Sorption and Distribution of Copper in Unsaturated Pseudomonas putida CZ1 Biofilms as Determined by X-Ray Fluorescence Microscopy

ABSTRACTThe spatial and temporal distribution of metals in unsaturatedPseudomonas putidaCZ1 biofilms was determined using synchrotron-based X-ray fluorescence microscopy (XRF). It was found that Fe, Mn, and Ca were mainly distributed near the air-biofilm interface of a biofilm grown on 40 mM citrate, while there were two Fe-, Mn-, and Ca-rich layers within a biofilm grown on 10 mM citrate. The sorption of copper by biofilm grown in medium containing 10 mM citrate was rapid, with copper being found throughout the biofilm after only 1 h of exposure. Copper initially colocalized with Fe and Mn element layers in the biofilm and then precipitated in a 40-μm-thick layer near the air-biofilm interface when exposed for 12 h. Cu K-edge X-ray absorption near edge structure (XANES) analysis revealed that Cu was primarily bound with citrate within the biofilm, and the precipitate formed in the biofilm exposed to copper for 12 h was most similar to copper phosphate. LIVE/DEAD staining revealed that cells at the biofilm-membrane interface were mostly alive even when the copper concentration reached 80.5 mg copper g−1biomass. This suggests that the biofilm matrix provided significant protection for cells in this area. These results significantly improve our understanding of metal acquisition, transportation, and immobilization in unsaturated biofilm systems.