Absorption,Combined Form And Detoxification Mechanism Of Pb In Cladophora

This study aimed to investigate the distribution and combined form of Pb,to explore the detoxification and absorption mechanism of Pb in Cladophora subcells.The experiment used air acetylene flame atomic absorption spectrometry researched subcellular distributions of Pb in the Cladophora under different Pb concentrations（0,0.5,1.0,2.5,5.0,7.5,and 10 mg/L）and different exposure times（0,2,4,6,8,10 and15d）;used transmission electron microscopy（TEM）,and energy dispersive X–ray（EDX）analysis the localization of Pb in subcellular.The chemical form analysis at different concentrations was determined by the stepwise extraction method,and the characterization of Pb（0,0.5,and 5.0 mg/L）at the subcellular was conducted via Fourier-transform infrared spectroscopy（FTIR）,Three dimensional excitation emission matrix spectroscopy（3D–EEM）,protein secondary structure fitting,X–ray photoelectron spectroscopy（XPS）,and Nuclear Magnetic Resonance（NMR）,to speculation the combined form of Pb²⁺in the Cladophora.The experimental results showed that:1.Pb was mainly distributed in the cell wall,followed by organelle fractions under low concentration of Pb（≤2.5 mg/L）,whereas its relative content in soluble fraction was higher than that in organelle when its concentration exceeded 5.0 mg/L,the content of Pb in the cell wall were reached 2757 mg/kg.TEM–EDX revealed that Cladophora could accumulate Pb intracellularly and extracellularly.2.At the first two days,the concentration of Pb in the cell wall increased significantly,soluble fraction increased a little,the organelle changes are relatively small.after 15d,the proportion of Pb in the cell wall,organelle and soluble fraction were 74.8%,9.11%and 16.51%,respectively.Pb²⁺was initially absorbed on the cell wall.Then,it entered the soluble fraction and the organelles.3.The most of the Pb²⁺（37%–76%）were integrated with oxalate and undissolved phosphate,which were important to the detoxification of Cladophora.Pb–polysaccharides（（C₆H₅）–OO–Pb–OH,C–O–Pb,and symmetric Pb–O–Pb）,Pb–organic acid（（C₆H₅）–COO–Pb and（C₆H₅）–P=O–Pb）,and Pb–protein complexes(OH–C₇H₆–CN–Pb–COOH,C₉H₁₀–NH–CN–C=O–Pb,Pb–S–C,and Pb–S)were formed in the cell wall;and produced contains S proteins,such as metallothionein（MT）and glutathione（GSH）,which bound and helped Pb²⁺enter the cell.After entering the soluble fraction,the Pb–organic acid（（C₆H₅）–COO–Pb,（C₆H₅）–O–Pb,and（C₆H₅）–P=O–Pb）and Pb–sulfhydryl compound（Pb–S–C/Pb–S）assumed the most important role in resisting the toxicity of Pb²⁺.Pb²⁺was absorbed in the organelle and formed（C₆H₅）–C–O–Pb and（C₆H₅）–P=O–Pb,and complexed with protein（Pb–C–N）when treated with 5.0 mg/L Pb.4.When Pb concentration was 0.5 mg/L,93.7%of Pb²⁺in the cell wall corresponded to Pb–N–protein.Furthermore,6.3%accounted for Pb–S（such as Pb–C–S）.A large number of Pb–N–protein in the organelle.When Cladophora was treated with a high Pb concentration（5.0 mg/L）,91.5%of Pb²⁺in the cell wall was Pb–N–protein,4.1%was Pb–C–polysaccharide,2.2%was Pb–S containing group,and 2.2%was Pb–O.In the soluble fraction,72.3%,24.2%and 3.5%of Pb²⁺accounted for Pb–N–protein,Pb–organic acid such as Pb–（OOC）₂and Pb–S.Pb²⁺entered the organelles and formed a large number of Pb–N–protein（53.2%）,Pb–N–protein（44.6%）and Pb–S（2.2%）.5.Pb combined with protein,polysaccharides and organic acids of the cell wall is the first step of detoxification of Pb by Cladophora.And the cell wall produced contain S proteins when treated with Pb,which bound and helped Pb²⁺enter the organelle and soluble fraction.Inside the cell,the binding of Pb²⁺with proteins and organic acids were the most important detoxification mechanisms in the soluble fraction.A mount of protein could directly bind with Pb²⁺to achieve detoxification when Cladophora was treated with Pb content.