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

Alejandro Gonzalez-Martinez,Mark C M Van Loosdrecht,Maria Victoria Martinez-Toledo,Alejandro Rodriguez-Sanchez,Belén Rodelas,Ben A Abbas,F Osorio,Jesus Gonzalez-Lopez

doi:10.1155/2015/892013

Alejandro Gonzalez-Martinez, Mark C M Van Loosdrecht + Show 6 more

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https://doi.org/10.1155/2015/892013

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Abstract

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.

Highlights

Anaerobic ammonium oxidizing bacteria belong to the Candidatus Brocadiales order first described in 1999 [1]
We found that primer 530F targeted the 16S rRNA genes of all the anammox Candidatus species known to date yielding a minimum of 90% perfect matches to sequences filed in the database
The results described here demonstrated that an annealing temperature of 44∘C was inappropriate to analyze the bacterial community structure of the bioreactors sampled in the study, due to a severe underestimation of the relative abundance of Brocadiales

Summary

Introduction

Anaerobic ammonium oxidizing (anammox) bacteria belong to the Candidatus Brocadiales order first described in 1999 [1]. Their ability to perform anaerobic ammonium oxidation has attracted the attention of many researchers due to the change it made for the understanding of the nitrogen cycle. They have been found as important bacteria for the ecology of nitrogen in oceanic environments [2,3,4] and have been proposed to play an important role in the nitrogen cycle at global scale [2]. Anammox bacteria have become of relevance in both natural and engineered systems, and more research about the ecology of the ecosystems where they develop is expected in the following years

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