重金属铅(Pb)污染问题由来已久，污染范围广且具有高生物毒性，对生态环境及人体健康构成严重威胁。由于Pb的不可降解性，利用功能微生物进行Pb吸附及固化稳定化，降低其生物毒性的修复思路越来越受到研究者的关注。值得注意的是，微生物修复技术往往受制于菌株重金属耐受性、去除效率等，因此在关注Pb去除效率的同时，菌株对Pb的形态转化及其耐受解毒机制的研究探索十分必要。本文以草酸青霉SL2(Penicillium oxalicum SL2)为研究对象，综合利用电子成像技术、光谱技术、同步辐射技术、蛋白组学及代谢组学联合分析等，进行重金属Pb的微生物修复研究。主要研究内容包括：Pb²⁺耐受性及吸附累积规律；Pb²⁺在P.oxalicum SL2作用下的微生物结晶及形态转化机制研究；Pb²⁺胁迫下P.oxalicum SL2的蛋白组学和代谢组学响应机制。主要研究结果如下： (1)弄清P.oxalicum SL2对Pb²⁺的耐受能力及Pb吸附累积规律。P.oxalicum SL2对Pb²⁺的最低抑制浓度(MIC)值为2500-2600 ㎎/L，半数有效浓度(EC₅₀值)为607.55㎎/L，具有较好的Pb耐受性。在Pb²⁺初始浓度为2500㎎/L，培养时间7d的条件下，最高Pb²⁺去除率为155.6±8.0㎎/g dw，且去除方式以胞外吸附为主。拟二级动力学方程和Freundlich等温吸附方程可分别较好的拟合P. oxalicum SL2对Pb²⁺的吸附动力学及等温吸附过程，限速步骤为化学吸附，具备较高的吸附容量，与Pb的亲和力较高，属于优惠型吸附。傅里叶红外表面基团分析显示，Pb²⁺胁迫下P.oxalicum SL2细胞表面多糖、糖蛋白、可溶性蛋白及磷脂大分子的含量升高，表面羧酸盐含量变化较大，表面多肽含量同样上升，部分解释了 P.oxalicum SL2菌丝体与Pb²⁺亲和力较高且吸附容量大的原因。 (2)阐明P.oxalicum SL2的Pb²⁺形态转化机制。在P. oxalicum SL2的Pb²⁺固定化过程中，通过光学显微镜及扫描电子显微镜-能谱(SEM-EDS)分析，发现菌丝体胞外形成了纳米级和微米级的含Pb次生矿物。进一步利用透射电镜-能谱分析(TEM-EDS)方法，确定了Pb可渗透到P.oxalicum SL2胞内，并形成含Pb复合物。为更深入解P.oxalicum SL2中Pb的形态转化机制，使用了X-射线吸收近边结构(XANES)技术，结果显示菌丝体中Pb形态主要为草酸铅、柠檬酸铅、磷酸氢铅及谷胱甘肽铅类似物。为进一步验证该结果的可信性，对胞内谷胱甘肽(GSH)及氧化型谷胱甘肽(GSSG)含量进行检测，发现Pb²⁺胁迫下胞内GSH和GSH/GSSG值显著提高，尤其是高浓度Pb²⁺处理组，GSH及GSSG含量显著提高。另外，通过离子色谱检测发现在高浓度Pb²⁺胁迫下柠檬酸分泌量显著增加，磷酸氢根含量随时间变化不断升高，而中低浓度Pb²⁺处理可显著提高草酸分泌量。胞内磷酸酶(AKP，ACP)含量在Pb²⁺处理下同样出现显著变化。判断GSH合成、有机酸分泌及有机磷水解作用的加强可能是P.oxalicum SL2进行Pb形态转化，抵御Pb²⁺生物毒性的重要手段。 (3)揭示P.oxalicum SL2在Pb²⁺胁迫下的蛋白质组响应机制。糖分解代谢与能量合成方面，P.oxalicum SL2在Pb²⁺胁迫下海藻糖通路被激活，进而打开糖酵解通路，提高了果糖二磷酸醛缩酶和磷酸甘油酸变位酶活性，为Pb²⁺胁迫下的细胞提供额外能量；氨基酸合成与信号传导方面，通过上调结合类、抗氧化类、分子伴侣及DNA损伤修复相关蛋白的表达，提高细胞抗逆性；抗氧化应激及防御机制方面，可通过几丁质合成通路的激活建立了第一道胞外防御机制，并促进胞内半胱氨酸合成相关蛋白及谷胱甘肽S-转移酶的表达，从而建立胞内Pb螯合解毒的第二道抗氧化应激机制，第三道防御机制则是通过转运蛋白的过表达来促进Pb螯合物的区室化或泵出。 (4)明确P.oxalicum SL2在Pb²⁺胁迫下的代谢组响应机制。通过非靶向代谢组学的鉴定分析，发现cAMP信号通路在应对Pb²⁺生物毒性过程中发挥重要作用，主要通过GPCR结合相关的代谢物实现下游信号通路的调控，并进一步证实了蛋白组中几丁质合成通路的激活；脯氨酸、组氨酸及谷氨酰胺的合成及其所在的ABC转运通路对于抵抗Pb²⁺毒性十分重要；TCA循环的激活可为该菌株抵抗Pb²⁺毒性提供更有效的物质及能量供应机制，包括柠檬酸分泌的显著增加，并再次验证了GSH系统在解毒过程中的关键作用；P. oxalicum SL2可通过糖酵解通路的激活抵抗Pb²⁺毒性并获得额外能量，这与蛋白组结果相符；此外，Pb²⁺胁迫下细胞分裂活性及DNA损伤修复能力增强。 关键词：铅；草酸青霉SL2；生物固定化；形态转化；谷胱甘肽；有机酸；蛋白质组学；代谢组学

 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 Pb²⁺ 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 ㎎/L, and the medium effective concentration (EC50) was 607.55 ㎎/L, which showing good Pb tolerance. When the initial concentration of Pb²⁺ was 2500 ㎎/L and the incubation time was 7 days, the highest Pb²⁺removal rate was 155.6 ± 8.0 ㎎/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 adsoiption with higher adsoiption capacity and appetency with Pb, which belongs to preferential adsoiption. 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. Keywords: Lead; Penicillium oxalicum SL2; bioimmobilization; speciation transformation; glutathione; organic acids; proteomics; metabonomics