Removal of lead , mercury and nickel using the yeast Saccharomyces cerevisiae

Objective. In this study the biomass of the yeast Saccharomyces cerevisiae was used to remove lead, mercury and nickel in the form of ions dissolved in water. Materials and methods. Synthetic solutions were prepared containing the three heavy metals, which were put in contact with viable microorganisms at different conditions of pH, temperature, aeration and agitation. Results. Both individual variables and the interaction effects influenced the biosorption process. Throughout the experimental framework it was observed that the biomass of Saccharomyces cerevisiae removed a higher percentage of lead (86.4%) as compared to mercury and nickel (69.7 and 47.8% respectively). When the pH was set at a value of 5 the effect was positive for all three metals. Conclusions. pH was the variable that had a greater influence on the biosorption of lead on the biomass of Saccharomyces cerevisiae. The affinity of the heavy metals for the biomass followed the order Pb>Hg>Ni.


INTRODUCTION
Low concentrations of heavy metals can be highly toxic to microorganisms, whereas high concentrations can be tolerated if they are provided with enough sulfur.The toxicity of metals such as lead is well known; however, for others such as copper, zinc and nickel, their effects and permissible limits in surface waters are still under discussion (1).Uptake mechanisms range from physical adsorption, ion exchange with the functional groups of the cell wall of the microorganism or they may penetrate into the cytoplasm and accumulate in the form of granules or intracellular inclusions (2).
Various microorganisms have been used for bioremediation purposes, such as bacteria, yeasts, fungi and microalgae; biosorbents based on macroalgae in non-viable state have also been prepared and have been effective in the removal of heavy metals from industrial effluents at concentrations ranging from 1 to 100 mg/L (2).
Growing cells may show a greater capacity for the removal of metals than non-viable biomass, especially in environments with enough nutrients (3) Sources of pollution may be natural such as the erosion and weathering of rocks; or anthropogenic erosion, manufacturing pigments, batteries, biocides, processing of ores and metals, industrial effluent from electroplating plants, effluents from tanneries and sanitary landfills (4).

S a c c h a r o m y c e s c e r e v i s i a e
o s s e s s e s phosphate, amino, carboxyl and hydroxyl groups in its cell wall, which are responsible for the removal of heavy metals (5).
The advantages of the use of Saccharomyces cerevisiae include the global boom in bioethanol production, which ensures the availability of a steady supply of residual biomass that could be used in the bioremediation of industrial effluents (6).Moreover, this yeast is considered as safe, which favors its use in practical applications that will thereafter be easily accepted by the population.S. cerevisiae has also been immobilized for recovering precious metals such as platinum (7), being considered as a cheap and plentiful source of biomass, making its application feasible in the
The brewing industry is another important source of low-cost yeast with flocculating properties, which facilitates the mechanical separation at the end of the biosorption process (8).
On the other hand, environmental conditions such as aeration, agitation, temperature and pH influence cell growth and an effect on the uptake of heavy metals associated with both the microorganism and the fluid dynamics of the medium could therefore be expected.
Based on the above, experiments were carried out in order to determine the influence of the variables pH, temperature, agitation and aeration on the biosorption of lead, mercury and nickel in the biomass of S. cerevisiae.

MATERIALS AND METHODS
Microorganism.Commercial dry active baker's yeast was used, which was kept in refrigeration at 4°C.

Culture medium.
A culture medium containing 20 g/L of glucose, 20 g/L of peptone and 10 g/L of yeast extract was prepared for the cultures per lot of S. cerevisiae.This medium was dosed with lead nitrate, nickel chloride and mercuric chloride solutions to obtain a concentration of 100 μM in each of the heavy metals, using a workload of 100 mL in each experiment.
Biosorption.Biosorption tests were carried out according to a 2 4 factorial design in which the levels of each factor were identified according to the coding (Table 1).
The tests were conducted by inoculating the medium with 0.2 g of dry yeast and incubated La industria cervecera es otra fuente importante de levadura de bajo costo con propiedades floculantes, lo cual facilita la separación mecánica al final del proceso de biosorción (8).
Con base en lo anterior se realizaron experiencias con el fin de determinar la influencia de las variables pH, temperatura, agitación y aireación sobre la biosorción de Plomo, Mercurio y Níquel en la biomasa de S. cerevisiae.

Medio de cultivo.
Se preparó un medio de cultivo que contenía 20 g/L de glucosa, 20 g/L de peptona y 10 g/L de extracto de levadura para los cultivos por lote de S. cerevisiae.
Determination of heavy metals.The atomic absorption spectroscopy method was selected to analyze the heavy metals, using the spectrophotometer UNICAM 969.All samples were analyzed in triplicate.
The lead and nickel were determined using the flame atomization technique, while the cold vapor technique was used for analyzing mercury, on the basis of the standardized methods compiled in the APHA in section 3112B (12).
Experiment design.A complete 2 4 factorial design was used.The experiments planned through the combination of the variables under study were performed randomly and in duplicate.
The removal percentages for each metal were reported as the mean of the experiments ± the standard deviation of the mean effect, calculated from the effects of third and fourth order interactions.
The software SPSS Statistics version 17.0 was used for the calculation of the effects.The effects calculated were compared with a reference distribution (F test) at a confidence level of 95%.

RESULTS
The biomass of S. cerevisiae adsorbed the metals under study in different proportions (Table 2), showing greater affinity for lead and lower affinity for nickel.
Los porcentajes de remoción para cada metal se reportaron como la media de los experimentos ± la desviación estándar del efecto medio, calculada a partir de los efectos de las interacciones de tercer y cuarto orden.
La biomasa de S. cerevisiae presentó una capacidad media para el mercurio de 10.25 mg/g.La capacidad de adsorción para el níquel fue 2.8 mg /g de levadura seca.
Análisis de los efectos sobre la biosorción del plomo.De los seis efectos significativos para este metal, el pH fue la variable que tuvo For average removed for lead was 86.4±3.8, for mercury 69.7±3.66 and for nickel 47.8±2.67.
The average biosorption capacity for lead was 8.9 mg/g of dry yeast.The only metal for which a removal of 100% was obtained was lead, particularly in experiments with a pH of 5, except the trial where the agitation and pH were at a high level.
The biomass of S. cerevisiae showed an average capacity for mercury of 10.25 mg/g.The adsorption capacity for nickel was 2.8 mg/g of dry yeast.
A significant influence was observed in the variables for each metal, represented by simple effects and second and third order interactions (Table 3).The effects calculated correspond to the coefficients of the linear model, which were considered significant when the likelihood value was less than 0.05 (p≤0.05).
El segundo efecto más significativo y favorable para la retención del plomo fue la aireación, este resultado indica que el estado viable y el metabolismo oxidativo de la levadura influyen de manera positiva en la captación de este metal.La interacción entre el pH y la agitación tuvo efecto negativo sobre la biosorción.
En cuanto a las interacciones de tercer orden, se presentó un efecto favorable para la interacción entre el pH, la agitación y la aireación, lo que indica que si el proceso de biosorción es agitado mecánicamente a pH 5, también deberá airearse con el fin de generar esta interacción positiva; mientras que la interacción entre el pH, la temperatura y la aireación fue negativa, estableciendo a la temperatura como una variable crítica en este proceso.
Análisis de los efectos sobre la biosorción del mercurio.Como se observa en la figura 2, la mayoría de los efectos resultaron significativos para la biosorción de mercurio: la temperatura, el pH, todas las interacciones de segundo orden excepto la que ocurrió entre el pH y la temperatura; así como las interacciones de tercer orden entre pH, temperatura y agitación y entre pH, agitación y aireación.Infante -Removal of lead, mercury and nickel

Analysis of the effects on the biosorption of lead.
Of the six significant effects for this metal, the pH was the variable that had the greatest influence on the uptake of lead by the biomass (Figure 1).
The second most significant and favorable effect for the retention of lead was aeration, this result indicates that the viable state and oxidative metabolism of yeast influence positively on the uptake of this metal.The interaction between pH and agitation had a negative effect on biosorption.
In terms of third-order interactions, a favorable effect was observed for the interaction

Effects
Treatments between pH, agitation and aeration, which indicates that if the biosorption process is mechanically agitated at a pH of 5, it also must be aerated in order to generate this positive interaction; while the interaction between pH, temperature and aeration was negative, setting the temperature as a critical variable in this process.
Analysis of the effects on the biosorption of mercury.As shown in Figure 2, most of the effects were significant for mercury biosorption: temperature, pH, all second-order interactions except for that between pH and temperature; as well as third-order interactions between pH, temperature and agitation and between pH, agitation and aeration.
In addition, a greater number of positive rather than negative effects was noted.The temperature and pH had positive effects as well as the interactions between pH and agitation as well as between agitation and aeration.
The interactions between temperature and agitation; temperature and aeration; as well as pH and aeration, showed a negative effect, the first being the stronger, indicating that the desorption of mercury is favored under these conditions.
The third-order interaction between pH, temperature and agitation was positive and had a strong effect comparable to the effects of pH and temperature separately on the uptake of mercury; while the interaction between pH, agitation and aeration had negative effect.
Analysis of the effects on the biosorption of nickel.Figure 3 shows that the biosorption of nickel displayed the highest number of negative effects, which is consistent with the lower removal percentage observed in this study.
All the variables under study influenced on the biosorption of nickel individually, temperature and pH with positive effects and agitation and aeration with negative effects but with a lower weight than the first two.
The most significant effect was the second-order interaction between temperature and agitation; it can be noticed that as in the case of mercury biosorption, it was negative and had a higher magnitude than the rest of the effects.
It is observed that the third-order interactions, which were significant for the biosorption of lead, were also significant in the case of nickel, but with the opposite sign.
La interacción de tercer orden entre el pH, la temperatura y la agitación fue positiva y tuvo un efecto fuerte comparable a los efectos del pH y de la temperatura por separado sobre la captación de mercurio; mientras que la interacción entre el pH, la agitación y la aireación tuvo efecto negativo.
Todas las variables estudiadas influyeron sobre la biosorción de níquel de forma individual, la temperatura y el pH con efectos positivos y la agitación y la aireación con efectos negativos pero de menor peso que las dos primeras.
El efecto más significativo fue la interacción de segundo orden entre la temperatura y la agitación; puede notarse que al igual que para el caso de la biosorción de mercurio, este fue negativo y de mayor magnitud que el resto de los efectos.

DISCUSSION
The higher affinity of biomass for lead was a result in line with what has been reported in other studies (13,14).
At a pH of 5, lead is a positive divalent ion that can interact with the negative functional groups of the cell wall of yeast (13).
Zhang et al (15) found an adsorption capacity of 6.52 Pb/g mg of dry yeast, lower to that obtained in this study (8.9 mg Pb/g of dry yeast).Skountzou et al (16) reported a capacity of 4 mg Pb/g based on a concentration of 20 mg/L, as that used in this study; in addition, these authors obtained a removal percentage for lead of 71.8%, lower to that obtained in this study.
Galedar and Younesi ( 17) reported a capacity of 1.2 mg Ni/g of dry yeast in experiments with S. cerevisiae immobilized at a pH of 8. Again, this capability was less than that obtained in this study.Another study found a removal rate for nickel of 72% from synthetic solutions, higher than that obtained in this study (18).
Suazo et al (19) found a direct relationship between the uptake of nickel and pH using S. cerevisiae var ellipsoideus; at a pH of 3.5, the adsorption capacity was 5.74 mg Ni/g of dry yeast for an initial concentration of 1000 µM.In this study, the capacity was approximately half of this value with an initial concentration ten times lower.
The repulsion between H + ions and metal ions at a low pH restricts access to ligands, but as the pH increases, more negatively charged ligands are exposed to capture heavy metals (13).
The effect of aeration can be explained taking into account that biosorption is performed with cells in a viable state with an aerobic metabolism, it is expected that a good air supply is reflected in a specific high growth rate and a better use of the nutrients in the culture medium.
The effect of agitation is related to the shear forces generated that could cause the desorption of the metal joined by weak bonds such as electrostatic attraction, hydrogen bonding and Van der Waals forces.
The positive effect of temperature for mercury and nickel can be attributed to an increase in ionic mobility.The relationship between the metal removal capacity and temperature was studied by Zhang et al (15) finding that the maximum
El efecto de la aireación puede explicarse teniendo en cuenta que la biosorción se realiza con células en estado viable con metabolismo aeróbico, es de esperar que un buen suministro de aire se refleje en una velocidad específica de crecimiento alta y un mejor aprovechamiento de los nutrientes del medio de cultivo.
El efecto positivo de la temperatura para mercurio y níquel puede atribuirse a un aumento de la movilidad iónica.La relación entre la capacidad de Infante -Removal of lead, mercury and nickel capacity was obtained at 30°C (20), which corresponds to the high level used in this study.
In conclusion, the pH was the variable that had the greatest influence on the biosorption of lead, mercury and nickel on the biomass of S. cerevisiae.The temperature was the variable that most influenced the biosorption of mercury and nickel.Lead was the metal with greater affinity for biomass, followed by mercury and nickel, which can be asserted by comparing their removal percentage and the effects of the interaction of temperature and agitation.Second and third order interactions were significant for the biosorption of lead, mercury and nickel.Aeration only had a positive effect on the biosorption of lead, which can indicate a relationship between the uptake of lead and the metabolic state of the cell.The strongest negative effect on the biosorption of mercury and nickel was represented by the interaction between temperature and agitation.

Agradecimientos
Los autores agradecen a la Universidad de Cartagena por su apoyo para la realización de este trabajo.

Figure 1 .
Figure 1.Comparison of the significant effects on the biosorption of lead.

Figure 2 .
Figure 2. Comparison of the significant effects on the biosorption of Mercury.

Figure 3 .
Figure 3.Comparison of the significant effects on the biosorption of Nickel.

Table 1 .
Levels of variables.

Table 2 .
Removal percentages of Pb, Hg and Ni by S. cerevisiae.

Table 3 .
Effects of the variables on the biosorption of lead, mercury and nickel.