Characterization of Saccharomyces cerevisiae Mutants Resistant to High Concentrations of Co 2+ : A Primary Step to Bioremediation by Removal and Recovery of Co 2+ from Waste Waters

attention and growing metal-resistant cells that accumulate

Heavy metals represent an important environmental problem due to their potential toxic effects, and their accumulation throughout the food chain leads to serious ecological and health problems.The increase of environment pollution by heavy metals generated the interest to the problem of live organisms resistance to these metals.Biosorption is one of the mechanisms of microorganisms resistance to heavy metals and yeasts as biosorbents are of special interest [1][2][3][4].
The most well-known and commercially significant yeasts are the related species and strains of Saccharomyces cerevisiae.Although this microorganism is commonly used as baker's yeast and for some type of fermentation, its potential as bioremediation effector is more and more studied [5][6].Saccharomyces cerevisiae is an excellent model system to study the uptake and accumulation of heavy metals by living cells.This simple organism can grown on low-cost media, can be manipulated easily, and is not pathogenic.Its genome has been entirely sequenced and many cellular processes have been identified at molecular level.
In this work we focused on obtaining Saccharomyces cerevisiae mutants that are resistant to high concentrations of Co 2+ in their environment, as primary step in designing a bioremediation system suitable for Co 2+ -contaminated environments.Although Co 2+ is an essential micronutrient as a cofactor of vitamin B 12 [7] and various other enzymes in animals, yeasts, bacteria, Archaea, and plants [8], exposure to inorganic Co 2+ is associated with various human diseases such as contact dermatitis [9], allergic asthma, leading to subsequent interstitial fibrosis [10] and lung cancer [11].Cobalt increases oxidative stress in cells by raising the concentration of reactive oxygen species [12] and mimics or replaces ions such as magnesium and calcium in various essential reactions [13].
Metal remediation through common physico-chemical techniques is expensive and unsuitable in case of voluminous effluents containing complexing organic matter and low metal contamination.Alternative biotechnological approaches received great deal of attention and growing metal-resistant cells that accumulate heavy metals can ensure better removal through a combination of bioprecipitation, biosorption and continuous metabolic uptake of metals after physical adsorption.
Here, we present the isolation of Co 2+ resistant mutants and the characterization of these mutants in terms of Co 2+ uptake, accumulation and cellular compartmentalization, aiming to select for cell lines that hyperaccumulate Co 2+ as a primary tool for bioremediation of wastewaters contaminated with Co 2+ .

Strains, media, and growth conditions
The Saccharomyces cerevisiae strain W303 1A (MATa trp1 leu2 ade2 ura3 his3 can1-100) was used throughout our experiments.Cell growth, manipulation, and genetic analysis were done as described [14].Cells were grown in YPD (yeast extract-polypeptone-dextrose) supplemented with adenine (400 µg/mL) and uracil (200 µg/mL).For solid medium, 2% agar was used.For liquid cultures, overnight pre-cultures were used for inoculation, then cells were incubated with shaking for at least two hours at 28 o C before NiCl 2 was added from sterile stocks.

Selection of Co 2+ -resistant mutant cells
The Saccharomyces cerevisiae cells were mutagenized by exposure for 10 minutes to non-lethal concentrations of the chemical mutagen ethyl methanesulfonate (EMS) [14].Cells were subsequently washed, and spread (approximately 10 6 cells/plate) onto YPD plates containing lethal concentrations of Co 2+ (10 mM).Plates were incubated at 28 o C before the first resistant colonies appeared.

Co 2+ accumulation
Co 2+ loading of cells was done essentially as described [15].Cells grown in media containing various concentrations of CoCl 2 , were harvested by centrifugation and were washed three times with 10 mM 2-(Nmorpholino)ethanesulfonic acid (MES)-Tris buffer, pH6, at 0 o C. All centrifugation (1 min, 5000 rpm) was done at 4ºC.Cells were finally suspended (10 9 cells/ml) and used for Co 2+ assay.The Co 2+ cellular content was normalized to total cellular proteins.

Differential extraction of Co 2+ soluble pools from the cytosol and vacuoles
We used DEAE-dextran to obtain cytosolic extracts, and 60% methanol to obtain vacuolar extracts, as described earlier [16,17].The absence of crosscontamination between the cytosolic and vacuolar extracts was monitored by assaying the activities of glucose-6-phosphate dehydrogenase (cytosolic marker) [18] and carboxypeptidase Y (vacuolar marker) [19].

Co 2+ assay
The amount of Co 2+ in biological material was determined using 1-(2-pyridylazo)-naphtol (PAN) assay [20] modified for aqueous solutions.100 µL solubilized biologic material (whole or partial cell extract) was added to 400 µL PAN solution, and left for two minutes at room temperature for color development.The formation of Co 2+ -PAN complex was detected at 560 nm, using a Shimadzu UV-Vis spectrophotometer, model UV mini-1240.

Protein assay
Cellular total protein was assayed using the method described by Bradford [21].

Results and discussions Selection of Co 2+ -tolerant mutants
The parental Saccharomyces cerevisiae strain (also called by us "wild type" strain) used by us can grow on YPD plates containing CoCl 2 up to 3-4 mM.To obtain Co 2+ -resistant mutants, we exposed the parental strain to the chemical mutagen ethyl methane sulfonate (EMS).The EMS-mutagenized cells were spread onto YPD plates that contained lethal concentrations of CoCl 2 (10 mM) at a density of approximately 10 6 cells/plate.After 6-8 days incubation at 28ºC, Co 2+ -resistant colonies appeared (1-3 colonies/plate).We selected 100 resistant colonies and we passed them five times on fresh YPD plates, checking each time for the Co 2+ -tolerant phenotype, to exclude the false-positive candidates.Out of the 100 initial colonies, only five retained the phenotype of interest.These cells were back-crossed with the wild type cells, the diploids were sporulated and tetrads were dissected.All five mutants exhibited 2:2 segregation of the Co 2+ -resistant phenotype, suggesting that this was probably the result of a mutation in a single gene.These mutants were denoted as cor1, cor2, cor3, cor4, and cor5 (for Co-Resistant), and selected for further analysis.

Growth characteristics of the Co 2+ -tolerant lines
The five mutants were tested for growth abilities in the presence of high concentrations of Co 2+ in their environment.Thus, it was shown that all five mutants could grow on YPD plates containing up to 8-10 mM CoCl 2 , conditions under which the parental line no longer survives (data not shown).The dynamics of mutant cells growth in liquid YPD containing Co 2+ was also determined.All five mutants exhibited improved growth properties in the presence of Co 2+ , when compared to the parental type (fig.1).

Co 2+ -accumulation by the mutant cells
The tolerance to high concentrations of Co 2+ can be acquired by at least two different mechanisms: 1) Exclusion of the ions from the cells by stimulating the export process.2) Intracellular accumulation of ions in non-toxic forms, usually by compartmentalization in the organelles (mainly vacuole).We therefore tested our mutant lines for the ability to accumulate Co 2+ from their culture media, and we found that only mutant cor5 accumulated more Co 2+ than the parental strain (fig.2).The cor1-cor3 clearly gained their tolerance to Co 2+ by acquiring a low levels of Co 2+ accumulation, while mutant cor4 was no much different from the parental line in terms of Co 2+ accumulation (fig.2).For bioremediation purposes, only the mutant lines that (hyper)accumulate the ions from the environment would be useful, therefore solely mutant cor5 was selected for further investigation.

Intracellular distribution of Co 2+ ions in mutant nir5
For bioremediation use, the tolerant mutants that accumulate the ions in organelles are more promising, as compartmentalized ions are not so easily mobilized for export and thus they can be retained within the cell for times long enough to allow various technologic processes.We therefore determined the intracellular distribution of Co 2+ ions in mutant cor5, following the uptake across cell membrane.While the cytosolic level of Co 2+ was similar in both wild type and in cor5 mutant, the latter exhibited higher vacuolar content than the parental line (fig.3), in good agreement with the overall cellular accumulation (fig.2).The absence of crosscontamination between the cytosolic and vacuolar extracts was monitored by assaying the activities of cytosolic enzyme glucose-6-phosphate dehydrogenase and vacuolar enzyme carboxypeptidase Y (data not shown).

Mutant cor5 can decrease the Co 2+ content of their environment
We further wanted to determine whether the mutant cells are capable of taking up enough Co 2+ to cause a detectable decrease of its concentration in the growth Fig. 1.Growth of the Co 2+ -tolerant mutants in Co 2+ -containing media.Cells were inoculated from an overnight culture into liquid YPD to density 10 6 cells/mL and incubated with shaking at 28°C for 2 h before CoCl 2 was added (final concentration 2 mM).Growth of cells was assessed at various times by measuring the absorbance of the cell suspension at 600 nm (OD 600 ).The growth was calculated relatively to the absorbance measured at the moment when CoCl 2 was added (considered time 0).The data represent the average of three distinct experiments.WT, wild type (parental strain); cor, isogen Co 2+ -resistant strain environment.We found that when adding non-toxic concentrations of Co 2+ to a log-phase culture (10 7 cells/ ml), an approximate 28% and 38% of the total medium Co 2+ was removed by the cor5 mutant cells, after 5 min and 30 min, respectively (fig.4).Further increase of culture time did not lead to any significant modification to the Co 2+ concentration in the culture medium (data not shown).

Conclusion
In recent years, strict environmental regulations compel industries to shift to cleaner production methods, demanding the development of environmental friendly, low-cost and efficient treatment technique for metal rich effluents.Under such circumstances, biotechnological approaches to clean contaminated environments received great deal of attention in the recent years.Growing metalresistant cells that accumulate heavy metals can ensure better removal through a combination of bioprecipitation, biosorption and continuous metabolic uptake of metals after physical adsorption.
The Saccharomyces cerevisiae cells develop an acidic environment (creating in their environment pH 5.5).Thus, the cell wall behaves like polyanionic aggregates having a high capacity to reversibly bind metallic ions from their environment.This is why when suspending Saccharomyces cerevisiae cells in a liquid medium that contains metallic ions, the latter will be initially sorbed onto the surface of the cells.Metabolically active cells will then transport the ions across the cell membrane into the cytosol, from where they are either compartmentalized (usually in the vacuole) or excluded back in the environment.Both processes are aimed to protect the cell from the harmful effect of the metallic ions.Engineering cell lines that would hyperaccumulate heavy metals can be an invaluable tool in removing such ions from aqueous environments.Subsequent removal of these cells (by centrifugation, decantation or filtration) would result in partial removal of contaminating ions.Repeating the process would decrease the metal concentration even more.The metallic ions taken up by the cells can be released through cell digestion, followed by metal separation.
In this study, we obtained one Saccharomyces cerevisiae mutant that was both tolerant to high concentrations of Co 2+ and that could also accumulate this metal within the vacuoles.Equally important, this mutant had the ability to decrease the Co 2+ concentration of the culture medium in a single culture cycle, making it good candidate as bioremediation effector.The possibility to use such cells for decontamination of Co 2+ -containing wastewaters is now under investigation.

Fig. 3 .Fig. 2 .Fig. 4 .
Fig. 3. Intracellular distribution of Co 2+ .Cells were inoculated from an overnight culture into liquid YPD to density 10 6 cells/mL and incubated with shaking at 28°Cfor two hours before CoCl 2 was added (final concentration 2 mM).Aliquots of cells were harvested after 30 min; cytosolic and vacuolar fractions were prepared and Co 2+ was determined as described in the Materials and Methods section.The data represent the average of three similar experiments.WT, wild type (parental strain); cor, isogen Co 2+ -resistant strain