Applied Microbiology and Biotechnology

, Volume 102, Issue 15, pp 6725–6737 | Cite as

The link between the microbial ecology, gene expression, and biokinetics of denitrifying polyphosphate-accumulating systems under different electron acceptor combinations

  • A. Vieira
  • A. Ribera-Guardia
  • R. Marques
  • M. T. Barreto Crespo
  • A. Oehmen
  • G. CarvalhoEmail author
Environmental biotechnology


The emission of the greenhouse gas nitrous oxide (N2O) can occur during biological nutrient removal. Denitrifying enhanced biological phosphorus removal (d-EBPR) systems are an efficient means of removing phosphate and nitrogen, performed by denitrifying polyphosphate-accumulating organisms (d-PAOs). The aim of this work was to study the effect of various combinations of electron acceptors, nitrate (NO3), nitrite (NO2), and N2O, on the denitrification pathway of a d-EBPR system. Batch tests were performed with different electron acceptor combinations, to explore the denitrification pathway. Reverse transcriptase-qPCR (RT-qPCR) and high-throughput sequencing, combined with chemical analysis, were used to study gene expression, microbial diversity, and denitrification kinetics. The potential for N2O production was greater than the potential for its reduction in most tests. A strong correlation was observed between the N2O reduction rate and the relative gene expression of nitrous oxide reductase per nitrite reductase (nosZ/(nirS + nirK)), suggesting that the expression of denitrifying marker genes is a strong predictor of the N2O reduction rate. The d-EBPR community maintained a core population with low variations throughout the study. Furthermore, phylogenetic analyses of the studied marker genes revealed that the organisms actively involved in denitrification were closely related to Thauera sp., Candidatus Accumulibacter phosphatis, and Candidatus Competibacter denitrificans. Moreover, Competibacter-related OTUs seem to be important contributors to the N2O reduction capacity of the system, likely scavenging the N2O produced by other organisms. Overall, this study contributes to a better understanding of the microbial biochemistry and the genetics involving biological denitrification removal, important to minimize N2O emissions in wastewater treatment plants.


Denitrification Enhanced biological phosphorus removal (EBPR) Nitrous oxide (N2O) Greenhouse gas (GHG) Wastewater treatment plants (WWTPs) Nitrous oxide reductase (nos



This work was also supported by the Portuguese Research Units UCIBIO and iNOVA4Health, which are funded by national funds (UID/Multi/04378/2013) and co-financed by the ERDF under the PT2020 Partnership Agreement (POCI-01-0145-FEDER-007728 and LISBOA-01-0145-FEDER-007344, respectively). Anabela Vieira acknowledges the PhD grant (SFRH/BD/97640/2013) provided by FCT/MCES, and Anna Ribera-Guardia the FPIPhD grant (BES-2012-052753) provided by the Spanish government.

Funding information

The authors acknowledge the Portuguese Fundação para a Ciência e Tecnologia (FCT/MCES) for funding (PTDC/AAC-AMB/120581/2010) and the European Commission by supporting the Water JPI project 196 (WaterWorks2014 ERA-NET Co-funded Call): “Smart decentralized water management through a dynamic integration of technologies (Watintech)” and the COST action ES1202 (Water 2020).

Compliance with ethical standards

This article does not contain any studies with human participants or animals performed by any of the authors.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

253_2018_9077_MOESM1_ESM.pdf (255 kb)
ESM 1 (PDF 254 kb)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.iBET − Instituto de Biologia Experimental e TecnológicaOeirasPortugal
  2. 2.ITQB − Instituto de Tecnologia Química e Biológica António XavierUniversidade Nova de LisboaOeirasPortugal
  3. 3.Instituto Catalán de Investigación del Agua, ICRA, Edificio H2O- Parque Científico y TecnológicoGironaSpain
  4. 4.UCIBIO-REQUIMTE, Departamento de Química, Faculdade de Ciências e TecnologiaUniversidade NOVA de LisboaLisbonPortugal

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