Table of Contents Author Guidelines Submit a Manuscript
BioMed Research International
Volume 2015 (2015), Article ID 892013, 12 pages
Research Article

454-Pyrosequencing Analysis of Bacterial Communities from Autotrophic Nitrogen Removal Bioreactors Utilizing Universal Primers: Effect of Annealing Temperature

1Department of Civil Engineering, University of Granada, Campus de Fuentenueva, s/n, 18071 Granada, Spain
2Institute of Water Research, University of Granada, C/Ramón y Cajal 4, 18071 Granada, Spain
3Department of Biotechnology, Technical University of Delft, Julianalaan 67, 2628 BC Delft, Netherlands

Received 20 October 2014; Accepted 26 February 2015

Academic Editor: Weixing Feng

Copyright © 2015 Alejandro Gonzalez-Martinez et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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.