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dc.contributorChieh-Chen Huangen_US
dc.contributorChiu-Yue Linen_US
dc.contributor.advisorFu-Shyan Wenen_US
dc.contributor.authorChen, Yung-Tienen_US
dc.identifier.citation洪國彦 (2005) 在不同水力停留時間條件下醱酵產氫系統中的微生物多樣性. 中興大學生命科學系. 碩士論文 張瑞仁 (2006) 以分子生物技術建構厭氧產氫系統之基礎研究. 中興大學生命科學系. 博士論文 Ackrell, B. A., R. N. Asato, and H. F. Mower. 1966. Multiple forms of bacterial hydrogenases. J Bacteriol 92:828-38. Beckers, G., L. Nolden, and A. Burkovski. 2001. Glutamate synthase of Corynebacterium glutamicum is not essential for glutamate synthesis and is regulated by the nitrogen status. Microbiology 147:2961-70. Behensky, P., V. Stenzl, F. Adamek, and V. Behal. 1999. Conversion of glutamate to glutamine by permeabilized Corynebacterium glutamicum. Folia Microbiol (Praha) 44:487-90. Both, B., W. Buckel, R. Kroppenstedt, and E. Stackebrandt. 1992. Phylogenetic and chemotaxonomic characterization of Acidaminococcus fermentans. FEMS Microbiol Lett 76:7-11. Bott, M., and A. Niebisch. 2003. The respiratory chain of Corynebacterium glutamicum. J Biotechnol 104:129-53. Buckel, W. 1980. 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dc.description.abstract氫氣燃燒後無污染的特性,使全球積極發展以氫能源作為替代石化能源的研究。在眾多氫氣產生方式中,由於生物性產氫技術實現了將有機廢棄物生質物轉為潔淨氫能源,其兼具廢棄物利用與能源再生的特性而受到重視。 本研究利用以糖蜜醱酵液作為基質來源的連續式厭氧產氫系統進行微生物產氫的研究。前人曾以產氫酶基因為目標進行反轉錄聚合酶連鎖反應(RT-PCR),發現系統中有Clostridium屬細菌的存在。因此本研究利用PYG培養基篩選分離系統中的Clostridium屬細菌,得到了Clostridium. tyrobutyricum F4及Clostridium pasteurianum F40兩個菌株。也利用VR培養基自糖蜜中篩到了Clostridium sporosphaeroides F52會利用味精產生氫氣的菌株。也確實有文獻提到Clostridium sporosphaeroides能以味精為單一碳源來產氫。實驗利用根據Clostridium屬細菌的產氫酶基因核苷酸序列上的保守性區域所設計的引子對,對HRT為8及4小時系統內的總RNA進行RT-PCR,另外也分別對作為殖種源的污水處理廠的污泥、作為基質的糖蜜、及HRT為8及4小時系統內的試樣總DNA進行聚合酶連鎖反應(PCR),產物經過定序比對後,顯示在HRT為8及4小時的系統中活躍表現的Clostridium屬細菌產氫酶基因都近似於C. acetobutylicum的產氫酶基因,而將污泥、糖蜜、HRT為8小時及4小時得到的clone,進一步作親緣關係樹的分析,發現來自糖蜜的clone和來自HRT為8小時及4小時的clone較相近,顯示系統中的主要產氫微生物,可能來自於糖蜜。進一步探討此產氫系統中微生物社會的組成,實驗以16s rDNA及16s rRNA為目標分別對不同HRT條件下的系統內總DNA及RNA試樣以變性梯度膠體電泳(DGGE)進行分析。結果顯示系統HRT條件的改變使系統內微生物族群的組成及相對的數量發生改變。DGGE分析的結果發現在HRT8及HRT4小時的系統中, Clostridium tyrobutyricum和Clostridium butyricum可能為主要的產氫微生物。由於糖蜜中含有醣類及味精,因此認為在本系統中,同時存在利用醣類和味精的兩株菌,可能可以最充分地利用榶蜜中的養分來生產氫氣。因此將自糖蜜中分離出的F4、F40及F52三株菌,兩兩共培養於糖蜜中,發現當F40及F52共培養時,果如推論,可得到最高的產氫量,約有96 ml的氫氣量。zh_TW
dc.description.abstractHydrogen is known as a clean energy resource. The recent surge in global H2 production research promises to make this clean fuel a replacement energy utilization of fossil fuels. The biological production of hydrogen by using wastewater and other biomass as raw materials has been attracting attention due to the characteristic of conversion of waste materials to energy resources. An anaerobically fermentative continuous culture system using condensed molasses fermentation solubles (CMS), which comes from a monosodium glutamate factory, as nutrient substrate was used to study hydrogen production. Previous results of reverse transcription polymerase chain reaction (RT-PCR) indicated that Clostridium spp. existed in the system. In fact, one Clostridium tyrobutyricum-like and one C. pasteurianum-like clostridia (F4 and F40) were substantially isolated from CMS by using PYG agar plate (glucose as sole carbon source). One C. sporosphaeroides-like clostridia (F52) were also isolated from CMS by using VR agar plate (monosodium glutamate as sole carbon source). C. sporosphaeroides was reported that they could produce hydrogen when using monosodium glutamate as sole carbon source. Primers were synthesized according to the consensus sequence of different clostridial hydrogenase genes. The results of PCR and RT-PCR indicated that those clostridial hydrogenase genes actively expressed under HRT 8 h and HRT 4 h were similar to the hydrogenase genes of C. acetobutylicum. Phylogenetic analysis of all the hydrogenase amino acid sequences obtained from RT-PCR, and PCR indicated that clostridial hydrogenase genes actively expressed under HRT 8 h and HRT 4 h were similar to hydrogenase genes of Clostridium sp. existed in CMS. The result reveal that the major hydrogen-producing clostridia may come from CMS. The hydrogen-producing rate of this system varied with different hydraulic retention time (HRT), probably due to the variation of microbial composition in this system. Indeed, this variation was clearly shown in the denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragment that were produced by PCR and RT-PCR from total cellular DNA and RNA at HRT 8 h and HRT 4 h, respectively. When those 16S rDNA fragments that significantly appeared on the DGGE gel were further cloned and sequenced, the results indicated that Clostridium tyrobutyricum and C. butyricum were probably the major hydrogen-producing microbes under HRT 8 h and HRT 4 h. Because CMS contain carbohydrate and monosodium glutamate, it was speculated that two strains of bacteria using glucose and monosodium glutamate, respectively, might efficiently use CMS to produce hydrogen. Hence co-culture of F4、F40、F52 using CMS medium, reveal that co-culture of F40 and F52 can have the highest H2 production up to 96 ml .zh_TW
dc.description.tableofcontents摘要 i Abstract ii 目錄 iv 第一章 前言 1 一、 氫氣 1 二、產氫微生物 1 三、氫酶 (Hygrogenase) 2 四、產氫醱酵系統 3 五、Clostridium屬細菌 3 六、Clostridium屬細菌氫酶基因(hydA)之表現 4 七、能利用Glutamate產氫的微生物 5 八、分子生物技術與環境微生物的研究 5 九、研究目的 6 第二章 材料與方法 7 第一節 實驗材料 7 一、厭氧醱酵產氫系統及菌液試樣的採收 7 二、細胞菌種及質體 7 三、藥品及酵素 7 四、培養基 8 五、產氫測試: 8 六、試劑與緩衝溶液 9 七、變性梯度膠體電泳法(Denature gradient gel electophoresis, DGGE)的設備與試劑 9 八、反轉錄聚合酶連鎖反應(RT-PCR)之試劑 10 九、實驗中所使用的引子對 10 第二節 實驗方法 11 一、厭氧醱酵產氫系統菌液試樣中染色體DNA之萃取 11 二、小量質體DNA之萃取 11 三、厭氧醱酵產氫系統菌液試樣中RNA之萃取 12 四、聚合酶連鎖反應 (PCR) 12 五、反轉錄聚合酶連鎖反應(RT-PCR) 13 六、洋菜膠體電泳分析(agarose gel electrophoresis)及DNA回收 14 七、變性梯度膠體電泳(DGGE)分析 14 八、變性膠體梯度電泳(DGGE)膠體中DNA片段的回收 16 九、DNA片段的選殖(cloning) 16 十、PYG及VR厭氧培養基的配製方法 17 十一、Molasses medium的配製方法 18 十二、VR medium的配製方法 19 十三、厭氧產氫菌之分離 20 十四、菌種鑑定 21 第三章 實驗結果 22 一、 以PCR偵測糖蜜廢水、污泥殖種源及不同水利停留時間下的厭氧醱酵產氫系統中Clostridium屬產氫酶基因 22 二、以RT-PCR偵測HRT為8小時及4小時的厭氧醱酵產氫系統中Clostridium屬產氫酶基因 22 三、不同HRT條件下厭氧醱酵產氫系統中微生物相的變化 23 四、厭氧醱酵產氫系統中主要產氫微生物之分離與鑑定 25 第四章 討論 29 表與圖 38 參考文獻 53zh_TW
dc.subjectCMS (condensed molasses fermentation solubles)en_US
dc.subjectRT-PCR (reverse transcriptase polymerase chain reaction)en_US
dc.subjectDGGE (denaturing gradient gel electrophoresis)en_US
dc.titleDetection and isolation of clostridial strains in a fermentative hydrogen-producing systemen_US
dc.typeThesis and Dissertationzh_TW
item.openairetypeThesis and Dissertation-
item.fulltextno fulltext-
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