Hiroki Tsukamoto’s research while affiliated with Tokyo University of Agriculture and Technology and other places

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.

A transition to ammonia recovery from wastewater has started; however, a technology for sustainable nitrogen retention in the form of ammonia and organic carbon removal is still in development. This study validated a microaerophilic activated sludge (MAS) system to efficiently retain ammonia from high-strength nitrogenous wastewater. The MAS is based on conventional activated sludge (CAS) with aerobic and settling compartments. Low dissolved oxygen (DO) concentrations (<0.2 mg/L) and short solids retention times (SRTs) (<5 days) eliminated nitrifying bacteria. The two parallel MASs were successfully operated for 300 days and had ammonia retention of 101.7 ± 24.9% and organic carbon removal of 85.5 ± 8.9%. The MASs mitigated N2O emissions with an emission factor of <0.23%, much lower than the default value of CAS (1.6%). A short-term step-change test demonstrated that N2O indicated the initiation of nitrification and the completion of denitrification in the MAS. The parallel MASs had comparable microbial diversity, promoting organic carbon oxidation while inhibiting ammonia-oxidizing microorganisms (AOMs), as revealed by 16S rRNA gene amplicon sequencing, the quantitative polymerase chain reaction of functional genes, and fluorescence in situ hybridization of β-proteobacteria AOB. The microbial analyses also uncovered that filamentous bacteria were positively correlated with effluent turbidity. Together, controlling DO and SRT achieved organic carbon removal and successful ammonia retention, mainly by suppressing AOM activity. This process represents a new nitrogen management paradigm.

A transition to ammonia recovery from wastewater has started; however, a technology for sustainable nitrogen retention in the form of ammonia is still in development. This study validated a microaerophilic activated sludge (MAS) system to efficiently retain ammonia from high-strength nitrogenous wastewater. The MAS is based on conventional activated sludge (CAS) with aerobic and settling compartments. Low dissolved oxygen (DO) concentrations (<0.1 mg/L) and short solid retention times (SRTs) (<5 d) eliminated nitrifying bacteria. The two parallel MASs were successfully operated for 300 d and had ammonia retention of 101.7 ± 24.9% and organic carbon removal of 85.5 ± 8.9%. The MASs mitigated N 2 O emissions with an emission factor of <0.23%, much lower than the default value of CAS (1.6%). A short-term step-change test demonstrated that N 2 O indicated the initiation of nitrification and the completion of denitrification in the MAS. The parallel MASs had comparable microbial diversity, promoting organic carbon oxidation while inhibiting ammonia-oxidizing microorganisms (AOMs), as revealed by 16S rRNA gene amplicon sequencing, qPCR of functional genes, and fluorescent in situ hybridization of β-Proteobacteria AOB. The microbial analyses also uncovered that filamentous bacteria were positively correlated with effluent turbidity. Together, controlling DO and SRT achieved successful ammonia retention, mainly by suppressing AOM activity. This process represents a new nitrogen management paradigm. Synopsis Moving from nitrogen removal to nitrogen recovery is critical for establishing a sustainable society. We provided proof-of-the-concept for a novel ammonia retention technology by retrofitting an activated sludge system.