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

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.


SUPPLEMENTARY FIGURES
Figure S1.Protein concentrations in the NO-reducing enrichment culture.Until day 616 (indicated by a red line), a protein quantification method based on the Bradford assay was used.From that day onwards, proteins were measured following the BCA method.Decreases observed in the protein concentrations were due to excessive biomass sampling.When the sampling periods ended and the culture was not disturbed, protein levels in the enrichment culture recovered.Methylophosphatis haderslevensis and Ca.Methylophosphatis roskildensis were not available and instead, accession numbers of their genome assembly from which the 16S rRNA gene sequence was extracted is provided.The genus Thauera (MH251633, KX953213, AB681853, AB681922, KY425607, AJ315677, AF123264, X77118) was used as outgroup.The tree was calculated based on maximum likelihood (1,000 iterations) using IQtree.Ultrafast bootstrap values 4 are indicated at the branch nodes.Scale bar indicates 0.01 estimated substitutions per nucleotide.
Figure S4.Visualization of Candidatus Nitricoxidivorans perseverans and Candidatus Nitricoxidireducens bremensis in sludge collected from the municipal wastewater treatment plant in Bremen, Germany.A) Cells hybridized with probe Nper205 (green) corresponded to Ca. Nitricoxidivorans perseverans while B) cells that hybridized with probe Nbre448 (green) corresponded to Ca. Nitricoxidireducens bremensis.All cells were stained with DAPI (blue).Experiments were performed in triplicate using 3 filtered wastewater treatment plant sludge samples.Scale bar: 20 µm.
Figure S5.Phylogeny of heme-copper oxidases (HCOs) families.Families of oxygen reductases are presented in red (A-type family; cytochromes aa3 and caa3), orange (B-type family; cytochrome ba3) and yellow (C-type family; cytochrome cbb3), and nitric oxide reductases are presented in blue (qNOR), and dark green (cNOR).NOR sequences from Candidatus Nitricoxidivorans perseverans and Candidatus Nitricoxidireducens bremensis are indicated in bold.The tree was calculated using 530 amino acid positions.The tree was calculated based on maximum likelihood (1,000 iterations) using IQtree.Ultrafast bootstrap values 4 are indicated at the branch nodes.Scale bar indicates 1 estimated substitutions per site.

Figure S2 .
Figure S2.Substrate reduction kinetics of the NO-reducing enrichment culture.Graphics on the left and right of the figure represent the results obtained from duplicate experiments.Closed circles indicate N-oxide reduction rates observed experimentally, while red lines indicate the fitted Monod equations (in a, b, c, d, g, h, I, j) or the fitted Haldane equation (e, f).Vmax (µmol N-oxide mg protein −1 h −1 ) indicates the maximum reduction rate, Km(app) (µM) indicates the apparent half saturation constant (both calculated with the Michaelis-Menten equation), and Ki (µM) indicates the substrate inhibition constant (calculated with the Haldane equation).

Figure S7 .
Figure S7.Phylogeny of NosZ.Sequences belonging to clade I and clade II are indicated on the right.NosZ sequences from Candidatus Nitricoxidivorans perseverans and Candidatus Nitricoxidireducens bremensis are indicated in bold.The tree was calculated with IQtree using 750 amino acid positions, based on maximum likelihood (1,000 iterations).Ultrafast bootstrap values 4 are indicated at the branch nodes.Scale bar indicates 0.1 estimated substitutions per site.

Table S1 .
Characteristics of metagenome and metatranscriptome samples obtained from the enrichment culture and included in this study.

Table S2 .
Equivalence between taxonomic classification of NCBI and GTDB of MAGs produced in this study.

Table S5 :
Genes of interest within the genome of Candidatus Nitricoxidivorans perseverans and their abundance in the metatranscriptome (RPKM: reads per kilo base per million mapped reads) and metaproteome (NSAF: normalized spectral abundance factor).

Table S6 :
Genes of interest within the genome of Candidatus Nitricoxidireducens bremensis and their abundance in the metatranscriptome (RPKM: reads per kilo base per million mapped reads) and metaproteome (NSAF: normalized spectral abundance factor).