Comammox Functionality Identified in Diverse Engineered Biological Wastewater Treatment Systems

Abstract

Complete ammonia oxidation (comammox) to nitrate by certain Nitrospira-lineage bacteria (CMX) could contribute to overall nitrogen cycling in engineered biological nitrogen removal (BNR) processes in addition to the more well-documented nitrogen transformations by ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), and anaerobic ammonia-oxidizing (anammox) bacteria (AMX). A metagenomic survey was conducted to quantify the presence and elucidate the potential functionality of CMX in 16 full-scale BNR configurations treating mainstream or sidestream wastewater. CMX proposed to date were combined with previously published AOB, NOB, and AMX genomes to create an expanded database for alignment of metagenomic reads. CMX-assigned metagenomic reads accounted for between 0.28 and 0.64% of total coding DNA sequences in all BNR configurations. Phylogenetic analysis of key nitrification functional genes amoA, encoding the α-subunit of ammonia monooxygenase, haoB, encoding the β-subunit of hydroxylamine oxidoreductase, and nxrB, encoding the β-subunit of nitrite oxidoreductase, confirmed that each BNR system contained coding regions for production of these enzymes by CMX specifically. Ultimately, the ubiquitous presence of CMX bacteria and metabolic functionality in such diverse system configurations emphasizes the need to translate novel bacterial transformations to engineered biological process interrogation, operation, and design.

Figure 1.

Nitrogen cycling in engineered biological nitrogen removal (BNR) systems. In conventional BNR, autotrophic ammonia-oxidizing bacteria (AOB) convert ammonium (NH4⁺) to nitrite (NO₂⁻) through hydroxylamine (NH₂OH), and nitrite-oxidizing bacteria (NOB) convert nitrite to nitrate (NO₃⁻). Ordinary heterotrophic denitrifiers (OHO) then convert nitrate to dinitrogen gas (N₂) through nitrite, nitric oxide (NO), and nitrous oxide (N₂O). Alternatively, anaerobic ammonia-oxidizing bacteria (AMX) convert ammonium and nitrite directly to dinitrogen gas through hydrazine (N₂H₄). Complete ammonia-oxidizing bacteria (CMX) have only recently been described and studied, and are capable of converting ammonium to nitrate through hydroxylamine and nitrite in a single organism.

Figure 2.

Phylogenetic relationships between published amoA, haoB, and nxrB sequences and CMX-assigned sequences from each BNR process. Unique merged contigs from each BNR process that were assigned with ≥30× coverage as CMX amoA, haoB, and nxrB were included in the phylogenetic analysis, along with amoA, haoB, and nxrB sequences from representative published sequences (see Table S2 for a complete list of accession numbers). (A) CMX (red) and AOB (blue) reference amoA sequences compared through phylogenetics to CMX-assigned amoA in each BNR process. (B) CMX (red), AOB (blue), and AMX (green) reference haoB sequences compared to CMX-assigned haoB from each BNR system. (C) CMX (red), NOB (orange), and AMX (green) reference nxrB sequences compared to CMX-assigned nxrB from each BNR system. Tree-scale units are average amino acid substitutions per site, and circles at each node are scaled to reflect P values at nodes with ≥50% support. Each BNR process contained at least one amoA, haoB, and nxrB sequence that clustered with CMX rather than AOB, NOB, or AMX, providing evidence of potential CMX functionality in each system.