and microbial communities associated to these substrates are only based on total
analysis of the metal in the whole sediment (e.g., Farag et al. 2007; Kohušová et al.
2011) or leaf litter (e.g., Sridhar et al. 2008; Schaller et al. 2011), including attached
biofilms. The analytical procedures commonly used include a first step of extraction
with aqua region or nitric acid, for instance, on fresh or dried sediment/leaf litter.
Then, metal concentrations in the extracts are measured by conventional analytical
methods (ICP-OES, ICP-MS, or AAS). To date, and to the best of our knowledge, no
study has been conducted to separate and specifically assess metal concentrations in
microbial communities from sediment or leaf litter.
Besides traditional fractionation and extraction methods, imaging techniques
have been developed to investigate interactions between metals and biofilms and
to visualize metals within the biofilm structure. Analytical electron microscopy
techniques such as transmission electron microscopy (TEM), often coupled with
energy-dispersive X-ray spectrometry, have been used to identify metal
bioaccumulation in microbial biofilms, in particular in bioremediation studies in
which biofilms are used as a sink to accumulate metals from contaminated waters
(Mattila et al. 1997; Miller et al. 2012). For example, this technique allowed Vilchez
et al. (2011) to show that Cr(III) bioaccumulated in the EPS matrix of microbial
biofilms, while Pb(II) was detected in both the EPS matrix and the microbial biofilm
cells. In order to determine macromolecules and metals composition in EPS from
microbial biofilms, TEM can also be coupled to electron energy loss spectrometry,
which is a complex technique for measuring atomic composition and chemical
binding and speciation, even for lower elements (C, O). The main drawback of
TEM is that sample preparation often requires dehydration, creating artifacts such as
particle shrinkage or aggregation (Dynes et al. 2006a). Furthermore, the high energy
of TEM causes radiation damage in biological samples, which leads to spectral
distortions, making high-resolution mapping difficult (Hitchcock et al. 2008).
Other imaging techniques such as confocal laser scanning microscopy (CLSM)
and scanning transmission X-ray microscopy (STXM) are especially well suited for
biofilm studies as they can be applied to fully hydrated biological materials and
reduce radiation damage (see Neu et al. 2010 for a comparative review on those
techniques applied to biofilms). In CLSM, metal binding to specific fluorescent
probes allows detection and localization of those contaminants within biofilms (for
a description of the different probes available, see the review by Hao et al. 2013). In
particular, the metal-sensitive probe Newport Green has been successfully used
to investigate Ni and Zn bioaccumulation in river biofilm (Wuertz et al. 2000;
Lawrence et al. 2019). The combination of different probes can be particularly
useful to compare localization of various metals on microbial aggregates (Hao
et al. 2016). STXM, which uses near-edge X-ray absorption fine structure as the
contrast mechanism, provides spatially resolved quantitative information on the
distribution of elements, macromolecules, and redox states in the biofilm matrix
(Lawrence et al. 2003; Behrens et al. 2012). According to Lawrence et al. (2016),
STXM is “capable of mapping the biochemical composition of bacteria and biofilms
at the subcellular scale [. . .] as well as speciation of metals.” Comprehensive reviews
on STXM applied to biofilms have found that it holds relevancy for investigating
Role of Biofilms in Contaminant Bioaccumulation and Trophic Transfer in Aquatic. . .
123
analysis of the metal in the whole sediment (e.g., Farag et al. 2007; Kohušová et al.
2011) or leaf litter (e.g., Sridhar et al. 2008; Schaller et al. 2011), including attached
biofilms. The analytical procedures commonly used include a first step of extraction
with aqua region or nitric acid, for instance, on fresh or dried sediment/leaf litter.
Then, metal concentrations in the extracts are measured by conventional analytical
methods (ICP-OES, ICP-MS, or AAS). To date, and to the best of our knowledge, no
study has been conducted to separate and specifically assess metal concentrations in
microbial communities from sediment or leaf litter.
Besides traditional fractionation and extraction methods, imaging techniques
have been developed to investigate interactions between metals and biofilms and
to visualize metals within the biofilm structure. Analytical electron microscopy
techniques such as transmission electron microscopy (TEM), often coupled with
energy-dispersive X-ray spectrometry, have been used to identify metal
bioaccumulation in microbial biofilms, in particular in bioremediation studies in
which biofilms are used as a sink to accumulate metals from contaminated waters
(Mattila et al. 1997; Miller et al. 2012). For example, this technique allowed Vilchez
et al. (2011) to show that Cr(III) bioaccumulated in the EPS matrix of microbial
biofilms, while Pb(II) was detected in both the EPS matrix and the microbial biofilm
cells. In order to determine macromolecules and metals composition in EPS from
microbial biofilms, TEM can also be coupled to electron energy loss spectrometry,
which is a complex technique for measuring atomic composition and chemical
binding and speciation, even for lower elements (C, O). The main drawback of
TEM is that sample preparation often requires dehydration, creating artifacts such as
particle shrinkage or aggregation (Dynes et al. 2006a). Furthermore, the high energy
of TEM causes radiation damage in biological samples, which leads to spectral
distortions, making high-resolution mapping difficult (Hitchcock et al. 2008).
Other imaging techniques such as confocal laser scanning microscopy (CLSM)
and scanning transmission X-ray microscopy (STXM) are especially well suited for
biofilm studies as they can be applied to fully hydrated biological materials and
reduce radiation damage (see Neu et al. 2010 for a comparative review on those
techniques applied to biofilms). In CLSM, metal binding to specific fluorescent
probes allows detection and localization of those contaminants within biofilms (for
a description of the different probes available, see the review by Hao et al. 2013). In
particular, the metal-sensitive probe Newport Green has been successfully used
to investigate Ni and Zn bioaccumulation in river biofilm (Wuertz et al. 2000;
Lawrence et al. 2019). The combination of different probes can be particularly
useful to compare localization of various metals on microbial aggregates (Hao
et al. 2016). STXM, which uses near-edge X-ray absorption fine structure as the
contrast mechanism, provides spatially resolved quantitative information on the
distribution of elements, macromolecules, and redox states in the biofilm matrix
(Lawrence et al. 2003; Behrens et al. 2012). According to Lawrence et al. (2016),
STXM is “capable of mapping the biochemical composition of bacteria and biofilms
at the subcellular scale [. . .] as well as speciation of metals.” Comprehensive reviews
on STXM applied to biofilms have found that it holds relevancy for investigating
Role of Biofilms in Contaminant Bioaccumulation and Trophic Transfer in Aquatic. . .
123