Skip to main content
Log in

Growth of Kluyveromyces marxianus and formation of ethyl acetate depending on temperature

  • Biotechnological products and process engineering
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

Conversion of lactose into ethyl acetate by Kluyveromyces marxianus allows economic reuse of whey-borne sugar. The high volatility of ethyl acetate enables its process-integrated recovery by stripping. This stripping is governed by both the aeration rate and the partition coefficient, K EA,L/G. Cultivation at elevated temperatures should decrease the K EA,L/G value and thus favor stripping. K. marxianus DSM 5422 as a potent producer of ethyl acetate was cultivated aerobically in whey-borne media for studying temperature-dependent growth and ester formation. Shake flask cultivation proved thermal tolerance of this yeast growing from 7 to 47 °C with a maximum rate of 0.75 h−1 at 40 °C. The biomass yield was 0.41 g/g at moderate temperatures while low and high temperatures caused distinct drops. The observed μ-T and Y X/S-T dependencies were described by mathematical models. Further cultivations were done in an 1-L stirred reactor for exploring the effect of temperature on ester synthesis. Cultivation at 32 °C caused significant ester formation (Y EA/S = 0.197 g/g) while cultivation at 42 °C suppressed ester synthesis (Y EA/S = 0.002 g/g). The high temperature affected metal dissolution from the bioreactor delivering iron for yeast growth and preventing ester synthesis. Cultivation at 32 °C with a switch to 42 °C at the onset of ester synthesis allowed quick and efficient ester production (Y EA/S = 0.289 g/g). The high temperature lowered the K EA,L/G value from 78 to 44 L/L which heightened the gas-phase ester concentration (favoring ester recovery) without increasing the liquid-phase concentration (avoiding product inhibition).

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Fig. 1
Fig. 2

Similar content being viewed by others

References

  • Abdel-Banat BMA, Hoshida H, Ano A, Nonklang S, Akada R (2010) High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Appl Microbiol Biotechnol 85:861–867

    Article  PubMed  CAS  Google Scholar 

  • Antoce O-A, Antoce V, Takahashi K (1997) Calorimetric study of yeast growth and its inhibition by added ethanol at various pHs and temperatures. Netsu Sokutei 24(4):206–213

    CAS  Google Scholar 

  • Arrhenius S (1908) Immunochemie. Ergeb Physiol 7:480–551

    Article  CAS  Google Scholar 

  • Aziz S, Memon HR, Shah FA, Rajoka MI, Soomro SA (2009) Production of ethanol by indigenous wild and mutant strain of thermotolerant Kluyveromyces marxianus under optimized fermentation conditions. Pak J Anal Environ Chem 10(1+2):25–33

    CAS  Google Scholar 

  • Banat IM, Marchant R (1995) Characterization and potential industrial applications of five novel, thermotolerant, fermentative, yeast strains. World J Microbiol Biotechnol 11:304–306

    Article  Google Scholar 

  • Banat IM, Nigam P, Singh D, Marchant R, McHale AP (1998) Review: ethanol production at elevated temperatures and alcohol concentrations: Part I—yeasts in general. World J Microbiol Biotechnol 14:809–821

    Article  CAS  Google Scholar 

  • Beal C, Louvet P, Corrieu G (1989) Influence of controlled pH and temperature on the growth and acidification of pure cultures of Streptococcus thermophilus 404 and Lactobacillus bulgaricus 398. Appl Microbiol Biotechnol 32:148–154

    Article  CAS  Google Scholar 

  • Brady D, Nigam P, Marchant R, McHale AP (1997) Ethanol production at 45 °C by alginate-immobilized Kluyveromyces marxianus IMB3 during growth on lactose-containing media. Bioprocess Eng 16:101–104

    Article  CAS  Google Scholar 

  • Cardona CA, Sánchez OJ (2007) Fuel ethanol production: process design trends and integration opportunities. Biores Technol 98:2415–2457

    Article  CAS  Google Scholar 

  • Christensen AD, Kádár Z, Oleskowicz-Popiel P, Thomsen MH (2011) Production of bioethanol from organic whey using Kluyveromyces marxianus. J Ind Microbiol Biotechnol 38:283–289

    Google Scholar 

  • Dragone G, Mussatto SI, Almeida e Silva JB, Teixeira JA (2011) Optimal fermentation conditions for maximizing the ethanol production by Kluyveromyces fragilis from cheese whey powder. Biomass Bioenergy 35:1977–1982

    Article  CAS  Google Scholar 

  • Duboc P, von Stockar U (1998) Systematic errors in data evaluation due to ethanol stripping and water vaporization. Biotechnol Bioeng 58:428–439

    Article  PubMed  CAS  Google Scholar 

  • Esener AA, Roels JA, Kossen NWF (1981) The influence of temperature on the maximum specific growth rate of Klebsiella pneumoniae. Biotechnol Bioeng 23:1401–1405

    Article  Google Scholar 

  • Etschmann MMW, Sell D, Schrader J (2003) Screening of yeasts for the production of the aroma compound 2-phenylethanol in a molasses-based medium. Biotechnol Lett 25:531–536

    Article  PubMed  CAS  Google Scholar 

  • Fatichenti F, Bernardi E (1987) Kluyveromyces fragilis SS-437: an associatively profiled thermotolerant yeast. Antonie van Leeuwenhoek 53:119–124

    Article  PubMed  CAS  Google Scholar 

  • Garcia D, Ramos AJ, Sanchis V, Marín S (2009) Predicting mycotoxins in foods: a review. Food Microbiol 26:757–769

    Article  PubMed  CAS  Google Scholar 

  • Ghaly AE, Kamal M, Avery A (2003) Influence of temperature rise on kinetic parameters during batch propagation of Kluyveromyces fragilis in cheese whey under ambient conditions. World J Microbiol Biotechnol 19:741–749

    Google Scholar 

  • Guimarães PMR, Teixeira JA, Domingues L (2010) Fermentation of lactose to bio-ethanol by yeasts as part of integrated solutions for the valorisation of cheese whey. Biotechnol Adv 28:375–384

    Article  PubMed  Google Scholar 

  • Grba S, Stehlik-Tomas V, Stanzer D, Vahčić N, Škrlin A (2002) Selection of yeast strain Kluyveromyces marxianus for alcohol and biomass production on whey. Chem Biochem Eng Q 16(1):13–16

    CAS  Google Scholar 

  • Guo X, Zhou J, Xiao D (2010) Improved ethanol production by mixed immobilized cells of Kluyveromyces marxianus and Saccharomyces cerevisiae from cheese whey powder solution fermentation. Appl Biochem Biotechnol 160:532–538

    Article  PubMed  CAS  Google Scholar 

  • Hinshelwood CN (1946) Influence of temperature on the growth of bacteria. In: The chemical kinetics of the bacterial cell. Clarendon, Oxford, pp 254–257

    Google Scholar 

  • Hortsch R, Löser C, Bley T (2008) A two-stage CSTR cascade for studying the effect of inhibitory and toxic substances in bioprocesses. Eng Life Sci 8:650–657

    Article  CAS  Google Scholar 

  • Hughes DB, Tudroszen NJ, Moye CJ (1984) The effect of temperature on the kinetics of ethanol production by a thermotolerant strain of Kluyveromyces marxianus. Biotechnol Lett 6:1–6

    Article  CAS  Google Scholar 

  • Johnson FH, Lewin I (1946) The growth rate of E. coli in relation to temperature, quinine and coenzyme. J Cell Comp Physiol 28:47–75

    Article  CAS  Google Scholar 

  • Kallel-Mhiri H, Engasser J-M, Miclo A (1993) Continuous ethyl acetate production by Kluyveromyces fragilis on whey permeate. Appl Microbiol Biotechnol 40:201–205

    Article  CAS  Google Scholar 

  • Kallel-Mhiri H, Miclo A (1993) Mechanism of ethyl acetate synthesis by Kluyveromyces fragilis. FEMS Microbiol Lett 111:207–212

    Article  CAS  Google Scholar 

  • Kourkoutas Y, Dimitropoulou S, Kanellaki M, Marchant R, Nigam P, Banat IM, Koutinas AA (2002) High-temperature alcoholic fermentation of whey using Kluyveromyces marxianus IMB3 yeast immobilized on delignified cellulosic material. Biores Technol 82:177–181

    Article  CAS  Google Scholar 

  • Longhi LGS, Luvizetto DJ, Ferreira LS, Rech R, Ayub MAZ, Secchi AR (2004) A growth kinetic model of Kluyveromyces marxianus cultures on cheese whey as substrate. J Ind Microbiol Biotechnol 31:35–40

    Article  PubMed  CAS  Google Scholar 

  • Löser C, Schröder A, Deponte S, Bley T (2005) Balancing the ethanol formation in continuous bioreactors with ethanol stripping. Eng Life Sci 5:325–332

    Article  Google Scholar 

  • Löser C, Urit T, Nehl F, Bley T (2011) Screening of Kluyveromyces strains for the production of ethyl acetate: design and evaluation of a cultivation system. Eng Life Sci 11:369–381

    Article  Google Scholar 

  • Löser C, Urit T, Förster S, Stukert A, Bley T (2012) Formation of ethyl acetate by Kluyveromyces marxianus on whey during aerobic batch and chemostat cultivation at iron limitation. Appl Microbiol Biotechnol 96:685–696

    Article  PubMed  Google Scholar 

  • Löser C, Urit T, Stukert A, Bley T (2013) Formation of ethyl acetate from whey by Kluyveromyces marxianus on a pilot scale. J Biotechnol 163:17–23

    Article  PubMed  Google Scholar 

  • Mussatto SI, Dragone G, Guimarães PMR, Silva JPA, Carneiro LM, Roberto IC, Vicente A, Domingues L, Teixeira JA (2010) Technological trends, global market, and challenges of bio-ethanol production. Biotechnol Adv 28:817–830

    Article  PubMed  CAS  Google Scholar 

  • Panesar PS, Kennedy JF, Knill CJ, Kosseva M (2010) Production of L(+) lactic acid using Lactobacillus casei from whey. Braz Arch Biol Technol 53:219–226

    Article  CAS  Google Scholar 

  • Pinheiro R, Belo I, Mota M (2002) Oxidative stress response of Kluyveromyces marxianus to hydrogen peroxide, paraquat and pressure. Appl Microbiol Biotechnol 58:842–847

    Article  PubMed  CAS  Google Scholar 

  • Pinheiro R, Belo I, Mota M (2003) Growth and β-galactosidase activity in cultures of Kluyveromyces marxianus under increased air pressure. Lett Appl Microbiol 37:438–442

    Article  PubMed  CAS  Google Scholar 

  • Pohl P (2007) What do metals tell us about wine? Trends Anal Chem 26:941–949

    Article  CAS  Google Scholar 

  • Ratkowsky DA, Olley J, McMeekin TA, Ball A (1982) Relationship between temperature and growth rate of bacterial cultures. J Bacteriol 149:1–5

    PubMed  CAS  Google Scholar 

  • Ratkowsky DA, Lowry RK, McMeekin TA, Stokes AN, Chandler RE (1983) Model for bacterial culture growth rate throughout the entire biokinetic temperature range. J Bacteriol 154:1222–1226

    PubMed  CAS  Google Scholar 

  • Rosso L, Lobry JR, Flandrois JP (1993) An unexpected correlation between cardinal temperatures of microbial growth highlighted by a new model. J Theor Biol 162:447–463

    Article  PubMed  CAS  Google Scholar 

  • Rosso L, Lobry JR, Bajard S, Flandrois JP (1995) Convenient model to describe the combined effects of temperature and pH on microbial growth. Appl Environ Microbiol 61:610–616

    Google Scholar 

  • Sá-Correia I, van Uden N (1982) Effects of ethanol on thermal death and the maximum temperature for growth of the yeast Kluyveromyces fragilis. Biotechnol Lett 4:805–808

    Article  Google Scholar 

  • Sá-Correia I, van Uden N (1983) Temperature profiles of ethanol tolerance: effect of ethanol on the minimum and the maximum temperature for growth of the yeast S. cerevisiae and K. fragilis. Biotechnol Bioeng 25:1665–1667

    Article  PubMed  Google Scholar 

  • Saerens SMG, Delvaux F, Verstrepen KJ, Van Dijck P, Thevelein JM, Delvaux FR (2008) Parameters affecting ethyl ester production by Saccharomyces cerevisiae during fermentation. Appl Environ Microbiol 74:454–461

    Article  PubMed  CAS  Google Scholar 

  • Sampaio JP, Spencer-Martins I (1989) Adaptive growth at high temperatures of the lactose-fermenting yeast Kluyveromyces marxianus var. marxianus. J Basic Microbiol 29:61–64

    Article  CAS  Google Scholar 

  • Schoolfield RM, Sharpe PJH, Magnuson CE (1981) Non-linear regression of biological temperature-dependent rate models based on absolute reaction-rate theory. J Theor Biol 88:719–731

    Article  PubMed  CAS  Google Scholar 

  • Sharpe PJH, DeMichele DW (1977) Reaction kinetics of poikilotherm development. J Theor Biol 64:649–670

    Article  PubMed  CAS  Google Scholar 

  • Silveira WB, Passos FJV, Mantovani HC, Passos FML (2005) Ethanol production from cheese whey permeate by Kluyveromyces marxianus UFV-3: a flux analysis of oxido-reductive metabolism as a function of lactose concentration and oxygen levels. Enzyme Microb Technol 36:930–936

    Article  CAS  Google Scholar 

  • Szczodr J, Szewczuk D, Rogalski J, Fiedurek J (1997) Selection of yeast strain and fermentation conditions for high-yield ethanol production from lactose and concentrated whey. Acta Biotechnol 17:51–61

    Article  Google Scholar 

  • Urit T, Löser C, Wunderlich M, Bley T (2011) Formation of ethyl acetate by Kluyveromyces marxianus on whey: studies of the ester stripping. Bioprocess Biosyst Eng 34:547–559

    Article  PubMed  CAS  Google Scholar 

  • Urit T, Löser C, Stukert A, Bley T (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

    Article  PubMed  CAS  Google Scholar 

  • Urit T, Manthey R, Bley T, Löser C (2013) Formation of ethyl acetate by Kluyveromyces marxianus on whey: influence of aeration and inhibition of yeast growth by ethyl acetate. Eng Life Sci 13:247–260

    Article  CAS  Google Scholar 

  • Vivier D, Ratomahenina R, Moulin G, Galzy P (1993) Study of physicochemical factors limiting the growth of Kluyveromyces marxianus. J Ind Microbiol 11:157–161

    Article  CAS  Google Scholar 

  • Ward C, Nolan AM, O’Hanlon K, McAree T, Barron N, McHale L, McHale AP (1995) Production of ethanol at 45 °C on starch-containing media by mixed cultures of the thermotolerant, ethanol-producing yeast Kluyveromyces marxianus IMB3 and the thermophilic filamentous fungus Talaromyces emersonii CBS 814.70. Appl Microbiol Biotechnol 43:408–411

    Article  CAS  Google Scholar 

  • Willetts A (1989) Ester formation from ethanol by Candida pseudotropicalis. Antonie van Leeuwenhoek 56:175–180

    Article  PubMed  CAS  Google Scholar 

  • Wolter H, Lietz P, Beubler A (1966) Influence of temperature and yeast strain on the formation of amyl alcohol, isobutanol and ethyl acetate in fermenting malt wort. Folia Microbiol 11:210–214

    Article  CAS  Google Scholar 

  • Zourari A, Accolas JP, Desmazeaud MJ (1992) Metabolism and biochemical characteristics of yogurt bacteria. A review. Lait 72:1–34

    Article  CAS  Google Scholar 

  • Zwietering MH, de Koos JT, Hasenack BE, de Wit JC, van’t Riet K (1991) Modeling of bacterial growth as a function of temperature. Appl Environ Microbiol 57:1094–1101

    PubMed  CAS  Google Scholar 

  • Zwietering MH, Wijtzes T, Rombouts FM, van’t Riet K (1993) A decision support system for prediction of microbial spoilage in foods. J Ind Microbiol 12:324–329

    Article  Google Scholar 

  • Zwietering MH, de Wit JC, Notermans S (1996) Application of predictive microbiology to estimate the number of Bacillus cereus in pasteurised milk at the time of consumption. Int J Food Microbiol 30:55–70

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

Thanet Urit would like to express his thank to the Rajabhat Nakhon Sawan University (Muang Nakhon Sawan, Thailand) for financial support. We are grateful to Mrs. E. Kneschke from our institute for general technical assistance, to Dipl.-Ing. M. Heller from the Sachsenmilch AG (Leppersdorf, Germany) for providing whey permeate, and to Dr. H.-J. Stärk from the Helmholtz-Zentrum für Umweltforschung GmbH – UFZ (Leipzig, Germany) for analyzing trace metals.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Christian Löser.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Urit, T., Li, M., Bley, T. et al. Growth of Kluyveromyces marxianus and formation of ethyl acetate depending on temperature. Appl Microbiol Biotechnol 97, 10359–10371 (2013). https://doi.org/10.1007/s00253-013-5278-y

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-013-5278-y

Keywords

Navigation