N2O-reducing Bacteria Research Articles

Diversity and Activity of Soil N2O-Reducing Bacteria Shaped by Urbanization.

Nitrous oxide (N2O) is a potent greenhouse gas with various production pathways. N2O reductase (N2OR) is the primary N2O sink, but the distribution of its gene clades, typically nosZI and atypically nosZII, along urbanization gradients remains poorly understood. Here we sampled soils from forests, parks, and farmland across eight provinces in eastern China, using high-throughput sequencing to distinguish between two N2O-reducing bacteria clades. A deterministic process mainly determined assemblies of the nosZI communities. Homogeneous selection drove nosZI deterministic processes, and both homogeneous and heterogeneous selection influenced nosZII. This suggests nosZII is more sensitive to environmental changes than nosZI, with significant changes in community structure over time or space. Ecosystems with stronger anthropogenic disturbance, such as urban areas, provide diverse ecological niches for N2O-reducing bacteria (especially nosZII) to adapt to environmental fluctuations. Structural equation modeling (SEM) and correlation analyses revealed that pH significantly influences the community composition of both N2O-reducing bacteria clades. This study underscores urbanization's impact on N2O-reducing bacteria in urban soils, highlighting the importance of nosZII and survival strategies. It offers novel insights into the role of atypical denitrifiers among N2O-reducing bacteria, underscoring their potential ecological importance in mitigating N2O emissions from urban soils.

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.

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.

Benthic clade II-type nitrous oxide reducers suppress nitrous oxide emissions in shallow lakes

Shallow lakes, recognized as hotspots for nitrogen cycling, contribute to the emission of the potent greenhouse gas nitrous oxide (N2O), but the current emission estimates for this gas have a high degree of uncertainty. However, the role of N2O-reducing bacteria (N2ORB) as N2O sinks and their contribution to N2O reduction in aquatic ecosystems in response to N2O dynamics have not been determined. Here, we investigated the N2O dynamics and microbial processes in the nitrogen cycle, which included both N2O production and consumption, in five shallow lakes spanning approximately 500 km. The investigated sites exhibited N2O oversaturation, with excess dissolved N2O concentrations (ΔN2O) ranging from 0.55 ± 0.61 to 53.17 ± 15.75 nM. Sediment-bound N2O (sN2O) was significantly positively correlated with the nitrate concentration in the overlying water (p < 0.05), suggesting that nitrate accumulation contributes to benthic N2O generation. High N2O consumption activity (RN2O) corresponded to low ΔN2O. In addition, a significant negative correlation was found between RN2O and nir/nosZ, showing that bacteria encoding nosZ contributed to N2O consumption in the benthic sediments. Redundancy analysis indicated that benthic functional genes effectively reflected the variations in RN2O and ∆N2O. qPCR analysis revealed that the clade II nosZ gene was more sensitive to ΔN2O than the clade I nosZ gene. Furthermore, four novel genera of potential nondenitrifying N2ORB were identified based on metagenome-assembled genome analysis. These genera, which are affiliated with clade II, lack genes responsible for N2O production. Collectively, benthic N2ORB, especially for clade II-type N2ORB, harnesses N2O consumption activity leading to low N2O emissions from shallow lakes. This study advances our knowledge of the role of benthic clade II-type N2ORB in regulating N2O emissions in shallow lakes.

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.

Natural chalcopyrite mitigates nitrous oxide emissions in sediment from coastal wetlands

Coastal wetlands are one of the most important natural sources of nitrous oxide (N2O). Previous studies have shown that copper-containing chemicals are able to reduce N2O emissions from these ecosystems. However, these chemicals may harm organisms present in coastal waters and sediment, and disturb the ecological balance of these areas. Here, we first investigated the physiological characteristics and genetic potential of denitrifying bacteria isolated from coastal wetlands. Based on an isolated denitrifier carrying a complete denitrification pathway, we tested the effect of the natural mineral chalcopyrite on N2O production by the bacteria. The results demonstrated that chalcopyrite addition lowers N2O emissions from the bacteria while increasing its N2 production rate. Among the four denitrification genes of the isolate, only nosZ gene expression was significantly upregulated following the addition of 2 mg L−1 chalcopyrite. Furthermore, chalcopyrite was applied to coastal wetland sediments. The N2O flux was significantly reduced in 50–100 mg L−1 chalcopyrite-amended sets relative to the controls. Notably, the dissolved Cu concentration in chalcopyrite-amended sediment remained within the limit set by the National Sewage Treatment Discharge Standard. qPCR and metagenomic analysis revealed that the abundance of N2O-reducing bacteria with the nosZ or nirK + nosZ genotype increased significantly in the chalcopyrite-amended groups relative to the controls, suggesting their active involvement in the reduction of N2O emissions. Our findings offer valuable insights for the use of natural chalcopyrite in large-scale field applications to reduce N2O emissions.

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.

NosZ–II–type N2O-reducing bacteria play dominant roles in determining the release potential of N2O from sediments in the Pearl River Estuary, China

The microbial reduction of N2O serves as a “gatekeeper” for N2O emissions, determining the flux of N2O release into the atmosphere. Estuaries are active regions for N2O emissions, but the microbial functions of N2O-reducing bacteria in estuarine ecosystems are not well understood. In this study, the 15N isotope tracer method, qPCR, and high-throughput sequencing were used to analyze N2O production, reduction, and emission processes in surface sediments of the Pearl River Estuary. The 15N isotope tracer experiment showed that the N2O production rates declined and the N2O reduction potential (Rr, the ratio of N2O reduction rates to N2O production rates) increased from upstream to downstream of the Pearl River Estuary, leading to a corresponding decrease of the N2O emission rates from upstream to downstream. The gene abundance ratio of nosZ/nir gradually increased from upstream to downstream and was negatively correlated with the water N2O saturation. The gene abundance of nosZ II was significantly higher than that of nosZ I in the estuary, and the nosZ II/nosZ I abundance ratio was positively correlated with N2O reduction potential. Furthermore, the community composition of NosZ-I- and NosZ–II–type N2O-reducing bacteria shifted from upstream to downstream. NosZ–II–type N2O-reducing bacteria, especially Myxococcales, Thiotrichales, and Gemmatimonadetes species, contributed to the high N2O reduction potential in the downstream. Our results suggest that NosZ–II–type N2O-reducing bacteria play a dominant role in determining the release potential of N2O from sediments in the Pearl River Estuary. This study provides a new insight into the function of microbial N2O reduction in estuarine ecosystems.

Clarifying Microbial Nitrous Oxide Reduction under Aerobic Conditions: Tolerant, Intolerant, and Sensitive.

One of the major challenges for the bioremediation application of microbial nitrous oxide (N2O) reduction is its oxygen sensitivity. While a few strains were reported capable of reducing N2O under aerobic conditions, the N2O reduction kinetics of phylogenetically diverse N2O reducers are not well understood. Here, we analyzed and compared the kinetics of clade I and clade II N2O-reducing bacteria in the presence or absence of oxygen (O2) by using a whole-cell assay with N2O and O2 microsensors. Among the seven strains tested, N2O reduction of Stutzerimonas stutzeri TR2 and ZoBell was not inhibited by oxygen (i.e., oxygen tolerant). Paracoccus denitrificans, Azospirillum brasilense, and Gemmatimonas aurantiaca reduced N2O in the presence of O2 but slower than in the absence of O2 (i.e., oxygen sensitive). N2O reduction of Pseudomonas aeruginosa and Dechloromonas aromatica did not occur when O2 was present (i.e., oxygen intolerant). Amino acid sequences and predicted structures of NosZ were highly similar among these strains, whereas oxygen-tolerant N2O reducers had higher oxygen consumption rates. The results suggest that the mechanism of O2 tolerance is not directly related to NosZ structure but is rather related to the scavenging of O2 in the cells and/or accessory proteins encoded by the nos cluster. IMPORTANCE Some bacteria can reduce N2O in the presence of O2, whereas others cannot. It is unclear whether this trait of aerobic N2O reduction is related to the phylogeny and structure of N2O reductase. The understanding of aerobic N2O reduction is critical for guiding emission control, due to the common concurrence of N2O and O2 in natural and engineered systems. This study provided the N2O reduction kinetics of various bacteria under aerobic and anaerobic conditions and classified the bacteria into oxygen-tolerant, -sensitive, and -intolerant N2O reducers. Oxygen-tolerant N2O reducers rapidly consumed O2, which could help maintain the low O2 concentration in the cells and keep their N2O reductase active. These findings are important and useful when selecting N2O reducers for bioremediation applications.

Mycorrhiza-mediated recruitment of complete denitrifying Pseudomonas reduces N2O emissions from soil

BackgroundArbuscular mycorrhizal fungi (AMF) are key soil organisms and their extensive hyphae create a unique hyphosphere associated with microbes actively involved in N cycling. However, the underlying mechanisms how AMF and hyphae-associated microbes may cooperate to influence N2O emissions from “hot spot” residue patches remain unclear. Here we explored the key microbes in the hyphosphere involved in N2O production and consumption using amplicon and shotgun metagenomic sequencing. Chemotaxis, growth and N2O emissions of isolated N2O-reducing bacteria in response to hyphal exudates were tested using in vitro cultures and inoculation experiments.ResultsAMF hyphae reduced denitrification-derived N2O emission (max. 63%) in C- and N-rich residue patches. AMF consistently enhanced the abundance and expression of clade I nosZ gene, and inconsistently increased that of nirS and nirK genes. The reduction of N2O emissions in the hyphosphere was linked to N2O-reducing Pseudomonas specifically enriched by AMF, concurring with the increase in the relative abundance of the key genes involved in bacterial citrate cycle. Phenotypic characterization of the isolated complete denitrifying P. fluorescens strain JL1 (possessing clade I nosZ) indicated that the decline of net N2O emission was a result of upregulated nosZ expression in P. fluorescens following hyphal exudation (e.g. carboxylates). These findings were further validated by re-inoculating sterilized residue patches with P. fluorescens and by an 11-year-long field experiment showing significant positive correlation between hyphal length density with the abundance of clade I nosZ gene.ConclusionsThe cooperation between AMF and the N2O-reducing Pseudomonas residing on hyphae significantly reduce N2O emissions in the microsites. Carboxylates exuded by hyphae act as attractants in recruiting P. fluorescens and also as stimulants triggering nosZ gene expression. Our discovery indicates that reinforcing synergies between AMF and hyphosphere microbiome may provide unexplored opportunities to stimulate N2O consumption in nutrient-enriched microsites, and consequently reduce N2O emissions from soils. This knowledge opens novel avenues to exploit cross-kingdom microbial interactions for sustainable agriculture and for climate change mitigation.C1gn2zbPQXat79TnoeAsNQVideo

Comparison of N2O-reducing abilities and genome features of two nosZ-containing denitrifying bacteria, Pseudomonas veronii DM15 and Pseudomonas frederiksbergensis DM22.

The study systematically compared the N2O-reducing functional performances and the genomic features of two N2O-reducing isolates, aimed to screen out effective N2O-reducing bacteria with strong environmental adaption, and explore the possible regulation. Two N2O reducers, namely, Pseudomonas veronii DM15 (DM15) and Pseudomonas frederiksbergensis DM22 (DM22), isolated from paddy soil were selected. Their N2O-reducing abilities, and nosZ gene transcript abundance were determined under different temperatures (20°C, 30°C, 40°C) and oxygen concentrations (0%, 10%, 21%), and the whole genomes were sequenced by Illumina sequencing. The results showed that both DM15 and DM22 exhibited the strongest N2O reducing activity at 30°C and under anaerobic conditions. In comparison, DM15 generally exhibited significantly higher N2O reducing abilities and nosZ gene expression than DM22 under all tested conditions. In addition, DM15 possessed obviously higher expression potentials (codon adaptation index (CAI) value) of nos genes than DM22, and the nos cluster of the former contained a transcriptional regulator gene of dnr, while the latter did not. The results indicate that DM15 showed obviously stronger N2O-reducing abilities than DM22 under various conditions, which might be closely associated with its dnr transcriptional regulator, and thus promoting the higher transcriptional activities of nos genes. Although anaerobic conditions were the optimal conditions for N2O reduction in both strains, DM15 still reduced a certain amount of N2O even under aerobic conditions.

Hydrochar amendments stimulate soil nitrous oxide emission by increasing production of hydroxyl radicals and shifting nitrogen functional genesin the short term: A culture experiment.

The application of waste biomass-derived hydrochar to soil may cause extremely intensive nitrous oxide (N2O) fluxes that can challenge our current mechanistic understanding of the global nitrogen cycle in the biosphere. In this study, two waste biomasses were used to prepare cyanobacterial biomas-derived hydrochar (CHC) and wheat straw-derived hydrochar (SHC) for short-term incubation experiments to identify their effects and mechanisms of waste biomass-derived hydrochar on soil N2O efflux, with time-series samples collected for N2O efflux and soil analysis. The results showed that CHC and SHC caused short-term bursts of N2O effluxes without nitrogen inputs. Moreover, the enrichment of exogenous organics and nutrients at the hydrochar-soil interface was identified as the key factor for enhancing N2O fluxes, which stimulated microbial nitrification (i.e., increased gene copy number of ammonia oxidizing bacteria) and denitrification (i.e., increased gene copy number of nitrate and N2O reducing bacteria) processes. The concentrations of Fe (II) and hydroxyl radicals (HO•) were 6.49 and 5.63 times higher, respectively, in the hydrochar layer of CHC than SHC amendment. Furthermore, structural equation models demonstrated that HO•, as well as soil microbiomes, played an important role in driving N2O fluxes. Together, our findings provide a deeper insight into the assessment and prognosis of the short-term environmental risk arising from agricultural waste management in integrated agriculture. Further studies under practical field application conditions are warranted to verify the findings.

Differential response of N2O emissions, N2O-producing and N2O-reducing bacteria to varying tetracycline doses in fertilized soil

Tetracyclines are the most widely used antibiotics worldwide. Their presence in soils could affect nutrient cycling, but our knowledge regarding how they affect soil microbial communities involved in greenhouse gas emissions is limited. The objective of the current study was to evaluate how tetracycline is affecting N2O emissions and the abundance of denitrifiers in fertilized soil. For this purpose, soil mesocosms were treated with only NH4NO3 (100 mg/kg) or NH4NO3 (100 mg/kg) plus three different doses of tetracycline (0.1, 0.5 and 2 mg/kg). Soils that did not receive tetracycline or NH4NO3 were used as controls. Nitrous oxide fluxes were monitored daily for 16 days. The total bacterial (16S rRNA), the abundance of N2O-reducing and -producing bacteria were quantified by qPCR at the end of the experiment. The application of NH4NO3 caused a significant increase of N2O emissions and AOB abundance but did not affect the abundance of denitrifiers and AOA compared to control soils. Different doses of tetracycline in fertilized soils did not mitigate these N2O emissions; instead, higher cumulative emissions were noticed in soils treated with the lowest dose. In these soils the total bacterial abundance was higher compared to soils received higher tetracycline concentration. The abundances of the N2O-producing and N2O-reducing communities were also differently affected by the addition of tetracycline, which was dose-dependent. Higher doses of tetracycline favored N2O-reducers within the total bacterial community, which could be important for mitigating N2O emissions in the long term.

Distinct Denitrifying Phenotypes of Predominant Bacteria Modulate Nitrous Oxide Metabolism in Two Typical Cropland Soils.

Denitrifying nitrous oxide (N2O) emissions in agroecosystems result from variations in microbial composition and soil properties. However, the microbial mechanisms of differential N2O emissions in agricultural soils are less understood. In this study, microcosm experiments using two main types of Chinese cropland soil were conducted with different supplements of nitrate and glucose to simulate the varying nitrogen and carbon conditions. The results show that N2O accumulation in black soil (BF) was significantly higher than that in fluvo-aquic soil (FF) independent of nitrogen and carbon. The abundance of most denitrifying genes was significantly higher in FF, but the ratios of genes responsible for N2O production (nirS and nirK) to the gene responsible for N2O reduction (nosZ) did not significantly differ between the two soils. However, the soils showed obvious discrepancies in denitrifying bacterial communities, with a higher abundance of N2O-generating bacteria in BF and a higher abundance of N2O-reducing bacteria in FF. High accumulation of N2O was verified by the bacterial isolates of Rhodanobacter predominated in BF due to a lack of N2O reduction capacity. The dominance of Castellaniella and others in FF led to a rapid reduction in N2O and thus less N2O accumulation, as demonstrated when the corresponding isolate was inoculated into the studied soils. Therefore, the different phenotypes of N2O metabolism of the distinct denitrifiers predominantly colonized the two soils, causing differing N2O accumulation. This knowledge would help to develop a strategy for mitigating N2O emissions in agricultural soils by regulating the phenotypes of N2O metabolism.

Exploring the Functions of Efficient Canonical Denitrifying Bacteria as N2O Sinks: Implications from 15N Tracer and Transcriptome Analyses.

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.

Nitrite- and N2O-reducing bacteria respond differently to ecological factors in saline lakes.

The distribution of nitrite- and N2O-reducing bacteria is key to potential N2O emission from lakes. However, such information in highland saline lakes remains unknown. Here, we investigated the abundance and community composition of nitrite- and N2O-reducing bacteria in the sediments of six saline lakes on the Qing-Tibetan Plateau. The studied lakes covered a wide range of salinity (1.0-340.0g/L). Results showed that in the studied saline lake sediments, nitrite-reducing bacteria were significantly more abundant than N2O-reducing bacteria, and their abundances ranged 7.14 × 103-8.26 × 108 and 1.18 × 106-6.51 × 107 copies per gram sediment (dry weight), respectively. Nitrite-reducing bacteria were mainly affiliated with α-, β- and γ-Proteobacteria, with β- and α-Proteobacteria being dominant in low- and high-salinity lakes, respectively; N2O-reducing bacterial communities mainly consisted of Proteobacteria (α-, β-, γ- and δ-subgroups), Bacteroidetes, Verrucomicrobia, Actinobacteria, Chloroflexi, Gemmatimonadetes and Balneolaeota, with Proteobacteria and Bacteroidetes/Verrucomicrobia dominating in low- and high-salinity lakes, respectively. The nitrite- and N2O-reducing bacterial communities showed distinct responses to ecological factors, and they were mainly regulated by mineralogical and physicochemical factors, respectively. In response to salinity change, the community composition of nitrite-reducing bacteria was more stable than that of N2O-reducing bacteria. These findings suggest that nitrite- and N2O-reducing bacteria may prefer niches with different salinity.

Organic carbon determines nitrous oxide consumption activity of clade I and II nosZ bacteria: Genomic and biokinetic insights

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.

Nitrous oxide-sink capability of denitrifying bacteria impacted by nitrite and pH

Nitrous oxide (N2O) is a highly potent greenhouse gas and ozone-depleting substance, produced and consumed during denitrification. Evaluation of the N2O production and consumption activities of complete denitrifying bacteria is essential for understanding their capacity to act as N2O sinks in engineered systems for cost-effective nitrogen removal via nitrite (NO2−). However, the physiologies of these N2O-reducing bacteria (N2ORB) are poorly understood. This study aimed to evaluate the physiologies of two N2ORB, Azospira sp. strain I13 and Alicycliphilus denitrificans strain I51. A 15N tracer method was applied to determine N2O production and consumption activities in the co-presence of NO2− and N2O. Both N2ORB displayed a higher N2O consumption rate (RN2O by Azospira sp. strain I13 and Alicycliphilus denitrificans strain I51, 23.85 and 7.60 μmol-N mg-biomass−1h−1, respectively) than N2O production rate (PN2O, 5.88 and 1.32 μmol-N mg-biomass−1h−1, respectively) at an initial NO2− concentration of 2.14 mmol-N L−1 with exogenous addition of N2O, indicating that these N2ORB acted as N2O sinks. On increasing the NO2− concentration from 0.36 to 7.14 mmol-N L−1, the net N2O consumption rate RO_N2O (=RN2O – PN2O) decreased for both N2ORB; the magnitude of the decrease was greater for Azospira sp. strain I13 than for Alicycliphilus denitrificans strain I51. The formation of free nitrous acid (FNA) from NO2− in acidic conditions noticeably affected the N2O sink activities of the N2ORB. A higher FNA concentration decreased RO_N2O for both N2ORB, creating the risk of N2O emission at pH 6 and high NO2− concentration. Our results show the ranges of pH and NO2− concentration where Azospira sp. strain I13 is promising for use as an N2O sink in shortcut nitrogen removal via NO2−.

Combination of 15N Tracer and Microbial Analyses Discloses N2O Sink Potential of the Anammox Community.

Although nitrogen removal by partial nitritation and anammox is more cost-effective than conventional nitrification and denitrification, one downside is the production and accumulation of nitrous oxide (N2O). The potential exploitation of N2O-reducing bacteria, which are resident members of anammox microbial communities, for N2O mitigation would require more knowledge of their ecophysiology. This study investigated the phylogeny of resident N2O-reducing bacteria in an anammox microbial community and quantified individually the processes of N2O production and N2O consumption. An up-flow column-bed anammox reactor, fed with NH4+ and NO2- and devoid of oxygen, emitted N2O at an average conversion ratio (produced N2O: influent nitrogen) of 0.284%. Transcriptionally active and highly abundant nosZ genes in the reactor biomass belonged to the Burkholderiaceae (clade I type) and Chloroflexus genera (clade II type). Meanwhile, less abundant but actively transcribing nosZ strains were detected in the genera Rhodoferax, Azospirillum, Lautropia, and Bdellovibrio and likely act as an N2O sink. A novel 15N tracer method was adapted to individually quantify N2O production and N2O consumption rates. The estimated true N2O production rate and true N2O consumption rate were 3.98 ± 0.15 and 3.03 ± 0.18 mgN·gVSS-1·day-1, respectively. The N2O consumption rate could be increased by 51% (4.57 ± 0.51 mgN·gVSS-1·day-1) with elevated N2O concentrations but kept comparable irrespective of the presence or absence of NO2-. Collectively, the approach allowed the quantification of N2O-reducing activity and the identification of transcriptionally active N2O reducers that may constitute as an N2O sink in anammox-based processes.

Revisiting the involvement of ammonia oxidizers and denitrifiers in nitrous oxide emission from cropland soils

Nitrous oxide (N2O), an ozone-depleting greenhouse gas, is generally produced by soil microbes, particularly NH3 oxidizers and denitrifiers, and emitted in large quantities after N fertilizer application in croplands. N2O can be produced via multiple processes, and reduced, with the involvement of more diverse microbes with different physiological constraints than previously thought; therefore, there is a lack of consensus on the production processes and microbes involved under different agricultural practices. In this study, multiple approaches were applied, including N2O isotopocule analyses, microbial gene transcript measurements, and selective inhibition assays, to revisit the involvement of NH3 oxidizers and denitrifiers, including the previously-overlooked taxa, in N2O emission from a cropland, and address the biological and environmental factors controlling the N2O production processes. Then, we synthesized the results from those approaches and revealed that the overlooked denitrifying bacteria and fungi were more involved in N2O production than the long-studied ones. We also demonstrated that the N2O production processes and soil microbes involved were different based on fertilization practices (plowing or surface application) and fertilization types (manure or urea). In particular, we identified the following intensified activities: (1) N2O production by overlooked denitrifying fungi after manure fertilization onto soil surface; (2) N2O production by overlooked denitrifying bacteria and N2O reduction by long-studied N2O-reducing bacteria after manure fertilization into the plowed layer; and (3) N2O production by NH3-oxidizing bacteria and overlooked denitrifying bacteria and fungi when urea fertilization was applied into the plowed layer. We finally propose the conceptual scheme of N flow after fertilization based on distinct physiological constraints among the diverse NH3 oxidizers and denitrifiers, which will help us understand the environmental context-dependent N2O emission processes.