1160 Guillaume Wille et al.
[e.g., fluorescent in situ hybridization, 4’,6-diamidino-2- phenylindol (DAPI)] and exopolymeric substance labeling (e.g., lectins for EPS, dichlorodimethylacridinone for eDNA) (Surman et al., 1996; Lawrence et al., 2003; Kämper et al., 2004; Zhang et al., 2015). Each approach has its specific advantages, but also its drawbacks, the main two being the potential impacts of sample preparation (fixation, drying) on native morphologies and cell structures and the difficulties observing abiofilm associated with its growth substratum (solid), often leading to the removal of biofilms from substrata before observation (Surman et al., 1996; Priester et al., 2007). To overcome the these drawbacks, the present study
investigates the possibilities of coupling several approaches, taking into account that sample preparation and labeling differ according to the type of microscopy (Wrede et al., 2008). The methodology was applied to an example of a multispecies biofilm interacting with nZVI. Biofilms were grown for several weeks either as flocs or on a solid surface [sand, polyvinyl chloride (PVC) tube]. The sampling, labeling, and treatment strategies were developed and adapted for each type of microscopy to access the surface or the inside of the biofilm, biofilm structure, and metal loca- tion. Special attention was given to the different problems caused by these specific samples, which are a mixture of “soft” hydrated biological samples and “high hardness”
mineral particles. Due to the size of the bacteria and EPS filaments in the biofilm (a few microns at most), their observation is limited with optical microscopy. Thus, we applied high-vacuum SEM, variable pressure field-emission scanning electron microscopy (VP-FE-SEM), cryo-SEM, scanning transmission electron microscopy in the SEM (STEM-in-SEM) and TEM techniques.
MATERIALS AND METHODS
Table 1 summarizes the approaches tested in this study, in terms of biofilm growth, sampling, labeling, and treatment according to the microscopy techniques used.
Biofilm labeling for fluorescence microscopy
Biofilms as flocs were first stained with DAPI for cell labeling and then with lectin for EPS detection (Michel et al., 2011).
Table 1. Sample Selection and Preparation and Observation Characteristics According to the Microscopy Used for Biofilm/nZVI Inter- action Analysis.
Microscopy Sample
Sample preparation Labeling Sample treatment Resolution Imaging mode(s) Chemistry
Fluorescence Floc
Incubation with nZVI. Washing step for non-fixed NPs elimination
DAPI + lectin (PNA or ConA) - FITC
None µm Cryo-SEM + EDS Floc or biofilm grown on sand grain
Incubation with nZVI. Washing step for non-fixed NP elimination
Lectin PNA-Au and Lectin ConA-Au (gold size 15 to 40nm)
Sample frozen in nitrogen slush at −210°C. Cryo-fracturation (if necessary)
nm
SE: morphology BSE: chemical composition
EDS STEM-in-SEM + EDS TEM + EDS
Incubation with nZVI. Washing step for non-fixed NP elimination
Biofilm attached on PVC tube
Lectin PNA-Au (40 nm) and Lectin ConA-Au (15 nm) Prefixation and postfixation. Ultrathin sectioning (80nm)
nm (better than cryo-SEM) <nm
SE: morphology BSE: chemical composition BF/DF
EDS
Transmitted e-BF HAADF
EDS EELS (data not shown)
SEM, scanning electron microscopy; EDS, energy dispersive X-ray spectrometry; STEM, scanning transmission electron microscopy; TEM, transmission electron microscopy; PVC, polyvinyl chloride; nZVI, zero-valent iron nanoparticles; NP, nanoparticle; DAPI, 4’,6-diamidino-2-phenylindol; SE, secondary electron; BSE, backscattered electron; BF, bright field; HAADF, high angle annular dark field; DF, dark field; EELS, electron energy loss spectroscopy.
Culture and sampling of biofilms
Biofilms were grown either in PVC tubes (inner Ø: 4.8mm) or a continuous up-flow fixed-bed bioreactor (laboratory- scale column) (20cm high; 2.5cm in diameter) filled with sand (d50=0.51mm) (Fig. 1). The main advantage of PVC tubes is the opportunity to easily and quickly obtain a large amount of biological material as flocs or biofilms attached to a smooth surface (i.e., the inner face of the PVC tube). The column approach was used in order for the biofilm to attach to the sand grains such as in a natural aquifer. However, the intrinsic characteristics of sand grains (rough and hard) limit the microscopic approaches that can be applied. For biofilm culture, a groundwater sample was used as the inoculum and microbial development was favored by supplementing the water with a nutritive solution (sodium acetate 10mM and yeast extract 0.2 g/L) and an electron acceptor (NO3
−, solution of NaNO3 10mM) (Hellal et al.,
2015). Anaerobic conditions (under N2 bubbling) were maintained as in a deep contaminated aquifer. The incuba- tion temperature was maintained at 20°C. First the biofilms were left to grow during 6 weeks
without nZVI. Then a nZVI suspension (1 g/L, NANOFER 25 S, mean particle size ~50–80nm; NanoIron, Rajhrad, Czech Republic) was continuously introduced into the PVC tube or column (1 g/L, 10 mL/min, for five poral volumes). Finally, biofilms were sampled as flocs in the PVC tube or as attached biofilms on the inner face of PVC tubes, or collected as attached to sand grains in the column. Biofilms attached to the inner face of the PVC tube were used either still attached to the PVC tubes, or detached from the tubes by removing the biofilm with a sterile scalpel (this allows to access the external face of the biofilm) and placing them on a SEM copper stub.
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