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Yeast continuous mixed cultures on whey permeate and hydrolysed starch
Process Biochemirny 29 (1994) 381-386 0 1994 Elsevier Science Liiited Printed in Great Britain. AU rights reserved 0260~8774/94/.$7.00 ELSEVIER
Yeast Continuous Mixed Cultures on Whey Permeate and Hydrolysed Starch H. Kallel-Mhiri,aC. Valance,b.T.M. Engasserb& A. Miclo a %aboratoirede BiologicJnctustrielle et Alimentaire, ENSAJA,bLaboratoire des Sciences du Wnie Chimique CNRSENSAJA, 2, avenuede la fotit de Haye54500 Vandocuvre&s-Nancy, France (Received30 April1993; accepted19 June1993)
Mixed cultures of Saccharomycescerevisiae CBS 80% and Kluyveromyces fragiliswereused for continuous biomass production on media containing whey permeate and glucose (WC) or whey permeate, maltose and glucose (WGM). Glucose and maltose were provided Jiom a totally or a partially hydrolysed starch. For each medium the effects of twoyeast extract concentrations (0. I and 0.5 g/ litre) were investigated. The yeast extract concentration markedly @ected the equilibrium of the co-culture and the cell yield which was also influenced by the dilution rate. The yield variedfrom 0.26 to 0.48 g/g. The results obtained were due to the dserent kinetics of glucose transport in Kluyveromycesfra8ihs and Saccharomycescerevisiae.Kluyveromycesfragilispossessed two glucose carriers, present at all the tested dilution rates, characterised by their af7inity constants which were much higher than those present in Saccharomyces cerevisiae .
INTRODUCTION Food processing industries produce different waste materials with a wide range of carbon composition (glucose, maltose, lactose, etc.). The microbial treatment of these substrates requires more than one kind of micro-organism and to overcome this problem many authors have proposed the use of mixed cultures.1-4 In addition to the simultaneous use of several carbon substrates, mixed cultures have other advantages such as better resistance to contamination and improvement in cell yield.5,6 Whey permeate is an abundant by-product of the dairy industry. The yeast Kluyveromyces fragiCorresponding author: Dr A Miclo.
381
Iti has been usually cultivated on whey permeate for biomass production. This strain produces a high cell yield,‘7 but the quality of the biomass obtained from whey permeate is less appreciated than that obtained from starch and its derivatives by Saccharomyces cerevLsiae culture.8 The production of biomass on the two substrates (whey permeate and starch derivatives) is possible with the use of the two strains of yeasts (Kluyveromyces fragilis and Succharomyces cerevisiae) in a mixed culture but it is necessary to determine the optimal conditions for maintaining a stable co-cultnre?~lo In this work, we have investigated the influence of substrate, dilution rate and nutrient supplementation on the stability of the continuous mixed culture of K. fragilis and S. cerevisiae on whey
382
H. Kallel-Mhiri,
C. Valance, J. M. Engasser, A. Miclo
permeate and hydrolysed starch. To explain the effects of these parameters, we have also studied glucose transport in K. fragilis. MATERIAEJ AND METHODS Micro-organisms S. cerevisiae CBS 8066 and K. ~gi1i.s were maintained on yeast malt agar slopes at 4°C. K. fragilis was isolated from a dairy product. It was obtained from the Laboratoire de Biologie Industrielle et Alimentaire. Substrate preparation Two types of hydrolysed starch were used. The first substrate was composed of a completely hydrolysed starch (hydrolysed starch ‘A’) containing 97% of glucose. The second substrate (hydrolysed starch ‘B’) contained 45% of glucose and 45% of maltose. These substrates were added separately to whey permeate and the mixtures obtained were supplemented with 1.5 g/litre ammonium sulphate, 0.5 g/litre yeast extract and 2 mg/litre FeCl,. The composition of the different growth media used is indicated in Table 1. Culture conditions S. cerevkiae and K. fragilis were grown continuously in a 2-litre Biolafitte fez-mentor (LSL Biolafitte, Saint-Germain-en-Laye, France) with a 1 litre working volume. Dissolved oxygen, measured by a polarographic probe, was maintained above 30% air saturation. The pH was regulated at 4.5 by automatic addition of 2 M NaOH. The temperature was maintained at 30°C. The continuous culture was preceded by a batch culture of S. cerevzXae on 5 g/litre of ethanol. When the ethanol was completely consumed, the continuous feed of the fermentor was started. At the steady state, an inoculum of K. pagi1i.swas added to the medium. Glucose transport To study glucose transport in K. fiagih, the yeast was grown continuously at different dilution rates, in a Biolafitte fermentor with 1 litre working volume on a yeast nitrogen base (Osibio, Paris, France) at 1.34% supplemented with 10 g/litre of glucose. The pH was regulated at 4.5 by automatic addition of 2 M NaOH. The temperature was maintained at 30°C and dissolved oxygen was kept above 30% of air saturation.
Table 1. Composition
starch media Medium
WG WGM
of whey permeate and hydrolysed
Composition
Whey permeate + hydrolysed starch ‘A’ Whey permeate + hydrolysed starch ‘B’
Concentration (gjlitre) Lactose
Glucose
Maltose
5
5
-
5
3
3
At each dilution rate studied, 10 ml of cell suspension harvested from the continuous culture was washed twice with distilled water, suspended in 50 ml of 0.1 Mphosphate buffer (pH- 6.5) and then exposed to difEerent concentrations of the labelled glucose. Transport assays were carried out at 30°C. The reaction was started by the addition of 0.1 ml of the labelled substrate 1 ,&i/ml D[U-‘“Cl glucose (Amersham) to 2.2 ml of cell suspension. The concentration of glucose varied from 0.5 to 100 mu. The final volume was 2.5 ml. Samples (0.5 ml) were taken every 20 s for 80 s. Under these conditions the glucose uptake was linear. The reaction was stopped by diluting the sample in 10 ml unlabelled glucose (200 mu) at 4°C. The cells were immediately filtered through a Millipore 0.22 pm filter and washed twice with a concentrated glucose solution. The filters were then dissolved with 0,5 ml SoluenelOO (Packard). Scintillation liquid ( 10 ml) (In&gel Packard) were added to the vials and the radioactivity was measured in a Packard Tri-Carb 460 CD benchtop scintillation counter. The parameters of glucose transport were determined using a numerical technique adapted for parametric estimation based on the use of artificial life for global optimisation, developed in the laboratory. Analytical techniques Dry weight Of the continuous culture 10 ml were harvested, centrifuged at 7000g for 10 min, washed with distilled water, centrifuged again then dried at 105°C for 24 h. Residual sugars Culture samples obtained during the steady state were filtered through a 0.22 pm Millipore fiker. Lactose, glucose and maltose were determined using enzymic kits (Boehringer, Meylan, France, ref. 986 119 and 1113950).
Yeast continuous
mixed culture on whey permeate and hydrolysed starch
383
Cells counis
cells of S. ceratisiae and K. fiagih were obtained by counting colonies on a solid medium contain@ yeast nitrogen base 6.7 g/litre; lactose 10 g/litre; glucose 10 g/line and 5-bromo-4chloro-3-indolyl &D galactopyranoside at 40 mg/ litre. Spread plates were incubated at 30°C for 48 h. Under these conditions, K. [email protected] hydrolyses 5-bromo-4-chloro-3-indolyl B-D galactopyranoside, hence the colonies of K. j?agiZis appear blue whereas the colonies of S. cerevisiae are white.
Viable
Analysis of vohtiles Ethanol and acetate were measured by gas chromatography using a chromatograph Delsi Di 200 with an Enica 2 1 integrator (Del& Argenteuil, France). The 2 m x 0.002 m stainless steel cohunn was packed with Rorapak Q SO/l00 mesh. The carrier gas used was nitrogen. The injector and the detector were set at 220°C and the oven temperature was 170°C. Methanol was used as an internal standard.
RESULTS Influence of the substrate nature The mixed cultures of S. cerevisiae and K. fragilis are produced continuously at D = 0.18 h - ’ on the two media (whey permeate + hydrolysed starch A WG and whey permeate+ hydrolysed starch B WGM) as described in Materials and Methods. Figure l(A) shows that for the two media tested, the levels of residual lactose and glucose were negligible. On the WG medium, the cell yield obtained was 0.44 g/g; on the WGM the cell yield was 0.48 g/g. Note that in the latter case, the level of residual maltose was relatively high (0.4 g/litre). Figure l(B) shows that on the WG medium, at the steady state, the yeast population was composed of 100% K. fragilis, S. cerevisiae having been washed out. On the other hand, when WGM was fed to the culture, the population percentages of K. fragiais and S. cerevtiiae stabilised respectively at 15 and 85%. Influence of dih&on rate Contimtous cultures were carried out on the medium WG at two dilution rates (0.18 and 0.3 h-l). Higher dilution rates were not tested because in such conditions S.ceretiiae CBS 8066
WG
WGM
Medium
WG
WGM
Medium
Fig. 1. Effect of the substrate on cell yield (Y,,&, residual sugars (A) and composition of the yeast population at the steady state (B), during the continuous mixed culture of Kluyveromyces fra@is and Saccharomyces ceretiiae at D=O.18 h-‘.
exhibits a fermentative metabolism resulting in a decrease in cell yield.’l Figure 2 shows that at all the dilution rates studied, the growth yield was equal to 0.44 g/g and both lactose and glucose were completely assimilated. Nevertheless at D = 0.3 h-l, slight increases in both residual lactose and glucose were noted. The evolution of S. cerevisiae and K. fragilis populations was also similar. S. cerevisiae was progressively washed out and at the steady state, the yeast population was made up of 100% K. fragilis. Influence of yeast extract For an economical process for biomass production, the nutrient supplementation should be minimised. To reach this goal the concentration of yeast extract was lowered from 0.5 to 0.1 g/litre. Figure 3 shows that on WGM medium, when the dilution rate was maintained at 0.18 h- ’ and yeast extract concentration was equal to 0.1 g/litre, the cell yield obtained was 0.44 g/g and only a slight accumulation of residual lactose and residual maltose was observed. The shift of yeast extract from 0.1 to 0.5 g/litre contributes to a
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H. Kallel-Mhiri, C. Valance, J. M. Engasser,A. Miclo
0.18
0.3 D (h-l)
0.18
0.3 D (h-1)
Fig. 2. Influence of dilution rate on cell yield ( Y&. residual sugars (A) and composition of the yeast population at the steady state (B), during the continuous mixed culture of Kluyveromyces j?agiIis and Saccharomyces cerevisiae at D-O.18 h-’ on diluted whey permeate and hydrolysed starch composed of 97% glucose (WG).
minimal level of residual lactose and an increase in the cell yield (0.48 g/g) but the concentration of residual maltose increased. The K. fregilis percentage in yeast population dropped from 60 to 15% whereas that of S.cerevisiae increased from 40 to 85%. On WG medium, when the yeast extract concentration was equal to 0.5 g/&e, the same yield (0.44 g/g) and percentages of K. jkrgiks and S. cerevkiae (100-O%) were reached for D = 0.18 and D = 0.3 h-r. Figure 4 shows that at D = 0.3 h-t, when the yeast extract was reduced from 0.5 to 0.1 g/litre, the cell yield fell to 0.26 g/g and a high residual lactose was noted. The percentage of K. frqiiis dropped to 70% while that of S. cerevisiae rose to 30%. Glucose transport To explain the results obtained, we have studied glucose transport in K. jkqiZi.s and compared it to that already mentioned by several authors for S.cerevi&e CBS 8066.12 Glucose transport in K. jkzgifis was only studied in batch culture13and in such conditions
0.1
0.5
Yeastnrtract(en) Fig. 3. Effect of yeast extract concentration on cell yield ( Yx, S), residual sugars (A) and composition of the yeast population at the steady state (B), during the continuous mixed culture of Kluyvemmyces fragiiis and Soccharomyces cerevisiae at D = 0.18 h-’ on diluted whey permeate and hydrolysed starch composed of 45% glucose and 45% maltose (wGM).
the glucose concentration was not constant. However many studies concerning different yeasts including Candidu utili.s and S. cerevisiue, had shown that sugar concentration is a significant parameter in the regulation of the synthesis of sugar carriers.12,r4The yeasts exhibit different kinetics of sugar transport depending on the sugar concentration present in the medium. To overcome the difficulties due to glucose concentration variation, we have studied glucose transport in K. jirrgi2i.sin a continuous culture. The fermentation was performed at different dilution rates and the results obtained are presented in Table 2. These results indicate that K. fragilis possesses two glucose carriers. A high afhnity glucose carrier characterised by a K, of 0.025 m and a low affinity carrier which has a Km of 4 mu. The presence of these glucose transporters depends on the dilution rate. At a low dilution rate (D < 0.26 h- ’) where the glucose concentration was low, the two transporters were synthesised by the yeast. At high dilution rates (D > 0.3 h- ‘) corresponding to
Yeast continuous
mixed culture on whey permeate and hydrolysed starch 5.0
0.1
DISCUSSION
n
4.0
Ii
3.0
5
2.0
% 1
1.0
1 ._
0.0
cl
45
Yeastextract (pn, Fig. 4. Effect of yeast extract concentration on cell yield ( Yx, S), residual sugars (A) and the yeast population composition at the steady state (B), during the continuous mixed culture of Kluyveromyces jkag% and Saccharomyces cerevisiae at D = 0.3 h- ’on diluted whey permeate and hydrolysed starch composed of 97% glucose (WG).
Table 2. Glucose fragiis
transport in continuous cultures of K.
Dilution rate (h-l)
Residual glucose (mu)
High afinify carrier K, Cm@
Low afinify carrier K, PW
o-12 0.26
0.16 0.15 13.9 36.6
0.025 0.026 -
4 4 4 4
.0.31 0.45
385
high levels of residual glucose, only the low affinity carrier was present. Similar studies were carried out in S. cerevisiae by Postma et al. l4 These authors have shown that S. ceretiiae also possesses two glucose transporters which are present at all the tested dilution rates (from 0.1 to 0.46 h-l). Compared to the results obtained for K. fmgiIti, the high affinity carrier of S. cemvkze is characterised by a K, 40 fold higher than the one of K. fiugi2i.s.The K, of the low affinity transporter of S. cerevisiue is also higher than that of K. jkzgilis (5 fold).
A comparison of the kinetics of glucose transport in the two yeasts indicates that the rate of glucose uptake in K. fragilis is faster than that of S. cerevikze. When the medium contains only lactose and glucose, K. fragiis is pre-eminent over S. cerevisiue at all dilution rates. The results obtained on WG at D = 0.3 h- l for a yeast extract concentration of 0.1 g/litre, are due to the growth limitation of K. ji-ugiiis. At this dilution rate this limitation causes an accumulation of lactose and glucose. The level of the accumulated glucose induces its use by S. cerevkze. This fact explains the appearance of S. cerevisiue in the medium. For the lower dilution rate (D-O.18 h-l) on the medium WGM and for 0.1 g/litre of yeast extract, the growth limitation of K. fiugiZi.s seems to be less severe because less lactose and glucose are accumulated in the medium. Under these conditions K. frugiZi.s grows on lactose and a major part of glucose, whereas S. cerevisiae used maltose and a minor part of glucose. However, the percentage of S. cerevisiae obtained on WGM and yeast extract at 0.5 g/litre is relatively high compared to the results expected according to glucose transport. These results show that it is possible to use mixed cultures for biomass production on whey permeate and different types of hydrolysed starch. In most the cases, the cell yield was high without a noticeable accumulation of residual sugars. Moreover, we have also obtained stable mixed yeast populations. The nature of the substrate used, the concentration of the yeast extract and the dilution rate are important parameters which influence the composition of yeast population and the performance of the process. When the medium contains a mixture of lactose, glucose, maltose and yeast extract at a concentration of 0.5 g/litre, Scerevisiue represents 85% of the yeast population and the cell yield obtained is high (0.48 g/g). However when the continuous culture is operated on the same medium but supplemented by only 0.1 g/litre of yeast extract, the yeast population was made up of 60% of K. j-iugik.s and the cell yield obtained was slightly decreased. On the other hand, if the medium contains only glucose, lactose and yeast extract at 0.5 g/litre, the yeast population is mainly composed of K. fragilis and the cell yield is equal to 0.44 g/g. These
386
H. Kalkd-Mhiri,
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results were obtained at all the tested dilutions rates. At D = 0.3 h- *, lowering the concentration of yeast extract to 0.1 g/l&e resulted in a decrease of the cell yield and in the appearance of Scerevisiue. RESERENCE!3 1. Fellows. P. J. & Worgan. J. T. Growth of Saccharomvces and Cam&a kilti in mix& culture on sic @digei materials. EnzwneMicrob. TechnoL.9 (198’7) 430-3. 2. Fellows, P. J. & Worgan, J. T. Growth of Sackwvmyces fibufiger and Candkia utilis in tied culture on apple processjng wastes. Enzyme Microb. TechnoL, 9 (1987) 434-7. 3. Grootjen, D. R J., Meijlink, H. H. M., Vleesenbeek, R, Van der Lans, R. G. J. M. & Luyben, K. Ch. A. M. Cofermentation of glucose and xylose with immobilized Pi&a stipitis in combination with Saccharomyces cerevi&e. Enzyme Microb. Technol., 13 (1991) 530-6. 4. Bodie, E. A., Anderson, T. M., Goodman, N. & Schwartz, R D. Propionic acid fermentation of ultrahigh-temperature sterilized whey using mono and ~4e$cultures. Appl. Microbial. Biotechnol., 25 (1987) 5. Harrison, D. E. F. Mixed cultures in iudustrial processes. In Advances in AppIied Microbiology, ed. D. Perhnan. Academic Press, London, 1978, pp.129-64. 6. Carlotti, A., Jacob, F., Bxrier, J. & Poncet, S. Yeast production from crude sweet whey by a mixed culture of
7. 8.
9.
10. 11.
12.
13. 14.
Candida kfyr LY496 and Candida valida LY497. Biotechnoi.Lett., 13 (1990) 437-40. Meyrath, J. & Bayer, K. Biomass from whey. In Ecunomic Microbiology, ed. A. H. Rose. Academic Press, London, 1979, pp.207-69. Alam, L. Effect of ferment&cm on lactose, ghxose, and galactose content in milk and suitability 01 fermented milk tmxiucts for Lactose intolerants individuals. J. Dairy Sci., 65 (1982) 346-52. Goochee, C. F., Hatch, R T. & Cadman, T. W. Evahration of Escherichia coli and Candida utilis as a model continuous. comnetitive mixed culture svstem. BiotechnoI.B&ngng,S29 (1987) 453-63. _ Van Breuseaem. V. & Bastin. G. Ootimal control of biomass grow& in’s mixed culture. kotechnol. Bioengng, 35 (1990) 349-55. Postma, E., Verduyn, C., Scheffers, W. A. % Van Dijken J. P. Enzymic an&&s of the Crabtree effect in glucoselimited chemostat cultures of Sacchuromyces cerevisiae. Appl. Environ. Mzkrobiol., 55 (1989) 468-77. Postma, E., Scheffers, W. A. & Van Dijken, J. P. Kinetics of growth and ghrcose transport in glucose-limited chemostat cultures of Sacc~myce-s cerevisiae CBS 8066. Yeast, 5 (1989) 159-65 Gasnier, B. Characterization of low- and hiah-affinitv glucose ~transporters in the yeast Kluyverom~es ma& ianus. Biochim. Biophys. Acm, 903 (1987) 425-33. pbstma, E., Verduyn, C., Scheffers, W. A. & Van Dijken, J. P. Adaptation of the kinetics of ghrcose transport to environmental conditions in the yeast Can&&r u&s CBS 62 1: a continuous culture study. J . Gen .Mictwbioi., 134 (1988) 1109-16.
Yeast Continuous Mixed Cultures on Whey Permeate and Hydrolysed Starch H. Kallel-Mhiri,aC. Valance,b.T.M. Engasserb& A. Miclo a %aboratoirede BiologicJnctustrielle et Alimentaire, ENSAJA,bLaboratoire des Sciences du Wnie Chimique CNRSENSAJA, 2, avenuede la fotit de Haye54500 Vandocuvre&s-Nancy, France (Received30 April1993; accepted19 June1993)
Mixed cultures of Saccharomycescerevisiae CBS 80% and Kluyveromyces fragiliswereused for continuous biomass production on media containing whey permeate and glucose (WC) or whey permeate, maltose and glucose (WGM). Glucose and maltose were provided Jiom a totally or a partially hydrolysed starch. For each medium the effects of twoyeast extract concentrations (0. I and 0.5 g/ litre) were investigated. The yeast extract concentration markedly @ected the equilibrium of the co-culture and the cell yield which was also influenced by the dilution rate. The yield variedfrom 0.26 to 0.48 g/g. The results obtained were due to the dserent kinetics of glucose transport in Kluyveromycesfra8ihs and Saccharomycescerevisiae.Kluyveromycesfragilispossessed two glucose carriers, present at all the tested dilution rates, characterised by their af7inity constants which were much higher than those present in Saccharomyces cerevisiae .
INTRODUCTION Food processing industries produce different waste materials with a wide range of carbon composition (glucose, maltose, lactose, etc.). The microbial treatment of these substrates requires more than one kind of micro-organism and to overcome this problem many authors have proposed the use of mixed cultures.1-4 In addition to the simultaneous use of several carbon substrates, mixed cultures have other advantages such as better resistance to contamination and improvement in cell yield.5,6 Whey permeate is an abundant by-product of the dairy industry. The yeast Kluyveromyces fragiCorresponding author: Dr A Miclo.
381
Iti has been usually cultivated on whey permeate for biomass production. This strain produces a high cell yield,‘7 but the quality of the biomass obtained from whey permeate is less appreciated than that obtained from starch and its derivatives by Saccharomyces cerevLsiae culture.8 The production of biomass on the two substrates (whey permeate and starch derivatives) is possible with the use of the two strains of yeasts (Kluyveromyces fragilis and Succharomyces cerevisiae) in a mixed culture but it is necessary to determine the optimal conditions for maintaining a stable co-cultnre?~lo In this work, we have investigated the influence of substrate, dilution rate and nutrient supplementation on the stability of the continuous mixed culture of K. fragilis and S. cerevisiae on whey
382
H. Kallel-Mhiri,
C. Valance, J. M. Engasser, A. Miclo
permeate and hydrolysed starch. To explain the effects of these parameters, we have also studied glucose transport in K. fragilis. MATERIAEJ AND METHODS Micro-organisms S. cerevisiae CBS 8066 and K. ~gi1i.s were maintained on yeast malt agar slopes at 4°C. K. fragilis was isolated from a dairy product. It was obtained from the Laboratoire de Biologie Industrielle et Alimentaire. Substrate preparation Two types of hydrolysed starch were used. The first substrate was composed of a completely hydrolysed starch (hydrolysed starch ‘A’) containing 97% of glucose. The second substrate (hydrolysed starch ‘B’) contained 45% of glucose and 45% of maltose. These substrates were added separately to whey permeate and the mixtures obtained were supplemented with 1.5 g/litre ammonium sulphate, 0.5 g/litre yeast extract and 2 mg/litre FeCl,. The composition of the different growth media used is indicated in Table 1. Culture conditions S. cerevkiae and K. fragilis were grown continuously in a 2-litre Biolafitte fez-mentor (LSL Biolafitte, Saint-Germain-en-Laye, France) with a 1 litre working volume. Dissolved oxygen, measured by a polarographic probe, was maintained above 30% air saturation. The pH was regulated at 4.5 by automatic addition of 2 M NaOH. The temperature was maintained at 30°C. The continuous culture was preceded by a batch culture of S. cerevzXae on 5 g/litre of ethanol. When the ethanol was completely consumed, the continuous feed of the fermentor was started. At the steady state, an inoculum of K. pagi1i.swas added to the medium. Glucose transport To study glucose transport in K. fiagih, the yeast was grown continuously at different dilution rates, in a Biolafitte fermentor with 1 litre working volume on a yeast nitrogen base (Osibio, Paris, France) at 1.34% supplemented with 10 g/litre of glucose. The pH was regulated at 4.5 by automatic addition of 2 M NaOH. The temperature was maintained at 30°C and dissolved oxygen was kept above 30% of air saturation.
Table 1. Composition
starch media Medium
WG WGM
of whey permeate and hydrolysed
Composition
Whey permeate + hydrolysed starch ‘A’ Whey permeate + hydrolysed starch ‘B’
Concentration (gjlitre) Lactose
Glucose
Maltose
5
5
-
5
3
3
At each dilution rate studied, 10 ml of cell suspension harvested from the continuous culture was washed twice with distilled water, suspended in 50 ml of 0.1 Mphosphate buffer (pH- 6.5) and then exposed to difEerent concentrations of the labelled glucose. Transport assays were carried out at 30°C. The reaction was started by the addition of 0.1 ml of the labelled substrate 1 ,&i/ml D[U-‘“Cl glucose (Amersham) to 2.2 ml of cell suspension. The concentration of glucose varied from 0.5 to 100 mu. The final volume was 2.5 ml. Samples (0.5 ml) were taken every 20 s for 80 s. Under these conditions the glucose uptake was linear. The reaction was stopped by diluting the sample in 10 ml unlabelled glucose (200 mu) at 4°C. The cells were immediately filtered through a Millipore 0.22 pm filter and washed twice with a concentrated glucose solution. The filters were then dissolved with 0,5 ml SoluenelOO (Packard). Scintillation liquid ( 10 ml) (In&gel Packard) were added to the vials and the radioactivity was measured in a Packard Tri-Carb 460 CD benchtop scintillation counter. The parameters of glucose transport were determined using a numerical technique adapted for parametric estimation based on the use of artificial life for global optimisation, developed in the laboratory. Analytical techniques Dry weight Of the continuous culture 10 ml were harvested, centrifuged at 7000g for 10 min, washed with distilled water, centrifuged again then dried at 105°C for 24 h. Residual sugars Culture samples obtained during the steady state were filtered through a 0.22 pm Millipore fiker. Lactose, glucose and maltose were determined using enzymic kits (Boehringer, Meylan, France, ref. 986 119 and 1113950).
Yeast continuous
mixed culture on whey permeate and hydrolysed starch
383
Cells counis
cells of S. ceratisiae and K. fiagih were obtained by counting colonies on a solid medium contain@ yeast nitrogen base 6.7 g/litre; lactose 10 g/litre; glucose 10 g/line and 5-bromo-4chloro-3-indolyl &D galactopyranoside at 40 mg/ litre. Spread plates were incubated at 30°C for 48 h. Under these conditions, K. [email protected] hydrolyses 5-bromo-4-chloro-3-indolyl B-D galactopyranoside, hence the colonies of K. j?agiZis appear blue whereas the colonies of S. cerevisiae are white.
Viable
Analysis of vohtiles Ethanol and acetate were measured by gas chromatography using a chromatograph Delsi Di 200 with an Enica 2 1 integrator (Del& Argenteuil, France). The 2 m x 0.002 m stainless steel cohunn was packed with Rorapak Q SO/l00 mesh. The carrier gas used was nitrogen. The injector and the detector were set at 220°C and the oven temperature was 170°C. Methanol was used as an internal standard.
RESULTS Influence of the substrate nature The mixed cultures of S. cerevisiae and K. fragilis are produced continuously at D = 0.18 h - ’ on the two media (whey permeate + hydrolysed starch A WG and whey permeate+ hydrolysed starch B WGM) as described in Materials and Methods. Figure l(A) shows that for the two media tested, the levels of residual lactose and glucose were negligible. On the WG medium, the cell yield obtained was 0.44 g/g; on the WGM the cell yield was 0.48 g/g. Note that in the latter case, the level of residual maltose was relatively high (0.4 g/litre). Figure l(B) shows that on the WG medium, at the steady state, the yeast population was composed of 100% K. fragilis, S. cerevisiae having been washed out. On the other hand, when WGM was fed to the culture, the population percentages of K. fragiais and S. cerevtiiae stabilised respectively at 15 and 85%. Influence of dih&on rate Contimtous cultures were carried out on the medium WG at two dilution rates (0.18 and 0.3 h-l). Higher dilution rates were not tested because in such conditions S.ceretiiae CBS 8066
WG
WGM
Medium
WG
WGM
Medium
Fig. 1. Effect of the substrate on cell yield (Y,,&, residual sugars (A) and composition of the yeast population at the steady state (B), during the continuous mixed culture of Kluyveromyces fra@is and Saccharomyces ceretiiae at D=O.18 h-‘.
exhibits a fermentative metabolism resulting in a decrease in cell yield.’l Figure 2 shows that at all the dilution rates studied, the growth yield was equal to 0.44 g/g and both lactose and glucose were completely assimilated. Nevertheless at D = 0.3 h-l, slight increases in both residual lactose and glucose were noted. The evolution of S. cerevisiae and K. fragilis populations was also similar. S. cerevisiae was progressively washed out and at the steady state, the yeast population was made up of 100% K. fragilis. Influence of yeast extract For an economical process for biomass production, the nutrient supplementation should be minimised. To reach this goal the concentration of yeast extract was lowered from 0.5 to 0.1 g/litre. Figure 3 shows that on WGM medium, when the dilution rate was maintained at 0.18 h- ’ and yeast extract concentration was equal to 0.1 g/litre, the cell yield obtained was 0.44 g/g and only a slight accumulation of residual lactose and residual maltose was observed. The shift of yeast extract from 0.1 to 0.5 g/litre contributes to a
384
H. Kallel-Mhiri, C. Valance, J. M. Engasser,A. Miclo
0.18
0.3 D (h-l)
0.18
0.3 D (h-1)
Fig. 2. Influence of dilution rate on cell yield ( Y&. residual sugars (A) and composition of the yeast population at the steady state (B), during the continuous mixed culture of Kluyveromyces j?agiIis and Saccharomyces cerevisiae at D-O.18 h-’ on diluted whey permeate and hydrolysed starch composed of 97% glucose (WG).
minimal level of residual lactose and an increase in the cell yield (0.48 g/g) but the concentration of residual maltose increased. The K. fregilis percentage in yeast population dropped from 60 to 15% whereas that of S.cerevisiae increased from 40 to 85%. On WG medium, when the yeast extract concentration was equal to 0.5 g/&e, the same yield (0.44 g/g) and percentages of K. jkrgiks and S. cerevkiae (100-O%) were reached for D = 0.18 and D = 0.3 h-r. Figure 4 shows that at D = 0.3 h-t, when the yeast extract was reduced from 0.5 to 0.1 g/litre, the cell yield fell to 0.26 g/g and a high residual lactose was noted. The percentage of K. frqiiis dropped to 70% while that of S. cerevisiae rose to 30%. Glucose transport To explain the results obtained, we have studied glucose transport in K. jkqiZi.s and compared it to that already mentioned by several authors for S.cerevi&e CBS 8066.12 Glucose transport in K. jkzgifis was only studied in batch culture13and in such conditions
0.1
0.5
Yeastnrtract(en) Fig. 3. Effect of yeast extract concentration on cell yield ( Yx, S), residual sugars (A) and composition of the yeast population at the steady state (B), during the continuous mixed culture of Kluyvemmyces fragiiis and Soccharomyces cerevisiae at D = 0.18 h-’ on diluted whey permeate and hydrolysed starch composed of 45% glucose and 45% maltose (wGM).
the glucose concentration was not constant. However many studies concerning different yeasts including Candidu utili.s and S. cerevisiue, had shown that sugar concentration is a significant parameter in the regulation of the synthesis of sugar carriers.12,r4The yeasts exhibit different kinetics of sugar transport depending on the sugar concentration present in the medium. To overcome the difficulties due to glucose concentration variation, we have studied glucose transport in K. jirrgi2i.sin a continuous culture. The fermentation was performed at different dilution rates and the results obtained are presented in Table 2. These results indicate that K. fragilis possesses two glucose carriers. A high afhnity glucose carrier characterised by a K, of 0.025 m and a low affinity carrier which has a Km of 4 mu. The presence of these glucose transporters depends on the dilution rate. At a low dilution rate (D < 0.26 h- ’) where the glucose concentration was low, the two transporters were synthesised by the yeast. At high dilution rates (D > 0.3 h- ‘) corresponding to
Yeast continuous
mixed culture on whey permeate and hydrolysed starch 5.0
0.1
DISCUSSION
n
4.0
Ii
3.0
5
2.0
% 1
1.0
1 ._
0.0
cl
45
Yeastextract (pn, Fig. 4. Effect of yeast extract concentration on cell yield ( Yx, S), residual sugars (A) and the yeast population composition at the steady state (B), during the continuous mixed culture of Kluyveromyces jkag% and Saccharomyces cerevisiae at D = 0.3 h- ’on diluted whey permeate and hydrolysed starch composed of 97% glucose (WG).
Table 2. Glucose fragiis
transport in continuous cultures of K.
Dilution rate (h-l)
Residual glucose (mu)
High afinify carrier K, Cm@
Low afinify carrier K, PW
o-12 0.26
0.16 0.15 13.9 36.6
0.025 0.026 -
4 4 4 4
.0.31 0.45
385
high levels of residual glucose, only the low affinity carrier was present. Similar studies were carried out in S. cerevisiae by Postma et al. l4 These authors have shown that S. ceretiiae also possesses two glucose transporters which are present at all the tested dilution rates (from 0.1 to 0.46 h-l). Compared to the results obtained for K. fmgiIti, the high affinity carrier of S. cemvkze is characterised by a K, 40 fold higher than the one of K. fiugi2i.s.The K, of the low affinity transporter of S. cerevisiue is also higher than that of K. jkzgilis (5 fold).
A comparison of the kinetics of glucose transport in the two yeasts indicates that the rate of glucose uptake in K. fragilis is faster than that of S. cerevikze. When the medium contains only lactose and glucose, K. fragiis is pre-eminent over S. cerevisiue at all dilution rates. The results obtained on WG at D = 0.3 h- l for a yeast extract concentration of 0.1 g/litre, are due to the growth limitation of K. ji-ugiiis. At this dilution rate this limitation causes an accumulation of lactose and glucose. The level of the accumulated glucose induces its use by S. cerevkze. This fact explains the appearance of S. cerevisiue in the medium. For the lower dilution rate (D-O.18 h-l) on the medium WGM and for 0.1 g/litre of yeast extract, the growth limitation of K. fiugiZi.s seems to be less severe because less lactose and glucose are accumulated in the medium. Under these conditions K. frugiZi.s grows on lactose and a major part of glucose, whereas S. cerevisiae used maltose and a minor part of glucose. However, the percentage of S. cerevisiae obtained on WGM and yeast extract at 0.5 g/litre is relatively high compared to the results expected according to glucose transport. These results show that it is possible to use mixed cultures for biomass production on whey permeate and different types of hydrolysed starch. In most the cases, the cell yield was high without a noticeable accumulation of residual sugars. Moreover, we have also obtained stable mixed yeast populations. The nature of the substrate used, the concentration of the yeast extract and the dilution rate are important parameters which influence the composition of yeast population and the performance of the process. When the medium contains a mixture of lactose, glucose, maltose and yeast extract at a concentration of 0.5 g/litre, Scerevisiue represents 85% of the yeast population and the cell yield obtained is high (0.48 g/g). However when the continuous culture is operated on the same medium but supplemented by only 0.1 g/litre of yeast extract, the yeast population was made up of 60% of K. j-iugik.s and the cell yield obtained was slightly decreased. On the other hand, if the medium contains only glucose, lactose and yeast extract at 0.5 g/litre, the yeast population is mainly composed of K. fragilis and the cell yield is equal to 0.44 g/g. These
386
H. Kalkd-Mhiri,
C. Valance, J. M. Engasser, A. Miclo
results were obtained at all the tested dilutions rates. At D = 0.3 h- *, lowering the concentration of yeast extract to 0.1 g/l&e resulted in a decrease of the cell yield and in the appearance of Scerevisiue. RESERENCE!3 1. Fellows. P. J. & Worgan. J. T. Growth of Saccharomvces and Cam&a kilti in mix& culture on sic @digei materials. EnzwneMicrob. TechnoL.9 (198’7) 430-3. 2. Fellows, P. J. & Worgan, J. T. Growth of Sackwvmyces fibufiger and Candkia utilis in tied culture on apple processjng wastes. Enzyme Microb. TechnoL, 9 (1987) 434-7. 3. Grootjen, D. R J., Meijlink, H. H. M., Vleesenbeek, R, Van der Lans, R. G. J. M. & Luyben, K. Ch. A. M. Cofermentation of glucose and xylose with immobilized Pi&a stipitis in combination with Saccharomyces cerevi&e. Enzyme Microb. Technol., 13 (1991) 530-6. 4. Bodie, E. A., Anderson, T. M., Goodman, N. & Schwartz, R D. Propionic acid fermentation of ultrahigh-temperature sterilized whey using mono and ~4e$cultures. Appl. Microbial. Biotechnol., 25 (1987) 5. Harrison, D. E. F. Mixed cultures in iudustrial processes. In Advances in AppIied Microbiology, ed. D. Perhnan. Academic Press, London, 1978, pp.129-64. 6. Carlotti, A., Jacob, F., Bxrier, J. & Poncet, S. Yeast production from crude sweet whey by a mixed culture of
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Candida kfyr LY496 and Candida valida LY497. Biotechnoi.Lett., 13 (1990) 437-40. Meyrath, J. & Bayer, K. Biomass from whey. In Ecunomic Microbiology, ed. A. H. Rose. Academic Press, London, 1979, pp.207-69. Alam, L. Effect of ferment&cm on lactose, ghxose, and galactose content in milk and suitability 01 fermented milk tmxiucts for Lactose intolerants individuals. J. Dairy Sci., 65 (1982) 346-52. Goochee, C. F., Hatch, R T. & Cadman, T. W. Evahration of Escherichia coli and Candida utilis as a model continuous. comnetitive mixed culture svstem. BiotechnoI.B&ngng,S29 (1987) 453-63. _ Van Breuseaem. V. & Bastin. G. Ootimal control of biomass grow& in’s mixed culture. kotechnol. Bioengng, 35 (1990) 349-55. Postma, E., Verduyn, C., Scheffers, W. A. % Van Dijken J. P. Enzymic an&&s of the Crabtree effect in glucoselimited chemostat cultures of Sacchuromyces cerevisiae. Appl. Environ. Mzkrobiol., 55 (1989) 468-77. Postma, E., Scheffers, W. A. & Van Dijken, J. P. Kinetics of growth and ghrcose transport in glucose-limited chemostat cultures of Sacc~myce-s cerevisiae CBS 8066. Yeast, 5 (1989) 159-65 Gasnier, B. Characterization of low- and hiah-affinitv glucose ~transporters in the yeast Kluyverom~es ma& ianus. Biochim. Biophys. Acm, 903 (1987) 425-33. pbstma, E., Verduyn, C., Scheffers, W. A. & Van Dijken, J. P. Adaptation of the kinetics of ghrcose transport to environmental conditions in the yeast Can&&r u&s CBS 62 1: a continuous culture study. J . Gen .Mictwbioi., 134 (1988) 1109-16.