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KMID : 0545120160260122116
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Journal of Microbiology and Biotechnology
2016 Volume.26 No. 12 p.2116 ~ p.2126
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Spatial Pattern of Copper Phosphate Precipitation Involves in Copper Accumulation and Resistance of Unsaturated Pseudomonas putida CZ1 Biofilm
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Chen Guang Cun
Lin Hui Rong
Chen Xin Cai
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Abstract
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Bacterial biofilms are spatially structured communities that contain bacterial cells with a wide range of physiological states. The spatial distribution and speciation of copper in unsaturated Pseudomonas putida CZ1 biofilms that accumulated 147.0 mg copper per g dry weight were determined by transmission electron microscopy coupled with energy dispersive X-ray analysis, and micro-X-ray fluorescence microscopy coupled with micro-X-ray absorption near edge structure (micro-XANES) analysis. It was found that copper was mainly precipitated in a 75 μm thick layer as copper phosphate in the middle of the biofilm, while there were two living cell layers in the air-biofilm and biofilm-medium interfaces, respectively, distinguished from the copper precipitation layer by two interfaces. The X-ray absorption fine structure analysis of biofilm revealed that species resembling Cu3(PO4)2 predominated in biofilm, followed by Cu-Citrate- and Cu-Glutathione-like species. Further analysis by micro-XANES revealed that 94.4% of copper were Cu3(PO4)2-like species in the layer next to the air interface, whereas the copper species of the layer next to the medium interface were composed by 75.4% Cu3(PO4)2, 10.9% Cu-Citrate-like species, and 11.2% Cu-Glutathione-like species. Thereby, it was suggested that copper was initially acquired by cells in the biofilm-air interface as a citrate complex, and then transported out and bound by out membranes of cells, released from the copper-bound membranes, and finally precipitated with phosphate in the extracellular matrix of the biofilm. These results revealed a clear spatial pattern of copper precipitation in unsaturated biofilm, which was responsible for the high copper tolerance and accumulation of the biofilm.
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KEYWORD
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Pseudomonas putida CZ1, unsaturated biofilm, copper phosphate, spatial pattern, speciation
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