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標題: 以分子生物方法探討活性污泥中紫色不含硫光合作用細菌
A Study of Purple Nonsulfur Bacteria in Activated Sludge Using Moleculr Biotechnology
作者: 梁志銘
關鍵字: Purole Nonsulfur Bacteria;紫色不含硫光合作用細菌;FISH;PCR;DGGE;螢光原位雜交;聚合脢連鎖反應;變性梯度凝膠電泳
出版社: 環境工程學系
為避免水質的優養化,以工程方法來減少廢水中磷的濃度便成為廢水處理的重要課題之一。若考慮經濟性、可靠、及高效率的特性,生物除磷被建議為最適合的除磷方式。在目前廣為使用的程序為EBPR(Enhanced Biological Phosphorus Removal)生物除磷程序中,雖然有幾類分離出的微生物菌株被認定為聚磷酸鹽累積菌,但實際參與此反應的微生物種類以及以生長特性至今尚未有所定論。而文獻中曾指出,某些以醋酸為基質之除磷反應槽的菌群中有80%為演化上接近Rhodocyclus (Rhodocyclus-related)的菌種,且由最近學者發表之研究中得知此類微生物在實廠活性污泥中所佔比例可達10%以上,但此類Rhodocyclus-related的菌種是否為紫色不含硫光合作用細菌之一,或是紫色不含硫光合作用細菌與Rhodocyclus-related的菌種在EBPR生物除磷程序中所扮演的角色及累積聚磷酸鹽的能力也尚未明瞭。
本研究欲瞭解紫色不含硫光合作用細菌或演化分類上接近紫色不含硫光合作用細菌之菌株在活性污泥生物除磷系統中所可能扮演的角色。實驗設計上,先以傳統培養純化並作菌株計數比較一般處理程序與EBPR處理程序中紫色不含硫光合作用細菌之菌數及菌相,再配合分子生物方法,包含螢光原位雜交(FISH)、聚合脢連鎖反應(PCR)、瓊脂糖電泳(agarose gel electrophoresis)、變性梯度凝膠電泳(DGGE)…等方法進行不同來源污泥槽中紫色不含硫光合作用細菌之比較分析。
初步結果發現,紫色不含硫光合作用細菌在活性污泥槽中之總菌數較文獻中提及之100,000 cells/mL少兩個數量級。光譜分析發現,分離出之純菌約90%以上較趨向於Rhodopseudomonas屬;經電泳分析結果發現,一般僅具曝氣程序之活性污泥中紫色不含硫光合作用細菌之種類較複雜,但總數較少,約1,000 cells/mL以下;而EBPR除磷程序中紫色不含硫光合作用細菌之種類較為單純,但總菌數較多,約3,000 cells/mL至6,000 cell/mL之間。
由PCR-DGGE的結果發現,一般僅具曝氣程序的反應槽活性污泥菌相,比厭氧好氧交替程序(如EBPR程序)的反應槽活性污泥菌相複雜。活性污泥中所純化出之紫色不含硫光合作用細菌菌種以Rhodopseudomonas palustris最常見,此外尚有Rhodoplanes elegans 、Rhodobacter blasticus及 Rhodobacter capsulatus,此外亦分離出紫色含硫光合作用細菌Allochromatium vinosum。而在生物除磷程序的反應槽活性污泥中,最易分離出的為Rhodopseudomonas palustris。但在純化分離純菌試驗中,並未成功分離出Rhodocyclus,故尚無法探究此類菌種累積聚磷酸鹽的能力。

Wastewater phosphorus removal using engineering strategies is one of the important subjects on eutrophication control. In all the available methods, biological phosphorus removal becomes very popular because its low cost, good reliability, and high efficiency. Even though several groups of microorganisms had been isolated from the enhanced biological phosphorus removal (EBPR) process and their ability on accumulating polyphosphate had been confirmed, there is no conclusion on what microorganism plays the major role on the biological phosphorus removal process. From the literatures, more than 80% bacteria in some acetate-feeding phosphorus removal reactors were found to be closely related to Rhodocyclus (belonging to purple non-sulfur phototropic bacteria). Furthermore, the number of these Rhodocyclus - related microorganisms were found to be above 10% in some full-scale wastewater treatment plant. However, the role of purple non-sulfur phototropic bacteria or these Rhodocyclus — related bacteria in the full-scale EBPR process as well as their ability on polyphosphate accumulating is still not clear.
The objectives of this study are to understand the possible roles of purple non-sulfur phototropic bacteria or these Rhodocyclus — related bacteria in the full-scale EBPR processes. Traditional isolation and numeration methods for purple non-sulfur bacteria were applied first to compare the difference between sludge samples from normal activated sludge process and EBPR process. After that, molecular methods including fluorescence in situ hybridization (FISH)、polymerase chain reaction (PCR)、agrose gel electrophoresis、denauring gradient gel electrophoresis (DGGE) were used for further bacterial community comparisons.
It was shown that the number of purple non-sulfur bacteria is 100 times lower than the previously reported 100,000 cfu/mL. Absorption spectrums of isolated pure cultures showed that more than 90% of purple non-sulfur bacteria isolated were similar to Rhodopseudomonas. Furthermore, numeration result combined with DGGE analysis demonstrated that sludge sample from EBPR process had more purple non-sulfur bacteria (3,000 cells/ml — 6,000 cells/ml) than from normal activated sludge (lower then 1,000 cells/ml). Furthermore, even though normal activated sludge sample had fewer purple non-sulfur bacteria, the composition of these bacteria were more complicated than sample from EBPR process.
Isolated purple non-sulfur bacteria were identified by gene extraction and sequencing. Four major species were found in the isolated pure cultures : Rhodopseudomonas palustris、Rhodoplanes elegans、Rhodobacter blasticus and Rhodobacter capsulatus. Among them, Rhodopseudomonas palustris is the most frequently isolated one. One purple sulfur bacteria identified as Allochromatium vinosum was also isolated by the same method used for culturing purple non-sulfur bacteria. However, no pure culture of Rhodocyclus or organisms closely relate to Rhodocyclus were isolated.
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