The biosorption capacity of Saccharomyces cerevisiae for Cadmium in Milk

This study aimed to evaluate the capacity of Saccharomyces cerevisiae for Cadmium absorption in Milk. Nowadays one of the most serious problems of the industrialized world is heavy metals pollution. Applying microorgaisms as a novel biotechnology is so useful especially in foodstuffs. Among the biosorbents used for heavy metals’ removal, Saccharomyces cerevisiae has got an increasing attention due to its popularity in food industry. In this regard, the effects of some important factors such as the initial metal concentration, biomass concentration and contact time on the biosorption capacity of Saccharomyces cerevisiae were studied. The biosorption was analyzed by the inductively coupled plasma mass spectrometer (ICP-MS). The maximum Cd removal (70%) was at 80 μg/L of Cd concentration in milk samples containing 30×108 CFU Saccharomyces cerevisiae at the end of storage time (the 4th day). There were no significant differences in sensory and physicochemical properties of milk samples during storage (p < 0.05). The isotherm studies followed by two popular models; Langmuir and Freundlich and the results showed a better fit to the Langmuir isotherm. Altogether, the results of this study demonstrated that the approach of using this valuable yeast, could be applied for foods’ detoxification and producing healthier foodstuffs.


Introduction
Toxic metal contamination is a serious environmental problem all around the world due to the fast development of industries such as fuel, pesticides and mining. Their wastes discharge metals into the environment directly or indirectly [1,2]. These toxic metals can enter to the food chain and then into our bodies [3]. Cadmium (Cd) is one of the high toxic metals in this regard [4]. Milk is a valuable food source for humans and animals. It has nearly all essential nutrients for growth [5]. According to World Health Organization (WHO), the maximal allowed concentration values for Cd in milk is less than 10 µg/L [6].
Some reports have shown Cd contamination of milk around the world and unfortunately in some places it is more than the permissible level: Turkey [7], China [8], Iraq [9] and Iran [10,11].
Common techniques for heavy metals removal from aqueous solution like ion exchange, chemical precipitation, membrane technologies, electrochemical treatment and using activated carbon which are expensive and also not effective for using in foodstuffs [12,13].
Biosorption as a green technology, is the process of metal binding from aqueous solution to the surface of microorganism. The mechanism occurred through the absorption of metal ions to functional groups which is on the cell wall of the biomass [12]. It is a cheap, eco-friendly and fast technique [14]. Biosorpton is process that the heavy metals trap into the cell wall's active site [12]. The heavy metal's removal takes place through various mechanisms. The functional groups of the cell wall of S . cerevisiae such as hydroxyl and carboxyl, are responsible for the biosorption technique.
They are the main agents for metals to be attached during the mechanism. Moreover, the heavy metals intracellular accumulation occurs in the cell wall and metals are able to attach to the cell molecules [14][15][16].
In bosorption method various microorganisms like yeasts, bacteria, algae and fungi are applied. They possess some advantages such as being cheap and practical for foodstuffs [15,16].
Our study aims to evaluate the capacity of S. cerevisiae for Cd absorption in Milk. So, the effects of three main factors; initial metal concentration, biomass concentration and contact time on the biosorption capacity of S. cerevisiae were studied. These factors were chosen through the previous studies of heavy metals bioremoval [20][21][22][23] and also based on the results of our research team. This technique would be useful in case of emergency in food and beverage industry.

Preparation of the biomass
The S. cerevisiae (PTCC-5020) was purchased from the Science Research and Technology Department, Tehran, Iran. Glucose, yeast extract, (NH4)2SO, K2HPO4, MgSO4 and KH2PO4 were combined as the yeast culture medium and then autoclaved at 121°C for 20 min. The medium was inoculated with S. cerevisiae after cooling followed by 20 h shaking at 70 rpm and then incubated at 30°C. The biomass colonies were counted and the mean of 30×10 8 CFU/mL was obtained through the dilution method; the seed culture (1 mL) was diluted in a ratio of 1:10 with NaCl with serial dilutions (10 times). Then the dilution (1 mL) was added to the nutrient agar medium by pour plate method and incubated for 72 h at 30°C for 72 h [24].

Chemicals
All chemicals were provided from Merck company (Germany) and Cd standard solution from Accu Trace company (USA). All the containers were acid-washed by HNO3 (15% v/v) overnight and then then rinsed with distilled water.

Physicochemical Analysis
The pH, acidity and density of milk samples were determined according to AOAC methods [24]. The pH value of milk samples was evaluated with a pH meter (Metrom, Switzerland) at room temperature. The titratable acidity was determined by titration method; milk sample (10 ml) was titrated by NaOH solution (0.1 N) and adding phenolphthalein as an indicator. The Lactodensimeter (Alla, France) was used to measure the density of milk samples [24].

Sensory Analysis
The sensory analysis was evaluated during storage time (1st to 4th day) by 10 trained panelists [25]. Milk samples were analyzed for consistency, color, odor and overall acceptability. The samples were scored in a 9-point hedonic scale. The scores were from 1(extremely dislike) to 9 (extremely like). Mean values (± SD) were calculated from the panelists scores of each sample.

Central Composite Design (CCD)
The 3 variables; initial Cd concentration, S. cerevisiae biomass and contact time, having significant effects on Cd removal. In this study, CCD was used to find the optimal conditions of Cd biosorption with the experimental factors levels as shown in the Table 1.

ICP-MS Analysis
The inductively coupled plasma mass spectrometer (ICP-MS, England) applied in this study, with a standard torch, a cross flow nebulizer and a quartz spray chamber. It was tuned before each experiment started. All the samples were put in microwave 1200W (Milestone Micro oven) to be digested with segmented rotor MPR-600 [26].

Removal Evaluation
The milk sample containing S. cerevisiae and Cd were digested in the microwave and then centrifuged (at 2000×g) for 15 min. The supernatant was injected to the ICP-MS for Cd residual determination. measured by using the ICP-MS. All the trials were repeated triple.
The Cd removal efficiency (%) was calculated by Eq. (1) [27]: where Co (μg/L): is the initial Cd concentration in solution; Cf (μg/L): is the final Cd concentration in solution.

Absorption Isotherm
The biosorption isotherm were evaluated by adding the biosorbent (S. cerevisiae) to the milk samples with initial Cd concentrations (20 -100 μg/L). After biosorption, the remained Cd was determined by ICP-MS. The biosorption experiments were repeated three times.
The effect of initial Cd concentration (40, 50, 60, 70, 80 μg/L) on the bioremoval efficiency was investigated (Figure 1a). The results showed that by increasing the Cd concentrations, the absorption improved. The highest Cd removal (70%) was observed at the initial metal concentration of 80 μg/L.

The effect of contact time
In this study, the Cd biosorption was evaluated during the contact times from 1 to 4 days. Figure  1(b) shows that Cd removal by S. cerevisiae increased as the time passed. As it shows the maximum removal of Cd was occurred in the 4th day. By increasing time up to 8 days, the yeast count was enhanced as the removal was nearly constant. Table 2 shows the yeast count and the bioremoval levels during 8 days of storage. Different letters are significantly different (p < 0.05).

The effect of biomass concentration
As shown in Figures 1 (a and b), by increasing S. cerevisiae biomass concentration from 10 up to 50×10 8 CFU/mL, the removal efficiency enhanced. The optimum level of S. cerevisiae biomass concentration was 30×10 8 CFU with the highest removal amount of 70%.

Physicochemical evaluation
There was a slight reduction in pH values and a rise in titratable acidity of the milk samples. Also the density level was nearly constant. However, the differences were not significant in the milk samples (p>0.05) ( Table 2). Table 3 also represents the results of sensory analysis during the storage of milk samples. There were no significant differences in consistency, smell and color of these milk samples during time intervals with control samples (p < 0.05). Also the overall acceptance of milk samples had no significant difference through storage period (p < 0.05). Physicochemical properties pH 6.70±0.01 a 6.67±0.05 a 6.78±0.01 a 6.67±0.07 a 6.80±0.07 a 6.71±0.07 a Acidity (% lactic acid) 0.14±0.01 a 0.15±0.05 a 0.14±0.07 a 0.15±0.07 a 0.14±0.07 a 0

Isotherm studies
The capacity of S. cerevisiae biomass concentration (10 8 CFU/mL) for Cd biosorption was determined at Cd initial concentrations (20, 40, Table 4. As Table 3 shows, both correlation coefficients were high in Langmuir and Freundlich isotherm models. By the comparison of calculated R 2 values, it was revealed that the Langmuir isotherm model showed better fit than Freundlich model.

Discussion
As shown in figure 1 (a and b) by rising the biomass concentration up to 30 × 10 8 CFU, the absorption rate increased. The yeast of S. cerevisiae has a high biosorption affinity for heavy metals [30,31]. This trend is due to the carboxyl, hydroxyl and amino groups of the cell wall as the main responsible for the heavy metals' absorption [32][33][34]. As the amount of metal ions increased, their absorption to the surface of the S. cerevisiae increases so, the higher biosorption would be observed [21, 35 and 36]. By enhancing the S. cerevisiae biomass concentrations, the biosorption increases that is because of the more available binding sites for metal ions and therefore more binding combinations [37].
Also by increasing Cd concentration from 40 to 100 μg/L, the biosorption yield increased ( Figure  1a). Similar to our studys' results; Hadiani et al. [21] reported that Cd removal by S. cerevisiae increased with rising the Cd level (25 to 80 μg/L). Ghorbani et al. [38] observed the Cd bioremoval by S. cerevisiae (2.13 g/L) at the concentration of 26.46 mg/L. Also Peng et al. [39] showed that Cu absorption by S. cerevisiae increased by increasing the metal from 40 to 120 mg/L. As shown in Figure  1(b), Cd absorption enhanced by rising contact time from 1-4 day. With time passing, more Cd ions would attach to S. cerevisiae receptor sites in the surface [40]. The findings of this study is in accordance with Hadiani et al. [21] observed the increasing Mercury biosorption by S. cerevisiae from 24-48 h and Hatami Fard and Mehrnia [35] reported more mercury absorption during 4 days. Like the above studies, in this study, the highest Cd removal efficiency (70%) was observed at the Cd concentration of 80 μg/L and the biomass of 30 × 10 8 CFU in the 4th day of storage. Prolongation of the experiment is recommended to evaluate more removal of heavy metal in longer exposure. Also, it should be taken into account that in the case of spoilage of milk with bacterial cells, or high initial microbial loading of milk the rate of bio-decontamination could be quite different with this report.
Also Table 3 shows that the absorption increased by increasing the initial concentration of Cd, as more initial concentration prepared more contact sites for absorbent and Cd [35]. Comparing both R 2 values in Langmuir and Freundlich isotherm models, it shows that Langmuir model has a better fit, which confirms that Langmuir equation is correct for monolayer absorption on surface with similar sites. The higher R 2 in Langmuir model confirm the Cd absorption by S. cerevisiae in our study obey this model.

Conclusions
In this study, three important variables; Cd and biomass concentration and the contact time for Cd bioremoval by S. cerevisiae were evaluated. Our findings showed the highest level of Cd biosorption (70%) observed in the S. cerevisiae concentration of 30 × 10 8 CFU and Cd amount of 80 μg/L in the 4th day. The ability of S. cerevisiae had been studied in high levels (ppm) of Cd and other heavy metals in effluents not in foodstuffs. This study shows the ability of this valuable yeast for Cd remediation in very low concentrations (ppb) from milk with no changes in physiochemical and sensorial acceptability. S. cerevisiae is a desirable and eco-friendly biosorbent for toxic metals bioremediation from food and water resources. These findings open the window for evaluating the capacity of heavy metals' binding by S. cerevisiae in milk. There is a need for more studies in this field to reduce the toxic effects of the heavy metals in food and drinks.