- 學位論文
活性污泥銨氧化基因多樣性之研究
The diversity of ammonia oxidization genes in activated sludge systems
摘要
由於銨氧化菌生長競爭力弱，且依其型態外觀，難以區分各個種之間的差異，再加上自營性銨氧化菌生長緩慢不易培養，為了解本研究室所馴養的SBMBR模廠中銨氧化菌之多樣性，本研究利用具銨氧化菌特殊功能之amoA基因，結合分子生物技術以了解SBMBR模廠中銨氧化菌之菌相。並針對SBMBR模廠污泥之16S rDNA基因做菌相分析，以了解SBMBR反應槽中菌種分佈及銨氧化菌所佔之比例。 研究結果顯示，具銨氧化菌特殊功能之amoA基因，僅擁有兩個copy number，因此DNA增幅不易，所以需做二次PCR才能獲得夠亮之band送定序。因而本研究僅於自營培養獲得利用amoA primer所增幅可送定序之PCR產物，定序比對後，皆與Nitrosmonas oligotropha菌種之amoA基因親源關係較近。本研究接著將銨氧化菌之16S rDNA基因定序比對後，發現具有同時硝化及脫硝功能之Bacillus之菌屬。 SBMBR模廠污泥及生物膜經基因轉殖後，以DGGE及RFLP分類，皆將銨氧化菌之amoA基因分為兩類，分別與Nitrosmonas europaea及Nitrosmonas oligotropha菌種親源關係較親近。因此本研究室馴養之SBMBR模廠，其硝化菌之amoA基因集中於Nitrosomonas屬。 SBMBR污泥經總菌落數培養，其16S rDNA基因之比對結果大多為具有脫硝功能之菌屬，而由基因轉殖之16S rDNA比對分析則大多與除磷及脫硝作用有關之菌屬，與本研究室SBMBR反應槽具有脫硝、除磷作用相符。 此外經培養所得之銨氧化菌比例甚低，其原因可能為銨氧化菌培養不易，且在SBMBR模廠所佔比例偏低，因此導致本研究經傳統培養法所得之銨氧化菌數目甚少。
並列摘要
Ammonia oxidization bacterium are not easy to grow, according to their shapes, it’s hard to distinguish the differences from each species. Autotrophic AOB (ammonia oxidization bacterium) not only grow slowly, but also difficultly culture. To realize the diversity of ammonia oxidization bacterium cultured in sequencing batch membrane bioreactor (SBMBR bioreactor) in laboratory. This research uses amoA gene which is ammonia oxidization bacterium’s special function to combine molecular biological technology and to realize the diversity of AOB, Aim directly at analysis of 16S rDNA gene in activated sludge, to know bacterium’s spreading. According to the research , amoA gene only has two copy numbers, therefore, PCR is not easy to amplify, it will obtain strength single in second time of PCR. Using amoA primer to obtain PCR products with autotrophic culture, then compare the sequence, it shows that the family relation is closed to amoA gene of Nitrosmonas oligotropha. Next, compare the sequence of 16S rDNA gene of ammonia oxidization bacteria, discovered genius of Bacillus has nitrification and denitrification at the same time. Activated sludge and biological membrane are cloned and classify amoA gene into two groups by DGGE and RELP. One of the two groups is closed to Nitrosmonas europaea and the other is closed to Nitrosmonas oligotropha. Therefore, the amoA gene of ammonia oxidization bacterium gathers around Nitrosomonas genius in SBMBR bioreactor. The result of comparison of 16S rDNA gene by SBMBR bioreactor, it shows part of genes have denitrifying. On the contrast, 16S rDNA gene by gene cloning , it shows that part of genes have denitrifying and phosphorus release. This experiment matches the result of SBMBR bioreactor. In addition, the percentage of AOB is pretty lower from cultured numbers. The reason perhaps the AOB are not easy to cultivate and they have lower percentage in SBMBR bioreactor. Consequenly, the result that mentions above leads to get lower ammonia oxidization bacterium in tradition cultivated way.