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微生物异化铁还原再氧化过程中活性氧的产生及其机理研究
Alternative TitleMechanism of the production of reactive oxygen species during microbial dissimilatory iron reduction and reoxidation process es
韩瑞霞
Subtype博士
Thesis Advisor张淑贞
2020-06
Degree Grantor中国科学院生态环境研究中心
Place of Conferral北京
Degree Name理学博士
Degree Discipline环境科学
Keyword异化铁还原 再氧化, 活性氧 奥奈达 希瓦氏菌 Mr 1 ,铁 氧化物 土 壤 d Issimilatory i Ron r Eduction And Reo Xidation, Reactive Oxygen Species , Shewanella Oneidensis Mr 1, i Ron ( o Xyhydr)o Xides, s Oil s
Abstract

    异化铁还原菌在土壤 和底泥 环境中广泛存在,厌氧条件下能氧化电子供体 并偶联胞外不溶性三价铁 Fe(II I 还原 生成亚铁( Fe(II) II))),进入好氧环境时 Fe(II)被氧化 并 伴随 活性氧 的产生 。环 境 介 质例如 土壤 和沉积物 中 富 含 铁氧化物 矿物因此 铁的氧化还原转化 是一重要的环境过程, 对碳、氮 和 磷等元素的生物地球化学循环以及污染物的 转化与 迁移 都会产生 重要的 影响 。因此, 微生物介导的 异化铁还原 再氧化过程也成为近 期的 研究热点之一。本研究以奥奈达希瓦氏菌M R 1 S hewanella oneidensis MR 1 简称 MR 1 为模式微生物, 分别以不同类型铁氧化物 、 不同晶面 暴露 的赤铁矿以及典型土壤为对象 ,系统研究了不同体系在 微生物还原 再 氧化过程中 铁还原、矿物转化以及活性氧生成 的 过程 ,旨在全面 揭示铁氧化物的 微生 物 还 原 再 氧化过程中羟基自由基 (· O H 的生成途径与机制,并 从 铁氧化物表面精细结构 角度 阐明 矿物结构 在 微生物异化铁还原 再氧化过程 中的作用 最终探讨实际 土壤 的 异化铁还原 再氧化 过程中 · O H 产生的关键控制因素 。
    分子探针法是·OH 定量分析 使用 最广泛 的方法,但是在复杂异相介质中利用 该方法 对· OH 进行定量分析还面临很多问题, 例 如探针分子的特异性、基质的干扰、矿物的吸附等。 因此 研究 首先 比较 了 目前常用 探针 分子 对苯二甲酸二钠( TPA 、 苯甲酸钠( BA )和 1,2 苯并芘喃酮 C OU 在检测 复 杂矿 物 微生物 体系中 · O H 的可行 性, 优化了定量方法 。 结果 表明 COU 对· OH 的响应灵敏度、响应线性和线性范围均优于 TPA 和 BA COU 的 羟基化 产物 7 羟基 1,2 苯并芘喃酮 7 hCOU 的检测限 也 低 于 TPA 和 BA 的 羟基化 产物 2 羟基对苯二甲酸 h TPA 和 2 羟基苯甲酸 S A 。 更为关键 的是 h TPA 和 S A 均 会被 水铁矿 大量吸附 最大吸附量分别为 2.49 和 2.09 mg/g 导致 测定结果 偏低, 因此 不适用于矿物特别是铁氧化物存在 时· O H 的测定 。 COU 的 羟基化 产物 7 hCOU 不 存在 矿物吸附 C OU 和· O H 的反应可在 1 5 m in 内完成 且 羟基化 产 物 7 hCOU 性质稳定 ,具有良好的 准确性和 重现性 。 综 上 所述, 本 研究发现 COU 更适合 作为定量 检测 复杂 异相 体系中 · OH 的探针分子 并被应用 于 后续研究中· OH 的 定量分析 。
    为阐明铁氧化物 的 微生物还原 再 氧化循环 过程 中· O H 的产生 途径与 机制,本研究利用 M R 1 与具有不同 结构的典型铁氧化物 研究 了 还原 氧化循环过程中铁还原、矿物转化和活性氧产生 的 过程 。 本研究 首次发现 MR 1 能 直接产生胞外超氧阴离子, 并且 通过 不依赖于外源铁 的途径进一步生成 · O H 。但 是· OH 产生的主要途径为 微生物异化还原 铁氧 化物 形成 F e(II) 矿物或吸附态 F e(II) II),其 在氧化过程中产生超氧 自由基 和 过氧化氢 ,进一步 通过类芬顿 反应 生 成 · OH 。 进而提出了微生物异化铁还原 再氧化循环过程中不 依赖 于外源铁 和 依 赖 于外源铁的· OH 产生机制,表明微生物 同时 参与了厌氧条件下 铁还原和氧化 产生 活性氧两个过程。 研究还发现 不同 类型 铁氧化物 被微生物 异化还原 产生 F e(II) 的 能力和F e(II) 再 氧化过程中产生 · OH 的 能力均存在差异,初始矿物结构以及转化过程中形成的二次矿物均 影响 · OH 的 产生 效率 。
    为揭示铁氧化物表面 结构 对 异化铁还原 再 氧化过程 中 · OH 产生 效率 的影响 考察了 { 和 100} 暴露 晶面 赤铁矿的微生物 异化铁还原 再氧化过程。 结果表明 两种 暴露晶面赤铁矿经过微生物介导的还原 氧化循环后形成相同 种类 的二次矿物。 随着还原 氧化循环 次数的增加 赤铁矿的还原程度不断增加, 表明 循环过程中产生的二次矿物更易被 MR 1 还原。 比表面积 归一化 的 Fe(II) 产率 为{ 晶面 大 于 { 晶面, 这 主要 由 不同 暴露 晶面铁原子 对 微生物 的可接近度存在 差异 所致 。 但是氧化过程中比表面积归一化或 Fe ( 归一化的 活性氧 产量大小却相反 通过理论计算发现铁氧化物表 面不同的原 子排布决定 了其表面 O 2分子还原反应的活化 能垒差异,从而影响 不同表面活性氧的产生效率,揭示了不同类型铁氧化物在微生物还原 再 氧化过程中· O H 产生效率 差异 的 微观机理 。
    为明确 真实环境介质中 是否存在这一环境 过程,进一步研究了多种不同类型土壤 的 微生物 异化还原 再氧化循环 。 结果发现在此过程中持续产生 Fe(II)和 · OH ,表明土壤的微生物还原 再 氧化 是 · OH 的 自然 来 源 之一 。微生物还原产生的 Fe(II) 量和氧化过程中 产生的 · OH 量均 不取决于 土壤总含铁量,而与土壤中游离态铁含量呈显著正相关 关 系 且 去除 游离态铁的土壤在还原 氧化过程中 · OH 的 产量显著降低, 表明 游离 态铁 是土壤在异化铁还原 再氧化过程中微生物利用和 · OH 产生 的 主要 贡献者 ,因此可通过游离态铁含量评估和预测土壤在异化铁还原 再氧化过程中产生 · OH 的潜力。此外,研究发现细颗粒中的铁更容易被微生物还原且在异化还原 再 氧化过程中产生 · OH 的效率更高,表明细颗粒对土壤的 异化铁还原 再氧化过程中 · OH 产生的贡献更大。
    综上所述,本论文从复杂矿物微生物体 系 · OH 的定量分析方法、微生物异化铁还原 再氧化过程中 · OH 的 产生 机理以及铁氧化物 类型和表 面精细结构对该过程的影响展开了系统研究,全面揭示了铁氧化物在微生物还原再氧化过程中·OH 的产生途径与界面分子机制。在此基础上,进一步研究了不同类型土壤在微生物异化铁还原 再氧化过程中 · OH 的产生 ,明确了土壤中 · OH 产生的关键因素。研究 为全面认识微生物介导的环境氧化还原界面活性氧的产生以及 由此引起的污染物或有机质界面转化反应提供 了 一定 的 理论基础。

Other Abstract

     Dissimilatory iron r educing bacteria are widespread in soils and sed iments ,which can utilize anaerobic respiration to produce electrons and then transfer them to insoluble ferric iron (Fe(III)) in the environmental matrices through direct contact or electron shuttles, leading to the generation of ferrous iron (Fe(II)). When converting to an aerobic environment, Fe(II) is easily oxidized to Fe(III), accompanied by the spontaneous production of reactive oxygen species ( affecting the biogeochemical cycles of nutrient eleme nts suc h as carbon, nitrogen and phosphorus,and transformation of a wide variety of inorganic and organic contaminants in the environment as consequences Therefore, there is a considerable effort underway to unders tand the microbial dissimilatory iron r eductio n and re oxidation process es . In this study , Shewanella oneidensis MR 1 (hereafter refer to as MR 1), a typical iron reducing bacteria was employed and iron (oxyhydr)oxides with different
mineralogical phases he matite with different exposed facets an d repre sentative soils were used to investigate the reduction of iron, transformation of iron (oxyhydr)oxides and production of reactive oxygen species during the microbial dissimilatory iron reduction and re oxidat ion process es . The aim s of the present stud y were to fully reveal the pathway s and mechan ism s of the hydroxyl radical ( (•OH) production in the microbial dissimilatory iron reduction and re oxidation proces s e s , to clarify the effects of mineral structure s of iron oxides on the proces s es at the fine structure level, and to expl ore the key factors which aff e ct the production of •OH in soils in the microbially mediated redox processes.
    In order to select the suitable probe for the quantification of •OH in heterogeneous systems , three common mole cular probes , including disodium tereph thalate (TPA), so dium benzoate (BA) and coumarin (COU) were assessed. The results showed that COU was much better than TPA and BA in terms of sensitivity,linearity and c o ncentration range responded to •OH , and 7 hyd roxycoumarin 7 hCOU), the transformati on prod uct of COU , ha d lower limit of detection than those of 2 hydroxyterephthalic acid ( and salicylic acid ( SA), the corresponding transformation products of TPA a n d BA , respectively . Most importantly, hTPA an d SA were observed to be adsorbed serio usly on ferrihydr ite with the maximum adsorption capacities of 2.49 and 2.09 mg/g, respectively , which resulted in the low measuring results and made them unsuitable for the us e in heterogeneous systems . Whereas there was no adsorption of 7 hCOU on fe rrihyd rite an d therefo re detection was not influenced in the heterogeneous systems . Reaction between COU and •OH could be finished within 15 min with the transformation pr oduct 7 hCOU having a high stability . Further, the method showed high accuracy and reprod ucibili ty . Based on all these we can conclude that COU is a promising candidate as a molecular probe for the det ermi na tion of •OH produced in heterogeneous systems a nd was therefore adopted in the following studies.
    Iron (oxyhydr)oxides with different mineralogical phases and MR-1 were employed to explore the pathway and mechanism of •OH production during the microbially mediated redox cycles. This study for the first time obs erved the direct production of extracellular superoxide by MR 1, which further produ ced •OH through the exogenetic iron independent pathway. Howeve r , in microbe iron (oxyhydr)oxide systems, the main pathway for •OH production was through Fenton-like reaction, in which superoxide and hydrogen peroxide were produced firstly upon the oxidation of the insoluble Fe(II) formed by microbial dissimilatory reduction. The amounts of Fe(II) and •OH produced in the microbially mediated iron redox cycles were different among the iron (oxyhydr)oxides with different mineralogical phases. Production of •O H was determined by both the mineralogical properties of iron (oxyhydr)oxides and the secondary minerals formed as well.
    To further reveal the influence of surface structu re of iron oxides on •OH production, hematite nanoplates with {001} face t s and hematite nanorods wit h {100} face t s were used to inve stigate the f acet mediated microbial dissimilatory iron redox processes . The results showed that after redox cycles hematite nanoplates and nanorods formed the same ki nd s of secondary minerals. With the increase of redox cycles, the reduction extent i ncrease d for both the hematite s , suggesting that the secondary minerals formed were more liable to be reduce d by MR 1. The production of Fe(II) normalized by specific surfa ce area by hematite nanoplates was higher than that by he matite nanorods , which was ascribed to the diff er ent acce ssibili ty of iron atoms with different exposed facets to microorganisms However, the production of ROS normalized by specific surface area or content of Fe(II) by nano rod s was higher than that by na no plate s Theoretical calcula tion results showed that the atomic arrangement on the surface of iron oxides controlled the activation energy barrier of oxygen reduction reaction and therefore etermined the production efficiency of ROS on different surfaces . This study revealed the key factor determining the difference in the prod uction of •OH by different iron (oxyhydr)oxides during the microbially mediated reduction and re oxidation processes.
    In order to explore the microbial dissimilatory iron redox proc ess es in the environment, a vari ety of soils were employe d to investiga te the reduction of iron and the production of ROS during microbially mediated iron reduction and reoxidation processes . The results showed that Fe(II) and •OH were produced sustainably in the redox cycles, indicating t hat microbially mediate d redox process es contribute s to the production of •OH in the nature . The production of Fe(II) under anaerobic conditions and the yield of •OH under aerobic conditions were unrelated to the soil iron content .Whereas both of them w ere positively correlated to the conten t of fr ee iron (oxyhydr)oxides (extracted with sodium dithionite A fter removing the free iron (oxyhydr)oxides from soils, the yields of •OH decreased significantly during redox processes , indicating that free iron (ox yhydr)oxides were the main components utilized by microorganisms to produce Fe(II) and further •OH in the microbially mediated redox processes . Therefore the content of free iron (oxyhydr)oxides can be used to ev aluate an d predict the potential of •OH produ ction in soils during microbial ly media ted red uction and re oxidation process es In addition soil fractions with smaller particle size were found to have higher production s of Fe(II) and •OH during the redox proc ess es , s uggesti ng that they have greater contri butions to the production of •OH than t he coar se fractions. 
    In conclusion,the method for the quantification of •OH in heterogeneous  microbe mineral systems, pathway for the pr oducti on of •OH during the microbially mediated iron reduction and re oxidation process es , a nd the effects of mineralogical phases and surface fine structure of iron (oxyhydr)oxides on the microbially mediated iron redox process es were investigated in this dissertation The findings reveal ed the pathway an d interfacial molecular mechanism for the pro duction of •OH during the microbially m ediated iron reduction and re oxidation process es T he production of •OH in different types of soils during the microbial ly mediated reduction and reoxidation proce ss es was fu rther studied, and the key determinants for t he production of •OH were identified. T hese fi ndings contribute substantially to our understanding of the generation mechanism of ROS during the microbially m ediated iron reduction and re oxidation pro cess es and will be hel p ful to further understand the tra nsformation of pollutants or organic ma tters a t the redox interface s in the environment.

Pages124
Document Type学位论文
Identifierhttp://ir.rcees.ac.cn/handle/311016/43575
Collection环境化学与生态毒理学国家重点实验室
Recommended Citation
GB/T 7714
韩瑞霞. 微生物异化铁还原再氧化过程中活性氧的产生及其机理研究[D]. 北京. 中国科学院生态环境研究中心,2020.
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