Abstract
The yeast Kluyveromyces marxianus has been pointed out as a promising microorganism for a variety of industrial bioprocesses. Although genetic tools have been developed for this yeast and different potential applications have been investigated, quantitative physiological studies have rarely been reported. Here, we report and discuss the growth, substrate consumption, metabolite formation, and respiratory parameters of K. marxianus CBS 6556 during aerobic batch bioreactor cultivations, using a defined medium with different sugars as sole carbon and energy source, at 30 and 37 °C. Cultivations were carried out both on single sugars and on binary sugar mixtures. Carbon balances closed within 95 to 101 % in all experiments. Biomass and CO2 were the main products of cell metabolism, whereas by-products were always present in very low proportion (<3 % of the carbon consumed), as long as full aerobiosis was guaranteed. On all sugars tested as sole carbon and energy source (glucose, fructose, sucrose, lactose, and galactose), the maximum specific growth rate remained between 0.39 and 0.49 h−1, except for galactose at 37 °C, which only supported growth at 0.31 h−1. Different growth behaviors were observed on the binary sugar mixtures investigated (glucose and lactose, glucose and galactose, lactose and galactose, glucose and fructose, galactose and fructose, fructose and lactose), and the observations were in agreement with previously published data on the sugar transport systems in K. marxianus. We conclude that K. marxianus CBS 6556 does not present any special nutritional requirements; grows well in the range of 30 to 37 °C on different sugars; is capable of growing on sugar mixtures in a shorter period of time than Saccharomyces cerevisiae, which is interesting from an industrial point of view; and deviates tiny amounts of carbon towards metabolite formation, as long as full aerobiosis is maintained.








Similar content being viewed by others
References
Bacci Júnior M, Siqueira CG, Antoniazi SA, Ueta J (1996) Location of the beta-galactosidase of the yeast Kluyveromyces marxianus var. marxianus ATCC 10022. Anton Leeuw 69:357–361
Bajpai P, Margaritis A (1987) The effect of temperature and pH on ethanol production by free and immobilized cells of Kluyveromyces marxianus grown on Jerusalem artichoke extract. Biotechnol Bioeng 30:306–313
Bellaver LH, de Carvalho NMB, Abrahão-Neto J, Gombert AK (2004) Ethanol formation and enzyme activities around glucose-6-phosphate in Kluyveromyces marxianus CBS 6556 exposed to glucose or lactose excess. FEMS Yeast Res 4:691–698
Belloch C, Fernández-Espinar T, Querol A, Dolores-García M, Barrio E (2002) An analysis of inter- and intraspecific genetic variabilities in the Kluyveromyces marxianus group of yeast species for the reconsideration of the K. lactis taxon. Yeast 19:257–268
Berkamp RJM, Bootsman TC, Toschka HY, Mooren ATA, Kox L, Verbakel JMA, Geerse RH, Planta RJ (1993) Expression of an alpha-galactosidase gene under control of the homologous inulinase promoter in Kluyveromyces marxianus. Appl Microbiol Biotechnol 40:309–317
Breunig KD (1989) Glucose repression of Lac gene expression in yeast is mediated by the transcriptional activator LAC9. Mol Gen Genet 216:422–427
Caballero R, Olguín P, Cruz-Guerrero A, Gallardo F, García-Garibay M, Gómez-Ruiz L (1995) Evaluation of Kluyveromyces marxianus as baker’s yeast. Food Res Int 28:37–41
Cai XP, Zhang J, Yuan HY, Fang ZA, Li YY (2005) Secretory expression of heterologous protein in Kluyveromyces cicerisporus. Appl Microbiol Biotechnol 67:364–369
Castrillo JI, Ugalde UO (1993) Patterns of energy metabolism and growth kinetics of Kluyveromyces marxianus in whey chemostat culture. Appl Microbiol Biotechnol 40:386–393
Cruz-Guerrero A, García-Peña I, Bárzana E, García-Garibay M, Gómez-Ruiz L (1995) Kluyveromyces marxianus CDBB-L-278: a wild inulinase hyperproducing strain. J Ferment Bioeng 80:159–163
de Bruijne AW, Schuddemat J, van den Broek PJA, van Steveninck J (1988) Regulation of sugar transport systems of Kluyveromyces marxianus: the role of carbohydrates and their catabolism. Biochim Biophys Acta 939:569–576
Fabre CE, Blanc PJ, Goma G (1998) Production of 2-phenylethyl alcohol by Kluyveromyces marxianus. Biotechnol Prog 14:270–274
Fiechter A, Fuhrmann GF, Kappeli O (1981) Regulation of glucose metabolism in growing yeast cells. Adv Microb Physiol 22:123–183
Fonseca GG, Gombert AK, Heinzle E, Wittmann C (2007) Physiology of the yeast Kluyveromyces marxianus during batch and chemostat cultures with glucose as the sole carbon source. FEMS Yeast Res 7:422–435
Fonseca GG, Heinzle E, Wittmann C, Gombert A (2008) The yeast Kluyveromyces marxianus and its biotechnological potential. Appl Microbiol Biotechnol 79:339–354
Furlan AS, Carvalho-Jonas MF, Merkle R, Bertoli GB, Jonas R (1995) Aplicação do sistema Microtiter Reader na seleção de microrganismos produtores de β-galactosidase. Arq Biol Tecnol 38:1261–1268
Gasnier B (1987) Characterization of low- and high-affinity glucose transports in the yeast Kluyveromyces marxianus. Biochim Biophys Acta 903:425–433
Goffrini P, Ficarelli A, Donnini C, Lodi T, Pulglisi PP, Ferrero I (1995) FOG1 and FOG2 genes, required for the transcriptional activation of glucose-repressible genes of Kluyveromyces lactis, are homologous to GAL83 and SNF1 of Saccharomyces cerevisiae. Curr Genet 29:316–326
Heinzle E, Dunn IJ (1991) Methods and instruments in fermentation gas analysis. In: Rehm HJ, Reed G, Pühler A, Stadler P (eds) Rehm biotechnology, vol. 4. VCH, Weinheim, pp 27–173
Hensing MC, Vrouwenvelder H, Hellinga C, Baartmans R, van Dijken JP (1994) Production of extracellular inulinase in high-cell-density fed-batch cultures of Kluyveromyces marxianus. Appl Microbiol Biotechnol 42:516–521
Hensing MC, Rouwenhorst RJ, Heijnen JJ, van Dijken JP, Pronk JT (1995) Physiological and technological aspects of large-scale heterologous-protein production with yeasts. Anton Leeuw 67:261–279
Johnston M (1999) Feasting, fasting and fermenting. Trends Genet 15:29–33
Kiers J, Zeeman AM, Luttik M, Thiele C, Castrillo JI, Steensma HY, van Dijken JP, Pronk JT (1998) Regulation of alcoholic fermentation in batch and chemostat cultures of Kluyveromyces lactis CBS 2359. Yeast 14:459–469
Kruse B, Schugerl K (1996) Investigation of ethanol formation by Pachysolen tannophilus from xylose and glucose/xylose co-substrates. Process Biochemistry 31:389–407
Møller K, Bro C, Piškur J, Nielsen J, Olsson L (2002a) Steady-state and transient-state analyses of aerobic fermentation in Saccharomyces kluyveri. FEMS Yeast Res 2:233–244
Monod J (1942) Diauxie et respiration au cours de la croissance des cultures de B. coli. Annales de L'Institut Pasteur 68:548–550
Nobre A, Lucas C, Leão C (1999) Transport and utilization of hexose and pentoses in the halotolerant yeast Debaryomyces hansenii. Appl Environ Microbiol 65:3594–3598
Olsson L, Nielsen J (1997) On-line and in situ monitoring of biomass in submerged cultivations. Trends Biotechnol 15:517–522
Ostergaard S, Olsson L, Johnston M, Nielsen J (2000) Increasing galactose consumption by Saccharomyces cerevisiae through metabolic engineering of the GAL gene regulatory network. Nat Biotechnol 18:1283–1286
Postma E, van der Broek PJA (1990) Continuous-culture study of the regulation of glucose and fructose transport in Kluyveromyces marxianus CBS 6556. J Bacteriol 172:2871–2876
Rech R, Cassini CF, Secchi AR, Ayub MAZ (1999) Utilization of protein-hydrolyzed cheese whey for the production of β-galactosidase by Kluyveromyces marxianus. J Ind Microbiol Biotechnol 23:91–96
Rocha SN, Abrahão-Neto J, Cerdán ME, González-Siso MI, Gombert AK (2010) Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus. Microbial Cell Fact 9:4
Rocha SN, Abrahão-Neto J, Cerdán ME, Gombert AK, González-Siso MI (2011) Heterologous expression of a thermophilic esterase in Kluyveromyces yeasts. Appl Microbiol Biotechnol 89:375–385
Ronnow B, Olsson L, Nielsen J, Mikkelsen JD (1999) Depression of galactose metabolism in melabiase producing baker’s yeast and distillers yeast. Biotechnology 72:213–228
Rouwenhorst RJ, Ritmeester WS, Scheffers WA, Van Dijken JP (1990) Localization of inulinase and invertase in Kluyveromyces species. Appl Environ Microbiol 56:3329–3336
Schulze KL, Lipe RS (1964) Relationship between substrate concentration, growth rate, and respiration rate of Escherichia coli in continuous culture. Arch Microbiol 48:1–20
Sonnleitner B, Kappeli O (1986) Growth of Saccharomyces cerevisiae is controlled by its limited respiratory capacity: formulation and verification of a hypothesis. Biotechnol Bioeng 28:927–937
Urit T, Stukert A, Bley T, Löser C (2012) Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch cultivation at specific trace element limitation. Appl Microbiol Biotechnol 96:1313–1323
van Dijken JP, Weusthuis RA, Pronk JT (1993) Kinetics of growth and sugar consumption in yeasts. Antonie van Leeuwenhoek 63:343–352
van Dijken JP, Bauer J, Brambilla L, Duboc P, Francois JM, Gancedo C, Giuseppin ML, Heijnen JJ, Hoare M, Lange HC, Madden EA, Niederberger P, Nielsen J, Parrou JL, Petit T, Porro D, Reuss M, van Riel N, Rizzi M, Steensma HY, Verrips CT, Vindeløv J, Pronk JT (2000) An interlaboratory comparison of physiological and genetic properties of four Saccharomyces cerevisiae strains. Enzym Microb Technol 26:706–714
van Hoek P, van Dijken JP, Pronk JT (1998) Effect of specific growth rate on fermentative capacity of baker’s yeast. Appl Environ Microbiol 64:4226–4233
van Zyl C, Prior BA, Kilian SG, Kock JL (1989) D-xylose utilization by Saccharomyces cerevisiae. J Gen Microbiol 135:2791–2798
Verduyn C (1991) Physiology of yeasts in relation to biomass yields. Anton Leeuw 60:325–353
Verduyn C, Postma E, Scheffers WA, van Dijken JP (1992) Effect of benzoic acid on metabolic fluxes in yeasts: a continuous-culture study on the regulation of respiration and alcoholic fermentation. Yeast 8:501–517
Wittmann C, Hans M, Bluemke W (2002) Metabolic physiology of aroma-producing Kluyveromyces marxianus. Yeast 19:1351–1363
Yazgan A, Ozcengiz G (1994) Subcellular-distribution of accumulated heavy metals in Saccharomyces cerevisiae and Kluyveromyces marxianus. Biotechnol Lett 16:871–874
Yazgan A, Ozcengiz G, Alaeddinoglu NG (1993) Studies on metal resistance system in Kluyveromyces marxianus. Biol Trace Elem Res 38:117–127
Yoshida Y, Yokoi W, Wada Y, Ohishi K, Ito M, Sawada H (2004) Potent hypocholesterolemic activity of the yeast Kluyveromyces marxianus YIT 8292 in rats fed a high cholesterol diet. Biosci Biotechnol Biochem 68:1185–1192
Yoshida Y, Yokoi W, Ohishi K, Ito M, Naito E, Sawada H (2005) Effects of the cell wall of Kluyveromyces marxianus YIT 8292 on the plasma cholesterol and fecal sterol excretion in rats fed on a high-cholesterol diet. Biosci Biotechnol Biochem 69:714–723
Zacchariae W, Breuning KD (1993) Expression of the transcriptional activator LAC9 (KlGAL4) in Kluyveromyces lactis is controlled by auto regulation. Mol Cell Biol 13:3058–3066
Acknowledgments
This work was financed by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, São Paulo, Brazil). We would also like to thank Prof. Marcos Morais Jr for providing us the strain used throughout this work.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Fonseca, G.G., de Carvalho, N.M.B. & Gombert, A.K. Growth of the yeast Kluyveromyces marxianus CBS 6556 on different sugar combinations as sole carbon and energy source. Appl Microbiol Biotechnol 97, 5055–5067 (2013). https://doi.org/10.1007/s00253-013-4748-6
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00253-013-4748-6


