Abstract

Anaerobic ammonium oxidation bacteria (anammox) have the metabolic ability to oxidize ammonium and reduce nitrite to form nitrogen gas (Strous et al., 1999). Its application is now a proven technology and constitute a robust, reliable and cost-effective system, to treat effluents with high concentration of nitrogen but a relatively low level of organic carbon (Lackner et al., 2014). DNA/ RNA-based molecular techniques are the most widely used approaches available for detecting this group of microorganisms belonging to Planctomycetes phylum, due to its very slowly growth rate and that have not been isolated in pure culture (Schmid et al. 2005). The aim of this work was to study the full bacterial community composition and in particular of anammox, from two granular-sludge anammox reactors, using different molecular approaches. One of the samples came from a reactor that was operated in batch mode with synthetic influent for over 5 years (Reino and Carrera, 2017) (AM sample), and the other, came from a reactor that was operated at mainstream conditions treating a low-strength real wastewater coming from a partial nitritation process of a municipal wastewater treatment plant (MWW), called S1 sample. Using FISH-CLSM technique in granule slices, a total of 42±10% of anammox bacteria was detected in AM and only a 7±3% in S1. With a specific set of fluorescent probes was also possible to detect at species taxonomic level, but considering the high autofluorescence of the sample, its quantification can be over or underestimated. Amplicon 16S rRNA gene sequencing was performed with two sets of universal bacterial primers 515F-906R and 515F-806R and different coverage of our target group Candidatus Brocadia genus, was detected, thus, from data analysis of mayor coverage primers (515F-806R), Operational Taxonomic Units (OTUs) abundance pointed out that Ca. Brocadia was the main genus of the AM sample while was almost not present in the reactor treating MWW. By contrast, the most abundant OTUs in S1 corresponded to Ignavibacteria (Chlorobi phylum). Using metagenome analysis, a total of 42 genomes were assembled with over 80% of completeness, 3 of them corresponded to Ca. Brocadia. According to functional gene annotation, the metabolic nitrogen pathways indicates that only in the Planctomycetes phylum genes involved in anaerobic ammonium oxidation were present while the Chlorobi phylum presented genes involved in nitrous-oxide reduction and nitrite reduction. According to our results, each molecular approach has a bias and the use of several techniques are necessary to recover more reliable information in terms of the dynamics of the bacterial community composition. A better understanding of the community structure of the reactor will contribute to understand the role played by the different microorganisms in the community and therefore would allow optimize a large-scale process design.