# Monitoring complete ammonia oxidizers bacteria: relevant players for nitrogen removal from wastewater

## TL;DR

This study explores the role of COMAMMOX bacteria in removing nitrogen from wastewater and how to monitor them effectively.

## Contribution

The study introduces specific genetic markers and methods to monitor COMAMMOX bacteria in wastewater treatment.

## Key findings

- COMAMMOX bacteria are present and abundant in wastewater treatment systems.
- Digital PCR and fluorescence techniques help quantify and locate COMAMMOX bacteria.
- Monitoring tools have biases that need to be considered for effective wastewater management.

## Abstract

COMAMMOX (Complete Ammonia Oxidizers) bacteria seem to play an important role in nitrification, being able to directly transform ammonia into nitrates. Due to the relevance of this process in WWTPs, different approaches were used in this work to monitor COMAMMOX bacteria, as well as other microorganisms involved in the biological removal of nitrogen occurring in the bioreactor of a full-scale WWTP. This facility operates with low dissolved oxygen concentrations and prolonged sludge retention times. Fluorescence in situ hybridization confirmed the presence of COMAMMOX bacteria showing their distribution within the activated sludge flocs. Primers targeting the amoA gene, which encodes the A subunit of the ammonia monooxygenase enzyme, were employed as genetic markers in PCR techniques to simultaneously detect and amplify both COMAMMOX clades A and B, as well as specifically clade A, clade B, or various COMAMMOX Nitrospira species (Ca. N. nitrosa, Ca. N. inopinata, and Ca. N. nitrificans). Using digital PCR, a set of these primers enabled the estimation of the proportion of amoA COMAMMOX genes versus other nitrifiers (AOB, AOA and NOB). The data obtained not only confirmed the presence of COMAMMOX bacteria, but also their abundance, comparable to that of NOB and AOB. The 16S rRNA gene-based meta-taxonomy analysis gave complementary data about the microbial diversity and community composition within the samples. The results also highlight the biases of each technology used as monitoring tools, which must be accurate for the effective management of wastewater treatment plants (WWTPs) and for exploiting the potential of COMAMMOX bacteria.

The online version contains supplementary material available at 10.1186/s13568-025-01878-6.