SEM/STEM Observation of Biofilm/Mineral Interface 1171
Advantages and drawbacks of the tested microscopy methods for biofilm/nZVI observation studies
A comparison of the advantages and drawbacks of the microscopy methods applied to the study of biofilms/nZVI interaction are summarized in Table 2. Our results show that coupling EDS analysis with fluorescent, SEM and TEM microscopy techniques offers the possibility to access the inside and outside of the biofilm and thus enables biofilm/nZVI characterization at (i) the biofilm level, with the location and nature of nZVI (here as aggregates) at the surface of the biofilm structure, (ii) the cell level with the absence of cells/nZVI interaction, and (iii) themolecular levelwith the lectin labeling strongly suggesting a major role of EPS in nZVI/biofilm
interaction.Moreover, the present study demonstrated that the three microscopy methods used are complementary approa- ches for a complete study of biofilm/nZVI interactions. Each microscopy technique has its advantages and draw-
backs that can be bypassed by the use of other techniques (Table 2). Moreover, preparing biofilm samples for accurate observations, especially when interacting with sand or metal elements such as nZVI, can prove difficult without modifying
their structure. Many studies have been carried out using bio- films developed on a smooth surface (glass or flow cell experi- ments) and protocols that are developed are thus not applicable to most biofilms/substrata directly taken from the environment. Working with flocs using fluorescent microscopy is also not easy. Indeed, samples are thick and water rich (compared with biofilm grown on a substratum). Observation of flocs between slide and cover slide can result to the flocs moving during ana- lysis (because of the presence of water inside the sample), and the thickness makes fluorescence detection in the deeper zones of the biofilm possible, thus leading to fluorescence that is out of focus. The coupling of several approaches in microscopy is thus an obligate approach and the best route to study biofilm/NP interaction with biofilms still attached to a “natural” substratum (sand grains in this study) that are usually porous, not flat, voluminous, and impossible to section.
CONCLUSION
visible in the biofilm, but showed rather a network with forms whose shape and size are similar to that of bacteria. We suggested that these forms may be bacteria embedded in an EPS network, as was shown by lectin labeling. This hypothesiswas confirmed by STEM-in-SEM observations on the microtome sections of the biofilm with lectin–gold labeling for EPS detection, which showed that the bacteria were encapsulated in a matrix of EPS. STEM-in-SEM
Understanding biofilm organization and interactions with surrounding substratum and pollutants or particles can benefit from the array of existing microscopy tools such as fluorescent microscopy, cryo-SEM, STEM-in-SEM or (S)TEM (in TEM), and EDS. Microscopy is indeed a very powerful approach to characterize biofilms and has pre- viously been used to study biofilm/nZVI interaction. Cryo-SEM samples exhibited a low number of bacteria
analyses also showed nZVI aggregates on or near the surface (up to 10 µm) of the biofilm, but not in direct contact with bacteria. Complementary (S)TEM (in TEM)/EDS analysis also revealed that nZVI are connected to the EPS matrix, as confirmed by EDS mapping of gold and iron which exhibit co-location of lectin–gold (EPS markers) with iron aggregates. Then, the combination of fluorescent microscopy, cryo-SEM and STEM-in-SEM, supplemented by TEM/EDS analysis appeared to be an excellent tool for understanding the biofilm/mineral interface and interaction. These observations enabled access to the inside and
outside of the biofilm at different scales of magnitude, allowing to understand the behavior and the internal struc- ture of biofilms in contact with mineral or metallic particles, and the ability of biofilms to fix nZVI or to bind to sand particles. This approach allows nZVI characterization (in terms of size distribution and aggregation) and location inside the biofilm (intracellular location, association with cells wall/membrane).
ACKNOWLEDGMENTS
This work is in the projects BIOMEB and BIOSORP sup- ported by BRGM Research Direction in collaboration with project NANOREM (FP7 2007–2013 Grand agreement No. 309517). The authors gratefully acknowledge the financial support provided to the PIVOTS project by the Région Centre—Val de Loire (ARD 2020 programand CPER 2015– 2020) and the French Ministry of Higher Education and Research (CPER 2015–2020 and public service subsidy to Brgm). Cryo-SEM (Hitachi) and TEM (CM20) observations were performed at the Electron Microscopy Center at the University of Orleans (France) by A.R. and A.S.. TEM and STEM preparation were carried out by MRic TEM Biosit electron microscopy platform at the University of Rennes (France). TEM /EDS/EELS (Jeol ARM200F) analysis were performed at the Centre Raymond Castaing (Toulouse, France) and at the demonstration center of JEOL Ltd (Tokyo, Japan).
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