3.1. Biofilm Culture Conditions
Culture media, which were composed of Geneva Lake water enriched with Hg, were characterized by a pH value of 8.2 ± 0.2, a DOC concentration of 1.4 ± 0.2 mg L
−1 and a Ca
2+ concentration of 1.14 ± 0.03 mM (
Table S1). The measured trace metal concentrations were low, with a Cu and Zn concentrations of 8.09 ± 0.49 nM and 9.55 ± 0.78 nM, respectively. These levels of the nutrients are in accordance with the mesotrophic, with a low-eutrophic tendency, condition of the Lake Geneva [
31] and was thus appropriate to serve as a culture media, in which the presence of high concentrations of pollutants was not expected. The addition of Hg into the microcosms allowed us to obtain three dissolved culture Hg concentrations, i.e., 11 ± 2 pM, 121 ± 9 pM, and 1454 ± 54 pM dissolved Hg. A factor of approximately 10 was used between each studied concentration, with the first two studied Hg concentrations representing those measured in freshwater lakes, 121 pM being found in rather highly contaminated systems [
32]. After 5 days of cultivation, almost all Hg in solution was depleted (
Figure S1). That phenomenon was possibly due to a combination of adsorption to microcosm walls and biofilm supports, volatilisation under Hg
0 form, and bioaccumulation, which was overcome with the renewing of the culture media or with its Hg spiking.
3.2. Hg Accumulation by Biofilms
The cultivation of biofilms in Hg led to the increase of bioaccumulated Hg (
Figure 1I) as compared with the unexposed control biofilm. Biofilms grown in the control microcosm already contained a natural background of 332 ± 17 pmol g
dw−1 of {THg}. Comparable {THg} was found in biofilms grown in 11 pM Hg (
p = 0.05;
n = 3) whereas at 121 pM Hg and 1454 pM Hg, {THg} was 2.2-times and 25-times higher than the one measured in control biofilms, respectively. The natural {THg} background measured in the present study is in good agreement with those previously measured in biofilms grown in Geneva Lake, in which {THg} was found to vary between 86 and 214 pmol.gdw
-1 depending on the biofilm age [
22]. Measured {THg} in biofilms collected in various Hg-impacted rivers range in a wide range between 269 pmol to 1.7 µmol g
dw−1 [
9]. The extremely high concentration of 1.7 µmol g
dw−1 of {THg} was measured in biofilms collected in the Idrijca River (Slovenia), close to a former Hg mine, where high concentrations of dissolved Hg (272 ng L
−1) were measured [
33]. The present studied biofilms are thus situated at the lower end of Hg bioaccumulation globally measured, suggesting that Hg is most probably not of concern for the water quality of the Geneva lake water surrounding our water sampling site, in agreement with the very low measured dissolved Hg concentrations.
Comparing Hg accumulation between biofilms to infer conclusions on the quality of their ambient water should, however, be taken with caution. Indeed, Hg accumulation is not only dependent on the concentration of Hg in the ambient water but also of their surrounding environmental parameters (substrata, light, current, temperature), water chemical variables (pH and DOC concentration), and composition [
9]. Indeed, taxonomic composition and biofilm thickness were recently shown to modulate Hg uptake kinetics in freshwater biofilms [
22]. Moreover, in contrast with other metals such as Cu, Zn, Pb or Cd, Hg could be further transformed by biofilms and be excreted under its methylated and elemental form. Such processes would decrease accumulated Hg and led to a misinterpretation of accumulated Hg values. Further studies are needed to identify and quantify the extent with which such processes take place in biofilms grown under our experimental conditions.
The measurement of the total accumulated Hg in biofilms included Hg adsorbed to particles and microorganisms, Hg bound to the EPS matrix and Hg intracellularly taken up by biofilms. Applying a rinsing-step with cysteine allowed us to remove the non-specifically bound Hg and to get closer to the actual Hg (intracellular) concentration that would impact the microbial communities. In the present study, {IHg}
n-ext, proxy of the intracellular fraction, measured in the control biofilm represented 39% of {THg} (130 ± 27 pmol g
dw−1). Similar {IHg}
n-ext was observed upon cultivation in 11 pM Hg with the percentage of {IHg}
n-ext being 89% (176 ± 98 pmol g
dw−1). At 121 pM Hg and 1454 pM, {IHg}
n-ext increased 3.9-times (non-significant) and 22.5-times (2.7 ± 1.2 nmol g
dw−1), respectively. Interestingly, the fraction of {IHg}
n-ext as compared to {THg} decreased up to 14% at the higher Hg concentration (that percentage is 65% for biofilms grown in 121 pM Hg). These measured {IHg}
n-ext in control and Hg-cultivated biofilms were found to be comparable with those measured in biofilms grown in ~150 pM Hg for 24 h (between 886 pmol g
dw−1 and 1.6 nmol g
dw−1) [
22] and collected in a chlor-alkali impacted river (between 167 pmol gdw
−1 (control reservoir) and 1 nmol gdw
−1 (highly affected reservoir) [
19]. The decrease of {IHg}
n-ext proportion as compared to {THg} with the increase of Hg in the culture media might be the result of the microbial community response to Hg exposure. Indeed, at the highest Hg concentration, microorganisms might have developed strategies to cope with higher ambient Hg concentration and thus accumulation. They might have regulated their metal transporters to limit Hg uptake [
34]. They might also have formed larger amounts of EPS or modified their EPS synthetized structure to more efficiently sequestered Hg [
35]. Finally, the culture conditions might have changed the microbial species communities with microbes less prone to accumulate Hg (see
Section 3.3). Further research would be needed to better understand the role of each process in the increase of adsorbed Hg in Hg-exposed communities.
No direct relationship between {MeHg}
n-ext and Hg concentrations in the culture media could be established and the maximal {MeHg}
n-ext (19.60 pmol g
dw−1) was found in the control biofilms, representing 5.9% of {THg} (
Figure 1II). A decrease in the fraction of {MeHg}
n-ext as compared to {THg} was observed with the increase of Hg cultivation concentration, with values of 1.6%, 0.29%, and 0.08% in biofilms exposed to 11 pM, 121 pM and 1454 pM, respectively, reflecting the large increase in {THg}. Such low percentage of {MeHg}
n-ext was also found in biofilms grown in Hg-contaminated reservoirs [
19]. That lack of {MeHg}
n-ext increase found in the present study with the increase of culture Hg concentrations contrasts with the large body of evidence reporting methylation occurring in biofilms [
15,
36,
37]. However, comparison is difficult to make since only the total bioaccumulated MeHg (adsorbed + absorbed) as well as the production of MeHg in the exposure media are usually measured.
Overall, our measured concentrations of bioaccumulated Hg were found to be representative of those encountered in natural environments, even at the highest dissolved Hg culture concentrations.