Molecular Mechanisms Of Heavy Metals Tolerance And Accumulation In Unsaturated Pseudomonas Putida CZ1Biofilm

Heavy metal pollution in soil is one of the global environmental issues. More than99%of microbes live as biofilm, which would paly more important role in the species, bioavailability and transportation processes of heavy metals in soil compared with plan tonic microbes. In the present study, a high heavy metal-resistant rhizobacterium Pseudomonas putida CZ1was studied. The macromolecular composition, surface morphology, spatial structure, metal resistance and accumulation, temporal and spatial distribution of metal elements, as well as the Cu speciation of unsaturated Pseudomonas putida CZ1biofilm were investigated using atomic force microscope (AFM), confocal laser scanning microscope (CLSM), transmission electron microscopy (TEM), X-ray absorption spectroscopy (XAS), X-ray fluorescence microprobe (XRF) combined with conventional physical-chemical methods. These results illustrated the molecular mechanisms of metal resistance, accumulation and transportation within the unsaturated biofilms, which could provide theory basis for risk evaluation and bioremediation of heavy metal in contaminated soils.It was found that the protein, carbohydrate and DNA in the cellular and extracellular fractions of biofilm were regulated by nutritional factors, while the extracellular polymeric substances (EPS) were more easily influenced. Low pH, high temperature and appropriate osmotic stress (120mM NaCl) distinctly stimulated EPS production, and the main component enhanced was extracellular protein. With the aging of biofilm, the surfaces as well as the valleys among cells were studded with15-50nm spheroid-like EPS. Cells in the biofilm adhere tightly together to maintain a hydrated microenvironment under high osmotic (328mM NaCl) stress. These results indicated the variations of EPS composition and the cooperation of cells in biofilms is important for the survival of Pseudomonas putida CZ1from environmental stresses in unsaturated environments such as rhizosphere.P. putida CZ1biofilm has high resistant and accumulation capacity for Cu, Zn, Pb, Fe, Mn, and Ni. It was found that biofilm cultures were2to8times more resistant to Cu and Zn than the planktonic bacteria, and bacterial cells in the biofilm were killed by metals only for long-term exposure. Furtherly, AFM, SEM and CLSM analyses revealed that the spatial structure as well as the EPS of biofilm was responsible for the high metal resistance of biofilm.The spatial and temporal distribution of metals in unsaturated Pseudomonas putida CZ1biofilms was determined using synchrotron-based X-ray fluorescence microscopy (XRF). It was found that Fe, Mn and Ca were primarily located near the air-biofilm interface of biofilms. The sorption of copper by biofilm was rapid, with copper being found throughout the biofilm after only1h of exposure. Copper initially colocalized with Fe and Mn element layers in the biofilm, and then precipitated as copper phosphate in a40μm thick layer near the air-biofilm interface when exposed for12h. The results indicate the heterogeneity of metal distribution within the biofilm is one of the most important mechanisms for high metal resistance and accumulation.Within the biofilm,60-67%of copper were located in the extracellular fraction of biofilms, with44.7-42.3%in the capsular EPS. Enhanced production of cellular proteins, extracellular DNA and alginate were found for Cu stressed biofilm. Additionally, with Cu treatment, the variations of absorption band of amide I,>P=O phosphodiester functional groups and carboxylate group in the FTIR spectra of cells and EPS were observed as well. These results indicated the important role of cellular protein, extracellular DNA and alginate in Cu binding of biofilm. Moreover, XAFS study furtherly revealed that Cu was primarilly bound with hydrosulfide, phosphate and carboxyl like ligands within the cell, cell wall and extracellular polymeric matrix, respectively.Extracellular DNA was also found to play a role in Cu binding. It was found that the composition of extracellular DNA was distinctly changed for Cu stress as revealed by RAPD analyses, and Cu was bound with oxygen ligands in the extracellular DNA with less oxygen and shorter radial distance compared with cellular DNA.