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標題: 偵測及篩選分離厭氧產氫系統中的微生物
Detection and isolation of clostridial strains in a fermentative hydrogen-producing system
作者: 陳永田
Chen, Yung-Tien
關鍵字: 產氫酶反轉錄聚合酶連鎖反應
CMS (condensed molasses fermentation solubles)
RT-PCR (reverse transcriptase polymerase chain reaction)
DGGE (denaturing gradient gel electrophoresis)
出版社: 生命科學系所
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摘要: 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。
Hydrogen 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 .
其他識別: U0005-0902200708514000
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