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標題: 暗醱酵產氫系統指標微生物組成及功能鑑定分析
Bacteria Community Composition and Their Possible Roles in Biohydrogen Dark Fermentation Systems
作者: 鄭景鴻
Cheng, Chin-Hung
關鍵字: Dark-fermentation;指標微生物;Indicator microorganism;Biotechnology;Real-Time PCR;Bacteria community composition;Metagenomics;分子生物技術;即時聚合酶鏈鎖反應;微生物族群;總體基因體學
出版社: 環境工程學系所
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國際上研究生物產氫技術已有數十年的歷史,對於厭氧暗醱酵產氫技術的開發已從基礎研究轉移至實際商業化的應用,而傳統的微生物分析法及一些基礎分子生物技術已經被大量的應用在生物暗醱酵產氫系統的微生物分析上,藉由分子生物技術的優勢,可探究反應器在各種情況下微生物種類變化。然而現有的基礎分子生物技術仍有僅能定性無法有效定量、若要定量分析則相當耗時等缺陷,並且缺乏一套可對於產氫系統中微生物族群快速且立即之監測方法及分析指標,故本研究即應用總體基因體學(Metagenomics)的概念找出暗醱酵產氫系統中的指標微生物族群,並建立一可立即針對產氫系統中微生物組成及產氫菌活性表現之分析方法,以即時聚合酶連鎖反應(Real-Time PCR)為基礎,發展快速菌種檢測技術,以此即時監測商業化實場操作過程中的微生物族群及產氫活性表現情況,可輔助縮短系統啟動時間,在系統遭遇失敗時能有效的針對問題提供故障排除之依據。
本研究初期以四種不同基質供給之暗醱酵產氫系統,利用聚合酶鏈鎖反應-變性梯度凝膠(PCR-DGGE)及螢光原位雜合(FISH)技術分析其中菌群結構與產氫效能間的相互關系,獲得五種對產氫系統具有影響性的微生物族群,並將之定義為產氫系統的指標微生物族群,這五種指標微生物分別為Clostridium sp.、Klebsiella sp.、Stretpcoccus sp.、Bifidobacterium sp.及Pseudomonas sp.。其中Clostridium sp.為系統中主司產氫的微生物,種類又區分為C. pasteurianum、C. butyricum及C. beijerinckii三種,分別對葡萄糖及蔗糖、木糖及澱粉、紡織廢水具有產氫優勢。
其他微生物族群對產氫效能具有輔助性質,但數量過多均會影響整個產氫系統的穩定度,其中Klebsiella sp.對氧氣敏感度高,可迅速消耗系統中的氧氣並使氧化還原電位(ORP)達到適於產氫的條件,同時可作為系統中氧氣影響與否的參考因素之一;而Streptococcus sp.則具有強化顆粒性污泥結構的功用;Bifidobacterium sp.可將複雜基質水解,提供產氫菌種有較容易利用之簡單糖類,提升複雜基質產氫系統的產氫效能;而Pseudomonas sp.則屬於其他有機物質分解菌,由於其對環境耐受性較強,可作為系統是否遭受有害物質影響的指標。
本實驗設計了針對五種指標微生物族群的分析引子,以real-time PCR為基礎,針對指標菌群進行定量分析;在分別操作蔗糖、糖蜜及紡織工廠棉布漿料廢水為基質之模場規模的反應系統中,此技術可成功的應用並判讀菌群結構與產氫效能間的正向變化,其中在蔗糖供給的模場系統中,菌群結構變化與實驗室規模系統相似,且只要Clostridium sp.的比例佔總細胞數量的50%以上,即可有穩定的產氫效能,但在判斷顆粒污泥的產生,物理因素可能較生物性因素更為重要,反觀Streptococcus sp.的存在與否對顆粒污泥的生成影響並不大。而在糖蜜廢水模場系統產氫的菌相結果顯示,系統中乳酸菌群生長旺盛,故如何有效的控制乳酸菌或水解菌種在產氫系統中的數量,為值得持續探討的課題之一。
整體而言,本研究中所發展之real-time PCR結合多重引子分析技術,可有效的應用在糖類醱酵產氫系統中,然而,在廢水產氫系統方面,由於各項廢水特性不一,會造成主要的微生物並非為此五種指標微生物而無法完整的判讀,故須進一步利用PCR-DGGE技術協助找出其他可能影響的菌群,加強目標分析的指標微生物種,此可持續且廣泛的應用於廢水產氫系統上。

The bio-hydrogen production technology has been developed for several decades. Currently, the scientists aim to build the hydrogen producing dark fermentation system from basic research to commercial application. The bacterial community composition is considered as one of the effective factor in the fermentation systems. Analytical methods of biotechnology have been performed on bacterial community structure identity and on monitoring the bacterial divergence, abundance and predominance in fermentation system operating under different conditions. However, in the existing biotechnological methods used in bio-hydrogen system there is a disadvantage of non-combination with quantitative analysis and qualitative analysis, as well as the time consuming quantitative analysis step. There is a lack of real-time analytical method for combined quantitative analysis and qualitative analysis as well as the indicator microorganism for the bio-hydrogen system. Therefore, the main purpose of this thesis is to develop a real-time analytical method, based on Real-Time PCR technique and the knowledge of metagenomics for bacterial community structure in hydrogen producing dark fermentation system. Further to use this method to study the diversity of the hydrogen producing species and non-hydrogen producing species as well as to identify the indicator microorganism for the system. The method will provide the reason for the failure of the system operation and can help to establish a successful operational strategy.
Firstly, the analysis of bacteria community composition by the methods of PCR-DGGE and FISH with cell counting was performed on four different carbon sources fed lab-scale hydrogen producing dark fermentation systems to identify the indicator microorganism. Five indicator microorganisms namely Clostridium sp., Klebsiella sp., Streptococcus sp., Bifidobacterium sp. and Pseudomonas sp. were obtained. The Clostridium sp. is the predominant hydrogen producing genus and three species distinguished as C. pasteurianum, C. butyricum and C. beijerinckii in our systems. These three species have great capacity of hydrogen production with degradation of glucose and sucrose (for C. pasteurianum), xylose and starch (for C. butyricum), and cotton pulp producing waste water (for C. beijerinckii) individually.
Other facultative anaerobic indicator microorganisms sometimes assist hydrogen production but affect the stability of the system if they become predominant. Klebsiella sp. has the ability for consuming the oxygen rapidly, and maintains low ORP as the hydrogen production condition of the system. It can also be one of the indicators for oxygen affinity. The Streptococcus sp. is known to form a net-like structure with Clostridium sp. in the granular sludge, thus strengthening the architecture of the biological granule. Bifidobacterium sp., a lactic acid producing bacteria hydrolyze the starch or complex substrate to produce the reducing sugar to enhance the utility ratio of substrate for hydrogen producing species and increase the hydrogen production. Pseudomonas sp. is a common species in the environment with high tolerance to environmental hazardous chemicals. This species can be used as an indicator for systems which are affected by hazardous compounds.
Five specific real-time PCR primers targeting the 16S rRNA gene of the five indicator microorganisms were designed and tested in this thesis to quantify the microorganisms in pilot-scale fermentative biohydrogen production systems. The systems fed three substrates individual as sucrose, C.M.S. and cotton pulp producing waste water. In the sucrose fed system, the diversion of bacteria community is similar with lab-scale system. The stable operation can be found on the Clostridium sp. cell count percentage of total cell count more than 50%. However, the negative correlation between the cell count of Streptococcus sp. and bio-granular formation indicated that the physical factors are more important than biological factors on formation of granular sludge in pilot-scale system. In the C.M.S. fed system, lactic acid producing bacteria Bifidobacterium sp. was a predominant species. Use of C.M.S. for hydrogen production not only promotes the growth of the predominant hydrogen-producing species but also controls the unwanted growth of Bifidobacterium sp.
The novel application of the method presented in this study was successfully performed on sugar based substrate fed and some waste water fed hydrogen producing dark fermentation systems. However, since the complex component of waste water varies the dominance of bacteria community, the predominant genus may be out of detection of those five indicator microorganisms, such as the results of C.M.S. fed system in this research. Use PCR-DGGE method to find the specific bacteria that dominate on other waste water fed system as additional indicator microorganism is the way to broaden the application of this method.
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