Effects of low dose silver nanoparticle treatment on the structure and community composition of bacterial freshwater biofilms.

The application of engineered silver nanoparticles (AgNPs) in a considerable amount of registered commercial products inevitably will result in the continuous release of AgNPs into the natural aquatic environment. Therefore, native biofilms, as the prominent life form of microorganisms in almost all known ecosystems, will be subjected to AgNP exposure. Despite the exponentially growing research activities worldwide, it is still difficult to assess nanoparticle-mediated toxicity in natural environments. In order to obtain an ecotoxicologically relevant exposure scenario, we performed experiments with artificial stream mesocosm systems approaching low dose AgNP concentrations close to predicted environmental concentrations. Pregrown freshwater biofilms were exposed for 14 days to citrate-stabilized AgNPs at a concentration of 600 µg l-1 in two commonly used sizes (30 and 70 nm). Sublethal effects of AgNP treatment were assessed with regard to biofilm structure by gravimetric measurements (biofilm thickness and density) and by two biomass parameters, chlorophyll a and protein content. The composition of bacterial biofilm communities was characterized by t-RFLP fingerprinting combined with phylogenetic studies based on the 16S gene. After 14 days of treatment, the structural parameters of the biofilm such as thickness, density, and chlorophyll a and protein content were not statistically significantly changed by AgNP exposure. Furthermore, t-RFLP fingerprint analysis showed that the bacterial diversity was not diminished by AgNPs, as calculated by Shannon Wiener and evenness indices. Nevertheless, t-RFLP analysis also indicated that AgNPs led to an altered biofilm community composition as was shown by cluster analysis and multidimensional scaling (MDS) based on the Bray Curtis index. Sequence analysis of cloned 16S rRNA genes further revealed that changes in community composition were related with the displacement of putatively AgNP-sensitive bacterial taxa Actinobacteria, Chloroflexi, and Cyanobacteria by taxa known for their enhanced adaptability towards metal stress, such as Acidobacteria, Sphingomonadales, and Comamonadaceae. This measurable community shift, even after low dose AgNP treatment, causes serious concerns with respect to the broad application of AgNPs and their potentially adverse impact on the ecological function of lotic biofilms, such as biodegradation or biostabilization.

Sublethal concentrations of silver nanoparticles affect the mechanical stability of biofilms.

Bacterial biofilms are most likely confronted with silver nanoparticles (Ag NPs) as a pollutant stressor in aquatic systems. In this study, biofilms of Aquabacterium citratiphilum were exposed for 20 h to 30 and 70 nm citrate stabilized Ag NPs in low-dose concentrations ranging from 600 to 2400 µg l-1, and the Ag NP-mediated effects on descriptive, structural, and functional biofilm characteristics, including viability, protein content, architecture, and mechanical stability, were investigated. Viability, based on the bacterial cell membrane integrity of A. citratiphilum, as determined by epifluorescence microscopy, remained unaffected after Ag NP exposure. Moreover, in contrast to information in the current literature, protein contents of cells and extracellular polymeric substances (EPS) and biofilm architecture, including dry mass, thickness, and density, were not significantly impacted by exposure to Ag NPs. However, the biofilms themselves served as effective sinks for Ag NPs, exhibiting enrichment factors from 5 to 8. Biofilms showed a greater capacity to accumulate 30 nm sized Ag NPs than 70 nm Ag NPs. Furthermore, Ag NPs significantly threatened the mechanical stability of biofilms, as determined by a newly developed assay. For 30 nm Ag NPs, the mechanical stability of biofilms decreased as the Ag NP concentrations applied to them increased. In contrast, 70 nm Ag NPs produced a similar decrease in mechanical stability for each applied concentration. Overall, this finding demonstrates that exposure to Ag NPs triggers remarkable changes in biofilm adhesion and/or cohesiveness. Because of biofilm-mediated ecological services, this response raises environmental concerns regarding Ag NP release into freshwater systems, even in sublethal concentrations.