metal (Cu, Fe, Mn, Ni) speciation in biofilm matrices (Neu et al. 2010; Behrens et al.
2012). Dynes et al. (2006a) used STXM to highlight the close association of Ni with
Mn-oxides and the role of EPS in the sequestration of metals in aquatic microbial
biofilms. This technique also allowed Lawrence et al. (2012, 2016) to follow the
dissolution and fate of Cu nanoparticles (Lawrence et al. 2012) and the fate and
speciation of Ce, TiO 2 , and Cu in river biofilms (Lawrence et al. 2016). Yang et al.
(2016) characterized the biotransformation of selenium oxyanions by biofilms
using STXM and X-ray fluorescence imaging (at higher energies than STXM).
Finally, STXM image sequences revealed that Fe localization (on the cell surface
or within the EPS matrix) was speciation-dependent in a monospecific biofilm of
Pseudomonas aeruginosa (Hunter et al. 2008). The main limitations of the STXM
techniques are their low sensitivity and limit of resolution (25–50 nm against 4 nm
for TEM techniques). Indeed, to our knowledge, very few STXM studies have been
conducted at environmentally relevant concentrations: one study reports results on
Mn, Fe, and Ni with water concentrations ranging from 0.01 to 0.02 mg Mn L
À1 ,
0.02 to 0.06 mg Fe L
À1 , and 1 to 10 mg Ni L
À1 , respectively (Hitchcock et al. 2009).
The combination of different imaging techniques remains essential to determine
metal distribution in biofilms and better understand the interactions between metals,
cellular components, and extracellular material (van Hullebusch et al. 2003). In a
recent study, Lawrence et al. (2019) combined CLSM with different fluorescent
probes, scanning electron microscopy, and X-ray microprobe analyses to show that
Ni was mainly associated to EPS in biofilm and was four times more concentrated
around specific microcolonies than in the rest of the microbial community.
2.2 Organic Contaminants
In contrast to the substantial research on metal distributions within periphytic
biofilms, there is a dearth of studies dealing with the accumulation in microbial
assemblages of organic contaminants. These studies mainly focus on pesticides,
polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and
more recently pharmaceuticals, hormones, and parabens. And the few studies
published fail to detail analytical methods for the quantification of organic contaminants in the biofilm following both laboratory and in situ exposure (Schorer and
Eisele 1997; Headley et al. 1998; Lawrence et al. 2001; Writer et al. 2011; Kohušová
et al. 2011; Ruhí et al. 2016). Due to the high complexity of the biofilm matrix,
nonselective solvent extraction methods generate analytical interferences; therefore
biofilm contamination by organic contaminants is preferably estimated indirectly
from contamination in water (Wang et al. 2002; Proia et al. 2013a, b). To directly
measure the concentration of organic contaminants in periphytic biofilms, samples
are first collected from the surfaces of stones or artificial substrates and then freezedried before analysis (Wang et al. 1999; Huerta et al. 2016). Organic contaminants
are commonly extracted from the whole biofilm matrix without fractionation to
avoid matrix destruction and cell lysis. However, Chaumet et al. (2019a) recently
124
C. Bonnineau et al.
2012). Dynes et al. (2006a) used STXM to highlight the close association of Ni with
Mn-oxides and the role of EPS in the sequestration of metals in aquatic microbial
biofilms. This technique also allowed Lawrence et al. (2012, 2016) to follow the
dissolution and fate of Cu nanoparticles (Lawrence et al. 2012) and the fate and
speciation of Ce, TiO 2 , and Cu in river biofilms (Lawrence et al. 2016). Yang et al.
(2016) characterized the biotransformation of selenium oxyanions by biofilms
using STXM and X-ray fluorescence imaging (at higher energies than STXM).
Finally, STXM image sequences revealed that Fe localization (on the cell surface
or within the EPS matrix) was speciation-dependent in a monospecific biofilm of
Pseudomonas aeruginosa (Hunter et al. 2008). The main limitations of the STXM
techniques are their low sensitivity and limit of resolution (25–50 nm against 4 nm
for TEM techniques). Indeed, to our knowledge, very few STXM studies have been
conducted at environmentally relevant concentrations: one study reports results on
Mn, Fe, and Ni with water concentrations ranging from 0.01 to 0.02 mg Mn L
À1 ,
0.02 to 0.06 mg Fe L
À1 , and 1 to 10 mg Ni L
À1 , respectively (Hitchcock et al. 2009).
The combination of different imaging techniques remains essential to determine
metal distribution in biofilms and better understand the interactions between metals,
cellular components, and extracellular material (van Hullebusch et al. 2003). In a
recent study, Lawrence et al. (2019) combined CLSM with different fluorescent
probes, scanning electron microscopy, and X-ray microprobe analyses to show that
Ni was mainly associated to EPS in biofilm and was four times more concentrated
around specific microcolonies than in the rest of the microbial community.
2.2 Organic Contaminants
In contrast to the substantial research on metal distributions within periphytic
biofilms, there is a dearth of studies dealing with the accumulation in microbial
assemblages of organic contaminants. These studies mainly focus on pesticides,
polychlorinated biphenyls (PCBs), polycyclic aromatic hydrocarbons (PAHs), and
more recently pharmaceuticals, hormones, and parabens. And the few studies
published fail to detail analytical methods for the quantification of organic contaminants in the biofilm following both laboratory and in situ exposure (Schorer and
Eisele 1997; Headley et al. 1998; Lawrence et al. 2001; Writer et al. 2011; Kohušová
et al. 2011; Ruhí et al. 2016). Due to the high complexity of the biofilm matrix,
nonselective solvent extraction methods generate analytical interferences; therefore
biofilm contamination by organic contaminants is preferably estimated indirectly
from contamination in water (Wang et al. 2002; Proia et al. 2013a, b). To directly
measure the concentration of organic contaminants in periphytic biofilms, samples
are first collected from the surfaces of stones or artificial substrates and then freezedried before analysis (Wang et al. 1999; Huerta et al. 2016). Organic contaminants
are commonly extracted from the whole biofilm matrix without fractionation to
avoid matrix destruction and cell lysis. However, Chaumet et al. (2019a) recently
124
C. Bonnineau et al.