Recently, floating membrane filter cultivation was adopted to simulate solid surface and enrich surface-adapted soil ammonia-oxidizing archaea (AOA) communities from agricultural soil, as opposed to the conventional liquid medium. Here, we conducted metagenomic sequencing to recover nitrifier bins from the floating membrane filter cultures and reveal their genomic properties. Phylogenomic analysis showed that AOA bins recovered from this study, designated FF_bin01 and FF_bin02, are affiliated with the Nitrososphaeraceae family, while the third bin, FF_bin03, is a nitrite-oxidizing bacterium affiliated with the Nitrospiraceae family. Based on the ANI/AAI analysis, FF_bin01 and FF_bin02 are identified as novel species within the genera “Candidatus Nitrosocosmicus” and Nitrososphaera, respectively, while FF_bin03 represents a novel species within the genus Nitrospira. The pan and core genome analysis for the 29 AOA genomes considered in this study revealed 5,784 orthologous clusters, out of which 653 were core orthologous clusters. Additionally, 90 unique orthologous clusters were conserved among the Nitrososphaeraceae family, suggesting their potential role in enhancing culturability and adaptation to diverse environmental conditions. Intriguingly, FF_bin01 and FF_bin02 harbor a gene encoding manganese catalase and FF_bin03 also possesses a heme catalase gene, which might enhance their growth on the floating membrane filter. Overall, the floating membrane filter cultivation has proven to be a promising approach for isolating distinct soil AOA, and further modifications to this technique could stimulate the growth of a broader range of uncultivated nitrifiers from diverse soil environments.
Denitrification and dissimilatory nitrate reduction to ammonium (DNRA) were thought to be carried-out by anaerobic bacteria constrained to anoxic conditions as they use nitrate (NO3-) as a terminal electron acceptor instead of molecular O2. Three soil bacilli, Neobacillus spp. strains PS2-9 and PS3-12 and Bacillus salipaludis PS3-36, were isolated from rice paddy field soil in Korea. The bacterial strains were selected as possible candidates performing aerobic denitrification and DNRA as they were observed to reduce NO3- and produce extracellular NH4+ regardless of oxygen presence at the initial screening. Whole genome sequencing revealed that these strains possessed all the denitrification and DNRA functional genes in their genomes, including the nirK, nosZ, nirB, and nrfA genes, which were simultaneously cotranscribed under aerobic condition. The ratio between the assimilatory and dissimilatory NO3- reduction pathways depended on the availability of a nitrogen source for cell growth, other than NO3-. Based on the phenotypic and transcriptional analyses of the NO3- reductions, all three of the facultative anaerobic strains reduced NO3- likely in both assimilatory and dissimilatory pathways under both aerobic and anoxic conditions. To our knowledge, this is the first report that describes coexistence of NO3- assimilation, denitrification, and DNRA in a Bacillus or Neobacillus strain under aerobic condition. These strains may play a pivotal role in the soil nitrogen cycle.
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