Gonzalez-Martinez Alejandro, Rodriguez-Sanchez Alejandro, Rodelas Belén, Abbas Ben A, Martinez-Toledo Maria Victoria, van Loosdrecht Mark C M, Osorio F, Gonzalez-Lopez Jesus
Department of Civil Engineering, University of Granada, Campus de Fuentenueva, s/n, 18071 Granada, Spain.
Institute of Water Research, University of Granada, C/Ramón y Cajal 4, 18071 Granada, Spain.
Biomed Res Int. 2015;2015:892013. doi: 10.1155/2015/892013. Epub 2015 Sep 3.
Identification of anaerobic ammonium oxidizing (anammox) bacteria by molecular tools aimed at the evaluation of bacterial diversity in autotrophic nitrogen removal systems is limited by the difficulty to design universal primers for the Bacteria domain able to amplify the anammox 16S rRNA genes. A metagenomic analysis (pyrosequencing) of total bacterial diversity including anammox population in five autotrophic nitrogen removal technologies, two bench-scale models (MBR and Low Temperature CANON) and three full-scale bioreactors (anammox, CANON, and DEMON), was successfully carried out by optimization of primer selection and PCR conditions (annealing temperature). The universal primer 530F was identified as the best candidate for total bacteria and anammox bacteria diversity coverage. Salt-adjusted optimum annealing temperature of primer 530F was calculated (47°C) and hence a range of annealing temperatures of 44-49°C was tested. Pyrosequencing data showed that annealing temperature of 45°C yielded the best results in terms of species richness and diversity for all bioreactors analyzed.
通过分子工具鉴定厌氧氨氧化（anammox）细菌以评估自养脱氮系统中的细菌多样性，受到为细菌域设计能够扩增anammox 16S rRNA基因的通用引物的困难所限制。通过优化引物选择和PCR条件（退火温度），成功地对包括五种自养脱氮技术、两个中试规模模型（MBR和低温CANON）和三个全规模生物反应器（anammox、CANON和DEMON）中的anammox菌群在内的总细菌多样性进行了宏基因组分析（焦磷酸测序）。通用引物530F被确定为总细菌和anammox细菌多样性覆盖的最佳候选引物。计算了引物530F的盐调整最佳退火温度（47°C），因此测试了44 - 49°C的一系列退火温度。焦磷酸测序数据表明，对于所有分析的生物反应器，45°C的退火温度在物种丰富度和多样性方面产生了最佳结果。
