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Nitrous Oxide Reduction Kinetics Distinguish Bacteria Harboring Clade I NosZ from Those Harboring Clade II NosZ
Abstract
Bacteria capable of reduction of nitrous oxide (N2O) to N2 separate into clade I and clade II organisms on the basis of nos operon structures and nosZ sequence features. To explore the possible ecological consequences of distinct nos clusters, the growth of bacterial isolates with either clade I (Pseudomonas stutzeri strain DCP-Ps1, Shewanella loihica strain PV-4) or clade II (Dechloromonas aromatica strain RCB, Anaeromyxobacter dehalogenans strain 2CP-C) nosZ with N2O was examined. Growth curves did not reveal trends distinguishing the clade I and clade II organisms tested; however, the growth yields of clade II organisms exceeded those of clade I organisms by 1.5- to 1.8-fold. Further, whole-cell half-saturation constants (Kss) for N2O distinguished clade I from clade II organisms. The apparent Ks values of 0.324 ± 0.078 μM for D. aromatica and 1.34 ± 0.35 μM for A. dehalogenans were significantly lower than the values measured for P. stutzeri (35.5 ± 9.3 μM) and S. loihica (7.07 ± 1.13 μM). Gen
... Measurements of whole-cell N 2 O reduction kinetics yielded significantly lower apparent half-saturation constants for the microorganisms harboring clade II nosZ, e.g., Dechloromonas spp. and Azospira spp., than those harboring clade I nosZ [16,18]. The microorganisms possessing clade II nosZ were selectively enriched in bioreactors of different configurations where biological reduction of low-concentration N 2 O was observed [19,20]. ...
... Identification of the active sinks of N 2 O is crucial for understanding of dynamics and emission of N 2 O in natural and built environments [17,18,66,67]. This study, by combining culturebased experiments with metagenome and metatranscriptome analyses, identified the groups of microorganisms that are likely to function as such crucial N 2 O sinks. ...
... These observations were consistent with the previous measurements of N 2 O consumption kinetics, where Dechloromonas spp. and Azospira spp., possessing nosZ belonging to NosZG5 consistently exhibited the lowest half-saturation constants among diverse N 2 O-reducing microorganisms [16,18,69]. ...
Microorganisms possessing N2O reductases (NosZ) are the only known environmental sink of N2O. While oxygen inhibition of NosZ activity is widely known, environments where N2O reduction occurs are often not devoid of O2. However, little is known regarding N2O reduction in microoxic systems. Here, 1.6-L chemostat cultures inoculated with activated sludge samples were sustained for ca. 100 days with low concentration (<2 ppmv) and feed rate (<1.44 µmoles h−1) of N2O, and the resulting microbial consortia were analyzed via quantitative PCR (qPCR) and metagenomic/metatranscriptomic analyses. Unintended but quantified intrusion of O2 sustained dissolved oxygen concentration above 4 µM; however, complete N2O reduction of influent N2O persisted throughout incubation. Metagenomic investigations indicated that the microbiomes were dominated by an uncultured taxon affiliated to Burkholderiales, and, along with the qPCR results, suggested coexistence of clade I and II N2O reducers. Contrastingly, metatranscriptomic nosZ pools were dominated by the Dechloromonas-like nosZ subclade, suggesting the importance of the microorganisms possessing this nosZ subclade in reduction of trace N2O. Further, co-expression of nosZ and ccoNO/cydAB genes found in the metagenome-assembled genomes representing these putative N2O-reducers implies a survival strategy to maximize utilization of scarcely available electron acceptors in microoxic environmental niches.
... Energy conservation via N 2 O reduction was implied in the observed cell growth in the W. succinogens (the nosZ + variant), A. dehalogenans, and B. vireti cultures fed N 2 O as the sole electron acceptor together with a non-fermentable electron donor (27,32,33). Apparent from these observations, N 2 O-reducing capability would benefit the DNRA-cat alyzing organisms by enabling them to utilize the fugitive N 2 O from DNRA, as well as N 2 O released from other organisms in their habitat (27,34,35). Perhaps, as the nosZ genes these organisms harbor mostly belong to the clade II, which, in general, tend to exhibit higher affinities to N 2 O, the possession of nosZ and the capability to capitalize on sub-micromolar N 2 O may even be crucial for their survival in environmental niches unfavorable for DNRA in competing with denitrifiers (27,34). ...
... Apparent from these observations, N 2 O-reducing capability would benefit the DNRA-cat alyzing organisms by enabling them to utilize the fugitive N 2 O from DNRA, as well as N 2 O released from other organisms in their habitat (27,34,35). Perhaps, as the nosZ genes these organisms harbor mostly belong to the clade II, which, in general, tend to exhibit higher affinities to N 2 O, the possession of nosZ and the capability to capitalize on sub-micromolar N 2 O may even be crucial for their survival in environmental niches unfavorable for DNRA in competing with denitrifiers (27,34). ...
Climate change and nutrient pollution are among the most urgent environmental issues. Enhancing the abundance and/or the activity of beneficial organisms is an attractive strategy to counteract these problems. Dissimilatory nitrate reduction to ammonium (DNRA), which theoretically improves nitrogen retention in soils, has been suggested as a microbial process that may be harnessed, especially since many DNRA-catalyzing organisms have been found to possess nosZ genes and the ability to respire N 2 O. However, the selective advantage that may favor these nosZ -harboring DNRA-catalyzing organisms is not well understood. Here, the effect of N 2 O on Nrf-mediated DNRA was examined in a soil isolate, Bacillus sp. DNRA2, possessing both nrfA and nosZ genes. The DNRA metabolism of this bacterium was observed in the presence of C 2 H 2, a NosZ inhibitor, with or without N 2 O, and the results were compared with C 2 H 2 -free controls. Cultures were also exposed to repeated oxic-anoxic transitions in the sustained presence of N 2 O. The NO 2 ⁻ -to-NH 4 ⁺ reduction following oxic-to-anoxic transition was significantly delayed in NosZ-inhibited C 2 H 2 -amended cultures, and the inhibition was more pronounced with repeated oxic-anoxic transitions. The possibility of C 2 H 2 involvement was dismissed since the cultures continuously flushed with C 2 H 2 /N 2 mixed gas after initial oxic incubation did not exhibit a similar delay in DNRA progression as that observed in the culture flushed with N 2 O-containing gas. The findings suggest a possibility that the oft-observed nosZ presence in DNRA-catalyzing microorganisms secures an early transcription of their DNRA genes by scavenging N 2 O, thus enhancing their capacity to compete with denitrifiers at oxic-anoxic interfaces.
IMPORTANCE
Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is a microbial energy-conserving process that reduces NO 3 ⁻ and/or NO 2 ⁻ to NH 4 ⁺ . Interestingly, DNRA-catalyzing microorganisms possessing nrfA genes are occasionally found harboring nosZ genes encoding nitrous oxide reductases, i.e., the only group of enzymes capable of removing the potent greenhouse gas N 2 O. Here, through a series of physiological experiments examining DNRA metabolism in one of such microorganisms, Bacillus sp. DNRA2, we have discovered that N 2 O may delay the transition to DNRA upon an oxic-to-anoxic transition, unless timely removed by the nitrous oxide reductases. These observations suggest a novel explanation as to why some nrfA -possessing microorganisms have retained nosZ genes: to remove N 2 O that may otherwise interfere with the transition from O 2 respiration to DNRA.
... In addition, a significantly increasing trend of nosZ II-type N 2 O reducers was found from upstream to downstream (one-way ANOVA, p < 0.01), which was similar to the findings in sediments of estuaries . In contrast to nosZ I-type N 2 O reducers, nosZ II-type N 2 O reducers have lower whole-cell half-saturation constants for N 2 O (low K s ), and thus have a higher competitive edge under low N 2 O conditions (Yoon et al., 2016). The higher N 2 O affinity allows nosZ II-type N 2 O reducers to occupy a lower N 2 O concentration niche downstream and contribute to a steady background rate of N 2 O reduction. ...
... The higher N 2 O affinity allows nosZ II-type N 2 O reducers to occupy a lower N 2 O concentration niche downstream and contribute to a steady background rate of N 2 O reduction. nosZ II-type N 2 O reducers produce more energy and biomass when reduced equal N 2 O (Yoon et al., 2016). In addition, the higher diversity of nosZ II type N 2 O reducers may increase their adaptability to unstable environments driven by freshwater and seawater exchange in estuarine ecosystems. ...
Denitrification is the dominant process of nitrogen removal and nitrous oxide (N2O) emissions in estuarine ecosystems. However, little is known regarding the microbial mechanism of the production and reduction of N2O in estuaries. We investigated in situ dissolved N2O as well as potential N2O production rate (NPR), reduction rate (NRR), and emission rate (NER), and key functional genes related to N2O transformation of denitrification in the Pearl River Estuary. Higher N2O emission potential was found in the upstream and midstream regions with higher NPR and lower NRR values. In contrast, higher NRR values were detected in downstream. Notably, nirS and nirK type N2O producers dominated the upstream zone, whereas abundant N2O reducers, especially nosZ II type N2O reducers, were observed in downstream. Most importantly, the gene abundance ratio (Rnir/nosZ) was significantly correlated with the N2O emission potential (Re). Niche differentiation between N2O producers and N2O reducers from upstream to downstream affected N2O emission potential. This study highlights the N2O emission potential in estuarine sediments is determined by an imbalance between N2O production and the reduction of multi-bacterial communities.
... Several studies have reported the physiological differences between the two clades. Yoon et al. (14) report that clade II bacteria (Dechloromonas aromatica and Anaeromyxobacter dehalogenans) showed high affinities to N 2 O but lower maximum reduction rates than those of clade I bacteria (Stutzerimonas stutzeri, formerly known as Pseudomonas stutzeri [15], and Shewanella loihica). In contrast, Suenaga et al. (3) found that the N 2 O reduction biokinetics could not be used to distinguish the clade I bacteria (S. stutzeri and Paracoccus denitrificans) and clade II bacteria studied (Azospira spp.). ...
... Moreover, the half-saturation coefficients for N 2 O under anaerobic and aerobic conditions agree with previously reported observations. Bacteria harboring clade II NosZ generally have lower K m values than those with clade I NosZ, suggesting differentiating ecological niches for these two groups of N 2 Oreducing bacteria (14). ...
Some bacteria can reduce N 2 O in the presence of O 2 , whereas others cannot. It is unclear whether this trait of aerobic N 2 O reduction is related to the phylogeny and structure of N 2 O reductase.
... Exploring the niche preference and driving factors of microorganisms containing nosZ I and nosZ II is essential for improving management strategies to mitigate N 2 O emissions (Shan et al., 2021). Microorganisms containing nosZ I and nosZ II were found to have different substrate affinities, with nosZ I-carrying microorganisms having a relatively lower substrate affinity (Yoon et al., 2016). Niche differentiation of nosZ I and nosZ II carrying microorganisms has been frequently found in agricultural soils, with nosZ II being more prevalent in soils, whereas nosZ I predominated on roots (Graf et al., 2016;Zhao et al., 2017;Graf et al., 2019). ...
... However, the niche preference of nosZ I and nosZ II carrying microorganisms was less studied (Shan et al., 2021). Some pioneer studies showed that nosZ I-carrying microorganisms had a lower substrate affinity than nosZ II (Yoon et al., 2016), which might restrict nosZ I-carrying microorganisms to the environments with higher N 2 O concentrations while nosZ IIcarrying microorganisms were better adapted to the habitats with a low N 2 O concentration (Shan et al., 2021). This is supported by the finding that the abundance of nosZ I, rather than nosZ II, was positively correlated to soil dissolved organic N (Juhanson et al., 2017), indicating a niche differentiation of nosZ I and nosZ II in agricultural soils. ...
Nitrous oxide (N2O) reducers are the only known sink for N2O and pivotal contributors to N2O mitigation in terrestrial and water ecosystems. However, the niche preference of nosZ I and nosZ II carrying microorganisms, two divergent clades of N2O reducers in coastal wetlands, is not yet well documented. In this study, we investigated the abundance, community structure and co-occurrence network of nosZ I and nosZ II carrying microorganisms and their driving factors at three depths in a subtropical coastal wetland with five plant species and a bare tidal flat. The taxonomic identities differed between nosZ I and nosZ II carrying microorganisms, with nosZ I sequences affiliated with Alphaproteobacteria and Betaproteobacteria while nosZ II sequences with Gemmatimonadetes, Verrucomicrobia, Gammaproteobacteria, and Chloroflexi. The abundances of nosZ I and nosZ II decreased with increasing soil depths, and were positively associated with salinity, total carbon (TC) and total nitrogen (TN). Random forest analysis showed that salinity was the strongest predictor for the abundances of nosZ I and nosZ II. Salinity, TC and TN were the major driving forces for the community structure of nosZ I and nosZ II carrying microorganisms. Moreover, co-occurrence analysis showed that 92.2 % of the links between nosZ I and nosZ II were positive, indicating that nosZ I and nosZ II carrying microorganisms likely shared similar ecological niches. Taken together, we provided new evidence that nosZ I and nosZ II carrying microorganisms shared similar ecological niches in a subtropical estuarine wetland, and identified salinity, TC and TN serving as the most important environmental driving forces. This study advances our understanding of the environmental adaptation and niche preference of nosZ I and nosZ II carrying microorganisms in coastal wetlands.
... Recent molecular analyses, including quantitative PCR (qPCR), an amplicon sequencing analysis of nosZ clade II, and a metagenomic analysis of soil DNA, revealed the abundant distribution of nosZ clade II in various types of soils, similar to nosZ clade I (Sanford et al., 2012;Jones et al., 2013;Jones et al., 2014;Orellana et al., 2014;Domeignoz-Horta et al., 2015;Samad et al., 2016;Juhanson et al., 2017). In addition to their phylogenetic differences, N 2 O reducers carrying nosZ clade II showed an affinity for N 2 O that was up to two orders of magnitude higher than those carrying nosZ clade I (Yoon et al., 2016;Suenaga et al., 2018), suggesting their significant contribution to N 2 O mitigation from soils because the concentration of N 2 O is generally low in soils (i.e., typically less than 1 μM) (Schreiber et al., 2012). A linear regression analysis (Domeignoz-Horta et al., 2015;Samad et al., 2016) and structural equation modeling and network analysis (Jones et al., 2014) of the gene abundance and diversity of nosZ clade II in terrestrial soils indicated the greater contribution of N 2 O reducers carrying nosZ clade II to the soil N 2 O sink capacity than those carrying nosZ clade I. ...
... (Takata et al., 2011). The Ks value for N 2 O (i.e., 4.4 μM) was in the range of those previously reported in cultures of denitrifiers and bacteria catalyzing dissimilatory nitrite reduction to ammonium (0.324-100 μM) (Betlach and Tiedje, 1981;Conrad, 1996;Yoon et al., 2016;Park et al., 2017;Suenaga et al., 2018) and using bulk agricultural soils (0.1-5.8 μM) (Holtan-Hartwig et al., 2000). N 2 O concentrations in agricultural soils were generally less than 1 μM, but increased up to 400 μM (Schreiber et al., 2012 and references therein) with nitrogenous fertilizer treatment, which was higher than the K s value of G. aurantiaca. ...
Agricultural soil is the primary N2O sink limiting the emission of N2O gas into the atmosphere. Although Gemmatimonadetes bacteria are abundant in agricultural soils, limited information is currently available on N2O reduction by Gemmatimonadetes bacteria. Therefore, the effects of pH and temperature on N2O reduction activities and affinity constants for N2O reduction were examined by performing batch experiments using an isolate of Gemmatimonadetes bacteria, Gemmatimonas aurantiaca (NBRC100505T). G. aurantiaca reduced N2O at pH 5-9 and 4-50°C, with the highest activity being observed at pH 7 and 30°C. The affinity constant of G. aurantiaca cells for N2O was 4.4 μM. The abundance and diversity of the Gemmatimonadetes 16S rRNA gene and nosZ encoding nitrous oxide reductase in agricultural soil samples were also investigated by quantitative PCR (qPCR) and amplicon sequencing ana-lyses. Four N2O-reducing agricultural soil samples were assessed, and the copy numbers of the Gemmatimonadetes 16S rRNA gene (clades G1 and G3), nosZ DNA, and nosZ mRNA were 8.62-9.65×108, 5.35-7.15×108, and 2.23-4.31×109 copies (g dry soil)-1, respectively. The abundance of the nosZ mRNA of Gemmatimonadetes bacteria and OTU91, OUT332, and OTU122 correlated with the N2O reduction rates of the soil samples tested, suggesting N2O reduction by Gemmatimonadetes bacteria. Gemmatimonadetes 16S rRNA gene reads affiliated with OTU4572 and OTU3759 were predominant among the soil samples examined, and these Gemmatimonadetes OTUs have been identified in various types of soil samples.
... Moreover, nosZ clade II has a wider distribution among microbial taxa (Chee- Sanford et al., 2020;Jones et al., 2014;Shan et al., 2021), and these microorganisms are widely found in different ecosystems (Bertagnolli et al., 2020;Domeignoz-Horta et al., 2016). Compared with nosZ clade I carrying N 2 O reducers, nosZ clade II organisms appear to have a higher affinity for N 2 O, which provides them with a selective advantage when competing for limited N 2 O in situ in the micro-environment of soil (Yoon et al., 2016). All these studies suggest that nosZ clade II-carrying microorganisms may have better N 2 O reduction potential than nosZ clade I-carrying microorganisms. ...
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.
... Nitricoxidivorans perseverans and Ca. Nitricoxidireducens bremensis using clade II NOS ( Supplementary Fig. 7), which often have higher affinity for N 2 O than clade I NOS, in line with the minute concentrations of N 2 O detected in the bioreactor [34][35][36][37] . Interestingly, both organisms encoded duplicate copies of nosZ genes that were identical to each other (that is, except for the nosZ copies in Ca. ...
Nitric oxide (NO) is a highly reactive and climate-active molecule and a key intermediate in the microbial nitrogen cycle. Despite its role in the evolution of denitrification and aerobic respiration, high redox potential and capacity to sustain microbial growth, our understanding of NO-reducing microorganisms remains limited due to the absence of NO-reducing microbial cultures obtained directly from the environment using NO as a substrate. Here, using a continuous bioreactor and a constant supply of NO as the sole electron acceptor, we enriched and characterized a microbial community dominated by two previously unknown microorganisms that grow at nanomolar NO concentrations and survive high amounts (>6 µM) of this toxic gas, reducing it to N2 with little to non-detectable production of the greenhouse gas nitrous oxide. These results provide insight into the physiology of NO-reducing microorganisms, which have pivotal roles in the control of climate-active gases, waste removal, and evolution of nitrate and oxygen respiration.
... The closest reference sequences to DechloroB were from Dechloromonas aromatica, a common soil and aquatic bacteria capable of denitrification. 34,35 The species level classification of DechloroB cannot be confirmed definitively, as DechloroB had a 97.6% identity match with D. aromatica reference sequences. DechloroA was generally more abundant than DechloroB in both the RAS fermenter and EBPR basin ( Figure 5). ...
... Soil nutrient status has been shown to increase nosZ-I community size . Yoon et al. (2016) suggested that nosZ-II communities should be more abundant in most environmental conditions, but also that nosZ-I organisms are predicted to be the dominant N 2 O consumers under conditions where N addition increases N 2 O emissions for a prolonged period. Since our study soils originated from unfertilized pastures with low background N levels, nosZ-II organisms were the most abundant and active N 2 O consumers following urine addition, and the short-lived N 2 O peaks did not provide conditions conducive to an increase in nosZ-I abundances during our short-term incubation. ...
AimIncorporating non-bloat legumes into grass pastures can reduce enteric methane and alter cattle urinary urea N output by increasing protein intake. Deposition of high urea N urine influences soil N-cycling microbes and potentially N2O production. We studied how urine urea N concentration affects soil nitrifier and denitrifier abundances, activities, and N2O production.Methods15N13C-labelled urea dissolved in cattle urine was added at 3.5 and 7.0 g L−1 to soils from a grazed, non-bloat legume pasture and incubated under controlled conditions. CO2, N2O, 13C-CO2, and 15N-N2O production were quantified over 240 h, along with nitrifier and denitrifier N-cycling genes and mRNA transcripts.ResultsHigh urea urine increased total N2O relative to the control; low urea was not significantly different from the control or the high urea treatment. As a result, N2O-N emission factors were not significantly different between the low urea treatment (1.17%) and high urea treatment (0.94%). Doubling urea concentration doubled CO2-Curea and N2O-Nurea but not total N2O-N. Urine addition initially inhibited then increased AOB transcripts and gene abundances. nirK and nirS transcript abundances indicated that denitrification by ammonia oxidizers and/or heterotrophic denitrifiers dominated N2O production. Urine addition increased nosZ-II vs. nosZ-I transcripts, improving soil N2O reduction potential.Conclusion
Characterizing this interplay between nitrifiers and denitrifiers improves the understanding of urine patch N2O sinks and source dynamics. This mechanistic information helps to explain the constrained short-term N2O emissions observed in response to excess urine N excretion from cattle consuming high protein diets, e.g. non-bloat legumes.
... Taken together, these results suggest that the abundance of N 2 O production and reduction-related genes in sediments can influence N 2 O saturation in water, with the nosZ II gene being primarily involved in N 2 O reduction and determining N 2 O emission potential. NosZ-II-type N 2 O-reducing bacteria were reported to have a higher affinity for N 2 O than NosZ-I-type N 2 O-reducing bacteria (Yoon et al., 2016). Thus, the environment with low N 2 O concentration was a favorable condition for the survival of NosZ-II-type N 2 O-reducing bacteria. ...
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.
... The new type of nitrous oxide reductase (nosZ II) is capable of catalyzing the final step of denitrification in which N 2 O is converted into N 2 (Wan et al., 2012) , showing a negative relationship with N 2 O emissions (Fig. 5). Compared to nosZ I, nosZ II has a selective advantage when competing for N 2 O for its lower whole-cell half-saturation constants (Ks) for N 2 O (Yoon et al., 2016). Elevated soil pH resulted in a decline in soil N 2 O emissions due to the promotion of the abundance of nosZ genes (Shaaban et al., 2018;Zurovec et al., 2021;Bleken and Rittl, 2022). ...
Soil acidification is a major land degradation process globally, and impacts soil nitrogen (N) transformation. However, it is still not well known how soil acidification affects net N mineralization and nitrification, especially N-cycling microbes and nitrous oxide (N2O) emissions. Hence, three soils characterized by different soil pH values (5.5, 6.3, and 7.7) were collected from the paddy fields, and experiments were conducted to evaluate the effect of soil acidification on net N mineralization and nitrification, and N2O emissions. Compared to those in the soils with pH 7.7 and 6.3, net N mineralization, net nitrification, and N2O emissions were decreased by 75-76 %, 89-91 %, and 19-48 %, respectively, in the soil with pH 5.5, while net N nitrification and N2O emissions decreased by 18 % in the soil with pH 6.3 when compared to those in the soil with pH 7.7. The significantly decreased net nitrification in the soils with pH 6.3 and 5.5 was mainly attributed to the limited N availability and abundance of nitrification-related microbes including ammonia-oxidizing bacteria and complete ammonia-oxidizers. The decrease in N2O emissions of soils with pH 6.3 and 5.5 had mainly resulted from decreasing nitrification and denitrification via suppressing microbes including nirS and fungal nirK and limiting N availability. Hence, this study provides new insights and improves our understanding of how soil acidification regulates N mineralization, nitrification, and N2O emissions in paddy soils, which gives guidance on developing N management strategies for sustainable production and N2O mitigation in acid soils.
... Pseudomonas spp. are fast-growing r-strategists enriched in nutrient-rich environments such as the rhizosphere [54] and hyphosphere [55]. In a similar fashion to the rhizosphere, the hyphosphere provides a unique niche in which microbial communities differ from those in the bulk soil due to hyphal exudates [53,56], as supported by the increased patch DOC concentrations in the +AMF treatment (Fig. S1C). ...
Background
Arbuscular 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 N 2 O emissions from “hot spot” residue patches remain unclear. Here we explored the key microbes in the hyphosphere involved in N 2 O production and consumption using amplicon and shotgun metagenomic sequencing. Chemotaxis, growth and N 2 O emissions of isolated N 2 O-reducing bacteria in response to hyphal exudates were tested using in vitro cultures and inoculation experiments.
Results
AMF hyphae reduced denitrification-derived N 2 O 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 N 2 O emissions in the hyphosphere was linked to N 2 O-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 N 2 O 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.
Conclusions
The cooperation between AMF and the N 2 O-reducing Pseudomonas residing on hyphae significantly reduce N 2 O 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 N 2 O consumption in nutrient-enriched microsites, and consequently reduce N 2 O emissions from soils. This knowledge opens novel avenues to exploit cross-kingdom microbial interactions for sustainable agriculture and for climate change mitigation.
... PICRUSt2 predictions of bacterial nosZ gene abundances based on 16S rRNA sequences were negatively correlated with ΔN 2 O (Fig. 6), supporting previous reports of elevated nosZ activity within the Saanich Inlet deep basin 7 . Organisms possessing the atypical nosZ variant are commonly associated with higher N 2 O affinities and lower O 2 sensitivities, and also typically lack additional genes in the denitrification pathway [93][94][95] . A conceptual model describing mutualistic N 2 O-cycling interactions has already been proposed in which N 2 O produced by SUP05 is used by nosZ-harboring Marinimicrobia ecotypes to store polysulfide and regenerate H 2 S 12 . ...
The mechanisms by which large-scale microbial community function emerges from complex ecological interactions between individual taxa and functional groups remain obscure. We leveraged network analyses of 16S rRNA amplicon sequences obtained over a seven-month timeseries in seasonally anoxic Saanich Inlet (Vancouver Island, Canada) to investigate relationships between microbial community structure and water column N2O cycling. Taxa separately broadly into three discrete subnetworks with contrasting environmental distributions. Oxycline subnetworks were structured around keystone aerobic heterotrophs that correlated with nitrification rates and N2O supersaturations, linking N2O production and accumulation to taxa involved in organic matter remineralization. Keystone taxa implicated in anaerobic carbon, nitrogen, and sulfur cycling in anoxic environments clustered together in a low-oxygen subnetwork that correlated positively with nitrification N2O yields and N2O production from denitrification. Close coupling between N2O producers and consumers in the anoxic basin is indicated by strong correlations between the low-oxygen subnetwork, PICRUSt2-predicted nitrous oxide reductase (nosZ) gene abundances, and N2O undersaturation. This study implicates keystone taxa affiliated with common ODZ groups as a potential control on water column N2O cycling and provides a theoretical basis for further investigations into marine microbial interaction networks.
... 51−53 Nevertheless, most of the nosZ genes found in high relative abundance across all biofilms belonged to the organisms with experimentally verified capability to utilize N 2 O as the sole electron acceptor. 26,54,55 Along with the consistent net nosZ abundance, the consistent abundance of N 2 O-respiring taxa, especially those affiliated or closely related to the genus Dechloromonas with high affinity to N 2 O, strongly suggests that an N 2 O-repairing potential can be sustained despite inconsistent N 2 O availability. 26−28 A common dilemma for biological environmental processes has been their vulnerability to low temperatures. ...
Wastewater treatment plants (WWTPs) are a major
source of N2O, a potent greenhouse gas with 300 times higher global warming potential than CO2. Several approaches have been proposed for mitigation of N2O emissions from WWTPs and have shown promising yet only site-specific results. Here, self-sustaining
biotrickling filtration, an end-of-the-pipe treatment technology, was
tested in situ at a full-scale WWTP under realistic operational conditions. Temporally varying untreated wastewater was used as
trickling medium, and no temperature control was applied. The off-gas from the covered WWTP aerated section was conveyed through the pilot-scale reactor, and an average removal efficiency of 57.9 ± 29.1% was achieved during 165 days of operation despite the generally low and largely fluctuating influent N2O concentrations (ranging between 4.8 and 96.4 ppmv). For the following 60-day period, the continuously operated reactor system removed 43.0 ± 21.2% of the periodically augmented N2O, exhibiting elimination capacities as high as 5.25 g N2O m−3·h−1. Additionally, the bench-scale experiments performed abreast corroborated the resilience of the system to short-term N2O starvations. Our results corroborate the feasibility of biotrickling filtration for mitigating N2O emitted from WWTPs and demonstrate its robustness toward suboptimal field operating conditions and N2O starvation, as also supported by analyses of the microbial compositions and nosZ gene profiles.
... True concentrations were probably higher in proximity around actively N 2 O-generating GR particles. Contemporary microbial N 2 O consumption is based on high-affinity enzymes adapted to low N 2 O steady-state concentrations 49 . For example, 30 nM N 2 O is readily metabolized in anoxic seawater 20 , but the minimum threshold for marine N 2 O respiration is probably much lower (S. ...
Microbial denitrification converts fixed nitrogen species into gases in extant oceans. However, it is unclear how such transformations occurred within the early nitrogen cycle of the Archaean. Here we demonstrate under simulated Archaean conditions mineral-catalysed reduction of nitrite via green rust and magnetite to reach enzymatic conversion rates. We find that in an Fe²⁺-rich marine environment, Fe minerals could have mediated the formation of nitric oxide (NO) and nitrous oxide (N2O). Nitrate did not exhibit reactivity in the presence of either mineral or aqueous Fe²⁺; however, both minerals induced rapid nitrite reduction to NO and N2O. While N2O escaped into the gas phase (63% of nitrite nitrogen, with green rust as the catalyst), NO remained associated with precipitates (7%), serving as a potential shuttle to the benthic ocean. Diffusion and photochemical modelling suggest that marine N2O emissions would have sustained 0.8–6.0 parts per billion of atmospheric N2O without a protective ozone layer. Our findings imply a globally distributed abiotic denitrification process that feasibly aided early microbial life to accrue new capabilities, such as respiratory metabolisms.
... The value estimated in the anoxic bottom water of upstream station CB1.5 (647 nM N2O)was higher than the range observed in oxic waters (40-177 nM N2O); however, both the anoxic and oxic were on the same order of magnitude as the measured at the oxic-anoxic interface from the eastern tropical north Pacific OMZ (334±258 nM N2O)(Sun et al., 2020). The anoxic value was also within the range of the of some soil bacteria with clade II nosZ , like Dechloromonas aromatica and Anaeromyxobacter dehalogenans (324-1340 nM N2O)(Yoon, Nissen, Park, Sanford, & Löffler, 2016). The observed difference in values may result from the different physiological states or activities of N2O-reducing microbial communities inhabiting oxygenated and anoxic waters. ...
Plain Language Summary
Nitrous oxide (N2O) gas affects Earth's climate system as a powerful greenhouse gas and ozone‐depleting agent. A large amount of N2O is released from estuarine environments into the atmosphere but the estimate of the total flux remains highly uncertain. To better estimate estuarine N2O flux, an improved understanding of the distribution and environmental controls on N2O production and consumption would be very useful. Previous studies have mainly focused on N2O production processes. Here we used isotope tracers to directly measure the N2O consumption rate and used molecular techniques to characterize N2O‐consuming microbes in the water column of the largest estuary in the United States—the Chesapeake Bay. We found a strong dependence of N2O consumption rate on oxygen and N2O substrate concentration: the N2O consumption rate decreases as oxygen rises and increases with higher N2O substrate concentration. N2O‐consuming microbes were detected across the water column in both fall and summer. Overall, these new observations will help to constrain estimates of net N2O emission from estuaries and guide mitigation efforts to reduce N2O emissions from anthropogenic perturbations.
... Further, other bacteria such as Dechloromonas 40 , Ardenticatena 41 and Melioribacter 42 also mediate iron reduction, an additional trait that could promote chemodenitrification by recycling Fe 2+ . Therefore, chemodenitrifiers may outcompete canonical denitrifiers in the studied peatlands (for example, Nitrospirillum) due to higher affinity under low levels of substrate 43 , and abundance patterns of the Myxococcales suggest a notable benefit for some chemodenitrifiers in soils associated with high abiotic N 2 O yields. ...
Atmospheric nitrous oxide (N2O) is a potent greenhouse gas thought to be mainly derived from microbial metabolism as part of the denitrification pathway. Here we report that in unexplored peat soils of Central and South America, N2O production can be driven by abiotic reactions (≤98%) highly competitive to their enzymatic counterparts. Extracted soil iron positively correlated with in situ abiotic N2O production determined by isotopic tracers. Moreover, we found that microbial N2O reduction accompanied abiotic production, essentially closing a coupled abiotic-biotic N2O cycle. Anaerobic N2O consumption occurred ubiquitously (pH 6.4–3.7), with proportions of diverse clade II N2O reducers increasing with consumption rates. Our findings show that denitrification in tropical peat soils is not a purely biological process but rather a ‘mosaic’ of abiotic and biotic reduction reactions. We predict that hydrological and temperature fluctuations differentially affect abiotic and biotic drivers and further contribute to the high N2O flux variation in the region. There are many open questions about biogeochemical function in peatlands. Here, the authors investigate the nitrogen cycle of tropical peatlands, finding that a surprisingly high fraction of nitrous oxide production is abiotic and that denitrification is a coupled abiotic-biotic process.
... The nosZ clade I gene was transcribed more actively even though the nosZ clade II gene was more abundant (e.g., the case in the BS shown in Fig. 3e and l). The higher growth yields of clade II-type N 2 O-reducing bacteria than those of clade I-type (Yoon et al., 2016) may lead to a preponderance of the nosZ clade II gene. However, a microbial culture of clade I-type N 2 O-reducing bacteria has been reported to have the capability of continually synthesizing N 2 O reductase enzymes under oxic conditions to allow for a rapid transition into anoxic environments (Lycus et al., 2018). ...
Nitrous oxide (N2O) is an important ozone-depleting
greenhouse gas produced and consumed by microbially mediated nitrification
and denitrification pathways. Estuaries are intensive N2O emission
regions in marine ecosystems. However, the potential contributions of
nitrifiers and denitrifiers to N2O sources and sinks in China's
estuarine and coastal areas are poorly understood. The abundance and
transcription of six key microbial functional genes involved in
nitrification and denitrification, as well as the clade II-type nosZ
gene-bearing community composition of N2O reducers, were investigated
in four estuaries spanning the Chinese coastline. The results showed that
the ammonia-oxidizing archaeal amoA genes and transcripts were more dominant in
the northern Bohai Sea (BS) and Yangtze River estuaries, which had low
nitrogen concentrations, while the denitrifier nirS genes and transcripts were
more dominant in the southern Jiulong River (JRE) and Pearl River estuaries,
which had high levels of terrestrial nitrogen input. Notably, the nosZ clade II
gene was more abundant than the clade I-type throughout the estuaries except
for in the JRE and a few sites of the BS, while the opposite transcript
distribution pattern was observed in these two estuaries. The gene and
transcript distributions were significantly constrained by nitrogen and
oxygen concentrations as well as by salinity, temperature, and pH. The nosZ clade
II gene-bearing community composition along China's coastline had a high level of
diversity and was distinctly different from that in the soil and in marine
oxygen-minimum-zone waters. By comparing the gene distribution patterns
across the estuaries with the distribution patterns of the N2O
concentration and flux, we found that denitrification may principally
control the N2O emissions pattern.
... The higher N 2 O production rates in barely roots compared to soil indicate higher availability of N 2 O. This could explain the relatively higher abundance of nosZ clade I in roots and nosZ clade II in soil as they have been suggested to have a lower apparent affinity for N 2 O (Yoon et al. 2016), although this is not conclusive (Conthe et al. 2018). However, this pattern was not observed for sunflower due to low or even inhibited activity of N 2 O producing microorganisms on sunflower roots. ...
The rhizosphere is a hotspot for denitrification. The nitrous oxide (N2O) reductase among denitrifiers and non-denitrifying N2O reducers is the only known N2O sink in the biosphere. We hypothesized that the composition of root-associated N2O-reducing communities when establishing on annual crops depend on soil type and plant species, but that assembly processes are independent of these factors and differ between nosZ clade I and II. Using a pot experiment with barley and sunflower and two soils, we analyzed the abundance, composition and diversity of soil and root-associated N2O reducing communities by qPCR and amplicon sequencing of nosZ. Clade I was more abundant on roots compared to soil, while clade II showed the opposite. In barley, this pattern coincided with N2O availability, determined as potential N2O production rates, but for sunflower no N2O production was detected in the root compartment. Root and soil nosZ communities differed in composition and phylogeny-based community analyses indicated that assembly of root-associated N2O reducers was driven by the interaction between plant and soil type, with inferred competition being more influential than habitat selection. Selection between clades I and II in the root/soil interface is suggested, which may have functional consequences since most clade I microorganisms can produce N2O.
... However, the only known N 2 O sink is the N 2 O reduction to N 2 by the N 2 O reductase encoded by the genes of nosZ I or nosZ II (Sanford et al., 2012;Jones et al., 2013). Although the nosZ II gene has been found to be a different type of N 2 O reductase gene (Sanford et al., 2012;Yoon et al., 2016), many research on the nosZ gene still ignored the differences between the two distinctive clades, especially in estuaries and oceans (Wang et al., 2019b;Aamer et al., 2020). In this study, the nosZ I and nosZ II genes were both widely distributed in the northern SCS surface sediments, with gene abundances ranging from 10 5 to 10 7 copies·g -1 and from 10 6 to 10 8 copies·g -1 , respectively (Figure 2 and Supplementary Table 3). ...
Denitrification is an important pathway for nitrogen sink and N2O emissions, but little is known about the ecological distribution of key functional genes of denitrification and their potential N2O emissions in marine sediments. In this study, we analyzed the abundance, ecological distribution, and diversity of key functional genes (nir and nosZ) for denitrification in the northern South China Sea (SCS) surface sediments. Our results showed that the gene abundances varied from 105 to 108 and from 106 to 107 copies·g-1 for the nirS and nirK, respectively. The nosZ II/nosZ I gene abundance ratios were 1.28–9.88 in shallow-sea and deep-sea sediments, suggesting that the nosZ II gene should play a dominant role in N2O reduction in the northern SCS sediments. Moreover, the significantly higher abundance ratios of nir/nosZ in deep-sea surface sediments implied that there might be stronger N2O emissions potential in deep-sea sediments than in shallow-sea sediments. The ecological distribution profiles of the nirS, nosZ I, and nosZ II gene communities varied with water depth, and denitrification genes in shallow-sea and deep-sea sediments differed in their sensitivity to environmental factors. Water temperature was the major factor affecting both the abundance and the community distribution of the nirS gene in deep-sea sediments. Nitrate was the major factor shaping the community of nosZ I and nosZ II genes in shallow-sea sediments. Our study provides a pattern of ecological distribution and diversity for the nir and nosZ genes and emphasizes the role of these key functional genes in potential N2O emissions of the northern SCS surface sediments.
... However, as the separation of nosZ between nosZ clade I and II has only recently been distinguished, uncertainty remains regarding their varying response to soil P and preferred soil conditions and habitat (Hallin et al. 2018;Jones et al. 2013). Despite this, several studies have demonstrated that, in agreement with our results, nosZ clade II is more responsive to environmental conditions, including soil properties and fertilisation practices (Domeignoz-Horta et al. 2016;Xu et al., 2020a;Yoon et al. 2016). Our results further support the hypothesis that, similar to nirS and nirK harbouring bacteria (Jones et al. 2013;Tang et al. 2016), the two nosZ also vary in their niche, with the abundance of nosZ clade II harbouring microorganisms possibly being favoured in high P content (Jones et al. 2013;Tang et al. 2016). ...
The influence of soil phosphorous (P) content on the N-cycling communities and subsequent effects on N 2 O emissions remains unclear. Two laboratory incubation experiments were conducted on soils collected from a long-term (est. 1995) P-addition field trial sampled in summer 2018 and winter 2019. Incubations were treated with a typical field amendment rate of N as well as a C-amendment to stimulate microbial activity. Throughout both incubations, soil subsamples were collected prior to fertiliser amendment and then throughout the incubations, to quantify the abundance of bacteria (16S rRNA), fungi (ITS) and Thaumarcheota (16S rRNA) as well as functional guilds of genes involved in nitrification (bacterial and archaeal amoA, and comammox) and denitrification (nirS, nirK, nosZ clade I and II) using quantitative PCR (qPCR). We also evaluated the correlations between each gene abundance and the associated N 2 O emissions depending on P-treatments. Our results show that long-term P-application influenced N-cycling genes abundance differently. Except for comammox, overall nitri-fiers' genes were most abundant in low P while the opposite trend was found for denitrifiers' genes. C and N-amendments strongly influenced the abundance of most genes with changes observed as soon as 24 h after application. ITS was the only gene correlated to N 2 O emissions in the low P-soils while microbes were mostly correlated to emissions in high P, suggesting possible changes in the organisms involved in N 2 O production depending on soil P-content. This study highlights the importance of long-term P addition on shaping the microbial community function which in turn stimulates a direct impact on the subsequent N emissions.
... These results showed that more N decreased nosZ I genes, and Li reported that N 2 O emission from sediments or soils was closely negatively correlated to nosZ abundance [45]. Therefore, N will increase N 2 O emissions from aquatic environment, which may give us a warning that higher N addition (>40 kg NH 4 Cl per month) may increase the potential for ozone layer destruction in climate warming [46]. However, it remains to be verified by subsequent metatranscriptomics whether the nosZ I gene copies were transcribed into a corresponding active enzyme that can catalyze NH 4 + to produce NO 3 − . ...
Excessive nitrogen (N) input is an important factor influencing aquatic ecosystems and has received increasing public attention in the past decades. It remains unclear how N input affects the denitrifying bacterial communities that play a key role in regulating N cycles in various ecosystems. To test our hypothesis—that the abundance and biodiversity of denitrifying bacterial communities decrease with increasing N—we compared the abundance and composition of denitrifying bacteria having nitrous oxide reductase gene (nosZ I) from sediments (0–20 cm) in five experimental ponds with different nitrogen fertilization treatment (TN10, TN20, TN30, TN40, TN50) using quantitative PCR and pyrosequencing techniques. We found that (1) N addition significantly decreased nosZ I gene abundance, (2) the Invsimpson and Shannon indices (reflecting biodiversity) first increased significantly along with the increasing N loading in TN10–TN40 followed by a decrease in TN50, (3) the beta diversity of the nosZ I denitrifier was clustered into three groups along the TN concentration levels: Cluster I (TN50), Cluster II (TN40), and Cluster III (TN10–TN30), (4) the proportions of Alphaproteobacteria and Betaproteobacteria in the high-N treatment (TN50) were significantly lower than in the lower N treatments (TN10–TN30). (5) The TN concentration was the most important factor driving the alteration of denitrifying bacteria assemblages. Our findings shed new light on the response of denitrification-related bacteria to long-term N loading at pond scale and on the response of denitrifying microorganisms to N pollution.
... There was, however, considerable variation in transient N 2 O accumulation between replicate experiments, also observed by Liu et al. (2013) with Thauera strains performing heterotrophic denitrification. The type of nitrous oxide reductase may influence the amount of N 2 O accumulation as clade II NosZ enzymes have higher apparent N 2 O affinity, higher biomass yield, and a more energy-efficient translocation mechanism compared with clade I NosZ (Yoon et al., 2016). Isolate H3 (HD) also differed in this regard from F76 (HD) and D110 (HD) , as it contained clade I nosZ, while the other tested strains harboured clade II. ...
Stimulating litho‐autotrophic denitrification in aquifers with hydrogen is a promising strategy to remove excess NO3−, but it often entails accumulation of the cytotoxic intermediate NO2− and the greenhouse gas N2O. To explore if these high NO2− and N2O concentrations are caused by differences in the genomic composition, the regulation of gene transcription or the kinetics of the reductases involved, we isolated hydrogenotrophic denitrifiers from a polluted aquifer, performed whole‐genome sequencing and investigated their phenotypes. We therefore assessed the kinetics of NO2−, NO, N2O, N2 and O2 as they depleted O2 and transitioned to denitrification with NO3− as the only electron acceptor and hydrogen as the electron donor. Isolates with a complete denitrification pathway, although differing intermediate accumulation, were closely related to Dechloromonas denitrificans, Ferribacterium limneticum or Hydrogenophaga taeniospiralis. High NO2− accumulation was associated with the reductases' kinetics. While available, electrons only flowed towards NO3− in the narG‐containing H. taeniospiralis but flowed concurrently to all denitrification intermediates in the napA‐containing D. denitrificans and F. limneticum. The denitrification regulator RegAB, present in the napA strains, may further secure low intermediate accumulation. High N2O accumulation only occurred during the transition to denitrification and is thus likely caused by delayed N2O reductase expression.
... Moreover, the distribution of nosZ clade I and II genes is significantly different in microbial species. nosZ clade I genes were mainly distributed in α-, β-, and γ-Proteobacteria, while nosZ clade II genes were found in Firmicutes, Bacteroidetes, Chloroflexi, Delta-Proteobacteria, Wolinella succinogenes, Campylobacter fetus, Anaeromyxobacter dehalogenans, Dechloromonas aromatica, and Bacillus vireti (Daniel et al. 2016;Kern and Simon 2015;Payne et al. 1982;Sanford et al. 2012;Yoon et al. 2016;Yoshinari 1980). In fact, nosZ clade II includes a large amount of nondenitrified N 2 OR, which is involved in the process of N 2 O consumption and has no significant contribution to N 2 O production (Domeignoz-Horta et al. 2016). ...
Nitrous oxide (N2O) is an important greenhouse gas that plays a significant role in atmospheric photochemical reactions and contributes to stratospheric ozone depletion. Soils are the main sources of N2O emissions. In recent years, it has been demonstrated that soil is not only a source but also a sink of N2O uptake and consumption. N2O emissions at the soil surface are the result of gross N2O production, uptake, and consumption, which are co-occurring processes. Soil N2O uptake and consumption are complex biological processes, and their mechanisms are still worth an in-depth systematic study. This paper aimed to systematically address the current research progress on soil N2O uptake and consumption. Based on a bibliometric perspective, this study has highlighted the pathways of soil N2O uptake and consumption and their driving factors and measurement techniques. This systematic review of N2O uptake and consumption will help to further understand N transformations and soil N2O emissions.
Graphical abstract
... In the study of Palmer et al (2012), numbers of bacteria capable to transform N 2 O into N 2 were significantly lower compared to nitrite reducers, which also explains the strong increase of N 2 O emissions in the cryoturbated soils after NO 3 − addition. However it must be considered that in the mentioned study only those bacteria were assessed which belong to the clade 1 of nosZ, and not those which harbor the nosZ genes of clade 2, due to the selection of the primers for analysis (Yoon et al 2016). Calderoli et al (2018) demonstrated the importance of clade 2 of the nosZ gene for N 2 O reduction in permafrost-affected soils. ...
The paradigm that permafrost-affected soils show restricted mineral nitrogen (N) cycling in favor of organic N compounds is based on the observation that net N mineralization rates in these cold climates are negligible. However, we find here that this perception is wrong. By synthesizing published data on N cycling in the plant-soil-microbe system of permafrost ecosystems we show that gross ammonification and nitrification rates in active layers were of similar magnitude and showed a similar dependence on soil organic carbon (SOC) and total nitrogen (TN) concentrations as observed in temperate and tropical systems. Moreover, high protein depolymerization rates and only marginal effects of C:N stoichiometry on gross N turnover provided little evidence for N limitation. Instead, the rather short period when soils are not frozen is the single main factor limiting N turnover. High gross rates of mineral N cycling are thus facilitated by released protection of organic matter in active layers with nitrification gaining particular importance in N-rich soils, such as organic soils without vegetation. Our finding that permafrost-affected soils show vigorous N cycling activity is confirmed by the rich functional microbial community which can be found both in active and permafrost layers. The high rates of N cycling and soil N availability are supported by biological N fixation, while atmospheric N deposition in the Arctic still is marginal except for fire-affected areas. In line with high soil mineral N production, recent plant physiological research indicates a higher importance of mineral plant N nutrition than previously thought. Our synthesis shows that mineral N production and turnover rates in active layers of permafrost-affected soils do not generally differ from those observed in temperate or tropical soils. We therefore suggest to adjust the permafrost N cycle paradigm, assigning a generally important role to mineral N cycling. This new paradigm suggests larger permafrost N climate feedbacks than assumed previously.
... This included a community of known denitrifiers that were found in all BMP types sampled and which clustered predominantly by BMP type. While emphasis was placed on denitrifiers that carried specific denitrification genes (nirK, nirS, and nosZ), it should be noted that there is recent evidence to indicate the presence of two nosZ clades (Sanford et al. 2012;Yoon et al. 2016). Clade I contains known denitrifiers that harbor an additional denitrification gene, such as nirS, in addition to nosZ (for example, Pseudomonas aeruginosa). ...
Stormwater best management practices (BMPs) are engineered structures that attempt to mitigate the impacts of stormwater, which can include nitrogen inputs from the surrounding drainage area. The goal of this study was to assess bacterial community composition in different types of stormwater BMP soils to establish whether a particular BMP type harbors more denitrification potential. Soil sampling took place over the summer of 2015 following precipitation events. Soils were sampled from four bioretention facilities, four dry ponds, four surface sand filters, and one dry swale. 16S rRNA gene analysis of extracted DNA and RNA amplicons indicated high bacterial diversity in the soils of all BMP types sampled. An abundance of denitrifiers was also indicated in the extracted DNA using presence/absence of nirS, nirK , and nosZ denitrification genes. BMP soil bacterial communities were impacted by the surrounding soil physiochemistry. Based on the identification of a metabolically-active community of denitrifiers, this study has indicated that denitrification could potentially occur under appropriate conditions in all types of BMP sampled, including surface sand filters that are often viewed as providing low potential for denitrification. The carbon content of incoming stormwater could be providing bacterial communities with denitrification conditions. The findings of this study are especially relevant for land managers in watersheds with legacy nitrogen from former agricultural land use.
Gemmatimonadota is a diverse bacterial phylum commonly found in environments such as soils, rhizospheres, fresh waters, and sediments. So far, the phylum contains just six cultured species (five of them sequenced), which limits our understanding of their diversity and metabolism. Therefore, we analyzed over 400 metagenome-assembled genomes (MAGs) and 5 culture-derived genomes representing Gemmatimonadota from various aquatic environments, hydrothermal vents, sediments, soils, and host-associated (with marine sponges and coral) species. The principal coordinate analysis based on the presence/absence of genes in Gemmatimonadota genomes and phylogenomic analysis documented that marine and host-associated Gemmatimonadota were the most distant from freshwater and wastewater species. A smaller genome size and coding sequences (CDS) number reduction were observed in marine MAGs, pointing to an oligotrophic environmental adaptation. Several metabolic pathways are restricted to specific environments. For example, genes for anoxygenic phototrophy were found only in freshwater, wastewater, and soda lake sediment genomes. There were several genomes from soda lake sediments and wastewater containing type IC/ID ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO). Various genomes from wastewater harbored bacterial type II RuBisCO, whereas RuBisCO-like protein was found in genomes from fresh waters, soil, host-associated, and marine sediments. Gemmatimonadota does not contain nitrogen fixation genes; however, the nosZ gene, involved in the reduction of N 2 O, was present in genomes from most environments, missing only in marine water and host-associated Gemmatimonadota. The presented data suggest that Gemmatimonadota evolved as an organotrophic species relying on aerobic respiration and then remodeled its genome inventory when adapting to particular environments.
IMPORTANCE
Gemmatimonadota is a rarely studied bacterial phylum consisting of a handful of cultured species. Recent culture-independent studies documented that these organisms are distributed in many environments, including soil, marine, fresh, and waste waters. However, due to the lack of cultured species, information about their metabolic potential and environmental role is scarce. Therefore, we collected Gemmatimonadota metagenome-assembled genomes (MAGs) from different habitats and performed a systematic analysis of their genomic characteristics and metabolic potential. Our results show how Gemmatimonadota have adapted their genomes to different environments.
Nitrous oxide (N 2 O) reductase is a copper enzyme that catalyzes the reduction of N 2 O to dinitrogen, the last step of the denitrification pathway. This enzyme has two copper centers: a CuA center that is the electron transfer center and the “CuZ center” where the catalysis occurs. This enzyme has been the center of several studies over the last 20 years, and many of its spectroscopic and catalytic properties have been determined, as well as its structure in different oxidation states. These studies have also revealed that the CuA center is similar to the one present in cytochrome c oxidase, being a binuclear copper center, while the CuZ center is unique in biology, being a tetranuclear copper center bridged by a sulfur atom. Moreover, these studies have also identified that the CuZ center can exist in two forms, CuZ*(4Cu1S) and CuZ(4Cu2S). The first has a high turnover number in the fully reduced state, while the CuZ(4Cu2S) form of CuZ center has a very small turnover number and cannot explain the high capacity of the whole cells in reducing N 2 O, from which the enzyme is isolated with CuZ center mainly in that form. This fact envisages an activation mechanism still to be unraveled that might involve one or more enzymes/proteins encoded by the nos genes and a putative sulfur‐displacement mechanism.
The biogenesis of these two centers has not been extensively studied, and at least with respect to the copper insertion, it has been postulated that there are alternative routes. The apo‐N 2 OR is synthesized in the cytoplasm in the apo form and is transported to the periplasmic space by either the Sec (unfolded state) or Tat system (dimer folded state), where the centers are assembled. Thus, CuA assembly has been proposed to be dependent on a copper chaperone, pCu A C, and Sco, a thiol‐disulfide reductase that reduces the disulfide bridge between the two cysteines in the apo‐CuA center. Relative to the CuZ center, the delivery of copper atoms is by NosL, an outer‐membrane protein, while the sulfur atoms are transported by the ABC transporter encoded by nosDFY genes. The other accessory proteins NosR and NosX have been assigned broader functions. NosR has been implicated in nosZ expression, as well as in maintaining the activity of N 2 OR, a function that has been shared by NosX.
In this chapter the main biochemical properties of N 2 OR, as well as the biogenesis of its two copper centers, will be reviewed.
Long-term anthropogenic nitrate (NO3-) enrichment is a serious threat to many coastal systems. Nitrate reduction coupled with the oxidation of reduced forms of sulfur is conducted by chemolithoautotrophic microbial populations in a process that decreases nitrogen (N) pollution. However, little is known about the diversity and distribution of microbes capable of carbon fixation within salt marsh sediment and how they respond to long-term NO3- loading. We used genome-resolved metagenomics to characterize the distribution, phylogenetic relationships, and adaptations important to microbial communities within NO3- enriched sediment. We found NO3- reducing sulfur oxidizers became dominant members of the microbial community throughout the top 25 cm of the sediment following long-term NO3- enrichment. We also found that most of the chemolithoautotrophic genomes recovered contained striking metabolic versatility, including the potential for complete denitrification and evidence of mixotrophy. Phylogenetic reconstruction indicated that similar carbon fixation strategies and metabolic versatility can be found in several phylogenetic groups, but the genomes recovered here represent novel organisms. Our results suggest that the role of chemolithoautotrophy within NO3- enriched salt marsh sediments may be quantitively more important for retaining carbon and filtering NO3- than previously indicated and further inquiry is needed to explicitly measure their contribution to carbon turnover and removal of N pollution.
The boreal spruce forest soil can assimilate atmospheric N 2 O through symbiotic relationships with mycorrhizae or with bacteria, especially during spring and autumn, when aerobic microsites to soil can form. In cold soils with large field capacity (FCD), high humidity and absence of fertilisation, a balance between absorption and emission of nitrous oxide and dinitrogen was observed to be close to zero, and even to assume negative values in some cases, thus suggesting that forest soils absorb more N 2 O than they emit. Furthermore, in the presence of cryptogamic coverings of mosses and lichens, the absorption value was observed to be greater than in forests with less coverage; although the main role in N 2 O absorption is played by soil and root system. However, the role played by epiphytic organisms in N 2 O absorption in the boreal forests has not been uncovered yet. We studied, N 2 O dynamics of the lichen, Platismatia glauca , showing that N 2 O is consumed especially at lower incubation temperatures. The quantitative analysis with real-time PCR of nitrous oxide reductase gene fragment nosZ, showed that enzyme is present in the lichen and the gene is more transcribed under lower incubation temperature. The presented results unveil that cryptogamic covers consume nitrous oxide (with values between 0.1 and 0.4 ng N 2 O-C/g (ww)/h) at the atmospheric concentration via complete dissimilatory denitrification when nitrogen is limited.
Dam construction is regarded as the greatest anthropogenic disturbance in aquatic ecosystems, and it promotes denitrification, through which large N2O emissions occur. However, the effect of dams on N2O producers and other N2O-reducing microorganisms (especially for nosZ II), and the associated denitrification rates remain poorly understood. This study systematically investigated the spatial variation of potential denitrification rates in dammed river sediments in winter and summer and the microbial processes driving N2O production and reduction. Sediments in the transition zone of dammed rivers were found to be critical for N2O emission potential, with lower potential denitrification rate and N2O production rate in winter than in summer. In dammed river sediments, the dominant N2O-producing microorganisms and N2O-reducers were nirS-harboring bacteria and nosZ I-harboring bacteria, respectively. Diversity analysis showed that diversity of N2O-producing did not differ significantly between upstream and downstream sediments, whereas the population size and diversity of N2O-reducing microbial communities in upstream sediments significantly decreased, leading to biological homogenization. Further ecological network analysis revealed that the ecological network of nosZ II microbes was more complex than that of nosZ I microbes, and both exhibited more cooperation in the downstream sediments than in the upstream sediments. Mantel analysis showed that the potential N2O production rate was mainly influenced by electrical conductivity (EC), NH4+, and TC content, and that higher nosZ II/nosZ I ratios contributed to improved N2O sinks in dammed river sediments. Moreover, the Haliscomenobacter genus from the nosZ II-type community in the downstream sediments contributed significantly to N2O reduction. Collectively, this study elucidates the diversity and community distribution of nosZ-type denitrifying microorganisms influenced by dams, and also highlights the non-negligible role played by nosZ II-containing microbial groups in mitigating N2O emissions from dammed river sediments.
Denitrifying woodchip bioreactors (WBRs) are increasingly used to manage the release of non-point source nitrogen (N) by stimulating microbial denitrification. Woodchips serve as a renewable organic carbon (C) source, yet the recalcitrance of organic C in lignocellulosic biomass causes many WBRs to be C-limited. Prior studies have observed that oxic-anoxic cycling increased the mobilization of organic C, increased nitrate (NO3 - ) removal rates, and attenuated production of nitrous oxide (N2 O). Here, we use multi-omics approaches and amplicon sequencing of fungal 5.8S-ITS2 and prokaryotic 16S rRNA genes to elucidate the microbial drivers for enhanced NO3 - removal and attenuated N2 O production under redox-dynamic conditions. Transient oxic periods stimulated the expression of fungal ligninolytic enzymes, increasing the bioavailability of woodchip-derived C and stimulating the expression of denitrification genes. Nitrous oxide reductase (nosZ) genes were primarily clade II, and the ratio of clade II/clade I nosZ transcripts during the oxic-anoxic transition was strongly correlated with the N2 O yield. Analysis of metagenome-assembled genomes revealed that many of the denitrifying microorganisms also have a genotypic ability to degrade complex polysaccharides like cellulose and hemicellulose, highlighting the adaptation of the WBR microbiome to the ecophysiological niche of the woodchip matrix.
Aerobic environments exist widely in wastewater treatment plants (WWTP) and are unfavorable for greenhouse gas nitrous oxide (N2O) reduction. Here, a novel strain Pseudomonas sp. YR02, which can perform N2O reduction under aerobic conditions, was isolated. The successful amplification of four denitrifying genes proved its complete denitrifying ability. The inorganic nitrogen (IN) removal efficiencies (NRE) were >98.0% and intracellular nitrogen and gaseous nitrogen account for 52.6-58.4% and 41.6-47.4% of input nitrogen, respectively. The priority of IN utilization was TAN > NO3--N > NO2--N. The optimal conditions for IN and N2O removal were consistent, except for the C/N ratio, which is 15 and 5 for IN and N2O removal, respectively. The biokinetic constants analysis indicated strain YR02 had high potential to treat high ammonia and dissolved N2O wastewater. Strain YR02 bioaugmentation mitigated 98.7% of N2O emission and improved 32% NRE in WWTP, proving its application potential for N2O mitigation.
Climate change and nutrient pollution are among the most urgent environmental issues. Enhancing the abundance and/or the activity of beneficial organisms is an attractive strategy to counteract these problems. Dissimilatory nitrate reduction to ammonium (DNRA), which theoretically improves nitrogen retention in soils, has been suggested as a microbial process that may be harnessed, especially since many DNRA-catalyzing organisms have been found to possess clade II nosZ genes and the ability to respire N 2 O. However, the selective advantages that may favor these nosZ -harboring DNRA-catalyzing organisms is not well understood. Here, the effect of N 2 O on Nrf-mediated DNRA was examined in a recently isolated soil bacterium, Bacillus sp. DNRA2, possessing both nrfA and nosZ genes. The DNRA metabolism of this bacterium was observed in the presence of C 2 H 2 , a NosZ inhibitor, with or without N 2 O, and the results were compared with C 2 H 2 -free controls. Cultures were also exposed to repeated oxic-anoxic transitions in the sustained presence of N 2 O. The NO 2 ⁻ -to-NH 4 ⁺ reduction following oxic-to-anoxic transition was significantly delayed in NosZ-inhibited C 2 H 2 -amended cultures, and the inhibition was more pronounced with repeated oxic-anoxic transitions. The possible involvement of C 2 H 2 was dismissed since the cultures continuously flushed with C 2 H 2 /N 2 mixed gas after initial oxic incubation did not exhibit a similar delay in DNRA progression as that observed in the culture flushed with N 2 O-containing gas. The findings provide novel ecological and evolutionary insights into the oft-observed presence of nosZ genes in DNRA-catalyzing microorganisms.
Importance
Dissimilatory nitrate/nitrite reduction to ammonium (DNRA) is a microbial energy-conserving process that reduces NO 3 ⁻ and/or NO 2 ⁻ to NH 4 ⁺ . Interestingly, many DNRA-catalyzing microorganisms possessing nrfA genes harbor nosZ genes encoding nitrous oxide reductases, i.e., the only group of enzymes capable of removing the potent greenhouse gas N 2 O. Here, through a series of physiological experiments examining DNRA metabolism in one of such microorganisms, Bacillus sp. DNRA2, we have discovered that N 2 O may delay transition to DNRA upon an oxic-to-anoxic transition, unless timely removed by the nitrous oxide reductases. These observations suggest a novel explanation as to why some nrfA -possessing microorganisms have retained nosZ genes that had probably been acquired via horizontal gene transfers: to remove N 2 O that may otherwise interfere with the transition from O 2 respiration to DNRA.
Nitrogen, an essential component of most biomolecules, used to be a scarce nutrient in many ecosystems since the beginning of life on Earth. The increasing use of artificial fertilizers has led to significant nitrogen pollution in many parts of the world. The resulting inorganic nitrogen species are interconverted by different microorganisms. The reactions involved are summarized in the biological nitrogen cycle. One of these reactions is the dissimilatory nitrate reduction to ammonium (DNRA), a pathway used by various bacteria and archaea for energy conservation. One of the key enzymes of DNRA is the pentahaem cytochrome c nitrite reductase (NrfA), which catalyses the reduction of nitrite to ammonium in a concerted six-electron reduction. NrfA is a particularity in the diverse family of cytochrome c proteins, as the catalytic haem is bound to an unusual CXXCK motif that requires an additional maturation system. Besides nitrite reduction to ammonium, many other reactions in the nitrogen cycle are catalysed by multihaem cytochromes (MCC). Most of these MCCs are evolutionary related, with NrfA as the common ancestor. The latest member of these related proteins is the octahaem hydroxylamine oxidoreductase (HAO). Several intermediate proteins from this transition have been identified and characterised to date. One such intermediate is represented by a family of proteins identified in the class of -proteobacteria. Because of their sequence homology with HAO, these enzymes were annotated as HAOs, although recent studies showed that they actually catalyse the reduction of nitrite to ammonium. The -proteobacterium Geobacter metallireducens, well known for its capability to utilise various metal ions as terminal electron acceptors, contains two genes encoding canonical NrfA proteins, as well as a protein assigned to be a member of the HAO-family. This octahaem cytochrome, called GmNOR, has previously been crystallised and the structure was solved by X-ray crystallography. Structural similarity shows the relation of GmNOR, to the HAOs but the investigation of its catalytic properties revealed low nitrite reductase activity. In this work, GmNOR was characterised as an enzyme that reduces nitric oxide to ammonium with high specificity and at high rates by UV-vis spectroscopy-based enzymatic activity assays. Although this reaction is a side reactivity of nitrite-reducing enzymes such as NrfA or HOAs, no enzyme with such a clear selectivity for the reduction of nitric oxide has been reported yet. Furthermore, NrfA from Geobacter metallireducens was heterologously expressed in E. coli, by a novel expression approach for the maturation of unusual haem-binding motifs. The enzyme’s structure was determined by X-ray crystallography to a resolution of 1.9 Å. The structure showed that GmNrfA is another member of the recently described family of Ca2+-independent NrfAs. Additionally, the active site revealed the presence of an unprecedented loop. This loop harbours an aspartate, an active site residue unseen in any previously characterised NrfA protein. The catalytic relevance of this aspartate was investigated by kinetic studies with the wild type and two variants of the enzyme.
Microorganisms acting as sinks for the greenhouse gas nitrous oxide (N2O) are gaining increasing attention in the development of strategies to control N2O emissions. Non-denitrifying N2O reducers are of particular interest because they can provide a real sink without contributing to N2O release. The bacterial strain under investigation (IGB 4-14T), isolated in a mesocosm experiment to study the litter decomposition of Phragmites australis (Cav.), is such an organism. It carries only a nos gene cluster with the sec-dependent Clade II nosZ and is able to consume significant amounts of N2O under anoxic conditions. However, consumption activity is considerably affected by the O2 level. The reduction of N2O was not associated with cell growth, suggesting that no energy is conserved by anaerobic respiration. Therefore, the N2O consumption of strain IGB 4-14T rather serves as an electron sink for metabolism to sustain viability during transient anoxia and/or to detoxify high N2O concentrations. Phylogenetic analysis of 16S rRNA gene similarity revealed that the strain belongs to the genus Flavobacterium. It shares a high similarity in the nos gene cluster composition and the amino acid similarity of the nosZ gene with various type strains of the genus. However, phylogenomic analysis and comparison of overall genome relatedness indices clearly demonstrated a novel species status of strain IGB 4-14T, with Flavobacterium lacus being the most closely related species. Various phenotypic differences supported a demarcation from this species. Based on these results, we proposed a novel species Flavobacterium azooxidireducens sp. nov. (type strain IGB 4-14T = LMG 29709T = DSM 103580T).
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.
Rice paddy surface soils emit substantial amounts of atmospheric nitrous oxide (N2O). However, the soil can also act as sites for the abiotic and biological consumption of some of the N2O produced in the soil profile, thus, reducing net N2O emissions. Determining the proportion of N2O consumed in the soil, as well as the amount of N2O microbially reduced to nitrogen (N2) is critical for developing N2O mitigating strategies in paddy soils. However, few studies have focused on these processes. Furthermore, the association of microorganisms containing nosZI and nosZII genes with variations in biological N2O consumption potential remains largely unexplored. Here, moisture-controlled (60% gravimetric water content, GWC) and completely anoxic (helium [He] system) microcosm experiments were conducted on paddy surface soils (0–5 cm deep) treated with N2O. During a 96-h incubation period, the N2O cumulative emissions were monitored, the total N2O consumption and N2 production were quantified, and the population sizes of nosZI- and nosZII-containing microorganisms were analyzed. The results showed that 97.87%–99.99% of the N2O that accumulated in the 5 cm soil profile were consumed before emission, and the N2 increase in these soils accounted for 64.50%–82.44% of the total N2O consumption, indicating that the total N2O consumption potential and biological N2O sink capacity of paddy surface soils were considerable under experimental conditions. Furthermore, the production of N2 from N2O seemed to be positively related to soil pH, with less N2 being produced in more acidic soil. In addition, nosZI gene abundance had a strong positive correlation with N2 production, while nosZII had a much weaker correlation. Considering the positive correlation between N2 production and soil dissolved organic carbon (DOC) consumption and available potassium content (AK), it is speculated that complete denitrification by nosZI-N2O reducers dominate the N2O biological sink capacity of the paddy surface soils investigated in this study.
Nitrous oxide is a highly potent greenhouse gas and one of the main contributors to the greenhouse gas footprint of wastewater treatment plants (WWTP). Although nitrous oxide can be produced by abiotic reactions in these systems, biological N2O production resulting from the imbalance of nitrous oxide production and reduction by microbial populations is the dominant cause. The microbial populations responsible for the imbalance have not been clearly identified, yet are likely responsible for strong seasonal nitrous oxide concentration patterns. Here, we examined the seasonal nitrous oxide concentration pattern in Avedøre WWTP alongside abiotic parameters, and the microbial community composition based on 16S rRNA gene sequencing and already available metagenome-assembled genomes (MAGs). We found that the WWTP parameters could not explain the observed pattern. While no distinct community changes between periods of high and low dissolved nitrous oxide concentrations were determined, we found 26 and 28 species with positive and negative correlations to the seasonal N2O concentrations, respectively. MAGs were identified for 124 species (approximately 31% mean relative abundance of the community), and analysis of their genomic nitrogen transformation potential could explain this correlation for four of the negatively correlated species. Other abundant species were also analysed for their nitrogen transformation potential. Interestingly, only one full-denitrifier (Candidatus Dechloromonas phosphorivorans) was identified. 59 species had a nosZ gene predicted, with the majority identified as a clade II nosZ gene, mainly from the phylum Bacteriodota. A correlation of MAG-derived functional guilds with the N2O concentration pattern showed that there was a small but significant negative correlation with nitrite oxidizing bacteria and species with a nosZ gene (N2O reducers (DEN)). More research is required, specifically long-term activity measurements in relation to the N2O concentration to increase the resolution of these findings.
Rice paddy fields are major sources of atmospheric methane (CH4) and nitrous oxide (N2O). Rice variety is an important factor affecting CH4 and N2O emissions. However, the interactive effects of rice metabolites and microorganisms on CH4 and N2O emissions in paddy fields are not clearly understood. In this study, a high greenhouse gas-emitting cultivar (YL 6) and a low greenhouse gas-emitting cultivar (YY 1540) were used as experimental materials. Metabolomics was used to examine the roots, root exudates, and bulk soil metabolites. High-throughput sequencing was used to determine the microbial community composition. YY 1540 had more secondary metabolites (flavonoids and isoflavonoids) in root exudates than YL 6. It was enriched with the uncultured members of the families Gemmatimonadanceae and Rhizobiales_Incertae_Sedis in bulk soil, and genera Burkholderia-Caballeronia-Paraburkholderia, Magnetospirillum, Aeromonas, and Anaeromyxobacter in roots, contributing to increased expression of pmoA and nosZ genes and reducing CH4 and N2O emissions. YL 6 roots and root exudates contained higher contents of carbohydrates [e.g., 6-O- acetylarbutin and 2-(3- hydroxyphenyl) ethanol 1′-glucoside] than those of YY 1540. They were enriched with genera RBG-16-58-14 in bulk soil and Exiguobacterium, and uncultured member of the Kineosporiaceae family in roots, which contributed to increased expression of mcrA, ammonia-oxidizing archaea, ammonia-oxidizing bacteria, nirS, and nirK genes and greenhouse gas emissions. In general, these results established a link between metabolites, microorganisms, microbial functional genes, and greenhouse gas emissions. The metabolites of root exudates and roots regulated CH4 and N2O emissions by influencing the microbial community composition in bulk soil and roots.
Herein, it was demonstrated that the different forms of methanobactin differentially enhance N 2 O emissions from Pseudomonas stutzeri strain DCP-Ps1 (harboring clade I nitrous oxide reductase) and Dechloromonas aromatica strain RCB (harboring clade II nitrous oxide reductase). This work contributes to our understanding of how aerobic methanotrophs compete with denitrifiers for the copper uptake and also suggests how MBs prevent copper collection by denitrifiers, thus downregulating expression of nitrous oxide reductase.
Bacteroidetes VC2.1 Bac22 (referred to as VC2.1) is an uncultured clade that is widely distributed in marine ecosystems, including hydrothermal vents, oxygen‐minimum zones and other anoxic, sulfide‐rich environments. However, the lack of cultured representatives and sequenced genomes of VC2.1 limit our understanding of its physiology, metabolism and ecological functions. Here, we obtained a stable co‐culture of VC2.1 with autotrophic microbes by establishing an autotrophy‐based enrichment from a hydrothermal vent chimney sample. We recovered a high‐quality metagenome‐assembled genome (MAG) that belonged to VC2.1. Phylogenetic analyses of both 16S rRNA genes and conserved protein markers suggested that VC2.1 belongs to a novel order in the Bacteroidetes phylum, which we named Candidatus Sulfidibacteriales. The metabolic reconstruction of this MAG indicated that VC2.1 could utilize polysaccharides, protein polymers and fatty acids as well as flexibly obtain energy via NO/N2O reduction and polysulfide reduction. Our results reveal the ecological potential of this novel Bacteroidetes for complex organic carbons mineralization and N2O sinks in deep‐sea hydrothermal vents. Furthermore, guided by the genome information, we designed a new culture medium in which starch, ammonium and polysulfide were used as the carbon source, nitrogen source and electron acceptor respectively, to isolate VC2.1 successfully.
Nitrous oxide (N2O) is an important greenhouse gas leading to global warming. For the purpose of achieving a clean and sustainable environment within this century, the adoption of technologies to mitigate the N2O emissions is warranted. Intensive management practices for N2O reduction have been implemented for decades. N2O generated from fuel production and biofuel combustion processes could reduce or even negate the global warming reduction by utilizing CO2 as commodities or reducing CO2 yields. In this chapter, the key biological N2O production pathways and the microbiomes involved are elucidated. The current N2O mitigation strategies are systematically summed up to assist future work regarding curbing N2O formation from various ecosystems. The impact of N2O on CO2-based biomaterials and biofuels is clarified, and the potential of nanotechnology for efficient nutrient recovery and N2O mitigation in both natural and industrial fields is discussed. The perspectives and the future challenges regarding the unrevealed N2O production pathways, the large-scale feasibility of current N2O mitigation approaches, and the potential strategies are then put forward with the aim to achieve high biofuel and biomaterial production efficiency while minimizing N2O emissions.
Diatoms are among the opaquest photosynthetic microorganism found in oceans, rivers, and freshwaters. They play a major role in reducing global warming as they fix more than 25% of atmospheric carbon di oxide (CO2). They are a reservoir of untapped potential with the multifaceted application including CO2 mitigation, play a vital role in the aquatic food web as primary producers, and wastewater remediation by quenching pollutants originating from diverse sources such as industries, agricultural, and human sources. Despite their abundance and diversity in nature, only a few species are currently used for biotechnological applications. Diatom biorefinery has gained importance in recent years as more and more algae are identified and explored as a source for lipids, pigments, and other biomolecules. In this chapter, the role of diatom biorefinery has been elaborated extensively displaying the potential of diatoms in carbon dioxide (CO2) mitigation, lipid production for biofuel, nutraceutical potential, and development of new-age drug molecules for therapeutic applications.
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.
Shewanella loihica strain PV-4 harbors both a functional denitrification (NO3 (-) → N2) and a respiratory ammonification (NO3 (-) → NH4 (+)) pathway. Batch and chemostat experiments revealed that NO2 (-) affects pathway selection and the formation of reduced products. Strain PV-4 cells grown with NO2 (-) as the sole electron acceptor produced exclusively NH4 (+). With NO3 (-) as electron acceptor, denitrification predominated and N2O accounted for ∼90% of reduced products in the presence of acetylene. Chemostat experiments demonstrated that the NO2 (-):NO3 (-) ratio affected the distribution of reduced products, and respiratory ammonification dominated at high NO2 (-):NO3 (-) ratios whereas low NO2 (-):NO3 (-) ratios favored denitrification. The NO2 (-):NO3 (-) ratios affected nirK transcript abundance, a measure of denitrification activity, in the chemostat experiments and cells grown at a NO2 (-):NO3 (-) ratio of 3 had ∼37-fold fewer nirK transcripts per cell than cells grown with NO3 (-) as the sole electron acceptor. In contrast, the transcription of nrfA, implicated in NO2 (-)-to-NH4 (+) reduction, remained statistically unchanged under continuous cultivation conditions at NO2 (-):NO3 (-) ratios below 3. At NO2 (-):NO3 (-) ratios above 3, both nirK and nrfA transcript numbers decreased and the chemostat culture washed out, presumably due to NO2 (-) toxicity. These findings implicate NO2 (-) as a relevant modulator of NO3 (-) fate in S. loihica strain PV-4, and, by extension, suggest that NO2 (-) may be a relevant determinant for N-retention (i.e., ammonification) versus N-loss and greenhouse gas emission (i.e., denitrification).
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Denitrification and respiratory ammonification are two competing, energy-conserving NO3-/NO2- reduction pathways that have major biogeochemical consequences for N retention, plant growth and climate. Batch and continuous culture experiments using Shewanella loihica strain PV-4, a bacterium possessing both the denitrification and respiratory ammonification pathways, revealed factors that determine NO3-/NO2- fate. Denitrification dominated at low carbon-to-nitrogen (C/N) ratios (that is, electron donor-limiting growth conditions), whereas ammonium was the predominant product at high C/N ratios (that is, electron acceptor-limiting growth conditions). pH and temperature also affected NO3-/NO2- fate, and incubation above pH 7.0 and temperatures of 30[thinsp][deg]C favored ammonium formation. Reverse-transcriptase real-time quantitative PCR analyses correlated the phenotypic observations with nirK and nosZ transcript abundances that decreased up to 1600-fold and 27-fold, respectively, under conditions favoring respiratory ammonification. Of the two nrfA genes encoded on the strain PV-4 genome, nrfA0844 transcription decreased only when the chemostat reactor received medium with the lowest C/N ratio of 1.5, whereas nrfA0505 transcription occurred at low levels ([le]3.4 [times] 10-2 transcripts per cell) under all growth conditions. At intermediate C/N ratios, denitrification and respiratory ammonification occurred concomitantly, and both nrfA0844 (5.5 transcripts per cell) and nirK (0.88 transcripts per cell) were transcribed. Recent findings suggest that organisms with both the denitrification and respiratory ammonification pathways are not uncommon in soil and sediment ecosystems, and strain PV-4 offers a tractable experimental system to explore regulation of dissimilatory NO3-/NO2- reduction pathways.
Nitrous oxide (N2O) is the predominant ozone-depleting
substance and contributes approximately 6% to overall global
warming1,2. Terrestrial ecosystems account for nearly 70%
of total global N2O atmospheric loading, of which at least
45% can be attributed to microbial cycling of nitrogen
in agriculture3. The reduction of N2O to nitrogen gas by
microorganisms is critical for mitigating its emissions from
terrestrial ecosystems, yet the determinants of a soil’s capacity
to act as a source or sink for N2O remain uncertain4. Here, we
demonstrate that the soilN2Osink capacity is mostly explained
by the abundance and phylogenetic diversity of a newly
described N2O-reducing microbial group5,6, which mediate the
influence of edaphic factors. Analyses of interactions and niche
preference similarities suggest niche di�erentiation or even
competitive interactions between organisms with the twotypes
of N2O reductase.We further identified several recurring communities
comprised of co-occurring N2O-reducing bacterial
genotypes that were significant indicators of the soil N2O sink
capacity across di�erent European soils.
Unlabelled:
Microbial activities in soils, such as (incomplete) denitrification, represent major sources of nitrous oxide (N2O), a potent greenhouse gas. The key enzyme for mitigating N2O emissions is NosZ, which catalyzes N2O reduction to N2. We recently described "atypical" functional NosZ proteins encoded by both denitrifiers and nondenitrifiers, which were missed in previous environmental surveys (R. A. Sanford et al., Proc. Natl. Acad. Sci. U. S. A. 109:19709-19714, 2012, doi:10.1073/pnas.1211238109). Here, we analyzed the abundance and diversity of both nosZ types in whole-genome shotgun metagenomes from sandy and silty loam agricultural soils that typify the U.S. Midwest corn belt. First, different search algorithms and parameters for detecting nosZ metagenomic reads were evaluated based on in silico-generated (mock) metagenomes. Using the derived cutoffs, 71 distinct alleles (95% amino acid identity level) encoding typical or atypical NosZ proteins were detected in both soil types. Remarkably, more than 70% of the total nosZ reads in both soils were classified as atypical, emphasizing that prior surveys underestimated nosZ abundance. Approximately 15% of the total nosZ reads were taxonomically related to Anaeromyxobacter, which was the most abundant genus encoding atypical NosZ-type proteins in both soil types. Further analyses revealed that atypical nosZ genes outnumbered typical nosZ genes in most publicly available soil metagenomes, underscoring their potential role in mediating N2O consumption in soils. Therefore, this study provides a bioinformatics strategy to reliably detect target genes in complex short-read metagenomes and suggests that the analysis of both typical and atypical nosZ sequences is required to understand and predict N2O flux in soils.
Importance:
Nitrous oxide (N2O) is a potent greenhouse gas with ozone layer destruction potential. Microbial activities control both the production and the consumption of N2O, i.e., its conversion to innocuous dinitrogen gas (N2). Until recently, consumption of N2O was attributed to bacteria encoding "typical" nitrous oxide reductase (NosZ). However, recent phylogenetic and physiological studies have shown that previously uncharacterized, functional, "atypical" NosZ proteins are encoded in genomes of diverse bacterial groups. The present study revealed that atypical nosZ genes outnumbered their typical counterparts, highlighting their potential role in N2O consumption in soils and possibly other environments. These findings advance our understanding of the diversity of microbes and functional genes involved in the nitrogen cycle and provide the means (e.g., gene sequences) to study N2O fluxes to the atmosphere and associated climate change.
Grazing systems represent a substantial percentage of the global anthropogenic flux of nitrous oxide (N2O) as a result of nitrogen addition to the soil. The pool of available carbon that is added to the soil from livestock excreta also provides substrate for the production of carbon dioxide (CO2) and methane (CH4) by soil microorganisms. A study into the production and emission of CO2, CH4 and N2O from cattle urine amended pasture was carried out on the Somerset Levels and Moors, UK over a three-month period. Urine-amended plots (50 g N m−2) were compared to control plots to which only water (12 mg N m−2) was applied. CO2 emission peaked at 5200 mg CO2 m−2 d−1 directly after application. CH4 flux decreased to −2000 μg CH4 m−2 d−1 two days after application; however, net CH4 flux was positive from urine treated plots and negative from control plots. N2O emission peaked at 88 mg N2O m−2 d−1 12 days after application. Subsurface CH4 and N2O concentrations were higher in the urine treated plots than the controls. There was no effect of treatment on subsurface CO2 concentrations. Subsurface N2O peaked at 500 ppm 12 days after and 1200 ppm 56 days after application. Subsurface NO3− concentration peaked at approximately 300 mg N kg dry soil−1 12 days after application. Results indicate that denitrification is the key driver for N2O release in peatlands and that this production is strongly related to rainfall events and water-table movement. N2O production at depth continued long after emissions were detected at the surface. Further understanding of the interaction between subsurface gas concentrations, surface emissions and soil hydrological conditions is required to successfully predict greenhouse gas production and emission.
Rice is staple food of half of mankind and paddy soils account for the largest anthropogenic wetlands on earth. Ample of research is being done to find cultivation methods under which the integrative greenhouse effect caused by emitted CH4 and N2O would be mitigated. Whereas most of the research focuses on quantifying such emissions, there is a lack of studies on the biogeochemistry of paddy soils. In order to deepen our mechanistic understanding of N2O and CH4 fluxes in rice paddies, we also determined NO3− and N2O concentrations as well as N2O isotope abundances and presence of O2 along soil profiles of paddies which underwent three different water managements during the rice growing season(s) in (2010 and) 2011 in Korea. Largest amounts of N2O (2 mmol m−2) and CH4 (14.5 mol m−2) degassed from the continuously flooded paddy, while paddies with less flooding showed 30–60 % less CH4 emissions and very low to negative N2O balances. In accordance, the global warming potential (GWP) was lowest for the Intermittent Irrigation paddy and highest for the Traditional Irrigation paddy. The N2O emissions could the best be explained (*P < 0.05) with the δ15N values and N2O concentrations in 40–50 cm soil depth, implying that major N2O production/consumption occurs there. No significant effect of NO3− on N2O production has been found. Our study gives insight into the soil of a rice paddy and reveals areas along the soil profile where N2O is being produced. Thereby it contributes to our understanding of subsoil processes of paddy soils.
The continuous increase of nitrous oxide (N2O) abundance in the atmosphere is a global concern. Multiple pathways of N2O production occur in soil, but their significance and dependence on oxygen (O2) availability and nitrogen (N) fertilizer source are poorly understood. We examined N2O and nitric oxide (NO) production under 21%, 3%, 1%, 0.5%, and 0% (vol/vol) O2 concentrations following urea or ammonium sulfate [(NH4)2SO4] additions in loam, clay loam, and sandy loam soils that also contained ample nitrate. The contribution of the ammonia (NH3) oxidation pathways (nitrifier nitrification, nitrifier denitrification, and nitrification-coupled denitrification) and heterotrophic denitrification (HD) to N2O production was determined in 36-h incubations in microcosms by (15)N-(18)O isotope and NH3 oxidation inhibition (by 0.01% acetylene) methods. Nitrous oxide and NO production via NH3 oxidation pathways increased as O2 concentrations decreased from 21% to 0.5%. At low (0.5% and 3%) O2 concentrations, nitrifier denitrification contributed between 34% and 66%, and HD between 34% and 50% of total N2O production. Heterotrophic denitrification was responsible for all N2O production at 0% O2. Nitrifier denitrification was the main source of N2O production from ammonical fertilizer under low O2 concentrations with urea producing more N2O than (NH4)2SO4 additions. These findings challenge established thought attributing N2O emissions from soils with high water content to HD due to presumably low O2 availability. Our results imply that management practices that increase soil aeration, e.g., reducing compaction and enhancing soil structure, together with careful selection of fertilizer sources and/or nitrification inhibitors, could decrease N2O production in agricultural soils.
Lactate, but not acetate oxidation was reported to support electron acceptor reduction by Shewanella spp. under anoxic conditions. We demonstrate that the denitrifiers Shewanella loihica strain PV-4 and Shewanella denitrificans OS217 utilize acetate as electron donor for denitrification, but not for fumarate or ferric iron reduction.
Agricultural and industrial practices more than doubled the intrinsic rate of terrestrial N fixation over the past century with drastic consequences, including increased atmospheric nitrous oxide (N(2)O) concentrations. N(2)O is a potent greenhouse gas and contributor to ozone layer destruction, and its release from fixed N is almost entirely controlled by microbial activities. Mitigation of N(2)O emissions to the atmosphere has been attributed exclusively to denitrifiers possessing NosZ, the enzyme system catalyzing N(2)O to N(2) reduction. We demonstrate that diverse microbial taxa possess divergent nos clusters with genes that are related yet evolutionarily distinct from the typical nos genes of denitirifers. nos clusters with atypical nosZ occur in Bacteria and Archaea that denitrify (44% of genomes), do not possess other denitrification genes (56%), or perform dissimilatory nitrate reduction to ammonium (DNRA; (31%). Experiments with the DNRA soil bacterium Anaeromyxobacter dehalogenans demonstrated that the atypical NosZ is an effective N(2)O reductase, and PCR-based surveys suggested that atypical nosZ are abundant in terrestrial environments. Bioinformatic analyses revealed that atypical nos clusters possess distinctive regulatory and functional components (e.g., Sec vs. Tat secretion pathway in typical nos), and that previous nosZ-targeted PCR primers do not capture the atypical nosZ diversity. Collectively, our results suggest that nondenitrifying populations with a broad range of metabolisms and habitats are potentially significant contributors to N(2)O consumption. Apparently, a large, previously unrecognized group of environmental nosZ has not been accounted for, and characterizing their contributions to N(2)O consumption will advance understanding of the ecological controls on N(2)O emissions and lead to refined greenhouse gas flux models.
Soils are the main sources of the greenhouse gas nitrous oxide (N2O). The N2O emission at the soil surface is the result of production and consumption processes. So far, research has concentrated on net N2O production. However, in the literature, there are numerous reports of net negative fluxes of N2O, (i.e. fluxes from the atmosphere to the soil). Such fluxes are frequent and substantial and cannot simply be dismissed as experimental noise.
Net N2O consumption has been measured under various conditions from the tropics to temperate areas, in natural and agricultural systems. Low mineral N and large moisture contents have sometimes been found to favour N2O consumption. This fits in with denitrification as the responsible process, reducing N2O to N2. However, it has also been reported that nitrifiers consume N2O in nitrifier denitrification. A contribution of various processes could explain the wide range of conditions found to allow N2O consumption, ranging from low to high temperatures, wet to dry soils, and fertilized to unfertilized plots. Generally, conditions interfering with N2O diffusion in the soil seem to enhance N2O consumption. However, the factors regulating N2O consumption are not yet well understood and merit further study.
Frequent literature reports of net N2O consumption suggest that a soil sink could help account for the current imbalance in estimated global budgets of N2O. Therefore, a systematic investigation into N2O consumption is necessary. This should concentrate on the organisms, reactions, and environmental factors involved.
Fossil fuel combustion and fertilizer application in the United States have substantially altered the nitrogen cycle, with serious effects on climate change. The climate effects can be short-lived, by impacting the chemistry of the atmosphere, or long-lived, by altering ecosystem greenhouse gas fluxes. Here we develop a coherent framework for assessing the climate change impacts of US reactive nitrogen emissions, including oxides of nitrogen, ammonia, and nitrous oxide (N(2)O). We use the global temperature potential (GTP), calculated at 20 and 100 y, in units of CO(2) equivalents (CO(2)e), as a common metric. The largest cooling effects are due to combustion sources of oxides of nitrogen altering tropospheric ozone and methane concentrations and enhancing carbon sequestration in forests. The combined cooling effects are estimated at -290 to -510 Tg CO(2)e on a GTP(20) basis. However, these effects are largely short-lived. On a GTP(100) basis, combustion contributes just -16 to -95 Tg CO(2)e. Agriculture contributes to warming on both the 20-y and 100-y timescales, primarily through N(2)O emissions from soils. Under current conditions, these warming and cooling effects partially offset each other. However, recent trends show decreasing emissions from combustion sources. To prevent warming from US reactive nitrogen, reductions in agricultural N(2)O emissions are needed. Substantial progress toward this goal is possible using current technology. Without such actions, even greater CO(2) emission reductions will be required to avoid dangerous climate change.
The effects of anthropogenic emissions of nitrous oxide (N(2)O), carbon dioxide (CO(2)), methane (CH(4)) and the halocarbons on stratospheric ozone (O(3)) over the twentieth and twenty-first centuries are isolated using a chemical model of the stratosphere. The future evolution of ozone will depend on each of these gases, with N(2)O and CO(2) probably playing the dominant roles as halocarbons return towards pre-industrial levels. There are nonlinear interactions between these gases that preclude unambiguously separating their effect on ozone. For example, the CH(4) increase during the twentieth century reduced the ozone losses owing to halocarbon increases, and the N(2)O chemical destruction of O(3) is buffered by CO(2) thermal effects in the middle stratosphere (by approx. 20% for the IPCC A1B/WMO A1 scenario over the time period 1900-2100). Nonetheless, N(2)O is expected to continue to be the largest anthropogenic emission of an O(3)-destroying compound in the foreseeable future. Reductions in anthropogenic N(2)O emissions provide a larger opportunity for reduction in future O(3) depletion than any of the remaining uncontrolled halocarbon emissions. It is also shown that 1980 levels of O(3) were affected by halocarbons, N(2)O, CO(2) and CH(4), and thus may not be a good choice of a benchmark of O(3) recovery.
Nitrous oxide (N(2)O) is a powerful atmospheric greenhouse gas and cause of ozone layer depletion. Global emissions continue to rise. More than two-thirds of these emissions arise from bacterial and fungal denitrification and nitrification processes in soils, largely as a result of the application of nitrogenous fertilizers. This article summarizes the outcomes of an interdisciplinary meeting, 'Nitrous oxide (N(2)O) the forgotten greenhouse gas', held at the Kavli Royal Society International Centre, from 23 to 24 May 2011. It provides an introduction and background to the nature of the problem, and summarizes the conclusions reached regarding the biological sources and sinks of N(2)O in oceans, soils and wastewaters, and discusses the genetic regulation and molecular details of the enzymes responsible. Techniques for providing global and local N(2)O budgets are discussed. The findings of the meeting are drawn together in a review of strategies for mitigating N(2)O emissions, under three headings, namely: (i) managing soil chemistry and microbiology, (ii) engineering crop plants to fix nitrogen, and (iii) sustainable agricultural intensification.
The widespread occurrence of nitrogen limitation to net primary production in terrestrial and marine ecosystems is something of a puzzle; it would seem that nitrogen fixers should have a substantial competitive advantage wherever nitrogen is limiting, and that their activity in turn should reverse limitation. Nevertheless, there is substantial evidence that nitrogen limits net primary production much of the time in most terrestrial biomes and many marine ecosystems. We examine both how the biogeochemistry of the nitrogen cycle could cause limitation to develop, and how nitrogen limitation could persist as a consequence of processes that prevent or reduce nitrogen fixation. Biogeochemical mechansism that favor nitrogen limitation include: the substantial mobility of nitrogen across ecosystem boundaries, which favors nitrogen limitation in the "source" ecosystem - especially where denitrification is important in sediments and soils, or in terrestrial ecosystems where fires is frequent; differences in
Nitrogen fixing and denitrifying bacteria, respectively, control bulk inputs and outputs of nitrogen in soils, thereby mediating nitrogen-based greenhouse gas emissions in an ecosystem. Molecular techniques were used to evaluate the relative abundances of nitrogen fixing, denitrifying and two numerically dominant ribotypes (based on the > or =97% sequence similarity at the 16S rRNA gene) of bacteria in plots representing 10 agricultural and other land-use practices at the Kellogg biological station long-term ecological research site. Quantification of nitrogen-related functional genes (nitrite reductase, nirS; nitrous oxide reductase, nosZ; and nitrogenase, nifH) as well as two dominant 16S ribotypes (belonging to the phyla Acidobacteria, Thermomicrobia) allowed us to evaluate the hypothesis that microbial community differences are linked to greenhouse gas emissions under different land management practices. Our results suggest that the successional stages of the ecosystem are strongly linked to bacterial functional group abundance, and that the legacy of agricultural practices can be sustained over decades. We also link greenhouse gas emissions with specific compositional responses in the soil bacterial community and assess the use of denitrifying gene abundances as proxies for determining nitrous oxide emissions from soils.
In agricultural cropping systems, crop residues are sources of organic carbon (C), an important factor influencing denitrification.
The effects of red clover, soybean, and barley plant residues and of glucose on denitrifier abundance, denitrification gene
mRNA levels, nitrous oxide (N2O) emissions, and denitrification rates were quantified in anoxic soil microcosms for 72 h. nosZ gene abundances and mRNA levels significantly increased in response to all organic carbon treatments over time. In contrast,
the abundance and mRNA levels of Pseudomonas mandelii and closely related species (nirSP) increased only in glucose-amended soil: the nirSP guild abundance increased 5-fold over the 72-h incubation period (P < 0.001), while the mRNA level significantly increased more than 15-fold at 12 h (P < 0.001) and then subsequently decreased. The nosZ gene abundance was greater in plant residue-amended soil than in glucose-amended soil. Although plant residue carbon-to-nitrogen
(C:N) ratios varied from 15:1 to 30:1, nosZ gene and mRNA levels were not significantly different among plant residue treatments, with an average of 3.5 × 107 gene copies and 6.9 × 107 transcripts g−1 dry soil. Cumulative N2O emissions and denitrification rates increased over 72 h in both glucose- and plant-tissue-C-treated soil. The nirSP and nosZ communities responded differently to glucose and plant residue amendments. However, the targeted denitrifier communities
responded similarly to the different plant residues under the conditions tested despite changes in the quality of organic
C and different C:N ratios.
The objective of this study was to investigate how changes in soil pH affect the N2O and N2 emissions, denitrification activity, and size of a denitrifier community. We established a field experiment, situated in
a grassland area, which consisted of three treatments which were repeatedly amended with a KOH solution (alkaline soil), an
H2SO4 solution (acidic soil), or water (natural pH soil) over 10 months. At the site, we determined field N2O and N2 emissions using the 15N gas flux method and collected soil samples for the measurement of potential denitrification activity and quantification
of the size of the denitrifying community by quantitative PCR of the narG, napA, nirS, nirK, and nosZ denitrification genes. Overall, our results indicate that soil pH is of importance in determining the nature of denitrification
end products. Thus, we found that the N2O/(N2O + N2) ratio increased with decreasing pH due to changes in the total denitrification activity, while no changes in N2O production were observed. Denitrification activity and N2O emissions measured under laboratory conditions were correlated with N fluxes in situ and therefore reflected treatment differences in the field. The size of the denitrifying community was uncoupled from in situ N fluxes, but potential denitrification was correlated with the count of NirS denitrifiers. Significant relationships were
observed between nirS, napA, and narG gene copy numbers and the N2O/(N2O + N2) ratio, which are difficult to explain. However, this highlights the need for further studies combining analysis of denitrifier
ecology and quantification of denitrification end products for a comprehensive understanding of the regulation of N fluxes
by denitrification.
Anaeromyxobacter spp. respire soluble hexavalent uranium, U(VI), leading to the formation of insoluble U(IV), and are present at the uranium-contaminated
Oak Ridge Integrated Field Research Challenge (IFC) site. Pilot-scale in situ bioreduction of U(VI) has been accomplished in area 3 of the Oak Ridge IFC site following biostimulation, but the susceptibility
of the reduced material to oxidants (i.e., oxygen) compromises long-term U immobilization. Following oxygen intrusion, attached
Anaeromyxobacter dehalogenans cells increased approximately 5-fold from 2.2 × 107 ± 8.6 × 106 to 1.0 × 108 ± 2.2 × 107 cells per g of sediment collected from well FW101-2. In the same samples, the numbers of cells of Geobacter lovleyi, a population native to area 3 and also capable of U(VI) reduction, decreased or did not change. A. dehalogenans cells captured via groundwater sampling (i.e., not attached to sediment) were present in much lower numbers (<1.3 × 104 ± 1.1 × 104 cells per liter) than sediment-associated cells, suggesting that A. dehalogenans cells occur predominantly in association with soil particles. Laboratory studies confirmed aerobic growth of A. dehalogenans strain 2CP-C at initial oxygen partial pressures (pO2) at and below 0.18 atm. A negative linear correlation [μ = (−0.09 × pO2) + 0.051; R2 = 0.923] was observed between the instantaneous specific growth rate μ and pO2, indicating that this organism should be classified as a microaerophile. Quantification of cells during aerobic growth revealed
that the fraction of electrons released in electron donor oxidation and used for biomass production (fs) decreased from 0.52 at a pO2 of 0.02 atm to 0.19 at a pO2 of 0.18 atm. Hence, the apparent fraction of electrons utilized for energy generation (i.e., oxygen reduction) (fe) increased from 0.48 to 0.81 with increasing pO2, suggesting that oxygen is consumed in a nonrespiratory process at a high pO2. The ability to tolerate high oxygen concentrations, perform microaerophilic oxygen respiration, and preferentially associate
with soil particles represents an ecophysiology that distinguishes A. dehalogenans from other known U(VI)-reducing bacteria in area 3, and these features may play roles for stabilizing immobilized radionuclides
in situ.
Nitrous oxide (N20) is a greenhouse gas, the third most significant contributor to global warming. As a key process for N20 elimination from the biosphere, N20 reductases catalyze the two-electron reduction of N20 to N2. These 2 x 65 kDa copper enzymes are thought to contain a CuA electron entry site, similar to that of cytochrome c oxidase, and a CuZ catalytic center. The copper anomalous signal was used to solve the crystal structure of N20 reductase from Pseudomonas nautica by multiwavelength anomalous dispersion, to a resolution of 2.4 A. The structure reveals that the CuZ center belongs to a new type of metal cluster, in which four copper ions are liganded by seven histidine residues. N20 binds to this center via a single copper ion. The remaining copper ions might act as an electron reservoir, assuring a fast electron transfer and avoiding the formation of dead-end products.
Unwelcome Dominance
Stratospheric ozone is depleted by many different chemicals; most prominently, chlorofluorocarbons (CFCs) responsible for causing the Antarctic ozone hole. Nitrous oxide is also an ozone-depleting substance that has natural sources in addition to anthropogenic ones. Moreover, unlike CFCs, its use and emission are not regulated by the Montreal Protocol, which has helped to reverse the rate of growth of the ozone hole. Surprisingly, Ravishankara et al. (p. 123 , published online 27 August; see the Perspective by Wuebbles ) now show that nitrous oxide is the single greatest ozone-depleting substance that, if its emissions are not controlled, is expected to remain the dominant ozone-depleting substance throughout the 21st century. Reducing nitrous oxide emissions would thus enhance the rate of recovery of the ozone hole and reduce the anthropogenic forcing of climate.
Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.
Biomass formation represents one of the most basic aspects of bacterial metabolism. While there is an abundance of information concerning individual reactions that result in cell duplication, there has been surprisingly little information on the bioenergetics of growth. For many years, it was assumed that biomass production (anabolism) was proportional to the amount of ATP which could be derived from energy-yielding pathways (catabolism), but later work showed that the ATP yield (YATP) was not necessarily a constant. Continuous-culture experiments indicated that bacteria utilized ATP for metabolic reactions that were not directly related to growth (maintenance functions). Mathematical derivations showed that maintenance energy appeared to be a growth rate-independent function of the cell mass and time. Later work, however, showed that maintenance energy alone could not account for all the variations in yield. Because only some of the discrepancy could be explained by the secretion of metabolites (overflow metabolism) or the diversion of catabolism to metabolic pathways which produced less ATP, it appeared that energy-excess cultures had mechanisms of spilling energy. Bacteria have the potential to spill excess ATP in futile enzyme cycles, but there has been little proof that such cycles are significant. Recent work indicated that bacteria can also use futile cycles of potassium, ammonia, and protons through the cell membrane to dissipate ATP either directly or indirectly. The utility of energy spilling in bacteria has been a curiosity. The deprivation of energy from potential competitors is at best a teleological explanation that cannot be easily supported by standard theories of natural selection. The priming of intracellular intermediates for future growth or protection of cells from potentially toxic end products (e.g., methylglyoxal) seems a more plausible explanation.
The kinetics of denitrification and the causes of nitrite and nitrous oxide accumulation were examined in resting cell suspensions of three denitrifiers. An Alcaligenes species and a Pseudomonas fluorescens isolate characteristically accumulated nitrite when reducing nitrate; a Flavobacterium isolate did not. Nitrate did not inhibit nitrite reduction in cultures grown with tungstate to prevent formation of an active nitrate reductase; rather, accumulation of nitrite seemed to depend on the relative rates of nitrate and nitrite reduction. Each isolate rapidly reduced nitrous oxide even when nitrate or nitrite had been included in the incubation mixture. Nitrate also did not inhibit nitrous oxide reduction in Alcaligenes odorans , an organism incapable of nitrate reduction. Thus, added nitrate or nitrite does not always cause nitrous oxide accumulation, as has often been reported for denitrifying soils. All strains produced small amounts of nitric oxide during denitrification in a pattern suggesting that nitric oxide was also under kinetic control similar to that of nitrite and nitrous oxide. Apparent K m values for nitrate and nitrite reduction were 15 μM or less for each isolate. The K m value for nitrous oxide reduction by Flavobacterium sp. was 0.5 μM. Numerical solutions to a mathematical model of denitrification based on Michaelis-Menten kinetics showed that differences in reduction rates of the nitrogenous compounds were sufficient to account for the observed patterns of nitrite, nitric oxide, and nitrous oxide accumulation. Addit