Kohei Oba’s research while affiliated with Tokyo University of Agriculture and Technology and other places

Nitrous oxide-reducing bacteria (N2ORB) are generally considered the only biological sink for the potent greenhouse gas N2O. Although N2O consumption activities by diverse heterotrophic N2ORB have been detected, knowledge gaps remain about the phylogenies, physiologies, and activities of N2ORB. Here, we successfully enriched a methylotrophic N2ORB consortium under intermittent oxygen and N2O supplies. 15N tracer analysis showed that the N2O consumption activity of the enriched consortium was higher than its N2O production activity in the presence of either a single or multiple electron acceptors (i.e., nitrogen oxides). The observed maximum N2O consumption was 80.7 μmol·g-biomass–1·h–1. Quantitative PCR results showed that clade I nosZ bacteria overwhelmed clade II nosZ bacteria at high (0.41 mmol·min–1) and low (0.08 mmol·min–1) N2O loading rates. The dilution rate and N2O loading rate affected the microbial community composition and activity. A higher N2O loading rate stimulated active and oxygen-tolerant N2ORB that boosted N2O consumption by approximately 50% in the presence of oxygen. Metagenomic analysis unraveled the predominance of a novel methylotrophic N2ORB, possessing entire denitrifying genes and high-affinity terminal oxidase genes, from the reactor with a high N2O loading rate. The unique physiological traits of the consortium enriched by methanol shed light on a novel function─aerobic N2O consumption by N2ORB─and pave the way for innovative N2O mitigation strategies applying powerful N2O sinks in engineered systems.

Shifting from ammonia removal to recovery is the current strategy in wastewater treatment management. We recently developed a microaerophilic activated sludge system for retaining ammonia whereas removing organic carbon with minimal N2O emissions. A comprehensive understanding of nitrogen metabolisms in the system is essential to optimize system performance. Here, we employed metagenomics and metatranscriptomics analyses to characterize the microbial community structure and activity during the transition from a microoxic to an oxic condition. A hybrid approach combining high-quality short reads and Nanopore long reads reconstructed 98 medium- to high-quality non-redundant metagenome-assembled genomes from the communities. The suppressed bacterial ammonia monooxygenase (amoA) expression was upregulated after shifting from a microoxic to an oxic condition. Seventy-three reconstructed metagenome-assembled genomes (>74% of the total) from 11 bacterial phyla harbored genes encoding proteins involved in nitrate respiration; 39 (~53%) carried N2O reductase (nosZ) genes with the predominance of clade II nosZ (31 metagenome-assembled genomes), and 24 (~33%) possessed nitrite reductase (ammonia-forming) genes (nrfA). Clade II nosZ and nrfA genes exhibited the highest and second-highest expressions among nitrogen metabolism genes, indicating robust N2O consumption and ammonification. Non-denitrifying clade II nosZ bacteria, Cloacibacterium spp., in the most abundant and active phylum Bacteroioda, were likely major N2O sinks. Elevated dissolved oxygen concentration inhibited clade II nosZ expression but not nrfA expression, potentially switching phenotypes from N2O reduction to ammonification. Collectively, the multi-omics analysis illuminated bacteria responsible for N2O reduction and ammonification in microoxic and oxic conditions, facilitating high-performance ammonia recovery.

Shifting from ammonia removal to recovery is the current strategy in wastewater treatment management. We recently developed a microaerophilic activated sludge (MAS) system for retaining ammonia while removing organic carbon with minimal N2O emissions. A comprehensive understanding of nitrogen metabolisms in the MAS system is essential to optimize system performance. Here, we employed metagenomics and metatranscriptomics analyses to characterize the microbial community structure and activity during the transition from a microaerophilic to an aerobic condition. A hybrid approach of high-quality Illumina short reads and Nanopore long reads recovered medium- to high-quality 98 non-redundant metagenome-assembled genomes (MAGs) from the MAS communities. The suppressed bacterial ammonia monooxygenase (amoA) expression was upregulated after shifting from a microaerophilic to an aerobic condition. The 73 MAGs (>74% of the total) from 11 bacterial phyla harbored genes encoding proteins involved in nitrate respiration; 39 MAGs (~53%) carried N2O reductase (nosZ) genes with the predominance of clade II nosZ (31 MAGs), and 24 MAGs (~33%) possessed nitrite reductase (ammonia forming) genes (nrfA). Clade II nosZ and nrfA genes exhibited the highest and second-highest expressions among nitrogen metabolism genes, indicating robust N2O consumption and ammonification. Non-denitrifying clade II nosZ bacteria, Cloacibacterium spp., in the most abundant and active phylum Bacteroioda, were likely major N2O sinks. Elevated dissolved oxygen (DO) concentration inhibited clade II nosZ expression but not nrfA expression, potentially switching phenotypes from N2O reduction to ammonification. Collectively, the multi-omics analysis illuminated vital bacteria responsible for N2O reduction and ammonification in microaerophilic and aerobic conditions, facilitating high-performance ammonia recovery.

N2O-reducing bacteria have been examined and harnessed to develop technologies that reduce the emission of N2O, a greenhouse gas produced by biological nitrogen removal. Recent investigations using omics and physiological activity approaches have revealed the ecophysiologies of these bacteria during nitrogen removal. Nevertheless, their involvement in‍ ‍anammox processes remain unclear. Therefore, the present study investigated the identity, genetic potential, and activity‍ ‍of N2O reducers in an anammox reactor. We hypothesized that N2O is limiting for N2O-reducing bacteria‍ ‍and an‍ ‍exogeneous N2O supply enriches as-yet-uncultured N2O-reducing bacteria. We conducted a 1200-day incubation of N2O-reducing bacteria in an anammox consortium using gas-permeable membrane biofilm reactors (MBfRs), which efficiently supply N2O in a bubbleless form directly to a biofilm grown on a gas-permeable membrane. A ¹⁵N tracer test indicated that the supply of N2O resulted in an enriched biomass with a higher N2O sink potential. Quantitative PCR and 16S rRNA amplicon sequencing revealed Clade II nosZ type-carrying N2O-reducing bacteria as protagonists of N2O sinks. Shotgun metagenomics showed the genetic potentials of the predominant Clade II nosZ-carrying bacteria, Anaerolineae and Ignavibacteria in MBfRs. Gemmatimonadota and non-anammox Planctomycetota increased their abundance in MBfRs despite their overall lower abundance. The implication of N2O as an inhibitory compound scavenging vitamin B12, which is essential for the synthesis of methionine, suggested its limited suppressive effect on the growth of B12-dependent bacteria, including N2O reducers. We identified Dehalococcoidia and Clostridia as predominant N2O sinks in an anammox consortium fed exogenous N2O because of the higher metabolic potential of vitamin B12-dependent biosynthesis.

Here, we report a genome sequence of Afipia carboxidovorans strain SH125 isolated from an anammox reactor. This facultative anaerobic strain possesses the clade I-type nitrous oxide (N2O) reductase gene, devoid of nitrite- and nitric oxide reductase genes. Deciphering the genome will help explore N2O reducers instrumental in N2O mitigation.

In denitrifying reactors, canonical complete denitrifying bacteria reduce nitrate (NO3-) to nitrogen via N2O. However, they can also produce N2O under certain conditions. We used a 15N tracer method, in which 15N-labeled NO3-/nitrite (NO2-) and nonlabeled N2O were simultaneously supplied with organic electron donors to five canonical complete denitrifying bacteria affiliated with either Clade I or Clade II nosZ. We calculated their NO3-, NO2-, and N2O consumption rates. The Clade II nosZ bacterium Azospira sp. strain I13 had the highest N2O consumption rate (3.47 ± 0.07 fmol/cell/h) and the second lowest NO3- consumption rate (0.20 ± 0.03 fmol/cell/h); hence, it is a N2O sink. A change from peptone- to acetate/citrate-based organic electron donors increased the NO3- consumption rate by 4.8 fold but barely affected the N2O consumption rate. Electron flow was directed to N2O rather than NO3- in Azospira sp. strain I13 and Az. oryzae strain PS only exerting a N2O sink but to NO3- in the Clade I nosZ N2O-reducing bacteria Pseudomonas stutzeri strain JCM 5965 and Alicycliphilus denitrificans strain I51. Transcriptome analyses revealed that the genotype could not fully describe the phenotype. The results show that N2O production and consumption differ among canonical denitrifying bacteria and will be useful for developing N2O mitigation strategies.

Harnessing nitrous oxide (N2O)-reducing bacteria is a promising strategy to reduce the N2O footprint of engineered systems. Applying a preferred organic carbon source as an electron donor accelerates N2O consumption by these bacteria. However, their N2O consumption potential and activity when fed different organic carbon species remain unclear. Here, we systematically compared the effects of various organic carbon sources on the activity of N2O-reducing bacteria via investigation of their biokinetic properties and genomic potentials. Five organic carbon sources—acetate, succinate, glycerol, ethanol, and methanol—were fed to four N2O-reducing bacteria harboring either clade I or clade II nosZ gene. Respirometric analyses were performed with four N2O-reducing bacterial strains, identifying distinct shifts in DO- and N2O-consumption biokinetics in response to the different feeding schemes. Regardless of the N2O-reducing bacteria, higher N2O consumption rates, accompanied by higher biomass yields, were obtained with acetate and succinate. The biomass yield (15.45 ± 1.07 mg-biomass mmol-N2O−1) of Azospira sp. strain I13 (clade II nosZ) observed under acetate-fed condition was significantly higher than those of Paracoccus denitrificans and Pseudomonas stutzeri, exhibiting greater metabolic efficiency. However, the spectrum of the organic carbon species utilizable to Azospira sp. strain I13 was limited, as demonstrated by the highly variable N2O consumption rates observed with different substrates. The potential to metabolize the supplemented carbon sources was investigated by genomic analysis, the results of which corroborated the N2O consumption biokinetics results. Moreover, electron donor selection had a substantial impact on how N2O consumption activities were recovered after oxygen exposure. Collectively, our findings highlight the importance of choosing appropriate electron donor additives for increasing the N2O sink capability of biological nitrogen removal systems.