- 學位論文
泡沫化薄膜生物反應槽中絲狀菌之研究
The Identification of Filamentous Bacteria in a Foaming Membrane Bioreactor
摘要
泡沫化及膨化問題常出現在國內外污水處理廠，對於處理效率有非常大的影響。而薄膜生物反應槽(SBMBR)為目前積極發展的一種污水處理程序，但由於其特殊的處理單元，常會導致泡沫化、膨化及薄膜阻塞的問題。本研究將利用Eikelboom及Jenkins所提出的絲狀菌型態鑑定方法，鑑定SBMBR程序中泡沫化污泥(foaming sludge)之絲狀菌。並以16S rDNA為基礎的分子生物技術(PCR-RFLP)針對純菌培養的泡沫污泥菌株進行篩選及分類，且結合螢光原位雜交法(FISH)對於100個菌株做進一步的分類，藉以加強泡沫化污泥鑑定的可靠性。 實驗結果顯示該模廠之DSVI介於200~300ml/g、Filament Index為5、Scum Index為14％等各指標結果發現本模廠為一複雜的泡沫化及膨化的反應槽且其污泥含有大量EPS。 在泡沫化污泥直接觀察的部分，依Eikelboom的絲狀菌鑑定方法，可發現存在於泡沫中的優勢菌種為Nostocoida limicola III及Actinomycetes菌屬，但顯微鏡觀察常會造成的人為的誤差，故本研究接著以純菌培養的方式培養泡沫中的微生物並進行型態觀察及染色試驗，以進一步確認鑑定結果是否正確。 另外以螢光原位雜交法分析泡沫污泥之菌相結構方面，利用四種不同之螢光探針進行雜交的結果分別為: Alphaproteobacteria綱(3.97％)、Betaproteobacteria綱(8.52％)、Gammaproteobacteria綱(8.33％)及針對革蘭氏陽性菌的High mol% G+C (54.8％)，由於放射菌屬於High mol% G+C，故可證明泡沫化污泥中含有大量的放射菌屬，且間接證明與前述之直接觀察結果相符。 在泡沫純菌的鑑定方面，本研究以PCR-RFLP法針對100株純菌進行篩選，在PCR成功之66個樣品中，以RFLP快速篩選分出11類並將其定序之，由定序結果縮減類別。由定序結果發現，分別為Firmicutes門(Low G+C)和Actinobacteria門(High G+C)之革蘭氏陽性菌，此結果與純菌染色試驗結果相符。 另外在FISH法篩選菌種方面，首先以HGC69a螢光探針篩選前述之100株純菌，發現有51株屬於HGC(High mol％ G+C)及26株為Non-HGC，而其中Non-HGC的部分再以Alphapoteobacteria綱、Betaproteobacteria綱及Gammaproteobacteria綱等三種探針進行篩選，發現此26株Non-HGC皆不屬於此三個division的菌種。在比對其定序結果，發現皆為Firmicutes門之菌屬，故以FISH的方式篩選菌種將可快速地獲得正確的鑑定結果。 最後比較各方法之鑑定結果發現，純菌以顯微鏡鑑定時常會導致兩種不同的結果，而以分子生物技術則相對可以得到較一致的結果。然而每一菌株的四項結果無法完全一致，但只要有某幾項結果相符，其菌種鑑定還是具有相當的可靠性。而本研究認為螢光原位雜交法對於絲狀菌的研究較為準確。
並列摘要
The foaming and bulking problems influence significantly on the treatment efficiency of wastewater treatment plant. Recently, the submerged membrane bioreactor (SBMBR) are being increasingly used in the treatment of municipal wastewater due to their compact size and energy efficiency compare with conventional systems. However, because of its special treatment unit, usually causes the problem of foaming , bulking and membrane fouling, etc. This study wound identify filamentous bacteria base on Eikelboom and Jenkins identification methods in a foaming membrane bioreactor. And the biotechnology of molecule (PCR-RFLP) based on 16S rDNA to screen and classify bacterial strain, and combine fluorescence in situ hybridization (FISH) to make further classification of 100 pieces of bacterial strain, by way of strengthening the dependability that the foaming sludge appraises. The experimental results show that DSVI of this pilot-scale mould between 200~300ml/g, Filament Index is 5, Scum Index find for 14%. Every indicator show that the pilot-scale is complicated foaming and bulking and its contain a large number of EPS . Observing directly in the foaming sludge, it shows advantage bacterial of foam belong to Nostocoida limicola III and Actinomycetes genus base on method of Eikelboom, but this widely used identification system does not distinguish different organisms with the same morphology and cannot deal with bacteria exhibiting variable morphology . so research this is it train by pure way that fungus train microorganism of foam and person who go on attitude observe and staining test, in order to confirm determining whether the result is correct further. The result showed that the Proteobacteria was the predominant bacteria in foaming sludge, that is Alphaproteobacteria (3.97%) , Betaproteobacteria (8.52%) , Gammaproteobacteria (8.33%) and High mol% G +C (54.8%),respectively. Because Actinomycetes belongs to High mol% G +C Gram positive, so it can prove that foaming sludge contains a large number of Actinomycetes, and prove indirectly that is in conformity with above-mentioned direct observation results. In identification of the pure culture, the study is screened 100 pure funguses with PCR-RFLP , at PCR screen not lasting 11 kinds and not making it by preface among 66 piece when it is successful sample fast by RFLP, made the preface result and reduced the classification . Made the preface result to find, the positive fungus in order that the leather on Firmicutes door (Low G +C ) and Actinobacteria door (High G +C ) is blue, dye the result of the test and conform in this result and pure fungus respectively. In addition, Screen the bacterial by FISH, screen 100 above-mentioned pure culture with HGC69a probe at first, find that 51 are HGC (High mol% G +C ) and 26 as Non-HGC, and the part of Non-HGC among them is and then in Alphapoteobacteria key link , three kinds of probes , such as Betaproteobacteria key link and Gammaproteobacteria key link ,etc. are screened , find that this 26 Non-HGCs do not all belong to the bacterial of this three divisions . Make result of preface than to it , find that all belongs to the fungus of Firmicutes, so screen the bacterial and can obtain the correct qualification result fast by way of FISH. Finally, the result of comparing every method , the pure fungus determines with the microscope that will often cause two kinds of different results, can relatively receive more unanimous result with molecule biotechnology . But it is unable to be totally unanimous that four items of every bacterial strain fruit, but only some several results agree, its bacterial qualification has suitable dependability . And this research shows that hybridization is comparatively accurate in research of the filamentous microorganism.