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 N2 O. To explore if these high NO2 - and N2 O 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, N2 O, 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 N2 O accumulation only occurred during the transition to denitrification and is thus likely caused by delayed N2 O reductase expression.
© 2022 The Authors. Environmental Microbiology published by Society for Applied Microbiology and John Wiley & Sons Ltd.