Surface displayed MerR increases mercury accumulation by green microalga Chlamydomonas reinhardtii.

Mercury is a highly toxic trace metal that can accumulate in aquatic ecosystems and when resent at high concentrations can pose risks to both aquatic life and humans consuming contaminated fish. This research explores the use of the metalloregulatory protein MerR, known for its high affinity and selectivity toward mercury, in a novel application. Through a cell surface engineering approach, MerR was displayed on cells of green alga Chlamydomonas reinhardtii. A hydroxyproline-rich GP1 protein was used as an anchor to construct the engineered strains GP1-MerR that expresses the fluorescent protein mVenus. The surface engineered GP1-MerR strain led up to five folds higher Hg2+ accumulation compared to the WT strain at concentration range from 10-9 to 10-7 M Hg2+. The binding of Hg2+ via MerR was specific and did not get significantly affected by major freshwater water quality variables such as Ca2+ and dissolved organic matter. The presence of other trace metals (Zn2+, Cu2+, Ni2+, Pb2+, Cd2+) in a same concentration range even resulted in 30-40 % increase in the accumulated Hg. Further, the engineered cells also demonstrated the ability to accumulate Hg2+ from the water extracts of the Hg-contaminated sediment samples. These results demonstrate a novel approach utilizing the cell surface display system in C. reinhardtii for its potential application in bioremediation.

Asymmetrical Flow Field-Flow Fractionation Methods for Quantitative Determination and Size Characterization of Thiols and for Mercury Size Speciation Analysis in Organic Matter-Rich Natural Waters.

Asymmetrical flow field-flow fractionation (AF4) efficiently separates various macromolecules and nano-components of natural waters according to their hydrodynamic sizes. The online coupling of AF4 with fluorescence (Fluo) and UV absorbance (UV) detectors (FluoD and UVD, respectively) and inductively coupled plasma-mass spectrometry (ICP-MS) provides multidimensional information. This makes it a powerful tool to characterize and quantify the size distributions of organic and inorganic nano-sized components and their interaction with trace metals. In this study, we developed a method combining thiol labeling by monobromo(trimethylammonio)bimane bromide (qBBr) with AF4-FluoD to determine the size distribution and the quantities of thiols in the macromolecular dissolved organic matter (DOM) present in highly colored DOM-rich water sampled from Shuya River and Lake Onego, Russia. We found that the qBBr-labeled components of DOM (qB-DOM) were of humic type, characterized by a low hydrodynamic size (d h < 2 nm), and have concentrations <0.3 µM. After enrichment with mercury, the complexes formed between the nano-sized components and Hg were analyzed using AF4-ICP-MS. The elution profile of Hg followed the distribution of the UV-absorbing components of DOM, characterized by slightly higher sizes than qB-DOM. Only a small proportion of Hg was associated with the larger-sized components containing Fe and Mn, probably inorganic oxides that were identified in most of the samples from river to lake. The size distribution of the Hg-DOM complexes was enlarged when the concentration of added Hg increased (from 10 to 100 nM). This was explained by the presence of small iron oxides, overlapping the size distribution of Hg-DOM, on which Hg bound to a small proportion. In addition, to provide information on the dispersion of macromolecular thiols in colored DOM-rich natural water, our study also illustrated the potential of AF4-FluoD-UVD-ICP-MS to trace or quantify dynamic changes while Hg binds to the natural nano-colloidal components of surface water.

Species-specific isotope tracking of mercury uptake and transformations by pico-nanoplankton in an eutrophic lake.

The present study aims to explore the bioaccumulation and biotic transformations of inorganic (iHg) and monomethyl mercury (MMHg) by natural pico-nanoplankton community from eutrophic lake Soppen, Switzerland. Pico-nanoplankton encompass mainly bacterioplankton, mycoplankton and phytoplankton groups with size between 0.2 and 20 µm. Species-specific enriched isotope mixture of 199iHg and 201MMHg was used to explore the accumulation, the subcellular distribution and transformations occurring in natural pico-nanoplankton sampled at 2 different depths (6.6 m and 8.3 m). Cyanobacteria, diatoms, cryptophyta, green algae and heterotrophic microorganisms were identified as the major groups of pico-nanoplankton with diatoms prevailing at deeper samples. Results showed that pico-nanoplankton accumulated both iHg and MMHg preferentially in the cell membrane/organelles, despite observed losses. The ratios between the iHg and MMHg concentrations measured in the membrane/organelles and cytosol were comparable for iHg and MMHg. Pico-nanoplankton demethylate added 201MMHg (~4 and 12% per day depending on cellular compartment), although the involved pathways are to further explore. Comparison of the concentrations of 201iHg formed from 201MMHg demethylation in whole system, medium and whole cells showed that 82% of the demethylation was biologically mediated by pico-nanoplankton. No significant methylation of iHg by pico-nanoplankton was observed. The accumulation of iHg and MMHg and the percentage of demethylated MMHg correlated positively with the relative abundance of diatoms and heterotrophic microorganisms in the pico-nanoplankton, the concentrations of TN, Mg2+, NO3-, NO2-, NH4+ and negatively with the concentrations of DOC, K+, Na+, Ca2+, SO42-. Taken together the results of the present field study confirm the role of pico-nanoplankton in Hg bioaccumulation and demethylation, however further research is needed to better understand the underlying mechanisms and interconnection between heterotrophic and autotrophic microorganisms.