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The aim of the study was to examine heavy metal tolerance (Cd, Zn, Ni and Cu) of single- and mixed-species biofilms (Rhodotorula mucilaginosa and Escherichia coli) and to determine metal removal efficiency (Cd, Zn, Ni, Cu, Pb and Hg). 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 Cu (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 Zn (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 Cd removal was shown by the E. coli biofilm (94.85%) and R. mucilaginosa biofilm (97.85%), individually. The highest efficiency in Cu (99.88%), Zn (99.26%) and Pb (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.

The presence of heavy metals (HMs) in the environment represents a serious environmental problem. In this regard, this work was conceived with the aim of finding, among indigenous microorganisms, the species and their combinations with the best biosorption activity for the following HMs: zinc, lead, cadmium, copper, and nickel. The experiment was carried out in several steps: (1) isolation and identification of microbial strains from the Central Effluent Treatment Plant’s wastewater; (2) studying the interaction of microorganisms and the ability to form biofilms in 96-well plates; (3) testing the resistance of biofilms to HMs; (4) testing the growth of biofilms on AMB media carriers in the presence of HMS; and (5) biosorption assay. The selected strains used in this study were: Enterobacter cloacae, Klebsiella oxytoca, Serratia odorifera, and Saccharomyces cerevisiae. The best biofilm producers in control medium were K. oxytoca/S. odorifera (KS), followed by K. oxytoca/S. odorifera/S. cerevisiae (KSC), and E. cloacae/K. oxytoca/S. odorifera (EKS) after 10 days of incubation. Mixed cultures composed of three species showed the highest resistance to the presence of all tested metals. The best biosorption capacity was shown by KSC for Cu2+ (99.18%), followed by EKS for Pb2+ (99.14%) and Cd2+ (99.03%), K. oxytoca for Ni2+ (98.47%), and E. cloacae for Zn2+ (98.06%). This research offers a novel approach to using mixed biofilms for heavy metal removal processes as well as its potential application in the bioremediation of wastewater.

ABSTRACT. This study investigated the antimicrobial activity as well as some phytochemical analysis of ethanol and diethyl ether extracts from plant species Vinca minor L. In vitro antimicrobial activity of extracts was studied on 20 strains of microorganisms (16 bacteria and four yeasts). Testing was performed by microdilution method and minimum inhibitory concentration (MIC) and minimum microbicidal concentration (MMC) were determined. The strongest antimicrobial activity was detected on G+ bacteria of the genus Bacillus. Tested G -bacteria and yeasts were not sensitive to the action of the extracts or the sensitivity was insignificant.Phytochemical analysis involved determining the amount of total phenolics, flavonoids and tannins, as well as the determination of antioxidant activity monitoring capability to neutralize free radicals (DPPH) and the reductive potential. Phytochemical examination indicates that the total phenolic compounds were more in the ethanolic extract and the content of flavonoids and tannins marginally higher in the diethyl ether extract. The antioxidant activity (DPPH) of the ethanolic extract of V. minor was significantly stronger as compared to the diethyl ether extract, and the reduction potential was approximately the same.

ABSTRACT. The aim of this study was to examine heavy metal tolerance (lead (Pb 2+ ) and mercury (Hg 2+ )) of single-and mixed-species biofilms, formed by yeast Rhodotorula mucilaginosa and bacteria Escherichia coli LM1. Single-and mixed-species biofilms were quantified by crystal violet test and the absorbance was measured using microplate reader (OD 570 ). The minimal inhibitory concentration (MIC) and the minimal lethal concentration (MLC) were determined and the results were confirmed by fluorescence microscopy.The significant difference in lead tolerance was observed between the mixed-and the single-species biofilms. The MIC of lead (Pb 2+ ) for the examined biofilms (E. coli LM1, R. mucilaginosa and R. mucilaginosa / E. coli) was recorded at concentrations of 4000 µg/ml, 4000 µg/ml and 16000 µg/ml, respectively. The MIC of mercury (Hg 2+ ) for the biofilms was noticed at concentrations of 31.25 µg/ml, 250 µg/ml and 250 µg/ml, respectively. Standard antibiotics (amphotericin B and tetracycline) were used as positive control. Results obtained for single-species biofilms were compared in between and with the results obtained for mixed-species biofilm.The tolerance of the mixed-species biofilm was higher in comparison to the singlespecies biofilms and the results were confirmed by a fluoresecence microscope. The obtained results suggest that the R. mucilaginosa / E. coli biofilm may have a potential to be used in bioremediation of wastewaters contaminated with lead and mercury.