of C. krusei to produce compatible intracellular glycerol. Generally high glucose concentration was used to provide extracellular hyperosmotic environment. But it would cause higher residual glucose in the end which troubled the purification of glycerol. Addition of other osmoregulators was an alternative. Different osmoregulators such as NaC1, PEG4OOO and glycerol were studied in this work. The time courses of biomass, residual glucose and glycerol concentrations in shaking flasks by C. krusei were depicted in Fig.2. As shown in Fig.2(a), the final cell densities with the addition of three osmoregulators were much lower than that without osmoregulators. It indicated that the growth of yeast would be restrained in some extent by osmoregulators. The biomass concentration in medium with initial NaCl concentration of 4Og.L-' was (4.74 & 0.24)g-L-' which was much lower than (7.49kO.37)g.L-I in medium without osmoregulators. The addition of glycerol resulted in the lowest negative effect on biomass concentration compared with another two osmoregulators. Fig.2(b) showed that glucose was almost exhausted at lO8h in medium without existing of PEG4OOO. The residual glucose was still as high as 42.4g.L-' after 120h when PEG4000 was added into medium as osmoregulator. It meant that PEG4000 inhibited the glucose consumption. In medium without
Influence of Osmoregulatorson OsmotolerantYeast Cundida krusei for the Production of Glycerol
osmoregulators, more energy was provided for cell leading to faster growth thus decreasing productivity of glycerol. As shown in Fig.2(c), a maximum glycerol concentration of 85g.L-I (excluding initial glycerol concentration of 2Og.L-') and a maximum glyceroVglucose yield of 46.4% were obtained under
373
2 0 g . ~ - ' glycerol as osmoregulator. The results indicated that glycerol was a very good osmoregulator which induced high activity of critical enzyme Gpdlp. From Fig.3, glyceroVglucose yield was 29.4%, 26.5%, 46.4% by addition of PEG4000, NaCl and glycerol in medium, respectively, compared with 21.8% without osmoregulators.
=
Ep;3glycerol concentration
-
2- ss"-'u 3 'Z
I
I
,
20
40
60
1
,
I
80
100
120
5.
(a)
!#
120
-a
80
z
40
0
20
40 60 80 cultivation time, h
100
120
(b) 120 I 7
5
40
33
20
A
B C different osmoregulators
D
3.2 Effects of initial glycerol concentration on glycerol production
M
7
$ 2.5
Figure 3 Maximum glycerol concentration, yield and productivity with different osmoregulators in medium (A- no osrnoregulators; B-20g.L-' glycerol; C- 4Og.L-' NaCl; D- 2Og.L-' PEG4000)
200 160
60
0
cultivation time, h
5
80
4
-g a2 4g M
0 productivity
.u-
;8 ay M.$
0
glycerollglucose yield
I00
I
100
Since the addition of glycerol as osmoregulator could greatly enhance the glycerol concentration and glyceroVglucose yield from the aforementioned results, it is necessary to study the effects of different initial glycerol concentrations on glycerol production. As shown in Fig.4(a), the final biomass concentration decreased with the increasing of initial glycerol concentration. If the initial glycerol concentration exceeded 20g-L-', the growth of cell would be severely inhibited. The final biomass concentration was 6.72g.L-' and 5.56g.L-' with initial glycerol concentration 2Og.L-' and 40g.L-', respectively. But the final biomass concentration was nearly the same when initial glycerol concentration was beyond 40g.L . The cell growth rate was decreased with increasing initial glycerol concentration especially in the earlier stage of cell growth. From the Fig.4(b), at initial 24h, the glucose consumption rate was equivalent for all experiments. Subsequently, the glucose consumption rate was quite different and increasing the initial glycerol concentration slowed the glucose consumption. From Fig.4(c), glycerol producing rate enhanced with the addition of initial glycerol. Glycerol concentration decreased somewhat at the earlier stage with initial ~
0
20
40
60
80
100
120
cultivation time, h (C)
Mgure 2 Time profdes of cell growth(a), residual glucose level (b) and glycerol concentration(c) with different osmoregulators in medium by C. krusei at 35C 0 no osmoregulator; 0 2%(mass concentration) glycerol; W 2%(mass concentration) PEG4000, 0 4%(massconcentration ) NaCl
'
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glycerol in medium, which was not observed without initial glycerol in medium. That means glycerol is not only an osmoregulator, but also incorporated into metabolic pathway. Glycerol concentration decreased at intermediate period, which possibly was caused by competition between production and glycerol trans-
20
0
40
60
80
100
120
cultivation time, h
(4 250 I
I
20
0
40
60
80
100
port. Different glycerol concentration would induce corresponding regulation mechanism. On one hand high activity of enzyme Gpdlp was induced, on the other hand tunnel of glycerol transport was activated. At the earlier stage, glycerol was transferred into cells and accumulated in the cells to balance the extracellular hyperosmotic pressure. If the glycerol accumulated in cell to a certain value, the glycerol began to permeate out of cell. It was a dynamic equilibrium of extracellular and intracellular osmotic pressure. The better glycerol production was obtained at initial glycerol concentration of 2OgL-' and 80 gL-'. From the Fig.5, a maximum glycerol concentration of 99g.L-l (excluding initial sglycerol concentration of 80gL-'), a maximum glycerol yield of 55% and a maximum glycerol productivity of 22g.L-'.d-' were obtained at initial glycerol concentration of 8Og.L-' . Table 1 was the comparison of average cell growth rate, glucose consumption rate and glycerol producing rate with initial addition of different osmoregulators and different concentration of glycerol.
120
cultivation time, h (b) 200
,
I
A
B
C D cultivation time, h
E
Figure 5 Maximum glycerol Concentration, yield and productivity with different initial glycerol concentration in medium (A-no glycerol; B-20g.L-I glycerol; C-4Og.L-' glycerol; D-6Og.L-l glycerol;E-8Og.L-' glycerol)
0
20
40
60
80
100
120
cultivation time, h (c)
Figure 4 Time profiles of cell growtb(a), residual sugar level (b) and glycerol concentration (c) with different initial glycerol concentrationin medium by C. krusei at 3572 W no glycerol; 0 2Og.L-' glycerol; A 4Og.L-' glycerol; 0 6Og.L-' glycerol; 0 8OgL-' glycerol
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3.3 Fed-batch culture in an air-lift internal loop reactor To investigate the effects of initial glycerol as osmoregulator on the production of glycerol, in this part five fed-batches of fermentation were carried out in an air-lift internal loop reactor. The initial glucose concentration was 200g.~-'.The initial glycerol concentration was 8Og.L-'. The cultivation time of each batch was 96h. At the end of each batch, 200ml fermentation medium was taken out and 2OOml fresh fer-
Influence of Osmoregulatorson Osmotolerant Yeast Cundida krusei for the Production of Glycerol Table 1 Comparison of average cell growth, glucose consumption and glycerol producing rate with the addition of different osmoregulatorsin medium Average cell growth rate, h-' A
Glucose consumption rate, g.L-'.h-'
0.065
Glycerolhiomass yield
1.812
5.74
B
0.038
1.821
11.2
C
0.05 1
1.242
7.58
D
0.057
1.625
12.63
E
0.046
1.518
12.95
F
0.044
1.646
8.99
G
0.044
1.593
18.68
8 I
250
6
200
UF 65
150
7 cl
2 4
100
E 3
',
4 CONCLUSIONS Different osmoregulators were added into the ini-
.?
y T+
'5g Mp 8 C
s2
50
1
0 100
0
200 300 cultivation time, h
400
500
-
M
$ 3
6
e 9
-g E M
Figure 6 Fed-batch fermentation for production of glycerol using C. krusei in air-lift reactor W biomass; 0 residual glucose concentration; A glycerol concentration 6o I
Note: A-no osmoregulatom; B--40g.L-' NaC1; C-2OgL-' PEG~OOO,D - ~ O ~ L -giyami; ~ E-IO~L-' glycerol; F - - 6 o g P g l y m d ; G8Og.L-l g l y m d in initial medim.
mentation medium including 50% glucose was added into reactor. Fig6 showed the time course of glycerol fermentation in air-lift reactor by C . krusei. In the first batch, glucose was consumed gradually for the low concentration of initial biomass growth. In the following batches, glucose was consumed rapidly and exhausted in 72h. Final biomass concentration was compared in five batches, with the final glycerol concentration of each batch 154 g.L-', 186 g.L-', 203 g.L-', 217g.L-' and 233g.L-' correspondingly. Fig.7 showed the glycerol yield and productivity for each batch of fermentation in air-lift reactor. The average yield and productivity of glycerol for five batches are 45.7% and 22.85g.L- 'd- respectively. From the results, initial addition of glycerol as osmoregulator was an effective method to increase the production of glycerol. Table 2 compared our results with the literature results.
375
0
=
glyceroVglucose yield
1
2
3 batch
4
5
6
Figure 7 The glycerol yield and productivity for 5 batches of fermentation in an air-lift reactor
tial medium to understand the effects of extracellular osmotic pressure on glycerol production. The results showed that osmoregulators actually could increase the synthesis of glycerol by yeast C. krusei. Compared with other osmoregulators, glycerol as an osmoregulator was more effective for production of glycerol and did not burden the downstream processing of glycerol. Glycerol was initially added into medium to obtain high extracellular osmotic pressure which could induce glycerol-3-phosphate dehydrogenase. High productivity of glycerol was obtained under different initial glycerol concentration. The maximal glycerol
Table 2 Comparison of some representative microorganismsused for glycerol production Yeast S. cerevisiae S. cerevisiae
Oxygen
anaerobic anaerobic C. glycerinogenes aerobic f! farinose aerobic C. krusei aerobic C. krusei aerobic
Fermentation time, d 4-5 5-7 3-5 4-5 3-5 3
in broth, GlyceroVglucose Average g b c erol productivRef. yield, g.L-l % ity, g.L-'.d-' - 80 25% 30 [I01 45 23% 9 [Ill 11-130 52%63% 28-32 [I1 300 NIA 75 PI 60 25% [ 12,131 19.4 90-120 45.7% this work 22.85
cgly
Processes fed-batch sulfite batch alkali batch osmotolerant fed-batch osmotolerant multistage osmotolerant fed-batch osmotolerant
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yield was 55% at initial concentration of glycerol 8% in shaking flasks. For fed-batch fermentation in 550ml air-lift loop reactor, the average yield and productivity of glycerol for five batches attained 45.7 % and 22.85 g.L-’.d-’. Glycerol played an essential role as compatible solute during osmoregulation in yeast^[^*'^] and glycerol permeated through the Fps 1p-channel from the cells with the absence of extracellular hyperosmotic tress['^''^'. According to our results, it was taken granted that initial addition of glycerol not only induced the high enzyme activity of GPDH, but also activated the other diffusion channels of glycerol. So initially addition of glycerol into medium was an effective way to increase glycerol production for the osmotolerant yeast C. krusei.
NOMENCLATURE Cglu Cglu(i) Cgly(i) Cgly(m)
mi mm t
residual glucose concentration, g.L-l initial glucose concentration,g.L-’ initial glycerol concentration, g ~ - ’ maximum glycerol concentration, g.L-’ initial dry cell mass, g.L-’ maximum dry cell mass, g.L-’ cultivation time, h
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301-3 12(2001). Liu, Y.Q., Liu, D.H., Su, Q., Xie, D.M., “Glycerol production by Candida krusei employing NaCl as an osmoregulator in batch and continuous fermentations”, Biotechnol. IRtt., 24, 1137-1 140(2002). 6 Liu, Y.Q., Liu, D.H., Su, Q., “Glycerol production by batch and continuous fermentation with NaCl as osmoregulator”, J. Chem. Ind. Eng. (China)., 54, 259263(2003). (in Chinese) 7 Blomberg, A., Adler, L., “Physiology of osmotolerance in fungi”, Adv. Microbiol. Physiol., 33, 145-212(1992). 8 Miller, GL., “Use of dinitrosalicylic acid reagent for determination of reducing sugars”, Anal. Chem., 31, 426428( 1959). 9 Ashwath, M.F.R., Newman, A.A., Analytical Methods for Glycerol, Academic Press, New York, 19-2 1( 1979). 10 Kalle, GP., Naik, S.C., “Continuous fed-batch vacuum fermentation system for glycerol from molasses by sulfite process”, J. Ferment. Technol.,63,411-414(1985). 11 Schade, A.L., Farber, E., “Glycerol”, U. S. Pat., 2414838( 1947). 12 Liu, H.J., Liu, D.H., Zhong, J.J., “Oxygen limitation improves glycerol production by Candih krusei in a bioreactor”, Process Biochem., 39, 1899-1902(2004). 13 Liu, Y.Q., Liu, D.H., Su, Q., Liu, J.R., Xie, D.M., “Critical influence of osmotic pressure on continuous production of glycerol by an osmophilic strain of Candida krusei in a multistage cascade bioreactor”, Process Biochem., 38,427-432(2002). 14 Blomberg, A., “Metabolic surprises in Saccharomyces cerevisiae during adaption to saline conditions: Question, some answers and a model”, FEMS Microbial. Lett., 182, 86-97(2000). 15 Tamas, M.J., Luyten, K., Sutherland, F.C.W., Hernandez, A., Albertyn, J., Valadi, H., Prior, B.A., Kilian, S.G. Ramos, J., Gustafsson, L., Thevelein, J.M., Hohmann, S., “Fpslp controls the accumulation and release of the compatible solute glycerol in yeast osmoregulation”, Mol. Microbiol., 31,1087-1 104(1999). 16 Hohmann, S., Bill, R.M., Kayingo, G, prior, B.A., “Microbial MIP channels”, TrendsMicrobiol.,8,33-38(2O00). 5