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Duffner, C. ; Kublik, S. ; Fösel, B. ; Frostegård,* ; Schloter, M. ; Bakken, L.* ; Schulz, S.

Genotypic and phenotypic characterization of hydrogenotrophic denitrifiers.

Environ. Microbiol. 24, 1887-1901 (2022)

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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.

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