Importance of denitrifiers lacking the genes encoding the nitrous oxide reductase for N2O emissions from soil

Analyses of the complete genomes of sequenced denitrifying bacteria revealed that approximately 1/3 have a truncated denitrification pathway, lacking the nosZ gene encoding the nitrous oxide reductase. We investigated whether the number of denitrifiers lacking the genetic ability to synthesize the nitrous oxide reductase in soils is important for the proportion of N₂O emitted by denitrification. Serial dilutions of the denitrifying strain Agrobacterium tumefaciens C58 lacking the nosZ gene were inoculated into three different soils to modify the proportion of denitrifiers having the nitrous oxide reductase genes. The potential denitrification and N₂O emissions increased when the size of inoculated C58 population in the soils was in the same range as the indigenous nosZ community. However, in two of the three soils, the increase in potential denitrification in inoculated microcosms compared with the noninoculated microcosms was higher than the increase in N₂O emissions. This suggests that the indigenous denitrifier community was capable of acting as a sink for the N₂O produced by A. tumefaciens. The relative amount of N₂O emitted also increased in two soils with the number of inoculated C58 cells, establishing a direct causal link between the denitrifier community composition and potential N₂O emissions by manipulating the proportion of denitrifiers having the nosZ gene. However, the number of denitrifiers which do not possess a nitrous oxide reductase might not be as important for N₂O emissions in soils having a high N₂O uptake capacity compared with those with lower. In conclusion, we provide a proof of principle that the inability of some denitrifiers to synthesize the nitrous oxide reductase can influence the nature of the denitrification end products, indicating that the extent of the reduction of N₂O to N₂ by the denitrifying community can have a genetic basis.     

施氮对水稻土N2O释放及反硝化功能基因(narG/nosZ)丰度的影响

以紫潮泥和红黄泥两种不同质地的水稻土壤作为研究对象,通过室内培养试验,分析施用硝态氮肥对N2O释放和反硝化基因(narG/nosZ)丰度的影响,并探讨反硝化基因丰度与N2O释放之间的关系。结果表明,施用硝态氮显著增加两种水稻土的N2O释放量。在72h培养过程中,施氮改变了紫潮泥反硝化基因(narG/nosZ)的丰度,但并未明显影响红黄泥反硝化基因(narG/nosZ)丰度。通过双变量相关分析发现,除了紫潮泥narG基因外,其它的反硝化基因丰度和N2O释放之间并没有显著相关性。     

Abundance and Structure Composition of nirK and nosZ Genes as Well as Denitrifying Activity in Heavy Metal-polluted Paddy Soils

Denitrification is an important microbial process in soils and leads to the emission of nitrous oxide (N₂O). However, studies about the microbial community involved in denitrification processes in polluted paddy fields are scarce. Here, we studied two rice paddies which had been polluted for more than three decades by metal mining and smelter activities. Abundance and community composition were determined using real-time polymerase chain reaction (PCR) assay and denaturing gradient gel electrophoresis of nitrite reductase and nitrous oxide reductase gene amplicons (nirK and nosZ), while denitrifying activities were assessed by measuring potential denitrifier enzyme activity. We found that the community structure of both nirK and nosZ containing denitrifiers shifted under pollution in the two rice paddies. All the retrieved nirK sequences did not group into either α- or β-proteobacteria, while most of the nosZ species were affiliated with α-proteobacteria. While the abundance of both nirK and nosZ was significantly reduced in the polluted soils at “Dexing” (with relatively higher Cu levels), these parameters did not change significantly at “Dabaoshan” (polluted with Cd, Pb, Cu, and Zn). Furthermore, total denitrifying activity and N₂O production and reduction rates also only decreased under pollution at “Dexing.” These findings suggest that nirK and nosZ containing denitrifier populations and their activities could be sensitive to considerable Cu pollution, which could potentially affect N₂O release from polluted paddy soils.     

Nitrous Oxide (N2O) Emissions by Termites: Does the Feeding Guild Matter?

In the tropics, termites are major players in the mineralization of organic matter leading to the production of greenhouse gases including nitrous oxide (N₂O). Termites have a wide trophic diversity and their N-metabolism depends on the feeding guild. This study assessed the extent to which N₂O emission levels were determined by termite feeding guild and tested the hypothesis that termite species feeding on a diet rich in N emit higher levels of N₂O than those feeding on a diet low in N. An in-vitro incubation approach was used to determine the levels of N₂O production in 14 termite species belonging to different feeding guilds, collected from a wide range of biomes. Fungus-growing and soil-feeding termites emit N₂O. The N₂O production levels varied considerably, ranging from 13.14 to 117.62 ng N₂O-N d^-1 (g dry wt.)^-1 for soil-feeding species, with Cubitermes spp. having the highest production levels, and from 39.61 to 65.61 ng N₂O-N d^-1 (g dry wt.)^-1 for fungus-growing species. Wood-feeding termites were net N₂O consumers rather than N₂O producers with a consumption ranging from 16.09 to 45.22 ng N₂O-N d^-1 (g dry wt.)^-1. Incubating live termites together with their mound increased the levels of N₂O production by between 6 and 13 fold for soil-feeders, with the highest increase in Capritermes capricornis, and between 14 and 34 fold for fungus-growers, with the highest increase in Macrotermes muelleri. Ammonia-oxidizing (amoA-AOB and amoA-AOA) and denitrifying (nirK, nirS, nosZ) gene markers were detected in the guts of all termite species studied. No correlation was found between the abundance of these marker genes and the levels of N₂O production from different feeding guilds. Overall, these results support the hypothesis that N₂O production rates were higher in termites feeding on substrates with higher N content, such as soil and fungi, compared to those feeding on N-poor wood.     

Reduction of the Powerful Greenhouse Gas N2O in the South-Eastern Indian Ocean

Nitrous oxide (N₂O) is a powerful greenhouse gas and a key catalyst of stratospheric ozone depletion. Yet, little data exist about the sink and source terms of the production and reduction of N₂O outside the well-known oxygen minimum zones (OMZ). Here we show the presence of functional marker genes for the reduction of N₂O in the last step of the denitrification process (nitrous oxide reductase genes; nosZ) in oxygenated surface waters (180–250 O₂ μmol.kg^-1) in the south-eastern Indian Ocean. Overall copy numbers indicated that nosZ genes represented a significant proportion of the microbial community, which is unexpected in these oxygenated waters. Our data show strong temperature sensitivity for nosZ genes and reaction rates along a vast latitudinal gradient (32°S-12°S). These data suggest a large N₂O sink in the warmer Tropical waters of the south-eastern Indian Ocean. Clone sequencing from PCR products revealed that most denitrification genes belonged to Rhodobacteraceae. Our work highlights the need to investigate the feedback and tight linkages between nitrification and denitrification (both sources of N₂O, but the latter also a source of bioavailable N losses) in the understudied yet strategic Indian Ocean and other oligotrophic systems.     

Association of Novel and Highly Diverse Acid-Tolerant Denitrifiers with N2O Fluxes of an Acidic Fen

Wetlands are sources of denitrification-derived nitrous oxide (N₂O). Thus, the denitrifier community of an N₂O-emitting fen (pH 4.7 to 5.2) was investigated. N₂O was produced and consumed to subatmospheric concentrations in unsupplemented anoxic soil microcosms. Total cell counts and most probable numbers of denitrifiers approximated 10¹¹ cells·g_DW⁻¹ (where DW is dry weight) and 10⁸ cells·g_DW⁻¹, respectively, in both 0- to 10-cm and 30- to 40-cm depths. Despite this uniformity, depth-related maximum reaction rate (v_max) values for denitrification in anoxic microcosms ranged from 1 to 24 and −19 to −105 nmol N₂O h⁻¹· g_DW⁻¹, with maximal values occurring in the upper soil layers. Denitrification was enhanced by substrates that might be formed via fermentation in anoxic microzones of soil. N₂O approximated 40% of total nitrogenous gases produced at in situ pH, which was likewise the optimal pH for denitrification. Gene libraries of narG and nosZ (encoding nitrate reductase and nitrous oxide reductase, respectively) from fen soil DNA yielded 15 and 18 species-level operational taxonomic units, respectively, many of which displayed phylogenetic novelty and were not closely related to cultured organisms. Although statistical analyses of narG and nosZ sequences indicated that the upper 20 cm of soil contained the highest denitrifier diversity and species richness, terminal restriction fragment length polymorphism analyses of narG and nosZ revealed only minor differences in denitrifier community composition from a soil depth of 0 to 40 cm. The collective data indicate that the regional fen harbors novel, highly diverse, acid-tolerant denitrifier communities capable of complete denitrification and consumption of atmospheric N₂O at in situ pH.Nitrous oxide (N₂O) is a potent greenhouse gas with a global warming potential that is 300-fold higher than that of CO₂, and its concentration increased from 270 ppb in 1750 to 319 ppb in 2005 (17). N₂O can be produced in soils during denitrification, nitrification, the dissimilatory reduction of nitrate to nitrite and/or ammonium (hereafter referred to as dissimilatory nitrate reduction), or the chemical transformation of nitrite or hydroxylamine (5, 7, 49). The percentage of N₂O produced in any of these processes is variable, depending mainly on the redox potential, pH, and C/N ratio (49). In anoxic ecosystems such as waterlogged soils, most of the N₂O is considered to be denitrification derived (7, 9). Complete denitrification is the sequential reduction of nitrate to dinitrogen (N₂) via nitrite, nitric oxide (NO), and N₂O (75). The main product of denitrification varies with the organism and in situ conditions and is usually either N₂O or N₂ (68). N₂O can occur as a by-product during dissimilatory nitrate reduction when accumulated nitrite interacts with nitrate reductase to form N₂O (59). The production of N₂O by dissimilatory nitrate reducers is favored in environments with large amounts of readily available organic carbon (65). Thus, their contribution to nitrate-dependent production of N₂O in soils is likely insignificant compared to that of denitrifiers.The oxidoreductases involved in denitrification are termed dissimilatory nitrate reductase (Nar, encoded by narGHJI, or Nap, encoded by napEDABC), nitrite reductase (Nir, encoded by nirK and nirS), NO reductase (cNor and qNor, encoded by norBC and norB, respectively), and N₂O reductase (Nos, encoded by nosZ) (75). Nitrate reductase is also found in dissimilatory nitrate reducers (60). narG can therefore be used as a molecular marker to assess both denitrifiers and dissimilatory nitrate reducers, whereas nosZ is specific for the assessment of denitrifiers (25, 43, 48).Denitrification in soils is regulated by temperature, pH, substrate (i.e., carbon) availability, and water content (10, 24, 66). Although denitrification increases with increasing temperature, it can still occur at temperatures below 0°C (10, 24). Low temperatures appear to limit the activity of N₂O reductase more severely than other enzymes involved in denitrification and thus yield higher relative amounts of denitrification-derived N₂O (24). Although denitrification activity usually decreases under acidic conditions, the relative percentage of N₂O to total denitrification-derived nitrogenous gases increases with increasing acidity, a result attributed to the sensitivity of N₂O reductase to low pH (27, 70). However, denitrifier communities can be adapted to the in situ pH of the system (40, 58, 73).Wetlands are ecosystems in which denitrification is likely a dominant source of emitted N₂O (7, 44, 45). The identification and analysis of main drivers for N₂O production (i.e., the microbiota catalyzing N₂O production and consumption) is thus of major concern in such environments. Fens are specialized wetlands characterized by soil acidity (67). However, information on acid-tolerant denitrifier communities of such wetlands is scarce. It is hypothesized that fens harbor a diverse, hitherto unknown, denitrifier community that is adapted to in situ conditions and associated with N₂O fluxes (i.e., fen denitrifiers are acid tolerant and have a high affinity for nitrate and N₂O). Thus, the main objectives of the present study were to evaluate the capacities of denitrifier communities of an N₂O-emitting fen (20) to produce or consume N₂O and to determine if a novel and diverse denitrifier community was associated with these capacities.     

Insights into the Effect of Soil pH on N2O and N2 Emissions and Denitrifier Community Size and Activity

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.Denitrification is the microbial reduction of NO₃⁻ via NO₂⁻ to gaseous NO, N₂O, and N₂, which are then lost into the atmosphere (36). It therefore results in considerable loss of nitrogen, one of the most limiting nutrients for crop production in agriculture (20). Denitrification is also of environmental concern since, together with nitrification, it is the main biological process responsible for N₂O emissions (7). N₂O is a potent greenhouse gas which has a global warming potential about 320 times greater than that of CO₂ and has a lifetime of approximately 120 years (32). In the stratosphere, N₂O can also react with O₂ to produce NO, which induces the destruction of stratospheric ozone (8). N₂O can be released into the atmosphere by incomplete denitrification due to the effect of environmental conditions on the regulation of the different denitrification reductases (14, 41, 51), but it has recently been suggested that it could also be due to lack of nitrous oxide reductase in some denitrifiers (19, 41). Since N₂O is an intermediate in the denitrification pathway, both the amount of N₂O produced and the N₂O/(N₂O + N₂) ratio are important in understanding and predicting N₂O fluxes from soils.The main environmental factors known to influence the N₂O/(N₂O + N₂) ratio are pH, organic carbon and NO₃⁻ availability, water content, and O₂ partial pressure (50). Soil pH is one of the most important factors influencing both denitrification and N₂O production (43). In general, the denitrification rate increases with increasing pH values (up to the optimum pH) while, in contrast, the N₂O/(N₂O + N₂) ratio decreases (50). This relationship has been characterized in laboratory experiments (9, 45), but it is not clear whether the same relationships exist in the field because of methodological limitations of in situ measurement of N₂ emissions (16). Nevertheless, ¹⁵N tracing experiments based on the addition of a labeled denitrification substrate to soil offer a useful tool to quantify emissions of both N₂O and N₂ in situ (47, 49). Soil pH is also an important factor influencing denitrifier community composition (35, 39), which can be an important driver of denitrification activity and N₂O emissions (5, 21). A recent study reported a negative relationship between the proportion of bacteria genetically capable of reducing N₂O within the total bacterial community and the N₂O/(N₂O + N₂) ratio, with both being strongly correlated with soil pH (38).The objective of the present study was to explore the effect of changes in soil pH on in situ N₂O and N₂ emissions, denitrifying enzyme activity (DEA), and potential N₂O production. In addition, we also investigated whether differences in N fluxes could be related to changes in the size of the microbial community possessing the different denitrification genes. A field experiment was conducted using replicated grassland plots in which the soil pH was modified by addition of either acid or hydroxide to the soil. A ¹⁵N tracer method was used to provide information on N emissions. In addition to measuring potential denitrification activity, the size of the denitrifier community was determined by real-time PCR quantification of the denitrification genes.     

Symbiotic relationships between soil fungi and plants reduce N2O emissions from soil

N₂O is a potent greenhouse gas involved in the destruction of the protective ozone layer in the stratosphere and contributing to global warming. The ecological processes regulating its emissions from soil are still poorly understood. Here, we show that the presence of arbuscular mycorrhizal fungi (AMF), a dominant group of soil fungi, which form symbiotic associations with the majority of land plants and which influence a range of important ecosystem functions, can induce a reduction in N₂O emissions from soil. To test for a functional relationship between AMF and N₂O emissions, we manipulated the abundance of AMF in two independent greenhouse experiments using two different approaches (sterilized and re-inoculated soil and non-mycorrhizal tomato mutants) and two different soils. N₂O emissions were increased by 42 and 33% in microcosms with reduced AMF abundance compared to microcosms with a well-established AMF community, suggesting that AMF regulate N₂O emissions. This could partly be explained by increased N immobilization into microbial or plant biomass, reduced concentrations of mineral soil N as a substrate for N₂O emission and altered water relations. Moreover, the abundance of key genes responsible for N₂O production (nirK) was negatively and for N₂O consumption (nosZ) positively correlated to AMF abundance, indicating that the regulation of N₂O emissions is transmitted by AMF-induced changes in the soil microbial community. Our results suggest that the disruption of the AMF symbiosis through intensification of agricultural practices may further contribute to increased N₂O emissions.     

N<Subscript>2</Subscript>O production by denitrification in an urban river: evidence from isotopes,functional genes,and dissolved organic matter

Rivers are important sources of N₂O emissions into the atmosphere. Nevertheless, N₂O production processes in rivers are not well identified. We measured concentrations and isotopic ratios of N₂O, NH₄ ⁺, NO₂ ^?, and NO₃ ^? in surface water to identify the microbial processes of N₂O production along the Tama River in Japan. We also measured the functional gene abundance of nitrifiers and denitrifiers (amoA-bacteria, nirK, nirS, nosZ clade I, nosZ clade II) together with concentrations of dissolved organic carbon (DOC) and fluorescence intensities of protein and humic components of dissolved organic matter (DOM) to support the elucidation of N₂O production processes. The observed nitrogen (δ¹⁵N) and oxygen (δ¹⁸O) of N₂O were within the expected isotopic range of N₂O produced by nitrate reduction, indicating that N₂O was dominantly produced by denitrification. The positive significant correlation between N₂O_Net concentration and nirK gene abundance implied that nitrifiers and denitrifiers are contributors to N₂O production. Fluorescence intensities of protein and humic components of DOM and concentrations of DOC did not show significant correlations with N₂O concentrations, which suggests that DOC and abundance of DOM components do not control dissolved N₂O. Measurement of isotope ratios of N₂O and its substrates was found to be a useful tool to obtain evidence of denitrification as the main source of N₂O production along the Tama River.     

Effects of heavy metals on aerobic denitrification by strain Pseudomonas stutzeri PCN-1

Effects of heavy metals on aerobic denitrification have been poorly understood compared with their impacts on anaerobic denitrification. This paper presented effects of four heavy metals (Cd(II), Cu(II), Ni(II), and Zn(II)) on aerobic denitrification by a novel aerobic denitrifying strain Pseudomonas stutzeri PCN-1. Results indicated that aerobic denitrifying activity decreased with increasing heavy metal concentrations due to their corresponding inhibition on the denitrifying gene expression characterized by a time lapse between the expression of the nosZ gene and that of the cnorB gene by PCN-1, which led to lower nitrate removal rate (1.67∼6.67 mg L⁻¹ h⁻¹), higher nitrite accumulation (47.3∼99.8 mg L⁻¹), and higher N₂O emission ratios (5∼283 mg L⁻¹/mg L⁻¹). Specially, promotion of the nosZ gene expression by increasing Cu(II) concentrations (0∼0.05 mg L⁻¹) was found, and the absence of Cu resulted in massive N₂O emission due to poor synthesis of N₂O reductase. The inhibition effect for both aerobic denitrifying activity and denitrifying gene expression was as follows from strongest to least: Cd(II) (0.5∼2.5 mg L⁻¹) > Cu(II) (0.5∼5 mg L⁻¹) > Ni(II) (2∼10 mg L⁻¹) > Zn(II) (25∼50 mg L⁻¹). Furthermore, sensitivity of denitrifying gene to heavy metals was similar in order of nosZ > nirS ≈ cnorB > napA. This study is of significance in understanding the potential application of aerobic denitrifying bacteria in practical wastewater treatment.

     

Symbiotic Bradyrhizobium japonicum Reduces N2O Surrounding the Soybean Root System via Nitrous Oxide Reductase

N₂O reductase activity in soybean nodules formed with Bradyrhizobium japonicum was evaluated from N₂O uptake and conversion of ¹⁵N-N₂O into ¹⁵N-N₂. Free-living cells of USDA110 showed N₂O reductase activity, whereas a nosZ mutant did not. Complementation of the nosZ mutant with two cosmids containing the nosRZDFYLX genes of B. japonicum USDA110 restored the N₂O reductase activity. When detached soybean nodules formed with USDA110 were fed with ¹⁵N-N₂O, they rapidly emitted ¹⁵N-N₂ outside the nodules at a ratio of 98.5% of ¹⁵N-N₂O uptake, but nodules inoculated with the nosZ mutant did not. Surprisingly, N₂O uptake by soybean roots nodulated with USDA110 was observed even in ambient air containing a low concentration of N₂O (0.34 ppm). These results indicate that the conversion of N₂O to N₂ depends exclusively on the respiratory N₂O reductase and that soybean roots nodulated with B. japonicum carrying the nos genes are able to remove very low concentrations of N₂O.     

Sub-Parts-Per-Billion Nitrate Method: Use of an N2O-Producing Denitrifier to Convert NO3− or 15NO3− to N2O

A more sensitive analytical method for NO₃⁻ was developed based on the conversion of NO₃⁻ to N₂O by a denitrifier that could not reduce N₂O further. The improved detectability resulted from the high sensitivity of the ⁶³Ni electron capture gas chromatographic detector for N₂O and the purification of the nitrogen afforded by the transformation of the N to a gaseous product with a low atmospheric background. The selected denitrifier quantitatively converted NO₃⁻ to N₂O within 10 min. The optimum measurement range was from 0.5 to 50 ppb (50 μg/liter) of NO₃⁻ N, and the detection limit was 0.2 ppb of N. The values measured by the denitrifier method compared well with those measured by the high-pressure liquid chromatographic UV method above 2 ppb of N, which is the detection limit of the latter method. It should be possible to analyze all types of samples for nitrate, except those with inhibiting substances, by this method. To illustrate the use of the denitrifier method, NO₃⁻ concentrations of <2 ppb of NO₃⁻ N were measured in distilled and deionized purified water samples and in anaerobic lake water samples, but were not detected at the surface of the sediment. The denitrifier method was also used to measure the atom% of ¹⁵N in NO₃⁻. This method avoids the incomplete reduction and contamination of the NO₃⁻ -N by the NH₄⁺ and N₂ pools which can occur by the conventional method of ¹⁵NO₃⁻ analysis. N₂O-producing denitrifier strains were also used to measure the apparent K_m values for NO₃⁻ use by these organisms. Analysis of N₂O production by use of a progress curve yielded K_m values of 1.7 and 1.8 μM NO₃⁻ for the two denitrifier strains studied.     

Peaks of in situ N2O emissions are influenced by N2O‐producing and reducing microbial communities across arable soils

Agriculture is the main source of terrestrial N₂O emissions, a potent greenhouse gas and the main cause of ozone depletion. The reduction of N₂O into N₂ by microorganisms carrying the nitrous oxide reductase gene (nosZ) is the only known biological process eliminating this greenhouse gas. Recent studies showed that a previously unknown clade of N₂O‐reducers (nosZII) was related to the potential capacity of the soil to act as a N₂O sink. However, little is known about how this group responds to different agricultural practices. Here, we investigated how N₂O‐producers and N₂O‐reducers were affected by agricultural practices across a range of cropping systems in order to evaluate the consequences for N₂O emissions. The abundance of both ammonia‐oxidizers and denitrifiers was quantified by real‐time qPCR, and the diversity of nosZ clades was determined by 454 pyrosequencing. Denitrification and nitrification potential activities as well as in situ N₂O emissions were also assessed. Overall, greatest differences in microbial activity, diversity, and abundance were observed between sites rather than between agricultural practices at each site. To better understand the contribution of abiotic and biotic factors to the in situ N₂O emissions, we subdivided more than 59,000 field measurements into fractions from low to high rates. We found that the low N₂O emission rates were mainly explained by variation in soil properties (up to 59%), while the high rates were explained by variation in abundance and diversity of microbial communities (up to 68%). Notably, the diversity of the nosZII clade but not of the nosZI clade was important to explain the variation of in situ N₂O emissions. Altogether, these results lay the foundation for a better understanding of the response of N₂O‐reducing bacteria to agricultural practices and how it may ultimately affect N₂O emissions.     

Intergenomic Comparisons Highlight Modularity of the Denitrification Pathway and Underpin the Importance of Community Structure for N2O Emissions

Nitrous oxide (N₂O) is a potent greenhouse gas and the predominant ozone depleting substance. The only enzyme known to reduce N₂O is the nitrous oxide reductase, encoded by the nosZ gene, which is present among bacteria and archaea capable of either complete denitrification or only N₂O reduction to di-nitrogen gas. To determine whether the occurrence of nosZ, being a proxy for the trait N₂O reduction, differed among taxonomic groups, preferred habitats or organisms having either NirK or NirS nitrite reductases encoded by the nirK and nirS genes, respectively, 652 microbial genomes across 18 phyla were compared. Furthermore, the association of different co-occurrence patterns with enzymes reducing nitric oxide to N₂O encoded by nor genes was examined. We observed that co-occurrence patterns of denitrification genes were not randomly distributed across taxa, as specific patterns were found to be more dominant or absent than expected within different taxonomic groups. The nosZ gene had a significantly higher frequency of co-occurrence with nirS than with nirK and the presence or absence of a nor gene largely explained this pattern, as nirS almost always co-occurred with nor. This suggests that nirS type denitrifiers are more likely to be capable of complete denitrification and thus contribute less to N₂O emissions than nirK type denitrifiers under favorable environmental conditions_. Comparative phylogenetic analysis indicated a greater degree of shared evolutionary history between nosZ and nirS. However 30% of the organisms with nosZ did not possess either nir gene, with several of these also lacking nor, suggesting a potentially important role in N₂O reduction. Co-occurrence patterns were also non-randomly distributed amongst preferred habitat categories, with several habitats showing significant differences in the frequencies of nirS and nirK type denitrifiers. These results demonstrate that the denitrification pathway is highly modular, thus underpinning the importance of community structure for N₂O emissions.     

Impacts of varying durations of passive oxygen exposure on freshwater denitrifier community structure and function

Fertilizer use has dramatically increased the availability of nitrate (NO₃ ^?) in aquatic systems. Microbe-mediated denitrification is one of the predominant means of NO₃ ^? removal from freshwaters, yet oxygenation (O₂)-induced disruptions—e.g., extreme precipitation events—can occur, resulting in a disproportional increase in nitrous oxide (N₂O) production and efflux as facultative anaerobic bacterial populations use of O₂ as a terminal electron acceptor increases. We examined the effects of 12- and 24-h passive O₂ exposure on previously anaerobic bacterial communities focusing on denitrification enzyme activity (DEA), N₂O production, and bacterial community 16S rRNA and nitrous oxide reductase gene (nosZ) profiles after 12, 24, and 48 h of anaerobic recovery. Treatments experiencing 24-h O₂ exposure had significantly higher DEA 12 h into anaerobic recovery than treatments undergoing 12-h O₂ exposure. Initial N₂O emissions were significantly lower in the 24-h O₂ exposure treatments although by 24 h a dramatic spike (tenfold relative to the 12-h O₂ exposure treatments) in N₂O concentrations was observed. However, within 6 h (30-h anaerobic recovery) these differences were gone. Community nosZ profiles experiencing 24-h O₂ exposure exhibited reduced diversity after 24-h recovery, which corresponded with an increase in N₂O emissions. However, after 48 h of anaerobic recovery, nosZ diversity had recovered. These observations highlight the effects of short-term aerobic disruption on denitrification, as well as the effects on the denitrifier community profile. Together, these data suggest that recovery to ambient N cycling is exacerbated by disturbance length due to increased lag time and subsequent loss of denitrifier community diversity.     

In-depth analysis of N2O fluxes in tropical forest soils of the Congo Basin combining isotope and functional gene analysis

Primary tropical forests generally exhibit large gaseous nitrogen (N) losses, occurring as nitric oxide (NO), nitrous oxide (N₂O) or elemental nitrogen (N₂). The release of N₂O is of particular concern due to its high global warming potential and destruction of stratospheric ozone. Tropical forest soils are predicted to be among the largest natural sources of N₂O; however, despite being the world’s second-largest rainforest, measurements of gaseous N-losses from forest soils of the Congo Basin are scarce. In addition, long-term studies investigating N₂O fluxes from different forest ecosystem types (lowland and montane forests) are scarce. In this study we show that fluxes measured in the Congo Basin were lower than fluxes measured in the Neotropics, and in the tropical forests of Australia and South East Asia. In addition, we show that despite different climatic conditions, average annual N₂O fluxes in the Congo Basin’s lowland forests (0.97 ± 0.53 kg N ha⁻¹ year⁻¹) were comparable to those in its montane forest (0.88 ± 0.97 kg N ha⁻¹ year⁻¹). Measurements of soil pore air N₂O isotope data at multiple depths suggests that a microbial reduction of N₂O to N₂ within the soil may account for the observed low surface N₂O fluxes and low soil pore N₂O concentrations. The potential for microbial reduction is corroborated by a significant abundance and expression of the gene nosZ in soil samples from both study sites. Although isotopic and functional gene analyses indicate an enzymatic potential for complete denitrification, combined gaseous N-losses (N₂O, N₂) are unlikely to account for the missing N-sink in these forests. Other N-losses such as NO, N₂ via Feammox or hydrological particulate organic nitrogen export could play an important role in soils of the Congo Basin and should be the focus of future research.Subject terms: Microbiology, Biogeochemistry     

Contrasting denitrifier communities relate to contrasting N2O emission patterns from acidic peat soils in arctic tundra

Cryoturbated peat circles (that is, bare surface soil mixed by frost action; pH 3–4) in the Russian discontinuous permafrost tundra are nitrate-rich ‘hotspots'' of nitrous oxide (N₂O) emissions in arctic ecosystems, whereas adjacent unturbated peat areas are not. N₂O was produced and subsequently consumed at pH 4 in unsupplemented anoxic microcosms with cryoturbated but not in those with unturbated peat soil. Nitrate, nitrite and acetylene stimulated net N₂O production of both soils in anoxic microcosms, indicating denitrification as the source of N₂O. Up to 500 and 10 μ nitrate stimulated denitrification in cryoturbated and unturbated peat soils, respectively. Apparent maximal reaction velocities of nitrite-dependent denitrification were 28 and 18 nmol N₂O g_DW⁻¹ h⁻¹, for cryoturbated and unturbated peat soils, respectively. Barcoded amplicon pyrosequencing of narG, nirK/nirS and nosZ (encoding nitrate, nitrite and N₂O reductases, respectively) yielded ≈49 000 quality-filtered sequences with an average sequence length of 444 bp. Up to 19 species-level operational taxonomic units were detected per soil and gene, many of which were distantly related to cultured denitrifiers or environmental sequences. Denitrification-associated gene diversity in cryoturbated and in unturbated peat soils differed. Quantitative PCR (inhibition-corrected per DNA extract) revealed higher copy numbers of narG in cryoturbated than in unturbated peat soil. Copy numbers of nirS were up to 1000 × higher than those of nirK in both soils, and nirS nirK⁻¹ copy number ratios in cryoturbated and unturbated peat soils differed. The collective data indicate that the contrasting N₂O emission patterns of cryoturbated and unturbated peat soils are associated with contrasting denitrifier communities.     

Four years of experimental climate change modifies the microbial drivers of N2O fluxes in an upland grassland ecosystem

Emissions of the trace gas nitrous oxide (N₂O) play an important role for the greenhouse effect and stratospheric ozone depletion, but the impacts of climate change on N₂O fluxes and the underlying microbial drivers remain unclear. The aim of this study was to determine the effects of sustained climate change on field N₂O fluxes and associated microbial enzymatic activities, microbial population abundance and community diversity in an extensively managed, upland grassland. We recorded N₂O fluxes, nitrification and denitrification, microbial population size involved in these processes and community structure of nitrite reducers (nirK) in a grassland exposed for 4 years to elevated atmospheric CO₂ (+200 ppm), elevated temperature (+3.5 °C) and reduction of summer precipitations (?20%) as part of a long‐term, multifactor climate change experiment. Our results showed that both warming and simultaneous application of warming, summer drought and elevated CO₂ had a positive effect on N₂O fluxes, nitrification, N₂O release by denitrification and the population size of N₂O reducers and NH₄ oxidizers. In situ N₂O fluxes showed a stronger correlation with microbial population size under warmed conditions compared with the control site. Specific lineages of nirK denitrifier communities responded significantly to temperature. In addition, nirK community composition showed significant changes in response to drought. Path analysis explained more than 85% of in situ N₂O fluxes variance by soil temperature, denitrification activity and specific denitrifying lineages. Overall, our study underlines that climate‐induced changes in grassland N₂O emissions reflect climate‐induced changes in microbial community structure, which in turn modify microbial processes.