Manganese contamination in water is one of the most serious problems in southeastern countries, including Vietnam, because the large amount of manganese provides the toxicity in cells. Bioremediation using microorganisms is expected to be a useful technique to remove manganese from contaminated water. We employed to breed a yeast strain having the manganese accumulation ability from the brewing yeast Saccharomyces cerevisiae. In this study, we indicate that the growth and the manganese accumulation of the isolated yeast strains cultured in manganese ion-containing media.
Strains used were S. cerevisiae BY4741 as the wild-type strain and some manganese accumulation mutants bred from BY4741. The used medium was YPD (2% glucose, 1% yeast extract, and 1% peptone) with various concentrations of manganese chloride, if necessary. To test the toxicity of the manganese ion, the growth profile of yeast was determined by turbidity measurement at 660 nm. The concentration of manganese in cells was measured by atomic absorption spectrophotometry after the digestion of yeast cells by the concentrated nitric acid and then an adequate dilution.
Manganese accumulation mutants were screened from the growing strains on YPD with the high concentration of manganese ion (Mn2+) in which BY4741 could not grow, as follows. Overnight cultured BY4741 was inoculated into YPD containing various concentrations of Mn2+ and tested for the growth. BY4741 could not grow more than 10 mM of Mn2+. To select manganese tolerant mutants, BY4741 was inoculated in YPD with 10 mM of Mn2+ and cultured at 30°C for 24 h. Survival strains were inoculated again in the same medium and cultured at the same condition. Then, the variants that accumulated more manganese than BY4741 were isolated by repeat screening of survivors in 10mM Mn2+-containing YPD media. Finally, nine mutants which accumulated manganese were isolated and named IM1 to IM9. One variant showing the highest manganese accumulation, IM3, accumulated over 4-fold more manganese than its parent strain BY4741, and could grow under 25 mM Mn2+. Effects of culture temperature and pH on manganese accumulation were analyzed in IM3. The maximum accumulation was shown at 30°C while the maximum growth was shown at 37°C. The manganese accumulation was recognized from pH 4.0-9.0 and was highest at pH 6.0. Interestingly, IM3 could grow a little at pH 9.0 when manganese was added to the culture media, while IM3 could not grow without manganese addition. This reason is unclear but it may show the possibility that Mn2+ uptake affects the intracellular pH in IM3. We try to analyze the effect of the other stress on manganese accumulation in IM3.