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Differential Sensitivity of Environmentally Relevant Microorganisms to Metal-containing Nanoparticles

  • Author(s): Reyes, Vincent C.
  • Advisor(s): Mahendra, Shaily
  • et al.
Abstract

Increasing applications of metal-containing nanoparticles (NPs), the largest class of consumer nanomaterials, has heightened the need evaluate their potential environmental ramifications. Of these, copper- and zinc-containing nanoparticles (Cu-NP and Zn-NP) are important due to their widespread use, low cost, and biological relevance of their respective metals. As microorganisms are at the foundation of all food webs and define available nutrients for ecological niches, understanding their interactions with metal-containing NPs is essential for addressing their inevitable release into the environment. Few studies, however, examine the impact of biological variables on the microbial response to metal-containing NPs. To address these gaps, high-throughput screening methods, which examine multiple conditions simultaneously, were combined with traditional toxicity diagnostics, which examine specific parameters in depth, to effectively explore NP-microbe interactions. Previous studies have also neglected to examine how NP sensitivity changes with exposure time. To this end, Zn-NP toxicity was assessed using a time-resolved high-throughput screening methodology in an arrayed Escherichia coli genome-wide knockout (KO) library to determine changes in sensitivity with time. Through sequential screening, this method identified sensitive clones from diverse biological pathways, which fell into two general groups: early and late responders. These results suggested bacterial toxicity mechanisms changed from pathways related to general metabolic function, transport, signaling, and metal ion homeostasis to membrane synthesis pathways and reflected different growth stages in E. coli. To ensure that the responses to NP exposures observed for the model laboratory strain, E. coli, translated to environmentally relevant microbes, nitrogen cycling microorganisms were studied further because of their role as primary drivers of biogeochemical nitrogen transformations. Previous assessments of NP interactions have ignored biofilms, the primary mode of growth for environmental microorganisms. Impacts of Cu-NP exposure to biofilms were compared to respective planktonic cultures of the ammonium oxidizing Nitrosomonas europaea, nitrogen-fixing Azotobacter vinelandii, and denitrifying Paracoccus denitrificans using a suite of independent toxicity diagnostics. When compared to unexposed controls, growth parameters in N. europaea and P. denitrificans biofilms were more resilient to inhibition than planktonic cultures. Likewise, physiological evaluation of ammonium oxidation and nitrate reduction and respective functional gene expression showed that biofilms were also less impacted by Cu-NPs than their respective planktonic cells. These results suggest biofilms reduced NP inhibition, and that nitrogen cycling bacteria in wastewater, wetlands, and soils are more resilient to NPs than planktonic-based assessments might suggest. To build upon trends observed for pure cultures, changes in nitrogen cycling microorganisms in wetland-derived microcosms following acute and chronic Cu-NP exposure were characterized using the functional microarray Geochip platform. Although shifts to nitrogen transformation activity occurred after acute exposure, significant changes to microbial ecology only occurred after long-term exposure. Among genes coding for various nitrogen cycling enzymes, anammox and nitrogen fixation genes decreased to the largest extent, while those for denitrification were least sensitive to changes. This study implies that sudden NP influxes into wetlands may impair nitrogen cycling initially, but chronic exposure may result in microbial changes, which promote net nitrogen fluxes out of the system. Taken together, these results highlight a methodological framework for using high-throughput and traditional assays in tandem to explore biological factors in NP-microbial assessments. Furthermore, the data suggest that biological factors significantly shape microbial sensitivity to NPs and must be considered while assessing environmental impacts of NP releases as a result of manufacturing, use, and disposal of nano-enabled products and applications.

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