1168 Guillaume Wille et al.


Figure 8. Energy dispersive X-ray spectrometry mapping on a bacteria (TEM 80kV – JEOL ARM): (a) Scanning trans- mission electron microscopy-bright field; (b) calcium Kα;(c) oxygen Kα; (d) phosphorus Kα.


interact with cells could potentially lead to a less toxic effect of nZVI on biofilms compared with those described for nZVAg (Choi et al., 2010; Peulen & Wilkinson, 2011; Mallerve et al., 2016). nZVI were found as aggregates embedded in the biofilm structure and located on the outside of the biofilm in contact with the medium. This is in agree- ment with results obtained by Choi et al. (2010), who showed that nZVAg aggregated with an average size of about 800nm could penetrate to ~40 µm in a thick biofilm after 1h expo- sure. In our study, nZVI aggregates were bigger in size (about 1–5 µm). This could explain the shorter distance of nZVI aggregates penetration [7–11 µm in this study compared with 40 µm in the study of Choi et al. (2010)]. Using STEM-in-SEM imaging only, it was not possible


to conclude—or exclude—a co-location of nZVI and gold labeling, that is, a connection and an interaction between nZVI and EPS. For this, elemental mapping appeared to be difficult or impossible due to the low gold content. EDS and/ or electron energy loss spectroscopy (EELS) in the TEM are required for such analyses. The presence of gold labeling together with nZVI was


confirmed by EDS spectra (not shown) andmapping on Fe and Au on a nZVI aggregate in the JEOL ARM200 (Fig. 12). This


co-location Fe/Au suggests an interaction between nZVI and EPS. Moreover, these results confirm that EPS are also present in intercellular spaces inside the biofilm.Asin previousstudies, this work underlined the role of EPS in nanoparticles/biofilm interaction (Moreau et al., 2007; Peulen & Wilkinson, 2011). Here, the role of EPS labeled by lectins ConA and PNA was confirmed, but the presence and the role in nanoparticle inter- actions with other extracellular components, such as exopoly- saccharides nontargeted by the two tested lectins or extracellular protein orDNA, can, of course, not be excluded. As an example, Moreau et al. (2007) demonstrated the role of extracellular proteins.No, or very few, individual iron particles were detected. It also suggests that nZVI were present in the biofilm as aggre- gates in contact with EPS, but not directly with bacteria. It also confirms that no direct interaction between nZVI and micro- organisms can be considered, because of the presence of a thick layer of EPS surrounding the bacteria. Biofilm/EPS composition thus plays an important role in biofilm/nanoparticle interac- tions, as well as biofilm structure. In particular, density of both exopolymers and bacteria has been demonstrated to be an important parameter controlling the diffusion of nanoparticles inside the biofilm (Peulen &Wilkinson, 2011). Growth condi- tions that can influence biofilm density could thus influence


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