Ecophysiological and Genomic Characterization of the Freshwater Complete Ammonia Oxidizer Nitrospira sp. Strain BO4

Appl Environ Microbiol. 2023 Feb 28;89(2):e0196522. doi: 10.1128/aem.01965-22. Epub 2023 Jan 25.


Complete ammonia oxidizers (comammox) are a group of ubiquitous chemolithoautotrophic bacteria capable of deriving energy from the oxidation of ammonia to nitrate via nitrite. Here, we present a study characterizing the comammox strain Nitrospira sp. BO4 using a combination of cultivation-dependent and molecular methods. The enrichment culture BO4 was obtained from the sediment of Lake Burr Oak, a mesotrophic lake in eastern Ohio. The metagenome of the enrichment culture was sequenced, and a metagenome-assembled genome (MAG) was constructed for Nitrospira sp. BO4. The closest characterized relative of Nitrospira sp. BO4 was "Candidatus Nitrospira kreftii." All genes for ammonia and nitrite oxidation, reductive tricarboxylic acid (TCA) cycle, and other pathways of the central metabolism were detected. Nitrospira sp. BO4 used ammonia and oxidized it to nitrate with nitrite as the intermediate. The culture grew on initial ammonium concentrations between 0.01 and 3 mM with the highest rates observed at the lowest ammonium concentrations. Blue light completely inhibited the growth of Nitrospira sp. BO4, while white light reduced the growth and red light had no effect on the growth. Nitrospira sp. BO4 did not grow on nitrite as its sole substrate. When supplied with ammonium and nitrite, the culture utilized nitrite after most of the ammonium was consumed. In summary, the genomic information of Nitrospira sp. BO4 coupled with the growth experiments shows that Nitrospira sp. BO4 is a freshwater comammox species. Future research will focus on further characterization of the niches of comammox in freshwater environments. IMPORTANCE Nitrification is a key process in the global nitrogen cycle. Complete ammonia oxidizers (comammox) were discovered recently, and only three enrichment cultures and one pure culture have been characterized with respect to activity and growth under different conditions. The cultivated comammox strains were obtained from engineered systems such as a recirculating aquaculture system and hot water pipes. Here, we present the first study characterizing a comammox strain obtained from a mesotrophic freshwater lake. In freshwater environments, comammox coexist with ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). Our results will help elucidate physiological characteristics of comammox and the distribution and niche differentiation of different ammonia oxidizers in freshwater environments.

Keywords: Nitrospira; ammonia oxidation; comammox; freshwater; nitrification.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Ammonia* / metabolism
  • Ammonium Compounds* / metabolism
  • Archaea / metabolism
  • Bacteria / metabolism
  • Fresh Water
  • Genomics
  • Nitrates / metabolism
  • Nitrification
  • Nitrites / metabolism
  • Oxidation-Reduction
  • Phylogeny


  • Ammonia
  • Nitrites
  • Nitrates
  • Ammonium Compounds