Springer Nature is making SARS-CoV-2 and COVID-19 research free. View research | View latest news | Sign up for updates

Membrane fluidity of halophilic ectoine-secreting bacteria related to osmotic and thermal treatment

  • 515 Accesses

  • 5 Citations


In response to sudden decrease in osmotic pressure, halophilic microorganisms secrete their accumulated osmolytes. This specific stress response, combined with physiochemical responses to the altered environment, influence the membrane properties and integrity of cells, with consequent effects on growth and yields in bioprocesses, such as bacterial milking. The aim of this study was to investigate changes in membrane fluidity and integrity induced by environmental stress in ectoine-secreting organisms. The halophilic ectoine-producing strains Alkalibacillus haloalkaliphilus and Chromohalobacter salexigens were treated hypo- and hyper-osmotically at several temperatures. The steady-state anisotropy of fluorescently labeled cells was measured, and membrane integrity assessed by flow cytometry and ectoine distribution. Strong osmotic downshocks slightly increased the fluidity of the bacterial membranes. As the temperature increased, the increasing membrane fluidity encouraged more ectoine release under the same osmotic shock conditions. On the other hand, combined shock treatments increased the number of disintegrated cells. From the ectoine release and membrane integrity measurements under coupled thermal and osmotic shock conditions, we could optimize the secretion conditions for both bacteria.

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

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8



Dimethyl sulfoxide



I :

Fluorescence emission intensity


Propidium iodide

r DPH :

Steady-state anisotropy

T m :

Phase transition temperature


  1. 1.

    Harishchandra RK, Wulff S, Lentzen G, Neuhaus T, Galla H-J (2010) The effect of compatible solute ectoines on the structural organization of lipid monolayer and bilayer membranes. Biophys Chem 150(1–3):37–46

  2. 2.

    Oren A (2002) Halophilic microorganisms and their environments. Kluwer Academic Publishers, New York

  3. 3.

    Russell NJ (1989) Adaptive modifications in membranes of halotolerant and halophilic microorganisms. J Bioenerg Biomembr 21(1):93–113

  4. 4.

    Simonin H, Beney L, Gervais P (2008) Controlling the membrane fluidity of yeasts during coupled thermal and osmotic treatments. Biotechnol Bioeng 100(2):325–333

  5. 5.

    Laroche C, Beney L, Marechal PA, Gervais P (2001) The effect of osmotic pressure on the membrane fluidity of Saccharomyces cerevisiae at different physiological temperatures. Appl Microbiol Biotechnol 56(1–2):249–254

  6. 6.

    Beney L, Mille Y, Gervais P (2004) Death of Escherichia coli during rapid and severe dehydration is related to lipid phase transition. Appl Microbiol Biotechnol 65(4):457–464

  7. 7.

    Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer, New York

  8. 8.

    Lentz BR (1993) Use of fluorescent probes to monitor molecular order and motions within liposome bilayers. Chem Phys Lipids 64(1–3):99–116

  9. 9.

    Petty HR, Niebylski CD, Francis JW (1987) Influence of immune complexes on macrophage membrane fluidity: a nanosecond fluorescence anisotropy study. Biochemistry 26(20):6340–6348

  10. 10.

    Vincent M, England LS, Trevors JT (2004) Cytoplasmic membrane polarization in Gram-positive and Gram-negative bacteria grown in the absence and presence of tetracycline. Biochim Biophys Acta 1672(3):131–134

  11. 11.

    Mykytczuk NCS, Trevors JT, Leduc LG, Ferroni GD (2007) Fluorescence polarization in studies of bacterial cytoplasmic membrane fluidity under environmental stress. Prog Biophys Mol Biol 95(1–3):60–82

  12. 12.

    Hosono K (1992) Effect of salt stress on lipid composition and membrane fluidity of the salttolerant yeast Zygosaccharomyces rouxii. J Gen Microbiol 138(1):91–96

  13. 13.

    Nichols DS, Olley J, Garda H, Brenner RR, McMeekin TA (2000) Effect of temperature and salinity stress on growth and lipid composition of Shewanella gelidimarina. Appl Environ Microbiol 66(6):2422–2429

  14. 14.

    López CS, Garda HA, Rivas EA (2002) The effect of osmotic stress on the biophysical behavior of the Bacillus subtilis membrane studied by dynamic and steady-state fluorescence anisotropy. Arch Biochem Biophys 408(2):220–228

  15. 15.

    Tymczyszyn EE, Gómez-Zavaglia A, Disalvo EA (2005) Influence of the growth at high osmolality on the lipid composition, water permeability and osmotic response of Lactobacillus bulgaricus. Arch Biochem Biophys 443(1–2):66–73

  16. 16.

    Huffer S, Clark ME, Ning JC, Blanch HW, Clark DS (2011) Role of alcohols in growth, lipid composition, and membrane fluidity of yeasts, bacteria, and archaea. Appl Environ Microbiol 77(18):6400–6408

  17. 17.

    Mueller S, Ullrich S, Loesche A, Loffhagen N, Babel W (2000) Flow cytometric techniques to characterise physiological states of Acinetobacter calcoaceticus. J Microbiol Methods 40(1):67–77

  18. 18.

    Shapiro HM (2003) Practical flow cytometry, 4th edn. Wiley, New York

  19. 19.

    Mueller S, Nebe-von-Caron G (2010) Functional single-cell analyses: flow cytometry and cell sorting of microbial populations and communities. FEMS Microbiol Rev 34(4):554–587

  20. 20.

    Bergmann S, David F, Franco-Lara E, Wittmann C, Krull R (2013) Ectoine production by Alkalibacillus haloalkaliphilus—bioprocess development using response surface methodology and model-driven strategies. Eng Life Sci 13 (in press)

  21. 21.

    Fallet C, Rohe P, Franco-Lara E (2010) Process optimization of the integrated synthesis and secretion of ectoine and hydroxyectoine under hyper/hypo-osmotic stress. Biotechnol Bioeng 107(1):124–133

  22. 22.

    Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37(8):911–917

  23. 23.

    Marangoni AG, Narine SS (2002) Physical properties of lipids. Marcel Dekker, New York

  24. 24.

    Fritze D (1996) Bacillus haloalkaliphilus sp. nov. Int J Syst Bacteriol 46(1):98–101

  25. 25.

    Jeon CO, Lim J-M, Lee J-M, Xu L-H, Jiang C-L, Kim C-J (2005) Reclassification of Bacillus haloalkaliphilus Fritze 1996 as Alkalibacillus haloalkaliphilus gen. nov., comb. nov. and the description of Alkalibacillus salilacus sp. nov., a novel halophilic bacterium isolated from a salt lake in China. Int J Syst Evol Microbiol 55(Pt 5):1891–1896

  26. 26.

    Sauer T, Galinski EA (1998) Bacterial milking: a novel bioprocess for production of compatible solutes. Biotechnol Bioeng 57(3):306–313

  27. 27.

    Pastor JM, Salvador M, Argandoña M, Bernal V, Reina-Bueno M, Csonka LN, Iborra JL, Vargas C, Nieto JJ, Cánovas M (2010) Ectoines in cell stress protection: uses and biotechnological production. Biotechnol Adv 28(6):782–801

  28. 28.

    Clejan S, Krulwich TA, Mondrus KR, Seto-Young D (1986) Membrane lipid composition of obligately and facultatively alkalophilic strains of Bacillus spp. J Bacteriol 168(1):334–340

  29. 29.

    Yumoto I (2002) Bioenergetics of alkaliphilic Bacillus spp. J Biosci Bioeng 93(4):342–353

  30. 30.

    Beney L, Simonin H, Mille Y, Gervais P (2007) Membrane physical state as key parameter for the resistance of the gram-negative Bradyrhizobium japonicum to hyperosmotic treatments. Arch Microbiol 187(5):387–396

  31. 31.

    Träuble H, Teubner M, Woolley P, Eibl H (1976) Electrostatic interactions at charged lipid membranes. Biophys Chem 4(4):319–342

  32. 32.

    Vargas C, Kallimanis A, Koukkou AI, Calderon MI, Canovas D, Iglesias-Guerra F, Drainas C, Ventosa A, Nieto JJ (2005) Contribution of chemical changes in membrane lipids to the osmoadaptation of the halophilic bacterium Chromohalobacter salexigens. Syst Appl Microbiol 28(7):571–581

  33. 33.

    Ventosa A (2004) Halophilic microorganisms. Springer, Berlin

Download references


The authors gratefully acknowledge the financial support granted by the German Research Foundation (DFG), No. FR 2596/2-1.

Conflict of interest

The authors have declared no conflict of interest.

Author information

Correspondence to Rainer Krull.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Bergmann, S., David, F., Clark, W. et al. Membrane fluidity of halophilic ectoine-secreting bacteria related to osmotic and thermal treatment. Bioprocess Biosyst Eng 36, 1829–1841 (2013).

Download citation


  • Membrane fluidity
  • Steady-state fluorescence anisotropy
  • Osmotic and thermal shock
  • Ectoine release
  • Halophile bacteria