Genotypic and phenotypic characterization of hydrogenotrophic denitrifiers

Stimulating litho-autotrophic denitrification in aquifers with hydrogen is a promising strategy to remove excess NO₃⁻, but it often entails accumulation of the cytotoxic intermediate NO₂⁻ and the greenhouse gas N₂O. To explore if these high NO₂⁻ and N₂O concentrations are caused by differences in the genomic composition, the regulation of gene transcription or the kinetics of the reductases involved, we isolated hydrogenotrophic denitrifiers from a polluted aquifer, performed whole-genome sequencing and investigated their phenotypes. We therefore assessed the kinetics of NO₂⁻, NO, N₂O, N₂ and O₂ as they depleted O₂ and transitioned to denitrification with NO₃⁻ as the only electron acceptor and hydrogen as the electron donor. Isolates with a complete denitrification pathway, although differing intermediate accumulation, were closely related to Dechloromonas denitrificans, Ferribacterium limneticum or Hydrogenophaga taeniospiralis. High NO₂⁻ accumulation was associated with the reductases' kinetics. While available, electrons only flowed towards NO₃⁻ in the narG-containing H. taeniospiralis but flowed concurrently to all denitrification intermediates in the napA-containing D. denitrificans and F. limneticum. The denitrification regulator RegAB, present in the napA strains, may further secure low intermediate accumulation. High N₂O accumulation only occurred during the transition to denitrification and is thus likely caused by delayed N₂O reductase expression.