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References

Task 2 - Laboratory experiments

Task 2b - Partner in charge: F. Jorand (LCPME-Nancy, partner 5)
Evaluation of the mercury bio-methylation of permafrost thaw lakes (EPOC-Bordeaux: Jörg Schäfer, Gérard Blanc, Cécile Bossy, Lionel Dutruch ; LCPME-Nancy: Frédéric Jorand, Hélène Guilloteau, Rémi Guyonneaud)

This sub-task aims at (i) quantifying the Hg methylation/demethylation potential of the different microbial biomass compartments (suspended micro-organisms, sediments and biofilms) and (ii) identifying the main microbial activity involved (i.e. sulfate reducing activities (SRB); iron reducing activities (IRB); and methanogens) and (iii) evaluating the role of both exo-cellular polymers and inorganic compounds of biofilms in Hg net methylation.

Mercury bio-methylation in aquatic environments: It is well known that the microbial biomass of aquatic anoxic environments hosts most of the mercury methylators/demethylators such as sulphate- and FeIIIreducing bacteria or methanogens (Compeau & Bartha, 1985). However, it remains unclear to what extent each group of anaerobic bacteria contributes to the net methylation (Avramescu et al., 2011). Bacteria mostly form mixed communities, e.g. in biofilms and sediments, where several species implied in methylation/demethylation activities may be closely associated. Indeed, co-cultures of bacteria may show much higher methylation potential than pure strains (Pak and Bartha, 1998). Therefore, the capacity of natural microbe communities to methylate/demethylate Hg needs to be appreciated as a whole. Moreover, the organic and inorganic matrixes constituting biofilms, suspended particles or sediments (e.g.: exocellular polymers, minerals resulting from anaerobic respiration, e.g. FeII-FeIII minerals, etc.) may influence the Hg biotransformation either by decreasing Hg bio-availability (Huguet et al., 2010) or by a direct reaction (O'Loughlin et al., 2003; Estrade et al., 2009). Accordingly, microbially-driven methylation/demethylation processes probably depend on community properties and on the type of natural aquatic system hosting them. The increasing intensity and length of permafrost thawing periods, suggest that the related aquatic systems will soon play a key role in arctic methylmercury production. Therefore, it is crucial to quantify and understand the control of each microbial compartment (biofilm, sediment or plankton cells, Huguet et al., 2010) on net Hg methylation, a key process of the Hg cycle in the changing Arctic environment.

Hg bio-methylation experiments: The material used for the incubation experiments will be suspended solids (SS), biofilms and sediments from anaerobic zones at the Whapmagoostui-Kuujjuarapik Site, i.e. where the bio-methylation potential is expected to be the highest. Adapted sampling and experimental protocols will allow maintaining the anaerobic conditions until the end of the incubation periods (Huguet et al., 2010). Biofilms will be collected from both natural surfaces on-site and artificial substrata (glass plates previously implanted during 12 or 18 months). Sediment incubations will be optimised, ideally at different water depth according to results obtained in Task 3 (e.g. different degree of sulfate depletion, AVS-zone, etc.; Schäfer et al. 2010) to focus on specific relations between methylation/demethylation rates and the dominant bacterial activity. Part of the incubation experiments will be performed on-site, whereas other experiments will be run at LCPME (partner 5), whenever useful (reduction of field trips, better infrastructure) or necessary (specific sample pre-reatments, e.g. preconcentration of microorganisms or sterilization). Dissolved and particulate Hg(II) and MMHg measurements will be done at EPOC by species-specific isotope-dilution GC-ICP/MS (e.g. Monperrus et al. 2004; Schäfer et al. 2010).

Briefly, precise quantities of isotopically labelled Hg species (199Hg(II); MM202Hg) will be spiked into hermetic Teflon(R) reactors (triplicate) containing filtered (sterile) and unfiltered lake water with and without suspended matter or biofilms (from natural and/or artificial supports; e.g. glass plates to preserve the biofilm structure; Huguet et al. 2010). After 3 days and 1 week of incubation time (20°C), the methylating/demethylating microbial activity is stopped by acidification and both the concentrations and the experimentally modified isotopic compositions of the present Hg species will be analyzed by speciesspecific isotope-dilution GC-ICP/MS (Schäfer et al. 2010). Additional incubations with previously iced samples (transport and conservation) will be performed in the laboratory by adding i) exo-cellular polymeric substances (EPS) previously extracted (Jorand et al., 1995) and lyophilized from freshly sampled biofilms, ii) biogenerated FeII-bearing minerals (products of the anaerobic respiration of iron obtained from pure iron reducing strains or/and from IRB enrichments), iii) a nutritive solution (diluted R2A medium; Huguet et al. 2010). These assays will focus on the most methylating samples and compared to Hg methylation by pure bacterial strains used as a positive control (e.g. the methylating strain Desulfobulbus propionicus). The nutritional conditions in the reactors will be monitored by dissolved organic carbon (DOC) analyses. The samples will be characterized in terms of cell density (SYBR II green numeration), dry matter and TOC/DOC, SRB and IRB (MPN estimation on specific culture medium; Huguet, PhD thesis, 2009).

Mercury methylating/demethylating potential of microbial communities enriched in either SRB, IRB or methanogens from the biofilms, sediment and SS will be evaluated. Furthermore we will apply specific inhibitors of SRB and methanogen activity (e.g. NaMoO4, bromoethane sulfonate, respectively; e.g. Avramescu et al. 2011), in the presence/absence of FeIII in co-cultures, to evaluate the role of each anaerobic metabolic activity in the net Hg methylation. Note that the incubation of SRB with the HgII, will be done with electron acceptors other than sulfate (or at very low SO4 2- levels) in order to avoid artifacts due to the interaction of HgII with sulfur (Benoit et al., 2001). All incubations will be performed at 20°C supporting microbial activity during the laboratory experiments and reflecting realistic water temperatures during the summer (up to 15-25°C at the Whapmagoostui-Kuujjuarapik Site - Laurion et al., 2010).

Contribution of the partners:
EPOC (partner 6): J. Schäfer: coordination of Hgtotal, MMHg, Hg(II) analyses, species-specific monoisotopic spikes (199Hg(II) and MM202Hg) and incubation; J. Schäfer and G. Blanc: interpretation and publication of results; supervision of graduate (Master) students and a PhD student in collaboration with LCPME; C. Bossy and L. Dutruch: Hgtotal, MMHg, Hg(II) analyses in natural and incubated samples.

LCPME (partner 5): coordination of experimentations with biofilms by F. Jorand. Supervision of PhD (MSER grant) and graduate (Master thesis) students for enrichments with iron reducing and sulfate reducing bacteria, FeII measurements, evaluation of methylation capacity of microbial compartments; interpretation and publication of results. Biofilm characterization and DOC analysis will be realized by the assistance of H. Guilloteau.

Risks: The partners EPOC and LCPME have previously applied, established and adjusted incubation protocols to quantify methylation potentials of biofilms, plancton cells, SS and estuary sediments in equatorial and moderate climate, respectively, within 2 PhD thesis supervised by J. Schäfer, G. Blanc and F. Jorand (Schäfer et al., 2010; Huguet et al., 2010). The main risk may arise from too weak microbial growth on artificial substrata (glass plates) related to arctic conditions. The biofilm supports will be installed for 12 to 24 months to maximize the mass of bacteria collected and iced by the Canadian researchers. Moreover, the collecting of biofilms from natural substrata will be done during the first sampling campaign, providing sufficient material for the optimization and performance of methylation/demethylation experiments, bacteria enrichments/characterization and EPS extraction. Finally, the use of pure bacterial strains isolated from the site (Lab. of Warwick Vincent) or culture collections (banks) will support additional experiments to evaluate potential effects of biofilm extracts on microbial net Hg methylation.