Process And Mechanism On The Migration And Transformation Of Heavy Metals At Root-soil Interface Mediated By Glomalin-Related Soil Protein

Glomalin-related soil protein（GRSP）is widely distributed in soils.As an important active component of soil organic carbon,GRSP has a long degradation cycle and can easily bind with heavy metals,affecting the conversion and absorption of metal ions by plants.The molecular speciation of heavy metals combined with GRSP in the rhizosphere is important for evaluating the pollution risks associated with the ecological environment and agricultural product safety,as well as the key to remediate the heavy metal-contaminated soil.However,most previous investigations have focused on the macro scale.The interface process of GRSP binding with heavy metals is rarely studied,and the molecular mechanism of the migration and transformation of metal cations mediated by GRSP in plant-soil systems remains unclear.In this study,batch adsorption,plant culture and long-term localization experiments in a lead-zinc mining area were employed to investigate the interactions between GRSP,soil minerals,and heavy metals at molecular scale.This was achieved by combining attenuated total reflection Fourier transform infrared（ATR-FTIR）spectroscopy,isothermal titration calorimetry（ITC）,X-ray photoelectron spectroscopy（XPS）,X-ray absorption fine structure（XAFS,μ-XAFS）analysis,microfocus X-ray fluorescence spectroscopy（μ-XRF）maps,NICA-Donnan model,and 2D correlation spectroscopy（2D-COS）analysis.The molecular species,spatial distribution,and bioavailability of heavy metals mediated by GRSP were also revealed through macroscopic and microscopic techniques.The main results are as follows.（1）The molecular mechanisms of Pb（II）binding to GRSP surface were revealed.The GRSP exhibited a large adsorption capacity for Pb（II）,which was higher than that of most soil minerals and microbes.The ATR-FTIR spectroscopy suggested that GRSP had the surface characteristics of both polysaccharides and proteins,containing carboxyl,hydroxyl,and amino functional groups.The carboxyl and hydroxyl groups in the carbohydrate structure were the main sites for GRSP binding with Pb（II）.The ITC results showed that Pb（II）adsorption on the GRSP surface was an entropy-driven exothermic process.The measured values ofΔH andΔS were 13.52 k J mol^-1and123.83 J mol^-1 K^-1,respectively,indicating that Pb（II）primarily formed stable inner-sphere complexes with oxygen-containing anion functional groups on the GRSP surface.The XPS and XAFS spectroscopy further confirmed that Pb（II）formed Pb–O–C bond with the carboxyl group of GRSP,accounting for approximately 77.7%of the total Pb forms.The monodentate and bidentate mononuclear carboxyl-Pb complexes predominantly contributed to the Pb（II）inner-sphere binding with GRSP.（2）The interaction mechanisms between GRSP and ferrihydrite via co-precipitation（Cor）and adsorption（Ads）were investigated.The Cor composite exhibited greater GRSP binding capacity.The total organic carbon content of the Cor composite was approximately 2.3 times that of the Ads composite under the high GRSP loading.Compared with the Ads composite,the micropores of the Cor composite were obviously fewer,and the change in the surface charge characteristics was more obvious.These results demonstrated the formation of inner-sphere complexes of GRSP and ferrihydrite,which bound more closely to each other under co-precipitation conditions.The FTIR spectra showed that hydroxyl groups on the surface of ferrihydrite participated in the formation of the complex through coordination,while the response of these functional groups was slight under adsorption conditions.The fluorescence spectra（FS）indicated that the binding constant of the Cor composite was 4.96×10⁵ ml mol^-1,and the number of binding sites was 0.93,which were higher than those of the Ads composite.This suggested that the affinity between GRSP and ferrihydrite was stronger under co-precipitation conditions.The XAFS analysis further confirmed the formation of inner-sphere complexes of GRSP and ferrihydrite through Fe–N bonds.The higher Fe–N coordination number of the Cor composite demonstrated that the binding of GRSP and ferrihydrite was closer under co-precipitation conditions.In addition,the transform of Fe–Fe bond length in the second coordination shell of approximately 3.1?and 3.5?indicated that the interaction between GRSP and ferrihydrite could promote the conversion of the Fe–Fe coordination structure from corner-sharing to edge-sharing under the co-precipitation condition.（3）The mechanisms of heavy metals binding with soil mineral-GRSP composites were clarified.The adsorption capacities of GRSP-goethite and GRSP-montmorillonite composites for Pb（II）were higher than those of pure minerals,respectively.The Pb（II）adsorption capacity of the GRSP-montmorillonite composite increased by 25.9%,which was higher than that of the GRSP-goethite composite（17.7%）.In the process of Pb（II）adsorption by the GRSP-montmorillonite composite,the montmorillonite fraction preferentially bound with heavy metal.The ion-exchange interaction contributed approximately 23.5%to Pb（II）adsorption,playing a dominant role.While in the process of Pb（II）adsorption by the GRSP-goethite composite,the GRSP fraction preferentially interacted with metal cations.The complexation of Pb（II）with the oxygen-containing functional groups on the surface of GRSP contributed approximately 31.0%to Pb（II）binding,playing a key role in the interactions of heavy metals and GRSP-goethite composite.（4）The molecular morphology and absorption pathways of Pb in plant roots mediated by GRSP were revealed through traditional chemical detection,TEM-EDS,and XAFS analysis.Compared with the free Pb treatment,the Pb content in plant roots under the GRSP-Pb complex treatment decreased by 94.7%-96.6%,indicating that GRSP could significantly inhibit the bioavailability of Pb,but the GRSP-Pb complex could still be slightly absorbed.The analysis of TEM-EDS and subcellular cell binding state of Pb showed that Pb deposited around the cell wall was dispersed to different parts of the cell.Under GRSP mediation,Pb transformed from the residual state and cell wall-bound state to phases bound to the cytoplasm and hydrophobic protein.Notably,Pb bound to the cell wall decreased by more than 90%.This result suggested that the phytotoxicity of Pb in the GRSP-Pb complex was weakened,thus more likely to enter the tissue cells.The XAFS analysis further showed that the entry of free Pb into plant roots was mainly in the form of binding with small molecular organic acids.Under GRSP mediation,some Pb was absorbed as the molecular phase of the GRSP-Pb complex.The results of uptake inhibition revealed that the main pathway of the GRSP-Pb complex into root tissues was the active transport of transporters,and the regulation of ATP energy synthesis and metabolism.（5）The speciation of Cu,Zn,Pb,and Cd in the phytoremediation of lead-zinc mining soil induced by GRSP were investigated using macroscopic and microscopic techniques.The GRSP content,average weight diameter,and stability of the soil aggregates significantly increased after phytoremediation.The PLS-PM analysis showed that GRSP could inhibit the bioavailability of heavy metals by affecting the particle size distribution and stability of the soil aggregates,which played a dominant role in inhibiting heavy metal availability（52.8%）.The total site density of the acid functional groups in the soil followed an apparent order in the magnitude of artemisia≈ryegrass>silphium>control,suggesting that the soils in the phytoremediation treatments were rich in acid functional groups to sequester heavy metals.The microfocus X-ray absorption/fluorescence spectroscopy analysis indicated evident transformation of the solid-phase speciation of heavy metals in the phytoremediation process.The phases associated with sulfur and clay minerals dominated heavy metal speciation in the phytoremediation treatments.The sulfur-containing sites of GRSP might have great potential for the inhibition of environmental risks closely related to heavy metal migration in soil-plant systems.These findings are valuable for providing a new strategy associated with GRSP application for the remediation of soils polluted by heavy metals.