The aim of the study was to examine heavy metal tolerance (Cd2+, Zn2+, Ni2+ and Cu2+) of single- and mixed-species biofilms (Rhodotorula mucilaginosa and Escherichia coli) and to determine metal removal efficiency (Cd2+, Zn2+, Ni2+, Cu2+, Pb2+ and Hg2+). Metal tolerance was quantified by crystal violet assay and results were confirmed by fluorescence microscopy. Metal removal efficiency was determined by batch biosorption assay. The tolerance of the mixed-species biofilm was higher than the single-species biofilms. Single- and mixed-species biofilms showed the highest sensitivity in the presence of Cu2+ (E. coli-MIC 4 mg/ml, R. mucilaginosa-MIC 8 mg/ml, R. mucilaginosa/E. coli-MIC 64 mg/ml), while the highest tolerance was observed in the presence of Zn2+ (E. coli-MIC 80 mg/ml, R. mucilaginosa-MIC 161 mg/ml, R. mucilaginosa-E. coli-MIC 322 mg/ml). The mixed-species biofilm exhibited better efficiency in removal of all tested metals than single-species biofilms. The highest efficiency in Cd2+ removal was shown by the E. coli biofilm (94.85%) and R. mucilaginosa biofilm (97.85%), individually. The highest efficiency in Cu2+ (99.88%), Zn2+ (99.26%) and Pb2+ (99.52%) removal was shown by the mixed-species biofilm. Metal removal efficiency was in the range of 81.56%–97.85% for the single- and 94.99%–99.88% for the mixed-species biofilm.

INTRODUCTION

In the natural environment, biofilms have a great impact on our daily life (Elias & Banin 2012). Our current understanding of the physiology and complexity of the mixed-species biofilms are still in progress. Although the mixed-species biofilms represent the dominant form in the environment (Elias & Banin 2012), previous studies were still based mainly on the studies of the individual laboratory biofilms (Hall-Stoodley et al. 2004; Høiby et al. 2010).

The single-species biofilms were up to 65 times more tolerant to the influence of heavy metals than planktonic cells (Harrison et al. 2005b, 2006). The mixed-species biofilm were more tolerant to stressors such as antibiotics, disinfectants, heavy metals etc., than the single-species biofilm (Golby et al. 2014; Jahid & Ha 2014). The ability of microbial communities to cooperate and survive the impact of antimicrobial agents explains the tolerance in the mixed-species biofilms (Elias & Banin 2012). This ability turns the mixed-species biofilm into a practical tool that has potential in bioremediation of contaminated environments (Golby et al. 2014).

One of the most serious environmental problems has been heavy metal pollution. Conventional methods of metal removal have proved to be ineffective when the concentration of the metal ions was low (1–100 mg/l) (Wang & Chen 2006). Biosorption presents a modern low-cost method that uses bacteria, algae, yeasts, filamentous fungi, etc. (planktonic form) (Fu & Wang 2011), but the use of biofilms is still in the process of evaluation. A successful bioremediation process relies on understanding the interactions between microbes and contaminants (Zhang et al. 1995). Biofilms that live in contaminated environments have usually developed effective multiple defence mechanisms for survival (Harrison et al. 2007). Regardless of the fact that heavy metals cause toxic effects, tolerance to heavy metals is a natural phenomenon that depends on the various conditions in which biofilms develop (Harrison et al. 2007). In recent years, attention has been devoted to examining the biofilms' biosorption potential (Harrison et al. 2006; Quintelas et al. 2008, 2009). To the authors' knowledge, one of the first studies on the effect of heavy metals on a mixed bacterial biofilm was published by Golby et al. (2014). In addition to bacteria, the efficiency of metal removal by yeast biofilm has been studied (Basak et al. 2014).

Rhodotorula mucilaginosa biofilm showed noticeably more resilience in the presence of heavy metals than corresponding planktonic cells (Grujić et al. 2017). For this reason, the aim of the study was to test the influence of heavy metals on single- and mixed-species biofilm, formed of R. mucilaginosa and Escherichia coli. The hypothesis was that with the establishment of the synergistic interaction between R. mucilaginosa and E. coli, the mixed-species biofilm will show better results. Pb2+ and Hg2+ tolerance of single- and mixed-species biofilm (R. mucilaginosa and E. coli) was tested (Buzejić et al. 2016). Except for Buzejić et al.’s (2016) study, the heavy metal tolerance of single- and mixed-species biofilms has not been tested in the presence of other metal ions. Therefore, the aim of our study was to examine the heavy metal tolerance of the single- and mixed-species biofilm formed by R. mucilaginosa and E. coli in the presence of heavy metals Cd2+, Zn2+, Ni2+ and Cu2+ and to determine Cd2+, Zn2+, Ni2+ Cu2+, Pb2+ and Hg2+ removal efficiency.

METHODS

Microorganisms

Two species of microorganisms isolated from the environment were used in this study (R. mucilaginosa and E. coli). The E. coli strain was a gift from the Institute for Public Health, Kragujevac, Serbia. 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 both strains (Adam et al. 2002).

Preparation of metal solutions

Metal tolerance of the R. mucilaginosa, E. coli and R. mucilaginosa/E.coli biofilms was tested in the presence of metal ions, Cd2+, Zn2+, Ni2+ and Cu2+, originating from CdSO4, ZnSO4, NiSO4 and CuSO4, salts (Sigma-Aldrich, St Louis, MO, USA). Biosorption potential was tested in the presence of two extra metal ions: Pb2+ and Hg2+, originating from Pb(NO3)2 and HgCl2 salts (Sigma-Aldrich). Working solutions were prepared in TSB medium from stock solutions, no more than 60 min before use. Based on previous research and the preliminary test, a range of tested concentrations was selected in which the lowest concentration does not lead to a significant response (compared to control) and the highest concentration causes a 100% test response of the organism. Standard antibiotics (amphotericin and tetracycline) were used as the positive control. Based on previous studies on microbial biosorption potential, the selected concentration was 100 μg/ml for each metal (Basak et al. 2014).

Biofilm formation

To test heavy metal tolerance, the R. mucilaginosa and E. coli biofilms were formed in polystyrene microtitre 96-well plates (Sarstedt, Germany). One hundred μl of suspension (OD520 0.8) was added in every well of the plate. To form the mixed R. mucilaginosa/E. coli biofilm, an equal amount of suspension was mixed immediately before use (Adam et al. 2002).

To test biosorption potential, the single- and mixed-species biofilms were formed on coverslips 22 × 22 mm, that were immersed in the wells of microtitre plates with the nutritive medium and suspension (McFarland 0.5 for bacteria; McFarland 1.0 for yeast; to form the mixed R. mucilaginosa/E. coli biofilm, an equal amount of suspension was mixed immediately before incubation) (Sternberg et al. 2014).

Heavy metal tolerance of tested biofilms and quantification

After the incubation period, the tested biofilms were treated with heavy metals and antibiotics. Quantification was performed using crystal violet (CV) assay according to the method described by Almeida et al. (2011) with certain modifications. The effect of the tested metals was followed during 72 hours. The contents of the plates (after 24 h, 48 h and 72 h), where the biofilms were formed, were removed and 50 μl of methanol 98% (vol/vol) was added. After 15 min the methanol content was removed and the plates were allowed to dry at room temperature. Then, to each well, 50 μl of CV was added (5 min). Plates were washed with sterile distilled water and 100 μl of glacial acetic acid, 33% (vol/vol) was added to each well of the plate. The minimal inhibitory concentration (MIC) and minimal lethal concentration (MLC) were determined by reading the optical density (OD570) using a microplate reader (Rayito, China). All tests were performed in triplicate and the mean value was calculated.

Fluorescence microscopy

Fluorescence microscopy was used to examine the influence of heavy metals on tested biofilms according to the method described by Kronvall & Myhre (1977) with certain modifications. In each well of a microtitre plate, 50 μl of methanol was added and incubated at room temperature until the methanol evaporated. After incubation, 50 μl of acridine orange stain (5 mg/ml) was added into the microtitre plate. After 2 min, the microtitre plate was washed with sterile distilled water. Tested biofilms were observed on the Olympus BX51 fluorescence microscope (Olympus, Shinjuku, Tokyo, Japan) and analysed using the Cytovision 3.1 software package (Applied Imaging Corporation, Santa Clara, CA, USA).

Batch biosorption assay

Batch assay was performed according to the method described by Basak et al. (2014). In order to study the effect of metal removal by biofilms, the batch experiments were conducted by immersing the coverslips (22 × 22 mm), with formed biofilms, in metal solution (100 μg/ml) at pH 6. Each day for 5 days, 1.5 ml of aliquots were 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: 
formula
1
where Ci is the initial concentration of metal ion (μg/ml) and Cr is the final concentration of the metal ion (μg/ml).

Dry weight determination of biofilms

Dry weight determination was chosen in order to examine and monitor the impact of the total biofilm mass of single- and mixed-species biofilms on metal removal. Each day for 5 days, the dry weight of biofilms was measured according to the method described by Pedersen (1982).

RESULTS AND DISCUSSION

Biofilm formation

R. mucilaginosa and E. coli formed a single- and mixed-species biofilm in the 96-well microtitre plate as well as on the coverslips. Biofilm production was quantified by CV and dry weight assay. The results of both tests showed that production of the R. mucilaginosa/E. coli biofilm was higher in relation to the single-species biofilms (E. coli < R. mucilaginosa < R. mucilaginosa/E. coli). The same results were obtained by Buzejić et al. (2016). Harrison et al. (2006) reported that Candida tropicalis could survive in unfriendly conditions due to its ability of biofilm formation. E. coli showed an ability of biofilm formation that was in accordance with the results of Harrison et al. (2005a), where different strains of E. coli (E. coli JM109, E. coli HM21 and E. coli HM22) showed potential for biofilm formation on an MBEC-HTP system. The formation of the mixed-species biofilm was influenced by numerous processes that determine the biofilm's shape and nature (Elias & Banin 2012).

Heavy metal tolerance of tested biofilms and quantification

The results of heavy metal tolerance of tested biofilms were expressed as the MIC and the MLC (Table 1). The MIC for the E. coli biofilm was determined after 24 h and the MLC after 48 h. The MIC for the R. mucilaginosa and R. mucilaginosa/E. coli biofilm was determined after 48 h and MLC after 72 h.

Table 1

Tolerance of single- and mixed-species biofilm in the presence of tested substances

Tested substances Single-species biofilms
 
Mixed-species biofilm
 
R. mucilaginosa
 
E. coli
 
R. mucilaginosa/E. coli
 
MIC MLC MIC MLC MIC MLC 
Cd 52 834 26 208 208 834 
Zn 161 645 80 161 322 645 
Ni 76 153 38 153 307 615 
Cu 64 64 128 
Amphotericin B 0.007 0.06 0.007 0.03 0.25 0.5 
Tetracycline 0.06 0.12 0.01 0.06 0.5 0.5 
Tested substances Single-species biofilms
 
Mixed-species biofilm
 
R. mucilaginosa
 
E. coli
 
R. mucilaginosa/E. coli
 
MIC MLC MIC MLC MIC MLC 
Cd 52 834 26 208 208 834 
Zn 161 645 80 161 322 645 
Ni 76 153 38 153 307 615 
Cu 64 64 128 
Amphotericin B 0.007 0.06 0.007 0.03 0.25 0.5 
Tetracycline 0.06 0.12 0.01 0.06 0.5 0.5 

MIC, minimal inhibitory concentration; MLC, minimal lethal concentration.

Values in the table are in mg/ml.

Heavy metal tolerance of the R. mucilaginosa biofilm was better than the E. coli biofilm in our work (Table 1). This was in accordance with the results of Buzejić et al. (2016) who had examined the biofilms' tolerance to the metal ions Pb2+ and Hg2+. Harrison et al. (2006) reported that MLC100 for the C. tropicalis biofilm in the presence of Cd, Zn, Ni and Cu was observed at concentrations >2.8 × 102 mM, 64 mM, >4.9 × 102 mM and 1.4 × 102 mM, respectively. In our study, the obtained MLC results for the R. mucilaginosa biofilm, in the presence of Ni, Cu, Zn and Cd, were obtained at concentrations of 153 mg/ml, 64 mg/ml, 645 mg/ml and 834 mg/ml, which were partially in accordance with the results of the mentioned study, since the authors did not determine the MLC (larger than the maximum used concentration). In our work, the MLC was determined in the presence of all tested substances. The results in the available literature suggest that heavy metal tolerance of biofilms was time dependent (Harrison et al. 2005a, 2005b). Harrison et al. (2005a) investigated the sensitivity of the E. coli JM109 biofilm in the presence of heavy metal ions including Cd, Zn, Ni, Cu and Hg. The MIC was obtained at concentrations of 2.3 mM, 31 mM, 17 mM, 16 mM, and 0.04 mM. In our study, the E. coli biofilm showed tolerance in the presence of Cd2+, Zn2+, Ni2+, Cu2+ and the MIC was obtained at concentrations of 26 mg/ml, 81 mg/ml, 38 mg/ml and 4 mg/ml. The MIC for Cu was in accordance with Harrison et al. (2005a).

Mixed-species biofilms can be established by one or several different microbial species, which are the dominant form in nature. Studies confirmed that the multispecies biofilm was extremely resistant to antimicrobial treatment in comparison to the single-species biofilm (Burmølle et al. 2006). Our results matched with the results of the mentioned studies. Heavy metal tolerance of single- and mixed-bacterial biofilms (isolates from the sludge tailings) in the presence of metal ions, including Cu, Zn, Ni and Pb, was examined by Golby et al. (2014). The obtained results showed that the mixed bacterial biofilm was extremely resistant to the applied metal ions. The reported tolerance values were the following: over 20 mg/l for Pb, 16 mg/l for Zn and 3.2 mg/l for Ni. Accurate concentrations of the heavy metals' tolerance for biofilms were not determined in the mentioned study. Hence, we could say that the results of the mentioned study were in accordance with ours (Table 1). The MIC of Zn and Ni for the mixed-species biofilm (R. mucilaginosa/E. coli) was 323 mg/ml and 308 mg/ml, which was several times larger than in the study of Golby et al. (2014).

Antibiotics (amphotericin B and tetracycline) were used as the control of our experiment. Mixed-species biofilm showed a higher tolerance to antibiotics in relation to single-species biofilm (Table 1), which was in accordance with the study carried out by Adam et al. (2002).

Fluorescence microscopy

The results obtained by fluorescence microscopy for the single- and mixed-species biofilm are presented in Figure 1.
Figure 1

The MIC results of heavy metals' tolerance for tested biofilms (E. coli, R. mucilaginosa and R. mucilaginosa/E.coli).

Figure 1

The MIC results of heavy metals' tolerance for tested biofilms (E. coli, R. mucilaginosa and R. mucilaginosa/E.coli).

The panels in the figure present the MIC of tested substances on the E. coli, R. mucilaginosa and R. mucilaginosa/E. coli biofilms. The results of MIC, which were obtained by reading the optical density using a microplate reader for the single- and mixed-species biofilm, were confirmed by fluorescence microscopy, which was used as visual confirmation.

Batch biosorption assay

The results of metal removal efficiency (Cd2+, Zn2+, Ni2+, Cu2+, Pb2+ and Hg2+) by single- and mixed-species biofilm, after 5 days, are presented in Figures 2 and 3.
Figure 2

The removal efficiency of (a) Cd2+, (b) Zn2+ and (c) Ni2+ by single- and mixed-species biofilms.

Figure 2

The removal efficiency of (a) Cd2+, (b) Zn2+ and (c) Ni2+ by single- and mixed-species biofilms.

Figure 3

The removal efficiency of (a) Cu2+, (b) Pb2+ and (c) Hg2+ by single- and mixed-species biofilms.

Figure 3

The removal efficiency of (a) Cu2+, (b) Pb2+ and (c) Hg2+ by single- and mixed-species biofilms.

The heavy metal removal efficiency by mixed-species biofilm was the best on all 5 days (concentrations of the heavy metal ions were reducing during this time), in comparison with the single-species biofilms. The best results showed the R. mucilaginosa/E. coli biofilm with a range of metal removal from 94.99% to 99.88% (the ranges of residual concentrations of metal ions were from 5.01% to 0.12%) (Figures 2 and 3). Concentrations of the heavy metal ions in the substrate were reducing during the testing time, whereby the microbial biofilms showed high efficiency, which was in accordance with the results of the study of Basak et al. (2014). Figures 2 and 3 show variations in the speed of removing metal ions by days. It is obvious that the process of metal removal occurs in two phases. The first phase was extremely fast due to the high initial activity of biofilms as biosorbents. It was observed that the highest efficiency during the treatment was in the first 2–3 days. 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). Quintelas et al. (2009) reported the percentage of Cd2+ and Ni2+ removal at different initial concentrations using the E. coli biofilm and kaolin. The percentage of Cd2+ removal on the initial concentration of 97 mg/l was 71.3%, which was in accordance with our results, where the percentage of Cd2+ removal efficiency after 48 h was 74.18%. The percentage of Ni2+ removal at the initial concentration of 101 mg/l was 45.3%. The same results were obtained in our work, where the percentage of Ni2+ removal efficiency after 24 h was 49.34%. Basak et al. (2014) investigated the removal of Zn2+ using the Candida rugosa and Cryptococcus laurentii biofilms. The removal of Zn2+ ions was found to be 88% and 74.2%. In our study, the percentage of Zn2+ removal efficiency for the R. mucilaginosa biofilm after 48 h was 74%, which was in accordance with the results of the mentioned study. The percentage of Zn2+ removal efficiency for the R. mucilaginosa/E. coli biofilm after 48 h was better (87%) than the percentage of removal efficiency of the R. mucilaginosa (74%) and E. coli (68.25%) biofilms individually. Similar results were obtained by White & Gadd (1998), who tested the accumulation and effects of cadmium on single- and mixed-species sulfate-reducing bacterial biofilms. In our work, both biofilms formed separately and together showed extremely high biosorption potential, but the absorption amount of single-species biofilms was slightly lower than in the mixed-species biofilm (Figures 2 and 3). The amount of single- and mixed-species biofilm (Figure 4) could affect the removal of metal ions (Cd2+, Zn2+, Ni2+, Cu2+, Pb2+ and Hg2+).
Figure 4

The amount of single- and mixed-species biofilm in the presence of (a) Cd2+, (b) Zn2+, (c) Ni2+, (d) Cu2+, (e) Pb2+ and (f) Hg2+.

Figure 4

The amount of single- and mixed-species biofilm in the presence of (a) Cd2+, (b) Zn2+, (c) Ni2+, (d) Cu2+, (e) Pb2+ and (f) Hg2+.

The increase in amounts of biofilms over the time significantly impacted the efficiency of metal removal. These observations were in accordance with a study conducted by Al-Garni et al. (2009) that investigated the biosorption characteristics of the filamentous fungi Aspergillus fumigatus in the removal of cadmium from aqueous solutions.

CONCLUSION

Based on the knowledge of detoxification mechanisms used by microorganisms to reduce 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 the effect of selected heavy metals in the context of single- and mixed-species biofilms used in development of biotechnologies suitable for metal removal. Based on the obtained results, a noticeable difference in the metal tolerance, as well as high biosorption potential between the single- and mixed-species biofilms occurs. The R. mucilaginosa and E. coli biofilms show significant potential in the process of elimination of heavy metals from contaminated environments, whereby the mixed-species biofilm (R. mucilaginosa/E. coli) has greater efficiency.

ACKNOWLEDGEMENT

This study was supported by the Serbian Ministry of Education, Science, and Technological Development.

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