HEAVY METAL TOLERANCE AND REMOVAL EFFICIENCY OF THE Rhodotorula mucilaginosa AND Saccharomyces boulardii PLANKTONIC CELLS AND BIOFILM

The impact of heavy metals, cadmium (Cd ), zinc (Zn) and nickel (Ni ) on planktonic cells and biofilm of Rhodotorula mucilaginosa and Saccharomyces boulardii was examined. The metal tolerance testing was perf ormed by MBEC-HTP assay. The minimum inhibitory concentration (MICp) and minimum lethal concentration (MLCp) were determined as well as the minimum biofi lm eradication concentration (MBEC). Biofilm was more tolerant on the presence o f heavy metals than the planktonic cells. The planktonic cells of R. mucilaginosa were tolerant to high concentrations of Cd, Zn and Ni, while the planktonic cells of S. boulardii tolerated Zn, exclusively. The R. mucilaginosa biofilm was tolerant to all of the tested metal co n entrations and the obtained results were confirmed by fluorescence mic roscopy. S. boulardii did not show ability of biofilm formation. Metal removal efficie ncy of the R. mucilaginosa planktonic cells and biofilm were also tested. The R. mucilaginosa biofilm showed higher efficiency in metals removing compared to the planktonic cells . Until now, the heavy metal tolerance and the removal efficiency (Cd , Zn and Ni) analyzes were performed solely on planktonic cells of Rhodotorula species. In this study, we investigated the metal removal efficiency of R. mucilaginosa planktonic cells and biofilm and compared the obtained results.


INTRODUCTION
An understanding of the nature of heavy metals, their relationships and toxicity or deficiency problems associated with them, is important for environmental protection. As more and more analytical data become available in the world literature, it is evident that considerable areas in many parts of the world have been contaminated with heavy metals, which present potential toxicity problems (ALLOWAY, 1995).
A wide range of methods for the heavy metals removal from contaminated environment are being used. Most of them are not efficient in removing low concentrations of metals, have high energy requests, lead to accumulation of toxic sludge and other waste products, therefore requiring a careful disposal of waste (AHALYA et al., 2003). With increasing ecological awareness, search for effective alternative technologies is essential. Microbial biomass is considered as an alternative for the heavy metals removal (ALLURI et al., 2007).
Some authors reported nickel tolerance of planktonic cells Rhodotorula mucilaginosa (SAN and DÖNMEZ, 2012) and Rhodotorula glutinis (SUAZO-MADRID et al., 2011). Cadmium tolerance was tested by planktonic cells of Rhodotorula Y11 YUAN 2006, 2008) and Rhodotorula rubra (SALINAS et al., 2000). The heavy metal tolerance of biofilms are taking a great attention, since they could be applied in bioremediation of polluted environments. HARRISON et al., (2006) reported that Candida tropicalis could survive in the most adverse environmental conditions, thanks to the ability to form a biofilm.
Heavy metal tolerance is associated with the ability to remove heavy metals from the environment (FAZLI et al., 2015). Recently, it was reported that R. rubra (planktonic cells) have a potential application in degradation and bioleaching of heavy metals (REZZA et al., 2001). The accumulation of lead and cadmium by R. rubra biomass was tested (SALINAS et al., 2000), as well as the removal of nickel by planktonic cells of Rhodotorula sp. (LI and YUAN, 2008).
In previous studies, the heavy metal tolerance and the removal potential for Cd 2+

Microorganisms and growth conditions
Two species of yeast were used -R. mucilaginosa (isolated from the environment) and S. boulardii (commercial probiotic). The R. mucilaginosa was identified by the test for rapid identification of yeast API 20 C AUX (Biomerieux, France). Tryptic soy broth (TSB) was chosen as the growth medium for all metal tolerance assays (HARRISON et al., 2006). For the metal removal assays, YPED medium was used (MUNEER et al., 2007). All serial dilutions were carried out using 0.9% saline.

Cultivation of biofilms
Growth of the selected yeasts in the presence of heavy metals was tested by quantitative assay in the MBEC-HTP device (MBEC BioProducts, Innovotech, Canada) as previously described (CERI et al., 1999). Plastic lid with 96 pegs that fits inside a standard 96-well microplate was used. The peg lid was immersed into a sterile solution of 1% L-lysine in distilled water (dH2O) and incubated at room temperature for 16 h.
Cryogenic stocks cultures of R. mucilaginosa and S. boulardii were streaked out twice on TSA and incubated at 26°C for 48 h. The growth was monitored throughout 48 h. This culture was used for inoculum preparation for setting MBEC-HTP device. Inoculum was prepared in TSB to match a 1.0 McFarland standard and diluted 30-fold in TSB. 150 µL of inoculum was transferred into each well of a 96-well microtiter plate. The dried, L-lysinecoated peg lids were then inserted into 96-well microtiter plate containing this inoculum, and placed for 48 h in incubator at 26 o C.

Preparation of metal solution
Tolerance of the planktonic cells and biofilms was tested in the presence of Cd 2+ , Zn 2+ , and Ni 2+ metal ions originating from the CdSO4, ZnSO4, NiSO4 salts (Sigma). All metal compounds were dissolved in the sterile distilled water. Stock solutions were filtered using the 0.2 μm syringe filter. Work solutions of metals were diluted in TSB from stock solutions, to prepare challenge media, no more than 60 minutes before the exposure. Used concentrations were in accordance with the concentrations used in the study of AL-ENZI and AL-CHARRAKH (2013). Range of concentrations for nickel was from 1.30 to 20.67 mM; for cadmium and zinc was 0.1, 1, 10 and 100 mM. Range of concentrations for amphotericin B was: 0.24; 0.47; 0.94; 1.89; 3.78; 7.57; and 15.15 µg/mL. This antimycotic was a control for yeast cells susceptibility, not to compare with metals. The Zn 2+ and Cd 2+ ions were neutralized using 10 mM reduced glutathione while the Ni 2+ ions were chelated with 0.5 mM reduced glutathione (HARRISON et al., 2006). Based on the previous studies on metal removal potential, selected concentration was 100 µg/mL for each metal (BASAK et al., 2014).

Tolerance of planktonic cells to heavy metals
The tolerance of the R. mucilaginosa and S. boulardii planktonic cells was determined according to the method described by CERI et al. (1999). The culture inoculum was prepared in McFarland 1.0 and diluted 30-fold in TSB for setting MBEC-HTP device. Each well of a 96-well microtitre plate was set with 150 μL with plastic lid with 96 pegs. After 48-h incubation at 26 °C, biofilm was formed on peg and planktonic cells left in wells were both used for metal challenge.

Tolerance of biofilms to heavy metals
Tolerance of biofilms was evaluated as previously described by HARRISON et al. (2006). The peg lid (with the formed biofilms) was immersed in the 96-well microtiter plates containing TSB with metal salt in the appropriate concentrations. The challenge plates were incubated at 26 o C for 48 h.
After exposure period, pegs with biofilm were removed from the challenge plates and washed twice with sterile 0.9% saline. Plastic lid with pegs, was transferred to a new plate with TSB containing neutralizer (200 µL per well). After neutralization, plastic lid with pegs was transferred to a plate with TSB and the entire plate was exposed to the ultrasonic waves, the frequency of 20 kHz to 400 kHz for 5 min in a water bath for sonification (Aquasonic 250 HT Ultrasonic Cleaner, VWR International, Radnor, PA, USA). This microtiter plate was marked as recovery plate and it was incubated for 48 h at 26 o C. After the incubation period, minimum biofilm eradication concentration (MBEC) was obtained using ELISA microplate reader (OD650) (Rayto, China).

Fluorescence microscopy
Fluorescence microscopy was used to evaluate the effect of metals on the R. mucilaginosa biofilm according to the method described by KRONVALL and MYHRE (1977) with some modifications. The content of the recovery microtiter plate was removed. 50 µL of methanol was added in each well of microtiter plate. Microtiter plate was incubated at room temperature until methanole vaporized. 50 µL of acridine orange (5 mg/mL) was added in each well. After 2 min., the microtiter plate was washed with sterile distilled water. The R. mucilaginosa biofilm was observed on the Olympus BX51 fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan) and analyzed using Cytovision 3.1 software package (Applied Imaging Corporation, Santa Clara, California, USA).

Metal removal efficiency using planktonic cell
Metal removal efficiency was analyzed according to the method described by MUNEER et al. (2007). The cells were grown in 250 ml Erlenmeyer flasks containing 100 ml of YPED medium. One flask was the control and other three contained YPED medium, suspension, and metals with concentration of 100 μg/ml. The flasks were incubated at 26 °C. Growth of the R. mucilaginosa planktonic cells was determined by reading optical density at 520 nm (OD520) after 12, 24, and 48 h. At the same time, from the flasks with tested metal, 5 ml of aliquots was taken out and cells were separated by centrifugation. The supernatant (samples and controls) were subjected to spectrophotometer (357.9 nm) analysis for residual metal concentration. All experiments were performed in triplicates and their mean value was calculated.
The metal removal percentage (%) was calculated from the following equation (1): (1) where Ci is the initial concentration of metal ion (µg/mL) and Cr is the final concentration of metal ion (µg/mL).

Metal removal efficiency using biofilm
Metal removal efficiency was analyzed according to the method described by BASAK et al. (2014). Biofilm was formed on 22 × 22 mm polyvinyl plastic coverslips placed in each well of a 6-well culture plate. Fifty microliters of suspension (McFarland 1.0) was added to each well with 5 ml YPED medium. Coverslips with formed biofilm were placed in the new 6-well plate that contained tested metals individually, with concentration of 100 μg/ml. After 12, 24, and 48 h incubation period, 1.5 mL aliquots were taken and centrifuged at 10000 rpm for 5 min. The supernatant (samples and controls) were subjected to spectrophotometer (520 nm) analysis for residual metal concentration. All experiments were performed in triplicates and their mean value was calculated.
The metal removal percentage (%) was calculated from the equation 1.

Tolerance of planktonic cells on heavy metals
Heavy metal tolerance of R. mucilaginosa and S. boulardii planktonic cells, for the exposure period of 48 h, was analyzed. The planktonic cells of R. mucilaginosa showed high tolerance in the presence of metals (Cd 2+ , Zn 2+ and Ni 2+ ), while S. boulardii showed tolerance toward Zn 2+ only. The results are presented in the Table 1. Cadmium tolerance of Rhodotorula sp. Y11 was reported by LI and YUAN (2006,2008), with the highest tolerated concentration of 0.1 mM. In our study, R. mucilaginosa tolerated cadmium concentration up to 10 mM. A possible reason for the disparity may be the species difference, even though they belong to the same genus. The R. mucilaginosa species in this study was isolated from environment, which also may influence the obtained results. In another study, R. rubra tolerated cadmium to concentration of 10 mM (SALINAS et al., 2000), which is in accordance with our results.
The tolerance of R. mucilaginosa to the presence of 50 mg/L nickel was previously reported by SAN and DÖNMEZ (2012). Furthemore, the tolerance of another species, R. glutinis to the presence of nickel under concentration range from 10 to 400 mg/L was reported by SUAZO-MADRID et al. (2011). In our study, the range of concentrations was significantly higher, ranging from 100 to 3200 mg/L. The MIC was observed at the 400 mg/L, which implies the similar metabolic response to heavy metal impact by two different species.

Tolerance of biofilms to heavy metals
The heavy metal tolerance of R. mucilaginosa and S. boulardii biofilms was analyzed. R. mucilaginosa formed the biofilm after 48 h of exposure, while S. boulardii did not exhibit the biofilm formation ability. The results of R. mucilaginosa heavy metal tolerance is presented in the Table 2. The obtained results showed a significant difference in metal tolerance between the R. mucilaginosa biofilm and planktonic cells. The R. mucilaginosa biofilm was more tolerant in the presence of all tested metals, compared to planktonic cells. This is due to the extracellular polymeric substances (EPS) that surround the cells in biofilms. Furthermore, it is confirmed, and our results were similar to the results of HARRISON et al. (2006), who examined the effect of heavy metal (AsO4 3-, Cd 2+ , Pb 2+ , Ni 2+ , SeO3 2-, CrO4 2-, Mn 2+ , Co 2+ , Cu 2+ , Ag + , Zn 2+ , Hg 2+ , Al 3+ , AsO2 -, SeO3 2-, Te3 2-) on Candida tropicalis biofilm.

Fluorescence microscopy
The fluorescence microscopy was used as visual confirmation of already obtained results through MBEC. The impact of heavy metals and amphotericin B on the R. mucilaginosa biofilm were observed and results were shown in Figure 1    The results of reading the optical density at microplate rider were in accordance with the results of fluorescence microscopy.

Metal removal efficiency using planktonic cells and biofilm
The percentage of heavy metals removal by R. mucilaginosa planktonic cells after 48 hours of incubation is shown in the  The metal removal efficiency of the R. mucilaginosa biofilm was better, compared to the planktonic cells. Obtained results showed that the R. mucilaginosa biofilm removed over 90% of every tested metal after 48 h. These results are in accordance with the results of BASAK et al. (2014), who reported 88% and 72.2% Zn 2+ removal by Candida rugosa and Cryptococcus laurentii biofilm, respectively, for 24 h. The percentage of Zn 2+ removal in our study after 24 hours was 85.04%, which was in accordance with results of mentioned studies.

CONCLUSION
Our findings suggest that biofilm and planktonic populations show different levels of tolerance to heavy metals. Understanding this difference is significant for understanding the microbial ecology of environments polluted with heavy metals, as well as the basics of biofilm tolerance to antimicrobial agents in general. This study gives an insight about the ability of R. mucilaginosa to form biofilm on coverslips and remove metal ions (Cd 2+ , Zn 2+ , Ni 2+ ) as an inexpensive and alternative method to traditional techniques for removal of heavy metals from waste waters. Our results indicate that biofilm has a higher ability to remove heavy metals compared to planktonic cells, which suggests that biofilm has a better potential for application in the environment remediation. The ability of the R. mucilaginosa biofilm to remove Cd 2+ , Zn 2+ and Ni 2+ ions could be used in some future examinations on real effluent.