UPTAKE OF CADMIUM BY DIFFERENTS YEASTS STRAINS

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1 2005 International Nuclear Atlantic Conference - INAC 2005 Santos, SP, Brazil, August 28 to September 2, 2005 UPTAKE OF CADMIUM BY DIFFERENTS YEASTS STRAINS Kézia Pereira de Oliveira, Maria Ângela de Barros Correia de Menezes, Ângela Maria Amaral, Carlos Augusto Rosa*, Maria José Neves Centro de Desenvolvimento da Tecnologia Nuclear Lab. Radiobiologia / Comissão Nacioanl de Energia Nuclear (CDTN / CNEN) Cx. Postal 941, CEP Belo Horizonte MG *Depto. Microbiologia / ICB / UFMG Belo Horizonte - MG ABSTRACT Within the past decade, the potential of metal biosorption has been well established. For economic reasons, of particular interest are abundant biomass types generated by product of scale industrial fermentations. Some of these high metal sorbing biomass types serve as a basis for newly developed metal biosorption processes as a competitive means to detoxification of metal bearing industrial effluents. Yeasts possess an acknowledged potential for accumulating a range of metal cations. This work, specifically, intend to select a yeast that can removal cadmium from contaminated effluents. First, the experiment was done in S. cerevisiae (laboratory strains) and later compared with 14 yeasts selected in regional environmental or isolates from cachaça distilleries were tested for maximum uptake of cadmium. Four differents genera (Saccharomyces, Picchia, Candida, Starmerella), were used. The cadmium has no biological roles on biological system, then, all the cadmium founded into the cells gone by uptake or biorsorption on the superficies of the cells. The presence of cadmium in culture medium in concentration as low as 2 ppm inhibits the growth of cells and stimulates the trehalose accumulation. Live biomass was more efficient than dead biomass in processed the cadmium uptake. Some yeast presented a high uptake of cadmium, theses results are encouragement and possibilities think about a process that use these yeast to remove cadmium from industrial effluents or polluted waters. 1. INTRODUCTION Due to the increasing of deleterious ecological effects of heavy metals, a number of studies of metal accumulation to remove its from aqueous solutions have been researched. Metallic species released into the environment tend to persist indefinitely, circulating and eventually accumulating throughout the food chain posing a serious threat to animals and man. Virtually any industrial activity using metal has a metal disposal problem. In Brazil, the CONAMA regulates the level of permissive metal presence in water. To drink water cadmium limit is 0.1 ppm. To meet the water quality standards consistent with environment protection, industrial wastewaters need removal the pollutants. Conventional technology for removing metals from solutions include precipitation and separation by settling. This approach has several limitations. Biological treatments arouse great interest because of their lower impact on the environmental with respect to chemical treatments. Many microorganisms are able to remove heavy metals from wasterwaters but to depend on the microorganism used. Strong biosorbent behavior of certain types of microbial biomass toward metallic ions is a function of microbial cells, so it is necessary know which cell should be employed. Our work try to understand which is the behavior of cadmium accumulation in a cells of S. cerevisiae laboratory strain. After known some

2 parameters in lab strains it will be possible to compare with yeasts isolates in the region and decide which is the best yeast to accumulate cadmium. The use of yeasts employed in cachaça s distilleries of the region is advantageous because is a cheaper biomass disposable in a environmental. Normally, the strains isolated in the environmental used to be more resistant to many aggressions when compared with the lab strains. It was determined the influence of cadmium in a growth curve, and the influence of the metal on the pool of trehalose. Trehalose is, besides glycogen, a reserve carbohydrate but also an unspecific marker of stress. In some adverse situations like a heat shock, liophylization, and presence of heavy metal [1, 3], the previous accumulation of intracellular trehalose by the cells can protect its of the nocuous effect. By neutronic activation was determined the concentration of cadmium accumulated by the yeasts cells. Viable and non-viable biomass were used. 2. METHODOLOGY Growth conditions: All yeast strains were grown on YPG medium (Yeast extract 1%, Peptone 2%, Glucose 2%). CdCl 2 was sterilized separately and added to the liquid media after autoclaving. Growth curve: The growths of cells were accompanied by OD increase at 660 nm. Trehalose determination: described in [2]. Table 1. Strains used Strains Distilleries/Sources City/Place Region Saccharomyces cerevisiae W303 Laboratory strain - - Saccharomyces cerevisiae 1011 Distillerie Seleta Boazinha Salinas Jequitinhonha Valley Saccharomyces cerevisiae 1978 Distillerie Galo Bravo Salinas Jequitinhonha Valley Saccharomyces cerevisiae 2049 Distillerie Sebastião Salinas Jequitinhonha Valley Saccharomyces cerevisiae 2057 Distillerie Derci Salinas Jequitinhonha Valley Saccharomyces cerevisiae 2062 Distillerie Deia Salinas Jequitinhonha Valley Saccharomyces cerevisiae 2464 Distillerie Seleta Boazinha Salinas Jequitinhonha Valley Saccharomyces cerevisiae 2089 Distillerie Preciosa Salinas Jequitinhonha Valley Saccharomyces cerevisiae 2097 Distillerie Seleta Boazinha Salinas Jequitinhonha Valley Pichia guilliermondii Isolated of Drosophila spp UFMG Rio Doce Park Pichia membranifaciens Isolated of Drosophila spp UFMG Rio Doce Park Pichia kluyveri Isolated of Drosophila spp UFMG Rio Doce Park Candida cylindracea Isolated of Drosophila spp UFMG Rio Doce Park Candida sorboxylosa Isolated of Drosophila spp UFMG Rio Doce Park Starmerela meliponinorum Isolated of honey produced by Tetragonisca angustula UFMG Rio Doce Park

3 Determination of intracellular cadmium: Yeast cells grown in YPG medium were collected by vacuum filtration and washed twice with distilled water and freezing. Samples of the freezing cellular pellet were weight and sent to the TRIGA 1 reactor of CDTN. The samples were submitted to the neutron flux for 8 hours. After the time necessary, the samples were analyzed by gamma spectroscopy. Table 2- Cadmium resistance to increases concentrations of CdCl 2 Strains / ppm CdCl Saccharomyces cerevisiae WT.303 Y Y Y N N N N N Saccharomyces cerevisiae 1011 Y Y Y Y Y Y N N Saccharomyces cerevisiae 1978 Y Y Y Y Y Y N N Saccharomyces cerevisiae 2041 Y Y Y Y Y Y N N Saccharomyces cerevisiae 2049 Y Y Y Y Y Y N N Saccharomyces cerevisiae 2057 Y Y Y Y Y Y N N Saccharomyces cerevisiae 2062 Y Y Y Y Y Y N N Saccharomyces cerevisiae 2464 Y Y Y Y Y Y N N Saccharomyces cerevisiae 2089 Y Y Y Y Y Y N N Saccharomyces cerevisiae 2097 Y Y Y Y Y Y N N Starmerela meliponinorum Y Y Y Y Y Y Y N Cândida cylindracea Y Y Y Y Y Y N N Candida sorboxylosa Y Y Y Y Y Y N N Pichia guilliermondii Y Y Y Y Y N N N Pichia kluyvera Y Y Y Y Y N N N Pichia membranifociens Y Y Y Y Y Y N N The cells were incubated on YPG (12 hours, 30 0 C, 160 rpm) transferred at new YPG medium. After 3 h, 1 x 10 6 cells/ml of each strain were transferred to the well of replicate plate. The upper part of replica plate stayed in contact with the cells by 2 minutes and were plated on agar-ypg plate supplemented with the indicates concentrations of CdCl 2. The plates were incubated at 30 0 C during 4 days and the growth analyzed by visual inspection. Y = growth, N = not growth Until 20 ppm all the strains tested grown. The only exception was the laboratory strain S.cerevisiae W-303, the other strain presented more resistance. The strain S. meliponinorum grown in 100 ppm of Cadmium Table 2. The presence of cadmium had influence on the level of trehalose (Fig. 1).The increase of trehalose was depend on the time and the concentration of cadmium used. There was no linear correlation. High level of cadmium like 850 and 1000 ppm were toxic to the cells.

4 Trehalose (µmoles of glucose liberaded/g wet weight) Control 50 ppm 100 ppm 175 ppm 350 ppm 500 ppm 750 ppm 850 ppm 1000 ppm Time (hours) Figure 1 Influence of increase of cadmium concentration on the level of trehalose Cells of strain S. cerevisiae W303 grown in YPG medium were collected in logaritimic phase, transferred to YPG in presence of the discriminate concentrations of cadmium. 8 µg Cd incorporation/g weight wet Control 100 ppm 500 ppm 750 ppm 1000 ppm 1500 ppm Time (hours) Figure 2 - Cadmium incorporation by yeast cells (S. cerevisiae W303) Cells of laboratory strain grown in presence of discriminate concentration of cadmium. Samples were collected on pre determined times. The incorporation of cadmium was dependent on the concentration of cadmium and the time that the cells stayed in contact with the metal Figure 2.

5 Table 3 - Cadmium incorporation by biomass prepared in different way Strain/Conditions (A) Addition Cd to Viable cells during 24 h (B) Viable cells grown 24 h in presence Cd (C) Nonviable cells Saccharomyces cerevisiae WT Saccharomyces cerevisiae 1011 ND Saccharomyces cerevisiae Saccharomyces cerevisiae Saccharomyces cerevisiae 2049 ND Saccharomyces cerevisiae 2057 ND Saccharomyces cerevisiae 2062 ND Saccharomyces cerevisiae 2089 ND Saccharomyces cerevisiae 2097 ND Saccharomyces cerevisiae 2464 ND Candida cylindracea Candida sorboxylosa Pichia guilliermondii Pichia kluyvera Pichia membranifociens Starmerela meliponinorum Control no cadmium was founded. ND = Not Determined A = Cells grown during 48 hour, after this time 50 ppm CdCl 2 was added. After 24h the cells were collected. B = Cells grown during 24 h in the presence of 50 ppm CdCl 2. After this time the cells were collected. C = Cells grown 24 h, after this time the cells was autoclaved and 50 ppm CdCl 2 was added. The cells were collected after 24h. Cadmium incorporation expressed in: µg/ g wet weight Non lived biomass was better cadmium incorporation when the incubation was processed in presence of 50 ppm of CdCl 2, Table 3. The results obtained indicated that yeast could be an alternative to clean up industrial effluents. 3. CONCLUSIONS The strains of yeasts isolated of the cachaça fermentation and region area, presented a larger tolerance to high concentrations of cadmium, when compared to laboratory strain. Theses yeasts tolerate, for its growth, a concentration of 50 ppm of chloride of cadmium, while, the yeast Starmerela meliponinorum grows at 100 ppm. The laboratory strain tolerates only 10 ppm of chloride cadmium for its growth. High concentrations of chloride of cadmium inhibit the growth of the cells in all the studied strains of yeasts. The incorporation of

6 cadmium by yeasts cells is dependent of the initial concentration of the metal in the medium and, of the period of time that the cells are exposed to the metal. The incorporation of cadmium by viable cells is dependent of the growth phase. Cells in stationary phase has smaller incorporation of cadmium when compared with cells in logarithmic phase. On the other hand, cells killed by autoclaving incorporated more cadmium when compared with living cells. The fact that non lived biomass has a greater incorporation of cadmium is an advantageous to use yeast cells because is a cycle independent mechanism. Microorganisms exposed to adverse situations presents an adaptable answer that qualifies the cells to survive to such stress. Usually, in theses situations the carbohydrate trehalose is synthesized. We have observed that the presence of chloride of cadmium induces the trehalose synthesis. ACKNOWLEDGMENTS This research has been supported by CDTN/CNEN(Centro de Desenvolvimento da Tecnologia Nuclear/ Comissão Nacional de Energia Nuclear) REFERENCES 1. Van Laere, A, Trehalose, reserve and/or stress metabolite? FEMS Microbiol. Rev.,63, pp (1989). 2. Neves, MJ, Terenzi, HF, Leone, FA, Jorge, JA, Quantification of trehalose in biological samples with conidial trehalase from thermophilic fungus Humicola grisea var. Thermoidea. World J. Microbiol. Biotechnol.,10, pp (1994) 3. Singer, MA, Lindquist, S. The thermotolerance in Saccharomyces cerevisiae: the Yin and Yang of trehalose. TIBTECH, 16, pp (1998)