Biofilm formation by selected microbial strains isolated from wastewater and their consortia: mercury resistance and removal potential.

Wastewater often contains an increased amount of mercury and, at the same time, resistant microorganisms. During wastewater treatment, a biofilm of indigenous microorganisms is often unavoidable. Therefore, the objective of this research is to isolate and identify microorganisms from wastewater and investigate their ability to form biofilms for possible application in mercury removal processes. The resistance of planktonic cells and their biofilms to the effects of mercury was investigated using Minimum Biofilm Eradication Concentration-High Throughput Plates. The formation of biofilms and the degree of resistance to mercury were confirmed in polystyrene microtiter plates with 96 wells. Biofilm on AMB Media carriers (Assisting Moving Bad Media) was quantified using the Bradford protein assay. The removal of mercury ions by biofilms formed on AMB Media carriers of selected isolates and their consortia was determined by a removal test in Erlenmeyer flasks simulating MBBR. All isolates in planktonic form showed some degree of resistance to mercury. The most resistant microorganisms (Enterobacter cloacae, Klebsiella oxytoca, Serratia odorifera, and Saccharomyces cerevisiae) were tested for their ability to form biofilms in the presence and absence of mercury, both in polystyrene plates and on ABM carriers. The results showed that among planktonic forms, K. oxytoca was the most resistant. A biofilm of the same microorganisms was more than 10-fold resistant. Most consortia biofilms had MBEC values > 100,000 µg/mL. Among individual biofilms, E. cloacae showed the highest mercury removal efficiency (97.81% for 10 days). Biofilm consortia composed of three species showed the best ability to remove mercury (96.64%-99.03% for 10 days). This study points to the importance of consortia of different types of wastewater microorganisms in the form of biofilms and suggests that they can be used to remove mercury in wastewater treatment bioreactors.

Finding the best combination of autochthonous microorganisms with the most effective biosorption ability for heavy metals removal from wastewater.

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.

Response of selected microbial strains and their consortia to the presence of automobile paints: Biofilm growth, matrix protein content and hydrolytic enzyme activity.

The goal of the current study was to examine the effects of pollutants (White color - CP; Metallic red color - FM; Thinner - CN; Thinner for rinsing paint - MF; Basic color (primer) - FH) originating from the automotive industry on the biofilm growth, matrix protein content, and activity of the hydrolytic enzymes of selected microbial strains in laboratory conditions that mimic the bioreactor conditions. The chosen microorganisms (bacteria, yeasts, and fungi) were isolated from automotive industry wastewater. Pure microbe cultures and their consortia were injected into AMB Media carriers and developed into biofilms. The use of AMB media carriers has been linked to an increase in the active surface area colonized by microorganisms. Afterwards, the carriers were transferred to Erlenmeyer flasks with nutrient media and pollutants at a concentration of 200 µL/mL. The current study found that, depending on the microbial strain, development phase, and chemical structure, the assessed pollutants had an inhibitory or stimulatory influence on the growth of single cultures and their consortia. Statistical analysis found positive correlations between the protein content in the matrix and the biofilm biomass of Rhodotorula mucilaginosa and consortia in CP and FH media, respectively. The proteolytic activity of Candida utilis was very pronounced in media with MF and CN. The best alkaline phosphatase activity (ALP) was achieved in the CN medium of R. mucilaginosa. Acid invertase activity was the highest in the FM and CP media of Escherichia coli and consortia, respectively, whereas the highest alkaline invertase activity was measured in the MF medium of E. coli. A positive correlation was confirmed between ALP and the biofilm biomass of R. mucilaginosa in CP and CN media, as well as between ALP and the biofilm biomass of Penicillium expansum in FM medium. The findings provide novel insights into the extracellular hydrolytic activity of the investigated microbial strains in the presence of auto paints, as well as a good platform for subsequent research into comprehensive biofilm profiling using modern methodologies.

Heavy metal tolerance and removal potential in mixed-species biofilm.

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.