以(NH4)2SO4和NaNO2作为基质,富集厌氧氨氧化污泥。提取厌氧氨氧化污泥中细菌总DNA,纯化后使用特异性引物对厌氧氨氧化菌16SrDNA进行PCR扩增。扩增产物连接到pMD19-T载体,将载体转化到感受态细胞大肠杆菌JM109中,并对其16SrDNA基因进行测序。将测序结果进行系统发育树分析,发现富集得到的厌氧氨氧化菌与Candidatus Anammoxoglobus propionicus进化关系比较接近,是一种尚未见报道的厌氧氨氧化菌。
Anaerobic ammonium oxidation (ANAMMOX) sludge was enriched with (NH4)2SO4 and NaNO2. Crude DNA of the total bacteria in cultivated anaerobic ammonium-oxidizing sludge was extracted and purified. Then partial 16SrDNA sequence of ANAMMOX bacterium was amplified by polymerase chain reaction (PCR) with a pair of specific primers. The PCR product was cloned into the vector pMD19-T and was transfected into competent cells E. coil JM109, then 16S rDNA sequence was determined. Phylogenetic analysis indicates that the cultivated ANAMMOX bacterium closes to Candidatus Anammoxoglobus propionicus, and has never been found.