Ammonia-Oxidizing Bacteria Maintain Abundance but Lower amoA-Gene Expression during Cold Temperature Nitrification Failure in a Full-Scale Municipal Wastewater Treatment Plant

Juliet Johnston, Zhe Du, Sebastian Behrens

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1 Scopus citations


In this study, we explore the relationship between community structure and transcriptional activity of ammonia-oxidizing bacteria during cold temperature nitrification failure in three parallel full-scale sequencing batch reactors (SBRs) treating municipal wastewater. In the three reactors, ammonia concentrations increased with declines in wastewater temperature below 15°C. We quantified and sequenced 16S rRNA and ammonia monooxygenase (amoA) gene fragments in DNA and RNA extracts from activated sludge samples collected from the SBRs during the warmer seasons (summer and fall) and when water temperatures were below 15°C (winter and spring). Taxonomic community composition of amoA genes and transcripts did not vary much between the warmer and colder seasons. However, we observed significant differences in amoA transcript copy numbers between fall (highest) and spring (lowest). Ammonia-oxidizing bacteria of the genus Nitrosomonas sp. could maintain their population abundance despite lowering their amoA gene expression during winter and spring. In spite of relatively low population abundance, an amoA amplicon sequence variant (ASV) cluster identified as most similar to the amoA gene of Nitrosospira briensis showed the highest amoA transcript-to-gene ratio throughout all four seasons, indicating that some nitrifiers remain active at wastewater temperatures below 15°C. Our results show that 16S rRNA and amoA gene copy numbers are limited predictors of cell activity. To optimize function and performance of mixed community bioprocesses, we need to collect high-resolution quantitative transcriptomic and potentially proteomic data to resolve the response of individual species to changes in environmental parameters in engineered systems. IMPORTANCE The diverse microbial community of activated sludge used in biological treatment systems exhibits dynamic seasonal shifts in community composition and activity. Many wastewater treatment plants in temperate/continental climates experience seasonal cold temperature nitrification failure. “Seasonal nitrification failure” is the discharge of elevated concentrations of ammonia (greater than 4 mg/liter) with treated wastewater during the winter (influent wastewater temperatures below 13°C). This study aims at expanding our understanding of how ammonia-oxidizing bacteria in activated sludge change in activity and growth across seasons. We quantified the ammonia monooxygenase (amoA) gene and transcript copy numbers using real-time PCR and sequenced the amoA amplicons to reveal community structure and activity changes of nitrifying microbial populations during seasonal nitrification failure in three full-scale sequencing batch reactors (SRBs) treating municipal wastewater. Relevant findings presented in this study contribute to explain seasonal nitrification performance variability in SRBs.

Original languageEnglish (US)
JournalMicrobiology Spectrum
Issue number2
StatePublished - Mar 2023

Bibliographical note

Funding Information:
J. Johnston was supported by National Science Foundation Graduate Research Fellowship 2015191729. The research was enabled by the Legislative-Citizen Commission on Minnesota Resources through a grant entitled “Wastewater Treatment Process Improvements” funded by the Environment and Natural Resources Trust Fund under legal citation M.L. 2016, Chp. 186, Sec. 2, Subd. 04 k. This research was carried out at Lawrence Livermore National Laboratory (LLNL) under Contract DE-AC52-07NA27344 and release No. LLNL-1055914.

Funding Information:
We thank the team at Brainerd Wastewater Treatment Facilities for sample collection and access to treatment plant performance data. Additional thanks to Laurel Hunt, Deirdre Manion-Fischer, and Michael Brown for their assistance during sampling trips. We thank the University of Minnesota Genomics Center for assistance with amplicon sequencing. We thank the National Science Foundation Graduate Research Fellows Program for providing Juliet Johnston with a fellowship opportunity, as well as the Legislative-Citizen Commission on Minnesota Resources for funding the project.

Publisher Copyright:
Copyright © 2023 Johnston et al.


  • AOB
  • activated sludge
  • ammonia oxidation
  • nitrification
  • wastewater treatment

PubMed: MeSH publication types

  • Journal Article


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