Shewanella oneidensis 厭氧狀態下針對吸附於非鐵礦物之二價汞的胞外還原作用;Extracellular reduction of Hg(II) adsorbed onto non-ferrous minerals by Shewanella oneidensis under anaerobic conditions

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Title:	Shewanella oneidensis 厭氧狀態下針對吸附於非鐵礦物之二價汞的胞外還原作用;Extracellular reduction of Hg(II) adsorbed onto non-ferrous minerals by Shewanella oneidensis under anaerobic conditions
Authors:	黃愈掄;Huang, Yu-Lun
Contributors:	環境工程研究所
Keywords:	鐵還原菌;胞外電子傳遞;吸附汞還原;氧化鋁礦;矽酸鹽礦;iron-reducing bacteria;extracellular eletron transfer;sorbed-Hg(II) reduction;γ-Al2O3;montmorillonite
Date:	2022-09-23
Issue Date:	2022-10-04 12:26:00 (UTC+8)
Publisher:	國立中央大學
Abstract:	過往的文獻已記載鐵還原菌可左右汞於厭氧含水層的移動性而造成地下水汞污染事件，但鐵環原菌與低濃度汞之間的互動機制卻仍未清楚。針對此議題，實驗室過去幾年在利用Shewanella oneidensis MR-1作為鐵還原模式菌株探究後發現：1) 在無鐵礦的條件下，Shewanella可利用外膜電子輸送蛋白以及不論是自身分泌或是環境現成的電子穿梭物質，將溶解態的二價汞於胞外還原成元素汞；2) 當鐵礦作為唯一的終端電子受體時，因Shewanella的呼吸作用所生成的吸附型與特定晶格型的亞鐵具有卓越的還原能力，足以使預吸附於鐵礦的二價汞迅速轉化成零價汞，進而增加汞的移動性。然而這些結果尚無法得知該菌對於普遍存在於非鐵礦物表面的二價汞，是否在無亞鐵的協助下依舊得以有效還原該形態的二價汞。有鑑於此，本研究試著模擬在天然含水層環境可預見的吸附於地殼成分含量最多的氧化鋁礦(γ-Al2O3)與矽酸鹽礦(montmorillonite)表層的二價汞的條件下，探討此二價汞“能否”、以及“如何”被Shewanella還原。實驗除使用野生型MR-1外，也包括將調控黃素類物質排出蛋白基因剔除後的變種株Δbfe，和將調控外膜表面多血紅素細胞色素蛋白基因剔除後的變種株ΔmtrC/omcA。實驗結果的確發現吸附在這些礦物上的二價汞可因Shewanella的胞外呼吸作用而還原：從ΔmtrC/omcA的實驗得知MtrC/OmcA蛋白參與胞外電子傳遞的重要性，因移除MtrC/OmcA蛋白的ΔmtrC/omcA不僅失去直接接觸的還原途徑，也失去活化黃素物質的能力，使得該變種菌株無法有效將礦物表面的二價汞還原；從Δbfe的實驗發現該變種依然具有還原吸附汞的能力，表明Shewanella可能主要是藉由直接接觸的方式將該汞還原。至於實驗過程中彼此表面電性相斥的礦物與菌株究竟是如何抵抗靜電斥力、最終靠著直接接觸的方式將汞還原，本研究推論可能是因菌株於異質環境中生成胞外聚合物質(EPS)所致，因EPS不僅具有保護與黏附的功能，且其成分含有多血紅素細胞色素蛋白與黃素類化合物等物質，故可使菌株黏附在礦物上，並讓Δbfe在無黃素物質的協助下，藉由多血紅素細胞色素蛋白以電子躍遷的方式將吸附於礦物上的汞還原。此外，本研究也發現相較於黏土(montmorillonite)，鋁礦不易導電的性質使得鋁礦系統中的二價汞被還原的程度不如黏土系統，意味著不同礦物似乎會影響著胞外電子的二價汞還原力。整體而言，本研究透過實驗進一步了解汞在異質環境中的生地化傳輸與轉化機制，並從結果推論在異質與均質系統中的Shewanella胞外呼吸作用有著不同的使用策略，且會受到環境介質的影響。;Previous literatures have documented that iron-reducing bacteria can control the mobility of mercury in anaerobic aquifers and cause mercury contamination events in groundwater, but the interaction mechanism between iron-reducing bacteria and low-concentration mercury is still unclear. In response to this issue, the laboratory has used Shewanella oneidensis MR-1 as an iron-reducing model strain in the past few years and found that: (1) In the absence of iron mineral, bacteria such as Shewanella can use the outer membrane electron transport protein and whether or not self-secreted or ready-made electron shuttle substance that reduces dissolved Hg(II) to Hg(0) outside the cell; (2) When Shewanella respires iron mineral as the sole electron acceptor, adsorbed ferrous and specific crystal ferrous both have excellent reducing ability will be generated, which can rapidly reduce Hg(II) that adsorbed on iron mineral to Hg(0), thereby increasing the mobility of mercury. Even so, these results still do not know whether the bacteria can effectively reduce Hg(II) in this general form without the assistance of ferrous. In view of this, this study attempts to simulate the condition of Hg(II) adsorbed on the surface of alumina (γ-Al2O3) and clay (montmorillonite) which are predictable in the natural aquifer environment, and to explore whether Shewanella can reduce Hg(II) in this form and how to occur. In addition to using wild-type MR-1, the experiment also included strain Δbfe that deleted the gene of flavin substances transport protein, and strain ΔmtrC/omcA that deleted the gene of the polyheme cytochrome protein on the outer membrane surface. This study results indeed found that Hg(II) adsorbed on these minerals could be reduced by extracellular respiration of Shewanella: from the experiment of ΔmtrC/omcA, the importance of MtrC/OmcA involved in extracellular electron transfer was known, because the removal of MtrC/OmcA. The strain ΔmtrC/omcA not only loses the reduction pathway of direct contact, but also loses the ability to activate flavin substances, so that this mutant can not effectively reduce Hg(II) on the mineral surface; from the experiment of Δbfe, it is found that this mutant still has the ability to reduce Hg(II) on the mineral surface, indicating that Shewanella may reduce Hg(II) by direct contact. As for how the minerals and strains that have negative charge on the surface of each other resist the electrostatic repulsion, and finally reduce Hg(II) by direct contact during the experiment, this study assumes that bacteria may generate extracellular polymeric substances (EPS) in a heterogeneous environment. EPS not only has the functions of protection and adhesion, but also contains many heme cytochrome proteins and flavin substances, which can make the strain adhere to minerals, and make Δbfe reduce Hg(II) by electron hopping through the heme cytochrome protein in the absent of flavin substances. In addition to compare with clay (montmorillonite), this study also found that the low conductivity of alumina makes the reduction of Hg(II) in the alumina system inferior to that in the clay system, which means different minerals seem to affect the ability of extracellular electron to reduce Hg(II). Overall, this study further understands the biogeochemical transport and transformation mechanism of mercury in heterogeneous environments through these experiments. And from the results, it is inferred that extracellular respiration of Shewanella in heterogeneous and homogeneous systems has different utilizing strategies and is affected by environmental media.
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