Unraveling the genetic potential of nitrous oxide reduction in wastewater treatment: insights from metagenome-assembled genomes.

This study explores the genetic landscape of nitrous oxide (N2O) reduction in wastewater treatment plants (WWTPs) by profiling 1,083 high-quality metagenome-assembled genomes (HQ MAGs) from 23 Danish full-scale WWTPs. The focus is on the distribution and diversity of nitrous oxide reductase (nosZ) genes and their association with other nitrogen metabolism pathways. A custom pipeline for clade-specific nosZ gene identification with higher sensitivity revealed 503 nosZ sequences in 489 of these HQ MAGs, outperforming existing Kyoto Encyclopedia of Genes and Genomes (KEGG) module-based methods. Notably, 48.7% of the total 1,083 HQ MAGs harbored nosZ genes, with clade II being predominant, accounting for 93.7% of these genes. Taxonomic profiling highlighted the prevalence of nosZ-containing taxa within Bacteroidota and Pseudomonadota. Chloroflexota exhibited unexpected affiliations with both the sec and tat secretory pathways, and all were found to contain the accessory nosB gene, underscoring the importance of investigating the secretory pathway. The majority of non-denitrifying N2O reducers were found within Bacteroidota and Chloroflexota. Additionally, HQ MAGs with genes for dissimilatory nitrate reduction to ammonium and assimilatory nitrate reduction frequently co-occurred with the nosZ gene. Traditional primers targeting nosZ often focus on short-length amplicons. Therefore, we introduced custom-designed primer sets targeting near-full-length nosZ sequences. These new primers demonstrate efficacy in capturing diverse and well-characterized sequences, providing a valuable tool with higher resolution for future research. In conclusion, this comprehensive analysis enhances our understanding of N2O-reducing organisms in WWTPs, highlighting their potential as N2O sinks with the potential for optimizing wastewater treatment processes and mitigating greenhouse gas emissions. IMPORTANCE: This study provides critical insights into the genetic diversity of nitrous oxide reductase (nosZ) genes and the microorganisms harboring them in wastewater treatment plants (WWTPs) by exploring 1,083 high-quality metagenome-assembled genomes (MAGs) from 23 Danish full-scale WWTPs. Despite the pivotal role of nosZ-containing organisms, their diversity remains largely unexplored in WWTPs. Our custom pipeline for detecting nosZ provides near-full-length genes with detailed information on secretory pathways and accessory nos genes. Using these genes as templates, we developed taxonomically diverse clade-specific primers that generate nosZ amplicons for phylogenetic annotation and gene-to-MAG linkage. This approach improves detection and expands the discovery of novel sequences, highlighting the prevalence of non-denitrifying N2O reducers and their potential as N2O sinks. These findings have the potential to optimize nitrogen removal processes and mitigate greenhouse gas emissions from WWTPs by fully harnessing the capabilities of the microbial communities.

Evaluating the role of high N2O affinity complete denitrifiers and non-denitrifying N2O reducing bacteria in reducing N2O emissions in river.

Freshwater rivers are hotspots of N2O greenhouse gas emissions. Dissolved organic carbon (DOC) is the dominant electron donor for microbial N2O reduction, which can reduce N2O emission through enriching high N2O affinity denitrifiers or enriching non-denitrifying N2O-reducing bacteria (N2ORB), but the primary regulatory pathway remains unclear. Here, field study indicated that high DOC concentration in rivers enhanced denitrification rate but reduced N2O flux by improving nosZ gene abundance. Then, four N2O-fed membrane aeration biofilm reactors inoculated with river sediments from river channel, estuary, adjacent lake, and a mixture were continuously performed for 360 days, including low, high, and mixed DOC stages. During enrichment stages, the (nirS+nirK)/nosZ ratio showed no significant difference, but the community structure of denitrifiers and N2ORB changed significantly (p < 0.05). In addition, N2ORB strains isolated from different enrichment stages positioned in different branches of the phylogenetic tree. N2ORB strains isolated during high DOC stage showed significant higher maximum N2O-reducing capability (Vmax: 0.6 ± 0.4 ×10-4 pmol h-1 cell-1) and N2O affinity (a0: 7.8 ± 7.7 ×10-12 L cell-1 h-1) than strains isolated during low (Vmax: 0.1 ± 0.1 ×10-4 pmol h-1 cell-1, a0: 0.7 ± 0.4 ×10-12 L cell-1 h-1) and mixed DOC stages (Vmax: 0.1 ± 0.1 ×10-4 pmol h-1 cell-1, a0: 0.9 ± 0.9 ×10-12 L cell-1 h-1) (p < 0.05). Hence, under high DOC concentration conditions, the primary factor in reducing N2O emissions in rivers is the enrichment of complete denitrifiers with high N2O affinity, rather than non-denitrifying N2ORB.

nosZ II/nosZ I ratio regulates the N2O reduction rates in the eutrophic lake sediments.

Nitrous oxide (N2O) is a more potent greenhouse gas with an atmospheric lifetime of 121 years, contributing significantly to climate change and stratospheric ozone depletion. Lakes are hotspots for N2O release due to the imbalance between N2O sources and sinks. N2O-reducing bacteria are the only biological means to mitigate N2O emission, yet their roles in lakes are not well studied. This study investigated the potential for N2O reduction, keystones of typical and atypical N2O-reducing bacterial communities, and their correlations with environmental factors in the sediments of Lake Taihu through microcosm experiments, high-throughput sequencing of the nosZ gene, and statistical modeling. The results showed that potential N2O reduction rates in sediments ranged from 13.71 to 76.95 µg N2O g-1 d-1, with lower rates in December compared to March and July. Correlation analysis indicated that the nosZ II/nosZ I ratio and the trophic lake index (TLI) were the primary factors influencing N2O reduction rates and N2O-reducing bacterial community structures. The genera Pseudogulbenkiania and Ardenticatena were identified as the most abundant typical and atypical N2O-reducing bacteria, respectively, and were also recognized as the keystone taxa. Quantitative real-time PCR (qPCR) results revealed that nosZ II was more abundant than nosZ I in the sediments. Partial least squares path modeling (PLS-PM) further demonstrated that atypical N2O-reducing bacteria had significant positive effects on N2O reduction process in the sediments (p < 0.05). Overall, this study highlights the crucial ecological roles of atypical N2O-reducing bacteria in the sediments of the eutrophic lake of Taihu, underscoring their potential in mitigating N2O emissions.

Aquatic nitrous oxide reductase gene (nosZ) phylogeny and environmental distribution.

Nitrous oxide (N2O) is a potent greenhouse gas and a major cause of ozone depletion. One-third of atmospheric N2O originates in aquatic environments. Reduction of N2O to dinitrogen gas (N2) requires the nitrous oxide reductase enzyme, which is encoded by the gene nosZ. Organisms that contain nosZ are the only known biological sinks of N2O and are found in diverse genera and a wide range of environments. The two clades of nosZ (Clade I and II) contain great diversity, making it challenging to study the population structure and distribution of nosZ containing organisms in the environment. A database of over 11,000 nosZ sequences was compiled from NCBI (representing diverse aquatic environments) and unpublished sequences and metagenomes (primarily from oxygen minimum zones, OMZs, where N2O levels are often elevated). Sequences were clustered into archetypes based on DNA and amino acid sequence identity and their clade, phylogeny, and environmental source were determined. Further analysis of the source and environmental distribution of the sequences showed strong habitat separation between clades and phylogeny. Although there are more Clade I nosZ genes in the compilation, Clade II is more diverse phylogenetically and has a wider distribution across environmental sources. On the other hand, Clade I nosZ genes are predominately found within marine sediment and are primarily from the phylum Pseudonomonadota. The majority of the sequences analyzed from marine OMZs represented distinct phylotypes between different OMZs showing that the nosZ gene displays regional and environmental separation. This study expands the known diversity of nosZ genes and provides a clearer picture of how the clades and phylogeny of nosZ organisms are distributed across diverse environments.

pH selects for distinct N2O-reducing microbiomes in tropical soil microcosms.

Nitrous oxide (N2O), a greenhouse gas with ozone destruction potential, is mitigated by the microbial reduction to dinitrogen catalyzed by N2O reductase (NosZ). Bacteria with NosZ activity have been studied at circumneutral pH but the microbiology of low pH N2O reduction has remained elusive. Acidic (pH < 5) tropical forest soils were collected in the Luquillo Experimental Forest in Puerto Rico, and microcosms maintained with low (0.02 mM) and high (2 mM) N2O assessed N2O reduction at pH 4.5 and 7.3. All microcosms consumed N2O, with lag times of up to 7 months observed in microcosms with 2 mM N2O. Comparative metagenome analysis revealed that Rhodocyclaceae dominated in circumneutral microcosms under both N2O feeding regimes. At pH 4.5, Peptococcaceae dominated in high-N2O, and Hyphomicrobiaceae in low-N2O microcosms. Seventeen high-quality metagenome-assembled genomes (MAGs) recovered from the N2O-reducing microcosms harbored nos operons, with all eight MAGs derived from acidic microcosms carrying the Clade II type nosZ and lacking nitrite reductase genes (nirS/K). Five of the eight MAGs recovered from pH 4.5 microcosms represent novel taxa indicating an unexplored N2O-reducing diversity exists in acidic tropical soils. A survey of pH 3.5-5.7 soil metagenome datasets revealed that nosZ genes commonly occur, suggesting broad distribution of N2O reduction potential in acidic soils.

NosZ I carrying microorganisms determine N2O emissions from the subtropical paddy field under elevated CO2 and strongly CO2-responsive cultivar.

Elevated CO2 (eCO2) decreases N2O emissions from subtropical paddy fields, but the underlying mechanisms remain to be investigated. Herein, the response of key microbial nitrogen cycling genes to eCO2 (ambient air +200 µmol CO2 mol-1) in four rice cultivars, including two weakly CO2-responsive (W27, H5) and two strongly CO2-responsive cultivars (Y1540, L1988), was investigated. Except for nosZ I, eCO2 did not significantly alter the abundance of the other genes. NosZ I was a crucial factor governing N2O emissions, especially under eCO2 and a strongly responsive cultivar. eCO2 affected the nosZ I gene abundance (p < 0.05), for instance, the nosZ I gene abundance of cultivar W27 increased from 1.53 × 107 to 2.86 × 107 copies g-1 dw soil (p < 0.05). In the nosZ I microbial community, the known taxa were mainly Pseudomonadota (phylum) (19.74-31.72 %) and Alphaproteobacteria (class) (0.56-13.12 %). In the nosZ I community assembly process, eCO2 enhanced the role of stochasticity, increasing from 35 % to 85 % (p < 0.05), thereby inducing diffusion limitations of weakly responsive cultivars to dominate (67 %). Taken together, the increase in nosZ I gene abundance is a potential reason for the alleviation of N2O emissions from subtropical paddy fields under eCO2.

Respiration and growth of Paracoccus denitrificans R-1 with nitrous oxide as an electron acceptor.

In the nitrogen biogeochemical cycle, the reduction of nitrous oxide (N2O) to N2 by N2O reductase, which is encoded by nosZ gene, is the only biological pathway for N2O consumption. In this study, we successfully isolated a strain of denitrifying Paracoccus denitrificans R-1 from sewage treatment plant sludge. This strain has strong N2O reduction capability, and the average N2O reduction rate was 5.10 ± 0.11 × 10-9 µmol·h-1·cell-1 under anaerobic condition in a defined medium. This reduction was accompanied by the stoichiometric consumption of acetate over time when N2O served as the sole electron acceptor and the reduction can yield energy to support microbial growth, suggesting that microbial N2O reduction is related to the energy generation process. Genomic analysis showed that the gene cluster encoding N2O reductase of P. denitrificans R-1 was composed of nosR, nosZ, nosD, nosF, nosY, nosL, and nosZ, which was identified as that in other strains in clade I. Respiratory inhibitors test indicated that the pathway of electron transport for N2O reduction was different from that of the traditional electron transport chain for aerobic respiration. Cu2+, silver nanoparticles, O2, and acidic conditions can strongly inhibit the reduction, whereas NO3- or NH4+ can promote it. These findings suggest that modular N2O reduction of P. denitrificans R-1 is linked to the electron transport and energy conservation, and dissimilatory N2O reduction is a form of microbial anaerobic respiration. IMPORTANCE: Nitrous oxide (N2O) is a potent greenhouse gas and contributor to ozone layer destruction, and atmospheric N2O has increased steadily over the past century due to human activities. The release of N2O from fixed N is almost entirely controlled by microbial N2O reductase activities. Here, we investigated the ability to obtain energy for the growth of Paracoccus denitrificans R-1 by coupling the oxidation of various electron donors to N2O reduction. The modular N2O reduction process of denitrifying microorganism not only can consume N2O produced by itself but also can consume the external N2O generated from biological or abiotic pathways under suitable condition, which should be critical for controlling the release of N2O from ecosystems into the atmosphere.

Contribution of Microorganisms with the Clade II Nitrous Oxide Reductase to Suppression of Surface Emissions of Nitrous Oxide.

The sources and sinks of nitrous oxide, as control emissions to the atmosphere, are generally poorly constrained for most environmental systems. Initial depth-resolved analysis of nitrous oxide flux from observation wells and the proximal surface within a nitrate contaminated aquifer system revealed high subsurface production but little escape from the surface. To better understand the environmental controls of production and emission at this site, we used a combination of isotopic, geochemical, and molecular analyses to show that chemodenitrification and bacterial denitrification are major sources of nitrous oxide in this subsurface, where low DO, low pH, and high nitrate are correlated with significant nitrous oxide production. Depth-resolved metagenomes showed that consumption of nitrous oxide near the surface was correlated with an enrichment of Clade II nitrous oxide reducers, consistent with a growing appreciation of their importance in controlling release of nitrous oxide to the atmosphere. Our work also provides evidence for the reduction of nitrous oxide at a pH of 4, well below the generally accepted limit of pH 5.

Greenhouse gases emissions from aquaculture ponds: Different emission patterns and key microbial processes affected by increased nitrogen loading.

Global aquaculture production is expected to rise to meet the growing demand for food worldwide, potentially leading to increased anthropogenic greenhouse gases (GHG) emissions. As the demand for fish protein increases, so will stocking density, feeding amounts, and nitrogen loading in aquaculture ponds. However, the impact of GHG emissions and the underlying microbial processes remain poorly understood. This study investigated the GHG emission characteristics, key microbial processes, and environmental drivers underlying GHG emissions in low and high nitrogen loading aquaculture ponds (LNP and HNP). The N2O flux in HNP (43.1 ± 11.3 µmol m-2 d-1) was significantly higher than in LNP (-11.3 ± 25.1 µmol m-2 d-1), while the dissolved N2O concentration in HNP (52.8 ± 7.1 nmol L-1) was 150 % higher than in LNP (p < 0.01). However, the methane (CH4) and carbon dioxide (CO2) fluxes and concentrations showed no significant differences (p > 0.05). N2O replaced CH4 as the main source of Global Warming Potential in HNP. Pond sediments acted as a sink for N2O but a source for CH4 and CO2. The △N2O/(△N2O + â–³N2) in HNP (0.015 ± 0.007 %) was 7.7-fold higher than in LNP (0.002 ± 0.001 %) (p < 0.05). The chemical oxygen demand to NO2-N ratio was the most important environmental factor explaining the variability of N2O fluxes. Ammonia-oxidizing bacteria driven nitrification in water was the predominant N2O source, while comammox-driven nitrification and nosZII-driven N2O reduction in water were key processes for reducing N2O emission in LNP but decreased in HNP. The strong CH4 oxidization by Methylocystis and CO2 assimilation by algae resulted in low CH4 emissions and CO2 sink in the aquaculture pond. The Mantel test indicated that HNP increased N2O fluxes mainly through altering functional genes composition in water and sediment. Our findings suggest that there is a significant underestimation of N2O emissions without considering the significantly increased △N2O/(△N2O + â–³N2) caused by increased nitrogen loading.

Quest for Nitrous Oxide-reducing Bacteria Present in an Anammox Biofilm Fed with Nitrous Oxide.

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.

Comparative analysis of three next-generation sequencing techniques to measure nosZ gene abundance in Missouri claypan soils.

Quantitative next-generation sequencing techniques have been critical in gaining a better understanding of microbial ecosystems. In soils, denitrifying microorganisms are responsible for dinitrogen (N2) production. The nosZ gene codes for nitrous oxide reductase, the enzyme facilitating the reduction of nitrous oxide (N2O) to N2. The objectives of this research were to: 1) understand how soil depth influences RNA concentration and nosZ gene abundance; 2) assess the spatial dependence of nosZ gene abundance in two claypan soil fields; and 3) compare and evaluate multiple RNA-based sequencing methods for quantifying nosZ gene abundance in soils in relation to dinitrogen (N2) production. Research sites consisted of two intensively studied claypan soil fields in Central Missouri, USA. Soil cores were collected from two landscape transects across both fields and analyzed for extractable soil RNA at two depths (0-15 cm and 15-30 cm). Measurements of nosZ gene abundance were obtained using real-time quantitative polymerase chain reaction (RT-qPCR), droplet digital polymerase chain reaction (ddPCR), and nanostring sequencing (NS). In both fields, soil RNA concentrations were significantly greater at 0-15 cm depth compared to 15-30 cm. These data indicated low overall soil microbial activity below 15 cm. Due to low quantities of extractable soil RNA in the subsoil, nosZ gene abundance was only determined in the 0-15 cm depth. Sequencing method comparisons of average nosZ gene abundance showed that NS results were constrained to a narrow range and were 10-20-fold lower than ddPCR and RT-qPCR at each landscape position within each field. Droplet digital PCR appears to be the most promising method, as it reflected changes in N2 production across landscape position.

Complete genome sequence of Afipia carboxidovorans strain SH125, a non-denitrifying nitrous oxide-reducing bacterium isolated from anammox biomass.

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.

Spatial distribution characteristics of denitrification functional genes and the environmental drivers in Liaohe estuary wetland.

Genes nirS, nirK, and nosZ are specific for the denitrification process, which is associated with greenhouse gas N2O emission. The abundances and diversities of community containing these three genes are usually used as a common index to reflect the denitrification process, and they would be affected by differences in environmental factors caused by changes from warm to cold conditions. The quantification of denitrification in natural wetlands is complex, and straightforward identification of spatial distribution and drivers affecting the process is still developing. In this study, the bacterial communities, gene diversities, and relative abundances involved in denitrification were investigated in Liaohe Estuary Wetland. We analyzed the relative abundances, diversities, and communities of bacteria containing the three genes at warm and cold conditions using Illumina MiSeq sequencing and detected the potential environmental factors influencing their distribution by using a random forest algorithm. There are great differences in the community composition of the bacteria containing genes nirS, nirK, and nosZ. All the abundant taxa of nirS and nirK communities belonged to phylum Proteobacteria. Compared with the community composition of bacteria containing nirS and nirK, the community of bacteria containing nosZ is more diverse, and the subdivision taxa of phylum Euryarchaeota was also abundant in the community containing nosZ. The distribution characteristics of the relative abundance of nirS and nirK showed obvious differences both at warm and cold climate conditions. The oxidation-reduction potential, nitrite nitrogen, and salinity were detected as potential variables that might explain the diversity of nirS. The total nitrogen and nitrite nitrogen were the important variables for predicting the relative abundance of nirS at warm climate condition, while oxidation-reduction potential and pH contributed to the diversity of nirS at cold condition. The bulk density of sediment was detected as a potential variable affecting the relative abundance of nirK at warm and cold conditions, and diversity of nirK at warm condition, while nitrite nitrogen was detected as an important environmental factor for predicting the diversity of nirK at cold condition. Overall, our results show that the key environmental factors, which affect the relative abundance, diversity, and community of bacteria containing the functional denitrification genes, are not exactly the same, and the diversities of nirS, nirK, and nosZ have a higher environmental sensitivity than their relative abundances.

Relationships between different types of biochar and N2O emissions during composting based on roles of nosZ-carrying denitrifying bacterial communities enriched on compost and biochar particles.

Biochar has demonstrated the potential in mitigating N2O emissions during composting. However, little is known about how microbial communities on biochar particles interact with N2O emissions. This study selected three types of biochar (corn stalk biochar (CSB), rape straw biochar (RSB), and bamboo charcoal (BC)) to investigate the relationship between N2O emissions and denitrifying bacterial communities on compost and biochar particles. The results showed that N2O emissions rate were higher in the thermophilic phase, and the average emissions rate of BC treatment were lower 40% and 26% than CSB and RSB, respectively. The nosZ-carrying denitrifying bacterial community played a key role in reducing N2O emissions, and the network indicated that Rhizobium and Paracoccus on compost particles may have played major roles in reducing N2O emissions, but only Paracoccus on biochar particles. Notably, BC enhanced the efficiency of N2O emission reduction by enhancing the abundance of these key genera.

Effect of different types of biochar on soil properties and functional microbial communities in rhizosphere and bulk soils and their relationship with CH4 and N2O emissions.

Biochar as an agricultural soil amendment plays vital roles in mediating methane (CH4) and nitrous oxide (N2O) emissions in soils. The link between different types of biochar, bulk soil, and rhizosphere microbial communities in relation to CH4 and N2O emissions is being investigated in this study. The rice pot experiment was conducted using biochar at two temperatures (300°C and 500°C) in combination with three biochar levels (0, 2, 10% w/w). Soil properties and the abundance of genes associated with CH4 and N2O emissions from both rhizosphere and bulk soils were investigated. The study also aimed to examine the structure of microbial communities (pmoA, nosZ) in rhizosphere and bulk soils whereas CH4 and N2O emissions were monitored while growing rice. Results showed that biochar at 300°C and 10% incorporation significantly increased the CH4 emissions by up to 59% rise compared to the control group. Random Forest analysis revealed that the ratio of mcrA/pmoA along with the abundance of mcrA from both rhizosphere and bulk soils, the abundance of AOA, TN, DOC, and the community composition of pmoA-harboring microorganisms from both bulk and rhizosphere soils were important predictors of CH4 emissions. Therefore, the ratio of mcrA/pmoA in rhizosphere soil and the abundance of AOA in bulk soil were the main factors influencing CH4 emissions. Variation Partitioning Analysis (VPA) results indicated that the effects of these factors on bulk soil were 9% of CH4 emissions variations in different treatments, which contributed more than rhizosphere soils' factors. Moreover, random forest analysis results indicated that the abundance of AOB in bulk soil was the most important predictor influencing N2O emissions. The VPA result revealed that the factors in rhizosphere soil could explain more than 28% of the variations in N2O emissions. Our study highlights that rhizosphere soil has a more significant effect than bulk soil on N2O production. Our findings further the understanding of the link between bulk and rhizosphere attributes, and their impact on CH4 and N2O emissions in paddy soils. In summary, we recommend the application of biochar at 500°C and 2% incorporation rate for agricultural production in the area.

Homogeneous environmental selection mainly determines the denitrifying bacterial community in intensive aquaculture water.

Nitrate reduction by napA (encodes periplasmic nitrate reductase) bacteria and nitrous oxide reduction by nosZ (encodes nitrous oxide reductase) bacteria play important roles in nitrogen cycling and removal in intensive aquaculture systems. This study investigated the diversity, dynamics, drivers, and assembly mechanisms of total bacteria as well as napA and nosZ denitrifiers in intensive shrimp aquaculture ponds over a 100-day period. Alpha diversity of the total bacterial community increased significantly over time. In contrast, the alpha diversity of napA and nosZ bacteria remained relatively stable throughout the aquaculture process. The community structure changed markedly across all groups over the culture period. Total nitrogen, phosphate, total phosphorus, and silicate were identified as significant drivers of the denitrifying bacterial communities. Network analysis revealed complex co-occurrence patterns between total, napA, and nosZ bacteria which fluctuated over time. A null model approach showed that, unlike the total community dominated by stochastic factors, napA and nosZ bacteria were primarily governed by deterministic processes. The level of determinism increased with nutrient loading, suggesting the denitrifying community can be manipulated by bioaugmentation. The dominant genus Ruegeria may be a promising candidate for introducing targeted denitrifiers into aquaculture systems to improve nitrogen removal. Overall, this study provides important ecological insights into aerobic and nitrous oxide-reducing denitrifiers in intensive aquaculture, supporting strategies to optimize microbial community structure and function.

Nitrogen fertilization promoted microbial growth and N2O emissions by increasing the abundance of nirS and nosZ denitrifiers in semiarid maize field.

Nitrous oxide (N2O) emissions are a major source of gaseous nitrogen loss, causing environmental pollution. The low organic content in the Loess Plateau region, coupled with the high fertilizer demand of maize, further exacerbates these N losses. N fertilizers play a primary role in N2O emissions by influencing soil denitrifying bacteria, however, the underlying microbial mechanisms that contribute to N2O emissions have not been fully explored. Therefore, the research aimed to gain insights into the intricate relationships between N fertilization, soil denitrification, N2O emissions, potential denitrification activity (PDA), and maize nitrogen use efficiency (NUE) in semi-arid regions. Four nitrogen (N) fertilizer rates, namely N0, N1, N2, and N3 (representing 0, 100, 200, and 300 kg ha-1 yr.-1, respectively) were applied to maize field. The cumulative N2O emissions were 32 and 33% higher under N2 and 37 and 39% higher under N3 in the 2020 and 2021, respectively, than the N0 treatment. N fertilization rates impacted the abundance, composition, and network of soil denitrifying communities (nirS and nosZ) in the bulk and rhizosphere soil. Additionally, within the nirS community, the genera Cupriavidus and Rhodanobacter were associated with N2O emissions. Conversely, in the nosZ denitrifier, the genera Azospirillum, Mesorhizobium, and Microvirga in the bulk and rhizosphere soil reduced N2O emissions. Further analysis using both random forest and structural equation model (SEM) revealed that specific soil properties (pH, NO3--N, SOC, SWC, and DON), and the presence of nirS-harboring denitrification, were positively associated with PDA activities, respectively, and exhibited a significant association to N2O emissions and PDA activities but expressed a negative effect on maize NUE. However, nosZ-harboring denitrification showed an opposite trend, suggesting different effects on these variables. Our findings suggest that N fertilization promoted microbial growth and N2O emissions by increasing the abundance of nirS and nosZ denitrifiers and altering the composition of their communities. This study provides new insights into the relationships among soil microbiome, maize productivity, NUE, and soil N2O emissions in semi-arid regions.

Ascription of nosZ gene, pH and copper for mitigating N2O emissions in acidic soils.

Soil nitrous oxide (N2O) emissions are alarming for global warming and climate change. N2O reduction is carried out only by nosZ gene encoded N2O-reductase, which is highly sensitive to acidic pH and copper (Cu) contents. Therefore, a microcosm study was conducted to examine the attribution of soil pH management, Cu supply and nosZ gene abundance for N2O emission mitigation. Cu was applied at the dose of 0, 10, 25 and 50 mg kg-1 to three acidic soils (Soil 1, 2 and 3) without and with dolomite (0 and 5 g kg-1). Cu application and soil pH increment substantially enlarged the abundance of nosZ gene, and consequently mitigated soil N2O emissions; highest reduction with 25 Cu mg kg-1. Decline in NH4+ and subsequently accumulation of NO3-, and large contents of MBC and DOC in dolomite treated soils led to a substantial N2O reduction. The cumulative N2O emissions were lowest in the treatment of 25 Cu mg kg-1 with dolomite application for each soil. Results suggest that soil pH increment, an adequate Cu supply, and nosZ gene abundance can potentially lower soil N2O emissions in acidic soils.

Using adaptive and aggressive N2O-reducing bacteria to augment digestate fertilizer for mitigating N2O emissions from agricultural soils.

Nitrous oxide (N2O) emitted from agricultural soils destroys stratospheric ozone and contributes to global warming. A promising approach to reduce emissions is fertilizing the soil using organic wastes augmented by non-denitrifying N2O-reducing bacteria (NNRB). To realize this potential, we need a suite of NNRB strains that fulfill several criteria: efficient reduction of N2O, ability to grow in organic waste, and ability to survive in farmland soil. In this study, we enriched such organisms by sequential anaerobic batch incubations with N2O and reciprocating inoculation between the sterilized substrates of anaerobic manure digestate and soils. 16S rDNA amplicon sequencing and metagenomics analysis showed that a cluster of bacteria containing nosZ genes encoding N2O-reductase, was enriched during the incubation process. Strains of several dominant members were then isolated and characterized, and three of them were found to harbor the nosZ gene but none of the other denitrifying genes, thus qualifying as NNRB. The selected isolates were tested for their capacities to reduce N2O emissions from three different typical Chinese farmland soils. The results indicated the significant mitigation effect of these isolates, even in very acidic red soil. In conclusion, this study demonstrated a strategy to engineer the soil microbiome with promising NNRB with high adaptability to livestock manure digestate as well as different agricultural soils, which would be suitable for developing novel fertilizer for farmland application to efficiently mitigate the N2O emissions from agricultural soils.

nZVI decreases N2O emission from pesticide-contaminated paddy soil.

The abundance and speciation of iron in farmland soils may affect the environmental fate of residual pesticides and their effects on soil N-cycling, which remains unclear. Herein, the roles of nanoscale zero-valent iron (nZVI) and iron oxidates (α-Fe2O3, γ-Fe2O3, and Fe3O4) as exogenous iron in reducing the adverse effect of pesticide pollution on soil N-cycling were firstly studied. It was found that the iron-based nanomaterials, especially nZVI, effectively reduced the N2O emission by 32.4-69.7 % at 5 g kg-1 from pentachlorophenol (PCP, as a representative pesticide, 100 mg kg-1) contaminated paddy soil, and remarkable N2O reduction (86.9 %) and PCP removal (60.9 %) were simultaneously achieved by applying 10 g kg-1 nZVI. Moreover, nZVI significantly mitigated the PCP-induced soil NO3--N accumulation and increased soil NH4+-N. Mechanistically, the nZVI restored the activities of nitrate- and N2O- reductases and the abundances of N2O-reducing microorganisms in the PCP-contaminated soil. Moreover, the nZVI suppressed N2O-producing fungi while promoting soil bacteria (especially nosZ-II bacteria) to increase N2O consumption in the soil. This study provides a strategy for adding iron-based nanomaterials to mitigate the adverse effects of pesticide residues on soil N-cycling and provides basic data for subsequent understanding of the effects of iron cycling in paddy soils on pesticide residues and N-cycling.