利用16S rDNA分子生物技術探討薄膜生物反應器處理ABS樹脂廢水之硝化菌菌群結構

Nitrifying Bacterial Diversity in the Membrane Bioreactor System Treating ABS Wastewater Revealed by 16S rDNA Molecular Approach

本研究之實驗室薄膜生物反應器處理ABS樹脂廢水，在水力停留時間0.75 day及污泥停留時間30 day之高污泥濃度操作下，對於COD及BOD之去除相當顯著，且硝化現象相當明顯，故本研究進一步採取此操作條件下之污泥進行DNA萃取、聚合酶鏈反應（PCR）、選殖（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。研究結果得知在氨氧化菌方面，以限制性片段長度多樣性樣態分類後為12株，且Clone NO.AOB-2在總數160個菌落中佔了121個，為本研究AOB之優勢菌株（dominate），並與Thauera mechernichensis相似度為96%，此菌在相關文獻中指出其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。而Clone NO.AOB-11、AOB-23、AOB-33、AOB-34、AOB-71及AOB-139）組成一個聚落，在總數160個菌落中佔了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在總數147個菌落中佔了59個，為本研究NOB1之第一優勢菌株，並與亞硝酸氧化菌相似度分別為99%，推估實驗室MBR系統之NOB1約有40%屬於此類菌種。Clone NO.NOB1-6在總數147個菌落中佔了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%，此菌已被證明指出生長在含有鹽分之養殖水內，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個菌落共佔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。本研究綜合討論結果得知，實驗室MBR系統其樣態較一般活性污泥法有所差異，可作為未來MBR反應槽設計、操作及菌相分析之依據。

Molecular biology techniques were applied to analyze nitrifying bacterial diversity in the laboratory-scale 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 0.75 days 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（superscript -1）. The results indicated that nitrification is a crucial process in the system. 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. 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. 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.