Acute effects of TiO2 nanomaterials on the viability and taxonomic composition of aquatic bacterial communities assessed via high-throughput screening and next generation sequencing

Abstract

The nanotechnology industry is growing rapidly, leading to concerns about the potential ecological consequences of the release of engineered nanomaterials (ENMs) to the environment. One challenge of assessing the ecological risks of ENMs is the incredible diversity of ENMs currently available and the rapid pace at which new ENMs are being developed. High-throughput screening (HTS) is a popular approach to assessing ENM cytotoxicity that offers the opportunity to rapidly test in parallel a wide range of ENMs at multiple concentrations. However, current HTS approaches generally test one cell type at a time, which limits their ability to predict responses of complex microbial communities. In this study toxicity screening via a HTS platform was used in combination with next generation sequencing (NGS) to assess responses of bacterial communities from two aquatic habitats, Lake Michigan (LM) and the Chicago River (CR), to short-term exposure in their native waters to several commercial TiO2 nanomaterials under simulated solar irradiation. Results demonstrate that bacterial communities from LM and CR differed in their sensitivity to nano-TiO2, with the community from CR being more resistant. NGS analysis revealed that the composition of the bacterial communities from LM and CR were significantly altered by exposure to nano-TiO2, including decreases in overall bacterial diversity, decreases in the relative abundance of Actinomycetales, Sphingobacteriales, Limnohabitans, and Flavobacterium, and a significant increase in Limnobacter. These results suggest that the release of nano-TiO2 to the environment has the potential to alter the composition of aquatic bacterial communities, which could have implications for the stability and function of aquatic ecosystems. The novel combination of HTS and NGS described in this study represents a major advance over current methods for assessing ENM ecotoxicity because the relative toxicities of multiple ENMs to thousands of naturally occurring bacterial species can be assessed simultaneously under environmentally relevant conditions.

Figure 1. Ratio of live/dead bacteria in Lake Michigan water after 1 hour exposure in the dark (D) or under simulated sunlight (L) to one of four nano-TiO₂ materials: rutile nanopowder (RNP), anatase nanopowder (ANP), Pigment White 6 (PW) or P25.

Values are reported as mean of 4 replicates ± standard deviation. Nt: no treatment control prior to exposure. Data points marked by asterisks are significantly different (p<0.05) from the 0 mg L⁻¹ nano-TiO₂ control.

Figure 2. Ratio of live/dead bacteria in Chicago River water after 1 hour exposure in the dark (D) or under simulated sunlight (L) to one of four nano-TiO₂ materials: rutile nanopowder (RNP), anatase nanopowder (ANP), Pigment White 6 (PW) or P25.

Values are reported as mean of 4 replicates ± standard deviation. Nt: no treatment control prior to exposure. Data points marked by asterisks are significantly different (p<0.05) from the 0 mg L⁻¹ nano-TiO₂ control.

Figure 3. nMDS analysis of composition of bacterial communities from Lake Michigan water (A) and Chicago River water (B) prior to treatment (no treatment) and after 1 hour exposure to 0 mg L⁻¹ nano TiO₂, 100 mg L⁻¹ Pigment White 6 (PW6) or 100 mg L⁻¹ P25 under simulated sunlight.

(C) nMDS for Chicago River water with 100 mg L⁻¹ P25 treatment excluded. Bacterial community composition was analyzed based on tag pyrosequencing of 16S rRNA genes.

Figure 4. Diversity of bacterial communities from Lake Michigan and Chicago River prior to treatment (no treatment) and after 1 hour exposure to 0 mg L⁻¹ nano TiO₂, 100 mg L⁻¹ Pigment White 6 (PW6) or 100 mg L⁻¹ P25 under simulated sunlight

. Bacterial community composition was analyzed based on tag pyrosequencing of 16S rRNA genes. ANOVA indicated significant treatment effects for Lake Michigan (p<0.001) and Chicago River (p<0.05) bacterial communities. Data points marked by different letters indicate significant differences between nano-TiO₂ treatments within Lake Michigan or Chicago River communities based on Tukey's post hoc test.

Figure 5. Mean relative abundances (in %; n = 4) of numerically dominant bacterial phyla and classes in Lake Michigan water prior to treatment (no treatment) and after 1 hour exposure to 0 mg L⁻¹ nano-TiO₂, 100 mg L⁻¹ Pigment White 6 (PW6) or 100 mg L⁻¹ P25 under simulated sunlight.

Figure 6. Mean relative abundances (in %; n = 4) of numerically dominant bacterial phyla and classes in Chicago River water prior to treatment (no treatment) and after 1 hour exposure to 0 mg L⁻¹ nano-TiO₂, 100 mg L⁻¹ Pigment White 6 (PW6) or 100 mg L⁻¹ P25 under simulated sunlight.