Enrichment and characterization of a nitric oxide-reducing microbial community in a continuous bioreactor

Abstract

Nitric oxide (NO) is a highly reactive and climate-active molecule and a key intermediate in the microbial nitrogen cycle. Despite its role in the evolution of denitrification and aerobic respiration, high redox potential and capacity to sustain microbial growth, our understanding of NO-reducing microorganisms remains limited due to the absence of NO-reducing microbial cultures obtained directly from the environment using NO as a substrate. Here, using a continuous bioreactor and a constant supply of NO as the sole electron acceptor, we enriched and characterized a microbial community dominated by two previously unknown microorganisms that grow at nanomolar NO concentrations and survive high amounts (>6 µM) of this toxic gas, reducing it to N₂ with little to non-detectable production of the greenhouse gas nitrous oxide. These results provide insight into the physiology of NO-reducing microorganisms, which have pivotal roles in the control of climate-active gases, waste removal, and evolution of nitrate and oxygen respiration.

Fig. 1. The enrichment culture reduced NO to N₂ and oxidized formate to CO₂ in a continuous bioreactor with virtually no accumulation of N₂O.

Values of N₂O are only shown when the measured concentration was above the detection limit (10–25 ppm). Formate was supplied as the reducing agent for energy conservation and carbon source for biomass growth. Source data

Fig. 2. Phylogenetic tree of the Sterolibacteriaceae family based on 120 concatenated single-copy marker genes.

Genomes of cultured representatives of the Sterolibacteriaceae family and MAGs from uncultured organisms closely related to Ca. Nitricoxidivorans perseverans (MAG1) and Ca. Nitricoxidireducens bremensis (MAG5) were obtained from GTDB and NCBI. Organisms obtained in this study are indicated in bold. The genus Thauera (GCF_001696715, GCF_002245655, GCF_001051995, GCF_000443165, GCF_001591165, GCF_000310205, GCF_003030465, GCF_001922305, GCF_000310185 and GCF_000310225) was used as the outgroup. The tree was calculated based on maximum likelihood (1,000 iterations) using IQ-TREE. Ultrafast bootstrap values above 95% are indicated at the branch nodes. The scale bar indicates 0.1 estimated substitutions per site. The set of bacterial single-copy marker genes is according to ref. .

Fig. 3. Visualization of Ca. Nitricoxidivorans perseverans and Ca. Nitricoxidireducens bremensis in the enrichment culture.

Double CARD–FISH was performed using probes Nper205 and Nbre448 to target cells of Ca. Nitricoxidivorans perseverans (green) and Ca. Nitricoxidireducens bremensis (pink), respectively, followed by DAPI staining of all cells (blue). Cell counts were performed in triplicate filter pieces from CARD–FISH and DAPI counts (n ≥ 1,000). Scale bar, 10 µm.