Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/22602
標題: 以螢光原位雜交技術偵測厭氧醱酵系統中產氫微生物產氫活性
Detection of the hydrogen-producing bacteria in an anaerobically fermentative culture system by fluorescence in situ hybridization
作者: 徐志華
Choi, Chi-Wa
關鍵字: Molasses fermentative fluid
產氫酶反轉錄聚合酶連鎖反應
Hydrogenase
RT-PCR
Co-culture
Clostridia
共培養
梭狀芽孢桿菌
螢光原位雜交技術
探針
出版社: 生命科學系所
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摘要: 在石油危機及全球暖化的威脅下,開發與使用環保再生能源已成為人類重要的共識。由於氫氣可經由然料電池轉為電能,將氫氣然燒也只會產生能量和水,氫氣已被認為是一種極為理想的替代能源,因此有關氫氣的生產、儲存和利用等,便成為各國積極研究的課題。本實驗室進行以微生物醱酵的方式從有機廢棄物生產氫氣的研究。本實驗使用略具選擇性的PYG(碳源是glucose)及VR(碳源是sodium glutamate)兩種培養基,分別從白蟻腸道、廢棄酵母粉醱酵產氫系統和濃縮糖蜜醱酵液醱酵產氫系統等篩選Clostridium屬細菌,得到的十八株菌株均經過16S rDNA定序法鑑定。由於本研究最終是要用稀釋的濃縮糖蜜醱酵液作為營養基質來進行醱酵產氫氣,因此挑選從濃縮糖蜜醱酵醱酵產氫系統篩選的菌株,以PYG、VR、稀釋的糖蜜醱酵液來測試產氫能力,結果顯示以PYG培養時,產氫最多的是C. tyrobutyricum F4,以VR培養時,C. sporosphaeroides F52的產氫最好,而以稀釋的糖蜜醱酵液培養時則以C. pasteurianum F40的產氫最好。為了充份利用濃縮糖蜜醱酵液所含的養分來產氫,則同時以偏好glucose 的F4菌株和偏好sodium glutamate的F52菌株進行共培養可達到較佳的產氫效果。為了營造適合Clostridium屬產氫菌生長的厭氧環境,進一步利用Bacillus和Clostridium屬細菌共培養,實驗的結果顯示,添加Bacillus會嚴重影響氫氣的產量,和單以Clostridium屬產氫菌株進行培養的結果比較,兩者產氫量最多可相差約3.5倍。以螢光原位雜合技術去計數共培養時Bacillus和Clostridium屬產氫菌的數量比例,發現產氫菌在總菌數上最多只佔17%,証明無論在好氧與厭氧的環境,Bacillus都會影響Clostridium屬細菌的生長與產氫。最後本研究針對Clostridium屬細菌產氫酶基因序列設計專一性探針,嘗試藉由RT-PCR放大mRNA之後,能偵測醱酵產氫系統內正在產氫的族群。
Be under the threat of oil crisis and global warming, increased research attention is being paid to new and recycled energy sources. Because the utilization of hydrogen, either via combustion or via fuel cells, results in pure water and no polluting emissions, hydrogen is thought as one of the most ideal energy resources for the purpose of replacing the utilization of fossil fuels, therefore the production, storage and utilization of hydrogen have become the hot subjects that are studied actively. Our laboratory is carrying out some research on producing hydrogen from organic waste by means of microbial fermentation. In this study selective media PYG (contains glucose as carbon source) and VR (contains sodium glutamate as carbon source), were used to isolate hydrogen-producing clostridia form the hindgut of termite and the fermentative culture of hydrogen-producing systems that used waste brewery yeast and molasses fermentative fluid as nutrient, respectively.18 clostridial isolates were obtained and identified by 16S rDNA sequencing method. Because the ultimate goal of this study was to use molasses fermentative fluid as nutrient substrate for studying hydrogen production, therefore three clostridial isolates from molasses fermentation system and named as Clostridium tyrobutyricum F4, C. sporosphaeroides F52, and C. pasteurianum F40 were further grown in PYG broth, VR broth, and molasses broth to compare their hydrogen production. The results showed that F4 produced more hydrogen than the other two isolates when grown in PYG broth ; when grown in VR broth, F52 produced hydrogen best ; when grown in molasses broth, F40 produced hydrogen best. Because molasses fermentative fluid contains both glucose and sodium glutamate, therefore a co-culture of F4 and F52 grown in molasses broth displayed even better hydrogen production than any other combination. In order to establish an anaerobic condition for the growth of clostridial hydrogen producer, a Bacillus isolate was added to the different culture combination of F4, F40 and F52, but the results exhibited that Bacillus significantly decreased hydrogen production 1.4 fold to 3.5 fold. Took the co-culture of Bacillus isolate and F4 as an example, fluorescent in situ hybridization ( FISH ) analysis indicated that the ratio of F4 population to the total bacterial population was never more than 17% during the growing period. Probably this was the reason that Bacillus would seriously affect hydrogen production. Finally, this study attempted to develope a technique to detect the active hydrogen-producing clostridia in a fermentation system by RT-PCR and hydrogenase gene-specific probe.
URI: http://hdl.handle.net/11455/22602
其他識別: U0005-3107200715062500
文章連結: http://www.airitilibrary.com/Publication/alDetailedMesh1?DocID=U0005-3107200715062500
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