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

Abstract

The spatial and temporal distribution of metals in unsaturated Pseudomonas putida CZ1 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-m-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 ؊1 biomass. 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.

Determination of the accumulation, spatial distribution and reduction of Cr in unsaturated Pseudochrobactrum saccharolyticum LY10 biofilms by X-ray fluorescence and absorption methods

“…Biofilms after exposured to Cr for 10 min, 30 min, 1 h, 4 h, 12 h and 24 h were cryoembedded, cryosected, and then scanned in vertical sections using the method of Chen et al [18]. The biofilms exposed for 24 h was further scanned directly in the horizontal area of the entire biofilms.…”

“…Unsaturated biofilms were cultured on the surfaces of Nuclepore polyester membranes as previously described [18,30]. P. saccharolyticum LY10 was incubated on the LB agar plate at 28°C for 24 h in a biochemical incubator.…”

“…Previous studies have focused on the adsorption and distribution of Cu, Zn and other inorganic metals in biofilms in aquatic systems under saturated conditions [17][18][19][20]. For example, Harrison et al [17] reported that a P. aeruginosa ATCC 27853 biofilm had more cell density compared to the free phytoplankton cells, and thus, it could better tolerate the shock loading of high concentrations of heavy metals and maintain the stability of the system.…”

“…Techniques based on synchrotron-derived X-rays such as X-ray fluorescence microprobe (XRF) and X-ray absorption spectroscopy (XAS) have been recently proven to be powerful for the analysis of metals [25][26][27][28][29]. The adsorption and distribution of copper in unsaturated P. putida CZ1 biofilms have also been determined by XRF and K-edge X-ray absorption near-edge structure (XANES) methods [18].…”

“…Thus, spatial distribution of toxic chemicals within the unsaturated biofilms is quite important for clarifying the biogeochemical cycling of Cr and other metals in soil environment [18]. But relatively little is known regarding the spatial distribution and speciation of Cr(III) after reduction in unsaturated systems.…”