Small changes in Cu redox state and speciation generate large isotope fractionation during adsorption and incorporation of Cu by a phototrophic biofilm

International audience; Despite the importance of phototrophic biofilms in metal cycling in freshwater systems, metal isotope fractionation linked to metal adsorption and uptake by biofilm remains very poorly constrained. Here, copper isotope fractionation by a mature phototrophic biofilm during Cu surface adsorption and incorporation was studied in batch reactor (BR) and open drip flow reactor (DFR) systems at ambient conditions. X-ray Absorption Spectroscopy (both Near Edge Structure, XANES, and Extended Fine Structure, EXAFS) at Cu K-edge of the biofilm after its interaction with Cu in BR experiments allowed characterizing the molecular structure of assimilated Cu and quantifying the degree of Cu II to Cu I reduction linked to Cu assimilation. For both BR and DFR experiments, Cu adsorption caused enrichment in heavy isotope at the surface of the biofilm relative to the aqueous solution, with an apparent enrichment factor for the adsorption process, e 65 Cu ads , of +1.1 ± 0.3‰. In contrast, the isotope enrichment factor during copper incorporation into the biofilm (e 65 Cu inc) was highly variable, ranging from À0.6 to +0.8‰. This variability of the e 65 Cu inc value was likely controlled by Cu cellular uptake via different transport pathways resulting in contrasting fractionation. Specifically, the Cu II storage induced enrichment in heavy isotope, whereas the toxicity response of the biofilm to Cu exposure resulted in reduction of Cu II to Cu I , thus yielding the biofilm enrichment in light isotope. EXAFS analyses suggested that a major part of the Cu assimilated by the biofilm is bound to 5.1 ± 0.3 oxygen or nitrogen atoms, with a small proportion of Cu linked to sulfur atoms (N S < 0.6) of sulfhydryl groups. XANES analyses showed that the proportion of Cu II vs Cu I , compared to the initial Cu II /Cu I ratio, decreased by 14% after the first hour of reaction and by 6% after 96 h of reaction. The value of e 65 Cu inc of the biofilm exhibited a similar trend over time of exposure. Our study demonstrates the complexity of biological processes associated with live phototrophic biofilms, which produce large and contrasting isotope fractionations following rather small Cu redox and speciation changes during uptake, storage or release of the metal, i.e., favoring heavy isotopes during complexation with carboxylate ligands and light isotopes during reduction of Cu II-O/N to Cu I-sulfhydryl moieties.