Identification of soil bacteria susceptible to TiO2 and ZnO nanoparticles

Abstract

Because soil is expected to be a major sink for engineered nanoparticles (ENPs) released to the environment, the effects of ENPs on soil processes and the organisms that carry them out should be understood. DNA-based fingerprinting analyses have shown that ENPs alter soil bacterial communities, but specific taxon changes remain unknown. We used bar-coded pyrosequencing to explore the responses of diverse bacterial taxa to two widely used ENPs, nano-TiO(2) and nano-ZnO, at various doses (0, 0.5, 1.0, and 2.0 mg g(-1) soil for TiO(2); 0.05, 0.1, and 0.5 mg g(-1) soil for ZnO) in incubated soil microcosms. These ENPs significantly altered the bacterial communities in a dose-dependent manner, with some taxa increasing as a proportion of the community, but more taxa decreasing, indicating that effects mostly reduced diversity. Some of the declining taxa are known to be associated with nitrogen fixation (Rhizobiales, Bradyrhizobiaceae, and Bradyrhizobium) and methane oxidation (Methylobacteriaceae), while some positively impacted taxa are known to be associated with the decomposition of recalcitrant organic pollutants (Sphingomonadaceae) and biopolymers including protein (Streptomycetaceae and Streptomyces), indicating potential consequences to ecosystem-scale processes. The latter was suggested by a positive correlation between protease activity and the relative abundance of Streptomycetaceae (R = 0.49, P = 0.000) and Streptomyces (R = 0.47, P = 0.000). Our results demonstrate that some metal oxide nanoparticles could affect soil bacterial communities and associated processes through effects on susceptible, narrow-function bacterial taxa.

Fig 1

Principal coordinates analysis (PCoA) to illustrate the shifts of soil bacterial communities exposed to nano-TiO₂ (a and b) and nano-ZnO (c and d) based on Bray-Curtis distance and weighted UniFrac distance. Con, control; TL, TM, and TH, low (0.5 mg g⁻¹ soil), medium (1.0 mg g⁻¹ soil), and high (2.0 mg g⁻¹ soil) doses of nano-TiO₂, respectively; ZL, ZM, and ZH, low (0.05 mg g⁻¹ soil), medium (0.1 mg g⁻¹ soil), and high (0.5 mg g⁻¹ soil) doses of nano-ZnO, respectively. Exposure time is indicated by the numerical suffix; e.g., Con15 represents the control at day 15.

Fig 2

Multivariate regression tree (MRT) to reveal the hierarchical environmental determinants on soil bacterial communities associated with exposure dose (0, 0.5, 1.0, and 2.0 mg g⁻¹ soil for TiO₂; 0.05, 0.1, and 0.5 mg g⁻¹ soil for ZnO) and exposure time (0, 15, and 60 days). In an MRT, each split is represented graphically as a branch that is labeled with the levels of the classification variable; bar plots show the multivariate means of OTUs at each branch; the numbers of samples included in the splits are shown under the bar plots. The heat map shows the normalized abundances of OTUs that occurred in more than 50 samples. Con, control; TL, TM, and TH, low (0.5 mg g⁻¹ soil), medium (1.0 mg g⁻¹ soil), and high (2.0 mg g⁻¹ soil) doses of nano-TiO₂; ZL, ZM, and ZH, low (0.05 mg g⁻¹ soil), medium (0.1 mg g⁻¹ soil), and high (0.5 mg g⁻¹ soil) dose of nano-ZnO. Exposure time is indicated by the numerical suffix (see the legend of Fig. 1).

Fig 3

Regression analysis to illustrate the systematic shifts of soil bacterial communities along dose gradients of nano-TiO₂ (a, b, e, and f) and nano-ZnO (c, d, g, and h) after 15-day and 60-day exposures. The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Error bars above and below the box indicate the 90th and 10th percentiles, respectively.

Fig 4

Soil bacteria susceptible to both nano-TiO₂ and nano-ZnO at both sampling times. Error bars indicate the standard error of the mean (n = 4).

Fig 5

Effects of nano-TiO₂ (a) and nano-ZnO (b) on protease activity. Error bars indicate the standard error of the mean (n = 4). Bars within the same bar cluster labeled by the same letter do not differ at a P value of <0.05.