title: 454-Pyrosequencing Analysis of Bacterial Communities from Autotrophic Nitrogen Removal Bioreactors Utilizing Universal Primers: Effect of Annealing Temperature.
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.
authors.affiliation: Department of Civil Engineering, University of Granada, Campus de Fuentenueva, s/n, 18071 Granada, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Gonzalez-Martinez
authors.affiliation: Institute of Water Research, University of Granada, C/Ramón y Cajal 4, 18071 Granada, Spain.
authors.forename: Alejandro
authors.initials: A
authors.lastname: Rodriguez-Sanchez
authors.affiliation: Institute of Water Research, University of Granada, C/Ramón y Cajal 4, 18071 Granada, Spain.
authors.forename: Belén
authors.initials: B
authors.lastname: Rodelas
authors.affiliation: Department of Biotechnology, Technical University of Delft, Julianalaan 67, 2628 BC Delft, Netherlands.
authors.forename: Ben A
authors.initials: BA
authors.lastname: Abbas
authors.affiliation: Institute of Water Research, University of Granada, C/Ramón y Cajal 4, 18071 Granada, Spain.
authors.forename: Maria Victoria
authors.initials: MV
authors.lastname: Martinez-Toledo
authors.affiliation: Department of Biotechnology, Technical University of Delft, Julianalaan 67, 2628 BC Delft, Netherlands.
authors.forename: Mark C M
authors.initials: MC
authors.lastname: van Loosdrecht
authors.affiliation: Department of Civil Engineering, University of Granada, Campus de Fuentenueva, s/n, 18071 Granada, Spain.
authors.forename: F
authors.initials: F
authors.lastname: Osorio
authors.affiliation: Institute of Water Research, University of Granada, C/Ramón y Cajal 4, 18071 Granada, Spain.
authors.forename: Jesus
authors.initials: J
authors.lastname: Gonzalez-Lopez
mesh_terms: D052818:Autotrophic Processes / Q000235:genetics*; D001419:Bacteria / Q000235:genetics*; D019149:Bioreactors / Q000382:microbiology*; D016000:Cluster Analysis; D003198:Computer Simulation; D017931:DNA Primers / Q000378:metabolism*; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D009584:Nitrogen / Q000302:isolation & purification*; D025341:Principal Component Analysis; D013045:Species Specificity; D013696:Temperature
chemical_list: D017931:DNA Primers; D009584:Nitrogen