Utilization and accumulation of compatible solutes in Halomonas pacifica: a species of moderately halophilic bacteria isolated from a saline lake in South Libya

Abstract

When grown in high salt concentrations, halophilic bacteria often accumulate compatible solutes, which have major applications in biotechnology because they stabilize cells and proteins. Four Gram-negative bacterial strains, belonging to the family Halomonadaceae, were isolated from Qaberoun and Um-Alma lakes in South Libya using high-salinity medium. The strains were identified using 16S rRNA gene sequencing as belonging to Halomonas pacifica (strain ABQ1), Halomonas venusta (ABQ2), Halomonas elongata (ABU1) and Halomonas salifodinae (ABU2). H. pacifica ABQ1 is a moderate halophile (salinity range 0.05 to 2.5 M NaCl), with a broad tolerance to pH (7 to 9) and temperature (25-37 °C). Addition of the compatible solutes glycine betaine (betaine) and ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidine carboxylic acid) to the medium had a positive effect on growth of H. pacifica at 2 M NaCl. In rich LB medium, betaine was the major compatible solute accumulated, with ectoine only being accumulated at salinities in excess of 1 M NaCl. In minimal M9 medium, betaine was not produced, but increasing amounts of ectoine were synthesized with increasing salinity, and hydroxyectoine [(4S,5S)-5-hydroxy-2-methyl-1,4,5,6-tetrahydropyrimidine-4-carboxylic acid] was also synthesized when the cells were grown in very high salt. We have thus identified H. pacifica as a producer of ectoine and hydroxyectoine, with more being produced at higher salinities. As industrial demand for these compatible solutes continues to increase, this system has biotechnological potential.

Keywords: Halomonas bacteria; compatible solutes; ectoine; extreme environments; halophilic bacteria.

Fig. 1.

Scanning electron micrographs showing the cell morphology of Halomonas pacifica (ABQ1) grown in LB medium (a) and M9 minimal salt medium (b) at 0.5 M NaCl. Bars, 5 µm.

Fig. 2.

Final OD₆₀₀ values after overnight growth of H. pacifica (ABQ1) in M9 minimal salt medium at pH 7.8, incubated in a 37 °C constant temperature room on an orbital shaker at 250 r.p.m. Data points are the means of triplicates. Note that the x-axis is not a linear scale.

Fig. 3.

Final OD values after overnight growth of H. pacifica (ABQ1) in M9 minimal salt medium at 0.5 M NaCl, incubated in a 37 °C constant temperature room on an orbital shaker at 250 r.p.m. overnight. Data points are the means of triplicates and show the standard error.

Fig. 4.

Effect of temperature on overnight growth of H. pacifica (ABQ1). Cells were grown in M9 minimal salt medium at 0.5 M NaCl and pH 9, incubated in a 37 °C constant temperature room on an orbital shaker at 250 r.p.m. Data are an average of three triplicates with error bars representing one standard error.

Fig. 5.

Effect of three different compatible solutes (final concentration 1 mM) on growth of H. pacifica (ABQ1) on M9 minimal salt medium with 2 M NaCl, at pH 8. Data presented are means of triplicate experiments with standard error. Optical density (OD₆₀₀) was measured every hour.

Fig. 6.

H NMR spectra of H. pacifica (ABQ1) cell extracts grown in LB medium at different concentrations of NaCl showing presence of betaine and ectoine.

Fig. 7.

H NMR spectra of H. pacifica (ABQ1) grown in M9 minimal salt medium at different concentrations of NaCl showing the presence of ectoine and hydroxyectoine.

Fig. 8.

H NMR spectra of H. pacifica (ABQ1) grown in M9 minimal salt medium at 0.5 and 2 M NaCl, at pH 8, with 17 mM betaine as the sole carbon source, showing the presence of betaine and ectoine.