Study On The Bioimmobilization,Speciation Transformation And Molecular Mechanism Of Pb²⁺by Penicillium Oxalicum SL2

The problem of heavy metal lead(Pb)pollution has been a long-standing issue,which is extensive and high biological toxicity,posing a serious threat to the ecological environment and human health.Using functional microorganisms for Pb adsorption and stabilization to reduce the biological toxicity of Pb has been received increasing attention because of the non biodegradability of Pb.Noteworthily,bioremediation is limited by the tolerance and removal efficiency,therefore,it is necessary for strains to explore the speciation transformation and tolerance mechanism of Pb while paying attention to the removal efficiency of Pb.In this paper,Penicillium oxalicum SL2 was used as a research object,comprehensively utilizing electron imaging technology,spectroscopic technology,synchrotron radiation technology,proteomics,and metabolomics analysis to carry out the microbial remediation of heavy metal Pb.The principal research contents include:Pb2+ tolerance and adsorption accumulation law of P.oxalicum SL2;The biomineralization and transformation mechanism of Pb2+ for P.oxalicum SL2;the proteomics response mechanism of P.oxalicum SL2 under PbrU stress;the metabolomics response mechanism of P.oxalicum SL2 under Pb2+ stress.The principal research findings as follow:(1)Find out the tolerance of P.oxalicum SL2 to Pb2+ and the adsorption and accumulation rule of Pb.The minimum inhibitory concentration(MIC)of P.oxalicum SL2 to Pb2+ was 2500-2600 mg/L,and the medium effective concentration(EC50)was 607.55 mg/L,which showing good Pb tolerance.When the initial concentration of Pb2+was 2500 mg/L and the incubation time was 7 days,the highest Pb2+removal rate was 155.6 ± 8.0 mg/g dw,and the mainly removal method was extracellular adsorption.Quasi second order kinetic equation and Freundlich isothermal adsorption equation can be used to fitting the Pb2+ adsorption kinetics and isothermal adsorption process of P.oxalicum SL2 respectively.The rate-limiting step of adsorption was chemical adsorption with higher adsorption capacity and appetency with Pb,which belongs to preferential adsorption.Fourier infrared surface group analysis showed that the content of polysaccharides,glycoproteins,soluble proteins and phospholipid macromolecules on the surface of P.oxalicum SL2 increased under Pb2+ stress.The carboxylate content changed markedly,and the polypeptides content on the surface of P.oxalicum SL2 also increased,which partly explained the higher affinity and adsorption capacity of P.oxalicum SL2 under Pb2+stress.(2)The Pb2+ transformation mechanisms of P.oxalicum SL2 was elucidated.The Pb-containing secondary minerals of nano-scale and micron scale was carried out by optical microscope and scanning electron microscopy-energy spectrum analysis(SEM-EDS)in the process of Pb2+ immobilization using P.oxalicum SL2.And proofing Pb can infiltrate into P.Oxalicum SL2 cells and form Pb-containing compounds using transmission electron microscopy-energy spectrum analysis(TEM-EDS).X-ray absorption near edge structure(XANES)technique based on synchronous radiation technology was utilized to further understand the Pb speciation transformation mechanism of P.oxalicum SL2,the results showed that the form of Pb in mycelium was lead oxalate,lead citrate,lead hydrogen phosphate and lead glutathione analogues.In order to further validate the results,the content of intracellular glutathione(GSH)and oxidized glutathione(GSSG)was detected,which showed that the intracellular GSH and GSH/GSSG value increased significantly under Pb2+ stress,especially in the group treated with high concentration of Pb2+.In addition,The citric acid and hydrogen phosphate secretion increased significantly under Pb2+ stress,and the oxalic acid production was improved significantly in low concentration of Pb2+ treatment by ion chromatography detection.Intracellular phosphatase(AKP,ACP)content also changed significantly under Pb2+ treatment.The above phenomenon indicates that the enhancement of GSH synthesis,organic acid secretion and organophosphorus hydrolysis may be an important means for P.oxalicum SL2 to carry out Pb speciation transformation and resist the Pb-initialed biotoxicity.(3)Reveal the proteomics response mechanism of P.oxalicum SL2 under Pb2+stress.In the aspect of sugar catabolism and energy synthesis,the trehalose pathway of P.oxalicum SL2 was activated under Pb2+ stress,and then the glycolytic pathway was opened up,which increased the activity of fructo-diphosphate aldolase and phosphoglycerate mutase and provide extra energy for cells under Pb2+ stress.For amino acid synthesis and signal transduction,the resistance of cells can be improved by up-regulating the expression of binding proteins,antioxidants,chaperones,and DNA damage repair related proteins.In terms of anti-oxidative stress and defense mechanisms,the first extracellular defense mechanism can be established through the activation of the chitin synthesis pathway and promoted the expression of intracellular cysteine synthesis-related proteins and glutathione S-transferases,to establish the second anti-oxidative stress mechanism of intracellular Pb chelation and detoxification,and the third defense mechanism was to promote compartmentalization or pumping of Pb chelates by over-expression of transporters.(4)Defining the response mechanism of P.oxalicum SL2 protein under the Pb2+stress.Through the identification and analysis of non-targeted metabolomics,it was found that the cAMP signaling pathway played an important role in coping with the biological toxicity of Pb2+,mainly through the use of GPCRs in combination with related metabolites to achieve downstream signal pathway regulation,and further confirmed the mass synthesis pathway activation of the protein group in chitin.The synthesis of valine,histidine and glutamine and the ABC transport pathway in which they are located are very important for resistance to Pb2+ toxicity.The activation of TCA cycle could provide more effective material and energy supply mechanism for Pb2+ toxicity of this strain,and once again verified the key role of GSH system in detoxification process.Biological toxicity of Pb2+ to P.oxalicum SL2 may include respiratory inhibition,and get additional energy through activation of glycolysis pathways;in addition,Pb2+ stress promoted cell division activity and enhanced DNA damage repair.