1 Introduction

The environmental pollution by metals is a serious problem which negatively affects natural ecosystems (Chipasa 2003). On the other hand, it represents great potential for studying the interactions between microorganisms and metals (Gadd 2004). Heavy metal polluted environments are a source of undiscovered microbial diversity and specific microbial strains and understanding the processes which are involved in natural biogeochemical cycles, already fundamental for environmentally friendly biotechnologies (Pieper and Reineke 2000; Gadd 2010).

Microbes and metals interact at all spatial scales for centuries (Ehrlich 1997; Gadd 2004). Metals support microbial growth by providing essential nutrients, microbial activity alters metal solubility and their oxidation state (Ehrlich 1997). Nowadays, when our reliance on metals increases, also does their amount in the environment and living systems. The dichotomy between metabolic requirements for metals by microorganisms and the potential associated toxicity has created an interesting and broad set of metal homeostasis and resistance systems required to maintain a delicate balance (Ehrlich 1997). Metal toxicity is significant to metal mobilizing microbes, which must resist toxic heavy metals released into the environment as a result of their metabolism (Gadd 2004). However, exposure to high toxic concentrations can trigger also various specific responses, involving metal resistance (Gadd and Griffiths 1977). Knowledge of metal-microbe interactions and the consequences of these interactions are necessary to understand metal resistance mechanisms of microorganisms (Haferburg and Kothe 2007).

Microbes, as predominant population in nature and environments, are present in two forms of life—as planktonic cells and biofilm. The most of microbes exist in surface-attached communities called biofilms, whose metabolism differ from their planktonic cells (Booth et al. 2011). Researches confirmed that biofilms were more resilient on antibiotic than their planktonic cells (Ceri et al. 1999). Teitzel and Parsek (2003) studied heavy metal resistance of biofilm and planktonic Pseudomonas aeruginosa and reported that biofilms were from 2 to 600 times more resistant to heavy metal stress than free-swimming cells. Harrison et al. (2004) examine the metalloid oxyanions toxicity against biofilm and planktonic cells of P. aeruginosa and Staphylococcus aureus and got the same results. The tolerance to antimicrobials depends on metabolic heterogeneity or the existence of a “persister” cell phenotype (Harrison et al. 2004). Also, differential metabolic shifts play a role in biofilm-specific multimetal resistance and tolerance (Booth et al. 2011). Persister cells of Escherichia coli biofilm, known for high tolerance on antibiotics, were also resistant to metal oxyanions (Harrison, et al. 2005). The comparison of tolerance between bacterial planktonic cells and biofilms to metal ions has been documented in a number of studies. No study to date has examined the susceptibility of yeast biofilms and planktonic cells to heavy metals, except for Candida tropicalis (Harrison et al. 2006).

Considering that yeasts are often predominant microbial species in environments contaminated with pollutants (Hagler and Mendonça-Hagler 1981) and that the planktonic cells of Rhodotorula mucilaginosa are known for high levels of resistance to the metal ions (Garza et al. 2016), we decided to test heavy metal susceptibility and removal efficiency of biofilm and planktonic cells of yeasts R. mucilaginosa isolated from environment and Saccharomyces boulardii (commercial probiotic) and make comparison of the obtained results.

2 Materials and Methods

2.1 Strains and Growth Media

Two species of microorganisms were used in this study. R. mucilaginosa (isolate from the environment, food spoilage, Serbia) and S. boulardii (commercial probiotic). The R. mucilaginosa strain was identified by the test for rapid identification of yeast API 20 C AUX (Biomerieux, France). Tryptic Soy Broth (TSB, Difco) was chosen as the growth medium for the both strains and for all metal susceptibility assays (Harrison et al. 2006). For metal removal studies, YPED medium was used (Muneer et al. 2007). All serial dilutions were performed using 0.9% saline.

2.2 Biofilm Cultivation

The R. mucilaginosa and S. boulardii biofilms were formed in the MBEC-HTP device (MBEC BioProducts) according to the manufacturer’s instructions and as described previously Ceri et al. (1999). Cryogenic stock cultures of R. mucilaginosa and S. boulardii were streaked out twice on TSA and incubated at 26 °C for 48 h. After incubation, period culture was used to prepare inoculum (McFarland 1.0 and diluted 30-fold in TSB) for setting MBEC-HTP device. One hundred fifty microliters of inoculum was transferred into each well of a 96-well microtitre plate, and plastic lid with 96 pegs was immersed into it. After 48-h incubation at 26 °C, biofilm was formed on peg, which was used for metal challenge.

Biofilm formation on peg lid was confirmed by sonication (Aquasonic 250 HT Ultrasonic Cleaner, VWR International, Radnor, PA, USA) of peg with lid in the new 96-well plate with fresh TSB media (biofilm formation confirmation). Evaluation of biofilm growth was obtained by reading the optical density of released biofilm at 650 nm (OD650) using ELISA microplate reader (Rayto, China).

2.3 Stock Metal Solutions

The susceptibility testing of the planktonic cells and biofilms was tested in the presence of ions; Hg2+, Cu2+, and Pb2+ originating from Pb(NO3)2, CuSO4, and HgCl2 (Sigma). All metal compounds were dissolved in sterile distilled water. Stock solutions were filtered using a 0.22-mm syringe filter into sterile glass vials and stored at fridge until use. Working solutions of metals were diluted in TSB from stock solutions, to prepare challenge media, no more than 60 min before exposure. Used concentrations for metal susceptibility assays were in accordance with the concentrations used in other study (Al-Enzi and Al-Charrakh 2015). Range of concentrations for lead (Pb2+) was from 0.11 to 7.03 mM; for copper (Cu2+) was from 0.40 to 12.81 mM and for mercury (Hg2+) was from 0.01 to 0.32 mM. Twofold dilutions were made in the wells of a sterile 96-well microtiter plate (the challenge plate), leaving the first wells of each row for sterility control (media only), and the second as a growth control (without metal ion).

Concentration for metal removal studies was 100 μg/ml, which was in accordance with the concentrations used in other studies (Li and Yuan 2006; Muneer et al. 2007; Basak et al. 2014).

2.4 Neutralization

The susceptibility testing of the planktonic cells and biofilms used neutralizing step as previously described Harrison et al. (2006). Pb2+ was neutralized using 10 mM reduced glutathione (TwinLab), Cu2+ was chelated with 0.5 mM sodium diethyldithiocarbamate (Sigma) and Hg2+ with 10 mM l-cysteine (Scitec Nutrition).

2.5 Metal Susceptibility Testing of Biofilm

Metal susceptibility testing of biofilm was performed as described previously (Harrison et al. 2006). The peg lid (with the formed biofilms) was immersed in the 96-well microtitre plates containing TSB with metal salt in the appropriate concentrations. The challenge plates were incubated at 26 °C for 48 h.

After 48 h exposure, plastic peg lid was removed from the challenge plate 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 biofilm was disrupted by the ultrasonic waves (frequency of 20 to 400 kHz for 5 min in a water bath for sonication) into recovery medium. After incubation, minimum biofilm eradication concentration (MBEC) was obtained by reading the optical density at 650 nm (OD650) of the recovery plate using ELISA microplate reader.

2.6 Metal Susceptibility Testing of Planktonic Cells

Experiments with R. mucilaginosa and S. boulardii planktonic cells were prepared according to the manufacturer’s instructions and as described previously (Ceri et al. 1999).

Challenge plates were prepared as described above. Fourteen-microliter aliquots of the planktonic cultures from challenge plate were transferred into the new 96-well microtitre plate (which contained neutralizing agents). Twenty-microliter aliquots of the neutralized planktonic cultures were placed into the 96-well microplate with TSB. MICp and MLCp values were obtained after 48- and 72-h incubation at 26 °C by reading the optical density at 650 nm (OD650) using a ELISA microplate reader.

2.7 Fluorescence Microscopy

Fluorescence microscopy was used to evaluate the effect of metals on the R. mucilaginosa biofilm as described previously by Kronvall and Myhre (1977) with certain modifications. The contents of the recovery microtiter plate were removed. Fifty microliters of methanol was added in each well. Microtiter plate was incubated at room temperature until methanol vaporized. Fifty microliters of acridine orange (0.1 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).

2.8 Metal Removal Study Using Biofilm

Metal removal study using biofilm was performed by batch biosorption assay according to the method described by Basak et al. (2014) with certain modifications. Biofilm was formed on 22 × 22 mm polyvinyl plastic coverslips placed in each well of a 6-well culture plate. Fifty microliter of suspension (McFarland 1.0) was added to each well with 5 ml YPED medium (Sternberg et al. 2014). 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 of aliquots was taken and centrifuged at 10,000 rpm for 5 min. The supernatant (samples and controls) was 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 following Eq. (1):

$$ E\left(\%\right)=\frac{\left( Ci- Cr\right)}{Ci}\times 100 $$
(1)

where Ci is the initial concentration of metal ion (μg/ml) and Cr is the residual concentration of metal ion (μg/ml).

2.9 Metal Removal Study Using Planktonic Cells

The R. mucilaginosa planktonic cells were used in metal removal study. Biosorption potential was tested in accordance to the method described by Muneer et al. (2007) with certain modifications. 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 (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 Eq. 1.

3 Results and Discussion

3.1 Metal Susceptibility Testing of Biofilm

Susceptibility of the R. mucilaginosa and S. boulardii survival preexisting biofilm and planktonic cells in the presence of heavy metal was analyzed. Hg2+, Pb2+, and Cu2+ metal ions were used for testing, and obtained results are presented in Table 1.

Table 1 Susceptibility of the R. mucilaginosa biofilm on heavy metals at exposure period of 48 h
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The obtained results indicated that R. mucilaginosa showed potential in biofilm formation. S. boulardii did not show potential in biofilm formation. The R. mucilaginosa biofilm showed noticeable resilience in the presence of all tested metals, which was in accordance with the results of Harrison et al. (2006), who reported the high tolerance of C. tropicalis biofilm to heavy metal toxicity. In mentioned study, MLCb for Hg2+ was obtained at 1.9±0.7 mM. MLCb for Pb2+ and Cu2+ was obtained at >77 mM and >1.3 × 102 mM. Our results were in accordance with the results of Harrison et al. (2006). Within a biofilm, regulatory processes occur (chemical, physical, physiological, and genetic), which allow microbes to develop better mechanism of tolerance and improve their resilience in the presence of metals (Harrison et al. 2007). Teitzel and Parsek (2003) reported Pb2+ and Cu2+ resistance of biofilm and planktonic P. aeruginosa, whereby the MBC for Pb2+ was obtained at 0.8 mM and MBC for Cu2+ was at 6 mM. In comparison with our results, we could suppose that R. mucilaginosa biofilm was more tolerant in the presence of Pb2+ and Cu2+ metal ions than P. aeruginosa biofilm.

3.2 Metal Susceptibility Testing of Planktonic Cells

Susceptibility of the R. mucilaginosa and S. boulardii planktonic cells was analyzed in the presence of Hg2+, Pb2+, and Cu2+ metal ions. The results of heavy-metal susceptibility testing are presented in Table 2.

Table 2 Susceptibility of the R. mucilaginosa and S. boulardii planktonic cells in the presence of heavy metals at exposure period of 48 h
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The R. mucilaginosa planktonic cells showed the highest tolerance in the presence of Cu2+ (MICp 6.40 mM) than other two metal ions, while the S. boulardii planktonic cells only tolerated Pb2+ (MICp 0.43 mM). Obtained results indicated that R. mucilaginosa was more tolerant to Pb2+ than S. boulardii. The highest sensitivity R. mucilaginosa showed in the presence of Hg2+ (MICp 0.08 mM).

Other authors reported the copper tolerance of the R. mucilaginosa LM9 (planktonic cells) (Rajpert et al. 2013). In correlation with available literature, Garza-Gonzalez et al. (2016) reported that R. mucilaginosa strain, isolated from the water streams, registered as UANL-001 L, was capable to survive heavy metal stress, whereby MIC for Cu2+ and Pb2+ were obtained at 0.8 g/l and 1 g/l. These results positively correlated with results obtained in our study in which MIC for Cu2+ and Pb2+ was noted at 6.40 mM (1.02 g/l) and 5.27 mM (1.74 g/l) (Table 2).

Mercury (Hg2+) tolerance of Rhodotorula rubra was literature supported and that strain showed tolerance at concentration of 16.8 μg/ml (Ghosh et al. 2006). Results in this study were approximate to our result where R. mucilaginosa tolerated mercury at 0.08 mM (21.7 μg/ml).

3.3 Differences Between Biofilm and Planktonic Response to Metal Susceptibility

Heavy metal susceptibility of biofilm and planktonic culture revealed that R. mucilaginosa biofilm was more tolerant to all tested metals than its planktonic cells (Fig. 1; Tables 1 and 2).

Fig. 1
figure 1

Comparison of the R. mucilaginosa biofilm and planktonic culture survival in percentage

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Fold toleranceFootnote 1 for Hg2+ was >1.93, for Pb2+ was >1, and for Cu2+ was >2.01. Biofilms, by their physiology, differ from their planktonic cells and they were up to 600 times more resistant to the effects of heavy metals (Teitzel and Parsek 2003; Harrison et al. 2006). C. tropicalis biofilm was up to 65 times more tolerant to metals than corresponding planktonic cultures. Fold tolerance for C. tropicalis biofilm for Cu2+ was >26; for Hg2+ 1.5; and for Pb2+, fold tolerance was not applicable (Harrison et al. 2006). Results of our work were in accordance with mentioned study. Biofilms were less susceptible to antimicrobials than planktonic form, because of structure dependent metabolic heterogeneity or because of the existence of a persister cell phenotype (Harrison et al. 2004). Booth et al. (2011) reported that differential metabolic shifts play a role in biofilm-specific multimetal resistance and tolerance. Biochemical and genetic regulatory processes that occur within a biofilm allow microbes to develop better mechanism of tolerance and improve their resilience in the presence of metals (Harrison et al. 2007).

3.4 Fluorescence Microscopy

The impact of heavy metals on the R. mucilaginosa biofilm was observed under fluorescence microscopy, and results are presented in Figs. 2, 3, 4, and 5.

Fig. 2
figure 2

The effect of copper (Cu2+) on the R. mucilaginosa biofilm

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Fig. 3
figure 3

The effect of lead (Pb2+) on the R. mucilaginosa biofilm

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Fig. 4
figure 4

The effect of mercury (Hg2+) on the R. mucilaginosa biofilm

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Fig. 5
figure 5

The effect of amphotericin B on the R. mucilaginosa biofilm

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Considering that impact of heavy metals was observed by fluorescence microscopy after 72 h, it was noticed that the biofilm was present at all tested metal concentrations in same quantity, as well as in growth control. Results of MBEC which were obtained by reading the optical density at microplate rider after 72 h, were in accordance with the results of fluorescence microscopy (which was used as visual confirmation).

3.5 Metal Removal Efficiency Using Biofilm and Planktonic Culture

Removal of Hg2+, Pb2+, and Cu2+ ions using R. mucilaginosa preexisting biofilm and planktonic cells was tested, and results are presented in the Tables 3 and 4.

Table 3 Metal removal using R. mucilaginosa biofilm (%)
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Table 4 Metal removal by R. mucilaginosa planktonic cells (%)
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Concentrations of the heavy metal ions in the medium were reducing during testing time, whereby the microbial biofilms showed high efficiency, which was in accordance with the results of Basak et al. (2014) study. Variations in the speed of removing metal ions during time periods are presented in Table 3. It is obvious that the process of the metal removing occurs in two phases. The first phase was extremely fast due to the high initial activity of biofilms as biosorbent. It was observed that the highest efficiency, during the treatment, was during the first day. After that, the slow phase of metal removal occurs, whereby the change in residual metal concentrations was insignificant. Our observation was in accordance with the study of Volesky (2003). Removal of Zn2+ using Candida rugosa and Cryptococcus laurentii biofims was examined by Basak et al. (2014). The removal of Zn2+ ions was found to be 88 and 74.2%. In our study, the percentage of metal removal efficiency for the R. mucilaginosa biofilm after 12 h was in the range from 75–80% which was in accordance with the results in mentioned study.

Percentage of heavy metals removal using R. mucilaginosa planktonic cells during 48 h are presented in Table 4.

The available literature contains information about copper (Cu2+) removal efficiency by planktonic cells of Candida spp., isolated from sewage, in which Candida spp. removed 22–52% Cu2+ ions after 8 days (Dönmez and Aksu 2001). The removal percentage of copper (Cu2+) for the Saccharomyces cerevisiae planktonic cells after 4 days exposure was 13–74% (Malik 2004). Our study reported that removal percentage of Cu2+ was 5.49 after 48 h/2 days exposure. The affinity of the metals for binding to the yeast cells could be explained by the fact that the cells of microorganisms have active nuclear sites for sorption of metal ions (Volesky 2003).

The obtained results showed a significant difference in metal removal efficiency between the biofilm and the planktonic cells. The best results in removal of all tested metals exhibited the R. mucilaginosa biofilm. Metal removal efficiency was in the range from 4.79–10.25% for planktonic cells and 91.71–95.39% for biofilm (Tables 3 and 4).

4 Conclusions

Based on the knowledge about microorganisms for reducing the heavy metal toxicity, it is possible to develop efficient and environmentally friendly (bio) technologies for metal remediation. The main aim of our work was to study metal-microbe interaction in the context of two microbial forms of life (biofilm and planktonic cells). This study also compared metal removal efficiency of the R. mucilaginosa planktonic cells and biofilm. The obtained results showed a significant difference in metal tolerance as well as in removal efficiency between the biofilm and their corresponding planktonic cells. The biofilm shows much better results than their planktonic cells, which suggests that biofilm could be used in the development of biotechnologies suitable for metal removal from the environment.