Penicillium Oxalicum Induced Lead Phosphate Precipitation Affects Cell Metabolism Regulation And Soil Pb(Ⅱ) Immobilization Mechanism

Microbial-induced phosphate（P）precipitation（MIPP）is regarded as a novel and promising approach to bioimmobilize lead（Pb）and enhance Pb stability by adding exogenous P minerals and P-solubilizing microorganisms（PSM）.Nevertheless,few studies discuss the change of Pb biotoxicity and the underlying mechanisms during MIPP process duo to the exogenous phosphate.Meanwhile,the effect and mechanism on MIPP in Pb-contaminated soil induced by PSM are still unclear.In this study,phosphate solubilizing fungi P.oxalicum was used as the model organism to explore its physiological characteristics and changes of intracellular regulation during the MIPP process.Then,it was inoculated to Pb-contaminated soil to explore the potential mechanism of P.oxalicum affecting soil P cycle and changing MIPP process.The main results were as follows:（1）Environmental Pb²⁺can induce the burst of membrane of P.oxalicum cell,leading to the leakage of intracellular substances and cell death.While TCP was solubilized by P.oxalicum,and then Pb²⁺was immobilized during MIPP process,the biotoxicity of Pb²⁺was significantly alleviated.Meanwhile,the growth of P.oxalicum at Pb²⁺stress also can profit from effectively elevation in biomass and antioxidant kinase bioactivity.The significant negative correlation between free Pb²⁺and soluble-P（R=-0.66,p<0.001）suggested the involvement of P radicals in Pb²⁺immobilization.Secondary biomineralization products were mainly Pb-P hydroxypyromorphite(Pb₁₀（PO₄）₆（OH）₂).（2）Using untargeted metabolomic and transcriptomics,we found that Pb²⁺exposure stimulated the deterioration of membrane integrity of P.oxalicum.Intracellular Pb²⁺directly reacted with pyrimidine,boosted the nucleotide metabolism obstruction,and restrained DNA/RNA synthesis.P.oxalicum could produce higher level of gamma-aminobutyric acid（GABA）to enhance the adaptive cellular machineries under Pb²⁺stress.During MIPP-induced Pb mineralization process,TCP improved extracellular Pb mineralization and decreased extracellular Pb²⁺concentrations,consequently alleviating the nucleotide metabolism inhibition and membrane deterioration.Multi-omics analysis suggested that GABA synthesis shunt pathway was inhibited and GABA degradation pathway was stimulated for arginine biosynthesis and TCA cycle after Pb mineralization.（3）The inoculation of P.oxalicum in Pb-contaminated soil remediated by HAP significantly stimulates the bioactivities of soil alkaline/acid phosphatase,and there is a conspicuous negative correlation between available P and available Pb（R=-0.78,p<0.001）,indicated that P.oxalicum stimulated the soil MIPP process.Moreover,the contents of more available P had significantly increased in P.oxalicum inoculation treatments,thus the bioavailability of soil P was greatly improved.Owing to the obvious increase in proportion of R-Pb,the mobility and availability of soil Pb were effectively reduced,indicated that P.oxalicum can achieve more stable and efficient Pb bioimmobilization via induced MIPP process.（4）Using shotgun sequencing,we found that the enhancedαdiversity and the composition of soil P-cycle function genes were all closely associated with available P.Inoculation of P.oxalicum may not directly participated in soil P cycling,but it can directly affect the relative abundances of indigenous PSMs,such as Bradyrhizobium,Mesorhizobium,Niastella,Streptomyces,etc.,to achieve indirectly participation for P cycling.Moreover,the P-cycle functional genes in P.oxalicum inoculation treatments possessed a more complex co-occurrence pattern,implying a more elastic and robust P-cycling network for responding Pb stress.Random forest model showed that genes involved in P transport and starvation response regulation（pst SABC,pho U）,organic P mineralization（pho X,phn A）and inorganic P solubilization（gcd,ppa,ppx）were considered as the important predictors for available P,their abundances increased to varying levels.Structural equation model indicated that P.oxalicum had a significant positive effect on the total abundance of genes involved in inorganic P solubilization and organic P mineralization,while only the former significantly positively affect soil available P.Briefly,we concluded that P.oxalicum could significantly stimulate soil P cycling and drive soil MIPP process enhancement by stimulating inorganic P solubilization potential,thus soil Pb immobilization was enhanced.