Applied Microbiology and Biotechnology

, Volume 64, Issue 3, pp 403–409 | Cite as

Metabolite profiles of the biocontrol yeast Pichia anomala J121 grown under oxygen limitation

  • E. Fredlund
  • A. Broberg
  • M. E. Boysen
  • L. Kenne
  • J. Schnürer
Original Paper


The biocontrol yeast Pichia anomala J121 prevents mould growth during the storage of moist grain under low oxygen/high carbon dioxide conditions. Growth and metabolite formation of P. anomala was analyzed under two conditions of oxygen limitation: (a) initial aerobic conditions with restricted oxygen access during the growth period and (b) initial microaerobic conditions followed by anaerobiosis. Major intra- and extracellular metabolites were analyzed by high-resolution magic-angle spinning (HR-MAS) NMR and HPLC, respectively. HR-MAS NMR allows the analysis of major soluble compounds inside intact cells, without the need for an extraction step. Biomass production was higher in treatment (a), whereas the specific ethanol production rate during growth on glucose was similar in both treatments. This implies that oxygen availability affected the respiration and not the fermentation of the yeast. Following glucose depletion, ethanol was oxidized to acetate in treatment (a), but continued to be produced in (b). Arabitol accumulated in the culture substrate of both treatments, whereas glycerol only accumulated in treatment (b). Trehalose, arabitol, and glycerol accumulated inside the cells in both treatments. The levels of these metabolites were generally significantly higher in treatment (b) than in (a), indicating their importance for P. anomala during severe oxygen limitation/anaerobic conditions.



We are grateful to Kalle Svensson for assistance with the statistical analysis of the data and to Inger Ohlsson for technical assistance. This work was financially supported by The Foundation for Strategic Environmental Research (MISTRA).


  1. Andlid T, Larsson C, Liljenberg C, Marison I, Gustafsson L (1995) Enthalpy content as a function of lipid accumulation in Rhodotorula glutinis. Appl Microbiol Biotechnol 42:818–825Google Scholar
  2. Argüelles JC (2000) Physiological roles of trehalose in bacteria and yeasts: a comparative analysis. Arch Microbiol 174:217–224PubMedGoogle Scholar
  3. Bellinger Y (1991) Salt tolerance and osmolyte composition of the yeast Hansenula anomala grown in the presence of fermentable or non-fermentable sources of carbon. Sci Aliment 11:37–48Google Scholar
  4. Björnberg A, Schnürer J (1993) Inhibition of the growth of grain-storage molds in vitro by the yeast Pichia anomala (Hansen) Kurtzman. Can J Microbiol 39:623–628Google Scholar
  5. Broberg A, Kenne L (2000) Use of high-resolution magic-angle spinning nuclear magnetic resonance spectroscopy for in situ studies of low-molecular-mass compounds in red algae. Anal Biochem 284:367–374CrossRefPubMedGoogle Scholar
  6. Broberg A, Kenne L, Pedersén M (1998) In situ identification of major metabolites in the red alga Gracilariopsis lemanieformis using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy. Planta 206:300–307CrossRefGoogle Scholar
  7. Caggia C, Restuccia C, Pulvirenti A, Giudici P (2001) Identification of Pichia anomala isolated from yoghurt by RFLP of the ITS region. Int J Food Microbiol 71:71–73CrossRefPubMedGoogle Scholar
  8. Cheng LL, Ma MJ, Becerra L, Ptak T, Tracey I, Lackner A, González RG (1997) Quantitative neuropathology by high resolution magic angle spinning proton magnetic resonance spectroscopy. Proc Natl Acad Sci USA 94:6408–6413CrossRefPubMedGoogle Scholar
  9. De Deken R (1966) The Crabtree effect: a regulatory system in yeast. J Gen Microbiol 44:149–156PubMedGoogle Scholar
  10. Druvefors U, Jonsson N, Boysen ME, Schnürer J (2002) Efficacy of the biocontrol yeast Pichia anomala during long-term storage of moist feed grain under different oxygen and carbon dioxide regimens. FEMS Yeast Res 2:389–394CrossRefPubMedGoogle Scholar
  11. Fitch WL, Detre G, Holmes CP, Shoolery JN, Keifer PA (1994) High-resolution 1H NMR in solid-phase organic synthesis. J Org Chem 59:7955–7956Google Scholar
  12. Fredlund E, Druvefors U, Lingsten K-J, Boysen ME, Schnürer J (2002) Physiological characteristics of the biocontrol yeast Pichia anomala J121. FEMS Yeast Res 2:395–402CrossRefPubMedGoogle Scholar
  13. Gancedo C, Serrano R (1989) Energy yielding metabolism. In: Rose AH, Harrison JS (eds) The yeasts, vol 3, 2nd edn. Academic Press, London, pp 206–259Google Scholar
  14. Garrod S, Humpfer E, Spraul M, Connor SC, Polley S, Connelly J, Lindon JC, Nicholson JK, Holmes E (1999) High-resolution magic angle spinning 1H NMR spectroscopy studies on intact rat renal cortex and medulla. Magn Reson Med 41:1108–1118CrossRefPubMedGoogle Scholar
  15. Griffin JL, Walker LA, Garrod S, Holmes E, Shore RF, Nicholson JK (2000) NMR spectroscopy based metabolic studies on the comparative biochemistry of the kidney and urine of the bank vole (Clethrionomys glareolus), wood mouse (Apodemus sylvaticus), white toothed shrew (Crocidura suaveolens) and the laboratory rat. Comp Biochem Physiol B 127:357–367CrossRefGoogle Scholar
  16. Kalathenos P, Sutherland JP, Roberts TA (1995) Resistance of some wine spoilage yeasts to combinations of ethanol and acids present in wine. J Appl Bacteriol 78:245–250Google Scholar
  17. Kitamoto HK, Hasebe A, Ohmomo S, Suto EG, Muraki M, Iimura Y (1999) Prevention of aerobic spoilage of maize silage by a genetically modified killer yeast, Kluyveromyces lactis, defective in the ability to grow on lactic acid. Appl Environ Microbiol 65:4697–4700PubMedGoogle Scholar
  18. Krainer E, Stark RE, Naider F, Alagramam K, Becker JM (1994) Direct observation of cell wall glucans in whole cells of Saccharomyces cerevisiae by magic-angle spinning 13C-NMR. Biopolymers 34:1627–1163PubMedGoogle Scholar
  19. Lanciotti R, Sinigaglia M, Gardini F, Guerzoni ME (1998) Hansenula anomala as spoilage agent of cream-filled cakes. Microbiol Res 153:145–148PubMedGoogle Scholar
  20. Martínez-Force E, Benítez T (1992) Changes in yeast amino acid pool with respiratory versus fermentative metabolism. Biotechnol Bioeng 40:643–649Google Scholar
  21. Martínez-Force E, Benítez T (1995) Effects of varying media, temperature, and growth-rates on the intracellular concentrations of yeast amino acids. Biotechnol Prog 11:386–392Google Scholar
  22. Meiboom S, Gill D (1958) Modified spin-echo method for measuring nuclear relaxation times. Rev Sci Instrum 29:688–691Google Scholar
  23. Mingorance-Cazorla L, Clemente-Jiménez JM, Martínez-Rodríguez S, Las Heras-Vázquez FJ, Rodríguez-Vico F (2003) Contribution of different natural yeasts to the aroma of two alcoholic beverages. World J Microbiol Biotechnol 19:297–304CrossRefGoogle Scholar
  24. Nobre MF, Da Costa MS (1985) The accumulation of polyols by the yeast Debaryomyces hansenii in the response to water stress. Can J Microbiol 31:1061–1064Google Scholar
  25. Petersson S, Schnürer J (1995) Biocontrol of mold growth in high-moisture wheat stored under airtight conditions by Pichia anomala, Pichia guilliermondii, and Saccharomyces cerevisiae. Appl Environ Microbiol 61:1027–1032PubMedGoogle Scholar
  26. Tokuoka K, Ishitani T, Chung WC (1992) Accumulation of polyols and sugars in some sugar-tolerant yeasts. J Gen Appl Microbiol 38:35–46Google Scholar
  27. Van Eck J, Prior B, Brandt E (1989) Accumulation of polyhydroxy alcohols by Hansenula anomala in response to water stress. J Gen Microbiol 135:3505–3514Google Scholar
  28. Van Eck J, Prior B, Brandt E (1993) The water relations of growth and polyhydroxy alcohol production by ascomycetous yeasts. J Gen Microbiol 139:1047–1054Google Scholar
  29. Viola A, Bortesi T, Pizzigoni R, Puglisi P, Goffrini P, Ferrero I (1986) The respiratory activities of four Hansenula species. Antonie Van Leeuwenhoek 52:295–308PubMedGoogle Scholar
  30. Walt JP van der, Yarrow D (1984) Methods for isolation, maintenance, classification, and identification of yeasts. In: Kreger-van Rij NJW (ed) The yeasts, a taxonomic study, 3rd edn. Elsevier, Amsterdam, pp 45–104Google Scholar
  31. Weybright P, Millis K, Campbell N, Cory D, Singer S (1998) Gradient, high-resolution, magnetic angle spinning 1H nuclear magnetic resonance spectroscopy of intact cells. Magn Reson Med 39:337–344Google Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • E. Fredlund
    • 1
  • A. Broberg
    • 2
  • M. E. Boysen
    • 1
    • 3
  • L. Kenne
    • 2
  • J. Schnürer
    • 1
  1. 1.Department of MicrobiologySwedish University of Agricultural SciencesUppsalaSweden
  2. 2.Department of ChemistrySwedish University of Agricultural SciencesUppsalaSweden
  3. 3.National Food AdministrationUppsalaSweden

Personalised recommendations