Molecular and microscopic assessment of the effects of caffeine, acetaminophen, diclofenac, and their mixtures on river biofilm communities.

The authors examined effects of three common contaminants, caffeine (CF), acetaminophen (AC), and diclofenac (DF), as well as their mixtures on the development, functioning, and biodiversity of river biofilm communities. Biofilms were cultivated in rotating annular reactors. Treatments included AC, CF, DF, AC + CF, AC + DF, CF + DF, AC + CF + DF at 5 µg/L, and their molar equivalent as carbon and nutrients. Incubations using ¹4C-labeled AC, DF, and CF indicated that 90% of the CF, 80% of the AC, and less than 2% of the DF were converted to CO2. Digital imaging revealed a variety of effects on algal, cyanobacterial, and bacterial biomass. Algal biomass was unaffected by AC or CF in combination with DF but significantly reduced by all other treatments. Cyanobacterial biomass was influenced only by the AC + DF application. All treatments other than AC resulted in a significant decrease in bacterial biomass. Diclofenac or DF + CF and DF + AC resulted in increases in micrometazoan grazing. The denaturing gradient gel electrophoresis of Eubacterial community DNA, evaluated by principal component analysis and analysis of similarity, indicated that relative to the control, all treatments had effects on microbial community structure (r = 0.47, p < 0.001). However, the AC + CF + DF treatment was not significantly different from its molar equivalent carbon and nutrient additions. The Archaeal community differed significantly in its response to these exposures based on community analyses, confirming a need to integrate these organisms into ecotoxicological studies.

Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems.

An assessment of lectin-binding analysis for the characterization of extracellular glycoconjugates as part of the extracellular polymeric substances in environmental microbial communities was performed using fully hydrated river biofilms. The applicability of the method was evaluated for single, dual and triple staining with a panel of fluor-conjugated lectins. It was shown that lectin-binding analysis was able to stain glycoconjugates within biofilm communities. Lectin staining also demonstrated spatial heterogeneity within the biofilm matrix. Furthermore, the application of two or even three lectins was possible if suitable combinations were selected. The lectin-binding analysis can be combined with general nucleic acid stains to collect both nucleic acid and glycoconjugate signals. The effects of incubation time, lectin concentration, fluor labelling, carbohydrate inhibition, order of addition and lectin interactions were studied. An incubation time of 20 min was found to be sufficient for completion of lectin binding. It was not possible to ascertain saturating concentration for individual lectins, therefore a standard concentration was used for the assay. Carbohydrate inhibition tests indicated that fluorescein isothiocyanate (FITC)-conjugated lectins had more specific binding characteristics than tetramethyl rhodamine isothiocyanate (TRITC)- or cyanine dye (CY5)-labelled lectins. The order of addition and the nature of the fluor conjugate were also found to influence the binding pattern of the lectins. Therefore the selection of a panel of lectins for investigating the EPS matrix must be based on a full evaluation of their behaviour in the biofilm system to be studied. Despite this necessity, lectin-binding analysis represents a valuable tool to examine the glycoconjugate distribution in fully hydrated biofilms. Thereby, chemical heterogeneities within extracellular biofilm locations can be identified in order to examine the role (e.g. sorption properties, microenvironments, cell-extracellular polymeric substance interactions) of the extracellular polymeric substances in environmental biofilm systems.