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標題: 結合螢光原位雜交技術與共軛焦雷射顯微鏡於冷卻水管管壁鐵氧化菌附著情形之研究
Investigation of the iron-oxidizing bacteria biofilm formation on the wall of cooling water system by in situ hybridization and confocal laser microscopy.
作者: 鄒文博
關鍵字: 生物腐蝕;microbially influenced corrosion(MIC);螢光原位雜交技術;共軛焦雷射顯微鏡;菌群結構;基因選殖;in situ flourescent hybridization(FISH);confocal laser scanning microscopy(CLSM);community structure;clone library
出版社: 環境工程學系
根據本實驗室先前的研究,由某火力發電廠廠用冷卻水泥濘物所分離出之純菌株Leptothrix sp.(TPH-2)、Denitrifying Fe-oxidizing Bacteria(TPH-7)、Nocardia farcinica(TPH-13-3)等,做為進行批次實驗時純菌株的來源,菌株經由calcofluor white進行胞外聚合物的染色,並以共軛焦雷射顯微鏡觀察,發現隨著菌株所產生胞外聚合物濃度的增加,而有助於鐵氧化菌形成生物膜的結構;藉由觀察碳鋼試片重量的變化,發現微生物的附著會降低碳鋼試片化學腐蝕的速率。此外,本研究為得知鐵氧化菌在環境樣品中附著的情形,針對兩株鐵氧化菌(菌株TPH-2、TPH-7)設計探針LEP445,並對架設於冷卻水系統中1、2、4、8、52週,且表面附著生物膜之TYGON管進行螢光原位雜交,並以共軛焦雷射顯微鏡觀察,發現鐵氧化菌約占總菌數的51.4%,且鐵氧化菌附著於TYGON管表面的數量,隨著胞外聚合物濃度上升而增加,並被包覆在胞外聚合物之中。本研究對火力發電廠廠用冷卻水泥濘物進行基因選殖(clone library),將選殖出的6株菌經定序後,藉由與NCBI資料庫進行16S rDNA序列之比對,發現其菌相大致上可分為Actinobacteria、Alphaproteobacteria、Betaproteobacteria三大類,其中Clone 1、Clone 10、Clone 15及Clone 21是屬於Mycobacterium sp.,且相似度皆為98%;Clone 12是屬於Sphingomonas subterranea,且相似度為100%;Clone 13是屬於Denitrifying Fe-oxidizing bacteria,且相似度為98%。

The role of biofilms in the deterioration of metals and their alloys in both freah water and marine environments has been of considerable interest to industry worldwide. Problems associated with microbially influenced corrosion(MIC)due to the formation of biofilms are widespread and have serious economic and safety implications. Most MIC takes place in the presence of microbial consortion in which many different physiological types of bacteria interact in complex ways within the structure of biofilms. The aim of this study, is to investigate the iron-oxidizing bacteria biofilm formation on the wall of cooling water system by in situ flourescent hybridization(FISH)and confocal laser scanning microscopy(CLSM).
Leptothrix sp.(TPH-2), Denitrifying Fe-oxidizing Bacteria(TPH-7), Nocardia farcinica(TPH-13-3)were inoculated for biofilm formation. The biofilms were stained with calcofluor white for extracellular polymeric substances(EPS)and to observe by CLSM. Batch experiments indicated that the biofilm depth increased with the EPS concentration. Weight loss was measured in batch experiments. The experimental results show that the formation of biofilm can decrease the corrosion rate of mild steel because the formation of a biofilm was crucial, as oxygen depletion under the biofilm was responsible for the corrosion protection. The community structure of biofilm on the wall of cooling water system was determined by in situ hybridization using fluorescently labelled 16S rRNA-targeted oligonucleotide probes LEP445-Cy3, which is specific for the iron-oxidizing bacteria(TPH-2 and TPH-7). In these experiments the iron-oxidizing bacteria accounted on average for 51.4% of the total CFU. Phylogenetic analysis of a bacterial 16S rDNA clone library from the biofilm on the wall of cooling water system showed that major phylogenetic three groups are Actinobacteria, Alphaproteobacteria, and Betaproteobacteria. Clone 1, 10, 15, and 21 belonged to Mycobacterium sp.(similarity 98%);Clone 12 Sphingomonas subterranean(similarity 100%); Clone 13 Denitrifying Fe-oxidizing bacteria(similarity 98%).
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