|摘要: ||本研究利用分子生物技術探討實驗室雙槽式薄膜生物反應器內之硝化菌樣態，研究背景為雙槽式MBR系統在水力停留時間0.75天及污泥停留時間30天之操作下，處理ABS樹脂廢水，兩槽MLSS污泥濃度範圍為28000~32000 mg/L，具有高COD及BOD之去除，且硝化現象相當顯著(高硝酸氮累積)，故本研究採取此操作條件下之污泥進行DNA萃取、聚合酶鏈反應(PCR)、Cloning、限制性片段長度多樣性(RFLP)、定序(Sequencing)及親源樹狀圖(Phylogenic tree)等分子生物實驗，探討硝化菌在實驗室兩槽式薄膜生物反應器內之種類及樣態分佈。
硝化作用之微生物多樣性，由氨氮氧化至亞硝酸氮稱為氨氧化菌(Ammonia-oxidizing bacteria，簡稱AOB)，再由亞硝酸氮氧化至硝酸氮稱為亞硝酸氧化菌(Nitrite-oxidizing bacteria，簡稱NOB1)。本研究在氨氧化菌方面，以RFLP樣態分類後為12株，且Clone NO.AOB-2在兩槽總數160個colonies中佔了121個，為本研究AOB之優勢菌株(dominate)，並與Thauera mechernichensis相似度為96%，相關文獻(86)指出菌重要特性是能在好氧及厭氧下都能進行脫硝作用，為硝化作用下被分離出來之菌株，故本研究氨氧化菌75.6%類似於此類菌種。其Clone NO.AOB-11、AOB-23、AOB-33、AOB-34、AOB-71及AOB-139)組成一個聚落，與已知氨氧化菌離群所居之聚落，在總兩槽總數160個colonies中佔了24個(15%)，且無相近已知菌株可知其此菌落之特性及生長條件，可能為新菌株。
亞硝酸氧化菌研究方面，以RFLP樣態分類後為17株，且Clone NO.NOB1-3在兩槽總數147個colonies中佔了59個，為本研究NOB1之第一優勢菌株，並與Nitrite-oxidizing bacterium相似度分別為99%，推估實驗室MBR系統之NOB1約有40%屬於此類菌種。Clone NO.NOB1-6在兩槽總數147個colonies中佔了44個，為本研究NOB1之第二優勢菌株，並與Uncultured Nitrobacter sp.(AY683484)相似度為97%，相關文獻(92)指出此菌為在含有鹽分之養殖水內分離出來，推估實驗室MBR系統之NOB1約有30%屬於此類菌種。Clone NO.NOB1-106及NOB1-132兩株在兩槽總數147個colonies共佔5個，且無相近已知菌株可知其此菌落之特性及生長條件，可能為新菌株。
Molecular biology techniques were applied to analyze nitrifying bacterial diversity in the laboratory-scale dual tank (biological treatment tank and membrane tank) type membrane bioreactor (MBR) system. The high removal efficiencies of COD and BOD were obtained under the steady state, at which hydraulic retention time (HRT) and sludge retention time (SRT) were 18 hours and 30 days, respectively. The MBR system was able to retain high concentrations of MLSS at tank in the range of 28000~32000 mg L-1. The results indicated that nitrification is a crucial process in the system (high nitrate concentration in the effluents). Therefore, the diversity of nitrifying bacteria in the system had been investigated by 16S rDNA molecular approach, including DNA extraction, polymerase chain reaction (PCR), cloning, restriction fragment length polymorphism (RFLP), sequencing and construction of phylogenic tree.
Nitrifying bacterial diversity was divided two steps: conversion from ammonia to nitrite by ammonia-oxidizing bacteria (AOB) and nitrite to nitrate by nitrite-oxidizing bacteria (NOB). The results of AOB in this study, 12 strains were classified from the results of RFLP. Besides, the clone NO.AOB-2 was dominate, the clone percentage of AOB-2 being 75.6% (121/160) in the system, similar to Thauera mechernichensis with the similarity of 96% by the sequencing data. The identified strain Thauera mechernichensis, characterized as nitrifying bacteria under aerobic and anaerobic condition, was isolated under the process of nitrification. In addition, Clone NO.AOB-11, AOB-23, AOB-33, AOB-34, AOB-71 and AOB-139) were clustered far away from the identified AOB in the gene bank, the percentage of the above mentioned clones being 15.0 % (24/160), could be novel bacteria due to the low similarity with identified AOB.
In terms of NOB, the clone NO.NOB1-3 was the first dominate, the clone percentage of NOB1-3 being 40.1% (59/147) in the system, similar to Nitrite-oxidizing bacterium with the similarity of 99% by the sequencing data. The clone NO.NOB1-6 was the second dominate, the clone percentage of NOB1-6 being 30% (44/147) in the system, similar to Uncultured Nitrobacter sp. with the similarity of 97% by the sequencing data. We proposed that 30% of NOB1 was affiliated to the Uncultured Nitrobacter sp, characterized as nitrifying bacteria in the high salinity breeding water. In addition, Clone NO. NOB1-106 and NOB1-132 were 5/147, could be novel bacteria due to the low similarity with identified NOB.
In the conclusion, the nitrifying bacterial diversity was not much different between biological and membrane tank of MBR system, but the dual tank system provides the advantage for hydraulic operation. The molecular data indicates that nitrifying bacterial diversity in the MBR system was different from nitrifying bacterial diversity in the traditional activated sludge process. The results of molecular data could be applied for designing, operation and analyses of bacteria in the MBR system.