Comparative Analysis of Denitrifying Activities of Hyphomicrobium nitrativorans, Hyphomicrobium denitrificans, and Hyphomicrobium zavarzinii

<jats:title>ABSTRACT</jats:title> <jats:p> <jats:named-content content-type="genus-species">Hyphomicrobium</jats:named-content> spp. are commonly identified as major players in denitrification systems supplied with methanol as a carbon source. However, denitrifying <jats:named-content content-type="genus-species">Hyphomicrobium</jats:named-content> species are poorly characterized, and very few studies have provided information on the genetic and physiological aspects of denitrification in pure cultures of these bacteria. This is a comparative study of three denitrifying <jats:named-content content-type="genus-species">Hyphomicrobium</jats:named-content> species, <jats:named-content content-type="genus-species">H. denitrificans</jats:named-content> ATCC 51888, <jats:named-content content-type="genus-species">H. zavarzinii</jats:named-content> ZV622, and a newly described species, <jats:named-content content-type="genus-species">H. nitrativorans</jats:named-content> NL23, which was isolated from a denitrification system treating seawater. Whole-genome sequence analyses revealed that although they share numerous orthologous genes, these three species differ greatly in their nitrate reductases, with gene clusters encoding a periplasmic nitrate reductase (Nap) in <jats:named-content content-type="genus-species">H. nitrativorans</jats:named-content> , a membrane-bound nitrate reductase (Nar) in <jats:named-content content-type="genus-species">H. denitrificans</jats:named-content> , and one Nap and two Nar enzymes in <jats:named-content content-type="genus-species">H. zavarzinii</jats:named-content> . Concurrently with these differences observed at the genetic level, important differences in the denitrification capacities of these <jats:named-content content-type="genus-species">Hyphomicrobium</jats:named-content> species were determined. <jats:named-content content-type="genus-species">H. nitrativorans</jats:named-content> grew and denitrified at higher nitrate and NaCl concentrations than did the two other species, without significant nitrite accumulation. Significant increases in the relative gene expression levels of the nitrate ( <jats:italic>napA</jats:italic> ) and nitrite ( <jats:italic>nirK</jats:italic> ) reductase genes were also noted for <jats:named-content content-type="genus-species">H. nitrativorans</jats:named-content> at higher nitrate and NaCl concentrations. Oxygen was also found to be a strong regulator of denitrification gene expression in both <jats:named-content content-type="genus-species">H. nitrativorans</jats:named-content> and <jats:named-content content-type="genus-species">H. zavarzinii</jats:named-content> , although individual genes responded differently in these two species. Taken together, the results presented in this study highlight the potential of <jats:named-content content-type="genus-species">H. nitrativorans</jats:named-content> as an efficient and adaptable bacterium that is able to perform complete denitrification under various conditions. </jats:p>