β-Galactosidase production by Kluyveromyces lactis on milk whey: batch versus fed-batch cultures

Proce.wBiochemisnyf9(1994) 565-568 Q 1994 Elsevier scimce Limited Britain. All rights reserved 0032-9592/94/$7.00

Printed in Great ELSEVIER

/If?-GalactosidaseProduction by KZuyveromyces Zactison Milk Whey: Batch versus Fed-Batch Cultures M. I. Gonzalez Siso Departamento de Biologia Celular y Molecular, Universidad de Coruiia, Spain (Received 26 April 1993; revised version received and accepted 8 September 1993)

/3-Galactosidase production by the yeast Kluyveromyces la&is on milk whey as culture medium was compared in batch andfed-batch cultures. Significant prolongation of the phase of maximum @galactosi&se production can be achieved by optimizing the feeding rate infed-batch cultures.

INTRODUCTION The most promising procedures for the economic use of milk whey are based on the hydrolysis of lactose by /S-galactosidase (EC 3.2.1.23). Kluyveromyces la& and K. fiagz7i.s are the main sources of this enzyme,’ but the industrial development of such procedures has been limited by the high costs associated with &galactosidase production.*T3 The intracellular /?-galactosidase synthesized by K. 1acfi.s is known to be inducible by lactose and galactose. Repression by glucose depends on the yeast strain employed. In some strains pgalactosidase synthesis is blocked by glucose but in K. lactis NRRL_-Y 1140, the strain used in this work, about three-fold repression occurs in 2% glucose-galactose media.4,5 In preliminary experiments we have observed that when this strain was batch-cultured on milk whey, /3-galactosidase activity decreased drastically after lactose depletion when the cells approached stationary phase.6 In this paper, we have evaluated the performance of fed-batch relative to batch cultures for improvCorresponding author: M. I. GonzPez Siso. Tel: 28 0788; Fax: 10 4129.

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ing B-galactosidase production by K. Eactis on milk whey. The main experimental aims were: (a) to maintain a lactose concentration in the cultures sufficient to prevent a decrease in /3-galactosidase activity without causing repression and, in consequence, to prolong the phase of maximum pgalactosidase production; (b) to maximize both the amount of total B-galactosidase per millilitre of culture and the /3-galactosidase activity content per cell, at the end of fermentation, so as to make downstream processing more economical.

MATERIALS

AND METHODS

Kluyveromyces la& NRRL-Y1140 was used throughout. The culture medium was sweet milk whey obtained from a local dairy plant. The chemical composition of the two types used, which were derived from different steps of cheese-making, was as follows: Wl: lactose 50 g/litre, proteins 7-5 g/litre, lipids 5 g/litre, chemical oxygen demand 79 g O,/litre; W2: lactose 30 g/litre, proteins 4-5 g/litre, lipids 3 g/litre. Wl was also used with 5 g/litre NH,Cl, 5 g/litre K,HPO., and 1 g/litre yeast extract, up to the initial level of nutrients reported

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M. I. GonzxilezSiso

earlier.’ Prior to use, proteins were removed by centrifugation (15 min at 10 000 r-pm) under sterile conditions after precipitation by autoclave treatment at 121°C for 15 mm. The protein content in Wl and W2 was thus reduced to 2-5 and 1.5 g/litre, respectively, as measured by Lowry’s method8 with bovine serum albumin as standard. Batch cultures were performed in 500-ml Erlenmeyer flasks with 100 ml of culture medium in an orbital shaker at 30°C and 150 rpm. Fedbatch cultures were performed in a temperaturecontrolled bioreactor with magnetic stirring, fresh milk whey being added by a peristaltic pump. The rate of substrate inflow was altered to compensate for the decrease in volume when samples were taken. An exponential phase preculture grown on the same medium was used as culture inoculum, adjusted to produce an initial population of 0.5 x lo6 cells/ml. The number of cells in the suspension was determined by optical density measurement at 600 mn following calibration with cell counts performed in a Neubauer chamber. /3-Galactosidase activity was measured as described by Guarente.9 One enzyme unit (EU) is defined as the quantity of enzyme that catalyses the liberation of 1 ,ug of orthonitrophenol from orthonitrophenyl-p-n-galactopyranoside per min under assay conditions (3O”C, gentle mixing). Units are expressed per milligram of yeast biomass or per millilitre of culture. Yeast biomass was determined by centrifugation of lo-ml culture aliquots (4000 rpm for 5 mm), washing the pellet twice with distilled water and weighing after overnight drying at 60°C. Total sugars were determined as described by Dubois ef aZ.‘Owith glucose as standard; lipids by the method of Mars and Weinstein’ with tripalmitine as standard; and ethanol using the Boehringher Manheim (Germany) enzymatic test kit.

00

0 0

15

10

Time

45

60

7s

(hl

Fig. 1. Batch culture of Kluyveromyces lactis on milk whey (W 1). EU = enzymatic units; B = biomass.

1.0

200 1.1

y5

II rk_7

look;

-_

:

4.0 40

5

d!!

-;-__. 0 10

g

2 .

5

g

P

=20

5 = B f

t f!

i

4 I

I

1 0

0

0 Time

(hl

Fig. 2. Batch culture of Khyveromyces lactti on milk whey (WZ). EU = enzymatic units: B = biomass.

RESULTS AND DISCUSSION Batch cultures

From the batch cultures of K. ZactisNRRL-Y 1140 on milk whey Wl and W2 (Figs 1 and 2), it can be outlined that: (a) maximum values of total pgalactosidase activity were reached at the end of the exponential growth phase, (b) both /?-galac-

tosidase/biomass and #I-galactosidase millilitre culture decrease drastically when lactose was exhausted and the yeasts began to utilize the ethanol previously produced, (c) such decreases in activity were attenuated when cultures were grown in milk whey supplemented as described in Materials and Methods (Fig. 3).

/I-Galactosidaseproduction

by Kluyveromyces lactis

4J:’

567

,‘o

,

r lo

‘0 1

o-

0 0

20

40

60

80

100

Time (h)

Fig. 3. Batch culture of Kluyveromyces lactti on milkwhey (Wl) supplemented with 5 g/litre NH&l, 5 g/litre K,HF’O, and 1 g/litre yeast extract. EU =enzymatic units; B= biomass.

Fed-batch cultures A fed batch has been defined as a bioreactor with inflowing substrate but without outflow. F,, the volumetric flow rate, is defined as the quotient dV/dt where V is the reactor volume and t is the time. The specific growth rate, p, is equal to F,/ V at each time of the fed-batch culture.‘* In this work, two types of fed-batch cultures were performed: (a) maintaining constant F, (,H variable) and (b) maintaining constant ,u (F, variable). Cultures were initiated in the batch mode but, when about 40% of the initial lactose had been utilized, the continuous addition of substrate was started. F,, was adjusted so as to equalize the specific growth rate of the batch culture at the time of switching, with the initial (cultures ,u variable) or the constant (cultures F, variable) specific growth rate of the fed-batch system. With such a strategy of switching, biological perturbation is diminished and quasi-steadystate conditions can be achieved from the beginning of the fed batch.12*13 Cultures at constant F, Two values of F, were assayed, O-2 1 and O-17 ml/ h, starting at about 20 h of culture and with a V, (initial volume of the cultures) of 10 ml. Culture medium was unsupplemented W 1. Corresponding results are represented in Figs 4 and 5. As can

0

20

40

60

80

1

r

400

100

Time (h)

Fig. 4. Fed-batch culture of Kluyveromyces lactis on milk whey (W 1). F, = 0.21 ml/h. EU = enzymatic units; B = biomass.

Time M

Fig. 5. Fed-batch culture of Kluyveromyces lactis on milk whey (Wl). F,=O.17 ml/h. EU=enzymatic units; B = biomass.

be observed, the kinetics of cultures progressively approached a batch pattern until lactose depletion (as V increases, ,u decreases in parallel, because p = F,/ V at each time of the culture). With a feeding rate equivalent to 0.21 ml/h, ethanol production decreased with respect to the

M. I. Gonzcilez Siso

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Table 1. Ethanol produced by Kluyveromyces Iactis during growth on milk whey C&me

Wl, batch W2, batch Supplemented W2, batch Wl, fed-batch (a21 ml/h) W 1, fed-batch (0.17 ml/h)

When lactose concentration was about 25% of the initial g ethanol/ g biomass

g ethanol/ g utilized lactose

1.5 1.9 4.0 0.9 1.7

o-2 0.2 0.2 0.1 0.3

batch culture but this did not occur with the lower feeding rate (see also Table 1). With F, = 0.17 ml/ h, biomass and enzymic activity were maintained at a level equivalent to the maximum of the batch culture in the same medium (about 5-6 g/litre and 500 EU/ml, respectively) for a greatly increased period of time (from 30 up to 100 h in fed-batch, from 35 to 50 h in batch culture). While the stationary state was not reached on employing fedbatch cultures with constant F,,, it was possible to prolong the phase of #%galactosidase production significantly. Cultures at constant p In this type of culture, with an exponential increase in F,,, ~1, sugar concentration and #lgalactosidase levels can be maintained invariable for a long period of time. F,, was adjusted so that p was maintained at the level corresponding to the period between 24 and 30 h of the batch culture (p = 004 h-l V, = 100 ml). In such conditions a value of 4.7 + 0.1 g/litre biomass with /3-galactosidase activity of 500 z!z50 EU/ml was maintained for at least 4 days. The lactose concentration was 9 *O-3 g/litre. In this case, a quasi-stationary state was reached whose duration could even be significantly greater than that mentioned above. In conclusion, the fed-batch cultures appear to be interesting systems for /3-galactosidase production by K. h&s on milk whey, because they permit the extension of the phase of maximum /%galactosidase production.

When ethanol concentration was the maximum g ethanol/ g biomass

;:;: ;:; 2.3

g ethanol/ g utilized lactose 0.3 0.3 0.4 ;::

REFERENCES 1. Castillo. F. J.. Lactose metabolism bv veasts. In Yeast Biotechnolo& and Biocatalysts, ed. H;b&t Verachtert & Rene De Mot. Marcel Dekker, New York, 1990, pp. 297-320. 2. Gekas, V. & Lbpez-Leiva, M., Hydrolysis of lactose: a literature review. Proc. Biochem., 20 (1985) 2-12. 3. Machado, K. M. G. & Linardi, U. R., Production of amylase and B-galactosidase by yeasts. Arch. Biol. Technol., 33 (1990) 247-53. 4. Dickson, R. C. & Markin, J. S., Physiological studies of /&galactosidase induction in Kluyveromyces lactis. J. Bacterial., 142 (1980) 777-8.5. 5. Breunig, K. D., Glucose repression of LAC gene expression in yeast is mediated by the transcriptional activator LAC9. Mol. Gen. Genet., 216 (1989) 422-7. 6. Siso, M. I. G., Cerdh, E., Freire, M. A., Ramil, E., Belmonte, E. R. & Torres, A. M. R., /?-Galactosidase production by Kluyveromyces Iactis on milk whey. Abstracts of the ZV Portuguese-Spanish Biochemistry Congress (1991) lOP99. 7. Mawson, M. W., Yeast biomass production from acid whey permeate. Biotechnol. Lett., 10 (1988) 503-8. 8. Lowry, 0. H., Rosenbrough, N. J., Farr, A. L. & Randall, R. J., Protein measurements with the Folin Phenol reagent. J. Biol. Chem., 193 (1951) 265-75. 9. Guarente, L., Yeast promoters and LacZ fusions designed to study expression of cloned genes in yeasts. Method in Enzymology, lOl(l983) 181-9. 10. Dubois, M., Giles, U. A., Hamilton, J. K., Rebers, P. A. & Smith, F., Calorimetric method for determination of sugars and related substances. Anal. Chem., 28 (1956) 350-6. 11. Marsch, J. B. & Weinstein, D. B., Simple charring method for determination of lipids. J. Lipid Res., 7 (1966) 574-6. 12. Dunn, I. J. & Mor, 5. R, Variable-volume continuous cultivation. Biotechnol. Bioeng., 17 (1975) 1805-22. 13. Shimizu, H., Araki, K., Shioya, S. & Suga, K., Optimal production of glutathione by controlling the specific growth rate of yeasts in fed-batch culture. Biotechnol. Bioeng., 38 (1991) 196-205.