(typically cyanobacteria, algae, or fungi). The appropriate method was selected
according to different criteria, such as accuracy, sensitivity, amount of sample
available, and the need or not to identify the location of the contaminant in the
microorganism structure (White and Gadd 1995). The democratization of inductively coupled plasma optical emission and inductively coupled plasma mass spectrometry (ICP-OES and ICP-MS) allowing high-sensitivity analysis of several
elements at the same time led to a large number of studies on Cu, Zn, Pb, and Cd
bioaccumulation in microbial biofilms (e.g., Meylan et al. 2003; Farag et al. 2007;
Bradac et al. 2009, 2010). The bulk of research on metal bioaccumulation in
microbial biofilms was conducted on periphytic biofilms collected in situ on rock
and/or gravel (e.g., Ancion et al. 2010) or on artificial substrates (low-density
polyethylene membranes (Fechner et al. 2012); glass discs (Ivorra et al. 1999);
glass slides (Morin et al. 2008)) beforehand immersed in the water column to
allow biofilm colonization. These artificial substrates were also used in microcosm
experiments to obtain enough biological material to combine metal analysis and
toxicity tests under controlled exposure conditions (Fechner et al. 2011; Kim et al.
2012; Lambert et al. 2012).
To assess total metal concentration in the microbial biomass, hot (100
C) concentrated nitric acid digestion (by using a heating plate or a microwave oven) is
commonly used to extract total metal content from samples that had previously
been oven-dried at 50
C or freeze-dried (e.g., Morin et al. 2008; Fechner et al.
2012). To better discriminate between intracellular and extracellular metal
bioaccumulation in microbial assemblage, biofilms are first flushed with a solution
of ethylenediaminetetraacetic acid (EDTA) at 4 mM during 10 min (e.g., Meylan
et al. 2004; Bradac et al. 2009; Arini et al. 2012; Fabure et al. 2015). This step
removes the metals adsorbed to cell membranes and a fraction of the inorganic
complexes in the biofilm structure (Meylan et al. 2003). The amount of intracellular
metal content in the microbial assemblage is then deduced by analyzing the two
fractions (raw and EDTA-washed) of a sample. To better identify metal (Al, Cu, Zn,
and Pb) site within the EPS matrix and better characterize the exposure of microbial
cells to metals, Aguilera et al. (2008) proposed subsequent extractions and centrifugation steps to separate first the “colloidal fraction” (extracted with distilled water),
then the “capsular fraction” (extracted with NaCl at 80
C, ultrapure water at 30
C,
Dowex at 4
C, or crown ether at 4
C), and finally the cellular debris from microbial
biofilms. Metal content can then be quantified independently in each of the three
collected fractions to determine the amount of intracellular metal (in the cellular
debris) as well as the metal concentration in the EPS matrix (in the colloidal and
capsular fractions). The colloidal fraction includes carbohydrates and proteins that
are loosely bound to microorganisms, whereas the capsular fraction contains tightly
bound compounds. However, Aguilera et al. (2008) showed that no single extraction
method was able to extract all the potential EPS components with the same
efficiency.
There have been several developments to assess metal accumulation and distribution in freshwater periphytic communities, but none in microbial communities
from sediment or from leaf litter. Indeed, studies dealing with metal contamination
122
C. Bonnineau et al.
according to different criteria, such as accuracy, sensitivity, amount of sample
available, and the need or not to identify the location of the contaminant in the
microorganism structure (White and Gadd 1995). The democratization of inductively coupled plasma optical emission and inductively coupled plasma mass spectrometry (ICP-OES and ICP-MS) allowing high-sensitivity analysis of several
elements at the same time led to a large number of studies on Cu, Zn, Pb, and Cd
bioaccumulation in microbial biofilms (e.g., Meylan et al. 2003; Farag et al. 2007;
Bradac et al. 2009, 2010). The bulk of research on metal bioaccumulation in
microbial biofilms was conducted on periphytic biofilms collected in situ on rock
and/or gravel (e.g., Ancion et al. 2010) or on artificial substrates (low-density
polyethylene membranes (Fechner et al. 2012); glass discs (Ivorra et al. 1999);
glass slides (Morin et al. 2008)) beforehand immersed in the water column to
allow biofilm colonization. These artificial substrates were also used in microcosm
experiments to obtain enough biological material to combine metal analysis and
toxicity tests under controlled exposure conditions (Fechner et al. 2011; Kim et al.
2012; Lambert et al. 2012).
To assess total metal concentration in the microbial biomass, hot (100
C) concentrated nitric acid digestion (by using a heating plate or a microwave oven) is
commonly used to extract total metal content from samples that had previously
been oven-dried at 50
C or freeze-dried (e.g., Morin et al. 2008; Fechner et al.
2012). To better discriminate between intracellular and extracellular metal
bioaccumulation in microbial assemblage, biofilms are first flushed with a solution
of ethylenediaminetetraacetic acid (EDTA) at 4 mM during 10 min (e.g., Meylan
et al. 2004; Bradac et al. 2009; Arini et al. 2012; Fabure et al. 2015). This step
removes the metals adsorbed to cell membranes and a fraction of the inorganic
complexes in the biofilm structure (Meylan et al. 2003). The amount of intracellular
metal content in the microbial assemblage is then deduced by analyzing the two
fractions (raw and EDTA-washed) of a sample. To better identify metal (Al, Cu, Zn,
and Pb) site within the EPS matrix and better characterize the exposure of microbial
cells to metals, Aguilera et al. (2008) proposed subsequent extractions and centrifugation steps to separate first the “colloidal fraction” (extracted with distilled water),
then the “capsular fraction” (extracted with NaCl at 80
C, ultrapure water at 30
C,
Dowex at 4
C, or crown ether at 4
C), and finally the cellular debris from microbial
biofilms. Metal content can then be quantified independently in each of the three
collected fractions to determine the amount of intracellular metal (in the cellular
debris) as well as the metal concentration in the EPS matrix (in the colloidal and
capsular fractions). The colloidal fraction includes carbohydrates and proteins that
are loosely bound to microorganisms, whereas the capsular fraction contains tightly
bound compounds. However, Aguilera et al. (2008) showed that no single extraction
method was able to extract all the potential EPS components with the same
efficiency.
There have been several developments to assess metal accumulation and distribution in freshwater periphytic communities, but none in microbial communities
from sediment or from leaf litter. Indeed, studies dealing with metal contamination
122
C. Bonnineau et al.